Characteristics of treatment-naïve HBV-infected individuals with HIV-1 coinfection: A cross-sectional study from South India

Purpose: Human immunodeficiency virus-1 (HIV-1) and hepatitis B virus (HBV) coinfection has become a major health problem across the globe. The increased life expectancy of HIV-1 patients due to antiretroviral therapy has led to the emergence of liver disease as a major mortality factor among them. The purpose of the study was to examine the baseline characteristics of HBV in treatment-naïve HBV/HIV coinfection from southern India compared to monoinfected individuals. Materials and Methods: The study was cross sectional in design, and samples were examined from 80 HIV-1, 70 HBV and 35 HBV/HIV-coinfected individuals using chemiluminescent microparticle immunoassay, real-time polymerase chain reaction and flow cytometry assays. Results: There was a significant increase in HBV DNA (P = 0.0001), higher hepatitis B e antigen percentage difference (P = 0.027) and lower CD4 counts (P = 0.01) among the HBV/HIV-coinfected individuals, but no difference in the HIV-1 viral load compared to HIV-1-monoinfected individuals. Also, the aspartate aminotransferase levels, prothrombin time and the international normalised ratio were significantly high among coinfected individuals. Conclusion: These findings conclude that HIV-1 coinfection can have serious implications on the outcome of HBV-related liver disease. To the contrary, HBV infection had no consequence on the progression of HIV-1 disease but distinctly lowered CD4+ T-cells.

BK virus characterisation among HIV-1-Infected individuals and its association with immunosuppression

Purpose: BK virus (BKV) is an opportunistic pathogen which causes significant morbidity and mortality in individuals who are immunodeficient. We aimed to quantitate and characterise BKV and to correlate with the degree of immunosuppression among human immunodeficiency virus (HIV)-1-infected individuals. Methods: BKV DNA detection was carried out using an in-house quantitative real-time polymerase chain reaction on paired whole-blood and urine samples collected from 187 antiretroviral therapy (ART)-naïve HIV-1-infected individuals and 93 healthy individuals who served as controls. Sequencing was performed for a proportion of high BK viral load (VL) samples to observe non-coding control region (NCCR) rearrangements. Results: BKV positivity in urine was 25.6% among HIV-infected individuals and 10.7% in control individuals (P = 0.03). The BK VL showed a significant negative correlation with CD4+ T-cell counts, a positive correlation with WHO clinical staging and no significant correlation with HIV-1 VL. Of 42 BKVs from urine samples sequenced, two showed rearrangements without clinically severe disease or high VL. Their NCCR and VP1 sequence-based genotyping revealed genotype I. In a small subset of individuals (n = 8) on ART who were being followed up, six individuals showed either decrease or complete clearance of virus with ART. Conclusion: There was a higher frequency of BK viruria in HIV-1-infected individuals than among healthy controls and the positivity correlated with the degree of immunosuppression. There was no association of high VL with NCCR rearrangements in urine.

Expression of cytokine‑mRNA in peripheral blood mononuclear cell of human immunodeficiency virus‑1 subtype C infected individuals with opportunistic viral infections from India (South).

Human immunodeficiency virus (HIV) disease progression is associated with a marked change in the level of plasma cytokines. The study reported here investigated the level of mRNA expression of different cytokines: Tumour necrosis factor‑alpha (TNF‑α), interferon (INF)‑gamma, interleukin‑10 (IL‑10) and IL‑21 in the peripheral blood mononuclear cell among the antiretroviral therapy naive subtype C HIV‑1 infected individuals and normal healthy controls by real time polymerase chain reaction. The mRNA expressions of all the 4 cytokines in HIV‑1 infected individuals were significantly higher compared to healthy controls (P value range 0.0004–0.01). The mean level of IL‑10, INF‑gamma and TNF‑α were higher in HIV infected individuals with low CD4 counts (<300 cells/μl). The IL‑10 expression showed a significant negative correlation with CD4 counts (r = −0.25, P = 0.04) while IL‑21 showed a positive correlation with CD4 counts (r = 0.26, P = 0.03). There was a significant negative correlation between the cytomegalovirus (CMV) viral load and IL‑21 expression. Cytokine levels by mRNA detection avoids the inherent problem of measuring plasma level and this study also provide information on the cytokine levels and CD4+ T cell level among HIV‑1 subtype C infected individuals with opportunistic viral infections like CMV.

Frequency of Epstein - Barr virus infection as detected by messenger RNA for EBNA 1 in histologically proven gastric adenocarcinoma in patients presenting to a tertiary care center in South India.

Background: Epstein–Barr virus (EBV)‑associated gastric carcinoma is a relatively uncommon entity detected in approximately 10% of gastric adenocarcinoma. Objective: The purpose of this study is to estimate the frequency of EBV‑associated gastric carcinoma and also to assess the nature of presentation, any significant difference between this subgroup and EBV‑negative gastric adenocarcinomas with respect to age and sex predilection, lymph nodal status, site of presentation. Materials and Methods: We prospectively analyzed 100 cases of gastric adenocarcinoma who underwent either a partial or total gastrectomy during the period from March 2010 to August 2011. The tumour and the corresponding normal gastric tissue from the same patient were analyzed for the presence of Epstein–Barr nuclear antigen 1 (EBNA1) messenger ribonucleic acid (mRNA) by real‑time polymerase chain reaction (PCR). Result: EBV was detected in 6% cases of gastric adenocarcinoma. All the positive patients were males. The majority of cases involved the proximal stomach and there was variable lymph nodal involvement. Conclusion: Our study endorses that there is an association between EBV infection and gastric adenocarcinoma in the Indian population. There was no significant difference between this subgroup and EBV‑negative gastric adenocarcinomas with respect to age and sex predilection, lymph nodal status and site of presentation. Short‑term follow‑up of this subgroup of patients seems to indicate a good overall prognosis after appropriate treatment. However, a larger study with long‑term follow‑up is needed to further establish the role of EBV in gastric adenocarcinoma in this study population.

Emergence of HIV‑1 drug‑resistant variants in women following antiretroviral prophylaxis for the prevention of mother to child transmission.

Purpose: Emergence of drug resistance following HIV prophylaxis has an important impact on ART program. Objective: To investigate the emergence of drug resistance in HIV‑1 infected pregnant women. Materials and Methods: Fifty‑three HIV‑1 infected pregnant women who had received 4‑12 weeks of antenatal AZT followed by Nevirapine during delivery and Combivir [AZT + 3TC] for 1 week postpartum (group‑1, n = 48) or who come at the time of delivery and received Nevirapine followed by Combivir for 1 week (group‑2, n = 5) were recruited. Samples were collected prior to the start of the prophylaxis and 5‑8 weeks postpartum. In addition, a third sample was collected between 26‑65 weeks postpartum from 7 women. Amplification of HIV‑1 pol gene and drug resistance analysis was done. Result: Two (3.8%) women in group‑1 showed transmitted drug resistance and they continued to show this even at 6 weeks postpartum. One (2%) woman from group‑1 showed a mutation after 6‑8 weeks of prophylaxis. Among the samples collected between 26‑65 weeks postpartum, 3/7 (43%) showed mutations and all these women belong to group‑1. Women belonging to group‑2 didn’t show mutation prior to or following prophylaxis. Conclusion: In contrast to the available data among pregnant women with ART prophylaxis, our data showed reduced frequency of mutations following 5‑8 weeks of postpartum but an emergence of mutation later (26‑65 weeks). The addition of Combivir with the single dose Nevirapine during delivery and the early stage of the disease with higher CD4 counts could be the reasons for this.

Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals.

Purpose: Opportunistic viral infections are one of the major causes of morbidity and mortality in HIV infection and their molecular detection in the whole blood could be a useful diagnostic tool. Objective: The frequency of opportunistic DNA virus infections among HIV-1-infected individuals using multiplex real-time PCR assays was studied. Materials and Methods: The subjects were in two groups; group 1: Having CD4 counts <100 cells/μl (n = 118) and the group 2: counts >350 cells/μl (n = 173). Individuals were classified by WHO clinical staging system. Samples from 70 healthy individuals were tested as controls. In-house qualitative multiplex real-time PCR was standardised and whole blood samples from 291 were tested, followed by quantitative real-time PCR for positives. In a proportion of samples genotypes of Epstein-Barr virus (EBV) and CMV were determined. Results: The two major viral infections observed were EBV and CMV. The univariate analysis of CMV load showed significant association with cryptococcal meningitis, oral hairy leukoplakia (OHL), CMV retinitis, CD4 counts and WHO staging (P < 0.05) while the multivariate analysis showed an association with OHL (P = 0.02) and WHO staging (P = 0.05). Univariate analysis showed an association of EBV load with CD4 counts and WHO staging (P < 0.05) and multivariate analysis had association only with CD4 counts. The CMV load was significantly associated with elevated SGPT and SGOT level (P < 0.05) while the EBV had only with SGOT. Conclusion: This study showed an association of EBV and CMV load with CD4+ T cell counts, WHO staging and elevated liver enzymes. These viral infections can accelerate HIV disease and multiplex real-time PCR can be used for the early detection. Genotype 1 and 2 of EBV and genotype gB1 and gB2 of CMV were the prevalent in the HIV-1 subtype C-infected south Indians.

Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South).

Purpose: The use of dried blood spots (DBS) for HIV-1 viral load determination could greatly enhance the management of HIV infected individuals in resource-limited countries. Objective: To compare the HIV-1 viral load values obtained between parallel collected plasma and DBS. Materials and Methods: DBS and plasma samples were collected from 62 HIV-1 infected individuals and were used for determination of HIV-1 RNA concentrations using the Abbot real-time HIV-1 PCR. Result: Mean of the log difference of viral load values between plasma and DBS was -0.41 log. DBS viral load values significantly correlated with plasma viral load (r = 0.9818, P < 0.0001). Conclusion: These results suggest that DBS samples can be used as an alternative to plasma for the estimation of HIV-1 viral load if samples are appropriately stored.

Evaluation of a dry format reagent for CD4+ and CD8+ T-cell enumeration with FACSCount and Guava polymerase chain reaction.

Purpose: In all CD4+/CD8+ T-cell estimation systems, the reagents used are liquid in nature and have to be transported and stored at 2°-8°C. This causes problems in countries where the ambient temperature is high for most parts of the year or where the laboratories are at remote places. Materials and Methods: We evaluated a dry format of CD4/CD8 reagents from ReaMetrix (Bangalore, India) against the existing liquid reagents from Becton Dickinson (San Jose, CA, USA) and Guava PCA system (Guava Technologies, Hayward, CA, USA). Blood samples collected during March 2009 through May 2009 from 102 HIV-infected individuals and 31 normal healthy individuals in a tertiary care centre in India (south) were tested by Guava; EasyCD4™ System (PCA) and FACSCount using the respective reagents and the corresponding ReaMetrix reagents. Results: Overall, the correlation (r) of the new Rea T Count and FACSCount reagents for the CD4+ T-cell estimation was 0.98, while with ReaPan 3 4 G reagent in the Guava PCA system with the Guava reagent was 0.97. The mean bias for CD4+ T-cell measurements between Rea T count and BD reagent was -6 cells/ml, while the same with ReaPan 3 4 G reagent in the Guava PCA system was 78 cells/ml. The mean bias for the Rea T count and the ReaPan 3 4 G reagent tested in the FACSCount and Guava PCA system was 17 cells. Conclusions: The dry reagents were found to be reliable and cheaper compared to the existing liquid reagents. This allows the transportation of reagents in the absence of cold chain and will facilitate a more user-friendly CD4+ T-cell testing system.

Immune responses to Epstein-Barr virus in individuals with systemic and organ specific autoimmune disorders.

Purpose: Autoimmune diseases usually manifest in genetically predisposed individuals following an environmental trigger. There are several viral infections including Epstein-Barr virus (EBV) implicated in the pathogenesis of autoimmune disorders. The aim of this study was to look at the antibody pattern to EBV proteins in the plasma of both systemic and organ specific autoimmune disorders, estimate pro-inflammatory plasma cytokines (IL-8 and TNF-á) among these autoimmune patients and compare the observations with those in normal healthy controls. Materials and Methods: Samples from 44 rheumatoid arthritis patients, 25 Hashimoto's thyroiditis patients, appropriately age and sex matched healthy controls were tested for EBV IgM antibodies by an immunoblot assay and two cytokines (IL-8 and TNF-á) by commercial assays. Results: Among the rheumatoid arthritis patients, 23 (52%) were positive for EBNA1 antibody, while 13 (52%) of the Hashimoto's thyroiditis patients and 12 (30%) of the healthy controls showed similar bands. The intensity of the bands was high in the autoimmune patients when compared to the bands seen in control samples. The difference in the EBNA1 reactivity between rheumatoid arthritis patients and controls were significant (P = 0.038). There was a significant difference in the IgM reactivity to VCAp19 protein between patients and controls (P = 0.011). Conclusion: Our study showed an increased EBV activation among the autoimmune patient groups compared to the normal healthy controls. Further studies are required to delineate the association between the aetiology of autoimmune disorders and EBV.

Detection of opportunistic DNA viral infections by multiplex PCR among HIV infected individuals receiving care at a tertiary care hospital in South India.

Purpose: Opportunistic viral infections cause increased morbidity and mortality among human immunodeficiency virus (HIV) infected individuals, especially those who are not on antiretroviral treatment. Early diagnosis of these opportunistic viruses will be able to reduce the risk of disease progression with appropriate intervention. Materials and Methods: Multiplex PCR was attempted to detect the opportunistic herpes viruses (HSV-1, HSV-2, VZV, EBV, and CMV), adenovirus and polyoma viruses (JC and BK) in three cocktails of PCR reactions. Subsequently, all the viruses detected were quantitated by testing using monoplex real time PCR. Whole blood samples collected between 2006 and 2007 from 68 treatment naοve HIV-1 infected and 30 normal healthy individuals were tested for these eight viruses. Among the 68 HIV -1 infected individuals 35 had CD4+ T cell count less than or equal to 200 while the other 33 had greater than 200 CD4+ T cells. Results: Among the 68 HIV-1 infected individuals, 49 (72%) were positive for EBV, 5 (7%) samples were positive for CMV. All the five CMV positive individuals had CD4+ T cell count of less than or equal to 200 cells/µL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/µL was 3.88 log 10 while among those with greater than 200 CD4+ T cells it was 3.75 log 10 . The mean CMV load was 6.98 log 10. Three samples were positive for both CMV & EBV. None of the samples was positive for HSV-1, HSV-2, VZV, Adenovirus, JC and BK viruses. Conclusions: In our study, multiplex PCR based detection system was found useful in detecting opportunistic viruses in HIV infected individuals. Though EBV is the most prevalent opportunistic viral infection among HIV infected individuals, there was no significant association between EBV load, CD4+ T cell counts and HIV-1 virus load. CMV was seen in HIV infected individuals with low CD4+ T cell counts (less than 200 cells/μL).