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

Challenges and Pragmatic Solutions for Assessing the Reliability of HIV-1 Viral Load Monitoring in Resource-Constrained Settings

Introduction: HIV-1 RNA detection is the most reliable method for monitoring treatment response among people living with HIV. Effective quality control measures that include internal quality control (IQC) are challenging in resource-constrained settings. Methods: We ascertained the utility of the kit low positive control (LPC) as an effective IQC to monitor the reliability of the HIV-1 viral load assay. Variations in LPC values were measured for 390 different runs over 10 years (2011–2021) and compared to in-house IQC data using Levey-Jennings control chart. Results: Overall, the Levey-Jennings analysis showed minimal variation (±0.5 log) for both the LPC and IQC data. The mean LPC value for first 20 runs (20 days) was 2.91. The mean LPC value for the 390 runs comprising 35 different lots was 3.01 ± 0.1 log. Conclusion: Our decadal data reveal that Abbott RealTime HIV-1 assay (Abbott Molecular Inc., IL, USA) LPC exhibited no significant biological variation over 390 runs distributed over 10 years. Hence, assay LPC can supplant the IQC for monitoring assay trends as a stable and commutable material in resource-constrained settings

Comprehensive Genetic and Epigenetic Analysis of Occult Hepatitis B from Liver Tissue Samples

Background. Occult infection with hepatitis B virus (HBV) is a type of chronic HBV infection that is characterized by the absence of a detectable hepatitis B surface antigen in the blood and by very low levels of HBV DNA in the blood and liver. The mechanisms leading to occult HBV infection remain poorly understood but include possible genetic mutations and deletions. Recently, it has been shown that HBV has CpG islands that are methylated, raising the possibility that epigenetic changes may also be important

Performance of a Rapid Immunochromatographic Screening Test for Detection of Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2: Experience at a Tertiary Care Hospital in South India

The performance characteristics of a rapid immunochromatographic-screening test, SD Bioline HIV-1/2 3.0 (Standard Diagnostics Inc., Kyonggi-do, South Korea) on 23,754 sera and 30 plasma samples are reported. The sensitivity and specificity for the assay on serum samples are 100% and 99.4%, respectively. The assay detected antibodies in individuals infected with human immunodeficiency virus type 1 (HIV-1) genotypes A and C and HIV-2. This straightforward assay is a reliable diagnostic tool for screening HIV in resource-poor settings

Role of polymerase chain reaction in the diagnosis of Trichomonas vaginalis infection in human immunodeficiency virus-infected individuals from India (South)

Background: Trichomonas vaginalis is a protozoan parasite and an etiological agent for trichomoniasis, a sexually transmitted infection (STI). Fifty to eighty percentage of women with trichomoniasis are asymptomatic and in the absence of treatment the infection persists longer. Aim: To evaluate the role of polymerase chain reaction (PCR) in the diagnosis of trichomoniasis and also to look at the frequency of infection among human immunodeficiency virus (HIV) infected women. Methods: A non-nested PCR was standardized to detect 102 bp size amplified product of the adhesin gene of T. vaginalis. The real time performance of this assay was performed with vaginal swab samples from 198 HIV-seropositive women who attended the infectious disease clinic and compared with wet mount and culture in Diamond′s modified media. Results: Among the prospectively studied 198 HIV-infected women, 1 (0.51%) was positive by wet mount, 6 (3.03%) were positive by culture and 10 (5.02%) were positive by the PCR. There was a significant observed agreement between the PCR and culture (k=0.74, Z=10.7, P<0.0000). Conclusion: Our study showed that the PCR assay for the amplification of adhesion gene is a highly sensitive method to screen the high risk group individuals like HIV-positive women for Trichomonas vaginalis compared to the culture. Testing algorithm should be, wet mount and if negative, test by PCR as it is rapid compared to culture which takes 7 days

Hepatitis B Virus Replication Induces Methylation of both Host and Viral DNA▿

Control of viral replication is a major therapeutic goal to reduce morbidity and mortality from chronic hepatitis B virus (HBV) infection. Recently, methylation has been identified as a novel host defense mechanism, and methylation of viral DNA leads to downregulation of HBV gene expression. To better understand the mechanisms of HBV methylation, cell lines were exposed to HBV using a model system that mimics natural infection and the expression of host DNA methyltransferase genes (DNMTs) was measured. DNMT1, DNMT2, and DNMT3 were all significantly upregulated in response to HBV. DNMT3 was further studied because of its known role in the de novo methylation of DNA. Cotransfection experiments with full-length HBV and DNMT3 led to the downregulation of viral protein and pregenomic RNA production. To investigate whether the upregulation of DNMTs could also have an effect on the methylation of host DNA, cell lines were exposed to HBV in two independent model systems, one that mimics natural infection and a second model with temporary transfection. Host DNA methylation was measured by DNA microarray analysis. Increased methylation of host CpG islands was detected in both experimental systems. Two CpG islands, corresponding to genes SUFU and TIRAP, were selected, and the downregulation of these genes in hepatocellular carcinomas was confirmed. In conclusion, hepatocytes respond to HBV infection by upregulating DNMTs. The DNMTs methylate viral DNA, leading to decreased viral gene expression and decreased viral replication. However, virus-induced overexpression of DNMTs also leads to methylation of host CpG islands