Includes various description of diagnostic techniques used for Malaria diagnosis.

1. LABORATORY
DIAGNOSIS OF
MALARIA
20 February 2013
Shridhan Anand Patil

2. • Introduction
• Light microscopy
• Antigen detection - Rapid malaria diagnosis tests
(RDTs)
• Serology
• Other
• QBC - PCR
• Summary

3. 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

4. Light microscopy
• Gold standard
• 0.0001% parasitemia detection(5-10/microL)
• Species identification
• Response to therapy
• Thin and thick smears

5. 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

6. Recognizing Malaria Parasites
Inside a red blood cell
One or more red chromatin dots
Blue cytoplasm

7. RING TROPHOZOITE
SCHIZONT GAMETOCYTE
Blue
Cytoplasm
Red
Chromatin
Brown
Pigment
Recognizing Erythrocytic Stages:
Schematic Morphology

8. Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes

9. Plasmodium falciparum
Rings: double chromatin dots; accole forms;
multiple infections in same red cell
Gametocytes: mature (M)and
immature (I) forms (I is rarely
seen 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

10. Plasmodium vivax
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-ovalSchizonts: 12-24 merozoites
Rings
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)

11. Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
“malariae - like parasite in vivax - like erythrocyte”
Rings
Trophozoites: compact
Schizonts: 6-14 merozoites;
dark pigment; (“rosettes”)
Gametocytes: round-oval

12. 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

13. 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 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

14. Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes

15. 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

16. 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

17. 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.

18. 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 and
severe 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 conventional
microscopy. Clin Lab. Haem. 21, 235-245.

19. Estimating Parasite Density
Alternate 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

20. Malaria Antigen Detection
• Cannot detect mixed
infections
• Cross reactivity with
rheumatoid factor reportedly
corrected

21. Malaria Serology – antibody detection
• Not routinely used for diagnosis
• Transfusion blood screening
• Investigation of cryptic malaria
• Epidemiological purposes
• Blood stage antigen used

22. 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

23. Quantitative buffy coat

26. 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

27. 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