Study design and setting
A cross-sectional study using purposive sampling method was conducted at the Southern Area Blood Center (SABC), Ghana from August 2016 to February 2017. The center is a satellite facility of the National Blood Service, Ghana (NBS). It is mandated to collect, screen, and distribute safe blood to various hospitals and clinics in the southern part of Ghana. The NBS recruits VBD using a questionnaire to first assess their behavioral risk factors and health status to ascertain their suitability to donate blood. Upon successful recruitment and blood donation, the blood is screened for HBsAg, HCV antibodies, HIV and antibodies for Treponema pallidum as procedures for safe blood. The test algorithm for HIV-1 at the blood bank is by the detection of p24 antigen using the HIV (Ag/Ab) 4thGen (Fortress Diagnostics Limited, Antrim, U.K). Currently, a more sensitive test tool such as the PCR and/or INNO-LIA is not employed.
Ethical approval was obtained from the Ethics and Protocol Review Committee of the College of Health Sciences, University of Ghana. Approval to select HIV positive samples was also obtained from the NBS.
A total of eighty-one (81) voluntarily donated blood samples that were rejected as been HIV sero-positive using the HIV (Ag/Ab) 4thGen (Fortress Diagnostics Limited, Antrim, U.K) were used. A data extraction sheet was used in obtaining information on age and gender from the donors’ records upon approval from the SABC. Study numbers were assigned to anonymize the blood samples.
Sample Collection and Confirmation
Plasma was obtained from the SABC and were transported in cold boxes with ice packs to the Virology Department of NMIMR and stored at -30°C until further processing. A confirmatory test (INNO-LIATM HIV-I/II score, Fujirebio, Gent, Belgium) was done on all plasma samples following manufacturer’s protocol.
RNA Extraction and Complementary DNA (cDNA) Synthesis
Viral RNA was extracted using the QIAamp® viral RNA mini kit (QIAGEN, Hilden, Germany) following manufacturer’s protocol. A two-step reverse transcription method was used to generate complementary DNA (cDNA) of HIV-1 from extracted RNA using Transcriptor High Fidelity cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). An initial reaction mix of 2.0µl random hexamer primer, 2.4µl nuclease-free water and 7.0µl of extracted RNA were incubated at 65°C for 10 minutes and immediately placed on ice. A second reaction mix made up of 4.0µl of 5X High fidelity reverse transcriptase (5X HFRT) buffer, 0.5µl protector RNAase inhibitor, 2.0µl deoxynucleotide triphosphates (dNTPs), 1.0µl dithiothreitol (DTT) and 1.1µl transcriptor HFRT enzyme was prepared. An aliquot of 8.6µl of the second mix was added to the first reaction, thoroughly mixed and incubated at 45°C for 30minutes followed by 85°C for 5 minutes.
Polymerase Chain Reaction (PCR) Amplification
Nested PCR was done to separately amplify the protease (PR) and reverse transcriptase (RT) genes from the cDNA synthesized using the Expand High Fidelityplus PCR kit (Roche Diagnostics, Mannheim, Germany) with specific primers and cycling conditions previously published . In the first round, 5.0µl of 5 buffer with MgCl2, 0.5µl of dNTPs, 1.0µl each of forward and reverse primers, 0.25µl of expand high fidelity polymerase and 12.25µl of nuclease free water were added to 5.0 l of cDNA. In the second round of the PCR, 5.0µl of 5X buffer with MgCl2, 0.5µl dNTPs, 0.5µl of each of forward and reverse primers, 0.25µl expand high fidelity polymerase and 15.25µl nuclease-free water were added to 3.0µl of round 1 product. A fragment of 463 base pairs (bp) and 887 bp for the PR and RT genes respectively, were generated and confirmed by agarose gel electrophoresis.
Purification of PCR Amplicons and Cycle Sequencing
Purification of nested PCR products was done using QIAquick PCR purification kit (QIAGEN, Hilden, Germany) following manufacturer’s protocol. Purified amplicons were eluted in 50µl of elution buffer for cycle sequencing. The BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, MA, U.S.A) was used to separately sequence the PR and RT genes of HIV-1 using primers and cycling conditions previously published . A total reaction volume of 10µl comprising of 2µl each of a primer, BigDye terminator, BigDye terminator buffer, nuclease free water and purified PCR product was used. Sequenced products were purified using the Agencourt® CleanSEQ® Dye-Terminator Removal system (Agencourt Bioscience Corporation, U.S.A) following manufacturer’s protocol. The purified product was loaded onto the ABI 3130xl genetic analyzer (Applied Biosystems, MA, U.S.A) to generate sequence data for HIV-1 DRM and HIV subtype analyses.
Sequence and Phylogenetic Analysis
Nucleotide sequences for each sample were assembled to form a contig using SeqManPro 13 (DNASTAR Incorporation, U.S.A). Consensus sequence obtained was aligned with an HIV reference sequence (B-HXB2-PRT_2253-3700) in BioEdit (http://www.mbio.ncsu.edu/Bioedit/bioedit.html). Sequences were submitted to the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/hivdb/by-sequences/) to assign subtypes detect HIV drug resistance mutations and. The subtypes were confirmed with the Los Alamos National Laboratory HIV Database (http://www.hiv.lanl.gov). Neighbor-joining tree with the Kimura’s 2-parameter distances was generated with the RT sequences using Molecular Evolutionary Genetic Analysis version 6.0 (MEGA 6).
Statistical analysis was done using SPSS version 23 (Armonk, USA) to describe patients’ demographics using frequencies and percentages.