Diagnosis Of Malaria - Infectious Diseases in Children

4th Pediatric Infectious Diseases Conference

ISSN 0973 - 0958

Diagnosis Of Malaria

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DIAGNOSIS OF MALARIA

Dr Ira Shah, Dr Vishal Dublish

Diagnosis of malaria involves identification of malarial parasite or its antigen or product in the blood of the patient. Following factors can have bearing on identification and interpretation of malarial parasite on a diagnostic test:

Endemicity of different species
Population movement
Different forms of malarial species
Different stages of erythrocytic schizogony
Immunity, recurrent malaria, drug resistance
Persisting viable or nonviable parasitemia
Sequestration of parasites in the deeper tissues
Treatment taken etc.

2 types of tests can be employed for diagnosis of malaria:

Microscopic tests
Non-microscopic tests

Microscopic tests: Careful examination of a well prepared and well stained blood film provides direct visualization of the parasite on thick and thin smear and is the gold standard.

Peripheral smear for MP (PS for MP): Blood smear is stained with Romanowsky stain / Giemsa / Leishman's stain and RBC examined for intracellular malarial parasite.
- Thick smear can identify up to 5 parasites / microlitre
- Thin smear can identify species and up to 200 parasites / microlitre.
In bone marrow aspirate, malarial pigment can be detected in neutrophils or monocytes.
Quantitative buffy coat test (QBC): It is developed by Becton and Dickenson Inc. Centrifuged and compressed red cells are stained with acridine orange dye and examined under ultraviolet light. Malarial parasite DNA takes up acridine orange stain and appears as bright specks of light among the non-fluorescent red cells. This test is fast and easy and considered more sensitive than thick films. Drawbacks include cost, artifacts being diagnosed as malarial parasite. Filaria worms can also be detected in QBC test.

Non-microscopic tests: Employs identification of parasite antigen, antiplasmodial antibodies or parasite metabolic products.

Rapid Diagnostic Tests (RDTs): These tests capture parasitic antigens from peripheral blood using either monoclonal or polyclonal antibodies against the malarial parasite antigen targets, e.g. histidine rich protein (hrp-2), aldolase or malaria parasite specific lactate dehydrogenase (LDH).
- Immunochromatographic test: If the target antigen is present in the blood, a labeled antigen antibody complex is formed which migrates up the test strip to be captured by the predeposited capture antibodies specific against the antigens and the labeled antibody.
- Parasight-F test: HRP-2 antigen is unique to plasmodium falciparum localized in several cell compartments of parasite, infected RBC cytoplasm and membrane.
  - Sensitivity is > 98.5% and specificity is 98.6%
  - Can detect 10-100 parasites / ml
  - Detects both viable and non-viable malarial parasite
  - Does not detect plasmodium vivax / ovale / malariae.
- OptiMAL (pLDH): LDH is an intracellular metabolic enzyme produced by each species as distinct isoforms, accumulates within cytoplasm of the host RBC and secreted by the RBC membrane.
- For P. vivax, sensitivity - 94%, specificity - 100%
- For P. Falciparum, sensitivity - 88%, specificity - 99%
- Can detect 100-200 parasite / ml.
- Only viable parasite are detected
- Tests become negative after 5 days of treatment
- Does not identify plasmodium ovale or malaria.
Limitations of RDTs :
- Parasight F can detect only P. falciparum and may miss more common non-falciparum malaria.
- Cross reactions of Parasight F with non-falciparum malaria
- False positive results: Gametocytemia, persistent viable asexual stage, parasitemia below the detection level of microscopy, antigen persistence because of sequestration and incomplete treatment, delayed clearance of circulating antigen, cross reactions with non-falciparum malaria or autoantibodies, rheumatoid factor (RF).
- False negative results: Some times in cases of severe malaria with > 40,000 parasites per microlitre because of prozone phenomenon.
- Sensitivity of test decreases with low density of parasites.
- These tests are unable to differentiate between sexual and asexual parasitemia.
- Persistence of antigens: All the antigen targeted by RDTs are expressed by the asexual and sexual forms of the malarial parasite. If schizonticidal drugs are used which have no effect on gametocytes of plasmodium falciparum then RDTs may not be so reliable to predict treatment response even after one month.
- Experience and level of training of staff can influence sensitivity and specificity of tests.
- High cost
- Following factors may influence RDT results:
  - Parasite type
  - Level of parasitemia
  - Type of test
  - Target antigen, capture antibodies.
  - Presence of gametocytemia.
  - Persistent antigenemia
  - Sequestration of parasites
  - Cross reaction with non-falciparum species and autoantibodies.
  - Prozone phenomenon
  - Previous treatment
Polymerase chain reaction (PCR): Highly sensitive and specific for diagnosis of all four species of malaria especially in low levels of parasitemia and mixed infections. It is considered ten times more sensitive than microscopy.
Detection of antimalarial antibodies: Antibodies to asexual blood stages appear within few days after malaria infection. Titres increase within few weeks and persist for months to years in semi-immune patients, residing in endemic areas where reinfection is frequent. In non-immune persons, antibodies subside rapidly within 3-6 months. Antibodies can be detected by immunofluorescence or enzyme immunoassays. Utility of antibodies detection is limited for epidemiological surveys, screening blood donors or occasionally as evidence of recent infection in non-immune individuals. In future it can be used for diagnosis of malarial protection antibodies in assessing response to malaria vaccine.
Intraleucocytic malarial pigment: One study from Nigeria showed that pigment containing neutrophil count is a simple marker of disease severity in childhood malaria in addition to the parasitic count.
Flow cytometry: Blood with malarial parasite can be run on automated analyzer and malaria can be suspected on scatter plots because in malaria there is abnormal cell clusters.
Mass spectrometry: Novel method and can detect as low as 10 parasites per microlitre.

But simplest and surest diagnosis of malaria is by peripheral smear. None of the other newer tests have surpassed the gold standard peripheral smear studies.

Points to remember:

Whenever high level of suspicion - always ask for PS for MP.
Can be done at any time. Do not wait for typical signs and symptoms or chills etc.
Negative test does not rule out malaria whenever in doubt - repeat PS for MP.
Widal may be positive even up to a dilution of 1:320 for 'O' and 'H' and low titres of AH and BH. So a positive widal in a patient with confirmed malaria should not be considered as suffering from typhoid fever.

Last updated on 01-05-2006 Vol 3 Issue 5 Art # 16

How to cite this url

Shah I,Dublish V.Diagnosis Of Malaria. Pediatric Oncall [serial online] 2006 [cited 2006 May 1];3. Art # 16. Available from:

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