Anusri Tripathi

Differential clinical symptoms among acute phase Indian patients revealed significant association with dengue viral load and serum IFN-gamma level.

BACKGROUND During last three decades, dengue (DF), an arthropod-borne viral disease is increasingly prevalent worldwide including India, resulting in serious public health problems. OBJECTIVE This study was carried out during 2012-2013 to evaluate the association of WHO-classified dengue disease symptoms with viral load (VL) and serum IFN-gamma levels in dengue patients from India. STUDY DESIGN Blood samples were collected from dengue symptomatic febrile patients (n=127). DEN-VL was determined by quantitative real-time RT-PCR using RNA, extracted from plasma and anti-DENV-IgM was detected by ELISA. Serum IFN-gamma level was detected by ELISA among DENV infected and age-matched control individuals. Association between DEN-VL and IFN-gamma level in patients sera with WHO-classified disease symptoms was statistically analyzed. RESULTS DENV RNA and anti-DENV-IgM was detected among 94% (n=73) and 36% (n=28) of dengue infected patients (n=78) respectively. Fever, nausea, rash, aches & pains, leucopenia and persistent vomiting were significantly correlated with DENV infection (P-value<0.05). Only patients with high-VL exhibited leucopenia, persistent vomiting, abdominal pain and clinical fluid accumulation, which were warning signs of dengue infection according to revised WHO-criteria (P-value<0.05). Clinical symptoms of DENV infected patients, viz. leucopenia, abdominal pain and persistent vomiting were significantly correlated to each other (P-value<0.05). Increased serum IFN-gamma level was detected among dengue patients compared to control individuals. DEN-VL and symptoms like nausea, leucopenia, persistent vomiting and abdominal pain were significantly negatively correlated with serum IFN-gamma level (P-value<0.05). CONCLUSION Serum IFN-gamma level and dengue viremia among acute stage patients might be used as early prognostic marker for disease severity prediction.

Copy number variation of chikungunya ECSA virus with disease symptoms among Indian patients

After a gap of three decades, from 2005 onwards, a series of Chikungunya virus (CHIKV) outbreaks occurred worldwide. This study was performed to detect CHIKV infection, its genotype among symptomatic Eastern Indian patients and to analyze any association between the presence of CHIKV genome in patient body with appearance of disease symptoms (n = 199). Plasma‐extracted viral RNA was reverse transcribed to cDNA and PCR‐amplified followed by agarose gel electrophoresis. Viral load among CHIKV‐positive patients was determined by real time RT‐PCR. CHIKV‐IgM in sera was detected by ELISA. Sequencing and phylogenetic analysis of plasma‐extracted PCR products was done. CHIKV genome and IgM were detected among 65.3% (n = 130) and 41.2% (n = 82) patients respectively. Joint swelling was significantly associated with CHIKV infection (P‐value: 0.0003). CHIKV PCR positive patients were grouped in two categories: Group‐I: viral load <104 copies/ml and Group‐II: viral load ≥104 copies/ml. Higher number of acute stage patients clustered in Group‐II. Fever and joint swelling were significantly more prevalent among Group‐II patients, whereas rash and diarrhoea among Group‐I patients (P‐value <0.05). Patient‐isolated CHIKV sequences clustered with CHIKV ECSA genotypes in the phylogenetic tree, with two types of CHIKV strains found to circulate among them—as indicated by their different nucleotide sequences. This is the first study detecting the presence of CHIKV ECSA genotype among Eastern Indian patients. Fever and joint swelling might have appeared first followed by rash, diarrhea during disease progression—as indicated by CHIK viral load in patients. Thus, viral load can be used as unique diagnostic and prognostic marker of Chikungunya disease pathogenesis. J. Med. Virol. 86:1386–1392, 2014.

Genetic and structural insights into plasmid-mediated extended-spectrum β-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients

Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum β-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated blaCTX-M, blaSHV and blaOXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of blaCTX-M-15 (29.8%), blaCTX-M-truncated (1.3%), blaCTX-M-27 (0.7%), blaSHV-1 (20.5%), blaSHV-11 (2.0%), blaSHV-42 (0.7%) and blaOXA-1 (9.9%), among which blaCTX-M variants and blaSHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all blaCTX-M variants, blaSHV-11 and blaSHV-42. The instability index and Gibbs free folding energy change (ΔΔG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A β-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (ΔG and Km) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.

Molecular characterization and in silico analysis of naturally occurring TEM beta-lactamase variants among pathogenic Enterobacteriaceae infecting Indian patients.

Cephalosporin resistance, particularly due to bla TEM encoded β-lactamases, among Enterobacteriaceae is, though, an increasing public health problem in India; their circulating genetic variants remain unknown. The present study deals with determination of bla TEM variants among 134 pathogenic Enterobacteriaceae of Indian origin. Their resistance profile against 3rd generation cephalosporins was determined. The presence of bla TEM variants among the bacterial plasmids was characterized by PCR followed by sequencing. Intergenic relations among the variants was determined by phylogenetic analysis. bla TEM protein were modeled by Modeller9v5 and verified. The catalytic pockets were characterized, and their interaction with cephalosporins was analyzed using AutoDock tools. More than 87% of isolates showed cephalosporin resistance with ESBL production among 57.8% of Escherichia coli and 50.6% of klebsiella pneumoniae. bla TEM-1 (84.21%), bla TEM-1 like (3.94%), bla TEM-33 (3.94%), bla TEM-116 (3.94%), bla TEM-169 (3.94%), and bla TEM-190 (7.89%) were detected in 76 isolates. Four variants, namely, bla TEM-1like, bla TEM-33, bla TEM-169, and bla TEM-190, coexisted in 3 isolates. The largest catalytic pocket of bla TEM-33 explained its expanded activity towards β-lactam-β-lactamase inhibitor combinations. Molecular docking indicated differential resistance pattern of bla TEM variants.

Association of toll-like receptor polymorphisms with susceptibility to chikungunya virus infection

Chikungunya virus (CHIKV) infection leads to activation of innate immune response by triggering Toll-like receptor (TLR) pathways resulting in elevated cytokines and type-I interferon levels. Genetic variations of these genes may influence human CHIKV-susceptibility and disease progression. Present study aimed to identify role of TLR polymorphisms in CHIKV-susceptibility and their association with cytokines and clinical symptoms. This is the first study illustrating certain genotypes of TLR-7 and TLR-8 SNPs viz. CT(p = 0.002)]; rs3853839[GC(p < 0.001), CC(p = 0.039)] and rs3764879[GC(p < 0.001)] were considerably associated with CHIKV susceptibility. Increased risk of CHIKV infection among male patients with CC-genotype (rs179010) (p = 0.028) and female patients with GT-genotype (rs5741880) (p = 0.019) was observed. Significant higher IFN-α (P = 0.002) levels among chikungunya TNF-α (P = 0.034) patients was reported. Chikungunya patients with rs179010-CC genotype showed significantly high IFN-α level(p = 0.003). Thus, these TLR variants might act as potential prognostic biomarkers for predicting CHIKV susceptibility among uninfected individuals.

An in silico approach to elucidate structure based functional evolution of oxacillinase

Bacterial Oxacillinases (OXAs), genetically being extremely diverse and highly versatile in hydrolyzing antibiotics of different classes, holds utmost significant clinical importance. Hence, to analyze functional evolution of this enzyme, plausible changes in drug profile, affinity and binding stability of different subclasses of OXA with their preferred drugs, viz. penicillin, ceftazidime, imipenem/meropenem were investigated. Maximum-Likelihood dendrogram was constructed and based on tree topology, the least and most divergent variants of each clade were selected. Pocket characterization, enzyme structural stability and mutational effect were analyzed in silico. Modes of interaction of selected OXA variants with respective antibiotics were analyzed by Autodock4.0 and LIGPLOT. Comparative mobility profiling and subsequent ΔG° and Km calculations of representative OXA variants revealed that after RSBL evolution, perhaps, two competitive strategies evolved among the OXA variants. Either loops flanking helix5 gets stabilized or it becomes more flexible. Therefore, while OXA variants (e.g. OXA-2, OXA-32, OXA-23, OXA-133, OXA-24, OXA-25, OXA-51 and OXA-75) with highly stabilized loops flanking helix5 exhibited improved binding stability and affinity towards carbapenems, especially meropenem, OXA variants (e.g. OXA-10, OXA-251, OXA-48 and OXA-247) possessing highly flexibile loops flanking helix5 revealed their catalytic proficiency towards ceftazidime. Moreover, LIGPLOT and PROMALS3D jointly identified ten consensuses/conserved residues, viz. P68, A69, F72, K73, W105, V120, W164, L169, K216 and G218 to be critical for drug hydrolysis. Hence, novel inhibitors could be designed to target these sites.

Chikungunya virus: genomic microevolution in Eastern India and its in-silico epitope prediction

This is the first study reporting whole genome sequences of two CHIKV strains (KJ679577 and KJ679578) isolated from Eastern Indian patients sera during 2010–2011 outbreak, both of which were of ECSA genotype, but from different subgroups: Indian Ocean outbreak and ECSA subtypes. Furthermore, viral sequences were analyzed using different in-silico approaches to identify potential genetic variations that might have functional implications on various aspects of virus replication, viral protein functionality, immunogenicity and transmission. Epitope prediction analysis revealed 70.9% increase in number of MHC Class-II interacting epitopes of KJ679578 and 25–28% increase in Class-I interacting epitopes of KJ679577 and KJ679578 compared to that of EF027141 (CHIKV of Asian genotype circulating in India during 1973, after which CHIKV infection disappeared from India for three decades). CHIKV peptides DLAKLAFKRSSKYDLECAQIPVHMKSDA and KVVLCGDPKQCGFFNMMQMKYNYNHNI were predicted to interact with maximum number of HLA Class-I (68 and 76.5%, respectively) and Class-II (47 and 100%, respectively) alleles present within Indian population with allele frequency of > 0.1 and were also recognized as predicted B-cell epitopes with BCPred score between 0.766 and 0.961 and with antigenicity ranging from 0.52 to 1.69; thus these peptides might be used to induce T- and B-cell-mediated immunity against CHIKV. Thus, the present study might help to bridge the gap between virus microevolution and its implication in host immunity by taking into account viral genetic and conformational changes. Predicted epitopes might be used as promising targets for peptide-based vaccine development and rapid diagnostics against CHIKV infection.