Aj Kandathil

Usefulness of Alternate Prognostic Serum and Plasma Markers for Antiretroviral Therapy for Human Immunodeficiency Virus Type 1 Infection

ABSTRACT In developing countries, the usability of peripheral blood constituents that are low-cost alternatives to CD4-positive (CD4+) T-cell and human immunodeficiency virus type 1 (HIV-1) RNA estimation should be evaluated as prognostic markers. The aim of our study was to investigate the use of plasma levels of dehydroepiandrosterone sulfate (DHEAS), albumin, and C-reactive protein (CRP) as alternate prognostic markers for antiretroviral treatment (ART) response in place of HIV-1 load measurements. Paired blood samples were collected from 30 HIV-infected individuals before and after initiation of ART, 13 HIV-infected individuals before and after completion of antituberculosis therapy (ATT), and 10 HIV-infected individuals not on either ATT or ART. Because of the nonavailability of samples, the CRP estimation was done for samples from only 19, 9, and 8 individuals in groups 1, 2, and 3, respectively. The measurements of all three markers, i.e., DHEAS, albumin, and CRP, were carried out with commercial assays. The differences in the albumin levels before and after ART or ATT were significant (P < 0.05), while the differences in DHEAS and CRP levels were not significant (P > 0.05). When levels of DHEAS among the individuals who were followed up were analyzed, 13 (44.8%) in the ART group and 9 (69%) in the ATT group showed an increase following treatment. Prior to treatment of HIV-infected individuals, there was a significant positive correlation of CD4+ T-cell counts and a negative correlation of viral load with albumin and DHEAS levels (P < 0.01). Among the three plasma markers we tested, plasma albumin and, to some extent, DHEAS show promise as prognostic markers in monitoring HIV infection.

Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4+ and CD8+ T-Cell Estimation

ABSTRACT An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation (r) of the two systems for CD4+ T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies.

Evidence for lower CD4+ T cell and higher viral load in asymptomatic HIV-1 infected individuals of India: Implications for therapy initiation

PURPOSE We have earlier documented that the south Indian population had lower CD4 counts. The aim of this study was to investigate a previous suggestion on a new CD4+ T cell cut off and association with HIV-1 RNA levels for decision on anti retroviral therapy in India (south). METHODS We evaluated a new methodology i.e., artus real-time PCR and CD4+ T cell count by Guava EasyCD4 system. From 146 HIV infected individuals seen at a tertiary care centre, blood was collected for CD4+ T cell and HIV-1 RNA estimation. RESULTS The receiver operating characteristic curve cut off value for the CD4 counts to distinguish between CDC clinical categories A and B was 243 cells/microL, and to distinguish B and C was 153 cells/microL. The RNA level that differentiated CDC A and B was 327473 RNA copies/mL, while for CDC B and C was 688543 copies/mL. There was a significant negative correlation (r = -0.55, P + T cell counts in HIV infected individuals. CONCLUSIONS A majority with CD4 counts of 201-350 cells/microL in our population had higher viral load than the treatment threshold suggested by the International AIDS society and the above two methodologies are useful in monitoring HIV infections.

Drug resistant mutations detected by genotypic drug resistance testing in patients failing therapy in clade C HIV-1 infected individuals from India

PURPOSE There has been an increase in the number of individuals administered antiretroviral therapy (ART) in India but treatment outcome is hampered by increasing development of drug resistance. Previous reports from India have shown M184V as the commonest mutation in treated individuals. However, there is no evidence for any protease mutations in these reports. This study was done to observe the common/unique mutational patterns observed in reverse transcriptase (RT) and protease (Pr) genes of clade C HIV-1 strains from individuals showing treatment failure in India. MATERIALS AND METHODS The assay was done by sequencing the Pr and RT genes of the HIV-1 strains from 18 individuals failing ART. Analysis was carried out using Stanford HIV drug resistance database (SHDB). The sequences were also submitted to the calibrated population resistance tool of SHDB and Rega HIV-1 sub typing tool. Phylogenetic analysis and quality control were performed with Mega 4. RESULTS Among the 20 strains, 19 showed resistance to both nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), one strain to NNRTIs and five strains showed protease inhibitors (PI) resistance and 3-class resistance. The most common mutation conferring NRTI resistance was M184V (90%) while K103N (45%) was the most common mutation conferring NNRTI resistance. The M46I mutation was seen in 20% of the Pr sequences. CONCLUSION Resistance testing to check the prevalence of drug resistance mutations that arise following failure of the first line regimen to establish guidelines for second line regimens in India is a must. Studies are needed to confirm if mutation patterns that arise among clade C following failure of ART are the same as for clade B strains.

Genotypic resistance profile of HIV-1 protease gene: A preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.

Human Immunodeficiency Virus Infection and Levels of Dehydroepiandrosterone Sulfate in Plasma among Indians

ABSTRACT The shift in cytokine profile during human immunodeficiency virus (HIV) disease progression is influenced by dehydroepiandrosterone sulfate (DHEAS) level. Radioimmunoassay was used to measure plasma DHEAS for 30 treatment-naïve HIV-infected and 30 uninfected individuals. There was a significant negative correlation of viral load with DHEAS level (P < 0.05). Further studies of the use of DHEAS levels for monitoring HIV patients economically are warranted.

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the ’sequence search interface‘ in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.

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.

HIV-1 with Predicted CXCR4 Genotype Identified in Clade C from India

AbstractBackground and objective: HIV-1 uses co-receptors CCR5 and CXCR4 in addition to CD4 for viral entry into cells. CCR5 is used in the early stages of HIV-1 infection, but viruses that utilize CXCR4 for viral entry emerge in the later stages. This is not common among clade C strains, with previous data from India showing the absence of the emergence of CXCR4-using strains. Sequence analysis has demonstrated that the V3 loop plays a very important role in determining the syncytium-inducing (SI) phenotype. The V3 region of the SI variants were observed to have positively charged amino acids at positions 11 and/or 25 and also a overall higher charge. This study looked at co-receptor usage among HIV-1 strains in India from individuals who were antiretroviral therapy (ART) naïve and those not responding to ART. Methods: Amplification and sequencing of the HIV-1 env gp120 V3 region was done on 40 ART-naïve individuals, who were selected for the study based on their CD4 counts, and eight patients who had not responded to ART. The sequences were submitted to Geno2Pheno and Web PSSM. The pol gene sequences of these strains were submitted to the REGA HIV-1 subtyping tool. Results: Forty-seven strains were identified as clade C and one strain as clade A1. Geno2Pheno identified three CXCR4-using strains, and the Web PSSM clade C matrix identified two. Conclusion: We report, for the first time, CXCR4-using strains among HIV-1 clade C strains circulating in India.

HIV reverse transcriptase: structural interpretation of drug resistant genetic variants from India.

The reverse transcriptase (RT) enzyme is the prime target of nucleoside/ nucleotide (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors. Here we investigate the structural basis of effects of drug-resistance mutations in clade C RT using three-dimensional structural modeling. Apropos the expectation was for unique mechanisms in clade C based on interactions with amino acids of p66 subunit in RT molecule. 3-D structures of RT with mutations found in sequences from 2 treatment naïve, 8 failed and one reference clade C have been modeled and analyzed. Models were generated by computational mutation of available crystal structures of drug bound homologous RT. Energy minimization of the models and the structural analyses were carried out using standard methods. Mutations at positions 75,101,118,190,230,238 and 318 known to confer drug resistance were investigated. Different mutations produced different effects such as alteration of geometry of the drugbinding pocket, structural changes at the site of entry of the drug (into the active site), repositioning the template bases or by discriminating the inhibitors from their natural substrates. For the mutations analyzed, NRTI resistance was mediated mainly by the ability to discriminate between inhibitors and natural substrate, whereas, NNRTI resistance affected either the drug entry or the geometry of the active site. Our analysis suggests that different mutations result in different structural effects affecting the ability of a given drug to bind to the RT. Our studies will help in the development of newer drugs taking into account the presence of these mutations and the structural basis of drug resistance.