Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

Background: India has the third largest HIV-1 epidemic with 2.4 million infected individuals. Molecular epidemiological analysis has identified the predominance of HIV-1 subtype C (HIV-1C). However, the previous reports have been limited by sample size, and uneven geographical distribution. The introduction of HIV-1C in India remains uncertain due to this lack of structured studies. To fill the gap, we characterised the distribution pattern of HIV-1 subtypes in India based on data collection from nationwide clinical cohorts between 2007 and 2011. We also reconstructed the time to the most recent common ancestor (tMRCA) of the predominant HIV-1C strains. Methodology/Principal Findings: Blood samples were collected from 168 HIV-1 seropositive subjects from 7 different states. HIV-1 subtypes were determined using two or three genes, gag, pol, and env using several methods. Bayesian coalescent-based approach was used to reconstruct the time of introduction and population growth patterns of the Indian HIV-1C. For the first time, a high prevalence (10%) of unique recombinant forms (BC and A1C) was observed when two or three genes were used instead of one gene (p<0.01; p = 0.02, respectively). The tMRCA of Indian HIV-1C was estimated using the three viral genes, ranged from 1967 (gag) to 1974 (env). Pol-gene analysis was considered to provide the most reliable estimate 1971, (95% CI: 1965-1976)]. The population growth pattern revealed an initial slow growth phase in the mid-1970s, an exponential phase through the 1980s, and a stationary phase since the early 1990s. Conclusions/Significance: The Indian HIV-1C epidemic originated around 40 years ago from a single or few genetically related African lineages, and since then largely evolved independently. The effective population size in the country has been broadly stable since the 1990s. The evolving viral epidemic, as indicated by the increase of recombinant strains, warrants a need for continued molecular surveillance to guide efficient disease intervention strategies.

Clinical and immunological studies on persons exposed toParthenium hysterophorus L

Studies on 300 persons subjected by occupational hazard to the allergenic weed, Parthenium hysterophorus L. for periods ranging from 3 to 12 months revealed that 4% of them developed contact dermatitis of the exposed parts of the body, while 56% of them got sensitized to the weed without apparently exhibiting any dermatitis. None of them suffered from allergic manifestations like rhinitis or bronchial asthma during the period of study which extended for 2 years.

Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and lsquoshortrsquo RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI+II) or subgroup I specificity and lsquolongrsquo RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.

A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Malaria causes a worldwide annual mortality of about a million people.Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition,our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.

Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples

Antibiotic resistance in 40 Staphylococcus aureus clinical isolates from 110 diabetic patients (36%) was evaluated. Of these, 32 (80%) of the isolates showed multidrug-resistance to more than eight antibiotics and 35% isolates were found to be methicillin resistant S. aureus (MRSA). All 40 S. aureus strains (100%) screened from diabetic clinical specimens were resistant to penicillin, 63% to ampicillin, 55% to streptomycin, 50% to tetracycline and 50% to gentamicin. Where as low resistance rate was observed to ciprofloxacin (20%) and rifampicin (8%). In contrast, all (100%) S. aureus strains recorded susceptibility to teicoplanin, which was followed by vancomycin (95%). Genotypical examination revealed that 80% of the aminoglycoside resistant S. aureus (ARSA) have aminoglycoside modifying enzyme (AME) coding genes; however, 20% of ARSA which showed non-AME mediated (adaptive) aminoglycoside resistance lacked these genes in their genome. In contrast all MRSA isolates possessed mecA, femA genetic determinants in their genome.

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant Staphylococcus aureus Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.

Molecular markers for discriminating Streptococcus pyogenes and S.dysgalactiae subspecies equisimilis

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus,a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Detection,amplification and sequence homology of sodC in clinical isolates of Salmonella sp

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain Salmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonellaenterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X.Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between thesodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028.Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Immediate hypersensitivity to Parthenium hysterophorus. II. Clinical studies on the prevalence of parthenium rhinitis

The airborne pollen of the South American weed, Parthenium hysterophorus (American feverfew), accidentally introduced into India was found to be responsible for severe allergic rhinitis. A random clinical survey conducted on 2035 residents of Bangalore city with the aid of questionnaires and skin tests revealed that 7.1% of the study population was suffering from allergic rhinitis due to exposure to Parthenium pollen. Skin-prick tests performed on 1294 clinic patients suffering from nasobronchial allergy during the past 4 years have also shown that 42.5% were sensitive to Parthenium pollen. IgE and IgG antibodies specific for Parthenium pollen allergens were demonstrable in the sera of Parthenium-sensitive rhinitis patients. The specificity of these antibodies to Parthenium allergens was established by ELISA. A 7- to 11-fold higher stimulation was observed when lymphocytes from two Parthenium-sensitive rhinitis patients were treated in vitro with Parthenium pollen extract. To our knowledge, nowhere in the world has such a high incidence of allergic rhinitis due to a single pollen ever been reported.

Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax. This study is an important step in providing insight into physiology of the parasite under clinical settings.

Immunomodulation using agonists and antagonists: potential clinical applications

Introduction: Extensive studies have gone into understanding the differential role of the innate and adaptive arms of the immune system in the context of various diseases. Receptor-ligand interactions are responsible for mediating cross-talk between the innate and adaptive arms of the immune system, so as to effectively counter the pathogenic challenge. While TLRs remain the best studied innate immune receptor, many other receptor families are now coming to the fore for their role in various pathologies. Research has focused on the discovery of novel agonists and antagonists for these receptors as potential therapeutics. Areas covered: In this review, we present an overview of the recent advances in the discovery of drugs targeting important receptors such as G-protein coupled receptors, TRAIL-R, IL-1 beta receptor, PPARs, etc. All these receptors play a critical role in the modulation of the immune response. We focus on the recent paradigms applied for the generation of specific and effective therapeutics for these receptors and their status in clinical trials. Expert opinion: Non-specific activation by antagonist/agonist is a difficult problem to dodge. This demands innovation in ligand designing with the use of strategies such as allosterism and dual-specific ligands. Rigorous preclinical and clinical studies are required in transforming a compound to a therapeutic.

IDH1 Mutations in Diffusely Infiltrating Astrocytomas Grade Specificity, Association With Protein Expression, and Clinical Relevance

IDH1 mutations are frequent genetic alterations in low-grade diffuse gliomas and secondary glioblastoma (GBM). To validate mutation frequency, IDH1 gene at codon 132 was sequenced in 74 diffusely infiltrating astrocytomas: diffuse astrocytoma (DA; World Health Organization WHO] grade II), anaplastic astrocytoma (AA; WHO grade III), and GBM (WHO grade IV). All cases were immunostained with IDH1-R132H monoclonal antibody. Mutational status was correlated with mutant protein expression, patient age, duration of symptoms, and prognosis of patients with GBM. We detected 31 (41.9%) heterozygous IDH1 mutations resulting in arginine-to-histidine substitution (R132H;CGT-CAT). All 12 DAs (100%), 13 of 14 AAs (92.9%), and 6 of 48 GBMs (12.5%) (5/6 83.3%] secondary, and 1/42 2.4%] primary) harbored IDH1 mutations. The correlation between mutational status and protein expression was significant (P < .001). IDH1 mutation status, though not associated with prognosis of patients with GBM, showed significant association with younger age and longer duration of symptoms in the whole cohort (P < .001). Our study validates IDH1 mutant protein expression across various grades of astrocytoma, and demonstrates a high incidence of IDH1 mutations in DA, AA, and secondary GBM.

Rationalization and prediction of drug resistant mutations in targets for clinical anti-tubercular drugs

Resistance to therapy limits the effectiveness of drug treatment in many diseases. Drug resistance can be considered as a successful outcome of the bacterial struggle to survive in the hostile environment of a drug-exposed cell. An important mechanism by which bacteria acquire drug resistance is through mutations in the drug target. Drug resistant strains (multi-drug resistant and extensively drug resistant) of Mycobacterium tuberculosis are being identified at alarming rates, increasing the global burden of tuberculosis. An understanding of the nature of mutations in different drug targets and how they achieve resistance is therefore important. An objective of this study is to first decipher sequence as well as structural bases for the observed resistance in known drug resistant mutants and then to predict positions in each target that are more prone to acquiring drug resistant mutations. A curated database containing hundreds of mutations in the 38 drug targets of nine major clinical drugs, associated with resistance is studied here. Mutations have been classified into those that occur in the binding site itself, those that occur in residues interacting with the binding site and those that occur in outer zones. Structural models of the wild type and mutant forms of the target proteins have been analysed to seek explanations for reduction in drug binding. Stability analysis of an entire array of 19 mutations at each of the residues for each target has been computed using structural models. Conservation indices of individual residues, binding sites and whole proteins are computed based on sequence conservation analysis of the target proteins. The analyses lead to insights about which positions in the polypeptide chain have a higher propensity to acquire drug resistant mutations. Thus critical insights can be obtained about the effect of mutations on drug binding, in terms of which amino acid positions and therefore which interactions should not be heavily relied upon, which in turn can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. The methodology can serve as a general framework to study drug resistant mutants in other micro-organisms as well.