malaria diagnostics

1. Malaria Diagnostics
Dr. B. K. Iyer

2. Malaria Diagnosis
• Clinical Diagnosis
• Malaria Blood Smear
• Fluorescent microscopy
• Antigen Detection
• Serology
• Polymerase Chain Reaction

3. 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

4. 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/t l
• thick film: 5 -20 parasites/t l
• Requirements:
– equipment, training, reagents, supervision
• Simple, inexpensive yet labor-intensive
• Accuracy depends on laboratory technician skill

5. Interpreting Thick & Thin Films
• THICK FILM • THIN FILM
– lysed RBCs – fixed RBCs, single layer
– larger volume – smaller volume
– 0.25 μl blood/100 fields – 0.005 μl blood / 100 fields
– blood elements more – good species differentiation
concentrated – requires more time to read
– good screening test – low density infections can be
– positive or negative missed
– parasite density
– more difficult to diagnose
species

6. Malaria Blood Smear
• Prepare smears as soon as possible after
collecting venous 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

7. Collection of Blood Smears
1. 4.
The second or third Slide must always be
finger is usually grasped by its edges.
selected and cleaned.
2. 5.
Puncture at the side Touch the drop of
of the ball of the blood to the slide
finger. from below.
3.
Gently squeeze
toward the puncture
site.

8. Preparing thick and thin films
1. 4.
Touch one drop of Carry the drop of blood
blood to a clean to the first slide and hold
slide. at 45 degree angle.
2. 5.
Spread the first Pull the drop of blood
drop to make a 1 across the first slide in
cm circle. one motion.
3. 6.
Touch a fresh drop Wait for both to
of blood to the edge dry before fixing
of another slide. and staining.

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

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

11. Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes

12. Plasmodium falciparum
Infected erythrocytes: normal size
M I
Gametocytes: mature (M)and
immature (I) forms (I is rarely
Rings: double chromatin dots; appliqué forms;
seen in peripheral blood)
multiple infections in same red cell
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)

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

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

15. Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)
Trophozoite: Trophozoite: Schizont: Gametocyte:
compact typical 6-12 merozoites; round; coarse,
band form coarse, dark pigment dark pigment

16. Species Differentiation on Thin
Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape round round, oval, round
distorted fimbriated
Stippling infected RBC Mauer clefts Schuffner Schuffner none
spots spots
Trophozoite shape small ring, large ring, large ring, small ring,
appliqui amoeboid compact compact
Chromatin dot often double single large single
Mature schizont rare, 12-30 12-24 4-12 6-12
merozoites merozoites merozoites merzoites
Gametocyte crescent shape large, large, compact,
round round round

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

18. Species Differentiation on Thick Films

19. 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/µl (or count it)
parasites/µl = parasites counted X WBC count/µl
WBC counted

20. Calculating Parasite Density - 2
• Count the number of parasitized and
nonparasitized red blood cells (RBCs) in
the same fields on thin smear
• Count asexual stages separately from
gametocytes
• Count 500-2000 RBCs (fewer RBCs if
parasitemia is high)
% parasitemia = # parasitized RBCs X 100
total # of RBCs

21. Calculating Parasite Density
• Can interconvert results in % parasitized
RBCs and parasites /µl if you know the
RBC or WBC counts
• If unknown, can assume
4,000,000 RBCs /µl or 8000 WBCs /µl

22. Parasitemia and clinical correlates
Parasitemia Parasites / µl 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.

23. Parasitemia and clinical correlates
Parasitemia Parasites/µl 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 /µl and severe anaemia
(haemoglobin < 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.

24. 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

25. 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%

26. 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%

27. 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

28. 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

29. 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

30. 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

31. Detection of Plasmodium antigens:
pLDH (parasite lactate dehydrogenase)

32. 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)

33. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Test Use of monoclonal (Ab) Use of monoclonal and polyclonal Ab
principle
Detects a parasite enzyme, lactate
Detects a histidine rich dehydrogenase
protein of P.f.
pLDH is found in sexual and asexual
Water-soluble protein is forms
released from parasitized
RBCs Differentiation between malarial
species is based on antigenic
Not present in mature differences between pLDH isoforms
gametocytes

34. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Advantages Threshold for parasite Threshold for parasite detection ≥ 100
detection as low as 10 parasites/µl
parasites/µl (but
sensitivity drops at < Can detect all species which infect humans
100 parasites /µl)
Can differentiate between P.f. and non-
falciparum malaria
Does not cross react
with other species –
P.v., P.o., P.m. Does not cross react with human LDH
Positive only in viable parasites, potentially
useful for monitoring success of treatment

35. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Disadvan- Some tests only detect P.f. Cannot differentiate between
tages non-falciparum species
Cannot detect mixed infections Cannot detect mixed infections
Sensitivity and specificity Sensitivity and specificity
decreases < 100 parasites/µl decreases < 100 parasites/µl
Can remain positive up to 14
days post treatment, in spite of
asexual and sexual parasite
clearance, due to circulating
antigens

36. Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Sensitivity/ Sensitivity 92-100% Sensitivity P.f. 88-98%
Specificity* Specificity 85- 100% P.v. 89-94%
Specificity P.f. 93-99%
P.v. 99-100%
Commercial Approximately US$ 0.60 –1.00 Approximately US$ 2.50
cost/test**
Commercial 1) PATH falciparum Malaria 1) OptiMAL® - Flow, Inc.
products IC Strip test – Program for 2) Binax NOW ®ICT
Appropriate Technology in Malaria - Binax, Inc.
Health
2) MAKROmed™ * Compared to microscopy, results
from multiple studies
3) Orchid ® ** Varies by size of order and vendor

37. Polymerase Chain Reaction (PCR)
• Molecular technique to identify parasite
genetic material
• Uses whole blood collected in
anticoagulated tube (200 µl) or directly onto
filter paper (5 µl)
– 100% DNA is extracted
– 10% blood volume used in PCR reaction

38. Polymerase Chain Reaction (PCR)
• Threshold of detection at CDC
– 0.1 parasite/µl if whole blood in tube
– 2 parasites/µl if using filter paper
• Definitive species-specific diagnosis now possible
• Can identify mutations – try to correlate to drug
resistance
• Parasitemia not quantifiable
• May have use in epidemiologic studies
• Requires specialized equipment, reagents, and
training

39. Real-Time PCR
• New technique based on
fluorescence
• Promising because it has
potential to quantify
parasitemia, decreases
contamination, may detect
multiple wavelengths in
same tube identifying
multiple species in one
run, saves hands-on time
• Needs further research and
validation for malaria

40. Real-Time PCR

41. Preventing Transfusion-Transmitted
Malaria (TTM) - Detection of Parasites/Parasite Products
100 parasites/unit PCR (0.05 to 0.1 parasites/Pl)
(25 X 10-5// l)
Microscopy (5 parasites/Ml)
10 parasites/unit Antigen detection
(2.5 X 10-5/( l) (10 to 100 parasites/ l)
Detection of 10
parasites/unit
requires a
sensitivity:
-4,000 times
better than
PCR 10-5 10-3 10-1 10 103
-200,000 times
better than Parasite densities (parasites/Pl)
microscopy

42. Detecting Thresholds
Malaria Parasites/Products:
Sensitivity Thresholds
HRP-2 pLDH
Kenya (children) Hospital for Trop. Diseases - London
Parasites/µl n= Sensit.(%) Parasites/µl n= Sensit.(%)
1-10 9/23 39 <5 13/22 60
11-60 17/21 81 50-500 9/11 81
61-100 14/16 88 500-1500 17/18 94
101-500 57/57 100 >1500 36/36 100
501-1000 12/12 100 Ref: Piper et al. Am J Trop Med Hyg 60: 109-118 (1999)
Ref: Beadle et al. Lancet 343: 564-568 (1994)
PCR
Vietnam (Serial dilution)
Detection limits for:
- P. falciparum: 0.02-0.08 parasites/µl
-P. vivax: 0.8-2.6 parasites/µl
Ref: Vu thi Ty Hang et al. Trans R Soc Trop Med Hyg 89: 44-47 (1995)

43. Mass Screening for Malaria
in Populations for Resettlement
• Blood smear examinations to detect
asymptomatic parasitemia
• Not useful for predicting individual risks
• undetectable parasitemias
• dormant liver phase parasites
• Can be used to make a decision about the
need for mass treatment of the entire group

44. Issues in application of diagnostics
• Roll Back Malaria objective – At least 60% of those
suffering from malaria have prompt access to and are
able to use correct, affordable and appropriate
treatment within 24 hours of the onset of symptoms
• Cost should not focus on unit cost alone
• Must put in context of case management
– Amount of drugs being inappropriately dispensed
– Increasing drug resistance
– Increasingly costly, complex, and toxic alternative drugs
– Epidemiology of malaria, populations served
– Provider and patient acceptability, esp. of negative results

45. Issues in application of diagnostics
• Rapid diagnostic tests have potential to complement
conventional microscopy or provide a diagnostic
modality when none is available
• Operational research is needed to evaluate best uses
and cost effectiveness
• Potential uses
– Epidemics and emergencies
– Inadequate or absent lab services, unskilled staff
– Mobile clinics
– Low transmission areas; areas with high levels of drug
resistance
– Epidemiologic surveys, seroprevalence data