Malaria Diagnostics

Gail Stennies, M.D., M.P.H.Medical Officer

Malaria Epidemiology BranchDPD/ NCID/ CDC

May, 2002

Malaria Diagnosis

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

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

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/l• thick film: 5 -20 parasites/l

• Requirements: equipment, training, reagents, supervision• Simple, inexpensive yet labor-intensive• Accuracy depends on laboratorian skill

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 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

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

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

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

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 total # of RBCs

X 100

Calculating Parasite Density

• Can interconvert results in % parasitized RBCs and parasites /l if you know the RBC or WBC counts

• If unknown, can assume 4,000,000 RBCs /l or 8000 WBCs /l

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 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

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

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

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)

Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsTest principle

Use of monoclonal (Ab)

Detects a histidine rich protein of P.f.

Water-soluble protein is released from parasitized RBCs

Not present in mature gametocytes

Use of monoclonal and polyclonal Ab

Detects a parasite enzyme, lactate dehydrogenase

pLDH is found in sexual and asexual forms

Differentiation between malarial species is based on antigenic differences between pLDH isoforms

Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsAdvantages Threshold for parasite

detection as low as 10 parasites/µl (but sensitivity drops at < 100 parasites /µl)

Does not cross react with other species – P.v., P.o., P.m.

Threshold for parasite detection ≥ 100 parasites/µl

Can detect all species which infect humans

Can differentiate between P.f. and non-falciparum malaria

Does not cross react with human LDH

Positive only in viable parasites, potentially useful for monitoring success of treatment

Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsDisadvan-tages

Some tests only detect P.f.

Cannot detect mixed infections

Sensitivity and specificity 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

Cannot differentiate between non-falciparum species

Cannot detect mixed infections

Sensitivity and specificity decreases < 100 parasites/µl

Malaria Antigen Detection - RDTsFeature PfHRP-2 tests pLDH testsSensitivity/Specificity*

Sensitivity 92-100%Specificity 85- 100%

Sensitivity P.f. 88-98% P.v. 89-94%Specificity P.f. 93-99% P.v. 99-100%

Commercialcost/test**

Approximately US$ 0.60 –1.00 Approximately US$ 2.50

Commercial products

1) PATH falciparum Malaria IC Strip test – Program for Appropriate Technology in Health

2) MAKROmed™3) Orchid ®

1) OptiMAL® - Flow, Inc.2) Binax NOW ®ICT

Malaria - Binax, Inc.

* Compared to microscopy, results from multiple studies** Varies by size of order and vendor

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

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

Real-Time PCRNew 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

Real-Time PCR

Quantitative Real-Time PCR

D e te c tin g M a la r ia P a ra s ite s /P ro d u c ts : S e n s itiv ity T h re s h o ld s

P C RV ie tna m (S e ria l d ilu tio n )D e te c tio n lim its fo r:

- P . fa lc ip a ru m : 0 .0 2 -0 .0 8 p a ra s ite s /µ l-P . v iva x : 0 .8 -2 .6 p a ra s ite s /µ l

R ef: V u th i T y H a n g e t a l. T ra n s R S o c T ro p M e d H yg 8 9 : 4 4 -47 (1 9 9 5 )

H R P -2K e n ya (ch ild re n )P a ra s ite s /µ l n = S e n s it.(% )1 -1 0 9 /2 3 3 91 1 -6 0 1 7 /2 1 8 16 1 -1 0 0 1 4 /1 6 8 81 0 1 -5 0 0 5 7 /5 7 1 0 05 0 1 -1 0 0 0 1 2 /1 2 1 0 0R ef: B ea d le e t a l. L a n c e t 3 43 : 56 4 -5 68 (19 9 4 )

p L D HH o sp ita l fo r T ro p . D ise a se s - L o n d o nP a ra s ite s /µ l n = S e n s it.(% )< 5 1 3 /2 2 6 05 0 -5 0 0 9 /1 1 8 15 0 0 -1 5 0 0 1 7 /1 8 9 4> 1 5 0 0 3 6 /3 6 1 0 0R ef: P ip er e t a l. A m J T ro p M e d H yg 6 0 : 10 9 -1 18 (1 9 9 9 )

Preventing Transfusion-Transmitted Malaria (TTM)Detection of Parasites/Parasite Products

Parasite densities (parasites/l)1031010-110-310-5

PCR (0.05 to 0.1 parasites/l)Microscopy (5 parasites/l)

Antigen detection(10 to 100 parasites/ l)

Preventing TTM:Detection of Parasites/Parasite Products

Parasite densities (parasites/l)1031010-110-310-5

PCR (0.05 to 0.1 parasites/l)Microscopy (5 parasites/l)

Antigen detection(10 to 100 parasites/ l)

10 parasites/unit(2.5 X 10-5/l)

100 parasites/unit(25 X 10-5/l)

Detection of 10 parasites/unit requires a sensitivity: -4,000 times better than PCR -200,000 times better than microscopy

Mass Screening for Malariain 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

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

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