Desire Takou

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon

Most commercial HIV-1 genotyping assays are hampered by high cost in resource-limited settings. Moreover, their performance might be influenced over time by HIV genetic heterogeneity and evolution. An in-house genotyping protocol was developed, and its sequencing performance and reproducibility were compared to that of ViroSeq™. One hundred ninety plasma samples from HIV-1-infected subjects in Cameroon, a resource-limited setting with a high HIV genetic variability, were processed for pol gene sequencing with an in-house protocol, ViroSeq™, or both. Only non-B subtypes were found. The in-house sequencing performance was 98.7% against 92.1% with ViroSeq™. Among 36 sequence pairs obtained using both assays, the overall rate of discordant amino acid positions was negligible (0.24%). With its high sensitivity and reproducibility, as well as its affordable cost (about half of ViroSeq™: 92 € vs. 217 €), this in-house assay is a suitable alternative for HIV-1 genotyping in resource-limited and/or in high-genetic-diversity settings.

Characterization of drug resistance mutations in naïve and ART-treated patients infected with HIV-1 in Yaounde, Cameroon.

Currently the prevalence of HIV‐1 infection in Cameroon is 5.1%, CRF02_AG subtype is responsible for about 50% of infections. Since an HIV‐1 drug resistance test is not yet available widely, accurate data on the prevalence of resistant viral strains are missing. The objective of this study was to determine HIV‐1 genetic diversity and to characterize HIV‐1 mutations conferring drug resistance among antiretroviral therapy (ART)‐naïve and ART‐treated patients. A cohort of 239 patients infected with HIV were followed‐up between January 2007 and July 2010 in Cameroon. Two hundred and sixteen plasma samples were sequenced for phylogenetic analysis and identification of drug resistance mutations in the HIV‐1 pol region. A significant genetic diversity was found: Seven pure subtypes (A1, A3, D, F1, F2, G, H), nine circulating recombinant forms (CRFs: 01_AE, 02_AG, 06cpx, 09cpx, 11cpx, 13cpx, 16cpx, 18cpx, 37cpx) and one new unique recombinant form (URF) (G/F2). The rate of transmitted drug resistance (TDR) in naïve patients was 8.2% (4/49). Around 80% of patients failing a first‐line ART harbored a virus with at least one resistance mutation to two antiretroviral (ARV) classes, and 36% of those failing a second‐line regimen carried a virus with at least one resistant mutation to three ARV classes. The high level of drug resistance observed in the cohort is alarming because this occurred as a result of only few years of treatment. Adherence to therapy, adequate education of physicians, and the appropriate use of genotypic resistance assay are critical points of intervention for the improvement of patient care. J. Med. Virol. 84:721–727, 2012.

Lipid peroxidation and total cholesterol in HAART-naïve patients infected with circulating recombinant forms of human immunodeficiency virus type-1 in Cameroon.

Background HIV infection has commonly been found to affect lipid profile and antioxidant defense. Objectives To determine the effects of Human Immunodeficiency Virus (HIV) infection and viral subtype on patient’s cholesterol and oxidative stress markers, and determine whether in the absence of Highly Active Antiretroviral Therapy (HAART), these biochemical parameters could be useful in patient’s management and monitoring disease progression in Cameroon. For this purpose, we measured total cholesterol (TC), LDL cholesterol (LDLC), HDL cholesterol (HDLC), total antioxidant ability (TAA), lipid peroxidation indices (LPI), and malondialdehyde (MDA) in HIV negative persons and HIV positive HAART-naïve patients infected with HIV-1 group M subtypes. Methods We measured serum TC, LDLC, HDLC, plasma MDA, and TAA concentrations, and calculated LPI indices in 151 HIV-positive HAART-naïve patients and 134 seronegative controls. We also performed gene sequence analysis on samples from 30 patients to determine the effect of viral genotypes on these biochemical parameters. We also determined the correlation between CD4 cell count and the above biochemical parameters. Results We obtained the following controls/patients values for TC (1.96±0.54/1. 12±0. 48 g/l), LDLC (0. 67±0. 46/0. 43±0. 36 g/l), HDLC (105. 51±28. 10/46. 54±23. 36 mg/dl) TAA (0. 63±0. 17/0. 16±0. 16 mM), MDA (0. 20±0. 07/0. 41±0. 10 µM) and LPI (0. 34±0. 14/26. 02±74. 40). In each case, the difference between the controls and patients was statistically significant (p<0.05). There was a positive and statistically significant Pearson correlation between CD4 cell count and HDLC (r = +0.272; p<0.01), TAA (r = +0.199; p<0.05) and a negative and statistically significant Pearson correlation between CD4 cell count and LPI (r = −0.166; p<0.05). Pearson correlation between CD4 cell count and TC, CD4cell count and LDLC was positive but not statistically significant while it was negative but not statistically significant with MDA. The different subtypes obtained after sequencing were CRF02_AG (43.3%), CRF01_AE (20%), A1 (23.3%), H (6.7%), and G (6.7%). None of the HIV-1 subtypes significantly influenced the levels of the biochemical parameters, but by grouping them as pure subtypes and circulating recombinant forms (CRFs), the CRF significantly influenced TC levels. TC was significantly lower in patients infected with CRF (0.87±0.27 g/l) compared to patients infected with pure HIV-1 subtypes (1.32±0.68 g/l) (p<0.017). MDA levels were also significantly higher in patients infected with HIV-1CRF01_AE (0.50±0.10 µM), compared to patients infected with CRF02_AG (0. 38±0. 08 µM) (p<0.018). Conclusion These results show that HIV infection in Cameroon is associated with significant decrease in TAA, LDLC, HDLC and TC, and increased MDA concentration and LPI indices which seem to be linked to the severity of HIV infection as assessed by CD4 cell count. The data suggests increased oxidative stress and lipid peroxidation in HIV-infected patients in Cameroon, and an influence of CRFs on TC and MDA levels.

Phylogenetics of HIV-1 subtype G env: Greater complexity and older origins than previously reported.

HIV-1 subtype G has played an early and central role in the emergent complexity of the HIV-1 group M (HIV-1M) epidemic in central/west Africa. Here, we analysed new subtype G env sequences sampled from 8 individuals in Yaoundé, Cameroon during 2007-2010, together with all publically available subtype G-attributed full-length env sequences with known sampling dates and locations. We inferred that the most recent common ancestor (MRCA) of the analysed subtype G env sequences most likely occurred in ∼1953 (95% Highest Posterior Density interval [HPD] 1939-1963): about 15 years earlier than previous estimates. We found that the subtype G env phylogeny has a complex structure including seven distinct lineages, each likely dating back to the late 1960s or early 1970s. Sequences from Angola, Gabon and the Democratic Republic of Congo failed to group consistently in these lineages, possibly because they are related to more ancient sequences that are poorly sampled. The circulating recombinant form (CRF), CRF06_cpx env sequences but not CRF25_cpx env sequences are phylogenetically nested within the subtype G clade. This confirms that the CRF06_cpx env plausibly was derived through recombination from a subtype G parent, and suggests that the CRF25_cpx env was likely derived from an HIV-1M lineage related to the MRCA of subtype G that has remained undiscovered and may be extinct. Overall, this fills important gaps in our knowledge of the early events in the spread of HIV-1M.

Comparative Evaluation of Subtyping Tools for Surveillance of Newly Emerging HIV-1 Strains

ABSTRACT HIV-1 non-B subtypes/circulating recombinant forms (CRFs) are increasing worldwide. Since subtype identification can be clinically relevant, we assessed the added value in HIV-1 subtyping using updated molecular phylogeny (Mphy) and the performance of routinely used automated tools. Updated Mphy (2015 updated reference sequences), used as a gold standard, was performed to subtype 13,116 HIV-1 protease/reverse transcriptase sequences and then compared with previous Mphy (reference sequences until 2014) and with COMET, REGA, SCUEAL, and Stanford subtyping tools. Updated Mphy classified subtype B as the most prevalent (73.4%), followed by CRF02_AG (7.9%), C (4.6%), F1 (3.4%), A1 (2.2%), G (1.6%), CRF12_BF (1.2%), and other subtypes (5.7%). A 2.3% proportion of sequences were reassigned as different subtypes or CRFs because of misclassification by previous Mphy. Overall, the tool most concordant with updated Mphy was Stanford-v8.1 (95.4%), followed by COMET (93.8%), REGA-v3 (92.5%), Stanford-old (91.1%), and SCUEAL (85.9%). All the tools had a high sensitivity (≥98.0%) and specificity (≥95.7%) for subtype B. Regarding non-B subtypes, Stanford-v8.1 was the best tool for C, D, and F subtypes and for CRFs 01, 02, 06, 11, and 36 (sensitivity, ≥92.6%; specificity, ≥99.1%). A1 and G subtypes were better classified by COMET (92.3%) and REGA-v3 (98.6%), respectively. Our findings confirm Mphy as the gold standard for accurate HIV-1 subtyping, although Stanford-v8.1, occasionally combined with COMET or REGA-v3, represents an effective subtyping approach in clinical settings. Periodic updating of HIV-1 reference sequences is fundamental to improving subtype characterization in the context of an effective epidemiological surveillance of non-B strains.

HIV-1 drug susceptibility to potential second- and third-line antiretroviral regimens among cameroonian patients: Evidence from a crosssectional design

Background: Scale-up of antiretroviral therapy (ART) and the growing number of longterm treated patients may favor multi-HIV drug resistance (HIVDR) in resource-limited settings. Understanding the burden of HIVDR with ART-exposure may provide new insights for an effective long-term management of infected patients. Methods: Sixty-six HIV-infected individuals (18 ART-naive, 24 failing first-line, 24 failing secondline ART) living in Yaounde-Cameroon were evaluated by sequencing protease-reverse transcriptase (PR-RT, n=62), envelope-V3 loop (V3, n=58) and integrase (IN, n=30) regions. Drug resistance mutations (DRMs) were interpreted using Stanford University HIV drug resistance database and geno2pheno, while viral tropism prediction was done using geno2pheno, position-specific scoring matrices (PSSM) and Net charge rule. Results: Participants, from naive, first- to second-line, had respectively 5.30, 4.85 and 4.66 log HIV RNA, and 532, 203 and 146 CD4 cells/mm 3 ), and infected with diverse HIV-1 non-B clades (58.1% CRF02_AG). Among ART-naive patients, 6.7% harbored K103N, 28.6% had IN accessory-mutations (L74I, E157Q) and 26.7% carried CXCR4-tropic viruses. At first-line failure, 79.2% harbored DRMs to nucleoside and non-nucleoside RT inhibitors, 33.3% had IN accessory-mutations (L68I, L74I, T97A, E157Q), and 47.4% carried CXCR4-tropic viruses. At second-line failure, 91.3% harbored multi-DRMs to PR-RT inhibitors (with 52.2% and 4.3% DRMs to second-generation NNRTIs and darunavir/ r, respectively), 27.3% had IN accessory-mutations (L74I, T97A, E157EQ), and 37.5% carried CXCR4-tropic viruses. Conclusion: Levels of PR-RT resistance increases with ART-exposure, with needs for new ART-options following second-line failure. IN inhibitors and darunavir/r are potentially suitable for a third-line regimen, while the use of maraviroc, etravirine or rilpivirine, requires individual genotypic testing.

Co-Infection of Hepatitis B Virus and Hepatitis Delta Virus in Yaounde-Cameroon

Aims: To determine the seroprevalence of HDV as well as the virological and clinical characteristics of HBV mono-infected and HBV/HDV co-infected patients. Study Design: The few studies on HDV in Cameroon have reported a high prevalence of this viral infection. This is a first step in describing the virological and clinical profile of HBV mono-infected and of HBV/HDV co-infected patients. Place and Duration of Study: Blood collection was carried out in the Gastroenterology Unit of the Yaounde University Hospital Centre, Yaounde General Hospital and “Centre Medical la Cathedrale”, from August 2012 to May 2013. Methodology: We included into this study treatment-naive HBV-infected patients from Yaounde irrespective of age and gender free of HIV and HCV infection. Blood samples were collected from each patient for laboratory analysis. Detection of HDV antibodies