INVESTIGATION OF DRUG RESISTANCE AGAINST PROTEASE, REVERSE TRANSCRIPTASE AND INTEGRASE INHIBITORS BY NEXT GENERATION SEQUENCE IN HIV POSITIVE PATIENTS
Project no. 88 · Status: Kapanmış · 2017
Summary
OBJECTIVE: HIV (Human Immunodeficiency Virus) infection has transformed, by using antiretroviral drugs in appropriate combinations (highly active antiretroviral therapy-HAART); from a fatal disease to a chronic disease that can be controlled. One of the biggest patient management problem is antiretroviral drug resistance. To determine the most appropriate treatment regimen, it is recommended that the drug susceptibility test be performed as soon as possible after diagnosis. The Sanger chain termination reaction is the gold standard for HIV drug resistance mutation detection. But the disadvantage is that it can not detect resistance mutations below 20%. In addition, Sanger-based commercial kits mainly detect protease and reverse transcriptase gene region mutations. It has become possible to detect >1% mutations with the new generation sequencing (NGS). Increasing use of integrase inhibitors as an initial treatment option has necessitated the monitoring of resistance mutations against these drugs. In our study, it was aimed to detect the protease, reverse transcriptase and integrase resistance mutations with NGS using a single amplicon. MATERIALS AND METHODS: In the Department of Medical Microbiology Laboratory of Ege University; drug resistance to protease, reverse transcriptase inhibitors has been routinely studied in HIV positive patient samples with Viroseq (Abbott, USA) based on the Sanger chain termination reaction since 2008. Forty samples of HIV positive patients with different resistance profiles were included from the archived samples. After viral RNA extraction, one step reverse transcription and nested PCR were performed. HIV pol gene region were sequenced by NGS technology. Sequence data were analyzed using 1% and 5% cut off with the CLC Genomics Workbench version 8.1 with the HXB2 reference sequence (GenBank accession # NC_001802). Mutation detection and subtyping was performed using Stanford University HIV-1 Drug Resistance Data Bank version 8.4 (http://hivdb.stanford.edu). RESULTS: Of the 40 patients, 27 were subtype B (67.5%), six were B + CRF 02_AG (15%), three were subtype A (7.5%), two were G + J (5%), one was CRF07_BC (%2,5) and one was B + F (2.5%). Seven of the samples (17.5%) had a resistance to at least one antiretroviral drug, ranging from a possible low to high level of resistance; the rate of resistance mutations of nucleotide reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were found to be 5%, 12.5% respectively. Protease inhibitor (PI) and integrase inhibitor (INSTI) resistance mutations were not detected. In one sample major K103N NNRTI mutation, in two samples M41L and T215D / S NRTI mutations, in two samples E138A NNRTI mutation, in two samples E138G NNRTI mutation were detected. Forty sample results in terms of genotype and resistance to protease and reverse transcriptase inhibitors were found to be concordant with the Sanger chain termination reaction (100%). There was no difference between 1% and 5% cut off. CONCLUSION: In our study, drug resistances to INSTI in addition to PI, NRTI, and NNRTI were detected using a single amplicon in 40 patients with NGS. In this method, integrase gene region sequence analysis has been made and 90% cost reduction was achieved by pooling and "multiplexing" of samples. Furthermore, detection of >1% drug resistant mutations will guide the treatment of patients. Keywords: HIV Drug Resıstance, Next Generatıon Sequencıng, Integrase gene region sequence.