Report on African trypanosomiasis (sleeping sickness) · research agenda for African trypanosomiasis, closely linked to control needs and open to the opportunities that science and

Report on

African trypanosomiasis(sleeping sickness)

4-8 June 2001Geneva, Switzerland

www.who.int/tdr

TDR/SWG/01Original: English

Scientific Working Group

Report of the Scientific Working Groupmeeting on African trypanosomiasis

Geneva, 4-8 June, 2001

TDR/SWG/01

Copyright ©World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases (2003)All rights reserved.

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Message from the Executive Director, CDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Message from the Director, TDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

1 Executive summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Overview and objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3 Epidemiology, disease surveillance and controlEMERGENCE AND RE-EMERGENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Reservoir Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Incidence and Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9SURVEILLANCE AND INTERVENTION FOR CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Changing Institutional Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10SOCIOECONOMIC AND BEHAVIOURAL ASPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4 Drug development, preclinical and clinical studies, and drug resistancePRECLINICAL STUDIESResistance to Arsenicals and Diamidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Role of CNS Trypanosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Drug Discovery and Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16CLINICAL STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Clinical Aspects of Treatment Failure and Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Application of Existing Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Early-stage drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Late-stage drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Other potential drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

New Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Contents

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Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Follow-up of Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Prevention and Management of Encephalopathy Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Coordination of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5 Pathogenesis, genomics and applied genomicsPATHOGENESISBackground . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Trypanosome Biological Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22APPLIED GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Trypanosoma brucei Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 TSETSE GENETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6 Cross-cutting issuesRESOURCE FLOW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27ADVOCACY AND MARKETING FOR SLEEPING SICKNESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28SOUTH-SOUTH COLLABORATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Annex 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31RESOURCE FLOW FOR AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Annex 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33A POSITION PAPER ON AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Annex 3EMERGENCE AND RE-EMERGENCE OF HUMAN AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . 42

I The situation in Angola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43II The situation in Tanzania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45III The situation in Uganda and Sudan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

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Annex 4EPIDEMIOLOGY, DISEASE SURVEILLANCE AND CONTROL, AND VECTOR CONTROL . . . . . . . . . . . 54

I Epidemiology and control of human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . 55II Vector control in relation to human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . 73III A basis for financial decision-making on strategies for the control of

human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Annex 5DRUG DEVELOPMENT, PRECLINICAL AND CLINICAL STUDIES, AND DRUG RESISTANCE . . . . . 90

I Drug development for African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91II Drug resistance in sleeping sickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96III Human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112IV Treatment and clinical studies: Working paper for the Scientific Working

Group on Human African Trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Annex 6PATHOGENESIS, GENOMICS AND APPLIED GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

I Discussion document on pathogenesis/applied genomics . . . . . . . . . . . . . . . . . . . . . . 121II Applied genomics: Prospects for control of African trypanosomiasis via

the tsetse vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140III Applied genomics and bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Annex 7INSTITUTIONAL CAPABILITY STRENGTHENING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163Institutional development and capacity building in countries endemic for sleeping sickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Annex 8 LIST OF PARTICIPANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

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Following the appointment of Dr Brundtland as Director-General of the World HealthOrganization (WHO) in 1998, functional restructuring placed both the control of infectiousdiseases and tropical diseases research under one cluster. This has permitted better identification of priorities and the linking of research with prevention and control activities,resulting in joint fundraising. The recent agreement with the drug industry on chemotherapy ofAfrican trypanosomiasis is a product of such joint activities. It is hoped that other agreementsemphasizing sleeping sickness will also be signed.

In addition, there has been a concerted effort to move infectious diseases higher in theeconomic development agenda in various world economic fora, resulting in political commit-ment by governments of both developed and disease endemic countries, and in financial com-mitment by members of the G8, in particular France and the United States of America, to theGlobal Fund for AIDS and Health. Currently, the Global Fund is directed at the three major(based on morbidity and mortality data) infectious diseases – namely malaria, tuberculosisand AIDS. This will lead to improved health delivery systems that will be better placed to dealwith other diseases such as sleeping sickness. It is envisaged that the health delivery systemswill undergo diversification, allowing governments, NGOs and the private sector to worktogether to deliver health services more effectively. This will be of particular importance fordiseases such as sleeping sickness, where NGOs have had to be depended on for continuedsupport during periods of decreased resources.

This meeting aims to draw up a well thought out research agenda, provide data that can be used to convince policy-makers to place infectious diseases at the forefront of development activities, and show the world that the job on infectious diseases is not yet done.

David L Heymann

World Health Organization

Geneva, June 2001

Message from the Executive Director, Communicable Diseases

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In 1998, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) underwent an external review. It was noted that, with the reorgani-zation of the Programme into functional units, certain aspects of disease focus had been neglected. It was decided, therefore, that TDR should hold scientific working group (SWG)meetings to address each of the ten diseases handled by the Programme. Two of these SWGmeetings, this SWG on African trypanosomiasis being the first, will be held each year in orderthat all ten diseases are covered in five years. It is expected that this first meeting will set aresearch agenda for African trypanosomiasis, closely linked to control needs and open to theopportunities that science and technology can provide, which will act as a guide not only to TDR but also to other parties interested in research on African trypanosomiasis.

Funding through TDR is on the increase, and a full-time disease research coordinator forAfrican trypanosomiasis is to be recruited. This is a reflection of growing donor interest in the disease, for which a significant amount of funding has already been assured.However, the funding available is largely restricted to particular projects and limited in geo-graphical coverage to a small proportion of the 250 known foci of sleeping sickness in Africa.Furthermore, the number and mix of donors is limited, as is the period covered by the currentfunding agreement, which is only for five years. Securing resources for the period followingthese five years is therefore a priority. Strategies to attract more funding will include creatinga supportive environment, joining forces with others of like mind, and using the voices ofaffected countries. The current matrix approach to programme management in TDR lays greatemphasis on accountability and gives a certain amount of funding security to diseases that areunlikely to receive additional funding from alternative sources.

Carlos M Morel

Director, TDR

Geneva, June 2001

Message from the Director, TDR

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During the last ten years, sleeping sickness has only been marginally recognized as a healthproblem and a research priority, but the recent re-emerging outbreaks and increasing drugresistance have painfully demonstrated the potential danger in any endemic area. Althoughsleeping sickness was brought to low endemic levels practically everywhere in Africa duringthe sixties, since then, most national sleeping sickness control programmes have graduallybeen sacrificed in favour of more prominent health needs. As a result, the core of nationalknow-how simply disappeared while dependency on external support was rendered evengreater. Recent outbreaks have opened eyes worldwide as to how serious the situation is, andincreasing awareness is manifest in scientific publications, the layman’s press, and televisionprogrammes. The donor community, as well as industry, has been alerted, and is now provid-ing unprecedented levels of support to both control and research. As this report states, it wasan opportune time to convene a Scientific Working Group.

This TDR Scientific Working Group was asked to provide technical guidance to donors,responsible officers at national level, and research institutions as to the concrete directionsresearch should take, in the Group’s opinion, and where resources should be made available.The relatively large group of approximately 30 participants permitted wide geographical rep-resentation and ample coverage in terms of scientific discipline. In order to guarantee that therecommendations issuing from the meeting were critical, the SWG decided to restrict these tothe two of highest priority for each subsection of the report.

This report briefly reviews the recent emergence and re-emergence of trypanosomiasis, andelaborates on as yet unanswered questions and issues such as the role of animal reservoirs inT. b. gambiense transmission, the need for epidemiological indicators for monitoring controlinterventions, and the lack of a “gold standard” for new and existing diagnostic tests. Oneresearch area pertinent to the identification of the reservoir of infection and transmission pat-terns would be the improvement and validation of the current bloodmeal analysis technology.New tools for vector control per se are not considered a priority at this moment – “the toolbox is full” – but research into the question of why relatively little use is being made of thecurrently available tools is indicated.

In the past, practically everywhere, trypanosomiasis control was in the hands of vertical pro-grammes, which meant a great deal of adaptation was needed, both mentally and logistically,to integrate personnel and equipment into the general health services. Although in a few coun-tries this took place gradually and more or less satisfactorily, in countries with a large num-ber of endemic foci and where large numbers of personnel and facilities were involved, inte-gration was, and remains, a complicated process. In such cases, local studies on new deliverypathways are urgently needed.

Preface

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The disability adjusted life year (DALY) has finally found grace in socioeconomic reasoningand is now being applied in several economic analyses for trypanosomiasis control. However,the data collected so far indicate that further refinement is needed to be able to compare thecost-effectiveness of the different control approaches. Also, little is yet known of the personalcosts a patient spends on seeking care, his/her participation in cost sharing, or the costs ofdeath and incapacity for the family.

The arsenal of trypanocidal drugs, being scarce already, is becoming more and more limiteddue to increasing drug resistance. As an immediate relief measure, applied research to helpdevelop standard protocols for optimal use of combinations of currently available compoundsis needed. As a long-range objective, the highest priority should be given to laboratoryresearch to develop new molecules.

When Mott published, in 1899, his classic report demonstrating perivascular infiltration to bethe main histopathological characteristic of sleeping sickness, he probably would not haveimagined how, a good hundred years later, specialists would still be busy following up thepathogenesis of these brain lesions. Although only a few research groups have been involvedup till now, this field of interesting research is relevant to the possible development of non-invasive tests for determining the stage of disease. Pathogenesis research could also lead toprevention and treatment of the fatal reactive encephalopathies which occur during treatment.

Much attention has been paid in this report to genomics as a means of refining parasite identi-fication, and to genetics for studies on the vectorial capacity of tsetse flies. The T. bruceigenome network of collaborating centres, initiated by TDR jointly with other institutions, isexpected to strengthen functional links at an international level. Emphasis is placed onstrengthening laboratories in Africa for participation in the work on bioinformatics andgenomics.

As a result of the lack of career openings, a high proportion of ex-trainees has left the try-panosomiasis field. One of the recommendations of the Group is to improve the position oflocal scientists in African research institutes by allowing for salary supplements. Makingresearch careers more attractive and stable should eventually result in establishing an accept-able core of scientists in the endemic countries. Although the drain of specific knowledge andexperience to elsewhere has been disappointing, the Group has no doubt that continued train-ing is the only solution to filling the gap in availability of scientists and technicians in endemiccountries.

Considering the improved funding perspectives for both control and research, it is now oppor-tune to make efforts to enhance collaboration between the two. In order to be able to generate

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valid disease data from each endemic country, it is recommended that a research nucleusgroup be identified and that inter-country collaboration and comparison of results betweenendemic countries be strongly advocated.

With this up-to-date review of the present epidemiological situation and the actual controlneeds, the Scientific Working Group calls for a response from the scientific community all overthe world. The improved funding possibilities that exist at the present time provide the neces-sary momentum for introducing new research and control projects and for strengthening rele-vant ongoing programmes.

Peter de Raadt,

SWG Chairperson

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The Scientific Working Group (SWG) meeting

on African trypanosomiasis brought together a

multidisciplinary group of scientists, partners

and collaborators from academia, public and

private sectors, sleeping sickness control pro-

grammes, and both disease endemic and disease

non-endemic countries. The objectives of the

meeting were to chart out a global research

agenda on African trypanosomiasis, closely

linked to control needs and open to opportunities

arising from basic science, to guide TDR and

other parties interested in research on African

trypanosomiasis and provide data that can be

used in advocacy to convince policy-makers and

donor agencies to place control of this disease

higher on their agenda.

The Group reviewed the current status of know-

ledge and made recommendations on what tools

are needed for appropriate and effective manage-

ment and control of sleeping sickness. Three

broad areas were considered:

• Epidemiology, disease surveillance and

control.

• Drug development, preclinical and clinical

studies, drug resistance.

• Pathogenesis and applied genomics.

Considerable progress has been achieved in

research on African trypanosomiasis in the fol-

lowing areas: diagnosis and development of

diagnostic tests; epidemiology, host-parasite-

vector relationships, animal reservoirs; develop-

ment of tsetse traps and screens; understanding

of the pathology of the disease; and, drug target-

ed biochemistry of trypanosomes. However, this

progress has not been matched in control of the

disease, due to lack of capacity to sustain

improved interventions and to civil disorder in

some endemic countries.

It was noted that recent scientific advances in

applied genomics and bioinformatics provide

opportunities that can be exploited to provide

new tools for control of disease, and recent sup-

port from the pharmaceutical industry and a pri-

vate foundation has given impetus to tackling the

problem of African trypanosomiasis. However,

the challenges of obtaining adequate donor sup-

port and commitment of governments of endemic

countries, and of personnel recruitment and

retention, are daunting. Research on African try-

panosomiasis is an international effort and the

need for partnerships cannot be overemphasized.

The meeting provided an opportunity to identify

knowledge that could be exploited for developing

new, and improving existing, tools for manage-

ment of disease and vectors, and to determine the

needs for research capability strengthening in

basic sciences in disease endemic countries.

TDR’s comparative advantage in enhancing exist-

ing, and developing new partnerships for maxi-

mal application of knowledge was highlighted.

The following are the highest priority recommen-

dations made by the SWG. The SWG:

• noted with concern that sleeping sickness is a

re-emerging disease which is not given due

attention by governments of endemic countries

or the international donor community until it

attains epidemic proportions, and recommend-

ed that the disease burden and cost effective-

ness of control strategies be calculated to show

that the social and economic consequences of

epidemics outweigh the cost of maintaining

surveillance;

• noted that lack of appropriate field applicable

diagnostic tools for disease detection, and

stage of disease, critically affect the control of

sleeping sickness, and recommended that sim-

ple non-invasive, single-format, field-applica-

ble tests for diagnosis and determination of

stage of disease be developed and validated;

1 Executive summary

Report of the Sc ient i f i c Working Group on Af r i can t rypanosomias is, 2001 • TDR/SWG/012

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• considered the small number of anti-try-

panosomal drugs available, and recommended

that synthetic and natural product libraries be

integrated for use in drug development;

• acknowledged that the development of drugs

for late-stage disease is hindered by the blood-

brain barrier, which prevents the delivery of

drugs to the central nervous system (CNS),

and recommended that CNS-penetration mod-

els be used in drug development strategies for

human African trypanosomiasis, and that

strategies which facilitate the delivery of drugs

across the blood brain barrier be developed;

• took into account the limited evidence suggest-

ing that combination therapy with late-stage

drugs has an additive effect, and, in view of

the urgent need to have alternative treatments

for melarsoprol refractory patients, recom-

mended that combination chemotherapy using

late-stage drugs be optimized;

• acknowledged the difficulties associated with

the treatment of sleeping sickness patients, and

the lengthy post-treatment follow-up, and rec-

ommended investigating and applying new

information on immune parameters to (i) the

determination of stage of disease, (ii) the pre-

vention and/or amelioration of the encephalitis

and encephalopathy associated with the dis-

ease and its treatment respectively, and (iii) the

development and validation of a non-invasive

protocol for determining cure and shortening

the duration of after-treatment follow-up;

• expressed concern that the limited number of

suitable centres in Africa created a situation

where research was increasingly compartmen-

talized, and recommended that the capacity of

laboratories/centres within Africa be strength-

ened in the basic sciences, including in bioin-

formatics, genomics and applied genomics,

drug discovery and development;

• noted the possible co-existence of T. b. rhode-

siense and T .b. gambiense sleeping sickness

patients in foci where refugees are settled,

the difficulties in differentiating the two

trypanosome species, and the different treat-

ment schedules for both forms of the disease,

and recommended that genomics be applied

to comparing T. brucei sub species, strains and

life cycle stages for their differentiation and

disease management;

• appreciated the important role that vector

control plays in reducing the transmission of

vector borne diseases, and recommended that

tsetse-trypanosome interactions be investigated

to determine the molecular basis of refractori-

ness for trypanosome transmission and mecha-

nisms for driving desirable genes into vector

populations;

• noted with concern the absence, within TDR,

of a full-time staff member responsible for

research activities on African trypanosomiasis,

and recommended recruitment of such a

person;

• recognized the inadequacy of infrastructure for

research in different endemic countries, and

recommended networking and cross country

comparison of research progress to assist in

capacity building and stimulate cross border

interest and advocacy;

• noted with concern the low priority given to

African trypanosomiasis by governments of

endemic countries, and recommended strong

advocacy to persuade disease endemic country

governments to accord priority attention to

research and control of African trypanosomia-

sis amidst their other health priorities.

Other high priority recommendations, with sug-

gestions for studies and/or actions that should be

undertaken to meet the objectives of the meeting,

are listed in the text.


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African trypanosomiasis is caused by protozoan

parasites, trypanosomes, which are transmitted

by tsetse flies (of the genus Glossina). The dis-

ease occurs in two forms: a chronic form caused

by Trypanosoma brucei gambiense, which occurs

in West and Central Africa; and an acute form,

caused by T. b. rhodesiense, which occurs in

Eastern and Southern Africa. The chronic infec-

tion lasts for years, whilst the acute disease may

last for only weeks before death occurs, if treat-

ment is not administered.

The epidemiology of sleeping sickness is com-

plex and transmission cycles are subject to

interactions between humans, tsetse flies and

trypanosomes, and signif icantly, in T. b.

rhodesiense sleeping sickness, domestic and

wild animals. In T. b. gambiense disease, the

classical human-fly-human transmission cycle

occurs in both endemic and epidemic situations.

Sleeping sickness is a re-emergent disease,

but does not get due attention, probably because

its impact is regional. The disease occurs in

36 sub-Saharan countries, within the area of

distribution of the tsetse fly. Over 60 million

people living in some 250 foci within this region

are at risk of contracting the disease (see figure 1).

2 Overview and objectives

Figure 1 - The focal distribution of human African trypanosomiasis

Left of dotted line: Trypanosoma brucei gambiense foci in West and Central Africa

Right of dotted line: T. b. rhodesiense fociin Eastern and South Africa

Source: WHO picture library


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The number of cases reported annually is over

40 000, but this is highly underestimated, due

to difficulties in diagnosis and remoteness of

affected areas. It has been estimated that the

actual number of cases is at least 300 000, the

vast majority of whom are not diagnosed or

treated (WHO 1998). These figures are relatively

small compared to other tropical diseases, but

African trypanosomiasis, without intervention,

has the propensity to develop into epidemics,

making it a major public health problem.

Furthermore, the case fatality rate in untreated

patients is 100%. This fact, combined with the

focal nature of the disease, means that the dis-

ability adjusted life years (DALYs) averted per

infection cured or prevented are very high. As a

result, control of this disease in areas at risk is

highly cost-effective, falling well below the

accepted threshold value for money of US$25

per DALY averted (Dr A. Shaw, personal

communication, see attached working document,

Shaw and Cattand, Annex 4 section III).

At the beginning of the last century, huge sleep-

ing sickness epidemics devastated large areas of

the continent. In the 1960s, the prevalence of the

disease had been successfully reduced to less

than 0.1% in all endemic countries, through his-

toric campaigns by the former colonial powers.

Soon after independence, however, national gov-

ernments failed to sustain such programmes due

to lack or diversion of resources to other more

pressing health problems. Breakdown of special-

ized mobile teams and health facilities in several

countries, as a consequence of war and civil

strife or change in health policy, resulted in dra-

matic resurgence of the disease, the distribution

of which corresponds closely with that of major

conflicts in sub-Saharan Africa.

The social and economic impact of sleeping

sickness is often underestimated. During epi-

demics, large proportions of communities are

affected, with loss of life and untold suffering.

These have serious social and economic conse-

quences, which far outweigh the cost of main-

taining surveillance. The disease has been a

major cause of depopulation of large tracts of

Africa. The fear it causes has led to abandon-

ment of fertile lands, and is an impediment to

development. Some affected countries have agri-

culture-based economies, and workers in cocoa

and coffee plantations are always at risk of con-

tracting the disease, consequently decreasing the

labour force. This is reinforced by the fact that

the disease mainly strikes the active adult popu-

lation.

Regular medical surveillance, involving accurate

case detection and appropriate treatment, and

tsetse control where applicable, is the backbone

of the strategy for the control of sleeping sick-

ness (WHO, 1998). With the available tools, con-

trol is a continuing effort rather than eradication.

Experience has shown that where control is inter-

rupted, for a variety of reasons, resurgence of the

disease occurs sooner or later.

Over recent years, human trypanosomiasis has

been the subject of renewed interest among the

donor community and scientists. Substantial vol-

untary contributions have been made by Belgium

and France for research and control in the

endemic countries, as well as by the pharmaceu-

tical industry, but these contributions only partly

cover the current needs, and the number of

donors is still very limited. Nongovernmental

organizations (NGOs) have clearly committed

efforts to participate in control. Special articles

on the trypanosomiasis problem have appeared


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in both the scientific press and public media

(Figaro, June 2001; New York Times, 9 February

2001).

The objectives of the scientific working group

(SWG) were to:

• Identify areas where there are gaps in knowl-

edge, and studies that are necessary to fill

these gaps.

• Identify research that is directly relevant to

control programmes and treatment centres as a

priority.

• Promote development of new tools for control

and new methods for use of old tools, and

effective approaches to disease control.

• Set objectives for research capability strength-

ening for basic science, genomics and applied

genomics, drug discovery and development in

disease endemic countries.

ReferencesWHO Expert Committee on Control and Surveillanceof African Trypanosomiasis. Geneva, World HealthOrganization, 1998 (WHO Technical Report Series,No. 881).


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The persistence and re-emergence of sleeping

sickness in Africa is attributable to various

factors including lack of surveillance, shortage

of drugs, and several other determinants which

operate at different levels. Means for regular

surveillance are often inadequate. At the individ-

ual and family levels, there may be inadequate

knowledge about disease symptoms, transmis-

sion dynamics and treatment. Population move-

ments, such as seasonal migration and refugees,

may increase human-fly contact and hinder

regular medical surveillance of the population

at risk. In rhodesiense sleeping sickness, cattle

movements also increase the risk of infection.

Agro-ecological changes may alter tsetse habitat

and increase human-fly contact. A significant

resurgence of the disease has occurred, notably

in Angola, the Democratic Republic of the

Congo, Sudan and Uganda. New foci have also

emerged in recent years.

3 Epidemiology, disease surveillance and control

Sleeping sickness Country 1995 1996 1997 1998 1999 2000

Angola 6 786 8 275 7 373 5 351 4 546

D.R. Congo 18 158 19 342 25 200 26 318

Sudan 157 737 1 800 18 684 16 975

N.W. Uganda 1 062 980 1 069 967 1 020

S.E. Uganda 497 271 287 299

Tanzania 400 531 421 588 627

T. b. gambiense

T. b. rhodesiense

Table 1 - Number of sleeping sickness patients treated by year per country

Source: Compiled from the working documents of the various countries.

Ministries of health, research organizations

and services often lack, or do not have, adequate

economic resources for sleeping sickness

control programmes due to competing health pri-

orities. Recruitment of medium-level personnel

is inhibited by lack of incentives and career

prospects. Ministries may lack funds for the

purchase of diagnostic tests and drugs, except

as part of externally-funded programmes.

Other factors that increase the risk of infection

and human-fly contact include agricultural devel-

opment such as coffee and cocoa plantations, and

the tourism industry.

Central governments often accord sleeping

sickness a low priority, until it assumes epidemic

proportions. In addition, political upheavals, civil

strife and wars lead to the breakdown of health

services and of control programmes.

The international community is prepared to

mobilize resources when epidemics occur but

is often unable to provide long-term support

for surveillance and preventive measures in

endemic situations. Until recently, the continued

production of anti-trypanosomal drugs was in

question. This problem has been resolved for

the next five years through generous donations

EMERGENCE AND RE-EMERGENCE


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Reservoir hosts play an important part in sus-

taining endemicity and in the re-emergence of

epidemics. In the case of T. b. rhodesiense

sleeping sickness, several wild and domestic

animal reservoir hosts have been identified and

certain outbreaks brought under control by treat-

ing nearby cattle. Although natural infections

with T. b. gambiense have been reported in

domestic animals such as pigs, dogs, sheep and

cattle, and may occur in wild animals as well,

the role of an animal reservoir in the epidemiol-

ogy of T. b. gambiense remains as yet undeter-

mined. Another important group of reservoir

hosts, especially in T. b. gambiense epidemiolo-

gy, are the human carriers: infected individuals

who remain undiagnosed due to inadequate sur-

veillance and/or the limitations of current diag-

nostic tools in demonstrating low levels of try-

panosome infections. The following areas for

research were identified:

• Assessment of the epidemiological sig-

nificance of an animal reservoir for

gambiense sleeping sickness using new

approaches such as the molecular

approach.

• Assessment of the epidemiological and

clinical significance of “unconfirmed

cases”, defined as individuals with clin-

ical signs, or as cases where indirect

evidence of infection such as sero-posi-

tivity or polymerase chain reaction

(PCR)-positivity exists but where the

parasite cannot be demonstrated by

microscopy.

EPIDEMIOLOGY

Reservoir Studies

by pharmaceutical companies. However, arrange-

ments need to be made to ensure a sustained

supply beyond this period.

In order to ensure that research results are com-

parable over a wide area, advantage should be

taken of the potential of networking (for more

general data collection) and using consortia of

investigators, as appropriate, including public/

private partnerships. Avenues should be explored

as to how to make better use of existing

networks, such as the Programme Against

African Trypanosomiasis (PAAT) managed by

the Food and Agriculture Organization (FAO),

Organization of African Unity (OAU) and

WHO, and new networks, such as the WHO

surveillance network, should be supported.


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To design optimal control strategies and

estimate the burden of disease, knowledge of

current prevalence and the impact of control

measures is required. This could be obtained by:

The validation of diagnostic tests is needed

to obtain more accurate estimates of the real

disease prevalence.

• Standardizing and extending reporting

systems using geographic information

systems (GIS).

• Collating and analysing existing

epidemiological data on the impact of

past control schemes on prevalence.

• Introducing a standardized protocol

for monitoring the impact of control

measures on epidemiological indicators.

• Epidemiological modelling to better

understand and predict the disease’s

behaviour, e.g. to obtain better estimates

of the ratio of unreported to reported

cases, or of the basic reproductive

number, Ro.

Currently, the screening tests used are only avail-

able in bulk presentation, which limits their use

where small numbers of people or individuals

need to be tested, e.g. in rural health centres, for

surveillance on a small scale. There is a need for

further development of simple, field applicable,

single test formats (such as dipsticks), with high

specificity. A multicentre validation of diagnos-

tics based on molecular techniques (e.g. PCR)

for epidemiological and clinical studies is strong-

ly recommended. The need for field applicable,

non-invasive diagnostic methods is recognized,

especially to avoid lumbar punctures.

The main constraint to the development and vali-

dation of diagnostic tests is the lack of a gold

SURVEILLANCE AND INTERVENTION FOR CONTROL

Diagnostics

Incidence and Prevalence


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The monitoring protocols recommended above

should be applied here.

standard. Statistical methods exist for the evalua-

tion of diagnostic tests in the absence of a gold

standard, but so far these have not been applied

to the diagnosis of human African trypanosomia-

sis. It is recommended that the use of these

methods should be explored.

For stage determination and follow-up, the

following investigations should be undertaken:

Vectors

A range of tools for the control of tsetse flies

have been developed over the last twenty years,

some of which can be applied by communities.

However, their adoption and application by com-

munities in endemic areas has not been sustain-

able. The exact role of the vector in relation to

transmission of the disease from the various ani-

mal reservoir hosts is unclear. The following

research areas were identified:

Changing Institutional Environment

Far-reaching institutional changes have occurred

during the last decade which have had a major

impact on the organization and implementation

of sleeping sickness control. These include

decentralization, integration of human African

trypanosomiasis (HAT) control into the primary

health care (PHC) system, cost recovery/sharing,

and varying degrees of community involvement

in surveillance and control. Capacity building in

policy and health systems, to help identify and

remedy the changes that have adversely affected

disease control, is recommended. The following

areas of research were identified:

• Multicentre validation of latex/IgM

and PCR tests on cerebrospinal fluid.

• Identification of new markers of

neuropathogenesis, e.g. cytokines.

• Evaluation of the use of antigen

detection tests for follow-up.

• Evaluation of the impact of changes in

delivery pathways for sleeping sickness

control on the effectiveness of control

measures.

• Investigation of the effects of changes

in cost-sharing arrangements on disease

detection rates, compliance, and effec-

tiveness of control.

• The constraints affecting sustained use

of vector control tools by affected com-

munities.

• Comparison, validation and improve-

ment of available tools for blood-meal

analysis.

Controlling sleeping sickness is a highly cost-

effective intervention, with the cost per DALY

comparing very favourably with other health

interventions, and falling well below the

accepted value of US$25 per DALY averted

(Dr A. Shaw, personal communication). This

reflects the focal nature of the disease, and the

fact that the case fatality rate in untreated

patients is 100%. Although the funding situation

has improved somewhat, and greater awareness

of sleeping sickness as a public health priority

exists, it is vital to reinforce and extend this by

generating appropriate socioeconomic informa-

tion in order to:

• Determine the financial resources that are

required for control.

• Choose the most appropriate and cost-effec-

tive control strategies.

• Promote advocacy through better understand-

ing of the economic burden of the disease.

• Provide guidelines for allocating resources

amongst competing health needs.

It is recommended that social science research

address the following issues:

When combined with the epidemiological infor-

mation outlined in the section above (on epi-

demiology), such studies would enable the bur-

den of disease to be estimated and the cost-

effectiveness of different interventions to be cal-

culated and compared.


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• Costing of the different control strategies

to cover both endemic and epidemic sit-

uations, NGO and national programmes,

and a range of countries.

• The direct and indirect costs to patients

and their families of obtaining diagnosis,

treatment, hospitalization, and follow-up

examinations, as well as the costs of

permanent disability.

• Refinement of the work done so far on

calculation of DALYs, and its extension

to other settings.

• Clarification of issues influencing

community and individual support of,

and involvement in, control measures

including:

- The development of approaches for

enhancing and sustaining community

participation in the control and surveil-

lance of sleeping sickness in endemic

areas.

- The possible existence of gender issues

in the diagnosis and treatment of sleep-

ing sickness, focusing on knowledge,

practice and health care seeking patterns.

SOCIOECONOMIC AND BEHAVIOURAL ASPECTS


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RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority

• Development and validation of non-invasive, field-applicable, single-test format diagnostics

tests, including tests for disease-stage determination.

• Calculation of burden of disease and cost-effectiveness of control strategies.

High priority

• Assessment of the epidemiological and clinical significance of ‘unconfirmed suspects’.

• Systematic monitoring of disease incidence and prevalence, especially in relation to control

measures.

• Identification of issues influencing individual and community participation in control

measures.


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The frequency and extent of use of the standard

drugs against African trypanosomiasis, melarso-

prol and pentamidine, is likely to lead to the

development of resistance. Indeed, there has

been an increase of late-stage cases refractory

to melarsoprol treatment in the past decade

(Legros et al, 1999). The availability of these

agents, and of eflornithine (DFMO), was not

assured until recently. Even now, it is only

assured for the next five years. With the excep-

tion of a new pentamidine-related drug (DB

289), which is due to enter phase II clinical

trials in 2001, no new candidate agents are

currently in the advanced stage of development.

All the drugs are expensive and require hospital-

ization for administration by the parenteral

route. In addition, melarsoprol is associated

with a 10% incidence of reactive encephalopa-

thy that is fatal in up to 5% of the victims.

Therefore, there is an urgent need for novel,

safe, rapidly-acting and inexpensive agents for

the treatment of human African trypanosomiasis

(HAT) in the 21st Century.

During the late stages of African trypanososmia-

sis, parasites lodge in privileged sites within the

central nervous system, causing encephalitis.

The biological nature of such parasites, the

mechanisms by which they, and the drugs that

cure late-stage infections, cross the blood-brain

barrier, are unknown. There is evidence that

eflornithine acts additively with non-permeating

drugs in late-stage infections and suppresses the

encephalitis. The nature of these drug interac-

tions is not known but understanding them is

critical to the development of new approaches to

chemotherapy.

In the past decade, drug discovery has proceed-

ed along biochemical target-based approaches

with little success. But with the advent of com-

binatorial chemistry and the sequencing of the

trypanosome genome, new techniques can now

be applied to the development of novel, specific

and non-toxic agents for HAT. In addition, to

foster continuation of research and development

of new drugs after the end of the currently

assured five-year period, capacity strengthening

of laboratories and/or centres within Africa for

drug discovery and development is recommend-

ed.

Resistance to Arsenicals and Diamidines

Laboratory studies have identified the P2

amino-purine transporter as one route of entry

into trypanosomes for both melarsoprol and

pentamidine. Loss or alterations to this trans-

porter, caused by genetic modifications to the

TbAT1 gene, can contribute to the development

of resistance. However, other transporters have

been implicated in the uptake of pentamidine,

and possibly other analogues, since parasites

lacking the P2 transporter have been shown to

be sensitive to pentamidine. Moreover, geneti-

cally modified parasites, lacking the TbAT1

gene, are only marginally less sensitive to

melarsoprol than wild type trypanosomes.

Treatment failure with melarsoprol may also be

attributed to the level of melarsoprol permeating

into the cerebrospinal fluid (CSF), which varies

considerably between individuals. Concentra-

tions of this drug within the central nervous

system (CNS) are close to the minimum

inhibitory concentration (MIC) of the drug

against T. b. gambiense. Hence, modest increas-

4 Drug development, preclinical and clinical studies and drug resistance

PRECLINICAL STUDIES


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es in MIC values in parasites can increase the

proportion of late-stage patients showing refrac-

toriness to treatment with melarsoprol.

Therefore, the tripartite relationship between

drug, host and parasite, and cross-resistance

between veterinary trypanocides, e.g. dimi-

nazene aceturate, and HAT drugs, needs consid-

eration in determining treatment failure in the

field.

Three areas of research are recommended:

• Identification of modes of uptake, and

of action and mechanisms of resistance

to arsenicals and diamidines in veteri-

nary and human use. The influence of

both parasite and host factors on treat-

ment failure requires consideration.

• Comparison of the mechanisms under-

lying treatment failure in field isolates

of the parasite with the mechanisms

underlying resistance in laboratory

strains of the parasite in which resist-

ance has been induced.

• Reliable diagnosis of drug resistance in

the field.

Blood-Brain Barrier

The development of therapeutic drugs for the

treatment of late stage HAT is limited by the

blood-brain barrier (BBB) that prevents the free

distribution of drugs into the CNS. An essential

component in the development of drugs for the

treatment of late stage HAT is to design or

select compounds that can cross the BBB. This

approach has been a priority in the development

of drugs for the treatment of Alzheimer`s dis-

ease, brain tumours and neuro-psychiatric con-

ditions, where novel in vitro methods to screen

for compounds that cross the BBB or deliver

drugs across the BBB have been utilized. It is

recommended that the relevance of these

approaches in the treatment of HAT be investi-

gated and applied as the first approach in the

drug screening pathway, followed by more

focused animal model studies.

The following investigations are recommended:

• The use of models of CNS penetration

(BBB models) for inclusion in the HAT

drug development pathway, in particu-

lar:

- In silico models that use the physico-

chemical properties of compounds to

determine which are most likely to

distribute from the blood to the brain

(primary screen).

- In vitro cell culture models (e.g.

MDCK and endothelial cells) that

enable the study of permeation of

drugs through a cell layer that has

"tight" junctions (secondary screen).

• Strategies that facilitate the delivery

of drugs across the blood-brain barrier

in animal models using known

trypanocides, including:

- A bradykinin agonist (RMP-7,

Cereport) that is on clinical trial for

delivery of anticancer and antiviral

drugs into the brain.

- The role of drugs that modulate the

CNS inflammatory response (for exam-

ple, DFMO and azathioprine) on drug

access and activity in animal models.


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Role of CNS Trypanosomes

Drug action and efficacy depend on the physio-

logical/metabolic state of the trypanosome, which

may be different for stages in the CNS and CSF

from those in the bloodstream. Metabolically

inactive and non-dividing forms tend to be less

sensitive to drugs, and, depending on the mode of

action of the drug, can even be completely insen-

sitive. Apart from parasites in the brain and CSF,

trypanosomes in other tissue niches that are less

accessible to the drugs, must be taken into

account. Research therefore should be directed at:

• Development of suitable models for stud-

ies on CNS and CSF trypanosomes,

including in vitro models and animal

models.

• Studies on the biology of CNS and CSF

parasites, their localization and means of

entry from the vascular sites, and their

inter-exchange.

• Studies on the metabolic state, and

susceptibility to drugs, of CSF and CNS

trypanosomes.

Drug Discovery and Drug Targets

While agencies funding scientific research devote

considerable resources to carrying out research

on the basic biology of trypanosomes, limited

funding goes to drug discovery and development

projects. Likewise, drug discovery efforts by the

pharmaceutical industry, directed specifically

towards new agents for the treatment of HAT, are

nearly non-existent. A wealth of knowledge about

the biology of the organism, and the promise of

new drug targets resulting from genome research,

will have little impact on the discovery of new

drugs without support for the synthesis and test-

ing of new molecules. The use of contemporary

drug discovery methods should be encouraged to

find new molecules for the treatment of HAT.

These methods should follow current drug dis-

covery practices utilized by the pharmaceutical

industry. Building facilities and training scientists

to carry out drug discovery and development

research in endemic countries should be strongly

encouraged.

The following areas of research are recommended:

• Drug discovery efforts which integrate

synthetic and natural product libraries

with structure-based modelling, computa-

tional chemistry and high-throughput

screening (HTS) both for efficacy and

toxicity. Target based libraries will initial-

ly require specific biochemical assays

while diverse synthetic and natural prod-

uct libraries will require whole cell

assays.

• High-throughput screening of synthetic

combinatorial libraries designed based on

existing leads or obtained from existing

combinatorial libraries in pharmaceutical

companies.

• Investigation of target enzymes or path-

ways which, when inhibited, render para-

sites non-viable. Examples of such tar-

gets are S-adenosylmethionine decar-

boxylase, myristate metabolism and vari-

able surface glycoprotein (VSG) synthe-

sis, and farnesyltransferase.

• Identification, validation by genetic

manipulation, and production of new tar-

gets based on exploitation of information

from the genome project. Validated tar-

gets should enter HTS screens.


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RECOMMENDATIONS


Highest priority

• Drug discovery efforts which integrate synthetic and natural product libraries with structure-

based modelling, computational chemistry and high-throughput screening, and whole cell

assays, both for efficacy and toxicity.

• Investigation of the use of models of CNS penetration for inclusion in the HAT drug devel-

opment pathway, and development of strategies that facilitate the delivery of drugs across the

blood-brain barrier.

High priority

• Characterization of modes of uptake and action of melarsoprol and diamidines, and identifi-

cation of mechanisms of treatment failure with these drugs in the field.

• Studies on the biology of CNS and CSF parasites, their localization and means of entry from

the vascular site, and their inter-exchange.


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Clinical Aspects of Treatment Failureand Monitoring

Treatment failures with melarsoprol have been

observed in up to 30% of patients in some foci

in north-western Uganda, southern Sudan and

northern Angola (Legros, 1999). The WHO

coordinated Sleeping Sickness Treatment and

Drug Resistance Network is conducting sentinel

surveillance for treatment failures. Reasons

underlying treatment failures should be investi-

gated.

Application of Existing Drugs

Early-stage drugs

1. PentamidineRecent data from pharmacokinetic studies

suggest that the half-life of pentamidine is suff-

iciently long to allow shorter treatment regimens.

2. Suramin The efficacy of a shorter course for the treatment

of early stage T. b. rhodesiense should be

explored.

Late-stage drugs

1. MelarsoprolCurrently, the 10-day (concise) melarsoprol

regimen is being reviewed in 17 centres in

7 countries. A full report is expected at the end

of 2002.

Preclinical and clinical evaluation of the concise

melarsoprol regimen for treatment of T. b.

rhodesiense infections is recommended.

The possibilities of a new formulation for melar-

soprol should be explored in order to avoid the

adverse extravascular effects at the site of admin-

istration caused by the currently used solvent

propylene glycol. CNS penetration of new for-

mulations should be equivalent or superior to the

current formulation.

2. EflornithineThe ongoing pharmacokinetic study of oral

eflornithine monotherapy should be completed,

and the results used in planning further develop-

ment. In addition, development of a new route

of synthesis should be considered in view of the

technical difficulties with the current route.

3. NifurtimoxNifurtimox is being used on a compassionate

basis for the treatment of melarsoprol refractory

cases. However, it is not registered for treatment

of sleeping sickness. Complete development of

the drug up to registration is recommended.

Currently available data suggest that nifurtimox

may be more suitable for use in combination

than as monotherapy.

Other potential drugs

1. Diminazene aceturateDiminazene aceturate has been used by sleeping

sickness control programmes in several coun-

tries, but has not been registered for human use.

Development for human use as well as an oral

preparation should be considered.

2. BenznidazoleLimited experimental data suggest that

benznidazole has some activity against strains

of T. brucei. It has an established safety record

in humans in the treatment of American

trypanosomiasis (Chagas disease). It should be

considered as a possible alternative compound

for use in combination therapy of sleeping

sickness.

New Compounds

1. DB 289An international consortium is conducting clini-

cal research to develop DB 289 for oral use

against first-stage sleeping sickness. Phase I tri-

als have been concluded and a Phase IIa (proof-

of-principle) trial is planned. Results of the

investigations will be made available to TDR.

CLINICAL STUDIES


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2. MegazolLimited studies in animal models suggest that

megazol is effective as monotherapy in first-

stage sleeping sickness and in combination with

suramin in second-stage T. gambiense infections.

Toxicity studies should be completed. Should the

data from these studies be favourable, further

investigations on the efficacy of monotherapy in

first- and second-stage disease, using appropriate

animal models, should be carried out.

Combination Therapy

There is experimental and limited clinical evi-

dence suggesting that combinations of presently

available late-stage drugs, melarsoprol, eflor-

nithine and/or nifurtimox, act additively

(Jennings, 1988, 1993). The current simultaneous

availability of these drugs gives an unprecedent-

ed opportunity to establish optimal combination

treatment regimens. Although there are indica-

tions that the early-stage drug suramin, in combi-

nation with several other compounds, can cure

sleeping sickness, priority should be given to

studies of combinations of late-stage drugs.

In view of the urgent need to have available alter-

native treatments for melarsoprol refractory

patients, short-term as well as longer-term solu-

tions are needed. To identify alternative treatment

for melarsoprol-refractory patients, the following

clinical trials are currently being conducted:

• Combination of melarsoprol and nifur-

timox vs. monotherapy with each of the

drugs. Preliminary results show that the

combination therapy with low-dose con-

secutive melarsoprol combined with

short-duration nifurtimox was superior to

monotherapy with either melarsoprol or

nifurtimox.

• Trials comparing melarsoprol and nifur-

timox, melarsoprol and intravenous (iv)

eflornithine, iv eflornithine and nifur-

timox, have recently started.

In the longer term, combination treatment regi-

mens should be optimized (i.e. maximum effica-

cy, minimum toxicity, shortest possible duration,

simplicity, with preferably oral administration,

and minimal cost). Further pharmacological and

preclinical investigations are necessary, including

experimental studies on the optimal proportions

of drugs in combination treatment regimens.

Those investigations should be followed by

appropriate clinical trials, preferably including

pharmacokinetic data collection. Oral eflor-

nithine would be preferred for combination ther-

apy as the current complicated iv regimen of

eflornithine is not suitable for large-scale treat-

ment in rural areas. Drug combinations with

melarsoprol should be based on the concise

melarsoprol treatment regimen.


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Follow-up of Treatment

One of the major problems of clinical trials and

case management is the long two-year follow-up

period, requiring multiple lumbar punctures.

Appropriately planned studies to determine the

interval between treatment and relapses, which

may allow a reduction of the follow-up period,

are recommended. Research on less invasive

markers for stage determination and cure should

be given high priority, with emphasis on their

applicability in the field.

Prevention and Management ofEncephalopathy Syndromes

Encephalopathy syndromes, which occur during

administration of late-stage drugs, are a major

problem in the treatment of sleeping sickness.

Research into the underlying mechanisms is criti-

cal for the development of improved strategies

for prevention and management of the complica-

tions of treatment.

Coordination of Clinical Trials

The current number of suitable centres (with ade-

quate equipment, trained staff, accessibility and

security) to conduct clinical trials in the field of

sleeping sickness is very limited. In view of the

anticipated number of clinical trials, coordination

will be necessary. It is recommended that a clini-

cal trial group be created within the WHO sleep-

ing sickness treatment and drug resistance net-

work. This group should coordinate clinical trials

and provide appropriate training (good clinical

practice and ethics).


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RECOMMENDATIONS


Highest priority

• Development and optimization of a protocol for combination therapy using late-stage drugs.

• Development of tools for shortening the duration of after-treatment follow-up and for dis-

ease-stage determination.

High priority

• Development of nifurtimox up to registration for use against African trypanosomiasis.

• Preclinical evaluation of the 10-day concise melarsoprol regimen for treatment of

T. b. rhodesiense (evaluation in an appropriate monkey model).

• If the toxicological data are favourable, appropriate preclinical studies on the efficacy of

megazol in first- and second-stage infections.

• Elucidation of the underlying mechanisms of encephalopathy syndromes in view of their

prevention and management.

• Exploration of the possibility of better formulations of melarsoprol.

ReferencesLegros D et al. [Therapeutic failure of melarsoprolamong patients treated for late stage T. b. gambiense human African trypanosomiasis in Uganda]. Bulletin de la Société de PathologieExotique, 1999, 92(3):171-2 [in French].

Jennings FW. Chemotherapy of trypanosomiasis: thepotentiation of melarsoprol by concurrent difluoro-methylornithine (DFMO) treatment. Transactions ofthe Royal Society of Tropical Medicine and Hygiene,1988, 82(4):572-3.

Jennings FW. The potentiation of arsenicals withdifluoromethylornithine (DFMO): experimental studies in murine trypanosomiasis. Bulletin de laSociété de Pathologie Exotique et de ses Filiales,1988, 81(3 Pt 2):595-607.

Jennings FW. Combination chemotherapy of CNS trypanosomiasis. Acta Tropica, 1993, 54(3-4):205-13.


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Background

Proper and safe chemotherapy of trypanosomia-

sis is acutely dependent upon accurately identify-

ing the clinical stage of infection (staging).

Staging has been difficult in the past because of

a lack of accurate, sensitive and relatively non-

invasive clinical or parasitological tools for diag-

nosis. However, recent advances in the immunol-

ogy of trypanosomiasis and in parasite cell biolo-

gy suggest new avenues for accurate staging.

Immunological results from both experimental

and clinical trypanosomiasis studies have shown

that infection may be broken down into three rel-

atively well-defined stages: early innate

response; early acquired immune response; late

acquired immune response. Each of these

responses exhibits distinguishing prognostic

characteristics.

The early innate response is triggered by the

presence of trypanosomes within tissues, in suf-

ficiently high numbers, leading to the release of

pro-inflammatory mediators including the

cytokines interleukin-1 (IL-1), IL-6, IL-12 and

tumour necrosis factor alpha (TNFa) as well as

nitric oxide (NO). The early release of these pro-

inflammatory factors helps set the stage for early

acquired immune responses to the parasite vari-

ant surface glycoprotein (VSG). The early

release of IL-12 promotes T helper (Th) to

release cytokines, particularly interferon-gamma

(IFNg), which is responsible for host resistance

against parasites spreading throughout host tis-

sues. This may be more important than anti-VSG

antibodies which are only found within blood

vessels. This early resistance is, however, super-

seded by later immune responses that are associ-

ated with the spread of trypanosomes to the

CNS. The late-stage acquired immune response

is typified by a loss of Th1 cells secreting IFNg

and the production of very high levels of the

anti-inflammatory cytokine IL-10. The high IL-

10 levels are associated with loss of tissue resist-

ance and spread of trypanosomes to the CNS.

Early- and late-stage trypanosomiasis can there-

fore be characterized by the levels of specific

cytokines in patients. High IFNg but low IL-10

levels are associated with the early non-invasive

disease, and high IL-10 but low IFNg levels are

associated with late-stage invasive disease. Since

preliminary clinical measurements show that

there is concordance with respect to serum and

CSF levels of these cytokines, the prospects for

determining early- or late-stage disease by test-

ing serum for IFNg or IL-10 levels, respectively,

is clear. Furthermore, examination for cytokine

levels, which are very short-lived in serum and

CSF, may help clarify whether a seropositive

individual has had a recurrence of disease.

Moreover, information concerning immune status

and trypanosome-derived inflammatory factors

(e.g. glycosyl phosphatidyl inositol) may impact

on how patients are treated for late-stage disease.

However, the picture is clouded by new informa-

tion about the trypanosome itself. The following

areas of study were identified:

• Validation of experimental and prelimi-

nary clinical results suggesting that stage

of disease can be determined by the

presence of specific cytokines including

IFNg, IL-10 and TNF.

• Definition of the pathogenesis of the

encephalitis associated with late-stage

sleeping sickness and the encephalopa-

thy associated with treatment, and

5 Pathogenesis and applied genomics

PATHOGENESIS


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investigation of ways to prevent or ame-

liorate these phenomena using in vitro

and in vivo disease models.

• Utilization of the data and technical

approaches of the human genome project

to unravel the composite immune

response to trypanosomes at each stage

of the disease process.

Trypanosome Biological Phenotype

Trypanosomes have evolved mechanisms to reg-

ulate their biological virulence phenotype at the

clonal level. There is evidence that clonally

derived “virulent variants” are capable of enter-

ing the CNS at an accelerated rate in non-human

primates, suggesting the possibility for develop-

ment of discrete potential for CNS invasion

within the host. The molecular mechanisms

behind such clonal changes are unknown. This

information might be useful in choosing the

appropriate clinical treatment at any stage of dis-

ease. For example, the presence of trypanosomes

expressing “CNS invasion markers” at any stage

of disease might be used to initiate late-stage

chemotherapy. In addition, such information has

the potential to detect drug resistant variants in

patients and aid the choice of drugs used to treat

such patients.

RECOMMENDATIONS


Highest priority

• Identification of parameters within the host immune system for disease-stage determination

and improved management of post treatment encephalopathy.

High priority

• Determination of the biological phenotypes of trypanosomes lodging in different tissue com-

partments to identify tissue tropism and drug-resistance characteristics.


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Trypanosoma brucei Genomics

The T. brucei genome network was formed by

TDR with a brief to coordinate the analysis and

sequencing of the nuclear genome. This huge

task can progress most efficiently if the commu-

nity works together with open sharing of data and

resources. The network has sufficient funds

(from the Wellcome Trust UK and the US

National Institute of Allergy and Infectious

Diseases/National Institutes of Health

[NIAID/NIH] USA) to complete the sequencing

of the megabase chromosomes, where the majori-

ty of genes are located, which will take until

2004. Approximately 85-90% of the genome is

currently available as fragmentary sequences in

databases in the sequencing centre websites, and

will ultimately be available in public web-based

databases.

The Wellcome Trust has provided funds to estab-

lish a genome database, at the Sanger Centre,

that will contain all data related to sequencing

and functional analysis of genes. Two resource

centres have been established: DNA-based

resources in Cambridge, and derivative and

mutant lines of trypanosomes in Glasgow. These

centres are funded to the end of the sequencing

phase, at which time a re-appraisal of resource

provision will be required.

The TDR planning meeting for the Parasite

Genome Initiative assigned some activities to

be undertaken in Africa, but these activities were

not sustained for various reasons which included

reduced funding for African trypanosomiasis

by TDR and changes in available technologies

and priorities within the genome networks. The

involvement of African scientists and TDR is

crucial in ensuring the application of information

from genomics into disease control. African insti-

tutions must develop the capacity to fully exploit

the resources from the genome projects. It is

anticipated that capacity strengthening in African

institutions will impact on the applications from

both the parasite and the vector genome projects.

APPLIED GENOMICS

RECOMMENDATIONS


Highest priority

• Strengthening of capacity of research laboratories/centres in Africa in bioinformatics,

genomics and applied genomics.

• Application of genomics to comparison of Trypanosoma brucei subspecies, strains, and life

cycle stages.

High priority

• Identification, application and database collation of DNA-based polymorphic markers for

species differentiation, reservoir identification and determination of drug resistance.

• Application of bioinformatics and experimental methods of determination of gene function

to identify novel drug targets.

• Use of resources available from the human genome project to investigate host response to

infection.


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Recent advances in molecular technologies, and

their application to insects, are being widely

explored because they have the potential to

result in the development of novel strategies for

control of vector borne diseases. The technolo-

gies needed to undertake such studies have been

developed for other insect vectors and thus can

facilitate rapid application to tsetse biology.

Limited field data indicate extensive genetic

sub-structuring in populations, which display

differences in vectoral capacity. Information on

population genetics would facilitate more effec-

tive and targeted disease management strategies

by identifying sub-populations of flies responsi-

ble for disease transmission. There exists a mid-

gut symbiont-based genetic transformation sys-

tem which allows expression of gene products

that confer trypanosome refractory phenotypes.

Candidate genes with either anti-trypanosomal

traits or which, when expressed, can block para-

site transformation and /or differentiation,

should be identified for expression in the tsetse

mid-gut. The eventual genetic engineering of

anti-trypanosomal traits into tsetse populations

would result in new vector control strategies.

The laboratory and field-based research recom-

mended below is necessary to develop the

applied approaches that can lead to novel vector-

based disease-control strategies.

TSETSE GENETICS

Tsetse-trypanosome interactions

• Determination of the molecular

basis of refractoriness for try-

panosome transmission.

• Engineering of trypanosome refrac-

tory traits into strains of tsetse fly.

• Investigation of gene spreading

mechanisms that can be used for

population replacement studies, e.g.

cytoplasm incompatibility mediated

by Wolbachia symbionts of tsetse.

Population genetics of tsetse

• Development of molecular polymor-

phic DNA markers and their appli-

cation to field flies to understand

the extent of genetic sub-structuring

existing in tsetse populations.

• Investigation of mating incompati-

bilities existing among field popula-

tions.

• Investigation of differences in vector

competence abilities of genetically

isolated sub-populations of flies.


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RECOMMENDATIONS


Highest priority

• Determination of the molecular basis of refractoriness for trypanosome transmission and

development of mechanisms for driving the desired genes/traits into tsetse populations.

High priority

• Development and application of molecular markers to determine genetic sub-structuring and

mating incompatibilities in tsetse populations, and vector competence of genetically isolated

sub-populations.

• Development of a tsetse-parasite genome network to obtain and coordinate information on

expressed sequence tags (EST), genomic sequences, physical map locations of selected

genes, and eventual proteomics approaches.

• Coordination of the network by TDR, whose comparative advantage is evidenced by the suc-

cessful management of the mosquito-parasite genome networks.


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Funding support for control, and for research

and development, requires long-term commit-

ment. Over the past 20 years, many countries

have provided support for control of, and

research on, African trypanosomiasis; in particu-

lar the governments of Belgium, France and UK

are major supporters. The European Union,

USA, Canada, the Organization of Petroleum

Exporting Countries (OPEC) Fund and China are

also partners. A list of countries and institutions

(by no means complete) that provide financial

support is attached (Annex 1).

Considerable sums of money are invested in

basic research on trypanosomes by institutions in

USA, and EU countries, because the try-

panosome is a good model for basic research on

cell biology. Consequently, there is probably

more information on the cellular structure, bio-

chemistry and molecular biology of try-

panosomes than any other non-mammalian cell

type, and a great deal is known about the differ-

ences between trypanosomes and mammalian

cells. However, only a small amount of this

knowledge is being applied directly in the man-

agement and control of the disease. Some of the

knowledge is exploitable for development of new

tools for disease and vector control as well as for

improved patient management. The SWG meet-

ing provided an opportunity to identify the

knowledge that could be exploited for develop-

ment of new tools and improvement of existing

ones for disease and vector management, as well

as to determine the needs for research capability

strengthening in disease endemic countries for

basic sciences. TDR’s comparative advantage in

enhancing existing, and developing new, partner-

ships for maximal application of the available

knowledge was highlighted.

The SWG was held at an opportune time when

the funding level through TDR is increasing and

when African trypanosomiasis is re-surfacing in

the global health agenda. The funding and assur-

ance of a five-year drug supply by the private

sector provides a unique opportunity to deal with

the current epidemic in Africa as well as to

develop protocols for combination chemotherapy

in the face of increased melarsoprol refractori-

ness. In addition, the five years provides a period

of grace during which TDR and the scientific

community can lay the foundations for ensuring

continued availability of medicaments beyond

the five years by investing in research and capa-

bility strengthening within Africa for drug dis-

covery and development as well as for genomics,

applied genomics and proteomics. The SWG

acknowledged the comparative advantage that

TDR has in coordinating and networking the

activities of various scientific groups, laborato-

ries and centres in different parts of the world.

Consequently, the group recommended recruit-

ment of a full-time professional staff member

within TDR to coordinate the various activities

on African trypanosomiasis.

6 Cross-cutting issues

RESOURCE FLOW


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The SWG noted with appreciation the increase in

funding coming to TDR for African trypanosomi-

asis. However, there was concern that, in the pre-

vious six years or so, there had been little interest

from the donor community to fund activities on

African trypanosomiasis. A need to present the

disease in a way that changes donor perception of

the problem was identified. The Group was

informed that one way of changing is to present

projects that are convincing, focused and concise,

emphasizing donor/researcher partnerships in

project management and follow-up and the bene-

fits to the target communities. Networking

appears to be more attractive to donors than sin-

gle, isolated projects.

The Group indicated a number of networks

already existed, both formal and informal (with

NGOs and national systems). A need to enhance

collaboration between the groups working in

research and those working in control, and also

between groups working in disease and non-dis-

ease endemic countries, was identified. This

would increase the much needed credibility when

requesting donor support. The SWG noted that

scientists working in disease endemic countries

often work in isolation and have difficulties

developing the required credibility for donor sup-

port, and identified a need to support good ideas

from young scientists, enabling them to seek

independent funding, i.e. to support them to a

point where donors have confidence in them. In

addition, a mechanism to assist young scientists

to develop their ideas into fundable proposals,

where their ideas are likely to provide vital infor-

mation, should be put in place.

ADVOCACY AND MARKETING FOR SLEEPING SICKNESS

Given the re-emergent nature of African try-

panosomiasis, it is necessary to identify and

strengthen a nucleus of research groups and

institutions in different endemic countries to

generate data, on disease burden and surveil-

lance, for advocacy. Where whole institutions

are difficult to maintain as disease specific

research institutes, their disease mandates could

be expanded to enable them to add on other dis-

ease research activities while retaining core

activities in the area of sleeping sickness.

The SWG commended TDR for the great num-

ber of scientists it has supported for post-gradu-

ate training. However, continued training of

research personnel in African trypanosomiasis,

and linking of this training to specific techno-

logical needs, which vary from country to coun-

try, was recommended. A need to train technical

staff for specific needs in research, disease sur-

veillance and management was also identified.

The Group further noted the difficulties of sus-

taining trained staff within the field of African

trypanosomiasis in disease endemic countries,

and suggested lack of career development

opportunities as one of the factors contributing

to this shortage.

Staff retention is a global problem, not peculiar

to the field of African trypanosomiasis. Those

working in this field are in the public sector,

where salaries are not attractive and career

advancement is slow. Lack of resources to carry

out research, and to attend short-term training

courses, is a disincentive to remain in the sector.

Funding for research, on a more sustainable

INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING


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basis and for longer periods, is a necessity; while

linking of national institutions with overseas

ones, with donor support, is an area for possible

expansion.

The SWG welcomed the recent TDR initiative of

institutional strengthening grants; a call for let-

ters of intent was already issued. TDR is also

working on strategies for strengthening whole

national health research systems in countries that

do not yet have a strong research culture, and a

mechanism is being worked out to give priority

to funding of research-strengthening proposals.

The commitment of African governments to sup-

port trypanosomiasis research and control, with

improved terms of service for those working in

the sector, is important. Very few countries in the

disease endemic areas have identified scientists

or technicians in scientific institutions as a

national resource with unique operational needs.

It was agreed that advocacy would be crucial,

and international forums, such as the

Organization of African Unity (OAU)

International Scientific Council for

Trypanosomiasis Research and Control (ISC-

TRC), would be used to send the message. The

presence of TDR at such forums, and in other

African national systems, would also be useful.

At the same time, there is a need to ensure

appropriate networking in order to avail the nec-

essary assistance in writing good proposals. The

possibility of providing salary support with

research grants should also be given considera-

tion by TDR.

In March 2001, WHO held a meeting in Harare,

Zimbabwe, to discuss an initiative aimed at

increasing discussion and scientific interaction

between investigators in disease endemic coun-

tries. The meeting was attended by scientists

from several countries in Africa, Asia and Latin

America, who discussed ways of increasing the

collaboration that already exists between investi-

gators in the South and collaborators in more

advanced laboratories of the North. The need to

develop more sustainable pathways for training,

that will make more efficient use of available

resources by exploiting opportunities for the

exchange of complementary expertise present

within laboratories in Africa, Asia and Latin

America, was emphasized. This networking will

enhance the application of molecular biology

techniques and genomics (functional and

applied) to developing solutions to public health

problems. Initiatives for multicentre projects,

which incorporate capacity building and training,

will be developed. Additionally, a number of

short-term training courses in basic and applied

biology, that incorporate the application of the

latest technologies, are planned. It is anticipated

that the results of this network will include

process indicators (academic qualifications,

highest quality publications), products (e.g.

tools, chemotherapeutic agents), and an impact

on alleviating disease.

SOUTH-SOUTH COLLABORATION


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RECOMMENDATIONS


• Recruitment by TDR of a full-time professional staff member to coordinate African try-

panosomiasis activities.

• Identification and strengthening of a nucleus of research groups and institutions in different

endemic countries to generate data on disease surveillance and advocacy.

• Networking and cross-country comparison of research progress to assist in capacity building

and stimulate cross border interest and advocacy.

• Strong advocacy to persuade DEC governments to give priority to research and control of

African trypanosomiasis amidst other health priorities.

• Payment of a salary component within TDR funded grants to enhance retention of disease

endemic country scientists in the field.


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Annex 1RESOURCE FLOW FOR AFRICAN TRYPANOSOMIASIS


BCT Bureau Central de la Trypanosomiose

BMS Bristol-Myers Squibb

CAR Central African Republic

DFID Department for International Development

DRC Democratic Republic of Congo

EU European Union

FAO Food and Agriculture Organization

FITCA Farming in Tsetse Controlled Areas

FOMETRO Fonds Médicale Tropicale

IBAR InterAfrican Bureau for Animal Resources

ICCT Instituto de Combate e Controlo das Trypanosomiases

IPMP Instituto Português de MedecinaPreventiva

IRD Institut de Recherche et Développement

ITM Institut Tropical de Médecin

KETRI Kenya Trypanosomiasis Research Institute

LIRI Livestock Research Institute

MEMISA Medische Missie Samenwerking

MSF Médecins sans Frontières

NIAID National Institute of Allergy andInfectious Diseases

OAU Organization of African Unity

OCCGE Organisation de Coordination et de Coopération pour la lutte contre les Grandes Endemies

OCEAC Organisation de Coordination pour la lutte contre les

Endemies en Afrique centrale

PATT Programme against African Trypanosomiasis

STI Swiss Tropical Institute

UN United Nations

UNC University of North Carolina

UNDP United Nations Development Programme

WHO World Health Organization


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Annex 2POSITION PAPER


A POSITION PAPER ON AFRICANTRYPANOSOMIASIS

Felix A.S. KuzoeWHO/TDR, Geneva

SUMMARYThis position paper provides an overview of themain features of African trypanosomiasis and theproblems relating to its control. It gives a historicalaccount of the disease, its epidemiology and socioe-conomic impact. It describes the strategy for control,namely case detection, chemotherapy and vectorcontrol, and the limitations. It discusses global fund-ing of basic research on African trypanosomes, andthe pivotal role of WHO/TDR in facilitating theevaluation of any spin-offs from the researchtowards development of public health tools.Considerable progress has been achieved inresearch on African trypanosomiasis, but thisprogress has not been reflected in the field due tolack of interest in development of new tools andlack of sustainability of control methods, resultinglargely from inadequate resources for public healthand wars and civil strife in some endemic countries.With the decrease in resources for African try-panosomiasis in TDR, which started in 1994, therehave been grave consequences in endemic coun-tries, both in terms of reduction in human resourcesand degrading of facilities for research and evalua-tion of new tools for African trypanosomiasis. Tomeet the challenge of African trypanosomiasis in the21st century, this position paper makes a number ofproposals.

AFRICAN TRYPANOSOMIASIS AS A PUBLIC HEALTH PROBLEMAt the beginning of the last century, sleeping sick-ness was perceived by the colonial powers as by farthe most important public health problem in Africa.Huge epidemics devastated large areas of the conti-nent. In the 1960s, the prevalence of sleeping sick-ness was successfully reduced in all endemic coun-tries to less than 0.1%, through historic campaignsby the former colonial powers. Soon after inde-pendence, however, national governments wereeither lacking in resources or had divertedresources to other pressing health problems.Breakdown of specialized mobile teams and healthfacilities in several countries, as a consequence ofwar and civil strife or change in health policy,resulted in dramatic resurgence of African try-

MEETING THE CHALLENGE1. New tools are needed for the control of African try-panosomiasis (sleeping sickness); however, these ontheir own will not improve the trypanosomiasis situa-tion. Factors that militate against the effective use ofexisting tools, such as continued worsening economiesand structural adjustment programmes in the affectedcountries, will remain.• Operational research should be conducted on the

effective use of available tools with existing capaci-ties, such as the health services, strong control pro-grammes in other diseases, NGOs, etc.

• Development of simple surveillance systems withavailable tools that can be integrated into availablecapacities to improve case detection and activescreening of populations at risk will permit earlydiagnosis of cases and increase the number of peo-ple under medical surveillance.

• Long-term commitment by the international com-munity to national sleeping sickness control pro-grammes, rather than support for crisis manage-ment, will ensure sustainability.

2. Social, economic and cultural factors enhance orhinder efforts to control sleeping sickness. There isneed for studies on social, socio-cultural and anthro-pological aspects of endemicity of sleeping sickness.The effects of decentralization of health services onAfrican trypanosomiasis need to be studied forimproved management of control programmes.3. The private sector has the expertise for drug devel-opment. However, for diseases such as African try-panosomiasis that have no market for drugs, develop-ment must be based on public sector financing. • The recent award of US$ 15 million by the Bill &

Melinda Gates Foundation to a consortium of scien-tists towards the development of drugs for Africantrypanosomiasis and Leishmaniasis is a welcomedevelopment that has no precedence in the historyof African trypanosomiasis. Furthermore, the WorldHealth Organization (WHO) and Aventis Pharmahave announced a major initiative to control Africantrypanosomiasis, whereby Aventis Pharma has com-mitted US$25 million to support WHO’s activities inthis field for a period of five years.

• TDR has links with the outside world with specialreference to product development. Over its lifetime,it has generated multiple partnerships for productdevelopment and has gained considerable experi-ence. Thus, it has been successful and taken someproducts up to registration in collaboration with theprivate sector, for example, mefloquine, eflornithine,AmBisome and artemether. More partners are need-ed in drug discovery research and drug develop-ment for African trypanosomiasis.

4. Basic research on trypanosomes continues to befunded with considerable sums of money in the North.No drugs have yet come out of basic research byrational drug design. Functional genomics, bioinfor-matics and proteomics provide new opportunities that

should be explored for drug discovery and develop-ment of tools for trypanosomiasis control and patientmanagement. 5. Institutions in the endemic countries should bestrengthened to enable implementation research.Clinical studies should be conducted according to theconcept of good clinical practice (GCP). A few clinicalresearch centres or treatment centre networks shouldbe identified and strengthened and clinical investiga-tors given appropriate training.


panosomiasis, the distribution of which corre-sponds closely with that of major conflicts in sub-Saharan Africa. In the Democratic Republic of theCongo (DRC), the number of cases being reportedyearly has now reached levels comparable to the1930s, and may result in as many deaths in adultsas HIV/AIDS (Ekwanzala et al 1996). And yetAfrican trypanosomiasis is curable. Other countriesaffected by the resurgence/epidemics are Angola,the DRC, Central African Republic, Sudan andUganda. Sleeping sickness due to T. b. rhodesienseseems to be quiescent at present and less wide-spread, with active foci occurring in Tanzania andUganda. African trypanosomiasis is a re-emergentdisease, but does not get due attention, probablybecause its impact is regional.

The disease occurs in some 36 sub-Saharan coun-tries, within the area of distribution of the tsetse fly.Over 60 million people living in some 250 foci with-in this region are at risk of contracting the disease.The number of cases reported annually is over 40000, but this is highly underestimated due to the dif-ficulty of diagnosis and remoteness of affectedareas. It has been estimated that the actual numberof cases is about 300 000 (WHO 1998). Though thesefigures are relatively small compared to other tropi-cal diseases, African trypanosomiasis, without inter-vention, has the propensity to develop into epi-demics. This characteristic makes it a major publichealth problem. During epidemics, large propor-tions of communities are affected with great loss oflife and untold human suffering. Epidemics haveserious social and economic consequences, whichfar outweigh the cost of maintaining surveillance.Sleeping sickness has perhaps been a major cause ofdepopulation of large tracts of Africa, and fear of thedisease has led to abandonment of fertile lands andis an impediment to development. The World BankReport (1993) estimated that, in 1990, there were 55000 deaths due to African trypanosomiasis and 1.8million disability adjusted life years (DALYs) lostdue to the disease. More recent estimates are rathersimilar, with 2.1 million DALYs lost and 66 000deaths in 1999. As a comparison, the number of mil-lions of DALYs lost is estimated at 45.0 for malaria,4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9for schistosomiasis, 1.1 for onchocerciasis and 0.7 forChagas disease (WHO 2000).

THE DISEASEAfrican trypanosomiasis is caused by protozoanhaemoflagellates, trypanosomes that are transmit-ted by tsetse flies (Glossina spp). The disease occursin two forms: the chronic form caused byTrpanosoma brucei gambiense, which occurs in Westand Central Africa; and the acute form, caused by

T. b. rhodesiense, which occurs in Central andSouthern Africa. The chronic infection lasts foryears, whilst the acute infection may last only forweeks.

Other forms of trypanosomiasis, called nagana,affect livestock, and are considered the most impor-tant infectious disease holding back the develop-ment of livestock production in much of Africa. Theannual losses in meat production attributed to try-panosomiasis are estimated to be US$5 billion(ILRAD 1993). No new drug has entered the marketfor over 30 years because the disease does not affectlivestock in Western developed countries.

EpidemiologyThe epidemiology of sleeping sickness is complexand the transmission cycles are subject to interac-tions between humans, tsetse flies, trypanosomesand, significantly in T. b. rhodesiense sleeping sick-ness, domestic and wild animals. In T. b. gambiensedisease, the classical human-fly-human transmis-sion cycle occurs in both endemic and epidemic sit-uations. Humans are the important reservoir andhence it is possible to reduce transmission throughdiagnosis and treatment of the infected population.Although it has been demonstrated by biochemicaland DNA techniques that trypanosomes identical tothose which cause T. b. gambiense disease in humansoccur in domestic animals and some game, the sig-nificance of these potential reservoir hosts on trans-mission is not clear. In T. b. rhodesiense disease, onthe other hand, it has been recognized that infec-tions in humans in endemic situations are acquiredfrom savannah species of tsetse flies, all of whichfeed preferentially on a wide variety of game. Thegame-fly-human cycle is typical. Endemic T. b.rhodesiense situations are sporadic in nature andpatchy in distribution, and adult men tend to bepredominantly infected. In epidemic T. b. rhode-siense, however, human-fly-human or domestic ani-mal-fly-human cycles predominate. There is equaldistribution of infection among men, women, andchildren. For T. b. rhodesiense disease, chemoprophy-laxis of the domestic animal reservoir has been rec-ommended as a new approach to control. Availableevidence suggests that HIV infection has had littleimpact on the epidemiology of T. b. gambienseAfrican trypanosomiasis (Louis et al 1991; Pepinet al 1992; Meda et al 1995).

Medical SurveillanceRegular medical surveillance, involving case detec-tion and treatment, and tsetse fly control, whereapplicable, is the backbone of the strategy for con-trol of sleeping sickness (WHO, 1998). With avail-able tools, control is a continuing effort rather than


eradication. Experience over the last 50 years hasshown that, where control efforts are interrupted,e.g. due to civil strife, political upheavals, economicconstraints, or out of complacency, sooner or later,there will be resurgence of the disease.

DiagnosisFor unequivocal diagnosis in humans, it is essentialto demonstrate trypanosomes in lymph node aspi-rate, blood or cerebrospinal fluid (CSF). A numberof tools exist for the diagnosis of patients. In T. b.gambiense areas, the Card Agglutination Test forTrypanosomiasis (CATT), a serological test detect-ing antibodies, is used for mass screening.Serologically positive cases are then confirmedusing parasitological tests. Since parasitemia variesbetween foci and disease stage, it is necessary toadopt blood concentration techniques, such as theHaematocrit Centrifugation technique (HCT), theMiniature Anion Centrifugation Technique(MAECT), or the Quantitative Buffy Coat (QBC)technique. A study on the treatment of serologicallypositive patients who are parasitologically negativewith one injection of pentamidine is in progress inDRC. The use of the CATT for screening popula-tions in T. b. gambiense areas has greatly improvedthe potential for community diagnosis. Despite theadvances, techniques, especially the CATT, haverarely been put into practice in endemic areas exceptas part of externally funded programmes. Thisrelates in part to the costs (Smith et al 1998). TheCard Indirect Agglutination Test forTrypanosomiasis (CIATT) is another serological testwhich detects antigens and therefore active infec-tion. However, the very high frequency of positiveresults in low endemic areas, indicates that it maynot be suitable for screening in control programmes.The role of the polymerase chain reaction (PCR) indiagnosis remains to be determined. A test is need-ed to diagnose late-stage disease, which presentlyrelies on lumber puncture that is painful and notwell accepted by people. A test is also needed todetermine cure after chemotherapy. The currentrequirement for a 2-year period of follow-up oftreated patients is cumbersome, costly, and leads toloss of many patients.

ChemotherapyThe chemotherapy of African trypanosomiasis isunsatisfactory, relying on a few drugs which haveadverse side effects. Pentamidine, a diamidinedeveloped in 1937, is used for early-stage T. b. gam-biese sleeping sickness. Suramin, a sulphanatednaphthylamine developed in 1922, is used to treatearly stage T. b. rhodesiense. Melarsoprol, a trivalentarsenical derivative developed in 1948, is used forthe treatment of late-stage of both T. b. gambiense and

T. b. rhodesiense sleeping sickness. All these drugshave adverse effects, melarsoprol causing reactiveencephalopathy in 5-10% of patients with a fataloutcome in 1-5%. Increasing numbers of patients -between 20-25% in certain foci - do not respond tomelarsoprol treatment, probably due to resistance ofthe parasite to the drug (Legros et al 1999). The term‘drug resistance’ covers host and parasite-relatedfactors. Host related factors include poor distribu-tion of drug to infected tissues and intracellularsites, variation in drug metabolism between indi-viduals, and diminished activity of drug. Parasiterelated factors that contribute to resistance includereduced drug accumulation in the parasite, a changein enzyme target through increase in enzyme levels,increase in metabolite production or retention, oruse of alternative pathways to bypass the site ofinhibition. A number of these factors could beinvolved and therefore the mechanism of resistanceneeds to be elucidated and strategies to combat it,such as the use of drug combinations, developed.The development of a simple test to quickly diag-nose resistance in parasite isolates is also needed forsuccessful chemotherapy. Clinical trials with a com-bination of melarsoprol and eflornithine, whosesynergism has been demonstrated in the mousemodel, should be given priority (Jennings 1988).Combinations of other drugs are also envisaged.The varying treatment schedules of available drugswere developed empirically and, therefore, opti-mization of current treatment regimens is needed.Burri et al (2000) have shown that, with a new regi-men of melarsoprol, it is possible to reduce the dura-tion of treatment from 40 days to 10 days. A studysponsored by TDR on treatment with pentamidine,7 days versus 3 days, in DRC has been interrupteddue to rebel forces taking over that part of the coun-try. This occurrence underscores the difficulties ofcarrying out field research on African trypanosomi-asis.

Eflornithine, developed in 1990, is the only availablealternative drug to treat T. b. gambiense patients whodo not respond to melarsoprol. It is not effective inT. b. rhodesiense sleeping sickness. The drug hasmany drawbacks: a complicated mode of adminis-tration (intravenously every 6 hours at a dose of100mg/kg/day for 14 days), which limits its use toa hospital setting; it cannot be used for mass treat-ment; and the cost of treatment is US$300-500 perpatient, which makes it unaffordable by the affectedcountries. To reduce costs, a shorter course of eflor-nithine (7-day treatment) was compared to the stan-dard 14-day treatment. However, the resultsshowed that the 7-day treatment is effective only inpatients who have relapsed on melarsoprol andthat, for new patients, the 14-day course is superior


(Pepin et al 2000). In recent years, there have beendoubts about the future availability of eflornithine.However, through the collaboration betweenAventis, the manufacturer, WHO and Médecinssans Frontières (MSF), potential manufacturers havebeen identified, and it is likely that a suitable pro-ducer will be selected to produce the drug and thatfunds will be provided through donor contributionsto guarantee production for the next five years.Nifurtimox, nitrofurasan, was used for acuteChagas disease. Although it was not registered forhuman African trypanosomiasis, it has been usedexperimentally and on a compassionate basis totreat T. b. gambiense sleeping sickness patients.Bayer, the manufacturer, has agreed to continue pro-duction for treatment of African trypanosomiasis;however, registration of the drug for this purpose isan urgent issue that needs to be addressed.

Vector controlA variety of traps and screens impregnated withinsecticide have been shown to be effective in reduc-ing tsetse populations by 99% in control pro-grammes and are suitable for rural community par-ticipation. In any outbreak of sleeping sickness,tsetse control in combination with diagnosis andtreatment should arrest transmission. Besides, thesedevices can also be used as preventive measures toreduce human-fly contact. In vector-borne diseases,vector control plays an important role in reducingtransmission. For example, Chagas disease causedby T. cruzi has been successfully controlled by vec-tor control as a result of national government com-mitment to a long-term programme. In onchocercia-sis endemic areas of West Africa, blindness is nolonger a public health problem as a result of vectorcontrol and donated drug (Mectizan) distribution.The effect of deforestation and climatic changes ontsetse populations in West Africa may be responsi-ble for the lull in sleeping sickness in countries likeGhana and Nigeria. Operational issues, such asmotivation of communities and recurrent costs,which militate against sustainability in the use ofimpregnated traps and screens on regular basis,need to be studied and solutions found.

DRUG DEVELOPMENTAn oral formulation of eflornithine has manyadvantages over the injectable form and will allowuse on a wider scale. A pharmacokinetic study oforal eflornithine is in progress in Côte d’Ivoire andit is anticipated that Phase 3 clinical trials will takeplace in 2001, to evaluate its safety and efficacy,toward eventual submission of data for registrationto a regulatory authority. The need for new drugs isfundamental. One of the obstacles to finding newdrugs is the difficulty of transport across the blood-

brain barrier in humans. The functional integrity ofthe blood-brain barrier and its role in pharmacoki-netics and pathogenesis is pivotal to understandingthe disease. Attention should be given to newapproaches to drug delivery, such as across theblood-brain barrier. In 1999, two leading com-pounds, both of them diamidines that gave satisfac-tory results against trypanosomes in animal modelsin acute infection, failed to cure the chronic infec-tion, due to inability to cross the blood-brain barrier.A number of lead compounds have suffered thesame fate in the past. The ideal trypanocide must besafe and effective, and have a simple mode ofadministration to allow its use under rural condi-tions, where health facilities are usually poor, andabove all should be affordable. The occurrence of T.b. rhodesiense sleeping sickness outside the tradition-al focus in south-eastern Uganda (Enyaru et al1999), justifies fears of mixing T. b. gambiense and T. b. rhodesiense due to human population move-ments between foci in Uganda, the DRC andTanzania (Kigoma), and underlines the need for anew drug that can treat both T. b. gambiense and T. b.rhodesiense. A multicentre clinical trial with eflor-nithine showed that eflornithine was relatively lesseffective in Uganda than in three other countries(Pepin et al 2000). It should be noted that Uganda isthe only country where both T. b. gambiense and T. b.rhodesiense sleeping sickness foci exist and, there-fore, this observation needs further investigation.

SOCIAL AND ECONOMIC IMPACT OF SLEEPING SICKNESSThe social and economic impact of sleeping sicknessis often underestimated. Some affected countrieshave agriculture-based economies, and workers oncocoa and coffee plantations are at risk of contract-ing the disease; consequently the labour force isreduced. At community and family levels, mentalconfusion, personality and behaviour changes,which often characterize central nervous systeminvolvement in late-stage disease, may lead todivorce and break up in homes and present anunfavourable climate for bringing up children. Insome cases, such people become mentally dis-turbed, suicidal and violent, and constitute a dangerto themselves and to the community. Aroke et al(1998) reported that, in the past, T. b. gambiensesleeping sickness in children had an influence ontheir physical growth and attainment of sexualmaturity. In Central Africa, there are importantproblems regarding treatment, particularly thesevere social consequences of long-term hospitaliza-tion. Important behavioural factors contributing tothe risk of death from sleeping sickness, such asnegative attitudes towards hospital treatment, haveoften led to the patient absconding and not com-


pleting treatment. In studies on the impact of try-panosomiasis on land occupancy systems, on popu-lation movements and social conditions in BurkinaFaso and Côte d’Ivoire, the extreme mobility of thepeople due to migrant labour was identified as oneof the major problems in case detection, case report-ing and control of the disease. There is need to con-tinue monitoring and mapping population move-ments, as well as incidence of trypanosomiasis, par-ticularly as the impacts of the infection are ratherlatent but extremely serious in the long run. Studiesin Uganda demonstrated that African trypanosomi-asis had an adverse impact on the functioning ofhouseholds at Iganga, south-east Uganda. Adverseimpacts included increased poverty, decline in agri-cultural activities often leading to famine or lack ofbasic food security, disruption of children ‘s educa-tion, and general reversal of role obligations, whichmore often than not enhanced women’s and chil-dren’s burdens. The debilitating nature of the dis-ease also poses more problems for women, whomay be stigmatized and/or rejected by their spous-es even after recuperation. The extent of disruptionof household social and economic activities is great-ly influenced by such factors as the household’s eco-nomic status, composition and level of organization(Kyomunhendo 1995, 1998).

GLOBAL RESEARCH ON AFRICAN TRYPANOSOMES The trypanosome has many unique biological char-acteristics that make it one of the most studied par-asites. It offers many opportunities for basicresearch: it is easy to cultivate and purify to yieldlarge amounts of protein and nucleic acid, it is aeukaryotic experimental model for research on thecontrol of gene expression, etc. Though it is difficultto get funds for the control of sleeping sickness,large sums of money are invested annually, particu-larly in the North, on basic research on African try-panosomes. There is probably more information onthe biochemistry and molecular biology of try-panosomes than on any other non-mammalian celltype, and a great deal is known about the differ-ences between trypanosomes and mammalian cells.Yet no drugs have come out of basic research. TDRprovides an essential link between research institu-tions in the North and endemic countries, throughaccess to a network of national field projects andcontrol programmes, where spin-offs from basicresearch can be evaluated as tools for the controland prevention of sleeping sickness. It is necessaryfor TDR to preserve this unique role (Kuzoe, 1993).

For institutions in the South, TDR was conspicuous-ly a major source of funding for research on Africantrypanosomiasis and institutional strengthening

activities. TDR’s initiatives in bringing together sci-entists in Scientific Working Groups, meetings andworkshops to deliberate on specific issues, paid offon several occasions. A few examples follow.

A Glossina trapping meeting held in Brazzaville,Congo, in March 1985, brought together scientists,from West, Central and Southern Africa, interestedin developing Glossina trapping technology. Thismeeting opened the way for better interactionbetween the scientists and gave impetus toimprovement in trapping technology for tsetse con-trol. Five years later, the pyramidal trap impregnat-ed with insecticide used at 10 traps per km2 waseffectively employed in reducing tsetse populationsby over 95 per cent within 3-4 months during theepidemics of sleeping sickness in Busoga, south-eastUganda, at an estimated cost of US$0.9 per head ofpopulation protected (Lancien, 1991).

The rational development of control and treatmentrequires thorough knowledge of the pathology ofthe disease. At the inception of TDR in 1975, therewere less than 25 autopsies of African trypanosomi-asis reported in the literature. The need for system-atic autopsy backed by expert histopathology ledTDR, in collaboration with the University ofGlasgow, to establish a network of clinical centresthat were provided with kits and protocols forautopsy. From this effort, evidence was providedfrom both laboratory and clinical data showing thatreactive encephalopathy, which occurs in 3-5% ofpatients treated with melarsoprol, points to a drug-related immune response rather than to toxicity(Haller et al 1986). A set of slides showing the fea-tures of neuropathology in African trypanosomiasiswas prepared and made available to universities forteaching purposes.

New strategies for managing patients with centralnervous system involvement in African trypanoso-miasis are needed. In 1986, TDR organized a work-shop in collaboration with the Institut de Neuro-logie Tropicale, Limoges, France, which broughttogether clinicians, neurologists, neuropathologistsand scientists. Following the recommendations ofthis group, a number of studies funded by TDR incollaboration with other institutions, resulted in sig-nificant progress in understanding the molecularmechanisms underlying pathogenesis, includingbrain dysfunction and neuropsychiatric symptomsassociated with the disease. The trypanosome lym-phocyte triggering factor, TLTF, a molecule whichbinds to CD8+ cells and triggers the production ofgamma interferon, which is growth factor for T. b.brucei, was reported by the Karolinska Institute,Sweden, in 1991(Olsson et al 1991). The gene for


TLTF has since been identified, cloned andsequenced, and a recombinant TLTF produced(Bakhiet et al 1993; Vadya et al 1997; Hamadien et al1999). The exploitation of TLTF for immunotherapyneeds to be followed up. Other studies elsewherehave shown that proinflammatory cytokines,including tumor necrosis factor (TNF), interleukins1 and 6 (IL-1, IL-6), and prostaglandins, play animportant role in the pathogenesis of central nerv-ous system disease (Hunter et al , 1992; Alafiatayoet al, 1994; Jennings et al 1997). Furthermore, thispathology has been shown to be prevented andameliorated by drugs such as eflornithine (Jenningset al, 1997) and megazole (Enanga et al, 1998).Consideration should be given to the use of suchdrugs in the management of African trypanosomia-sis. Further, preclinical research with such drugs isneeded.

The availability of clinical centres in endemic coun-tries supported by TDR facilitated the evaluation ofthe diagnostic tests MAECT, CATT and CIATT, aswell as clinical trials of eflornithine which produceddata that were presented to the US FDA for registra-tion of the drug in 1990.

Under the auspices of TDR and its collaborators,considerable progress was made in research onAfrican trypanosomiasis in: diagnosis and develop-ment of diagnostic tests; epidemiology, host-para-site-vector relationships, animal reservoirs; devel-opment of tsetse traps and screens; better under-standing of the pathology of the disease, and drugtargeted biochemistry of trypanosomes. However,this progress has not been matched in the control ofthe disease, due to lack of capacity to sustainimproved interventions as well as civil disorder insome endemic countries. These factors were largelyresponsible for the current problems of African try-panosomiasis.

From 1994, when reorganization took place in TDRalong disciplines instead of diseases, the resourcesallocated to African trypanosomiasis decreased, andcontinued to decrease tremendously in subsequentyears, up to 64% in 2000. In view of TDR’s uniquerole in research on African trypanosomiasis, the con-sequences of this lack of funds were grave. Severaltrained researchers left trypanosomiasis research forHIV/AIDS and malaria, where they could getresearch funds. It is not surprising that the currentchairman of the Task Force on AfricanTrypanosomiasis now works on a project onHIV/AIDS. One tsetse trap expert has turned hisingenuity to making and supplying bednets for amalaria control project. Clinical research centres arerundown and have reduced capacity to conduct clin-

ical studies. One should, however, not overlook thesupport, no matter how small, that has been provid-ed by other institutions in the North to these centres.

The Bill & Melinda Gates Foundation, in December2000, awarded US$15.1 million to treat African try-panosomiasis and leishmaniasis to an internationalconsortium of researchers led by Dr Richard R.Tidwell, a scientist at University of North Carolinaat Chapel Hill, to develop new drugs to fightAfrican sleeping sickness and leishmaniasis. Thiswelcome news is without precedence in the historyof African trypanosomiasis. Some of the scientistsinvolved in this consortium already collaborate withTDR in drug discovery research and drug develop-ment. TDR should establish links and maintain col-laboration with this consortium. The primate facili-ty at the Kenya Trypanosomiasis Research Institute(KETRI), that was established with TDR’s supportmany years ago, is an asset that will be available forthe evaluation of potential lead compounds in pri-mates. There are few clinical research centres inendemic areas where clinical trials can be conductedand, therefore, these will be in demand for drugcombination trials, oral eflornithine phase 3 clinicaltrials, and trials with any potential candidate com-pounds. Two years ago, TDR started an initiative totrain clinical investigators and monitors worldwideto conduct clinical trials according to the good clini-cal practice (GCP) concept. Training will be requiredfor clinical investigators in the clinical centres thatwill be involved in clinical trials. TDR has a com-parative advantage in institutional strengtheningand can make a significant contribution to the workof this consortium towards the evaluation of candi-date compounds. TDR has links with the outsideworld, with special reference to product develop-ment, which should be maintained.

BIOINFORMATICSDuring 1993/94, TDR initiated a number of parasitegenome networks - for T. b. brucei, T. cruzi, Leishma-nia major, Schistosoma mansoni, Brugia malayi. Thenetworks are now oriented towards post genomics,and bioinformatics networks are being expandedfor data mining annotation and in-depth analysis.The T. b. brucei network should be assessed and thenecessary inputs provided to move it forward in thegenome analysis of T. b. brucei.

COORDINATION NETWORKA Human African Trypanosomiasis Treatment andDrug Resistance Network was formed in 1999 inWHO (Communicable Disease Surveillance andResponse unit). It has as objectives: to assess theeffectiveness of current treatment regimens; col-lect/disseminate information on refractoriness to


treatment; ensure availability and affordability ofexisting drugs; provide guidelines for treatment;promote research on causes of treatment failures,drugs and treatments (WHO 1999). The Go-vernment of France provides funds for running thesecretariat of the network. Participants in the net-work come from WHO (Communicable Diseasescluster, the Regional Office for Africa); MSF; theUnited States Centers for Disease Control andPrevention (CDC), Atlanta; the Swiss Tropical Insti-tute, Basel; the Institute of Tropical Medicine, Ant-werp. TDR should play an active role in the networkand participate in its meetings.

AcknowledgementsI wish to thank Professor David H. Molyneux,Professor of Tropical Health Sciences, LiverpoolSchool of Tropical Medicine, Pembroke Place,Liverpool, and Dr Jacques Pepin, AssociateProfessor, Infectious Diseases Division, Centre forInternational Health, University of Sherbrooke,Quebec, Canada, for their criticisms and commentson this article. I also wish to thank Dr Paul Nunn,Dr Johannes Sommerfeld and Dr Charity Gichuki,WHO, for reviewing the paper.

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Annex 3THE EMERGENCE AND RE-EMERGENCE OF HUMAN TRYPANOSOMIASIS (SLEEPING SICKNESS) IN AFRICA


I THE SITUATION IN ANGOLA∗Theophile JosenandoInstituto de comabate e controlo de Tripanosomiase,Luanda, Angola

Angola is a country situated at the interface betweenCentral Africa and Southern Africa and has an esti-mated population of 12 million. Sleeping sickness isa major public health problem in the country today,with more than 32 000 new cases having beendetected during the last five years. In contrast, in1974, when surveillance was more active and betterorganized, there were only three new cases.

Tsetse flies, or Glossina, the vectors of the disease, arepresent in 14 of the country’s 18 provinces. Two mil-lion out of the 4.5 million people living in the sevenprovinces affected - Zaire, Uige, Kwanza Norte,Malanje, Bengo, Kwanza Sul and the peripheralareas of Luanda - are exposed to the risk of directinfection. The number of patients has been risingcontinuously for more than ten years. The number ofnew cases identified in 1997 was 8275, the highestfigure ever reported in the history of the disease inAngola. The figures reported reflect the case detec-tion and treatment activities introduced over the lastfew years. Paradoxically, the rapid increase in thenumber of patients identified is witness to the effortsmade to manage the disease by the national healthservices with the help of non-governmental organi-zations (NGOs). In the period 1996-2000, 32 445patients were treated (Table 1).

Table 1. The number of patients treated in the years 1996-2000

Since African trypanosomiasis is invariably fatalwhen left untreated, the increase in number of treat-ed patients means a decrease in mortality. So the 32331 patients treated durng the period 1996-2000escaped from certain death. The treatment centresare in areas of military confrontation (war front) andthis might explain the fluctuations in number ofpatients treated each year. In addition, this has led tolow surveillance coverage and inadequate screeningof the population at risk, suggesting that the truenumber of patients is very high.

The main reasons for the persistence of sleepingsickness in Angola are inaccessibility of most of theterritory affected by trypanosomiasis, the scant sur-veillance coverage of endemic areas, for reasons

already stated, and the continual movement of thepopulation, which includes carriers of the parasitewho act as a reservoir for its dissemination. In addi-tion, socioeconomic factors including poverty arewithout doubt implicated in the spread and persist-ence of sleeping sickness.

At the symposium organized by Fundanga, theAngolan Foundation for Solidarity and Deve-lopment, and held in September 1998 on the prem-ises of the Angolan National Assembly in Luanda,sleeping sickness was clearly identified as a majorpublic health problem. As a result, the AngolanGovernment committed itself financially to takingaction against the disease, with annual financing ofabout $1 million in local currency (Kwanza) and $1million in convertible currency (dollars) since 1998for activities of the National Programme for controlof human trypanosomiasis.

The National Programme has been graduallystrengthened in its leadership role, and its effortshave borne fruit with the transformation of theNational Programme into the Institute for theControl of Human and Animal Trypanosomiasis(Instituto de Combate e Controlo das Trypano-somiases, or ICCT) by Government decree 2/00 of14 January 2000. The newly created Institute enjoysautonomy in management and its position haschanged in the structural chart of the Ministry ofHealth. This autonomy first found expression in thedelegation of management of salaries of personnelworking at ICCT headquarters and at the VianaReference Centre.

Control of trypanosomiasis addresses one of fivepriority endemic diseases (AIDS, trypanosomiasis,tuberculosis, leprosy, malaria) for the Ministry ofHealth in 2001. In Angola, the inclusion of ICCTnational staff in the activities of NGOs is effective,enabling it to be present at most places wherepatients are managed.

This year, the Ministry of Health assigned fivephysicians to the ICCT and is presently trying toobtain greater participation by the State in thefinancing of the Programme. There is also effectivefinancial participation by the autonomous provin-ces, especially the province of Zaire, which ensuressupplies of specific drugs for the disease.

Since the refurbishment of the screening and treat-ment centre and the Viana Reference Centre, somestaff have been transferred from the headquarters ofICCT to the Viana Reference Centre, including onephysician in charge, two clinicians, one head of lab-oratory, and one clinical assistant. This doubling of

* The orginal manuscript is in French

1996 1997 1998 1999 2000

6786 8275 7373 5351 4546


staff has enabled us to open a reference treatmentcentre, supported by the research projects of theSwiss Tropical Institute. We can therefore say that, inaddition to the refurbishment and the equipment ofthe laboratories, functional practice and expertisehave been developed.

A large number of NGOs operate in Angola. Most ofthem were originally working independently ofeach other, applying different methodologies to thecontrol of African trypanosomiasis, but numerousterritorial disputes arose and it became necessaryfor the Ministry of Health to take over direction oftrypanosomiasis control. As a result, coordination ofthe national and international NGOs through ICCTwas initiated. Many meetings were held for consul-tation and coordination (on average, one every threemonths), while project visits by ICCT staff alsohelped to enhance the legitimacy and credibility ofthe Institute, facilitating the coordination of inter-ventions. Currently, there is no more rivalry and therole of the ICCT is understood by all, such that thenumber of visits to partner treatment centres hassubstantially increased. The NGOs have acceptedstandardization of the methods for case detection,treatment and data collection, while a growingnumber of technical and medical staff belonging toICCT now work with NGOs, where they are replac-ing expatriate staff.

The NGOs that have worked, and are still working, inAngola are shown in Table 2.

Table 2. NGO presence in endemic areas between1996 and 2000

RECOMMENDATIONS FOR ACTION• Combat poverty at all levels. This is one of the

major objective, poverty being one of the factorsthat aggravate the socioeconomic status of thepopulation, including those suffering from try-panosomiasis.

• Adopt a strategy of permanent, regular, epidemi-ological surveillance, consisting of active diagno-sis of new cases of the disease, with a view toearly detection, treatment and effective follow-upof sufferers.

• Mobilize resources to combat the disease, such asdrugs, reagents, equipment and traps.

• Strengthen vector control actions, using the mostappropriate techniques to combat tsetse flies.

• Draw up an information, education and commu-nication (IEC) plan for the population involvingpolitical leaders, leaders of civil society and healthofficials in order to take concrete steps to controlthis disease.

• Draw up a plan of work for animal trypanosomi-asis and update Glossina mapping of the country.

• Strengthen cooperation between the ICCT and itsnational and international partners.

• Prepare coherent and feasible projects that areguaranteed to receive national and internationalfunding.

• Look for ways and means to motivate ICCT work-ers by improving working and social conditions.Organization Location 1996 1997 1998 1999 2000

MSF-B N’dalatanado + + + +

MSF-F Maquela do Zombo +Quiculungo +

Caritas M’banza-Congo + + +UigeQuitexe + + + + +Lucala + + +Negage + + +

APN Dondo + + + + +Casuala + + + + +

MMC Quiçama + + + +

FUNDANGA Caxito + +

ADRA Cacuso


II THE SITUATION IN TANZANIA

Stafford Kibona,National Institute of Medical Research, Tabora, Tanzania

GENERAL OVERVIEWIn the United Republic of Tanzania, sleeping sick-ness, also known as human African trypanosomiasis(HAT), is one of the major public health problems.Sleeping sickness was first recorded in 1922 inMaswa district, south of Lake Victoria (Kilama et al1981). It then spread throughout mainland Tanzaniaand is currently endemic in eight regions, namelyArusha, Lindi, Ruvuma, Kagera, Kigoma, Tabora,Mbeya, Rukwa. The annual average number ofcases for the last decade was 264. There are isolatedfoci, most of which have been producing cases formany years. Heavy foci exist in Kigoma Region(Table 1).

Table 1. Number of sleeping sickness incidences diag-nosed from district hospitals in six regions of Tanzaniafor the past five years.

Although sleeping sickness was present in eightregions during the past ten years, the disease wasmore concentrated in Kasulu and Kibondo districtsof Kigoma Region, which accounted for about 90%of the cases. These figures are underestimates due tolack of accurate diagnosis and under-reporting.

KIGOMA SITUATIONAn outbreak of sleeping sickness is currently beingexperienced in Kigoma Region of Western Tanzaniaalong Lake Tanganyika. The disease represents acontinuing threat to the health and morale of manycommunities in the area. All the districts of KigomaRegion are affected by sleeping sickness, withKibondo and Kasulu districts producing a great pro-portion of the total cases over the past few years.The whole of Kigoma Region is on the Western FlyBelt, a portion of a large forest which extends, withfew breaks, over an area of 10 368 sq. km. and liesbetween 31°E and 24.7°W, 3.5°N and 5.2°S. Here,game is fairly abundant, and the people regardsleeping sickness as an old disease.

The worst affected area is along the MalagarasiValley, where the vegetation is characterized by bigtrees overhanging open grassland. The populationin this large area is, for the most part, concentratedin villages on the Kigoma–Kibondo trunk road.Sleeping sickness infections are contracted mainlyby those going into the bush to hunt, fish, collecthoney, beeswax, etc. There are few cases of perido-mestic infection.

Each village has a population of over 2000. Some ofthe villages are remote from the main road, forexample Kagera and Mvinza in the north-east andKitanga and Heru-Ushingu in the extreme north-west.

Villages Affected by Sleeping Sickness inKasulu District Thirty-two villages are exposed to the risk of infec-tion in the endemic district of Kasulu. Of these, thefollowing are regarded as highest risk areas: Heru,Kitagata, Kitanga, Makere, Mugombe, Mvugwe,Mwali, Nyachenda, Nyakitonto, Nyamidaho,Nyarugusu, Shingu. Others include Kagera,Kaguruka, Kitema, Mvinza, Rungwe, Mpya, Titye.Overall, the estimated population at risk of infectionis 230 000. Each village has a health clinic. There isone health centre for every six villages.

Villages in Kibondo DistrictSleeping sickness occurs in the following villages inthis district: Bitare, Biturana, Busunzu, Kanembwa,Kazira mihunda, Kifura, Kilemba, Kingoro,Kitahana, Kumbanga, Kumhasha, Kumshindwi,Malagarasi, Mkabuye, Mvugwe, Nduta, Nyankwi,Nyaruyoba, Rusohoko. Each village has a dispensa-ry. Each health centre serves six or seven villages.

Refugee Camps in KigomaThere are about 280 000 refugees, mainly from theEastern part of the Democratic Republic of Congo(DRC), settled in camps in Kigoma region. Sleepingsickness cases have already been detected in thesecamps and there is concern that an overlap of gam-biense and rhodesiense sleeping sickness exists inthe region.

CAUSES OF EMERGENCE AND RE-EMERGENCEThe current situation of sleeping sickness inTanzania, especially the outbreak in Kigoma region,is caused by the following factors:• Poor surveillance due to inadequate funding and

staff.• Inadequate and erratic supply of specific try-

panocidal drugs.• Poorly equipped field laboratories for diagnosis.

Region District 1996 1997 1998 1999 2000

KIGOMA Kibondo 212 286 206 410 376

ARUSHA Kasulu 155 198 172 156 191

Babati 12 19 15 12 34

Monduli 19 6 2 9

Hanang 5 3 8 2

TABORA Urambo 1 7 2 9

RUKWA Nkansi 4 1 7 8 6

Mpanda 5 1 - - -

MBEYA Chunya 6 4 - - -

TOTAL 400 531 421 588 627


• Opening up of new unauthorized settlements andfarms in the endemic areas of sleeping sickness. Inthe past, the people in Nyakitonto area, for exam-ple, had wished to live in the valley (Kitome area),which is wooded and well watered but also heavi-ly infested with tsetse fly. Permissions to do so hadbeen consistently denied. However, a few familieshad occupied the area by 1983 and, as was perhapsinevitable, many infections occurred thereafter.

• Lack of monitoring and health education cam-paigns.

• Increased forest activities including cultivationoutside the villages or on the buffer zone. Some ofthe planned villages were, unfortunately, locatedin a rather dry and infertile area causing the vil-lagers to open new farmlands outside the villages.

• Apparent bush regeneration with resultant tsetseencroachment.

• The highly probable introduction of a virulentstrain of T. b. rhodesiense.

The serious outbreaks of sleeping sickness in thedistrict are a disappointing setback in the diseasecontrol efforts of the country, and indicate a neces-sity for review of sleeping sickness control meas-ures in the area. A study in Kigoma region sug-gests the following factors are important in thecurrent outbreak:

• Farming activities carried on outside the protect-ed area.

• Inter-village visits through tsetse infested bushesin search of the basic necessities of life.

• Increased forest activities – honey and beeswaxcollection, fishing, hunting, etc.

• Peridomestic activities e.g. firewood gathering,fetching water, cutting poles for building.

• Visits into the wildlife areas where no human set-tlement is allowed.

The problem that now confronts Tanzania is thatsleeping sickness is still endemic and there is lax-ity of control measures. Compared to otherendemic diseases, the number of human deathsfrom sleeping sickness appears insignificant, buteven temporary exacerbation of the disease fright-ens the local people.

While it is impossible to predict the future, thepossibility of a larger outbreak must be consid-ered. With the evidence currently available, itwould be a reasonable precaution to step up con-trol measures.

IDENTITY OF THE HUMAN INFECTIVETRYPANOSOME IN TANZANIAThe occurrence of the chronic syndrome of sleepingsickness, together with the marked variation in effi-cacy of chemotherapeutic treatments, in Tanzania,may be cited as indications that the trypanosomesconstitute a heterogeneous complex of organismsincluding perhaps a mixture of T. b. rhodesiense andT. b. gambiense.

Old records show that the Malagarasi focus(Kigoma region), for example, in north-westernTanzania, is an old gambiense sleeping sicknessfocus with the last case of gambiense infection hav-ing been reported in 1958 (Kihamia et al. 1991). Thearea is now considered endemic for rhodesiensesleeping sickness. In Tanzania there are considerabledifferences in the clinical types of human try-panosomiasis, with the severity of disease varyingaccording to geographical location and becomingless virulent the further one goes south. While thismay be attributed to the heterogeneity of T. b. rho-desiense strains (Komba et al. 1997), there is concernthat it may be due to the occurrence of the twospecies of trypanosome. Furthermore, the presenceof large numbers of refugees from the DRC, a coun-try known to be endemic to gambiense sleepingsickness, increases this possibility.

Further studies are required to:• Investigate why the disease is localized in specif-

ic foci despite the tsetse fly and reservoir animalsbeing present in large areas of the country.

• Investigate the distribution of T. b. rhodesiensestrains in Tanzania.

• Investigate the possibility of an overlap of rhode-siense and gambiense sleeping sickness, especial-ly in Kigoma focus, which has an influx ofrefugees from the DRC.

ReferencesKilama WL, Mtera KNM, Paul RK. Epidemiology ofhuman trypanosomiasis in Tanzania. In: Proceedingsof the 17th Meeting of the International ScientificCouncil of Trypanosomiasis Research and Control, 1981,Publication No. 112, pg. 187.

Kihamia CM et al. Trypanosomiasis. In: Health anddiseases in Tanzania. 1991, Harper Collins Academic,UK.

Komba EK, et al. Genetic diversity amongTrypanosoma brucei rhodesiense isolates fromTanzania. Parasitology, 1997, 115:571-579.


III THE SITUATION IN UGANDAAND SUDAN

D.B. Mbulamberi,Ministry of Health, P.O. Box 7272, Kampala, Uganda.

SUMMARYSleeping sickness is a disease of the rural poorwhich tends to emerge and re-emerge in epidemicsin some 36 sub-Saharan African countries, whereclose to 50 million people are at risk of contractingthe disease. Together, these 36 countries report onlyabout 25 000 new cases of the disease to WHO annu-ally. This is an obvious underestimate attributed topoor reporting, difficulty in diagnosing the disease,and poor accessibility of the affected areas. The truefigure is currently estimated to exceed 300 000 newcases annually.

In the recent past, Uganda and Sudan have not beenspared epidemics of this disease. A devasting epi-demic of T. b. rhodesiense sleeping sickness hasoccurred in south-eastern Uganda and one of T. b.gambiense in the West Nile region of the country(Uganda). In Southern Sudan, along the border withUganda, there is another epidemic of T. b. gambiensesleeping sickness.

The causes of the emergence and re-emergence ofepidemics of this disease are varied, but can be con-veniently grouped into political, economic, behav-ioural factors, and the effects of climate and vegeta-tion on tsetse fly distribution.

To limit the impact on human lives in these twocountries, external support will be required toimplement strategies for disease and vector control.On the one hand, donor agencies, NGOs and mis-sion organizations could play an important role insupporting these control efforts. On the other hand,national authorities will need to control and coordi-nate these efforts with assistance from WHO and theinternational community.

This paper presents a general introduction to thedisease (sleeping sickness) and a brief account offactual epidemic outbreaks in Uganda and theSudan. The paper then proceeds to discuss, in gen-eral terms, possible factors for the emergence andre-emergence of epidemics of the disease.

INTRODUCTION

Historical Aspects of the DiseaseTrypanosoma brucei, and the role of the tsetse fly(Glossina spp.) as its vector, was identified in game

animals in Zululand by David Bruce two centuriesago (WHO, 1995). Later, morphologically identicaltrypanosomes were identified in the blood of aEuropean from The Gambia, West Africa (Dutton,1902), and transmission by riverine tsetse (Glossinapalpalis) was confirmed. Subsequent studiesrevealed the extensive, often focal, distribution ofthe disease, the substantial endemicity and thechronic progressive nature of human infection inWest and Central Africa.

The disease was called Gambian trypanosomiasisand the parasite, T. gambiense (later T. brucei T. bruceigambiense). In 1908, a severe rapidly fatal trypanoso-mal infection was identified in the Luangwa valley,Zambia. Further investigation confirmed its clinicalseverity, the distinct epidemiology with transmis-sion via savannah tsetse, and the zoonotic nature ofinfection from game animals harbouring T. brucei.This led to the description of Rhodesian trypanoso-miasis due to T. rhodesiense (T. b. rhodesiense). Othermembers of the T. brucei T. brucei group, that arenon-infective to humans and occur in game anddomestic animals, were designated T. brucei (subse-quently T. b. brucei).

The General Epidemiology of the DiseaseTrypanosomes, the parasites which cause the dis-ease, occur in the blood of man and animals as thetrypomastigote.

All members of the T. brucei group are morphologi-cally identical. The parasites have evolved mecha-nisms for evading host immune responses throughvariation of their surface antigen glycoproteins. Thezoonotic nature of T. b. rhodesiense was initiallyestablished by inoculation of ‘volunteers’ with para-sites from a bushbuck (Heish et al, 1958), and laterfrom domestic cattle.

Subsequently, the blood incubation infectivity test(BIIT) was developed to assess the human infectivepotential of parasites from a range of wild anddomestic animals. More recently, a number ofmolecular techniques, especially isoenzyme analy-sis (Stevens and Godfrey, 1992) and restriction frag-ment length polymorphism (RFLP), have been usedas markers for parasite strains to explore the molec-ular epidemiology of this complex group of para-sites. These techniques allow T. b. gambiense to bedistinguished from T. b. rhodesiense.

In West and Central Africa, sleeping sickness istransmitted by riverine species of tsetse fly (palpalisgroup), which require sustained levels of humidityand prefer dense riverine habitats. These tsetse fliesfeed preferentially on man, especially where man-


fly contact is high, such as at water collection andbathing points, river crossings and sacred groves.For the riverine species of tsetse fly, man providesthe reservoir of infection, although both wild anddomestic animals may play a minor role in particu-lar foci. Throughout most of the range, T. b. rhode-siense transmission is effected by savannah speciesof tsetse (morsitans group). This group of tsetse fliessurvives in drier, more open areas of woodland,savannah and acacia thickets, and prefers to feed ongame animals and domestic stock. Human infectionoccurs sporadically in individuals coming into con-tact with the zoonotic cycle, for example poachers,hunters, honey gatherers, firewood collectors andtourists. A wide spectrum of animals, notably gameanimals and domestic cattle, provide a reservoir ofinfection. However, in East Africa, the epidemiologyis different in that T. b. rhodesiense is transmitted bya riverine species of tsetse, namely G. fuscipesfuscipes, and domestic cattle are the main reservoir.This situation creates a lot of potential for epidemicoutbreaks of the disease.

Clinical Manifestation of the DiseaseIn most endemic areas, T. b. gambiense causes a pro-tracted, often initially unrecognized, illness withepisodes of fever, headache and malaise, accompa-nied by progressive lymphadenopathy and followedlater by the development of a progressive, fatal,meningoencephalitis. This contrasts with the acute,severe, febrile disease observed with T. b. rhodesiense,with rapid progression to meningoencephalitis.There is relentless deterioration to a stuporous state,with cachexia, wasting and progressive malnutrition,deepening coma and death, within a few months inthe case of T. b. rhodesiense and extending for monthsor even years in the case of T. b. gambiense.

AVAILABLE OPPORTUNITIES FOR THECONTROL OF SLEEPING SICKNESSThe principle of control and prevention of sleepingsickness relies on an integrated strategy of continu-ous surveillance, involving diagnosis and treatmentof the population at risk, and vector control whereapplicable (de Raadt, 1986). A number of tools fordiagnosis and vector control have been developedthrough research during the past decade and are,indeed, field applicable by national health services.These include the card agglutination test for try-panosomiasis (CATT) (Magnus et al, 1978) for sero-diagnosis, and the miniature anion-exchange cen-trifugation technique (MAECT) (Lumsden et al,1979) for parasitological diagnosis. The antigenELISA, developed by Nantulya (1989) and evaluat-ed for detection of gambiense (Nantulya et al, 1992)and rhodesiense (Komba et al, 1992) sleeping sick-ness, was subsequently modified into a latex agglu-

tination test. Lancien in Uganda (1991) confirmedthat insecticide impregnated traps can be used, withcommunity participation, to control sleeping sick-ness epidemics.

Until recently, the treatment of sleeping sicknessrelied essentially on three drugs, namely pentami-dine, suramin and melarsoprol. Pentamidine, adiamidine introduced in 1937, is currently availableas pentamidine isethionate and is effective againstearly infections of T. b. gambiense. Suramin, whichwas introduced in 1922, is effective against earlyinfections of both T. b. gambiense and T. b. rhode-siense. Melarsoprol, a trivalent arsenical introduced in1949, was the only drug available, until 1990, for thetreatment of late infections of both T. b. gambienseand T. b. rhodesiense. All these drugs have adverseside effects, melarsoprol causing reactive encepha-lopathy in 5—10% of patients treated, with a fataloutcome in 1 -5%. Resistance of T. b. gambiense to pen-tamidine, and of both T. b. gambiense and T. b. rhode-siense to melarsoprol, occurs. Nifurtimox, a 5—nitro-furan, is currently being used, either singly or in com-bination with other drugs, to treat late-stage gambi-ense infections on a compassionate basis.

Eflornithine (DFMO), a potent inhibitor ofpolyamine synthesis, was developed for the treat-ment of sleeping sickness through collaborationbetween Marion Merrel Dow Inc., USA, and theUNDP/World Bank/WHO Special programme forResearch and Training in Tropical Diseases (TDR).However, while this drug provides the best alterna-tive treatment to melarsoprol for gambiense sleepingsickness, alone it is ineffective against T. b. rhodesienseinfection. Therefore, no alternative treatment forlate-stage rhodesiense infection is yet available.

The availability of all these drugs is currently high-ly uncertain, with the various manufacturing firmseither threatening to stop, or having alreadystopped, their production. It is evident that thetreatment of sleeping sickness is still unsatisfactory.The ideal trypanocide should be safe and effective.It must have a simple mode of administration toallow its use under rural conditions where healthfacilities are usually of a poor standard, and, aboveall, it should be affordable.

THE PAST AND PRESENT SLEEPINGSICKNESS SITUATION IN UGANDA AND SUDAN

UgandaThe sleeping sickness epidemic which devastatedthe shores of Lake Victoria at the beginning of the


last century is a famous event in the annals of tropi-cal medicine. It is famous because an estimated onequarter to one third of a million people lost theirlives (Langlands, 1967). The same epidemic broughtcontroversy as to whether Dr Castellani or ColonelBruce first identified the trypanosome as the causeof the epidemic.

The cause of the epidemic was attributed to T. gam-biense introduced to this part of the country by aparty accompanying the explorer Lugard from theCongo basin on relief of the Emin Pasha expeditionin 1894 (Christy, 1903). However, it is more accurateto say that the cause lay in the general increase insocial, commercial and military mobility whichdeveloped throughout tropical Africa in the latenineteenth and early twentieth centuries.

Another outbreak involving about 2500 personsoccurred in the same area from Jinja eastwards tothe border with Kenya between 1939 and 1945(Mackichan, 1944-45). The most striking feature ofthis epidemic was the virulence and rapidity of thefatal course of the disease observed on animal inoc-ulation. Thus, it is believed, the epidemic wascaused by T. rhodesiense. The first cases detectedwere among migrant workers employed on Kakirasugar estates. Since these immigrants came fromareas of reasonable proximity to the infected areas ofTanganyika (now Tanzania), this epidemic wasthought to have been introduced by them.

Since that outbreak, cases continued to be reportedfrom within the infected area, though not in epi-demic numbers. In 1971, infection spilled north ofthe usual focus and involved up to 169 persons.

Following the control of the small epidemic of 1971,surveillance programmes were not institutedbecause of the prevailing political and economicatmosphere in the country at that time. There wasindiscriminate and haphazard movement of peopleand livestock across the traditional trypanosomiasisbarrier zone. Besides, smuggling of commodities,including cattle, between Kenya and Uganda, acrossthe zone, became a means of livelihood. It was there-fore difficult for the Ministry of Health teams toenforce surveillance measures. The Tsetse ControlDepartment could not carry out control programmesdue to lack of insecticide, transport and humanresources. Thus, there was total breakdown of controlmeasures and hence, by 1976, the stage was set foranother epidemic outbreak of the disease in the area.

This new outbreak started in Luuka County ofIganga district in August 1976. Unfortunately, inJune 1977, the East African Community collapsed

and EATRO, the department probably in the bestposition to help contain this epidemic during itsoutset, lost valuable logistics to a partner state andbecame helpless. The Ministry of Health postedmicroscopists to the area and later opened up treat-ment centres in the area. However, the epidemiccontinued to increase in magnitude from 52 cases in1976 to over 8000 cases in 1980 (Fig. 1).

In the West Nile region, a new outbreak of T. b. gam-biense sleeping sickness occurred along the Dachariver. During the first year of the outbreak, a total of12 cases were recorded. In the following year (1958),this new focus produced 7 cases and it seemed as ifthe outbreak had ceased. However, the 1959 inci-dence of 30 cases, the highest annual figure since thebig epidemics had been finally checked 12 yearsbefore, made it evident that the outbreak was by nomeans under control.

However, this outbreak was finally brought undercontrol and the situation remained largely stablethereafter until the early 1980s, when the currentoutbreak in the region started. This current outbreakis associated with the war of liberation against IdiAmin in 1979, when most local residents in theregion fled into exile in Southern Sudan where therewas a ravaging epidemic of T. b. gambiense sleepingsickness, then as now. When these local residentsreturned to the West Nile region in the early 1980s,some of them had the infection, which they intro-duced into the area. The epidemiological trend ofthe disease in the region (West Nile), over the years,is shown in Fig. 2.

Figure 2. Annual incidence of sleeping sickness due to

T. b. gambiense in North Western Uganda, 1981-1999

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SudanSleeping sickness due to T. b. gambiense has beenknown to occur in the Southern Sudan since theearly 20th century. Epidemics of the disease haveoccurred mainly in the Southern and South-westernparts of the country bordering Uganda, theDemocratic Republic of the Congo (DRC), and theCentral African Republic. In addition, cases of thedisease have been reported along the Ethiopian bor-der, in Raga since 1909, in Yei since 1910, in Kajokajisince 1914, in Nimule since 1915, in Tambura since1918, and in Yambio since 1924.

Almost all the aforementioned foci are still active.Duku (1979) attributed these epidemic outbreaks to:the presence of active foci in neighbouring coun-tries, particularly those where tribal settlementsstraddled the borders; widespread distribution ofthe vectors; and political upheavals, instability andcivil disturbance.

The current flare-up of the disease started after thesigning of the Addis Ababa peace agreement in1972. The relative peace which followed the signingof this agreement made it possible for some controlmeasures to be instituted with external assistancefrom WHO and the Government of the Kingdom ofBelgium between 1974 and 1978, hence the avail-ability of the information shown in Fig. 3. Otherwiseinformation on the current epidemic in the SouthernSudan is not easily available.

FACTORS RESPONSIBLE FOR THEEMERGENCE AND RE-EMERGENCE OFSLEEPING SICKNESS

Factors responsible for the emergence and re-emer-gence of sleeping sickness are varied and diverse.Mbulamberi (1989) gave an outline of these factors.A brief account, a modified version of these factors,is given below.

Civil Disturbance and WarCivil disturbance and war cause extensive and often

uncontrolled movement of people into areas thatmay have been previously abandoned because ofepidemics, thereby promoting circulation of the par-asite in the population and the risk of contactbetween people and the tsetse flies. Civil distur-bance and war will, in the final analysis, lead to abreakdown in vital social services including med-ical and vector surveillance programmes. This isobviously the most important factor in the case ofthe current epidemics in both Uganda andSouthern Sudan, as is the case in most other sub-Saharan African countries, e.g. Angola, Mozambi-que, the DRC.

Declining EconomiesDeclining economies, as is the case in most sub-Saharan African countries, will dictate reducedfinancing of disease control programmes. This, inturn, will affect field control activities as well as theimplementation of advances so far made in diagno-sis, treatment and vector control. This is mainlybecause these new advances are not available on thelocal market and therefore require importing intothe country, for which foreign currency is required.In addition, reduced financing is likely to lead to thetemptation of progressively dismantling verticaldisease control programmes in preference for inte-grated, community-based programmes, thus lead-ing to loss of focus.

Behavioural FactorsOne of the factors to be considered here is the lowpriority rating accorded to sleeping sickness on thepart of both donors and national governments. Thisis despite the negative impact of the disease ondevelopment. One possible explanation is thatsleeping sickness control programmes do not havemuch appeal for international aid donors due tovarious factors including: the regional distributionof the disease and mainly rural nature of the prob-lem; the relatively small number of new casesreported annually compared to other diseases; therequirement for long-term input to control pro-grammes for sustainability in the absence of theprospect of eradication. Paradoxically, when epi-demics of the disease occur, financial support ismade easily available in amounts which are usuallydisproportionately higher than those required forthe regular preventive measures (Kuzoe, 1993).

Another pertinent behavioural factor, in this respect,is the population density of the tsetse flies and theirfeeding behaviour. A tsetse fly feeding on a numberof animals, and possibly also on man, may becomeinfected with many different strains of try-panosome. Most of these strains will be non-patho-genic for man and, even if a man-infective strain is

Figure 3. Self-reporting cases of sleeping sickness in Yambio district,

S. Sudan, 1974-1978

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acquired by the fly, the tendency will be for it to beso diluted by the non-pathogenic strains that it willnot be passed on in sufficient number to cause infec-tion in man.

Another important behavioural factor is increasedman-fly contact. This phenomenon occurs mostcommonly during the hot, dry season with theresult that transmission is enhanced. Indeed, a peri-od of drought almost invariably means an increasein the number of infections because the few sourcesof water are shared by man, tsetse flies and gameanimals, in close association; there is also morehunting and more searching for wild forest productsat times when crops are bad. This phenomenon isparticularly applicable to T. rhodesiense infections(Willet, 1965, and many other workers).

The presence of domestic and wild animal reservoirhosts is another important factor. The pig in the caseof T. gambiense, and cattle in the case of T. rhodesiense,have been incriminated as domestic animal reser-voir hosts, while the kob and hartebeest in the caseof T. gambiense and the bushbuck in the case of T.rhodesiense have been incriminated as wild gamereservoir hosts. The bushbuck is particularly impor-tant because it tends to live in thickets near humanhabitation, which puts it in close contact with man.

The appearance of different forms of the parasite isanother important factor. The appearance of suchparasites may be due either to the parasites beingintroduced from outside the area or to geneticchanges in the parasite. There is at least a suspicion,based on field observations, that zymodemes of try-panosome introduced into fresh localities mayexhibit an enhanced ability to spread through thecommunity. Scott (1961) reported two instances inwhich the introduction of infected persons from anestablished epidemic area resulted in outbreaks ofthe disease in endemic localities far removed fromthe original focus of infection. There are other simi-lar observations suggesting that severe local out-breaks which quickly follow the introduction ofinfected persons to fresh localities are, in some way,connected with enhanced ability of the zymodemeto spread. Indeed, the possible existence of epidem-ic trypanosome zymodemes has been advanced bysome workers.

Another factor of importance are the changes inpopulation movements and population growth. It isgenerally supposed that population movements areliable to precipitate epidemics. Refugees displacedas a result of war, famine, earthquakes and othersimilar occurrences are notoriously prone to diseasein epidemic form, as are immigrant labour forces

recruited for large-scale construction work (tropicalaggregation of labour) and pilgrims attending majorreligious festivals.

A new population in an area may spark off an epi-demic outbreak of sleeping sickness as a result ofimported cases among them, which may be suffi-ciently large to increase the reservoir of infectionavailable to the insect vector and so, in a quantita-tive manner, promote transmission. An importedstrain may also show quantitative differences suchas enhanced virulence or ability to spread, or maybe one to which the indigenous population has notbeen previously exposed and to which no resistancehas been acquired. This phenomenon can also oper-ate vice versa. Further, as with some other diseases,the periodicity of epidemics of sleeping sicknessmay be associated with the growing up of a newgeneration of people with no previous experience ofthe disease.

The occurrence of sub-acute cases of the disease isyet another important factor. The presence of anundetected and perhaps unsuspected reservoir ofinfection in the form of human “healthy carriers” ofthe disease, which has been reported by severalworkers (Buyst, 1977; Rickman, 1974; Woodruffet al, 1982), has important epidemiological implica-tions.

Under conditions in which man-biting tsetse arecommon and where people congregate, the ambu-lant human carrier assumes a powerful potential forthe onward transmission and spread of sleepingsickness. The occurrence of asymptomatic carriersof rhodesiense sleeping sickness is certainly low.However, sleeping sickness cases with non-specificsymptoms (fever, headache) who remain ambulantfor several weeks are common, and they too may beimportant reservoirs of infection where man-flycontact is intense (Wurapa et al, 1984). This threat isalso present among many early cases of the gambi-ense disease, in which the initial stages are general-ly relatively mild and the victim may continue towork for many months or even years before he iseventually driven, by increasing illness, to seektreatment or to retire to his home. During this time,he is a constant source of infection to tsetse so thatthe very nature of the illness provides great oppor-tunities for its spread. In both the Ugandan andSouthern Sudan situation, the question of delayeddiagnosis and treatment is a big factor.

Another critical factor in this category is humanbehaviour and activities in the fly’s habitat. Often,man becomes infected during travel, hunting, fish-ing, collection of honey or when working in the bush


which the fly inhabits. Fishing and “honey-hunting”are particularly hazardous occupations. While fish-ing in riverine pools surrounded by thickets, peoplemay be in close contact with tsetse flies for manydays at a time, a situation in which the associationbetween humans, bushbuck and tsetse fly is likely tobe significant and conducive to transmission andspread of the disease. Wyatt et al. (1985), working innorth-east Zambia, found fishing to be more com-mon among cases of sleeping sickness than controls.Fishing represented a hazard both while walking tothe stream and while engaged in the activity itself.

Climate, Vegetation and Tsetse FlyDistribution FactorsClimate appears to be of more than ordinary impor-tance. At higher temperatures, there is an increasedsalivary gland infection rate in the tsetse fly, and, inaddition to this direct effect, there are many ways inwhich climate influences a closer associationbetween man and fly.

In the current epidemic of sleeping sickness in south-eastern Uganda, heavy rains coupled with the abun-dant growth of Lantana camara thickets provided G.f. fuscipes with suitable conditions well outside itsusual riverine habitat, so it was able to live and breedin the vegetation surrounding homesteads.

Climate also has an influence on where peoplechoose to live, and on the population density of bothflies and human beings, as discussed by Ford (1971).This is relevant to the proper use and full develop-ment of land, which is the ultimate aim of eradicat-ing the tsetse fly and trypanosomiasis.

Infection rates in tsetse flies, and their infectivity, areaffected by climate. Wijers (1960) observed thatinfection rates were highest in flies taking an infec-tive blood-meal on the day on which they emerged,somewhat lower on the second day after emergence,and did not occur thereafter. Thus, the fact that fliesemerging during the hot season are likely to feedearly in their adult life means that infection rates inthe fly are maximal during the hot, dry season.However, the number of trypanosomes inoculatedby an infected tsetse fly varies greatly, even amongflies infected from the same host and in the same flyat different times.

Climate also affects tsetse longevity. Flies emergingat the end of the hot, dry season are particularlyreceptive to trypanosome infection since they willfeed early in adult life. With the onset of the rainyseason, the expectation of life of a tsetse fly is maxi-mal, so that a combination of these factors producesa situation in which infected flies are liable to sur-

vive for protracted periods. This enhances thepotential for these flies to transmit the disease, ofcourse depending on their infection rates.

ReferencesChristy C. The Epidemiology and Etiology of thesleeping sickness epidemic in the Equatorial EastAfrica with clinical observations. Report of theSleeping Sickness Commission of the Royal Society,1903, 3:3-32.

Buyst H. The epidemiology of sleeping sickness inthe historical Luangwa Valley. Annales de la SociétéBelge de Médecine Tropicale, 1977, 57(4-5):349-359.

De Raadt P. Integrated sleeping sickness control. In:Proceedings of the CEC International Symposium, Ispra,Italy, 1986, 4:147-151.

Duku MO. Human trypanosomiasis in the SouthernSudan: Present situation and control measures in:International Scientific Council for TrypanosomiasisResearch and Control 16th meeting, Yaounde Cameroon,1979, 139-145.

Dutton JE. Preliminary note upon a trypanosomeoccurring in the blood of man. Thomas YatesLaboratory Report, 1902, 4:455.

Ford J. The role of the trypanosomes in African ecology -a study of the tsetse fly problem. Oxford, Clarendonpress, 1971, 568 pp.

Heisch RB, McMahon JP, Manson Bahr PEC. Theisolation of Trypanosoma rhodesiense from a bush-buck. British Medical Journal, 1958, ii:1203.

Komba EK, et al. Multicentre evaluation of an anti-gen-detection ELISA for the diagnosis of Trypanosomabrucei rhodesiense sleeping sickness. Bulletin of theWorld Health Organization, 1992, 70:57-61.

Kuzoe FAS. Current situation of African trypanoso-miasis. Acta Tropica, 1993, 54:153-162.

Lancien J. Lutte contre la maladie du sommeil dansle sud-est Ouganda par le piégeage des glossines.Annales de la Société Belge de Médecine Tropicale, 1991,71 (Suppl.1): 35-47.

Langlands BW. The sleeping sickness epidemic inUganda 1900-1920: a study in historical geography.Department of Geography, Makerere UniversityCollege, Kampala, 1967 (unpublished document).

Lumsden WGR et al. Trypanosoma brucei: miniatureanion exchange centrifugation for detection of low


parasitemias: adaptation for field use. Transactions ofthe Royal Society of Tropical Medicine and Hygiene,1979, 73:312-317.

Mackichan IW. Rhodesian sleeping sickness in east-ern Uganda. Transactions of the Royal Society ofTropical Medicine and Hygiene, 1944-45, 38:49-60.

Magnus E, Vervoort T, Van Meirvenne N. A cardagglutination test with stained trypanosomes(CATT) for the serological diagnosis of T. b. gambi-ense trypanosomiasis. Annales de la Société Belge deMédecine Tropicale, 1978, 58:169-176.

Mbulamberi DB. Possible causes leading to an epi-demic outbreak of sleeping sickness: facts andhypotheses. Annales de la Société Belge de MédecineTropicale, 1989, 69 (Suppl. 1): 173-179.

Nantulya VM. An antigen detection enzymeimmuno assay for the diagnosis of rhodesiensesleeping sickness. Parasite Immunology, 1989, 11:69-75.

Nantulya VM, Doua F, Moilisho S. Diagnosis ofTrypanosoma brucei gambiense sleeping sickness usingan antigen detection enzyme-linked immunosor-bent assay. Transactions of the Royal Society of TropicalMedicine and Hygiene, 1992, 86:42-45.

Rickman KR. Investigations into an outbreak of thelower Luangwa valley, Eastern Province, Zambia.East African Medical Journal, 1974, 51:467-487.

Scott D. A recent series of outbreaks of human try-panosomiasis in Northern Ghana (1957-59). WestAfrican Medical Journal, 1961, 10:122-139.

Stevens JR, Godfrey DG. Numerical taxonomy oftrypanozoon based on polymorphism in reducedrange of enzymes. Parasitology, 1992, 104:75-86.

Wijers DBJ. The importance of the age of Glossinapalpalis at the time of the infective feed withTrypanosoma gambiense. In: International ScientificCommittee for Trypanosomaisis Research and Control.7th meeting, Bruxelles, 1958. London, Commission forTechnical Cooperation in Africa South of the Sahara,1960, 319-320.

Willet KC. Some observations on the recent epi-demiology of sleeping sickness in Nyanza region,Kenya, and its relation to the general epidemiologyof Gambian and Rhodesian sleeping sickness inAfrica. Transactions of the Royal Society of TropicalMedicine and Hygiene, 1965, 59:374-386.

Woodruff AW, Evans DA, Owino NO. A healthy car-rier of African trypanosomiasis. Journal of Infection,1982, 5:89-92.

World Health Organization. Planning overview oftropical disease control. Division of Control of TropicalDiseases, Geneva, 1995.

Wurapa FK et al. A healthy carrier of Trypanosomarhodesiense: a case report. Transactions of the RoyalSociety of Tropical Medicine and Hygiene, 1984, 78:349-350.

Wyatt GB, Boatin BA, Wurapa FK. Risk factors asso-ciated with the acquisition of sleeping sickness innorth-east Zambia: A case-control study. Annals ofTropical Medicine and Parasitology, 1985, 79(4):385-392.


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Annex 4EPIDEMIOLOGY, DISEASE SURVEILLANCE AND CONTROL, AND VECTOR CONTROL


I EPIDEMIOLOGY AND CONTROL OF HUMAN AFRICANTRYPANOSOMIASIS*

Honoré A. Méda1 and Jacques Pépin 2

1 Project SIDA 2, Benin (ACDI-Canada) B.P. 900 TriPostal, Cotonou, Republique du Benin.2 University of Sherbrook, Infectious Diseases Division,Centre for International Health, 3001, 12th Avenue,Sherbrook, Quebec, Canada.

INTRODUCTIONHuman African trypanosomiasis (HAT) is caused byhemoflagellates of the Trypanosoma genus,Trypanozoon subgenus and brucei species, whichclassically includes three subspecies: Trypanosomabrucei brucei, T. b. gambiense and T. b. rhodesiense.These subspecies are morphologically identical butdiffer in their ability to infect various hosts. T. b. bru-cei is essentially a parasite of domestic and gameanimals and is not pathogenic to humans because itis lysed by a haptoglobin-like molecule (Smith et al,1995). Only T. b. rhodesiense and T. b. gambiense areconsidered to be human pathogens. There are twoclinical variants: an acute syndrome attributed to T.b. rhodesiense and a chronic one caused by T. b. gam-biense. Both diseases result from complex interac-tions between the parasite and its tsetse fly(Glossina) vector and vertebrate hosts.

During the last couple of decades, considerableprogress has been made toward the improvement ofepidemiological knowledge. This has led to thedevelopment of new tools suitable for control. Arecent paper by Pépin et and Méda (2001) provides detailsof the advances in the epidemiology and control ofHAT. However, these advances have not been suffi-ciently used in the field for what they were intended.The aim of this paper is to try to summarize what weknow of the epidemiology and control and to identi-fy some of the most important gaps that need to beurgently tackled by the scientists and programmemanagers involved in research and control activities.

CURRENT EPIDEMIOLOGICAL SITUATION AND DISEASE BURDENHAT is the only vector-borne parasitic disease whosegeographical distribution is limited to the Africancontinent. T. b. gambiense is seen in West and CentralAfrica, and T. b. rhodesiense in East and SouthernAfrica. Uganda is the only country where both sub-species are found: T. b. gambiense in the north-westand T. b. rhodesiense in the south-east. This distribu-tion has probably remained constant over time.

According to the World Health Organization (1998),there remain within the “tsetse belt” more than 200active foci, located between latitudes 15° North and15° South. Within this area, 60 million individuals liv-ing in 36 countries are exposed to the infection. Dueto shortages of financial and human resources, lessthan 4 million benefit from an adequate surveillanceand control programme; all endemic countries arecharacterized by shortages of the financial andhuman resources necessary to implement or sustain acomprehensive control programme. Reports fromnational control programmes can only give a roughidea of the epidemiological situation, because of thedifficult security situation and the decay of commu-nication systems in many parts of high-incidencecountries. Year-to-year incidence for the 1977-1994period in all endemic countries can be found in arecent WHO report (WHO, 1998), where Figure 1shows the geographical distribution of the cumula-tive number of new cases reported between 1977 and1997 for the endemic countries. T. b. gambiense try-panosomiasis is now a major public health problemin Central Africa, specially in the DemocraticRepublic of Congo (DRC), Angola and SouthernSudan, where the ongoing civil war hampers controlefforts to such an extent that national statistics giveonly a very incomplete view of the problem. In DRC,where relatively better information is available, thetotal number of people at risk is estimated, by thenational control programme, to be 12 500 000. Thenumber of new cases reported each year has nowreached levels comparable to those seen in the early1930s, despite substantial underdiagnosis due toinadequate coverage of endemic regions; this situa-tion may result in the death of as many adults asAIDS (Ekwanzala et al, 1996). Underdiagnosis is alsoexacerbated by the poor sensitivity of diagnosticmethods. The number of cases reported annuallyincreased dramatically from about 10 000 in 1980 tomore than 27 000 in 1998. In the most endemicregions (e.g. Equateur and Bandundu), many com-munities have been found to have a prevalence ofover 10% during recent case-finding surveys. In 1994,an extra-ordinary prevalence of 72% in a small villageof the Bandundu region was reported.

Angola is the country with the second highest inci-dence of HAT, respectively 8275 and 6610 new caseswere reported in 1997 and 1998 by the national con-trol programme. Variations in the annual number ofreported cases must be interpreted with caution dueto the impact of the civil war on case-finding. Thedisease is endemic in the north-west provinces. Theprevalence rates reported vary between 1.3% and

* The contents of this paper are drawn largely from Pépin J. Méda AH. Theepidemiology and control of human African trypanosomiasis. Advances in

Parasitology, 2001, 49:71-132, by permission of the publisher AcademicPress.


9.7%. Uganda is the only country where both T. b.gambiense and T. b. rhodesiense are found, the formerin the north-west of the country, near the Sudaneseborder, and the latter in the south-east, without over-lap. Between 1000 and 2000 T. b. gambiense cases werereported annually in 1990-1994, but this has at leaststabilized with 978 cases reported in 1998, more thanhalf of them from Arua district. A major epidemic ofRhodesian HAT devastated SE Uganda from themid-1970s, with a cumulative total of about 40 000new cases over 15 years (Smith et al, 1998; Hide,1999). There has been some improvement recently,after many years of efforts by national authoritieswith substantial external support. Only 271 caseswere reported in 1998 from the SE of the country(Busoga). In Sudan, where reliable statistics are notavailable, the foci of T. b. gambiense HAT are locatedin the southern part of the Equatoria region, west ofthe Nile, within 100 kilometres of the borders withCentral African Republic (CAR), the DRC andUganda. Extrapolations suggest that there must be atleast a few thousand cases per year. In other coun-tries of Central Africa, HAT is more or less an emerg-ing public health problem. Elsewhere in East andSouthern Africa, the incidence of T. b. rhodesiense try-panosomiasis remains low. In West Africa, the dis-ease has regressed or disappeared from severalcountries as ecological changes reduced the intensityof man-fly contact. Only Côte d’Ivoire and Guineastill report a significant number of cases. Only a fewdozen cases are seen each year in Burkina Faso andMali, corresponding both to importation of cases andlocal transmission. The situation is less well knownin other West African countries.

It is difficult to estimate the overall burden of HAT.There are about 100 000 new cases per year, withbetween one third and one half of cases remainingundetected and untreated. Rhodesian trypanosomi-asis represents less than 5% of the overall burden ofthe disease. Recent estimates (WHO, 2000) arerather similar, with 2.05 million disability adjustedlife years (DALYs) lost, and 66 000 deaths, in 1999due to HAT. As a comparison, the number of mil-lions of DALYs lost is estimated at 45.0 for malaria,4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9for schistosomiasis, 1.1 for onchocerciasis, 0.7 forChagas disease.

EPIDEMIOLOGY OF T. B. GAMBIENSEHUMAN AFRICAN TRYPANOSOMIASIS

The Life Cycle of T. bruceiThe complex life cycle undergone by T. brucei in itstsetse fly vector and human host is described in var-ious standard textbooks. The whole cycle lasts about

30 days on average; it varies, according to speciesand the ambient temperature, between one andeight weeks. When Glossina takes a blood-meal froman infected host, it ingests bloodstream trypo-mastigote forms in its salivary glands. Trypanoso-mes then move to the fly’s midgut where, over a fewdays, they transform into the procyclic stage. Thevariant surface glycoprotein (VSG), the dominantconstituent of the surface of bloodstream try-panosomes, is replaced by an invariant surface pro-tein. After two to three weeks of maturation andmultiplication, trypanosomes migrate to the sali-vary gland, where other transformations lead totheir development into metacyclic trypanosomes,which require VSG to be infective for a susceptiblehuman or animal host during the next blood-meal.These forms are the metacyclic trypomastigotes, theonly stage which is infective to vertebrates. Onceinfected, a tsetse fly remains so for the rest of its lifespan of between three and four months. While tak-ing subsequent blood-meals, the fly is capable ofinoculating another vertebrate host with the meta-cyclic trypanosomes. Within the human host, theparasite multiplies at the site of inoculation, where achancre might develop. Inoculation chancres arerarely recognized on African skin. From this site, theparasite gets into the bloodstream and lymph nodesand multiplies through binary scissiparity withthree morphological forms: the short and stumpy,the intermediate, and the long and slender forms.

After infection of a human host, there is a switchfrom the expression of metacyclic VSG to blood-stream VSG. To evade the host’s immune response,trypanosomes can successively express differentVSG, but only one at a time. This antigenic variationis a unique feature of trypanosomes (Barry, 1997); ithas direct consequences for the epidemiology of thedisease. Up to a thousand different VSGs are genet-ically encoded, and it is thought that the VSG cur-rently expressed protects invariant constituentsfrom the host’s immune response. The mechanismsthrough which trypanosomes switch to expressing adifferent VSG are complex, but it allows the parasiteto escape from antibodies directed against the pre-vious VSG. This complex process explains the inter-mittent parasitaemia and the very long, largelyasymptomatic, incubation period. Thus, there is analternation between periods of higher parasitaemiafollowing expression of a new VSG, during whichthe human host might be more infectious, and peri-ods of lower or undetectable parasitaemia, duringwhich infectivity must be lower. Variations in thevirulence of T. b. gambiense strains were noted earlyby Van Hoof (1947). More recently, biochemical,molecular and immunological methods have beendeveloped for the identification of trypanosomes by


examining their genetic material (Gibson, 1994).These methods include two-dimensional gel elec-trophoresis and isoenzyme, chromosome and DNAanalysis using polymerase chain reaction (PCR)techniques, which have substantially contributed toa better understanding of the parasite’s taxonomyand the epidemiology of the disease, especially instudies on animal reservoirs of human trypano-somes. Cellular and molecular characteristics ofhuman trypanosomes have been reviewed else-where (El-Sayed and Donelson, 1997; Barry 1997).

The Vectors

Vector species and sub-speciesThe tsetse fly is a vector of trypanosomes that infectman as well as wild (antelopes, giraffes, etc.) anddomestic (pigs, cattle, sheep, goats, dogs, horses,etc.) animals. It belongs to the genus Glossina, whichincludes about 30 known species divided into threegroups: the Glossina sub-genus which includesspecies of the morsitans group; the Nemorhina sub-genus (palpalis group) and the Austenina sub-genus(fusca group). A software has been developed for theidentification of glossinids by species and sub-species and the determination of their epidemiolog-ical importance (Brunhes, 1994). Several factorsrelated to the vectors determine the transmission oftrypanosomes to humans: vector biology and ecolo-gy, vectorial capacity, man-fly contacts, longevity,dispersal, feeding behaviours, etc.

Distribution and ecology of GlossinaA large variety of traps have been tested for tsetsesampling and control, a dozen of which werereviewed by Leak (1999). The favourite techniquecurrently used to study Glossina habitats, densitiesand ecodistribution is the biconical trap developedby Challier and Laveissière (1973). Turner (1980)proposed the “marking-release-recapture” tech-nique using a radioactive compound (e.g. Fe59) andscintillometer to study its bioecological characteris-tics such as Glossina habitats, behaviour and dynam-ics, population size, densities, dispersal, survivalrates, resting sites.

The palpalis group contains two excellent vectorspecies of T. b. gambiense and of animal trypanoso-miases in West and Central Africa: G. palpalis palpalisin forest areas and G. p. gambiensis in savannahareas. The former inhabit the forest areas and themoist savannah, whereas the latter is found in thesemi-arid savannah. G. tachinoides and G. fuscipesfuscipes are related to the palpalis group and are vec-tors of sleeping sickness in the savannahs of Westand Central Africa while G. pallidipes is found in the

savannah of Central Africa. The savannah is theexclusive habitat of G. morsitans in various regionsof Africa. Its geographical distribution is limited togallery forests bordering streams, where optimalsurvival conditions are found. In a given geograph-ical area, the distribution of tsetse flies variesdepending on the species and is mainly determinedby the climate, presence of water, vegetation andavailability of sources of blood-meals (humans andanimals). Studies conducted in the forest zone ofWest Africa have shown that G. p. palpalis is a verymobile vector found in different biotopes and thatits distribution is closely linked to human occupa-tion patterns (Baldry, 1980; Challier and Gouteux,1980; Gouteux and Laveissière, 1982). Highestapparent densities per trap (ADT) per day wereobserved on the edges of villages where the swinepopulation provides an abundant, easily accessiblefood source. High densities have also been foundnear sources of potable water, in coffee and cocoaplantations, especially those located on the edge offorests or gallery forests, and along paths separatingplantations from the remaining forest used byhumans and some wild animals, especially thebushbuck, which are food sources for Glossina.

The tsetse fly is only active for a short time whenlooking for blood-meal (35 minutes on average perday). It spends most of the time resting to digest orgestate. The amount of activity of each speciesdetermines its chances of encountering a host onwhich a blood-meal may be obtained and variesdepending on climatic factors (temperature, humid-ity, amount of light, wind, rain), olfactory and visu-al stimuli (smelling and seeing a potential feedinghost), and on intrinsic factors (physiological age,nutritional status, gravidity).

Vectorial capacity, competence and susceptibility of GlossinaThe vectorial capacity of Glossina is determined byits ability to infect itself while feeding on a verte-brate host, and to subsequently develop an infectionand transmit the trypanosome to another verte-brate host (Challier, 1982). According to these crite-ria, only the palpalis and morsitans groups containspecies and sub-species that are vectors of T. b. gam-biense. It has been demonstrated that not all flies in agiven area have the same capacity to transmit theparasite. It is therefore important to determine whe-ther or not there are local conditions that mayreduce or increase this capacity. The tsetse fly’s abil-ity to infect itself while feeding on a parasitized hostdepends on several poorly understood factors. Thenumber of trypanosomes ingested by the fly duringits blood-meal could be one such factor. However, ithas been demonstrated that a single trypanosome


can infect G. m. morsitans (Maudlin and Welburn,1989; Baker, 1991). The concept that a blood-mealtaken from an individual with low-level para-sitaemia is less infectious for the vector than onetaken from a host with high-level parasitaemianeeds to be investigated. Under natural conditions,the infection rate among tsetse flies is quite low. Onaverage, less than 1% of tsetse flies are infected withT. brucei ssp (Molyneux, 1980b). Teneral flies aremore susceptible to infection by trypanosomes.

As reviewed by Leak (1999), it has been suggestedthat lectins present in the haemolymph and midgutof the fly are responsible for increasing the resist-ance of the tsetse fly, with age, to infection withT. brucei. Lectins are absent or blocked in unfed ten-eral flies, thus permitting infection, while in olderflies lectin production seems to be stimulated byblood-meals, preventing subsequent infections. Thetrypanosome also has effects on Glossina. Jenni et al(1980) have shown that the infected insect has a ten-dency to bite more often and feed more voraciously,which can have a substantial impact on its transmis-sion potential. According to Maudlin et al (1998), therisk of death increases with age, but much morequickly in infected than non-infected tsetse flies.Infected flies are much more susceptible to insecti-cides than non-infected flies (Nitcheman, 1990).

Feeding behaviour and trophic preferencesThe first concern of the teneral Glossina is to find ahost on which a blood-meal may be taken. Theyoung adult needs a blood-meal to complete itsmaturation. This first meal takes place between 24and 72 hours or even later, and depends on intrinsic(diurnal rythm, sex, gravidity, species, etc.) andenvironmental (climatic conditions, visual, mechan-ical and olfactory stimuli) factors (Colvin andGibson, 1992). The tsetse’s feeding ground may berestricted by the relatively limited dispersion ofpotential sources of blood-meals. In forest areas,G. p. palpalis confines itself to the outskirts of vil-lages where swine hide, along paths through plan-tations, near sources of potable water, and aroundfarm camps and other places used by man(Laveissière and Hervouët, 1981).

Most studies on the trophic preferences of Glossinawere conducted in West Africa and based on analysesof blood-meals collected by dissecting the insect.Methods with different degrees of sensitivity havebeen reviewed by Leak (1999), including the precip-itin test, the agglutination inhibition test, the comple-ment fixation test, direct and indirect ELISA assays,tests based on the latex agglutination technique, and,more recently, one based on electrophoresis of super-oxide dismutase (Diallo et al, 1997) which can only

distinguish between meals of human and animal ori-gin. Glossinids of the palpalis group are notable fortheir eclecticism and opportunism: they feed indis-criminately on many species and are therefore verydangerous to man. In the forest zone of Côte d’Ivoire,Gouteux et al (1982) found that at least 75% of theblood-meals were from suidae around the villages. Inplantations in the same zone, however, Laveissièreet al (1985) found that 46% of the blood-meals werefrom man. This observation was confirmed by recentresults of Sané et al (2000).

A comparison of the results of analyses of blood-meals collected in the five main foci in Côte d’Ivoireshowed that the proportion of human blood-mealsvaries significantly from one focus to the other,although the socio-geographic conditions in thesefoci appear to be identical: a human origin corre-sponded to 42% of blood-meals of G. p. palpalis atVavoua, 73% at Daniafla, 55% at Zoukougbeu, 91% atGagnoa and 27% at Sinfra. Humans, therefore, seemto be the favourite host, in differing degrees, ofG. p. palpalis in plantation zones of Côte d’Ivoire. Thispreference for man was greater in the foci with lowtransmission rates (Daniafla, Gagnoa) than in themore active foci (Sinfra, Vavoua and Zoukougbeu).Domestic animals appear to play an important role infeeding tsetse flies at Vavoua and Zoukougbeu, themost active foci, whereas, in the low-incidence foci,the percentage of animal blood-meals is insignificant.At Zoukougbeu, apart from on humans, G. p. palpalisfeeds freely on domestic swine (30% of meals),whereas at Vavoua it prefers the bushbuck whichprovides an equal percentage of its food as man(42%). The diversity of the feeding regimen of G.p. palpalis might explain to some extent the varia-tions in levels of incidence of human disease. In fociwhere transmission is more intense, G. p. palpalisfeeds on both man and animals and proper case-find-ing and treatment of infected humans is not sufficientto control the disease. With a diversified regimenalternating between man and animals as sources ofblood-meals, tsetse flies are able not only to transmittrypanosomes to humans, but also to maintain theputative animal reservoir. In contrast, in low trans-mission foci, the tsetse fly depends on man, trans-mission to and from animals is rare, and case-findingprevents the accumulation of human cases. This WestAfrican paradigm should probably not be systemati-cally extrapolated to high-incidence foci of CentralAfrica, where the role of animals is less clear. If oneof its usual mammal hosts is not available, G.tachinoides replaces it with reptiles as observed in thegallery forest of the savannah: snakes or lizardsaccount for between 54% and 67% of the blood-mealsof G. tachinoides, while only 8% of its meals are fromman (Laveissière and Boreham, 1976). The feeding


preferences of this vector also vary seasonallybecause of changes in the availability of hosts. In thehot season, 30%-55% of its meals come from mammalhosts (mainly humans and bushbucks) whereas inthe cold season, the only animals available are rep-tiles, and these provide over 50% of its meals(Laveissière and Boreham, 1976).

Transmission cycles of T. b. gambienseIn the forest zone of Côte d’Ivoire, man-fly contactsoccur in almost all biotopes but especially in planta-tions and peridomestic areas where it is easier forthe fly to find hosts (Challier and Gouteux, 1980;Laveissière et al, 1985). Some authors attribute thepersistence of residual foci of HAT to the existenceof other cycles adjacent or parallel to the commonman-fly-man cycle (Molyneux, 1980a; WHO, 1998),i.e. cycles where the parasite travels betweendomestic and/or wild animals (swine, sheep, goats,bushbuck, etc.) and man. They identify two suchtransmission cycles: a) a domestic cycle where theGlossina transmits the parasite between humanhosts and domestic animals; b) a sylvatic cyclewhere the vector transmits the parasite betweenwild animals, sometimes with humans or domesticanimals entering this cycle, resulting in sporadiccases or even epidemic outbreaks. The domesticcycle hypothesis is supported by similaritiesobserved between parasites isolated in humans, ani-mals and the vectors (Gibson et al, 1978; Mehlitzet al, 1982), whereas there is little evidence that wildanimals are infected with T. b. gambiense.

Tsetse flies and the epidemiology of Africantrypanosomiasis The transmission of infectious trypanosomes tohumans depends on many factors: the density ofGlossina populations, Glossina longevity, the vector’ssusceptibility to infection, Glossina infestation ratesand the factors that influence these, and humanbehaviours and activities in the biotopes of the fliesthat determine the frequency of man-fly contacts. Asdescribed in the previous sections, the vector’s biol-ogy, ecology and feeding behaviour have direct con-sequences on transmission of the parasite. Thus,through its biological cycle, the Glossina allows thematuration, development, multiplication, transmis-sion and dissemination of the parasite. The feedingbehaviour of various species of Glossina determinestheir epidemiological contribution to the transmis-sion of T. b. gambiense to the humans, and to someextent the animals, that constitute the parasite’sreservoir. The female G. p. palpalis, because of itsmore aggressive feeding behaviour and longer lifespan, plays an essential role in the transmission ofthe parasite.

The number of trypanosomes inoculated into themammal host during a blood-meal is probably oneof the factors that determine the probability of trans-mission. It has been estimated that, to infect man,the inoculum must contain between 300 and 500 try-panosomes (Challier, 1982). Transmission is alsoinfluenced by factors intrinsic to the vector (physio-logical age, nutritional status, gravidity, etc.) and byclimatic and other environmental conditions. Thelow infection rate naturally observed among tsetseflies limits the devastating epidemic potential ofAfrican trypanosomiasis, and explains the apparentparadox between the abundance of the G. p. palpalisand G. morsitans vectors, and the relative rarity ofhuman disease. There is no direct correlationbetween the densities of G. p. palpalis, the major vec-tor living in close contact with man, and the inci-dence of sleeping sickness.

In endemic foci, the nature, frequency and intensityof man-fly contacts are the major determinants ofthe risk of African trypanosomiasis. In the forestzone of Côte d’Ivoire, there is virtually no ecologicalzone where humans are safe from being bitten byG. p. palpalis. Man-fly contacts can occur in all botan-ical zones and are affected not only by the vegetalenvironment but also, and especially, by humanhosts. Multidisciplinary studies have shown thathuman activities and behaviours have an importantimpact on the epidemiology of sleeping sickness,through an increase in the frequency and intensityof man fly-contact (Laveissière et al, 1985, 1986a,1986b; Hervouët and Laveissière, 1987; Méda et al,1993). For example, coffee-growing, which requiresthe planter to spend more time in the plantationthan if growing cocoa or food crops, is associatedwith a high risk of infection due to the increasedcontact time with the vector. This risk is heightenedby residing in farm camps and by procuring waterfrom natural water sources located near edges ofplantations and gallery forests. Many other activi-ties, such as collecting firewood, washing and fish-ing, bring human hosts into contact with tsetse flies.The availability of other sources of blood-meals (e.g.pigs) reduces the chances of transmission.

The Human ReservoirHumans are the main reservoir of T. b. gambiense.Four factors potentially influence man’s potential intransmitting T. b. gambiense: the duration of infec-tion, the degree of parasitaemia, the number anddistribution of individuals who are infected, and theintensity of contact with the vectors.

The duration of infection in humansGiven that the tsetse fly is a relatively ineffectivevector, the very long duration of infection (from a


few months to several years) during which thehuman host can maintain normal activities, andprovide blood-meals for the vectors, must be thekey determinant behind the endemic features andepidemic potential of Gambian HAT (Baker, 1974).Fortunately, this long duration of infection offers agolden opportunity for intervention. Control pro-grammes focusing on the identification and treat-ment of asymptomatically infected humans, andthus on a shortening of the duration of infection, areextremely effective if an overwhelming majority ofthe population shows up during case-finding sur-veys and if sensitive diagnostic methods are used.

The distribution of infection in human popula-tions Prevalence of trypanosome-infected individuals inhuman populations varies tremendously accordingto socio-demographic factors. Trypanosomiasis isnot found equally in males and females. The appar-ent preponderance of cases among females wouldnot correspond to substantial differences in inci-dence or prevalence if reliable denominators wereavailable (which is rarely so). Females often partici-pate more regularly in case-finding sessions, whichcan also lead to a higher number of cases (Ason-ganyi and Ade, 1994). Variations in incidence andprevalence between age and ethnic group have alsobeen noted. This probably relates to differences inoccupational exposure and other determinants ofman-fly contact rather than to genetically deter-mined susceptibility (Laveissière et al, 1986a; Her-vouët and Laveissière, 1987; Méda et al, 1993). Otherrisk factors have been described, which are probablyall markers of exposure to infective tsetse flies: lackof formal education, absence of pigs (an alternativesource of blood-meals for tsetse) in the habitat, etc.(Méda et al, 1993; Méda et al, 1995).

Occupation is also related to exposure and to inci-dence. For instance, in Côte d’Ivoire, HAT is morefrequent in coffee and cocoa plantation workers orpeople who fetch water than in other inhabitants(Laveissière et al, 1986a and b; Méda et al, 1993).More than 80% of cases occur in people who notonly work but also live in small plantation settle-ments; a case-control study showed that such peo-ple were five times more likely to develop try-panosomiasis than their counterparts who residedin villages (Méda et al, 1993). In some foci of theDRC, a higher prevalence was found in fishermen,while elsewhere, farmers were more likely to getinfected (Henry et al, 1982; Mentens et al, 1988).

Human population density influences the risk ofepidemics in gambiense sleeping sickness (Scott,1970). If population density is very light, tsetse flies

take most blood-meals on non-human sources andtransmission to humans is unlikely. On the otherhand, high human population density results inmodifications of the habitat that reduce the tsetsepopulation. Thus, an intermediate population den-sity is optimal for Gambian trypanosomiasis toprosper. A recent study in Côte d’Ivoire showed astrong correlation between the epidemiological riskand settlement density (Laveissière and Méda,1999). Whether or not there are secular or seasonalvariations in the frequency with which humansbecome infected with T. b. gambiense is unknown butplausible through changes in tsetse densities, man-fly contact, presence of alternative sources of blood-meals, etc.

Familial aggregationFamilial clustering of trypanosomiasis has been rec-ognized since the beginning of the century. Compa-red to children of mothers without a past history ofHAT, the risk of a child having had trypanosomiasiswas four times higher if the mother had had the dis-ease, while it was two times higher in brothers andsisters of a case than in their half-brothers and half-sisters. Such clustering could be due to either genet-ic susceptibility or to shared exposure to the vector.Several arguments reviewed elsewhere (Khondeet al, 1997) suggest that the latter is the most plausi-ble explanation. Shared exposure could result fromsimultaneous contact with an infective tsetse whoseblood-meal on a first individual is interrupted andresumed on a nearby relative, or from members of asame family sharing an ecological microcosm andbeing similarly but not simultaneously exposed tothe vector bites (Gouteux et al, 1989).

The influence of human behaviourHuman behaviour plays an important role in theepidemiology of Gambian trypanosomiasis. Healthseeking behaviour might delay the recognition of anepidemic and enhance transmission of the parasiteif symptomatic individuals wait for months beforereaching a health facility where trypanosomes canbe detected, maybe because they first attributed thedisease to other, supra-natural, causes and soughttraditional treatment. Participation in case-findingsurveys varies from place to place and over time,and is a key determinant in the success or failure ofsuch programmes.

Migrations have contributed to trypanosomiasisepidemics, as they favour the circulation of try-panosomes from high-incidence to low-incidenceareas where the population is more susceptible(Prothero, 1963). Although this remains controver-sial, the explosive epidemics seen in Uganda andthe Congo a century ago have been attributed by


many authors to the large-scale circulation of workers organized by the colonizers to suit theirneeds (Leak, 1999). A recent example of the impactof migrations is the epidemic in NW Uganda, whichresulted from the exodus of Ugandan refugees toSudan and Zaire where they got infected, followeda few years later by the migration to Uganda ofinfected Sudanese refugees and the return ofUgandans back home (Paquet et al, 1995).

Immunity It was generally thought that no immunity followeda first diagnosis of HAT, because of the parasite’santigenic variation and its repertoire of VSG whichincludes hundreds of different antigens. However,observational and experimental studies in animalmodels have shown these animals to be more resist-ant to homologous trypanosomes when re-chal-lenged after adequate treatment of a previous infec-tion. This protection resulted from exposure tometacyclic trypanosomes rather than to blood-stream forms (Nantulya et al, 1984; Akol andMurray, 1985; Vos et al, 1988). The existence of pro-tective immunity in humans was investigated in avery-high incidence community of the DRC where38% of adults had a past history of trypanosomiasis(Khonde et al, 1995). The results suggest that a firstepisode of trypanosomiasis confers to adults about85% protection against subsequent reinfection.

The impact of HIV Little is known about the interactions between thehuman immunodeficiency virus (HIV) and Gam-bian trypanosomiasis. Three studies performed inCentral Africa (Louis et al, 1991; Pépin et al, 1992)and Côte d’Ivoire (Méda et al, 1995) suggest thatHIV infection has so far had little impact on the epi-demiology of Gambian trypanosomiasis, to someextent because the prevalence of HIV remains rela-tively low in rural communities where HAT isendemic. No data are available for T. b. rhodesienseareas. Given that HIV infection is getting moreprevalent in rural areas, it is worthwhile undertak-ing a well designed nested case control study to fur-ther investigate these interactions. It has beenobserved that HIV co-infected HAT patientsrespond less well to eflornithine treatment thanseronegatives (Milord et al, 1992), but this is unlike-ly to have any impact on transmission.

The Animal ReservoirThe existence of an animal reservoir of T. b. gambi-ense has been investigated for a long time(Makumyaviri et al, 1989; Leak, 1999). Earlier stud-ies showed that many species of domestic animalcould be experimentally infected with T. b. gambi-ense: pigs, dogs, goats, sheep and even chickens

(Van Hoof, 1947; Schutt and Mehlitz, 1981). Theseinfections generally resulted in low parasitaemiathat lasted less than a year. The study of naturallyoccurring infections in animals was facilitated bythe development of appropriate laboratory meth-ods: the blood incubation infectivity test (BIIT),isoenzyme electrophoresis, and DNA analysis.Domestic animals were found to be infected withparasites enzymatically identical to T. b. gambiense.Pigs have generated more interest because they area frequent source of blood-meal for tsetse flies, andhave been found infected with T. b. gambiense inLiberia (a country with little human disease), Côted’Ivoire, Congo and the DRC (Gibson et al, 1978;Schutt and Mehlitz, 1981; Mehlitz et al, 1982;Noireau et al, 1989; Truc et al, 1991). In Côte d’Ivoire,52 sympatric T. brucei strains were characterised byisoenzyme electrophoresis: among 12 zymodemesrevealed, the most frequent was found both inhumans and pigs (Penchenier et al, 1997). In Congo,sheep were also found to be infected with T. b. gam-biense, and the prevalence of Trypanozoon infection indomestic animals was estimated to be 0.5%(Noireau et al, 1989; Truc et al, 1991). A dog wasfound infected with T. b. gambiense in Liberia(Zillmann et al, 1984). A more recent studyusing PCR showed the simultaneous presenceof T. b. gambiense in humans and animals (a dog anda pig) from the Lower Congo province of the DRC(Schares and Mehlitz, 1996). However, the epidemi-ological significance of the animal reservoir isunknown. Whether or not animals may become athreat for reintroduction or persistence of the para-site in foci where near elimination of HAT has beenachieved remains an important research question(Molyneux, 1980a).

EPIDEMIOLOGY OF T. B. RHODESIENSETRYPANOSOMIASISHAT caused by T. b. gambiense and T. b. rhodesiensediffers in its epidemiological features. We will onlystress these diffences. T. b. rhodesiense is a zoonosissporadically transmitted to humans when they ven-ture into bush infested by tsetse fly vectors whoseblood-meals are normally taken on game animals.Humans enter this sylvatic cycle as an incidentalhost. Except during epidemics, human-fly-humanor domestic animal-fly-human transmission isthought to be rare. The epidemics usually involveG. f. fuscipes, a peridomestic fly, and transmissionoccurs around the villages. The disease, character-ized by an acute or subacute malaria-like syndromewith high parasitaemias, progresses over weeks ormonths rather than months or years. The vectorsdiffer from those of T. b. gambiense. The parasite istransmitted mostly by three species of the morsitansgroup: Glossina morsitans morsitans and G. m. cen-


tralis in East Africa, G. pallidipes in East andSouthern Africa and G. swynnertoni in Kenya andTanzania. G. fucipes fucipes, which belongs to the pal-palis group, is a vector of human and animal try-panosomiasis in Central and East Africa.

The existence of animal reservoirs was establishedin the 1950s. Cattle are the major reservoir (Hideet al, 1996), and played a major role in an epidemicin south-east Uganda, with 23% of cattle carryinghuman infective strains. Other domestic (dog,sheep, maybe pig) and various game (warthog,bushbuck, hartebeest, impala, lion, zebra, hyena)animals can harbour the parasite in their blood;some of these animals are well adapted to the para-site and can remain infected for more than two yearswithout overt disease. Conversely, some otherspecies (e.g. dog) rapidly succumb from the infec-tion. In contrast to T. b. gambiense trypanosomiasiswhich results from peridomestic infections inCentral Africa, rhodesiense trypanosomiasis is, inendemic situations, acquired far from the village,where the animal reservoir lives. It is mainly anoccupational disease of adult men: hunters, fisher-men, firewood collectors and honey gatherers havebeen found to be at higher risk. Tourists visitinggame parks are also exposed to the infection.However, in epidemic situations, when transmis-sion becomes peridomestic, all groups, includingmales and females are at similar risk. Indeed, it hasbeen observed that an increase in the number ofcases in children and women is an indication that anoutbreak is developing (Apted, 1970). As it is in gam-biense sleeping sickness, familial aggregation hasbeen noted (Okia et al, 1994). In contrast to Gambiantrypanosomiasis, there is a marked seasonality ofdisease occurrence, with a higher incidence duringthe warmer season (Smith et al, 1998). Populationmovements and political upheavals played animportant role in development of recent epidemicin south-east Uganda (Mbulamberi, 1989a; Smithet al, 1998). Changes in agricultural practices alsoled to more favourable conditions for the develop-ment of G. fucipes, and resulted in the recent perido-mestic epidemic, which was brought into control bythe surveillance and early diagnosis through sleep-ing sickness orderlies and vector control (Smithet al, 1998; Okoth, 1999)

CONTROL OF HUMAN AFRICAN TRYPANOSOMIASISSleeping sickness control relies on two principles:reduction of the parasite reservoir through casedetection and treatment, and reduction of man-fly contact through vector control. In the case ofT. b. gambiense, reduction of the human reservoir canbe achieved through case-finding and treatment.

The limiting factor is the relatively low sensitivity ofthe standard parasitological techniques. A more fre-quent use of finer serological and parasitologicaltechniques could lead to a rapid and sustainablereduction of the human reservoir. In order to reduceman’s exposure to infectious bites, the tsetse fliesmust be destroyed. This must be carried out withthe active involvement of the community, who cantake on trap laying, spraying, bush clearing, etc.There are a variety of vector control techniques, thechoice of which depends on financial and humanresources, the epidemiological situation, and theprogramme duration. Within the African context,vector control must be carried out with cheap, cost-effective and easy-to-use methods. Control integrat-ed into primary health care and targeted on groupsat risk has been found feasible, cost-effective andcheaper, provided village health workers (VHWs)are motivated and the beneficiary population partic-ipates fully. Community involvement, both in casefinding and vector control, through the implicationof VHWs, has been successfully tested on a largescale in various epidemiological contexts.

Case DetectionCase detection has been the cornerstone of HAT con-trol. For many years, specialized case-finding mobileteams have relied essentially on the presence ofswollen cervical lymphs nodes. Lymph node fluid,when present, is examined for evidence of try-panosomes. In some very active foci, the mobileteams carry out wet film and giemsa-stained thicksmear examinations. Because of the low sensitivity ofthese methods, better serological and parasitologicaltools have been developed over the last two decades.

Serological methodsVarious serological assays were developed to helpcase-finding teams identify a small number of anti-body carriers on whom to concentrate efforts for try-panosome detection using parasitological methods.In the 1970s, the indirect fluorescent antibody test(IFAT) was deemed the most reliable technique forepidemiological surveillance of T. b. gambiense try-panosomiasis. However, it required relatively expen-sive equipment and qualified staff, and its imple-mentation was possible only in laboratories. Delaysin obtaining results were such that some seropositivesuspects could not be located again to undergo theparasitological confirmation test. From the 1980sonwards, the IFAT was supplanted by the cardagglutination test for gambiense trypanosomiasis(CATT) (Magnus et al, 1978), the advent of whichconsiderably enhanced detection of cases of Gam-bian trypanosomiasis. The CATT is a latex aggluti-nation test relying on the detection of antibodiesusing the variable antigen type (VAT) LiTat 1.3 anti-


gen, which is expressed by several T. b. gambiensestocks. This is the only serological assay currentlyused by control programmes. It can be performed inthe field, without electricity and without specializedstaff, and the results are available within 10 minutes.It is cheap, at approximately 0.40 USD per test, andis generally performed on whole blood. Its sensitivi-ty varies between 92% and 100% when assessed inpatients parasitologically confirmed in hospital orother settings, and its specificity is thought to be 94-97% (Noireau et al, 1988; Miézan et al, 1991). TheCATT proved highly specific during testing in non-endemic areas (Bafort et al, 1986). Some strains ofT. b. gambiense do not express the LiTat 1.3 antigen,but their distribution seems fairly limited (Dukeset al, 1992). The positive predictive value of theCATT depends on the prevalence of disease in thepopulation. This prevalence ranges between 1 and5% in most endemic foci and the positive predictivevalue generally varies between 14% and 40%(Miézan et al, 1991). The CATT can be performed onfilter paper, using smaller volumes of reagents, thusreducing costs (Miézan et al, 1991). However, thereliability of this micro-CATT is less satisfactoryunder field conditions than in a research setting. TheCATT can also be used on diluted serum rather thanwhole blood, resulting in higher specificity at theexpense of lower sensitivity (WHO, 1998).

The card indirect agglutination trypanosomiasis test(CIATT) is an indirect assay that was newly pro-posed for the detection of circulating trypanosomeantigens in patients’ blood (Nantulya, 1997). Theparasite antigen detected is an internal, invariantmolecule that is common to both T. b. gambiense andT. b. rhodesiense. This test can be used to detect cur-rent infection in both forms of the disease. It hasbeen found to be highly sensitive in endemic areas(Asonganyi et al, 1998). The test was shown to beeasy to use in field conditions and does not needchain maintenance for the storage of reagents. Arecent evaluation in non-endemic areas revealed aspecificity ranging from 61% to 98% depending onthe proximity of the endemic zone (Meda et al, sub-mitted for publication). Specificity is improved bytitration of seropositive specimens. Apart from theirdiagnostic potential, the CATT and CIATT have alsobeen found to have potential for use in patient fol-low-up to determine chemotherapeutic cure.Further operational studies aimed at testing the use-fulness of the CIATT in clinical settings and for fielduse by national control programmes should be car-ried out.

Parasitological methodsConfirmation of diagnosis among seropositive indi-viduals depends on demonstrating the trypano-

some in biological fluids. This confirmation can beobtained through the examination of wet blood orgiemsa-stained blood smears, or by direct examina-tion of the lymph node aspirate when a typical lym-phadenopathy is palpable (Cattand and de Raadt,1991; Miézan et al, 1994). These classical methodshave a rather low sensitivity in gambiense try-panosomiasis. Demonstrating the presence of try-panosomes in blood and cerebrospinal fluid (CSF)has been considerably facilitated by the develop-ment of concentration methods. The micro-haemat-ocrit centrifugation technique (Woo’s test) is muchmore effective in the rhodesiense disease (and in vet-erinary medicine) than in T. b. gambiense HAT. Theminiature anion-exchange centrifugation technique(mAECT) is deemed at present to be the most sensi-tive parasitological method for the detection ofblood parasites (Miézan et al, 1994). Because of thecost (about US$2) and its relative complexity, it isused selectively to test CATT-positive suspectsamong whom the diagnosis could not be confirmedby classical methods. Two new parasitological tech-niques have recently been added to the diagnosticarsenal: the quantitative buffy coat technique (QBC)(Bailey and Smith, 1992) and the kit for in vitro iso-lation of trypanosomes (KIVI) (Aerts et al, 1992).Neither the QBC, which requires relatively expen-sive equipment, nor the KIVI, which is a parasiteisolation method rather than a screening test, hasproved to be superior to the mAECT (Truc et al,1994).

In the CSF, the most sensitive technique is that ofdouble centrifugation (Cattand et al, 1988; Miézanet al, 1994). A variation of the latter, the single cen-trifugation of cerebrospinal fluid in a sealed Pasteurpipette, has been recently developed (Miézan et al,2000). It makes detection of trypanosomes in theCSF simpler, quicker and more sensitive, and is spe-cially suitable for passive diagnosis in suspects whoconsult in health facilities with symptoms sugges-tive of sleeping sickness. The combination of allthese seroparasitological methods ensures increasedsensitivity, but falls short of perfectly reliable para-sitological diagnosis in all seropositive persons,hence the need to pursue efforts to develop moresensitive parasitological assays and more specificserodiagnostic techniques.

Treatment and Drug ResistancePentamidine remains the standard treatment forearly-stage patients and melarsoprol for late-stagecases. The treatment for HAT is selected by firstestablishing the stage of infection. The diagnosis oflate-stage trypanosomiasis is based on at least one ofthe following criteria (WHO, 1998): CSF white cellcount (WCC) > 5/mm3 or CSF proteins >37 mg/100


ml (as measured by dye-binding protein assay), oron both criteria, with or without the presence of try-panosomes in CSF. Miézan et al (1998) reported thatthe CSF WCC is, by itself, as sensitive for diagnosisof central nervous system involvement as is thecombination of the above criteria. Therefore, theWCC was recommended in patients with confirmedinfection, especially in poorly equipped facilities.Advances have been made in the treatment of HAT.Between 5% and 10% of late-stage patients treatedwith melarsoprol, an arsenical derivative, succumbto its undesirable effects. Until recently, melarsoprolwas the only available treatment for late-stagepatients. A new drug, eflornithine, has been devel-oped (Pépin and Milord, 1994). Results obtainedwith eflornithine are excellent, but its future avail-ability remains doubtful.

Drug resistance has been a relatively uncommonphenomenon in Gambian trypanosomiasis, despitesuramin, pentamidine and melarsoprol having beenused for five decades (Pépin and Milord, 1994), and,as a consequence, has not had any impact on theepidemiology of the disease. Suramin is little usedin the treatment of Gambian trypanosomiasis.Suramin and Pentamidine are given throughoutAfrica to patients with early-stage of T. b. rhodesienseand T. b. gambiense disease respectively. So far, thetreatment failure rate remains fairly low. The situa-tion is quite different for melarsoprol. There are atleast two foci where melarsoprol resistance is morefrequent than elsewhere; clearly this is an issue thatwill need better investigation and monitoring overthe next few years. One is the Mbanza Kongo focusof northern Angola, close to the border with Lower-Congo: a 40% failure rate was reported 25 years ago(Ruppol and Burke, 1977) and similar failure rateshave been observed in recent years. Limited epi-demiological data suggest that there has been littlespread of resistant strains over time. In the Arua dis-trict of northern Uganda, a 27% failure rate hasrecently been reported among new cases treatedwith melarsoprol (Legros et al, 1999); a 10-foldlower failure rate has been documented in the adja-cent focus of Adjumani. In the DRC, where melarso-prol failures are not specially frequent, statisticallysignificant differences in failure rates were found. Ithas been also observed that HIV co-infected HATpatients respond less well to eflornithine treatmentthan seronegatives (Milord et al, 1992), but this isunlikely to have any impact on transmission aseflornithine is little used and relapsing cases of HATare often not parasitaemic.

Vector ControlThe tsetse fly is one of the rare insects for which sev-eral control methods have been developed, based on

bioecological and epidemiological studies. Beforethe advent of insecticides, vector control dependedprimarily on elimination of the wooded vegetationwhich constitutes the habitat of Glossina. Nowadays,insecticides are applied to various types of traps andscreens to destroy the vector. A number of improvedbiconical, monoconical and pyramidal traps,inspired by the Challier-Laveissière biconical trap(Challier and Laveissière, 1973), have been tested forthe control of G. p. palpalis, G. morsitans andG. f. quanzensis in West and Central Africa. Lanciendesigned the monoconical trap (Lancien, 1981),which was followed by the pyramidal trap(Gouteux and Lancien, 1986), and later by theVavoua trap (Laveissière and Grébaut, 1990). Toenhance trap efficacy, especially against G. morsi-tans, olfactory attractive baits are attached to them(carbonic gas, acetone, urine phenols and host skinsecretions) (Leak, 1999). These act at a greater dis-tance than purely visual baits.

Pilot studies conducted in various epidemiologicalsettings have shown that trapping is effective.Efficacy is measured in terms of the apparent densi-ty of flies captured per trap per day (ADT), andvaries according to the type of trap, the species orsub-species of Glossina, the environmental and cli-matic conditions, etc. (Laveissière, 1988). In the for-est areas of Côte d’Ivoire, it was demonstrated thatthe black/blue/black screen is about twice as effi-cient as the simple blue one (Laveissière et al, 1987).In the West African savannah, the ADT of G. tachi-noides and G. p. gambiensis populations was reducedby 88-92% using the blue screens (Mérot et al, 1984).The same screens reduced the ADT of G. tachinoidesby 98% in only 15 days (Laveissière and Couret,1981). In contrast, in the forest areas of Congo, thescreens did not yield satisfactory results. The ADTwas more drastically reduced (99%) after fivemonths in the forest areas of Côte d’Ivoire usingbiconical traps (Laveissière and Hervouët, 1981).Later on, in the same areas, about 16 000 bluescreens sited in coffee and cocoa plantationsreduced the ADT of G. p. palpalis by 90% in oneweek, and by 98% at the end of five months(Laveissière et al, 1986c).

Traps and screens have thus replaced insecticidespraying. These vector control methods have gener-ated much interest: they are effective, simple, envi-ronmentally friendly and suitable for use by thecommunities themselves. Pilot projects in BurkinaFaso (Mérot et al, 1984), Côte d’Ivoire (Laveissièreet al, 1994b), Uganda (Lancien, 1991) and Congo(Gouteux and Sinda, 1990) have demonstrated thefeasibility and efficiency of traps and screens usedwith the participation of rural communities under


the supervision of specialized teams. The number oftraps and/or screens required for a particular loca-tion depends on the type of vegetation in the tsetsehabitats, and on the presence or number of edges,water sources, paths and encampments whichdetermine the frequency and intensity of man-flycontacts. The communities are in a position to pro-vide information on locations where the villagersare most often bitten. The life span of a trap dependson the quality of materials and of maintenance, andon the environmental conditions. In experimentsconducted in West Africa, it was estimated thatabout 10% and 20% of the traps or screens needed tobe renewed each year in the forest and savannahzones respectively (Laveissière, 1988). It is not pos-sible to give the precise number of traps or screensto be installed per hectare, since each microcosmwill have its own features. The number of traps orscreens to be installed does not depend on the den-sity of the human population to be covered. In theVavoua pilot study, traps and screens were installedas follows: one screen every 100 metres, one or twoscreens per water-point or encampment, one screenat each working place in the plantation, one trapevery 300 metres in forest areas and one every 100metres around villages (Laveissière et al, 1994).

Traps are preferable to screens if re-impregnationand surveillance cannot be carried out by the popu-lation. Traps need to be re-impregnated once a year,at the end of the rainy season. Screens have to be re-impregnated once during the first year of the cam-paign, and twice a year later on. Traps should beinstalled as far beyond the endemic area as possible,so as to provide an effective barrier. In the galleryforest of savannah areas, open and sunny places fre-quently visited by people, such as washing andbathing places, water collection points, bridges andbanks of rivers, are the preferable sites. In forestareas, the tsetse flies must be intercepted at theinterfaces between different ecological patterns, i.e.the edges that are considered as epidemiologicallydangerous areas (Laveissière et al, 1986b), such asareas around villages, paths separating woodedareas and other types of ecological patterns, espe-cially cocoa or coffee plantations, etc.

Vector control with community involvement, as partof a comprehensive sleeping sickness control pro-gramme, was organized in the forest areas ofVavoua (Côte d’Ivoire) where Laveissière et al(1985) recorded a 90% reduction of ADT in the vil-lages and farms a week after setting up the trapsand screens, and over 99% after three months. Theseresults remained stable for the first six months.Subsequently, ADTs have increased. This phenome-non was linked to the gradual abandonment of

equipment maintenance, especially during thefarming season, and to lower efficacy due to theinsecticide being washed away by rains and thescreens being concealed by weeds which growquickly during the rainy season. Two years later, theimpact of the control programme on the incidence ofhuman disease was nevertheless obvious: no newpatient had been detected during the case-findingsurvey carried out in the study area where the over-all prevalence was initially higher than 1%. Globally,this experiment had shown that about a year’s vec-tor control was needed to substantially reduce trans-mission. Similar results were recorded in Congo(Gouteux and Sinda, 1990) and Uganda (Lancien,1991). Clearly, efficient case-finding must be con-ducted simultaneously with vector control, other-wise the persistence of the human reservoir willlead to a rapid resurgence of disease when vectorcontrol is pursued less vigorously. The cost of ascreen and a trap (Vavoua type) was estimated to beUS$3 and US$6 respectively (Laveissière and Méda,1992). The cost of one hectare protected was esti-mated at US$1 for the first year, and much less forthe second year. Costs vary depending on the typeof trap or screen used and on whether or not theequipment is insecticide-impregnated.

Genetic control by the release of sterile males isunsuitable for control in epidemic situations; itposes a series of technical, financial and logisticproblems which do not facilitate its implementationin endemic countries.

GAPS IN KNOWLEDGE AND RESEARCH NEEDSOver the last two decades, new and much moreeffective tools for HAT control have been developedcompared to those that existed some forty years agowhen the disease was almost eliminated. Thisprogress contrasts with the present epidemiologicalsituation. The disease remains a serious emergingpublic health problem in Central, East and WestAfrica. WHO, industrial firms, bilateral and multi-lateral organizations have decided to mobilize ade-quate resources to combat it. Many gaps in knowl-edge relevant to control of the disease need to beurgently filled. Some of these gaps have been iden-tified as priority areas by the Working Group onOperational Research on African Trypanosomiasis(Geneva, 1997) and the International Colloquium onOperational research priorities on African try-panosomiasis (Antwerp 1998). Scientists and pro-gramme managers are invited to pay much moreattention to the multiple questions related to thedevelopment of new tools for control or better use ofexisting ones. Most perspectives in HAT researchand control proposed by Kuzoe (1991) still need to


be considered. In the short and intermediate terms,some of the research priorities include the followingfields.

Improvement of EpidemiologicalKnowledge and Disease Surveillance andControlLittle is known of the basic epidemiology of HATdisease in many endemic countries, especiallyTanzania, Southern Sudan, Angola, Chad, CentralAfrican Republic and Guinea. It has been speculat-ed that T. rhodesiense sleeping sickness occurs amongtourists in East Africa; if this is true, it does meanthat maybe the disease is rising to an epidemic levelamong local populations. Improvement in knowl-edge of the host-parasite and vector-parasite rela-tionships is of great interest for the development ofnew tools and products for control, including newdrugs for treatment. The zoonotic nature of T. rhode-siense was confirmed about forty years ago. The sit-uation is completely different for T. gambiense; it hasbeen shown that domestic animals such as pigs,dogs, sheep and cattle can, or do, carry parasiteswhose isoenzyme features match those of parasitesinfecting humans. An important question arises asto the epidemiological importance of these animalreservoirs in T. b. gambiense trypanosomiasis. Atpresent, the exact role of these reservoirs isunknown; it is difficult to establish the relativeimportance of transmission between animals andhumans. Some studies suggest that such transmis-sion could play a significant role by maintaining aminimum level of transmission when the level ofendemicity is low. If this transmission is epidemio-logically significant, what are the factors that influ-ence the infective contacts between animals andhumans? Is there a sylvatic cycle which contributesto the persistence of Gambian trypanosomiasis?How can this cycle be interrupted? Several methodshave been proposed for blood-meal analysis. Whatis the most reliable, easy to use in field conditions,and inexpensive, technique for the determination ofblood-meal origin of the vectors?

Surveillance and Management ofSleeping SicknessThe card agglutination test for trypanosomiasis(CATT) is a simple, easy to use, sensitive and rela-tively effective tool for the diagnosis of T. b. gambi-ense trypanosomiasis, which has been available fortwo decades. However, there is no equivalent testfor T. b. rhodesiense sleeping sickness; it has beenfound that T. b. gambiense in Central Africa does notexpress the antigens used in the CATT. Thereforethere is need to improve the reagents presentlyused. More recently, another simple test, the cardindirect agglutination trypanosomiasis test (CIATT),

was evaluated. This test has been found to be high-ly sensitive and sufficiently specific for both types ofHAT. Its suitability and predictive value in clinicalmanagement need to be assessed in different epi-demiological settings. The test is easy to use in fieldconditions and does not need cold chain mainte-nance for storage; therefore, it is worthwhile evalu-ating its suitability for screening in primary healthcare programmes. The last meeting of the Task Forceon Operational Research on African Trypano-somiasis elaborated guidelines for designing the fol-lowing studies on the CATT and CIATT:• Assessment of chemotherapeutic cure of sleeping

sickness using the CIATT in T. b. gambiense andT. b. rhodesiense HAT.

• Comparison of the CIATT and CATT in the diag-nosis of Trypanosoma brucei gambiense sleepingsickness in a clinical setting.

• Applicability of the CIATT in the diagnosis ofTrypanosoma brucei rhodesiense sleeping sickness ina clinical setting.

• Operationality of the CIATT versus CATT in afield survey of T. b. gambiense sleeping sickness.

The question of the treatment of CATT-positive butaparasitaemic individuals is under study in the DRCand NE Uganda. The significance of the results ofthese studies need to be assessed for impact on theepidemiology of T. b. gambiense infections in humans.

The usefulness of remote sensing as a tool for sup-porting vector control and surveillance needs to beexplored further (Kuzoe, 1991). Is it a reliable toolfor the identification of high risk areas for humantrypanosomiasis? Is it an adequate tool for surveil-lance of the disease? Some questions, specificallyrelated to surveillance and treatment and requiringinvestigation, are given in the handbook by Leak(1999), including: Is there a level of vector densitythat ensures a low endemic level of trypanosomia-sis? Is trypanosome prevalence, as determined bypassive surveillance, a reliable indication that moreresources are needed for active surveillance? Whatshould be the interval between visits of mobileteams in the context of active surveillance?

PCR has recently been introduced as a new tool forthe identification of trypanosomes; however its use-fulness in the diagnosis and management of the dis-ease in humans, and its application in studies onanimal reservoirs, remain unsatisfactory. The tech-nology needs to be refined, adapted for field use,and transferred to field workers who need to betrained in its application.

Geographical information system (GIS) and map-ping technology offers a cost-effective and rational


tool for monitoring and control of disease, and isnow available for spatial analysis of data collected inendemic foci. This tool has been successfully appliedin the control of animal trypanosomiasis in savannahareas. In the context of limited resources, GIS is moreand more perceived as an efficient tool for the iden-tification and mapping of high risk areas where con-trol efforts should be directed. However, its applica-tion in HAT has been very limited so far. Therefore,there is a need to provide opportunities for multidis-ciplinary research teams, including geographers, tofurther use GIS in pilot control programmes.

Another gap mentioned by some young researchersis that there is no functional network for scientistsworking in the field of trypanosomiasis at regionalor sub-regional levels. Such a network would linksenior and young scientists together, allow efficientuse of human resources and strengthening of thescientific capabilities of young researchers.

Research on Existing and New Drugs,and Drug ResistanceThe ideal drug is one that is safe, effective, afford-able, and can be taken orally. Scientists and indus-trialists are invited to collaborate in the develop-ment of new effective compounds for the treatmentof HAT. Pharmacokinetic studies have shown thatit is worthwhile evaluating pentamidine adminis-tered for tree days versus seven days in the firststage of the disease. Studies in laboratory animals,using several combinations of existing drugs suchas pentamidine and eflornithine, suramin sodiumand melarsoprol, or melarsoprol and nifurtimox,have shown them to be synergistic. However, fewdata on the effects of drug combinations on eithertype of African trypanosomiasis are available. Inview of the increasing development of resistance tomelarsoprol monotherapy in some areas, studies onthe available drugs, given separately or in combi-nation (eflornithine/melarsoprol, nifurtimox/me-larsoprol, etc.), should be undertaken in humans,based on the latest pharmacokinetic information.Relapses following treatment with melarsoprol,with or without pentamidine, occur on a large scalein the DRC and Angola. Studies on factors associat-ed with treatment failure with melarsoprol shouldbe designed to identify preventive measures. Trialson alternative regimens of melarsoprol (e.g. 14 daysversus 10 days) might improve its efficacy and tol-erance. Many hypothetical reasons have been givenfor treatment failure, such as possible reinfection,misclassification of the disease, lack of compliancewith treatment, and individual variation in thepharmacokinetics of the drug. Studies on the mag-nitude of the phenomenon, and on the risk factorsand biological and environmental conditions deter-

mining the epidemiology of resistance to the drug,are lacking. Nifurtimox is a synthetic nitrofuranwhich was primarily used for treatment of Chagasdisease due to T. cruzi. In HAT, it has only beenused in the former Zaire and in Sudan, mostly inpatients with infection refractory to melarsoprol, incombination with other trypanocidal drugs. Fur-ther studies using an improved protocol should becarried out in T. b. gambiense areas where malarso-prol resistance is expanding (the DRC, Angola,Sudan) in order to identify effective combinationsof drugs. Controlled clinical trials on prednisoneversus placebo, to assess reduction of incidence ofmelarsoprol-associated encephalopathy, might alsobe interesting. No data are available on the efficacyof new drugs (nifurtimox, steroids) in T. b. rhode-siense sleeping sickness.

Research on Tsetse Flies and Vector Control The biotopes most suitable for tsetse flies are knownfor almost every biogeographical area, except man-grove swamps. There is a need to collect basic epi-demiological information on types of sites in order todetermine which areas present the greatest risk tohumans in West and Central Africa. A better under-standing of the variability of vectorial capacity of thetsetse fly, including age, population structure, andfeeding patterns, might lead to improvement of con-trol; populations that are genetically different mighthave different vectorial capacities in different epi-demiological contexts. What are the sociological fac-tors that affect man-fly contact and are responsiblefor the outbreak of epidemics? A recent meeting heldin Harare recommended setting up a quality controlsystem for new insecticides to be used for tsetse con-trol. What is the role of population mobility in theemergence and maintenance of epidemics of sleep-ing sickness in various epidemiological settings?

Data collected from catching tsetse flies with trapsare classically analysed using latin squares. Howe-ver, this method is questionable; there is a need toidentify more adequate statistical techniques for theanalysis of these particular types of data.

Social and Economic Impact of Sleeping SicknessLittle information is available in the literature on thesocial and economic impact of HAT. Therefore, thereis a need for social and economic studies to evaluatethe sustainability and long-term cost-effectiveness ofintegrated control programmes (Kuzoe, 1991). Someevidence suggests that sleeping sickness can reducephysical attributes and socio-economic potential atindividual, family and community levels; but physi-cians, demographers, social scientists and econo-mists have so far been unable to quantify and quali-


fy the effects of the disease. Operational research isalso needed to assess the impact of the current struc-tural health sector reforms on sleeping sickness con-trol programmes. Community involvement in tsetsecontrol and disease surveillance is of crucial impor-tance. However, human factors which motivate com-munities to participate may vary considerably(according to socio-cultural factors, areas) and needfurther investigations. What kind of message andwhat is the best way to communicate information torural communities? Multidisciplinary researchteams including social scientists, economists andbasic scientists should be involved in such studies.

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II VECTOR CONTROL IN RELATION TO HUMAN AFRICANTRYPANOSOMIASIS

I. Maudlin, E. M. Fèvre, P. G. Coleman,Centre for Tropical Veterinary Medicine, The Universityof Edinburgh, Easter Bush Veterinary College,Midlothian, EH25 9RG, UK

CONTROLLING VECTOR-BORNE DISEASES Controlling vector-borne diseases represents a spe-cial challenge to societies in developing countries.Apart from the sheer size of the public health prob-lems posed by diseases such as malaria and sleepingsickness, vector-borne diseases are especially diffi-cult to control. One of the main reasons for this isthat vector-borne diseases are quantitatively differ-ent from directly transmitted diseases in that thebasic reproductive number (R0 - the number of newinfections which arise from a single current infec-tion, introduced into a population of susceptibles,during the period of its infectiousness) for vector-borne diseases is often an order of magnitudegreater than for directly transmitted diseases. Thismeans that, generally, vector-borne diseases aremore difficult to control and may bounce back fromcontrol faster than directly transmitted diseases.However, it follows from our theoretical under-standing that targeting control activities at the vec-tor has potentially the greatest impact on diseasetransmission (see below).

CONTROLLING SLEEPING SICKNESS It is over a century since it was first recognized thattsetse flies were responsible for transmitting try-panosomes from wild animals to domestic livestock(Bruce, 1895). When it subsequently became clearthat tsetse were also responsible for the transmis-sion of human sleeping sickness (Bruce andNabarro, 1903), control of these insects assumedmassive importance for the colonial powers becauseof the threat the disease posed to the economicdevelopment of Africa south of the Sahara.Subsequently the largely anglophone countries ofEast, Central and Southern Africa aimed a large partof their research and control efforts at elimination ofthe vector combined with passive case detection. Bycontrast in West Africa, and particularly among thefrancophone countries, the more direct approach ofcontrolling the disease in humans became estab-lished practice. This followed from the success ofJamot, who pioneered large-scale case detection andtreatment with the arsenical drug Atoxyl (de Raadtand Jannin 1999). By combining large-scale arsenicalchemotherapy with institutional interventions suchas regulating the movement of people, T. b. gambi-

ense epidemics were brought under control in thefrancophone region of West Africa. This francopho-ne approach was also adopted during the 1930s tocontrol epidemics in Nigeria and Ghana with thenewer arsenical tryparsamide. The different colo-nial administrations appear to have became lockedinto ‘their’ strategies of either treating the cases ordealing with the vector (de Raadt and Jannin, 1999).It is, nonetheless, clear that both approaches wereeffective.

In the case of sleeping sickness, as with other vector-borne diseases, the first line of defence in the face ofan epidemic would appear to be tsetse control –why then, was this approach not universally adopt-ed across colonial Africa? And why were the alter-native strategies adopted in francophone Africa suc-cessful? It is instructive to examine the reasoningbehind the decisions governing interventions forsleeping sickness control, not only to explain theevents of history but also to inform policy-makersfaced with the resurgence of sleeping sickness(World Health Organization 2001).

TWO DISEASES – TWO APPROACHESTO CONTROLIn retrospect, we tend to view this division betweenfrancophone and anglophone, in approaching whatwas apparently the same problem, as merely anoth-er example of the failure of communicationsbetween two competitive colonial powers. This may,however, be simplistic. Assuming that there wassome logic to this dichotomy of approach, it is prof-itable to first consider how this position arose on thetwo sides of the continent, as this may influencehow we might best proceed to control sleeping sick-ness in the future.

The reasons for the differing approaches to diseasecontrol have their origins in the biology of two quitedistinct diseases. These differences may be expres-sed within a theoretical framework of vector-bornedisease transmission, as discussed below.

AN EPIDEMIOLOGICAL FRAMEWORKTO EVALUATE SLEEPING SICKNESSCONTROL STRATEGIES The basic reproductive number, R0, is central tounderstanding how the parasite spreads through,and is maintained, in a population. A threshold con-dition of R0>1 must be achieved if a parasiticspecies “is capable of invading, and establishingitself within, a host population” (Anderson andMay 1992). Ideally, disease control will force R0<1,and so eventually lead to the eradication of the par-asite from the population. Determination of thisthreshold condition for transmission is, therefore,


important for the design and implementation ofcontrol strategies. (If the threshold condition cannotbe met, then control activities should aim to main-tain R0 at as low a value as possible.)

The study of vector-borne disease epidemiologyand the design of control programmes has benefitedfrom the development of a basic mathematical theo-ry by Macdonald (based on Ronald Ross’ earlierwork), which captures the essentials of vector trans-mission (Macdonald, 1957). From this theory, thereproductive number of a vector borne infection, R0,is defined as:

where m is the ratio of vectors to humans; a is thefeeding rate of vectors, such that the average timebetween feeds is 1/a; c is the probability that a sus-ceptible vector acquires infection after feeding on aninfectious human; b is the probability that a suscep-tible human acquires infection after being bitten byan infected vector; r is the rate at which humansrecover from infection, with the average duration ofinfection being 1/r; T is the time taken for the para-site to mature in the vector; and u is the vector mor-tality, such that 1/u is the average vector lifeexpectancy. A fuller explanation of how this expres-sion is derived, both mathematically and intuitively,is given by Anderson and May (1992).

The epidemiological impact of a particular controlintervention may be investigated by assigning itseffects to one or more of the parameters in theexpression for R0. For example, vector control willkill vectors, and so increase u and decrease m; whileactive case detection and treatment would increase r.Thus the expression for R0 provides insights intovector-borne disease epidemiology and a frame-work in which to evaluate the relative impact of dif-ferent interventions. An important qualitative con-clusion from the simple expression for R0 is thatkilling adult vectors is more effective than the earlytreatment of cases. The time a case is infected entersinto the expression for R0 linearly via the parameterr. In contrast, adult vector mortality enters in a non-linear fashion via the term exp(-uT).

This general model has subsequently been devel-oped to specifically model the African trypanosomi-ases, in both animals and humans, by Rogers (1988).The model provides a theoretical basis to investigatehow differences in the natural history of T. b. gambi-ense and T. b. rhodesiense will effect the epidemiologyof sleeping sickness caused by the two pathogens,and how these differences will impact on the relativeeffectiveness of the same control interventions.

Two well recognized differences between sleepingsickness caused by T. b. gambiense and T. b. rhodesienseare the duration of infection and the role of the animalreservoir. T. b. rhodesiense infections are characteristi-cally acute, with death often occurring within a fewmonths of infection (Odiit et al, 1997; Apted, 1970;Wellde et al, 1989). The role of a non-human animalreservoir in T. b. rhodesiense transmission has longbeen recognized (Heisch et al, 1958; Onyango et al,1966). Indeed, movement of cattle from the sleepingsickness endemic areas of south-east Uganda has beenimplicated in the origins of a T. rhodesiense outbreak inan area previously free from the disease (Fèvre et al,2001). By contrast, T. b. gambiense infections tend to bechronic, with the time between onset of symptoms,development of late-stage disease and subsequentdeath often occurring over several years (Apted,1970). Also, humans are generally considered to be themain host for T. b. gambiense infections with the animalreservoir less important than in T. b. rhodesiense.However, the role of the animal reservoir in gambienseinfections is still unresolved. Rogers (1988) arguedthat domestic animals may be essential in the mainte-nance of T. b. gambiense as R0 in humans alone mayfall below unity, leading to suggestions that the exis-tence of a non-human animal reservoir in T. b. gambi-ense infections may have contributed to the failure ofhuman population surveillance and treatment cam-paigns to eradicate sleeping sickness in certain set-tings (Morris, 1946; Rogers, 1988). However, it hasalso been argued that the existence of an animal pop-ulation on which tsetse flies preferentially feed mayresult in humans receiving infectious bites at a lowerrate than if flies only feed on humans (Goutex,Laveissièrre and Breham, 1982).

These differences in the natural history of the dis-eases may be incorporated into the expression forR0, and their impact on the relative effectiveness(expressed in terms of reducing R0) of strategies tocontrol the two types of sleeping sickness demon-strated (Welburn et al, 2001). It was shown thatdetection and treatment of cases profoundly reducesthe transmission of T. b. gambiense but has relativelylittle impact on the transmission of T. b. rhodesiense.This is because the duration of T. b. rhodesiense is rel-atively short, and the animal reservoir is a relativelyimportant component of R0.

In the light of these theoretical and biological con-siderations, the different approaches adopted by thecolonial authorities of the time are seen to be quitelogical.

CONTROL IN THE 21ST CENTURYWhile, theoretically, vector control will proportion-ally have the greatest impact on disease transmis-


sion, it may not always be the approach of choice inthe field when other variables are taken intoaccount:• Treatment of infected people has to be carried out

for humanitarian reasons, and funding may usu-ally being found for such emergency interven-tions.

• In the war zones of Africa, sustaining a tsetse con-trol operation is very difficult and logisticallymore demanding than case finding. Tsetse controloperations in these circumstances may be merelytoken, having little impact when carried out bypoorly equipped and under-trained personnel.Given limited resources and societal instability(e.g. as in the current T. b. gambiense outbreaks inSouthern Sudan, Congo and Angola), it may notbe appropriate to divert resources from case find-ing towards tsetse control.

• Targeted treatment of the animal reservoir, in thecase of T. b. rhodesiense, is a potentially effective,inexpensive and sustainable control option(Welburn et al, 2001).

Tsetse control operations, whether top-down orenacted by the community, will always suffer fromproblems of sustainability, which have been consid-ered in great depth elsewhere (Brightwell et al,2001). While trapping programmes run by the com-munity are appealing to donors, they may simply beineffective in the face of an epidemic. Under suchcircumstances, alternative actions (including aerialspraying) may have to undertaken, which mayrequire very large inputs from the donor communi-ty. Moreover, sleeping sickness control has unfortu-nately become a supply driven endeavour. As moreand more smart technology has become availablefor the control of tsetse, so the need has been felt toapply it to the control of sleeping sickness epidemics– but this may not always be appropriate.

By contrast, treatment of sleeping sickness casesand the domestic animal reservoir form part of thenormal activities of the medical and veterinaryservices. When these services break down, forwhatever reason, it is usual for aid agencies, the pri-vate sector and even communities themselves tostep in to the breach. Therefore, these activities arelikely to be more sustainable in the medium andlong-term.

CONCLUSION AND FUTURERESEARCHFrom a theoretical perspective, killing tsetse flies isa preferred strategy. However, epidemiologicaleffects are only one side of the story. Financial andlogistical resources needed to implement and sus-tain control interventions also have to be consid-

ered, as in reality these resources are always limited.Indeed, in countries affected by sleeping sickness,the many resources necessary for implementation ofcertain control strategies are often non-existent orseverely constrained. Control policies should bebased on the optimal utilization of the availableresources. To help determine this optimal utiliza-tion, cost-effectiveness analysis is an important tool(Murray et al, 2000; Shaw et al, 2001). Such analysismust be based on a sound understanding of the nat-ural history of the disease, to best predict the likelyepidemiological effects of the same resources invest-ed into alternative interventions.

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Murray CJL et al. Development of WHO guidelineson generalized cost-effectiveness analysis. HealthEconomics, 2000, 9:235-251.

Odiit M, Kansiime F, Enyaru JCK. Duration ofsymptoms and case fatality of sleeping sicknesscaused by Trypanosoma brucei rhodesiense in Tororo,Uganda. East African Medical Journal, 1997,74:792-795.

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III A BASIS FOR FINANCIAL DE-CISION-MAKING ON STRATEGIESFOR THE CONTROL OF HUMANAFRICAN TRYPANOSOMIASIS

APM Shaw,1 P Cattand,2 PG Coleman,3 M John4

1AP Consultants, Upper Cottage, Abbotts Ann,Hampshire, SP11 7BA, UK.2Association against Trypanosomiasis in Africa, 1 rue del’Hôtel Dieu, F-74200 Thonon, France.3CTVM, University of Edinburgh, Easter Bush, Roslin,Midlothian, EH25 9RG, UK.4Le Kerrio, 56230, Berric, France.

INTRODUCTIONThis discussion paper seeks to identify the mainissues involved in designing cost-effective strategiesfor the control of human African trypanosomiasis(HAT) and to highlight areas where more informa-tion needs to be compiled or new research initiated.

Some form of control of the disease has been ongo-ing in most parts of Africa since the beginning of thelast century. Thus many schemes have been fundedand budgeted for. However, the economics of thedifferent approaches and their cost-effectiveness

have only been studied sporadically. Thus, whilstmost people working on the disease have a veryclear idea of what the most cost-effective strategiesare in their locality, given their resources and pricestructure, little has been done to bring together theinformation gained in order to help decision-makersdesign strategies and prioritize in new areas orunder new circumstances.

This paper is the first step in an ongoing exercise to: • identify the main factors influencing the cost-

effectiveness of the different approaches to con-trolling the disease.

• review the current literature on this subject.• identify potential research areas and information

needs.

For 1999, the burden of HAT was estimated at 66 000deaths and 2 million DALYs lost (World HealthOrganization, 2000). Although only 45 000 newcases were reported (World Health Organization,2001), the likely number of people affected is proba-bly ten times greater, thus approaching half a mil-lion. Of the 60 million people thought to be ‘at risk’,only 3–4 million are covered by some form of dis-ease surveillance.

TREATMENT

of patients found- 1st/2nd stage

rhodesiense / gambiense

Control of Disease Reservoirs Control of the vectorPoint of contact

TSETSE CONTROL

Traps, targets, screens, aerial spraying,

insecticide treatment of cattle

TREATMENT OF LIVESTOCK

cattle and, in the past, some wildlife control

DETECTION

finding patients:passive/fixed-post

surveillance,active surveillance

Point of contact

Human Animal

Figure 1. Control options


The control of HAT is based on combinations of thefour different approaches illustrated in Figure 1. Itinvolves: • treating those human patients diagnosed with the

disease.• trying to improve diagnosis, by some level of sur-

veillance, to find patients and ensure that they aretreated earlier, hopefully while still in the firststage of the disease.

• proceeding to more active forms of case-detec-tion, aimed not only at finding and diagnosingpatients but also at reducing the size of the humanreservoir of the disease. This applies to the gambi-ense form of the disease.

• trying to reduce the chance of people picking upthe infection from domestic animals or wildlife, asin Uganda, where cattle are routinely treatedaround new outbreaks to control the rhodesienseform of the disease (personal communication, IMaudlin).

• controlling tsetse fly populations so as to reducetransmission of both forms of the disease.

Finally, as indicated in Fig. 1 by the two boxesmarked ‘point of contact’, control of HAT involves: • avoiding areas likely to lead to infection - a strat-

egy that people have used since time immemori-al. This strategy applies, above all, to avoidingcontact with the tsetse fly and is an approach thathas mainly been employed by livestock keepers toprevent their cattle becoming infected and eitherdying or becoming less productive. As peoplebecome aware of the dangers of working in cer-tain tsetse-infested thickets, they avoid them. Inthe past it was thought that people could avoidcontracting the rhodesiense form of the diseasesimply by avoiding areas containing wildlife. Inthe early stages of an epidemic of sleeping sick-ness, the disease tends to be found among peoplewhose occupations put them at risk by bringingthem into contact with infected flies, particularlyat certain times of the day (e.g. when collectingwater, washing clothes, entering game reserves -as in the case of beekeepers, hunters, parkrangers). Thus, avoidance, whilst not specificallydiscussed here, is a control strategy of sorts,although in practice it has mainly been used bycattle herders to protect their stock.

FINDING AND TREATING PATIENTSAs cited above, WHO estimates are that only about10% of sleeping sickness patients are correctly diag-nosed and receive treatment. This proportion maybe larger in endemic foci where active surveillanceis undertaken, but it can also be a great deal smaller.Typically, patients who are detected passively havesuffered from symptoms for some time, possibly

years in the case of gambiense sleeping sickness, andhave made several attempts to have their symptomstreated and obtain a correct diagnosis. Usually thiswill have involved several trips to their rural healthcentre, possibly also to a nearby hospital or treat-ment centre, a visit to a local healer, and being treat-ed for malaria and other diseases before being diag-nosed as having sleeping sickness. Both during tripsto the health centre and while the patient is hospi-talized, it will usually be necessary for a relative toaccompany and look after the patient. As the diseaseprogresses, the patient in search of a diagnosis willhave become more and more of a burden on his fam-ily, requiring care and being unable to undertakenormal activities.

A very rough estimate of the possible cost topatients trying to obtain a diagnosis was made byLandell Mills (2000) for the rhodesiense situation inUganda. This came to US$25 per patient, includingestimates for the cost of transport to rural healthcentres and hospitals, treatments for malaria,painkillers, and the time taken by relatives toaccompany and care for the person. For those rhode-siense patients who are never correctly diagnosedbut who do receive some treatments, this cost wouldrise to a minimum of US$50, including consultationswith a local healer, further trips to treatment centres,possibly a short stay in hospital, and care by rela-tives.

For those correctly diagnosed, the costs of treat-ment, as estimated by WHO in 1998, are given inTable 1. These figures are based on the then currentdrug costs, treatment regimes in use, and estimatesof the cost of hospitalization.

Table 1 Costs of treatment

However, these costings will need to be re-evaluat-ed in the light of current plans to make drugs avail-able free of cost from WHO for a specified numberof years. The costs of transport and administrationwill remain, but, for the relevant drugs, the cost paidby recipient countries or programmes will consistonly of transport and drug administration. As thetable indicates, treatment costs could be reduced bysignificant percentages, but nevertheless, in eco-nomic as against purely financial terms, the use ofthese drugs will still represent a resource cost whichshould be taken into account. Furthermore, drug

Item First-stage disease Second-stage disease

Pentamidine Suramin Melarsoprol Eflornithine

Estimate of total cost 107 114 253 675

Cost excluding drug cost 87 79 190 367Drug cost as %of total cost 19 31 25 46

Source: WHO, 1998


availability on these terms will alter with time.Taken together therefore, this implies that the aver-age cost for patients (a high proportion of whomwill already be in the second stage of the disease)passively diagnosed and correctly treated would bein the order of US$50–300 each.

In order to analyse this aspect further, there is a needto:• analyse case histories of individuals being treated

for the disease in order to determine by whatprocess they obtained a diagnosis, how long ittook, and how much it cost them and the healthservices.

• collate information on currently used treatmentregimes and the costs of hospitalization at try-panosomiasis treatment centres.

ECONOMICS OF CONTROLLING THE HUMAN RESERVOIR:GAMBIENSE FORM OF THE DISEASETurning next to active surveillance, which has beenthe mainstay of programmes to combat the gambi-ense form of the disease, calculated costs are givenin the World Health Organization (WHO) ExpertCommittee report of 1998 (see series of tables inAnnex 9 of that report). The discussion below isbased on these calculations, as originally present-ed by Shaw et al (1995) and following the sameapproach as those prepared for the previous WHOExpert Committee on HAT (Shaw and Cattand,1985). In order to produce a coherent set of cost-ings, it was necessary to use a real situation as abasis while making some adjustments to produce ascenario in line with generally accepted norms,and the figures and prices used were based main-ly on work conducted in the Moyo District ofUganda (John, 1995). The figures used in 1985 werebased on WHO’s work in the Daloa area of Côted’Ivoire.

WHO’s calculations of 1998 were used to create aspreadsheet (Microsoft Excel®) model, so allowingresults to be calculated for any starting prevalence.The population covered, number of units involvedin surveillance, sampling intensity, sensitivity andspecificity of screening and diagnostic tests, as wellas all prices and other costs, can also be varied. Theresults of repeated runs of this model enable the rel-ative cost-effectiveness of different sampling strate-gies at different prevalences to be analysed. Theseare presented and discussed in the series of graphsbelow.

The original analyses were based on five alternativesurveillance strategies, including the classic mobileteams, fixed-post surveillance, and the less widely

used innovative techniques of filter paper samplingby trained community health workers who eithervisit the community or are based at rural health cen-tres: • Fixed-post or passive surveillance. Using this

strategy, patients who present with symptomsthat are difficult to diagnose, or who don’trespond to treatments e.g. for malaria, are eventu-ally referred to a treatment centre and tested for avariety of diseases, including trypanosomiasis, sothat those with the disease are eventually diag-nosed. The initial screening test is performed onwet blood.

• Filter paper sampling at rural health centres.Under this strategy, community health workers(CHWs) based at rural health centres receivesome training in collecting samples on filter paperand routinely test all new patients presentingthemselves at the health centre for whatever rea-son.

• Filter paper sampling by community healthworkers. CHWs trained in collecting filter papersamples spend 20% of their time collecting sam-ples and following up seropositive individuals -as based on experience in Uganda (John, 1995)and Côte d’Ivoire (Laveissière et al, 1995).

• Monovalent mobile teams. These are the classicsurveillance teams. The card agglutination test fortrypanosomiasis (CATT) is performed on wholeblood, and all the parasitological tests, exceptlumbar punctures, are conducted in the field.Monovalent teams work only on trypanosomia-sis.

• Polyvalent mobile teams. These operate in thesame way as monovalent teams except that only athird of their work consists of screening for try-panosomiasis.

In order to standardize the results, calculations werebased on areas with a population of 100 000 people,containing 10 rural health centres, and where 20community health workers were operating. Thenumbers screened by each strategy were assumed tobe as given in Table 2.

Table 2 Numbers screened for trypanosomiasis usingdifferent surveillance strategies in an area with a pop-ulation of 100 000

The cost calculations covered:• initial screening using the CATT test.• parasitological confirmation using gland punc-

tures, the capillary tube centrifugation technique

Surveillancestrategy

Numberscreened per

annum

Fixed-postsurveillance

300 3000

10 ruralhealthcentres

24 000

20 communityhealth

workers

36 000

Onemonovalent

mobile team

20 000

Onepolyvalent

mobile team


(CTC) test, the miniature anion centrifugationtechnique (m-AECT), and lumbar punctures.

• training of staff in specialized techniques neededfor diagnosing trypanosomiasis.

• administrative overheads.• depreciation (annual cost) of capital items (vehi-

cles, laboratory equipment, etc.).• travel allowances and a share of salaries of all staff

in proportion to the amount of time they spend ontrypanosomiasis control.

• running costs for vehicles, specialized equipmentand other recurrent costs for each surveillancestrategy.

Firstly, the relative performance of the different sur-veillance strategies was analysed in terms of the costof finding gambiense patients. The results, in terms ofUS$ per trypanosomiasis patient found, are given inFigure 2. Obviously the cost of finding a patientdeclines very rapidly as the prevalence increases,since a higher proportion of those screened areinfected. This clearly does not reflect any increase inefficiency, simply that more and more of thosescreened are infected, so the costs of the operationaverage out over a larger number of individuals.Fig. 2 is given in three sections so that the differen-tials between the costs of each sampling strategy canbe seen more clearly.

At very low prevalences (of 1% or less, see Fig. 2a),such as those encountered in past years in areaswhere surveillance was reasonably regular and thedisease was considered to be under control, thecosts (at a 0.05% prevalence) are well over US$2000per patient identified using rural health centres ormobile teams, and drop to just under US$2000 usingCHWs. Passive or fixed-post detection costs only US$50 per patient identified since there are virtually nooverheads. When the prevalence reaches 1%, thecost of passive detection falls to just over US$20 per

person, while surveillance using CHWs costs justunder US$100 per person, and using mobile teamsor rural health centre costs between US$120 andUS$140.

At medium level prevalences (from 1% to 5%, seeFig. 2b), the costs per patient found continue to fall,and the differentials between strategies narrow fur-ther, with the cost of passive detection being US$14,of detection by CHWs being US$22, and of the otherthree options being about US$30.

3000

2500

2000

1500

1000

500

0

US$ PERPATIENT

FOUND

PREVALENCE (%)

160

140

120

100

80

60

40

20

0

US$ PERPATIENT

FOUND

PREVALENCE (%)

Rural Health centre

Communityhealth worker

Fixed postsurveillance

Monovalentmobile team

Polyvalentmobile team

0 0 1 2 3 4 50.5 1

Figure 2a. Cost of detection per

patient at low prevalences: 0.01% to 1%

Figure 2b. Cost of detection per patient

at medium prevalences: 1% to 5%


At high level prevalences (over 10%, see Fig. 2c), thecost of patients found passively falls to aroundUS$10, as it does for all surveillance strategies at aprevalence of 20%. When the prevalence reaches50%, the cost of detection becomes very low, aroundUS$5 for all surveillance strategies, except passivedetection where it remains at about US$10.

Given the way in which the figures were calculated,by independently building up the cost of each strat-egy using local norms and prices, it is surprisinghow similar the costs for finding patients using dif-ferent strategies are. Setting aside passive detection,of the four active detection strategies, CHWs usingfilter paper is consistently the most cost-effective.

The purpose-built mobile team concerned only withtrypanosomiasis surveillance is consistently themost costly.

The almost total convergence of costs at high preva-lences is due to the fact that, at higher prevalences,more than half of all costs are diagnostic costs forinitial screening and parasitological examinations(ranging between US$3.50 and US$2.50 per person).At these prevalences, most individuals are sero-pos-itive and have to be re-examined, so the runningcosts and overheads associated with each strategyare spread over a large number of patients.

In order to further examine the relative effectivenessof the different surveillance strategies, Fig. 3 shows

what proportion of the population could be sam-pled in a year using the different approaches andreflecting the assumptions made about the possibleworkload that each form of active surveillance cantackle. The figure shows the situation in the hypo-thetical area with a human population of 100 000, afixed number (10) of rural health centres, and a fixednumber (20) of CHWs who can assign a significantproportion of their time to active case detection forsleeping sickness. In such a situation, only theinputs by the mobile teams can be varied, for exam-ple increased as illustrated, from spending half theirtime in the area to having two teams working therefull time. Based on experience, it was also assumedthat mobile teams were able to undertake furtherexaminations on a higher proportion of CATT-posi-

Figure 2c. Cost of detection per patient

at high prevalences: 1% to 5%

35

30

25

20

15

10

5

0

US$ PERPATIENT

FOUND

PREVALENCE (%)

Rural Health centre

Communityhealth worker

Fixed postsurveillance

Monovalentmobile team

Polyvalentmobile team

0 10 20 30 40 60 7050

Figure 2c. Cost of detection per patient

at high prevalences: 5% and over


0

10 000

20 000

30 000

40 000

50 000

60 000

US $

10 Rura

l Health

Centre

s

20 Community

Health

Worke

rs

Fixed P

ost Surve

illance

1 Monova

lent Mobile

Team

1 Polyv

alent Mobile

Team

10% Prevalence1% Prevalence

tive patients than was possible under the surveil-lance strategies based on CHWs. The filter paperbased sampling strategies involve a delay before theresults are known, after which individuals testingpositive are recalled and taken to a treatment centrefor further testing. In contrast, mobile teams canundertake many of the parasitological tests them-selves, and transport the suspected patients to atreatment centre for final confirmation of diseasestatus.

Under these conditions, as illustrated in Fig. 3, find-ing and treating a majority of the patients in thepopulation can only be done using one or moremonovalent mobile teams. The other active surveil-lance strategies could be effective in detecting thepresence of the disease, or in gradually eroding thesize of the human reservoir, provided that the inci-dence is not high.

Turning from the costs per individual found with thedisease to the total investment required for findingtrypanosomiasis patients, Fig. 4 illustrates these forthe five different surveillance strategies for preva-lences of 1% and 10%. As would be expected, thecosts largely reflect the proportion of populationscreened by each strategy (Fig. 3 and Table 2).Although the costs would vary from country tocountry, and have probably increased somewhatsince these figures were published in 1998, they givean idea of the orders of magnitude involved, rangingfrom US$50 000 to US $60 000 for a monovalentmobile team, to a minimal investment for fixedpost/passive detection.

10 H

ealt

h C

entr

es

20 C

HW

's

Fix

ed P

ost

0.5

Mo

no

vale

nt

Tea

ms

0.5

Po

lyva

len

t T

eam

s

1 M

on

ova

len

t T

eam

1 P

oly

vale

nt

Tea

m

2 M

on

ova

len

t T

eam

s

2 P

oly

vale

nt

Tea

ms

% patients

% people

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

%

Fig. 3Percentage of population sampled and

percentage of all trypanosomiasis patients found according to surveillance strategy used

% people

Fig. 4 Total trypanosomiasis specific costs for

case-finding by surveillance strategy


In order to better understand how the situationevolves at high prevalences, details of the calcula-tion for intervention by a mobile team are shown inFig. 5. Here, the cost of treating the identifiedpatients has been added to produce the total costs,which have been broken down into four categories.The costs of surveillance (logistics, share of salaries,etc.), which are given as ‘sampling strategy’, and thecosts of diagnostic tests, which cover both initialscreening and parasitological confirmation, domi-

nate the total costs at very low prevalences. But oncea prevalence of 1% is reached, all other costs aredwarfed by the cost of treating patients. Given theuncertainty about how to value drug costs, thesehave been included at their recent commercial costbut separated from other treatment costs (adminis-tration of drugs and hospital care). Thus the graphscan be read so as to exclude the cost of drugs. Anotional figure for transport, or for their economiccost, could be included in subsequent analyses.

0

100,000

200,000

300,000

400,000

US$

0.1 0.5 1 2 3 4 5

% Prevalence

Cost of Drugs

Other treatment cost

Diagnostic Tests

Sampling Strategy

0500 000

1 000 0001 500 0002 000 0002 500 0003 000 0003 500 0004 000 0004 500 000

US$

10 20 30 40 50 60 70% Prevalence

Cost of DrugsOther treatment costDiagnostic TestsSampling Strategy


Fig. 5aBreakdown of costs of detection and treatment of patients

at low to medium prevalences (one mobile team sampling 36 000 people)

Fig. 5bBreakdown of costs of detection and treatment of patients

at high prevalences (one mobile team sampling 36 000 people)



The costs of controlling the disease in the area pos-tulated, with one monovalent mobile team screen-ing 36% of the population in a year, thus range fromjust over US$50 000 where the prevalence is 0.1%, toover US$4 million where the prevalence is 70%.

This analysis, based on conditions in Uganda andextrapolation of the situation encountered there,thus examines the ways in which the costs of con-trolling the human reservoir vary with the samplingstrategy, sampling intensity, and prevalence. Thespreadsheet model produced provides a basis forextending this analysis to cover other surveillanceprotocols, price sets, test sensitivities, etc. It is likelythat the costings for the extremely high prevalencesneed revising upwards, since in the model, labourand time requirements were not fully adjusted to asituation where virtually all individuals test positiveto the screening test and need parasitological confir-mation.

To update and validate this analysis in different cir-cumstances, what is required is: • collation and examination of data from budgets

and actual expenditures from a range of surveil-lance activities in different countries.

• to find out what protocols are used in differentfield situations and what results are obtained,particularly in terms of what sequence of tests areused to obtain parasitological confirmation andhow many patients are detected by each test atdifferent prevalences.

ECONOMICS OF CONTROLLING THEANIMAL RESERVOIR: RHODESIENSEFORM OF THE DISEASE Turning to the rhodesiense form of the disease, itsmore acute course means that patients usually pres-ent with symptoms shortly after infection.Nevertheless, diagnosis is often slow and inaccurate.Control of this form of the disease has relied less onactive surveillance and more on vector control.

Recent research results have added a powerful andcost-effective tool to the armoury of control methodsfor this form of the disease. The proof that cattlehave now become the main reservoir of the disease(Hide et al, 1996) in south-eastern Uganda has, as itscorollary, shown that treating cattle would controlthe reservoir, and, if at suitable level, would stoptransmission to humans. Although T. b. rhodesiense isnot pathogenic to cattle, the drugs used to control italso kill T. vivax and T. congolense, which are patho-genic to cattle and are prevalent among the cattle inthe area. Thus, treating cattle generates an econom-ic benefit which is independent of the control of thedisease in humans.

A preliminary and very approximate economicanalysis of the control of rhodesiense disease insouth-eastern Uganda was undertaken as part of aDFID-commissioned review of its research work(Landell Mills, 2000). Although based on extremelyapproximate assumptions, this analysis highlightedthe potentially very favourable situation where it ispossible to control the disease by treating cattle.Drug treatments cost US$1.75 to US$2 per dose, andit is thought that between 5% and 20% of cattle carrycattle pathogenic trypanosomes (personal commu-nication, Paul Coleman). Based on work done on theimpact of trypanosomiasis on livestock production,and current milk and cattle prices in Uganda, it wasestimated that treating 1000 cattle around a diseasefocus could yield a benefit of between US$500 toUS$3000, as compared to a cost of US$750 toUS$2000. Furthermore, if this expenditure on cattletreatment was successful in reducing the incidencein humans, the financial benefit in terms of sleepingsickness treatment cost avoided would probablymore than justify the expenditure.

In this preliminary analysis of the economics of dis-ease control over the past decade in south-easternUganda, four categories of costs were considered:research, vector control, medical surveillance andcattle treatment (Landell Mills, 2000). The benefitswere calculated by considering three likely alterna-tive scenarios for what the disease incidence mighthave been if there had been no control activities. Themonetary benefits consisted of costs saved for treat-ing patients, benefits to cattle production, andpatients’ costs incurred while seeking treatment (seesection Finding and treating patients above). The non-monetary benefits were calculated in terms ofDALYs averted. The monetary benefits producedbenefit-cost ratios ranging from 1.08 to 1.98. Becausethe project produced a net financial gain, the costper DALY was actually negative, ranging from -US$1.50 to -US$7.50. These results should be treatedwith caution, as they depend on very roughassumptions. However, they do point to the likeli-hood that this control approach, where it is feasible,could be extremely cost-effective.

Research into the involvement of cattle as a reser-voir of T. b. rhodesiense is ongoing. From the eco-nomic point of view, one key issue is, what propor-tion of the cattle population will need to be treated,and with what frequency, in order to generate finan-cial benefits which cover the costs of controlling thedisease in humans? Another important variable isthe ratio of reported to unreported cases of the dis-ease in humans. Obtaining this informationdepends on the results of epidemiological studies.The economic gains to cattle production need to be


more accurately estimated, using data on the preva-lence of cattle pathogenic trypanosomes and theirimpact on livestock productivity.

VECTOR CONTROLAt this stage of the work, it has not been possible toreview existing information about the costs of vec-tor control in detail. Recent years have seen fewinitiatives that use vector control to deal withhuman trypanosomiasis. Costs are available fromrecent work in Uganda, where pyramidal trapswere very effective in reducing tsetse density anddisease incidence; these costs (personal communi-cation R. Floto) amounted to ECU 781 000, exclud-ing technical assistance - at today’s prices, aboutUS $1.1 million.

The costs of vector control will tend to be very spe-cific to the situation, varying with terrain (especial-ly for target and spraying operations), type oforganization (especially for targets, traps orscreens), and type of settlements and rural economy(being affected by the availability of labour formaintaining traps and targets, and the presence ofcattle where these are to be treated with insecti-cides). Estimates of the cost of vector control opera-tions, such as those cited below, almost invariablyinclude only the marginal costs, i.e. the extra costsinvolved in field work, but neglect the considerableoverheads, which can double or triple the costs persq. km. Vector control operations include: • aerial spraying, using fixed wing aircraft and the

sequential aerosol technique, which typicallyinvolves spraying the area in a cycle of five atcarefully timed intervals. Apart from the sterileinsect technique, aerial spraying tends to be themost expensive control method. It has the greatadvantage of achieving a very rapid reduction inthe fly population. Costs are likely to be wellupwards of US$500 per sq. km.

• traps, screens and/or targets. The cost of theseoperations is far more variable than for aerialspraying, depending on the number deployed persq. km. or per linear km. and on the way in whichthey are deployed and serviced. The costs for tar-get operations probably range between US$300and US$400 per sq. km., and low-cost trap- orscreen-based operations could cost as little asUS$100 to US$150 per sq. km.

• treating cattle with insecticides (not to be con-fused with treating cattle with trypanocides, asdiscussed above). Here, various ‘pour-on’ formu-lations are used, but again the costs are very diffi-cult to estimate since the pour-on formulationsalso protect against tick-borne diseases, and thenumber of cattle to be treated per sq. km. is also avariable as is the number of treatments per year.

The cost of pour-on formulations currently rangesaround US$1.50 to US$2 for an adult bovine.Insecticide treatment of cattle reduces fly density,but whether or not this would be of use in pre-venting HAT depends entirely on the local epi-demiology of the disease.

A useful series of comparative costings for tsetsecontrol methods for one country, along with detailsof how they were calculated, can be found in Barrett(1997) for the case of Zimbabwe. Current researchand information needs include updating costingsfor the various strategies, and collating informationon the impact that vector control operations under-taken in the past have had on the incidence of sleep-ing sickness. The extent to which communityinvolvement and inputs, especially of labour, can besustained over long periods of time also needs to berevisited.

It is difficult, especially for gambiense disease, toseparate the effects of controlling the human reser-voir from those of vector control, since the two areusually undertaken at the same time. The cost-effectiveness of vector control versus case-findingand treatment was modelled by Shaw (1989). At thistime, it appeared that case-finding and treatmentwas the more cost-effective at lower incidences andwhen dealing exclusively with a human reservoir.The conclusion, then as now, was that epidemiolog-ical models and economic models need to be inte-grated in order to be of use in decision-making.

DALYS AND THE COST-EFFECTIVENESSOF SLEEPING SICKNESS CONTROLFinally, in order to examine the wider economics ofcontrolling sleeping sickness in relation to the con-trol of other diseases, an estimate of the cost perDALY averted is needed. So far there have been veryfew comprehensive attempts to calculate the actualand potential DALYs lost due to either form ofsleeping sickness.

Recent work in Uganda has produced calculationsof DALYs for rhodesiense (Odiit et al, 2000a and2000b). This work will be integrated into morecomprehensive economic analyses. The authorspoint out that the age distribution of trypanosomi-asis patients very closely follows that of the activeadult population, so that the disease tends to hitthe most economically productive group of societyhardest, affecting family livelihoods and communi-ty prosperity very much. These data confirm obser-vations made throughout Africa for both forms ofthe disease. Odiit et al estimate that, at the time ofdiagnosis, patients will have been suffering fromsymptoms of the disease for an average of 61 days


and will then require hospitalization for an averageof 34 days. For patients correctly diagnosed andtreated, the case fatality rate is 5.3%, but for unre-ported cases, the outcome is assumed to beinevitably fatal. Based on the age distribution ofpatients, Odiit et al estimate that the number ofDALYs lost for unreported, and thus untreated,patients is just over 20 years (personal communica-tion Dr Paul Coleman). This figure was used tounderpin the economic analysis discussed in thesection on Economics of controlling the animal reser-voir above. In this case, because controlling the ani-mal reservoir reduced the rate of transmission tohumans, and it was assumed that for every report-ed case there was one unreported case, the fulltwenty years applied to half of the cases averted.This DALY figure thus also means that the cost-effectiveness of controlling rhodesiense sleepingsickness compares very favourably with that ofother high priority health control activities, such asmalaria (Goodman et al, 2000), the expanded pro-gramme on immunization (EPI), and humanimmunodeficiency virus (HIV).

For gambiense disease, no published DALY figuresbased on detailed field records are available.However, ongoing work in Southern Sudan indi-cates that a similar situation probably exists (per-sonal communication Dr Anne Moore). An attempt

to look at the economics of alternative treatments forsecond-stage gambiense patients was based on theage-at-death distribution calculated for rhodesiensepatients in Uganda (Politi et al, 1995). These authorsalso concluded that the standard treatment for sec-ond-stage patients represented a very attractive costper DALY averted, ranking with the most cost-effec-tive interventions such as childhood immunizationand blood-screening for HIV.

Figure 6 takes this discussion a bit further by mod-elling how the situation could be analysed if moredata were available. The cost of treatment anddetection of patients using a mobile team at differ-ent prevalences, as shown in Figure 2, is divided bya conservative estimate of the number of DALYsaverted. The ‘zero’ baseline figure, which gives thehighest cost, is based on each patient treated, i.e.each premature death prevented, representing 15DALYs averted. This figure was selected as a con-servative estimate, taking into account the longasymptomatic period for gambiense disease, and cal-ibrated to be rather lower than the figure for rhode-siense. On this estimate, once the prevalence hasreached 1%, the cost per person found and treatedfalls to US$330, and thus the cost per DALY avertedfalls below the ‘good value for money’ threshold ofUS$25. At higher prevalences, the cost per DALYaverted stabilizes at between US$10 and US$12.

Figure 6. US$ per DALY averted at different prevalences

(Surveillance for gambiense using a monovalent mobile team)

200

175

150

125

100

75

50

25

0

US$

PREVALENCE

0

0.5

1

1.5

0.05 0.5 2 4 10 30 4020 50 60 70

Multiplier applied to DALYs averted

Figure 6. US$ per DALY averted at different prevalences

(Surveillance for gambiense using a monovalentmobile team)


In order to add another dimension to the analysis,three further lines have been drawn showing whatthe effect would be if the number of DALYs avertedper patient found and treated was increased by 0.5,1 or 1.5. This analysis needs to be developed whenmore data have been analysed or collected to show:• what the actual figure for DALYs averted per

patient treated is likely to be.• how the prevalence evolves from year to year in

gambiense foci.The latter brings the discussion back to epidemiolo-gy, since, at low prevalences, this multiplier can beseen to be a measure of Ro, the basic reproductiverate of the disease.

There is thus a clear agenda for analysing existingdata on the progress of epidemics and for collectingnew data in order to add to the knowledge of theepidemiology of the disease. The low proportion ofall cases actually recorded has meant that knowl-edge of the year-to-year changes in prevalence isoften patchy or anecdotal. Nevertheless, for fociwhich have been the subject of more intensive con-trol work over a number of years, data do exist. Inthis context, epidemiological models (e.g. Rogers,1988) have an important role to play.

CONCLUSIONS This paper has tried to cover the main issuesinvolved in the economics of controlling both gambi-ense and rhodesiense sleeping sickness. Some aspects,notably vector control, have only been superficiallytreated. Nevertheless, it is hoped that the paper pro-vides a sufficient basis for discussion on whatresearch and information gathering is needed tohelp plan funding and resource allocation andgauge what returns to expect from these invest-ments.

Returning to the information requirements thatwere noted at the end of each section, these fall intoone of two categories: economic and epidemiologi-cal. Some of the issues and needs identified havebeen included in the table below, which provides asimple framework for categorizing the informationrequired.


Table 3Framework for identifying information requirements and sources

From the point of view of those allocating fundswithin the health sector, the recent emergence ofDALY calculations for sleeping sickness has made itvery clear that control of this disease represents anextremely cost-effective investment. This is linked totwo factors. The first is the inevitably fatal outcome ofthe disease, which has been discussed above. The sec-ond is the focal nature of the disease, which meansthat although the population at risk is large, the dis-ease is nevertheless location specific, so that controloperations can target circumscribed geographicalareas where the disease is known to be present.

AcknowledgementsThis paper reflects: the inputs and ideas providedover many years by Pierre Cattand, the joint workon the cost of surveillance undertaken with MichèleJohn, and Paul Coleman’s many suggestions andgenerous inputs into the analysis of the economics

of controlling rhodesiense. Any errors remain theresponsibility of the first author,who would also liketo thank Ian Maudlin, Anne Moore, Jean Jannin andMartin Odiit for their helpful comments andencouragement.

ReferencesBarrett JC. Economic Issues in TrypanosomiasisControl. Bulletin 75, Natural Resources Institute,Chatham Maritime, UK, 1997.

Goodman C, Coleman P, Mills A. EconomicAnalysis of Malaria Control in Sub-Saharan Africa.Strategic research series. Geneva, Global Forum forHealth Research, WHO, 2000.

Hide G et al. The origins, dynamics and generationof Trypanosoma brucei rhodesiense epidemics in EastAfrica. Parasitology Today, 1996, 12: 50-55.

Epidemiological

• Analyse changes in preva-lence over time, relating tocontrol strategies (includingvector control) being used.

• Collate diagnostic protocolsused in differentcountries/situations.

• Collate information on sensi-tivity and specificity of dif-ferent tests in the field.

• Determine the ratio ofreported to unreportedcases.

• Determine changes in preva-lence over time and withrespect to control workundertaken.

Economic

• Look at finances of past con-trol programmes, budgets,expenditure, overheads.

• Compare prices and costs ofsurveillance between coun-tries and in different epi-demiological situations.

• Update costings on vectorcontrol.

• Cost out different surveil-lance strategies, and refineexisting spreadsheet modelor develop new ones.

• Cost out different treatmentprotocols, determine appro-priate cost for drugs, esti-mate hospitalization costs.

• Determine the costs of thedisease to the local economy.

Analyse existing data

Initiate new data collection


John M. Utilisation of testryp catt applied to samples ofdried blood for sleeping sickness screening (T. b. gambi-ense trypanosomiasis) of the population in villages andhealth units, Moyo District, Uganda 1993-1994. Dis-sertation for the MSc in Public Health in TropicalCountries, London School of Hygiene and TropicalMedicine, University of London, UK, Sept 1995.

Laveissière C et al. Intégration de la surveillance de lamaladie du sommeil aux soins de santé primaires dansle foyer de Sinfra (Côte d’Ivoire). O.C.C.G.E., InstitutPierre Richet, Bouaké, Côte d’Ivoire, 1995.

Landell Mills. Evaluation of selected livestock researchthemes. Report prepared for DFID by Landell MillsLtd, researched and written by APM Shaw and LSibanda, 2000.

Odiit M et al. Incorporating the burden of human sleep-ing sickness in an economic impact assessment of try-panosomiasis. ISVEE, Colorado, 2000(a).

Odiit M et al. The burden of sleeping sickness inSoutheastern Uganda – geographical variations. Paperpresented to the British Society of Parasitology,Oxford, 2000(b).

Politi C et al. Cost-effectiveness analysis of alterna-tive treatments of African gambiense trypanosomia-sis in Uganda. Health Economics, 1995, 4:272-287.

Rogers DJ. A general model for the African try-panosomiases. Parasitology, 1988, 97:193-212.

Shaw APM, Cattand P. The costs of different approach-es towards the control of human African trypanosomia-sis. Paper Prepared for the WHO Expert Committeeon the Epidemiology and Control of African Try-panosomiasis, Ref. TRY/EC/WP/85.17, 1985.

Shaw APM. Comparative analysis of the costs andbenefits of alternative disease control strategies: vec-tor control versus human case finding and treat-ment. Annales de la Société Belge de Médecine Tropicale,1989, 69 (Suppl. 1):237-253.

Shaw APM, John M, Cattand P. The financial implica-tions of different approaches to the detection and treat-ment of human African trypanosomiasis. PaperPrepared for the WHO Expert Committee on theEpidemiology and Control of African Trypanoso-miasis, Ref. TRY/EC/WP/95.14, 1995.

WHO Expert Committee on control and surveillance ofAfrican trypanosomiasis. Geneva, World HealthOrganization, 1998 (Technical Report Series 881).

World Health Organization. The world health report.WHO, Geneva, WHO, 2000.

World Health Organization. African trypanosomiasisor sleeping sickness. Geneva, WHO, 2001, Fact SheetNo. 269 (March 2001).


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Annex 5DRUG DEVELOPMENT, PRECLINICAL AND CLINICAL STUDIES AND DRUG RESISTANCE


I DRUG DEVELOPMENT FORAFRICAN TRYPANOSOMIASIS

Cyrus BacchiPace University, Haskins Laboratories, 41 Park Row,New York, NY 10038, USA

INTRODUCTIONThere is an urgent and obvious need for novel, effec-tive, non-toxic drugs for treatment of humanAfrican trypanosomiasis (HAT). This is evidentfrom:• Dramatic recent increases in incidence of the dis-

ease due to civil unrest, warfare, and generalbreakdown of health monitoring and deliveryinfrastructure in large areas of Africa.

• An increase in resistance to standard trypanocidalagents.

• Toxicity of standard agents.

Of the standard agents available, pentamidine isused for early-stage Trypanosoma brucei gambienseinfections but is not generally recommended forearly-stage T. b. rhodesiense. Pentamidine has been inuse since 1940, is available from Aventis, and is alsoused for treatment of Pneumocytis carinii in AIDS.Resistance is due in part to inability to transport theagent. Suramin, a polysulphonated naphthylurea,has been in use since 1920 and is presently used forearly-stage T. b. rhodesiense. It does not penetrate theblood-brain barrier and is not used for the centralnervous system (CNS) disease. It has the longesthalf-life of any routinely used trypanocide due tobinding to serum proteins and there is no consistentappearance of resistance. Melarsoprol (Mel B,Arsobal) is the first choice for treatment of late-stageCNS infection. Developed in 1949, it has saved mil-lions of lives but suffers from toxicity problems(encephalopathic syndrome) in about 10% ofpatients, which are life-threatening in about 5% ofpatients. Recently, there has been an increase in thenumber of patients who are refractory to treatment,which can largely be attributed to lack of uptake bythe parasite (Burri et al, in press).

In a recent development, Aventis and WHO havesigned an agreement in which Aventis will supplypentamidine and melarsoprol free of charge for fiveyears. There are indications that Bayer will enterinto a similar agreement for suramin (FAS Kuzoe,personal communication).

APPROVED NEW AGENTDL-αα-difluoromethylornithine (DFMO, Orni-dyl®‚) is an inhibitor of ornithine decarboxylase, thelead enzyme of polyamine biosynthesis. Initially

developed in the late 1970s (at the Merrell Dowresearch laboratory) as an antitumour agent, itproved to be minimally active in that context but waseffective against laboratory infections of T. b. brucei,and later against T. b. gambiense in clinical trials. Itcured late-stage and melarsoprol-resistant CNS infec-tions, but was not active against T. b. rhodesiense infec-tion. Its side effects are generally mild and reversibleupon withdrawal of the drug (Van Nieuwenhove1992; Pepin and Milord 1994; Burri et al, in press). Itwas approved by the US Food and Drug Admini-stration (FDA) in November 1990. Three problemsarise with its use: expense (about US$500 perpatient), availability, and dosing (four times a day,intravenously, for 14 days). Aventis (current holder ofthe patent) now has a production agreement withBristol-Myers Squibb for the synthesis of DFMO to beused in a depilatory cream (Vaniqa).

In a recent development, WHO and Aventis haveentered into an agreement in which Aventis willsupply 60 000 vials of eflornithine each year for fiveyears. WHO will continue to explore avenues tomaintain production after the five-year period.WHO is conducting clinical studies to determine theefficacy and safety of oral dose eflornithine, whichwould allow its use on a wide scale (FAS Kuzoe,personal communication).

New agent in Phase I clinical trials: DB 075/289A number of 2,5-bis(4 amidinophenyl)furan caba-mates are reported to have activity againsPneumocysitis carinii (Rahmathhulla, et al 1999). Oneof these compounds (DB 075/289) has nearly com-pleted Phase I testing (toxicity, pharmacokinetics),aimed at development against Pneumocystis cariniipneumonia (PCP) and African trypanosomiasis. Incase of a favourable outcome, first field trials (PhaseIIA: proof of principle in patients) are to be con-ducted starting in May 2001. Studies in try-panosome infected monkeys are ongoing and arethus far promising (C. Burri, Pesonal communica-tion). DB075/289 does not pass the blood-brain bar-rier and is only active against the first phase of thedisease. However, as an orally dosed agent, it wouldbe ideal for treatment of actue disease as an alterna-tive to pentamidine and potential additional meansfor disease control in high transmission areas. Thedevelopment of DB 075/289 is conducted by aninternational consortium lead by Dr RichardTidwell of the University of North Carolina, andfunded by a 15.1 million grant from the Bill andMelissa Gates Foundation.


LEADS TO OTHER AGENTSAntagonists of Polyamine Metabolism. Inaddition to DFMO, a number of other agents target-ing polyamine metabolism have shown promise.These include MDL 73811 (5’-{[(Z)-4-amino-2-butenyl]methylamino}-5’-deoxyadenosine), an enzy-me-activated inhibitor of S-adenosylmethionine(AdoMet) decarboxylase which supplies decarboxy-lated AdoMet, the source of aminopropyl groups forspermidine and spermine synthesis. This agent wasdeveloped by Marion Merrell Dow in the late 1980s.It cures acute laboratory infections of T. b. brucei andT. b. rhodesiense clinical isolates (Bitonti et al, 1990). Itis active against late-stage model infections whenused in combination with low dose DFMO (Bacchiet al, 1994). MDL-73811 is not toxic to mice at >10times the curative dose levels used. It is rapidlytransported by the parasite through the P2 adeno-sine site (Bitonti et al, 1990; Goldberg et al, 1998). It isgiven intraperitoneally once a day at 10-25 mg/kg.Supplies are now limited. However, the absence oftoxicity and the strong activity against T. b. rhode-siense isolates indicate steps should be taken toobtain further supplies and initiate preclinical trials.

CGP 40215 is a bicyclic analogue of methylglyoxalbis(guanylhydrazone) (MGBG), an inhibitor ofAdoMet dc. This agent also resembles the diamidinesberenil and pentamidine. It was the most active, bothin vitro and in vivo, of a series of derivatives producedby Ciba-Geigy in the 1990s, curing laboratory infec-tions of T. b. brucei, T. b. rhodesiense, T. congolense, andT. b. gambiense - a total of 19 isolates (Brun et al, 1996;Bacchi et al, 1996). Used singly, CGP 40215 was notcurative to a CNS model, but was curative when usedin combination with DFMO. Unfortunately, whentested in vervet monkeys with a CNS infection, thecompound was not curative and pharmacokineticstudies indicated that it did not cross the blood-brainbarrier (BBB) (Keiser et al, 2001).

Methionine Recycling. Methylthioadenosinephosphorylase (MTA-Pase) is the lead enzyme of asalvage pathway which regenerates adenine andmethionine from MTA, the by-product of amino-propyl group transfer from decarboxylated AdoMet.African trypanosomes have an MTA-Pase with abroad substrate specificity. The MTA substrate ana-logue hydroxyethylthioadenosine (HETA) is cleavedby MTA-Pase and the ribose moiety is metabolized,possibly to a keto acid, which appears to be theactive agent (Bacchi et al, 1999). HETA is able to cureacute T. b. brucei infections and infections caused by6 of 11 T. b. rhodesiense isolates in mice. HETA was 500times less toxic to mammalian cells in culture than totrypanosomes (Sufrin et al, 1996; Bacchi et al, 1997).In mice, it has given no evidence of toxicity at doses

of 150 mg/kg for seven days (infusion pumps).HETA costs about US$2/g to synthesize under labo-ratory conditions. It deserves further study andshould be examined in preclinical trials.

Trypanothione Reductase Inhibitors.Trypanosomes produce a unique glutathione ana-logue, N1,N8-bis(glutathionyl)spermidine, for useas a redox defence mechanism in combination witha specific trypanothione reductase, which restoresoxidized trypanothione to the reduced state. The lat-ter is thus a logical drug target. Many inhibitors ofthis enzyme have been developed, and some haveproven highly effective in vitro against bloodstreamform trypanosomes, yet none have been shown tohave significant activity in model infections. Classesof compounds include phenothiazines, tricycliccompounds, diphenylsulphides, phenylpropyl andnaphthylmethyl β-substituted polyamines. Dif-ficulties with bioavailability, pharmacokinetics, andmetabolism of these compounds need to beaddressed (Werbovetz, 2000; Keiser et al, 2001).

Cysteine Protease Inhibitors. Cysteine pro-teases have been detected in most parasitic proto-zoans and are considered important potential tar-gets for drug development. Initial studies examinedcruzain, the major cysteine protease activity in T.cruzi, while more recently trypanopain-Tb (themajor lysosomal cysteine protease of T. b. brucei) hasbeen the subject of extensive study. In this context,treatment with the cysteine protease inhibitor Cbz-Phe-Ala-CHN2 at 250 mg/kg/day for three daysreduced parasitemia to undetectable levels inT. b. brucei infected mice (Scory et al, 1999).However, the parasitemia returned and animalsrelapsed after treatment had ceased. This study, cou-pled with similar results obtained with T. cruzi, indi-cate these inhibitors have potential, but will needextensive refinement to adjust the availability andpharmacokinetics (Werbovetz, 2000).

Nitro-imidazoles. Megazole is a 5-nitroimida-zole (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]-1,3,4-thiadiazole) which was first synthesized in1968, but not studied because of risk of mutagenic-ity (Keiser et al, 2001). Recent studies, however,have demonstrated significant activity of megazoleagainst acute murine infections of T. b. brucei and T.b. gambiense. Megazole was also effective in elimi-nating a murine model CNS infection when used incombination with suramin. Suramin prolongedmegazole’s elimination half-life and altered otherpharmacokinetic parameters including the reten-tion time (doubled) and serum profile (extendedpeak). In most studies, single-dose oral administra-tion of megazole was used (Enanga et al, 1998). In


a primate model, cerebrospinal fluid (CSF) levelsin an animal dosed with a combination of mega-zole and suramin were found to be 5-10% of plas-ma levels after a single 100 mg/kg dose - a levelabout 10 times that of the MIC100 for T. b. gambi-ense. This animal was cured of a CNS infection(Enanga et al, 2000). Excretion of megazole is pri-marily (80%) in the urine in the primate model, andfour metabolites were consistently found in signif-icant amounts (Enanga et al, 1999). Althoughpromising, further preclinical studies will be need-ed to determine the structure of the metabolitesand their overall mutagenic potential before clini-cal studies can begin.

Inhibition of Glycolysis. The predominantform of African trypanosomes in the blood is thelong slender trypomastigote, which depends on gly-colysis for energy production. Recent molecularmodelling studies on glyceraldehyde-3-phosphatedehydrogenase (GAPDH) have detected differencesin the nicotinamide adenine dinucleotide (NAD)+

binding site between the trypanosome and humanenzyme, and an overall sequence identity of only28.5% with the trypanosome enzyme (Suresh et al,2000). From these observations, adenosine ana-logues modified in the C2’ ribose and N6 adeninepositions have been synthesized. The most active ofthese analogues was an N6 derivative, N6-(1-napthalenemethyl)-2’-(3-methoxygenzamido)adenosine, with an IC50 value of 12 µM againstT. b. brucei and inhibition of pyruvate excretion inin vitro incubations. This compound did not inhibithuman GAPDH at 50 µM, but had an IC50 of 0.28 µM against L. mexicana GAPDH (Aronov et al,1999). Although promising, no in vivo studies havebeen reported with these compounds.

SIPI 1029. This agent is a triazine derivative(Trybizine.HCl) which is used against T. evansi inbuffaloes in China. SIPI 1029 was effective bothin vitro and in acute mouse model infections againstT. b. brucei, T. b. gambiense and T. b. rhodesiense(Bacchi et al, 1998; Kaminsky and Brun, 1998). Incombination with low-dose DFMO, it cured a modelCNS infection (Bacchi et al, 1998), although usedsingly it was not active in this model, or in a vervetmonkey CNS model (Keiser et al, 2001). Pharma-cokinetic studies in the latter model revealed onlylow concentrations of the agent in CSF.

Over 200 analogues of SIPI 1029 were tested at theSwiss Tropical Institute against African try-panosomes in vitro. Over 40 were tested in an acutemouse model and the most active compounds (5-10)were examined for activity in a CNS model. Noneshowed activity in this model and no additional

studies are planned with this series (JRL Pink andFAS Kuzoe, personal communication).

VSG Synthesis. African trypanosomes incorpo-rate myristic acid into the glycosyl phosphatidyli-nositol (GPI), which serves as an anchor for the vari-ant surface glycoproteins (VSGs) which cover thetrypanosome. Myristate is obtained by try-panosomes either by scavenging from the host bymyristate exchange, or by fatty acid remodelling.The critical nature of VSG in the trypanosome’s exis-tence in the host makes the myristoylation step anattractive chemotherapeutic target (Werbovetz,2000). Fatty acid analogues of myristate gave IC50values <100 nM in vitro against T. b. brucei andT. b. rhodesiense bloodform trypanosomes. Theseanalogues were inactive in vivo however, becausethey were rapidly metabolized by the host(Werbovetz et al, 1996). The second pathway is onein which other fatty acids are remodelled to myris-tate and then preferentially incorporated into GPIand not into other lipids (Morita et al, 2000). Thispathway can be inhibited by the antibiotic thiolacto-mycin, which kills trypanosomes in vitro with anIC50 concentration of 150 µM. However, in pharma-cokinetic studies in mice, thiolactiomycin was foundto be rapidly excreted. Peak serum levels wereobtained 15 minutes after intramuscular injectionand then rapidly declined until, at 60 minutes, theywere 1/10th that of peak levels. This compoundcould be given orally with similar rapid elevationand decline of serum levels and tissue distributionpatterns (Miyakawa et al, 1982). It would be impor-tant to pursue these lines of study and developeffective myristate analogues which are not metabo-lizable by the host and/or thiolactomycin analogueswhich have an extended serum half-life.

Amidine Prodrugs and Cis-platinumDerivatives. A number of 2,5-bis (4 amidino-phenyl) furan carbamates are reported to have activ-ity against Pneumocystis carinii, and one is undergo-ing Phase I clinical trials for this target. As an orallydosed diamidine, it would be of significant interestto examine these agents for treatment of acute T. b.gambiense infections as an alternative to pentami-dine (Rahmathhulla et al, 1999).

A group of organometallic complexes derived frompentamidine have been evaluated for activity in vivoagainst acute T. b. brucei in mouse and sheep modelinfections. Cis-platinum pentamidine-bromide,–thiocyanate and -seleniocyanate were curative in asingle dose of 1-3 mg/kg. The bromide derivativewas curative at 1 mg/kg and had a chemotherapeu-tic index of 200 compared to pentamidine, whichwas curative at 3.5 mg/kg and had a chemothera-


peutic index of 13. Further studies are planned witha CNS model infection and with pentamidine-resist-ant isolates (Loiseau et al, 2001).

Combination Chemotherapy. A number ofcompounds have been identified in the past seven-eight years which are excellent trypanocides bothin vitro and in vivo, with the exception that they donot penetrate the BBB and hence do not cure modelCNS infections. In light of the lack of lead com-pounds, and the fact that even current promisingcompounds have not undergone extensive preclini-cal toxicology studies, which may very well elimi-nate them from consideration, it would be prudentto examine drug combinations for CNS activity.Which compounds however, should be the startingpoints? In light of the many studies and clinicalactivity, eflornithine (DFMO) at low dose levels hasproven active in curing model CNS infections incombination with: suramin, melarsoprol, SIPI 1029,MDL 73811, HETA, CGP 40215, 9-deazainosine.Clearly, the ability of DFMO to act synergisticallywith so many chemically distinct agents does notimply a common biochemical basis of action. Rather,the literature on artificially induced BBB injury indi-cates that polyamine metabolism is significantly outof balance after injury and that DFMO has a role incorrecting this (Croft, 1999). A recent review indi-cates that a number of different types of injuries tothe brain result, after a complex cascade of events, inthe enhanced synthesis, degradation and release ofpolyamines in brain tissue. Ultimately, damage maybe attributable to formation of toxic reaction prod-ucts as a result of the oxidative degradation ofpolyamines (Seiler, 2000). DFMO may amelioratethis injury, and in so doing a situation may developwhich allows passage of molecules impervious tothe BBB. Since, in most combination studies withDFMO, only one or two doses of the other agentneed be given, usually within in three-four days ofthe onset of the DFMO, there appears to be a shortwindow during which the other agent will pass theBBB. There is a real need to explore these combina-tions and the basis for their activity in primate mod-els using low-dose DFMO in combination withknown agents, e.g. suramin and melarsoprol. It maybe that other polyamine antagonists, e.g. MDL 73811which blocks AdoMet dc and hence spermidine pro-duction, may have similar synergistic activity.

Transport of Nucleosides. Recently, a signifi-cant effort has been made by a number of investiga-tors concerned with the mechanism of drug uptakeby African trypanosomes. While the normal func-tions of P1 and P2 transporters in bloodstream try-panosomes were determined to be nucleoside andnucleobase transport, the ability of P2 to take up

diamidines and melarsoprol has been the focus ofmuch study (Hasne and Barrett, 2000; Barrett andFairlamb, 1999). Thus P2 carries adenosine and ade-nine, while melarsopol and pentamidine interferewith their uptake. Trypanosomes resistant to me-larsoprol and to berenil have lost P2. Megazole, a5-nitromidazole active against African trypano-somes (see above) is also transported through P2(Hasne and Barrett, 2000). It now appears that thereare other transporters, in addition to P2, for dia-midines and melarsoprol (DeKoning, 2001).

Another aspect of trypanosome nucleoside uptakeis the demonstration of an AdoMet transporter sep-arable from P1 and P2 (Goldberg et al, 1997).AdoMet transporter is not a usual component ofmammalian cell nucleoside uptake. Its appearancein trypanosomes should be exploited in terms oftransport of currently active agents and in develop-ment of novel agents with the ability to transverse awide range of transporters. For example, the MTA-Pase analogue HETA is taken up by both P2 and theAdoMet transporter (Goldberg et al, 1998). Overall,the function and substrate specificity of try-panosome nucleoside/drug transporters should bemade a priority for study and drug development.

AcknowledgementsI thank Michael Barrett, Reto Brun, Christian Burri,Felix Kuzoe, and Karl Werbovetz for supplyingrecent references, helpful discussions, and/or otherinformation essential for this document.

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II DRUG RESISTANCE IN SLEEPING SICKNESS

Michael P Barrett,Institute of Biomedical and Life Sciences, University ofGlasgow, Glasgow G12 8QQ, UK

INTRODUCTIONNo vaccines exist against sleeping sickness, and theprospects of prophylactic immunization are poorsince the parasites change their surface coat period-ically in a process known as antigenic variation.Drugs remain the principal means of intervention.Five drugs are currently used against HumanAfrican Trypanosomiasis (HAT) (Pépin and Milord,1994), the drug of choice depending on which sub-species of T. brucei is involved and on whether thedisease is diagnosed before or after the parasiteshave become established within the cerebrospinalfluid (CSF). Problems associated with the currenttherapies for sleeping sickness include toxicity,resistance, and a lack of a guaranteed supply(Barrett, 2000) (although it seems likely that licenseddrugs will be available for at least the next fiveyears). It is unlikely that new formulations will beavailable for at least ten years. This report dealswith the problem of drug resistance.

Antimicrobial drug resistance is widespread. Manyantibiotics are obsolete and the antimalarial drugchloroquine has been rendered useless in manyparts of the world due to the emergence of resist-ance. Drug resistance has also become a seriousproblem in the treatment of animal trypanosomias-es (Geerts and Holmes, 1998). The epidemiology ofsleeping sickness and recommended regimens forthe administration of drugs against this diseaseappear to be unfavourable for the development ofresistance. HAT is relatively uncommon whencompared to malaria (estimates of 300 thousandcases of sleeping sickness compared with 300 mil-lion of malaria). No drugs are currently used pro-phylactically against sleeping sickness and admin-istration of all drugs is parenteral and should occurin a clinical setting and not through self-adminis-tration. Therefore the conditions which have nor-mally been implicated in the selection of drug-resistance, i.e. widespread use, significant under-dosing associated with self-administration andimproper prophylactic use, do not appear to be rel-evant to sleeping sickness. In addition, the zoonot-ic nature of the trypanosomes which cause humandisease could mean that drug selection would berelieved as parasites are transmitted to untreatedanimals, thus diminishing the pressure to maintainresistance genes in the parasite population.

However, reports have been mounting that manycases in the current epidemics of sleeping sicknessare not responding to melarsoprol, the principal drugused against stage II disease. This report aims to dis-cuss the current status of drug resistance in humaninfective trypanosomes, to consider mechanisms bywhich parasites become resistant to drugs, and to dis-cuss how resistance is detected and how detectionmay be improved. Suggestions as to how to deal withdrug resistant parasites and how to delay the onsetand spread of resistance are also made.

EPIDEMIOLOGY OF SLEEPING SICKNESS: IMPLICATIONS FOR THESPREAD OF RESISTANCEA full understanding of the epidemiology and pop-ulation structures of human infectious try-panosomes, and their relationship with non-humaninfectious parasites, is crucial in understanding pos-sible routes to the spread of drug resistance.Currently the epidemiology of the disease is not suf-ficiently well understood to make solid conclusions.However, recent data have started to clarify the sit-uation in the field, allowing room for some specula-tion on this topic (MacLeod et al, 2001).

T. brucei parasites are able to undergo geneticexchange inside the tsetse fly, although this processis not obligatory. It has recently been shown thatmany infected tsetse flies in the field carry mixedpopulations of trypanosome (MacLeod et al, 1999),thus genetic exchange is possible and studies on thepopulation genetics of trypanosomes have revealedthat genetic exchange does occur sufficiently fre-quently to be an important determinant of geneticdiversity.

However, it is not yet clear whether the humaninfectious Trypanosoma brucei gambiense does engagein genetic exchange. Moreover, in East Africa itappears that T. b. rhodesiense may have a predomi-nantly clonal structure (MacLeod et al, 2000), sug-gesting that it does not frequently engage in geneticexchange, although this does not mean that it can-not do so. While T. b. gambiense (type I) appears to begenetically distinct from other T. brucei sub-species,T. b. brucei and T. b. rhodesiense may simply be con-sidered as host-range variants of local T. b. bruceipopulations that become genetically isolated. TypeII T. b. gambiense found in West and Central Africaalso appear to closely resemble T. b. brucei (MacLeodet al, 2001).

Studies on trypanosome population genetics are stillin their infancy and it cannot be ruled out thathuman infectious parasites can engage in geneticexchange with non-human infectious organisms.


Moreover, human infectious parasites are not limitedin host-range to humans, and many trypanosomes(around 20%) isolated from cattle in Uganda andWestern Kenya are human infectious (Hide, 1999). InWest Africa it appears that T. b. gambiense may alsohave a host range beyond humans, with pigs repre-senting a particularly important reservoir(Penchenier et al, 1999). A critical corollary of theessential zoonotic nature of trypanosomes is thatresistant strains selected by drug use in animalscould be transferred to humans (this will be dis-cussed later). In addition, human infectious parasitescould also acquire resistance from non-human infec-tious parasites as a result of genetic exchange duringmixed transmission in tsetse flies. It is important tostress, however, that this has yet to be demonstratedin experimental or population genetic studies.

However, it is clear that even in instances where try-panosomes in humans are not exposed to the classi-cal risk factors for the development of drug resist-ance, the possibility exists that human infectiousparasites are exposed to these classic factors in ani-mals. Domestic animals do receive large doses ofvarious trypanocides, often administered at the dis-cretion of farmers, and frequently given over pro-longed periods as prophylaxis. While trypanocidesused to treat humans and animals do differ, thepotential to develop cross-resistance betweenhuman and animal trypanocides does exist. Forexample, diminazene is used extensively for thetreatment of cattle. Cross–resistance between dimi-nazene and the related diamidine pentamidine canbe induced in trypanosomes (Barrett and Fairlamb,1999). Cross-resistance between diminazene andmelarsoprol can also be selected with relative ease(reviewed in Barret and Fairlamb, 1999). This isbecause all of the drugs can enter trypanosomes viathe P2 amino-purine transporter. Loss of this trans-porter can diminish uptake of these drugs into par-asites, which decreases sensitivity. Although the sit-uation with respect to cross-resistance is not entire-ly straightforward, as outlined in the sections aboutresistance with respect to each of the human drugs,it should be considered when looking at treatmentof animal trypanosomiases in regions where thehuman disease is endemic.

THE STATUS OF RESISTANCE TO CURRENTLY USED DRUGS IN SLEEPING SICKNESS

Melarsoprol

Summary points on melarsoprol resistance• Melarsoprol has been widely used in the field

since the 1950s.

• Treatment failure is increasingly being reportedfrom epidemic loci (30% failure in northernUganda compared with 5-10% normally).

• T. b. gambiense isolates from treatment failures innorth-west Uganda have recently been shown tohave reduced sensitivity to the drug.

• While plasma levels of melarsoprol appear toremain at levels high enough to kill the parasites,the same may not be true in the CSF and otherextravascular compartments.

• The amino-purine transporter P2, encoded by theTbAT1 gene, is at least partially responsible foruptake of melamine-based arsenicals. Loss of thetransporter can contribute to resistance. Onestudy has shown that removal of the TbAT1 generenders parasites only fourfold less sensitive tomelarsen oxide than wild-type trypanosomes.However, this degree of resistance might beenough to allow survival in the CSF (and relapsesappear to depend on CSF involvement).

• Many of the isolates from treatment failures havean altered TbAT1 gene.

• It is possible that a combination of reduced drugsensitivity (in part due to reduced uptake becauseof changes to the P2 transporter) and variability inaccumulation of drug in the CSF can explain cur-rent treatment failures in Africa.

The drug and its useMelarsoprol (Mel B) is a melaminophenyl-basedorganic arsenical which was introduced as an anti-trypanosomiasis reagent in 1949 (Friedheim, 1949).It was Paul Ehrlich who promoted the idea thatarsenicals could be useful drugs for use againstsleeping sickness at the turn of the century. His com-pound, salvarsan or ‘606’, developed for use againstsyphilis, is often considered as the prototypicchemotherapeutic reagent. Ernst Friedheim devel-oped the melamine-based arsenicals in the late1940s after the dangers of serious side effects associ-ated with tryparsamide and high rates of treatmentfailure decreased confidence in this product.Interestingly, melarsoprol has recently been used inclinical trials against leukaemia (Soignet et al, 1999).The trials were abandoned when it became clearthat patients were suffering from seizures at more orless the same rate as in sleeping sickness treatment.However, different regimes might be tried.

Melarsoprol itself is amphipathic and will diffuseacross cellular membranes. However, the drug isvery rapidly converted to the highly hydrophilicmelarsen oxide in plasma (96% clearance of melar-soprol within 1 hr) (Burri et al, 1993). Melarsenoxide levels peak within 15 minutes and have a half-life of 3.9 hours. Relatively little melarsoprol, or itsmetabolites, accumulate across the blood-brain bar-


rier, with maximum levels being equal only toaround 1-2% of maximum plasma levels. The newpharmacokinetic information (Burri et al, 1993;Keiser et al, 2000) has been very useful in establish-ing a new regimen for administration of the drugwhich is likely to engender better patient compli-ance (Burri et al, 2000). Detailed information onpharmacokinetics may also be crucial in permittingan understanding of the factors that underlie clinicaldrug failure. It seems that levels of drug that reachthe CSF might be insufficient to kill parasites thatare only a few fold less sensitive to drug than wild-type trypanosomes.

Of all the trypanocides, toxic effects are worst withmelarsoprol; up to 10% of patients suffer a frequent-ly fatal reactive encephalopathy (Pépin and Milord,1994). A high proportion of leukaemia patients suf-fered neurological seizures when treated withmelarsoprol (Soignet et al, 1999), demonstrating thatmelarsoprol is itself responsible for the reactiveencephalopathy associated with drug treatment.

Exfoliative dermatitis has also been observed.Hypersensitivity, renal and hepatic dysfunction arealso known. Myocardial damage, albuminuria andhypertension can also occur. Headache, fever, gen-eral malaise, urticaria, abdominal pains, vomitingand acute diarrhoea are all less severe but commonside effects (Pépin and Milord, 1994).

Mode of uptake and actionIt has been proposed that both the melaminophenylarsenicals and diamidine classes of drug enter T.brucei by the P2 amino-purine transporter (Carterand Fairlamb, 1993). Trypanosomes selected forresistance to sodium melarsen had lost the P2 trans-porter (Carter and Fairlamb, 1993), and a T. equiper-dum line selected for resistance to diminazene,which displayed some cross-resistance to arseni-cals, had a P2 transporter with markedly reducedaffinity for substrate (Barrett et al, 1995). These datasuggested that the P2 transporter is involved inuptake of arsenicals (Carter and Fairlamb, 1993)and diamidines including diminazene (Barrett et al,1995) (the situation for pentamidine is more com-plicated, as described later). A simplistic model pro-posing that loss of the P2 transporter was necessaryand sufficient to induce resistance tomelaminophenyl arsenicals and diamidines wasdeveloped (Barrett and Fairlamb, 1999; Carter andFairlamb, 1993; Barrett et al, 1995). In vitro, unme-tabolized melarsoprol is likely to cross the mem-brane by passive diffusion (Scott et al, 1997)although melarsen oxide does not. The possibilityof additional modes of uptake should not beexcluded.

Trypanosomes exposed to arsenicals lyse very rap-idly. A mode of action has yet to be established. Lossof ATP due to inhibition of glycolysis could under-lie lysis caused by the drug, as bloodstream formtrypanosomes depend solely upon glycolysis forATP production. However, it seems that the cellslyse before ATP supplies are seriously depleted,leading several workers to question whether glycol-ysis is a target for arsenical action (Van Schaftingenet al, 1987).

Another suggested target of melarsoprol was try-panothione, a key low molecular weight thiol foundin trypanosomatids but not mammalian cells. Sincearsenic is known to form stable interactions withthiols, trypanothione was also proposed as thedefinitive target for these compounds (Fairlambet al, 1989). Arsenicals, however, interact more tight-ly with other thiols including lipoic acid and, at thepoint of arsenical-induced lysis, only a small frac-tion of trypanothione is conjugated with the drug(Fairlamb et al, 1992). Thus it seems unlikely thattrypanothione is the in situ target of these drugs.

ResistanceTreatment failure with melarsoprol has been report-ed in the field. There has always been a cohort of5–10% of treated patients who relapsed after treat-ment, although it was never clear what factors wereresponsible for this. However, in northern Ugandarelapse rates after melarsoprol treatment of around30% have been reported (Legros et al, 1999).Anecdotal reports of similar failure rates in northernAngola are also in circulation. The incidence ofrelapse after treatment does not, at present, seem tobe as serious in southern Sudan, although around16% failure rate has also been reported here. Therecent build up of data on the pharmacokinetics ofthe drug (Burri et al, 1993; Keiser et al, 2000), cou-pled with advances in understanding of the molec-ular and biochemical basis of resistance and ananalysis of field isolates from relapse cases inUganda (Matovu et al, in press), has allowed thedevelopment of a model for the likely causes oftreatment failure in the field.

Precise quantification of drug levels in a patient’splasma and CSF is difficult, although best resultshave been obtained using a bioassay for trypanoci-dal activity in fluids containing melarsoprol or itsactive metabolites (Burri and Brun, 1992). The logis-tics of drug delivery in primary health care centresis difficult, so administration of the drug can differfrom patient to patient in terms of both total deliv-ered dose and timing between doses. Moreover, themetabolism of the drug and also its distributionwithin the body will vary from patient to patient


(i.e. accumulation of the drug in the CSF and otherextravascular compartments can be highly vari-able).

These variables can be crucial to the success of thedrug. Absolute quantities vary between patients;however, maximum serum levels following fourinjections of the drug according to the “classic” pro-tocol were 5 – 6 µg ml-1 (Burri et al, 1993). This fallsto 0.22 µg ml-1 120 hours after the final injection inthe series. Drug detected in the CSF 24 hours afterthe final injection varied between individuals. Themaximum concentration was 260 ng ml-1 but thisdescended to undetectable quantities in somepatients and average levels in the CSF are estimatedto be in the order of 50-fold lower than those in plas-ma (Burri et al, 1993). Melarsoprol is rapidly con-verted to melarsen oxide (and possibly othermetabolites) in plasma (Keiser et al, 2000).

There is also some variability in intrinsic sensitivityto melarsoprol (or melarsen oxide) among differentisolates of trypanosome. Sensitive parasites appearto have minimal inhibitory concentration (MIC) val-ues of around 1-30 ng ml-1 (De Koning, 2001).

The quantity of active melamine-based arsenicalreaching extravascular parasites, combined with theMIC of the trypanosomes, will determine whetherthey are killed by the drug. The MIC values of mostparasites are only marginally lower than estimatedconcentrations of drug that are achieved in the CSFof most patients. Data regarding melarsoprol inother extravascular compartments that also harbourtrypanosomes are absent. The 5-10% of melarsoprolrefractory cases could conceivably represent acohort of individuals in whom extravascular accu-mulation of the drug is at the lower end of a nor-mally distributed curve and does not reach steriliz-ing levels, thus allowing relapse.

Since the achievable CSF dose is close to the MIC ofnormal sensitive trypanosomes, a mere halving insensitivity of a line of parasite could mean that alarger number of individuals fail to accumulate ster-ilizing levels of arsenical in the CSF. As the degree ofsensitivity declines, the number of refractory caseswill rise in proportion.

According to this model, a combination of parasitedrug-sensitivity and host factors, including the per-meability of the blood-brain barrier to the drug, willdetermine whether a case is sensitive or refractoryto arsenicals. This model is supported by recent datafrom northern Uganda which indicate that alter-ations to the TbAT1 gene that encodes the P2 trans-porter correlate with resistance to melarsoprol(Matovu et al, 2001).

Experiments that led to the identification of a rolefor the P2 transporter in resistance (Carter andFairlamb, 1993) involved laboratory derived isolatesselected for high level resistance to sodiummelarsen (which, like other melaminophenyl arseni-cals, is probably rapidly metabolized to melarsenoxide in vivo). However, recent data involvinggenetic manipulation of trypanosomes, removingthe TbAT1 gene, revealed that loss of the P2 trans-porter yields only around fourfold reduced sensitiv-ity to melarsen oxide (Matovu et al, 2001).Additional mechanisms must therefore be at play indetermining high level resistance in laboratoryderived lines. The modest change in sensitivityrelating to impairment of P2 function, however,could render parasites resistant to levels of melarsenoxide accumulating in the CSF and other extravas-cular compartments. It appears that the majority ofrelapses reported in Uganda do involve small num-bers of CSF parasites re-invading the bloodstreampost-treatment (Matovu et al, 2001). It is also note-worthy that genetic removal of TbAT1 had noimpact on parasite viability.

Many of the drug resistant T. b gambiense isolatesfrom northern Uganda have mutations in the TbAT1gene that encodes the P2 transporter (Matovu et al,2001). Many of these mutations are common withmutations found in laboratory derived drug resist-ant isolates (Maser et al, 1999). Although some of theresistant isolates apparently do not have changes tothe sequence of TbAT1, it cannot be ruled out thatother genetic alterations, beyond the open readingframe, would downregulate expression of that gene.One example of a T. b gambiense line lacking theTbAT1 gene has been reported (Matovu et al, 2001).A multi-drug resistant T. b rhodesiense isolate fromsouth-east Uganda (Matovu et al, 1997) had an iden-tical set of mutations as the T. b gambiense series andan Angolan isolate (Matovu et al, 2001).

Many instances of cross-resistance between mela-mine-based arsenicals and diamidines have beenreported (reviewed in Barrett and Fairlamb, 1999).The fact that the P2 transporter appears to beresponsible for the uptake of both of these classes ofdrug has suggested that transporter alterationscould be the basis of cross-resistance. The fact thatsome lines of pentamidine resistant parasite have noreduction in P2 transporter activity was explainedby the discovery of additional pentamidine trans-porters (De Koning, 2001) and by the fact that other,non-transport, related events could underlie pen-tamidine resistance (Berger et al, 1995). The recentdiscovery that loss of the P2 transporter throughgene knockout rather than drug selection leads to arather modest decrease in sensitivity to melamine-


based arsenicals suggests that other biochemicalchanges must be occurring in resistant cells. Theseother factors contributing to resistance remain to beidentified.

Pentamidine

Summary points on pentamidine resistance • Use of the drug in the field against early-stage

sleeping sickness has been extensive, although itswithdrawal from use as a prophylactic in the mid-twentieth century might have slowed the appear-ance of resistance.

• Anecdotal reports about resistance in the field areon the increase.

• Laboratory isolates resistant to the drug havebeen selected.

• There can be cross-resistance to other drugs,including melamine-based arsenicals and otherdiamidines, which may to be related to loss ofdrug uptake via the P2 transporter.

• Resistance need not necessarily involve cross-resistance. For example, one laboratory derivedline selected for melarsen resistance which hadlost the P2 transporter was not cross resistant topentamidine. Moreover, another line selected forpentamidine resistance was not cross resistant toarsenicals or other diamidines.

• Pentamidine can enter T. brucei via several trans-porters (P2, HATP1, LAPT1) and it accumulates tohigh intracellular levels. Resistance in some casescorrelates to reduced uptake, but in othersreduced uptake does not appear to be involved.

• The mode of action is not known.

The drug and its usePentamidine is an aromatic diamidine that has beenin use for treatment of trypanosomiasis for over fiftyyears (Sands et al, 1985). It is supplied as a whitepowder in 200 mg ampoules. It was developed afterthe observation that a related compound, synthalin,which induces hypoglycaemia in mammals, hadprolific anti-trypanosomal activity. Diamidinesactually work directly against the parasites, inde-pendently of their physiological action on the host.Pentamidine is active against early stages of thegambiense form of sleeping sickness. It is also usedagainst antimony refractory leishmaniasis andPneumocycstis carinii pneumonia (Sands et al, 1985).

Maximum plasma concentrations are reached with-in an hour of intramuscular injection. Extensivevariation in plasma concentration is found betweenindividuals (0.2-4.4 mg l-1 following a 4 mg kg-1

injection have been reported). Each daily dose leadsto an increase in residual drug concentration.

Elimination is slow, with estimates of 70-80% of thedrug reported binding to plasma proteins (Sandset al, 1985). The plasma half life is 12 days (butvaries), and the drug is probably metabolized byhumans since only around 11% is eliminated in theurine. The drug is thought not to cross the blood-brain barrier, although there are reports suggestingthat small amounts may enter the CSF.

Intramuscular injection can cause reactions at thesite of administration. Other reactions includehypotension, abdominal pain, hypersalivation, ver-tigo, nausea and chest pain. Nephrotoxicity is com-mon; hypoglycaemia is also seen in significant num-bers of patients. Diabetes mellitus may ensue sever-al months after therapy. Rapid intravenous injectionshould be avoided as it induces a number of effectsincluding hypotension, tachycardia, nausea andvomiting (Anon, 1998).

Mode of uptake and actionPentamidine is concentrated to high levels by theparasites. It seems that pentamidine can enter T. bru-cei via the same P2 amino-purine transporter whichaccumulates melaminophenyl arsenicals (Carteret al, 1995). Loss of this transporter can render para-sites cross-resistant to both diamidines and arseni-cals. However, some parasites without P2 remainsensitive to pentamidine (Carter and Fairlamb,1993). In T. brucei, three transporters that can carrypentamidine into the cell have been identified(De Koning, 2001). In addition to P2, a high affinitytransporter HAPT1 (Km for pentamidine = 36nM)and a low affinity transporter LAPT1 (Km for pen-tamidine = 56 µM) are responsible for the uptake ofpentamidine. This could explain why the P2 defi-cient line, RU15, which is resistant to melamine-ba-sed arsenicals and some diamidines, was not resist-ant to pentamidine (Carter and Fairlamb, 1993).Moreover, another line selected for pentamidineresistance was not resistant to other diamidines(Berger et al, 1995); a better understanding of thedifferent routes of uptake for different diamidines isdesirable.

The mode of action of the drug has not been estab-lished. As a polycation, the molecule interacts elec-trostatically with cellular polyanions, including theunique intercatenated network of circular DNAmolecules which make up the mitochondrialgenome of all kinetoplastid flagellates termed thekinetoplast. In the case of African trypanosomes, aninteraction with the kinetoplast may appear to be oflimited interest as these organisms do not have aclassical mitochondrial metabolism. However, it iswrong to consider the mitochondrion as inert sincesome kinetoplast genes are known to be expressedand the membrane is maintained in an energized


form indicating that several mitochondrial enzymesystems must be active.

T. brucei can retain viability when the kinetoplasthas been removed (dyskinetoplastidy). On the otherhand, dyskinetoplastid parasites have been shownto be somewhat less sensitive than wild-type cells todiminazene (Agbe and Yielding, 1995). Fluorescentanalogues of the diamidines, e.g. DB75 and stil-bamidine, accumulate rapidly in the kinetoplast andhave also been shown to accumulate in small vesic-ular structures in the cytosol.

Numerous other potential targets have been pro-posed but none have been verified. Given that thedrug reaches millimolar concentrations within try-panosomes, it could be that its toxic effect arisesfrom inhibition of multiple cellular targets, althoughthe fact that one resistant line (Berger et al, 1995)does accumulate drug to millimolar concentrationswithout adverse effects might indicate that there is aspecific target that has yet to be identified.

Resistance Pentamidine resistance did not emerge duringlarge-scale chemoprophylaxis campaigns in themiddle part of the twentieth century in west Africa,possibly because the drug was withdrawn fromwidescale use in the 1950s thus removing selectionpressure. In this regard, it is perhaps of note that onelaboratory derived line resistant to pentamidine hadsubstantially diminished viability in mammals(Berger et al, 1995).

It is not clear, nor easy to ascertain from available lit-erature, whether melarsoprol resistant field isolatesare pentamidine cross-resistant. However, it seemsthat both pentamidine and melarsoprol enter try-panosomes via the P2 transporter and anecdotal evi-dence has indicated that treatment failures withpentamidine are growing more common as they arewith melarsoprol. Possibly the presence of trans-porters, in addition to P2, which can accumulatepentamidine means that changes to P2 will notunderlie pentamidine resistance in the field.Mechanisms for resistance to pentamidine are cur-rently not known.

Suramin

Summary points on suramin resistance • Use of the drug in the field against sleeping sick-

ness is not extensive.• Few reports about resistance in the field have

been published.• Veterinary use was more widespread than human

use in the mid to late twentieth century.

• Suramin resistance in T. evansi lines appears to bestable in the field.

• Numerous laboratory isolates (T. brucei and T.evansi) have been selected for suramin resistance.

• Most laboratory resistant isolates are not cross-resistant to other drugs.

• Neither a mode of action nor mechanisms ofresistance are known.

The drug and its useSuramin, a colourless polysulphonated symmetricalnaphthalene derivative, was first used againstsleeping sickness in 1922 (Voogd et al, 1993). It isuseful for the treatment of early-stage infection dueto either T. b. gambiense or T. b. rhodesiense, whenthere is no central nervous system involvement.Other naphthalene dyes, including trypan red andtrypan blue, were initially developed for theirmarked trypanocidal activity. The drug has recentlybeen used in clinical trials against hormone-refrac-tory prostate cancer and has also been used in thechemotherapy of some helminth infections. Duringthe 1950s, there were reports that treatment failurein patients infected with T. b gambiense was relative-ly high (around 30% [Neujean and Evens, 1958]).Consequently the drug lost favour as a treatment forgambiense sleeping sickness although the factorsunderlying these treatment failures were neveridentified with any certainty.

Poor intestinal absorption, and a local irritation ifgiven intramuscularly, mean that the drug should beadministered by slow intravenous injection (Anon,1998). Dosing at 1 g per week over six weeks main-tains levels at 150-200 mg l-1. Most of the drug bindsto serum proteins. It does not cross the blood-brainbarrier to levels capable of killing trypanosomes inthe CSF at doses given during treatment of early-stage disease. Plasma concentrations decline expo-nentially with a half-life of up to 60 days. About 80%of the dose is eliminated in the urine.

Nausea, vomiting, urticaria and loss of consciousnesscan be immediate side-effects (Anon, 1998). Fever (upto 40oC within a few hours) is common as are photo-phobia and lacrimation. Renal damage can occur sev-eral days after treatment. Other adverse reactionsinclude exfoliative dermatitis, stomal ulceration,agranulocytosis and, rarely, haemolytic anaemia.

Mode of uptake and actionThe drug is a highly charged molecule containingsix negative charges at physiological pH and itbinds with high avidity to many serum proteinsincluding low density lipoprotein (LDL), for whichtrypanosomes have a receptor (Vansterkenburget al, 1993). Suramin accumulates in trypanosomesrelatively slowly and may be taken up bound to


LDL by receptor-mediated endocytosis, althoughdefinitive evidence proving this is absent. The sameexperiments that showed that uptake might dependon LDL also showed that suramin inhibits LDLuptake and that this inhibitory effect could be thecause of suramin’s toxic effect on trypanosomes(these organisms get most of their lipids from thebreakdown of exogenous LDL). The highly chargednature of the drug enables it to bind to many pro-teins through electrostatic interaction.Consequently, when the drug is tested for inhibitionof a variety of purified enzymes, it shows activity.This has led to many hypotheses regarding its modeof action.

Treatment of trypanosomes with the drug does leadto a reduction in the glycolytic rate. This inspired astudy into its inhibitory effect against the enzymeglycerol phophate oxidase, which is present in try-panosomes but absent from the mammalian host(Fairlamb and Bowman, 1977). Suramin inhibitedthe enzyme and some textbooks mistakenly reportthat this enzyme is the target. The discovery of clus-ters of positively charged amino-acids within sever-al of the T. brucei glycolytic enzymes led to thehypothesis that the drug’s action was dependentupon this interaction. However, none of these spec-ulative ideas have been proven and, at this stage, itshould be emphasized that the drug’s mode ofaction is not known.

The drug is highly active against bloodstream formsof the parasite in vitro, but around a hundredfoldless active against procyclic forms of the organisms(Scott et al, 1996). This indicates that either itsuptake, or its direct or indirect targets, are differen-tially regulated in the different forms of the parasite.Early suggestions that receptor mediated endocyto-sis did not occur at all in procyclic forms haveproven to be incorrect as this form of the parasitedoes endocytose a number of macromolecules (Liuet al, 1999). However, specific receptors may be dif-ferent. The fact that procyclic form organisms,unlike bloodstream forms, are not totally dependentupon glycolysis has also been used to fuel ideas onmodes of action.

Fang et al (1994) recently re-introduced the oldnotion that the immune system may play a role inthe action of suramin, since immunosuppressedmice needed higher doses (around threefold) toclear T. evansi infections.

Resistance Field reports on sleeping sickness resistant tosuramin are rare. In animal diseases, however, para-sites of the brucei group resistant to this drug have

been reported. Information from these species (par-ticularly T. evansi) might be useful in helping toascertain resistance mechanisms in man.

The relative scarcity of reports of suramin resistancein sleeping sickness has made it difficult to compilefield data on this subject. The large incidence ofreported treatment failures of T. b gambiense in westAfrica in the 1950s could have been due to multiplefactors of which parasite resistance is just one(Neujean and Evens, 1958). Many laboratoryderived lines have been selected for resistance tosuramin over the years (since the 1930s). Severalstudies have focused on T. evansi (Mutugi et al,1995). One laboratory study pointed to the fact thatresistant lines were more difficult to clone and growthan wild-type T. evansi. This prompted suggestionsthat resistance might not be a stable phenotype(Mutugi et al, 1995). However, T. evansi isolatedfrom the Sudan some twenty years after suraminhad been withdrawn from use due to the advent ofresistance (El Rayah, 1999) was still highly resistantto this drug, indicating that resistance can be verystable.

No evidence for a reduction in drug uptake associ-ated with resistance has been reported and mecha-nisms of resistance are not known. Most laboratorystudies, stretching back 70 years or more, havefailed to identify cross-resistance between suraminand other trypanocides. One study did show that aline selected for resistance to melarsen oxide (33-fold resistance) (Fairlamb et al, 1992) had a nearlysixfold decrease in susceptibility to suramin.However, most other studies have concluded thatthere is no cross-resistance between suramin and themelamine based arsenicals or other drugs.

EflornithineSummary points on eflornithine resistance• Use of the drug in the field against sleeping sick-

ness has not been extensive.• No reports about resistance in the field have been

published.• T. b. rhodesiense is innately refractory to eflor-

nithine, possibly due to a shorter half life of thetarget enzyme ornithine decarboxylase comparedto the susceptible T. b. gambiense.

• Laboratory isolates resistant to the drug havebeen derived.

• Model organisms (e.g. Leishmania, Neurospora)resistant to the drug have also been derived.

• In model organisms, resistance has been shown torelate to different phenomena e.g.:- Increase in ornithine decarboxylase levels (geneamplification).


- Decreased drug uptake (loss of basic amino acidtransporter in Neurospora; unknown mechanismbehind reduced uptake in procyclic T. brucei).• T. brucei resistant to DFMO have not been report-

ed to be cross-resistant to other drugs.• The mode of action of the drug relates to its inhi-

bition of ornithine decarboxylase. A functionalimmune system is required to kill cells in vivo.Whether it is simply loss of polyamines or otherindirect effects, e.g. increase in decarboxylated S-adenosyl methionine and inappropriate methy-lation, that is behind cytotoxicity is not clear.Uptake has been reported to be via passive diffu-sion in bloodstream forms and via carrier mediat-ed uptake in procyclic forms, although furtherstudies on this question are needed.

The drug and its useEflornithine, or D,L-α-difluoromethyl ornithine(DFMO), is an analogue of ornithine which acts as aspecific suicide inhibitor of the enzyme ornithinedecarboxylase (ODC). It was developed as an anti-cancer reagent, however, it remains at the trial stageagainst neoplastic disease. The drug also has activi-ty against sleeping sickness caused by T. b gambiense,even in the late CNS-involved stage. It was regis-tered in the USA in 1990 and the UK in 1991 (Pépinand Milord, 1994). By 1995 it was registered in sevenAfrican countries. Fourteen daily intravenous injec-tions of 400 mg per kg of body weight are recom-mended (Anon, 1998). It has also been licensed foruse as a topical application to prevent facial hairgrowth and is now marketed for this purpose(Hickman et al, 2001). Fifty four per cent of the dosebecomes bioavailable after oral administration(Anon, 1998). The mean half-life in plasma follow-ing intravenous injection is 3 hours, with 80% of thedrug excreted unchanged in urine after 24 hours.Little of the drug binds to serum proteins.Immediately after a 14-day course, the CSF to plas-ma ratio is 0.91 in adults and 0.58 in children.Children retain less of the drug than adults.

Few side effects are apparent although anaemia andother blood cell reductions (leukopenia, thrombocy-topeania) are known. Diarrhoea is a common prob-lem to those on oral eflornithine. Convulsions, feverand vomiting have also been reported in low num-bers of cases (Anon, 1998).

Mode of uptake and actionEflornithine is a specific suicide inhibitor of theenzyme ornithine decarboxylase (ODC), which is akey enzyme in the biosynthesis of polyamines(McCann and Pegg, 1992). Some early studies inmammalian cells indicated that DFMO uptake wasa passive process involving simple diffusion across

the membrane (Erwin and Pegg, 1982). It has beenreported that uptake of DFMO in T. brucei alsooccurs via passive diffusion across the plasma mem-brane (Bitonti et al, 1986; Bellofatto et al, 1987).These observations were based on the fact thatuptake appeared to be unsaturable over a widerange of DFMO concentrations and that internalconcentration of drug equilibrated with externalconcentration. Other features reported in these stud-ies, e.g. temperature sensitivity of uptake, might beinterpreted as evidence for transport although theauthors did not consider this. A separate report didnote a saturable process typical of transport-associ-ated uptake in procyclic organisms with a Km of244 µM (Phillips and Wang, 1987). In the yeastNeurospora crassa, a basic amino-acid transporter hasbeen implicated in uptake of DFMO (since thistransporter is lost in strains selected for resistance tothe drug) (Davis et al, 1994). None of the studies intrypanosomes have indicated that DFMO shares anuptake mechanism with ornithine, arginine orlysine. The mode of uptake into trypanosomesremains uncertain.

DFMO has similar affinity for both the mammalianand trypanosomal ornithine decarboxylases. Itsspecificity against the parasite apparently arisesbecause T. b. gambiense ODC is degraded within thecell and replenished at a rate much slower than itsmammalian counterpart (Phillips et al, 1987). Thus,a pulse of DFMO can deprive trypanosomes of ODCand polyamine synthesis for a prolonged periodcompared with mammalian cells, leading to a cessa-tion of growth.

Inhibition of ornithine decarboxylase has otherresults besides a reduction in putrescine and furtherpolyamine biosynthesis. For example, it leads to anincrease in cellular levels of S-adenosyl methionine,which might have toxic effects (Byres et al, 1991).Inappropriate methylation of proteins, nucleicacids, lipids and other cell components is thus beingimplicated.

Trypanothione is a glutathione-spermidine conju-gate unique to trypanosomatids and it plays a criti-cal role in maintenance of cellular redox potential(Fairlamb and Cerami, 1992). Trypanothione levelsare diminished after DFMO treatment, which mightrender parasites more vulnerable to oxidative stress.A functional immune system is required to kill thegrowth-arrested trypanosomes (De Gree et al, 1983).It has also been reported that T. brucei lackspolyamine transporters, rendering the parasite aux-otrophic for polyamines (Fairlamb and Le Quesne,1997). Conversely, many mammalian cells can scav-enge polyamines from plasma using transporters,allowing them to bypass the lack of endogenous


biosynthesis while T. brucei cannot tolerate this situ-ation.

In order to be effective against sleeping sickness, thedrug needs to be given in large doses. An addition-al drawback is the drug’s lack of activity againstrhodesiense sleeping sickness (Iten et al, 1995), whichappears to contain an ornithine decarboxylase thatis relatively quickly turned over (Iten et al, 1997). Aseven-day course appears to be somewhat less effi-cacious than the 14-day course; however, the cost-benefit ratio appears to favour the shorter course(Pépin et al, 2000).

Resistance No reports of resistance in the field were found inthe literature, which is not surprising since the drughas not been widely used in very large-scale treat-ment regimes. T. b. rhodesiense appears to be innate-ly less susceptible to the drug than T. b. gambiense(Iten et al, 1995) since it has a higher overall ODCactivity and the enzyme has a shorter half life thanthe gambiense counterpart (Iten et al, 1997). Anotherexplanation involving relative levels of S–adenosylmethionine, which accumulate to lower levels inrefractory but not sensitive cells treated withDFMO, has been proposed. Alternative explana-tions related to drug uptake, or polyamine uptakeallowing bypass of the inhibitory effect, have notbeen subject to extensive analysis.

In procyclic cells selected for DFMO resistance,putrescine uptake was noted to be three-four timeshigher than in wild-type lines (Phillips and Wang,1987). Putrescine at >1 mM allowed parasites to sur-vive DFMO treatment in vitro while 0.1 mM did not(Phillips and Wang, 1987). Moreover, T. brucei para-sites from which the ornithine decarboxylase genehad been removed were also viable and capable ofgrowth provided external putrescine was abundant(Li et al, 1988) (far more abundant than in mam-malian serum, where it is around 220 nM [Cooperet al, 1978]). A similar result was obtained withLeishmania parasites from which ODC had beenknocked-out, i.e. putrescine enabled bypass ofDFMO inhibition of polyamine synthesis, and alsoenabled the cells to dispose of accumulatedS–adenosyl methionine (Jiang et al, 1999).

Lines selected in the laboratory for resistance to thedrug have been studied. Reduced drug accumula-tion was noted in procyclic parasites resistant toeflornithine (Bellofatto et al, 1987). However,whether this was due to decreased uptake orincreased efflux was not determined.

A Leishmania line selected for resistance to DFMOwas shown to have an increase in ornithine decar-

boxylase activity associated with an amplification incopy number of the gene (probably associated withamplification of an episome) (Sanchez et al, 1997).Elevated putrescine uptake in L. infantum exposedto DFMO has also been reported (Balana-Fouce et al,1991).

Increased ornithine decarboxylase activity (associat-ed with a rise in transcript levels but not, this time,gene copy number) has also been reported in arsen-ite resistance, associated with increased trypanoth-ione biosynthesis in Leishmania tarentolae (Haimeuret al, 1999). The significance of this observation liesin the fact that, should a similar route to arsenicalresistance be possible in T. brucei, the possibility ofcross-resistance to DFMO would materialize.However, it should be stressed that so far a similarmechanism has not been noted in trypanosomes,and to date, lines selected for resistance to DFMOwere not cross-resistant to other trypanocides.

Nifurtimox

The drug and its useNifurtimox was originally licensed for use againstSouth American trypanosomiasis. The drug con-tains a nitro group which is central to its activity. Ithas also been used in trials, with only limited suc-cess (50-80% cure), against T. brucei gambiense inWest Africa, although since it is apparently activeagainst melarsoprol refractory parasites it may stillbe used and as treatment failures with arsenicalincrease (Pépin et al, 1992).

Serum levels are reportedly low when given orally,peaking one-three hours after administration. Thedrug can accumulate across the blood-brain barrier(Anon, 1998).

Toxic effects to the central nervous system andperipheral nervous system have been reported.

Mode of uptake and actionNo reports about the mechanism of action againstT. brucei could be found in the literature, althoughreports relating to activity against T. cruzi exist.Uptake of nifurtimox into Trypanosoma cruzi hasbeen reported to occur via passive diffusion acrossthe plasma membrane (Tsuhako, et al.,1991). Studieshave not yet been extended to T. brucei but it is alsolikely to enter these cells via passive diffusion.

Nifurtimox is a nitrofuran compound. One electronreduction of the nitro-group generates a potent freeradical which may interact with cellular con-stituents or generate reduced oxygen metabolites


believed to cause death of the parasite (Docampoand Moreno, 1984). The reduction potential of thecompound (-260 mV) is such that it is relatively eas-ily reduced in many cell types. The specificitytowards the parasite (which is not great, nifurtimoxbeing quite toxic to mammals) is thought to be asso-ciated with its being more readily reduced by theparasite than the host cells. Moreover, mammaliancells may have better protection against oxidativedamage. Specific targets or enzymes capable ofreducing the drug cannot be ruled out. Some path-ways which might lead to the preferential reductionof these compounds in trypanosomes have beenstudied. An intriguing hypothesis was that trypan-othione reductase might be responsible for thereduction (Henderson et al, 1988). Certainly, a num-ber of nitro-containing compounds can act as “sub-versive-substrates” for the enzyme. However, nifur-timox was one of the less successful substrates(Henderson et al, 1988). and it is unlikely that try-panothione reductase-mediated nitro-reductionunderlies the activity of the clinically used nitro-heterocyclics.

ResistanceAfrican trypanosome lines selected for resistance tonifurtimox have not been reported in the literature.It is not clear why treatment failure is high. Furtherpharmacokinetic studies should be made to deter-mine the degree to which the drug reaches the CSF.

Trypanosoma cruzi isolates show various levels ofsensitivity to the drug (Filardi and Brener, 1987).There appears to be a correlation between druguptake and sensitivity (with lines accumulatingleast drug being least sensitive to it). No systematicstudy on susceptibility of different sub-species orstrains of T. brucei have yet been conducted.Interestingly, another nitroheterocycle called mega-zol (discussed below) was equally active againstlines of T. cruzi showing different sensitivities tonifurtimox (Filardi and Brener, 1987).

Megazol

The drug and its useMegazol is a 5-nitroimidazole which has good effi-cacy against both T. cruzi (Filardi and Brener, 1987)and T. brucei (Enanga et al, 2000). Its synthesis wasfirst reported in 1968 (Berkelhammer and Asato,1968). The activity of the drug against African try-panosomes is striking. A single dose clears parasitesfrom the blood of rodents and a primate model.Administration of the drug following a single doseof suramin cleared parasites from the CSF of aninfected primate (B. Enanga, personal communica-tion).

The drug can be given orally. Peak plasma levels fol-lowing a 100 mg kg-1 dosing (Enanga et al, 2000) inprimates yielded plasma levels of between 0.2 µg mland 46 µg ml-1 24 hr after dosing. The drug or itsmetabolites can be found in the CSF at levels 5.5%-10.6% of those in plasma. The elimination half timewas around 2.5 hours. Suramin substantiallyincreases the half-life of the drug and also increasesthe amounts which can accumulate across theblood-brain barrier.

No published reports of the effect of megazol onhumans exist. When the drug was administered totwo patients with Chagas’ disease, anecdotal evi-dence indicated there was little toxicity. However,the compound is positive in Ames’ tests (Ferreiraand Ferreira, 1986) and this fact alone appears tohave served as a deterrent to further development,in spite of the excellent safety record of anotherAmes’ test positive anti-protozoal 5-nitroimidazole,metronidazole. Toxicological studies in mammalsshould be performed under recognized good labo-ratory practice (GLP) conditions as a matter ofurgency. If toxicity proves to be no worse than fornifurtimox, then the case for pursuing the drug as apotential reagent for clinical use would be strong.

Mode of uptake and actionMegazol possesses part of the motif recognized bythe P2 amino-purine transporter which is responsi-ble for the uptake of several anti-trypanosomaldrugs (Barrett et al, 2000). Should this drug share theP2 transporter as a portal of entry, it would be oflimited use against arsenical resistant parasites.However, strains of parasite lacking the P2 trans-porter and resistant to other drugs which use thisportal of entry into trypanosomes, were not cross-resistant to megazol drugs (Barrett et al, 2000).Uptake of radiolabelled megazol revealed that thisdrug, although capable of interacting with the P2transporter, enters cells predominantly via passivediffusion (Barrett et al, 2000).

The mode of action of the drug is not clear. The factthat a nitro group is central to its function does notnecessarily imply that the mode of action will be thesame as for nifurtimox. Indeed, its reduction poten-tial of -438 mV (Viodé et al, 1999) is far lower thanthat of nifurtimox (-260 mV), and 5-nitroimidazolesare not normally reduced by aerobic cells. However,megazol is susceptible to nitro-reduction in thepresence of several enzymatic systems includingsome found in T. cruzi extracts. How it exerts a lethalaction against parasites, however, is not certainalthough it seems likely that trypanosomes possessa specific enzyme capable of reducing this com-pound.


Resistance Both procyclic and bloodstream forms of the para-sites have been selected for resistance to megazol inthe laboratory (Enanga & Barrett, unpublishedresults) (procyclic forms with >100-fold resistance,bloodstream forms with about 20-fold resistance tothe drug). Modest levels of cross-resistance to theother nitroheterocycle, nifurtimox, were apparent(about 7-fold in both cases). Even more modest lev-els of cross-resistance to diamidines (2 to 4-fold) andmelamine based arsenicals (3 to 5-fold) were alsoobserved. Preliminary evidence suggests that thereis not a role for an efflux pump or for increased lev-els of trypanothione in the procyclic lines. Themechanism of resistance is currently unknown butunder investigation.

DETECTION OF DRUG RESISTANCETreatment failure and drug resistance are by nomeans synonymous. Resistance is best defined as“the heritable, temporary or permanent loss of theinitial sensitivity of the population of microorgan-isms against the active substance” (Schnitzer andGrunberg 1957). It is essential that drugs are admin-istered according to the recommendations put for-ward based on regimens optimal for activity,although further studies on optimizing the dose arerequired for most drugs. This is particularly impor-tant in late-stage sleeping sickness where the quan-tity of drug that accumulates in the extravascularcompartment may not extend far beyond the MICrequired to kill the parasites. Treatment failure cancome about for a number of reasons includingadministration of sub-curative doses of drug or hostfactors including metabolism and distribution with-in the body.

It is important to determine whether patients whohave not cleared all parasites after treatment actual-ly carry drug resistant trypanosomes. Thereforetests for resistance should be performed.

Isolation of Parasites, Propagation andDrug Sensitivity Testing in RodentsBlood (or CSF) can be taken from infected patientsand injected directly into a suitable rodent model. Inthe case of T. b. rhodesiense, standard laboratorywhite mice or rats are adequate for this process.High parasitemias are readily achieved in thesehosts and highly parasitemic blood isolated fromthese hosts can be isolated, mixed with a suitablecryopreservant, and frozen in liquid nitrogen.Preserved stocks can be re-injected into rodents andthen treated with trypanocidal drugs over a range ofconcentrations to determine the MIC and effectivedose 50 (ED50) of drug useful against these para-sites in vivo. Protocols must be standardized for thispurpose.

T. b. gambiense is much more difficult to grow inrodents, although Mastomys rats do allow prolifera-tion of some isolates (only 20% of T. b. gambiense iso-lates from a recent study in northern Uganda couldbe grown in Mastomys - Matovu, personal commu-nication). Standardization of rodent protocols isimportant, i.e. similar parasite numbers should beinoculated, similar drug dosing post-inoculationshould be given, and there should be similar followup with regard to checking mice for parasitemia.

The protocols established at the Swiss TropicalInstitute for each of these procedures may be rec-ommended as the standards which should be fol-lowed, although consensus agreement is requiredfor this and other procedures may be considered(for example those recommended for testing of vet-erinary trypanocides in a recent PAAT document)(Geerts and Holmes, 1998).

Drug Sensitivity Testing in VitroIt is also possible to cultivate some lines in vitrousing established culture media. Laboratory adapt-ed lines are relatively easy to establish in culture.However, many field isolates, particularly ofT. b gambiense, do not adapt readily to in vitro cultureand few reports of axenically cultured T. b. gambiensecould be found. Lines which have successfully beenpassed through Mastomys can proliferate in richmedium (containing human serum) over a mono-layer of Mastomys embryonic fibroblast cells (Brunet al, 1989). Until better conditions for the in vitrocultivation of T. b. gambiense are established, it isimportant that all drug tests against all isolatesshould be performed under the same conditions. IfT. b. gambiense is to be compared with T. b. rhode-siense, it is of limited use to use different cultivationsystems to determine drug sensitivity. This is partic-ularly the case when using mammalian cell mono-layers as part of the culture system since these mayaffect the drug and its activity against parasites.

Molecular Approaches to the Identification of Drug ResistanceAlterations to the gene TbAT1 that encodes the P2transporter have been identified, (Maser et al, 1999)and many parasite lines which are less sensitive thannormal to melarsen-based arsenicals possess similarmutations (Matovu, personal communication).Particular mutations appear to have been selected onmultiple independent occasions. This opens the pos-sibility of using a polymerase chain reaction (PCR)based approach to detect the presence of particularmutant alleles which correlate to drug resistance.This approach will be limited if different types ofmutation affect the status of expression of the TbAT1gene. More studies to investigate the frequency of


particular mutations that correlate to resistance areneeded if such a test is to be useful. The possibilitythat different types of mutation could affect the P2transporter in such a way as to reduce sensitivity todrug, and the possibility that other biochemicalchanges not related to the P2 transporter could alsoinduce resistance to arsenicals, means that a simplePCR based test might produce an unacceptably highnumber of false negative results.

GUIDELINES ON THE DELAY OF THEDEVELOPMENT OF DRUG RESISTANCERecent evidence indicates that the rise in the num-ber of sleeping sickness cases proving refractory tomelarsoprol treatment (Legros et al, 1999) is at leastpartially caused by the emergence and spread ofparasites resistant to the drug in the field (Matovuet al, in press). However, the global quantities ofmelarsoprol administered are far lower than, forexample, those for chloroquine in malaria prophy-laxis, or for many of the antibiotics to which resist-ance is now widespread. It is also recommendedthat melarsoprol is administered in a clinical setting,thus improving the likelihood that a full curativedose is given. As well, the dynamics of transmissionvia tsetse flies make it far less likely that human try-panosomes will be transmitted between humanhosts at the same frequency as are Plasmodium para-sites by anopheline mosquitoes. Therefore, it is per-haps surprising that resistance to the drug hasemerged, albeit apparently with a substantiallyslower time of onset than with chloroquine (chloro-quine was introduced in 1945 and melarsoprol in1949). These factors also need to be set against thefact that the quantities of melarsoprol reaching theparasites within the CSF are only marginally higherthan the MIC of the drug, so that parasites which areonly slightly less sensitive than wild-type to drugmay be selected with relative ease. While the param-eters that can lead to selection of resistance are com-plex, there is no doubt that it is critical to ensurethat the recommended dose of the drug is given toevery patient.

The potential of cross-resistance between pentami-dine and melarsoprol must also be considered. Thisis because both of these trypanocides can enterT. brucei via the P2 nucleoside transporter (Barrettand Fairlamb, 1999). Selection of resistance to onedrug could therefore, in principle, lead to cross-resistance to the other. Interestingly, the relativelywidescale use of pentamidine as a prophylactic inwest Africa up until the 1950s appears not to haveselected for resistance. Some laboratory derivedlines have been shown to be pentamidine-melarso-prol cross-resistant but this has not been shown tobe so in all cases. This could be due to the fact that

pentamidine appears to have alternative routes ofentry into the cells (De Koning, 2001). Nevertheless,pentamidine should also be administered prudent-ly, and curative doses ensured, to restrict the oppor-tunities for selection of resistance to this drug whichmight cause cross-resistance to melarsoprol.

A Possible Role for VeterinaryTrypanocide Use in the Selection ofMelarsoprol Resistance in SleepingSicknessMicrobes selected for resistance to drugs duringunsupervised treatment of livestock have become amajor route of introduction of resistance and resist-ance genes into human pathogens.

It seems that cross-resistance between diminazeneand melamine based arsenicals occurs more consis-tently than between the latter and pentamidine(Barrett and Fairlamb, 1999; Barrett et al, 1995). Thiscould be because diminazene appears to enter pre-dominantly via the P2 transporter and not via thealternative transporters that carry pentamidine(De Koning, 2001). The emergence of diminazene-melarsoprol cross-resistance can have profoundimplications for the development of drug resistantsleeping sickness in the field.

To date, no studies have been conducted to assesswhether a drug resistant parasite selected in an ani-mal can be transferred to humans – this is a specu-lative scenario. However, it is clear that diminazeneis administered to trypanosome infected cattle(Geerts and Holmes, 1998) and that several genericversions of this product have appeared on the mar-ket which are of highly variable quality. Ad-hoc,unsupervised administration of sub-curative dosesof diminazene to cattle is reportedly widespread,and resistance to diminazene (in T. congolense andpossibly other trypanosome species) is rife in partsof Kenya and elsewhere.

Up to 20% of trypanosomes isolated from cattle inwestern Kenya and parts of Uganda are infectious tohumans (MacLeod et al, 2001). Therefore, humaninfectious trypanosomes are present in areas whereconditions for selection of resistance to diminazeneare prevalent. Indeed, one isolate from south-easternUganda was clearly resistant to diminazene andisometamidium (Matovu et al, 1997). It has yet to bedemonstrated whether resistance is associated withloss of the P2 transporter, or whether cross-resistanceto melarsoprol occurs. In spite of this lack of data,there are reasons for concern about the selection ofdrug resistant parasites in animals which can then betransferred to human infections. It should be stressedat this point that the wider host range of T. b. rhode-


siense makes it more likely that such a scenario mightarise for this form of sleeping sickness more readilythan for the gambiense form of the disease.

Studies should be conducted to assess the degree towhich drug resistance selected in animals can betransferred to humans. In the meantime, effortsaimed at limiting the spread of drug resistanceamong trypanosomes in cattle should be givenadditional impetus so as to minimize the risk of thisoccurring.

Widespread implementation of the rationallyderived ten-day administration protocol for melar-soprol in gambiense patients (Burri et al, 2000)might also reduce the probability of transmission ofresistant lines.

GUIDELINES ON THE CONTROL OFDRUG RESISTANCE ONCE PRESENTMelarsoprol resistance appears to be on the increase.However, the only other drug licensed to treat late-stage sleeping sickness, namely eflornithine, is notcurrently available, although it seems that Aventiswill produce it for the coming five years so melarso-prol refractory patients should be treated with thisdrug if they are infected with gambiense parasites.If eflornithine is not available, then melarsoprolrefractory patients can be treated with recently rec-ommended doses of nifurtimox (Bisser et al, 2000).Combination chemotherapy can also be consideredand, in this regard, melarsoprol/eflornithine, melar-soprol/nifurtimox and nifurtimox/eflornithine haveall been used to treat drug resistant T. b gambiense(Jennings, 1993).

For early-stage disease, resistance to suramin hasnot yet been identified. It needs to be confirmedwhether contemporary lines of T. b gambiense aresusceptible to suramin but, if there is no pentami-dine-suramin cross-resistance, then one drugshould be suitable to treat parasites resistant to theother drug. Melarsoprol can also be used to treatearly-stage disease which is resistant to suramin, ascan eflornithine, if available. Caution should beexercised in treating melarsoprol refractory para-sites with pentamidine, or pentamidine refractoryparasites with melarsoprol, since cross-resistancedue to the lack of the P2 transporter is a possibility.

AcknowledgmentsI am grateful to Dr Enock Matovu for sharingunpublished results, also to Drs Reto Brun, MikeTurner, Harry De Koning, Dominique Legros andAnne Moore for critical reading of this manuscript.

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III HUMAN AFRICAN TRYPANOSOMIASIS(Original manuscript in French)

Felix DouaClinical Research Project into Trypanosomiasis, BP 1425Daloa, Côte d’Ivoire

INTRODUCTIONIn the last 20 years there has been a resurgence ofinterest in human African trypanosomiasis (HAT)following a recrudescence of the disease in mostcountries of sub-Saharan Africa. Although there arethousands of new cases every year in countries suchas the Democratic Republic of the Congo (DRC),Angola and Uganda, no noteworthy progress hasbeen recorded in the development of new drugs tofight the condition.

The drugs currently used in the treatment of sleep-ing sickness were first marketed in the 1950s.Pentamidine and suramin are the drugs of choicefor the early-stage disease, while melarsoprol(arsobal), despite its toxicity, is the drug of choice forthe late-stage disease, when the central nervous sys-tem is involved. However, a rise in treatment fail-ures with melarsoprol has been noted in the last fewyears (Ginoux et al, 1984; De Gros et al, 1999), there-by hampering efforts to combat sleeping sicknessdespite the development of sensitive diagnostictools and versatile low-cost anti-vectoral controlmethods such as insecticide impregnated screens.

Difluoromethylornithine (DFMO, ornidyl, eflor-nithine) has been undergoing clinical trials in thelast two decades but is not yet on the marketbecause its cost is considered prohibitive for usercountries. There is therefore an urgent need to usebetter the available trypanocides and to developnew ones that are cheap, well tolerated and effectiveat every phase of the disease.

TREATMENT AND CLINICAL TRIALSCONDUCTED ON THE T. B. GAMBIENSEFORM OF SLEEPING SICKNESS

Treatment and Clinical Trials using Pentami-dinePentamidine, an aromatic diamidine, has a successrate of 95% when used for early stages of T. b. gam-biense HAT (Doua et al, 1993), and of 94% whenused for patients suffering from the early nervoussystem phase (Doua et al, 1996). These results indi-cate that pentamidine passes the blood-brain barri-er and is capable of halting the multiplication oftrypanosomes in cerebrospinal fluid. This suggests

that more than 15% of all patients in the nervoussystem phase of the disease, treated in Côted’Ivoire, can be excluded from the fatal side-effectsof arsobal therapy.

Treatment and Clinical Trials usingMelarsoprolTreatment regimens using melarsoprol in countrieswhere sleeping sickness is endemic date back tocolonial times and consist of the administration ofthree to four series of two to four injections of melar-soprol spaced out at seven to ten day intervals.These regimens, developed on an empirical basis,have helped control HAT epidemics but could bethe cause of the growing number of recorded treat-ment failures. Recent pharmacokinetic data onarsobal, obtained by bioassay and computer simula-tions (Burri et al, 1993), have shown that an alterna-tive, more rational, therapy regimen, consisting often injections at low doses, can be used withoutincreasing the risk of fatal accidents. The new regi-men (Burri et al, 1995) is currently under clinicalevaluation in seven African countries.

Treatment and Clinical Trials using DFMODFMO (ornidyl, eflornithine) is an irreversible spe-cific inhibitor of ornithine-decarboxylase, a keyenzyme in the synthesis of polyamines, which arephysiological substances implicated in cell multipli-cation (Sjoerdsma et al, 1984).

Clinical trials carried out using DFMO show thata seven-day regimen is effective in treatment ofT. b. gambiense HAT. The side effects, recorded inparenteral DFMO administration, are generally re-versible (Doua et al, 1987; Taelman et al, 1987) andthe relapse rates after treatment are normally about1% (Doua et al, 1993). However, the cost of DFMOremains a limiting factor to its wider use. Clinicaltrials are ongoing in Côte d’Ivoire, to determine theefficacy after oral administration, and the pharma-cokinetics. The intended result is to develop an oralformulation of the product at an affordable cost topatients.

Treatment and Clinical Trials usingNifurtimoxThis use of nifurtimox for the treatment of T. b. gam-biense HAT has not been studied in large-scale clini-cal trials, although results obtained with four late-stage patients in the DRC show that the product iseffective but very toxic (Jansens et al, 1977).

CONCLUSIONS AND RECOMMENDATIONS In the last 20 years there has been rapid develop-ment of both serological and parasitological HAT


diagnostic techniques, but in 2001, treatment of thedisease is still based only on pentamidine andsuramin for the early stages of the infection, and onmelarsoprol for the central nervous system stages.Treatment schedules with these trypanocides werederived empirically, without pharmacokinetic stud-ies. Thus, the treatments applied to patients varyfrom one country to the next and sometimes fromone treatment centre to another in the same country,which only aggravates the high number of failuresobserved, in particular with melarsoprol, the drugof choice for HAT.

DFMO, which has given rise to great hopes for thetreatment of T. b. gambiense HAT, still has not beencommercialized because of its cost. To make effortsto control HAT more effective, and to make forrational treatment of patients, we recommend thefollowing:• Encouragement of research aimed at developing

new, effective, well-tolerated, cheap trypanocides. • Harmonization of treatment schedules for HAT,

particularly those for melarsoprol.• Conduct of multicentre clinical trials using pen-

tamidine for patients in the early nervous systemstage of HAT, with a view to evaluating its effica-cy in different HAT foci.

• Conduct of multicentre clinical trials using orallyadministered DFMO, after the current study onpharmacokinetics is completed.

• Conduct of multicentre clinical trials using melar-soprol-DFMO combination in patients sufferingrelapse after treatment with melarsoprol.

• Introduction of nifurtimox for the treatment ofHAT cases that are resistant to melarsoprol.

ReferencesBurri C et al. Pharmacokinetic properties of the try-panocidal drug melarsoprol. Chemotherapy, 1993,39:225-234.

Burri C et al. Alternative application of melarsoprolfor treatment of T.b. gambiense sleeping sickness.Annales de la Société belge de médecine tropicale, 1995,75:65-71.

De Gros et al. Echecs thérapeutiques du melarsoprolchez des patients traités au stade avancé de la tryponoso-mose humaine africaine à T. b. gambiense [Therapeuticfailures of melarsoprol among patients treated at anadvanced stage of T. b. gambiense HAT in Uganda].25th CISRLT, programme provisoire 1999, pp. 3.

Doua et al. Treatment of human late-stage gambiensetrypanosomiasis with alpha-difluoromethylorni-thine (eflornithine). Efficacy and tolerance in 14 ca-ses in Côte d’Ivoire. American journal of tropical med-icine and hygiene, 1987, 37:525-533.

Doua et al. Human tryponosmiasis in the IvoryCoast: therapy and problems. Acta tropica, 1993,54:163-168.

Doua et al. Efficacy of pentamidine in the treatmentof early late stage trypanosoma brucei gambiense try-panosomiasis. American journal of Tropical Medicineand Hygiene, 1996, 55:586-588.

Ginoux et al. Les échecs du traitement de la try-panosomiase à T. b. gambiense au Congo. [Failures ofT.b. gambiense trypanosomiasis treatment in theCongo]. Médecine tropicale, 1984, 22:149-154.

Jansens et al. Clinical trials with Nifurtimox inhuman trypanosomiasis. Annales de la Société Belgede Médecine Tropicale, 1977, 57:475-479.

Sjoerdsna A, Schecter PJ. Chemotherapeutic impli-cations of polyamine biosynthesis inhibition. Cli-nical Pharmacology and Therapeutics, 1984, 35:287-300.

Taelman H et al. Difluoromethylornithine, a new ef-fective treatment of Gambian trypanosomiasis. Re-sults in five patients. The American Journal ofMedicine, 1987, 82:607-614.


IV TREATMENT AND CLINICAL STUDIES

Martin OdiitLivestock Health Research Institute, P.O. Box 96, Tororo,Uganda

SUMMARY Diagnosis and treatment of sleeping sicknessremains the major control strategy of the disease.The current diagnostic kits are insensitive, the diag-nostic and treatment centres inadequately equippedand staffed, and the patients poor and often in inac-cessible rural locations. The drugs used for the treat-ment of human African trypanosomiasis are toxic,necessitating a positive demonstration of parasites,including the invasive, painful lumbar puncturesfor disease stage determination. The treatment ofthe disease is lengthy, parenteral and costly, and thedrugs used toxic and not readily available. Thesetraditional problems of sleeping sickness are com-pounded by the emergence of drug resistance tomelarsoprol in T. b. gambiense patients, the increas-ing uncertainty of sustained production of drugs forsleeping sickness due to economic justification, andthe increasing prices of some drugs due to theirnewly found use for HIV treatment. Clinical studiesthat address these problems which face the majorcontrol strategy for sleeping sickness are urgentlyneeded. For melarsoprol treatment of T. b. gambienselate-stage patients, studies on reduction in length oftreatment schedules, and therefore of costs, andimproved compliance, are in the advanced stages.Research into cost-effective and sustainable strate-gies of improving diagnosis and treatment are nec-essary to reduce the level of under-reporting that isassociated with certain death of sleeping sicknesspatients if untreated. This scientific working paperreviews some of the current problems of treatmentof sleeping sickness and suggests clinical studiesthat may address these problems in the near future.

INTRODUCTIONHuman African trypanosomiasis (sleeping sickness)is found in the tsetse belt of tropical Africa, where itis estimated that over 50 million people live (WHO,1998). Approximately 45 000 cases are reportedannually, though it is believed that 300 000 personsare infected each year (WHO, 1998). There are twotypes of sleeping sickness, caused by: Trypanosomabrucei gambiense, found in west and central Africa,and by T. b. rhodesiense, reported in east and south-ern Africa. The gross national product per capita forthe sleeping sickness affected countries (< US$500)ranks them as amongst the least developed coun-tries of the world (World Bank, 2000). However, thecosts of treatment for sleeping sickness are estimat-

ed to be more than US$100 per adult patient (WHO1998). In addition, sleeping sickness patients arealmost always from poor rural backgrounds withintheir countries (Okia et al 1994). Drugs for the treat-ment of sleeping sickness are not readily available indrug stores and are most often procured by the gov-ernments and non-governmental organizationsthrough the assistance of the World Health Organi-zation. Stocks of drugs are carefully monitored atnational levels. However, it is conceivable that, inthe event of an epidemic, it may not be possible tohave enough drugs immediately available, a con-straint already experienced by some African coun-tries during sleeping sickness epidemics. Most drugcompanies appear to be reluctant to produce drugsfor sleeping sickness because they will not obtainreturns that are adequate to finance further drugdevelopment (Barrett, 2000; Stephenson and Wiselka, 2000).

SLEEPING SICKNESS CONTROLSleeping sickness control has traditionally been car-ried out through case detection and treatment, andthrough vector control when epidemics occur(WHO, 1998). Early diagnosis and treatment is thegoal of control programmes, but is often hamperedby the paucity of resources required to implementthese programmes. Most programmes of sleepingsickness control are donor dependent because thecosts of sleeping sickness interventions are beyondthe budgets of the affected countries. The need forcheaper control strategies is therefore a priority.Decentralization of services, including health servic-es, is being advocated. In some countries, planningand implementation of the sleeping sickness controlprogrammes has been moved to the district level.However, current sleeping sickness control strategiesare expensive and often complicated, requiring qual-ified personnel. Research into the roles of the nation-al and sub-national levels in the decentralization ofsleeping sickness control should be reviewed.

DIAGNOSISDetection of the parasite in gland or lymph nodeaspirate or blood is always followed by a lumbarpuncture to determine whether there is involvementof the central nervous system (CNS) (also known asthe late stage), because drugs used when the CNS isinvolved are different from those used when theparasite is still restricted to the haematolymphaticsystem (also known as the early stage). However,most of the currently used confirmatory diagnostictechniques such as the thick blood smear and thehaematocrit centrifugation test have poor sensitivity(WHO 1998). The polymerase chain reaction (PCR)has been found to be very sensitive and specific(Kyambadde et al, 2000; Penchenier et al, 2000), but


it is rather complicated and expensive for applica-tion as a routine diagnostic test, which restricts itsuse to research laboratories and referral hospitals.

The criteria used for determination of CNS involve-ment are as follows: cell counts above 5 cells/mm3

or the demonstration of trypanosomes or proteinlevels above normal (25mg per cent) in the cere-brospinal fluid (CSF). The first two criteria are gen-erally used because they are simpler and do notrequire reagents.

CHEMOTHERAPY OF SLEEPING SICKNESSMost of the affected countries use broad guidelinesfor the treatment of sleeping sickness as recommend-ed by the World Health Organization (WHO, 1998).However, there is an urgent need for novel approach-es in sleeping sickness treatment and control.

Pentamidine is used for treating the early stage ofsleeping sickness caused by T. b. gambiense. It iseffective for treatment so long as there is nodetectable involvement of the CNS. The dosageused is 4mg of pentamidine base per kg of bodyweight. A total of seven injections are given daily,intramuscularly. Drug resistance appears not to bean important problem in the use of pentamidine forthe treatment of T. b. gambiense. The side effectsinclude pain and induration or sterile abscess at thesite of injection, vomiting, abdominal pain, hypo-tension, syncope, hypoglycaemia, and peripheralneuritis. Treatment failure attributable to resistanceto pentamidine appears not to be an important pub-lic health problem. Studies of the pharmokinetics ofpentamidine are required to determine if the lengthof the prescribed treatment regime can be reduced.

Suramin is used for the treatment of the early stageof T. b. rhodesiense because, though the duration oftreatment is longer than that for pentamidine and itis given by intravenous treatment, no primaryresistance to suramin has been reported forT. b. rhodesiense. The dosage used is 20mg/kg bodyweight. Intravenous injections - a maximum of se-ven - are given every seven days. Side effects obser-ved include pyrexia, pains in the joints and soles ofthe feet, skin rash and desquamation, hypersensitiv-ity reactions. Pharmacokinetic studies of suraminshould be undertaken to determine if the treatmentregime can be shortened.

Melarsoprol is the drug used to treat late-stagesleeping sickness due to both T. b. rhodesiense andT. b. gambiense in Uganda. The maximum dosage foreach injection is 3.6 mg/kg body weight. Currently,two regimes for the treatment, comprising several

series of injections each separated by an interval ofone week, are used (WHO, 1998). One regime startswith 2.5ml and the other with 0.5ml, but both total35 mls of a 36g/litre solution of melarsoprol.

The most serious side effect of melarsoprol is reac-tive encephalopathy occurring between the thirdinjection and the beginning of the second course.The onset may be sudden or the condition maydevelop slowly with fever, headache, tremor, slur-ring of speech, convulsions, and finally coma. Itoccurs in 5% of patients, and the incidence of fatali-ty due to these reactions is approximately 1% (Odiitet al, 1997). However, the fatality of untreated dis-ease is believed to be 100%, making the decision totreat an ethical necessity. The treatment of thesereactions includes the use of corticosteroids, hyper-tonic solutions to combat cerebral oedema, rapidlyacting anticonvulsants, and subcutaneous adrena-line. There are a few cases of diarrhoea, jaundice anddermatitis, but these are generally not life threaten-ing.

PATIENT FOLLOW-UPIt is mandatory to systematically follow up all treat-ed patients to ascertain that they have been cured. Itis recommended that patients be seen every sixmonths over a two-year period. In addition to a fullclinical and parasitological examination, a lumbarpuncture should be carried out on the patients, toallow the CSF to be examined for possible increasein leukocyte counts or presence of trypanosomesthat would indicate a relapse. However, due to thepainful lumbar puncture procedure, most patientsresent returning for follow-up once they begin tofeel better, while active follow-up to ensure compli-ance is not affordable. Therefore, less invasive andpainful methods of ascertaining cure need to bedeveloped. Changes in CATT titres or CIATT titresand haematological parameters such Hb, ESR etc.are suggested for investigation in assessing cure. Inaddition, the duration of post-therapeutic follow-upshould be reviewed, especially for T. b. rhodesiense,in the expectation that it may be shorter than thatrequired for T. b. gambiense.

DRUG RESISTANCEThere is no recent evidence for resistance of T. b. rho-desiense to melarsoprol. In the early 1970s, melarso-prol treatment failure of 12 (3.4%) out of 358 treatedcases was reported (Ogada, 1974). It is possible that,with low treatment failure rates and lack of system-atic follow-up, patients with treatment failure maybe missed. There should be periodic active follow-up of cohorts of sleeping sickness cases to monitordrug efficacy.


Treatment failure of melarsoprol in T. b. gambiensedisease is emerging. Legros et al (1999a and 1999b)reported a melarsoprol treatment failure rate of26.9% among 428 patients treated in north westernUganda. Melarsoprol treatment failure rates needsto be monitored and documented regularly to estab-lish the magnitude of the problem. The cause of thistreatment failure is under investigation to deter-mine whether it is due to variation of melarsoprolpharmacokinetics between individuals or if it isassociated with reduced susceptibility of try-panosomes to melarsoprol. Legros et al (1999b)emphasize the need for second-line drugs to treatpatients that have already received one or severalfull course(s) of melarsoprol. In Uganda, melar-soprol treatment failure is a more significant prob-lem to the west of the river Nile than to the east, inthe T. b. gambiense affected area in the north-west ofthe country. Possible risk factors explaining this dif-ference in geographical distribution here and else-where on the continent should be examined. Sub-clinical, inadequate treatments in the history ofthese melarsoprol treatment failure foci could be anexplanation.

HEALTH SYSTEMS AND SLEEPINGSICKNESS CASE MANAGEMENTDemonstration of the trypanosome is mandatorybefore treatment can be administered to a sleepingsickness case, due to the toxicity of the drugs used.Due to the costs of staffing and equipping sleepingsickness treatment facilities, few health units areequipped for treating the disease. The endemicarea for sleeping sickness in most countries is vast,with poor coverage by fixed post health units fordiagnosis and treatment of the disease. Therefore,most sleeping sickness patients travel more than5km to receive treatment. The problem of pooraccessibility to diagnosis and subsequent treat-ment may be alleviated by the use of mobile teams.However, this is more rewarding where a validscreening test is commercially available. The cardagglutination test for trypanosomiasis (CATT) issuch a test. It has good sensitivity and specificity(Magnus et al, 1978) but it is only useful for thegambiense form of sleeping sickness. A similar testfor the rhodesiense form of the disease is requiredbefore mobile teams can provide a rewarding strat-egy for routine diagnosis and treatment. A newtest, the card indirect agglutination trypanosomia-sis test (TrypTectCIATT®), is reported to have goodsensitivity for both forms of sleeping sickness(Nantulya, 1997) and is to undergo further fieldevaluation (TDR, 1999). In Uganda, mobile teamsare used in the north-west with support from anon-governmental organization (Médecins sansFrontières, France); during outbreaks in the south-

east, the teams are used to increase the awarenessof the population and detect cases who may not yethave sought health care. The costs of maintainingmobile teams are not affordable by the countriesaffected by sleeping sickness, and therefore theteams are often used in epidemic situations andwith donor support. Operational research isrequired to optimize the distribution of fixed postdiagnosis and treatment. This strategy may be amore sustainable strategy, especially in betweenepidemics of sleeping sickness.

NEWLY DESIGNED TREATMENT REGIMESPharmacokinetic data from studies carried out onT. b. gambiense patients indicate that the course ofmelarsoprol treatment in an adult can be reducedfrom a period of 25-36 days (WHO, 1998) to a peri-od of just ten days, greatly cutting the costs of hos-pitalization (Burri et al, 1995). A randomized trialcomparing the standard treatment schedule withthis new concise regimen showed the same levels ofparasitological cure (100%) and adverse effects (16-17%) as well as better compliance with the newregime (Burri et al, 2000). Adaptation of the newconcise regime is to be monitored. It would not beadvisable to adapt the model to treatment of late-stage T. b. rhodesiense infection because the pharma-cokinetics may be dissimilar given that this diseaseis much more severe than the gambiense form. Theblood-brain barrier may be more affected, allowingfor higher levels of melarsoprol in the central nerv-ous system and possible neurological side effects. Astudy of the pharmacokinetics of melarsoprol inlate-stage T. b. rhodesiense infections is therefore nec-essary. However, a seven-day course of eflornithinefor the treatment of late-stage T. b. gambiense infec-tion was found to be inferior to the standard 14-dayregimen (Pépin et al, 2000).

There have been attempts to adjust the criteria fortreatment and so reduce the number of patientsreceiving melarsoprol treatment that is very toxic.To determine CNS involvement, it has been pro-posed that the number of cells be raised to 20cells/mm3 (Doua et al, 1996). There is not yetenough evidence to change the cut-off point for thenumber of cells/mm3 in the CSF (Doua et al, 1996);therefore the WHO recommended criteria for stag-ing are still maintained.

Chemoprophylaxis is not currently used as a sleep-ing sickness control strategy. A multicentre evalua-tion of the positive predictive value of increasingdilutions of the CATT (Magnus et al, 1978) is beingcarried out; results of this study will be used to seeif mass chemoprophylaxis is indicated in instancesof high sero-prevalence.


Eflornithine (difluoromethylornithine, or α-DFMO,or Ornidyl®) is not effective against T. b. rhodesiense(Iten et al, 1995; Matovu et al, 1997) but is useful forthe treatment of late-stage T. b. gambiense infection(Doua et al, 1987). It is presently available for thetreatment of relapse following melarsoprol treat-ment, but the production of this drug was recentlysuspended. It has been suggested that nifurtimox beused in desperate situations where DFMO is notavailable for treating melarsoprol refractory cases.Nifurtimox is very toxic but is reported to be effec-tive for curing some T. b. gambiense patients, includ-ing those with late-stage disease refractory to melar-soprol treatment (WHO, 1998).

Nifurtimox (Lampit®, Bayer) has been tested for thetreatment of sleeping sickness with conflictingresults (Pépin et al, 1992; Van Niewenhove, 1992;Doua and Yapo, 1993). It is a cheap drug and easy toadminister but is not yet registered for use in thetreatment of this disease. With the current emer-gence of melarsoprol resistance, the efficacy andsafety of nifurtimox in the treatment of sleepingsickness caused by T. b. gambiense should be re-eval-uated.

REDUCTION OF DRUG PRESSUREIn areas where drug resistance to melarsoprol exists,the need to use the drug can be reduced by puttingemphasis on preventive control strategies such asvector control and mass chemotherapy of thedomestic animal reservoir of the disease.

TSETSE CONTROLAlthough tsetse control is reported to reduce thetransmission of sleeping sickness, it is rarely main-tained because of the costs of the methods applied.Currently, tsetse traps and targets are considered thecheapest, and an environmentally friendly option,applicable through community participation, butthey are still not widely used to control sleepingsickness. Pour-on insecticide for cattle is anothervector control option that may involve communityparticipation. However, use of pour-on insecticidesis dependent on the distribution and movement oflivestock.

MASS CHEMOTHERAPY OF LIVE-STOCK IN T. B. RHODESIENSE AREASResearch on the role of the livestock reservoir in theepidemiology of T. b. rhodesiense sleeping sicknessindicates that chemotherapy of livestock in endemicareas is an important policy for the control of sleep-ing sickness. There is molecular evidence for thesimilarity of the parasites of man and livestock(Enyaru et al, 1992; Hide and Tait, 1991; Hide et al,1994), suggesting that the parasite population circu-

lating in humans and livestock is similar. The cost-effectiveness and acceptability of the strategy tocontrol T. b. rhodesiense sleeping sickness by masschemotherapy of livestock should be investigated,taking into account other potential benefits to thefarmer.

In conclusion, the current problems in the treatmentof sleeping sickness in Africa include: melarsoproltreatment failure of T. b. gambiense sleeping sick-ness; unavailability of alternative drugs; unafford-able treatments; poor coverage by diagnostic andtreatment facilities. Studies to identify practicalsolutions to these problems are urgently required toreduce the current high fatality due to sleepingsickness, a disease that is regarded as always fatal ifnot treated.

SUGGESTED STUDIES• Research into new, safe, effective and cheap drug

combinations that are active for all stages of sleep-ing sickness. Suggested drug combinations tocompare are melarsoprol and eflornithine, melar-soprol and nifurtimox, nifurtimox and eflor-nithine.

• Continued screening of new compounds that areless toxic, easy to administer, cheaper and effec-tive for all stage of sleeping sickness.

• Research on cost-effective means to prevent out-breaks of sleeping sickness through methods suchas targeted vector control, and to avoid the highcosts of treatment, problems of drug manufacture,and complications associated with treatment fail-ure. Identification of markers for determining theprobability of occurrence of sleeping sickness out-breaks in space and time for use as a basis for dis-ease prevention.

• Active follow-up of cohorts of sleeping sicknesscases to accurately monitor drug efficacy, anddocument the results. Mapping of foci of melarso-prol treatment failure to study the possible riskfactors of its evolution.

• Research into other methods of control to reducethe pressure on human trypanocidal drugs, giventhe development of drug resistance. Evaluation ofthe cost-effectiveness of mass chemotherapy of live-stock in the control of T. b. rhodesiense epidemics.

• Operational research to optimize the geographicaldistribution of sleeping sickness diagnostic andtreatment facilities, taking into account factorssuch as current disease distribution, populationdistribution, availability of health infrastructure,


and the effects of distance on case detection andearly diagnosis.

• Diagnosis of suspect referrals, not positive by tra-ditional methods and requiring immediate atten-tion, by equipping research institutes and referralcentres with PCR technology. PCR can serve as agold standard for evaluating diagnostic tests.

• Pharmacokinetics studies of melarsoprol andsuramin in T. b. rhodesiense patients and of pen-tamidine in T. b. gambiense patients.

• Continued monitoring of the ten-day melarsoprolprotocol for treatment of sleeping sickness due toT. b. gambiense, making the results readily avail-able to all health policy-makers in affected coun-tries.

• Market research aimed at creating interest in theprivate sector for the manufacture of eflornithine.

• Review of the roles of national and sub-nationalcontrol programmes.

• Review of the parameters for, and duration of,post-treatment follow-up of sleeping sicknesspatients.

ReferencesBarrett MP. Problems for the chemotherapy of hu-man African trypanosomiasis. Current Opinion inInfectious Diseases, 2000, 13:547-651.

Burri C et al. Efficacy of new, concise schedule formelarsoprol in treatment of sleeping sickness cau-sed by Trypanosoma brucei gambiense: a randomisedtrial. The Lancet, 2000, 355:1419-1425.

Burri C, Blum J, Brun R. Alternative application ofmelarsoprol for treatment of T. b. gambiense sleepingsickness - Preliminary results. Annales de la SociétéBelge de Médecine Tropicale, 1995, 75: 65-71.

Doua F et al. Treatment of human late stage gambi-ense trypanosomiasis with alpha-difluoromethyl-ornithine (eflornithine): efficacy and tolerance in 14cases in Côte d’Ivoire. American Journal of TropicalMedicine and Hygiene, 1987, 37(3):525-533.

Doua F, Yapo FB. Human trypanosomiasis in IvoryCoast. Experimental Parasitology, 1993, 77:306-14.

Doua F et al. The efficacy of pentamidine in thetreatment of early-late stage Trypanosoma brucei gam-biense trypanosomiasis. American Journal of TropicalMedicine and Hygiene, 1996, 55:586-588.

Enyaru JCK et al. Characterisation by isoenzymeelectrophoresis of trypanozoon stocks from slee-ping sickness endemic areas of south east Uganda.Bulletin of the World Health Organization, 1992, 70:631-636.

Hide G, Tait A. The molecular epidemiology of par-asites. Experentia, 1991, 47:128-142.

Hide G et al. Epidemiological relationship of Try-panosoma brucei stocks from South East Uganda; evi-dence for different population structures in humanand non-human trypanosomes. Parasitology, 1994,109:95-111.

Iten M et al. Innate lack of susceptibility of UgandanTrypanosoma brucei rhodesiense to DL-alpha-difluo-romethyl ornithine (DFMO). Tropical Medicine andParasitology, 1995, 46:190-194.

Kyambadde JW et al. Detection of trypansomes insuspected sleeping sickness patients in Uganda usingthe polymerase chain reaction. Bulletin of the WorldHealth Organization, 2000, 78:119-124.

Legros D et al. Therapeutic failure of melarsoprolamong patients treated for late stage of T. b. gambi-ense human African trypanosomiasis in Uganda.Bulletin de la Société de Pathologie Exotique , 1999 (a),92:171-172.

Legros D et al. Risk factors for treatment failure ofmelarsoprol for Trypanosoma brucei gambiense try-panosomiasis in Uganda. Transactions of the RoyalSociety of Medicine and Hygiene, 1999 (b), 93:439-442.

Magnus E, Vervoort T, Van Meirvenne N. A cardagglutination test with stained trypanosomes(CATT) for the serological diagnosis of T. gambiensetrypanosomiasis. Annales de la Société Belge de Méde-cine Tropicale, 1978, 58:169-176.

Matovu E et al. Susceptibility of Ugandan Trypa-nosoma brucei rhodesiense isolated from man and ani-mal reservoirs to diminazene, isometamidium andmelarsoprol. Tropical Medicine and International He-alth, 1997, 2:13-18.

Nantulya VM. TrypTectCIATT® - a card indirectagglutination trypanosomiasis test for the diagnosisof Trypanosoma brucei gambiense and T. b. rhodesienseinfections. Transactions of the Royal Society of TropicalMedicine and Hygiene, 1997, 91:551-553.

Odiit M, Kansiime F, Enyaru JCK. Duration ofsymptoms and case fatality of sleeping sicknesscaused by Trypanosoma brucei rhodesiense in Tororo,


Uganda. East African Medical Journal, 1997, 74:792-795.

Ogada T. Clinical Mel B resistance in Rhodesiansleeping sickness. East African Medical Journal, 1974,51:56-59.

Okia M, Mbulamberi DB, De Muynk A. Risk-Factorsfor Trypanosoma brucei rhodesiense sleeping sicknessacquisition in SE Uganda- A case control study.Annales de la Société Belge de Médecine Tropicale, 1994,74:105-112.

Penchenier L et al. Diagnosis of human trypanoso-miasis, due to Trypanosoma brucei gambiense in cen-tral Africa, by the polymerase chain reaction.Transactions of the Royal Society of Tropical Medicineand Hygiene , 2000, 94:392-394.

Pepin J et al. High dose nifurtimox for arseno-resist-ant Trypanosoma brucei gambiense sleeping sickness.An open trial in central Zaire. Research in VeterinaryScience, 1992, 52:292-8.

Pepin J et al. Short-course eflornithine in Gambiantrypanosomiasis: a multicentre randomised con-trolled trial. Bulletin of the World Health Organiziation,2000, 78:1284-1295

Stephenson I, Wiselka M. Drug treatment of tropicalparasitic infections – Recent achievements. Drugs,2000, 60:985-995.

Van Niewenhove S. Advances in sleeping sicknesstherapy. Annales de la Société Belge de MédecineTropicale, 1992, 72: (Suppl. 1) 7-12.

TDR. Workplan for intervention research on Africantrypanosomiasis. No.58. p. 10, 1999.

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Annex 6PATHOGENESIS, GENOMICS AND APPLIED GENOMICS


I DISCUSSION DOCUMENT ONPATHOGENESIS / APPLIEDGENOMICS

John M. MansfieldDepartment of Bacteriology, University of Wisconsin-Madison, 1925 Willow Drive, FRI Building Madison, WI53706, USA

For the purposes of group discussion, I have arbi-trarily selected several research areas relevant tohuman African trypanosomiasis (HAT)-associatedpathogenesis; these areas are outlined below. Somebackground information on trypanosome immunol-ogy and cell biology is provided in the text that fol-lows (excerpted from recent reviews by Mansfieldand Olivier (2001), Mansfield et al. (2001), Paulnockand Coller (2001), and related resources (Imbodenet al, submitted for publication, 2001; Mansfield et al,in press; Paulnock et al, 2000). It is anticipated thatMelville and colleagues will provide more detailedand appropriate information on the trypanosomegenomics project and its practical applications.

DISCUSSION OUTLINE

1) Stage-specific immunobiological changes thatoccur during infection, with an emphasis on the fol-lowing subtopics: • Early pro-inflammatory events that trigger innate

immune system responses: - Glycosylphosphatidylinositol (GPI) substituents

of the variant surface glycoprotein (VSG) mole-cule shed into host tissues.

- Trypanosome lymphocyte triggering factor (TLTF)release from trypanosomes and induction of in-terferon-gamma (IFN-γ) production.

- Macrophage activation events associated withexposure to parasite activation molecules andhost activation factors.

• Early-stage acquired immune mechanisms associ-ated with parasite control in the vascular andextravascular tissue sites:- Distinct roles of Th1 cells and B cells in provid-

ing tissue specific resistance to trypanosomes.- Macrophage release of trypanocidal factors/

cytokines.

• Interrelationship of host innate and acquiredimmune responses to host pathology throughoutinfection:- Elucidation of immunological events that pro-

mote tissue specific pathology at all stages ofinfection.

• Late-stage anti-inflammatory and immunosup-pressive events that modulate the protectiveeffects of host T and B cell responses: - Elucidation of mechanisms that promote type 2

cytokine responses, and that suppress host tis-sue specific resistance.

- Role of these modulatory events in controllingor exacerbating tissue pathology.

2. Changes in parasite cell biology during infectionthat impact on host resistance, with an emphasis on:• Molecular basis of changes in trypanosome viru-

lence for the host - Relatedness to other clonal differentiation events.- Association with tissue specific residence/tro-

pism.- Identification and targeting of parasite genes

and molecules that are differentially expressedin highly virulent trypanosomes.

• Molecular basis for the resistance and susceptibil-ity of certain trypanosomes to the cytolytic effectsof human serum high density lipoproteins (HDL)and of tumour necrosis factor-alpha (TNFα).

3. Identification of stage-specific parasite moleculesthrough genomics and proteomics approaches thatmight be exploited, with an emphasis on:• Potential trypanosome molecules/cell biological

systems that could be targeted by highly specificchemotherapeutic agents:- BSF trypomastigote specific.- Tsetse fly stage specific.

• Highly conserved invariant antigens that could beused for either more accurate immunodiagnosisor for targeted immunotherapy.

BACKGROUND INFORMATION

The background information that follows is an opin-ionated view of trypanosomiasis, excerpted fromthis author’s writings and other resources, that ismeant to serve only as background information forgroup discussion on immunology and selectedaspects of parasite biology. As pointed out in thepassages below, there may be some disagreement asto the biological significance of certain observations.Additional background information on the geno-mics projects will be provided by others.

Stage-Specific Immunobiological Changesthat Occur During InfectionMacrophages and innate immunityCells of the macrophage lineage provide the first lineof host defense against infectious diseases, and also


modulate downstream events that impact on thedevelopment of acquired immunity. Macrophagesare present in all tissues and possess the ability torecognize and eliminate many microbes. It is wellestablished that recognition of microbes bymacrophages results in cellular activation followingthe uptake or binding of microbial components tospecific membrane receptors (Hoffman et al, Mosser,1992; Mosser and Karp, 1999). Receptor-mediatedactivation of macrophages represents one of the firstevents in the innate immune response to manymicrobial infections, leading to the production ofpro-inflammatory cytokines that initiate an inflam-matory response and affect the downstream devel-opment of activated T cells as well as other parame-ters of host immunity. Cytokines produced by acti-vated T cells, primarily IFN-γ, provide additionalactivation signals for macrophages, unleashing effec-tor functions that can destroy a wide range of intra-and extracellular microorganisms (Bendelac andFearon, 1997; Fearon and Locksley, 1996; Medzhitovet al, 1997). Thus, the processes of innate resistanceand acquired immunity are intimately interdepend-ent, with macrophages playing a dual role as the ini-tiators of acquired responses and as a major effectorcomponent of cell-mediated immunity.

Macrophage activation in trypanosomiasisMacrophage activation is one of the hallmarks ofinfection with the African trypanosomes (Askonas,1984; Askonas, 1985; Bancroft et al, 1983; Beschinet al, 1998; Borowy et al, 1990; Clayton et al, 1979;Darji et al, 1992; De Gee et al, 1985; Fierer andAskonas, 1982; Fierer et al, 1984; Grosskinsky andAskonas, 1981; Grosskinsky et al, 1983; Mayor-Wi-they et al, 1978; Murray and Morrison, 1979;Paulnock and Coller, 2001; Paulnock et al, 1989;Sacks et al, 1982; Schleifer and Mansfield, 1993,Sileghem et al, 1989; Wellhausen and Mansfield,1979). There is extensive evidence that the numbersand activity of macrophages increase dramaticallyin the tissues of trypanosome infected animals, andare associated with tissue pathology. Within the firsttwo weeks of experimental Trypanosoma brucei rhode-siense infection, for example, a large percentage ofcells in the enlarged spleen exhibit membrane andfunctional characteristics associated with activatedmacrophages. These include: increases in the releaseof interleukin-12 (IL-12) and IL-18, known to beimportant in the development of the early polarizedTh1 cell responses to trypanosome antigens(Mansfield, 1994; Mansfield et al, submitted for pub-lication; Schleifer et al, 1993; Schopf et al, 1998), anenhanced ability to serve as antigen processing cellscoupled to increases in expression of membrane I-Aα, B7-1 and B7-2 (Imboden, submitted for publica-

tion, 2001; Paulnock et al, 1989); and, upregulationof mRNA or proteins for other markers that includeTNFα, IL-1, IL-6, inducible nitric oxide synthase(iNOS), prostaglandins and IL-10 (Beschin et al,1998; Darji et al, 1992; Imboden et al, submitted forpublication, 2001; Magez et al, 1999; Mathias et al,1990; Schleifer and Mansfield, 1993; Sileghem et al,1989). More importantly, the expression or release ofseveral of these activation markers is associatedwith modulation of host immunity and resistance.For example nitric oxide (NO), prostaglandins andTNFα have been implicated in the suppressor cellactivity exhibited by macrophages at different timepoints of infection (Beschin et al, 1998; Borowy etal, 1990; Hertz and Manfield, 1999; Schleifer andMansfield, 1993; Sileghem et al, 1991; Sileghem etal, 1989; Sternberg and McGuigan, 1992; Sternbergand Mabbot, 1996); although NO and TNFα havebeen shown to kill trypanosomes in vitro and arethought to be important for trypanosome control inextravascular tissue sites (more below on this topic)(Lucas et al, 1994; Magez, et al 1997; Magez et al,1999; Mnaimneh et al, 1997; Vincendeau andDaulouede, 1991; Vincen-deau et al, 1992), neitherfactor alone has been linked definitively to protec-tion in vivo (Hertz and Mansfield; 1999; Magez et al,1999) and the expression of these factors may belinked to pathological changes during infection(Mabbott and Sternberg, 1995; Mabbott et al, 1994;Sternberg and Mabbott, 1996). However, the pro-inflammatory pattern of macrophage activationappears to change over the course of infection tobecome a counter-inflammatory pattern of activa-tion in which IL-10 predominates and Type 2cytokine responses appear to emerge (Namangalaet al, 2000; Namangala et al, 2000; Namangala et al,2001); these events have been associated with latestage disease (Imboden et al, 2001; Paulnock andColler, 2001; Sternberg, 2001).

Figure 1. Glycosylphosphatidylinositol (GPI) mem-brane anchor substituents of the trypanosome variantsurface glycoprotein (VSG) molecule. GPIs have beentermed “…one of the most potent microbial pro-inflam-matory agents known”(Almeida et al, 2000). The GPIanchor of the Trypanosoma brucei rhodesiense LouTat 1 VSG


molecule is depicted in this figure, showing glycosylinos-itolphosphate (GIP) substituents associated with sVSGafter cleavage from membrane-anchored GPI-mfVSG by atrypanosome membrane-associated phospholipase C(GPI-PLC), as well as dimyristoylglycerol (DMG) sub-stituents that remain associated with the trypanosomemembrane. Figure adapted from Menon (1999 and 1994),Magez (1998) and others (Varela-Nieto et al, 1996).

Role of the variant surface glycoproteinGPI anchor in macrophage activationThe source(s) and mode of action of activating fac-tors delivered to macrophages during trypanosomeinfection are only partially understood. One majoractivation factor is of parasite origin; this is the gly-cosylphosphatidylinositol (GPI) membrane anchorof the trypanosome VSG molecule (see Figure 1).The GPI anchor precursor is synthesized in theendoplasmic reticulum and subsequently is cova-lently attached to newly synthesized VSGs afterproteolytic cleavage of a VSG C-terminal GPIattachment signal sequence (Bangs et al, 1988;Cross, 1990; Doering et al, 1989; Doering et al, 1990;Englund, 1993; Ferguson et al, 1988; Mastersonet al, 1989; Menon, 1999; Menon, 1994, Menon et al,1997; Menon et al, 1990; Menon et al, 1990; Patnaiket al, 1993; Raper et al, 1993; Sharma et al, 1999;Vidugiriene and Menon, 1995; Werbovetz andEnglund, 1997). After further modifications in boththe glycoprotein and GPI anchor residue, themature VSG is transported to and anchored in thetrypanosome plasma membrane as membrane-formVSG (GPI-mfVSG). During the course of infection, atrypanosome membrane-associated phospholipaseC (GPI-PLC) becomes activated and cleaves the GPIanchor as shown in Figure 1; this results in therelease of substantial soluble VSG (GIP-sVSG) thatretains only the glycosylinositolphosphate (GIP)substituent of the original GPI anchor and leaves thedimyristoylglycerol (DMG) lipid componentremaining behind in the membrane (Armah andMensa-Wilmot, 2000; Bulow et al, 1989; Butikoferet al, 1996; Hereld et al, 1988; Hereld et al, 1986;Mensa-Wilmot and Englund, 1992; Mensa-Wilmot,1995; Paturiaux-Hanocq et al, 2000). Parasite num-bers routinely fluctuate during infection, both in theblood and extravascular tissues, primarily as theresult of host B and T cell responses to variant deter-minants of the VSG molecule. Since a) trypanosomenumbers may approach 108 organisms per ml bloodand may also be quite high in the extravascular tis-sues during peak parasitemias, b) there are approx-imately 107 VSG molecules per cell, c) GIP-sVSG isclipped and released from both viable andstressed/damaged trypanosomes, and d) try-panosomes are episodically destroyed by antibody-(Ab-) and Th1 cell/macrophage-dependent effector

mechanisms, the amounts of GPI substituents (GPI-mfVSG, GIP-sVSG and DMG) saturating host tis-sues during infection are quite substantial.Conservative calculations estimate that experimen-tally infected animals may be exposed to 15-20 µMVSG with each wave of parasitemia, which is aninordinate amount of parasite material with intrin-sic macrophage activation potential to be releasedinto host tissues.

Limited in vitro studies by several labs have begunto characterize the activating effects of GPI sub-stituents on macrophages. It appears that GIP-sVSGand GPI-mfVSG (containing the DMG lipid sub-stituent) have similar macrophage activating capa-bilities in terms of TNFα, IL-6 and NO production,but that there may be subtle differences in the abili-ty of GPI-mfVSG to more effectively induce IL-1 andIL-12 production (Magez et al, 1998; Schofield et al,1996; Schofield and Tachado, 1996; Tachado et al,1996; Tachado and Schofield, 1994). An extension ofthese initial studies regarding the ability of GIP-sVSG to interact with macrophages demonstratesthat the GIP-sVSG component binds directly tomacrophages and induces expression of a specificsubset of activation genes in an IFN-γ independentmanner (Imboden et al, submitted for publication2001; Paulnock and Coller, 2001). Studies have alsoshown that GPI substituents exhibit signaling activ-ities (Tachado et al, 1997). That specific signal(s) aredelivered to the cell is apparent from the resultantactivation phenotype of macrophages exposed toGPIs, and by recent in vitro studies showing thatGIP substituents (specifically the core glycansequence) activate a specific protein tyrosine kinase,while the DMG substituent may independently acti-vate a protein kinase C isoform in macrophages(Tachado et al, 1997). However, it is not yet knownhow GPIs interact with the macrophage membranenor what receptor(s) may be important in deliveringGPI-mediated activation signals to the cell nucleus.

During trypanosome infection, however, it is clearthat host cells are exposed to biologically active lev-els of GIP-sVSG, DMG and GPI-mfVSG, althoughthe timing of exposure to these molecules and thenature of their delivery to the macrophage mem-brane could be very different (see Figure 2). Whilethe effects of such GPI substituents on uninfectedmacrophages have been partially characterizedin vitro, the impact of tissue saturation with theseactivating agents in vivo during infection is justbeing fully appreciated (Imboden et al, submittedfor publication 2001; Paulnock and Coller 2001).Also, macrophages are not the only cell type thatcan be targeted by GPIs; recent evidence demon-strates that natural killer (NK) 1.1 T cells may


express T cell receptors (TCR) specific for GPI deter-minants and that B cells may be stimulated to under-go cell differentiation by GPIs (Bento et al, 1996; Scho-field, et al, 1999). Thus, GPI substituents released bypathogens such as the African trypanosomes mayhave broad effects on the host immune system thatsurpass central activating effects on macrophages.

Role of IFN-γ in macrophage activationand early host protection and/or pathologyThe other major macrophage activation factor pro-duced during trypanosomiasis is IFN-γ, which is ofhost origin. Small amounts of IFN-γ may be pro-duced very early during infection as the result ofTLTF activation of CD8 T cells (Bakhiet et al, 1996;Bakhiet et al, 1993; Schleifer and Mansfield, submit-ted for publication 2001; Vaidya et al, 1997). Firstdescribed by Bakhiet, Olsson and colleagues(Bakhiet et al, 1993; Bakhiet et al, 1996; Bakhiet et al,1990; Olsson et al, 1991; Olsson et al, 1993; Olssonet al, 1992) and subsequently cloned by the Do-nelson laboratory (Vaidya et al, 1997), TLTF is a 453amino acid protein with potentially important bio-logical effects. TLTF was discovered when resear-chers noted that rodents injected with T. b. brucei, orlymphoid cells cultured with trypanosomes in vitro,exhibited an increase in the number of antigen non-specific IFN-γ secreting cells; depletion of CD8+ Tcells in animals or cultures abrogated the effect and,interestingly, also resulted in less trypanosomegrowth (Bakhiet et al, 1990). Use of a chamber sys-tem separating lymphoid cells and trypanosomesshowed that a soluble factor was responsible forinduction of IFN-γ synthesis (Olsson et al, 1991).

Several Trypanosoma species appear to express TLTFbut may possess different IFN-γ stimulating abilitiesas measured by the relative increase of IFN-γ pro-ducing cell numbers in the presence of extracts orculture filtrates of species including T. evansi, T. b. rho-desiense, and T. b. gambiense (Bakhiet et al, 1996). Subse-quent characterization of CD8+ T cell IFN-γ activa-tion by TLTF showed that tyrosine protein kinasesare necessary for activation but protein kinase C andprotein kinase A specifically are not (Bakhiet et al,1993). Interestingly, TLTF may stimulate other cellsto release IFN-γ, such as rat dorsal root ganglia, andthis secretion apparently also is dependent on tyro-sine kinase(s) (Eltayeb et al, 2000). These types ofstudies and their experimental extension over thepast decade have led investigators to posit the fol-lowing hypotheses with respect to the role of TLTF:trypanosomes secrete TLTF which binds to CD8molecules expressed on CD8+ T cells, thereby in-ducing antigen non-specific activation and produc-tion of IFN-γ; TLTF-induced release of IFN-γ subse-quently serves as a growth factor that promotes try-

panosome growth (Bakhiet et al, 1996; Bakhiet etal, 1990; Hamadien et al, 1999; Olsson et al, 1991;Olsson et al, 1993; Vaidya et al, 1997). Thus a factorsecreted from the parasite, TLTF, is visualized asinducing an essential trypanosome growth factor,IFN-γ, from host cells.

Experiments partially in conflict with these pro-posed hypotheses exist, however. First there hasbeen no independent confirmation that IFN-γ servesas a growth factor for African trypanosomes;unpublished studies from several labs have failed tosubstantiate the claim that IFN-γ serves as a growthfactor and no critical biochemical studies of IFN-γbinding to or utilization by trypanosomes have yetbeen published. Furthermore, parasitemias arehigher in IFN-γ knockout mice, which are highlysusceptible rather than more resistant to infectionwith T. b. rhodesiense (see discussion below on therole of IFN-γ in early host resistance [Hertz et al,1998]). One might reasonably expect that parasi-temias would be lower in IFN-γ deprived animals ifthis cytokine served in any substantial manner as agrowth factor. Additionally, TLTF expression isidentical in trypanosomes expressing high and lowvirulence attributes (Mansfield, 2001), suggestingthat there is no modulation of the gene or protein inorganisms known to exhibit rapid growth character-istics and virulence attributes for mammalian hosts.

Initial studies asserted that TLTF was a secreted pro-tein (Vaidya et al, 1997), but subsequent characteri-zations showed that the amino acid sequence doesnot contain a hydrophobic region typical of mem-brane-transported trypanosome proteins, though itdid appear to be targeted to the flagellar pocketregion through an apparent conformational signalwithin a specific 144 amino acid domain (Hill et al,1999). To date there is no clear evidence that TLTF isa secreted protein, though the predicted proteindoes possess unique internal targeting sequences.More recent studies on the cell biology of TLTF havesuggested an alternate (or coincident) role for theprotein. The gene sequence identified as TLTF isexpressed in both insect and bloodstream forms ofT. b. brucei and the protein appears to be tightly asso-ciated with the flagellar cytoskeleton (present indetergent-resistant and Ca2++-resistant cytoskeletalfractions of trypanosome extracts) (Hill et al, 2000);modification of TLTF gene expression in the pro-cyclic form resulted in an unusual motility defect,suggesting that TLTF may be an integral part of thetrypanosome cytoskeletal architecture. Surprisingly,TLTF-like genes are present in a number of diver-gent eukaryotes including Drosophila and zebra fish.Notably, the human growth arrest specific gene(GAS)11, closely related to TLTF (Vaidya et al, 1997),


is a possible tumor suppressor molecule with a sub-molecular region that may localize to cellular micro-tubules.

Thus, it is difficult to see how TLTF, a tightly boundcytoskeleton-associated molecule, would be secret-ed or released in biologically active levels duringinfection or in cell cultures containing viable try-panosomes to affect the release of IFN-γ from hostcells. Yet, it is clear that trypanosome infections andtrypanosome extracts are capable of inducing IFN-γrelease from naive host lymphoid cells in an antigennonspecific manner; the levels are low and occurindependently of antigen specific induction of IFN-γ from host Th1 cells (Mansfield, 2001; Schopf et al,1998). Thus, IFN-γ secretion induced by parasitematerial(s) has been a repeatable phenomenon andis clearly of some interest; there is the distinct possi-bility that release of biologically active TLTF (or asimilar molecule with closely related effects) occursduring periods of cataclysmic elimination of try-panosome variant antigen types (VATs) by host Aband Th1/macrophage cell responses throughoutinfection (see below), rather than by an active secre-tory pathway, to induce IFN-γ. Given that try-panosomes / trypanosome extracts are capable ofinducing nonspecific IFN-γ release from host cells,and that IFN-γ may be an early and critical factor inhost protection (probably important in regulatingparasite numbers in the extravascular tissues—seeFigures 2 and 3), it may be that TLTF is important ina different context than originally suggested: byinducing low levels of IFN-γ that control an earlypotentially explosive spread of trypanosomes ininfected host tissues, regardless of the genetically-based resistance status of the host. Such an earlycontrol over parasite expansion and, in susceptibleanimals or individuals, a delay in host death, wouldpermit the host to survive for a period of time sothat the possibility of trypanosomes being taken upin a blood-meal and transmitted to new hosts couldoccur. Regardless of one’s view of TLTF, the hardwork of providing functional and genetic linkagesbetween TLTF expression and biological effects onthe host remain.

Clearly, however, the major source of IFN-γ duringinfection appears to be parasite antigen stimulatedTh1 cells, which appear in significant numberssomewhat later than early TLTF induced IFN-γresponses. Th1 cell responses to VSG (and to invari-ant antigens of the parasite) have now been charac-terized and result in strong IFN-γ responses ingenetically more resistant animals but not in sus-ceptible animals (Hertz et al, 1998; Hertz andMansfield, 1999; Schleifer et al, 1993; Schopf et al,1998). The IFN-γ response has been definitively

linked to early host resistance during T. b. rhodesienseinfection, and is thought to be associated withmacrophage activation characteristics responsiblefor control of the parasites in extravascular tissuesites (De Gee et al, 1985; Hertz et al, 1998; Imbodenet al, submitted for publication, 2001; Paulnock andColler, 2001).

Figure 2. Macrophage activation in African try-panosomiasis is mediated by exposure to host andparasite factors.

(Figure adapted from Paulnock and Coller, 2001)

The theoretical timing of exposure to host- and parasite-derived macrophage activating factors during an earlypeak of parasitemia in trypanosome infection is shownhere. GIP-sVSG homodimers, released from the try-panosome plasma membrane by the action of GPI-PLC,are detectable within several days of infection throughoutthe body (Diffley and Jayawardena, 1982; Diffley andStraus, 1986; Diffley et al, 1980; Norton et al, 1986).Although DMG is liberated by the same GPI-PLC cleav-age event as GIP-sVSG, host tissues may be exposed ear-lier to GIP-sVSG since effective membrane transfer ofDMG would largely be dependent on high concentrationsof parasites in contact with macrophage membranes. GIP-sVSG can activate macrophages independently of residualDMG (Imboden et al, submitted for publication 2001;Magez et al, 1998; Paulnock and Coller, 2001; Schofieldand Tachado, 1996; Tachado et al, 1997). During immuneclearance of trypanosomes, macrophages take up para-sites and presumably are also exposed to intact GPI-mfVSG as well as DMG remaining from GPI-PLC cleav-age of the anchor. Low levels of IFN-γ may also serve asan early activating agent during infection, presumablyderived from TLTF-stimulated CD8 T cells. Cytokine lev-els subsequently rise substantially over time due to CD4Th1 cell stimulation by VSG and other parasite antigens.The patterns shown here may differ markedly later ininfection.

Trypanosomes

0 1 2 3 4 5 6 7 8 9 10

DAY OF INFECTION

RE

LA

TIV

E L

EV

EL

S

DMG

GIP-sVSG

IFN-γ


The IFN-γ/IFN-γ receptor interaction and down-stream subcellular signaling pathways have alreadybeen well characterized in other model systems, andmacrophage activation events triggered by IFN-γhave been extensively studied (Adams andHamilton, 1984; Adams and Hamilton, 1987; Adamsand Hamilton, 1986; Hamilton, 1989; Paulnock,1992; Paulnock, 1994; Paulnock-King et al, 1985). InAfrican trypanosomiasis, it is clear that somemacrophage activation events are dependent onIFN-γ exposure and others on exposure to try-panosome-derived molecules, including GIP-sVSG(Imboden et al, submitted for publication 2001;Paulnock and Coller, 2001). The activation patternsobserved in the presence of both factors are differentfrom either one alone, are dependent on the geneticbackground of the infected host, and may be impor-tant in the control of infection. Why trypanosomesinduce such broad macrophage activation effects isunknown. It may be important for trypanosomes toinduce early temporal protection against the infec-tion regardless of the genetically-based resistancestatus of the host (e.g. so the host isn’t killed byinfection before natural transmission of the diseasecan effectively occur). Alternatively, it might belinked to the early generation of suppressormacrophage activity so as to depress host T cellresponses to parasite antigens. Or it may result inderegulation of IFN-γ-induced activation events inmacrophages in order to avoid parasite elimination.Clearly, a goal of unraveling the cellular and molec-ular basis of macrophage activation by try-panosome-derived antigens or other factors (e.g.DNA released from dead trypanosomes [Shodaet al, 2001]) in the context of overall host resistanceto infection and tissue pathology is important.

Tissue specific immune control mechanisms in early infectionThere are clear differences in the ability of varioushost species, and strains within species, to displayrelative resistance to African trypanosomiasis(Levine and Mansfield, 1981; Mulligan, 1970).Studies over the past twenty years have revealedthat the host Ab response plays only a partial role insuch relative resistance against trypanosomes.While VSG-specific Ab clearly is responsible for thecataclysmic elimination of VATs from the blood-stream of infected hosts, it is now known that thisevent is not linked, functionally or genetically, tooverall host resistance (De Gee et al, 1988; De Geeand Mansfield, 1984; Mansfield, 1995; Mansfield,1990; Mansfield and Olivier, 2001). The seminalstudies were those in which H-2 compatible radia-tion chimera mice, reconstituted with reciprocalbone marrow cell transplants from relatively resist-ant or susceptible donors, revealed the following:

that susceptible mice, which normally do not makea sufficient Ab response to VSG and do not clearVATs from the blood, were afforded by donor cellsfrom resistant mice a functional B cell response thatenabled them to clear parasitemia during infection;however, despite the ability to eliminate try-panosomes from the blood, these animals were justas susceptible as mice receiving susceptible donorbone marrow cells that failed to make protectiveVSG-specific B cell responses (De Gee andMansfield, 1984). Subsequent genetic studies withcrosses between Ab+ resistant and Ab- susceptiblemouse strains showed that the F1 hybrids all wereable to make VAT-specific Ab responses and controlparasitemias, but all such hybrids were as suscepti-ble as the susceptible parental strain (De Gee et al,1988). Taken together, these types of results showedthat the VSG-specific B cell response, althoughlinked to trypanosome clearance from the blood,was not by itself functionally or genetically linked tooverall host resistance.

Th cell responses to trypanosome antigensThis information led the way to studies that firstelucidated Th cell responses to VSG and other try-panosome antigens during infection (Hertz et al,1998; Hertz and Mansfield, 1999; Mansfield, 1994;Schleifer et al, 1993; Schopf et al, 1998). T cellresponses to trypanosome antigens were not discov-ered previously because of several interesting char-acteristics of trypanosome infections. First, a non-specific immunosuppression of T cell responses intrypanosomiasis had been recognized for manyyears (Mansfield and Wallace, 1974), and, althoughearlier studies revealed that T cell responses to try-panosome antigens could be induced in immunizedanimals (Campbell et al, 1982; Finerty et al, 1978),such responses were not readily detectable in infect-ed animals (Mansfield and Kreier, 1972; Paulnocket al, 1989). For example, not only were spleen orlymph node T cells from infected mice unable toproliferate in response to mitogens or antigen, theyalso failed to produce significant amounts of IL-2 orIL-4, and these events could be shown to impact onT-dependent B cell responses to a variety of antigens(Mansfield and Bagasra, 1978). This generalizedimmunodeficiency was shown to result in part fromthe presence of macrophage “suppressor cells” inlymphoid tissues (Sacks et al, 1982; Wellhausen andMansfield, 1980; Wellhausen and Mansfield, 1979;Wellhausen and Mansfield, 1980); in fact,macrophages from infected mice had the capacity toactively suppress the proliferative responses of nor-mal T cells to mitogens and antigens in vitro and invivo. A breakthrough in recognizing that Th cellresponses to trypanosome antigens occurred duringinfection came with the finding that functional com-


partmentalization of such responses occurred(Schleifer et al, 1993). It was revealed that Th cellsreactive with VSG were predominant in the peri-toneal T cell population; when stimulated withVSG, these cells made a substantial IL-2 and IFN-γcytokine response but failed to proliferate. Subse-quently, it was discovered that Th cells in theperipheral lymphoid tissues also made an IFN-γresponse (but little IL-2) when stimulated with VSG.Thus, it was apparent that VSG-reactive T cells werepresent in infected animal tissues but that theyexhibited a restricted cytokine response and mini-mal evidence for clonal expansion (Schleifer et al,1993). Since these VSG-reactive T cells displayed aCD4+ αβ TCR+ membrane phenotype, expressedType 1 cytokines, were major histocompatibilityclass II antigen (MHC II) restricted and antigen pre-senting cell (APC) dependent (Hertz et al, 1998;Schleifer et al, 1993; Schopf et al, 1998), it was clearthat they represented a classical Th1 subset of T cellsthat recognized VSG during infection. More recentwork has begun to elucidate the submolecular tar-gets of VSG-reactive Th cells. In preliminary studiesit has been shown that Th cell specificities are direct-ed against a defined hypervariable subregion ofVSGs that is not exposed when VSG homodimersare assembled into the surface coat structure(Mansfield, 2001; Mansfield and Olivier, 2001), ful-filling earlier predictions that VSG sequence vari-ability in nonexposed regions of the molecule mightbe driven by T cell selection (Blum et al, 1993; Fieldand Boothroyd, 1996; Reinitz et al, 1992).

The extreme polarization of the Th1 cell cytokineresponses seen in some experimental systems is duein part to the early production of IL-12 bymacrophages exposed to trypanosome GPI sub-stituents (Mansfield et al, submitted for publica-tion). That IL-12 is not the only polarizing factor isseen from preliminary studies with IL-12 knockout(KO) mice and mice exposed to Abs against IL-12; ineach case, early temporal depression of the Type 1cytokine response did not result in a compensatoryType 2 cytokine response and, after a period of 10days or so, the Th1 cell response emerged in bothgroups (Mansfield et al, submitted for publication). That IL-12 is not the only polarizing factor is seenfrom preliminary studies with IL-12 knockout (KO)mice and mice exposed to Abs against IL-12; in eachcase, early temporal depression of the Type 1cytokine response did not result in a compensatoryType 2 cytokine response and, after a period of 10days or so, the Th1 cell response emerged in bothgroups (Mansfield et al, submitted for publication).Thus, there are complex features of infection thatpromote the production of Type 1 cytokines and theoutgrowth of antigen-reactive Th1 cells. While rea-

sons for the relative tissue compartmentalization ofTh cell cytokine responses (e.g., IL-2 and IFN-γ pro-duction by peritoneal Th cells, but mostly IFN-γpro-duction by Th cells in the peripheral lymphoid tis-sues) have not been elucidated, the reason for inhi-bition of T cell clonal expansion has now beenresolved. Suppressor macrophages were shown toelaborate several factors that inhibited the prolifera-tive (but not the cytokine) responses of VSG activat-ed Th1 cells: NO, prostaglandins and TNFα (Darji etal, 1996; Hertz and Mansfield, 1999; Schleifer andMansfield, 1993; Sternberg and McGuigan, 1992).Macrophages were activated to produce these sup-pressive factors primarily as the result of exposureto GIP-sVSG and to IFN-γ released by parasite anti-gen stimulated Th cells (Hertz and Mansfield, 1999;Mansfield et al, submitted for publication; Schleiferand Mansfield, 1993). The full impact of NO andprostaglandins on host immunity to trypanosomeshas not been completely resolved, but studies withiNOS KO mice have shown that, although NO is themain “suppressor” factor that limits clonal expan-sion of T cells (and maybe also modulates cytokineresponses to a degree) the absence of NO did notaffect overall host resistance (Hertz and Mansfield,1999).

Early and strong trypanosome-specific Th1 cellresponses may provide an essential component ofhost resistance; this realization emerged from stud-ies with cytokine gene knockout mice. The centralfinding in one study was that mice with a resistantgenetic background but which lacked a functionalIFN-γ gene were as susceptible as scid mice to try-panosome infection, even though those mice pro-duced Abs sufficient to control parasitemia (Hertz etal, 1998). In contrast, the same genetic strain ofmouse with the IL-4 instead of the IFN-γ geneknocked out were as resistant as wt mice to infection(Hertz and Mansfield, 1999). These results under-scored earlier studies demonstrating that the VAT-specific Ab response and control of parasitemiawere not capable of providing resistance alone, andthat the production of a single cytokine, IFN-γ, inresponse to infection was found to be a critical ele-ment in host resistance. The mechanism(s) associat-ed with IFN-γ-mediated resistance are not yet clear,but seem to involve macrophage factors induced byIFN-γ activation. Several candidate factors havebeen proposed, such as NO and TNFα, both ofwhich have been shown to kill trypanosomes in vitro(Lucas et al, 1994; Lucas et al, 1993; Magez et al,1997; Magez et al, 1999; Mnaimneh et al, 1997;Vincendeau et al, 1992). Recent studies suggest,however, that neither factor alone is capable ofmediating resistance in vivo; results with try-panosome infected iNOS KO mice and TNFα KO


mice showed that such mutations on a resistantmouse genetic background do not significantlyaffect the course of infection (Hertz and Mansfield,1999; Magez et al, 1999; Millar et al, 1999), althoughit is possible that the combination of NO and TNFαis required for functional resistance. Clearly, IFN-γinducible events in macrophages must carefully beevaluated for their impact on trypanosomes duringearly stages of infection. Since these events occurindependently of B cell mediated resistance mecha-nisms that are known to control trypanosomes inthe vasculature, and since IFN-γ activatedmacrophage control mechanisms are presumed tobe important in regulating trypanosome numbers inthe extravascular tissue spaces (but this by itself isinadequate to provide protection [Mansfield, 2001]),it appears that multiple arms of the host immunesystem are required to control trypanosomes and toprovide relative resistance during infection.

Figures 3A and 3B. Innate and acquired immune elements in early- versus late-stage trypanosomiasis.

These figures portray the cell and molecular elementsimportant in controlling trypanosome infection of host tis-sues. In early infection stages, IFN-g cytokine responsesactivate macrophages to produce factors cytotoxic for try-panosomes, limiting the spread or survival of parasitesoutside the vasculature. B cell responses appear to selec-tively control trypanosomes within the vasculature. Inlate-stage infections, there appears to be a shift from the

phenotype of pro-inflammatory responses and parasitici-dal macrophage activation to a phenotype of counter-inflammatory responses and Type 2 cytokine responsesthat do not promote parasite elimination from tissues andperhaps also from the blood. Adapted from Mansfield andOlivier (2001).

Thus, relative resistance to African trypanosomesmay be mediated by two major components of hostimmunity, neither one of which by itself is adequateto provide resistance (Figure 3A). First, VSG specif-ic Ab responses control trypanosomes present in theblood. Second, T cell production of IFN-γ and sub-sequent macrophage activation events are necessaryto control trypanosomes in the extravascular tissues.Animals that make weak B cell and/or T cellresponses to trypanosome variant antigens invari-ably will demonstrate relative susceptibility; in con-trast, animals making pronounced B and T cellresponses (including appropriate macrophage acti-vation events) will display relative high resistance.However, this picture is evolving considerably dueto evidence that the later stages of experimentalinfection display a different pattern, a pattern that isassociated with loss of resistance to trypanosomes(see Figure 3B).

OverviewEvents that impact on B, T or macrophage cellresponses during infection can be expected to causemodulations in host resistance and tissue pathology.These events need to be clarified in both animalmodel systems and in clinical HAT.

Changes in Parasite Cell Biology duringInfection that Impact on Host ResistanceThe African trypanosomes display considerable bio-logical variation during their life cycle. This biolog-ical variation is directed by specific patterns of geneand protein expression. For example, bloodstreamtrypomastigote forms display a number of differentsurface antigenic phenotypes during infection oftheir mammalian hosts; this phenotypic variationhas as its basis the differential expression of VSGgenes and molecules (Borst et al, 1998; Borst andRudenko, 1994; Cross, 1990; Donelson, 1987;Van der Ploeg et al, 1992; Vickerman and Luckins,1969). Additionally, the differentiation of long slen-der (LS) trypomastigote forms to short stumpy (SS)trypomastigotes during infection results in pro-found morphological and functional changes inthese cells. Such changes include mitochondrial bio-genesis and the acquisition of new metabolic path-ways; it is the differential expression of specificgenes and proteins that presage these biologicalchanges (Bienen et al, 1983; Bohringer and Hecker,1975; Hua et al, 1997; Mulligan, 1970; Mutomba and


Wang, 1998; Tschudi, 1995; Vanhamme and Pays,1995; Vickerman, 1971; Vickerman et al, 1993).Furthermore, the differentiation of SS forms to pro-cyclic forms in the insect vector also results from thedifferential expression of specific genes and proteins(Butikofer et al, 1997; Roditi, 1996; Ruepp et al, 1997;Vanhamme and Pays, 1995; Vickerman et al, 1988).Finally, the transformation of insect forms to meta-cyclic trypomastigotes results in different morpho-logical and functional changes in the parasites thatpermit infection of a new mammalian host; thesechanges also occur as the result of differential geneand protein expression (Turner et al, 1986;Vanhamme and Pays, 1995; Vickerman, 1985;Vickerman et al, 1993). Thus, all trypanosomesexhibit considerable clonal plasticity in terms oftheir antigenicity, morphology and biological func-tion during the life cycle; this plasticity occurs as thedirect result of differential expression of specificsubsets of genes and proteins. It follows that the fail-ure of trypanosomes to undergo biological variationat critical points in the life cycle may result in elimi-nation by the mammalian host, failure to differenti-ate within the intermediate host and vector, orinability to establish new animal infections.

The molecular mechanisms that regulate changes inVSG phenotype and stage of cellular differentiationare shared in common among African trypano-somes; however these are not the only mechanismsthat regulate biological differences in these para-sites. New evidence is emerging that differentialsusceptibility of brucei group trypanosomes to hostfactors such as trypanosome lytic factor (TLF)(De Greef and Hamers, 1994; Hager et al, 1994;Hajduk et al, 1992; Rickman and Robson, 1970;Rifkin, 1978; Smith et al, 1995); may occur as theresult of clonal variation among trypanosomes(Hager and Hajduk, 1997; Hajduk et al, 1995); thebasis for such changes has not yet been defined, butis believed to encompass specific clonal modifica-tions in gene or protein expression. Host specificitymay also be defined by the clonal expression of dif-ferent transferrin receptor genes (Bitter et al, 1998).While these examples reflect on trypanosome infec-tivity for a host species, related observations andmathematical modeling predict that considerablebiological variation occurs among trypanosomesduring infection that is not related directly to infec-tivity or cyclical differentiation in the life cycle(Turner et al, 1995; Vassella et al, 1997). For example,it is well known that different isolates, species andsubspecies of trypanosomes exhibit remarkablevariation in pathogenicity and virulence for geneti-cally defined host species (Mulligan, 1970). A keyquestion has been whether such differences in viru-lence are immutable characteristics associated with

genetically distinct populations of trypanosomes, orwhether there is intraclonal biological variationwithin trypanosome populations that impacts onthe course of disease in a genetically defined host.

This question was addressed in an earlier study inwhich mice of a relatively resistant phenotype wereinfected with a single trypanosome of T. b. rhode-siense clone LouTat 1 (Inverso and Mansfield, 1983).Several different VATs were isolated from para-sitemic peaks at intermediate and late time pointsduring infection of a single mouse; these VATs weresubcloned and characterized as to VSG phenotype.Three different VATs, which represented antigeni-cally distinct daughter cell populations clonallyderived from a single LouTat 1 parental cell, wereused to infect the same mouse strain; the courses ofinfection were monitored in comparison with miceinfected with LouTat 1. The interesting result wasthat each of the daughter cell populations exhibiteda different virulence profile compared to theparental clone (Inverso and Mansfield, 1983). Forexample, LouTat 1 caused death in approximately62 days post-infection, while LouTat 1.3, LouTat 1.4and LouTat 1.5 caused death in approximately 44,30, and 28 days, respectively. These results demon-strated that VATs arising during infection expressedvirulence phenotypes different from the infectingVAT. In essence, daughter cells arising within a try-panosome population expressed the capacity totranscend host genetic resistance characteristics andrender a relatively resistant animal into a more sus-ceptible one.

These seminal observations have been repeated withconsistent results, and related observations on clonalheterogeneity among trypanosomes have been madein other studies (Diffley and Mama, 1989; Inversoet al, 1988; Joshua, 1990; Mamman et al, 1995; Ortizet al, 1994; Reinitz and Mansfield, 1988; Sacks et al,1980; Seed, 1978; Seed and Sechelski, 1996; Turner,1990; Turner et al, 1995). Additionally, the generalobservation has been made that many other daugh-ter cells/VATs arising naturally from LouTat 1 alsoexpressed differences in virulence (see Table 1,below), and that the most virulent VATs seemed toarise at later time points in infection, just prior tohost death. Thus, the apparent result of infectionwith relatively low virulence trypanosomes is a pro-gressive increase in population virulence with time,rather like the turning up of a “virulence rheostat”.Based on these observations, it was speculated thatthe longer a trypanosome population existed in amammalian host, the more virulent that populationmight become. Such a virulence rheostat may haveevolved as a programmed mechanism to overcomedifferent levels of host resistance that might be


encountered in nature, where there is a pool ofgenetically disparate mammalian hosts available forinfection. Implicit in this speculation, however, is theidea that a virulence rheostat must somehow bereset, perhaps upon cyclical passage through theintermediate host and vector, the tsetse fly.

Since virulent trypanosomes seem to arise after aprolonged period of replication in an infected host,it may be possible to generate highly virulent try-panosomes by rapid subpassage of low virulencetrypanosomes through mice for a substantial timeperiod; in essence, the effect would be one mimick-ing a single, prolonged course of infection. This wasachieved by infecting irradiated mice with LouTat 1and subpassaging the trypanosomes into differentmice every three days for approximately six months.At the end of this time, trypanosome stabilates weremade from sublines and subclones, and wereassessed for their virulence characteristics. One rep-resentative subclone, designated LouTat 1A, wasexamined in some detail. These trypanosomes dis-played a single uncontrolled peak of parasitemiaand were able to kill a resistant mouse strain (as wellas all other resistant or susceptible strains of mice) inapproximately four days post-infection; in contrastthe parental clone LouTat 1 gave rise to multiplepeaks of parasitemia and a prolonged survival timeof over 60 days in the same mouse strain (Inversoet al, 1988). Thus a model system of comparativetrypanosome virulence was developed from thisapproach, in which the relatively low virulenceclone LouTat 1 and the relatively high virulence sub-clone LouTat 1A represent different ends of a viru-lence spectrum, with the virulence of other natural-ly arising VATs existing somewhere between thesetwo extremes (Table 1).

A natural question that arose from these types ofstudies was whether the VSG molecules expressedby virulent VATs acted as virulence factors, with spe-cific VSG isotypes exerting defined biological effectson the host. This idea was not unfounded since sev-eral biological traits associated with VSG moleculeshave been described in the literature (Mathias et al,1990; Musoke and Barbet; 1977; Schofield et al, 1999;Tachado et al, 1997; Tizard et al, 1978). Alternatively,one could also speculate that expression site-associ-ated genes (ESAGs) co-transcribed with certain VSGgenes at specific chromosomal expression sites maybe responsible for virulence expression or regulation.This idea was based on observations of others con-cerning potential growth or differentiation regulato-ry roles associated with ESAGs (Cross, 1990;Vickerman et al, 1993). However, an analysis of theLouTat 1/LouTat 1A model system revealed thatboth organisms displayed the same antigenic surface

coat structure (Inverso et al, 1988), transcribed iden-tical VSG genes (Reinitz et al, 1992; Uphoff et al, sub-mitted 2001) and expressed their VSG genes by aduplicative transposition event from the same chro-mosomal telomeric expression site (Uphoff et al, sub-mitted 2001).

Table 1. Virulence phenotypes of LouTat 1-derived VATs.

Therefore, it is unlikely that either VSG molecules orthe active VSG gene expression site are importantfor virulence regulation in trypanosomes (seeFigures 4-6). Confirmation that VSG genes and VSGgene expression sites are not involved in virulencecame from additional experimental approaches.New sublines and subclones were derived by rapidsubpassage of LouTat 1 through irradiated mice, asabove, and examined for VSG phenotype and geneexpression. Many of the subclones expressed thesame VSG gene as LouTat 1 and all were highly vir-ulent like LouTat 1A (Table 1). In another approach,LouTat 1A was used to infect rabbits and goats;although these animals exhibited pathology soonerthan LouTat 1 infected controls, they did not die asearly as mice and trypanosomes were able to under-go antigenic variation. The VATs generated fromLouTat 1A in these animals were isolated and sub-sequently used to infect mice; the results showedthat the LouTat 1A-derived VATs were as virulentfor mice as LouTat 1A (Inverso et al, 1988). Thushigh levels of virulence, once expressed in mam-malian hosts, appear to be a constitutive trait that isunaffected by further VSG gene switching.

Variant Antigenic Types Virulence (Mean Survival Time)

(A) VATs isolated from natural infections*

LouTat 1 (parental clone) 62LouTat 1.9 46LouTat 1.3 44LouTat 1 4 30LouTat 1.5 28LouTat 1.7 25LouTat 1.2 13LouTat 1.6 11LouTat 1.10 6

(B) VATs derived by subpassage**

LouTat 1A 4LouTat 1D 4LouTat 1X 4LouTat 1n 6-60


Figure 4. Amino acid sequence of the T. b. rhodesiense LouTat 1/ LouTat 1AVSG molecule. The protein sequence was deduced from the iden-tical full-length nucleotide sequences (Accession no. X56643)(Reinitz et al, 1992) as well as from partial amino acid sequencingdata (Inverso et al, 1988). The N–terminal signal sequence and theC-terminal hydrophobic extension are underlined; threonine-27is the start site of the mature protein.

Figure 5. Southern blot analysis of LouTat 1 and 1A VSG genes. Genomic DNA from LouTat 1, 1.5 and 1A was digested withXmnI and hybridized in a Southern blot with a labeled LouTat 1VSG cDNA probe (BC, basic copy of VSG gene; ELC, expressionlinked copy).

Figure 6. Restriction map of the LouTat 1 and LouTat 1A VSG gene expression sites. The expressed copies are in a telomeric site while the basic copyof the VSG gene is found at an internal chromosomal site that isthe same for both trypanosomes. The solid bar denotes the VSGgene.

Subsequently LouTat 1 and 1A were examined fornon-VSG related cellular differences (Mansfield,2001). Comparative analysis of several traits re-vealed significant differences between the two

clones. A few chromosomes were altered in size, asdetermined by pulsed field gel electrophoresis;however, there was no net loss of cellular DNA norwere any differences apparent in restriction frag-ment length polymorphism (RFLP) patterns utiliz-ing a number of random and known non-VSGcDNA probes. Thus, the chromosomal size varia-tions observed may largely be subtelomeric or sim-ply do not involve chromosome regions to whichthe probes hybridized. Two-dimensional gel elec-trophoresis of 35S-labeled proteins showed notonly that different proteins were expressed inLouTat 1A compared to LouTat 1, but also thatthere were different proteins expressed in LouTat 1compared to LouTat 1A. Competitive Northernanalyses in which labeled total cDNA from LouTat1A, in the presence of excess unlabeled LouTat 1competitive total cDNA, was hybridized to mRNAfrom LouTat 1A showed that that there werenumerous mRNA species unique to LouTat 1A.Taken together, these observations suggested that asubset of genes and proteins was being expressedin LouTat 1A that was not being expressed at thesame level in LouTat 1.

Overall, preliminary observations on the biologicalbehaviour of LouTat 1 and LouTat 1A, and on thesubcellular differences detectable between LouTat 1and LouTat 1A, as well as biological variationobserved with other subspecies of T. b. brucei interms of TLF susceptibility or TNFα sensitivity, haveled to the hypothesis that African trypanosomeshave the capacity to regulate clonal expression ofvirulence. Specifically, it has been proposed (a) thattrypanosomes have evolved the programmedcapacity for clonal upregulation of virulence as ameans to successfully subvert host resistance mech-anisms, regardless of host genetic background; (b)that this capacity to modify virulence phenotypeoccurs independently of changes in VSG geneexpression; and (c) that the level of virulenceexpressed is determined by differential gene and/orprotein expression during trypanosome growth inan infected host.

OverviewIt seems therefore an important goal to characterizethe molecular mosaic associated with the virulencephenotype and to determine whether or not specificsubsets of genes or proteins linked to virulence canbe identified; the prediction is that such approachesmay open new doors in the understanding of try-panosome-mediated pathogenesis, and candidatevirulence factors could be targeted for specificimmuno- or chemotherapies.


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II APPLIED GENOMICS: PROSPECTSFOR CONTROL OF AFRICAN TRYPANO-SOMIASIS VIA THE TSETSE VECTOR

Serap Aksoy1, Wendy Gibson2, Ron H. Gooding3,Elliot Krafsur4, Anna Malacrida5, Michael J.Lehane6, Scott L. O’Neill1, Terry Pearson7, Alan S.Robinson8, Philip Solano9, Antigone Zacharo-poulou10 and Liangbiao Zheng.1

1Department of Epidemiology and Public Health, Sectionof Vector Biology, Yale University School of Medicine, 60College St., 606 LEPH, New Haven, CT 06510, USA.2School of Biological Sciences, University of Bristol,Bristol BS8 1UG, U.K.3Department of Biological Sciences, University ofAlberta, Edmonton, Alberta, Canada.4Department of Entomology, Iowa State University, 407Science 2, Ames, Iowa 50011, USA.5Dipartimento di Biologia Animale, ZoologicalLaboratory, Universita di Pavia, Pavia, Italy.6School of Biological Sciences, University of Wales,Bangor LL57 2UW, U.K.7Department of Biochemistry and Microbiology,University of Victoria, Victoria, BC V8W3P6, Canada.8Entomology Unit, FAO/IAEA Agricultural andBiotechnology Laboratory, A-2444 Seibersdorf, Austria.9Institut Pierre Richet, 01 BP 1500 Bouke 01, Côted’Ivoire.10University of Patras, Department of Biology,Laboratory of Genetics, Patras, Greece.

INTRODUCTIONDespite many decades of research on vector-borneparasites and their development in mammalianhosts, effective strategies have yet to materialize forcontrol of any of the diseases with which they areassociated. At the same time, a heavy reliance oninsecticides and therapeutic drugs has resulted inthe spread of insecticide resistance in many vectorsand the emergence of drug resistance in parasites,threatening the availability of effective tools to com-bat these diseases. This scenario is also apparent forsleeping sickness in Africa where there are current-ly at least half a million people estimated to havecontracted the fatal disease. Antigenic variation inthe mammalian host has hampered efforts for vac-cine development. The current strategies for man-agement of trypanosomiasis depend on active sur-veillance and treatment of infected hosts, and onlimited vector control measures. These efforts are,however, restricted by the lack of effective drugs,their high cost, adverse side effects, and the emer-gence of drug resistance in patients (McNeil, 2000).Thanks to recent international effort, there are nowseveral programmes in place that will allow contin-ued provision of the existing drugs for treatment

and for research on the development of new drugs.While this is encouraging news, treatment alone fora zoonosis for which there is no evidence of acqui-red immunity is likely to be extremely costly in thelong run even if effective drugs were to be available.The fact that the parasite relies on a single insect forits transmission opens up many avenues for controlvia the control of its vector. It is important to notethat trypanosomiasis is a disease based on interac-tions among at least three organisms, the human,the parasite and the tsetse fly. Interference with anyof these interactions can prevent disease. In fact,tsetse control strategies have been widely used formanagement of animal diseases. Most of theseefforts however have been on a small scale, involv-ing trapping and the use of insecticide sprays, andhave been costly and difficult to sustain. A recentstudy looking into the cost/benefit analysis of vari-ous strategies clearly identifies large-scale, area-wide methods as being far more efficient and affor-dable in the long run for tsetse control (Budd, 1999).In Lome, Togo, July 2000, the Organization ofAfrican Unity (OAU) endorsed a strategy of tsetsefly and trypanosomosis eradication on the Africancontinent by using an integrated approach includ-ing area-wide control strategies. The recent advan-ces in molecular technologies and their applicationto insects have revolutionized the field of vectorbiology although progress in the tsetse field hasbeen slow. In this paper, we review the current sta-tus of knowledge on the molecular aspects of tsetsewith particular attention given to interaction withtrypanosomes. We highlight areas where researchefforts are needed and are likely to contribute eitherto the development of new vector control strategiesor to the improvement of the efficacy and afford-ability of existing approaches.

TSETSE GENOMIC STUDIES The trypanosome has been most studied in efforts todevelop a mammalian-based vaccine. While theseresearch efforts have turned trypanosomes into amodel eukaryote to study novel mechanisms ofgene expression and cell biology, there are no effec-tive products forthcoming for disease control in theforeseeable future. There are now genome sequenc-ing programmes for the parasite, and the genome ofthe human host is being deciphered. In contrast, todate, only a handful of tsetse genes and proteinshave been characterized. This is also in sharp con-trast to the vast level of knowledge available formosquitoes such as Anopheles gambiae, for which aninternational genome sequencing effort is now inplace, and Aedes aegypti, where a similar genomeproject is currently being planned. The many molec-ular markers developed in A. gambiae have formedthe basis of a rapidly increasing number of popula-


tion genetics and ecological studies. The availabilityof contemporary genomics information will provideus with essential tools for investigating parasite-host interactions as well as vector population struc-tures. This information is central for vector-basedcontrol strategies to be effectively developed.

TSETSE POPULATION GENETICS Population StructureThe recent availability of several microsatellite lociand their application to population analysis hasrevealed extensive genetic structuring in tsetse pop-ulations of the morsitans and palpalis groups at vari-ous geographical scales (Krafsur, et al, 2000; Krafsurand Griffiths, 1997; Krafsur, et al, 2000; Luna, et al,2001; Solano, et al, 2000; Wohlford, 1999). In the caseof G. palpalis gambiensis, these populations wereshown to transmit different species of trypanosomeswith different efficiencies (Solano, et al, 2000; Solano,et al, 1997). Additional microsatellite loci and thedevelopment of efficient oligonucleotide primers arerequired to achieve the level of genetic resolutionnecessary to measure accurately the differentiationof taxa and populations and gene flow within andamong them. In addition, randomly amplified poly-morphic DNAs (RAPDs) have been used successful-ly to examine paternal lines of gene flow, an impor-tant tool in studying historical lines of dispersion(Malacrida, et al, 1999). These studies are importantfor vector control approaches such as the sterileinsect technique (SIT), which is being considered foruse in upcoming tsetse eradication campaigns. Forthe successful application of SIT, information is re-quired on the tsetse species represented in the area,the degree of reproductive isolation among fieldpopulations, the existence of natural barriers to dis-persion, and the locations of dense, stable popula-tions from which immigration is likely.

Hybrid Sterility Hybrid sterility has been considered as a potentialcontrol method for tsetse ever since Vanderplankeradicated an isolated population of G. swynnertoniby release of G. m. centralis into its habitat (Vander-plank, 1948; Vanderplank, 1944). In the two subgen-era of Glossina that contain the most important try-panosome vectors, Glossina sensu stricto and Nemo-rhina, there are several groups of closely related taxathat hybridize readily both in the field and in thelaboratory (Gooding, 1990). Hybridization of tsetsealmost always results in production of hybrid fema-les with lower than normal fecundity and males thatare completely sterile (Gooding, 1990). The sterilityin males is attributable mainly to incompatibilitybetween sex chromosomes from two taxa, but auto-somal genes are also involved in some cases. In thesubgenus Glossina, but not in the subgenus Nemorhi-

na, maternally inherited factors appear to be in-volved in hybrid male sterility (Gooding, 1990). Suc-cess in hybridizing members of the subgenus Glos-sina is often highly asymmetric, with one cross beingsignificantly more productive than the reciprocalcross. Gooding (1987) attributed this asymmetry tomaternally inherited factors, which are nowbelieved to be Wolbachia (O’Neil, et al, 1993). Thesefactors can lead to unusual situations. For example,using G. m. morsitans and G. m. centralis it has beenpossible to produce backcross males with maternal-ly inherited factors from G. m. morsitans and chro-mosomes from G. m. centralis, and yet such malescan fertilize G. m. morsitans but not G. m. centralis(Gooding, 1987). It was suggested that such fliescould be used as genetic control agents againstG. m. centralis, but the phenotypic expression ofthese maternally inherited factors is unstable(Gooding, 2000; Gooding, 1990). However, if the sta-bility problem can be overcome, this approach maybe useful for control of certain members of the sub-genus Glossina.

Most hybridization studies on tsetse have used clo-sely related species and subspecies. There have beenfew “intra-taxon crosses”, using flies from coloniesthat were established from widely separated geo-graphic areas. Without exception, the latter studiesfound that F1 males were fertile and this has beeninterpreted as indicating that, within a nominaltaxon, there are no genetic barriers between geo-graphically separated populations. However, hy-brid breakdown has recently been found in tsetse(Gooding, unpublished). When G. p. palpalis fromcolonies that originated from widely separated geo-graphic areas were crossed, the first generation off-spring had normal fertility, but a high proportion ofmales in the second and subsequent generations,and in backcrosses to the parental lines, were sterile.The molecular basis for the hybrid breakdown isunknown, but its elucidation will be most helpful ifhybrid breakdown is to be exploited for tsetse con-trol. Hybrid breakdown may be a useful geneticcontrol approach for tsetse, especially if its manifes-tations include reduced fecundity of females and/orreduced longevity of adult flies. In any case, theexistence of hybrid breakdown in tsetse suggeststhat there may be cryptic species of tsetse, a possi-bility that could have implications for evaluatingwhat now appears to be simply geographic varia-tion in vector competence. Further work with otherwidely distributed species is clearly needed.

Polytene Chromosomes Polytene chromosomes have proven especially va-luable in studies of chromosome structure and func-tion. They provide a means for the accurate map-


ping of chromosome rearrangements and for theprecise localization of genes by using both chromo-some rearrangement analysis and the technique ofin situ hybridization. This has been technically chal-lenging in tsetse, but photographic polytene mapshave now been constructed for three species,G. austeni, G. m. submorsitans, and G. pallidipes. Com-parison of the homologous chromosomes betweenthe three species indicates that, in addition to simi-larities in their banding patterns, there are also var-ious major differences, especially between G. austeniand the other two species (Dr A. G. Papalexiou,University of Patras, unpublished). Studies can nowbe undertaken to identify and characterize chromo-somal rearrangements in field populations tounderstand genetic variation. The development ofin situ hybridization using FISH analysis withcloned tsetse genes and selected molecular probessuch as microsatellites will facilitate the eventualmapping of genes of interest (e.g. genes affectingvector competence, refractoriness, etc.).

VECTORAL CAPACITY OF NATURAL TSETSEPOPULATIONS Trypanosome infections with T. brucei ssp. complexparasites are typically detected in less than 1% of thefield population of tsetse flies (Lehane, et al, 2000;Msangi, et al, 1998; Woolhouse, et al, 1994). Evenunder ideal conditions in the laboratory, transmis-sion rates are between 1-10% depending on the flyspecies/strain and parasite strain (Moloo, et al,1994; Moloo and Kutuza, 1988; Moloo, et al, 1992).The basis for this refractoriness is not known, butdepends on tsetse species/strains and their symbi-otic bacteria as well as the genotype of the strain ofthe parasite acquired. In laboratory infections, theage and sex of the fly and the source of the blood-meal also contribute to the outcome of the infection.Field data are limited, however infections with mul-tiple parasites are common, indicating that theywere acquired in different blood-meals, hence flyage may not be as significant a factor as oncethought.

The life cycle of T. brucei in the tsetse fly beginswhen it feeds from an infected mammalian host.The non-proliferating short stumpy parasites thatare pre-adapted for life in tsetse fly rapidly differen-tiate into procyclic forms in the gut lumen, lose theirvariant surface glycoprotein, and express a new coatcomposed of procyclin proteins. The procyclin coatcontributes to the establishment of infections in thefly (Ruepp, et al, 1997). It has recently been shownthat procyclic cells express different procyclin coatsduring establishment in the gut and that the N-ter-minal domain of all procyclins are quantitativelyremoved by proteolysis in the fly, but not in culture

(Acosta-Serrano, et al, 2001). It has also been shownthat the binding of a lectin (concavanalin A) to theprocyclin coat molecules of the procyclic formsinduces multinucleation, a disequilibrium betweennuclear and kinetoplast replication and a uniqueform of cell death (Pearson, et al, 2000). Those sur-viving procyclic cells eventually proliferate in thegut (establishment phase) and flies can be scoredwith infections seven-ten days after acquiring aninfectious meal. The subsequent maturation phaseoccurs in the salivary glands for T. brucei (Vicker-man, et al, 1988). Here, they first differentiate intoattached proliferating epimastigote forms whichthen yield the infective, free-living metacyclic formswhich are transmitted to the next host during blood-feeding by the fly (Vickerman, et al, 1988). It is atthis stage that parasites are thought to undergogenetic exchange (Gibson and Whittington, 1993).The factors triggering this differentiation step areunknown. There is believed to be a critical periodfor maturation between days 8 and 11 after infection(Ruepp, et al, 1997; Sinkins, et al, 1995). One sugges-tion is that lectins have a role, since feeding lectininhibitory sugars can block maturation (Sinkins, etal, 1995). However, it is clearly crucial to investigatethe role of other components of the immune system.

The first physical barrier to ingested parasites in thegut is the peritrophic matrix (PM), which is a promi-nent feature of the digestive tract of insects. While inmany adult insects, PM components are secreted bymidgut cells in response to a blood-meal, tsetseadults have a PM constitutively synthesized fromcells in the proventriculus (cardia) in the foregutprior to feeding (Lehane, et al, 1996; Lehane andMsangi, 1991). How trypanosomes cross the PM tomove from the endo- to the ectoperitrophic space ofthe gut has been controversial, with penetration ofthe thick chitinous PM and migration around itsopen posterior end in the hindgut both having beenproposed (Ellis and Evans, 1977; Welburn andMaudlin, 1999). It is generally thought that duringnormal development in the fly there are no intra-cellular stages, although reports of intracellularT. b. rhodesiense (Ellis and Evans, 1976; Evans andEllis, 1975) and T. congolense (Ladikpo and Seureau,1988) in anterior midgut cells have been published.It is also thought that during normal infection, try-panosomes do not cross an epithelial barrier in thefly, although once again there are several reports oftrypanosomes in the hemolymph of flies (Mshel-bwala, 1972; Otieno, 1973). Also unknown is howprocyclic parasites move from the gut to the mouthparts and/or the salivary glands of the fly. The clas-sical route involves crossing back into the gut lumenacross the proventriculus in the foregut and thenceto the mouthparts and salivary glands (Ruepp, et al,


1997; Turelli and Hoffmann, 1991). This is indeed anincredible journey, which few trypanosomes appar-ently embark on or complete (Ruepp, et al, 1997).Presumably the increased length and motility of thetrypanosomes observed en route are adaptationsenabling them to succeed (Ruepp, et al, 1997).

TSETSE VECTOR COMPETENCEAt the center of vector competence in insects areimmune reactions, which include a diverse set ofmechanisms ranging from phagocytosis, activationof proteolytic cascades, such as coagulation andmelanization and production of various antimicro-bial peptides (Barillas-Mury, et al, 2000; Dimo-poulos, et al, 2001). While much of this immuneresponse is initiated in the fat body, effector mole-cules expressed in the midgut are increasingly beingrecognized as playing a role in immune reactions(Dimopoulos, et al, 1997; Dimopoulos, et al, 1998;Lehane, et al, 1997). Discrimination between patho-gen groups is well known in Drosophila and mosqui-toes, where it is explained by the use of differentreceptor/signalling pathways. Because dipteransincluding mosquitoes and tsetse are largely refracto-ry to parasite transmission, much effort has goneinto understanding the immune mechanisms ofmosquitoes. If the genetic basis of this refractorinesscould be understood, then flies might be engineeredto completely block parasite transmission using var-ious recombinant approaches currently being devel-oped. Immunity genes are also being studied as theyare strong candidates to confer such transmissionblocking phenotypes for inducing refractoriness. Asa result of the application of molecular approaches,a good deal of information is now available aboutthe response of mosquitoes to Plasmodium species.

There is little known about the genetic basis forrefractoriness in tsetse or the molecular basis for thewide ranging differences observed in parasite trans-mission rates of different tsetse species. Lectins,which are known immune molecules, might play arole in the attrition of trypanosomes during estab-lishment (Welburn and Maudlin, 1999). There is alsosome information demonstrating antimicrobialactivity (Kaaya and Darji, 1988; Kaaya, et al, 1987;Kaaya, et al, 1986), the presence of the phenoloxi-dase cascade (Nigam, et al, 1997) in hemolymph.Preliminary results indicate that the immune re-sponse to different pathogens in tsetse is specific.These results also suggest that trypanosomes mayuse unprecedented novel mechanisms to achievetheir transmission through the fly by blocking theexpression of some of its immune responsive genesearly in the infection process (Zhengrong, et al,2001). When the immune system is upregulatedprior to an infectious meal, the transmission of try-

panosomes can be significantly reduced (Zhen-grong, et al, 2001). Further characterization of theseimmune mechanisms or products and their interac-tion with trypanosomes are needed. In addition, astudy of other tsetse molecules, such as receptorsinvolved in interactions with parasites, or moleculesthat are simply required for survival of tsetse or thatinfluence their fecundity, will be important fordeveloping strategies to interfere with transmissionof trypanosomes. These studies are of applied inter-est as they support the development of strategies toblock parasite transmission in vivo via transgenicapproaches.

TRANSGENESIS IN TSETSE Much effort has gone into developing genetic trans-formation systems for medically and agriculturallyimportant insect vectors. There is no doubt that theavailability of this technology will revolutionize in-sect genetics by allowing basic studies relating tothe functional characterization of various genes andtheir products. It might also allow for the develop-ment of alternative control strategies such as the useof transgenic refractory insects. It is thought thatsuch genetically engineered refractory insects can bedriven into natural populations to replace their sus-ceptible counterparts and hence reduce disease tran-smission. While the efficacy and feasibility of thisstrategy are being widely debated among scientistsat large, in the case of tsetse, the development of try-panosome-refractory strains will have an immediateapplication for at least one effective control strategy,the sterile insect technique (SIT), as described below.

At the core of transgenesis is the process of genetictransformation, which in many insects relies on themicroinjection of transposable elements that insertthemselves into insect DNA, resulting in germ-linetransformation. Marker genes carried by the trans-posable element help identify transgenic individu-als. It has now been possible to introduce foreigngenes into several important insect vectors includ-ing one important malaria vector in Asia, Anophelesstephensi (Cateruccia, et al, 2000), and others are like-ly to follow using similar technologies. Tsetse, how-ever, have an unusual reproductive biology. There isno free egg stage, females retaining each egg withinthe uterus. Following hatching and in utero develop-ment of the larva, one young larva matures and isfinally expelled as a fully developed third instarlarva. Each female can deposit four-six offspringduring its five-eight week average life span in thefield. This viviparous reproductive biology wouldundoubtedly complicate any attempts to transformtsetse through egg microinjection. However, tsetseflies naturally harbor a number of symbiotic micro-organisms that have been exploited to express for-


eign gene products (Aksoy, 2000). Through such anapproach, the insect cells are not transformed as ingerm-line transformation, but instead, foreign genesare expressed in the symbiotic bacteria. Since thesymbionts naturally live in close proximity to thedeveloping trypanosomes, anti-pathogenic geneproducts introduced and expressed in these cellscould adversely affect trypanosome transmission.

TSETSE SYMBIONTS AND EXPRESSION OF FOREIGN GENES IN VIVOMany insects with limited diets such as blood, plantsap or wood, rely on symbiotic microorganisms tofulfill their nutritional requirements. It has beenshown that tsetse harbour three distinct microor-ganisms (Aksoy, 2000). Two of these are present inthe gut tissue: genus Wigglesworthia (Aksoy, 1995;Aksoy, et al, 1995) and genus Sodalis, (Aksoy, et al,1995; Cheng and Aksoy, 1999; Dale and Maudlin,1999) and both are closely related to Escherichia coli.The third symbiont harboured in certain tsetsespecies is Wolbachia, an obligate intracellular bacteri-um which is closely related to the genus Ehrlichia(O’Neill, et al, 1993). During its intrauterine life, thetsetse larva receives nutrients along with the twogut symbionts from its mother, via milk-gland secre-tions (Aksoy, et al, 1997; Ma and Denlinger, 1974),while Wolbachia is vertically transmitted by transo-varial transmission.

It has been possible to culture the Sodalis symbiontin vitro (Beard, et al, 1993; Welburn, et al, 1987) anda genomic transformation system has been devel-oped (Beard, et al, 1993). Recently, a homologousrecombination approach has also been establishedso that foreign genes can now be directly insertedinto the symbionts chromosome (Dale, et al, 2001). Ithas also been possible to reconstitute tsetse with therecombinant Sodalis, which has been found to besuccessfully acquired by the intrauterine progenywhen microinjected into the haemolymph of thefemale parent (Cheng and Aksoy, 1999). It is nownecessary to identify effective gene products whichhave anti-trypanosomal effects when expressed inSodalis in tsetse midgut. Using a similar symbiont-based insect transformation approach, it has beenpossible to block the transmission of Trypanosomacruzi in Rhodnius prolixus in vivo by expressing theantimicrobial peptide cecropinA (cecA) in its sym-biont, Rhodococcus rhodnii, in the hindgut of the bugs(Durvasala, et al, 1997). It has also been possible toexpress a single-chain antibody gene fragment inthe bacterial symbionts of R. prolixus (Durvasala,et al, 1999). If the tsetse immune-responsive mole-cules, which the trypanosomes apparently down-regulate to achieve their transmission, can be identi-fied, they could be constitutively expressed in the

symbionts to prevent parasite survival in the mid-gut. The identification of monoclonal antibodies(mAbs) with parasite transmission blocking charac-teristics and their expression as single-chain anti-body gene fragments in the symbionts provides analternative approach. Towards this end, severaltransmission-blocking antibodies targeting themajor surface protein of the insect stage procyclictrypanosomes have already been reported (Nantu-lya and Moloo, 1988). Recently, midgut-specific mo-noclonals with transmission blocking activity havebeen characterized from A. gambiae (Lal, et al, 2001).It seems likely that similar molecules can be identi-fied in tsetse and ultimately expressed in the gutsymbionts. The relative ease of transformation andgene expression in bacteria, and the multitude ofpotential antiparasitic targets which can be explo-red, make this a desirable system for transgenicapproaches. Should resistance develop in parasitesagainst any of the expressed foreign gene products,it could be relatively easy to switch to express a dif-ferent gene product. Alternatively, several targetgenes can potentially be expressed simultaneouslyin the symbionts to prevent the development ofresistance against any one individual target.

FIELD APPLICATIONS OF TRANSGENESISIn order to interfere with disease transmission, theeventual goal of any transgenic approach is toreplace the naturally susceptible population withtheir engineered refractory counterparts in the field.At present, there are no proven mechanisms toachieve this spread. One powerful potential drivingsystem involves the use of Wolbachia symbionts,which confer a reproductive advantage to theirhosts, including the engineered females.

The functions of Wolbachia in their various hosts arevariable. One common reproductive abnormalitythey induce has been termed cytoplasmic incompat-ibility (CI). This, when expressed, results in embry-onic death due to disruptions in early fertilizationevents (Bandi, et al, 2001). In an incompatible cross,the sperm enters the egg but does not successfullycontribute its genetic material to the potentialzygote. In most species, this results in very few eggshatching. Wolbachia infected females have a repro-ductive advantage over their uninfected counter-parts because they produce progeny after matingwith both infected and uninfected males. This repro-ductive advantage allows Wolbachia to spread intopopulations. In Drosophila simulans in the centralCalifornia valley, a natural Wolbachia infectioninvading naive uninfected populations has spreadat a rate of over 100 km per year simply through theexpression of CI (Turelli and Hoffmann, 1991). Tounderstand the functional role of Wolbachia in


insects, most insects can be cured of their Wolbachiainfections by administering antibiotics in the diet.This approach, however, is not feasible in tsetsesince antibiotic treatment results in the clearing ofall bacterial symbionts, including the obligate sym-biont Wigglesworthia, in the absence of which theflies become sterile. In order to study CI expressionin tsetse, uninfected flies need to be collected fromthe field and colonized so that appropriate matingexperiments can be performed in the laboratory. AsWolbachia infected insects replace naive populationsby virtue of the CI phenomenon, they can driveother maternally inherited elements of tsetse, suchas the maternally inherited gut symbionts Sodalis,into that same population (Beard, et al, 1993). It hasbeen proposed that multiple Wolbachia infections, inwhich an insect contains two or more different Wol-bachia strains that are incompatible with each other,could be used to generate repeated population re-placements or to spread Wolbachia into target speciesthat already contain an existing infection (Sinkins,et al, 1995; Sinkins, et al, 1997). The analysis ofWolbachia strain types infecting different species oftsetse has shown that they are different, and as suchrepresent independent acquisitions (Cheng, et al,2000).

In addition to CI, certain Wolbachia strains, such aswMelPop characterized from Drosophila melanogaster,induce an age-shortening effect in their host (Minand Benzer, 1997). This age-shortening effect wasreversed when infected flies were cured of theirWolbachia infections with antibiotics (Min andBenzer, 1997). It remains to be seen whether suchWolbachia infections can be documented in tsetse. Ithas also been possible to introduce Wolbachia intoinsect species which do not harbour natural infec-tions, and these approaches can be pursued intsetse. Since the T. brucei group parasites require 14-30 days to complete their developmental cycle in thefly, tsetse flies need to be at least this age to transmitdisease. Reductions in the life span of individualflies might have a large effect on disease transmis-sion in the field (Sinkins and O’Neill, 2000).

STERILE INSECT TECHNIQUE AND TRANSGENIC INSECTS SIT is a genetic population suppression approachand involves sustained, systematic releases of irra-diated sterile male insects among the wild popula-tion. The sterile males fertilize wild females, whichare then unable to produce progeny. By continuallyreleasing sterile males in high numbers over a peri-od of three-four generations, after having previous-ly reduced the population density by other tech-niques (trapping, insecticide spraying, etc.), the tar-get population can be eradicated (Politzar and

Cuisance, 1984; Vreysen, et al, 2000). Improvementsin two aspects of current tsetse SIT technology havethe potential to enhance the efficacy of future pro-grammes (Aksoy, et al, 2001).

The first is the development of parasite refractorystrains. Since the large numbers of male flies re-leased can potentially contribute to a temporaryincrease in disease transmission, the incorporationof refractory traits into the SIT release strains willgreatly enhance the efficacy of this approach, espe-cially in human disease endemic foci. In the currentfield SIT programmes, male tsetse are provided,before release, with a blood-meal containing a try-panocide; no infections have so far been found inreleased sterile males caught in traps. The second isthe use of Wolbachia mediated CI, or hybrid sterility,as a method of inducing sterility - as an alternativeto irradiation. With CI, the released strain of tsetsewould carry a Wolbachia infection that would induceCI when males mate with wild females. The com-petitiveness of these males would be expected to bemuch higher compared with irradiated males, and,as a result, fewer insects would need to be releasedin order to achieve the same level of sterility in thewild population. This strategy is dependent on theuse of a very efficient sexing system. If Wolbachia-infected females were released in sufficient quanti-ties, then Wolbachia would have the opportunity toinvade the target population, which would rendersubsequent releases ineffective. If it were impossibleto guarantee extremely low quantities of releasedfemales, then it would be possible to incorporatelow levels of irradiation with Wolbachia inducedsterility to prevent released females from successful-ly reproducing. This approach has been successfullytested in Culex mosquitoes (Shahid and Curtis,1987).

CONCLUSIONS• Understanding the molecular/cellular basis of

trypanosome transmission in tsetse is of funda-mental significance, and will allow developmentof new applications for vector control. Since thesymbiont-based transformation system can beused to express gene products in tsetse midgut,such will aid in the identification of candidategenes that can be expressed to confer refractori-ness in tsetse.

• A research plan should be developed to coordi-nate information on expressed sequence tag (EST)sequences, genomic sequences and the physicalmap locations of selected genes. For EST analysis,the cDNAs to be sequenced can be obtained fromtsetse exposed to infected blood-meals containingeukaryotic pathogens, including trypanosomes,


and from tissue specific (midgut, fat body andsalivary glands) normalized libraries as well asfrom different developmental stages (larvae). TheESTs deemed to be of interest should be physical-ly mapped to metaphase chromosomes using insitu hybridization (FISH) technology. A bacterialartificial chromosome (BAC) library could be con-structed in order to obtain single pass sequences.Some of these resources are already available andthese efforts would promote and foster collabora-tion and intellectual input from the internationaltsetse community. The availability of the completeand annotated D. melanogaster genome, and theanticipated genome projects for A. gambiae and A.aegypti, will constitute an important resource forgene discovery efforts in Glossina. It follows that aproteomics approach to protein identification willallow further exploitation of the genomic infor-mation. This aspect of molecular entomology willbe a powerful approach in the post-genomic era.

• Molecular approaches for population geneticsstand to improve our understanding of vectorpopulations and are of significance for the suc-cess of area-wide control strategies. These field-based studies could easily be coupled withefforts to better understand the prevalence andphenotypic effects of natural Wolbachia infectionsin tsetse.

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III APPLIED GENOMICS AND BIOINFORMATICS

Sara E. Melville1 and Daniel Masiga2.1Cambridge University, Department of Pathology,Microbiology and Parasitology Division, University ofCambridge,CB2, IQP, U.K.2International Centre of Insect Physiology and Ecology(ICIPE), Molecular Biology and Biotechnology Unit, POBox 30772, Nyayo Stadium (00506), Nairobi, Kenya.

OVERVIEW OF THE T. BRUCEI GENOME AND THE HISTORY OF TRYPANOSOMEGENOMICS

The African trypanosome genome network wasformed by the UNDP/World Bank/WHO SpecialProgramme for Research and Training in TropicalDiseases (TDR) with a brief to coordinate the ana-lysis and sequencing of the nuclear genome. It con-sists of research laboratories worldwide, contribut-ing to different aspects of genome analysis. At thetime of its formation, some aspects of genomestructure and organization were known, especiallyfrom studies on antigenic variation and transcrip-tion, but only one group had actively initiatedglobal genomic analysis. In the last five years,much has been learnt about the organization of thenuclear genome of T. brucei, which consists of atleast eleven diploid megabase chromosomes (1-6Mb), a variable number of intermediate-sized chro-mosomes of indeterminate ploidy (200-900 Kb),and 50-100 minichromosomes (25-100 Kb)(Melville, et al, 1998).

With the exception of some variant surface glyco-protein (VSG) genes, most expressed genes arelocated in the megabase chromosomes. These areinherited in a Mendelian fashion (Turner, et al, 1990)and have been assigned Roman numerals in orderof increasing size in TREU927/4 (Turner, et al, 1997).Many, perhaps most, genes are expressed in poly-cistronic transcripts (Vanhamme and Pays, 1995).Some chromosome ends carry VSG expression sites:only one is active at one time, resulting in a uniformsurface protein coat. Gene- or promoter-switchingresults in antigenic variation (Barry and McCulloch,2001), which is an effective way for the parasite toevade destruction by the host immune system andresults in the characteristic fluctuating parasitemiaand accompanying fever. The intermediate chromo-somes also carry expression sites (Rudenko, et al,1998), and most mini-chromosomes contain non-transcribed VSG and simple repeat sequences(Weiden, et al, 1991). The network has prioritizedsequencing of the megabase chromosomes, as they

contain the majority of genes, but the DNA contentof the mini- and intermediate chromosomes shouldbe investigated in the future.

Most megabase chromosomes differ in size fromtheir homologues by up to 15%, but homologouschromosomes in different stocks vary more - con-siderably more than reported in other organisms.Nevertheless, mapping studies show that syntenicgroups are maintained in all stocks studied (Mel-ville, et al, 1998). Despite remarkable genomic plas-ticity (Melville, et al, 1999), studies reveal a signifi-cant level of conservation of genome structure.Initial apprehension that the genome was so poly-morphic as to make sequencing of one strain of littleuse for cross-comparison to other strains has beenassuaged – although further studies could investi-gate the role this plasticity is playing in the survivalstrategy of the trypanosome.

There is no perfect isolate that should form the basisof all molecular experiments on T. brucei. It is indeedpreferable that some genomic analyses are carriedout on multiple stocks, for example karyotypingand random gene sequencing. However, we canonly aim to sequence one complete genome in theimmediate future. T. brucei stock TREU927/4(GPAL/KE/70/EATRO1534) was cloned from apopulation of trypanosomes isolated from a tsetsefly in Kenya and was chosen as the referencegenome for a variety of reasons, some optimal andsome pragmatic. The original stock exists and thehistory of its isolation is documented. It was not iso-lated from an infected human, but laboratory testsindicate that it has intermediate resistance to humanserum (CMR Turner, personal communication). It ispleomorphic and may be replicated as the blood-stream or procyclic form in laboratory animals,tsetse flies or culture. It has been used as a parent inlaboratory-controlled genetic crosses with otherstocks, and cloned hybrids have been isolated(Turner, et al, 1990; Tait, et al, in press), allowing thecreation of a genetic map. A high quality, arrayedgenomic library of the megabase chromosomalDNA of TREU927/4 already existed (Melville, et al,1996), and was used to determine the structure of amegabase chromosome (Melville, et al, 1999).

Participation in the network is dynamic because, aspriorities change, researchers with different skillsare required. Many contribute without direct fund-ing, and the network is dependent on the supportand active participation of the research community.It has been vital to promote universal commitmentto the idea that this huge task can progress most effi-ciently if the community works together with opensharing of data and resources.


The availability of the DNA sequence will benefitmany research programmes. Some benefits are al-ready obvious, some will only become clear as moreresearchers begin to use the available data. This isour task: to be forward-looking and imaginative inour plans for the use of this tremendous resource.

CURRENT STATUS OF THE TRYPANOSOMA BRUCEI GENOME PROJECTExpressed Sequence Tags (ESTs)Rapid gene discovery was achieved in the earlyphase of the genome project by sequencing of ran-domly selected cDNA clones (El-Sayed, et al, 1995;Djikeng, et al, 1998). At the time of writing, thereare 5133 T. brucei ESTs in the public databases fromfour cloned stocks of T. brucei (dbEST:http://www.ncbi.nlm.nih.gov/dbEST/index.html).Most EST sequences were generated from cDNAclones of bloodstream-form mRNA. There has beenno concerted effort to produce large EST datasetsfrom each of the life-cycle stages as it was decided todivert effort and resources to sequencing of genom-ic DNA. The determination of life-cycle stage-spe-cific expression will be undertaken by other meth-ods (see section 3.5). In addition to discovery ofmany novel T. brucei genes, the ESTs have provideda rich source of markers (Melville, et al, 1998) andaid sequence annotation.

Genome Survey Sequences (GSSs) for GeneDiscoveryIn a pilot project, approximately 500 randomgenomic clones were sequenced (El-Sayed andDonelson, 1997) to show that this led to equally effi-cient gene discovery, due to the lack of introns andthe close spacing of genes in the T. brucei genome.Therefore, it was decided that a portion of the fundsobtained for high-throughput sequencing at TheInstitute for Genomic Research (TIGR) should beallocated to sequencing of random, short pieces ofDNA (GSSs). Sequencing of both ends of almost25000 clones (almost 50 000 short sequences) pro-vided a total of 29 Mb of the TREU927 genome(http://www.tigr.org/tdb/mdb/tbdb/status.html).A proportion of these sequences derive from theends of large genomic clones (in bacteriophage P1and bacterial artificial chromosome (BAC) vectors),and these contribute to the mapping and sequenc-ing of whole chromosomes (see section 2.4) by pro-viding paired markers of about 500 base pairs every2.5Kb across the chromosomes (excepting theminichromosomes). Many researchers have report-ed finding T. brucei homologues of known genes inthe GSSs, and this generated great enthusiasm forthe rapid provision of more such sequences. Inresponse to requests from the community, theSanger Institute has provided a further 47 000 sin-

gle-pass sequence reads to aid gene discovery andfacilitate the completion of contiguous chromosomesequences (http://ftp.sanger.ac.uk/pub/databas-es/T.brucei-sequences).

The EST and GSS sequences have been clusteredby collaborators at the Sanger Institute to providecontigs. In total, 96 474 sequences (~45.87Mb)were used for the clustering, achieved using thesequence assembly programme Phrap - 12 251contigs were generated and 8242 sequences couldnot be placed in a contig (singletons). One of thecontigs has 1926 constituent members (contiglength 9.342Kb), but this is probably due torepeated DNA and is an example of the care thatmust be taken in interpreting these automaticallygenerated data. The cluster data are available as asearchable subcomponent of the T. brucei BLASTserver (http://www.sanger.ac.uk/Projects/T_bru-cei Toolkit/blast_server.shtml) and from the ftp siteas a gzip file (hftp://ftp.sanger.ac.uk/pub/databas-es/T.brucei_sequences/GSS/clusters).

Physical and Genetic MappingOne chromosome was mapped thoroughly and tocompletion (Melville, et al, 1999) prior to submittingsequencing grants. However, rapid progress inmethods for completion of genome sequences hasreduced the requirement to produce prior contigu-ous physical maps of each chromosome (El-Sayed,et al, 2000). Nevertheless, mapping information isrequired for seeding BAC sequencing and to aidfinal reconstruction of the chromosome. Manyhybridization data have been amassed by theCambridge group and also by the communitythrough using centralized resources. So far, thesedata have been made freely available but only byemail request. A database is being established tomake the data web-accessible (see 4.4).

TDR and The Wellcome Trust have also supportedthe creation of a genetic map using classical geneticanalysis of F1 hybrids (Tait and Turner, Glasgow).F1 hybrids have been isolated following simultane-ous passage of genotypically distinct stocks throughtsetse flies. This group has developed amplifiedrestriction fragment polymorphism (AFLP), mini-and micro-satellite markers for use in genetic analy-sis and, together with the genomics group(Melville), has been able to combine some of thedata with chromosome maps. The aims are to deter-mine crossover frequency, estimate the physical sizeof the recombination unit (Centimorgan), and inves-tigate variation in crossing-over in different genom-ic regions. Initial data of this type indicate that it isindeed feasible to obtain sufficient hybrid progenyand genetic markers to aim towards using a genetic


map for positional cloning of genes underlying com-plex phenotypes (Tait, et al, in press).

Genomic SequencingSequencing of chromosome I of T. brucei strain 927/4GUTat 10.1 commenced at the Sanger Institute in1998. Sequence was obtained from shotgun clones ofchromosomal DNA eluted from a pulsed field gel(PFG) (> 8 X coverage) and from mapped P1 clones(1 X coverage), a combination of methodologies pio-neered by the malaria sequencing consortium. Whileapproximately 75% of the chromosome sequencewas contiguous by year two, 25% of the chromo-some has proved difficult due to repetitive DNA:genes in VSG expression sites, gene families, retro-transposons, and some tandem repeats (Melville,et al, 1999). Following the random sequencing phase(see 2.3), TIGR commenced the sequencing of chro-mosome II in 1998. This sequence is derived fromBAC clones, mapped to chromosome II usingcDNAs from the EST project (Melville, et al, 1998;El-Sayed, et al, 1995) and sequenced to ca. 7 X cover-age. The end-sequences determined in the firstphase of the project (section 2.2) allow the selectionof BACs with minimum overlap for maximum effi-ciency. The progress on chromosome II has been sim-ilar to that on chromosome I, with similar problemsin regions of clustered multicopy sequences.Chromosomes IV and VI are currently approximate-ly 75% completed at TIGR. Preliminary (and there-fore in parts inaccurate and incomplete) annotationis provided by the sequencing centres prior to com-pletion. It is best to follow progress by regularlymonitoring both sequencing centre websites( h t t p : / / w w w. s a n g e r. a c . u k / P r o j e c t s / T- b r u c e i / ;http://www.tigr.org/tdb/mdb/tbdb/index.html).

The funds to complete chromosomes IX, X and XIwere awarded to Barrell (Sanger Institute), Melvilleand the network in 2000. Chromosome X shotgunsequence is now available (http://ftp.sanger.ac.uk/pub/databases/T.brucei-sequences) and chromo-somes IX and XI are in library preparation. Thefunds to complete chromosomes III, V, VII and VIIIwere awarded to El-Sayed at TIGR (collaboratorsDonelson, Ullu, Melville) in 2001. Each sequencingcentre will therefore provide approximately 50% ofthe megabase chromosome DNA sequence. The proj-ects run until 2004 and all the sequence is likely to bein the databases by 2003 at the latest; however it can-not be foreseen how long it will take to completecontiguation.

To make full use of the substantial investment ingenome sequencing it is necessary to complete thetask, to ensure there is complete information on allenzyme pathways. It is also absolutely imperative toinvest substantial effort into bioinformatics to make

the data accessible and informative, and to stimulatenew ideas in the search for novel approaches to com-bat African trypanosomiasis.

Access to DataThe fields of genomics, databases and bioinformaticsare dynamic and researchers may find it difficult tolearn how best to access the most recent data, orwhere to find the most innovative new programmesfor analysis. The field is developing rapidly and herewe aim to provide an outline of where data may befound at different stages of the sequencing projects.Complementary DNA (cDNA) sequencing has beencarried out in individual research laboratories andthe sequences are made available via the databasefor expressed sequence tags (dbEST) at the NationalCenter for Biotechnology Information (NCBI). Allgenomic sequence data are made available immedi-ately via the websites at the respective sequencingcentres (TIGR and Sanger Institute). These are sin-gle-pass sequences and, although no error correctionor annotation are offered at this stage, this is animportant resource for researchers who are lookingfor genes in T. brucei that have homologues in otherorganisms. The clustering data provided by theSanger Institute are also generated automaticallyand any errors will be incorporated. Therefore, useof such data always requires verification by theresearcher. Search engines are provided, allowingresearchers to look for sequences with high similari-ty to the gene they seek (http://www.ebi.ac.uk/b l a s t 2 / p a r a s i t e s . h t m l ;http://www.tigr.org/tdb/mdb/tbdb/seq_search.html). Data on significant similarities to genes in thedatabases are provided but researchers should notethat new information becomes available in the cen-tral databases (European Molecular Biology Labora-tory, EMBL/GENBANK/Database of Japan,DDBJ) all the time, and should check when themost recent BLAST searches were performed. Atintervals, sequences are submitted in batches to thepublic databases at the European BioinformaticsInstitute (EBI) and NCBI. Random genomic shotgunsequences and end-sequences of genomic clones aresubmitted to the database for genome surveysequences (dbGSS) (http://www.ncbi.nlm.nih.gov/dbGSS/index.html). Sequences of shotgunclones derived from whole BACs are submitted tothe database for high-throughput genomic sequences (dbHTGS) (http://www.ncbi.nlm.nih.gov/dbHTGS/index.html) in three stages (at 3 Xand 7 X coverage, and at completion). All these datawill eventually be mirrored in the GENBANK,EMBL and DDBJ databases, ensuring that all avail-able T. brucei sequences may be found in a singledatabase. However, there are some advantages inlooking at the data in the specialized genome data-


bases, as annotation is more extensive and sequencesimilarity assignments are provided.

On completion of a chromosome sequence, the mostcareful annotation is carried out. Great emphasis islaid on ensuring the sequence is accurate and con-tiguous (some areas of ambiguity may be tolerated inthe final sequence, if too many resources are requiredto provide final clarification), and on trying to identi-fy all open reading frames (ORFs). Researchersshould be aware that, however careful the annota-tion, some protein coding genes may not be predict-ed from sequence analysis alone and a few of thosepredicted may not be transcribed. The analysis ofthe chromosome is published in a peer-reviewedjournal and the sequence, with full annotation, canthen be viewed with the Entrez browser at NCBI(http://www.ncbi.nlm.nih.gov/Entrez/Genome/org.html). The sequencing centre responsible for thesequencing of the chromosome also places the samedata on its website.

In an ideal world it would be possible to carry outsearches of all data at a single site but the curation ofsuch a database would necessitate delay in access tothe data. Immediate access to raw, unfinished se-quence is highly prized by a research communitywaiting impatiently for gene sequences, and thesequencing centres have responded to this need.This is only really possible via deposition on homewebsites prior to batch submission to NCBI. It istherefore important for researchers to considerwhich databases contain the most valuable data (e.g.the public genome databases because of their exten-sive annotation, or the sequencing centre databasesbecause they may contain sequences not yet pro-cessed for transfer to NCBI) and, in some cases, toperform searches at several sites. Long-term curationof interrelated genomic and functional data is a dif-ferent consideration (section 3.1).

Summary and Predicted Timeline.The following sequence data are available in 2001:• 5133 EST sequences, providing approximately

2000+ unique genes.• 96 474 GSS sequences (~45.87Mb), including 10

990 BAC and P1 end-sequences.• Clustering data of all GSS and EST sequences,

with data on significant similarities.• The complete sequence of chromosomes I and II

(to be fully annotated in 2001).• 75% of chromosomes IV and VI as complete BAC

sequences.• Chromosome X shotgun sequence (04/01); chro-

mosomes IX and XI shotgun sequence to follow.

Predicted timeline to 2002-2004:• 1 x sequencing of IX, X and XI-specific BACs.• Chromosomes III, V, VII and VIII to be sequenced

BAC by BAC and appearing in the databasespiecemeal over the next two years.

• Contiguation and annotation of chromosomes III– XI over at least three years.

The timeline to complete sequencing is the most dif-ficult to predict, and depends to some extent on thedetermination to complete subtelomeric regions,VSG arrays, etc.

CURRENT STATUS OF FUNCTIONALAND APPLIED GENOMICS PROJECTSThe African trypanosome genome network has ini-tiated discussions on the “post-genomic” agenda.This requires careful coordination and the involve-ment of appropriately skilled researchers. At the1999 and 2000 network meetings, lists of prioritiesdrawn up (http://parsun1.path.cam.ac.uk/net-work.htm) included microarrays for transcriptionanalysis, techniques for gene knockouts, generationof mutants, RNAi and phenotype characterization,and use of the genetic map for positional cloning ofgenes (some to proceed in collaboration with otherkinetoplastid genome networks). Recommendationsof interest to this discussion include identification oftrypanosome-specific genes essential for infectionand development, characterization of molecularstructures and parasite-specific metabolic path-ways, elucidation of unique trypanosome-specificmechanisms of gene regulation and RNA process-ing, and analysis of the protein profile of the para-site to identify functionally important genes (pro-teomics). Perhaps one or more of these approachescould lead to the identification of novel drug tar-gets, and/or vaccine candidates, but only if thoseresearchers skilled in, and committed to, their deve-lopment join the global collaboration and contributeto the post-genomic projects of the future.

Sequence Analysis and Relational DatabasesThe Wellcome Trust Functional Genomics Initiativehas provided funds to create a trypanosome data-base (genome databases for Schizosaccharomycespombe, Leishmania major and T. brucei ) to Drs BBarrell, Sanger Institute; M Rajandream (S. pombe),A Ivens (L. major); M Carrington and S Melville(T. brucei). These databases will contain all genomicinformation, e.g. primary sequence annotation fromthe sequencing centres, secondary (ongoing) se-quence annotation, references, expression data, pro-tein characterization, knockout and RNAi pheno-type analyses. They will be relational databasesusing SQL (structured query language) and hostedby the Sanger Institute. The T. brucei database will


be developed in close collaboration with TIGR, andis overseen by a management committee. Genomedatabases of this type are long-term projects, pro-viding a single site to review ongoing genomic andfunctional genomic analyses long after sequencingof the reference strain is complete, and as such servea different function to the rapid access sequencedeposition on the sequencing centre websites or theparasite genome BLAST server (see 2.5). News onprogress will be posted on relevant websites.

The importance of ensuring that this database isreadily available to African scientists hardly needsexplaining - they should not need to rely on collab-orations with laboratories in Europe or the Americasto gain direct access to such comprehensive data. Inthe long-term, the enabling of molecular biologicalresearch on trypanosomes will require that Africanlaboratories have secure Internet connections ofwide band-width. Until such time as this is possible,we should consider providing CD versions of thedatabase at regular intervals. This may be proposedto the database committee via Dr Melville, and willrequire some coordination.

Bioinformatics ProjectsThe field of bioinformatics is changing rapidly andthe databases described here will not remain static.NCBI, EBI, the sequencing centres (and many oth-ers, including academic institutions) are activelydeveloping better analysis tools, and it is necessaryto watch their websites for innovations and devel-opments.

In addition, there is certainly scope for specificbioinformatic analysis projects, either based on theexamples from more advanced genome projects(yeast, C. elegans) or on novel approaches based onparasite-specific interests. For example, investiga-tion of the components of metabolic enzyme path-ways (requiring skilled biochemists, but also novelbioinformatics approaches to reduce the timerequired for the largely manual approach currentlyused); comparison of metabolic pathways to thehomologous pathways in humans and livestock(again requiring biochemists and informaticians);searching for commonalities in the biochemistry ofthe kinetoplastid parasites, that differ from themammalian host; analysis of targeted or global/sin-gle-pass comparative sequencing of other trypano-some strains and species.

Biological Characteristics of the Reference StrainA minimally culture-adapted line of the originalstock TREU927/4 has been generated with greaterstability of variant antigen types (TREU927/4GUTat 10.1). This grows to a higher density in cul-

ture (M Turner, personal communication). The kary-otypes are identical. The P1 library is prepared from927/4 and the BAC library from 10.1. The primarysequencing substrate is 10.1 procyclic genomicDNA.

Stock 927 has the smallest nuclear genome of allT. b. brucei or rhodesiense stocks examined so far withapproximately 10 Mb less DNA (Melville, et al,1998), reducing the amount of funding and sequenc-ing required quite considerably. It is capable of com-pleting the life cycle, indicating that all vital genesare present and it is likely that much of the extraDNA consists of expansion of multicopy DNA –gene familes and repeats. Nevertheless we mustalways remember that trypanosomes are phenotyp-ically variable, and that the basis of this variationmust lie in the genome. It will require thought andinnovation to find methods to compare the geno-types of phenotypically variable strains.

Both 927/4 and the derivative 10.1 may be trans-formed with foreign DNA at the procyclic andbloodstream-form life cycle stages. Transformationof the bloodstream-form is less efficient than instrain 427, but this is common to most strains thathave not been replicated in vitro for long periods. Aline of GUTat10.1 expressing the tetracycline (TET)repressor has also been produced and tested. This isuseful for any experiment involving inducibleexpression or inducible ablation of transcription/translation, and this line is currently available fromProfessor Christine Clayton, Heidelberg (van Deur-sen, et al, 2001).

As stated in the introduction to this section, stock927/4 is found to be ‘intermediate’ in its resistanceto lysis by human serum (Turner, personal commu-nication). The SRA (serum resistance-associated)gene has been found in the genome databases (Pays,personal communication), although it is not yetknown if it is expressed in the correct form. The-refore, bearing in mind that T. b. brucei and T. b. rho-desiense may only be genotypic variants of a singlespecies and that the basis of serum resistance maynot lie exclusively in SRA expression, it should beassumed by all researchers using 927/4 and itsderivatives as bloodstream forms in experimentsthat there may be human-infective cells in the po-pulation.

Transformation TechnologyTransformation technology for genetic analysis ofT. brucei is in routine use (Clayton, 1999). There aremultiple vectors, several types of transformationmarkers (drug resistance, fluorescence) and a sys-tem of inducible expression. Foreign DNA inserts


into the genome by homologous recombination andthis can be precisely targeted if the exact sequence ofthe target site is known or determined, as recombi-nation occurs preferentially at sites of exact homolo-gy. Expression from episomal vectors is less success-ful. At Tri-Tryp 2000, there was some discussion ofthe need for technology development. There was aproposal that enhanced transformation efficiency ofbloodstream forms is one requirement for high-throughput analysis (e.g. of gene knockouts orRNAi mutants) of the mammal-infective form, astransformation of the procyclic forms (which ismore efficient) followed by transformation of thecells to bloodstream forms requires passage throughthe tsetse fly. Of course, the ability to culture otherlife cycle stages, e.g. metacyclic forms, would alsorepresent a considerable improvement in the toolsavailable for genetic analysis.

MicroarraysSome doubts have been expressed regarding theusefulness of expression analysis in the Kinetopla-stidae. The observed polycistronic transcriptionsuggests that control of gene expression is entirelypost-transcriptional, a possibility supported byreports of protein products of genes within the samepolycistrons that were found to be up/down-regu-lated at different life cycle stages. Nevertheless,there clearly is some life cycle stage-specific tran-scription and this is useful data for investigation ofprocesses such as infectivity, etc. It is easy to obtainlarge amounts of cultured procyclic RNAs frommany strains, but perhaps prudent to perform someexperiments to compare cultured procyclics withthose extracted from tsetse flies. It is possible toobtain sufficient RNA from bloodstream formsreplicating in laboratory rodents, although slow-growing (e.g. less virulent) strains can prove moreproblematic (but the rate of improvement inmicroarray techniques is still in the exponentialphase). Unfortunately, it is very difficult to obtainsufficient DNA from epimastigotes or the infectivemetacyclic forms. The metacyclic life cycle stage isarguably the most relevant for studies of infectivity,and for vaccine development. With the involvementof researchers with tsetse fly colonies, it may be pos-sible to perform some careful experiments withmetacyclic DNA after full optimization of the arraytechnology.

Still, for the examination of wild-type/referencestrains, it remains necessary to carry out specificexperiments that detect changes in levels of individ-ual mRNAs after processing of polycistronic tran-scripts, and to compare these to the concomitant lev-els of the protein products. This will provide data onthe relative importance of post-transcriptional and

post-translational controls on gene expression, andwill provide a firmer basis for the design of manyexperiments. For example, if transcription levels arefound to be relevant in at least some cases, microar-ray technology could be very useful to comparetranscription in strains of different clinical pheno-type, or to examine mutant strains, to observe notonly the ablation of the targeted transcript but alsoany “knock-on” effect on other genes. For now, thediscriminative power of microarray technology insome respects remains unproven. It also remains anopen question whether an array based on a singlestrain (927) is sufficient to detect all relevant differ-ences; it is possible that strains carry environment-specific genes and that strain 927 lacks the genes ofinterest. Nevertheless, if arrays are made availablefor use by the community, such preliminary (”look-and-see”) investigations could be carried out at littlecost. No doubt as production costs become less pro-hibitive, the number of strains from which arraysare available will increase.

The materials currently available for the creation ofstandard DNA-spot microarrays are the ESTs andGSS clones. The ESTs almost all derive from blood-stream forms. Two GSS libraries have been used:one with average inserts of 2 Kb and the other withinserts of of 4 Kb. The GSS libraries may prove asuseful as EST libraries for microarray analysis dueto the high gene density in the genome, and they arecertainly more representative of all the genes in thegenome than are EST libraries. However, the clonesmay contain fragments of more than one gene, thuscomplicating full analysis of the dataset. The useful-ness of GSS arrays is being investigated (Clayton,Heidelberg). Meanwhile, El-Sayed (TIGR) has pro-duced the first ORF-specific array by amplifyingfragments of ORFs identified from the sequence ofchromosome II (El-Sayed, et al, 2000). A UK group(Melville, Matthews, Turner, Sanger Institute) isdeveloping a pilot project to test the relationshipbetween post-transcriptional and post-translationalprocessing. The long-term aim, if the usefulness ofmicroarrays is proven, is to establish whole genomeORF-specific microarrays within four years as a col-laboration between TIGR and the UK group. Thesewould have to be made available as a resource -either the arrays themselves, the DNA, the oligonu-cleotides, or simply the oligonucleotide sequence. Itis hard to predict at the present time what thedemand will be or how the technology will develop.

An additional aim of the network is to develop stan-dard operating procedures for microarray analysisand presentation of data in collaboration with otherorganizations, including the EBI, where they areestablishing a public repository for microarray-based


gene expression data (http://www.ebi.ac.uk/array-express/). These organizations are, in turn, mem-bers of the Microarray Gene Expression DatabaseGroup (MGED), whose aims are to facilitate theadoption of standards for DNA-array experimentannotation and data representation, as well as tointroduce standard experimental controls and datanormalization methods (http://www.mged.org/).

ProteomicsProteomics is a rapidly developing field thatencompasses small-scale 2-D gel analysis to high-throughput techniques such as mass spectrometrythat are not available to all. Analysis of the T. bruceiproteome is ongoing in several laboratories (e.g.Wellcome News issue 26, Q1, 2001, p. 19). However,for a coordinated network approach to global pro-teomic analysis, it is necessary to develop standardoperating procedures and common marker proteinsbefore any proteomic data are presented on the web.This was discussed at some length at Tri-Tryp 2000,and the first step towards this goal in the T. bruceinetwork is to combine proteomic and array analysisas discussed in section 3.5.

Novel DNA-Based Markers for Epidemiology,Diagnosis and/or Positional CloningDNA-based markers have become increasinglyimportant in epidemiology and diagnosis in the lastdecade. The availability of genome sequence offersthe capacity to identify a whole range of new mark-ers (microsatellites, minisatellites, single nucleotidepolymorphisms or SNPs, indels) at much greaterefficiency than before. Previously, satellite markerswere identified by cloning and hybridization ofindividual oligonucleotides, or by serendipity,whereas now they are identified using informatics.The identification of SNPs and indels requires se-quence from different strains, but may provide theadvantages of isoenzyme analysis – stability overmillennia - at less cost in terms of money and resear-cher time. There is no doubt that numerous researchgroups are considering using the DNA sequence inthis way, and we may see more papers appearingusing recently identified polymorphic markers.

It seems, then, timely to suggest that these datacould usefully be collated at a central site ratherthan leaving individual researchers to draw up theirseparate lists from the literature. This may also pro-ve especially useful to scientists in institutions withless access to journals. This is not to subvert the useof publication, which is vital to all researchers, butrather to suggest that, on publication, all markersshould be submitted to a central database and thatthis database should be available to all – on the weband, if necessary, on CD (see section 3.1). Such a

database could include:• unique marker name/identifier.• type of marker.• oligonucleotide primer sequences.• polymerase chain reaction (PCR) parameters.• size(s) of PCR product in genome project refer-

ence strain 927.• brief description of population studied and

results.• list of allele variants in named isolates with

details of isolate history.• references.• coordination with physical mapping/sequencing

data to provide information on genomic location,etc.

Over time, sufficient information should accumu-late to facilitate the choice of suitable markers forgiven areas and defined experiments, withoutextensive literature searching or unnecessary collab-orations.

RNA Interference as a Method to Investigate Gene FunctionA common approach to the investigation of genefunction is to remove the gene from the genome(gene knockout) or to prevent its transcription andtranslation. Gene knockout requires two rounds oftransformation and gene knockout, as each gene ispresent as diploid alleles. RNAi functions by trans-formation of a plasmid encoding a double-strandedRNA that is homologous to part of the transcriptunder study. By an unknown mechanism, the pres-ence of the exogenous dsRNA prevents the transla-tion of the endogenous mRNA (Ngo, et al, 1998).This requires only one round of transformation andis useful for the analysis of function of single-locusand multi-locus genes. To date, RNAi is most suc-cessful in procyclic forms, although many groupsare now working to improve efficiency in blood-stream forms.

A group of UK researchers (M Field, ImperialCollege, et al) has recently been awarded a grantfrom the Wellcome Trust Functional GenomicsCommittee to begin analysis of gene function inT. brucei by RNAi . This will involve analysis of phe-notype following systematic disruption of individ-ual genes identified on the sequenced chromosomesI and II. It is the requirement of these types of grantsthat both the data and the biological resources aremade available to the community. The data will beweb-accessible and prepared in collaboration withthe functional genomics database (see section 3.1).Again, it may be necessary to consider how thisinformation may be accessed from areas with inse-cure access to the Internet. The DNA resources (con-


structs of vector plus gene sequence for transforma-tion) will be available from Melville/Cambridge(see section 4.5). It is hoped that the transformed celllines (i.e. 927 mutants) will be available fromTurner/Glasgow (see section 4.6).

Applied Genomics: Discovery of Novel Drug TargetsThe authors are not experts in drug target validationand, to avoid charges of naivety, first wish to ack-nowledge the considerable work by leading groupsof biochemists on parasite-specific pathways thatmay represent valid targets for design of drugs ofgreater specificity. No doubt such projects will bebrought to the discussion table by other contribu-tors. However, under the maxim that no singlemethod guarantees the discovery of a better drug,and that all methods (rational design and globalapproaches) have potential for both success and fal-se leads, we wish to pose the questions: is it neces-sary to expand our efforts now to increase the num-ber of leads on the table? and how can we best usethe limited funds available? This discussion needsto take place across a wide range of researchers withdifferent skills. As observed by members of thepharmaceutical industry when faced with the massof new genomic information, we may yet need fur-ther breaking down of the barriers between scientif-ic disciplines (Browne and Thurlby, 1996).

”Until recently genes have been cloned andexpressed usually as the result of targeted pro-grammes of research, their biochemistry and phar-macology evaluated, high-throughput mechanism-based screens devised and leads identified for opti-mization by chemists ... In this traditional model ofpharmaceutical research, gene identification hadoften been considered rate limiting. The paradigmhas shifted.... We need to be able to predict whichgenes may be useful and why... there are potentiallymany, many more proteins to work with (and) theabundance of opportunities (must) impact on con-ventional research strategies”... (extracted fromBrowne and Thurlby, 1996).

The following observations need critical assess-ment: • The mass of genomic sequence provides the pri-

mary data for discovery of new proteins and newmetabolic pathways. Initially, we will be facedwith thousands of novel genes for which we haveno function. At this stage, bioinformatic analysis(of metabolic pathways, for example) and system-atic (preferably high-throughput) analysis of genefunction (as attempted in the RNAi analysis de-scribed in 3.8) will be important. We would expectthat these kinds of analyses will provide new

leads for biochemists to investigate further at amolecular level and/or for design of high-throughput combinatorial screening program-mes. However, the latter will require funding forthe considerable work involved in such analyses,even in the preliminary stages.

• At this stage, the retention of such work in theacademic sector due to lack of interest from phar-maceutical companies could be exploited to en-sure that such studies are coordinated, data sha-red, basic functional data all web-accessible, andeffort is not wasted. (Although, future problemswith intellectual property rights have to be con-sidered.)

• The establishment of the genome network hasprovided one paradigm of how the research com-munity can pull together to collaborate ratherthan compete. There are already well-establishedgroups with an interest in increasing funding forthe discovery of new drugs for diseases of thepoor, e.g. TDR working groups, the Access toMedicine Campaign. Is it now possible to bring(sections of) these disparate groups together todiscuss a new “network”?

• Currently in the UK there are possibilities toapply for funding for “functional genomics”, afundamentally different activity to basic, hypoth-esis-driven research. Are funding agencies inother countries initiating such programmes? Isthis a good moment to use these openings to con-sider an “applied genomics” application, aimeddirectly at drug discovery? If Wellcome Trust-based, this would involve UK researchers, but theTrust recognizes the need to involve internationalgroups.

• It is possible that an early-stage drug would bemore attractive to manufacturers if it were usefulagainst several parasites, for example all kineto-plastids. Some effort should be put into bioinfor-matics comparison of kinetoplastid genomes asthey become available. This could provide infor-mation on common parasite pathways that differsubstantially from those found in humans andmammals.

Applied Genomics: High-throughput Testing of Vaccine CandidatesDiscussion of the identification and testing of vaccinetargets following analysis of genomic information iseven more subject to charges of naivety than feared insection 3.9. To the limited knowledge of the authors,attempts have been made by numerous skilled work-ers to identify trypanosome proteins that may serveas vaccine candidates, only to be thwarted by thedense coat of variable surface protein that preventsaccess by antibodies to invariant proteins, and theefficiency of antigenic variation. It is not immediately


obvious how the availability of genome sequenceand the discovery of novel genes and pathways mayprovide new approaches, given these results. Theonly high-throughput parasite vaccine testing projectknown to the authors is that of Professor J Blackwell,who is testing pooled DNA vaccines against leishma-niasis. All the resources for such a project applied toinfection with T. brucei are available, but any assess-ment of applicability should involve a wider range ofresearchers with appropriate skills.

SHARING OF INFORMATION AND ACCESSTO DATA AND RESOURCES: SUMMARIESThe T. brucei Genome ‘Network’It can seem difficult to obtain information from, orto know how to “join”, the genome network. This isnot in any way due to exclusivity as such, but be-cause it works as any other network – people whoknow each other and who have common interestscome together in subgroups to seek funding. Inevi-tably this is also driven by the availability of suitablefunding for functional/applied genomics in differ-ent countries – for example, the Wellcome Trust(UK) is currently the most forward planning in pro-viding funds for global analyses, reducing its insis-tence on hypothesis-driven research etc., so, pre-dictably, recent groupings have been UK-driven. Forthis reason it is very important that WHO continuesto direct some funds towards helping those Africanscientists with an interest in this work to travel tonetwork meetings, to make contacts, to set up col-laborations and to talk about priorities. This latterpoint is very important: there are many very goodbiologists working on the fascinating basic biologyof T. brucei, but their priorities may differ somewhatfrom those of the endemic countries and only thepresence of outspoken scientists whose interests liein controlling the disease will ensure that we do notlose sight of the necessity to direct funds towardsdisease-applied genomics.

Obtaining InformationUntil now, information dissemination has beenunderfunded and very dependent on the commit-ment of certain individuals to spreading the word,maintaining websites, and being approachable andamenable to email requests for information and ex-planations. But it is increasingly recognized that col-lected information and properly run databases arebecoming vital in our brave new world of collabora-tive “big science”, and as more resources are madeavailable towards these aims, it should become eas-ier to obtain up-to-date information on the progressof genome network-based projects. However, it isalso inevitable that these information resources willbe web-based. It is therefore vital to consider now

how we can ensure that the entire research commu-nity has access to information. In the near future thismay necessitate distribution of CDs, in which casehow should this be coordinated? In the more distantfuture, WHO and those institutions who see a rolefor such web-based information must seek a path toimproved institutional infrastructure and electronicresources.

Access to Unannotated and Annotated Sequence DataAll raw unannotated sequence data are made avail-able immediately on the websites of the sequencingcentres, and then in the public databases. However,access to annotated data is more problematic. TheSanger Institute is unable to annotate fully untilclose to completion of a chromosome, due to theirshotgun approach. Their provision of preliminaryannotation of chromosome I caused considerableconfusion as researchers did not fully appreciatethat the chromosome was discontiguous. TIGR isable to provide some annotation of completed BACsas they are finished, but this approach does not pro-vide as many raw sequences as early on in the proj-ect. This problem is unresolved and subject to muchdiscussion at network meetings. One interim solu-tion is provided by the clustering and BLAST analy-sis of discontinuous sequence data (GSS, shotgun,ESTs)(http://www.sanger.ac.uk/Projects/T_bru-cei/Toolkit/blast_server.shtml; hftp://ftp.sanger.ac.uk/pub/databases/T.brucei_sequences/GSS/clusters/). Many researchers annotate the small re-gions of immediate interest to them, but for some sci-entists this requires some bioinformatics training. Wehope that some of these problems will be addressedby the creation of the relational genome database.

Access to Comprehensive Genome Information in Relational DatabasesAs stated many times above, access to all databasescurrently under development will ultimately requiresecure access to the Internet, although provision ofCDs may be an interim solution. In addition to thegenome database to be hosted at the Sanger Centre(section 3.1), a separate Wellcome Trust-funded proj-ect (Melville, see 4.5) will also provide genome map-ping data in a relational database that will be com-munity-interactive and will facilitate some analysesbefore it is superceded by the complete genomesequence. Once again, this will be web-accessibleand, while it is possible to provide copies on CDs,personnel resources are limited to providing fullexplanations and guidance on a website.

Obtaining DNA Resources for Use in Research ProjectsAll biological resources used by the genome net-


work are available to researchers on request, includ-ing the TREU927/4 stock and 10.1 derivative, high-density filters of genomic libraries, genomic DNAclones, cDNAs and karyotype blots (Melville, et al,1998; http://parsun1.path.cam.ac.uk). The genomewebsite provides addresses and email links. Thegenome network insists that hybridization andsequence data derived using its resources are re-turned to the database curator for inclusion in thegenome database (http://parsun1.path.cam.ac.uk).These resources were provided with funding fromWHO/TDR for five years. Since the beginning of 2001,the Wellcome Trust Functional Genomics Initiative hasprovided a four-year programme grant to expand thisfacility to assist the growth of functional genomicsprojects within the network (Melville, Cambridge).

Obtaining Reference and Mutant Strains for Use in Research ProjectsAs part of the above project, a collection of referencestrains, transformed lines and mutants will be main-tained and provided to researchers on request (Tur-ner, Glasgow). Current funds are not sufficient toconsider curating large field collections. The sharingof well-curated field collections may be of value incertain comparative genomic projects and could beoffered up for discussion. Many institutions havegood collections of primary or very limited passageisolates that could be harnessed as a valuableresource. WHO is supporting efforts to establishgood quality databases in a number of places, inclu-ding Kenya Trypanosomiasis Research Institute(KETRI).

INVOLVEMENT OF SCIENTISTS FROM TRYPANOSOMIASIS-ENDEMIC COUNTRIESIt is with some disappointment that we report thelow level of participation of African institutions inthe genome network to date. There may be a rangeof reasons for this: inappropriateness of the tasks toinstitutional aims or facilities, competition fromother laboratories with access to materials and thefacilities to perform high-throughput tasks withgreater efficiency, disinterest in genomics given themore pressing problems of control of livestock infec-tion and human epidemics or research on pathogen-esis etc., or lack of sufficient contacts and encourage-ment. Maybe we need not be too disappointed thatAfrican laboratories did not play a central role in theprocess of sequence generation itself (other than theESTs, the majority of which were generated inKenya; and we should also note the employment ofAfrican scientists in European and US laboratories).But we should consider it a scandal if this tremen-dous resource, initiated by TDR, is not made avail-able to the entire global research community and isnot applied to the full to problems of control and

treatment of trypanosome infection. All scientistsshould have access to such data as will simplifyhypothesis generation and experimentation, whe-ther their interests lie in basic biology or in appliedscience. In addition to the promotion of good scien-tific experimentation, it seems likely at the currenttime that African scientists must help persuade inter-national funding bodies and the scientific communi-ty to apply the data for the development of novelepidemiological tools, drugs and vaccines.

The area of genomics and applied genomics hasexpanded considerably over the past decade or so;the driving force in this being the development ofbioinformatics tools that make access to the avail-able data possible. Quite important also is the widerange of publicly accessible tools developed to makeuse of these data. Yet many African scientists whocould use such information cannot, or do not, formany reasons. While this Scientific Working Group(SWG) will address the problem of African try-panosomiasis, there is no doubt that the opportuni-ties and impediments are generic, and hence appli-cable to virtually all disease problems in endemiccountries in Africa, and perhaps in most developingcountries. Increasing the ability of African scientiststo participate in the application of data derived fromgenome projects will stimulate research activitiesnot only in this area, but also in a multidisciplinarymanner, since such data have to be evaluated inprocesses that require broad-based competencies.

We propose here ways in which participation in A-frica, outside of international institutions, can bedeveloped and strengthened.

Bioinformatics (Present Competence and Training Needs)Although there is some competence in bioinformat-ics within the science community in Africa, it islargely fragmented and inadequate. To quantify itwould be virtually impossible. However, it is knownthat this capacity is mainly concentrated in interna-tional institutes, a situation that is inappropriate,and efforts should be made to extend this to univer-sities and national research centres. One way ofbeginning to do this is to develop a series of trainingcourses in bioinformatics, either continent-wide orregionally, to develop Africa’s capacity in bioinfor-matics. The value of this is two-fold:• It will equip participants with knowledge on what

information is available, where it is, how it can beaccessed and utilized to develop tools and meth-ods to address public health problems.

• It would begin to expand the human resourcebase in bioinformatics among African scientists,hence contributing to the expansion of the


research activities in African trypanosomiasis andother major disease situations. This would createan environment for continuous in-house training.

It is anticipated that such courses would bring to-gether expertise from within Africa and elsewhere(in or outside the African trypanosomiasis commu-nity). Doing this would help build the necessarypartnerships (within and across diseases) to sustainthe knowledge base in bioinformatics. It may be pos-sible to bring in experts in computing who have lit-tle biological sciences training but who are willing touse their expertise in biology.

Capacity building in bioinformatics can also beenhanced through providing funding support (byTDR and other donors) for Internet service provider(ISP) and access charges within projects. Addressedas a budget item in relevant proposals, this couldinclude visits to laboratories with expertise in bioin-formatics (north or south). We refer you also to theproposal from the Pathogenesis and Applied Geno-mics Committee for regional bioinformatics courses.If the funding is secure and consistent, these may gosome way to meeting these aims.

Infrastructural CapacityWorldwide, the capacity of personal computers(processor speed and storage space) continues toimprove phenomenally. Africa is no exception. Elec-tronic access to information is often limited due topoor communication infrastructure; good, fast anddedicated connections are few and far between, andoften only reliable in international institutions. Withproper training, however, good quality informationcan still be obtained by email via a telephone line,even where institutions have a very limited numberof computer terminals with on-line access (even ifonly one terminal with limited time slots). Im-provement of institutional capacity should continueto be an important objective; admittedly, maximalaccess to databases is only achievable in an environ-ment that recognizes the value of bioinformatics.

TDR can help by giving small grants, perhaps aboutUS$2000 annually, to national institutes to fund elec-tronic access. Such funds would be used to pay ISPand connection charges to individual scientistswithin national institutes and universities. Suchfunding would not necessarily be linked to specificproject funding, although applicants could beencouraged to budget for this within projects.

Institutional LinkagesAfrican scientists who have undertaken some train-ing in bioinformatics have experience that can beshared. However, there is not enough linkage with-

in the continent for within-continent exchange ofexpertise. In some cases, such limitations exist evenwithin countries. One reason for this is that thevalue of bioinformatics is not well recognized bymanagers of national institutions. Sharing of infor-mation across areas of disease interest is also notgood, especially in African trypanosomiasis, wheremany institutes have a single disease mandate.Thus:• there could be closer interaction between national

and the international institutions where there isgreater capacity to apply data derived from geno-me projects, to use the available resources moreefficiently.

• there should be greater effort to promote broadmultidisciplinary projects constituted in part bythe use of applied genomics. Such linkages willenhance the use of available information fromvarious genome projects to address different bio-logical questions.

Retention of Trained PersonnelIt may be prudent to say that no single agency canguarantee that good quality personnel are retained inresearch, and particularly in national systems withindisease endemic countries. However, important con-tributions towards stemming the outflow of person-nel can be made, even by a single stakeholder.

Perhaps the flight of personnel largely accounts forthe paucity of good quality funding applicationsfrom Africa to address the problem of African try-panosomiasis. In the terms of reference for thisSWG, TDR recognizes that “many well trained per-sonnel in African trypanosomiasis have left the fieldfor others, such as HIV/AIDS”. Personnel haveindeed left in many directions, including to othercountries, primarily in the north, to other researchareas within Africa, or even out of science.

While it has been suggested that TDR could followa strategy of choosing a few studies and the centresto carry them out, the success of this will depend onthe retention of good quality personnel in the field.Within Africa, the SWG should consider:• allowing African investigators working in their

own countries to draw salaries or salary supportfrom projects funded by TDR, since poor remu-neration is a major impediment to creating a criti-cal mass for research.

• promoting collaborative initiatives between coun-tries in the south, where this is scientificallysound, hence expanding the collaborations thatexist currently.

• encouraging collaborative projects (north-south;south-south; inter-institutional within countries)that have a bioinformatics (and bioinformatics


training) component in order to strengthen thecapacity to apply genomics data to address publichealth problems.

FUNDING AND HUMAN RESOURCES:REQUIREMENTS AND OPPORTUNITIESThe genome sequence will be a valuable resource forthe research community, but there is much to bedone to turn its potential for new directions and newdiscoveries into reality. No researcher need feel thereis no opportunity to become involved. However, in-volvement will depend heavily on having access tothe available information and contact with othergroups. One way to become involved in the genomenetwork is to develop an interest group, investigatethe resources available, then look for funding to usethe genomic data for a purpose – such a group maythen be “hooked in”. The RNAi group is one exam-ple, a drug discovery group may be another.

The next phase of the sequencing revolution will be“comparative genomics”. Technologies that aim tosimplify the resequencing of different genotypes(strains) are in development. Although it hardlyseems possible that we might be able to sequencemore than one genome, only five years ago wedidn’t believe we could sequence even one. Now isthe time to identify those species and strains thatwould provide vital information to researchers ifcomparative sequence information were available.

Funding agencies are increasingly realizing the va-lue of large collaborative groupings and high-throu-ghput techniques. It is timely to consider the poten-tial of novel applications of genomic methods tocurrent problems, as the openings are there. TheWellcome Trust is in the forefront of such develop-ments. The Trust also has an active internationalprogramme in (inter alia) tropical disease research(http://www.wellcome.ac.uk/en/1/biosfgintintfu-nunkcrg.html) and has recently indicated its desireto achieve a higher profile for this programme.Therefore, at this point in time, north-south col-laborations may be seriously considered: both fullinter-institute collaborations and relatively minorcollaborations that facilitate technology transferand the kind of training and contacts that enablescientists to develop an independent career.

Some projects mooted here are large and costly,requiring coordination and commitment. WhileTDR funds may not be sufficient to underpin coor-dinated applied genomics projects, it is importantthat working groups such as this take a lead role indefining the problems that need to be addressed, toensure that the genomics revolution does not bypassthose directly affected by trypanosomiasis.

RECOMMENDATIONS• Improve institutional capacity in Africa to access

data from genome projects by acquiring comput-ing hardware suitable for bioinformatics (throughprojects or independently). In the long term, thiswill be vital for full involvement in molecular bio-logical research on trypanosomes.

• Identify possibilities for adding value in the colla-tion of data, for example in a central database forDNA-based markers for use in epidemiologicalstudies.

• Identify areas of bioinformatic analysis that lacksupport, e.g. identification of metabolic path-ways, comparison of pathways across the Kineto-plastidae. Identify the researchers required forsuch projects.

• Identify now the requirement for further geno-mic analysis and sequencing of other strains(e.g. gambiense and other clinical variants) andspecies (T. congolense, T. vivax). This may becomplete genomic sequencing or shotgun se-quencing to a given level (e.g. 3x, 7x, 10x) andcould be based, once again, in high-throughputor local centres.

• Consider the techniques available for comparisonof strains, including the possibility of compara-tive sequencing (e.g. of gambiense).

• Consider inclusion of genomics experts in ongo-ing drug development working groups.

• Ensure that the voices of African scientists andapplied scientists are heard within the genomenetwork. Bring a drug development working par-ty into the network.

• Encourage the dissemination of information thatmay lead to the formation of new interest groups(where possible within existing groupings, e.g.TDR, the Programme Against African Trypano-somiasis (PAAT), Global Forum on AgriculturalResearch (GFAR), to discourage proliferation) toaccess the genomic resources for new projects.

• Consider current possibilities for funding of geno-mics-based projects leading to drug target identi-fication and validation; in this context, reevaluatethe potential for inclusion of scientists from try-panosomiasis-endemic countries.

• Facilitate training in bioinformatics to increasecompetency and promote the utilization of geno-mics data in Africa. We take note of the call byTDR for proposals in bioinformatics and appliedgenomics to develop research and training cen-tres/networks in Africa, Asia and Latin America.

• Encourage the establishment of contacts betweenscientists/institutions (in Africa and elsewhere),through exchange visits, training courses and jointproposals, to maximize additive competencies.

• Improve the remuneration of African scientistsworking on TDR-supported projects in order to


retain trained personnel, hence improving institu-tional and national research capacity.

• Promote collaboration between countries (South-South; North-South) to take advantage of sharedexperiences and of possibilities for shared funding.

AcknowledgementsSome of the text included here is extracted from:Melville SE. Characterisation and sequencing of theAfrican trypanosome genome. In: Guidelines andissues for the discovery and development of drugs againsttropical parasitic diseases, Vial H, Fairlamb A, RidleyR, eds. World Health Organization (2001). We thankall our colleagues for discussion of the points pre-sented here and especially the members of thegenome network for their open sharing of informa-tion and progress.

ReferencesBarry JD, McCulloch R. Antigenic variation in try-panosomes: enhanced phenotypic variation in aeukaryotic parasite. Advances in Parasitology, 2001,49:1-70.

Browne MJ, Thurlby PL, eds. Genomes, molecular biol-ogy and drug discovery. Academic Press Limited,1996.

Clayton CE. Genetic manipulation of kinetoplastida.Parasitology Today, 1999, 15:372-378.

Djikeng A et al. Generation of expressed sequencetags as physical landmarks in the genome ofTrypanosoma brucei. Gene, 1998, 221:93-106.

El-Sayed N et al. The African trypanosome genome.International Journal for Parasitology, 2000, 30:329-345.

El-Sayed NMA, Donelson JE. A survey of theTrypanosoma brucei rhodesiense genome using shot-gun sequencing. Molecular and Biochemical Parasi-tology, 1997, 84:167-178.

El-Sayed N et al. cDNA expressed sequence tags ofTrypanosoma brucei rhodesiense provide new insightsinto the biology of the parasite. Molecular andBiochemical Parasitology, 1995, 73:75-90.

Melville SE, Gerrard CS, Blackwell JM. Multiplecauses of size polymorphism in African try-panosome chromosomes. Chromosome Research,1999, 7:191-203.

Melville SE, Majiwa P, Donelson J. Resources avail-able from the African trypanosome genome project.Parasitology Today, 1998, 14:3-4.

Melville SE et al. The molecular karyotype of themegabase chromosomes of Trypanosoma brucei andthe assignment of chromosome markers. Molecularand Biochemical Parasitology, 1998, 94:155-173.

Melville SE et al. Selection of chromosome-specificDNA clones from African trypanosome genomiclibraries. In: Analysis of non-mammalian genomes,Birren B, Lai E, eds. New York, Academic Press,1996, pp 257-293.

Ngo H et al. Double-stranded RNA induces mRNAdegradation in Trypanosoma brucei. Proceedings of theNational Academy of Sciences, USA, 1998, 95:14687-92.

Rudenko G et al. Selection for activation of a newvariant surface glycoprotein gene expression site inTrypanosoma brucei can result in deletion of the oldone. Molecular and Biochemical Parasitology, 1998,95:97-109.

Tait A et al. Genetic analysis of phenotype in try-panosoma brucei: a classical approach to potentiallycomplex traits. Proceedings of the Royal Society (in press).

Turner CMR, Melville SE, Tait A. A proposal forkaryotype nomenclature in T. brucei. ParasitologyToday, 1997, 13:5-6.

Turner CMR et al. Evidence that the mechanism ofgene exchange in Trypanosoma brucei involves meio-sis and syngamy. Parasitology, 1990, 101:377-386.

van Deursen FJ et al. Characterisation of thegrowth and differentiation in vivo and in vitro ofbloodstream form Trypanosoma brucei strain TREU927. Molecular and Biochemical Parasitology, 2001,112:163-171.

Vanhamme L, Pays E. Control of gene expression intrypanosomes. Microbiological Reviews, 1995, 59:223-240.

Weiden M et al. Chromosome structure: DNAnucleotide sequence elements of a subset of theminichromosomes of the protozoan Trypanosoma bru-cei. Molecular and Cellular Biology, 1991, 11:3823-3834.

for example, see Wellcome News issue 26, Q1, 2001, p. 19.


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Annex 7INSTITUTIONAL CAPABILITY STRENGTHENING


INSTITUTIONAL DEVELOPMENTAND CAPACITY BUILDING IN COUNTRIES ENDEMIC FOR SLEEPING SICKNESS

J. Mathu Ndung’uKenya Trypanosomiasis Research Institute (KETRI),P.O. Box 362 Kikuyu, Kenya

INTRODUCTIONThis report is intended to stimulate discussion on thepossible solutions available for improving researchand training in, and strengthening institutions for,effective delivery of services in the 37 countriesaffected by trypanosomiasis and the tsetse vector.

The financial crisis that occurred in Africa in the 1980sand 1990s had an almost paralysing effect, leading to:• lack of funds to maintain physical infrastructure,

vehicles and equipment, or to replace outdatedand broken-down equipment.

• decline or lack of funding, at both national andinstitutional levels, to carry out field surveillanceand control of tsetse, and field and laboratoryresearch in African animal trypanosomiaisis andsleeping sickness.

• poor remuneration of staff, hence internal andexternal brain drain of trained manpower and,therefore, paucity of leadership.

Given this background, and considering that deter-mined efforts made over the years to solve the prob-lems have not had the desired impact, a more ration-al approach to research, training, and institutionaldevelopment is indicated. Issues arising include:number and size of available research and traininginstitutions; appropriateness or otherwise of any re-search and training undertaken; incentives for staff toremain on the job, e.g. career development, adequateremuneration and an enabling environment.

INSTITUTIONAL DEVELOPMENT One of the recommendations made at the 1974 FAOExpert Consultation on Animal Production andHealth Research in Copenhagen, Denmark, was toprovide facilities for training research workers ”intheir own environment” and to strengthen existingnational and regional institutes. Presently, most re-search institutes established pre-independence orimmediately post-independence in Africa are in adeplorable state due to breakdown of physical infra-structure, equipment and vehicles, lack of funds forlaboratory and field work, and severe depletion ofresearch and field staff.

Rehabilitation and strengthening of existing nationaland regional institutions to carry out appropriate

training, research and control work, must remain apriority. The first step would be to carry out an in-ventory of existing research and training institutions,and identify areas of immediate and long-term con-cern. This should assist in the identification of nation-al and regional institutions in Africa which could bestrengthened by the international community in col-laboration with national institutions and govern-ments. Linkage arrangements, between national andoverseas institutions with donor support, need to befurther explored, strengthened and extended.

TRAININGIn the past, training specifically addressed the prob-lem of tsetse and trypanosomosis in the laboratoryand field with a view to ensuring long-term com-mitment and contribution towards solving the prob-lem. However, this training did not take fully intoaccount some vital concerns, e.g. that African try-panosomiasis is just one of the many health prob-lems Africans are exposed to, the solution of whichdemands a broad understanding and approach.

To retain the interest and commitment of personnelwho have been trained, there is a need for careerdevelopment. In recent times therefore, efforts havebeen directed at broadening of training. Yet muchremains to be done, especially in light of the devel-opments towards privatization of services, includ-ing in tsetse control. It is in this light, and in light ofthe competing demands by different sectors of theeconomy for limited funds, that institutional devel-opment and training in disease endemic countriesshould be viewed.

Four types of training are generally recognized:• Training at sub-professional level, leading to the

award of a diploma or certificate, e.g. animalhealth assistant, animal health technician.

• Training at professional level, leading to theaward of professional degrees and diplomas.

• Training leading to postgraduate degrees anddiplomas.

• Short-term training leading to specialization.

To these must be added the training of:• auxiliary personnel, e.g. clinical officers and field

assistants, who need short courses and/or in-ser-vice training for field work.

• field agents, whose activities are coming moreand more into the limelight because of their clo-seness to the communities in community-basedprimary health care.

• contacts and farmers (general training).

For the first four types of training listed, a prerequi-site for admission is formal education at secondary


school level, while on-the-job and in-service trainingare essential to develop and sharpen skills. In thecase of training of auxiliaries, the requirement is thatthey should have attended a secondary school, butmay have dropped out along the way. In the case offield agents, the ideal requirement is basic primaryschool education, emphasizing numeracy andenabling them to follow simple instructions andtraining and to be selected by the community whomthey will serve.

Training NeedsThe priority given to training of the different cadreswill vary from country to country but will largelydepend on:• the prevalence of tsetse and trypanosomiasis.• the impact of tsetse and trypanosomiasis on the

national economy. • the human, material and financial resources avail-

able for dealing with the problem.

While there is a great need for experienced profes-sional laboratory and field staff, there is, at the pres-ent time, much greater demand for specialized mid-dle-level personnel to carry out the arduous task offield and laboratory operations, and for auxiliary andfield agents who are crucial to the success of com-munity-based health care and field programmes.

Training InstitutionsWhile there is no problem finding institutions totrain personnel at professional and postgraduatelevels, there are serious problems with training formiddle-level specialized personnel. In the last fewyears, many national and regional training institu-tions which give emphasis to tsetse and trypanoso-mosis have either been reduced in size or closeddown completely because of funding difficulties.Yet this is the training area of great need. The num-ber of trainees that can be deployed to serve in thefield depends largely on the number and size of thetraining institutions available, and whereas concernhas been expressed, it appears that no solution tosustaining the necessary institutions is in sight.

Although there are certain advantages of tyingtraining in institutions to ongoing projects, e.g. withregard to the funding and training of personnel, thedisadvantage is that the lifespan and funding ofprojects is uncertain. What is needed is a traininginstitution with its own lifespan and funding, andwith participation from ongoing projects.

What then are other viable options? Would a donorconsider supporting two or more training institu-tions in Africa on a sustainable basis? Another sug-gestion, which at first may appear remote, is to

explore whether there is a place for private initiativein training.

STAFF RETENTIONThe problem of staff retention is a global one, notpeculiar to the field of tsetse and trypanosomosis.What makes the case of those in the field of tsetseand trypanosomosis different, however, is thatcareer advancement can be painfully slow and frus-trating in the public sector. Among the causes ofinability to retain staff are:• slow career advancement and lack of career

prospects.• poor salary and working conditions.• delay or non-payment of field allowances.• lack of transport.• lack of self-worth.• few or no opportunities to take part in short-term

and/or in-service training programmes, whichwere a major incentive in the past but which arenow scarce due to lack of funds.

The low remuneration and lack of career prospectsmakes recruitment of good staff for training verydifficult. Young, talented people tend to declineappointment unless they lack other job opportuni-ties. Trained staff, on the other hand, either leavetheir employment in favour of more lucrative eco-nomic activities and/or political appointmentstotally unrelated to their training, or, because of theneed to supplement their meagre pay through otherunrelated economic activities, tend not to devoteattention to the job.

SUMMARYTraining is needed at all levels, but more so at themiddle personnel and auxiliary levels. It is impor-tant to address the career prospects and other bene-fits of trainees, e.g. short courses and in-servicetraining that could encourage staff to remain in thejob for which they are trained.

The issue of funding research and training institu-tions on a sustainable basis must be addressed, forwhich it may be necessary to review the presenttrend of guaranteeing funding of research projectsfor a period of 12 months only. The possibility ofinvolving private initiative in training, and how thiscan be implemented without sacrificing quality,should be examined. South-south research andtraining should be encouraged. There is an urgentneed to find out the present status of nationalresearch and training institutions with a view toadvising national governments on the role of suchinstitutions in tsetse and trypanosomosis researchand control.


In order to provide an enabling environment forfield and laboratory research and training, insti-tutional development needs to be given new impe-tus. A possible starting point would be to carry outan inventory of the existing research and training in-stitutions to provide insight as to what type of stren-gthening may be needed. Linking national institu-tions and overseas institutions with donor supportis a possible area for expansion.


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Annex 8LIST OF PARTICIPANTS


Dr Serap Aksoy, Department of Epidemiology andPublic Health, Section of Vector Biology, YaleUniversity School of Medicine, 60 College St. 606LEPH New Haven CT06510 USA. Tel: 1 203 737 2180,Fax: 1 203 785 4782, E-mail: [email protected].

Professor Cyrus Bacchi, Pace University, HaskinsLaboratories, 41 Park Row, New York, NY 10038–1598, USA. Tel: 1 212 346 1246, Fax: 1 212 346 1586,E-mail: [email protected].

Dr MP Barrett, University of Glasgow Institute ofBiomedical and Life Sciences, Division of Infectionand Immunity, Glasgow G12 8QQ, Scotland. Tel &Fax; 44 141 330 6904, E-mail: [email protected].

Dr Kwabena Bosompem, University of Ghana, No-guchi Memorial Institute for Medical Research, P.O.Box LG581, Legon. Tel: 233 21 500 374, Fax: 233 21502182, E-mail: [email protected], [email protected].

Dr Reto Brun, Swiss Tropical Institute, Postfach,Socinstrasse 57, Basel, Switzerland. Tel: 41 61 284 8231, Fax: 41 61 271 86 54, E-mail: [email protected].

Dr Christian Burri, Swiss Tropical Institute, Po-stfach, Socinstrasse 57, Basel, Switzerland. Tel: 41 61284 82 47, E-mail: [email protected]

Dr Philippe Büscher, Instituut voor Tropische Genee-skunde, 155 Nationalestraat, 2000 Antwerpen, Bel-gium. Tel: 32 3 2476371, Fax: 32 3 2476373, E-mail:[email protected].

Dr Simon L Croft, London School of Hygiene andTropical Medicine, Department of Infectious andTropical Diseases, London WC1E 7HT. Tel: 44 171927 23 45, Fax: 44 171 636 87 39, E-mail:[email protected].

Dr Peter de Raadt, Bruglaan 11, 3743 J.B. Baarn, TheNetherlands. Tel: 31 35 541 21 21, E-mail:[email protected]

Dr Felix Doua, Projet des Recherches Cliniques surla Trypanosomiase (P.R.C.T.), B.P. 1425, Daloa, Côted’Ivoire. Tel: 225 32 78 36 10, Fax: 225 32 78 3021: E-mail: [email protected]

Dr John K Enyaru, Livestock Health Research In-stitute, P.O. Box 96, Tororo, Uganda. Tel: 256 4545050, Fax: 256 422 1070, [email protected].

Dr J Josenando, Instituto de Combate e Controlodas Tripanosomiases, 168, Kwenha, Ingombota, CP2657, Luanda, Angola. Tel: 244 2 399610, Fax: 244 2399611, E-mail: [email protected].

Professor Krister Kristensson, Division of Neurodege-nerative Disease Research, Department of Neuro-science, Retzius vag 8, B2:5, Karolinska Institutet, SE-171 77 Stockholm. Tel: 46-8-728 7825; Fax: 46-8-32 53 25,E-mail: [email protected].

Dr Grace B Kyomuhendo, Department of Womenand Gender Studies, Makerere University, P. O. Box7062, Kampala, Uganda. Tel: 256 774 71 600 or 256 41531484, Fax: 256 41 543539, E-mail: [email protected], or [email protected].

Dr Francis J Louis Institut de médecine tropicale, BP 46Le Pharo, 13007 Marseille, France. Tel : 33 4 91 15 01 46,Fax : 33 4 91 15 01 43, E-mail: [email protected].

Professor John Mansfield, Department of Bacte-riology, University of Wisconsin-Madison, 1925Willow Drive/FRI Bldg Madison, WI 53706 USA. E-mail: [email protected].

Dr Daniel Masiga, Kenya Trypanosomiasis Re-search Institute, P.O. Box 362, Kikuyu, Kenya. E-mail: [email protected].

Professor Ian Maudlin, Centre for Tropical Veteri-nary Medicine, the University of Edinburgh, EasterBush Veterinary Centre, Roslin, Midlothian, UKEH25 9RG. Tel: 44 131 650 7347, Fax: 44 131 650 7348,E-mail: [email protected].

Dr Dawson B Mbulamberi, Ministry of Health, P.O.Box 7272, Kampala, Uganda. Tel: 256 41 43087, E-mail: [email protected].

Dr Honoré Meda, Projet SIDA 2, Benin (ACDI-Ca-nada), 08 B. P. 900 Tri Postal, Cotonou, Républiquedu Benin. Tel: 229 31 36 02, Mobile: 229 957 169, Fax: 229 31 36 05, E-mail: [email protected].

Dr Sara E Melville, Cambridge University, De-partment of Pathology, Microbiology and Parasito-logy Division, Cambridge, CB2 1QP, UK. Tel: 441223 333335, Fax: 44 1223 333346, E-mail:[email protected]

Dr C Miaka Mia Bilengé, Ministère de la Santé,Secrétariat Général à la Santé, B.P. 3040 Kinshasa,République Démocratique du Congo. E-mail:[email protected].

Dr V Nantulya, Harvard School of Public Health,665 Huntington Avenue, Boston, 02115 MA, USA.Tel: 1 617 432 0659 , E-mail: [email protected].


Dr Joseph Ndung’u, Kenya Trypanosomiasis Re-search Institute (KETRI), P.O. Box 396, Kikuyu,Kenya. Tel: 254 154 32960-4, Fax: 254 154 32397, E.-mail: [email protected] or [email protected] [email protected].

Dr Martin Odiit, Livestock Health Research Insti-tute, P.O. Box 96 Tororo, Uganda. Tel: 256 45 45050,Fax: 256 45 45052, [email protected].

Dr Alexandra PM Shaw, A. P. Consultants, UpperCottage, Abbotts Ann, Andover SP11 7BA, UK. Tel:44 1264 710 238, Fax: 44 1264 710 759, E-mail:[email protected].

Dr Richard R Tidwell, Department of Pathology &Laboratory Medicine, University of North Carolina,Chapel Hill, NC, USA. Te1: 1 919-966-4294, E-mail:[email protected].

Dr Philipe Truc, Institut de Recherche pour le Déve-loppement UMR 035 “Trypanosomoses Africaines”,OCEAC, BP288, Yaoundé, Cameroun. Tel: 237 23 2232 or 237 84 60 57 (mobile), Fax: 237 23 00 61, E-mail:[email protected]

Collaborators and partnersDr Alain Aumonier, Director, International Affairs,Aventis Pharma International, Tri E1/385 - 20Avenue Raymond Aron, F-92165 Antony Cedex,France. Tel: 33 1 5571 6087, Fax: 33 1 5571 4185 E-mail: [email protected].

Dr Jean-Pierre Helenport, Consultant, Medécinssans Frontières/World Health OrganizationEflornithine Project, 2 Impasse des Bambous, VillaMaéva, 13600 La Ciotat, France. Tel: 33 4 42 981042,E-mail: [email protected].

Dr Anne Moore, Division of Parasitic Diseases, F22,Centers for Disease Control and Prevention, 4770Buford Highway, Atlanta GA 30341, USA. Tel: 1 770488 7776, Fax: 1 770 488 7761, E-mail: [email protected].

Dr TC Nchinda, Senior Public health Specialist,Global Forum for Health Research, c/o WHO,Geneva, Switzerland. Tel: 41 22 791 3808, E-mail:[email protected].

Dr Bernard Pecoul, Project Director, MSF Access toEssential drugs Project, Médecins sans Frontières, 12rue du Lac, C.P. 6090, 1211 Genève 6, Switzerland. E-mail [email protected].

Dr Michaleen Richer, Medical Coordinator, SudanProgram, International Medical Corps, P. O. Box67513, Nairobi, Kenya. Tel: 254 2 574386/88/89, Fax:254 2 573973, E-mail: [email protected].

Dr M Schottler, Head, Health Policy, Bayer A.G.,Business Group Pharma, Europe and Overseas, D-1368 Leverkusen, Germany.

Dr J Slingenbergh, Senior Officer, Animal Pro-duction and Health, FAO, Rome, Italy. E-mail:[email protected].

WHO secretariatDr Mary Bendig, PRD/TDRMr Erik Blas, Programme Manager, PPMDr Charity Gichuki, OrganizerDr Melba Gomes TDR/IDEDr Win Gutteridge, Team Coordinator PRDDr Beatrice Halpaap, TDR/PRDDr David Heymann, EXD/CDSDr Jean Jannin, CSR/EDCDr J Karbwang, TDR/PRDDr Jane Kengeya-Koyondo Team CoordinatorTDR/IDEDr Deborah Kioy, TDR/PRDMr Felix Kuzoe TDR/PRDDr Maryinez Lyons CAH/FCHDr Carlos Morel, Director WHO/CDS/TDRDr MA Mouries, TDR/PRDDr Paul Nunn, TDR TB & LEP Disease CoordinatorDr Ayoade Oduola Team Coordinator TDR/STRDr Mark Perkins TDR/PRDDr Richard Pink, TDR/PRDDr J Sommerfeld, TDR/STRDr J Vanreas, TDR/PRDDr Simon Van Nieuwenhove, Regional Adviser,WHO Regional Office for Africa, B.P 1899, Kinshasa1, Republique Démocratique du Congo. Tel: 243 8803204, Fax: 1 321 953 9097, E-mail: [email protected] or [email protected] S Wayling, TDR/RCSDr F Zicker, Team Coordinator, TDR/RCS

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Documents

Report on African trypanosomiasis (sleeping sickness) · research agenda for African trypanosomiasis, closely linked to control needs and open to the opportunities that science and