J Sachithanandham

Association of Neurotropic Viruses in HIV-Infected Individuals Who Died of Secondary Complications of Tuberculosis, Cryptococcosis, or Toxoplasmosis in South India

ABSTRACT The frequencies of 10 opportunistic DNA viruses were determined by multiplex real-time PCR in paired cerebrospinal fluid (CSF) and brain tissue of HIV-infected individuals. In the CSF, viruses were detectable in 45/55 cases: JC virus (JCV) in 62%, Epstein-Barr virus (EBV) in 44%, cytomegalovirus (CMV) in 25%, varicella-zoster virus (VZV) in 3.6%, herpes simplex virus 1 (HSV-1) in 1.8%, and human herpesvirus 6 (HHV-6) in 1.8% of cases. A single virus was detectable in 20 cases, 19 cases had coinfection with two viruses, and 6 cases were positive for three viruses. JCV was detectable in the CSF of 62% of cases and in 42% of brain tissues, with higher loads in progressive multifocal leukoencephalopathy (PML) (P < 0.05).

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 naïve 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/microL. The mean EBV load among the individuals with a CD4+ T cells of less than or equal to 200 cells/microL 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/microL).

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.

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-alpha) among these autoimmune patients and compare the observations with those in normal healthy controls. MATERIALS AND METHODS Samples from 44 rheumatoid arthritis patients, 25 Hashimotos 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-alpha) by commercial assays. RESULTS Among the rheumatoid arthritis patients, 23 (52%) were positive for EBNA1 antibody, while 13 (52%) of the Hashimotos 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.

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.

Daily quality control in CD3+ and CD4+ T cell estimation by the FACSCount system at a tertiary care center in south India.

ABSTRACT CD4+ T cell count estimations are subject to high variations; hence, in this study, the previous days tested samples were included routinely as the internal quality controls. The percentages of variation of the 2-day values were analyzed for 280 observations and the mean variation for CD4+ and CD3+ T cell counts ranged from 5.21% to 9.66%. This method is a good internal quality control (IQC) procedure for the estimation of CD3+ and CD4+ T cell counts in resource-poor settings.

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.

Experience with a fourth generation human immunodeficiency virus serological assay at a tertiary care centre in south India

VDRL and TPHA test was 1.3% (2/150). This is lower than the prevalence rates reported in studies carried out in Delhi (6.09%),[2] Germany (3.3%) and Bangladesh (23%).[3] The possible explanation for this could be the difference in the subgroup of population studied and the diagnostic tests employed. In addition, region, gender, ethnic factors and socioeconomic factors which inß uence the development of sexual behaviour do play a big role in the prevalence of syphilis.

The performance of reverse transcriptase assay for the estimation of the plasma viral load in HIV-1 and HIV-2 infections

Abstract Viral load testing for human immunodeficiency virus 1 (HIV-1) in resource-poor settings continues to be a challenge. Although antiretroviral therapy (ART) is being made available in developing countries, monitoring of viral load is not being done on a regular basis. The purpose of this study was to assess the utility of Cavidi version 3.0, which measures the plasma reverse transcriptase (RT) activity and compare its performance with molecular HIV viral load assays. In all, 125 HIV-1 and 13 HIV-2 positive samples were analyzed. The overall sensitivity of the assay was 86.8% and 94.1% for viral load >1000 copies/ml measured by Qiagen Artus HIV-1 RG RT PCR and Abbott RealTime HIV-1 PCR assays, respectively. Compared with the routine molecular viral load assays, Cavidi version 3.0 is inexpensive, user-friendly, the expenditure on infrastructure is minimal, and it can be used for monitoring of both HIV types.