Avelin F. Aghokeng

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Extensive survey on the prevalence and genetic diversity of SIVs in primate bushmeat provides insights into risks for potential new cross-species transmissions ☆

To evaluate the risk of cross-species transmissions of SIVs from non-human primates to humans at the primate/hunter interface, a total of 2586 samples, derived from primate bushmeat representing 11 different primate species, were collected at 6 distinct remote forest sites in southeastern Cameroon and in Yaoundé, the capital city. SIV prevalences were estimated with an updated SIV lineage specific gp41 peptide ELISA covering the major part of the SIV diversity. SIV positive samples were confirmed by PCR and sequence analysis of partial pol fragments. The updated SIV ELISA showed good performance with overall sensitivity and specificity of 96% and 97.5% respectively. The overall SIV seroprevalence was low, 2.93% (76/2586) and ranged between 0.0% and 5.7% at forest sites, and reached up to 10.3% in Yaoundé. SIV infection was documented in 8 of the 11 species with significantly different prevalence rates per species: 9/859 (1.0%) in Cercopithecus nictitans, 9/864 (1.0%) Cercopithecus cephus, 10/60 (16.7%) Miopithecus ogouensis, 14/78 (17.9%) Colobus guereza, 15/37 (40.5%) Cercopithecus neglectus, 10/27 (33.3%) Mandrillus sphinx, 6/12 (50%) Cercocebus torquatus, and 3/6 (50%) Chlorocebus tantalus. No SIV infection was identified in Cercopithecus pogonias (n=293), Lophocebus albigena (n=168) and Cercocebus agilis (n=182). The SIV prevalences also seem to vary within species according to the sampling site, but most importantly, the highest SIV prevalences are observed in the primate species which represent only 8.5% of the overall primate bushmeat. The phylogenetic tree of partial pol sequences illustrates the high genetic diversity of SIVs between and within different primate species. The tree also showed some interesting features within the SIVdeb lineage suggesting phylogeographic clusters. Overall, the risk for additional cross-species transmissions is not equal throughout southern Cameroon and depends on the hunted species and SIV prevalences in each species. However, humans are still exposed to a high diversity of SIVs as illustrated by the high inter and intra SIV lineage genetic diversity.

Monitoring of HIV viral loads, CD4 cell counts, and clinical assessments versus clinical monitoring alone for antiretroviral therapy in rural district hospitals in Cameroon (Stratall ANRS 12110/ESTHER): a randomised non-inferiority trial

BACKGROUND Scaling up of antiretroviral therapy in low-resource countries is done on the basis of decentralised, integrated HIV care in rural facilities; however, laboratory monitoring is generally unavailable. We aimed to assess the effectiveness and safety of clinical monitoring alone (CLIN) in terms of non-inferiority to laboratory and clinical monitoring (LAB). METHODS We did a randomised, open-label, non-inferiority trial in nine rural district hospitals in Cameroon. Eligible participants were adults (≥18 years) infected with HIV-1 group M (WHO disease stage 3-4) who had not previously received antiretroviral therapy, and were followed-up for 2 years by health-care workers in routine activities. We randomly assigned participants (1:1) to CLIN or LAB (counts of HIV viral load and CD4 cell every 6 months) groups with a computer-generated list. The primary outcome was non-inferiority of CLIN to LAB in terms of increase in CD4 cell count with a non-inferiority margin of 25%. We did all analyses in participants who attended at least one follow-up visit. This trial is registered with ClinicalTrials.gov, number NCT00301561. FINDINGS 238 (93%) of 256 participants assigned to CLIN and 221 (93%) of 237 assigned to LAB were eligible for analysis. CLIN was not non-inferior to LAB; the mean increase in CD4 cell count was 175 cells per μL (SD 190, 95% CI 151-200) with CLIN and 206 (190, 181-231) with LAB (difference -31 [-63 to 2] and non-inferiority margin -52 [-58 to -45]). Furthermore, in the predefined secondary outcome of treatment changes, 13 participants (6%) in the LAB group switched to second-line regimens whereas no participants in the CLIN group did so (p<0·0001). By contrast, other predefined secondary outcomes were much the same in both groups-viral suppression (<40 copies per mL; 465 [49%] of 952 measurements in CLIN vs 456 [52%] of 884 in LAB), HIV resistance (23 [10%] of 238 participants vs 22 [10%] of 219 participants), mortality (44 [18%] of 238 vs 32 [14%] of 221), disease progression (85 [36%] of 238 vs 64 [29%] of 221), adherence (672 [63%] of 1067 measurements vs 621 [61%] of 1011), loss to follow-up (21 [9%] of 238 vs 17 [8%] of 221), and toxic effects (46 [19%] of 238 vs 56 [25%] of 221). INTERPRETATION Our findings support WHOs recommendation for laboratory monitoring of antiretroviral therapy. However, the small differences that we noted between the strategies suggest that clinical monitoring alone could be used, at least temporarily, to expand antiretroviral therapy in low-resource settings. FUNDING French National Agency for Research on AIDS (ANRS) and Ensemble pour une Solidarité Thérapeutique Hospitalière En Réseau (ESTHER).

Global epidemiology of drug resistance after failure of WHO recommended first-line regimens for adult HIV-1 infection: A multicentre retrospective cohort study

Summary Background Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART. Methods The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase (RT) gene. Findings We included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count <100 cells per μL). Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions (OR 1·48, 95% CI 1·20–1·82). Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma viral load at virological failure was similar in individuals with and without tenofovir resistance (145 700 copies per mL [SE 12 480] versus 133 900 copies per mL [SE 16 650; p=0·626]). Interpretation We recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial. Funding The Wellcome Trust.Summary Background Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART. Methods The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase ( RT ) gene. Findings We included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count Interpretation We recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial. Funding The Wellcome Trust.

Inaccurate Diagnosis of HIV-1 Group M and O Is a Key Challenge for Ongoing Universal Access to Antiretroviral Treatment and HIV Prevention in Cameroon

Background Increased access to HIV testing is essential in working towards universal access to HIV prevention and treatment in resource-limited countries. We here evaluated currently used HIV diagnostic tests and algorithms in Cameroon for their ability to correctly identify HIV infections. Methods We estimated sensitivity, specificity, and positive and negative predictive values of 5 rapid/simple tests, of which 3 were used by the national program, and 2 fourth generation ELISAs. The reference panel included 500 locally collected samples; 187 HIV -1 M, 10 HIV-1 O, 259 HIV negative and 44 HIV indeterminate plasmas. Results None of the 5 rapid assays and only 1 ELISA reached the current WHO/UNAIDS recommendations on performance of HIV tests of at least 99% sensitivity and 98% specificity. Overall, sensitivities ranged between 94.1% and 100%, while specificities were 88.0% to 98.8%. The combination of all assays generated up to 9% of samples with indeterminate HIV status, because they reacted discordantly with at least one of the different tests. Including HIV indeterminate samples in test efficiency calculations significantly decreased specificities to a range from 77.9% to 98.0%. Finally, two rapid assays failed to detect all HIV-1 group O variants tested, with one rapid test detecting only 2 out of 10 group O specimens. Conclusion In the era of ART scaling-up in Africa, significant proportions of false positive but also false negative results are still observed with HIV screening tests commonly used in Africa, resulting in inadequate treatment and prevention strategies. Depending on tests or algorithms used, up to 6% of HIV-1 M and 80% of HIV-1 O infected patients in Cameroon do not receive ART and adequate counseling to prevent further transmission due to low sensitivities. Also, the use of tests with low specificities could imply inclusion of up to 12% HIV negative people in ART programs and increase budgets in addition to inconveniences caused to patients.

Low Levels of Antiretroviral-Resistant HIV Infection in a Routine Clinic in Cameroon that Uses the World Health Organization (WHO) Public Health Approach to Monitor Antiretroviral Treatment and Adequacy with the WHO Recommendation for Second-Line Treatment

A cross-sectional study, performed at a routine human immunodeficiency virus (HIV)/AIDS clinic in Cameroon that uses the World Health Organization public health approach, showed low rates of virological failure and drug resistance at 12 and 24 months after initiation of antiretroviral therapy. Importantly, the cross-sectional study also showed that the World Health Organization recommendation for second-line treatment would be effective in almost all patients with HIV drug resistance mutations.

Scale-up of antiretroviral treatment in sub-Saharan Africa is accompanied by increasing HIV-1 drug resistance mutations in drug-naive patients.

Objectives:To evaluate the frequency and progression over time of the WHO-defined transmitted HIV-1 drug resistance mutations (DRMs) among antiretroviral treatment (ART)-naive HIV-1-infected patients in Cameroon. Design:We analyzed HIV-1 DRM data generated from 369 ART-naive individuals consecutively recruited between 1996 and 2007 in urban and rural areas in Cameroon. Methods:HIV-1 drug resistance genotyping was performed in the pol gene using plasma samples and surveillance DRMs were identified using the 2009 WHO-DRM list. Results:We observed in Yaounde, the capital city, an increasing prevalence of DRMs over time: 0.0% (none of 61 participants) in 1996–1999; 1.9% (one of 53 participants) in 2001; 4.1% (two of 49 participants) in 2002; and 12.3% (10 of 81 participants) in 2007. In the rural areas with more recently implemented ART programs, we found DRMs in six of 125 (4.8%) ART-naive individuals recruited in 2006–2007. DRMs identified in both areas included resistance mutations to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs) that might impair the efficacy of available first-line and second-line treatments. Conclusion:This report showed an increase in transmitted DRMs in areas where antiretroviral drugs were introduced earlier, although other factors such as natural viral polymorphisms and acquired DRMs through exposure to antiretroviral cannot be totally excluded. Further surveillances are needed to confirm this evolution and inform public health policies on adequate actions to help limit the selection and transmission of drug-resistant HIV, while scaling up access to ART in developing countries.

Characterization of a new simian immunodeficiency virus strain in a naturally infected Pan troglodytes troglodytes chimpanzee with AIDS related symptoms

BackgroundData on the evolution of natural SIV infection in chimpanzees (SIVcpz) and on the impact of SIV on local ape populations are only available for Eastern African chimpanzee subspecies (Pan troglodytes schweinfurthii), and no data exist for Central chimpanzees (Pan troglodytes troglodytes), the natural reservoir of the ancestors of HIV-1 in humans. Here, we report a case of naturally-acquired SIVcpz infection in a P.t.troglodytes chimpanzee with clinical and biological data and analysis of viral evolution over the course of infection.ResultsA male chimpanzee (Cam155), 1.5 years, was seized in southern Cameroon in November 2003 and screened SIV positive during quarantine. Clinical follow-up and biological analyses have been performed for 7 years and showed a significant decline of CD4 counts (1,380 cells/mm3 in 2004 vs 287 in 2009), a severe thrombocytopenia (130,000 cells/mm3 in 2004 vs 5,000 cells/mm3 in 2009), a weight loss of 21.8% from August 2009 to January 2010 (16 to 12.5 kg) and frequent periods of infections with diverse pathogens.DNA from PBMC, leftover from clinical follow-up samples collected in 2004 and 2009, was used to amplify overlapping fragments and sequence two full-length SIVcpzPtt-Cam155 genomes. SIVcpzPtt-Cam155 was phylogenetically related to other SIVcpzPtt from Cameroon (SIVcpzPtt-Cam13) and Gabon (SIVcpzPtt-Gab1). Ten molecular clones 5 years apart, spanning the V1V4 gp120 env region (1,100 bp), were obtained. Analyses of the env region showed positive selection (dN-dS >0), intra-host length variation and extensive amino acid diversity between clones, greater in 2009. Over 5 years, N-glycosylation site frequency significantly increased (p < 0.0001).ConclusionsHere, we describe for the first time the clinical history and viral evolution of a naturally SIV infected P.t.troglodytes chimpanzee. The findings show an increasing viral diversity over time and suggest clinical progression to an AIDS-like disease, showing that SIVcpz can be pathogenic in its host, as previously described in P.t.schweinfurthii. Although studying the impact of SIV infection in wild apes is difficult, efforts should be made to better characterize the pathogenicity of the ancestors of HIV-1 in their natural host and to find out whether SIV infection also plays a role in ape population decline.

Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

ABSTRACT Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at −20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of ≥1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 real-time PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory performance should be monitored.

Genetic diversity among human immunodeficiency virus-1 non-B subtypes in viral load and drug resistance assays

The tremendous diversity of human immunodeficiency virus (HIV)-1 strains circulating worldwide has an important impact on almost all aspects of the management of this infection, from the identification of infected persons, through treatment efficacy and monitoring, and prevention strategies such as vaccine design. The areas where HIV-1 genetic diversity is highest are those where the majority of patients in need of treatment and biological monitoring live. With increased access to treatment in these areas, it is expected that the demand for monitoring tools such as viral load assays and resistance tests will also increase, and their reliability will be critical. Regular updates of these assays during the last two decades have aimed at improving their performances in different ways that include their reliability with different HIV-1 strains. We here review to what extent HIV-1 genetic diversity still limits or not the use of currently available viral load and resistance tests.