Manual for the Laboratory Diagnosis of Malaria

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MANUAL FOR THE LABORATORYDIAGNOSIS OF MALARIA

First Edition

ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (EHNRI)

ETHIOPIAN FEDERAL MINISTRY OF HEALTH

SEPTEMBER, 2012

ADDIS ABABA


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MANUAL FOR THE LABORATORYDIAGNOSIS OF MALARIA

First Edition

ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (EHNRI)

ETHIOPIAN FEDERAL MINISTRY OF HEALTH

SEPTEMBER, 2012

ADDIS ABABA


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FOREWORD

Malaria is one of the leading public health diseases in Ethiopia with predominant unstable

transmission. Approximately 52 million people (68%) live in malaria-endemic areas in Ethiopia,chiefly at altitudes below 2,000 meters. Malaria is mainly seasonal in the highland fringe areas andof relatively longer transmission duration in lowland areas, river basins and valleys. Althoughhistorically there have been an estimated 10 million clinical malaria cases annually, cases havereduced since 2006 due to improved prevention and control strategies. As outlined in the NSP 2011-2015, Ethiopia has a target of 100% access to effective and affordable malaria treatment. Thisrequires improving diagnosis of malaria cases using microscopy or using multi-species RDTs, andproviding prompt and effective malaria case management at all health facilities in the country.

This manual is developed based on the recommendations of experts working in Malaria Programs atthe Federal Ministry of Health, Regional Health Bureaus,, National and Regional Reference

Laboratories, and partners with the aim of standardizing Malaria Laboratory Diagnosis trainings andstrengthening the quality of laboratory testing procedures for the diagnosis of malaria in the healthfacilities in Ethiopia.

The manual is divided into nine chapters : Introduction, Scope and purpose of the manual, Malariasituation in Ethiopia, Parasitological Diagnosis of Malaria using Microscopy, Parasitological Diagnosisof Malaria using RDTs, Quality Assurance of Malaria Laboratory Diagnosis, Laboratory safety, Supplyand logistics Management in Malaria Laboratory Diagnosis, and Annexes of formats, registers andStandard operating procedures.

EHNRI believes that this manual will be useful for laboratory personnel and other health workersduring routine laboratory work and as a reference material for trainers and supervisors onlaboratory diagnosis of malaria during in-service trainings, practical attachments, and supportivesupervisions and for Quality Control and Quality Assurance purposes. The manual could be useful asa reference material for clinicians too, mainly to understand the use and interpretation of laboratorytests for malaria case management. The manual is also helpful for health facility managers to enablethem in determining essential laboratory commodity requirements for malaria laboratory diagnosisand the need for their timely availability to ensure uninterrupted laboratory diagnostic services. Thismanual should also be of interest to those non-governmental organizations and funding agenciesthat are involved in the support for malaria laboratory diagnosis improvement and quality assuranceprograms.

Finally, I would like to express my sincere appreciation and thanks to all professionals andorganizations who have contributed their expertise and resources for the preparation of this manual

Amha Kebede, PhDActing Director General,EHNRI.


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ACKNOWLEDGMENTThe development of this Manual for Laboratory Diagnosis of Malaria was made possible through thecontribution of the professionals and institutions listed below:

Core Group Members: Organization

Getachew Belay EHNRIHabtamu Asrat EHNRIMarkos Sileshi EHNRIHussien Mohammed EHNRISindew Mekasha EHNRIMoges Kassa EHNRIBereket Hailegiorgis CU-ICAP New YorkTesfay Abreha CU-ICAP Ethiopia

Sintayehu G/Sellasie CU-ICAP EthiopiaLeykun Demeke CU-ICAP EthiopiaSamuel Girma CU-ICAP EthiopiaMicheal Aidoo CDC Atlanta

Contributors:Gudeta Tibesso EHNRIGonfa Ayana EHNRIAshenafi Assefa EHNRIAbinet Abebe EHNRI

Yenew Kebede CDC EthiopiaZenebe Melaku CU-ICAP EthiopiaAbebe Tadesse CU-ICAP EthiopiaFanuel Zewdu CU-ICAP EthiopiaMeseret Habtamu CU-ICAP EthiopiaMekonnen Tadesse CU-ICAP EthiopiaJoseph Malone CDC/PMI EthiopiaRichard Reithinger USAID/PMI EthiopiaHiwot Teka USAID/PMI Ethiopia

Institutions

Federal Ministry of Health Federal Hospitals I-TECH

Regional Health Bureaus The Carter Center Malaria Consortium

Regional Reference Laboratories JHU USAIDPHSP

I would like to express my appreciation and thanks to all professionals and institutions for theirvaluable contributions to make this manual a reality.


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The generous financial and technical support of Columbia University ICAP in Ethiopia throughfunding obtained from PMI USAID Ethiopia was of paramount importance to hold serial expert andnational consultative meetings to develop this manual. We are indebted to PMI for covering the costof printing the manual.

Gonfa Ayana, BSc, MSc,Acting Director, Regional Laboratories Capacity Building Directorate,EHNRI.


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TABLE OF CONTENTSPageForeword ....................................................................................................................................................... ii

Acknowledgment ......................................................................................................................................... iiiTable of contents .......................................................................................................................................... vList of Tables .............................................................................................................................................. viiiList of Figures ............................................................................................................................................... ixAcronyms ...................................................................................................................................................... xGlossary of Terms........................................................................................................................................ xii1 Introduction ..................................................................................................................................... 1

1.1 Malaria Etiology .......................................................................................................... 1 1.2 Life Cycle and Transmission of Malaria ..................................................................... 1 1.3 Overview of methods for malaria diagnosis ............................................................... 2

1.3.1 Clinical Diagnosis of Malaria .......................................................................... 2 1.3.2 Laboratory Diagnosis of Malaria ..................................................................... 4

2 Scope and Purpose of the manual ................................................................................................... 62.1 Purpose ........................................................................................................................ 6 2.2 Target Audience .......................................................................................................... 6

3 Malaria Situation in Ethiopia ........................................................................................................... 73.1 Burden of the Disease ................................................................................................. 7 3.2 Eco-epidemiological Strata of Malaria Transmission ................................................. 7 3.3 The National Strategic Plan for Malaria Prevention, Control and Elimination .......... 9 3.4 Goal and Objectives of 2011-2015 strategic plan ....................................................... 9 3.5 Levels of Health facilities and types of diagnostic tests in Ethiopia ......................... 10

3.5.1 National and Regional Reference Laboratories ............................................. 10 3.5.2 Hospitals and health centers ........................................................................... 11 3.5.3 Health posts .................................................................................................... 11

3.6 Case Management Practices ...................................................................................... 11 3.6.1 Treatment Approach ....................................................................................... 11 3.6.2 Case management of uncomplicated malaria ................................................. 12 3.6.3 General approach to management of Severe Malaria .................................... 13

4 Parasitological Diagnosis of Malaria Using Microscopy ................................................................. 144.1 Care and Handling of Microscope ............................................................................ 14

4.1.1 Microscope maintenance and storage conditions ........................................... 14 4.1.2 Maintenance of the microscope...................................................................... 15 4.1.3 Cleaning a Microscope ................................................................................... 16 4.1.4 Troubleshooting ............................................................................................. 17

4.2 Parasitological Procedures of Microscopy ................................................................ 19 4.2.1 Specimen collection and blood film preparation............................................ 19 4.2.2 Staining........................................................................................................... 24 4.2.3 Microscopic Examination and Species Identification .................................... 25 4.2.4 Reporting Blood Film Results ........................................................................ 42

5 Parasitological Diagnosis of Malaria using Rapid Diagnostic Tests (RDTs) .................................... 475.1 RDTs and their Significance ..................................................................................... 47 5.2 RDT versus Microscopy............................................................................................ 47 5.3 Malaria RDT Formats ............................................................................................... 48

5.4 Basic Principles of RDTs .......................................................................................... 49


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5.5 RDTs Mode of Action ............................................................................................... 50 5.6 General Procedures of Malaria RDTs ....................................................................... 51 5.7 RDT Kit Selection and Handling .............................................................................. 53

5.7.1 The Plasmodium species to be detected ......................................................... 53 5.7.2 Accuracy (Sensitivity and Specificity) ........................................................... 54

5.7.3 Shelf Life and Stability .................................................................................. 54 5.7.4 Ease of Use ..................................................................................................... 54 5.7.5 Cost................................................................................................................. 54

6 Quality Assurance of Malaria Laboratory Diagnosis ...................................................................... 556.1 What is Quality Assurance? ...................................................................................... 55 6.2 The Need for Accurate Malaria Laboratory Diagnosis ............................................. 56 6.3 Errors compromising quality laboratory diagnosis ................................................... 56 6.4 Objectives of Quality Assurance Programs .............................................................. 57 6.5 Challenges in Malaria laboratory Diagnosis ............................................................. 57 6.6 Setting up a QA system ............................................................................................. 57 6.7 Principles and Concepts of Quality Assurance in Malaria Diagnosis ....................... 58 6.8 Quality Assurance of Malaria Microscopy ............................................................... 58

6.8.1 Internal Quality Control (IQC) ....................................................................... 58 6.8.2 External Quality Assessment (EQA).............................................................. 60 6.8.3 Quality Indicators for Malaria Microscopy .................................................... 61

6.9 Quality Assurance (QA) of Malaria RDTs ............................................................... 62 6.10 Quality Assurance of Malaria RDTs in Remote Areas ......................................... 64

6.10.1 Ensuring Quality of RDTs.............................................................................. 64 6.10.2 External Quality Assessment of Malaria RDTs ............................................. 65 6.10.3 Quality Indicators of Malaria RDT ................................................................ 65

7 Laboratory Safety ........................................................................................................................... 667.1 General Safety Guidelines ......................................................................................... 66 7.2 Safety and Exposure Control Measures .................................................................... 67 7.3 Testing Infrastructure and Equipment Management ................................................. 70 7.4 Waste Disposal .......................................................................................................... 71

8 Supply and Logistic Management in Malaria Laboratory Diagnosis .............................................. 728.1 Logistics Management .............................................................................................. 72 8.2 Stock Management .................................................................................................... 72 8.3 Storage of Malaria Laboratory Commodities............................................................ 79

8.3.1 Storage of Reagents and Equipment .............................................................. 79 8.3.2 Handling Damaged or Expired Stocks ........................................................... 79

8.4 Supply List for Malaria Microscopy ......................................................................... 80

8.5

Supply list of Malaria RDT ....................................................................................... 81

References .................................................................................................................................................. 82ANNEXES ..................................................................................................................................................... 84

Annex 1: Microscope: Types, Parts, Care and Handling ..................................................... 84 Annex 2: SOP For Capillary Blood Collection And Preparation of Malaria Blood Films . 90 Annex 3: SOP Preparation of Giemsa Working Solution .................................................... 96 Annex 4: SOP for Preparation of Buffered Water ............................................................... 97 Annex 5: SOP for Examinination of Malaria Blood Films And Estimation of Parasitemia 98 Annex 6: SOP for Recording And Reporting of Malaria Blood Film Results .................. 101 Annex 7: SOP for Malaria Blood Film Slide Storage And Selection for BlindedRechecking ......................................................................................................................... 102

Annex 8: SOP for Care And Preventive Maintenance of Microscopes ............................. 104


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Annex 9: Monthly Malaria Case Report Format ............................................................... 106 Annex 10: Exposure Reporting Form ................................................................................ 107


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LIST OF TABLES

Table 1 Most common technical mistakes in collection and preparation of blood smears ................. 23Table 2 Characteristics of thick and thin blood films ............................................................................ 25

Table 3 Species differentiation on thin films ........................................................................................ 30

Table 4 Species differentiation on thick films ....................................................................................... 30

Table 5 Species differentiation of malaria parasites by cytoplasmic pattern of trophozoites inGiemsa-stained thick blood films .......................................................................................................... 42

Table 6.Comparison of RDT use versus Malaria Microscopy ................................................................ 48

Table 7. Comparison of Rapid Diagnostic Tests for Malaria Antigens .................................................. 49

Table 8 Limitations of RDT results ........................................................................................................ 52

Table 9 Safety precautions for chemicals used in malaria microscopy ................................................ 67

Table 10 Example of a stock book ......................................................................................................... 75

Table 11 Example of Stock card ............................................................................................................ 75

Table 12 Example of a Quarterly Supplies Request and Report, Requirement Form ........................... 77

Table 13 Example of a Quarterly RDT Supplies Requirement Form ..................................................... 78


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LIST OF FIGURES

Figure 1 Life Cycle of Malarial Parasites ................................................................................................. 1

Figure 2 Malaria Epidemiological Strata in Ethiopia ............................................................................... 8

Figure 3 Cabinet box ............................................................................................................................. 15

Figure 4 Example of well-made and correctly labeled thick and thin films .......................................... 21

Figure 5 Badly positioned blood film .................................................................................................... 21

Figure 6 Too much blood for both thin and thick films ........................................................................ 22

Figure 7 Too small blood for both thin and thick films ......................................................................... 22

Figure 8 The effect of unclean slide on blood films .............................................................................. 22

Figure 9 The effect of chipped edge spreader on thin and thick films ................................................. 23Figure 10 Basic components of a malaria parasite inside a red blood cell ........................................... 27

Figure 11 Trophozoite stage of the malaria parasite ............................................................................ 27

Figure 12 Stages of schizont growth ..................................................................................................... 28

Figure 13 Gametocytes of Plasmodium falciparum and Plasmodium malariae ................................... 29

Figure 14 Blood elements, artefacts and contaminants that cause confusion .................................... 29

Figure 15 Appearance of different species of Plasmodium in a thin blood film .................................. 35

Figure 16 Appearance of different species of Plasmodium in a thick blood film same as above ........ 35

Figure 17 Different formats of Malaria RDT: A-cassette; B-Dipsticks; and C-Card test ........................ 48

Figure 18 Mode of action of antigen-detecting malaria rapid diagnostic tests (RDTs). ...................... 51

Figure 19 The Quality Assurance Cycle ................................................................................................. 55


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ACRONYMS

ACT Artemisinin Based Combination Therapy

AMU Average monthly usageAO Acridine orangeAQ AmodiaquineBF Blood filmCQ ChloroquineDNA Deoxyribose Nucleic AcidECP Exposure control planEDTA Ethylene diamine tetra acetic acidEHNRI Ethiopian Health and Nutrition Research InstituteELISA Enzyme linked immune-sorbent assayEQA External quality assessmentHEWs Health extension workersHIV Human immunodeficiency virusHMIS Health management information systemHRP2 Histidine rich protein 2HSDP Health Sector Development ProgrammeIQC Internal quality controlIRS Indoor Residual sprayITNs Insecticide Treated Nets

LMIS Logistic Management Information SystemNEQAS National external quality assessment schemePEP Post-exposure prophylaxisPf Plasmodium falciparumPfHRP2 Plasmodium falciparum histidine rich proteinPHL Public health laboratorypLDH Plasmodium lactate dehydrogenasePMA Pan-malaria antigenPMI Presidents Malaria Initiative

PPE Personal protective equipmentPT Proficiency testPv Plasmodium vivaxQA Quality assuranceQBC Quantitative Buffy CoatQC Quality controlQI Quality improvementRBC Red blood cellRDT Rapid Diagnostic TestREQAS Regional External Quality Assessment SchemeSDPs Service delivery points


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SOPs Standard operating proceduresSP Sulphadoxine pyrimethaminel Micro literUSAID The United States Agency for International DevelopmentWBC White blood cellWHO World Health Organization


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GLOSSARY OF TERMS

Antibody A specialized serum protein (immunoglobulin or gamma globulin) producedby B lymphocytes in the blood in response to an exposure to foreign proteins(antigens ). The antibodies specifically bind to the antigens that induced theimmune response. Antibodies help defend the body against infectious agents,including bacteria, viruses, or parasites.

Antigen Any substance that stimulates the immune system to produce antibodies.Antigens are often foreign substances: invading bacteria, viruses, or parasites.

Asexual cycle The life-cycle of the malaria parasite in host from merozoite invasion of redblood cells to schizont rupture (merozoite ring stage trophozoite schizont merozoites). Duration approximately 48 h in Plasmodium

falciparum, P. ovale and P. vivax ; 72 h in P. malariae .

Asexualparasitaemia

The presence in host red blood cells of asexual parasites. The level of asexualparasitaemia can be expressed in several different ways: the percentage ofinfected red blood cells, the number of infected cells per unit volume ofblood, the number of parasites seen in one microscopic field in a high-powerexamination of a thick blood film, or the number of parasites seen per 200 1000 white blood cells in a high power examination of a thick blood film.

Control Reduction of disease incidence, prevalence, morbidity or mortality to a locallyacceptable level as a result of deliberate efforts.

Drug resistance The ability of a parasite strain to survive and/or to multiply despite theadministration and absorption of a medicine given in doses equal to or higherthan those usually recommended but within the tolerance of the subject,provided drug exposure at the site of action is adequate. Resistance toantimalarials arises because of the selection of parasites with geneticmutations or gene amplifications that confer reduced susceptibility (WHO).

Efficacy The power or capacity to produce a desired effect.

Elimination The interruption of local mosquito-borne malaria transmission in a definedgeographical area, creating a zero incidence of locally contracted cases.

Imported cases will continue to occur and continued intervention measuresare required.

Elimination ofdisease

Reduction to zero of the incidence of a specified disease in a definedgeographical area as a result of deliberate efforts.

Elimination ofinfection

Reduction to zero of the incidence of infection caused by a specified agent ina defined geographical area as a result of deliberate efforts.

Endemic Where disease occurs consistently.

Epidemic The occurrence of more cases of disease than expected in a given area or

among a specific group of people over a particular period of time.


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Epidemiology The study of the distribution and determinants of health-related states orevents in specified populations; the application of this study to control healthproblems.

Eradication Permanent reduction to zero of the worldwide incidence of infection causedby a specific agent as a result of deliberate efforts;

Erythrocytic stage A stage in the life cycle of the malaria parasite found in the red blood cells.Erythrocytic stage parasites cause the symptoms of malaria.

Exoerythrocyticstage

A stage in the life cycle of the malaria parasite found in liver cells(hepatocytes). Exoerythrocytic stage parasites do not cause symptoms.

External qualityAssessment

A system whereby a reference laboratory sends stained blood films to alaboratory for examination. The laboratory receiving the slides is not informedof the correct result of the slides until the laboratory has reported theirfindings back to the reference laboratory

False negative slide A positive smear that is misread as negative.False positive slide A negative smear that is misread as positive.Feedback The process of communicating results of external quality control to the

original laboratory, including identification of errors and recommendations forremedial action.

G6PD deficiency An inherited abnormality that causes the loss of a red blood cell enzyme.People who are G6PD deficient should not take the antimalarial drugprimaquine.

Gametocyte The sexual stage of malaria parasites. Male gametocytes (microgametocytes)and female gametocytes (macrogametocytes) are inside red blood cells in thecirculation. If a female Anopheles mosquito ingests them, they undergo sexualreproduction, which starts the extrinsic (sporogonic) cycle of the parasite inthe mosquito. Gametocytes of Plasmodium falciparum are typically banana orcrescent-shaped (from the Latin falcis = sickle).

Hypnozoite Dormant form of malaria parasites found in liver cells. Hypnozoites occur onlywith Plasmodium vivax and P. ovale . After sporozoites (inoculated by themosquito) invade liver cells, some sporozoites develop into dormant forms(the hypnozoites), which do not cause any symptoms. Hypnozoites canbecome activated months or years after the initial infection, producing arelapse.

Hypoglycemia Low blood glucose; can occur with malaria. In addition, treatment withquinine and quinidine stimulate insulin secretion, reducing blood glucose.

Immune system The cells, tissues, and organs that help the body resist infection and diseaseby producing antibodies and/or cells that inhibit the multiplication of theinfectious agent.

Immunity Protection generated by the body s immune system, in response to previousmalaria attacks, resulting in the ability to control or lessen a malaria attack.

Incubation period The interval of time between infection by a microorganism and the onset ofthe illness or the first symptoms of the illness. With malaria, the incubation is

between the mosquito bite and the first symptoms. Incubation periods range


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from 7 to 40 days, depending on the species.

Indigenous malaria Mosquito-borne transmission of malaria in a geographic area where malariaoccurs regularly.

Infection The invasion of an organism by a pathogen, such as bacteria, viruses, orparasites. Some, but not all, infections lead to disease.

Introduced malaria Mosquito-borne transmission of malaria from an imported case in ageographic area where malaria does not regularly occur.

Malaria pigment(haemozoin)

A dark brown granular pigment formed by malaria parasites as a by-productof haemoglobin catabolism. The pigment is evident in maturetrophozoites and schizonts. They may also be present in white blood cells(peripheral monocytes and polymorphonuclear neutrophils) and in theplacenta.

Merozoite A daughter-cell formed by asexual development in the life cycle of malaria

parasites. Liver-stage and blood-stage malaria parasites develop intoschizonts, which contain many merozoites. When the schizonts are mature,they (and their host cells!) rupture, the merozoites are released and infect redblood cells.

Microscopist A person who uses a microscope to read blood films to aid or confirm thediagnosis of malaria and reports on their findings. The term is used in thismanual to include personnel at all levels of a malaria programme involved inthis work, from professors involved in teaching and research to village healthvolunteers specifically trained in malaria microscopy.

Oocyst A stage in the life cyle of malaria parasites, oocysts are rounded cysts locatedin the outer wall of the stomach of mosquitoes. Sporozoites develop insidethe oocysts. When mature, the oocysts rupture and release the sporozoites,which then migrate into the mosquito s salivary glands, ready for injectioninto the human host.

Outbreak An epidemic limited to a localized increase in disease incidence, e.g. in avillage, town or closed institution.

Pandemic An epidemic occurring over a very wide area, crossing internationalboundaries and usually affecting a large number of people.

Parasite Any organism that lives in or on another organism without benefiting the hostorganism; commonly refers to pathogens, most commonly to protozoans andhelminthes.

Parasitemia The presence of parasites in the blood. The term can also be used to expressthe quantity of parasites in the blood (for example, a parasitemia of 2

percent ).

Paroxysm A sudden attack or increase in intensity of a symptom, usually occurring atintervals.

Pathogen Bacteria, viruses, parasites, or fungi that can cause disease.

Plasmodium The genus of the parasite that causes malaria. The genus includes four speciesthat infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium


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ovale, and Plasmodium malaria e.

Pre-erythrocyticdevelopment

The life-cycle of the malaria parasite when it first enters the host. Followinginoculation into a human by the female anopheline mosquito,sporozoites invade parenchyma cells in the host liver and multiply within thehepatocytes for 5 12 days, forming hepatic schizonts. These then burstliberating merozoites into the bloodstream, which subsequently invade redblood cells

Presumptivetreatment

Treatment of clinically suspected cases without, or prior to, results fromconfirmatory laboratory tests.

Panel testing The process by which laboratories (known as the test laboratories)performs malaria microscopy on a set of prepared slides received from theNational and Regional Laboratories. This exercise can check both thelaboratories staining quality as well as the ability of technicians to recognizeand identify malaria parasites present.

Quality assurance The monitoring and maintenance of high accuracy, reliability and efficiency oflaboratory services. Quality assurance addresses all factors that affectlaboratory performance including test performance (quality control, internaland external) equipment and reagent quality, workload, workplaceconditions, training and laboratory staff support.

Quality control Measures the quality of a test or a reagent. For malaria microscopy, the mostcommon form of quality control (QC) is the cross-checking of routine bloodslides to monitor the accuracy of examination. Quality control alsoencompasses external quality control and reagent quality control. Cross-checking QC is a system whereby a sample of routine blood slides are cross-checked for accuracy by a supervisor or the regional/national laboratory.

Reagent QC is a system of formally monitoring the quality of the reagentsused in the laboratory.

QualityImprovement

A process by which the components of microscopy and RDT diagnosticservices are analyzed with the aim of identifying and permanently correctingany deficiencies. Data collection, data analysis, and creative problem solvingare skills used in this process

Radical cure (alsoradical treatment)

Complete elimination of malaria parasites from the body; the term appliesspecifically to elimination of dormant liver stage parasites (hypnozoites) foundin Plasmodium vivax and P. ovale.

Recrudescence A repeated attack of malaria (short-term relapse or delayed), due to thesurvival of malaria parasites in red blood cells. Radical treatment: see radicalcure .

Relapse Recurrence of disease after it has been apparently cured. In malaria, truerelapses are caused by reactivation of dormant liver stage parasites(hypnozoites) found in Plasmodium vivax and P. ovale.

Residual insecticidespraying

Spraying insecticides that have residual efficacy (that continue to affectmosquitoes for several months) against houses where people spend nighttimehours. Residual insecticide spraying is done to kill mosquitoes when theycome to rest on the walls, usually after a blood meal.

Resistance The ability of an organism to develop strains that are impervious to specific


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threats to their existence. The malaria parasite has developed strains that areresistant to drugs, such as chloroquine. The Anopheles mosquito hasdeveloped strains that are resistant to DDT and other insecticides.

Ring stage Young usually ring-shaped intra-erythrocytic malaria parasites, before malariapigment is evident under microscopy.

Schizogony Asexual reproductive stage of malaria parasites. In red blood cells, schizogonyentails development of a single trophozoite into numerous merozoites; asimilar process happens in infected liver cells.

Schizont A developmental form of the malaria parasite that contains many merozoites.Schizonts are seen in the liver-stage and blood-stage parasites.

Serology The branch of science dealing with the measurement and characterization ofantibodies and other immunological substances in body fluids, particularlyserum.

Slide positivity rate The proportion of positive slides, detected by microscopy, among all thoseexamined within a laboratory over a defined period of time.

Sporozoite A stage in the life cycle of the malaria parasite. Sporozoites, produced in themosquito, migrate to the mosquito's salivary glands. They can be inoculatedinto a human host when the mosquito takes a blood meal on the human. Inthe human, the sporozoites enter liver cells where they develop into the nextstage of the malaria parasite life cycle (the liver stage or exo-erythrocyticstage).

Trophozoite A developmental form during the blood stage of malaria parasites. Aftermerozoites have invaded the red blood cell, they develop into trophozoites

(sometimes, early trophozoites are called rings or ring stage parasites );trophozoites develop into schizonts.

Vector An organism (for example, Anopheles mosquitoes) that transmits aninfectious agent (for example, malaria parasites) from one host to the other(for example, humans).


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1 INTRODUCTION

1.1 Malaria Etiology

Malaria is a disease caused by blood parasites of the genus Plasmodium . There are approximately156 named species of Plasmodium which infect various species of vertebrates. Four are known toinfect humans: P. falciparum , P. vivax , P. ovale , and P. malariae. Recently, a new malaria parasitespecies named P. knowlesi is identified in Asia affecting both humans and animals. Malaria can bevery severe and can lead to death if left untreated. Malaria parasite is transmitted from an infectedperson to another by the bite of a female anopheline mosquito. This can occur only after theparasite has been inside the mosquito for at least a week.

1.2 Life Cycle and Transmission of MalariaThe malaria life cycle takes place in humans and in the female Anopheles mosquito. The malariaparasite life cycle involves two hosts, the female anopheles mosquito as definitive host and thehuman as intermediate host. Malaria parasites are usually transmitted by the bite of an infectedfemale Anopheles mosquito. Malaria trophozoites may also be transmitted through bloodtransfusion and trans-placentally (congenital malaria). The life cycle follows three stages: the exo-erythrocytic, erythrocytic and sporogonic cycle.

Human infection begins when an infected female Anopheline mosquito inoculates plasmodialsporozoites from its salivary gland during a blood meal. The mosquito becomes infected by ingestinghuman blood containing the sexual forms of the parasite (gametocytes). In the mosquito gut wall thesporogonic cycle starts with the gametocytes fusing and forming zygote which further develop intoookinete and oocyst. The oocysts grow, rupture, and release sporozoites, into the mosquitossalivary glands.

Source: http://www.dpd.cdc.gov/dpdxFIGURE1 LIFECYCLE OFMALARIALPARASITES


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When the mosquito next feeds on humans, it injects sporozoites into the blood stream thateventually infect liver cells (hepatocytes). Within the liver cells, sporozoites are transformed intomerozoites. The stage of the life cycle from sporozoite injection to the liver schizont stage is termedthe pre-erythrocytic stage. The erythrocytic stage follows when merozoites released into the bloodstream infect red blood cells. Subsequent parasitic development in the red blood cells (blood

schizogony) results in the following parasitic forms: the asexual forms (trophozoites, schizonts &merozoite as well as the two sexual forms of gametocytes.Red blood cell lysis during schizontrupture and release of merozoites , initiates the typical clinical manifestations of malaria, fever,shiver & sweating paroxysm. Themerozoites immediately invade new red blood cells to repeat thecycle several times over the course of weeks. However, in P. vivax and P. ovale infections, somesporozoites become dormant hypnozoites upon invading hepatic cells. Reactivation of thehypnozoites can occur up to 6-8 months later, initiating either a delayed primary infection orrelapse.

1.3 Overview of methods for malaria diagnosisPrompt and accurate diagnosis of malaria is part of effective disease management. The diagnosis ofmalaria is based on clinical suspicion and on the detection of parasites in the blood (parasitologicalor confirmatory diagnosis). High sensitivity of diagnosis in malaria endemic areas is particularlyimportant for the most vulnerable population groups, such as young children and the non-immunepopulation, in whom the disease can be rapidly fatal, while high specificity will reduce unnecessarytreatment with anti-malarial drugs and improve diagnosis of other febrile illnesses in all settings.Thus, high quality malaria diagnosis is important in all settings.

1.3.1 Clinical Diagnosis of Malaria

A clinical diagnosis entails making a clinical assessment by taking an accurate history of the illnessand performing a physical examination. Clinical diagnosis of malaria is made in a patient who hasfever or history of fever in the last 48 hours and lives in malaria-endemic areas or has a history oftravel within the last 30 days to malaria-endemic areas. Basing the diagnosis on clinical featuresalone is not recommended, as this often has low specificity and increases the chances of the patientbeing misdiagnosed. Unless there is an ongoing malaria epidemic, or is a peak malaria transmissionseason, careful laboratory testing typically reveals confirmed malaria parasites in fewer than half ofclinically suspected malaria in most situations in Ethiopia. Malaria treatment based on clinicaldiagnosis must be the last option when there is no availability of RDTs or microscopy. WHOrecommends universal parasitological diagnosis of malaria to ensure targeted use of antimalarialdrugs for those who actually have malaria. The health worker examining a suspected malaria caseshould perform differential diagnosis to look for other causes of fever (e.g., typhoid fever, relapsingfever, acute respiratory tract infections, meningitis, etc) and manage the case accordingly. Malariashould still be considered, even if the individual has another obvious cause for the fever. Thenational algorithm of the Integrated Management of Neonatal and Childhood Illness (IMNCI) andCommunity-based Case Management (CCM) should also be employed for the management of thesick child presenting with fever.

The clinical course of malaria infection may be uncomplicated or severe. Because of its frequent andsevere complications, P. falciparum is the most serious malaria-causing parasite and cause of death.Patients under the age of five, pregnant women, non-immune individuals of all ages and peopleliving with HIV are particularly at risk for severe and complicated malaria and death.


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Uncomplicated malaria is characterized by fever and other features including chills, profusesweating, muscle pains, joint pains, headache, abdominal pain, diarrhoea, nausea, vomiting, loss ofappetite, irritability, and refusal to feed (in infants). These features may occur singly or incombination and are due to the presence of parasites in the peripheral blood.

Severe and complicated malaria is a life threatening condition, defined as the detection of P. falciparum in peripheral blood together with any of the following clinical or laboratory features(singly or in combination):

Inability to or difficulty in sitting upright;standing or walking without support; orinability to feed (in an infant)

Alteration in the level of consciousness(ranging from drowsiness to deep coma)

Cerebral malaria (unarousable coma notattributable to any other cause, otherneurological signs)

Respiratory distress Multiple generalised convulsions (2 or

more episodes within a 24 hour period) Circulatory collapse (shock, septicaemia) Pulmonary oedema Abnormal bleeding (Disseminated

Intravascular Coagulation DIC) Jaundice

Haemoglobinuria (black water fever) Acute renal failure presenting as oliguria

(passing scanty urine) or anuria (notpassing urine)

Severe anaemia (haemoglobin 200,000/l - in patients from hightransmission areas; or 100,000/l inpatients from low transmission areas)

Hyperlactataemia (whole blood lactate >5mmol/l)

Examples of illnesses that may present with symptoms and signs similar to malaria include:

Meningitis Otitis media Pharyngo-tonsillitis Pneumonia Acute gastroenteritis Typhoid fever Urinary tract infection Viral infections (e.g. mumps, measles) Hepatitis


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1.3.2 Laboratory Diagnosis of Malaria

Once malaria is suspected on clinical grounds, it is mandatory to obtain the laboratory confirmation ofthe presence of malaria parasites. Clinicians could request for diagnostic test for malaria to confirm thediagnosis of malaria in a patient with symptoms and signs suggestive of malaria disease; to rule outmalaria infection in a patient with other known causes of fever; to confirm malaria in febrile infantsunder 3 months of age; to look for treatment failure; and to investigate causes of anaemia, jaundice orsplenomegaly.

1.3.2.1 Common Diagnostic Methods

The two laboratory diagnostic methods or tools most often used for confirming a diagnosis of malariaare:

a. Rapid Diagnostic Tests RDTs: RDTs detect antigens (proteins produced by malaria parasite) inthe blood of a patient with malaria .

b. Light Microscopy : Good quality microscopy is the most acceptable method for detecting andidentifying malaria parasites from the blood of a suspected patient. The procedure consists ofcollecting a finger-prick blood sample; preparing a thin and thick blood films; staining the filmswith Giemsa or other stains such as Field stain and examining the film through a microscope forthe presence of malaria parasites.

1.3.2.2 Alternative Lab Diagnostic Methods

Although alternative malaria diagnostic methods exist, they are not as suitable for wide application inthe field as microscopy or RDTs. They are unsuitable for use in routine disease management in resource-limited settings and are often used for research purposes. These are:

a. Quantitative Buffy Coat (QBC)This technique is a qualitative method for rapidly detecting malaria parasites in centrifuged capillary orvenous blood. QBC utilizes density gradient layering of stained blood cells, together with mechanicalexpansion of the haematocrit buffy coat. The parasites are detected by fluorescent microscopy usingacridine orange stain. It is fast, easy and may be more sensitive than the traditional thick filmexamination. Its main advantages are faster result delivery within 15-30 minutes, and a potential foraccidentally detection of filarial worms. However, it may provide false positive results due to artifacts,species differentiation can be difficult, and per test cost is expensive.

b. Thin film acridine orange technique/ Microscopy using Kawamotos fluorochrometechnique

Fluorescence microscopy combined with fluorochrome staining of thin blood films with acridine orange(AO) has been reported to be more sensitive than the Romanowsky technique for the detection ofmalaria parasites and emits two fluorescence colors, green (530 nm) and red (650 nm) when excited at430 nm and 492~495 nm, respectively. Therefore, AO staining permits differential coloration of green(nuclei) and red (cytoplasm) in stained parasites; the outlines of the parasites stained by these dyes arewell preserved and the general morphology is comparable to specimens stained by Giemsa.


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c. Immunological tests (Anti-malarial Antibody Test)

Antibodies to the asexual blood stages appear a few days after malarial infection, increase in titer overthe next few weeks, and persist for months or years in semi-immune patients in endemic areas, wherere-infection is frequent. The antibody tests can be done using either indirect immunofluorescence (IFA)tests or an enzyme-linked immunosorbent assay (ELISA). Because of the time required for developmentof antibodies and also the persistence of antibodies, serologic testing is not practical for routinediagnosis of acute malaria but instead used to determine past exposure.

d. Polymerase Chain Reaction (PCR)

This technique is used to detect parasite nucleic acids. The principle is based on the extraction ofparasite DNA and amplification by polymerase chain reaction using specific primers to yield a productthat can easily be visualized in ethidium bromide stained agarose gel. As little as one parasite permicrolitre of blood can be detected by this method. It is highly specific and sensitive (10 times moresensitive than microscopy) in detecting the plasmodium species, particularly in cases of low level

parasitemia and mixed infections, with a sensitivity of 1.35 to 0.38 parasites/L for P.falciparum and0.12 parasites/L for P.vivax. However, it requires expensive laboratory equipment in specializedlaboratory settings and often used in reference laboratories to confirm malaria parasite species (if indoubt); to validate Rapid Diagnostic Tests (RDTs) as part of planned quality assurance programmes; andfor research purposes.

e. Flowcytometry

Flowcytometry and automated hematology analyzers have been found to be useful in indicatingdiagnosis of malaria during routine blood counts. In cases of malaria, abnormal cell clusters and smallparticles with DNA fluorescence, probably free malarial parasites, have been seen on automatedhematology analyzers and it is suggested that malaria can be suspected based on the scatter plots

produced on the analyzer. Automated detection of malaria pigment in white blood cells may alsosuggest a possibility of malaria with a sensitivity of 95% and a specificity of 88%.


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2 SCOPE AND PURPOSE OF THE MANUAL

2.1 Purpose

The purpose of this manual is to guide professionals and stakeholders responsible for malaria control

and prevention programs on the best ways of ensuring quality laboratory diagnosis. The manualdescribes overview of malaria epidemiology, laboratory procedure, quality assurance and supplymanagement; and outlines the technical knowledge needed for laboratory diagnosis of malaria.

The aim of this manual is to help to ensure that malaria diagnosis at national, regional, district andcommunity levels are efficiently and effectively organized to allow early diagnosis and prompt, effectivetreatment. The manual provides basic information for the successful operation of malaria laboratorydiagnosis and defines the skills required in the following areas:

Implementation of quality assured malaria laboratory diagnosis through standard procedure Planning training and conducting quality assurance program Planning effective lab diagnosis and identifying the technical and managerial elements that

require revision Logistical organization to ensure regular supplies Planning supervision, monitoring and evaluation Coordinating and integrating malaria diagnosis with other laboratory programs

2.2 Target Audience

The manual is intended for use in particular by health professionals and stakeholders working on malarialaboratory diagnosis program, and in general for multidisciplinary teams involved in managing nationalmalaria control program, including program managers, epidemiologists, program supervisors, health

educators, logistics officers and trainers. Health project managers dealing with malaria at national,district and community levels, including those responsible for private health services, will also find thismanual useful.

The manual will be a useful resource in Ministry of Health or in projects supported by international andmultilateral cooperation agencies or nongovernmental organizations, in medical, nursing, laboratory andpublic health schools for training in effective malaria case management.


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3 MALARIA SITUATION IN ETHIOPIA

3.1 Burden of the Disease

Malaria is a serious public health problem in many parts of the world, exacting an unacceptable toll on

the health and economic welfare of the worlds poorest communities. Based on the WHO report in2011, reductions in reported malaria cases of more than 50% have been recorded between 2000 and2010 in 43 of the 99 countries with ongoing transmission, while downward trends of 25% 50% wereseen in 8 other countries. There were an estimated 216 million episodes of malaria in 2010, of whichapproximately 81%, or 174 million cases, were in the African Region. There were an estimated 655 000malaria deaths in 2010, of which 91% were in Africa. Approximately 86% of malaria deaths globally wereof children under 5 years of age. The estimated incidence of malaria globally has reduced by 17% since2000 and malaria-specific mortality rates by 26%. These rates of decline are lower than internationallyagreed targets for 2010 (reductions of 50%) but nonetheless, they represent a major achievement

Malaria is the leading cause of morbidity and mortality in Ethiopia. Malaria is ranked as the leadingcommunicable disease in Ethiopia, accounting for about 30% of the overall Disability Adjusted Life Yearslost. Approximately 68% of the total population of 78 million lives in areas at risk of malaria. Accordingto Ethiopias Federal Ministry of Health (FMOH), in 2008/2009, malaria was the leading cause ofoutpatient visits, health facility admissions and inpatient deaths, accounting for 12% of reportedoutpatient visits and nearly 10% of admissions. In 2009, 3 million suspected malaria cases were seenand nearly 2.3 million (77%) were tested. The number of malaria cases decreased from an annualaverage of 3 million during 2000 2005 to 1.75 million cases in 2009 (41% decline). In the same periodthe malaria admissions decreased from an average of 44,000 to 30,102 in 2009 (33% decline). Inpatientmalaria deaths fell by 43% in all age groups and by 60% in children


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3.3 The National Strategic Plan for Malaria Prevention, Control and Elimination

The 2011-2015 National strategic plan (NSP) will focus on sustained control and moving towards malariaelimination through an integrated community health approach, especially in areas of unstable malariatransmission, building on Scale Up for impact (SUFI) achieved by the 2006-2010 strategic plan.

WHO recommends indicative epidemiological milestones for determining when a low- or medium-transmission country has an incidence low enough to begin the rigorous surveillance required duringelimination. When the slide positivity rate (SPR) of all febrile patients with suspected malaria is less than5% or the incidence is less than 5 per 1000 people at risk, the country, or district in some cases, couldconsider transitioning into pre -elimination if other factors are in place as well.

SUFI and sustained control are similar to the attack phase, when malaria infection prevalence is reducedto less than 5 per 1,000 populations per year in risk areas. Pre-elimination and elimination areequivalent to the consolidation phase, when interruption of transmission is achieved. In the finalmaintenance phase, prevention of introduction and local transmission are maintained for 3 consecutiveyears, at which time a country can be certified by WHO as malaria free.

3.4 Goal and Objectives of 2011-2015 strategic plan

Goals:

By 2015, achieve malaria elimination within specific geographical areas with historically lowmalaria transmission.

By 2015, achieve near zero malaria death in the remaining malarious areas of the country.

Objectives

The objective of the 2011-2015 National Strategic Plan is to consolidate the achievements of the 2006-2010 National Strategic Plan, and sustain its impacts. This overall objective will be attained through thefollowing specific objectives:

1. 100% of suspected malaria cases are diagnosed using RDTs and/or microscopy within 24 hoursof fever onset

2. 100% of positive malaria diagnosis are treated according to national guidelines3. 100% of households in malarious areas own one LLIN per sleeping space4. At least 80% of people at risk of malaria use LLINs properly and consistently5. IRS coverage is increased and maintained to 90% of households in IRS-targeted areas.6. 100% of health posts in malarious Kebeles provide the full malaria prevention and treatment

package, including outreach services.7.

Achieve a high quality, broadly-based malaria infection detection

and situational awareness

The National strategic plan provides a detailed account on the status and direction of the major malariaprevention and control strategies which includes:


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A. Community Empowerment and Mobilisation

Community empowerment and mobilization are central to malaria prevention and control. EthiopiasHealth Extension Program educates, mobilizes and involves the community in all aspects and stages ofmalaria control and leads to increased ownership of the program.

B. Diagnosis and Case Management

Since 2005, there has been a major shift from clinical diagnosis to confirmatory diagnosis following thewide-scale use of RDTs in peripheral health facilities. To improve the quality of malaria diagnosis andtreatment at peripheral health facilities (health posts) panspecific RDTs are now being introduced. HEWswill be trained on the use of multi-species RDTs in the integrated refresher training (IRT).

C. Prevention

The main major vector control activities implemented in the country include IRS, LLINs andenvironmental control.

D. Active Surveillance and Epidemic Control

Aims to achieve a high quality, broadly based malaria infection detection, investigation and responseSurveillance System to further reduce malaria transmission and improve the detection and timelyresponse to malaria epidemics. Malaria detection, investigation, response and elimination activities willachieve a high quality, broadly based malaria infection detection, investigation and responsesurveillance System to further reduce malaria transmission, prevent and stop epidemics and eliminatemalaria especially in targeted areas that are prone to outbreaks. There will be a transition fromepidemic detection and response to surveillance and infection response as transmission declines to nearzero.

E. Health system strengthening and capacity buildingThe health system strengthening and capacity building includes monitoring and Evaluation activities anddevelopment of Human Resources.

3.5 Levels of Health facilities and types of diagnostic tests in Ethiopia

3.5.1 National and Regional Reference Laboratories

The national and regional reference laboratories are performing specialized laboratory diagnostic testsmainly for operational researches and trainings. Malaria parasite molecular, serological tests, drug leveldeterminations and RDT evaluations are conducted at the national reference laboratory. Malariamicroscopy is mainly used at the national level for research, large surveys, quality control and trainingpurposes. At the regional reference laboratories, malaria microscopy is mainly conducted for thepurposes of training and external quality assessment schemes.


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3.5.2 Hospitals and health centers

In accordance with the National Malaria guidelines of 2012, malaria microscopy is the sole techniqueemployed in hospital and health center levels. Therefore, it is critical that these facilities are equippedwith standard microscopes, have adequate supplies and skilled microscopists.

3.5.3 Health posts

The basis of suspicion for malaria infection is fever (rise in body temperature) from the patients historyand verified by touching or recording the temperature with a thermometer. Rapid diagnostic tests (RDTs)shall be used by the health extension workers who have received training in its use.

3.6 Case Management Practices

3.6.1 Treatment Approach

Ensuring prompt and effective treatment will prevent most cases of uncomplicated malaria from

progressing to severe and fatal illnesses. To avoid this progression, treatment must begin as soon aspossible, generally within 24 hours after symptoms onset. Effective malaria treatment requiresimproved diagnosis of malaria (i.e. laboratory-based microscopy or use of multi species RDTs); welltrained health workers in both the public and private health sectors; constant availability of highlyefficacious medicines as close to the patient as possible to ensure prompt access. Communities shouldbe aware of the importance to seek early diagnosis and treatment and to adhere to prescribed drugregimens for malaria.

Treatment of malaria should be based upon a parasitologically confirmed diagnosis whenever thesituation permits. Laboratory evidence providing confirmation of malaria (i.e. microscopy or RDT) bymalaria species requires prompt treatment with the appropriate antimalarial medications. If the RDT ormicroscopy test indicates P. falciparum , then the patient should be treated with appropriate doses ofArthemisisin-Lumefantrine ( AL), ensuring the patient is able to swallow the medication, and not vomit.If the RDT or microscopy reveals P. vivax only (and no P. falciparum ), then chloroquine treatment shouldbe dispensed, also ensuring that oral medicine is tolerated. Radical treatment with Primaquine isrecommended at health center and hospital level for patients who are vivax positive & are not living inmalaria endemic areas. Health workers should be vigilant to detect side effect of Primaquine. Mixedinfection of P. falciparum and P.vivax should be treated with AL. Pregnant women with P. falciparum inthe first trimester and children weighing less than five kilograms need to be treated with oral Quinine,an alternative to AL. When there is a negative laboratory result by RDT or microscopy for malaria, nomalaria medications need be provided, but a thorough search for other causes of acute febrile illnessshould continue, such as pneumonia; in such cases, referral to Health Centers or hospital Is advised. Bytesting as many clinically suspected malaria patients as possible with RDTs or microscopy, and by

treating them according to their malaria lab test result, the waste of antimalarial medications can bereduced and eliminated.


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3.6.2 Case management of uncomplicated malaria

Uncomplicated malaria is defined as symptomatic malaria without signs of severity or evidence of vitalorgan dysfunction. It is important that uncomplicated malaria is treated well, because if left untreated, itcan progress rapidly to severe disease and death.

The most important principles of malaria management are:

early and accurate diagnosis prompt and effective treatment adherence to treatment Advice and follow-up.

Uncomplicated malaria is mainly characterized by clinical symptoms such as fever, chills, shivering,headache, loss of appetite,& rarely joint pains (arthralgia)and generalized muscle ache (myalgia) in thepresence of asexual forms of malaria parasites in blood sample. Based on the Ethiopian national malariacase management guidelines, the treatment approach is as described below:

First Line Treatment

Artemether-Lumefantrine: The recommended first-line treatment of all clinically and parasitologicallydiagnosed uncomplicated Plasmodium falciparum malaria in Ethiopia is an Artemisinin-basedCombination Therapy (ACT) called Artemether-Lumefantrine (AL). This is currently available as co-formulated tablets containing 20 mg of artemether and 120 mg of lumefantrine. The totalrecommended treatment is a 6-dose regimen of artemether-lumefantrine (i.e. twice a day for 3 days).

Note that both of the above, including other ACTs, are contraindicated in:

Children


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3.6.3 General approach to management of Severe Malaria

Severe and complicated malaria (SCM) is a clinical emergency and time is of the essence in preventinglong term complications and death. The main objective in the treatment of severe malaria is to preventdeath. Secondary objectives include prevention of disability, recrudescence and the development ofresistance, Every staff member has a potential role to play in patient management especially those withSCM, whether it is to carry out selection of very sick patients and alerting qualified staff, puttingunconscious patients in the right position to protect their airway or ensuring that blood tests are takenand prioritized so that appropriate treatment is commenced in a speedy fashion.

At peripheral health post level it is important that some form of triage system is in place. The wordTRIAGE comes from the French verb to sort and it is the process of ra pidly screening sick patientswhen they arrive at any health facility. These patients should be prioritized at all stages of managementfrom the initial assessment, to admission, investigation and treatment. Health centers and hospitals willdiagnose and treat severe malaria, Health centers shall refer all cases of severe malaria which cant betreated at that level. The recommended drug treatment for severe malaria is IV or IM Artesunate.Alternatives are IV Quinine and IM Artemether. Health posts will diagnose, give pre-referral treatment

to suspected SCM patients and speedily refer them to the appropriate Health Centres or Hospitals.

Pre-referral Treatment

The risk of death from severe malaria is greatest in the first 24 hrs, it is recommended that patients betreated with the first dose of one of the recommended treatments before referral. At health post level,the preferred pre-referral treatment is rectal artesunate. Artemether IM, Quinine IM are alternativesand will be available for pre-referral treatment.


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4 PARASITOLOGICAL DIAGNOSIS OF MALARIA USING MICROSCOPY

Microscopy is the science of investigating small objects using a microscope. It has an essential role forthe diagnosis and management of many infectious diseases such as malaria, tuberculosis, intestinal

parasites, etc. through examination of clinical specimen.

4.1 Care and Handling of Microscope

Good working knowledge and proper care of the microscope are critical to good diagnostic work. Thereare only a few absolute rules to observe in caring for the microscopes you will use. Taken care of, theseinstruments will last many decades and continue to work well. Please report any malfunctionsimmediately.

1. Always use two hands to carry the microscope - one on the arm and one under the base.Never carry the microscope upside down, for the ocular can and will fall out.

2. Never expose it to sharp knocks, vibrations, moisture, dust or direct sunlight.3. Use lens paper to clean all lenses before and after using the oil immersion lens. Other

papers are too impure and will scratch the optical coating on the lenses. Also, do not useany liquids when cleaning the lenses use lens paper only!

4. Always use the proper focusing technique to avoid ramming the objective lens into a slide -this can break the objective lens and/or ruin an expensive slide.

5. Always turn off the light when not in use.6. Always carefully place the wire out of harms way. Wires looped in the leg spaces invite a

major microscope disaster. Try sliding the wire down through the drawer handles besideyour bench space.

7. Always replace the cover on the microscope when you put it away

4.1.1 Microscope maintenance and storage conditions

Never attempt to disassemble any part of the microscope for repair. If there is any problem with themicroscope, contact the microscope companys technical support unit or thier local agents, or consultwith a qualified technician, around.

Humidity causes fungal growth on the surface of lenses and prisms. This can cause cloudiness of the viewfield and rusting of metal parts of the microscope. To protect the microscope from fungus, always keepthe glass surface as clean as possible and free of dirt and fingerprints. Reduce the growth of fungus bycontinuously using an air conditioner to lower humidity. The use of air-conditioning in the daytime onlywill lead to condensation on the microscope once it is turned off, again favoring growth of fungus.Alternatively, drying the microscope within a temperature-controlled cabinet, silica gel (desiccant), oranti-mold strips may be useful.

Cabinet Box (for humidity and temperature control) (see Figure 11.2) Store a microscope in a cabinet box with air inlets and outlets for air circulation and a 20-watt bulb forkeeping a dry, stable environment.
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4.1.3 Cleaning a Microscope

Anti-Mold Strips

Anti-mold strips can be also applied to prevent mold. Replace these strips every 3 years. Always keep thefour optical parts of the microscope (see Figure 11.1) clean. Remove dust attached to the microscopewith a blower or other towels/tissue paper.

Use only immersion oil with the proper clearness, viscosity, and refractive index for the immersion lens.Cedar oil and other types of oil such as baby oil, cooking oil and liquid paraffin are not acceptable for thispurpose as they will damage the lens.

Before putting the microscope away, wipe off the immersion oil by rubbing the surface of the immersion(100 objective) lens gently with a washed soft gauze or lens paper which is lightly moistened with ethylether/alcohol (80/20 vol/vol). This can also be used to remove fingerprints or grease. Remove dust bysoftly brushing the surfaces. For cleaning lenses and filters, wipe the object from the center, winding aspiral to the periphery.

Microscope Cleaning Process

Cleaning the eye piece Cleaning the objectives Cleaning the microscope stage Cleaning the microscope body Cleaning the condenser

Cleaning the eye piece

Blow to remove dust before wiping lens Clean the eyepieces with a cotton swab moistened with lens cleaning solution Clean in a circular motion inside out Wipe the eyepieces dry with lens paper Repeat cleaning and drying if required

Cleaning the Objectives

Objectives are cleaned while attached to the microscope- Moisten the lens paper with the cleaning solution- Wipe gently the objective in a circular motion from the inside out

- Wipe with a dry tissue or lens cleaning paper Objectives should never be removed from the nosepiece

Cleaning the Microscope Stage

Wipe the microscope stage using the cleaning solution on a soft cloth Thoroughly dry the stage Repeat the above steps, if required


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Cleaning the Microscope Body

Unplug the microscope from the power source Moisten the cotton pad with a mild cleaning agent (please give examples) Wipe the microscope body to remove dust, dirt and oil Repeat steps1 3, if required

Cleaning the Condenser

Unplug the microscope from the power source Clean the condenser lens and auxiliary lens using lint-free cotton swabs moistened with lens

cleaning solution Wipe with dry swabs

4.1.4 Troubleshooting

There are several conditions that can affect the proper functioning of the microscope. Review these

problems and their solutions.

1. The brightness of the viewing field is poor

Problem SolutionThe condenser is too low. Raise the condenser to correct its position.The condenser iris diaphragm is closed. Open the diaphragm properly.

2. There are dark shadows in the field which move as you turn around the eyepiece.

Problem SolutionThe surface of the eyepiece has scratches. Replace the eyepiece.The eyepiece is dirty. Clean the eyepiece.

3. The image with the high power objective is not clear.

Problem SolutionThe slide is upside down. Turn the slide over.There is an air bubble in the oil. Move 100x lens quickly from side to side.There is dirt on the objective. Clean the lens.The oil is too sticky. Use thinner immersion oil or specified

immersion oil.4. The image is not clear with the low power objective.

Problem SolutionThere is oil on the lens. Clean the lens.There is a layer of dust on the upper surface ofthe objective.

Clean the lens.


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If the view field is still dim and cloudy, consider the following possible causes: Massive growth of fungus on the lenses or prisms due to storage in a high humidity environment Penetration of immersion oil between the lenses of the objective through damaged lens cement

(due to use of poor-quality oil such as cedar oil or misuse of xylene). This is likely the cause if acompletely hazy field becomes clear after changing the objective.

A damaged objective (due to careless focusing, dropping, rough changing of slides)

Frequently-encountered operational errors include the following: Focusing the first slide using the 100x immersion objective without passing through a low power

objective. Changing slides from under the immersion objective without turning it away first. Wiping lenses without first blowing away dust and sand. Cleaning lenses or other parts with xylene. Using cedar wood oil, liquid paraffin, or xylene-diluted oil instead of pure synthetic immersion

oil. Keeping the microscope in a confined space without ventilation in a humid climate.

Log Book

A microscope log book should be maintained to enter problems encountered in the operation of themicroscope, maintenance schedule, repairs done on the microscope and availability of spares like bulbs,fuses, anti-mold strips etc.


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4.2 Parasitological Procedures of Microscopy

4.2.1 Specimen collection and blood film preparation

4.2.1.1 Blood Sample Collection

Capillary Blood Collection

Capillary blood is preferred to venous blood. Blood obtained by pricking a fingertip is the ideal samplebecause the density of developed trophozoites or schizonts is greater in blood from capillary-rich areas.For adults, the best site to prick is the lateral side of the third or fourth finger of the non-dominant hand(left hand unless the patient is left-handed) and the big toe is preferred for infants. The skin area to bepunctured should be warm so that blood flow will be adequate. Depending on the physical settings andthe patients condition, warming the hand with warm water, covering the hand with a hot, wet towel orbriskly rubbing the hand may be used to warm the hands prior to the finger prick. For routine malariamicroscopy, thin and thick blood films are prepared on the same slide.

Apply gentle pressure again (do not squeeze the finger too tightly) to transfer more blood and collecttwo or three larger drops on the slide, about 1 cm from the drop intended for the thin film or 1 cm fromthe end of the slide.

Venous Blood Collection

Venous blood is not collected for routine use in Malaria laboratory diagnosis. The venipunctureprocedure is complex, requiring both knowledge and skill to perform. Each phlebotomist generallyestablishes a routine that is comfortable for her or him. Several essential steps are required for everysuccessful collection procedure and venipuncture site selection. Although the larger and fuller mediancubital and cephalic veins of the arm are used most frequently, the basilic vein on the dorsum of the armor dorsal hand veins are also acceptable for venipuncture. Palpate and trace the path of veins with theindex finger. Arteries pulsate, are most elastic, and have a thick wall. Thrombosed veins lack resilience,feel cord-like, and roll easily.

If superficial veins are not readily apparent, you can force blood into the vein by massaging the armfrom wrist to elbow, tap the site with index and second finger, apply a warm, damp wash cloth to thesite for 5 minutes, or lower the extremity over the bedside to allow the veins to fill. Foot veins are a lastresort because of the higher probability of complications. One should recognize complicationsassociated with the phlebotomy procedure, assess the need for recollection and/or rejection of sampleand perform proper labeling of the specimen.


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4.2.1.2 Blood Film Preparation

Types of Blood Films

Two types of blood films, thick and thin, are used in the microscopic diagnosis of malaria. Both thick andthin films should be prepared and examined in all cases of suspected malaria.

Thick Blood Film:

Thick blood film consists of a thick layer of lysed erythrocytes. The blood elements (including parasites, ifany) are more concentrated (~30x) than in an equal area of a thin smear, allowing a greater volume ofblood to be examined. Because a larger volume (6 l) of blood is examined in the thick film, it is muchbetter than the thin film for detection of low levels of parasitemia and reappearance of circulatingparasites during infection, recrudescence or relapse.

Thick film is therefore the most suitable method for the rapid detection of the parasite, but it does notpermit an optimal review of parasite morphology for species identification. If the thick smear is positivefor malaria parasites, the thin smear should be used for species identification. Thus, the thick films areperformed to detect and quantify (parasite density) malaria parasites in routine malaria microscopicdiagnosis.

Thin Blood Film

Thin blood film consists of blood spread in a layer such that the thickness decreases progressivelytoward the feathered edge. In the feathered edge, the red blood cells should be in a single layer, nottouching one another. Thin blood smear should be fixed with methanol so that the parasites are foundintact inside the RBCs.

The morphological identification of the parasite to the species level is much easier and provides greaterspecificity than the thick film examination. However , low-density infections can be missed and require

more time to read . Thin blood film is used to assist in the identification of the malaria species after theparasites have been seen in the thick film.

Both thick and thin films must be thoroughly dry . Allow the slide with the thin and thick films to dryinside a folder rack in a flat, level position (which allows the thick film to dry with even thickness),protected from flies, dust and extreme heat. Insufficiently dried blood film (and/or blood films that aretoo thick) can detach from the slides during staining. Thin smears will dry and be ready to fix and stain inabout 15 minutes . Thick smears will dry in a minimum of 30 minutes at room temperature. You canaccelerate the drying by using a fan or hair dryer (set on cool). Do not dry in an incubator or by exposureto heat or sunlight as this will fix the blood cells and interfere with lysing the red blood cells prior tostaining.


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Too much blood

After staining films made with too much blood, the background of the thick film will be too blue. Therewill be too many white blood cells per thick film field, and these could obscure or cover up any malariaparasites that are present. If the thin film is too thick, red blood cells will be on top of one another and itwill be impossible to examine them properly after fixation.

FIGURE6 TOO MUCH BLOOD FOR BOTH THIN AND THICK FILMS

Too little blood

If too little blood is used to make the films, there will not be enough white cells in the thick film field andyou will not be able to examine enough blood in the standard examination. The thin film may be toosmall for use as a label for patient identification.

FIGURE7 TOO SMALL BLOOD FOR BOTH THIN AND THICK FILMS

Blood films spread on a greasy slide

On a greasy slide blood films will spread unevenly, making the examination very difficult. Some of thethick film will probably come off the slide during the staining process.

FIGURE8 THE EFFECT OF UNCLEAN SLIDE ON BLOOD FILMS

Chipped edge of spreader slideWhen the edge of the spreader slide is chipped, the thin film spreads unevenly, is streaky and has manytails. The spreading of the thick f ilm may also be affected.


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FIGURE9 THE EFFECT OF CHIPPED EDGE SPREADER ON THIN AND THICK FILMS

Thin film too large, thick film in the wrong place

If the thin film is too large, the thick film will be out of place and may be so near the edge of the slidethat it cannot be seen through the microscope. During staining or drying, portions of the thick film willprobably be scraped off by the edges of the staining trough or drying rack. It may be very difficult orimpossible to position the thick film on the microscope stage for examination.

Table 1 Most common technical mistakes in collection and preparation of blood smears

Mistake Effect

Pricking of non dried finger The parasites and host cells may be fixedby the alcoholic detergent solution.

Use of unclean or contaminated slides The blood smear will not be spread evenly.Generates artifacts commonly mistaken for malariaparasites, including bacteria, fungi, stainprecipitation, and dirt and cell debris.

Excessive use of blood The blood smear will not be spread evenly due to thebeginning coagulation process.

Too large a drop of blood used for thin films Erythrocytes are laid on multiple layers.Observation is impossible.

Too little blood used for thin films Parasites may be virtually absent if parasitemia islow.

Labeling the slide (Improper or no labeling) Confusion may arise leading to slides that areunidentifiable and cannot be linked to a patient.

Slides are wrapped together before all thethick films are properly dried

The slides stick to one another and become unusable.

Excessive time elapses between bloodcollection and preparation of thick films

Autofixation occurs and hemolysis is impossible.

Exposure of thick films to excessive heat Autofixation occurs and hemolysis is impossible.

Thick films are dried too slowly P. falciparum gametocytes may exflagellate.


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4.2.2 Staining

4.2.2.1 Principles of Romanowsky Stains

Stains used to stain blood cells and parasites in blood are called Romanowsky stains. Romanowsky stainscomprise two staining components: azures (oxidation products of methylene blue) and eosin. Examplesof Romanowsky stains include Fields stain, Giemsas stain, Leishmans stain and Wrights stain. Giemsastain is regarded as the best stain for malaria microscopy; however Fields stain is use ful in healthfacilities with a low patient workload as it is rapid, economical and easy to use. All Romanowsky stainscan be used to stain thick and thin blood films once the staining principles are understood.

4.2.2.2 Giemsa Stain

The Giemsa stain must be diluted for use with water buffered to a pH 7.2, depending on the specifictechnique used. The stain should be tested for proper staining reaction before use. The stock is stable,but it must be protected from moisture because of the staining reaction. Giemsa stain will not functionas expected if stock is mixed with even small amounts of water or moisture solution during its

preparation or storage.

To control the quality of Giemsa stain for proper staining results, a known positive smear should beincluded with each new batch of working Giemsa stain. Control slides may be prepared from a patientsblood and stored for future use. From a patient known to have a malaria infection, collect a bloodsample in an EDTA (ethylene diamine tetra acetic acid) or citrated blood tube, if it requires multipleblood film preparations, or needs further diagnosis at a molecular level. An ideal blood sample has atleast one parasite in every 2 3 fields on a thin blood smear. Make as many thin smears as possible,preferably within one hour of drawing the blood from the patient.

Allow the smears to dry quickly, using a fan or blower at room temperature. Fix the smears in absolute(100%) methanol and allow them to dry. Place them, touching back to back, in a box with separating

groo ves. Label the outside of the box with the species, date and Giemsa control slides. The slides canbe stored at room temperature but will last longer if stored at -20C or -70C. Just before use, removethe slide from the box and allow the condensation t o evaporate; label the slide with the date and +control. The smear can then be stained and examined to check that the working solution of Giemsastain is of good quality.

4.2.2.3 Fields Stain

Fields stain is useful for rapid detection of malaria parasites par ticularly for thick films. However,Schuffeners dots are not always stained with this procedure. It is made up of Fields stain A and Fieldsstain B as both are used in the staining procedure.

4.2.2.4 Buffer Solution for Malaria Staining

A phosphate buffer solu tion, correctly balanced to pH 7.2, is essential for Giemsa and Fields staining formalaria parasites. Check the pH level using narrow-range pH papers or a pH meter and store the buffersolution at room temperature. The buffer is stable for several months. To check its quality, the pH ofbuffered water should be checked, and appropriate correcting fluid should be added.


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Buffer tablets

Buffer tablets that produce a solution of pH 7.2 when dissolved are readily available from laboratorysuppliers but are rather expensive.

Quality Control of Buffered Water

Prepare a buffer reagent carefully; weighing accurately the dry chemicals and checking the pH level.Alternatively, use buffer tablets. Store buffered reagents at 2-8 C in a tightly stoppered (preferablyplastic) bottle; when in use, avoid leaving the reagents exposed to sunlight (which encourages thegrowth of algae) and check for contamination (cloudiness) at regular intervals.

4.2.3 Microscopic Examination and Species Identification

Microscopy is the accepted standard method for detecting malaria parasites in blood. Microscopyrequires trained laboratory personnel and equipment (functional microscope) and other logistics.Stained thick or thin blood films are examined for the presence of malaria parasites, using an electricbinocular microscope. In the absence of electricity, alternative power sources must be used to ensure

quality microscopy.

4.2.3.1 Examining blood films for mala

Documents

Manual for the Laboratory Diagnosis of Malaria