Plasmodium falciparum - Laboratory diagnosis

Laboratory diagnosis of Plasmodium falciparum

Specimen

Blood

* Blood samples should be collected before starting antimalarial treatment. The collection of blood samples is done from peripheral areas such as the earlobe, finger, and from the greater toe in infants during the time of fever.

Microscopy

The laboratory diagnosis of Plasmodium falciparum infection depends upon the demonstration of parasites in the blood.

Blood smears should be examined at least twice daily – until the malarial parasite is detected. If microscopy gives out negative results, it does not mean the infection is ruled out. In only 50% of cases, even on repeated examinations, the smear comes out as positive.

The numerous methods of microscopy include light microscopy, fluorescence microscopy, and QBC.

Light microscopy

• the gold standard for confirmation of Plasmodium falciparum infection

• stained peripheral blood is viewed under a microscope to detect the presence of ring form and gametocytes of the parasite

• both thin and thick blood smear is prepared and stained with one of Romanosky’s stains such as Giemsa’s, JSB, Leishmans’, Wrights’, or Fields’ stain

Thick blood smear

• it is based on the principle that the concentrated RBCs are lysed with distilled water during preparation which releases the intact parasites

• the smear slides are not fixed but rather dried thoroughly and stained

• since the concentration of RBC is high, sensitivity is increased

• this method is followed for the detection of parasites, demonstrating the pigments, and quantitating parasitemia

• this method is not useful for species diagnosis – rather thin blood smears are used to distinguish malarial species after positive thick blood smear

• as quantitative parasitemia has prognostic value, it is done to determine if parasitemia is increasing or decreasing during treatment

• the baseline to report negative thick smear examination includes visualization of 100-200 fields with each field containing 20 WBCs

Thin blood smear

• this method is followed for the detection of Plasmodium falciparum malaria parasites and for determining the species of parasite observed

• in this method, the thin blood smears are air-dried rapidly, fixed with alcohol, and stained

• the tail end of the smear is examined under oil-immersion

Fluorescence microscopy

• Kawamoto technique of fluorescence microscopy is used for the detection of the Plasmodium parasite in blood samples

• this method involves staining of blood smears with acridine orange (differential staining)

• cytoplasmic RNA is stained red while nuclear DNA is stained green

• examined under a fluorescence microscope

QBC

• this method is based on the ability of acridine orange to stain the nucleic acid of Plasmodium

• a blood sample is collected in a capillary tube coated with a fluorescent dye and centrifuged to microhaematocrit centrifugation

• after centrifugation, the buffy coat in the capillary tubes is visualized under a fluorescent microscope

• the acridine orange stains the malaria parasites green

• high sensitivity as this method can detect 3-4 parasites/μl in blood

• however, it is costly and does not differentiate species of Plasmodium

Serodiagnosis

The serological tests in the case of Plasmodium falciparum are mostly done for epidemiological purposes. Common serological tests used are:

• Indirect haemagglutination (IHA)

• Indirect fluorescent antibody (IFA)

• Enzyme-linked immunosorbent assay (ELISA) – detects circulating antibodies

• Dipstick test

Dipstick test

• a type of enzyme immunoassay

• rapid diagnostic test- result given within 5 minutes

• detects the metabolic product histidine-rich protein-2 (PfHRP-2) antigen which is produced by Plasmodium falciparum

• monoclonal antibodies produced against PfHRP-2 antigen are used to detect PfHRP-2 antigen in the serum or urine

• only used to detect Plasmodium falciparum infections as it does not detect other malaria species

• high sensitivity or specificity

• cannot detect in low parasitemia (below 100 parasites/ml)

• useful for cerebral malaria diagnosis

Molecular Diagnosis

• DNA, RNA probes (can detect <10 parasites/μl of blood)

• PCR (can detect a single parasite in 20 μl of blood and is highly species-specific)