ART = antiretroviral therapy, NNRTI: EFV = efavirenz, NVP = nevirapine; NRTI: 3TC = lamivudine, AZT = zidovudine, TDF = tenofovir; PI: LPV/r = lopinavir/r

ART = antiretroviral therapy, NNRTI: EFV = efavirenz, NVP = nevirapine; NRTI: 3TC = lamivudine, AZT = zidovudine, TDF = tenofovir; PI: LPV/r = lopinavir/r. 3.2. For drug resistance screening, nucleic acids were extracted from plasma and peripheral blood mononuclear cells. The reverse transcriptase and protease genes of HIV-2 were amplified, sequenced and analyzed for drug resistance mutations and HIV-2 group. HIV-2 viral weight was detected in 9 of 16 patients. Six of these experienced quantifiable viral loads (range: 2.62C5.45 log IU/mL) while 3 experienced viral loads below the limit of quantification. Sequences were generated from 7 out of 16 samples. Five of these were classified as HIV-2 group B and 2 as HIV-2 group A. HIV-2 drug resistance mutations (M184V, K65R, Y115F) were recognized in 1 individual. This study is the first to statement HIV-2 viral INH154 weight and drug resistance mutations in HIV-2 strains from Ghana. The results indicate the need for continuous monitoring of drug resistance among HIV-2- infected patients to improve their clinical management. value of 1 1.96 at 95% confidence level and 0.05 confidence interval.[38] Clinical histories were INH154 retrieved from hospital folders of study patients. The study was conducted in accordance with procedures approved by the Institutional Review Table of the Noguchi Memorial Institute for Medical Research (NMIMR-IRB CPN 063/14-15) and the Ethical and Protocol Review Committee of the University or college of Ghana Medical School (MS-Et/M.4-P4.3/2014-2015). 2.2. Whole blood processing into plasma and peripheral blood mononuclear cells Blood was processed into plasma and peripheral blood mononuclear cells (PBMC) using a sucrose-gradient based protocol INH154 with Histopaque 1077 (Sigma Aldrich Organization; Darmstadt, Germany). Plasma was stored at ?80oC while PBMCs were stored in freezing medium, consisting of 1% dimethyl sulfoxide (DMSO) (Sigma Aldrich, Germany) in fetal bovine serum (Sigma Aldrich), at ?80oC until use. 2.3. Nucleic acid extraction and purification Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were extracted from plasma and PBMCs, respectively using the QIAamp viral RNA mini kit and the DNAeasy Blood and Tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. 2.4. HIV-2 viral weight and genotyping HIV-2 viral weight was performed on 0.2?ml of plasma following a published protocol.[37] Viral weight testing was completed at the Wadsworth Center, New York State Department of Health according to the approved IRB protocol (#17-039). The HIV-2 viral weight assay had a lower limit of detection of 32 international models (IU)/ml (1.50 log IU/ml) and a lower limit of quantification (LLOQ) of 225?IU/ml (2.35 log IU/ml). For genotyping, reverse transcriptase (RT) and protease (PR) genes of HIV-2 were amplified separately using specific primers.[35,36] A nested polymerase chain reaction (PCR) for the RT gene from PBMC portions was carried out to amplify a genomic region of 1050 base pairs (bp) encoding the RT gene (Table ?(Table1).1). The reactions were carried out in a total volume of 25?l containing 5?l of DNA template, 12.5?l of Supermix (Life Technologies, Invitrogen; Austin, TX) and 0.5?l of 20?M of primers (Table ?(Table1).1). The first round PCR conditions were: 94C for 2 moments, 40 cycles of 94C for 30?seconds, 55C for 1 minute and 72C for 1 minute 30?seconds, and then an elongation step at 72C for 7 moments. A nested PCR was carried out using 5?l of the first-round PCR product under the following cycle conditions: 94C for 2 moments, 40 cycles of 94C for 30?seconds, 56C for 1 minute and 72C for 1 minute, and an extension at 72C for 5?moments. The entire coding region for the PR gene (297 bp) was amplified by nested PCR using primers (Table ?(Table1).1). The cycling conditions for PR gene PCRs were the same as that for RT INH154 gene. Table 1 Details of primers used to amplify HIV-2 sub-genomic fragments previously published. Open in a separate windows The RT and PR genes in plasma were amplified by nested PCR using QIAGEN OneStep RT-PCR Kit (Qiagen, Germany) for the first round in a total reaction volume of 25?l containing 5?l of RNA, 5?l of 5 buffer, 0.75?l of 20?M of primers, 1?l of enzyme mix and dNTPs followed by nested PCR with Amplitaq Platinum master mix kit (Applied Biosystems, USA) in 25?l reaction volume containing 12.5?l Amplitaq Platinum, 0.5?l of Rabbit Polyclonal to SYT11 20?M of primers (Table ?(Table1).1). The first round PCR experienced reverse transcription at 50C for 30 minutes, and enzyme degradation at 95C for 15 minutes followed by 40 amplification cycles (94C for 30?seconds, 55C for 1 minute, 72C for 1 minute 30?seconds) and then an extension at 72C for 7 moments. The second round PCR conditions were: 94C for 2 moments, followed by 40 amplification cycles (94C for 30?seconds, 56C for 1 minute and 72C for 1 minute) and extension at 72C for.