Description
In areas with repeated & frequent parasite inoculations, individuals may be infected with more than one parasite species at a time. Despite having a routine surveillance system, data on the burden of mixed Plasmodium spp. infections is scarce. Further, the presence of multiple species within the host can impede diagnosis and thus treatment because different species give rise to distinct clinical features, and require specific treatments. Therefore, it is critical to examine parasite biology during mixed infections. Evidence suggests that coexistence affects the biology of individual species, but the nature, extent, and implications of these effects are unknown. During mixed infections, the presence of multiple species in the same host provides a window & ample opportunity for sexual interactions, which may be favoured by drug and shrinking population pressures and result in the emergence of an unknown species.
Considering these challenges, the current study aimed (i) To estimate the prevalence & study the clinical profile and complications of P. falciparum-vivax mixed infections, (ii) To develop a method to separate each species from mixed P. falciparum-vivax culture and, (iii) To investigate hetero-species interactions between P. falciparum and P. vivax gametes.
In the current study blood samples were collected in the year 2020 from fever patients at 4 different government settings in India. Samples were screened for P. falciparum-vivax mixed infections by microscopy, bivalent RDT and PCR (qPCR & nested). Clinical features & socio-demographic characteristics of patients have been recorded. Individuals with P. falciparum-vivax mixed infections were followed telephonically to identify & study associated complications & recurrences. The study reported 8% (86/1030) prevalence of P. falciparum-vivax mixed infections along with 18% (188/1030) mono P. falciparum and 0.58% (6/1030) mono P. vivax, accounting a total malaria prevalence 27% (280/1030). During follow-up no subsequent incidences of fever or other complaints were reported. Aside from that, the study developed a novel flow-cytometry based method to separate P. falciparum & P. vivax from mixed culture using synchronized populations. Schizont-specific gates were made for both P. falciparum & P. vivax, independently. As per our best knowledge, there has been no earlier research that used flow cytometry approach to compare P. vivax and P. falciparum infected RBCs simultaneously that allows sorting of these two species at a particular stage.
Furthermore, to investigate inter-species fertilization between P. falciparum & P. vivax a novel-approach was established. P. falciparum gametocytes (enriched from genetically modified cell line NF54HT GFP-Luc) were treated with a sex-specific drug that prevents male gametocyte activation. P. vivax gametocytes were purified using magnetic-based purification. Separated cross-species opposite-sex gametocytes were cultured in ookinete media to investigate cross-fertilization and zygote/ookinete formation through fluorescence microscopy. Mixed cultures, were supposed to yield both inter-& intra species zygotes/ookinetes. But with P. vivax, no zygote/ookinete observed. P. vivax gametocytes could not survive both in controls as well as in mixed cultures (with P. falciparum). But, this research attempts to open up a whole bunch of new questions and challenges as well as opening avenues for further exploration in the field of malaria research.
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