Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in India Using Next-Generation Sequencing.

Background: Tracking the emergence and spread of antimalarial drug resistance has become critical to sustaining progress towards the control and eventual elimination of malaria in South Asia, especially India. Methods: An amplicon sequencing protocol was used for high-throughput molecular surveillance of antimalarial drug resistance in a total of 158 isolates at three sites in India: Chennai, Nadiad and Rourkela. Five genes of the Plasmodium falciparum implicated in antimalarial resistance were investigated here; Pfcrt for chloroquine resistance, Pfdhfr for pyrimethamine resistance, Pfdhps for sulfadoxine resistance, Pfk13 for artemisinin resistance and Pfmdr1 for resistance to multiple antimalarials. Results: Mutations in the propeller domain of PfK13 were observed in two samples only, however these mutations are not validated for artemisinin resistance. A high proportion of parasites from the P. falciparum dominant site Rourkela showed wild-type Pfcrt and Pfdhfr haplotypes, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type PfDHPS haplotype was predominant across all study sites. Finally, we observed the largest proportion of suspected multi-clonal infections at Rourkela, which has the highest transmission of P. falciparum among our study sites. Conclusion: This is the first simultaneous high-throughput next generation sequencing of five complete P. falciparum genes from infected patients in India.

Antibody responses within two leading Plasmodium vivax vaccine candidate antigens in three geographically diverse malaria-endemic regions of India.

BACKGROUND: Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India. METHODS: A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed. RESULTS: The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119. CONCLUSION: These data suggest that natural acquired antibody response is higher for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.

The burden of submicroscopic and asymptomatic malaria in India revealed from epidemiology studies at three varied transmission sites in India.

Malaria in India, while decreasing, remains a serious public health problem, and the contribution of submicroscopic and asymptomatic infections to its persistence is poorly understood. We conducted community surveys and clinic studies at three sites in India differing in their eco-epidemiologies: Chennai (Tamil Nadu), Nadiad (Gujarat), and Rourkela (Odisha), during 2012-2015. A total of 6,645 subject blood samples were collected for Plasmodium diagnosis by microscopy and PCR, and an extensive clinical questionnaire completed. Malaria prevalence ranged from 3-8% by PCR in community surveys (24 infections in Chennai, 56 in Nadiad, 101 in Rourkela), with Plasmodium vivax dominating in Chennai (70.8%) and Nadiad (67.9%), and Plasmodium falciparum in Rourkela (77.3%). A proportional high burden of asymptomatic and submicroscopic infections was detected in community surveys in Chennai (71% and 71%, respectively, 17 infections for both) and Rourkela (64% and 31%, 65 and 31 infections, respectively). In clinic studies, a proportional high burden of infections was identified as submicroscopic in Rourkela (45%, 42 infections) and Chennai (19%, 42 infections). In the community surveys, anemia and fever were significantly more common among microscopic than submicroscopic infections. Exploratory spatial analysis identified a number of potential malaria hotspots at all three sites. There is a considerable burden of submicroscopic and asymptomatic malaria in malarious regions in India, which may act as a reservoir with implications for malaria elimination strategies.

Dengue, chikungunya, and scrub typhus are important etiologies of non-malarial febrile illness in Rourkela, Odisha, India.

BACKGROUND: We conducted a diagnostic surveillance study to identify Plasmodium, dengue virus, chikungunya virus, and Orientia tsutsugamushi infections among febrile patients who underwent triage for malaria in the outpatient department at Ispat General Hospital, Rourkela, Odisha, India. METHODS: Febrile patients were enrolled from January 2016-January 2017. Blood smears and small volumes or vacutainers of blood were collected from study participants to carry out diagnostic assays. Malaria was diagnosed using rapid diagnostic tests (RDT), microscopy, and PCR. Dengue, chikungunya, and scrub typhus infections were identified using rapid diagnostic test kits and ELISA. RESULTS: Nine hundred and fifty-four patients were prospectively enrolled in our study. The majority of patients were male (58.4%) and more than 15 years of age (66.4%). All 954 enrollees underwent additional testing for malaria; a subset of enrollees (293/954) that had larger volumes of plasma available was also tested for dengue, chikungunya and scrub typhus by either RDT or ELISA or both tests. Fifty-four of 954 patients (5.7%) were positive for malaria by RDT, or microscopy, or PCR. Seventy-four of 293 patients (25.3%) tested positive for dengue by either RDT or ELISA, and 17 of 293 patients (5.8%) tested positive for chikungunya-specific IgM by either ELISA or RDT. Ten of 287 patients tested (3.5%) were positive for scrub typhus by ELISA specific for scrub typhus IgM. Seventeen patients among 290 (5.9%) with results for ≥3 infections tested positive for more than one infection. Patients with scrub typhus and chikungunya had high rates of co-infection: of the 10 patients positive for scrub typhus, six were positive for dengue (p = 0.009), and five of 17 patients positive for chikungunya (by RDT or ELISA) were also diagnosed with malaria (p < 0.001). CONCLUSIONS: Dengue, chikungunya and scrub typhus are important etiologies of non-malarial febrile illness in Rourkela, Odisha, and comorbidity should be considered. Routine febrile illness surveillance is required to accurately establish the prevalence of these infections in this region, to offer timely treatment, and to implement appropriate methods of control.

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium.

The technical challenges of working with the sexual stages of the malaria parasite Plasmodium have hindered the characterization of sexual stage antigens in the quest for a successful malaria transmission-blocking vaccine. One such predicted and largely uncharacterized group of sexual stage candidate antigens is the CPW-WPC family of proteins. CPW-WPC proteins are named for a characteristic domain that contains two conserved motifs, CPxxW and WPC. Conserved across Apicomplexa, this family is also present earlier in the Alveolata in the free-living, non-parasitophorous, photosynthetic chromerids, Chromera and Vitrella. In Plasmodium falciparum and Plasmodium berghei blood stage parasites, the transcripts of all nine cpw-wpc genes have been detected in gametocytes. RNA immunoprecipitation followed by reverse transcriptase-PCR reveals all P. berghei cpw-wpc transcripts to be bound by the translational repressors DOZI and CITH, and thus are likely under translational control prior to transmission from the rodent host to the mosquito vector in P. berghei. The GFP tagging of two endogenous P. berghei genes confirmed translational silencing in the gametocyte and translation in ookinetes. By establishing a luciferase transgene assay, we show that the 3' untranslated region of PF3D7_1331400 controls protein expression of this reporter in P. falciparum gametocytes. Our analyses suggest that cpw-wpc genes are translationally silenced in gametocytes across Plasmodium spp. and activated during ookinete formation and thus may have a role in transmission to the mosquito.

A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

Functional complementation of yeast ribosomal P0 protein with Plasmodium falciparum P0.

A complex of three phosphoproteins (P0, P1 and P2) constitutes the stalk region at the GTPase center of the eukaryotic large ribosomal subunit, amongst which the protein P0 plays the most crucial role. Earlier studies have shown the functional complementation of the conditional P0-null mutant of Saccharomyces cerevisiae (W303dGP0) with orthologous P0 genes from fungal and mammalian organisms, but not the protozoan parasite Leishmania infantum. In this paper we show that the PfP0 gene from the protozoan malaria parasite Plasmodium falciparum can functionally complement the conditional P0-null W303dGP0 mutant of S. cerevisiae. Unlike the above orthologous genes, PfP0 gene could also rescue the D67dGP0 strain, which in addition to being a conditional null for ScP0 gene, is a null-mutant for both ScP1alpha and beta genes. However, under stress conditions such as high temperature, salt and osmolarity, PfP0 gene could not rescue D67dGP0 strain. Ribosomes purified from W303dGP0 carrying PfP0 gene did not contain ScP1 protein, indicating a lack of binding of ScP1 to PfP0 protein. Yeast 2-hybrid analysis further confirmed the lack of binding of ScP1 to PfP0 protein. The polymerizing activities of ribosomes with ScP0 or PfP0 protein, in the absence of ScP1 protein, were found to be about 40-45% that of ribosomes with all the yeast P-proteins. In its sensitivity to the inhibitor sordarin, PfP0 was similar to the P0 protein from the fungus Aspergillus fumigatus. These results indicate a closer functional relationship of P. falciparum P0 gene to fungal P0 genes.