Diversity of hepatitis C virus infection among HIV-infected people who inject drugs in India.

The availability of generic direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has prompted many low-and-middle-income countries to launch HCV elimination programs. Because the efficacy of some of these generic DAAs varies by HCV viral subtype, information on subtype distribution can contribute important information to these elimination programs. We conducted a cross-sectional serosurvey to characterize HCV subtype diversity among HIV positive people who inject drugs (PWID) across 14 cities in India. Of 801 HIV positive PWID sampled, 639 tested HCV antibody positive (78.9%). Among 105 samples sequenced, genotype 3 (58.1%) was the most commonly observed followed by genotype 1 (36.2%) and genotype 6 (5.7%). Of the genotype 3 infections, 65% were subtype 3a and 35% were subtype 3b. Of the genotype 1 infections, 94% were subtype 1a and 6% were subtype 1b. All genotype 6 samples were subtype 6n. There was some variability in genotype diversity depending on geographic region and PWID epidemic stage with greater diversity observed in older PWID epidemics. One sequence, HY018, did not cluster with any known reference sequences in phylogenetic analysis. Nearly 80% of HIV infected PWID across India are co-infected with HCV, and subtype prevalence and genetic diversity varied by region and PWID epidemic stage. HCV elimination programs in India will need to consider HCV subtype.

Lenalidomide as a novel therapy for gastrointestinal angiodysplasia in von Willebrand disease.

INTRODUCTION: Lenalidomide is a thalidomide analog with anti-angiogenic properties. Previous case reports suggest its efficacy in preventing gastrointestinal bleeding (GIB) secondary to angiodysplasia (AD) in hereditary haemorrhagic telangiectasia and potentially in reversing AD. We present the first case series to explore lenalidomide as a treatment for AD-related GIB in patients with von Willebrand disease (VWD). METHODS: A retrospective chart review was conducted to include patients with VWD, who were evaluated from 2010 to 2013 and who had received lenalidomide to treat recurrent GIB secondary to AD. All patients had failed single-agent use of antifibrinolytic agents. Patients were observed for at least 2 years on therapy. RESULTS: Five patients (3 males; 68.2 ± 4.9 years) with VWD (3 with type 3 and 1 each with types 1 and 2a) and AD were found. Sites of AD included the stomach, duodenum, jejunum and colon. Lenalidomide was started at 5 mg oral daily. Uptitration to 10 and 15 mg in 1 patient each was necessary due to recurrence of GIB. The mean number of endoscopies performed for control of GIB post lenalidomide was significantly lower compared to pretherapy (0.25 vs 5.50; P = .001). Mean bleed-free duration on lenalidomide was 12.6 ± 4.7 months. Three patients have reported no GIB on lenalidomide. CONCLUSION: This case series demonstrates significantly reduced number of endoscopies and increased bleed-free duration with lenalidomide treatment in selected patients with VWD and recurrent GIB from AD. Prospective multicenter trials are needed to further define the role of lenalidomide in the management of GIB from angiodysplasia and VWD.

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications.

Directly observed therapy of sofosbuvir/ribavirin +/- peginterferon with minimal monitoring for the treatment of chronic hepatitis C in people with a history of drug use in Chennai, India (C-DOT).

We assessed the feasibility of field-based directly observed therapy (DOT) with minimal monitoring to deliver HCV treatment to people with a history of drug use in Chennai, India. Fifty participants were randomized 1:1 to sofosbuvir+peginterferon alfa 2a+ribavirin (SOF+PR) for 12 weeks (Arm 1) vs sofosbuvir+ribavirin (SOF+R) for 24 weeks (Arm 2). SOF+R was delivered daily at participant chosen venues and weekly peginterferon injections at the study clinic. HCV RNA testing was performed to confirm active HCV infection and sustained virologic response 12 weeks after treatment completion (SVR12). No baseline genotyping or on-treatment viral loads were performed. Median age was 46 years. All were male and 20% had significant fibrosis/cirrhosis. All self-reported history of injection drug use, 18% recent noninjection drug use and 38% alcohol dependence. Six discontinued treatment (88% completed treatment in each arm). Of 22 who completed SOF+PR, all achieved SVR12 (22/25=88%); 15 of 22 who completed SOF+R achieved SVR12 (15/25=60%; P=.05). Among those completing SOF+R, SVR12 was significantly less common in participants reporting ongoing substance use (36% vs 100%) and missed doses. Active substance use and missed doses did not impact SVR with SOF+PR. Field-based DOT of HCV therapy without real-time HCV RNA monitoring was feasible; however, achieving 100% adherence was challenging. SOF+PR appeared superior to SOF+R in achieving SVR12, even when doses were missed with no discontinuations due to side effects. Further exploration of short duration treatment with peginterferon plus direct-acting antivirals is warranted.

A rapid and low-cost microscopic observation drug susceptibility assay for detecting TB and MDR-TB among individuals infected by HIV in South India.

BACKGROUND: The converging epidemics of HIV and tuberculosis (TB) pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. OBJECTIVE: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS) assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB) directly from sputum specimens, in the Indian setting. MATERIALS AND METHODS: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. RESULTS: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P<0.001). CONCLUSION: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.

Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India.

PURPOSE: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. MATERIALS AND METHODS: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. RESULTS: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. CONCLUSIONS: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.

TNF-alpha inhibits insulin action in liver and adipose tissue: A model of metabolic syndrome.

Several studies suggest that TNF-alpha contributes to the development of insulin resistance (IR). We compared transcriptional profiles of rat H-411E liver cells exposed to insulin in the absence or presence of TNF-alpha. We identified 33 genes whose expression was altered by insulin, and then reversed by TNF-alpha. Twenty-six of these 33 genes created a single network centered around: insulin, TNF-alpha, p38-MAPK, TGFb1; SMAD and STAT1; and enzymes and cytokines involved in apoptosis (CASP3, GADD45B, IL2, TNF-alpha, etc.). We analyzed our data together with other data of gene expression in adipocytes and found a number of processes common to both, for example, cell death and inflammation; intercellular signaling and metabolism; G-Protein, IL-10 and PTEN signaling. Moreover, the two datasets combined generated a single molecular network that further identified PTEN (a phosphatase) as a unique new link between insulin signaling, IR, and apoptosis reflecting the pathophysiology of "metabolic syndrome".

Factors associated with anaemia in HIV-infected individuals in southern India.

Anaemia accelerates disease progression and increases mortality among HIV-infected individuals. Few studies have characterized this problem in developing countries. Haemoglobin values of adults presenting to an HIV tertiary care center in India between 1996 and 2007 were collected (n = 6996). Multivariate logistic regression analysis was performed to examine associations among anaemia, HIV progression and co-morbidities. Overall, anaemia prevalence was 41%. Twenty percent of patients with CD4 counts >500 cells/microL were anaemic, compared with 64% of those with CD4 counts <100 cells/microL (P < 0.001). In multivariate analysis, CD4 count <100 cells/microL (odds ratio [OR]:5.0, confidence interval [CI]:4.0-6.3), underweight body mass index (OR:4.8, CI:3.6-6.5), female gender (OR:3.1, CI:2.8-3.6) and tuberculosis (TB) (OR:1.6, CI:1.4-1.8) were significantly associated with anaemia. In this setting, management of anaemia should focus on antiretroviral therapy, nutritional supplementation and TB control. The high anaemia prevalence among patients meeting criteria for antiretroviral therapy highlights the need for increased access to non-zidovudine nucleoside reverse transcriptase inhibitors in developing countries.

Establishment of T-lymphocyte subset reference intervals in a healthy adult population in Chennai, India.

Estimation of CD4+ T-lymphocytes continues to be an important aspect for monitoring HIV disease progression and response to antiretroviral therapy. Most of the diagnostic laboratories often rely on western text books for CD4+ T-lymphocyte reference values, which could, often be unreliable for usage in local settings. Therefore, we attempted to establish the reference values for T-lymphocyte subsets among healthy adults in a cross-sectional study carried out at the YRG Centre for AIDS Research and Education (YRG CARE) in Chennai, south India, in 213 (84 female and 129 male) healthy, HIV-1/2 seronegative adults as volunteers. Whole blood specimens were processed for CD4+, CD8+ T-lymphocyte estimation and haematological parameters. The established range of CD4+ T-lymphocyte counts for men and women were 383-1347 cells/microl (mean 865 and median 845 cells/microl) and 448-1593 cells/microl (mean 1021 and median 954 cells/microl), respectively. Women had significantly higher absolute CD4+ Tlymphocyte counts (P<0.001) and CD4+:CD8+ T-lymphocyte ratio as compared to men. The established normal range of CD4+ T-lymphocyte % was 21-59 (mean 40.2 and median 40.1). The influence of age was not observed in any of the parameters except CD4+/CD8+ T-lymphocyte ratio with the >45 yr age group. Further studies with greater sample size may be required to define the staging of HIV disease in relation to the normal CD4 T-lymphocyte count in the general population.

Comorbidities among HIV-infected injection drug users in Chennai, India.

BACKGROUND & OBJECTIVE: HIV-infected injection drugs users (IDUs) are known to have high rates of co-infections. A few reports exist on comorbidities among HIV-infected IDUs in India. We carried out a retrospective study to analyse data on comorbidities in India and treatment challenges faced when treating HIV-infected IDUs in India. METHODS: A retrospective chart review of 118 HIV-infected IDUs who accessed care at the YRG Centre for Substance Abuse-Related Research, Chennai, between August 2005 and February 2006 was done. Demographic, laboratory and clinical information was extracted from medical records. Descriptive demographic and clinical characteristics and distributions of comorbidities across CD4 cell count strata were analysed. RESULTS: All IDUs were male with a median age of 35.5 yr. The majority were married with average monthly income less than INR 3000 per month. The prevalence of hepatitis B and C infections were 11.9 and 94.1 per cent, respectively. Other common co-morbidities included oral candidiasis (43.2%), tuberculosis (33.9%), anaemia (22.9%), lower respiratory tract infections (16.1%), cellulitis (6.8%), herpes zoster (9.3%) and herpes simplex (9.3%). Among participants with CD4+ < 200 cells/microl, the prevalence of TB was 60 per cent. INTERPRETATION & CONCLUSION: IDUs in Chennai were commonly co-infected with HBV, HCV and tuberculosis, complicating use of antiretroviral and anti-tuberculous therapy. The current regimens available for the management of HIV and TB in India may need to be re-assessed for IDUs given the potential for increased rates of hepatotoxicity.

Ethnic variation in certain hematological and biochemical reference intervals in a south Indian healthy adult population.

BACKGROUND: We established the biochemical and hematological reference intervals among a south Indian healthy adult population attending an HIV referral centre in Chennai, southern India. METHODS: In a cross sectional study, 213 study subjects (129 male and 84 female) were studied between March and August 2005. All of the parameters were analyzed using standard hematological and biochemical techniques. RESULTS: Certain biochemical (viz. total bilirubin, alanine transaminase, albumin, creatinine, total protein, lipid profile, creatine phosphokinase, uric acid and lactate) and hematological (mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and lymphocyte levels) parameters presented higher upper limits. In addition, the upper limits of white blood cell count, platelet count, hematocrit, red blood cell count and hemoglobin level were low in comparison to the currently reported ranges. CONCLUSION: Ethnic variation in reference intervals was observed in certain biochemical and hematological analytes in a south Indian adult population.

HIV-1 drug resistance among untreated patients in India: Current status.

HAART has dramatically improved survival and quality of life among people living with HIV and AIDS globally. However, drug resistant mutations of HIV are a great challenge to the benefits of HAART. Antiviral resistance can be mediated either by changes in the molecular target of therapy (the primary mechanism observed in HIV-1) or in other viral proteins that indirectly interfere with a drug's activity. Drug resistant mutations easily evolve in the presence of sub-optimal adherence. With the introduction of generic HAART, there has been a steep increase in the number of patients put on HAART in India. It should also be noted that since most patients pay for medications out of their own pockets, interruptions in therapy due to monetary constraints are not uncommon. There is little information on HIV drug resistance in resource constrained settings like India where the predominant circulating HIV-1 sub-type is C. The transmissibility of drug-resistant forms of the virus is also a major concern especially when formulating treatment guidelines. This article reviews published data available on the patterns of HIV-1 drug resistance among treatment naïve in India.

Trends in HIV-related morbidity among patients admitted to a South Indian tertiary hospital between 1997 and 2003.

This paper describes trends in HIV-related morbidity among people living with HIV/AIDS (PLWHA) admitted to a tertiary hospital in Chennai, South India, between 1997 and 2003. Patients comprised HIV-infected men, women and children who had been admitted at least once to YR Gaitonde Centre for AIDS Research and Education (YRGCARE). A non-parametric trends analysis was conducted to observe trends in clinical and demographic parameters and diagnoses at admission over the seven-year period. Among clinical and demographic parameters, we identified a significantly increasing time trend in the use of antiretroviral therapy (p<0.001) and a significant decrease in the mean hemoglobin level (p=0.01). Among diagnoses at admission, we identified a decreasing time trend for admissions due to pulmonary tuberculosis (p<0.001) and increasing trends for admissions due to extra pulmonary tuberculosis (p<0.01), toxoplasmosis (p<0.01), Pneumocystis carinii pneumonia (p=0.02) and anemia (p<0.001). The results indicate a changing pattern among the clinical conditions requiring admission. With increasing proportions of patients initiating highly active antiretroviral therapy (HAART), it is probable that adverse events due to HAART will account for larger proportions of admissions in the years to come, as is being seen in the industrialized countries.

Dried blood spots are an acceptable and useful HIV surveillance tool in a remote developing world setting.

Enzyme-linked immunosorbent assay and Western blot analysis of dried blood spots (DBS) on filter paper have been shown to be as sensitive and specific as analysis of serum, and therefore may be a cost-effective and culturally appropriate HIV seroprevalence tool in remote areas. This study examines the acceptability of DBS in a tropical, rural population from an outpatient clinic in Andhra Pradesh, India, where participation was offered to every fifth patient seeking general medical care between March and April 2001. All 1413 patients approached for the study agreed to participate and provide a DBS for examination. The overall HIV seroprevalence in this sample was 2.8%. Of the participants, 51.7% were male, 93.2% were between the ages of 18 and 40, 85.3% were married, 29.7% were employed, 47.6% had no education and 73.1% resided in a rural setting. In the univariate analysis, history of genital warts (P = 0.01), sexually transmitted disease (P = 0.001), premarital sexual intercourse (P = 0.002), sexual contact with a commercial sex worker (P = 0.003), being employed (P = 0.011) and having more than 10 injections for medical purposes (P = 0.006) all correlated with being HIV-infected. Given the uniform willingness of these clinic attendees to be tested, we conclude that DBS is a useful, cost-effective tool in HIV serosurveillance in a rural, tropical setting.

Mechanisms involved in tumor necrosis factor-alpha induction of insulin resistance and its reversal by thiazolidinedione(s).

Insulin resistance (IR) remains one of the major pathogenic mechanisms for non-insulin-dependent type 2 diabetes mellitus. We have previously modelled IR in H-411E liver cells in culture. In past experiments, we used both labeled glucose uptake, lipogenesis, and stimulation of calmodulin gene expression to quantify the ability of the antidiabetic drugs (pioglitazone and metformin) to reverse tumor necrosis factor-alpha (TNF-alpha)-induced IR in these insulin-treated cells. In these current experiments, H-411E liver cells were rendered IR by a combination of TNF-alpha and insulin. In other experiments, the ability of C2 ceramide (Cer) to inhibit insulin action and induce IR was assessed as well as the phospholipase C inhibitor D609 to reverse IR induced by these TNF-alpha-like agents. C2 Cer, like TNF-alpha, inhibited insulin action. D609 reversed TNF-alpha induced--and to a lesser extent, C2 Cer-induced--IR. At selected times, the cells were also treated with troglitazone (TRG) in 2 groups: (1) 1-time exposure and (2) chronic exposure followed by acute exposure. TRG concentrations ranged from 0.015 to 15.0 micromol/L. Our data demonstrate a powerful effect of TRG in reducing IR and restoring insulin sensitivity in TNF-alpha-treated H-411E cells. Furthermore, pretreatment with TRG, reflecting chronic exposure, as in human clinical use, was more potent than 1-time acute exposure. These data support the efficacy of using thiazolidinediones (TRG) in human type 2 diabetes, and support the use of this cell culture model to further study the effects of thiazolidinediones on TNF-alpha-induced insulin resistance.

Insulin deprivation leads to deficiency of Sp1 transcription factor in H-411E hepatoma cells and in streptozotocin-induced diabetic ketoacidosis in the rat.

Members of the family of Sp transcription factors include Sp1, Sp3, and Sp4 and are important regulators of eukaryotic gene expression. We previously reported that Sp1 mediated stimulation of rat calmodulin I gene expression in response to insulin. To test whether other members of the Sp family are direct targets of insulin action, we compared the levels of Sp1 and Sp3 proteins from nuclear extracts obtained from both insulin-treated and untreated rat hepatoma (H-411E) cells. We demonstrated by Western blot analysis that levels of Sp1 and Sp3 proteins were increased more than 2-fold in the insulin-treated group. Additionally, the up-regulation of both Sp1 and Sp3 transcription factors by insulin was antagonized by tumor necrosis factor-alpha, a known inhibitor of insulin action. Immunohistochemical analysis demonstrated that H-411E cells treated with insulin (10,000 microU/ml) had a marked increase in demonstrable Sp1 in the nucleus compared with cells incubated in insulin-free medium. We extended these in vitro observations to in vivo studies in the streptozotocin-diabetic rat model. We demonstrated in rat liver tissue by both Western blot and immunohistochemical staining with anti-Sp1 antibody that 1) livers of fully diabetic streptozotocin rats have low levels of Sp1 transcription factor; and 2) insulin treatment of the diabetic rat rapidly reversed this process by markedly stimulating accumulation of Sp1 in rat liver. Studies of the signal transduction mechanisms involved in insulin's effect on Sp1 demonstrate a facilitating role for phosphoinositol 3-kinase and an inhibitory role for cyclic nucleotides. In summary, insulin stimulates Sp1 protein, a transcription factor that is shown to regulate calmodulin gene expression and most likely other, as yet untested, genes.

Members of the Sp transcription factor family regulate rat calmodulin gene expression.

We have previously demonstrated that insulin positively regulates transcription of the rat calmodulin (CaM) I gene and that both basal and insulin stimulation of this gene are critically dependent on Sp1. Furthermore, a 392 bp CaM promoter was stimulated by insulin equal to the full promoter but lost activity with deletion of any of the three Sp1 sites (Solomon SS, Palazzolo MR, Takahashi T, Raghow R. Endocrinology 1997;138:5052-5054). Herein we document that Sp1 preferentially binds to the upstream sites Sp1(2) and Sp1(3) but not Sp1(1). Furthermore, gel-mobility super-shift assays demonstrate that both Sp1 and Sp3 protein are found in these complexes. When pPac-Spl, pPac-Sp3, pPac-USp3, and pPac-Sp4 were cotransfected with rCaM 1-392 promoter into Drosophila SL2 cells and challenged with 10,000 microU/mL insulin, we discovered that (1) Sp1 enhanced both basal and insulin-stimulated CaM I gene expression; (2) USp3, a "long" form of the Sp3 molecule, had a stimulatory effect on CaM I gene expression; (3) Sp1 or USp3 is involved in mediating insulin-stimulation of the CaM I gene in SL2 cells; and (4) Sp3, a "short" form of the Sp3 molecule, and Sp4 inhibited Spl-stimulated and insulin-stimulated Sp1-mediated CaM I gene expression. Together these data corroborate and extend our previous observations on Sp1 and elucidate that other members of the Sp family of transcription factors may also be involved in regulating the activity of the CaM promoter.