Malaria diagnosis by PCR revealed differential distribution of mono and mixed species infections by Plasmodium falciparum and P. vivax in India

Abstract

Malaria is a vector-borne infectious disease, caused by five different species of the genus Plasmodium, and is endemic to many tropical and sub-tropical countries of the globe. At present, malaria diagnosis at the primary health care level in India is conducted by either microscopy or rapid diagnostic test (RDT). In recent years, molecular diagnosis (by PCR assay), has emerged as the most sensitive method for malaria diagnosis. India is highly endemic to malaria and shoulders the burden of two major malaria parasites, Plasmodium falciparum and P. vivax. Previous studies using PCR diagnostic assay had unraveled several interesting facts on distribution of malaria parasites in India. However, these studies had several limitations from small sample size to limited geographical areas of sampling. In order to mitigate these limitations, we have collected finger-prick blood samples from 2,333 malaria symptomatic individuals in nine states from 11 geographic locations, covering almost the entire malaria endemic regions of India and performed all the three diagnostic tests (microscopy, RDT and PCR assay) and also have conducted comparative assessment on the performance of the three diagnostic tests. Since PCR assay turned out to be highly sensitive (827 malaria positive cases) among the three types of tests, we have utilized data from PCR diagnostic assay for analyses and inferences. The results indicate varied distributional prevalence of P. vivax and P. falciparum according to locations in India, and also the mixed species infection due to these two species. The proportion of P. falciparum to P. vivax was found to be 49:51, and percentage of mixed species infections due to these two parasites was found to be 13% of total infections. Considering India is set for malaria elimination by 2030, the present malaria epidemiological information is of high importance.

Fig 1. Agarose gel electrophoresis pictures showing bands of 15 representative PCR products.

The first lane (lane L) contains ladder (100 bp marker) for comparison of PCR products and for determination of product size. The second lane (Lane 1) contains PCR products from negative control, lane 2 contains product of negative control with human DNA. Lanes 3–6 display 120 bp PCR product signifying P. vivax mono infection and lanes 7–11 show 205 bp PCR product testifying mono infection of P. falciparum. Lanes 12–13 and 15 present both the bands of 120 bp (P. vivax) and 205 bp (P. falciparum) size in a single sample, indicating mixed species infections due these two species of malaria parasites.

Fig 2. Visual representation on the comparative assessment (in number and percentage) of efficacy of three different malaria diagnostic methods (microscopy, RDT and PCR assay).

To be noted that out of the total 2333 collected malaria-symptomatic individuals (outer-most circle of the middle circles, in grey), PCR assay could identify 827 (35.44%) as positive for malaria parasite infection out of which 42% was P. vivax, 45% P. falciparum and 12.69% mixed infection due to these two species. In comparison, the RDT (third circle from out) and microscopy (4th circle from out) could identify less number of infections.

Fig 3. Map of India showing malaria sample collection site.

Each site is represented by a pie-chart three different kinds of infection (two types of mono infections and a mixed species infection due to P. falciparum and P. vivax). To be noted here that locations in all the four directions (peripheral populations) (north, east, west and south) are majorly dominated by P. vivax, but in northeast, south-west and middle Indian locations P. falciparum was found to be in higher abundance than P. vivax. Mixed parasitic infections majorly are restricted to middle of India.