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

Polymorphisms of TNF-enhancer and gene for FcγRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population

<p>Abstract</p> <p>Background</p> <p>Susceptibility/resistance to <it>Plasmodium falciparum </it>malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the <it>TNF </it>and <it>FCGR2A </it>genes in determining severity/resistance to <it>P. falciparum </it>malaria in Indian subjects.</p> <p>Methods</p> <p>Allelic frequency distribution in populations across India was first determined by typing genetic variants of the <it>TNF </it>enhancer and the <it>FCGR2A </it>G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfo™ version 3.4.</p> <p>Results</p> <p>A novel single nucleotide polymorphism (SNP) at position -76 was identified in the <it>TNF </it>enhancer along with other reported variants. Five <it>TNF </it>enhancer SNPs and the <it>FCGR2A </it>R131H (G/A) SNP were analyzed for association with severity of <it>P. falciparum </it>malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. <it>TNF </it>-1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcγRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of <it>P. falciparum </it>severity/resistance in the Indian population.</p> <p>Conclusion</p> <p>Association of specific <it>TNF </it>and <it>FCGR2A </it>SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.</p

Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India

<p>Abstract</p> <p>Background</p> <p>Molecular techniques have facilitated the studies on genetic diversity of <it>Plasmodium </it>species particularly from field isolates collected directly from patients. The <it>msp-1 </it>and <it>msp-2 </it>are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the <it>msp-1 </it>gene and the central repetitive domain (block3) of the <it>msp-2 </it>gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among <it>Plasmodium falciparum </it>population of India, this report evaluates the extent of genetic diversity in the field isolates of <it>P. falciparum </it>in eastern and north-eastern regions of India.</p> <p>Methods</p> <p>A study was designed to assess the diversity of <it>msp-1 </it>and <it>msp-2 </it>among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa.</p> <p>Results</p> <p><it>P. falciparum </it>isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of <it>msp-1 </it>and <it>msp-2</it>. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the <it>P. falciparum </it>populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of <it>msp-1 </it>was observed to be monomorphic with an allele size of 150/160 bp. The observed 80–90% sequence identity of Indian isolates with data of other regions suggests that Indian <it>P. falciparum </it>population is a mixture of different strains.</p> <p>Conclusion</p> <p>The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of <it>msp-1 </it>(block 2) and <it>msp-2 </it>(central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.</p

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Anaplastic lymphoma kinase immunocytochemistry in fine needle aspiration diagnosis of anaplastic large-cell lymphoma

Background: Anaplastic large-cell lymphoma (ALCL) is a rare subtype of non-Hodgkin's lymphoma (NHL) characterized by the presence of unusual giant cells. It is a CD30+lymphoma of T-cells lineage, which shows anaplastic lymphoma kinase-nucleophosmin (ALK-NPM) rearrangement. ALCL on fine needle aspiration cytology (FNAC) shows unusually large and bizarre tumor cells. Materials and Methods: All aspirates seen over a 6-year period from November 2009 to November 2015 in which a diagnosis of ALCL or Hodgkin's lymphoma (HL) with bizarre giant cells were suspected on cytomorphology were prospectively selected. Twenty such aspirates were subjected to CD-30 and ALK-1 immunocytochemistry (ICC). Subsequent biopsy was available in all cases. Results: Out of 20 cases, seven cases, suspected to be ALCL on FNAC, were confirmed on biopsy. ALK-1 was positive in both cytology and biopsy of 6/7 of these. Two cases suspected to be ALCL on cytomorphology were HL (1) and diffuse large B-cell lymphoma (DLBCL) (1) on biopsy, both of which were ALK-1 negative on cytology. Eight cases of HL and three cases of large-cell NHL, which were all ALK negative on cytology, were confirmed on biopsy. Conclusion: ICC for ALK and CD30 is useful in aspiration cytodiagnosis of ALCL. One CD30 positive DLBCL and one ALK negative ALCL showed concordant results of ICC on cytology and histology

Polymorphism in drug resistance genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India

Abstract Background Sulfadoxine-pyrimethamine (SP) combination drug is currently being used in India for the treatment of Plasmodium falciparum as partner drug in artemisinin-based combination therapy (ACT). Resistance to sulfadoxine and pyrimethamine in P. falciparum is linked with mutations in dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes respectively. This study was undertaken to estimate the prevalence of such mutations in pfdhfr and pfdhps genes in four states of India. Methods Plasmodium falciparum isolates were collected from two states of India with high malaria incidence i.e., Jharkhand and Odisha and two states with low malaria incidence i.e., Andhra Pradesh and Uttar Pradesh between years 2006 to 2012. Part of sulfadoxine-pyrimethamine (SP) drug resistance genes, pfdhfr and pfdhps were PCR-amplified, sequenced and analyzed. Results A total of 217 confirmed P. falciparum isolates were sequenced for both Pfdhfr and pfdhps gene. Two pfdhfr mutations 59R and 108N were most common mutations prevalent in all localities in 77 % of isolates. Additionally, I164L was found in Odisha and Jharkhand only (4/70 and 8/84, respectively). Another mutation 51I was found in Odisha only (3/70). The pfdhps mutations 436A, 437G, 540E and 581G were found in Jharkhand and Odisha only in 13, 26, 14 and 13 % isolates respectively, and was absent in Uttar Pradesh and Andhra Pradesh. Combined together for pfdhps and pfdhfr locus, triple, quadruple, quintuple and sextuple mutations were present in Jharkhand and Odisha while absent in Uttar Pradesh and Andhra Pradesh. Conclusion While only double mutants of pfdhfr was present in low transmission area (Uttar Pradesh and Andhra Pradesh) with total absence of pfdhps mutants, up to sextuple mutations were present in high transmission areas (Odisha and Jharkhand) for both the genes combined. Presence of multiple mutations in pfdhfr and pfdhps genes linked to SP resistance in high transmission area may lead to fixation of multiple mutations in presence of high drug pressure and high recombination rate

Molecular Tools for Early Detection of Invasive Malaria Vector Anopheles stephensi Mosquitoes

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species

Chemotypical variations in Withania Somnifera Lead to Differentially Modulated Immune Response in BALB/c Mice

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10–100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3–30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10 mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses

Mutant <it>pfcrt </it>"SVMNT" haplotype and wild type <it>pfmdr1 </it>"N86" are endemic in <it>Plasmodium vivax </it>dominated areas of India under high chloroquine exposure

<p>Abstract</p> <p>Background</p> <p>Chloroquine resistance (CQR) phenotype in <it>Plasmodium falciparum </it>is associated with mutations in <it>pfcrt </it>and <it>pfmdr-1 </it>genes. Mutations at amino acid position 72-76 of <it>pfcrt </it>gene, here defined as <it>pfcrt </it>haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of <it>pfcrt </it>gene and N86Y <it>pfmdr-1 </it>mutation were studied in blood samples collected across 11 field sites, inclusive of high and low <it>P. falciparum </it>prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated.</p> <p>Methods</p> <p>Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low <it>P. falciparum </it>prevalent areas. For <it>pfcrt </it>haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the <it>pfcrt </it>gene. For <it>pfmdr-1 </it>N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme.</p> <p>Results</p> <p>In 240 <it>P. falciparum </it>isolates with reported <it>in vivo </it>CQ therapeutic efficacy, the analysis of mutations in <it>pfcrt </it>gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 <it>P. falciparum </it>isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low <it>P. falciparum </it>prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high <it>P. falciparum </it>prevalent areas. Investigation of <it>pfmdr-1 </it>N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low <it>P. falciparum </it>prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high <it>P. falciparum </it>prevalent areas (42.76%).</p> <p>Conclusions</p> <p>The wild type <it>pfcrt </it>gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher <it>P. vivax </it>prevalance and chloroquine exposure, whereas, diversity is maintained in <it>pfcrt </it>gene at high <it>P. falciparum </it>prevalent areas.</p