Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana.

Background: The successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. Method: From a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. Results: Sequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). Conclusion: The study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Resistance to protease inhibitors among persons living with HIV in Ghana: a case for viral load and drug resistance monitoring.

OBJECTIVE: Routine viral load and drug resistance testing are well supported in most resource-rich settings and provide valuable benefits in the clinical care of PLWH in these communities. Undoubtedly, there exist financial and political constraints for the scale-up of viral load and drug resistance testing in Sub-Saharan Africa. To achieve the global UNAIDS 95/95/95 targets, there is the need to bridge this inequity in patient care and allow for a universal approach that leaves no community behind. METHODS: Venous blood from 96 PLWH on second-line ART from Korle-Bu Teaching Hospital were collected and processed into plasma for CD4+ T- cell and viral load assessments. Ribonucleic acid (RNA) was extracted from stored plasma and the protease gene amplified, sequenced and analyzed for subtype and drug resistance mutations using the Stanford HIV drug resistance database. RESULTS: Out of the 96 PLWH, 37 experienced virological failure with 8 patients' samples successfully sequenced. The predominant HIV-1 subtype identified was CRF02_AG (6/8, 75.0%) with 12.5% (1/8) each of CFR06_cpx infection and one case unable to subtype. The major PI resistance mutations identified were; M46I, I54V, V82A, I47V, I84V and L90M. CONCLUSIONS: Persons living with HIV who had experienced virologic failure in this study harboured drug resistance mutations to PI, thus compromise the effectiveness of the drugs in the second line. Resistance testing is strongly recommended prior to switching to a new regimen. This will help to inform the choice of drug and to achieve optimum therapeutic outcome among PLWH in Ghana.

Hepatitis C virus (HCV) infection among patients with sickle cell disease at the Korle-Bu teaching hospital.

BACKGROUND: Hepatitis C virus (HCV) infection is a blood borne infection that remains potentially transmissible through blood transfusions. Sickle cell disease (SCD) is a common inheritable haemoglobinopathy in Ghana that requires multiple blood transfusions as part of its management. The SCD patient is therefore at a high risk of HCV infection; however, data on the occurrence of HCV in SCD patients has not been documented in Ghana. This study sought to determine the prevalence and genotypes of HCV infection in SCD patients. MATERIALS AND METHODS: This was a cross-sectional study which enrolled 141 sickle-cell disease patients from the Ghana Institute for Clinical Genetics, Korle-Bu Teaching Hospital (KBTH). Patient information was obtained through a structured questionnaire. Aliquots of the plasma obtained was used for both serology with Advanced Quality Rapid Anti-HCV Test Strip and molecular testing by RT-PCR with primers targeting the HCV core gene. The amplified DNA were purified and subjected to phylogenetic analysis to characterize HCV genotypes. RESULTS: Twelve (9%) out of the 141 patients were sero-positive for HCV total antibodies. HCV RNA was amplified from 8 (6%) out of the total number of patients' samples. One of the 12 sero-positives was HCV RNA positive. Five (63%) out of the 8 HCV RNA positive samples were successfully sequenced. The phylogenetic tree constructed with the study and GenBank reference sequences, clustered all five study sequences into HCV genotype 1. CONCLUSION: The HCV seroprevalence of 9% among sickle cell disease patients is higher than reported for the general Ghanaian population which is 3%. Genotype 1 is the common HCV genotype infecting SCD patients. Sickle cell disease is likely to be a high-risk group for HCV inapparent infections in Ghana as seroprevalence does not correlate with viremia. However, even with higher seroprevalence, the group must be given priority in resource allocation for preventive, diagnostic and therapeutic strategies.

Transmitted drug resistance mutations and subtype diversity amongst HIV-1 sero-positive voluntary blood donors in Accra, Ghana.

BACKGROUND: Detection of HIV-1 transmitted drug resistance (TDR) and subtype diversity (SD) are public health strategies to assess current HIV-1 regimen and ensure effective therapeutic outcomes of antiretroviral therapy (ART) among HIV-1 patients. Globally, limited data exist on TDR and SD among blood donors. In this study, drug resistance mutations (DRMs) and SD amongst HIV-1 sero-positive blood donors in Accra, Ghana were characterized. METHODS: Purposive sampling method was used to collect 81 HIV sero-positive blood samples from the Southern Area Blood Center and confirmed by INNO-LIA as HIV-1 and/or HIV-2. Viral RNA was only extracted from plasma samples confirmed as HIV-1 positive. Complementary DNA (cDNA) was synthesized using the RNA as a template and subsequently amplified by nested PCR with specific primers. The expected products were verified, purified and sequenced. Neighbour-joining tree with the Kimura's 2-parameter distances was generated with the RT sequences using Molecular Evolutionary Genetic Analysis version 6.0 (MEGA 6.0). RESULTS: Out of the 81 plasma samples, 60 (74%) were confirmed as HIV-1 sero-positive by INNO-LIA HIVI/II Score kit with no HIV-2 and dual HIV-1/2 infections. The remaining samples, 21 (26%) were confirmed as HIV sero-negative. Of the 60 confirmed positive samples, (32) 53% and (28) 47% were successfully amplified in the RT and PR genes respectively. Nucleotide sequencing of amplified samples revealed the presence of major drug resistance mutations in two (2) samples; E138A in one sample and another with K65R. HIV-1 Subtypes including subtypes A, B, CRF02_AG and CRF09_cpx were found. CONCLUSION: This study found major drug resistance mutations, E138A and K65R in the RT gene that confer high level resistance to most NNRTIs and NRTI respectively. CRF02_AG was most predominant, the recorded percentage of subtype B and the evolutionary relationship inferred by phylogenetic analysis may suggest possible subtype importation. However, a more prospective and detailed analysis is needed to establish this phenomenon. The data obtained would inform the selection of drugs for ART initiation to maximize therapeutic options in drug-naïve HIV-1 patients in Ghana.