Occult Hepatitis B Virus infection in ART-Naive HIV-Infected Patients seen at a Tertiary Care Centre in North India

<p>Abstract</p> <p>Background</p> <p>Co-infections of hepatitis B and C viruses are frequent with HIV due to shared routes of transmission. In most of the tertiary care health settings, HIV reactive patients are routinely tested for HBsAg and anti-HCV antibodies to rule out these co-infections. However, using the routine serological markers one can only detect active HBV infection while the occult HBV infection may be missed. There is insufficient data from India on HIV-HBV co-infection and even scarce on occult HBV infection in this group.</p> <p>Methods</p> <p>We estimated the burden of HBV infection in patients who were tested positive for HIV at a tertiary care centre in north India. We also attempted to determine the prevalence and clinical characteristics of occult HBV infection among these treatment-naïve patients and compare their demographic features with other HIV patients. During a period of 6 years between January 2002 to December 2007, 837 HIV positive patients (631 males and 206 females (M: F :: 3.06:1) were tested for serological markers of HBV (HBsAg) and HCV (anti-HCV antibodies) infections in our laboratory. For comparison 1000 apparently healthy, HIV-negative organ donors were also included in the study. Data on demographics, sexual behaviour, medical history, laboratory tests including the serum ALT and CD4 count of these patients were recorded. A sub-group of 53 HBsAg negative samples from HIV positive patients were assessed for anti-HBs, anti-HBc total (IgG+IgM) and HBV-DNA using a highly sensitive qualitative PCR and analysed retrospectively.</p> <p>Results</p> <p>Overall, 7.28% of HIV positive patients showed presence of HBsAg as compared to 1.4% in the HIV negative control group. The prevalence of HBsAg was higher (8.55%) in males than females (3.39%). The study revealed that occult HBV infection with detectable HBV-DNA was prevalent in 24.5% of patients positive for anti-HBc antibodies; being 45.5% in HBsAg negative patients. Most importantly the occult infection was seen in 20.7% patients who were positive for anti-HBs antibodies. However, in none of the seronegative patient HBV-DNA was detected. Five of the nine HBV-DNA positive (55.6%) patients showed raised alanine aminotransferase levels and 66.7% had CD4⁺T cell counts below 200 cells/cumm.</p> <p>Conclusions</p> <p>High prevalence of HIV-HBV co-infection was found in our patients. A sizeable number of co-infected patients remain undiagnosed, if only conventional serological markers are used. Presence of anti-HBs antibodies was not a reliable surrogate marker to rule out occult HBV infection. The most reliable method to diagnose occult HBV co-infection in HIV seropositive patients is the detection of HBV-DNA.</p

Evaluation of anti-leishmanial and antibacterial activity of Waldheimia tomentosa (Asteraceae), and chemical profiling of the most bioactive fraction

Purpose: To evaluate the anti-leishmanial and antibacterial activities of a relatively unexplored whole plant of Waldheimia tomentosa (Asteraceae) and the chemical profiling of its most bioactive fraction.Methods: The whole plant material was extracted with methanol - water (9 : 1) and fractionated into nhexane (C6H14 or n-Hex), dichloromethane (CH2Cl2 or DCM), ethyl acetate (C4H8O2 or EtOAc) fractions and aqueous residue. The fractions were screened for leishmanicidal activity against promastigotes and intracellular amastigotes of L. donovani, while antibacterial activity was evaluated against four multidrug resistant (MDR) clinical isolates by bioassay guided fractionation. Scanning Electron Microscopy (SEM) performed on S. aureus at the minimum inhibitory concentration (MIC). Chemical profiling of the most bioactive fraction was performed using gas chromatography-mass spectrometry (GC-MS).Results: The most significant leishmanicidal activity was exhibited by n-Hex fraction against promastigotes (DD8 strain) with half maximal inhibitory concentration (IC50 of 89.85 ± 0.84 μg/mL) and intracellular amastigotes (IC50 of 48.3 ± 0.40 μg/mL). The same fraction also exhibited maximum potency against S. aureus and E.coli at MIC of between 62.5 and 125 μg/mL. The fraction comprised mainly fatty acids and alkyl ketones. SEM examination performed on S. aureus at MIC revealed swelling and multiple blisters on cell surface compared to untreated control.Conclusion: The profound antibacterial activity of Waldheimia tomentosa justifies the use of the plant in traditional medicine for stomach ache and food preservation.Keywords: Waldheimia tomentosa, Antibacterial activity, Leishmanicidal activity, Stomach ache, Food preservatio

Evolution of M. bovis

BCG vaccine is usually considered to be safe though rarely serious complications have also been reported, often incriminating contamination of the seed strain with pathogenic Mycobacterium tuberculosis. In such circumstances, it becomes prudent to rule out the contamination of the vaccine seed. M. bovis BCG can be confirmed by the absence of nitrate reductase, negative niacin test, and resistance to pyrazinamide and cycloserine. Recently in India, some stocks were found to be niacin positive which led to a national controversy and closer of a vaccine production plant. This prompted us to write this review and the comparative biochemical and genotypic studies were carried out on the these contentious vaccine stocks at the Indian vaccine plant and other seeds and it was found that some BCG vaccine strains and even some strains of M. bovis with eugenic-growth characteristics mainly old laboratory strains may give a positive niacin reaction. Most probably, the repeated subcultures lead to undefined changes at the genetic level in these seed strains. These changing biological characteristics envisage reevaluation of biochemical characters of existing BCG vaccine seeds and framing of newer guidelines for manufacturing, production, safety, and effectiveness of BCG vaccine

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

<p>Abstract</p> <p>Background</p> <p>Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance.</p> <p>Objective</p> <p>To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM.</p> <p>Methods</p> <p>A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM).</p> <p>Results</p> <p>Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). <it>M. mucogenicum </it>was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 – 100%.</p> <p>Conclusion</p> <p>Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.</p

Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India

Objectives: Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis. Methods and results: Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate. Conclusions: Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions

Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis sensitive and resistant Beijing type from a patient with pulmonary tuberculosis.

Abstract Aim & objective In India, tuberculosis (TB) is a foremost health problem, and the emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis ( M. tuberculosis ) has further complicated the situation. Although various mechanisms have been proposed to elucidate the emergence of resistance, our knowledge remains insufficient. The formation of a very complex network and drugs of proteins are countered by their efflux/modification or target over-expression/modification. The analysis of the over-expressed proteins and their qualitative and phenotypic evaluation before and after the development of drug-resistance may be the most appropriate tool to understand the mechanisms of the mechanism of development of drug-resistance. Most studies are performed on distinct strains. Therefore, the objective of this study was to compare the proteomic information of sequential isolates of M. tuberculosis Beijing type from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy. Methods In this study, a clinical isolate of M. tuberculosis was grown in Middlebrook 7H9 broth medium for 2 weeks, and the cell lysate of isolates was prepared by sonication and centrifugation. We compared and analyzed the whole cell lysate proteins of M. tuberculosis sequential clinical isolate from a patient with pulmonary TB before and after the development of drug resistance using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and bioinformatics tools. Results The genotypes of both isolates remained homologous, showing no re-infection. The first isolate (before treatment) was sensitive to all the first-line drugs, sequential isolate was found resistant to rifampicin (RIF) and isoniazid (INH) and developed mutations in r poB , katG and inhA . The concentrations of 17 protein spots were found to be consistently over-expressed in RIF- and INH-resistant isolates. The most prominent and over-expressed proteins found during the development of drug resistance were wag31, Rv2714, GarA, SSB, FabG4, Probable lipase, Rv3924c, Rv3204A, Rv2031c, Rv3418c and GroES. The InterProScan and homology searches generated insights into the possible functions and essential domains of the proteins. Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c showed significant in silico interaction with RIF and INH; thus, the over-expression in the drug-resistant isolates could be compensating the inhibited/modulated molecules. Other proteins, which are over-expressed but do not unveil good binding with drug, might be indirectly associated with RIF and INH. Conclusions This proteomic study provides an understanding about the proteins that are over-expressed during the development of drug resistance. These over-expressed proteins, identified here, could prove useful as vaccine candidate, immunodiagnostic and possibly drug-resistant or chemotherapeutic markers in future

Proteomic analysis of heparin-binding proteins from human seminal plasma: a step towards identification of molecular markers of male fertility

Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identified these proteins. Forty different types of proteins were identified. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identified 8% of proteins with unknown functions, although their expression in seminal fluid has been documented. Proteins of seminal fluid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future

Isolation and identification of Concanavalin A binding glycoproteins from human seminal plasma: A step towards identification of male infertility marker proteins

Abstract. Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma

Genome-wide Analysis of the Host Intracellular Network that Regulates Survival of Mycobacterium tuberculosis

SummaryWe performed a genome-wide siRNA screen to identify host factors that regulated pathogen load in human macrophages infected with a virulent strain of Mycobacterium tuberculosis. Iterative rounds of confirmation, followed by validation, identified 275 such molecules that were all found to functionally associate with each other through a dense network of interactions. This network then yielded to a molecular description of the host cell functional modules that were both engaged and perturbed by the pathogen. Importantly, a subscreen against a panel of field isolates revealed that the molecular composition of the host interface varied with both genotype and the phenotypic properties of the pathogen. An analysis of these differences, however, permitted identification of those host factors that were invariantly involved, regardless of the diversification in adaptive mechanisms employed by the pathogen. Interestingly, these factors were found to predominantly function through the regulation of autophagy

Seroprevalence of HIV in pregnant women in North India: a tertiary care hospital based study

Abstract Background Estimating the seroprevalence of HIV in a low risk population such as pregnant women provides essential information for an effective implementation of AIDS control programmes, and also for the monitoring of HIV spread within a country. Very few studies are available from north India showing the current trend in HIV prevalence in the antenatal population;which led us to carry outthis study at a tertiary care hospital in north India Methods Blood samples from pregnant women attending antenatal clinics at the All India Institute of Medical Sciences, New Delhi were collected after informed consent and pre-test counseling. The samples were tested for HIV antibodies as per the WHO guidelines, over a period of four years from January 2003 to December 2006. Results Of the 3529 pregnant women tested in four years, 0.88% (CI 0.5 – 1.24) women were found to be HIV seroreactive. Majority of the seroreactive pregnant women (41.9%) were in the age group of 20–24 years followed by the 30–34 yrs (25.8%) and 25–29 years (22.6%) age group. The mean age of the HIV positive women was 24.9 years (SD ± 1.49 yrs). The HIV seroprevalence rates showed an increasing trend from 0.7% (CI 0.14 – 2.04) in 2003–2004 to 0.9% (CI 0.49 – 1.5) in 2005–2006. This prevalence rate indicates concern, as Delhi and its adjoining states are otherwise considered as 'low prevalence states'. Conclusion Seroprevalence of HIV infection was found to be increasing in the last four years amongst pregnant women of North India. These findings are in contrast to the national projections.</p