Study location
This cross sectional study was carried out in University of Calabar Teaching Hospital, Cross River State, Nigeria. Cross River State is located in Southern Nigeria [26] in the Niger Delta Region. It is bounded in the north by Benue State, the west by Ebonyi and Abia State, the east by Cameroon Republic and the south by Akwa Ibom State and the Atlantic Ocean [27]. Cross River State has an area of 21,787km2 and a population of 2,892,988 (2006 census) [28]. University of Calabar is sited in Calabar Metropolis which is a fusion of Calabar south local government and Calabar municipality. Calabar has a geographical coordinates of 8019’37.02E with an estimated population of 375,196 (2006 census). [28,29]. The map showing the study area is in indicated in Figure 1.
Study population
One hundred HIV-infected clients attending antiretroviral therapy (ART) clinic of University of Calabar Teaching Hospital were purposively recruited for this study. Positive subjects who have been confirmed and were on ART treatment who gave their consent were recruited. HIV negative and HIV positive subject who declined were excluded.
DNA extraction
DNA was extracted using gDNA mini prep DNA extraction kit, Zymo Research supplied by Inqaba Biotechnological South Africa. One hundred microliter (100µL) of whole blood was pipetted into a 1.5 ml. Four hundred microliters (400µL) of the genomic lysis buffer were added. The samples were mixed by votexing for 5 seconds and were allowed to stand at room temperature for 10 minutes. The mixtures were transferred to a zymo-spin column in a collection tube. It was then centrifuged at 12,000 rpm for 1 minute. The flow through and collection tubes were discarded. The zymo-spin columns were transferred to a new collection tube and 200µL of DNA pre-wash buffer was added and centrifuged at 12,000 rpm for 1 minute. Five hundred microliter (500µL) of gDNA was buffer was added to the spin column and centrifuged at 12,000 rpm for 1 minute. The spin column was then transferred into another micro-centrifuge tube and 50µL of DNA elution buffer added to the spin column and incubated at room temperature for 5 minutes followed by centrifugation for 30 seconds to elute the DNA. The eluted DNA was then used for PCR.
CCR5 gene amplification
The CCR5 gene was amplified by polymerase chain reaction (PCR) utilizing primers that flank the 32-base pair deletion; primary: P1: P1:5’CAAAAGGTCTTCATTACACC3’ and secondary: P2: P2:5’CCTGTGCCTCTTCTCATTTC-3’ on a 9700 ABI thermal cycler. The PCR mixture included 1X master mix (which contains Tq polymerase, DNTPS, MgCl2), forward and reverse primers at concentration of 0.4 M and 3 µL of the extracted DNA. DNAse free water was used to make up the PCR to a final volume of 20 µL. The PCR conditions used for the amplification of the gene was as follows: 5 cycles of 940C for 3 minutes (initial denaturation), 940C for 45 seconds (denaturation), 550C for 45 seconds (annealing), 720C for 1.5 seconds (extension) and and a final extension of 720C for 5 minutes and 30 cycles at 940C for 30 seconds (denaturation), 600C for 30 seconds (annealing) and 720C for 30 seconds (extension) and a final extension of 720C for 3 minutes. A 5µl aliquot of each amplicon were resolved on 4% agarose gel at 120V for 20 minutes and visualized in an ultraviolet trans-illuminator. The presence of a single fragment of 185 bp was considered to represent homozygote for CCR5 gene.
HIV-V3 region amplification
A two-step PCR amplification, first with outer primers and then with nested or inner primers, was performed to detect the presence of HIV-1 in infected patients’ PBMC. The DNA oligonucleotide primers HIV-19: 5’-AATGTCAGCACAGTACAATGTACA-3’ and HIV-20: 5’-CAGTAGAAAAAATTCCCCTCCACAATT-3’ and HIV-21 : 5’-CTGCTGTTAAATGGCAGTCTAGC-3’, and HIV-22: 5’-TCTGGGTCCCCTCCTGAGGA-3’ were used The PCR components comprised of 5 PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), 200 mM each dATP, dCTP, dGTP, and TTP, 0.2 to 1.0 mM each HIV-1 outer primer pair, The reactions were carried out at 94 0C for 1.5 min, 450C for 2 min, and 720C for 3 min for 35 cycles. The amplified DNA products were analyzed by electrophoresis on a 1.2% agarose gel. Negative controls consisting of DNA from PBMC of seronegative individuals were included in each set of reactions, which were negative in all assays. After the first round PCR, 1 ml of the product was amplified for 25 cycles with the corresponding inner primers at 940C for 1.5 min, 500C for 2 min, and 720C for 3 min. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel.
Sequencing of CCR5 and HIV-V3 gene
The sequencing of the V3 gene and CCR5 was done using the Big Dye Terminator kit on ABI 3500 sequence by Inquaba, South Africa.
Phylogenic analysis
Obtained sequences were edited using the Bioinformatic software Bioedit. Similar sequences were downloaded from the National Centre for Bioinformatics Information (NCBI) using Blast N. Downloaded sequence were aligned using the alignment software MAFT and the evolutionary relationship was eliminated at 500 bootstrap using MEGA.