Malaria Molecular Epidemiology Unit...Malaria Molecular Epidemiology Unit, Scientific Activity Report 2013 5 | P a g e especially in areas of low transmission which are seen as prime

Prepared by: Didier Menard, Head, Malaria Molecular Epidemiology Unit Email: [email protected]; Tel +855 23 426 009 Institut Pasteur du Cambodge, 5, Bvd. Monivong P.O Box 983 - Phnom Penh, Cambodia

Malaria Molecular Epidemiology Unit

2013 The scientific activity report of the Malaria Molecular Epidemiology Unit of the Institut Pasteur du Cambodge for the period January 1st to December 31st, 2013.

Scientific

activity

report

Malaria Molecular Epidemiology Unit, Scientific Activity Report 2013

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RESEARCH PROGRAM Challenges and key issues on malaria elimination: P. falciparum artemisinin resistance and knowledge on P. vivax malaria epidemiology

The Malaria Molecular Epidemiology Unit is a research structure, which enhances Pasteur Institute of

Cambodia actions’ in Public Health and scientific researches, alongside Cambodian national actors and

regional/international partners. Founded in 2001, this unit is currently composed by 23 permanent staff: one

senior scientist (Charge de recherché IP, head of the unit), 2 IPC research assistants (PhD), 3 PhD students,

three research engineers, one unit administrator, one quality manager, one field/samples coordinator and

twelve laboratory technicians.

1. Context

In Cambodia, malaria with an incidence of 4.07 per 1,000 population and 135 deaths in 2012 continues to be a major cause for public health and economic burden. Its control is given high priority by the government and development partners. Forest villagers in the eastern and northern provinces are at high risk of malaria, with all age groups suffering infection; children under the age of five years are at highest risk of severe disease due to their lack of immunity. Elsewhere, malaria is an occupational disease with specific high-risk groups, including forestry workers, new settlers and mobile/migrant populations who have come into forested areas, and soldiers, and their families, serving in the forests. The five Plasmodium species known to cause malaria in humans (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi) have already been described. Currently, P. falciparum remains the most frequent cause of malaria infections (prevalence of 63 % in 2012). However, distributions of Plasmodium species are changing since several years, with a particularly significant rising of P. vivax malaria cases (from 8% in 2000 to 37% in 2012). Moreover, in areas of low transmission, a proportion of P. vivax infections up to 50% is commonly found. This trend, probably related to various effective strategies implemented in Cambodia against P. falciparum malaria, shows clearly that we had significantly underestimated the burden of other simian Plasmodium species.

Although there has been a steady reduction in the total number of clinically diagnosed and treated malaria cases as well as in the severe case fatality rate over the last thirteen years, morbidity and mortality due to malaria remain high compared to other countries in the region. Malaria in Cambodia is also a key contributor to anaemia, complications during pregnancy, low-birth weight and poor child growth.

In addition, multi-drug resistant strains of Plasmodium falciparum are common, particularly in the west of the country. As with earlier antimalarials, we are now facing to the emergence of artemisinin resistance in western Cambodia while no suitable alternative currently exists for first-line treatments of P. falciparum malaria. As previously observed with chloroquine resistance in the last century, artemisinin-resistant parasites represent a major threat to worldwide goals of malaria eradication and the potential to devastate sub-Saharan Africa by increasing childhood mortality.

2. Major Areas of Research since 2010

Scientific projects conducted in the unit rely on performant technical platforms including equipment for cellular

culture, molecular biology and immunology. Built around the control/elimination concept in South East Asia,


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projects are conducted in close collaboration with the Cambodian National Malaria Control Programme, WHO

and others regional and international partners (RIIP, IPP, European and US Universities).

They are mainly focused on three major areas of

research (figure 1):

1. Supporting and evaluating the impact of strategies against malaria implemented by National Malaria Control Programmes

2. Conduct researches focused on P. falciparum artemisinin resistant parasites.

3. Conduct researches on vivax malaria & other emerging Plasmodium sp.

3. Major Achievements (2010-2013)

3.1. Research Area 1. Supporting and evaluating the impact of the strategies implemented by NMCPs

Investigation of undifferentiated febrile illnesses in rural Cambodia. In the past decade, control of falciparum

malaria has been successfully implemented in Cambodia, resulting to a significant decrease of reported

malaria. The introduction and wide-use of malaria rapid diagnostic tests (RDTs) within this program has

revealed a large burden of clinically suspected malaria cases in which no malaria parasites are detected. As a

first step towards developing an algorithm for malaria-RDT negative fever management, a 3-year cross-

sectional prospective observational study was designed to investigate the causes of acute malaria-negative

febrile illness in Cambodia. A total of 1193 febrile patients and 282 non-febrile individuals were recruited from

three sites in rural Cambodia. Whole venous blood, blood smear and nasopharyngeal throat swabs were

collected. The samples were screened for malaria parasites by RDT, microscopy and PCR, for Leptospira,

Rickettsia and O. tsutsugamushi by PCR, for Dengue- and Influenza virus by RT-PCR as well as for community

acquired septicaemia by blood culture. At least one pathogen was identified in 73.2% of febrile patients. Most

frequent pathogens detected by molecular diagnostics were P. vivax (33.4%), P. falciparum (26.5%), Leptospira

(11.3%), Influenza viruses (7.7%), Dengue viruses (5.4%), O. tsutsugamushi (3.7%), Rickettsia (0.2%), and P.

knowlesi (0.1%). A potential pathogen was identified in 873/1193 febrile patients and 114/282 non-febrile

subjects. Pathogens, particularly malaria parasites and leptospirosis, were also identified in asymptomatic

individuals. Clinic-based diagnosis of malaria RDT-negative cases was found to poorly predict for pathogen and

appropriate treatment.

G6PD deficiency issues: prevalence studies, diagnosis and recommendation for the safe introduction of the

Primaquine in National treatment guidelines. Controlling malaria remains a significant global health challenge,


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especially in areas of low transmission which are seen as prime areas for malaria elimination. In this context,

the WHO has been urging countries for many years to use primaquine for both transmission blocking of

Plasmodium falciparum, because it kills mature gametocytes, and as anti-relapse treatment against

Plasmodium vivax by killing liver hypnozoites. But Primaquine is not used widely because of anxiety over its

well-known propensity to cause acute haemolytic anaemia in individuals with G6PD deficiency, coupled with

the current logistical and fiscal impossibility of offering G6PD screening to all malaria patients. In this two-year

survey, the prevalence of the G6PD deficiency and haemoglobinopathies were assessed by quantitative

enzyme activity assay and haemoglobin electrophoresis, in samples collected from 2,408 confirmed malaria

patients in 19 health centres throughout Cambodia. Plasmodium falciparum was present in 1,443 (59.9%) and

P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb, G6PD deficiency was

present in 13.9% of all patients (335/2,408) and severe G6PDd (including WHO Class I and II variants) was more

common in western (158/1,732, 9.1%) versus eastern (21/414, 5.1%) Cambodia (P=0.01). Of 997/2,408 (41.4%)

had a haemoglobinopathy.

In addition, we assessed a rapid diagnostic test under research and development called CareStart™ G6PD

deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD

enzyme activity method ('gold standard'). Blood samples (n=903) were collected from Cambodian adults living

in Pailin province, western. Based on a normal haemoglobin concentration and wild-type G6PD gene, the

normal values of G6PD enzymatic activity for this population ranged from 3.6 to 20.5 U/g Hg (95th percentiles

from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient.

Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was

analysed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang

genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0,

respectively. Its detection threshold was < 2.7 U/g Hg, well within the range of moderate and severe enzyme

deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities < 2 U/g Hg were

falsely classified as "normal" by RDT. We concluded that this experimental RDT test here evaluated outside of

the laboratory for the first time showed real promise, but safe application of it will require lower rates of

falsely "normal" results.

Following two meeting dedicated to G6PD deficiency issues, the discussions and conclusions were reported in 2

publications. The first one from a workshop conducted in Incheon, Korea in May 2012 described the key

knowledge gaps in G6PD deficiency detection and proposed certain research priorities and an action plan. The

second meeting, held in Bangkok, Thailand in October 2012, was focused on challenges to the development

and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing

G6PD testing in support of radical cure.

An innovative tool for moving malaria PCR detection of parasite reservoir into the field. To achieve the goal of

malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput

diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will

enable a switch from passive to active malaria case detection in the field. In this study, we described an

innovative approach developed by our unit to detect malaria parasites carriers by PCR. DNA extraction and

real-time PCR assays (real-time PCR screening and species identification) were performed in a mobile

laboratory, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and


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treat positive cases within 24–48 hours after sample collection. An average of 240 clinical samples (and 40

quality control samples) was tested every day, six/seven days per week. 97.7 % of the results were available

<24 hours after the collection. The operational success of this diagnostic set-up proved that molecular testing

and subsequent treatment is logistically achievable in field settings, allowing the detection of clusters of

asymptomatic carriers and to provide useful epidemiological information. We concluded that the concept of

the mobile laboratory could be extended to other countries for the molecular detection of malaria or other

pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection

and culture.

3.2. Research Area 2. P. falciparum artemisinin resistance

Novel phenotypic assays for the detection of artemisinin resistant Plasmodium falciparum malaria in

Cambodia: in vitro and ex vivo drug-response studies. In this study, we aimed to assess whether the in vitro

ring-stage survival assay (RSA) can identify culture-adapted P. falciparum isolates from patients with slow-

clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to

dihydroartemisinin in the in vitro RSA, and to assess whether an ex vivo RSA can identify artemisinin-resistant

P. falciparum infections.

We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done

in Pursat, Cambodia, in 2010 and used new in vitro survival assays to explore the stage-dependent

susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the

RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia, to measure

the ex vivo responses of parasites from patients with malaria.

Our results showed that in vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-

clearing infections differed significantly when assays were done on 0–3 h ring-stage parasites (10.88% vs

0.23%; p=0.007). Ex vivo survival rates significantly correlated with in vivo parasite clearance half-lives (n=30,

r=0.74, 95% CI 0.50–0.87; p<0.0001). We concluded that the in vitro RSA of 0–3 h ring-stage parasites provides

a platform for biochemical and molecular characterization of artemisinin resistance and the ex vivo RSA can be

easily implemented where surveillance for artemisinin resistance is needed.

A molecular marker of artemisinin resistant Plasmodium falciparum malaria. Following our previous findings,

we were able in this study, by using whole-genome sequencing of an artemisinin-resistant parasite line from

Africa and clinical parasite isolates from Cambodia, to associate mutations in the PF3D7_1343700 kelch

propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Parasites bearing mutant

K13-propeller alleles were observed in Cambodian provinces where artemisinin resistance is prevalent. We also

found that the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent

spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele

(C580Y), in vitro parasite survival rates and in vivo parasite clearance rates indicated that K13-propeller

mutations are important determinants of artemisinin resistance. Our conclusion was that K13-propeller

polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin

resistance in the Greater Mekong Subregion and prevent its global spread.


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3.3. Research Area 3. P. vivax malaria challenges & other emerging Plasmodium sp.

Whole genome sequencing of field isolates provides robust characterization of genetic diversity in

Plasmodium vivax. Here, we reported results from whole genome sequencing of five P. vivax isolates obtained

from Malagasy and Cambodian patients, and of the monkey-adapted Belem strain. We obtained an average

70–400 X coverage of each genome, resulting in more than 93% of the Sal I reference sequence covered by 20

reads or more. Our study identified more than 80,000 SNPs distributed throughout the genome which will

allow designing association studies and population surveys. Analysis of the genome-wide genetic diversity in P.

vivax also revealed considerable allele sharing among isolates from different continents. This observation

seems to be consistent with a high level of gene flow among parasite strains distributed throughout the world.

Moreover, we demonstrated the feasibility to perform whole genome sequencing of P. vivax from field isolates

and the rigorous characterization of their genetic diversity. We concluded that the catalogue of polymorphisms

generated will enable large-scale genotyping studies and contribute to a better understanding of P. vivax traits

such as drug resistance or erythrocyte invasion, partially circumventing the lack of laboratory culture that has

hampered vivax research for years.

Whole genome sequencing of field isolates reveals a common duplication of the Duffy Binding Protein gene

in Malagasy Plasmodium vivax strains. Until recently the Duffy-negative blood group phenotype was

considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that

P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax

malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human

erythrocytes. Through recent whole genome sequencing we obtained ≥ 70X coverage of the P. vivax genome

from five field-isolates. Combined with sequences from one additional Malagasy field isolate and from five

monkey-adapted strains, we described identification of DNA sequence rearrangements in the P. vivax genome,

including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy

patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in

Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that

the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of

non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking

treatment of vivax malaria upon returning home. We also observed that PvDBP duplication prevalence was

highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative

people were reported. The highly conserved nature of the sequence involved in the PvDBP duplication

suggested that it has occurred in a recent evolutionary time frame. Our conclusion hypothesized that PvDBP, a

merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints

imposed by erythrocyte Duffy negativity in some human populations.

De novo assembly of a field isolate genome reveals a novel Plasmodium vivax Erythrocyte-Binding Protein

gene. Recent sequencing of Plasmodium vivax field isolates and monkey-adapted strains enabled

characterization of SNPs throughout the genome. These analyses relied on mapping short reads onto the P.

vivax reference genome generated from a monkey-adapted strain. Any locus deleted in this genome would be

lacking in the reference sequence and missed in previous analyses. In this study, we reported de novo assembly

of a P. vivax field isolate genome. Out of 2,857 assembled contigs, we identify 362 contigs each containing

more than 5 kb of contiguous DNA sequences absent from the reference genome sequence. These novel P.


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vivax DNA sequences accounted for 3.8 million nucleotides and contained 792 predicted genes. Most of these

contigs contained members of multigene families and likely originated from telomeric regions. Interestingly, we

identified two contigs containing predicted protein coding genes similar to Plasmodium red blood cell invasion

proteins. One gene encoded the reticulocyte-binding protein gene orthologous to P. cynomolgi RBP2e and P.

knowlesi NBPXb. The second gene harbored all the hallmarks of a Plasmodium erythrocyte-binding protein but

clustered separately from all known Plasmodium Duffy-binding protein genes. Additional analyses showed that

this gene was present in most P. vivax genomes and transcribed in blood-stage parasites. The result of this

study complemented previous genomic analyses and took full advantage of sequence data to provide a

comprehensive characterization of genetic variations in this important malaria parasite.

4. On-going projects

4.1. Research Area 1. Supporting and evaluating the impact of the strategies

implemented by NMCPs

4.1.1. Major objectives

As Cambodia move towards malaria elimination, activities which aim to measure how public health programs

operate over time and achieve their goals will need to shift from measuring reductions in morbidity and

mortality, to detecting infections especially in asymptomatic parasite carriers and measuring transmission.

Thus, the monitoring and evaluation researches needs to develop tools that will replace passive surveillance of

morbidity with active and prompt detection of infection, including confirmation of interruption of transmission

by detecting present and past infections, particularly in mobile populations.

In this context, the projects currently conducted in the unit aim at:

developing and implementing track and treat strategy by using high throughput real time PCR in the field (mobile laboratory unit) in hotspots areas and among populations at risk.

(re)defining the malaria epidemiology in low transmission areas by developing molecular approaches using high volume of blood sampling.

evaluating the structure of the parasite population to understand the parasite genes flow and monitor the spread of antimalarial drugs resistant parasites in Cambodia.

developing tools to evaluate the malaria transmission, including serological markers and detection of gametocytes carriers.

improving point-of-care tools to detect G6PD deficiency to facilitate the safety use of primaquine to treat falciparum malaria (gametocytes) or vivax malaria (hypnozoites).

4.1.2. Awarded on-going grants

Title: Towards malaria elimination: effective strategies against transmission. The new challenges in South East Asia. Sponsor: Initiative 5% (AP-5PC-2012-02) – French Government


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Period: July 2013 - July 2015

Title: Repellents as added control measure to long lasting insecticidal nets to target the residual transmission in Southeast Asia: a step forwards to malaria elimination Sponsor: Bill & Melinda Gates Foundation via the Prince Leopold Institute of Tropical Medicine

Period: October 2011 - September 2015

Title: Developing the Evidence for and assessing the Malaria Elimination Efforts among Mobile Migration Workers in Plantation Settings in Cambodia and Myanmar”. Sponsor: Bill & Melinda Gates Foundation via Population Services International

Period: December 2013 - February 2015

Title: Developing a screening algorithm to optimize identification of asymptomatic malaria among migrants crossing Cambodian borders (Cross Border Project) Sponsor: USAID via Malaria Consortium

Period: July 2013 - April 2014

4.2. Research Area 2. P. falciparum antimalarial drug resistance and treatment

4.2.1. Major objectives: One step beyond K13

While we have achieved some important steps in our understanding in artemisinin resistance (ART-R),

especially with the development of two major tools (in vitro phenotype with the RSA and molecular signatures

of ART-R parasites with K13 mutations), many questions remain unanswered. They are mainly focused around

three axes:

To better understand the phenomenon of “dormancy” at cellular and molecular levels, and its relationship

with the biological role of the K13 gene.

Avenues of investigations are now opened by the discovery of the K13 gene.

- We need to identify the metabolic pathway in which it operates, especially the biological partners involved in the cell and to investigate the fitness cost of the different K13 alleles in the absence of artemisinin pressure. We have still initiated these studies with several collaborators (David Fidock, University of Columbia) to assess the impact of the most frequent K13 mutations on the biology of the parasite, its responses to oxidative stress and other antimalarials.

- We also need to develop imaging methods to quantify the number of dormant parasites in cultures treated by artemisinin derivatives. Our data have already identified that 0-3 hours ring-stages are those who best survived to artemisinins and showed that this resistance is stable. To better understand the phenomenon of dormancy, we are planning to explore various strategies for labeling parasites with fluorescent dyes to quantify by flow cytometry the percentage of artemisinin-exposed survival parasites.

- We will investigate the level of RNA expression of certain genes involved in the response to artemisinin derivatives exposure. Our work will be particularly focused on the role of the mitochondria and the apicoplast. Quantitative PCR will be developed to explore gene amplification among our samples collection


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of Cambodian isolates. This work will allow us to identify new molecular mechanisms that may be involved in dormancy and resistance to artemisinin derivatives.

- Finally, we are also planning to study the relationship between resistance to artemisinins, dormancy and oxidative stress defense mechanisms. We have already observed a cross resistance between artemisinin derivatives and other oxidant molecules like methylene blue. It seems that the same cellular mechanism associated to dormancy is involved. This work will be done in coordination with imaging observations and genomic/transcriptomic studies.

To better understand the emergence and the spread of artemisinin resistant parasites by studying the

overtime selection of parasite populations maintained under drug pressure.

Our recent observations indicate that resistance to artemisinins and high survival rates in in vitro RSA assays

are not only associated with a single mutation in K13 gene. Beyond the analysis of the different K13 mutant-

type parasites, we have to understand how and where these mutations emerge and to investigate their ability

to further spread. Is that all C580Y mutant-type parasites (the most frequent mutant allele found in Cambodia)

come from the same parasite population or are they from several sub-populations which have acquired

independently the same mutation? This important issue should enable us to understand the natural evolution

of field parasite populations but also improve the development of effective tools used to monitor and control

malaria endemicity. Finally, by analyzing neutral SNPs/microsatellite sequences around the K13 gene we will

determine how many independent events have taken place to select mutants K13. This will open the door to

the analysis of biological, epidemiological and environmental factors that contributed to the selection of these

mutations. We will also analyze the speed of propagation of mutations in populations. These data help predict

potential outbreaks and deploy measures accordingly.

To improve the surveillance of the ACT efficacy with the evaluation of the spatial distribution of ART-R

parasites. The mutant type K13 alleles are markers will allow us to easily map the presence or absence of ART-

R parasites in different endemic areas, alert WHO and local health authorities on the need to recommend new

antimalarials regimens. Recently, it has been decided by WHO that Institut Pasteur leads a WHO reference

center for the global mapping of K13 alleles. This multi-site platform, implemented in Paris, in Cambodia and in

French Guiana, will be in charge to develop a central data base collecting information on K13 mutant

prevalence and SOPs for detecting K13 mutations and to implement a quality assurance system in association

with partners in malaria endemic countries involved in this global network. The aim will be to combine efforts

to make a world map of this new molecular marker and to inform in real-time decision-makers of the best

therapeutic option.

In addition, the Malaria Molecular Epidemiology Unit as head of the in vitro group of wWARN project will be in

charge to organize workshops and train staff from research centers (both in Phnom Penh or locally) to facilitate

the implementation of in vitro and ex vivo susceptibility testings for artemisinin derivatives and partner drug in

countries aiming to control or eliminate malaria. We will provide support to implement quality assurance of in

vitro susceptibility testings by providing reference strains through MR4 repository and support for external

quality controls (exchange of P. falciparum parasite isolates for internal or external QC), update detailed SOPs

and support for the interpretation of the results of the in vitro assays.


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Lastly, in collaborations with Medicines for Malaria Venture, we will be also actively involved in projects aiming

to screen new molecules. By using a panel of parasites with different in vitro susceptibility level and different

genetic background, we will assess different “Late Lead Molecules” and “preclinical Molecules” in 2014.

4.2.2. Awarded active grants

Title: IMMERSE: Innovative Malaria M&E, Research and Surveillance towards Elimination Sponsor: CDC/PMI/USAID via Malaria Consortium

Period: November 2013 - October 2015

Title: Leading In vitro module of wWARN project Sponsor: Bill & Melinda Gates Foundation via World Wide Antimalarial Resistance Network

Period: January 2014 - March 2015

Title: Artemisinin in vitro resistance Sponsor: Medicines for Malaria Venture

Period: January 2014 - December 2014

Title: MaPI: Lead optimisation of original anti-malaria compounds:a synergic multi-target approach Sponsor: ANR (ANR-2011-RPIB-002-01)

Period: March 2012 - February 2016

Title: SOREMA: Public Interventions and Health Inequalities in Recomposed Natural and Social Ecosystems of the Mekong Sub-Region Sponsor: ANR

Period: January 2012- January 2015

4.3. Research Area 3. P. vivax genetic diversity, resistance and biology challenges

4.3.1. Major objectives

Malaria threatens a quarter of the 13.6 million Cambodians who live near forested areas. While Plasmodium

falciparum is the most frequent reported cause of malaria in Cambodia, the proportion of vivax malaria has

significantly increased in the last decade. The extensive use of antimalarial drugs in the region, most notably

chloroquine (CQ), has led to the emergence of resistant P. vivax parasites which constitutes one of the greatest

challenges on malaria control in Southeast Asia. Assessment of drug resistance in P. vivax presents unique

challenges and must be considered separately from P. falciparum. Implementation of in vitro assays to

determine drug susceptibility is complicated by the difficulty to routinely culture P. vivax. In vivo assessments

of drug resistance in P. vivax are also problematic since recurrence of malaria after treatment can be caused by

i) parasite multiplication after incomplete elimination, ii) re-infection by new parasites and iii) release of

dormant parasites from the liver, a mechanism not observed in P. falciparum. These difficulties, combined with


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limited information on the genetic diversity of P. vivax, greatly limit our understanding of drug resistance in this

parasite.

We have recently sequenced the entire P. vivax genomes from field isolates from Cambodia. Using recent

development in sequencing technologies, we obtained 70-400 X coverage of the genomes and >93% of the

nucleotides covered by more than 20 reads. Our data provided a first characterization of the genome-wide

genetic diversity in P. vivax, at SNPs and sequence rearrangements. In addition, we identified the presence of

multiple P. vivax strains in each infected blood sample and were able to differentiate them by reconstructing

bioinformatically individual haplotypes at highly polymorphic loci. We propose to build on this experience to

characterize the genetic diversity of P. vivax in Cambodia and identify DNA polymorphisms underlying drug

susceptibility and resistance. In collaboration with David Serre (Cleveland Clinic), we will combine modern

fieldwork with population genomic analyses to address the following outstanding issues:

To characterize the genetic diversity of P. vivax in Cambodia. Understanding the structure and dynamics of

the parasite population is essential for monitoring the emergence and spread of drug resistance. We propose

to study isolates from symptomatic and asymptomatic P. vivax-infected individuals recruited in sites across

Cambodia. First, we will characterize the number and diversity of P. vivax strains present in each individual by

very deep re-sequencing (>20,000 X) of five highly polymorphic loci, including the Duffy binding protein and the

circumsporozoite surface protein genes. These data will allow us to reliably differentiate P. vivax strains and to

test i) whether the parasite population is geographically stratified, ii) whether vivax infections show seasonal

patterns, and iii) whether the number of strains in symptomatic and asymptomatic individuals differs. Second,

we will sequence the entire genome from field isolates at high coverage (>50 X) to characterize the genome-

wide patterns of genetic diversity. These data will allow us to perform state-of-the-art population genomic

analyses to confirm our stratification analyses and determine the amount of gene flow among Cambodian P.

vivax. We will also scan the P. vivax genome for recent positive selection events that may identify drug

resistant loci.

To identify genetic polymorphisms associated with drug resistance. Despite alarming reports of CQ resistant

P. vivax parasites in Cambodia and other Southeast Asian countries, we know very little about the genetic

bases of this resistance. We will follow infected solely with P. vivax for 42 days after CQ treatment and examine

the evolution of the parasitemia. First, we will monitor changes in the relative proportion of the strains in each

patient using our deep re-sequencing assay. Combined with microscopic determination of the parasitemia, this

analysis will provide quantitative estimates of the CQ susceptibility of each P. vivax strain. Second, we will

collect additional blood samples from patients who showed evidence of treatment failure, and use our deep re-

sequencing assay to differentiate recrudescence from new infections. We will also measure the drug

concentration in blood to confirm drug resistance, and perform short-term cultures of P. vivax directly in the

field, using a mobile laboratory, to assess ex vivo the susceptibility of the parasites to CQ. We will then link

these different estimates of drug resistance with genome-wide diversity data to identify DNA polymorphisms

statistically associated with drug resistance.

In addition, a second project on Plasmodium vivax will be conducted in collaboration with Peter Zimmerman

(Case Western University, Cleveland, Ohio) and Arsene Ratsimbasoa (National Malaria Control Programme in


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Madagascar). The study will aim to understand how Plasmodium vivax has gained capacity to infect

erythrocytes from Duffy-negative people based on our recent findings, to leading to the following hypotheses:

(1) In Madagascar Duffy (-) and Duffy (+) individuals are equally susceptible to P. vivax infection, but

differentially susceptible to different P. vivax strains, or (2) P. vivax has evolved alternative erythrocyte invasion

pathways to enable infection of Duffy (-) erythrocytes, or (3) P. vivax strains exhibit differences in erythrocyte

binding and invasion efficiency.

Our hypotheses will be tested through the following specific aims.

Identify associations between P. vivax strains and susceptibility of Duffy (-) people to P. vivax blood-stage

infection and clinical malaria. We will determine the relative susceptibility of Duffy (-) and Duffy (+) people to

P. vivax infection and disease in communities where our preliminary study found the highest prevalence of P.

vivax infections in Duffy (-) individuals.

Assess interactions between P. vivax erythrocyte binding ligands and human erythrocytes that influence

merozoite attachment and invasion of host red cells. Studies will first focus on the PvDBP allele(s) (single-copy

and duplicated) observed to be present in P. vivax-infected Duffy (-) and Duffy (+) people (Malagasy and

Cambodian). Following cloning, over-expression and purification of recombinant variant PvDBP alleles

(Malagasy and Cambodian), we will evaluate erythrocyte antigen binding of variant PvDBP alleles across a

range of concentrations. Through previously developed assays we will test erythrocyte binding of PvDBP

variants to Duffy (-) and Duffy (+) red blood cells that have been screened for standard blood group

specificities. Alternative invasion ligands of interest will include the P. vivax reticulocyte binding proteins

(PvRBP), apical membrane antigen-1 (PvAMA-1) and other micronemes and rhoptery proteins.

Classify in vitro invasion pathways of Malagasy P. vivax patient isolates for Duffy (-) and Duffy (+)

erythrocytes. In vitro P. vivax invasion studies will follow methods used with success by Grimberg et al. P. vivax

strains will first be classified as Independent or Dependent on PvDBP following exposure to PvDBP antibodies

(mono- and poly-clonal). Further classification P. vivax invasion pathways will include exposure of parasite

isolates to Duffy antigen variants (e.g. Fya vs Fyb), red cell enzyme treatments, and additional P. vivax antigen-

specific antibodies.

4.3.2. Awarded active grants

Title: Madagascar P. vivax Invasion of Duffy-Negative Red Cells Sponsor: NIH/NIAID (R01 AI097366)

Period: November 2013 - October 2018

Title: Genomic Analyses of Plasmodium vivax Responses to Antimalarial Drugs in Cambodia Sponsor: NIH/NIAID (R01 AI103228)

Period: May 2013 - April 2018


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COLLABORATIONS & SCIENTIFIC PARTNERSHIPS

National level

CNM C. CHAR MENG, K. SIM, C. NGUON, S. SIV, R. LEANG, D. LEK,

WHO national office R. ABDUR, D. MEY BOUTH

PFD/London School of Tropical Medicine P. GUYANT

Malaria Consortium S. MEEK, A. ROCA

IRD F. BOURDIER

Université des Sciences de la Santé P. MILLET

Regional level

WHO regional office E. CHRISTOPHEL

Eijkman-Oxford Clinical Research Unit K. BAIRD, F. AISYAH YUDHAPUTRI

Shoklo Malaria Research Unit F. NOSTEN, G. BANCONE

Mahidol Oxford Research Unit A. DONDORP, R. TRIPURA, C. WOODROW, N. WHITE

wWARN Bangkok J. SMITH

Mahosot Hospital, Vientiane P. NEWTON, M. MAYXAY

Hospital for Trop Diseases, Ho Chi Minh T. TINH HIEN, J. FARRAR

Menzies Sch. of Health Research, Darwin R. PRICE, S. AUBURN

Australian Army Malaria Institute, Enoggera Q. CHEN

International level

Europe

Institut Pasteur O. MERCEREAU-PUIJALON, JC. BARALE, F. ARIEY, C. BOUCHIER

WHO Geneva R. NEWMAN, P. RINGWALD, J. CUNNINGHAM, A. BOSMAN

FIND H. HOPKINS, I. GONZALES, S. INCARDONA

London School Hygiene & Trop Medicine C. DRAKELEY, S. YEUNG

WWARN P. GUERIN, C. SIBLEY

MMV T. WELLS, X. DING

Africa

Institut Pasteur de Madagascar C. ROGIER

North America

CWR University, Cleveland P. ZIMMERMAN, B. GRIMBERG

Cleveland Clinic, Cleveland, D. SERRE, E. CHAN

University of Columbia D. FIDOCK

Lab. Malaria & Vector Research. NIH R. FAIRHURST, C. ARAMARATUNGA, P. LIM

CDC, Global Health J. HWANG,

South America

Institut Pasteur de Guyane Française E. LEGRAND, L. MUSSET


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PUBLICATIONS LIST 2013 – 19 publications Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P, Leang R, Duong S, Sreng S, Suon S, Chuor CM, Bout DM, Ménard S, Rogers WO, Genton B, Fandeur T, Miotto O, Ringwald P, Le Bras J, Berry A, Barale JC, Fairhurst RM, Benoit-Vical F, Mercereau-Puijalon O, Menard D. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2013 ; Dec 18.

Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, Lim P, Mao S, Sopha C, Sam B, Anderson JM, Duong S, Chuor CM, Taylor WR, Suon S, Mercereau-Puijalon O, Fairhurst RM, Menard D. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis. 2013; 13(12):1043-9.

Hester J, Chan ER, Menard D, Mercereau-Puijalon O, Barnwell J, Zimmerman PA, Serre D. De Novo Assembly of a Field Isolate Genome Reveals Novel Plasmodium vivax Erythrocyte Invasion Genes. PLoS Negl Trop Dis. 2013 ; 5;7(12):e2569.

Menard D, Chan ER, Benedet C, Ratsimbasoa A, Kim S, Chim P, Do C, Witkowski B, Durand R, Thellier M, Severini C, Legrand E, Musset L, Nour BY, Mercereau-Puijalon O, Serre D, Zimmerman PA. Whole Genome Sequencing of Field Isolates Reveals a Common Duplication of the Duffy Binding Protein Gene in Malagasy Plasmodium vivax Strains. PLoS Negl Trop Dis. 2013 Nov 21;7(11):e2489.

Gryseels C, Uk S, Erhart A, Gerrets R, Sluydts V, Durnez L, Muela Ribera J, Hausmann Muela S, Menard D, Heng S, Sochantha T, D'Alessandro U, Coosemans M, Peeters Grietens K. Injections, cocktails and diviners: therapeutic flexibility in the context of malaria elimination and drug resistance in northeast Cambodia. PLoS One. 2013; 11;8(11):e80343.

Canier L, Khim N, Kim S, Sluydts V, Heng S, Dourng D, Eam R, Chy S, Khean C, Loch K, Ken M, Lim H, Siv S, Tho S, Masse-Navette P, Gryseels C, Uk S, Van Roey K, Grietens KP, Sokny M, Thavrin B, Chuor CM, Deubel V, Durnez L, Coosemans M, Menard D. An innovative tool for moving malaria PCR detection of parasite reservoir into the field. Malar J. 2013; 9;12(1):405.

Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, Suon S, Mao S, Sopha C, Sam B, Ashley EA, Miotto O, Dondorp AM, White NJ, Su XZ, Char MC, Anderson JM, Amaratunga C, Menard D, Fairhurst RM. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers. Antimicrob Agents Chemother. 2013; 57(11):5277-83.

Leang R, Ros S, Duong S, Navaratnam V, Lim P, Ariey F, Kiechel JR, Menard D, Taylor WR. Therapeutic efficacy of fixed dose artesunate-mefloquine for the treatment of acute, uncomplicated Plasmodium falciparum malaria in Kampong Speu, Cambodia. Malar J. 2013 ; 23;12(1):343.

Woodrow CJ, Dahlström S, Cooksey R, Flegg JA, Le Nagard H, Mentré F, Murillo C, Menard D, Nosten F, Sriprawat K, Musset L, Quashie NB, Lim P, Fairhurst RM, Nsobya SL, Sinou V, Noedl H, Pradines B, Johnson JD, Guerin PJ, Sibley CH, Le Bras J. High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool. Antimicrob Agents Chemother. 2013; 57(7):3121-30.

Bouillon A, Giganti D, Benedet C, Gorgette O, Pêtres S, Crublet E, Girard-Blanc C, Witkowski B, Menard D, Nilges M, Mercereau-Puijalon O, Stoven V, Barale JC. In Silico screening on the three-dimensional model of the Plasmodium vivax SUB1 protease leads to the validation of a novel anti-parasite compound. J Biol Chem. 2013; 21;288(25):18561-73.

Taylor JE, Pacheco MA, Bacon DJ, Beg MA, Machado RL, Fairhurst RM, Herrera S, Kim JY, Menard D, Póvoa MM, Villegas L, Mulyanto, Snounou G, Cui L, Zeyrek FY, Escalante AA. The evolutionary history of Plasmodium vivax as inferred from mitochondrial genomes: parasite genetic diversity in the Americas. Mol Biol Evol. 2013; 30(9):2050-64.


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Menard D, Ariey F, Mercereau-Puijalon O. Plasmodium falciparum susceptibility to antimalarial drugs: global data issued from the Pasteur Institutes international network. Med Sci (Paris). 2013;29(6-7):647-55.

Khim N, Benedet C, Kim S, Kheng S, Siv S, Leang R, Lek S, Muth S, Chea N, Chuor CM, Duong S, Kerleguer A, Tor P, Chim P, Canier L, Witkowski B, Taylor WR, Menard D. G6PD deficiency in Plasmodium falciparum and Plasmodium vivax malaria-infected Cambodian patients. Malar J. 2013 ; 28;12(1):171.

von Seidlein L, Auburn S, Espino F, Shanks D, Cheng Q, McCarthy J, Baird K, Moyes C, Howes R, Menard D, Bancone G, Winasti-Satyahraha A, Vestergaard LS, Green J, Domingo G, Yeung S, Price R. Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report. Malar J. 2013; 27;12:112.

Das D, Tripura R, Phyo AP, Lwin KM, Tarning J, Lee SJ, Hanpithakpong W, Stepniewska K, Menard D, Ringwald P, Silamut K, Imwong M, Chotivanich K, Yi P, Day NP, Lindegardh N, Socheat D, Nguon C, White NJ, Nosten F, Dondorp AM. Effect of high-dose or split-dose artesunate on parasite clearance in artemisinin-resistant falciparum malaria. Clin Infect Dis. 2013 ; 56(5):e48-58.

Leang R, Barrette A, Bouth DM, Menard D, Abdur R, Duong S, Ringwald P. Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010. Antimicrob Agents Chemother. 2013 ; 57(2):818-26.

Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, Chy S, Duong S, Leang R, Ringwald P, Dondorp AM, Tripura R, Benoit-Vical F, Berry A, Gorgette O, Ariey F, Barale JC, Mercereau-Puijalon O, Menard D. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrob Agents Chemother. 2013; 57(2):914-23.

Andriantsoanirina V, Khim N, Ratsimbasoa A, Witkowski B, Benedet C, Canier L, Bouchier C, Tichit M, Durand R, Menard D. Plasmodium falciparum Na+/H+ exchanger (pfnhe-1) genetic polymorphism in Indian Ocean malaria-endemic areas. Am J Trop Med Hyg. 2013; 88(1):37-42.

Domingo GJ, Satyagraha AW, Anvikar A, Baird K, Bancone G, Bansil P, Carter N, Cheng Q, Culpepper J, Eziefula C, Fukuda M, Green J, Hwang J, Lacerda M, McGray S, Menard D, Nosten F, Nuchprayoon I, Oo NN, Bualombai P, Pumpradit W, Qian K, Recht J, Roca A, Satimai W, Sovannaroth S, Vestergaard L, Von Seidlein L. G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests. Malar J. 2013; 4; 12(1):391.

2012 – 10 publications

Ratsimbasoa A, Ravony H, Vonimpaisomihanta JA, Raherinjafy R, Jahevitra M, Rapelanoro R, Rakotomanga Jde D, Malvy D, Millet P, Menard D. Management of uncomplicated malaria in febrile under five-year-old children by community health workers in Madagascar: reliability of malaria rapid diagnostic tests. Malar J. 2012 ; 25;11:85.

Menard D, Andriantsoanirina V, Khim N, Ratsimbasoa A, Witkowski B, Benedet C, Canier L, Mercereau-Puijalon O, Durand R. Global analysis of Plasmodium falciparum Na+/H+ exchanger (pfnhe-1) allele polymorphism and its usefulness as a marker of in vitro resistance to quinine. International Journal For Parasitology-Drugs and Drug Resistance Volume: 3 Pages: 8-19.

Hoyer S, Nguon S, Kim S, Habib N, Khim N, Sum S, Christophel EM, Bjorge S, Thomson A, Kheng S, Chea N, Yok S, Top S, Ros S, Sophal U, Thompson MM, Mellor S, Ariey F, Witkowski B, Yeang C, Yeung S, Duong S, Newman RD, Menard D. Focused Screening and Treatment (FSAT): a PCR-based strategy to detect malaria parasite carriers and contain drug resistant P. falciparum, Pailin, Cambodia. PLoS One. 2012;7(10):e45797.

http://www.ncbi.nlm.nih.gov/pubmed/23714236





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Acestor N, Cooksey R, Newton PN, Menard D, Guerin PJ, Nakagawa J, Christophel E, González IJ, Bell D. Mapping the aetiology of non-malarial febrile illness in Southeast Asia through a systematic review--terra incognita impairing treatment policies. PLoS One. 2012;7(9):e44269.

Chan ER, Menard D, David PH, Ratsimbasoa A, Kim S, Chim P, Do C, Witkowski B, Mercereau-Puijalon O, Zimmerman PA, Serre D. Whole genome sequencing of field isolates provides robust characterization of genetic diversity in Plasmodium vivax. PLoS Negl Trop Dis. 2012;6(9):e1811.

Chou M, Kim S, Khim N, Chy S, Sum S, Dourng D, Canier L, Nguon C, Menard D. Performance of "VIKIA Malaria Ag Pf/Pan" (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections. Malar J. 2012; 24;11:295.

Lin JT, Juliano JJ, Kharabora O, Sem R, Lin FC, Muth S, Menard D, Wongsrichanalai C, Rogers WO, Meshnick SR. Individual Plasmodium vivax msp1 variants within polyclonal P. vivax infections display different propensities for relapse. J Clin Microbiol. 2012 ; 50(4):1449-51.

Khim N, Kim S, Bouchier C, Tichit M, Ariey F, Fandeur T, Chim P, Ke S, Sum S, Man S, Ratsimbasoa A, Durand R, Menard D. Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene. Antimicrob Agents Chemother. 2012 ; 56(2):863-8.

Ratsimbasoa A, Ravony H, Vonimpaisomihanta JA, Raherinjafy R, Jahevitra M, Rapelanoro R, Rakotomanga Jde D, Malvy D, Millet P, Menard D. Compliance, safety, and effectiveness of fixed-dose artesunate-amodiaquine for presumptive treatment of non-severe malaria in the context of home management of malaria in Madagascar. Am J Trop Med Hyg. 2012 Feb;86(2):203-10.

Yalcindag E, Elguero E, Arnathau C, Durand P, Akiana J, Anderson TJ, Aubouy A, Balloux F, Besnard P, Bogreau H, Carnevale P, D'Alessandro U, Fontenille D, Gamboa D, Jombart T, Le Mire J, Leroy E, Maestre A, Mayxay M, Menard D, Musset L, Newton PN, Nkoghé D, Noya O, Ollomo B, Rogier C, Veron V, Wide A, Zakeri S, Carme B, Legrand E, Chevillon C, Ayala FJ, Renaud F, Prugnolle F. Multiple independent introductions of Plasmodium falciparum in South America. Proc Natl Acad Sci U S A. 2012; 10;109(2):511-6.

2011 – 5 publications Kim S, Nguon C, Guillard B, Duong S, Chy S, Sum S, Nhem S, Bouchier C, Tichit M, Christophel E, Taylor WR, Baird JK, Menard D. Performance of the CareStart™ G6PD deficiency screening test, a point-of-care diagnostic for primaquine therapy screening. PLoS One. 2011;6(12):e28357.

Khim N, Siv S, Kim S, Mueller T, Fleischmann E, Singh B, Divis PC, Steenkeste N, Duval L, Bouchier C, Duong S, Ariey F, Menard D. Plasmodium knowlesi infection in humans, Cambodia, 2007-2010. Emerg Infect Dis. 2011; 17(10):1900-2.

Andriantsoanirina V, Durand R, Pradines B, Baret E, Bouchier C, Ratsimbasoa A, Menard D. In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum. Malar J. 2011; 27;10:283.

Howes RE, Patil AP, Piel FB, Nyangiri OA, Kabaria CW, Gething PW, Zimmerman PA, Barnadas C, Beall CM, Gebremedhin A, Menard D, Williams TN, Weatherall DJ, Hay SI. The global distribution of the Duffy blood group. Nat Commun. 2011;2:266.

Andriantsoanirina V, Menard D, Tuséo L, Ratsimbasoa A, Durand R. Resistance of Plasmodium falciparum to antimalarial drugs: impact on malaria pre-elimination in Madagascar. Med Trop. 2011 Jun;71(3):298-304. 2









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Malaria Molecular Epidemiology Unit...Malaria Molecular Epidemiology Unit, Scientific Activity Report 2013 5 | P a g e especially in areas of low transmission which are seen as prime