Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances. AIM OF THE STUDY: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity. MATERIALS AND METHODS: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4+/CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC50 values in the nanomolar range. RESULTS: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 µM (IC50: 0.49 µM, CC50: 3089 µM) and 1343 µM (IC50: 2.3 µM, CC50: 3089 µM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC50 values of 11.11 µg/ml and 16.86 µg/ml, respectively. CONCLUSION: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.

High level of HIV-1 drug resistance mutations in patients with unsuppressed viral loads in rural northern South Africa.

BACKGROUND: Combination antiretroviral therapy (cART) has significantly reduced HIV morbidity and mortality in both developed and developing countries. However, the sustainability of cART may be compromised by the emergence of viral drug resistance mutations (DRM) and the cellular persistence of proviruses carrying these DRM. This is potentially a more serious problem in resource limited settings. METHODS: DRM were evaluated in individuals with unsuppressed viral loads after first or multiple lines of cART at two sites in rural Limpopo, South Africa. Seventy-two patients with viral loads of >1000 copies/ml were recruited between March 2014 and December 2015. Complete protease (PR) and partial Reverse Transcriptase (RT) sequences were amplified from both plasma RNA and paired proviral DNA from 35 of these subjects. Amplicons were directly sequenced to determine subtype and DRM using the Stanford HIV Drug Resistance Interpretation algorithm. RESULTS: Among the 72 samples, 69 could be PCR amplified from RNA and 35 from both RNA and DNA. Sixty-five (94.2%) viruses were subtype C, while one was subtype B (1.4%), one recombinant K/C, one recombinant C/B and one unclassified. Fifty-eight (84%) sequences carried at least one DRM, while 11 (15.9%) displayed no DRM. DRM prevalence according to drug class was: NRTI 60.8% NNRTI 65.2%, and PI 5.8%. The most common DRMs were; M184V (51.7%), K103N (50%), V106M (20.6%), D67N (13.3%), K65R (12%). The frequency of the DRM tracked well with the frequency of use of medications to which the mutations were predicted to confer resistance. Interestingly, a significant number of subjects showed predicted resistance to the newer NNRTIs, etravirine (33%) and rilpivirine (42%), both of which are not yet available in this setting. The proportion of DRM in RNA and DNA were mostly similar with the exception of the thymidine analogue mutations (TAMs) D67N, K70R, K219QE; and K103N which were slightly more prevalent in DNA than RNA. Subjects who had received cART for at least 5 years were more likely to harbour >2 DRM (p < 0.05) compared to those treated for a shorter period. DRM were more prevalent in this rural setting compared to a neighbouring urban setting. CONCLUSION: We found a very high prevalence of NRTI and NNRTI DRM in patients from rural Limpopo settings with different durations of treatment. The prevalence was significantly higher than those reported in urban settings in South Africa. The dominance of NNRTI based mutations late in treatment supports the use of PI based regimens for second line treatment in this setting. The slight dominance of TAMs in DNA from infected PBMCs compared to plasma virus requires further studies that should include cART subjects with suppressed virus. Such studies will improve our understanding of the pattern of drug resistance and dynamics of viral persistence in these rural settings.

Evaluating Adherence to Antiretroviral Therapy Using Pharmacy Refill Records in a Rural Treatment Site in South Africa.

Optimal adherence to combination antiretroviral therapy (cART) is critical to maintain virologic suppression, thereby ensuring the global success of HIV treatment. We evaluated adherence to cART using pharmacy refill records and determined the adherence threshold resulting in >90% virologic suppression in a community run treatment site in South Africa. Additionally, we analysed factors associated with adherence using univariable and multivariable logistic regression models. Logistic regression was also performed to determine the relationship between adherence and virologic suppression and the adherence threshold resulting in <10% virologic failure. The overall median (interquartile range) adherence was 95% (88.6-98.4%). Out of the study participants, 210/401 (52.4%) had optimal (≥95%) adherence while only 37/401 (9.2%) had poor (≤80%) adherence. The majority (90.5%) of patients with optimal adherence had virologic suppression. Having TB at registration into care was found to be negatively associated with adherence (adjusted odds ratio [AOR], 0.382; p ≤ .05). Compared to nonadherent individuals, optimally adherent participants were more likely to achieve virologic suppression (OR 2.92; 95% CI: 1.63-5.22). Only adherence rates above 95% were observed to lead to <10% virologic failure. cART adherence measured by pharmacy refill records could serve as a useful predictor of virologic failure; adherence rates >95% are needed to maintain optimal virologic suppression.

Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon.

PURPOSE: Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. METHODS: Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. RESULTS: Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. CONCLUSIONS: Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI.

Serologic and genotypic characterization of hepatitis B virus in HIV-1 infected patients from South West and Littoral Regions of Cameroon.

BACKGROUND: HBV and HIV share similar transmission routes. Concurrent infection with the two viruses usually results in more severe and progressive liver disease, and a higher incidence of cirrhosis, liver cancer and mortality. Further, this co-infection may lead to cross-resistance between HIV and HBV drugs and increased liver injury, either due to direct hepatotoxicity or drug-related immune-reconstitution hepatitis. These challenges necessitate continuous surveillance for HBV among HIV infected individuals to guide patient management. We conducted this study to understand the serologic and genotypic characteristics of HBV among HIV/HBV infected patients in South West and Littoral Regions of Cameroon. METHODS: Plasma samples were screened for HBsAg, HBeAg, Anti-HBs and anti-HBc using ELISA followed by DNA extraction from all HBsAg positive samples. A 366 bp region covering the overlapping surface/polymerase gene was amplified by a nested PCR and the product sequenced using Big Dye sequencing chemistry. The resulting sequences were then analyzed for genotypes and both escape and drug resistance mutations. RESULTS: Of the 455 samples in this study, 25.5 % (n = 116) were HBsAg positive and 46 of these had their DNA successfully amplified. Genotype E was found in 32 samples (69.6 %) and genotype A in the rest of the samples. Escape mutations associated with failure of diagnosis (Y100C, R122K and Q129H) and with vaccine escape (Q129R and T131N) were detected in varying frequencies in the population. Polymerase mutations implicated in resistance to lamivudine and other ʟ-nucleoside analogues were detected in seven patients (15.2 %), while all the samples lacked mutations associated with resistance to adefovir and tenofovir. CONCLUSIONS: These findings suggest the endemicity of HBV and the predominance of genotypes A and E in the study population. Also, drug resistance findings support the use of tenofovir based ART regimens among HIV/HBV co-infected persons. There is need for continuous HBV screening and monitoring in HIV infected individuals in these regions.

Molecular epidemiology of HIV in two highly endemic areas of northeastern South Africa.

There is paucity of data on the genetic landscape of HIV-1 viruses circulating in the Limpopo Province of northeastern South Africa. Here, we examine the genetic diversity of viruses from Bela-Bela and Musina, two towns with high HIV prevalence. Between June 2007 and March 2008, blood samples were collected from antiretroviral-drug-naïve individuals. Viruses were analyzed for genetic subtypes and drug resistance mutations. All of the viruses in these samples were shown by phylogenetic analysis based on gag p17, gag p24, reverse transcriptase, protease and envelope C2-C3 gene regions to belong to HIV-1 subtype C. Two of 44 reverse transcriptase sequences (4.5%) contained N rather than the consensus K at position 103. The K103N mutation is normally associated with resistance to NNRTIs. No major mutations were observed in the protease gene. However, several polymorphisms and amino acid changes normally considered to be minor drug resistance mutations were observed in the protease sequences. These results suggest that HIV-1 subtype C remains the predominant variant responsible for the epidemic in northeastern South Africa and that the prevalence of drug-resistant viruses among the naïve population is low.

Prevalence of antiretroviral drug resistance mutations and HIV-I subtypes among newly-diagnosed drug-naïve persons visiting a voluntary testing and counselling centre in northeastern South Africa.

Data on antiretroviral drug resistance among drug-naive persons are important in developing sentinel and surveillance policies. This study was conducted to determine the prevalence of antiretroviral drug resistance mutations among drug-naïve HIV-infected individuals attending a voluntary testing and counselling centre at the Mankweng Hospital in northeastern South Africa. In total, 79 drug-naïve HIV-positive individuals were sequentially recruited during February 2008-December 2008. Drug resistance mutations were determined using the calibrated population resistance tool available on the Stanford HIV drug resistance database. Viral DNA was obtained from 57 (72%) of the 79 individuals. Reliable nucleotide sequences were obtained for 54 reverse transcriptase (RT) and 54 protease (PR) gene regions from 54 individuals. Overall, five sequences (9.3%) harboured drug resistance mutations (95% confidence interval -1.53 to 16.99). Four (7.4%) of these were nucleoside RT inhibitor mutations (D67G, D67E, T69D, and T215Y), and one (1.9%) was a PR inhibitor mutation (M46I). No major non-nucleoside RT resistance mutation was detected. Several minor resistance mutations and polymorphisms common in subtype C viruses were observed in the PR and RT genes. Phlyogenetic analysis of the partial pol sequences showed that 52 (96%) of the 54 isolates were HIV-1 subtype C. One isolate (08MB08ZA) was HIV-1 subtype B while another (08MB26ZA) was related to HIV-1 subtype J. HIV-1 subtype recombination analysis with REGA assigned the pol sequence to HIV subtype J (11_cpx) with a bootstrap value of 75%. The prevalence of drug resistance mutations observed in the population studied was relatively higher than previously reported from other parts of South Africa. In addition, this is apparently the first report of an HIV-1 subtype J-like virus from northeastern South Africa.

Prevalence of drug-resistant mutations in newly diagnosed drug-naïve HIV-1-infected individuals in a treatment site in the waterberg district, limpopo province.

AIM: We studied the prevalence of resistance mutations in drug-naïve HIV-infected individuals at the Bela-Bela treatment site to gather information on the presence of antiretroviral (ARV) drug-resistant viruses in drug-naïve populations, so as to improve treatment guidance. SUBJECTS AND METHODS: Drug-naïve HIV-l1-infected individuals were sequentially recruited between February 2008 and December 2008 from individuals visiting the voluntary counselling and testing (VCT) services of the Bela-Bela HIV/AIDS Wellness Clinic. Viral subtyping was done by phylogenetic analysis; drug-resistant mutations were determined according to the Stanford HIV Drug Resistance Interpretation and the International AIDS Society-USA Guidelines. RESULTS: A drug-resistant mutation prevalence of 3.5% (95% confidence interval 0.019796-0.119077) comprising Y181C and L33F was observed; 98% of the viruses were HIV-1 subtype C on the protease (PR) and reverse transcriptase (RT) gene regions. CONCLUSION: The prevalence of drug-resistant mutations in drug-naïve persons may be low in Bela-Bela after 8 years of access to antiretroviral treatment (ART), and resistance testing before initiating treatment may not be needed.

Genetic analysis of the near full-length genome of an HIV type 1 A1/C unique recombinant form from northern South Africa.

Human immunodeficiency virus type 1 (HIV-1) has a high propensity for recombination. The epidemic in South Africa is predominantly driven by HIV-1 subtype C with occasional description of non-subtype C and intersubtype recombinant viruses. This report presents the genetic analysis of a unique recombinant variant from northern South Africa comprised exclusively of subsubtype A1 and subtype C parental viruses. Boot scanning analysis of the near full-length genome with the jumping profile Hidden Markov Model revealed a genomic arrangement with seven breakpoints of recombination alternating between subsubtype A1 and subtype C. Apparently, this is the first report of a unique HIV-1 A1/C recombinant form from northern South Africa and probably the fifth from South Africa. The epidemiologic implication of this variant is unknown.