MANUAL FOR THE LABORATORY

DIAGNOSIS OF MALARIA

First Edition













ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (EHNRI)
ETHIOPIAN FEDERAL MINISTRY OF HEALTH

SEPTEMBER, 2012
ADDIS ABABA

MANUAL FOR THE LABORATORY
DIAGNOSIS OF MALARIA

First Edition













ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (EHNRI)
ETHIOPIAN FEDERAL MINISTRY OF HEALTH

SEPTEMBER, 2012
ADDIS ABABA



ii

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 and
of relatively longer transmission duration in lowland areas, river basins and valleys. Although
historically there have been an estimated 10 million clinical malaria cases annually, cases have
reduced 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. This
requires improving diagnosis of malaria cases using microscopy or using multi-species RDTs, and
providing 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 at
the Federal Ministry of Health, Regional Health Bureaus,, National and Regional Reference
Laboratories, and partners with the aim of standardizing Malaria Laboratory Diagnosis trainings and
strengthening the quality of laboratory testing procedures for the diagnosis of malaria in the health
facilities in Ethiopia.

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

EHNRI believes that this manual will be useful for laboratory personnel and other health workers
during routine laboratory work and as a reference material for trainers and supervisors on
laboratory diagnosis of malaria during in-service trainings, practical attachments, and supportive
supervisions and for Quality Control and Quality Assurance purposes. The manual could be useful as
a reference material for clinicians too, mainly to understand the use and interpretation of laboratory
tests for malaria case management. The manual is also helpful for health facility managers to enable
them in determining essential laboratory commodity requirements for malaria laboratory diagnosis
and the need for their timely availability to ensure uninterrupted laboratory diagnostic services. This
manual should also be of interest to those non-governmental organizations and funding agencies
that are involved in the support for malaria laboratory diagnosis improvement and quality assurance
programs.

Finally, I would like to express my sincere appreciation and thanks to all professionals and
organizations who have contributed their expertise and resources for the preparation of this manual
Amha Kebede, PhD
Acting Director General,
EHNRI.


iii

ACKNOWLEDGMENT
The development of this Manual for Laboratory Diagnosis of Malaria was made possible through the
contribution of the professionals and institutions listed below:

Core Group Members: Organization
Getachew Belay EHNRI
Habtamu Asrat EHNRI
Markos Sileshi EHNRI
Hussien Mohammed EHNRI
Sindew Mekasha EHNRI
Moges Kassa EHNRI
Bereket Hailegiorgis CU-ICAP New York
Tesfay Abreha CU-ICAP Ethiopia
Sintayehu G/Sellasie CU-ICAP Ethiopia
Leykun Demeke CU-ICAP Ethiopia
Samuel Girma CU-ICAP Ethiopia
Micheal Aidoo CDC Atlanta


Contributors:

Gudeta Tibesso EHNRI
Gonfa Ayana EHNRI
Ashenafi Assefa EHNRI
Abinet Abebe EHNRI
Yenew Kebede CDC Ethiopia
Zenebe Melaku CU-ICAP Ethiopia
Abebe Tadesse CU-ICAP Ethiopia
Fanuel Zewdu CU-ICAP Ethiopia
Meseret Habtamu CU-ICAP Ethiopia
Mekonnen Tadesse CU-ICAP Ethiopia
Joseph Malone CDC/PMI Ethiopia
Richard Reithinger USAID/PMI Ethiopia
Hiwot 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 their
valuable contributions to make this manual a reality.




iv

The generous financial and technical support of Columbia University ICAP in Ethiopia through
funding obtained from PMI USAID Ethiopia was of paramount importance to hold serial expert and
national consultative meetings to develop this manual. We are indebted to PMI for covering the cost
of printing the manual.
Gonfa Ayana, BSc, MSc,
Acting Director, Regional Laboratories Capacity Building Directorate,
EHNRI.


v

TABLE OF CONTENTS
Page
Foreword ....................................................................................................................................................... ii
Acknowledgment ......................................................................................................................................... iii
Table of contents .......................................................................................................................................... v
List of Tables .............................................................................................................................................. viii
List of Figures ............................................................................................................................................... ix
Acronyms ...................................................................................................................................................... x
Glossary of Terms........................................................................................................................................ xii
1 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 ................................................................................................... 6
2.1 Purpose ........................................................................................................................ 6
2.2 Target Audience .......................................................................................................... 6
3 Malaria Situation in Ethiopia ........................................................................................................... 7
3.1 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 ................................................................. 14
4.1 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) .................................... 47
5.1 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 ...................................................................... 55
6.1 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 ........................................................................................................................... 66
7.1 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 .............................................. 72
8.1 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 .................................................................................................................................................. 82
ANNEXES ..................................................................................................................................................... 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 Blinded
Rechecking ......................................................................................................................... 102
Annex 8: SOP for Care And Preventive Maintenance of Microscopes ............................. 104


vii

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


viii

LIST OF TABLES

Table 1 Most common technical mistakes in collection and preparation of blood smears ................. 23
Table 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 in
Giemsa-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


ix

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 ................................................. 23
Figure 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



x

ACRONYMS

ACT Artemisinin Based Combination Therapy
AMU Average monthly usage
AO Acridine orange
AQ Amodiaquine
BF Blood film
CQ Chloroquine
DNA Deoxyribose Nucleic Acid
ECP Exposure control plan
EDTA Ethylene diamine tetra acetic acid
EHNRI Ethiopian Health and Nutrition Research Institute
ELISA Enzyme linked immune-sorbent assay
EQA External quality assessment
HEWs Health extension workers
HIV Human immunodeficiency virus
HMIS Health management information system
HRP2 Histidine rich protein 2
HSDP Health Sector Development Programme
IQC Internal quality control
IRS Indoor Residual spray
ITNs Insecticide Treated Nets
LMIS Logistic Management Information System
NEQAS National external quality assessment scheme
PEP Post-exposure prophylaxis
Pf Plasmodium falciparum
PfHRP2 Plasmodium falciparum histidine rich protein
PHL Public health laboratory
pLDH Plasmodium lactate dehydrogenase
PMA Pan-malaria antigen
PMI Presidents Malaria Initiative
PPE Personal protective equipment
PT Proficiency test
Pv Plasmodium vivax
QA Quality assurance
QBC Quantitative Buffy Coat
QC Quality control
QI Quality improvement
RBC Red blood cell
RDT Rapid Diagnostic Test
REQAS Regional External Quality Assessment Scheme
SDPs Service delivery points


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SOPs Standard operating procedures
SP Sulphadoxine pyrimethamine
l Micro liter
USAID The United States Agency for International Development
WBC White blood cell
WHO World Health Organization


xii

GLOSSARY OF TERMS

Antibody

A specialized serum protein (immunoglobulin or gamma globulin) produced
by B lymphocytes in the blood in response to an exposure to foreign proteins
(antigens). The antibodies specifically bind to the antigens that induced the
immune 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 red
blood 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.
Asexual
parasitaemia
The presence in host red blood cells of asexual parasites. The level of asexual
parasitaemia can be expressed in several different ways: the percentage of
infected red blood cells, the number of infected cells per unit volume of
blood, the number of parasites seen in one microscopic field in a high-power
examination 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 locally
acceptable level as a result of deliberate efforts.
Drug resistance The ability of a parasite strain to survive and/or to multiply despite the
administration and absorption of a medicine given in doses equal to or higher
than those usually recommended but within the tolerance of the subject,
provided drug exposure at the site of action is adequate. Resistance to
antimalarials arises because of the selection of parasites with genetic
mutations 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 defined
geographical area, creating a zero incidence of locally contracted cases.
Imported cases will continue to occur and continued intervention measures
are required.
Elimination of
disease
Reduction to zero of the incidence of a specified disease in a defined
geographical area as a result of deliberate efforts.
Elimination of
infection
Reduction to zero of the incidence of infection caused by a specified agent in
a 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 or
events in specified populations; the application of this study to control health
problems.
Eradication Permanent reduction to zero of the worldwide incidence of infection caused
by 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.
Exoerythrocytic
stage
A stage in the life cycle of the malaria parasite found in liver cells
(hepatocytes). Exoerythrocytic stage parasites do not cause symptoms.
External quality
Assessment

A system whereby a reference laboratory sends stained blood films to a
laboratory for examination. The laboratory receiving the slides is not informed
of the correct result of the slides until the laboratory has reported their
findings 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 for
remedial 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 drug
primaquine.
Gametocyte The sexual stage of malaria parasites. Male gametocytes (microgametocytes)
and female gametocytes (macrogametocytes) are inside red blood cells in the
circulation. If a female Anopheles mosquito ingests them, they undergo sexual
reproduction, which starts the extrinsic (sporogonic) cycle of the parasite in
the mosquito. Gametocytes of Plasmodium falciparum are typically banana or
crescent-shaped (from the Latin falcis = sickle).
Hypnozoite Dormant form of malaria parasites found in liver cells. Hypnozoites occur only
with Plasmodium vivax and P. ovale. After sporozoites (inoculated by the
mosquito) invade liver cells, some sporozoites develop into dormant forms
(the hypnozoites), which do not cause any symptoms. Hypnozoites can
become activated months or years after the initial infection, producing a
relapse.
Hypoglycemia Low blood glucose; can occur with malaria. In addition, treatment with
quinine and quinidine stimulate insulin secretion, reducing blood glucose.
Immune system The cells, tissues, and organs that help the body resist infection and disease
by producing antibodies and/or cells that inhibit the multiplication of the
infectious agent.
Immunity Protection generated by the bodys immune system, in response to previous
malaria 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 of
the illness or the first symptoms of the illness. With malaria, the incubation is
between the mosquito bite and the first symptoms. Incubation periods range


xiv

from 7 to 40 days, depending on the species.
Indigenous malaria Mosquito-borne transmission of malaria in a geographic area where malaria
occurs regularly.
Infection The invasion of an organism by a pathogen, such as bacteria, viruses, or
parasites. Some, but not all, infections lead to disease.
Introduced malaria

Mosquito-borne transmission of malaria from an imported case in a
geographic area where malaria does not regularly occur.
Malaria pigment
(haemozoin)
A dark brown granular pigment formed by malaria parasites as a by-product
of haemoglobin catabolism. The pigment is evident in mature
trophozoites and schizonts. They may also be present in white blood cells
(peripheral monocytes and polymorphonuclear neutrophils) and in the
placenta.
Merozoite A daughter-cell formed by asexual development in the life cycle of malaria
parasites. Liver-stage and blood-stage malaria parasites develop into
schizonts, which contain many merozoites. When the schizonts are mature,
they (and their host cells!) rupture, the merozoites are released and infect red
blood cells.
Microscopist

A person who uses a microscope to read blood films to aid or confirm the
diagnosis of malaria and reports on their findings. The term is used in this
manual to include personnel at all levels of a malaria programme involved in
this work, from professors involved in teaching and research to village health
volunteers specifically trained in malaria microscopy.
Oocyst A stage in the life cyle of malaria parasites, oocysts are rounded cysts located
in the outer wall of the stomach of mosquitoes. Sporozoites develop inside
the oocysts. When mature, the oocysts rupture and release the sporozoites,
which then migrate into the mosquitos salivary glands, ready for injection
into the human host.
Outbreak An epidemic limited to a localized increase in disease incidence, e.g. in a
village, town or closed institution.
Pandemic An epidemic occurring over a very wide area, crossing international
boundaries and usually affecting a large number of people.
Parasite Any organism that lives in or on another organism without benefiting the host
organism; commonly refers to pathogens, most commonly to protozoans and
helminthes.
Parasitemia The presence of parasites in the blood. The term can also be used to express
the 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 at
intervals.
Pathogen Bacteria, viruses, parasites, or fungi that can cause disease.
Plasmodium The genus of the parasite that causes malaria. The genus includes four species
that infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium


xv

ovale, and Plasmodium malariae.
Pre-erythrocytic
development
The life-cycle of the malaria parasite when it first enters the host. Following
inoculation into a human by the female anopheline mosquito,
sporozoites invade parenchyma cells in the host liver and multiply within the
hepatocytes for 512 days, forming hepatic schizonts. These then burst
liberating merozoites into the bloodstream, which subsequently invade red
blood cells
Presumptive
treatment
Treatment of clinically suspected cases without, or prior to, results from
confirmatory 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 the
National and Regional Laboratories. This exercise can check both the
laboratories staining quality as well as the ability of technicians to recognize
and identify malaria parasites present.
Quality assurance

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

Measures the quality of a test or a reagent. For malaria microscopy, the most
common form of quality control (QC) is the cross-checking of routine blood
slides to monitor the accuracy of examination. Quality control also
encompasses 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 reagents
used in the laboratory.
Quality
Improvement
A process by which the components of microscopy and RDT diagnostic
services are analyzed with the aim of identifying and permanently correcting
any deficiencies. Data collection, data analysis, and creative problem solving
are skills used in this process
Radical cure (also
radical treatment)
Complete elimination of malaria parasites from the body; the term applies
specifically to elimination of dormant liver stage parasites (hypnozoites) found
in Plasmodium vivax and P. ovale.
Recrudescence A repeated attack of malaria (short-term relapse or delayed), due to the
survival of malaria parasites in red blood cells. Radical treatment: see radical
cure.
Relapse Recurrence of disease after it has been apparently cured. In malaria, true
relapses are caused by reactivation of dormant liver stage parasites
(hypnozoites) found in Plasmodium vivax and P. ovale.
Residual insecticide
spraying
Spraying insecticides that have residual efficacy (that continue to affect
mosquitoes for several months) against houses where people spend nighttime
hours. Residual insecticide spraying is done to kill mosquitoes when they
come to rest on the walls, usually after a blood meal.
Resistance The ability of an organism to develop strains that are impervious to specific


xvi

threats to their existence. The malaria parasite has developed strains that are
resistant to drugs, such as chloroquine. The Anopheles mosquito has
developed strains that are resistant to DDT and other insecticides.
Ring stage Young usually ring-shaped intra-erythrocytic malaria parasites, before malaria
pigment is evident under microscopy.
Schizogony Asexual reproductive stage of malaria parasites. In red blood cells, schizogony
entails development of a single trophozoite into numerous merozoites; a
similar 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 of
antibodies and other immunological substances in body fluids, particularly
serum.
Slide positivity rate

The proportion of positive slides, detected by microscopy, among all those
examined within a laboratory over a defined period of time.
Sporozoite A stage in the life cycle of the malaria parasite. Sporozoites, produced in the
mosquito, migrate to the mosquito's salivary glands. They can be inoculated
into a human host when the mosquito takes a blood meal on the human. In
the human, the sporozoites enter liver cells where they develop into the next
stage of the malaria parasite life cycle (the liver stage or exo-erythrocytic
stage).
Trophozoite A developmental form during the blood stage of malaria parasites. After
merozoites 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 an
infectious agent (for example, malaria parasites) from one host to the other
(for example, humans).



1

1 INTRODUCTION
1.1 Malaria Etiology

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

1.2 Life Cycle and Transmission of Malaria
The malaria life cycle takes place in humans and in the female Anopheles mosquito. The malaria
parasite life cycle involves two hosts, the female anopheles mosquito as definitive host and the
human as intermediate host. Malaria parasites are usually transmitted by the bite of an infected
female Anopheles mosquito. Malaria trophozoites may also be transmitted through blood
transfusion 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 plasmodial
sporozoites from its salivary gland during a blood meal. The mosquito becomes infected by ingesting
human blood containing the sexual forms of the parasite (gametocytes). In the mosquito gut wall the
sporogonic cycle starts with the gametocytes fusing and forming zygote which further develop into
ookinete and oocyst. The oocysts grow, rupture, and release sporozoites, into the mosquitos
salivary glands.

Source: http://www.dpd.cdc.gov/dpdx
FIGURE 1 LIFE CYCLE OF MALARIAL PARASITES


2

When the mosquito next feeds on humans, it injects sporozoites into the blood stream that
eventually infect liver cells (hepatocytes). Within the liver cells, sporozoites are transformed into
merozoites. The stage of the life cycle from sporozoite injection to the liver schizont stage is termed
the pre-erythrocytic stage. The erythrocytic stage follows when merozoites released into the blood
stream 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 schizont
rupture and release of merozoites , initiates the typical clinical manifestations of malaria, fever,
shiver & sweating paroxysm. Themerozoites immediately invade new red blood cells to repeat the
cycle several times over the course of weeks. However, in P. vivax and P. ovale infections, some
sporozoites become dormant hypnozoites upon invading hepatic cells. Reactivation of the
hypnozoites can occur up to 6-8 months later, initiating either a delayed primary infection or
relapse.

1.3 Overview of methods for malaria diagnosis
Prompt and accurate diagnosis of malaria is part of effective disease management. The diagnosis of
malaria is based on clinical suspicion and on the detection of parasites in the blood (parasitological
or confirmatory diagnosis). High sensitivity of diagnosis in malaria endemic areas is particularly
important for the most vulnerable population groups, such as young children and the non-immune
population, in whom the disease can be rapidly fatal, while high specificity will reduce unnecessary
treatment 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 illness
and performing a physical examination. Clinical diagnosis of malaria is made in a patient who has
fever or history of fever in the last 48 hours and lives in malaria-endemic areas or has a history of
travel within the last 30 days to malaria-endemic areas. Basing the diagnosis on clinical features
alone is not recommended, as this often has low specificity and increases the chances of the patient
being misdiagnosed. Unless there is an ongoing malaria epidemic, or is a peak malaria transmission
season, careful laboratory testing typically reveals confirmed malaria parasites in fewer than half of
clinically suspected malaria in most situations in Ethiopia. Malaria treatment based on clinical
diagnosis must be the last option when there is no availability of RDTs or microscopy. WHO
recommends universal parasitological diagnosis of malaria to ensure targeted use of antimalarial
drugs for those who actually have malaria. The health worker examining a suspected malaria case
should perform differential diagnosis to look for other causes of fever (e.g., typhoid fever, relapsing
fever, acute respiratory tract infections, meningitis, etc) and manage the case accordingly. Malaria
should still be considered, even if the individual has another obvious cause for the fever. The
national algorithm of the Integrated Management of Neonatal and Childhood Illness (IMNCI) and
Community-based Case Management (CCM) should also be employed for the management of the
sick child presenting with fever.
The clinical course of malaria infection may be uncomplicated or severe. Because of its frequent and
severe 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 people
living with HIV are particularly at risk for severe and complicated malaria and death.


3

Uncomplicated malaria is characterized by fever and other features including chills, profuse
sweating, muscle pains, joint pains, headache, abdominal pain, diarrhoea, nausea, vomiting, loss of
appetite, irritability, and refusal to feed (in infants). These features may occur singly or in
combination 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; or
inability to feed (in an infant)
Alteration in the level of consciousness
(ranging from drowsiness to deep coma)
Cerebral malaria (unarousable coma not
attributable to any other cause, other
neurological 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 (not
passing urine)
Severe anaemia (haemoglobin <5g/dl or
haematocrit < 15%)
Hypoglycaemia (blood glucose level < 2.2
mmol/l)
Hyperparasitaemia (parasitaemia of
>200,000/l - in patients from high
transmission areas; or 100,000/l in
patients from low transmission areas)
Hyperlactataemia (whole blood lactate >5
mmol/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












4

1.3.2 Laboratory Diagnosis of Malaria
Once malaria is suspected on clinical grounds, it is mandatory to obtain the laboratory confirmation of
the presence of malaria parasites. Clinicians could request for diagnostic test for malaria to confirm the
diagnosis of malaria in a patient with symptoms and signs suggestive of malaria disease; to rule out
malaria infection in a patient with other known causes of fever; to confirm malaria in febrile infants
under 3 months of age; to look for treatment failure; and to investigate causes of anaemia, jaundice or
splenomegaly.
1.3.2.1 Common Diagnostic Methods
The two laboratory diagnostic methods or tools most often used for confirming a diagnosis of malaria
are:
a. Rapid Diagnostic Tests RDTs: RDTs detect antigens (proteins produced by malaria parasite) in
the blood of a patient with malaria.
b. Light Microscopy: Good quality microscopy is the most acceptable method for detecting and
identifying malaria parasites from the blood of a suspected patient. The procedure consists of
collecting a finger-prick blood sample; preparing a thin and thick blood films; staining the films
with Giemsa or other stains such as Field stain and examining the film through a microscope for
the 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 in
the 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 or
venous blood. QBC utilizes density gradient layering of stained blood cells, together with mechanical
expansion of the haematocrit buffy coat. The parasites are detected by fluorescent microscopy using
acridine orange stain. It is fast, easy and may be more sensitive than the traditional thick film
examination. Its main advantages are faster result delivery within 15-30 minutes, and a potential for
accidentally 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 fluorochrome
technique
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 of
malaria parasites and emits two fluorescence colors, green (530 nm) and red (650 nm) when excited at
430 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 are
well preserved and the general morphology is comparable to specimens stained by Giemsa.



5


c. Immunological tests (Anti-malarial Antibody Test)
Antibodies to the asexual blood stages appear a few days after malarial infection, increase in titer over
the next few weeks, and persist for months or years in semi-immune patients in endemic areas, where
re-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 development
of antibodies and also the persistence of antibodies, serologic testing is not practical for routine
diagnosis 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 of
parasite DNA and amplification by polymerase chain reaction using specific primers to yield a product
that can easily be visualized in ethidium bromide stained agarose gel. As little as one parasite per
microlitre of blood can be detected by this method. It is highly specific and sensitive (10 times more
sensitive 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 and
0.12 parasites/L for P.vivax. However, it requires expensive laboratory equipment in specialized
laboratory settings and often used in reference laboratories to confirm malaria parasite species (if in
doubt); to validate Rapid Diagnostic Tests (RDTs) as part of planned quality assurance programmes; and
for research purposes.
e. Flowcytometry

Flowcytometry and automated hematology analyzers have been found to be useful in indicating
diagnosis of malaria during routine blood counts. In cases of malaria, abnormal cell clusters and small
particles with DNA fluorescence, probably free malarial parasites, have been seen on automated
hematology 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 also
suggest a possibility of malaria with a sensitivity of 95% and a specificity of 88%.



6

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 manual
describes overview of malaria epidemiology, laboratory procedure, quality assurance and supply
management; 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 and
community levels are efficiently and effectively organized to allow early diagnosis and prompt, effective
treatment. The manual provides basic information for the successful operation of malaria laboratory
diagnosis 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 malaria
laboratory diagnosis program, and in general for multidisciplinary teams involved in managing national
malaria 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 this
manual useful.
The manual will be a useful resource in Ministry of Health or in projects supported by international and
multilateral cooperation agencies or nongovernmental organizations, in medical, nursing, laboratory and
public health schools for training in effective malaria case management.





7

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 in
2011, reductions in reported malaria cases of more than 50% have been recorded between 2000 and
2010 in 43 of the 99 countries with ongoing transmission, while downward trends of 25%50% were
seen in 8 other countries. There were an estimated 216 million episodes of malaria in 2010, of which
approximately 81%, or 174 million cases, were in the African Region. There were an estimated 655 000
malaria deaths in 2010, of which 91% were in Africa. Approximately 86% of malaria deaths globally were
of children under 5 years of age. The estimated incidence of malaria globally has reduced by 17% since
2000 and malaria-specific mortality rates by 26%. These rates of decline are lower than internationally
agreed 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 leading
communicable disease in Ethiopia, accounting for about 30% of the overall Disability Adjusted Life Years
lost. Approximately 68% of the total population of 78 million lives in areas at risk of malaria. According
to Ethiopias Federal Ministry of Health (FMOH), in 2008/2009, malaria was the leading cause of
outpatient visits, health facility admissions and inpatient deaths, accounting for 12% of reported
outpatient visits and nearly 10% of admissions. In 2009, 3 million suspected malaria cases were seen
and nearly 2.3 million (77%) were tested. The number of malaria cases decreased from an annual
average of 3 million during 20002005 to 1.75 million cases in 2009 (41% decline). In the same period
the malaria admissions decreased from an average of 44,000 to 30,102 in 2009 (33% decline). Inpatient
malaria deaths fell by 43% in all age groups and by 60% in children <5 years.
Malaria transmission in Ethiopia varies widely with the complex topography, which ranges from high
altitude mountain terrains to low altitude fertile valleys or semi-arid plains. Although relatively intense
in the western lowlands and some river basins, transmission is seasonal in most parts of the country in
relation to rainfall patterns and is characterized by inter-annual changes in meteorological factors. The
main transmission season follows the June to September rains and occurs between September and
December, while the minor transmission season occurs between April and May, following the February
to March small rains. The peak period of malaria transmission often coincides with peak periods of
agricultural activity such as planting and harvesting, producing a negative economic impact in the
country. Because of the unstable and seasonal nature of transmission, malaria occurs predominantly in
epidemic forms, resulting in serious morbidity and mortality in both adults and children.

3.2 Eco-epidemiological Strata of Malaria Transmission

Five main malaria eco-epidemiological strata are recognized in the country: The map (Figure 2 below)
shows the main eco-epidemiological strata in Ethiopia.



8












FIGURE 2 MALARIA EPIDEMIOLOGICAL STRATA IN ETHIOPIA


1. Stable, year-round transmission in the western lowlands and river basins areas of Gambella
2. Seasonal transmission in lowland areas below 1,500 meters
3. Epidemic-prone areas in highland fringes between 1,500 and 2,500 meters (i.e. Upland malaria
comprising areas under B 2000-2500m, D 1750-2000 &E 1500-1750m )
4. Arid areas where malaria is only found near semi-permanent water bodies
5. Malaria-free highland areas above 2,500 meters

Major epidemics occur every 5 to 8 years. Though malaria has been the leading cause of outpatient
consultation, admission and death for many years, the recent rapid scale up of interventions has
brought about a significant decline in the malaria burden, now ranking as sixth cause of outpatient
consultations.
All four human malaria parasite species are known to be present in the country. P. falciparum and P.
vivax, however, are the most dominant and prevalent in all malarious areas of the country. P.
falciparum is a major cause of morbidity and mortality, accounting for 60-70% of malaria cases, while P.
vivax causes about 30-40% of cases.
The major malaria-transmitting vector in Ethiopia is Anopheles arabiensis. Anopheles pharoensis,
Anopheles funestis and Anopheles nili are also incriminated as secondary vectors of malaria in the
country, the last one being a local vector in the Nile basin, in South western Ethiopian lowlands. .




9

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 malaria
elimination through an integrated community health approach, especially in areas of unstable malaria
transmission, 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 during
elimination. When the slide positivity rate (SPR) of all febrile patients with suspected malaria is less than
5% or the incidence is less than 5 per 1000 people at risk, the country, or district in some cases, could
consider 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 reduced
to less than 5 per 1,000 populations per year in risk areas. Pre-elimination and elimination are
equivalent to the consolidation phase, when interruption of transmission is achieved. In the final
maintenance phase, prevention of introduction and local transmission are maintained for 3 consecutive
years, 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 low
malaria 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 the
following specific objectives:
1. 100% of suspected malaria cases are diagnosed using RDTs and/or microscopy within 24 hours
of fever onset
2. 100% of positive malaria diagnosis are treated according to national guidelines
3. 100% of households in malarious areas own one LLIN per sleeping space
4. At least 80% of people at risk of malaria use LLINs properly and consistently
5. 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 malaria
prevention and control strategies which includes:






10


A. Community Empowerment and Mobilisation
Community empowerment and mobilization are central to malaria prevention and control. Ethiopias
Health Extension Program educates, mobilizes and involves the community in all aspects and stages of
malaria 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 the
wide-scale use of RDTs in peripheral health facilities. To improve the quality of malaria diagnosis and
treatment at peripheral health facilities (health posts) panspecific RDTs are now being introduced. HEWs
will 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 and
environmental control.
D. Active Surveillance and Epidemic Control
Aims to achieve a high quality, broadly based malaria infection detection, investigation and response
Surveillance System to further reduce malaria transmission and improve the detection and timely
response to malaria epidemics. Malaria detection, investigation, response and elimination activities will
achieve a high quality, broadly based malaria infection detection, investigation and response
surveillance System to further reduce malaria transmission, prevent and stop epidemics and eliminate
malaria especially in targeted areas that are prone to outbreaks. There will be a transition from
epidemic detection and response to surveillance and infection response as transmission declines to near
zero.
E. Health system strengthening and capacity building
The health system strengthening and capacity building includes monitoring and Evaluation activities and
development 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 tests
mainly for operational researches and trainings. Malaria parasite molecular, serological tests, drug level
determinations and RDT evaluations are conducted at the national reference laboratory. Malaria
microscopy is mainly used at the national level for research, large surveys, quality control and training
purposes. At the regional reference laboratories, malaria microscopy is mainly conducted for the
purposes of training and external quality assessment schemes.


11

3.5.2 Hospitals and health centers
In accordance with the National Malaria guidelines of 2012, malaria microscopy is the sole technique
employed in hospital and health center levels. Therefore, it is critical that these facilities are equipped
with 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 history
and 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 as
possible, generally within 24 hours after symptoms onset. Effective malaria treatment requires
improved diagnosis of malaria (i.e. laboratory-based microscopy or use of multi species RDTs); well
trained health workers in both the public and private health sectors; constant availability of highly
efficacious medicines as close to the patient as possible to ensure prompt access. Communities should
be aware of the importance to seek early diagnosis and treatment and to adhere to prescribed drug
regimens for malaria.
Treatment of malaria should be based upon a parasitologically confirmed diagnosis whenever the
situation permits. Laboratory evidence providing confirmation of malaria (i.e. microscopy or RDT) by
malaria species requires prompt treatment with the appropriate antimalarial medications. If the RDT or
microscopy test indicates P. falciparum, then the patient should be treated with appropriate doses of
Arthemisisin-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 should
be dispensed, also ensuring that oral medicine is tolerated. Radical treatment with Primaquine is
recommended at health center and hospital level for patients who are vivax positive & are not living in
malaria endemic areas. Health workers should be vigilant to detect side effect of Primaquine. Mixed
infection of P. falciparum and P.vivax should be treated with AL. Pregnant women with P. falciparum in
the 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, no
malaria medications need be provided, but a thorough search for other causes of acute febrile illness
should continue, such as pneumonia; in such cases, referral to Health Centers or hospital Is advised. By
testing 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 be
reduced and eliminated.


12

3.6.2 Case management of uncomplicated malaria
Uncomplicated malaria is defined as symptomatic malaria without signs of severity or evidence of vital
organ dysfunction. It is important that uncomplicated malaria is treated well, because if left untreated, it
can 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 the
presence of asexual forms of malaria parasites in blood sample. Based on the Ethiopian national malaria
case management guidelines, the treatment approach is as described below:

First Line Treatment
Artemether-Lumefantrine: The recommended first-line treatment of all clinically and parasitologically
diagnosed uncomplicated Plasmodium falciparum malaria in Ethiopia is an Artemisinin-based
Combination 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 total
recommended 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 <3 months of age or weighing less than 5 kg body weight,
During the first three months (1
st
trimester) of pregnancy
For these particular groups, oral quinine is used as an alternative first line treatment.
Chloroquine: The first line treatment for P. vivax is oral chloroquine . Chloroquine is recommended for
all patient groups including children under 3 months and pregnant women in first trimester of
pregnancy
Primaquine: In malaria-free areas and where compliance can be ensured, in order to eliminate
hypnozoite forms (relapsing stages) of P.vivax from the liver and to bring about radical cure, primaquine
may be administered daily for 14 days at health center and hospital level, starting after chloroquine
treatment is completed. Since the level of glucose-6-phosphate dehydrogenase deficiency is not known
in Ethiopia, clinicians should closely follow patients on primaquine chemotherapy for hemolysis.
Treatment should be discontinued if the patient develops evidence of hemolytic adverse effects.

Second Line Treatment
The recommended second-line antimalarial in Ethiopia is oral quinine for all patients. It should be
emphasized here that currently, there is almost no resistance to ACTs such as AL in Ethiopia; and
treatment failure is very highly unlikely to be due to the efficacy of the medicine itself.


13

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 preventing
long term complications and death. The main objective in the treatment of severe malaria is to prevent
death. Secondary objectives include prevention of disability, recrudescence and the development of
resistance, Every staff member has a potential role to play in patient management especially those with
SCM, whether it is to carry out selection of very sick patients and alerting qualified staff, putting
unconscious patients in the right position to protect their airway or ensuring that blood tests are taken
and 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 word
TRIAGE comes from the French verb to sort and it is the process of rapidly screening sick patients
when they arrive at any health facility. These patients should be prioritized at all stages of management
from the initial assessment, to admission, investigation and treatment. Health centers and hospitals will
diagnose and treat severe malaria, Health centers shall refer all cases of severe malaria which cant be
treated 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 be
treated 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 alternatives
and will be available for pre-referral treatment.



14

4 PARASITOLOGICAL DIAGNOSIS OF MALARIA USING MICROSCOPY

Microscopy is the science of investigating small objects using a microscope. It has an essential role for
the 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. There
are only a few absolute rules to observe in caring for the microscopes you will use. Taken care of, these
instruments will last many decades and continue to work well. Please report any malfunctions
immediately.

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 use
any 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 beside
your 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 the
microscope, contact the microscope companys technical support unit or thier local agents, or consult
with a qualified technician, around.

Humidity causes fungal growth on the surface of lenses and prisms. This can cause cloudiness of the view
field and rusting of metal parts of the microscope. To protect the microscope from fungus, always keep
the glass surface as clean as possible and free of dirt and fingerprints. Reduce the growth of fungus by
continuously using an air conditioner to lower humidity. The use of air-conditioning in the daytime only
will 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), or
anti-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 for
keeping a dry, stable environment.


15


FIGURE 3 CABINET BOX

Silica Gel
Place dry blue silica gel (about 250 g) in a shallow plate and place it in the bottom of the sealed
microscope box. Silica gel is blue when it is dry, but turns pinkish when it becomes wet. As soon as the
silica gel becomes pink, replace it. Alternatively, heat the gel in crucibles until I the absorbed water
evaporates & turns blue again before using it.
4.1.2 Maintenance of the microscope
Replacing the Microscope Bulb
Unplug the microscope from the power source
Find the location of the bulb
Follow manufacturers instructions to remove the bulb
Use tissue paper or an appropriate device to remove the bulb from the microscope
Check the model number on the bulb to ensure the use of a correct replacement bulb
Replace the bulb by holding it with lens paper or an appropriate device
NB: Never touch the bulb with your fingers.
Microscope Repair
Never disassemble the microscope
- Optics: eyepieces and objectives
- Mechanics: stage and focus adjustments
Repair of these items requires a service engineer
Khler Adjustment
August Khler invented the procedure for optimum illumination of an object in a light microscope.
Khler illumination is also known as double diaphragm illumination because it employs both a field and
an aperture iris diaphragm to set up the illumination. If the light path is set up properly, you will have
the advantages of an evenly illuminated field, a bright image without glare and minimum heating of the
specimen. Refer to the appendix for instructions on how to adjust the Khler illumination.
Note: In certain microscopes, the field diaphragm is usually not present and the Khler adjustment does
not apply.


16

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 the
four optical parts of the microscope (see Figure 11.1) clean. Remove dust attached to the microscope
with 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 this
purpose 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 ethyl
ether/alcohol (80/20 vol/vol). This can also be used to remove fingerprints or grease. Remove dust by
softly brushing the surfaces. For cleaning lenses and filters, wipe the object from the center, winding a
spiral 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


17

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 steps13, 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 Solution
The 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 Solution
The 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 Solution
The 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 Solution
There is oil on the lens. Clean the lens.
There is a layer of dust on the upper surface of
the objective.
Clean the lens.




18

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 a
completely 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 the
microscope, maintenance schedule, repairs done on the microscope and availability of spares like bulbs,
fuses, anti-mold strips etc.



19

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 sample
because 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 be
punctured should be warm so that blood flow will be adequate. Depending on the physical settings and
the patients condition, warming the hand with warm water, covering the hand with a hot, wet towel or
briskly rubbing the hand may be used to warm the hands prior to the finger prick. For routine malaria
microscopy, 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 collect
two or three larger drops on the slide, about 1 cm from the drop intended for the thin film or 1 cm from
the end of the slide.
Venous Blood Collection
Venous blood is not collected for routine use in Malaria laboratory diagnosis. The venipuncture
procedure is complex, requiring both knowledge and skill to perform. Each phlebotomist generally
establishes a routine that is comfortable for her or him. Several essential steps are required for every
successful collection procedure and venipuncture site selection. Although the larger and fuller median
cubital and cephalic veins of the arm are used most frequently, the basilic vein on the dorsum of the arm
or dorsal hand veins are also acceptable for venipuncture. Palpate and trace the path of veins with the
index 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 arm
from wrist to elbow, tap the site with index and second finger, apply a warm, damp wash cloth to the
site for 5 minutes, or lower the extremity over the bedside to allow the veins to fill. Foot veins are a last
resort because of the higher probability of complications. One should recognize complications
associated with the phlebotomy procedure, assess the need for recollection and/or rejection of sample
and perform proper labeling of the specimen.







20

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 and
thin 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, if
any) are more concentrated (~30x) than in an equal area of a thin smear, allowing a greater volume of
blood to be examined. Because a larger volume (6 l) of blood is examined in the thick film, it is much
better than the thin film for detection of low levels of parasitemia and reappearance of circulating
parasites during infection, recrudescence or relapse.
Thick film is therefore the most suitable method for the rapid detection of the parasite, but it does not
permit an optimal review of parasite morphology for species identification. If the thick smear is positive
for malaria parasites, the thin smear should be used for species identification. Thus, the thick films are
performed to detect and quantify (parasite density) malaria parasites in routine malaria microscopic
diagnosis.
Thin Blood Film
Thin blood film consists of blood spread in a layer such that the thickness decreases progressively
toward the feathered edge. In the feathered edge, the red blood cells should be in a single layer, not
touching one another. Thin blood smear should be fixed with methanol so that the parasites are found
intact inside the RBCs.
The morphological identification of the parasite to the species level is much easier and provides greater
specificity 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 the
parasites 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 dry
inside 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 are
too thick) can detach from the slides during staining. Thin smears will dry and be ready to fix and stain in
about 15 minutes. Thick smears will dry in a minimum of 30 minutes at room temperature. You can
accelerate the drying by using a fan or hair dryer (set on cool). Do not dry in an incubator or by exposure
to heat or sunlight as this will fix the blood cells and interfere with lysing the red blood cells prior to
staining.


21


FIGURE 4 EXAMPLE OF WELL-MADE AND CORRECTLY LABELED THICK AND THIN FILMS

Qualities of Good Thick and Thin Films
A thin smear should
Be uniformly spread over the slide
Be thin enough so that it is tongue shaped
Consist of a single layer of RBCs with a feathered end

A thick smear should
Be 10 mm away from the edge of the slide
Be round in shape with a diameter of about 10 mm
Have a thickness containing 10 layers of RBCs
Have 10-12 WBCs visible per oil immersion field of microscope

Common Mistakes in Making Blood Films
Badly positioned blood films
Care should be taken that the blood films are correctly sited on the slide. If they are not, it may be
difficult to examine the thick film. Also portions of the films may even be rubbed off during the staining
or drying process.


FIGURE 5 BADLY POSITIONED BLOOD FILM




22

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

FIGURE 6 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 and
you will not be able to examine enough blood in the standard examination. The thin film may be too
small for use as a label for patient identification.

FIGURE 7 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 the
thick film will probably come off the slide during the staining process.

FIGURE 8 THE EFFECT OF UNCLEAN SLIDE ON BLOOD FILMS

Chipped edge of spreader slide
When the edge of the spreader slide is chipped, the thin film spreads unevenly, is streaky and has many
tails. The spreading of the thick film may also be affected.


23


FIGURE 9 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 slide
that it cannot be seen through the microscope. During staining or drying, portions of the thick film will
probably be scraped off by the edges of the staining trough or drying rack. It may be very difficult or
impossible 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 fixed
by the alcoholic detergent solution.
Use of unclean or contaminated slides The blood smear will not be spread evenly.
Generates artifacts commonly mistaken for malaria
parasites, including bacteria, fungi, stain
precipitation, and dirt and cell debris.
Excessive use of blood The blood smear will not be spread evenly due to the
beginning 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 is
low.
Labeling the slide (Improper or no labeling) Confusion may arise leading to slides that are
unidentifiable and cannot be linked to a patient.
Slides are wrapped together before all the
thick films are properly dried
The slides stick to one another and become unusable.
Excessive time elapses between blood
collection 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.




24

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 stains
comprise two staining components: azures (oxidation products of methylene blue) and eosin. Examples
of Romanowsky stains include Fields stain, Giemsas stain, Leishmans stain and Wrights stain. Giemsa
stain is regarded as the best stain for malaria microscopy; however Fields stain is useful in health
facilities with a low patient workload as it is rapid, economical and easy to use. All Romanowsky stains
can 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 specific
technique 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 function
as 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 be
included with each new batch of working Giemsa stain. Control slides may be prepared from a patients
blood and stored for future use. From a patient known to have a malaria infection, collect a blood
sample in an EDTA (ethylene diamine tetra acetic acid) or citrated blood tube, if it requires multiple
blood film preparations, or needs further diagnosis at a molecular level. An ideal blood sample has at
least one parasite in every 23 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
grooves. Label the outside of the box with the species, date and Giemsa control slides. The slides can
be stored at room temperature but will last longer if stored at -20C or -70C. Just before use, remove
the slide from the box and allow the condensation to evaporate; label the slide with the date and +
control. The smear can then be stained and examined to check that the working solution of Giemsa
stain is of good quality.
4.2.2.3 Fields Stain
Fields stain is useful for rapid detection of malaria parasites particularly for thick films. However,
Schuffeners dots are not always stained with this procedure. It is made up of Fields stain A and Fields
stain B as both are used in the staining procedure.
4.2.2.4 Buffer Solution for Malaria Staining
A phosphate buffer solution, correctly balanced to pH 7.2, is essential for Giemsa and Fields staining for
malaria parasites. Check the pH level using narrow-range pH papers or a pH meter and store the buffer
solution at room temperature. The buffer is stable for several months. To check its quality, the pH of
buffered water should be checked, and appropriate correcting fluid should be added.


25

Buffer tablets
Buffer tablets that produce a solution of pH 7.2 when dissolved are readily available from laboratory
suppliers 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 (preferably

plastic) bottle; when in use, avoid leaving the reagents exposed to sunlight (which encourages the
growth 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. Microscopy
requires 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 electric
binocular microscope. In the absence of electricity, alternative power sources must be used to ensure
quality microscopy.
4.2.3.1 Examining blood films for malaria parasites
Microscopic examination of Giemsa stained thick and thin blood film is the most acceptable method for
detecting malaria parasites in blood. This technique requires trained and experienced laboratory
personnel and equipment such as a microscope.
The thick film used for rapid detection of malaria parasites enhances sensitivity for the detection of low
levels of parasitemia while the thin film is used to confirm plasmodium species and also to assist in the
identification of mixed infections.
Both thick and thin films should initially be examined completely with low power magnification to avoid
missing large organism such as Microfilaria and Trypanosomes.
Table 2 Characteristics of thick and thin blood films
Thick blood film Thin blood film
- Lysed RBCs - Fixed RBCs
- Many layers - Single layer
- Large volume - Smaller volume
- Good screening test - Good species differentiation
- Low density infection can be detected - Low density infection can be missed
- More difficult to diagnose species - Requires more time to read




26

4.2.3.2 Systematic Approach to Examining Thick and Thin Blood Films
A. Examining the Thick Film
Thick films are performed to detect parasites and measure parasite density (quantification), and can be
used to monitor response to treatment. Parasites are quantified by counting ring forms (trophozoites)
against white blood cells. The results are expressed as parasite count per 200 white blood cells (WBC) or
parasite count per microlitre of blood, assuming a total white blood cell count of 8000/l if a measured
white blood cell count is not available. This method of quantitation is useful in low and moderate
parasitaemia.
Examination of a thick film is based on examination of 100 good fields. The blood film can be
pronounced negative only after no parasites have been found in at least 100 fields of the blood film. If
parasites are found, a further 100 fields should be examined before a final identification of species is
made. This ensures that there is little possibility of a mixed infection being overlooked. Since the
erythrocytes have been lysed and the parasites are more concentrated, the thick film is useful for
screening for parasites and for detecting mixed infections.
Determination of "No Parasites Found" (NPF)
According to the WHO, at least 100 fields, each containing approximately 20 WBCs, should be screened
before reporting a thick blood film negative. Assuming an average WBC count of 8,000/l of blood, this
gives a threshold of sensitivity of 4 parasites per microliter of blood.
In non-immune patients, symptomatic malaria can occur at lower parasite densities, and screening more
fields (e.g., 200, 300, or even the whole smear) might be necessary, depending on the clinical context
and the availability of laboratory personnel and time.
B. Examining the Thin Film
Thin films are useful for species identification of plasmodium species if this is not clear from the thick
film. Examination of thin film greatly assists in the identification of mixed infections and is also used for
quantification purposes.
Examination of thin films is recommended in the following circumstances:
When it is not possible to examine a thick film because it is too small, has become auto fixed, or
is not examinable for other reasons.
When it is necessary to confirm the identification of species.
4.2.3.3 Recognition of Malaria Parasite Species, Other Blood Parasites and Artifacts
A. Morphology, stages and distinctive diagnostic features of the plasmodium species
The simplest guide to distinguishing between the four species of malaria is the effect the parasite has on
infected red blood cells. Features to concentrate on include the size of the red blood cell (whether or
not it is enlarged) and whether or not staining reveals Schffners dots or Maurers dots (also known as
Maurers clefts) within the cell. Schffner's dot refers to a hematological finding that is associated with
malaria, exclusively found in Plasmodium ovale and Plasmodium vivax. Plasmodium vivax induces


27

morphologic alterations in infected host erythrocytes that are visible by light microscopy in
Romanovsky-stained blood smears as multiple brick-red dots. These morphologic changes, referred to as
Schffner's dots, are important in the identification of this species of malarial parasite and have been
associated by electron microscopy with caveolavesicle complexes along the erythrocyte plasmalemma.
Maurer's dots are any of the fine granular precipitates or irregular cytoplasmic particles usually present
in red blood cells infected with the trophozoites of Plasmodium falciparum.

FIGURE 10 BASIC COMPONENTS OF A MALARIA PARASITE INSIDE A RED BLOOD CELL

Malaria parasites pass through a number of developmental stages. In all stages, the structural features
of the parasite stain the same colour using Romanowsky stains, as follows:
Chromatin (part of the parasite nucleus) is usually round in shape and stains red.
Cytoplasm occurs in a number of forms, from a ring shape to a totally irregular shape. It is always
stained blue, although the shade of blue may vary between malaria species.
Malaria pigments stain in various shades from yellow-gold through brown to black.
Stippling stains in shades of pink which vary among different species.

B. Identification of Malaria Parasite Species and Stages
The trophozoite stage
This stage is the most commonly seen; it is often called the ring stage (although sometimes it takes the
form of an incomplete ring).

FIGURE 11 TROPHOZOITE STAGE OF THE MALARIA PARASITE



28

Because the trophozoite stage is a growing stage, the parasite within the red blood cell may vary in size
from small to quite large. Pigment appears as the parasite grows. Malaria pigment is a byproduct of the
growth or metabolism of the parasite. It does not stain, but has a color of its own, which may range
from pale yellow to dark brown or black.

The schizont stage
At the schizont stage the malaria parasite starts to reproduce. This reproduction is referred to as asexual
because the parasite is neither male nor female but reproduces itself by simple division. There are
several obvious phases in this stage, ranging from parasites with two chromatin pieces to parasites with
a number of chromatin dots and definite cytoplasm. These are seen clearly in the diagram below.


FIGURE 12 STAGES OF SCHIZONT GROWTH
Note: The process of forming schizonts, which takes place in the liver and in blood, is referred to
as schizogony.


29

The gametocyte stage
The gametocyte stage is the sexual stage, in which parasites develop into either male or female forms in
preparation for the next stage, which takes place in the mid-gut of the female Anopheles mosquito.
Gametocytes may be either banana-shaped (P.falciparum) or round (other species), and are either male
(microgametocytes) or female (macrogametocytes).



FIGURE 13 GAMETOCYTES OF PLASMODIUM FALCIPARUM AND PLASMODIUM MALARIAE

C. Appearance of different malaria parasite species
In thick blood films
In thick blood films, the staining process ruptures non-nucleated red cells but keeps white cells,
nucleated red cells and parasite structures intact. Parasites and white blood cells can therefore be seen,
although they may appear smaller and less well-defined than in thin blood films.
In thick blood films, the fine rings of cytoplasm of trophozoites may appear incomplete or broken. The
absence of red blood cells makes Schuffners dots (usually seen in Plasmodium ovale or vivax) more
difficult to see. The ghosts of red cells may be seen surrounding the parasites in the thinner part of the
film.
In thin blood films
Malaria parasites appear more well-defined in thin blood films, although it may be more difficult to
detect parasites in blood with low parasitaemia. Some species of malaria parasites have an effect on the
appearance of red blood cells in thin blood films: for example, enlargement of red cells in P. vivax
infection; or oval red cells in P. ovale infection. Staining may also reveal Schffners dots, or Maurers
clefts within the cells.




30

The tables below provide a reference for both thin and thick film species differentiation.
Table 3 Species differentiation on thin films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape Round round, distorted oval, fimbriated Round
Stippling infected RBC Maurers clefts Schffners spots Schffners spots None
Trophozoite shape small ring, appliqu
large ring,
amoeboid
large ring,
compact
small ring,
compact
Chromatin dot often double Single Large Single
Mature schizont
rare, 12-30
merozoites
12-24 merozoites 4-12 merozoites 6-12 merzoites
Gametocyte Crescent shape large, round large, round compact, round

Table 4 Species differentiation on thick films
Feature P. falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +*
Fragmented trophozoites ++* +
Compact trophozoites + +
Pigmented trophozoites +
Irregular cytoplasm + +
Stippling (RBC ghosts) + +
Schizonts visible very rarely often Often Often
Gametocytes visible Occasionally usually Usually Usually
*Note: Refer to the semi-quantitative scale below

D. Artefacts and contaminants confusing malaria parasites
Artifacts found on slides may include the following:
Vegetable spores, yeast, pollen, algae and bacteria in the stain or on the slide
Platelets
Howell-jolly bodies in anaemic patients
Ghosts of immature red cells mimicking Schffner's stippling



31


FIGURE 14 BLOOD ELEMENTS, ARTEFACTS AND CONTAMINANTS THAT CAUSE CONFUSION


32

4.2.3.4 Microscopic Differentiation
Microscopic differentiation of species depends on host cell and parasite characteristics.
1. Features of infected red cells and ghosts
- Change in size, shape and colour
- Presence of dots, Maurer's clefts (not on ghost cell) on infected red cells
- Single or multiple infection of each cell
2. Parasite morphology at specific stages
- Number and size of chromatin beads
- Shape and size of cytoplasm
- Degree of pigmentation within cytoplasm
- Stages of parasite seen together

Diagnostic Points of Malaria Parasites
For P. falciparum
Red Cells are not enlarged
Maurers dots may be present
No Schffners dots
Rings appear fine and delicate, there may
be several in one cell
Some rings with 2 chromatin dots
Presence of marginal or appliqu forms
Gametocytes have a crescent/banana
shape
Usually trophozoites and gametocytes are
seen
Schizonts are rarely seen
Plate 1 Diagnostic stages of P. falciparum

For P. vivax
Red cells are usually enlarged
Schffners dots are frequently present in
the red cells as shown above
The mature ring forms tend to be large and
coarse
Trophozoites schizonts and gametocytes
Developing forms are frequently present
Plate 2 Diagnostic stages of P. vivax



33


For P. ovale
Red cells are enlarged
Comet forms are common (top right)
Rings are large and coarse
Schffners dots, when crescent, may be
prominent
Mature schizonts similar to those of P.
malariae but larger and more coarse
Plate 3 Diagnostic stages of P. ovale



For P. malariae
RBCs are not enlarged
Ring forms may have a square-like
appearance
Band forms are a characteristic of this
species
Mature schizonts may have a typical daisy
head appearance with up to ten
merozoites
Plate 4 Diagnostic stages of P. malariae



The appearance of Plasmodium stages in the different malaria species are shown on Plates 1 4.
Source: www.rph.wa.gov.au/malaria/diagnosis.html





34




















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


36


Plate 5 Plasmodium falciparum stages in Giemsa-stained in thin and thick film
Source: WHO (1991). Malaria training module



37


Plate 6 Plasmodium malariae stages in Giemsa stained thin and thick film
Source: WHO (1991). Malaria training module


38


Plate 7 Plasmodium ovale stages in Giemsa-stained thin and thick film
Source: WHO (1991). Malaria training module



39


Plate 8 Plasmodium vivax stages in Giemsa stained thin and thick film
Source: WHO (1991). Malaria training module



40

4.2.3.5 Malaria Parasite Counting Methods
The determination of the number of circulating parasites is exceedingly important for clinical purposes
to monitor the evolution of the disease and the efficacy of therapy.
Different methods have been proposed:
1. Number of parasites/L of blood (thick film):
Accurate parasite density estimation based on parasites per micro liter or white cell count is necessary
when parasite density determination is important for clinical decision-making (for example in severe
malaria or where monitoring of treatment efficacy is required) and in clinical trials. It is recommended in
routine practice that parasite quantitation be performed against 200 or 500 WBCs. If, after counting 200
WBCs, 100 or more parasites are found, record the results in terms of the number of parasites per 200
WBCs. If less than 100 parasites are found after counting 200 WBCs, parasite quantification should be
continued until 500 WBCs are counted. (This gives a probability <5% of chance variation greater than
25% of true parasite density using a x100 oil immersion objective and an eyepiece with a field number of
18). All parasites in the final field are counted, even if the count exceeds 500 WBCs. To determine
parasite density, the parasite count is adjusted against the true WBC where available. If unavailable, a
common practice is to assume a WBC count of 8000/L.

Example: Patient WBC = 8000 l
Parasite count against 200 WBCs = 650

Parasite count/ul = 650 x 8000/l
200

= 26 000 parasites/l

2. Number of parasites/L of blood (thin film):
This method requires the preliminary determination of the number of erythrocytes present in the
average microscopic field. The number of asexual parasites is counted in at least 25 microscopic fields.
The number of RBCs in the average microscopic field is about 200, so total RBCs counted in 25 fields is
roughly 200*25 = 5000. If the hemogram is not available, RBCs/L is assumed at 5,000,000 for males and
4,500,000 for females




41

Example: Parasite counted against 5000 RBCs/25 fields/= 50
# of RBCs in 25 fields = 5000
RBC count = 5,000,000/Male patient/

Parasites/l = 50 x 5,000,000
5000

= 50,000/l of blood
3. .Proportion of parasitized erythrocytes/total RBC count (thin film):
This method will indicate the percentage of erythrocytes that are infected by malaria parasites. The
percentage of infected red cell is determined by counting the number of red cells and that of parasitized
red cells. This method of quantification is useful in high parasitemia. Since it takes almost 10 times as
long to examine a thin film as to examine a thick film, routine examination of thin films is not
recommended. The number of parasitized erythrocytes (asexual forms) present in 25 microscopic fields
is counted and divided by the total number of erythrocytes present in these fields (about 5,000), and
multiplied by 100.

Example: Average # of RBCs/25 fields=5000
# parasitized RBCs/25 fields=100

% of parasitized RBCs = 100 X 100
5000

= 2%
In this example 2% of the RBCs are infected with asexual forms of malaria parasites.
4. Semi-quantitative count (thick film):
A simpler method of counting parasites in thick blood films is to use the plus system. This system is very
quick but less accurate and less satisfactory. Therefore, it should be used only when it is not possible to
carry out the more acceptable count of parasites per micro liter of blood.
The following semi-quantitative scale is used
+ 1-10 asexual parasites per 100 thick film fields
++ 11-100 asexual parasites per 100 thick film fields
+++ 1-10 asexual parasites per single thick film field
++++ > 10 asexual parasites per single thick film field


42

4.2.4 Reporting Blood Film Results
Reporting of malaria positive blood film should include the species, stage and, if necessary, the density
of the parasite.
Species, stage and approximate number of parasites
1. P. falciparum
- Ring only + Number(per l)
- Ring and gametocyte
- If schizonts seen, report immediately and mark with a red pen!
2. P. vivax, P. malariae, and P. ovale all stages can be found.
3. Mixed infection report appropriately
4. No parasites found (NPF) or Negative if you do not find malaria and other hemo-parasite after
examination of a minimum of 100 oil immersion fields.
Diagnostic quality control depends upon the:
Compliance with standards
Availability of supplies, equipment, infrastructure
Condition of the microscope
Training of lab personnel
Regular supervision
Quality of reagents and stains
Cleanliness
Work load
Technical ability and type of techniques used
Table 5 Species differentiation of malaria parasites by cytoplasmic pattern of trophozoites in
Giemsa-stained thick blood films
Species
Stage of parasite in peripheral blood
Trophozoite Schizont Gametocyte
P. falciparum



Young,
growing
trophozoites
Size: small to medium
Number: often numerous
Shape: ring and comma forms
Usually associated with
many young ring forms.
Size: small, compact
Immature pointed-end forms
uncommon.
Mature forms: banana-shaped or


43

and/or
mature
gametocytes
usually seen
common
Chromatin: often two dots
Cytoplasm: regular, fine to
fleshy
Mature forms: sometimes
present in severe malaria,
compact with pigment as few
coarse grains or a mass
Number: few,
uncommon, usually in
severe malaria
Mature forms: 12-30 or
more merozoites in
compact cluster
Pigment: single dark
mass
rounded
Chromatin: single, well defined
Pigment: scattered, coarse, rice-
grain-like; pink extrusion body
sometimes present. Eroded
forms with only chromatin and
pigment often seen
P. vivax



All stages
seen;
Schffners
stippling in
ghost of
host red cells,
especially at
film edge
Size: small to large
Number: few to moderate
Shape: broken ring to irregular
forms common
Chromatin: single, occasionally
two
Cytoplasm: irregular or
fragmented
Mature forms: compact, dense
Pigment: scattered, fine
Size: large
Number: few to
moderate
Mature forms: 12-24
merozoites, usually 16,
in irregular cluster
Pigment: loose mass
Immature forms difficult to
distinguish from mature
trophozoites.
Mature forms: round, large
Chromatin: single, well defined
Pigment: scattered, fine. Eroded
forms with scanty or no
cytoplasm and only chromatin
and pigment present
P. ovale




All stages
seen;
prominent
Size: may be smaller than P.
Vivax
Number: usually few
Size: rather like p.
malariae
Number: few
Immature forms difficult to
distinguish from mature
trophozoites.


44

Schffners
stippling in
ghost of
host red cells,
especially at
film edge.
Shape: ring to rounded,
compact forms
Chromatin: single, prominent
Cytoplasm: fairly regular, fleshy
Pigment: scattered, coarse
Mature forms: 4-12
merozoites, usually 8,
in loose cluster
Pigment: concentrated
mass
Mature forms: round, may be
smaller than p. vivax
Chromatin: single, well defined
Pigment: scattered, coarse.
Eroded forms with only
chromatin and pigment present.
P. malariae




All stages
seen.
Size: small
Number: usually few
Shape: ring to rounded,
compact forms
Chromatin: single, large
Cytoplasm: regular, dense
Pigment: scattered, abundant,
with yellow tinge in older
forms
Size: small, compact
Number: usually few;
Mature forms: 6-12
merozoites, usually 8,
in loose cluster, some
apparently without
cytoplasm
Pigment: concentrated
Immature and certain mature
forms difficult to distinguish
from mature trophozoites
Mature forms: round: compact
Chromatin: single, well defined
Pigment: scattered, coarse, may
be peripherally distributed.
Eroded forms with only
chromatin and pigment present
Source: WHO(1991). Malaria training module

4.2.4.1 Essential Elements of Record Keeping
Accurate record keeping in the malaria laboratory is essential. Recording means keeping the register up-
to-date. Accurate record keeping is based around four elements:
Completeness
Consistency
Credibility
Timeliness
Laboratory supervisors should regularly review specimen request forms, the Laboratory Register and the
reporting of results to ensure that laboratory record keeping meets the four elements above.
4.2.4.2 Laboratory Request and Report Forms
In many countries, the laboratory request form and the microscopy report form are combined into a
single sheet of paper. This enables better tracking of reports and reduces the time to transcribe the
patient- and sample-related information on separate report forms and hence reduces transcription
errors.


45

A Laboratory request form must be submitted with the patient information and it must match the
information on the slides exactly. If the form is incomplete but the patient is available, ask the patient
for the required information.
A completed Laboratory Request Form should contain the following information:
Name of the health facility
Date (day, month, year)
Patients name, address, age and sex
Type of specimen
Patient ID/ Specimen ID number (laboratory serial number)
Clinical impression
Signature of person requesting the exam

Microscopy Report
After the blood film has been read, immediately write the result in the result form. Check that the
specimen ID (laboratory serial numbers) on the slide matches that on the laboratory request form.
Subsequently, write the results in the Laboratory Register, again checking to make sure that the
laboratory serial number matches exactly.
The microscopy report should include the following information:
Specimen ID number (laboratory serial number)
Date (day, month, year)
Blood film test result (presence of parasite, species, stage and parasite count/density)
Name and signature of the technician who performed the microscopy

Once completed, the microscopy report should be made available as soon as possible. Send the report
to the referring health facility or clinician. Ensure that the health facility or clinician receives the result.
4.2.4.3 Entry of Data into the Laboratory Register
Laboratory Register Book
The laboratory register book is a record book maintained by the laboratory personnel responsible for
blood film examination of patients with suspected malaria. The health facility laboratory register must
include the following data for each patient with suspected malaria:
Laboratory serial number
Date of specimen testing (day, month, year)
Patients name, sex, age, and address
Type of test
Test result(s)
Signature of person responsible for tests
Make sure all necessary columns are completed accurately. Reset the laboratory register number to one
on July 1
st
(which corresponds to the start of the Ethiopian fiscal year). DO NOT reset at the end of each


46

day, week or month. Enter patients successively, increasing the line number by one each time. The line
number is sufficient for identification of the request form.
4.2.4.4 Consequences of Incorrect Reporting
False Negatives: A false negative is a result that was reported negative when it was truly positive. Patients with
malaria who receive a false negative result may not be treated, resulting in ongoing disease, disease transmission
and possibly death.
False Positives: A false positive is a result that was reported positive when it was truly negative. Patients who
receive a false positive result for malaria will be treated unnecessarily, given the wrong medications (malaria
medications) and in turn medications will be wasted.
4.2.4.5 Importance of Malaria Data
Malaria data are usually compiled from a laboratory register on a weekly basis and further summarized
to receive monthly, quarterly and yearly data. Errors in reporting will affect:
Accurate determination of malaria cases at specific localities
Determination of work load in specific laboratories
Epidemic detection
Malaria transmission trends based on confirmed cases

4.2.4.6 Laboratory Confirmed Malaria Case Report Form
Compilation of microscopically confirmed cases can be done by reviewing the malaria register
and counting the number of blood films performed with their results reported as positive with
species name and negative slides.
The report is usually submitted to the health facility manager and to the respective health
offices. This will assist to:
- Determine the work performed at the end of each week, month, quarter and year.
- Determine consumption of supplies based on the workload.
- Provide epidemiological data on microscopically confirmed cases of malaria at the
health facility.


47

5 PARASITOLOGICAL DIAGNOSIS OF MALARIA USING RAPID
DIAGNOSTIC TESTS (RDTS)
5.1 RDTs and their Significance
Rapid Diagnostic Tests (RDTs) or malaria rapid diagnostic devices detect malaria parasite antigens in a
small amount of blood by immunochromatography. In this assay, with monoclonal antibodies directed
against the target parasite antigen and impregnated on a test strip. The result, usually a colored test
line, is obtained in 5 to 20 minutes. Malaria RDTs have the potential to greatly improve the quality of
management of malaria infections in areas where the main alternative form of diagnosis, high quality
microscopy, is not readily available.
In Ethiopia as in other malaria-endemic countries there is increasing support for parasite-based rather
than clinical diagnosis in places where microscopy is unavailable, for instance at health posts. This is
particularly the case due to the implementation of ACT and the extension of health services to remote
areas. RDTs are important for at least three reasons to distinguish fevers caused by malaria parasites
from those caused by other illnesses:
Many life-threatening illnesses, such as meningitis and acute lower respiratory infections, cause
symptoms similar to malaria (fever, chills, malaise, aches, etc.). Treating all febrile cases as
malaria means that patients with these other conditions may not get the treatment they really
need. When an RDT shows that a febrile patient does not have malaria, the health worker will
manage the other febrile illness accordingly.
RDTs can help target patients to proper treatment. Those who are diagnosed to have falciparum
or mixed malaria infection should be treated with ACT. ACT is currently much more expensive
than older antimalarials such as chloroquine (CQ), sulfadoxine-pyrimethamine (SP) and
amodiaquine (AQ).
Avoiding unnecessary use of ACTs on patients who do not have malaria may help prevent or
delay drug resistance; making ACTs effective for a longer period.

5.2 RDT versus Microscopy
Antigen-based Rapid Diagnostic Tests (RDTs) can be performed by a wider range of health providers at
all levels of health care with minimal training. In addition, the results are available immediately to the
health provider. The tests have an important role at the periphery of health services capability because
none of the rural clinics has the ability to diagnose malaria on-site due to a lack of microscopes and
trained technicians to evaluate blood films. However, RDTs for malaria are more costly per test than
microscopy and should only be used where microscopy is not available. It is not recommended to use
RDTs where there is a well functioning laboratory, as microscopy provides additional useful information
such as parasite density, species identification, and support to patient follow-up.
Currently most RDTs in routine use are HRP2/pLDH based. HRP2 antigen which uses to detect P.
falciparum can persist in patients peripheral blood after effective treatment cannot be used for follow-
up of patients after treatment. RDTs cannot be used to determine parasite density and parasite stage.


48

Table 6.Comparison of RDT use versus Malaria Microscopy
Characteristics Microscopy RDT
Format Slides with blood smear Test device
Equipment Microscope Kit only
Training Trained microscopist 'Anyone with a little training'
Test duration 20-60 minutes or more 5-30 minutes
Test result Direct visualization of the parasites Color changes on antibody coated lines
Capability
Detects and differentiates all
plasmodia at different stages
Detects malaria antigens (PfHRP2/
PMA/pLDH) from asexual and/or sexual
forms of the parasite
Detection threshold 5-10 parasites/L of blood
1 00-500/L for P. falciparum,
higher for non-falciparum
Species differentiation Possible
No clear result between P. falciparum
and mixed infections (P. falciparum and
non-falciparum); appear as P.
falciparum
Quantification Possible Not possible
Differentiation between
sexual and asexual
stages Possible Not possible
Limitations
Availability of equipment and
skilled microscopists, particularly at
remote areas and odd hours
Unpredictable efficiency at low and
very high parasitemia; cross reactions
among plasmodial species and with
auto-antibodies; persistence of
antigens

5.3 Malaria RDT Formats
RDTs are commonly prepared in three different formats: strips, cassettes and cards.
Dipsticks are impregnated nitrocellulose strips; it can be done by dipping in wells containing blood.
Cassettes are plastic cases containing nitrocellulose paper with a test and control area. They are easy
and safe to use and handle.
Card-flaps containing nitrocellulose strips. Undergo sample wicking up the nitrocellulose strip after
application of the blood and reagent.


A B C


FIGURE 17 DIFFERENT FORMATS OF MALARIA RDT: A-CASSETTE; B-DIPSTICKS; AND C-CARD TEST


49

5.4 Basic Principles of RDTs
Rapid diagnostic tests are immune-chromatographic tests that detect specific parasite antigens mainly
Histidine Rich Protein 2 (HRP2) or Plasmodium lactate dehydrogenase (pLDH). Plasmodium aldolase is
another antigen that is used in some tests. Some RDTs can detect only one species (Plasmodium
falciparum) while others detect one or more species of malaria parasites that infect humans. Antigen s
Detected by currently used RDTs:
1. Histidine Rich Protein II (HRP-2): is a protein produced by trophozoites and young gametocytes of P.
falciparum. A substantial amount of HRP 2 is secreted by the parasite in to the host bloodstream
and the antigen can be detected in erythrocytes, serum, plasma, cerebrospinal fluid and even urine
as a secreted water-soluble protein. Tests based on HRP-2 detect only P. falciparum. HRP-2 has been
shown to persist and may be detectable for more than a month after clinical symptoms of malaria
have disappeared and parasites are cleared from the host. HRP-2 based tests are relatively more
stable at high ambient temperatures and humidity, and usually less costly.
2. Plasmodium Lactic Acid (Lactate) Dehydrogenase (pLDH) is produced by both trophozoites and
gametocytes of malaria parasites. The pLDH antigen is present in and released from parasite-
infected erythrocytes. pLDH is found in all 4 human malaria species, and different isomers of pLDH
for each of the 4 species exist. Currently available pLDH RDTs detect pLDH specific to Plasmodium
falciparum, P. vivax or are pan-specific detecting all Plasmodium species that infect humans. Some
pLDH RDTs are specific for P. vivax. Since pLDH is disappeared from the circulation within five days
of successful antimalarial therapy, this test has the ability to differentiate untreated from treated
malaria, and may therefore be used for patient follow up, although pLDH is also produced by
gametocytes. Tests based on pLDH are less stable at high ambient temperatures and humidity, and
are more costly.
3. Plasmodium aldolase is an enzyme produced in the glycolytic pathway by all species of human
Plasmodium parasites (pan-specific) and has been used in a combined 'P.f/P.v' immune-
chromatographic test. Tests that detect aldolase appear to be less sensitive than tests that detect
the other parasite products. Aldolase behaves in much the same way as pLDH.
The various RDTs appear to be similar; they vary considerably in their functioning due to the intrinsic
character of the critical components employed and their final result.









50

Table 7. Comparison of Rapid Diagnostic Tests for Malaria Antigens

PfHRP2 tests PfHRP2 and PMA test pLDH test
Target antigen
Histidine rich protein 2 of
P. falciparum, water
soluble protein expressed
on RBC membrane
Pan-specific Plasmodium
aldolase. parasite
glycolytic enzyme
produced by all species
and PfHRP2
Parasite lactate
dehydrogenase. parasite
glycolytic enzyme
produced by all species
General test format 2 lines 3 lines 3 lines
Capability
Detects P. falciparum
only
Can detect all 4 species Can detect all 4 species
Non-falciparum species Not detected
Detected; differentiation
between the 3 not
possible
Detected; differentiation
between the 3 not
possible
Mixed infections of P.
falciparum with non-
falciparum species
Appear as P. falciparum;
differentiation not
possible
Appear as P. falciparum;
differentiation not
possible
Appear as P. falciparum;
differentiation not
possible
Detection limit >40-100 parasites/L
Higher for P. vivax and
other non-falciparum
species
> 100-200 parasites/L
for P. falciparum and P.
vivax; may be higher for
P. malariae and P. ovale
Post-treatment
persistence of antigens
Reported up to 31 days
Reported; longer for pan
specific antigenemia than
for PfHRP2
Reported up to 1 -3
weeks
Cross-reactivity between
malarial species
Reported Reported Reported
Cross-reactivity with
auto antibodies
Reported, high (up to
83% with rheumatoid
factor)
Not known
Reported, low (3.3% with
rheumatoid factor)
Indication of viability of
parasites
No No
Positive test indicates
presence of viable
parasitemia

5.5 RDTs Mode of Action
Though variations occur among malaria RDT products, RDTs are lateral flow immune-chromatographic
antigen-detection tests, which rely on the capture of dye-labeled antibodies to produce a visible band
on a strip of nitro-cellulose. With malaria RDTs, the dye-labeled antibody first binds to a parasite
antigen, and the resultant complex is captured on the strip by a band of bound antibody, forming a
visible line (test line). A control line gives information on the integrity of the antibody-dye conjugate, but
does not confirm the ability to detect parasite antigen. The modes of action are indicated below:
a. A dye-labeled antibody, specific for targeting antigen, is present on the lower end of the
nitrocellulose strip or in a plastic well provided with the strip. Antibody, also specific for the
target antigen, is bound to the strip in a thin (test) line, and either antibody specific for the
labeled antibody, or antigen, is bound at the control line.


51

b. Blood and buffer, which have been placed on the strip or in the well, are mixed with
labeled antibody and are drawn up on the strip across the lines of bound antibody.
If antigens are present, some labeled antibody will be trapped on the test line. Excess-
labeled antibody is trapped on the control line.


FIGURE 18 MODE OF ACTION OF ANTIGEN-DETECTING MALARIA RAPID DIAGNOSTIC TESTS (RDTS).
Source: WHO/TDR, Ensuring quality and access for malaria diagnosis: how can it be achieved,
www.nature. com/reviews/micro
5.6 General Procedures of Malaria RDTs
Malaria RDT's are designed and standardized primarily for testing specimen obtained from fresh
capillary whole blood, and blood correctly collected through vein puncture. Venous blood collected in
appropriate anticoagulants, when stored at 2-8
0
C may be stable for up to 72 hours provided they are
not contaminated.
The test procedure varies between the different test kits. Therefore, reference should always be made
to the specific manufacturers instruction. In general, the blood specimen mixed with a buffer solution
that contains a hemolyzing compound and a specific antibody that is labeled with a visually detectable
marker such as colloidal gold. In some kits, labeled antibody is pre-deposited during the manufacturing
process and only a lysing or washing buffer is added. If the target antigen is present in the blood, a
labeled antigen-antibody complex is formed and it migrates up the test strip to be captured by the pre-


52

deposited capture antibodies specific for the antigens and against the labeled antibody (as a procedural
control). A washing buffer is then added to remove the hemoglobin and permit visualization of any
colored lines formed by the immobilized antigen-antibody complexes.
Result Reading and Interpretation
a. For PfHRP2 only test strips
The PfHRP2 test strips have 2 lines, one for the control and the other for the PfHRP2 antigen.
Invalid test result: no color change on the control line with or without a color change on the test
line.
Negative: color change only on the control line and no color change on the test lines.
Positive: test for P. falciparum malaria with the PfHRP2 test: color change on both test and
control lines.
b. For PfHRP2/pan-specific test strips
The PfHRP2/pan-specific PLDH or aldolase test strips have 3 lines, 1 for control, and the other 2
for P. falciparum (PfHRP2 or pLDH specific for P. falciparum) and non-falciparum antigens ( pan
specific pLDH or aldolase), respectively.
With the PfHRP2/ the pan-pLDH tests, a color change on the control line and the pan specific
line indicates non-falciparum infection.
A color change on all 3 lines indicates the presence of P. falciparum infection, either as mono
infection or as a mixed infection with non-falciparum species.
If the PfHRP2 line is visible when the pan-specific line is not, the test is interpreted as positive
for P. falciparum infection.
Note: Mixed infections of P. falciparum with the non-falciparum species cannot be
differentiated from pure P. falciparum infections.

C. P. falciparum pLDH/Panspecific pLDH or aldolase
The P.falciparum pLDH/pan-specific PLDH or aldolase test strips have 3 lines, 1 for control, and
the other 2 for P. falciparum ( pLDH specific for P. falciparum) and non-falciparum antigens ( pan
specific pLDH or aldolase), respectively.
With the Pf PLDH/ the pan-pLDH or aldolase tests, a color change on the control line and the
pan specific line indicates non-falciparum infection.
A color change on all 3 lines indicates the presence of P. falciparum infection, either as mono
infection or as a mixed infection with non-falciparum species.
If the Pf PLDH line is visible when the pan-specific line is not, the test is interpreted as positive
for P. falciparum infection.



Table 8 Limitations of RDT results



53

Negative Result Positive Result
Sometimes a negative test result does not
exclude malaria with certainty, because:
- There may be insufficient parasites to
give a positive result
- The RDT may have been damaged,
reducing its sensitivity
- Illness may be caused by another species
of malaria parasite which the RDT is not
designed to detect.
A positive result does not always signify
malaria illness, because:
- Antigen may sometimes be detected
after the infecting parasites have died
(i.e., after treatment) or due to the
persistence of malaria gametocytes which
do not cause illness
- Presence of other substances in the blood
may occasionally produce a false-positive
result

Repetition of the test after 1 to 2 days may therefore be indicated if illness persists or if the patients
condition deteriorates.
5.7 RDT Kit Selection and Handling
The specific performance requirements of a test will vary depending on the intended usage. When
considering whether or not to use malaria rapid diagnostic test in a particular setting, it is important to
consider their strengths and plan how to manage the challenges.
Important considerations in choosing an RDT include:
The plasmodium species to be detected.
Accuracy (sensitivity and specificity).
Shelf-life and temperature stability during transport, storage and use.
Ease of use
Cost per test.
5.7.1 The Plasmodium species to be detected
The appropriateness of P. falciparum-specific, pan-specific and non-falciparum RDTs varies with the
relative prevalence of the different human malaria species in the intended area of use. These areas are
categorized by the WHO as:
Zone 1. P. falciparum only, or with non-falciparum species occurring almost always as co-
infections with P. falciparum. This includes most areas of sub-Saharan Africa and lowland Papua
New Guinea.
Zone 2. Falciparum and non-falciparum infections occurring commonly as single-species
infections. This includes most endemic areas in Asia and the Americas as well as isolated areas in
Africa, particularly the Ethiopian highlands.
Zone 3. Areas with non-falciparum malaria only; this includes mainly vivax-only areas of East
Asia and central Asia and some highland areas elsewhere.



54

5.7.2 Accuracy (Sensitivity and Specificity)
A test method is said to be accurate when it measures what it is supposed to measure. Accuracy of RDTs
are expressed through several measures, the most widely used being sensitivity and specificity. These
measures complement each other.
Sensitivity: is the ability of a test to correctly identify individuals who have a given disease or
disorder. A high sensitivity of a malaria RDT means that it will produce a true positive result when used
in a population infected with malaria as compared to the reference malaria gold standard test,
microscopy. The sensitivity of an RDT for detecting malaria parasitemia (or recent parasitemia) depends
on the concentration of circulating antigens in the patients blood, and the ability of the labeled
antibody on the RDT to bind the antigen and accumulate to form a visible line.
Specificity: Specificity is the ability of a test to correctly exclude individuals who do not have a
given disease or disorder. A high specificity of a malaria RDT means that it will produce a negative result
when used in a population not infected with malaria as determined by the reference malaria gold
standard test of microscopy. In other words, it measures how often the test is negative when malaria is
absent.
Both sensitivity and specificity are influenced by product storage and conditions of use. In general it is
recommended that at least 95% of P. falciparum infections should be detected at 100 parasites/l and
higher parasite densities, which is likely similar to good field microscopy.
5.7.3 Shelf Life and Stability
To ensure that a product will retain its quality, it should be stored and transported within the
transporting requirements and necessary precautions. A longer shelf-life reduces the pressure on the
supply chain and the likelihood of wasting expired tests. A minimum of 18 months of shelf life (e.g., at
least 15 months after purchase) is recommended since the RDT can be used at least for one year period
which means for one major and minor transmission season in Ethiopian context.
5.7.4 Ease of Use

The intended conditions of use must be considered when choosing an RDT. If the RDTs are to be used in
a remote area without temperature-controlled storage, stability (temperature requirements and shelf
life) will be of great importance, compared to storage and use in temperature-controlled laboratories. In
Ethiopia, for example, RDTs are recommended to be used at health posts by health extension workers,
since an easy-to-use format is more important in settings where laboratories are absent.
5.7.5 Cost
RDTs are often more costly than microscopy and this should be taking into consideration when deciding
purchase in quantities and the level of use in the health care system.


55

6 QUALITY ASSURANCE OF MALARIA LABORATORY DIAGNOSIS
6.1 What is Quality Assurance?
Quality Assurance (QA) is a system designed to improve the reliability and efficiency of laboratory
services, which are critical to the success of malaria control programs. All parts of the testing system
must be monitored to ensure the quality of the overall process, to detect and reduce errors, and to
improve consistency between testing sites. To ensure reliability and to reduce errors, a quality system
must address all parts of laboratory testing.
The components of a quality assurance program for laboratory diagnosis of malaria are the following:
Quality Control (QC): QC is systematic internal monitoring of work practices, technical procedures,
equipment and materials, including the quality of stains.

External Quality Assessment (EQA): EQA is a schematic assessment by an external entity of a
laboratorys performance in testing of known and standardized but undisclosed content and comparing
the results with those of other participating laboratories to assess laboratory practices and identify
problems and weaknesses. EQA includes onsite evaluation of laboratories, proficiency panel tests and
blinded smear rechecking.

Quality Improvement (QI): QI is a process by which the components of blood film microscopy
diagnostic services are analyzed with the aim of identifying and permanently correcting any deficiencies.
Data collection, data analysis, and creative problem solving are skills used in this process

Generally quality assurance has three phases: pre-analytical, analytical and post analytical phases as
summarized in the figure below.

FIGURE 19 THE QUALITY ASSURANCE CYCLE
Source: http://wwwn.cdc.gov/mpep/labquality.aspx Accessed on 04/09/2012


56

6.2 The Need for Accurate Malaria Laboratory Diagnosis
Parasitological confirmation of the diagnosis of malaria provided by high-quality microscopy or, where
this is not available, by RDTs is recommended for all suspected cases of malaria. In settings where
malaria incidence is very low, parasitological diagnosis for all fever cases may lead to considerable
expenditure to detect only a few patients who are actually suffering from malaria. In such settings,
health workers should be trained to identify patients that may have been exposed to malaria (e.g.
recent travel to a malaria endemic area, or lack of effective preventive measures) and have symptoms
that may be attributable to malaria before they conduct a parasitological test. A parasitological
confirmation of malaria in stable high-transmission settings improves the differential diagnosis of fever,
improves fever case management, and reduces unnecessary use of antimalarial medicines.
A high-quality microscopy service is one that is cost-effective, provides results that are consistently
accurate and timely such that they have a direct impact on treatment. To achieve this, a comprehensive
and active quality assurance (QA) program is required. The primary aim of malaria microscopy QA
programs is to ensure that microscopy services are strengthened by competent and motivated staffs,
supported by effective training and supervision that maintain a high level of staff competency and
performance, and by a logistics system that provides and maintains an adequate supply of reagents and
equipment.
6.3 Errors compromising quality laboratory diagnosis
Laboratory errors can be seen at three different vital steps of the procedure. The forecast and
recognition of each source of error may create a way to correct inaccurate results.
The following are common sources of errors by phase:
a. Pre-analytical phase
- Incorrect test request or test selection (vague or ambiguous requests)
- Incomplete request forms
- Poor or inadequate patient preparation
- Poor method of specimen collection, labeling and transportation
- Use of dirty slides
- Defective equipment (microscope, weighing balances) and/or improper use of equipment
- Substandard or expired reagents, and poor reagent preparation and storage
b. Analytical phase
- Poor procedure in blood film preparation and staining
- Inaccurate reading by unqualified or incompetent laboratory staff
- Lack of adherence to SOPs
c. Post- analytical phase:
- Poor reporting and recording
- Inaccurate calculations, computation or transcription
- Delay in reporting results to the clinician
- Incorrect results or misinterpretation of results



57

6.4 Objectives of Quality Assurance Programs
To improve the overall performance of laboratory personnel at each level of the laboratory
system.
To sustain the highest level of accuracy (in sensitivity and specificity) in confirming the presence
of malaria parasites.
To systematically monitor Malaria laboratory diagnostic procedures, reagents and equipment.

The quality of Malaria Diagnosis Depends Upon
Agreement with quality standards
Availability of supplies, equipments and infrastructure
Good working condition of the microscope
The training of laboratory personnel
Regular supervision
Quality of reagents and stains
Cleanliness
Optimal work load, and
Technical ability of laboratory technician and type of techniques used.

6.5 Challenges in Malaria laboratory Diagnosis
It has been difficult to maintain good quality microscopy, especially at the periphery of the health
system where most patients are treated presumptively in spite of the fact that the importance of light
microscopy is well recognized. The current challenges of malaria microscopy include the:
Poor quality of microscope, particularly at the peripheral level.
Difficulties in maintaining microscopy facilities in good order, logistic problems and high costs of
maintaining adequate supplies and equipment.
Lack of adequate training and retraining of laboratory staff.
Poor QA system and supportive supervision of laboratory services.

6.6 Setting up a QA system
To set up a national QA system, the following should be considered
1. Description of the tiered laboratory network and responsibilities of each level
2. An inventory of available resources: The aim of this step is to establish the minimum acceptable
level of microscopy resources, including properly trained technicians, functional microscopes,
supplies, means of communication and program supervision. Future and historic resources from
national and international stakeholders need to be included in this assessment.
3. Analysis of the annual number of slides collected at each laboratory, and an estimate of the slide
positive rate. Availability of the data.
4. Evaluation of the status and effectiveness of current QA activities.


58

5. Comparative perspective: consider the adaptation of QA program of other, comparable
countries.
6.7 Principles and Concepts of Quality Assurance in Malaria Diagnosis
Immediate and long term clinical, public health and health planning decisions for malaria control are
based on laboratory test results. Incorrect, delayed or misinterpreted tests can have serious
consequences for patients and communities, undermine confidence in the service and waste scarce
resources.
Achieving reliable and valid laboratory test results is dependent on:
Understanding common causes of inaccuracy and imprecision in performing tests.
Understanding consequences of delay or misinterpretation of test results.
Taking necessary steps to prevent and minimize errors.
Enhancing a good relationship with clinicians on their work practices to enable the laboratory to
provide efficient, safe, cost effective and reliable services.
Maintaining good lines of communication among the health facility management, clinicians and
laboratory staff.
Maintaining complete and accurate laboratory records.
Quality Assurance includes:
Internal quality control (IQC)
External Quality Assessment (EQA)
Quality Improvement (QI)

(For more details refer to Malaria Laboratory Diagnosis External Quality Assessment Scheme
Guidelines, EHNRI, September 2009, pp 52.)
6.8 Quality Assurance of Malaria Microscopy
6.8.1 Internal Quality Control (IQC)
IQC comprises those measures implemented by the laboratory during each test run to verify that the
test or procedure is correct and working properly, for example, checking the quality of the stain by use
of a control blood film.
QC helps to ensure that the results produced by a laboratory are accurate, reliable and reproducible.
The IQC program should be performed regularly and, to be effective, the process must be practical and
readily included in standard laboratory reporting practices. All laboratory professionals are responsible
for performing, recording and reporting results of QC. Many components of QC are either performed in
conjunction with routine testing or as part of the regular quality management of the laboratory.
IQC in malaria includes correct specimen collection by qualified laboratory staff; preparation of good
quality blood films; staining using quality reagents from a reliable source; examination using a quality
Microscope; and correct result interpretation, recording and reporting.


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The quality of Giemsa working solution should be checked before its use. Therefore, IQC is performed
for every new batch of Giemsa stock solution, when there is a doubt in the quality of the reagent, and
on a regular basis using control slides.
Preparation of control slides
Collect a blood sample in an EDTA tube from a patients blood known to have malaria infection
(an ideal blood sample has at least one parasite in every 2-3 fields on thin smears).
If we cant get known patient having malaria infection we can prepare panel slides from
negative patient.
Make as much blood films as possible from collected blood samples, preferably within one hour
of drawing the blood from the patient.
Allow the blood film to air dry at room temperature.
Fix the thin blood film in absolute (100%) methanol and allow them to dry.
Label the slide with date and positive or Negative control
Place them back to back in a slide box with separating grooves.
Label the box with the species, date of preparation and Giemsa Control Slides.
Cover the slide box containing control slides with plastic envelop and store in -70 refrigerators
for longer storage.
The slides can be stored at room temperature for a maximum of one week.

Staining of control slides
If frozen, remove the slide from the box before use and allow the condensation to evaporate at room
temperature. The blood film can be then stained and examined to check that the working solution of
Giemsa stain is working properly or not using the criteria listed below.

Evaluation
The background of a well-stained thin film should be clean and free from debris; the color of
erythrocytes is a pale green pink and do not appear pink to red; Neutrophil leukocytes will have deep
purple nuclei and well-defined granules; the chromatin of malaria parasites is a deep purplish red and
cytoplasm a clear purplish blue; Stippling should show up as Schuffners dots in erythrocytes containing
P. vivax or P. ovale, and Maurers spots in erythrocytes containing the larger ring forms of P. falciparum.

In a well stained thick blood film, above 90% of the red cells should be completely lysed. The
background should be clean and free from debris, with a pale mottled-grey color derived from the lysed
erythrocytes; Leukocytes nuclei are observed as a deep, rich purple; Malaria parasites are well defined
with deep-red chromatin and pale purplish blue cytoplasm. In P. vivax and P. ovale infections the
presence of Schuffners stippling in the ghost of the host erythrocyte can be seen especially at the
edge of the film. If the explained criteria are not met, the reagent preparation techniques and the pH of
the distilled water used for working solution preparation should be checked. Stained blood film that is
too pinkish suggests low pH or over-staining and too bluish or purplish suggests high pH or under-
staining. All QC results should be documented.


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6.8.2 External Quality Assessment (EQA)
EQA refers to a system of objective checks of laboratory results by means of an external agency. EQA
schemes are effective means of assessing a laboratorys performance. The objective of EQA is to help
laboratories identify errors and improve practices for better performance.
Effective EQA is a collaboration of laboratories at every level. The performance of each laboratory is
determined, areas of weakness are identified and corrective measures undertaken. Intra-laboratory
performance can be compared. Data or information collected can be used for accreditation purposes
and to evaluate the use of certain laboratory equipment or techniques in the field.
Involvement in an EQA activity should not be seen as a threat, but rather as an opportunity to
strengthen skills. Most laboratory technicians want to provide accurate testing. Good performance in
EQA activities reassures them that their results are contributing to accurate malaria diagnosis and
control
There are three EQA methods for evaluating performance of malaria laboratory diagnosis: proficiency
panel testing blinded rechecking and on-site supervision.
A. Panel Testing
Panel testing refers to the process by which the laboratory (known as the test laboratory) performs
malaria microscopy on a set of prepared slides received from the reference laboratory. This exercise can
check both the laboratorys staining procedure as well as the ability of the laboratory professional to
recognize and identify malaria parasites present.
B. Blinded Rechecking
Blinded rechecking refers to the process by which a random selection of slides collected from the
testing laboratory is reexamined at a higher level laboratory. Slides are checked for quality of blood
film preparation (appropriate size and thickness), quality of staining and accuracy of the result.
Rechecking reflects the true performance of laboratories offering routine diagnostic services at health
facility level. The purpose of the exercise is to allow a statistically valid assessment of the proficiency of
the peripheral laboratory.
Rechecking may detect malaria misdiagnosis in routine work and assess the overall quality of testing.
This should not be considered a criticism of the person who performed the routine examination.
Misdiagnosis in routine examination is more frequently caused by different reasons such as high
workload, poor equipment and not necessarily lack of skill by the reader.
Each round of rechecking must be followed by feedback in the form of a written report, showing details
of incorrect scoring, if applicable, and offering suggestions for quality improvement (corrective action).
For the purpose of blinded rechecking, slides are selected from those stored in the health facility.
A. O n-Site Supervision
On-site supervision of malaria microscopy and RDT requires regular supervisory visits to obtain a
realistic picture of laboratory conditions and practices for malaria microscopy. On-site supervision
includes a comprehensive assessment of laboratory organization, equipment, adequacy and storage of
supplies, reagent quality, availability and usage of SOPs, reading and reporting of results, and infection


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control measures guided by the use of a supervisory checklist. On-site supervision is the ideal way to
obtain a realistic assessment of the skills practiced in the testing laboratory or facility, to provide
problem solving strategies and corrective action, and assess the need for training. Supportive
supervision includes assessment of test performance, provision of on-site training and strengthening of
services.
Malaria microscopy on-site supervision is conducted in accordance with the National EQA Program three
times a year by EHNRI, Regional Laboratories, quality officers and malaria experts at different levels, as
well as other partners working on malaria quality improvement.
6.8.3 Quality Indicators for Malaria Microscopy
The following are quality indicators for malaria laboratory diagnosis:
Laboratories should have SOPs, Job Aids and Bench Aids for malaria microscopy diagnosis, and
adhere to the procedures.
Qualified staff
- Laboratories should have laboratory personnel trained on malaria microscopy and RDTs
Functional Equipment
- Microscope is in good working order (electrical binocular microscope)
- Availability of maintenance and cleaning records
- Functional timer and tally counter
Reagent preparation and storage
- Daily preparation of fresh working reagent from stock solution
- Storage according to the manufacturer instructions (Giemsa stain should be stored in
brown bottle)
- Reagents are labeled clearly with day of preparation/opening and expiry dates
Quality control
- Check regularly the quality of every new batch of reagents using known positive and
negative blood films.
- RDT pre and post procurement lot testing
- RDT selection based on WHO recommended criteria
EQA (External Quality Assessment)
- Laboratory participation in EQA
- Mechanisms for implementing corrective action, including retraining
Correct blood film specimen
- Completed request
- Labeled with unique ID and matching the request
- The thin film should consist of a single layer of RBCs with a feathered end, uniformly
spread on the slide
- The thick film should be:
round in shape
10mm away from the edge of the slide
10mm in diameter
have 10-12 WBC per single 100x objective field
newsprint or hands of a watch can be seen through the film
Proper storage of slides for rechecking


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in a slide box
away from excessive heat and humidity
consecutively according to laboratory register number
Free from immersion oil.
Clearly labeled laboratory numbers
Results should not be written on slides
Safety and safe waste disposal procedures (see Laboratory safety section for details)

6.9 Quality Assurance (QA) of Malaria RDTs
A QA process for malaria RDTs aims to ensure high accuracy of test results in the hand of end-users. This
will include both monitoring of the technical standard of the RDTs, processes to minimize environmental
biosafety, and training and monitoring of preparation and interpretation by end-users.
1. Planning for RDT introduction
This requires a strategic plan with clear timelines to ensure that the various components of the RDT
program are in place at the right time. A Quality Assurance coordinator (or coordinators) should be
designated to oversee the overall implementation plan and ensure that all agencies involved understand
the process and their particular roles, and that none are neglected. WHO has produced a document
Good Practices for Selecting and Procuring rapid diagnostic tests for Malaria to guide country
programs through the process of choosing, procuring and planning for RDT rollout.
2. Procurement
Procurement from manufacturers with ISO 13485:2003 compliance is recommended.
Sensitivity and specificity are difficult to assess, as they are dependent on quality of the test, the
parasite density and other characteristics of the testing population, on RDT preparation and
interpretation, and on the quality of the reference standard. Data on test accuracy should be obtained
from the manufacturer and interpreted with caution. In order to evaluate test sensitivity and specificity,
lot-testing and field monitoring are essential.
Thermal stability data should be obtained from the manufacturer and compared with conditions of
intended transport, storage and use. The parasite density (antigen concentration) of the standard used
to assess stability should be noted, as a heat-damaged RDT may still detect samples with high parasite
density.
Staggered delivery is useful policy (splitting delivery from the manufacturer into 2 or 3 batches several
months apart), as it reduces the burden on central storage facilities and allows new products to be
received closer to the expected time of use, shortening storage duration and effectively lengthening the
shelf-life of the overall procurement.
3. Lot Testing: Pre- and Post-Purchase
It is recommended that all lots (batches) of RDTs be tested before deployment to the field. A lot to be
tested is normally defined as a production run using a particular batch of monoclonal antibodies and


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nitrocellulose. Lots are usually defined by a lot number provided by the manufacturer, and usually
consist of 40,000 to 80,000 tests.
Lot testing can be done either
Before purchase, directly arranged with the manufacturer and a lot-testing center, or
After purchase, before distribution to the field.
Lot testing is done
To identify inter and intra-lot variation in specific lots of products
To ensure no reduction in performance has occurred as a result of inadequate storage
conditions during transport to the country,
To convince stakeholders (clinicians, users, regulatory authorities) that tests are in working
order.

4. Monitoring Performance in the Field
Field monitoring is difficult, partly due to the inherent problems of accuracy of field microscopy, with
which RDTs must be compared.
At present, the following procedure is recommended:
Compare RDT results with expert light microscopy. RDTs and blood films should be taken from
the same patients in selected health facilities where RDTs have undergone typical storage,
distribution and usage.
E.g., every month, 40 RDTs (20 positive and 20 negative) should be cross-checked against the
corresponding 40 BF obtained from the same patients and examined by an expert laboratory
technician. Where >10% discordant results occur, a more detailed field evaluation should be
rapidly performed or the remaining RDTs should be returned for laboratory testing (see lot-
testing above).
It is important that the microscopists selected for the evaluation of RDT performance are highly
competent.
In addition, it is important to supervise the health workers performing RDTs on a regular basis at
least every 3 months in order to evaluate the laboratory personnels capacity of interpreting a
set of prepared RDTs.
Regular review of diagnosis and treatment records.
Ensure that good blood safety practices are maintained.
Ensure that sufficient supplies are in place for management of malarial and non-malarial fever.
5. Training and Instructions for Users
Appropriate training of health workers prior to the introduction of RDTs is necessary. Teaching
instructions and instructions provided as job aids need to be clear, in a locally-appropriate language, if
required, and tested.



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6. Use of Results and Community Education
There is extensive evidence that RDT (and microscopy) results are frequently ignored when treatment
decisions are made. To address this problem, it is essential to:
Ensure and demonstrate the accuracy of the RDTs (through the quality assurance processes
described above),
Provide management algorithms for appropriate management of parasite negative cases (non-
malarial febrile illness) and train health workers in their use,
Provide health workers with the means to manage parasite positive and negative cases
appropriately,
Educate (sensitize) the community on the importance of parasite-based diagnosis.
7. Storage and Transport
Standard supply management procedures should be applied to minimize storage times and exposure to
extremes in temperature, similar to those for the handling of drugs. These include staggered delivery of
large purchases, First Expiry, First Out stock management principles, a temperature-controlled
centralized storage, and minimizing of storage in peripheral facilities lacking temperature control. Direct
exposure to sunlight should be avoided and transport coordinated to minimize exposure to
temperatures exceeding the manufacturers recommended storage temperature.
6.10 Quality Assurance of Malaria RDTs in Remote Areas
The QA focus at this level should concentrate on initial training, supervision and continuous education
so that personnel working in remote areas achieve and retain competence and motivation. Training
should not only include test procedure methodology but also trouble shooting guidelines, especially on
how to suspect RDT failure, and operating procedures for reporting suspected failed tests and recall
procedures of all proven failed batches of tests to the first referral level or distribution point.
6.10.1 Ensuring Quality of RDTs
a. Pre-analytical phase
Important points to consider are:
- Ensure the quality of batches or lots for RDTs as they come into the country.
- Store and transport RDTs within temperature ranges recommended by the manufacturer.
- Check expiration date of RDTs prior to use.
- Check integrity of packages prior to use.
b. Analytical phase
- Ensure packages are opened only immediately prior to use.
- Ensure product instructions are accessible and tests are performed as instructed by the
manufacturer.
- Read results within the time frame stipulated by the manufacturer.
c. Post-analytical
- Ensure that RDTs are not re-used.
- Ensure that all used RDTs and accessories are discarded in a safe place for incineration.


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6.10.2 External Quality Assessment of Malaria RDTs
External quality assessment is one of the methods used to ensure that the RDTs are handled and used in
a correct way to provide valid and reliable results. Of all the EQA methods, on-site evaluation is
commonly used in our situation to assess the storage conditions, observe how the test procedure is
performed and on bio-safety methods, using standardized checklist. Corrective measures are taken
based on the gaps identified and trainings organized when found necessary.
On-site supervision for malaria RDT should be performed three times a year by supervisors of health
extension workers and other partners working on malaria RDT quality improvement. On-site supervision
provides an opportunity for assessment of RDT supply storage area and temperature, inventory, basic
procedures including sample collection, the RDT performance skills of the health extension worker,
internal quality control, result interpretation, reporting and recording, safety and waste disposal, and
need of retraining, by using a supervisory checklist (see Annex 11).
A major advantage of on-site supervision is the ability to identify sources of errors and provide on-site
corrective action to improve the quality of result output and implement appropriate measures to
resolve problems.
Standardized checklists should be developed to assist supervisors during on-site visits and to allow for
the collection and analysis of standard data for subsequent remedial action. Checklists should be
reviewed and revised as needed to capture all aspects of the testing process including laboratory related
matters in order to improve the entire process.
6.10.3 Quality Indicators of Malaria RDT
- RDTs in use have been checked for the quality of batches or lots as they come into the country.
- Storage and transport of RDTs is within the manufacturers recommended temperature ranges.
- SOP and Job Aid are in place and the adherence to the procedure.
- Expiration date is checked and recorded prior to use of RDT.
- Integrity of the packages is checked prior to use (ensure that packages are opened only prior to
testing). An RDT should be discarded if its envelope is punctured or badly damaged. If the
procedure is delayed beyond the recommendation of the manufacturer after opening the
envelope/package, humidity can damage the RDT.
- Tests are performed by personnel trained on malaria RDT and manufacturers instructions are
strictly followed.
- Test results are read only within the time limit specified by the manufacturer. Test lines may
become positive several hours after preparation.
- RDTs are not re-used.
- Participation in the EQA scheme (onsite supervision).
- Mechanisms for implementing corrective actions, including retraining, are in place.
- All used RDTs are discarded in a safe place for incineration.



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7 LABORATORY SAFETY
The management of the health facility is responsible for providing a safe healthy working environment
and to proactively maintain a well-documented and safe workplace. Employees are responsible for
adhering to all safety guidelines and regulations for demonstrating competency in implementing
laboratory safety techniques. Each blood sample drawn or handled in a health facility carries the risk of
occupational exposure to HIV and other blood-borne infectious agents and other biological samples are
also potentially infectious.
7.1 General Safety Guidelines
Standard operating procedures (SOPs) that cover all steps should be clearly written and carried out
which also ensures safety measures. Generally, the following safety precautions should be implemented
at all times.
Wear a laboratory coat when in the working room and remove any protective clothing before
leaving the laboratory.
Wear gloves when taking and handling blood specimens.
Do not touch your eyes, nose or other exposed membranes or the skin with gloved hands.
Change gloves between patients and remove the gloves before touching objects and surfaces
e.g. door handles and other objects not usually touched by gloved hands; wash your hands and
put on new gloves.
Cover broken skin with water resistant wound covers before wearing gloves
Wash your hands with soap and water immediately after any contamination and after the work
is completed. If gloves are worn, wash your hands with soap and water after removing gloves.
Puncture wounds, cuts and skin contaminated by spills or splashes of blood should be
thoroughly washed with soap and water. Bleeding from the wound should be encouraged.
Dispose of used lancets in a sharps container.
Disinfect work surface areas when blood collection procedures are completed and at the end of
each working day.
Do not eat, drink or smoke in the working area.
For use on all surfaces, use 0.5% solution of bleach.
Prepare fresh working solutions of bleach daily.
Carefully handle all chemicals and reagents according to accepted standards (refer the table
below)






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Table 9 Safety precautions for chemicals used in malaria microscopy
Chemical Main hazard Safety precautions
Giemsa stock
stain
- Highly flammable with flash
point 12 C
- Keep away from sources of ignition
- Avoid inhaling fumes and contact with
skin
Giemsa Powder
- Harmful if inhaled or
swallowed
- May cause irritation to
respiratory tract
- Contact with strong oxidizers
may cause fire or explosion.
- Fire or excessive heat may
produce hazardous
decomposition products.
- Keep container tightly closed in a cool,
well-ventilated place.

Methanol
- Highly flammable with
flashpoint 12 C
- Volatile and hygroscopic
- Toxic if ingested or inhaled
- Can cause dermatitis and
damage to the optic nerve
and central nervous system
- Keep away from sources of ignition,
sodium hypochlorite, nitric acid
chloroform, hydrogen peroxide
- Avoid breathing vapor, protect skin and
eyes
- Use in a well-ventilated area or
preferable in a fume hood
Xylene
- Harmful if inhaled, may cause
dermatitis if in contact with
skin
- Flammable with flashpoint
12 C
- Protect from skin contact and use in a
well-ventilated area
- Do not keep in plastic containers unless
they are made of polypropylene
- Do not use caps with rubber liners
Material Safety Data Sheets (MSDS) for all chemicals used in the lab should be available to laboratarians
for quick reference.

7.2 Safety and Exposure Control Measures

Application of safety procedures in the laboratory is crucial to minimize accidental exposure to
infectious agents achieved by
Applying universal safety precautions: Treat all biological samples as infectious
Wearing PPE
Training
Procedures to limit risk of infection should be instituted during blood collection, sample handling,
testing and disposal. Even though there are a variety of different microorganisms that may put


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laboratory personnel at risk while doing their job, the most important microorganisms to consider are
hepatitis B and C, and HIV.
There are 3 main routes of pathogen entry into the body:
1. Non-intact skin: Naturally intact skin provides a good barrier, this barrier is lost when skin is not
intact.
2. Mucous membrane exposure in eyes, nose and mouth
3. Percutaneous injury (through the skin): Needle sticks, cuts and punctures

The transmission of HIV through needle stick injury ranges from 0.01-0.06%. If in case needle-stick injury
occurs, the following procedures help to avoid immediate infection:
Personal hygiene and preventive measures: wash, wash, wash
o Mucous membranes: flush thoroughly with plenty of clean water
o Skin: apply soap and water for 5 minutes
o Percutaneous: apply soap and water for 5 minutes
Notify supervisor immediately
Get completed exposure Report and
Consult with local senior management in the health facility regarding possible treatment and
follow-up

The major elements of safety measures are:
A. Hand washing and First Aid
B. Exposure Control Plan
C. Hazard Communication
D. Exposure Determination
E. Methods of Compliance
F. Review of Safety Procedures and Training

A. Hand washing and First Aid
Needle stick injury is the most common injury faced by laboratory personnel during blood draws. Use
the following preventive measures:
Wash the punctured hand with running water and soap
Encourage bleeding by trying to squeeze out the excess blood
Notify and consult senior staff at the facility regarding possible treatment and follow-up.
Hand washing is the number one preventive measure against the spread of infection. Therefore, wash
hands before and after handling patients and after handling all materials known or suspected to be
contaminated.
B. Exposure Control Plan (ECP)
As part of the Laboratory Safety Manual, an Exposure Control Plan (ECP) addresses blood-borne
pathogen exposure and should include procedures for:
Hazard communication
Exposure determination
Methods of compliance


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Exposure evaluation
Post-evaluation for exposure occurrences
Annual review of procedures and training
Conduct regular safety audit
Along with a laboratory safety manual, a laboratory manager should have policies and procedures for an
exposure control plan.
C. Hazard Communication
The laboratory manager should provide graphics, warning signs and labels for general hazard safety and
bio-safety issues, Personal Protective Equipment (PPE) and practices which are not allowed in the
laboratory. General hazard safety information such as toxic or carcinogenic reagents, poisons,
flammable, combustible or radioactive reagents, and volatile solvents should be provided. In addition, a
file of material safety data sheets (MSDS) specific for all chemicals used in the laboratory should be
available for reference.
D. Exposure Determination
The laboratory manager or Safety Officer should identify the laboratory working areas which are at risk
of exposure, and should those in the ECP.
There are various levels of exposure
Those working directly with blood borne pathogens are at increased risk.
Those working in the laboratory but not necessarily with the blood borne pathogens are
at secondary risk.
The laboratory manager needs to determine and document the risk level of potential occupational
exposure for all laboratory personnel.
E. Methods of Compliance
Employers need to implement administrative, engineering and PPE controls as a means to protect
against employee exposure to bio-hazardous blood borne pathogens.
Administrative Control
o Use SOPs to limit employee exposure to blood borne pathogens
o Usage of appropriate safety signs
Engineering Controls
o Employ procedures for use of safety devices used in the laboratory (safety
cabinets, safety needle devices, sharps containers, etc.)
Personal Protective Equipment (PPE)
o Train laboratory personnel on the use of PPEs

F. Review of Procedures and Training
All procedures and policies should be reviewed annually. Procedures for training laboratory personnel
on potential hazards and exposure precautions should be established. Post evaluation procedures for


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treatment, counseling and follow-up, if exposure occurs, should be established. Training of laboratory
personnel should be done at commencement of employment and on a yearly basis thereafter. All
training should be documented for each employee.
7.3 Testing Infrastructure and Equipment Management
Applying safety practices in the laboratory requires both infrastructure and trained human resources.
The following are infrastructure requirements:
Waste disposal facilities
Adequate light, water, sewage, ventilation and electrical facilities
Appropriate laboratory design (superstructure, furniture and space)
Appropriate storage facilities
Restricted access to the laboratory
Safety devices and facilities are important to operate malaria diagnosis in compliance with general
safety standards and universal precautions. Some of the safety facilities are personal protective
equipments (PPE), sharp containers, hand washing and eye wash stations, emergency showers,
incinerators and others.
A. Personal Protective Equipment (PPE)
During phlebotomy, exposure to contaminated sharps presents the major risk. During biological sample
preparation in the laboratory, exposure to the skin or mucous membranes presents the major risk.
Perform the following during sample preparation:
Wear gloves and a laboratory coat
o Do not wear gloves or a laboratory coat in common areas or at home
o Change gloves regularly and never re-use gloves (change between patients)
o Change gloves whenever it is contaminated
Dont wear open shoes or slipper
Draw blood only in dedicated areas
Avoid crowded areas or pathways
Cover broken skin with water resistant wound covers even when wearing gloves

B. Sharp Container
Sharps injury is the most risky route of exposure. Sharps include: needles, blood lancets, pipettes,
broken test tubes,
Use biohazard-labeled sharps containers that are leak proof.
Use single-use disposable lancets, needles and scalpels.
Use phlebotomy equipment with built-in safety features.

C. Eye wash
Use eye washes for splashes to the eye
o Flush for 5 minutes for pathogens
o Flush at least 15 minutes for most chemicals


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7.4 Waste Disposal
Wastes should be segregated according to their types. Usually solid and liquid wastes are collected and
separately. Additionally segregation can be made for infectious and non infectious wastes. The solid
wastes should further be segregated into sharp and non sharp wastes. Liquid wastes can be classified
into chemical and biological types. In general, to protect the surrounding population, the laboratory
must dispose of wastes safely. Burning waste in an incinerator is usually the most practical way for safe
destruction of laboratory waste. If safe burning cannot be arranged, discard the waste in a deep pit of at
least 1.5 meters depth. Access to the disposal site should be restricted by building a fence around the
site to keep animals and children away. The burial site should be lined with a material of low
permeability (e.g., clay), if available and the location of the site should be selected at least 50 meters
away from any water source to prevent contamination of the water table. The site should have proper
drainage, be located downhill from any wells, free of standing water and not in an area that floods.
If an autoclave is available, place infected materials inside and follow procedures for safe and adequate
sterilization.
In addition, the underneath measures should be followed for waste disposal:
Dispose of all biohazardous waste appropriately.
Use dedicated leak-proof biohazard bags and bins for all potentially infectious material.
Discard biohazardous waste daily.
Incinerate all solid waste after recommended disinfection.
Liquid contaminated waste (e.g., human tissue, blood, feces, urine and other body
fluids) requires special handling. Carefully pour wastes down a utility sink drain or into a
flushable toilet and rinse the toilet or sink carefully and thoroughly with water to
remove residual wastes. If a sewage system doesnt exist, dispose of liquids in a deep,
covered hole, not into open drains. Decontaminate specimen containers by placing
them in a 0.5% chlorine solution for 10 minutes before washing them.




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8 SUPPLY AND LOGISTIC MANAGEMENT IN MALARIA LABORATORY
DIAGNOSIS

8.1 Logistics Management
The establishment of an effective supply chain is essential to foresee and provide all the equipment and
supplies that are needed to sustain an uninterrupted flow of reliable malaria diagnoses. Three essential
data items are required to run a logistics system
1. Stock on Hand: Quantities of usable stock available at a particular point in time.
2. Consumption/Usage Data: The quantity of laboratory commodities used during the reporting
period (every two months).
3. Losses and Adjustments:
Losses are the quantities of products removed from the stock for anything other than the
provision of laboratory services to patients or those issued to another facility (e.g., expiry, lost,
theft or damage) and are recorded as negative (-) numbers.
Adjustments are quantities of a product received from any source other than Pharmaceutical
fund and Supply Agency (PFSA) or issued to anyone other than your facilitys laboratory. An
adjustment may also be a correction due to an error in mathematics. An adjustment may be a
negative (-) or a positive (+) number.
There are only three activities that happen to laboratory commodities within a logistics system:
Stored in inventory,
Moved between facilities
Used to provide laboratory services to patients.
8.2 Stock Management

Stock management is properly maintaining adequate supplies to ensure uninterrupted service. It
involves:
Performing a stock count (physical inventory)
Maintaining proper inventory records
Determining how much and when to order
Placing orders properly
Inspecting and verifying supplies received
Ensuring proper storage of items on stock.

Stock management ensures the availability of staining reagents, supplies and RDT kits which avoids the
use of old reagents or expired RDT kits, and minimizes waste. It is important not to under-stock or over-
stock supplies at the testing site.
Under-stocking will result in insufficient supplies while testing clients, which interrupts the
testing process.


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Over-stocking can result in storing of laboratory commodities for much longer periods leading
to deterioration or expiring of reagents and RDT kits before use.
A. Inventory Control
An inventory control system is to inform personnel when and how much of a commodity to order and to
maintain an appropriate stock level to meet the needs. A well-designed and well-operated inventory
control system helps to prevent shortage, oversupply, and expiry of commodities.
The inventory control system designed for the laboratory logistic system is a forced ordering Maximum
and Minimum inventory control system. Therefore, every service delivery point (SDP) in the system is
required to report at the end of every other month and order all laboratory commodities back up to the
maximum level. If stock levels ever fall below 2 weeks (0.5 months) of stock before the end of the
reporting period, an emergency order should be placed.
To maintain adequate stock levels, the maximum months of stock, minimum months of stock and an
emergency order point have been established for each service delivery point in the system.
The maximum months of stock signifies the largest amount of each laboratory commodity a
facility should hold at any one time.
The minimum months of stock is the approximate level of stock on hand at the end of the
reporting period when an order is placed.
The emergency order point is the level where the risk of stocking out is likely, and an
emergency order should be placed immediately.

B. Assessing Stock Status
A maximum/minimum inventory control system is a system designed to ensure that quantities of stock
fall within an established range a maximum level and a minimum level. In order to know if the stocks
are within that range, you must assess your stock status.
When you review the stock status, you determine how much of each laboratory commodity you have
available at your facility and how long these stocks will last. You can review your stock status by
counting the stock available, as you do during a physical count. When you finish, you will have an
absolute quantity of stock available. But it is much more important to know how long the stocks will last
and if you have enough stock available until you receive your next order.
Months of stock is the number of months laboratory commodities will last, based on the present
consumption rate. When you review your stock status, you need to determine how many months of
stock you have in your facility. Three months of stock means that your stock will last three months, as
long as consumption remains at the current rate.








74

Determining Months of Stock
By calculating the months of stock, a facility can determine if the right quantities of laboratory
commodities are stocked. To determine how long stock will last, the following simple formula can be
used:
How long supplies will last
(months of stock on hand) =
How much we have
(stock on hand)
How much we have dispensed
(average monthly usage)

Before calculating Months of Stock on Hand, you need to know
Stock on Hand
Average Monthly Usage
To determine the Average Monthly Usage (AMU), add the latest three months usage of a particular
product, then divide by three.

Average monthly usage =
(AMU)
Current months usage + previous
two months usage
3 (three)

If you do not have three months of data, use whatever data you have to calculate the average monthly
usage.
C. Record Keeping
Efficient stock management depends upon accurate record keeping. Keeping accurate records ultimately
saves time.
Stock book
The stock book contains a list of all items in the store. It must be updated routinely when orders are
placed and received. It also serves as a reference to track orders that have been placed and not
received. The information recorded in the stock book regarding when orders are placed and when they
arrive may help a site to adjust the reserve quantities of supplies that are kept on-site to ensure
uninterrupted testing.






75

Table 10 Example of a stock book
Item Physical
Count
(Units)
Date
Physical
Count
Performed
Quantity
Requested
(Units)
Date
Requested
Quantity
Reserved
Date
Received
Total
Stock in
Hand
Expiry
date




Table 11 Example of Stock card
Item
required
Quantity
(units)
requested
Date
requested
Quantity
received
Date
received
Lot
number
and expiry
date
Quantity
used
Balance





D. Calculation of Required Supplies
Calculations of supplies required for a malaria microscopy and RDT can be based on the actual number
of patients performed during a quarter and a stock count of supplies on hand. This will give you more
accurate information about the actual condition. It is performed with a spreadsheet (see example
worksheet below).
How to calculate required supply levels:
1. Determine the number of blood films or RDT performed in a quarter (A).
2. Determine the amount of each item required for a single blood film examination or RDT (B).


76

3. Multiply the two values (A x B = C).
4. Add a reserve quantity (D) of each item (C+D).
Note: The reserve quantity can be a fixed amount equal to the quantity of each item required for
one quarter of operation.
5. From that estimate, subtract the supplies you already have on hand, E ((C+D) E).
6. The result will be the amount of items you must order to ensure uninterrupted testing during
the next quarter of operation.

Key A = the number of blood films examined in the quarter
B = the amount of material required per test
C = A x B
D = Reserve quantity
E = Stock at hand
Stock needed = (C+ D) - E
Example; what is the number of slide required for the coming quarter?
The # of blood films done in the quarter = 500 = (A)
The # of slides required per test = 1 = (B)
The # of reserve slide required = 500 = (D)
The # of slide at hand = 100 = (E)
Therefore: A x B = C 500 x 1 = 500
C + D E 500 +500 - 100 = 900
The required # of slides for the coming quarter is 900 pieces.


77

Table 12 Example of a Quarterly Supplies Request and Report, Requirement Form
Quarterly Supplies Requirement for a Microscopy Center
Region:
Zone: Supply Quarter:
Woreda: Year:
Health center/ Hospital: Facility Name:
Total Blood films examined in previous quarter(A)=500
Item Quantity
needed
per
blood
film(B)
Calculated
requireme
nt for one
quarter
(C)=A X B
Reserve
quantity for
one
quarter(D)=
C
Stock
on
hand
(E)
Calculated
request
(F)=C+D-E
Actual
request
(rounded)*
Order
Unit
Microscope
slides
1 pcs 500 500 100 900 18 pack Pack /50
pcs/
Blood
Lancet
1 pcs 500 500 200 800 4 pack Pack /200
Pcs/
Stock
Giemsa/ml
0.66 ml 330 330 100 560 1 bottle of
500 ml
Bottle
/300ml /
Immersion
oil/ml
0.05 ml 25 25 10 40 1 bottle of
100ml
Bottle/ 1
liter/
Filter
paper/day
2 pcs 180 180 40 320 1 pack Pack/100
pcs/
*Round up to the next indent unit




78

Table 13 Example of a Quarterly RDT Supplies Requirement Form
Quarterly Supplies Requirement for a Malaria RDT
Region:
Zone: Supply Quarter:
Woreda: Year:
Health Post/ center: Facility Name:
Total Malaria RDT performed in previous quarter (A)=300
Item Quantity
needed
per
patient (B)
Calculated
requireme
nt for one
quarter
(C)=A X B
Reserve
quantity for
one
quarter(D)=
C
Stock on
hand
(E)
Calculated
request
(F)=C+D-E
Actual
request
(rounded)
*
Order
Unit
RDT device 1 300 300 50 450 18 boxes 1 Box
/50 pcs/
Note: The RDT device described in the example is composed of all accessories for the single patient
You should assess your stock status any time you suspect that the stock levels do not fall within the
recommended maximum and minimum stock levels for your facility. This may occur if there is a loss of
supplies due to damage, expiry or theft or if there is an unexpected increase or decrease in
consumption.
E. Conducting a Physical Count
A physical count of the products is done to verify that the stock balance found on the stock card shows
the correct number of usable commodities that are available in the storeroom. If the quantity on the
stock card does not match the quantity on the shelf, the stock card should be updated and an
adjustment entered.
A physical count of laboratory supplies should be conducted ONLY at the end of every month and the
stock cards should be updated.
F. Conducting a Visual Inspection
A visual inspection should be completed each time products are handled: when receiving, issuing or
dispensing supplies, or when conducting a physical count. When conducting a visual inspection, be sure
to check the following:
Package and product integrity: check for supply, missing or illegible identification information.
Labeling: make sure that products are labeled with the date of manufacture or expiration, lot
number and manufacturers name.
Storage condition how the reagent is placed, temperature, humidity and store area.


79

8.3 Storage of Malaria Laboratory Commodities
Storage conditions will affect the quality of the laboratory products being stored. Rooms that
are too hot, stacks of cartons that are too high, and other poor storage conditions can cause
damage or cause a reduction in shelf life.
A well-organized storeroom will simplify a facilitys work; time will not be wasted trying to find
needed supplies.
Each commodity has a shelf life that is specified by the manufacturer. When the commodity
reaches the end of its shelf life, it has expired and should not be distributed to patients or used
in the laboratory. Some laboratory products have short shelf lives. Because of these short shelf
lives, it is important that proper storage procedures are followed, so that the shelf life is
protected. Always check the expiry dates before issuing or using, and do not use products that
have expired.
In some cases, a product will not have an expiration date on it, but it will have the
manufacturing date. By knowing the date it was manufactured and the shelf life of the product,
one is able to determine the expiration date of the product..
8.3.1 Storage of Reagents and Equipment
In order to manage storage of reagents and equipment:
Keep staining reagents in well-closed bottles and out of direct sunlight.
Kept tightly stoppered and free of moisture; stock Giemsa stain is stable at room temperature
indefinitely (stock stain improves with age).
Make working Giemsa stain fresh daily. If a large number of smears are made, the working stain
may need to be changed throughout the day.
Label all stock bottles containing staining reagents by name, date of preparation and person
who prepared it.
When storing new microscope slides, make sure they are as dry as possible to prevent fungus
growth.
Store microscopes and their spare parts in a well-ventilated, dry, dark and safe place. Spare
bulbs should always be available at the laboratory, while objectives, eyepieces, and other less-
frequently required parts can be stored at regional level. Optical parts must be kept in a dry
place to prevent damage from fungus.
In general, supplies should be protected from sun, heat and water. Follow manufacturer
recommendations for storing supplies. This information is usually printed on the product carton
and boxes.

8.3.2 Handling Damaged or Expired Stocks
If expired or damaged stocks are found at any time during a visual inspection or a physical count (or
upon receipt of a consignment), these items should be removed from the laboratory. These items also
need to be moved to a separate place so they cannot be dispensed or used. Damaged items should be
safely disposed off.



80

8.4 Supply List for Malaria Microscopy
Equipment and Materials:
1. Slide box
2. Staining jar (to hold 20 slides, placed back to back).
3. Drying rack
4. Forceps
5. Measuring cylinder
6. Slide boxes
7. Binocular microscope- Microscope spare bulb: 1 per microscope for 1 year, Microscope
8. Spare mirror, fuses, eyepiece, and oil immersion objective: 1 per microscope for 10 years
9. Tally counter(s)
10. Funnel
11. Dropper (with rubber bulb)
Consumables
1. Blood lancets
2. Absorbent cotton wool/cotton
3. 70% alcohol
4. Disposable gloves
5. Clean glass slides
6. Pencil/pen/marker
7. Sharps container
8. Biohazard containers
9. Distilled water/ buffered water
10. Lens paper
11. Immersion oil (Type A)
12. Lens cleaning solution
13. Filter paper (e.g., Whatman #1)
14. Measuring cylinder, capacity 100-500 ml (depending on the number of slides to be stained)
15. Measuring cylinder, capacity 10-25 ml (depending on the amount of stock stain to be measured)
16. Timer
Reagents
1. Absolute methanol
2. 10% Giemsa working solutions
3. Giemsa stock solution
Documents and records
1. Laboratory register
2. Request & result Form
3. Material safety data sheet



81

8.5 Supply list of Malaria RDT

1. RDT Device
2. Sterile Blood Lancet
3. Alcohol Swab
4. Pipette
5. Buffer
6. Glove
7. Timer
8. Sharp container
9. Biohazard bag
10. Labeling pensile / Pen/ Marker
11. West Bin



82

REFERENCES
1. CDC. DPDx: Laboratory Identification of Parasites of Public Health Concern/Parasites and
Parasitic Diseases; Blood-borne Parasites: Malaria http://www.dpd.cdc.gov/dpdx, accessed
on March 15, 2009.
2. Chansuda W, Mazie JB, et.al. (2007). Review of Malaria Diagnostic Tools: Microscopy and
Rapid Diagnostic Test (RDT) American journal of tropical medicine and hygiene, 77, pp. 119
127.
3. Cheesbrough M (1998). District laboratory Practice in Tropical Countries. Part 1, Cambridge
University Press, UK.239-258.
4. Chotivanich K, Silamut K, Day N (2007). A Short review of methods; Laboratory Diagnosis of
malaria infection, New Zealand Journal of medical laboratory science, 61 (1):4-7.
5. EHNRI (2008). Acid fast bacilli sputum smear microscopy training module.
6. EHNRI (2008).National AFB participant manual (Draft), April 2008.
7. EHNRI (2009). Malaria Laboratory Diagnosis External Quality Assessment Scheme
Guidelines. pp62.
8. EHNRI/TLCP (2008).Guidelines for Quality Assurance of Smear Microscopy for Tuberculosis
Diagnosis EHNRI/ TLCP - Federal Ministry of Health Ethiopia(DRAFT)
9. FMOH (1999). Health and Health Related Indicators.
10. FMOH (2008). Ethiopia National Malaria indicator survey 2007 technical summary- Federal
Democratic Republic of Ethiopia Ministry of Health 2008
11. FMOH/ANVER/WHO (2007). Entomological profile of malaria in Ethiopia , September 2007.
12. http://chsr.aua.am/malaria/eng/diagnostics.php. Accessed on April 25, 2012
13. http://en.wikipedia.org/wiki/Sch%C3%BCffner's_dots. Accessed on April 25, 2012
14. http: //www.rph.wa.gov.au/malaria/diagnosis.html. Accessed on April 10, 2009.
15. IMaD (2008). Draft of National Guideline for Laboratory diagnosis of malaria, Ghana
Ministry of health.
16. IMaD (2008). Draft of SOPs for Laboratory diagnosis of malaria, Ghana Ministry of health.
17. Kakkilaya BS. (2003). Rapid Diagnosis of Malaria. Malaria site Lab Medicine. 8(34):602-608.
18. Lawrence M. Tierney Jr., Stephen J. McPhee., Maxine A., Papadakis (2006). Current Medical
Diagnosis and Treatment, 15th edition (Kindle Edition).
19. Mandy FF (2004) General Laboratory Safety Issues, National HIV Immunology Laboratory,
Health Canada.
20. MOH (2006). National Five-Year Strategic Plan for Malaria Prevention and Control in
Ethiopia: 20062010 Federal Democratic Republic of Ethiopia Ministry of Health, Addis
Ababa.
21. MOH/EHNRI(2009).Master plan for the public health Laboratory System, Second Edition ,
2009-2013 Ethiopian Health and Nutrition Research Institute Federal Democratic Republic of
Ethiopia Ministry of Health
22. National Strategic Plan for Malaria Prevention, Control and Elimination in Ethiopia, 2010
2015
23. PMI (2009). President malaria initiative, malaria operational plan, Ethiopia.
24. SCMS (2007). Standard operating procedures manual for the management of the national
laboratory logistics system to support HIV/AIDS prevention, treatment and support
programs, December.


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25. SNNPR (2008). Implementation Guideline for malaria microscopic Diagnosis Quality
Assurance, SNNPRS Regional Health Bureau in collaboration with Malaria Consortium,
September 2008- Hawasa.
26. Transfusion, 2008; The American Red Cross
27. WHO (1991) Basic malaria microscopy. Part I. Learners guide. WHO, Geneva (Switzerland).
28. WHO (2000) Bench aids for the diagnosis of malaria infection.
29. WHO (2006). The use of malaria rapid diagnostic tests, second edition.
30. WHO (2008) Methods Manual for Laboratory Quality Control Testing of Malaria Rapid
Diagnostic Tests, Version 5(a), Geneva, Switzerland.
31. WHO (2008). Malaria QA updates: Quality Assurance of Malaria Rapid Diagnostic Tests
buying well and maintaining accuracy.
32. WHO (2008).How to use Rapid diagnostic Test (RDT) - A Guide for training at a village level
modified for training in the use of generic Pf- pan test for falciparum and Non falciparum
malaria.
33. WHO (2009). Malaria microscopy quality assurance manual. Version 1
34. WHO SEARO/WPRO (2005). Malaria Light Microscopy Creating A Culture of Quality Report
of Who SEARO/WPRO Workshop on Quality Assurance For Malaria Microscopy Kuala
Lumpur, Malaysia, 1821 April 2005
35. WHO(1999).New perspectives Malaria Diagnosis report of a joint WHO/USAID informal
consultation - 25-27 October 1999 World Health Organization Geneva
36. WHO. Malaria slide bank project protocol (WHO regional office for the western pacific)
37. WHO/TDR/FIND (2008). Methods Manual for Laboratory Quality Control Testing of Malaria
Rapid Diagnostic Tests. Version Five.
38. WHO/USAID/FIND/TDR/. Malaria Rapid Diagnostic Tests.
Available at http://www.wpro.who.int/sites/rdt/home.htm
39. World Malaria Report 2010.
Available at http://whqlibdoc.who.int/publications/2010/9789241564106_eng.pdf
40. WHO (2010). Guidelines for the treatment of Malaria. 2
nd
edition.



84

ANNEXES
Annex 1: Microscope: Types, Parts, Care and Handling

A. Types of Microscope for Malaria Diagnosis
There are two types of microscopes:
a. Simple microscope
The simple microscope is an ordinary magnifying glass which may have a magnification of 5x,
10x, 20x or more.

b. Compound microscope
A compound microscope has a much higher magnification than the simple microscope. The
typical compound light microscope is capable of increasing our ability to see details 1000 times
enlarged, so that objects as small as 0.1 micrometer (m) or 100 nanometers (nm) can be seen.
This microscope uses at least two lenses positioned at different places. A magnified image of the
object is first produced by one lens and this image is further enlarged by a second lens to give a
more highly magnified object. These two lenses are placed one at the end of each tube. The first
lens which is near to the object is known as the objective lens. While the second lens which is
near the eye is known as the eyepiece lens.
Types of Compound Microscope
Based on the type of illumination system, different types of compound microscopes are available:
Light microscope
Fluorescent microscope
Dark field microscope
Phase contrast microscope
Based on the available number of eyepieces, we can have at least two types of compound microscopes:
a. Monocular microscopes
- Have a single eyepiece
- Are convenient for use by beginners, for field work where there is no electricity and for
photographing clinical specimens.
b. Binocular microscopes
- Have two eyepieces
- Are recommended where much microscopic work has to be done, i.e. in routine
examinations.
The total magnification power of a microscope is the magnification of its objective multiplied by that of
its eyepiece. For example using a 10x objective and 10x eyepiece, the total magnification of microscope
is 100x.
The resolving power of a microscope is the ability of an objective to distinguish the dots separately and
distinctly. It is the limit of usable magnification.


85

For example
The human eye can separate /Resolve/ dots that are 0.25 mm in diameter
A light microscope can separates dots that are 0.25m apart
The electron microscope can separate dots that are 0.5 nm apart.
Parts of a Compound Microscope
1. Microscope Stand
The stand of a basic microscope includes
Tube
- Holds the eyepiece and objectives in line and at the correct distance
Stage
- Is a flat surface where the specimen to be examined is placed.
- In the center of the stage there is circular hole that allows the light from the mirror or
lamp to pass through
Mechanical stage
- This enables the slide on which the specimen is mounted to be moved in a controlled
way, vertically or horizontally.
Sub stage
- Immediately below the stage is the sub stage which holds a condenser lens with an iris
diagraph and a holder for light filters and stops.
Foot/Base
- This ensures microscope stability on the laboratory bench.


Compound microscope


86

2. The Mechanical Adjustment System
Coarse adjustment
- Usually used to focus using low-power objectives
- Controlled by a pair of large knobs positioned one on each end of the body
- Rotation of these knobs moves the tube with its lenses or, in some microscopes, the
stage up or down fairly rapidly.
Fine adjustment
- Use to focus objectives for high-power objectives because they require a fine
adjustment
- Moves the objectives or stage up or down very slowly.
- Controlled/moved by two smaller knobs on each side of the microscope.
Condenser adjustment
- The condenser has an adjustment system for its focusing light onto the specimen on the
stage. This is done by opening and closing of its aperture .
- It can also be swung aside to remove it or to exchange it with another.
- The condenser is usually focused by rotating a knob to one side of it.

3. Optics of a Light Microscope
Objectives
- Objectives are the most important parts of a microscope because the quality and most
of the magnification of the image depend on them.
- Modern objectives are described according to their magnification and older objectives
are often described according to their equivalent focal length (EFL)

Objective
Description in
Diameter Equivalent focal length (EFL)
10x 16 mm or 2/3 inch
40x 4mm or 1/6 inch
100x 2mm or 1/12 inch
- For most routine medical laboratory work, 10x, 40x and 100x objectives are required.
The low power objective: 10x
Used for initial scanning and observation in most microscopic works.
Used for initial focusing and light adjustment of the microscope.
The high power objective: 40x
Used for more detailed study as the total magnification with 10x eyepiece is 400.
Used for the diagnosis of intestinal protozoal parasites, urine sediments/cells, casts
crystals, and histological sections.
The oil immersion objectives: 100x
This lens has a very short focal length and working distance.
The objective lens rests almost on a microscopic slide when in use.
Known as oil immersion objective since a special grade oil must be placed between the
objective and the slide.
Oil is used to increase the numerical aperture and the resolving power of the objective.



87

Ocular (Eyepiece)
- A lens that magnifies the image formed by the objectives.
- The usual magnification of the ocular is 10x, others are 4x, 6x, 7x, 15x and sometimes as
high as 20x.
- The higher the power, the greater the total magnification of the microscope. The lower
the power of the eyepiece, however, the brighter and sharper is the image.
Condenser
- A large lens with an iris diaphragm placed below the stage.
- It directs and focuses the beam of light from the light source, lamp or mirror, to the
specimen under examination.
- Usually consists of two or sometimes three lenses
- The lenses are curved so that the light can pass to the objectives at a sufficiently wide
angle.
- The condenser position is adjustable; it can be raised and lowered beneath the stage
and the light must be correctly focused on the material to be examined.
Iris diaphragm
- It controls the amount of light passing through the specimen under examination.
- Located at the bottom of the condenser, under the lenses, but within the condenser
body.
- It can be opened or closed as necessary to adjust the light intensity.
Mirror
- Used in the microscope without built in illumination
- It reflects the beam of light from the light source upwards through the iris in to the
condenser.
The illumination system
- The modern compound microscope most often has a built-in illumination system with a
controller to adjust the amount of light comfortable for the microscopist.

B. Routine Use of a Basic Microscope Procedure:
1. Place the microscope on a firm bench and make sure it is not exposed to direct sunlight.
2. Select the source of light. If it is a built-in source, switch it on.
3. Place the specimen slide to be examined on the stage. Make sure the underside of the slide
is completely dry.
4. Select the objective to be used.
It is usually better to begin examination with low power (10X) objectives. Once in
focus, all the other objectives also will be in focus provided that they are par focal.
5. Focus the objectives
Move the objectives carefully downwards using the coarse adjustment knob and
looking at it from the side until the lens is near the specimen but not touching it.
Then while looking through the eyepiece, move the objectives slowly upwards, still
with the coarse adjustment, until the image comes into view and is sharply focused.
6. Focus the condenser.
Open the iris of the condenser fully and, using the condenser adjustment knob,
focus the condenser on the detail of the light source until the image of the
diaphragm appears sharp.


88

7. Adjust the opening of the condenser iris according to the specimen examined.
Specimen like stained smears give off a little glare and for these the condenser iris
should be opened more widely giving a well-illuminated image with fine details.
Unstained specimen like urine and saline preparations of stool give off a lot of glare
and require a reduced condenser iris to increase the contrast.
8. Examine the specimen using the mechanical stage to move it.
9. For a higher magnification, swing the 40x into place. Focus the 40x objectives using the fine
adjustment.
10. For the highest magnification, add a drop of immersion oil to the specimen and swing the
100x oil immersion objectives in to place. Open the iris fully to fill the objectives with light.
Note: If examining a stained smear directly with the oil immersion lens and it is not possible
to focus it, remove the slide and check that the oil has been placed on the smear side of the
slide.
C. Setting of the Khler Illumination for Light Microscope
1. Plug in the microscope and turn on the illuminator. Rotate the nosepiece so that the 10X
objective is locked into place.
2. Put the specimen slide on the stage and center it under the10X objective.
3. If there is a swing out (flip) condenser, be sure it is in the light path. Adjust the intensity of the
light to a comfortable level with the transformer.
4. Open the field diaphragm all the way and close the condenser diaphragm all the way.
5. Move up (rack up) the stage to its highest position.
6. Adjust the oculars for interpupillary distance so that when looking with both eyes only one circle
of light is seen.
7. Rack up the condenser as high as possible with the height adjustment knob.
8. Close the field diaphragm half way and focus on the specimen at 10X using the coarse
adjustment knob.
9. Close the field diaphragm until the diameter of the illuminated image is smaller than the field of
view. Note: If there is a flip condenser, it may need to be swung out at this time to achieve this
view of the illuminated image.
10. Lower the condenser with the positioning knob until a sharp, focused image of the edges of the
field diaphragm is achieved.
11. Using the centering screws on the side of the condenser, adjust the condenser so that the circle
of light is centered in the field.
12. Open the field diaphragm until the illuminated image is just larger than the field of view. If
more light is needed, use the transformer. Khler illumination is now set.

D. Microscope Specifications
Microscope must be completely UL*, CSA* and CE* tested, listed, and approved to ensure fire
and/or shock safety. Only UL listed components or line cords are not acceptable.
Must have10x/18mm eye pieces.
Must have auto compensating Siedentopf style binocular with diopter scale for interpupillary
distance (must have visible diopter scales).
Must have 4-position reversed nosepiece of metal construction with internal ball bearing stops.
External clip system not acceptable.


89

Must have 4x HI-Plan, 10x HI-plan, 40x HI-plan, and 100x oil HI-plan parfocal and parcentered
infinity corrected objectives.
Mechanical stage must be of built-in design with metal rack and pinion X-Y drives. No polymer
belts, metal cables, timing belt systems or nonmetallic components are acceptable in the drive
mechanism. Coaxial controls must be low mounted for ease of use.
Pre-aligned Abbe condenser with graduated iris diaphragm wheel with markings to show where
iris aperture should be set for each objective magnification.
Focus drive must be a self-tensioning, three ball design of all metal construction. Fine focus
must have graduations of 100 divisions and 3 microns per division. Focusing knobs on both sides
must have these markings.
All gears throughout the microscope: mechanical stage, focus, condenser rack and pinion must
be made of metal, brass, stainless steel or aluminum no plastic components.
Illumination system must be designed for 12v/35w tungsten halogen 2,000 hour average life
bulb.
Microscope must have hinged lamp door that is angled to help prevent breakage. Sliding
drawer type bulb covers not acceptable for safety reasons.
Must have blue filter fixed into its mount, not loose. In Koehler kits, lollipop filters have locking
slots to prevent them from falling out when tilted.
Microscope base temperature must not exceed 37 degrees centigrade using a 12v/20w halogen
lamp at full voltage for 6 hours.
Power supply must be voltage sensing 85-265 volts with surge suppression and soft start lamp
control.
Lamp intensity must be conveniently located in stand armrest and controlled via an illuminated
rotating wheel.
Stage finger assembly is to be slide friendly that does not damage or break slides.
Microscope must have ergonomic design.
*UL: Underwriters Laboratories Inc.
*CSA: Canadian Standards Association
*CE: Conformance European



90

Annex 2: SOP For Capillary Blood Collection And Preparation of Malaria
Blood Films


Purpose

This SOP provides instructions for capillary blood collection from the finger (or earlobe or heel in infants)
and preparing good quality thick and thin malaria blood films (MBFs).

Principle

Capillary blood obtained by direct pricking of the finger (in adults), or the earlobe or heel of the foot (in
infants). The blood is used immediately to make thin and thick blood film therefore it does not need
anticoagulant.
Materials and supplies

1. Alcohol (70% ethanol)
2. Disposable sterile lancets
3. Absorbent cotton
4. Disposable gloves
5. Clean frosted end glass slides
6. Lead Pencil/Glass writing pencil
7. Slide drying Tray
8. Biohazard containers (for infectious waste)
9. Sharp Container
10. Patient Register

Safety Precaution

1. Wear protective gloves when handling or taking blood samples.
2. Cover any cuts or abrasions on your hands with adhesive dressing.
3. Always wash your hands with soap and water after handling blood sample.
4. If blood gets on to your skin, wipe it off quickly with cotton wool soaked with alcohol and wash the
affected area with soap and water as soon as possible.
5. Take care not to accidentally prick yourself.
6. Never use disposable lancets more than once.

Procedure of blood collection using capillary

1. Label the frosted end of the slide with the patient ID number and date.
2. Disinfect the finger (in adults) or the earlobe or side of heel (in infants) thoroughly with an alcohol
swab.
3. Let the alcohol air dry.
4. Prick the finger/earlobe/heel with a disposable sterile lancet, deep enough for the blood to flow
freely.


91

5. Wipe the first drop of the blood with dry cotton.
6. Apply gentle pressure to the finger/earlobe/heel for the blood to flow
7. Discard used lancets directly into the sharps disposal container.
8. From the pricked finger/earlobe/heel, collect blood directly in to the pre-labeled glass slides
9. Make both thick and thin blood films on the same slide(See Appendix 1:Figure 3b) as follows:
10. By touching the slide on the blood, place a small drop(2l) of blood in the middle portion of the slide
and 1 bigger drop(6l) on the portion next to the frosted end. Allow some space between the thick
and thin films to be made on the same slide (See Appendix Figure 3a).


Procedure for preparation of the thin film (See Figure 3c, Illustration 1 and 2 below).
1. Working quickly, obtain a second clean and polished slide (spreader) and place in front of the small
blood drop at a 30 - 45 angle. Pull back the slide and hold until the blood is evenly spread along
the edge of the slide. Do not delay between applying and spreading the drop.
2. Rapidly push the slide forward in a single, smooth, continuous motion. Avoid hesitation or jerky
motions when spreading the blood. (A feathered end of the film should have red blood cells that are
lying individually without overlapping and relatively evenly distributed).

Procedure for Preparation of thick blood film (See Figure 3c, Illustration 3 below).
1. With one corner of the spreader slide, in a circular motion, spread the blood out to make a circle
with approximately 1cm (1/3 inch) in diameter, finishing off at the center.
2. The ideal thickness of the smear should allow for printed text to be readable when it is placed on it.
3. Discard the spreader into an appropriate slide container and DONT re-use it for another patients
blood sample.
4. Allow both blood films to air dry in a horizontal position on a slide tray . Slow drying prevents
cracking. Avoid using a fan or blow dryer to dry these slides.


Procedural Notes

A number of errors are common in making blood films. These can affect the labeling, the staining or the
examination.
a) Badly positioned blood films
Care should be taken that the blood films are correctly sited on the slide. If they are not, it may be
difficult to examine the thick film. Also, portions of the films may even be rubbed off during the staining
or drying process.
b) Too much blood
After staining films made with too much blood:
The background to the thick film will be too blue.
There will be too many white blood cells per thick film field, and these could obscure or cover up
any malaria parasites that are present.


92

If the thin film is too thick, red blood cells will be on top of one another and it will be impossible to
examine them properly after fixation.

c) 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 and you will not examine enough
blood in the standard examination.

d) Edge of spreader slide chipped
When the edge of the spreader slide is chipped:
The thin film spreads unevenly, is streaky and has many tails.
The spreading of the thick film may also be affected.

e) Thin film too big & thick film in the wrong place
The thick film will be out of place and may be so near the edge of the slide that it cannot be seen
through the microscope.
During staining or drying, portions of the thick film will probably be scraped off by the edges of
the staining trough or drying rack.
It may be very difficult, or impossible, to position the thick film on the microscope stage so that
it cannot be fully examined.

Quality Control

Monitor the quality of the preparation of thick and thin smears
1. Follow proper collection procedures.
2. Glass slides must be clean and free from grease.
3. Thick films and thin films must be prepared properly while drying protects blood films from dust,
flies and insects.
4. Do not dry expressed to direct sun light.
5. Too thin a film may not have adequate quantity of blood for detection of parasite.
6. Blood film spread unevenly on a greasy slide makes examination difficult.
7. Thin film too long, leaves less space for thick film.
8. When fixing the thin film, be careful not to let methanol touch the thick film.
9. Wet slides are wrapped together and the slides stick to one another.
10. Never add a pinch of EDTA powder directly to the sample tubes. High concentration of EDTA leads to
shrinking of RBC and destroys the structure of WBC and platelets
11. Never add the blood before the EDTA solution is completely dried. It will dilute the blood





93

Illustrations

Illustration of Blood film preparation
a. Template for Thick and Thin Malaria Blood Films




b. Preparation of thin and thick blood films


The larger blood drop on the left side is for the thick film. The smaller drop represents what would be appropriate
for a thin film.

c. Thick and Thin Malaria Blood Films





The edge of a clean slide is placed at about 45 angle in front of the smaller blood drop for thin film (see
Illustration below).
Slowly pull this second slide back into the drop while securing the sample slide with the forefingers of the
other hand.
Barely touch the drop of blood and, as the blood spreads laterally along at least two thirds of the edge of
the spreader slide,
rapidly push the spreader slide forward in a smooth, continuous and rapid motion, not stopping until the
clean slide leaves the bloody slide.
A properly prepared thin film is thick at the beginning end and thin or "feathered" at the other end. The
feathered end of the smear should not reach to the end of the glass slide. The feathered end should have
areas optimal for microscopy that are only one cell layer thick.
The thin smear is best prepared immediately after applying the drop of blood, before any drying occurs.

P
a
t
i
e
n
t

I
D

m
m
/
d
d
/
y
y

P
a
t
i
e
n
t

I
D

m
m
/
d
d
/
y
y

Thin

Thick



94






d. Illustration of thin Blood film making
The clean slide was placed just before the blood drop (to the right) then pulled back (to the left) and pushed
forward to the right leaving a feather edged thin film. The blood for the thick film remains untouched at this stage.

Use the corner of the same clean slide to make the thick film by gently swirling the drop of blood to form an even
circle of approximately 10mm diameter using the paper template over which the slide is placed during slide
preparation. Once the drop(s) are evenly spread, lift the corner of the clean slide out of the center of the smear,
trying not to leave any bubbles. If bubbles are present, stir again with the corner of the slide until no bubbles
remain, and/or break the bubbles with the sharp corner of the spreading slide.



and pushed forward forming a
feathered edge.

Clean slide is initially placed in front of
the blood drop.
It is then moved back to the center of
the slide to touch the blood drop.


95



e. Illustration of making Thick blood film
Once the thin film area has been produced, use the corner of the clean slide to make the thick blood film.


Allow the blood smears to dry in a horizontal position before staining in order to obtain the best staining
quality.



96

Annex 3: SOP Preparation of Giemsa Working Solution

Purpose
This SOP provides instructions for preparation of Giemsa working solutions from Giemsa stock.

Principle
Light microscopy, usually applying the Giemsa staining technique, is the established method for the
laboratory diagnosis of malaria.

Giemsa is a Romanowsky stain used for staining blood films. Romanowsky stains contain Eosin Y, an
anionic acidic dye, and Azure B, a cationic basic thiazine dye obtained by oxidation of methylene blue.
When the dyes are diluted in a buffer, the anionic dye stains the acidic components (nucleus) of cells
red, and the cationic dye stains the basic components (cytoplasm) of cells blue.

Materials and Reagents
1. Giemsa stock solution
2. Buffered Distilled water
3. Measuring cylinder 10 and 100ml capacity
4. Filter paper
5. Funnel

Special Safety Precaution
Highly flammable with flash point 12
0
c and Keep away from sources of ignition
Avoid inhaling fumes and contact with skin

Procedure of Preparing 10% Giemsa working solution
1. Pour 90 ml of buffered water (pH 7.0 7.2) into the measuring cylinder.
2. Add 10 ml of filtered Giemsa stock into the measuring cylinder
3. Mix well before using.

Procedure of Preparing 3% Giemsa working solution
1. Pour 97 ml of buffered water (pH 7.2) into the measuring cylinder.
2. Add 3 ml of filtered Giemsa stock into the measuring cylinder.
3. Mix the stain well before using.

Quality Control
Check the staining quality using known QC slides for every batch of Giemsa stain solution.

References
1. Cheesbrough M. District laboratory Practice in Tropical Countries. Part 1, Cambridge University Press, UK.
1998:239-258.
2. Methods Manual for Laboratory Quality Control Testing of Malaria Rapid Diagnostic Tests. Version Five A.
3. WHO Bench Aids for the Diagnosis of Malaria Infections.
4. WHO Basic Malaria Micrrosocpy, Learners Guide, 1991.




97

Annex 4: SOP for Preparation of Buffered Water

Purpose
This SOP provides instructions for preparation of buffered water (pH 7.2)

Principle
The importance of buffering the Giemsa stain solution resides in creating the optimal PH environment
for staining.

Materials and Reagents
1. Beaker, 250ml capacity
2. Graduated cylinder , 1000ml capacity
3. Buffer tablet
4. Distilled water

Procedure
5. Add 150ml of distilled water to beaker
6. Add one tablet
7. Shake the water until the tablets dissolve.
8. When dissolved add the fluid from the beaker to the measuring cylinder.
9. Fill the fluid in the measuring cylinder with distilled water until it is made up to 1L mark.

Quality control
Check expiry date of buffer tablet

References
1. WHO Publication: Bench Aids for the Diagnosis of Malaria Infection
2. WHO Basic Malaria Microscopy, Learners Guide, 2007 (revised edition)
3. Manufacturer instruction.



98

Annex 5: SOP for Examinination of Malaria Blood Films And Estimation of
Parasitemia

Purpose
This SOP provides instructions for the proper detection, identification and quantification of malaria
parasites in Giemsa-stained MBFs.

Principle
Examination of both thick and thin blood film is used to detect & identify malaria parasite respectively
and estimation of parasitemia.

In the thick blood film the red blood cells (RBCs) are lyses and dehemoglobinized while the malaria
parasites are left intact and concentrated and used as a screening test to detect the presence of malaria
parasite.

In the thin blood film, when fixed with absolute methanol, enables the RBCs to retain their original
morphology with malaria parasites, if present, visible inside the RBCs, is used to identify the species and
stages of malaria parasites.

Materials, Reagents and Equipment

Materials
1. Patient Register
2. Pen
3. Lens paper

Reagents
1. Immersion oil
2. Lens cleaning solution (80/20 Ethyl Ether solution)

Equipments
1. Binocular microscope
2. Tally counter(s) / Differential counter
3. Slide boxes


Procedure for Focusing and scanning blood films

1. Place the MBF on the microscope stage, switch on the light and adjust the light source optimally by
looking through the ocular and the 10X/40X objectives.
2. Place a drop of immersion oil on the dry stained slide. To avoid cross contamination, ensure that the
tip of immersion oil dropper never touches the slide.
3. Slowly change to the oil immersion objective, and a thin film of oil will form between the slide and
the lens.


99

4. Adjust the light source optimally by looking through the 10x ocular (eyepiece) and the100X objective
and use the fine adjustment knob to focus the field; the lens should not be allowed to touch the
slide.
5. Examine the slide in a systematic fashion. Start at the left end of the thick film and begin reading at
the periphery of the field and finish at the other end. When the field is read, move the slide right to
examine adjacent fields.

Procedure for examining the thick blood film

1. Scan the thick film under oil immersion objective (x100) and ascertain whether a smear is positive or
negative.
2. Use the WHO Bench Aids in the Diagnosis of Plasmodium Infections for the characteristics and
illustrations of Plasmodium species.
3. If positive, determine all species and stages present.
4. Read a minimum of 200 oil immersion fields before declaring the slide as negative. If time permits,
scan the whole thick film.

Procedure for examining the thin blood film

1. If the blood film is positive for malaria parasite on the thick blood film a careful examination of the
parasite morphology should continue on the thin blood film for verification of species.
2. If different species are observed, all types should be recorded.

Procedure for Estimating Parasite density
A. Parasites/l of blood by counting parasites against 200 WBCs in the thick film
1. Select a part of the thick film, under oil immersion objective, where the white cells are evenly
distributed and the parasites are well stained.
2. Using a piano-type tally counter (or 2 single tally counters), count parasites while simultaneously
counting WBCs in each field covered.
3. Count asexual parasites on the thick film against 200 or 500 WBCs.
4. Stop counting after counting 200 WBCs if the asexual parasites counted are greater than 150.
5. Continue counting up to 500 WBCs if parasites are less than 150 after 200 WBCS have been
counted.
6. All parasites in the final field must be counted even if a count of 200 or 500 WBCs has been
exceeded. Record actual number parasites and WBCs counted.
7. Compute for the number of parasites/l of blood using the formula:
= Number of parasites counted x 8000 WBC/l blood
200 WBCs

8. If an actual WBC count is available, use the formula:
= Number of parasites counted x actual WBC/ l blood
200 WBCs






100


B. Proportion of parasitized erythrocyte / 5000 RBCs count in thin film

1. This method will indicate the percentage of erythrocytes that are infected by malaria parasites
2. The number of parasitized erythrocyte (asexual forms) present in 25 microscopic fields is
counted divided by the total number of erythrocyte present in these fields (about 5000), and
multiplied by 100

% Parasitemia = Number of parasite RBCs x 100
Total RBCs counted in 25 fields

For example
Average number of RBCs/25 fields=5000
Number of parasitized RBCs /25 fields=100

% of parasitized RBCs = 100 x 100
5000
= 2% of RBCs are infected with asexual form of malaria parasite

Note: Estimation of parasitemia is done in case of severe P.falciparum, estimated values infected
RBCs of 2-3% or above or whenever a physician requested

Quality control
Before reading the slide, examine the thick and thin films grossly under 40 x objectives to check the
quality of the slide as follows and ensure the following:
a. Thick film is >90% intact and red cells should be completely lysed, except around the edges.
b. WBCs in the thick and thin films are properly stained (i.e., purple granules visible within the
cytoplasm of the neutrophils).
c. RBCs in the thin film do not appear pink to red.
d. Thin film has RBCs that are in one single, distinctive layer.
e. Thick or thin films have no significant debris.

If these criteria are not met, aim to collect another specimen from the patient.

Related Procedures and Documents
1. Patient Register
2. Laboratory request form

Reference
1. WHO Basic Malaria Microsocopy, Learners Guide, 2007 (revised edition).
2. Methods Manual For Laboratory Quality Control Testing of Malaria Rapid Diagnostic Tests. Version Five
3. RITM, Parasitology Manual of SOPs, August 2007.






101

Annex 6: SOP for Recording And Reporting of Malaria Blood Film Results

Purpose
This SOP provides instructions for interpretation, recording and reporting of results of MBFs.

Materials
1. Pen
2. Laboratory Request Form
3. Patient Register

Procedure of Recording of MBF Results
1. All MBFs examined, whether for routine diagnosis, referrals, confirmation or validation,
should be recorded accurately in the Patient Register.
2. MBFs for research, projects and trials should be recorded separately in study-specific
logbooks.

Procedure of Reporting of MBF Results
Report all species and stages seen and if necessary provide parasite count, according to the
table below:
Species Stages % parasites
P. falciparum
Trophozoites, schizonts (asexual
stages)
Gametocytes (sexual stages)
As required
P. vivax
P. malariae
P. ovale
No malaria parasites
seen

Examples:
a) P. falciparum Trophozoites stage : 2% of RBCs are infected.
b) P. vivax, Trophozoites Shizonts and gametocytes stages are found.
c) No malaria parasites seen



References
1. WHO Bench Aids for the Diagnosis of Malaria Infections.
2. WHO Basic Malaria Microscopy, Learners Guide, 2007 (revised edition).
3. RITM, Parasitology Manual of SOPs, August 2007.



102

Annex 7: SOP for Malaria Blood Film Slide Storage And Selection for
Blinded Rechecking

Purpose

This SOP provides instructions to ensure that malaria blood films (MBFs) are properly stored
and readily accessible. MBFs and their associated data records must be stored for blinded
rechecking.

Materials

For storing Malaria blood films:
1. Slide boxes
2. Tissue paper
3. cabinet

Precautions

1. Store slides by protecting from dust, direct sun light
2. Wear protective gloves when handling slides.

Procedure for Labeling and Storage of Malaria blood for External Quality Assessment
(EQA)


1. All MBFs collected for blinded rechecking must be placed in slide boxes labelled on the outside
with the short title, collection site, month and date.
2. Example of box label: Malaria EQA Program, -----HC/Hospital, 30/09/ 2003 EC
3. Store slides consecutively according to laboratory number so there is a direct link between the
results in the laboratory register and the slide location.
4. Stored slides should be free from immersion oil. Remove the oil by either gently wiping the film
with lens tissue or leaving the slides overnight with the smear side facing down on lint free
tissue paper.
5. Slides must have laboratory numbers clearly visible. Slides without laboratory numbers cannot
be used for validation purposes.
6. Results should not be written on slides; these slides cannot be used for validation purposes


Procedure for Selection of MBFs for blinded rechecking

1. Ten stained malaria slides are selected each month to determine accuracy: 5 positive slides and
5 negative slides.
2. If less than 10 slides are examined in the facility, select all slides for rechecking.
3. If the number of positive slides examined is less, make up the difference with negative slides.


103

4. Ideally malaria slides should be stored for 1 month and the selection made before discarding the
slides. The slide selection procedure will be conducted on monthly basis by the laboratory
head/quality officer using the procedure described above (if number of examined blood films
>1000/month selection will be conducted in weekly basis)
5. Select slide from registration book and note the serial number - put a mark on the register book
to identify the selected slides.
6. During collection of selected slides, the supervisors should counter check the conformity of the
selected slides with the laboratory registration book.
7. The laboratory number and results of the selected slides from the registration book should be
recorded on the format of Annex 1.


References

1. Malaria Laboratory Diagnosis External Quality Assessment Scheme Guidelines, EHNRI, 2009
2. KEMRI Kisumu Malaria SOPs, 2006.
3. RITM, Parasitology Manual of SOPs, August 2007.




104

Annex 8: SOP for Care And Preventive Maintenance of Microscopes

Purpose
This SOP provides guidelines for the proper use and preventive maintenance of microscopes.

Principle
A microscope magnifies minute objects making them visible to the eye. The microscope consists of
mechanical components, a system of lenses that magnify the specimen placed on the microscope stage,
and a light source that illuminates the specimen.

Materials, Reagents and Equipment
1. Lens cleaning solution (80/20 Ethyl Ether Alcohol)
2. Lens paper
3. Microscope
4. Plastic cover
5. Wooden storage Box

Procedure for Installation of Microscope
1. Place the microscope on a firm bench, free from vibration, near an electric power outlet and
away from direct sunlight.
2. During installation of new microscopes, follow manufacturers instructions.

Procedure for using microscope

1. Always follow the manufacturers instructions.
2. Connect to the power supply, and switch on the light source
3. Adjust the eyepieces by sliding them horizontally until both eyes fit comfortably and the two
fields merge.
4. Centre the condenser as follows:
Swing the x10 objective into position.
Raise the condenser to the uppermost position.
Open the iris diaphragm fully.
Open the light diaphragm to illuminate the whole field.
5. Clean and dry the underneath of a glass slide by wiping with cotton gauze.
6. Rotate the nosepiece so that the lowest power objective is in position. Slight resistance is felt as
the objective moves into the correct position.
7. Place the slide carefully on the stage.
8. Never place the slide on the stage when the x 40 or x 100 objectives are in position, to prevent
scratching of the lenses.
9. Adjust the illumination:
Open the lamp rheostat fully to obtain a bright light.
Reduce the iris diaphragm to control brightness.
10. Focus the specimen by racking the stage carefully upwards with the x10 objective in position.
11. Using the coarse adjustment knob, rack downwards slowly using the coarse adjustment knob
until the image comes into view. Use the fine adjustment knob to focus the image sharply.


105

12. Swing the x40 and x100 objectives into position to examine in more detail using the fine
adjustment knob to focus.
13. After examination, lower the stage or swing the lowest power objective into position before
removing the slide.
Never remove the slide when the x 40 and x 100 objectives are in position as this may
scratch the lenses.
14. Wipe off any oil from the lenses and microscope stage using a piece of lint free cotton gauze
soaked in lens cleaning solution (80/20 Ethyl Ether solution). Clean with lens tissue.
15. Switch off the microscope, disconnect from the power source and cover to protect from dust.

Procedure for Care and maintenance of Microscope
1. Always follow the manufacturers instructions carefully.
2. Clean the lenses with lens tissue and not a cloth or ordinary paper. Use lens cleaning solution
(80/20 Ethyl Ether solution) and not use xylene, methylated spirit or acetone; these may
dissolve the cement holding the lenses.
3. For removal of heavy contamination from the instrument surface, use a mild soap solution
never use acetone.
4. At the end of every day, disconnect the power source by switching off at the wall socket and
removing the plug, or disconnecting the battery terminals.
5. Cover the instrument after use.
6. To protect against fungus in humid climates, place the microscope in a
7. Small cabinet or cupboard that is heated continuously from below by a low watt bulb. Do not
store the microscope in its carrying case or under a plastic hood in humid climates.
8. Protect the microscope from power surges using a voltage stabilizer.
9. Replace blown bulbs, following the manufacturers instructions.
10. If the equipment is faulty, consult a qualified biomedical engineer.
11. All microscopes in the laboratory must be scheduled for routine cleaning and check-up daily
using daily microscope maintenance chart.(Appendix 1)

Troubleshooting
1. Always refer to the operations manual.
2. If the microscope fails to switch on, check the electric socket outlet, plug and fuse or the battery
terminals.
3. Do not dismantle any part of the microscope. If the microscope is not
4. functioning properly, consult a qualified biomedical engineer

Related procedures and documents
Microscope user manual

References
1. KEMRI, Kisumu Malaria SOPs, February 2006.
2. WHO Documents (CD). EQAS, September 2007
3. RITM, Parasitology Manual of SOPs, August 2007.



106

Annex 9: Monthly Malaria Case Report Format

Region ____________________ Zone ____________________
Wereda / District ______________
Health facility ____________________________
Month ____________________ Year _________
Age group Blood film
performed
Negative Positive P. falciparum P. vivax Mixed
(P.f. and P.v.)
< 1
1-4
5-15
>15
Total
Need separate column for persons suspected of malaria (total tested) and duplicates, and RDT results (P.
vivax or P. falciparum), number tested with both RDT and microscopy. Total suspected malaria (total
tested) = (Blood smear Pos + Blood smear negative) + (RDT Negative +RDT positives) (Repeat
Microscopy + repeat/duplicate RDT).




107

Annex 10: Exposure Reporting Form

Dear colleague here is just part of our care for your wellbeing. The hospital is committed to create a
healthy working environment. We always advise universal precautions in all your efforts in caring for
others. But in case you happen to be exposed to any suspicious body fluid that may put you at risk of
HIV infection please fill this exposure reporting form and call or get to the PEP focal person. In case you
start PEP drugs please report to your prescriber if you have any side effects in addition to your
recommended (scheduled) visits.
Exposed Person information
Name: . Age ------Sex: Male Female
Profession: MD HO Nurse Laboratory professional Other: Specify
Department: Emergency Regular OPD Inpatient OR Labor Ward Other: (Specify)
Date of exposure: // time of exposure
Where the exposure occurs: with in Hospital/ outside Hospital HC private health facility
other: Specify.
Type of exposure: Percutaneous Mucous membraneSkin..
Body part exposed: ..
Circumstance of injury: ..

Source patient information
HIV status of the source patient: Unknown Known HIV -ve Known HIV+ve
If source patient is HIV +: WHO stage.CD4 countViral load ARV status : On ARV
Not on ARV
If on ARV: RegimenDuration on ARV .

Risk assessment Result:
Exposure status
Source for HIV infection
The Source patient is
HIV Positive but is
asymptomatic and
has reasonably good
immune status
The Source patient is HIV
Positive and is symptomatic ,
may have AIDS or has other
evidence of advanced illness (
Low CD4 or High viral load )
The HIV status of the source
patients is unknown ( either the
patient has refused HIV testing
or died or discharged before
HIV testing ) or The source
patient is unknown ( e/g
Unlabeled blood sample in a
laboratory )
Is a minor mucocutanous exposure to
small volume of blood for short period (
Few Seconds to minutes ) ?
PEP may not be
needed
Consider Basic Regimen
(2 Drug Regimen)
PEP not recommended
Is a Major mucocutanous exposure to
large volume of blood for longer
duration ( Several minutes ) or
Mild Percutaneous exposure ( with Solid
needle or superficial scratch or injury)?
Recommend
Basic Regimen
(2 Drug Regimen)
Recommend Expanded
Regimen(3 Drug
Regimen)
Consider Basic Regimen
(2 Drug Regimen)
Sever Percutaneous exposure (Large
bore hollow needle , Deep puncture
,Visible blood on devise , Needle used in
patient artery/vein )
Recommend Expanded
Regimen
(3 Drug Regimen)


108

Is the exposed person willing to be tested for HIV: Yes No
If yes, test Result: HIV+ HIV
IS the staff eligible for PEP: Yes No
If eligible for PEP:
Other base line lab done and results: WBC.Hgb.ALT.
Regimen provided:
How long after exposure did the HCW start PEP medication: < 4 hours 4-24hours 24- 72
hours