NEWER DIAGNOSTIC METHODS OF MALARIA
Dr. S.A. Krishna*, Dr. A.K. Jaiswal**, Dr. Anil Kumar***
HOD, Dept of Pediatrics, NMCH, Patna.*, Asso. Professor , Dept of Pediatrics, NMCH, Patna.**, Senior Resident Dept of Pediatrics, NMCH, Patna.***
Definition
Malaria is an acute and chronic illness characterized by paroxysm of fever, chills, sweating, fatigue, anemia and splenomegaly.
Etiology
Malaria is caused by intracellular Plasmodium protozoa, transmitted to human by female Anopheles mosquitoes. Four species of Plasmodium cause malaria in human beings :
  1. P. Falciparum
  2. P.Malariae
  3. P. Ovale
  4. P. Vivax

Malaria also can be transmitted by blood transfusion, use of contaminated needles, by vertical transmission from a pregnant women to her fetus.
EPIDEMIOLOGY
WORLD:
    At present about hundred countries in the world are considered malarious,almost half of which is Sub-Sahara Africa.

INDIA:
    During 2003, about 1.65 million cases were reported with 943 deaths. Malaria has been a serious problem in North-eastern states mainly Assam, Nagaland, Tripura and West Bengal. Contributes 8.5 to 11% of total malaria cases and 13 to 15 percentage of mortality.
LIFE CYCLE
Sexual form: In female Anopheles
Asexual form: In human beings
LABORATORY DIAGNOSIS OF MALARIA
Various method for diagnosis of malaria can be classified as under:
  1. CONVENTIONAL METHODS
  2. NEWER TECHNIQUES
CONVENTIONAL METHODS
  1. Peripheral blood film
    It is an oldest method. Two types of smear (thick and thin) are prepared from peripheral blood:
    1. Thick film.
      1. Thick smears have more quantity of blood in small area.
      2. Chances of detection of malaria parasites are more in thick smear.
      3. Since morphology of the parasite is not clear, so species identification can not be done.
      4. Malarial pigment is easily detected in thick film.
      5. The thick film concentrates the parasites up to 40-times.
      6. So it increases the sensitivity of diagnosis.
    2. Thin film.
      1. Level of parasitaemia and malarial species can be detected.
      2. Count number of RBC containing asexual parasites per 1000 RBC.
      3. Mild -1 to 10%.
      4. Serious - 10 to 20%.
      5. Grave - 20 to 30%.
      6. Exceptionally Grave - more than 30%.
      7. In severe malaria, stages of parasite development can be detected.
      8. Neutrophils containing malarial pigment can be counted.
      9. It provides prognostic information.
    3. Parasite count
      1. Parasite count denotes their concentration in blood at a particular point of time.
      2. A rough estimate of parasite concentration is as follows:
        + - 1 to 10 parasites/100 thick film field.
        ++ - 11 to 100 parasites/100 thick film field.
        +++ - 1 to 10 parasites/thick film field.
        ++++ - more than 10/thick film field.
  2. Complete blood count
    1. WBC - count
      1. Normal or low WBC count. Elevated in severe infection.
    2. RBC count
      1. Low RBC count with decreasing Hemoglobin % Normocytic, Normochromic anemia is the rule.
    3. Platelets counts
      1. Platelet count is almost always decreased-Thrombocytopenia.
    4. ESR
      1. Increased.
    5. Plasma viscosity
      1. Increased.

  3. In severe infection - PT, PTT prolonged, thrombocytopenia is severe. CRP, CSF pressure, protein level is usually normal or elevated.
  4. Electrolyte level
    1. mild cases.
      1. Normal
    2. In severe cases
      1. Lactic acidosis.
    3. Decreased plasma concentration
      1. Glucose, Na, HCO3, Calcium, Phosphate.
    4. Increased concentration
      1. Lactate, Blood urea nitrogen,Creatinine, Urate etc.

NEWER TECHNIQUES
  1. FLUORESCENT MICROSCOPY
    Fluorescent dye binds to parasite nucleic acids. Methods used are:
    1. Direct Acridine Orange staining.
      1. Acridine Orange stains cytoplasm orange and DNA green.
    2. Quantitative Buffy Coat (QBC) Technique
      1. In this method, due stains DNA green and RNA orange.
    3. BPC(Benzothiocarboxypurine) method.
  2. IMMUNOLOGICAL METHODS
    1. Antibody detection
      1. Immunofluorescent Antibody absorption test (IFAT) - The main method for routine diagnosis.
      2. ELISA Test.
      3. Radioimmunoassay.
      4. Latex agglutination test.
      5. Solid phase dipstick and membrane dot blot.
    2. Antigen detection
      1. Based on the detection of circulating antigen specific for P. Falciparum in whole blood.
    3. PfHRP-II deep stick or ICT, Immunochromatographic card test
      1. This test utilizes affinity to purified sheep polyclonal antibody specific for P. Falciparum circulating antigen PfHRP-II.
      2. A drop of blood is placed on stick 01 card.
      3. Monoclonal antibodies captures the parasite antigen and read out as a colored band.
      4. Rapid, sensitivity is 0.001% parasitemia.
    4. Plasmodium LDH dipstick or card test
      1. A drop of blood is placed on stick or card.
      2. Monoclonal antibodies capture the parasite and read out a colored bands.
      3. Two bands-one band is genus specific and other is specific for P. Falciparum.

      4. Rapid, sensitivity similar to thick film up to 0.001% parasitemia.
  3. MOLECULAR BIOLOGICAL DETECTION TESTS
    1. Genetic probes.
      1. Two types of probes used are based on hybridization of probe DNA with genomic DNA or ribosomal RNA of parasites.
    2. In severe cases
      1. Lactic acidosis.
    3. Gene amplification technique or polymerase chain reaction (PCR).
      1. This technique is based on amplification of gene by PCR.
      2. More sensitive and specific than genetic probing.
      3. PCR is capable of detecting parasitemia of < 0.00002%.
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Krishna S D, Jaiswal A D, Kumar A D.. Available From : http://www.pediatriconcall.com/fordoctor/ Conference_abstracts/report.aspx?reportid=116
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