LABORATORY DIAGNOSIS OF MALARIA20 February 2013Shridhan Anand Patil

• Introduction

• Light microscopy

• Antigen detection - Rapid malaria diagnosis tests

(RDTs)

• Serology

• Other

• QBC - PCR

• Summary

Introduction• Accurate diagnosis of malaria

• Chloroquine-resistant malaria

• Ideal test

• Rapid - Easy to use - Reproducible

• Minimum equipment

• Detection of all species

• Quantification of infection

• Response to therapy

• Revert on treatment - Clinical diagnosis

Light microscopy

• Gold standard

• 0.0001% parasitemia detection(5-10/microL)

• Species identification

• Response to therapy

• Thin and thick smears

Interpreting Thick and Thin Films• THICK FILM

• lysed RBCs

• larger volume

• 0.25 μl blood/100 fields

• 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

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

Plasmodium falciparum

Rings: double chromatin dots; accole 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

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)

Plasmodium ovaleInfected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)

“malariae - like parasite in vivax - like erythrocyte”

RingsTrophozoites: compact

Schizonts: 6-14 merozoites; dark pigment; (“rosettes”)

Gametocytes: round-oval

Infected erythrocytes: size normal to decreased (3/4X)

Plasmodium malariae

Trophozoite:compact

Trophozoite:typical band form

Schizont:6-12 merozoites;coarse, dark pigment

Gametocyte:round; coarse,dark pigment

Species Differentiation on Thin FilmsFeature 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

Species Differentiation on Thick FilmsFeature 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

Calculating Parasite Density - 1

• Using 100X oil immersion lens, select area with 10-

20 WBCs/field

• Count the number of asexual parasites and white

blood cells in the same fields on thick smear

• Count ≥ 200 WBCs

• Assume WBC is 8000/l (or count it)

parasites/l = parasites counted WBC counted X WBC count/l

Parasitemia and clinical correlatesParasitemia 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 correlatesParasitemia 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

Malaria Antigen Detection• Cannot detect mixed

infections• Cross reactivity with

rheumatoid factor reportedly corrected

Malaria Serology – antibody detection

• Not routinely used for diagnosis

• Transfusion blood screening

• Investigation of cryptic malaria

• Epidemiological purposes

• Blood stage antigen used

Polymerase Chain Reaction

• Specific amplification of malaria DNA

• Excellent sensitivity and specificity

• Detects as low as ≥ 1 parasite/µL of blood

• Useful in identifying drug resistence

• Low parasitemia, therapeutic response

• Species identification – P. knowlesi

Quantitative buffy coat

Summary

• Conventional peripheral smear examination is the gold

standard

• RDTs are costly andrequire quality control

• Serological tests are suitable for epidemiological purpose

• Molecular-biological techniques are suitable for research

labs, quantification, drug resistence detection and species

detection

References• UpToDate – Malaria diagnosis• Lab diagnosis of Malaria. J Clin Pathol 1996;49:533-538• Malaria Diagnosis: A Brief Review. Korean J Parasitol.

Vol. 47, No. 2: 93-102, June 2009 DOI: 10.3347/kjp.2009.47.2.93

• Rapid Diagnostic Tests for Malaria Parasites. CLINICAL MICROBIOLOGY REVIEWS,66–78.2002. Jan. 2002, p. 66–78