Malaria Diagnostics

Malaria Diagnosis

• Clinical Diagnosis

• Malaria Blood Smear

• Fluorescent microscopy

• Quantitative Buffy coat

• Antigen Detection

• Serology

• Other tests

Clinical Diagnosis

• Hyperendemic and holoendemic areas

• Laboratory resources not needed

• Fever or history of fever

• Sensitivity ranges from poor to high

• Often has poor specificity and predictive values

• Overlap with other syndromes

• Clinical case description:

Fever case with any of the following:

Chills, Sweating, Jaundice, Splenomegaly

Convulsions, Coma, Shock, Pulmonary edema & Death (in severe cases)

• Case classification:

SUSPECT : Any case of fever

PROBABLE : Case that meets the clinical

case definition

CONFIRMED: A suspected/probable case that is laboratory confirmed

Malaria Blood Smear• Remains the gold standard for diagnosis

• Giemsa stain• distinguishes between species and life cycle stages• parasitemia is quantifiable

• Threshold of detection• thin film: 100 parasites/Field• thick film: 5 -20 parasites/Field

• Requirements: equipment, training, reagents, supervision

• Simple, inexpensive yet labor-intensive• Accuracy depends on laboratorian skill

• Jaswant Singh Battacharya (JSB) Stain for Jaswant Singh Battacharya (JSB) Stain for thick and thin films:thick and thin films: This is the standard method used by the laboratories under the National Malaria Eradication Programme in India. 

• Preparation of the stainPreparation of the stain::• JSB I stain:JSB I stain: Medicinal methylene blue (0.5 g) is dissolved in 500

ml of distilled water and 3 ml of 1% sulphuric acid (H2SO4) is gradually added, followed by 0.5 g of potassium dichromate (K2Cr2O7) when a purple precipitate forms. 3.5 g of disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) is next added and when the precipitate has dissolved, the solution is boiled in a flask with a reflex condenser for 1 hour. The stain is ready for immediate use.

• JSB II stain:JSB II stain: 1 g Eosin is dissolved in 500 ml tap water. • Buffered water:Buffered water: 0.22 g of disodium hydrogen phosphate

dihydrate (Na2HPO4.2H2O) and 0.74 g of potassium acid phosphate (KH2PO4) are added to 1000 ml of distilled water or filtered tap water.

• Staining:Staining:• After dehemoglobinisation, dip the thick smear in JSB II stain

two to three times. Wash it by dipping in buffer water two to three times. Then keep the thick film dipped in JSB I stain for 40-60 seconds. Wash it with buffer water. Drain, dry and examine.

Interpreting Thick and Thin Films

• THICK FILM– lysed RBCs– larger volume– 0.25 μl blood/100 fields– blood elements more

concentrated– good screening test– positive or negative– parasite density– more difficult to diagnose

species

• THIN FILM– fixed RBCs, single layer

– smaller volume

– 0.005 μl blood/100 fields

– good species differentiation

– requires more time to read

– low density infections can be missed

Malaria Blood Smear

• Prepare smears as soon as possible after collecting capillary blood to avoid

• Changes in parasite morphology• Staining characteristics

• Take care to avoid fixing the thick smear • Risk of fixing thick when thin is fixed with

methanol if both smears on same slide• Let alcohol on finger dry to avoid fixing thick• Be careful if drying with heat

Collection of Blood Smears

5.Touch the drop of blood to the slide from below.

4.Slide must always be grasped by its edges.

2.Puncture at the side of the ball of the finger.

3.Gently squeeze toward the puncture site.

1.The second or third finger is usually selected and cleaned.

Preparing thick and thin films

1.Touch one drop of blood to a clean slide.

2.Spread the first drop to make a 1 cm circle.

3.Touch a fresh drop of blood to the edge of another slide.

6.Wait for both to dry before fixing and staining.

5.Pull the drop of blood across the first slide in one motion.

4.Carry the drop of blood to the first slide and hold at 45degree angle.

Recognizing Malaria Parasites

Inside a red blood cell

One or more red chromatin dots

Blue cytoplasm

RING TROPHOZOITE

SCHIZONT GAMETOCYTE

BlueCytoplasm

RedChromatin

BrownPigment

Recognizing Erythrocytic Stages:Schematic Morphology

Malaria Parasite Erythrocytic Stages

Ring form

TrophozoiteSchizont

Gametocytes

• Diagnostic Points for Plasmodium falciparum• Red Cells are not enlarged. • Rings appear fine and delicate and there may be

several in one cell. • Some rings may have two chromatin dots. • Presence of marginal or applique forms. • It is unusual to see developing forms in peripheral

blood films. • Gametocytes have a characteristic crescent shape

appearance. However, they do not usually appear in the blood for the first four weeks of infection.

• Maurer's dots may be present.

Plasmodium falciparum

Rings: double chromatin dots; appliqué forms;multiple infections in same red cell

Gametocytes: mature (M)andimmature (I) forms (I is rarelyseen in peripheral blood)

Trophozoites: compact(rarely seen in

peripheral blood)

Schizonts: 8-24 merozoites(rarely seen in peripheral blood)

Infected erythrocytes: normal size

M I

• Diagnostic Points for P. vivax• Red cells containing parasites are usually enlarged. • Schuffner's dots are frequently present in the red cells as

shown above. • The mature ring forms tend to be large and coarse. • Developing forms are frequently present.

• Diagnostic Points for P. malariae• Ring forms may have a squarish appearance. • Band forms are a characteristic of this species. • Mature schizonts may have a typical daisy head appearance

with up to ten merozoites. • Red cells are not enlarged. • Chromatin dot may be on the inner surface of the ring.

Plasmodium vivax

Trophozoites: ameboid; deforms the erythrocyte

Gametocytes: round-oval Schizonts: 12-24 merozoites

Rings

Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)

• Diagnostic Points for P. ovale• Red cells enlarged. • Comet forms common .• Rings large and coarse. • Schuffner's dots, when present, may be prominent. • Mature schizonts similar to those of P. malariae but

larger and more coarse.

• Quantifying parasites: % parasitemia = (parasitized RBCs/total RBCs) × 100

Feature P. falciparum P. vivax P. ovale P. malariae

Enlarged infected RBC + +

Infected RBC shape round round, distorted

oval, fimbriated

round

Stippling infected RBC Mauer clefts Schuffner spots

Schuffner 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

Species Differentiation on Thin Films

P. falciparum P. vivax P. ovale P. malariae

Rings

Trophozoites

Schizonts

Gametocytes

Parasitemia and clinical correlates

Parasitemia Parasites /l Remarks

0.0001-0.0004% 5-20 Sensitivity of thick blood film

0.002% 100 Patients may have symptoms below this level, where malaria is seasonal

0.2% 10,000 Level above which immunes show symptoms

2% 100,000 Maximum parasitemia of P.v. and P.o.

Parasitemia and clinical correlates

Parasitemia Parasites/l Remarks

2-5% 100,000-250,00

Hyperparasitemia/severe malaria*, increased mortality

10% 500,000 Exchange transfusion may be considered/ high mortality

*WHO criteria for severe malaria are parasitemia > 10,000 /l andsevere anaemia (haemaglobin < 5 g/l).Prognosis is poor if > 20% parasites are pigment containing trophozoites and schizonts (more mature forms) and/or if > 5% of neutrophils contain visible pigment.Hänscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventionalmicroscopy. Clin Lab. Haem. 21, 235-245.

Estimating Parasite DensityAlternate Method

• Count the number of asexual parasites per high-power field (HPF) on a thick blood film

+ 1-10 parasites per 100 HPF++ 11-100 parasites per 100 HPF+++ 1-10 parasites per each HPF++++ > 10 parasites per each HPF

Fluorescent Microscopy• Modification of light microscopy • Fluorescent dyes detect RNA and DNA that is

contained in parasites• Nucleic material not normally in mature RBCs• Kawamoto technique

– Stain thin film with acridine orange (AO)– Requires special equipment – fluorescent microscope– Staining itself is cheap– Sensitivities around 90%

Quantitative Buffy Coat (QBC ®)

• Fluorescent microscopy after centrifugation

• AO-coated capillary is filled with 50-100 µl blood

• Parasites concentrate below the granulocyte layer in tube

• May be slightly more sensitive than light microscopy but some reports of 55-84%

Quantitative Buffy Coat (QBC ®)

• Useful for screening large numbers of samples

• Quick, saves time• Requires centrifuge, special stains• 3 main disadvantages

– Species identification and quantification difficult

– High cost of capillaries and equipment– Can’t store capillaries for later reference

The QBC TestThe QBC Test

                                          

                                 

                                           

                                        

                                           

                                

                                          

                                 

                                           

                                        

                                           

                                

Malaria Serology – antibody detection

• Immunologic assays to detect host response

• Antibodies to asexual parasites appear some days after invasion of RBCs and may persist for months

• Positive test indicates past infection

• Not useful for treatment decisions

Malaria Serology – antibody detection

• Valuable epidemiologic tool in some settings• Useful for

– Identifying infective donor in transfusion-transmitted malaria

– Investigating congenital malaria, esp. if mom’s smear is negative

– Diagnosing, or ruling out, tropical splenomegaly syndrome

– Retrospective confirmation of empirically-treated non-immunes

Malaria Antigen Detection• Immunologic assays to detect specific antigens• Commercial kits now available as immunochromatographic

rapid diagnostic tests (RDTs), used with blood• P. falciparum histidine-rich protein 2 (PfHRP-2)• parasite LDH (pLDH)

• Monoclonal and polyclonal antibodies used in antigen (Ag) capture test

• Species- and pan-specific Ab• Cannot detect mixed infections• Cross reactivity with rheumatoid factor reportedly

corrected

RDT Test FormatRDT Test Format

Examples of RDTs:

                                     

                                      

                 

                                          

Para Sight F testOptiMal

Assay KitOptiMal assay

Result

Detection of Plasmodium antigens:pLDH (parasite lactate dehydrogenase)

Detection of Plasmodium antigens

A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow)C: HRP-2 (histidine-rich protein 2) (PATH)

OTHER TESTS

• Polymerase chain reaction (PCR)

• Detection of Anti-malarial antibodies

• Intraleucocytic malaria pigment

• Flowcytometry

• Mass spectrometry

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