Malaria, a widely prevalent parasitic disease affects 500 million people each year and is associated with 2-5 million deaths [1]. One of the most pronounced problems in controlling the morbidity and mortality is limited access to effective diagnosis and treatment in areas where malaria is endemic [2]. Microscopic examination of blood smears is the widely used method for detection of malaria parasites and remains the gold standard for malaria diagnosis [3]. But microscopic examination is laborious and time consuming and requires considerable expertise for its interpretation particularly at low levels of parasitemia [4]. Rapid and early detection of malarial parasite and early treatment of infection still remains the most important goals of disease management [5]. A key feature of the World Health Organization global malaria control strategy is the rapid diagnosis of malaria at the village and district level so that effective treatment can be administered quickly to reduce morbidity and mortality. There is therefore an urgent need for a field test which is simple, rapid and accurate. These RDT’s have a number of important limitations, including suboptimal sensitivity at low parasite densities, to quantify infection rate and a higher unit cost relative to microscopy [6].

This study was conducted in the department of microbiology, Kasturba Medical College Hospital, Ambedkar circle, Mangalore, during the period from July 2005-2007. The study was cleared by the Institutional ethics committee. Patients attending the hospital, with symptoms and signs suggestive of malaria formed the study group. A total of 100 patients were included in the study. Blood sample collected from the patients were subjected to thick and thin smear (Traditional microscopy), Quantitative buffy coat (QBC) and Immuochromatographic test (ICT) Falcivax. Thick and thin smear were stained with Giemsa stain and observed under 100 X microscopy. Thick smear was used for the identification and thin smear for the speciation of the parasite. According to standard practice, thin smear was examined for 15 minutes and thick smear 200 fields were visualized.

Quantitative buffy coat

The QBC capillary tubes were filled with blood by capillary action and were centrifuged at the rate of 1200g for 5 min after proper balancing. The tubes were examined under fluorescence microscope. The ring forms appeared as apple green with or without an orange dot at one side, schizonts as dark brown in colour, and gametocytes as yellowish green sickle shaped bodies.

Immunochromatographic test

Falcivax [Tulip diagnostics pvt ltd, Goa, India], is a rapid self performing, qualitative, immunoassay used for the detection of P.falciparum specific histidine rich protein-2 (HRP-2) antigen and P.vivax specific lactate dehydrogenase (PLDH).The test was performed according to the manufacturer’s instructions, all the kit components were brought to room temperature, the whole blood was centrifuged, and 2-3 drops of serum was dispensed into the sample port, followed by 5 drops of buffer solution provided along with the kit. The results were read at the end of 15 minutes. A pink purple band appeared at the region ‘Pv’ in the test window ‘T’ in addition to the control band it was considered as P.vivax positive. A pink purple band appeared at the region ‘Pf ’ in the test window ‘T’ in addition to the control band, it was considered as P.falciparum positive.

To measure the agreement between Blood smears, QBC and Falcivax, Kappa statistics was used and statistical significance was assessed.

A total of 100 samples were examined for malaria parasites by quantitative buffy coat and Falcivax and the results were compared with peripheral blood smear examination. Blood smear results indicated that 74 cases were found to be positive for malaria parasites and the rest 24 were negative. Among the positive patients P.vivax was detected in 55 cases (75%) and P.falciparum in 19 cases (25%).

Correspondingly QBC method detected, 80(80%) of total malaria cases, of which 60 (75%) cases were positive for P.vivax and 20 (25%) cases were positive for P.falciparum (Table 1). QBC detected five cases of P.vivax and one case of P.falciparum that were negative by blood smear.

Table 1: Comparison of peripheral blood smears with other methods for the detection of malaria parasites

Falcivax indentified 71(71%) of total malaria cases, of which 53(74%) and18 (25%) cases were positive for P.vivax and P.falciparum infections (Table 1). Two cases of P.vivax and one case of P.falciparum positive by blood smears were not detected by Falcivax.

Sensitivity, specificity, positive and negative predicative value of QBC for P.vivax were 91.6,100, 100 and 88.8% respectively and for P.falciparum were 95,100, 100 and 8.7% where that of Falcivax were 100, 95.7, 96.3 and 100% for P.vivax and 100, 98.7, 94.7 and 100% for P.falciparum (Table 2).

Table 2: Comparison of sensitivity and specificity of various methods in the identification of malarial parasites

On comparing, QBC test with blood smear examination for P.vivax (K=0.898, P<0.0001) and for P.falciparum (K=0.968, P <0.0001) which is statistically significant. Comparison of Falcivax with peripheral blood smear examination for P.vivax (K=0.960, P< 0.0001) and for P.falciparum (K=--, P <0.0001) which is also statistically significant.

Malaria is a well-known disease and it continues to be a major public health problem at the start of new millennium. Reliable diagnosis of malaria requires laboratory confirmation of the presence of malaria parasites in the blood of a febrile patient [7]. Although microscopic examination of blood smear continues to be the gold standard, it has a drawback that it is time consuming and requires an expert microscopist and less sensitive in cases of low parasitemia [8]. Various sensitive methods have been employed for the simple, reliable, and rapid diagnosis of malaria, the most promising of these is the rapid diagnostic test and quantitative buffy coat [9]. We employed these tests and compared with Giemsa stained peripheral blood smear for the diagnosis of P.vivax and P.falciparum infections.

The QBC and RDT identified 80% and 71% as malaria positive while blood smears detected 74% of the positive cases. Five cases of P.vivax and one case of P.falciparum negative by blood smear were detected by QBC indicating a higher sensitivity and specificity of QBC. High sensitivity of QBC might be due to concentration of parasites below the buffy coat. Parzy et al found QBC to be more sensitive than blood smear examination and advocated its use for urgent diagnosis [10]. In our study the sensitivity and specificity of QBC for P.vivax was74.3% and 80.7% and for P.falciparum was100% and 98.67% respectively our results are in agreement with the results reported by various studies. Study by (Ye Htut et al. 2002) had a sensitivity of 82.8% and 100% for P.falciparum and P.vivax and specificity of 97.1% and 98.6% [11].

One of the major advantages of the QBC technique is rapidity and reliability in diagnosis of malaria even under field conditions. In addition, it requires less training and experience than blood smears. Its chief drawback is its high cost and in the identification of Plasmodium species. Ring stages of P. falciparum and P. vivax are difficult to distinguish by the QBC. This problem is particularly important in endemic areas where P.falciparum coexists with P. vivax [12].

Falcivax failed to detect two cases of P.vivax and one case of P.falciparum which were positive by blood smears. The sensitivity and specificity of falcivax was 96.3% and 100% for P.vivax and 94.7% and100% for P.falciparum. The low sensitivity of the Falcivax can be explained by the fact that it detects enzyme pLDH produced by live parasites and the parasites might have been killed and not cleared from the host4 and also due to low parasitemic levels as observed by Iqbal et al. who observed 75% sensitivity at parasitemia < 100/μl. However, the rapid diagnostic test was found to be user friendly and interpretation was more objective as compared to smear and QBC [13]. Although no single test can replace the conventional method of peripheral blood smear examination, these newer diagnostic tests can be used as supplement to microscopic examination of peripheral blood smear where the diagnosis cannot be made on microscopy and an experienced microscopists are not available. The high cost of the test may prevent routine use in many laboratories. However it is a valuable adjuvant at the time of emergency for rapid diagnosis, although microscopy remains the main stay for the diagnosis of malaria.