Malaria Diagnostics

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Malaria Diagnostics

• Gail Stennies, M.D., M.P.H.
• Medical Officer
• Malaria Epidemiology Branch
• DPD/ NCID/ CDC
• May, 2002

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Malaria Diagnosis

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

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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

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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

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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

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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

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Collection of Blood Smears
1. The second or third finger is usually selected
and cleaned.
4. Slide must always be grasped by its edges.
5. Touch the drop of blood to the slide from
below.
2. Puncture at the side of the ball of the finger.
3. Gently squeeze toward the puncture site.
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Preparing thick and thin films
4. Carry the drop of blood to the first slide and
hold at 45?degree angle.
1. Touch one drop of blood to a clean slide.
5. Pull the drop of blood across the first slide
in one motion.
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.
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Recognizing Malaria Parasites
Blue cytoplasm
Inside a red blood cell
One or more red chromatin dots
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Recognizing Erythrocytic StagesSchematic
Morphology
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Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes
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Plasmodium falciparum
Infected erythrocytes normal size
M
I
Gametocytes mature (M)and immature (I) forms (I
is rarely seen in peripheral blood)
Rings double chromatin dots appliqué
forms multiple infections in same red cell
Schizonts 8-24 merozoites (rarely seen in
peripheral blood)
Trophozoites compact (rarely seen in peripheral
blood)
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Plasmodium vivax
Infected erythrocytes enlarged up to 2X
deformed (Schüffners dots)
Rings
Trophozoites ameboid deforms the erythrocyte
Gametocytes round-oval
Schizonts 12-24 merozoites
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Plasmodium ovale
Infected erythrocytes moderately enlarged (11/4
X) fimbriated oval (Schüffners
dots) malariae - like parasite in vivax - like
erythrocyte
Trophozoites compact
Rings
Gametocytes round-oval
Schizonts 6-14 merozoites dark pigment
(rosettes)
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Plasmodium malariae
Infected erythrocytes size normal to decreased
(3/4X)
Trophozoite typical band form
Trophozoite compact
Schizont 6-12 merozoites coarse, dark pigment
Gametocyte round coarse, dark pigment
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Species Differentiation on Thin Films
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Species Differentiation on Thin Films
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Species Differentiation on Thick Films
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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 counted WBC counted
parasites/?l
X WBC count/?l
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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)

parasitized RBCs total of RBCs
parasitemia
X 100
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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

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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.
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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
  gt 10,000 /?l and
• severe anaemia (haemaglobin lt 5 g/l).
• Prognosis is poor if gt 20 parasites are pigment
  containing
• trophozoites and schizonts (more mature forms)
  and/or if gt 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.

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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 gt 10 parasites per each HPF
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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

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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

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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
• Cant store capillaries for later reference

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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

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Malaria Serology antibody detection

• Valuable epidemiologic tool in some settings
• Useful for
• Identifying infective donor in transfusion-transmi
  tted malaria
• Investigating congenital malaria, esp. if moms
  smear is negative
• Diagnosing, or ruling out, tropical splenomegaly
  syndrome
• Retrospective confirmation of empirically-treated
  non-immunes

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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

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Detection of Plasmodium antigens pLDH (parasite
lactate dehydrogenase)
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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)
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Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Test 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
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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 lt 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
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Malaria Antigen Detection - RDTs
Feature PfHRP-2 tests pLDH tests
Disadvan-tages Some tests only detect P.f. Cannot detect mixed infections Sensitivity and specificity decreases lt 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 lt 100 parasites/µl
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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
Commercialcost/test Approximately US 0.60 1.00 Approximately US 2.50
Commercial products PATH falciparum Malaria IC Strip test Program for Appropriate Technology in Health MAKROmed Orchid OptiMAL - Flow, Inc. Binax NOW ICT Malaria - Binax, Inc.
Compared to microscopy, results from multiple
studies Varies by size of order and vendor
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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

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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

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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
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Real-Time PCR
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Quantitative Real-Time PCR
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(No Transcript)
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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
10-1
10-3
10-5
103
Parasite densities (parasites/?l)
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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)
10 parasites/unit (2.5 X 10-5/?l)
Antigen detection (10 to 100 parasites/ ?l)
10
10-1
10-3
10-5
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
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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

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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

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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