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Malaria Diagnostics Gail Stennies, M.D., M.P.H. Medical Officer Malaria Epidemiology Branch DPD/ 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 1. The second or third finger is usually selected and cleaned.

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

3. Gently squeeze toward the puncture site.

4. Slide must always be grasped by its edges.

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


Preparing thick and thin films 1.

4.

Touch one drop of blood to a clean slide.

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

2.

5.

Spread the first drop to make a 1 cm circle.

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

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

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


Recognizing Malaria Parasites Inside a red blood cell

One or more red chromatin dots

Blue cytoplasm


Recognizing Erythrocytic Stages: Schematic Morphology Blue Cytoplasm RING

Red Chromatin

TROPHOZOITE

Brown Pigment SCHIZONT

GAMETOCYTE


Malaria Parasite Erythrocytic Stages

Ring form

Schizont

Trophozoite

Gametocytes


Plasmodium falciparum Infected erythrocytes: normal size

M

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

Trophozoites: compact (rarely seen in peripheral blood)

I

Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Schizonts: 8-24 merozoites (rarely seen in peripheral blood)


Plasmodium vivax Infected erythrocytes: enlarged up to 2X; deformed; (Schßffner’s dots)

Rings Schizonts: 12-24 merozoites

Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-oval


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; dark pigment; (“rosettes”)

Gametocytes: round-oval


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

Trophozoite: compact

Trophozoite: typical band form

Schizont: 6-12 merozoites; coarse, dark pigment

Gametocyte: round; coarse, dark pigment


Species Differentiation on Thin Films Feature

P. falciparum

P. vivax

P. ovale

+

+

round

round, distorted

oval, fimbriated

round

Mauer clefts

Schuffner spots

Schuffner spots

none

Enlarged infected RBC Infected RBC shape Stippling infected RBC Trophozoite shape Chromatin dot Mature schizont Gametocyte

small ring, appliqu often double rare, 12-30 merozoites crescent shape

large ring, large ring, amoeboid compact single

large

12-24 4-12 merozoites merozoites large, round

large, round

P. malariae

small ring, compact single 6-12 merzoites compact, round


Species Differentiation on Thin Films P. falciparum Rings Trophozoites

Schizonts Gametocytes

P. vivax

P. ovale

P. malariae


Species Differentiation on Thick Films Feature Uniform trophozoites

P. falciparum

P. vivax

P. ovale

++

+

P. malariae

+

Fragmented trophozoites Compact trophozoites

+

Pigmented trophozoites

+ +

Irregular cytoplasm

+

+

Stippling (“RBC ghosts�)

+

+

very rarely

often

often

often

occasionally

usually

usually

usually

Schizonts visible Gametocytes visible


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


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


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


Parasitemia and clinical correlates Parasitemia

Parasites /Âľl 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/Âľl Remarks

2-5%

100,000250,00

Hyperparasitemia/severe malaria*, increased mortality

10%

500,000

Exchange transfusion may be considered/ high mortality

*WHO criteria for severe malaria are parasitemia > 10,000 /¾l 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.


Estimating Parasite Density Alternate Method â&#x20AC;˘ 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 - RDTs Feature

PfHRP-2 tests

pLDH tests

Test principle

Use of monoclonal (Ab)

Use of monoclonal and polyclonal Ab

Detects a histidine rich protein of P.f.

Detects a parasite enzyme, lactate dehydrogenase

pLDH is found in sexual and Water-soluble protein is released from parasitized RBCs asexual forms Not present in mature gametocytes

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


Malaria Antigen Detection - RDTs Feature

PfHRP-2 tests

pLDH tests

Advantages

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

Threshold for parasite detection ≥ 100 parasites/µl Can detect all species which infect humans

Does not cross react with other Can differentiate between P.f. species – P.v., P.o., P.m. 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 - RDTs Feature

PfHRP-2 tests

pLDH tests

Disadvantages

Some tests only detect P.f.

Cannot differentiate between non-falciparum species

Cannot detect mixed infections Cannot detect mixed infections Sensitivity and specificity decreases < 100 parasites/Âľl Can remain positive up to 14 days post treatment, in spite of asexual and sexual parasite clearance, due to circulating antigens

Sensitivity and specificity decreases < 100 parasites/Âľl


Malaria Antigen Detection - RDTs Feature

PfHRP-2 tests

pLDH tests

Sensitivity/ 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%

Commercial Approximately US$ 0.60 –1.00 cost/test** Commercial 1) products 2) 3)

Approximately US$ 2.50

PATH falciparum Malaria 1) IC Strip test – Program for 2) Appropriate Technology in Health MAKROmed™ Orchid ®

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

OptiMAL® - Flow, Inc. Binax NOW ®ICT Malaria - Binax, Inc.


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 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


Real-Time PCR


Quantitative Real-Time PCR


Detecting Malaria Parasites/Products: Sensitivity Thresholds HRP-2

pLDH

Kenya (children) Parasites/µl 1-10 11-60 61-100 101-500 501-1000

n= 9/23 17/21 14/16 57/57 12/12

Hospital for Trop. Diseases - London Sensit.(%) 39 81 88 100 100

Parasites/µl <5 50-500 500-1500 >1500

n= 13/22 9/11 17/18 36/36

Sensit.(%) 60 81 94 100

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)


Preventing Transfusion-Transmitted Malaria (TTM) Detection of Parasites/Parasite Products PCR (0.05 to 0.1 parasites/ l) Microscopy (5 parasites/l) Antigen detection (10 to 100 parasites/ l)

10-5

10-3

10-1

10

103

Parasite densities (parasites/l)


Preventing TTM: Detection of Parasites/Parasite Products 100 parasites/unit (25 X 10-5/l)

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)

10-5

10-3

10-1

10

103

Parasite densities (parasites/l) Detection of 10 parasites/unit requires a sensitivity: -4,000 times better than PCR -200,000 times better than microscopy


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


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

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