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.

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.

Perspective and Potential of A2A and A3 Adenosine Receptors as Therapeutic Targets for the Treatment of Rheumatoid Arthritis.

Adenosine is a purine nucleoside which is an effective controller of inflammation. The inflammatory effect of adenosine is expressed via its four receptor subtypes viz. A1, A2A, A2B and A3. The various inflammatory conditions including rheumatoid arthritis (RA) are initiated by adenosine receptors of which A2A and A3 play a vital role. RA primarily is an auto-immune disorder which is manifested as chronic inflammation in the synovial lining of joints. In order to develop an effective treatment, the role of cytokines, IL-1, TNF-α and IL-6 is crucial. Besides, the knowledge of PI3K-PKB/Akt and NF-kB signaling pathway is also important to understand the antiinflammatory targets. Methotrexate along with various other molecules like, NSAIDs and DMARDs are presently used as treatment lines for controlling RA. The enhanced knowledge of the preclinical stages and pathogenesis along with recent potent therapeutics raises the hopes that RA can be prevented in the near future.

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.