SWATH-MS analysis of plasma proteins among Indian HIV-1 infected patients.

The identification and characterization of plasma proteins in drug resistant and drug sensitive in HIV-1 infected/AIDS patients were carried out using the SWATH-MS protocol. In total, 204 proteins were identified and quantified, 57 proteins were differentially expressed, out of which 25 proteins were down regulated and 32 proteins were up regulated in drug resistant patients. Six proteins such as complement C4-A, immunoglobulin heavy variable 1-2, carboxylic ester hydrolase, fibulin-1, immunoglobulin lambda constant7, secreted phosphoprotein 24 were differentially expressed in individuals with drug resistant HIV as compared to individuals with drug sensitive HIV. Gene ontology of 57 differentially expressed proteins was analysed and documented.

Toll-like receptor 2 (-196 to -174) del and TLR1 743 A > G gene polymorphism-a possible association with drug-resistant tuberculosis in the north Indian population.

Objectives: The objective of this study is to analyze the association between TLR2 deletion (-196 to -174) and TLR1 743 A > G gene polymorphism with drug resistant tuberculosis (PTB, MDR-TB, and XDR-TB) in a population from Agra, Uttar Pradesh. Methods: The present case-control study included 101 pulmonary TB patients, 104 multidrug-resistant TB patients, 48 extremely drug-resistant TB patients, and 130 healthy and unrelated controls residing in the same locality. The genotyping method for TLR2 deletion (-196 to -174) was carried out by allele-specific polymerase chain reaction (PCR), and TLR1 743 A > G gene polymorphism was performed by hybridization probe chemistry in Roche Real-Time PCR. Genotype and allele frequencies were analyzed by the chi-square test. Cytokine levels were measured by ELISA and compared using Mann-Whitney and Kruskal-Wallis tests. Results: The frequency of heterozygous (Ins/del) genotypes for TLR2 (-196 to -174) polymorphism was predominant in XDR-TB patients (0.57), whereas heterozygous A/G genotype for TLR1 743 A > G single nucleotide polymorphism (SNP) was predominant in healthy controls (0.57) for TLR1 743 A > G gene polymorphism. The heterozygous genotype of TLR2 deletion polymorphism was found to be significantly higher in XDR-TB (p = 0.0001). TLR1 743 A > G SNP, AG genotypes were found to be significantly associated with healthy controls than PTB (p = 0.047). The level of serum cytokines (IL-6, TNF-α, and IFN-γ) was also found to be significantly different among TB patients and healthy controls. Conclusion: The findings suggested that in the present population, the heterozygous (Ins/Del) genotype and deletion allele of TLR2 deletion (-196 to -174) polymorphism are associated with the risk for the development of drug-resistant TB. Furthermore, for TLR1 743 A > G gene polymorphism, A/G genotype, and G allele are found associated with healthy controls, suggesting the protective role against TB.

Genomic signatures of protease and reverse transcriptase genes from HIV-1 subtype C isolated from first-line ART patients in India.

Genomic signatures of the protease and reverse transcriptase gene of HIV-1 from HIV infected North Indian patients who were under ART from 1 to ≤ 7 years were analyzed. The DNA from plasma samples of 9 patients and RNA from 57 patients were isolated and subjected to amplification for the protease and reverse transcriptase gene of HIV-1 subtype C. Then sequencing was carried out following the WHO dried blood spot protocol. The drug resistance mutation patterns were analyzed using the HIV Drug Resistance Database, Stanford University, USA. Lamivudine-associated drug-resistance mutations such as M184V/M184I, nevirapine-associated drug resistance mutations Y181C and H221Y, and efavirenz-associated drug resistance mutations M230I were observed in reverse transcriptase gene of archived DNA of two HIV-1 infected patients. No mutation was observed in the remaining 7 patients. Various computational tools and websites like viral epidemiological signature pattern analysis (VESPA), hyper mutation, SNAP version 2.1.1, and entropy were utilized for the analysis of the signature pattern of amino acids, hyper mutation, selection pressure, and Shannon entropy in the protease and reverse transcriptase gene sequences of the 9 archived DNA, 56 protease gene and 51 reverse transcriptase gene from the HIV-1 DNA amplified sequences of RNA. The HIV-1 Subtype-C (Gene bank accession number: AB023804) and first isolate HXB2 (Gene bank accession number: K03455.1) was taken as reference sequence. The signature amino acid sequences were identified in the protease and reverse transcriptase gene, no hyper mutation, highest entropy was marked in the amino acid positions and synonymous to non-synonymous nucleotide ratio was calculated in the protease and reverse transcriptase gene of 9 archived DNA sequences, 56 protease and 51 reverse transcriptase gene sequences of HIV-1 Subtype C isolates.

Detection of Drug Resistance Mutations in the Reverse Transcriptase Gene of HIV-1-Infected North Indian Population Failing First-Line Antiretroviral Therapy "A Follow-Up Cohort Study".

We aim to characterize the drug resistance mutations in reverse transcriptase gene of HIV-1 subtype C-infected North Indian population in those who are failing first-line antiretroviral therapy (ART) and if these mutations are associated with mortality. We also attempted the assessment of switch over to second-line antiretroviral therapy in these patients. Based on the immunological marker CD4 count (<350 cubic/mm), 192 HIV/AIDS patients were selected and viral load was estimated in those who were enrolled from December 2009 to November 2016. Based on viral load, genotyping was carried out in 57 HIV-1 isolates (VL ≥1,000 copies/mL) by sequencing and drug resistance mutations were examined through the Stanford HIV Drug Resistance Database, USA. Among them, 21 (36.84%) first-line ART failure patients were shifted to second-line ART. These patients were followed for a period wide ranging from 10 months to 11 years. Drug resistance mutation M184V (ATG to GTA) (63.15%) associated with lamivudine and abacavir and K103N (AAG or AAA to AAU) (36.84%) associated with efavirenz and nevirapine were predominantly identified in first-line ART failure patients. During follow-up, it was observed that 3 out of 21 who were in second-line ART died, whereas 9 out of 36 died who were in the first-line ART. No mutation could be associated with mortality although TAM-2 mutations were absent in patients who died. This study indorses the need for a facility for viral load estimation and resistance monitoring in each treatment failure patient and availability of appropriate antiretroviral therapies.

Identification and differential expression of serotransferrin and apolipoprotein A-I in the plasma of HIV-1 patients treated with first-line antiretroviral therapy.

BACKGROUND: Plasma proteins are known to interfere the drug metabolism during therapy. As limited information is available regarding the role of plasma proteins in HIV drug resistance during ART in HIV/AIDS patients, the present study aimed to identify and characterize the differentially expressed plasma proteins in the drug resistant and drug respondent groups of HIV-1 infected patients with > 6 years of first line ART. METHODS: Four-drug resistant (treatment failure) and four-drug respondent (treatment responder) patients were selected for plasma proteomic analysis based on viral load and drug resistance associated mutations from a cohort study designed on the first line ART patients who were enrolled in the antiretroviral therapy center, Sarojini Naidu Medical College, Agra, India from December 2009 to November 2016. After depleting high abundant proteins, plasma proteins were resolved using two-dimensional gel electrophoresis on IPG strips, pH range of 3-10. Spots were selected in the gel based on the density of staining which was common in the drug resistant and drug respondent groups separately. The fold change of each spot was calculated using image-J. Each protein spot was identified using the matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) after tryptic digestion. Peptide peaks were identified through flex analysis version 3.3, and a search against a protein data base using the internal Mascot. Gene ontology study was completed through STRING v.11 and Panther15.0. RESULTS: Out of eight spots from 2D gel samples analyzed by MALDITOF/TOF, two proteins were found to have significant score (> 56) after Flex analysis. These two proteins were identified to be apolipoprotein A1 and serotransferrin. The fold change expression of these two proteins were analyzed in drug resistant and drug respondent group. Apolipoprotein-A1 and serotransferrin were observed to be expressed 1.76 and 1.13-fold more respectively in drug respondent group compared to drug resistant group. The gene ontology analysis revealed the involvement of these two proteins in various important physiological processes. CONCLUSION: Apolipoprotein A-I and serotransferrin were found to be expressed more in drug respondent group compared to drug resistant group.