Characterization of HIV-1 Reservoirs in Children and Adolescents: A Systematic Review and Meta-Analysis Toward Pediatric HIV Cure.

OBJECTIVE: To conduct a comprehensive, systematic review of the profile of HIV-1 reservoirs in children and adolescents with perinatally acquired HIV infection. STUDY DESIGN: Randomized and nonrandomized trials, cohort studies, and cross-sectional studies on HIV reservoirs in pediatric populations, published between 2002 and 2022, were included. Archived-drug resistance mutations (ADRMs) and the size of reservoirs were evaluated. Subgroup analyses were performed to characterize further the data, and the meta-analysis was done through random effect models. RESULTS: Overall, 49 studies from 17 countries worldwide were included, encompassing 2356 perinatally infected participants (48.83% females). There are limited data on the quantitative characterization of viral reservoirs in sub-Saharan Africa, with sensitive methodologies such as droplet digital polymerase chain reaction rarely employed. The overall prevalence of ADRMs was 37.80% (95% CI 13.89-65.17), with 48.79% (95% CI 0-100) in Africa, 42.08% (95% CI 6.68-82.71) in America, 23.88% (95% CI 14.34-34.90) in Asia, and 20.00% (95% CI 10.72-31.17) in Europe, without any difference between infants and adolescents (P = .656). Starting antiretroviral therapy (ART) before 2 months of age limited the levels of HIV-1 DNA (P = .054). Participants with long-suppressed viremia (>5 years) had lower levels of HIV-1 DNA (P = .027). Pre- and post-ART CD4 ≤29% and pre-ART viremia ≥5Log were all found associated with greater levels of HIV-1 DNA (P = .038, P = .047, and P = .041, respectively). CONCLUSIONS: The pooled prevalence of ADRMs is high in perinatally infected pediatric population, with larger proviral reservoir size driven by delayed ART initiation, a shorter period of viral suppression, and immunovirological failures. Thus, strategies for pediatric HIV functional cure should target children and adolescents with very early ART initiation, immunocompetence, and long-term viral suppression.

Evaluation of archived drug resistance mutations in HIV-1 DNA among vertically infected adolescents under antiretroviral treatment in Cameroon: Findings during the COVID-19 pandemic.

BACKGROUND: With the success of antiretroviral therapy (ART), children born with HIV are more likely to reach adolescence. However, frequent non-adherence to ART in adolescents living with HIV (ALHIV) leads to viral replication. Notably, a viraemic infection might lead to archived drug resistance mutations (ADRMs). Hence, within the context of the COVID-19 pandemic, we aimed to compare the patterns of ADRMs in viraemic and non-viraemic vertically infected ALHIV and to assess their immunity to and diagnosis of SARS-CoV-2. METHODS: A comparative study was conducted among COVID-19-unvaccinated ALHIV receiving ART in Yaoundé-Cameroon over the period October 2021 to March 2022. Plasma HIV-RNA was measured using Abbott® m2000rt; HIV-1 genotyping was performed on buffy-coat (HIV-1 DNA) and ADRMs were interpreted using HIVdb.v9.0.1. Patterns of HIV-1 ADRMs were compared between viraemic (≥ 1.60 log10 HIV-1 RNA copies/ml) and non-viraemic (< 1.60 log10 copies/ml) individuals. SARS-CoV-2 antibodies were assessed on whole blood using Abbott Panbio COVID-19 immunoglobulin G/M (IgG/IgM) rapid test and COVID-19 polymerase chain reaction test was performed using nasopharyngeal swab samples. RESULTS: Of the 60 ALHIV [aged 17 (16-19) years, 51.6% female], median ART duration was 14 (12-16) years; 31/55 (56.3%) were exposed to nonnucleoside reverse transcriptase inhibitor (NNRTI)-based first-line ART (of whom 19/31 transitioned to dolutegravir-based ART in 2020) and 24/55 (43.6%) were on second-line ART. Forty-two out of 60 (70.0%) ALHIV were non-viraemic; 43/60 (71.6%) were successfully sequenced. Overall the ADRM rate was 62.7% (27/43), with 69.2% (9/13) viraemic and 60.0% (18/30) non-viraemic (p = 0.56). NNRTI-ADRMs were significantly higher among viraemic ALHIV (69.2% vs. 46.7%, p = 0.030). Regarding immunity, those with CD4 nadir < 350 cells/µl had significantly higher rates of ADRMs [adjusted odds ratio (aOR) = 3.20 (1.36-95.53), p = 0.03]. In relation to COVID-19 immunity, overall SARS-CoV-2 IgG seropositivity was 28.3% (17/60), whereas 0% (0/60) were seropositive to IgM; in particular, those with CD4 count nadir ≥ 350 cells/µl had higher odds of SARS-CoV-2 IgG seropositivity [OR =7.85 (2.03-30.28), p < 0.01]. No significant association was found between SARS-CoV-2 IgG seropositivity and HIV-RNA (non-viraemic, 33.3%; viraemic, 16.7%; p = 0.18). SARS-CoV-2 RNA prevalence was 4.5% (2/44). The two positive participants were with low-levels of viral load (Ct > 30) and seropositive to IgG. CONCLUSION: In the context of virological success, the majority of ALHIV harbour ADRMs, essentially driven by NNRTI mutations and low CD4 nadir. During the current pandemic, about one-third of ALHIV were previously exposed to SARS-CoV-2. However, some children might have been exposed and uninfected and others might have been infected but showed no serological response at sampling. These findings support the use of NNRTI-sparing regimens and the implementation of COVID-19 barrier measures targeting ALHIV during such a pandemic.

Evaluation of HIV-1 capsid genetic variability and lenacapavir (GS-6207) drug resistance-associated mutations according to viral clades among drug-naive individuals.

OBJECTIVES: We evaluated the HIV-1 capsid genetic variability and lenacapavir drug resistance-associated mutations (DRMs) among drug-naive individuals across HIV-1 clades. METHODS: A total of 2031 HIV-1 sequences from drug-naive patients were analysed for capsid amino acid modification and the prevalence of lenacapavir DRMs. Amino acid positions with <5% variability were considered as conserved and variability was analysed by HIV-1 clades. RESULTS: Overall, 63% (148/232) of amino acid positions were conserved in the capsid protein. Of note, conservation was consistent in specific binding residues of cellular factors involved in viral replication [CypA (G89, P90), CPSF6 (Q4, N57, N74, A77, K182) and TRIM-NUP153 (R143)], while N183 (12.31%) was the only non-conserved lenacapavir binding residue. The overall prevalence (95% CI) of lenacapavir DRMs was 0.14% (0.05-0.44) (3/2031), with M66I (0.05%) and Q67H (0.05%) observed in subtype C, and T107N (0.05%) observed in CRF01_AE. Moreover, polymorphic mutations M66C (n = 85; 4.18%), Q67K (n = 78; 3.84%), K70R (n = 7; 0.34%), N74R (n = 57; 2.81%) and T107L (n = 82; 4.03%) were observed at lenacapavir resistance-associated positions. CONCLUSIONS: The low level of lenacapavir DRMs (<1%) supports its predicted effectiveness for treatment and prevention, regardless of HIV-1 clades. The established conserved regions hence serve as a hallmark for the surveillance of novel mutations potentially relevant for lenacapavir resistance.

Archiving of mutations in HIV-1 cellular reservoirs among vertically infected adolescents is contingent with clinical stages and plasma viral load: Evidence from the EDCTP-READY study.

INTRODUCTION: Globally, HIV-related adolescent deaths have increased about 50%, especially for those who are vertically infected. This could be driven by archived drug resistance mutations (DRMs) as children grow up, which might jeopardize antiretroviral therapy (ART). Our objective was to compare HIV-1 genotypic variation between plasma RNA and proviral DNA of vertically infected adolescents (aged 10-19 years) failing ART. METHODS: A comparative study was conducted in 2019 among 296 adolescents with perinatal HIV infection (ALPHI) failing ART in health facilities of the Centre Region of Cameroon. The WHO clinical stage, CD4 count and plasma viral load (PVL) were measured. For those failing ART (PVL ≥ 1000 copies/mL), RNA (plasma) and proviral DNA (buffy coat) were sequenced in the pol gene at Chantal BIYA International Reference Centre (CIRCB), Yaoundé, Cameroon. HIV-1 subtypes and DRMs were interpreted using Stanford HIVdb v.8.8 and MEGA-X. RESULTS: Of the 30% (89/296) failing ART, 81 had both RNA and DNA sequences generated and three were excluded for APOBEC mutations: the mean age was 16 ± 3 years; female-to-male ratio was 3:5; median PVL was 46 856 copies/mL [interquartile range (IQR): 19 898-271 410]; median CD4 count was 264 cells/µL (IQR: 131-574); and 42% were at WHO clinical stage 3/4. Subtype concordance between RNA and DNA viral strains was 100%, with CRF02_AG being predominant (65%) and two potential new recombinants found (A1/G/K; F1/G). Adolescents with DRMs were significantly higher in plasma than in proviral DNA (92% vs. 86%, p < 0.0001). Prevalent DRMs by drug class (RNA vs. DNA respectively) were at position M184 (74% vs. 67%) for nucleoside reverse transcriptase inhibitors (NRTIs), K103 (63% vs. 59%) for non-NRTIs, and V82, L76 and M46 (2% vs. 2%) for protease inhibitors. A total of 35% (27/78) of adolescents had concordant DRM profiles in RNA and DNA, while 27% (21/78) had DRMs only in proviral DNA. The presence of archived DRMs was associated with advanced clinical stage 3/4 (OR = 0.14, p = 0.0003) and PVL < 5 Log (Copies/mL) (OR: 4.88, p = 0.006). CONCLUSIONS: Although plasma RNA remains more sensitive for detecting HIV-1 DRMs, about a quarter of ALPHI experiencing ART failure in an African setting might have archived DRMs in viral reservoirs, indicating clinically occult resistance. Thus, to ensure effective ART success, proviral DNA profiling (alongside RNA genotyping) would provide additional DRMs for adolescents with advanced clinical stages and/or moderate PVL.

Baseline integrase drug resistance mutations and conserved regions across HIV-1 clades in Cameroon: implications for transition to dolutegravir in resource-limited settings.

BACKGROUND: Transition to dolutegravir-based regimens in resource-limited settings (RLS) requires prior understanding of HIV-1 integrase variants and conserved regions. Therefore, we evaluated integrase drug resistance mutations (DRMs) and conserved regions amongst integrase strand transfer inhibitor (INSTI)-naive patients harbouring diverse HIV-1 clades in Cameroon. METHODS: A cross-sectional study was conducted amongst 918 INSTI-naive patients from Cameroon (89 ART-naive and 829 ART-experienced patients). HIV-1 sequences were interpreted regarding INSTI-DRMs using the Stanford HIVdb v8.9-1 and the 2019 IAS-USA list. Amino acid positions with <1% variability were considered as highly conserved. Subtyping was performed by phylogeny. RESULTS: Overall prevalence (95% CI) of INSTI-DRMs was 0.8% (0.4-1.7), with 0.0% (0.0-4.0) amongst ART-naive versus 0.9% (0.5-1.9) amongst ART-experienced patients; P = 0.44. Accessory mutations (95% CI) were found in 33.8% (30.9-37.0), with 38.2% (28.1-49.1) amongst ART-naive versus 33.4% (30.4-36.7) amongst ART-experienced patients; P = 0.21. Of 288 HIV-1 integrase amino acid positions, 58.3% were highly conserved across subtypes in the following major regions: V75-G82, E85-P90, H114-G118, K127-W132, E138-G149, Q168-L172, T174-V180, W235-A239 and L241-D253. Wide genetic diversity was found (37 clades), including groups M (92.3%), N (1.4%), O (6.2%) and P (0.1%). Amongst group M, CRF02_AG was predominant (47.4%), with a significantly higher frequency (95% CI) of accessory mutations compared with non-AG [41.4% (36.8-46.0) versus 27.1% (23.3-31.2) respectively; P < 0.001]. CONCLUSIONS: The low baseline of INSTI-DRMs (<1%) in Cameroon suggests effectiveness of dolutegravir-based regimens. In spite of high conservation across clades, the variability of accessory mutations between major circulating strains underscores the need for monitoring the selection of INSTI-DRMs while scaling up dolutegravir-based regimens in RLS.

Programme quality indicators of HIV drug resistance among adolescents in urban versus rural settings of the centre region of Cameroon.

BACKGROUND: The high rate of mortality among HIV-vertically infected adolescents might be favoured by HIV drug resistance (HIVDR) emergence, which calls for timeous actions in this underserved population. We thus sought to evaluate program quality indicators (PQIs) of HIVDR among HIV-vertically infected adolescents on antiretroviral therapy (ART). METHODS: A study was conducted in the Centre region of Cameroon among adolescents (10-19 years) receiving ART in two urban (The Mother-Child Centre of the Chantal BIYA Foundation, the National Social Welfare Hospital) and three rural (Mfou District Hospital, Mbalmayo District Hospital and Nkomo Medical Center) health facilities. Following an exhaustive sampling from ART registers, patient medical files and pharmacy records, data was abstracted for seven PQIs: on-time drug pick-up; retention in care; pharmacy stock outs; dispensing practices; viral load coverage; viral suppression and adequate switch to second-line. Performance in PQIs was interpreted following the WHO-recommended thresholds (desirable, fair and/or poor); with p < 0.05 considered significant. RESULTS: Among 967 adolescents (888 urban versus 79 rural) registered in the study sites, validated data was available for 633 (554 in urban and 79 in rural). Performance in the urban vs. rural settings was respectively: on-time drug pick-up was significantly poorer in rural (79% vs. 46%, p = 0.00000006); retention in care was fair in urban (80% vs. 72%, p = 0.17); pharmacy stock outs was significantly higher in urban settings (92% vs. 50%, p = 0.004); dispensing practices was desirable (100% vs. 100%, p = 1.000); viral load coverage was desirable only in urban sites (84% vs. 37%, p < 0.0001); viral suppression was poor (33% vs. 53%, p = 0.08); adequate switch to second-line varied (38.1% vs. 100%, p = 0.384). CONCLUSION: Among adolescents on ART in Cameroon, dispensing practices are appropriate, while adherence to ART program and viral load coverage are better in urban settings. However, in both urban and rural settings, pharmacy stock outs, poor viral suppression and inadequate switch to second-line among adolescents require corrective public-health actions to limit HIVDR and to improve transition towards adult care in countries sharing similar programmatic features.

Determinants of periodontitis according to the immunological and virological profiles of HIV-infected patients in Yaoundé, Cameroon.

BACKGROUND: HIV infection is associated to different oral manifestations (including periodontal diseases), which have decreased with the advent of antiretroviral therapy (ART). Yet, the occurrence of periodontitis is still consistent among patients with HIV living in sub Saharan-Africa, with limited evidence on the driven factors and mitigating measures in these settings. We aimed at evaluating the occurrence of periodontitis and its associated immunological and virological factors in patients with HIV living in Yaoundé, Cameroon. METHODS: We included 165 (44 ART-naïve and 121 ART-experienced) patients > 18 years old attending the Yaoundé Central Hospital and the Chantal BIYA International Reference Centre, from January-April 2018. The periodontal status was assessed by measuring the clinical attachment loss, periodontal pocket depth, plaques index and gingival bleeding index. CD4+/CD8+ cells and viremia were measured using the fluorescence-activated cell sorting method (FACS Calibur) and the Abbott m2000 RT HIV-1 RNA kit respectively. A standard-questionnaire concerning participants' medical records and oral hygiene methods was filled. Data was analyzed and p < 0.05 considered statistically significant. RESULTS: There was a significantly high prevalence of periodontitis in the ART-naïve (53.2%) compared to the ART-experienced group (37.3%), with a twofold increased risk of the ART-naïve population presenting with periodontitis than the ART-experienced population (OR 2.06, p = 0.03). More importantly, ART-naïve, patients with CD4 < 200 cells presented with higher risk of having periodontitis compared to those with higher CD4-values, with a threefold difference (OR 3.21). Worth noting, males presented with a higher risk of having clinical attachment loss (OR 6.07). There was no significant association between the occurrence of periodontitis and the CD8 (p = 0.45) or viremia (p = 0.10). CONCLUSION: In the Cameroonian context, a considerable number of adults infected with HIV suffer from periodontitis regardless of their treatment profile. Nonetheless, ART-naïve patients have a higher risk, indicating the protective role of ART. Interestingly, severely immune-compromised patients and men are vulnerable to periodontitis, thereby highlighting the need for clinicians to refer patients for regular periodontal screening especially male patients and those with low CD4. Such measures could greatly improve the quality of life of the population living with HIV in Cameroon.

HIV-1 drug resistance testing is essential for heavily-treated patients switching from first- to second-line regimens in resource-limited settings: evidence from routine clinical practice in Cameroon.

BACKGROUND: With the phase-out of stavudine (d4T), change to first-line regimens with zidovudine (AZT) or tenofovir (TDF) in resource-limited settings (RLS) might increase risks of cross-resistance to nucleos(t) ide reverse transcriptase inhibitors (NRTI). This would restrict the scope of switching to the World Health Organisation (WHO)-recommended standard second-line combinations (SLC) without HIV drug resistance (HIVDR)-testing in routine clinical practice. METHODS: An observational study was conducted among 101 Cameroonian patients (55.4% male, median [IQR] age 34 [10-41] years) failing first-line antiretroviral therapy (ART) in 2016, and stratified into three groups according to NRTIs exposure: exposure to both thymidine analogues AZT "and" D4T (group-A, n = 55); exposure to both TDF and AZT "or" D4T (group-B, n = 22); exposure solely to D4T (group-C, n = 24). Protease-reverse transcriptase HIVDR was interpreted using the HIVdb penalty scores (≥60: high-resistance; 20-59: intermediate-resistance; < 20: susceptible). The acceptable threshold for potential-efficacy was set at 80%. RESULTS: The median [IQR] CD4, viral RNA, and time on ART, were respectively 129 [29-466] cells/µl, 71,630 [19,041-368,000] copies/ml, and 4 [2-5] years. Overall HIVDR-level was 89.11% (90/101), with 83.2% harbouring M184 V (high-level 3TC/FTC-resistance) and only 1.98% (2/101) major HIVDR-mutations to ritonavir-boosted protease-inhibitors (PI/r). Thymidine-analogue mutations (TAMs)-1 [T215FY (46.53%), M41 L (22.77%), L210 W (8.91%)], with cross-resistance to AZT and TDF, were higher compared to TAMs-2 [D67N (21.78%), K70R (19.80%), K219QE (18.81%)]. As expected, K65R was related with TDF-exposure: 0% (0/55) in group-A, 22.72% (5/22) group-B, 4.17% (1/24) group-C (p = 0.0013). The potential-efficacy of AZT vs. TDF was respectively 43.64% (24/55) vs. 70.91% (39/55) in group-A (p = 0.0038); 63.64% (14/22) vs. 68.28% (15/22) in group-B (p = 1.0000); and 37.50% (9/24) vs. 83.33% (20/24) in group-C (p = 0.0032). CRF02_AG was the prevailing subtype (63.40%), followed by CRF11.cpx (8.91%), A1 (7.92%), G (5.94%); without any significant effect of the subtype-distribution on HIVDR (92.2% in CRF02_AG vs. 83.8% in non-AG; p = 0.204). CONCLUSION: First-line ART-failure exhibits high-level NRTI-resistance, with potential lower-efficacy of AZT compared to TDF. Significantly, using our 80% efficacy-threshold, only patients without NRTI-substitution on first-line could effectively switch to SLC following the WHO-approach. Patients with multiple NRTI-substitutions (exposed to both thymidine-analogues and TDF) on first-line ART would require HIVDR-testing to select active NRTIs for SLC.

Viral suppression in adults, adolescents and children receiving antiretroviral therapy in Cameroon: adolescents at high risk of virological failure in the era of "test and treat".

BACKGROUND: After the launching of the « Test & Treat ¼ strategy and the wider accessibility to viral load (VL), evaluating virological success (VS) would help in meeting the UNAIDS targets by 2020 in Cameroon. SETTING AND METHODS: Cross-sectional study conducted in the Chantal BIYA International Reference Centre for research on HIV/AIDS prevention and management (CIRCB), Yaoundé, Cameroon; data generated between October 2016 and August 2017 amongst adults, adolescents and children at 12, 24, 36 and ≥ 48 months on ART. VS was defined as < 1000 copies/mL of blood plasma and controlled viremia as VL < 50 copies/mL. Data were analysed by SPSS; p < 0.05 considered as significant. RESULTS: 1946 patients (70% female) were enrolled (1800 adults, 105 adolescents, 41 children); 1841 were on NNRTI-based and 105 on PI-based therapy; with 346 patients at M12, 270 at M24, 205 at M36 and 1125 at ≥ M48. The median (IQR) duration on was 48 months (24-48). Overall, VS was 79.4% (95% CI 77.6-81.2) and 67.1% (95% CI 64.9-69.1) had controlled viral replication. On NNRTI-based, VS was 79.9% vs. 71.4% on PIs-based, p = 0.003. By ART duration, VS was 84.1% (M12), 85.9% (M24), 75.1% (M36) and 77.2% (≥ M48), p = 0.001. By age, VS was 75.6% (children), 53.3% (adolescents) and 81.1% (adults), p < 0.001. CONCLUSIONS: In this sub-population of patients receiving ART in Cameroon, about 80% might be experiencing VS, with declining performance at adolescence, with NNRTI-based regimens, and as from 36 months on ART. Thus, improving VS may require an adapted adherence support mechanism, especially for adolescents with long-term treatment in resource-limited settings.

Evaluation of treatment response, drug resistance and HIV-1 variability among adolescents on first- and second-line antiretroviral therapy: a study protocol for a prospective observational study in the centre region of Cameroon (EDCTP READY-study).

BACKGROUND: Sub-Saharan Africa (SSA) alone has nine out of every 10 children living with HIV globally and monitoring in this setting remains suboptimal, even as these children grow older. With scalability of antiretroviral therapy (ART), several HIV-infected children are growing towards adolescence (over 2.1 million), with the potentials to reach adulthood. However, despite an overall reduction in HIV-related mortality, there are increasing deaths among adolescents living with HIV (ADLHIV), with limited evidence for improved policy-making. Of note, strategies for adolescent transition from pediatrics to adult-healthcare are critical to ensure successful treatment response and longer life expectancy. Interestingly, with uptakes in prevention of mother-to-child transmission, challenges in ART programs, and high viremia among children in SSA, the success rate of paediatric ART might be quickly jeopardised, with possible HIV-1 drug-resistance (HIVDR) emergence, especially after years of paediatric ART exposure. Therefore, monitoring ART response in adolescents and evaluating HIVDR patterns might limit disease progression and guide on subsequent ART options for SSA ADLHIV. OBJECTIVES: Among Cameroonian ADLHIV receiving ART, we shall evaluate the rate of immunovirologic failure, acquired HIVDR-associated mutations, HIV-1 subtype distribution, genetic variability in circulating (plasma) versus archived (cellular) viral strains, and HIVDR early warning indicators (EWIs) at different time-points. METHODS: A prospective and observational study will be conducted among 250 ADLHIV (10-19 years old) receiving ART in the centre region of Cameroon, and followed-up at 6 and 12 months after enrollment. Following consecutive sampling at enrolment, plasma viral load and CD4/CD8 count will be measured, and genotypic resistance testing (GRT) will be performed both in plasma and in buffy coat for participants experiencing virological failure (two consecutive viremia > = 1000 copies/ml). Plasma viral load and CD4/CD8 will be monitored for all participants at 6 and 12 months after enrolment. HIVDR-EWIs will be monitored and survival analysis performed during the 12 months follow-up. Primary outcomes are rates of virological failure, acquired-HIVDR, and mortality. DISCUSSION: Our findings will provide evidence-based recommendations to ensure successful transition from paediatrics to adult ART regimens and highlight further needs of active ART combinations, for reduced morbidity and mortality in populations of ADLHIV within SSA.

Rates of HBV, HCV, HDV and HIV type 1 among pregnant women and HIV type 1 drug resistance-associated mutations in breastfeeding women on antiretroviral therapy.

BACKGROUND: HBV, HCV, HDV and HIV are blood borne and can be transmitted from mother-to-child. Reports of HBV infection rates show up to 11.9% in Cameroon while for HCV, the rate is less than 2%. More so, as pregnant women get enrolled in the HIV PMTCT Programme and stay in the care continuum, selection of HIV-1 drug resistant strains is evident. We sought to determine the seroprevalence of HBV, HCV, HDV and HIV among pregnant women, assess their knowledge, attitudes and practices on transmission and prevention of HBV infection, and determine HIV drug resistance profile of breastfeeding women. METHODS: A serosurvey of HBV, HCV, HDV and HIV was carried out among 1005 pregnant women in Yaounde, Cameroon. In 40 HIV-infected breastfeeding women enrolled in the PMTCT Programme, HIV-1 genotypes and HIV-1 resistance to NRTIs, NNRTIs and PIs, were determined by phylogeny and the Stanford University HIV Drug Resistance interpretation tool, respectively. RESULTS: Among the pregnant women, the rates of HIV-1, HBV, HCV and HDV infections were 8.5, 6.4, 0.8 and 4.0%, respectively. About 5.9% of the women knew their HBV status before pregnancy unlike 63.7% who knew their HIV status. Although 83.3% reported that vaccination against HBV infection is a method of prevention, and 47.1% knew that HBV could be transmitted from mother-to-child, only 2.5% had received the Hepatitis B vaccine. Of the 40 women on antiretroviral therapy (ART), 9 had at least one major resistance-associated mutation (RAM, 22.5%) to NRTI, NNRTI or PI. Of these M184 V (12.5%), K70R (10.0%), K103 N (12.5%), Y181C (10.0%), M46 L (2.5%) and L90 M (2.5%) were most frequently identified, suggesting resistance to lamivudine, nevirapine, efavirenz and zidovudine. Eighty four percent were infected with HIV-1 recombinant strains with CRF02_AG predominating (50%). CONCLUSIONS: The rates of HBV and HIV-1 infections point to the need for early diagnosis of these viruses during pregnancy and referral to care services in order to minimize the risk of MTCT. Furthermore, our results would be useful for evaluating the HIV PMTCT Programme and Treatment Guidelines for Cameroon.

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

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.

Evaluation of HIV-1 DNA levels among adolescents living with perinatally acquired HIV-1 in Yaounde, Cameroon: A contribution to paediatric HIV cure research in Sub-Saharan Africa.

Background: With the advent of antiretroviral therapy (ART), most children living with HIV in sub-Saharan Africa (SSA) are growing toward adolescence, with scarcity of evidence on the size of viral reservoirs to enhance paediatric cure research strategies. This study aims to compare HIV-1 proviral DNA levels according to virological response among adolescents living with perinatally acquired HIV-1 (ALPHIV) and identify associated-factors in the Cameroonian context. Methods: In this observational cohort study, HIV-1 RNA viremia and CD4+ T-cell count were assessed through RT-PCR and flow cytometry respectively at three time-points over 18 months of observation. At the third time-point, 80 randomly-selected participants were classified as with viremia (≥50 HIV-1 copies/mL; n = 40) or without viremia (<50 HIV-1 copies/mL; n = 40); immune-competent (≥500 CD4+ T cells/mm3) or immunocompromised (<500 CD4+ T cells/mm3). Among these participants, total HIV-1 DNA load was quantified through droplet digital PCR using Bio-Rad QX200. Results: Of the 80 randomly-selected adolescents, median [IQR] age was 15 (13-17) years, 56.2% were female, duration on ART was 9.3 [5.4-12.2] years. Among the 40 viremic ones (median viremia 7312 [283-71482]) HIV-1 copies/ml, 75.0% (30/40) were in virological failure (≥1000 HIV-1 copies/ml), while median of CD4 T cells were 494 [360-793] cell/mm3 with 48.8% (39/80) immunocompromised. No significant variation in HIV-1 RNA viremia and CD4 T cell count was observed between the three time-points, and 13.7% (11/80) adolescents remained aviremic and immune-competent throughout (stable adolescents). A positive and moderate correlation (r = 0.59; p < 0.001) was found between HIV-1 DNA levels and HIV- 1 RNA viremia. Regarding the CD4 T cell count, a negative and weak correlation (r = -0.28; p = 0.014) was found with HIV-1 DNA loads only among adolescents with viremia. Starting ART within the first year of life, ART for over 9 years and aviremia appear as predictors of low HIV-1 DNA loads. Conclusion: Among ALPHIV, high HIV-1 RNA indicates an elevated viral reservoir size, representing a drawback to cure research. Interestingly, early ART initiation and longer ARTduration lead to sustained viral control and limited HIV-1 reservoir size. As limited size of viral reservoir appears consistent with viral control and immune competence, adolescents with sustained viral control (about 14% of this target population) would be candidates for analytical ART interruptions toward establishing paediatric post-treatment controllers in SSA.

Current knowledge of human Mpox viral infection among healthcare workers in Cameroon calls for capacity-strengthening for pandemic preparedness.

Introduction: An increased incidence of human Monkeypox (Mpox) cases was recently observed worldwide, including in Cameroon. To ensure efficient preparedness and interventions in the health system, we sought to assess the knowledge of Mpox's transmission, prevention, and response among healthcare workers (HCWs) in Cameroon. Methods: A cross-sectional online survey was conducted among HCWs in Cameroon using 21-item questions adapted from the United States Centers for Disease Control and Prevention (US-CDC) standard questionnaire on Mpox. The overall knowledge of Mpox was assessed by cumulative score and categorized as excellent (≥80%, 17/21) or good (≥70%, ≥15/21) knowledge. The regression analysis was used to identify the predictors of Mpox knowledge. Results: The survey enrolled 377 participants, but only responses from 342 participants were analyzed. Overall, 50.6% were female participants, and 59.6% aged 30 years or younger. The majority of the participants were medical doctors (50.3%); most worked in central-level hospitals (25.1%) and had 1-5 years of experience (70.7%). A total of up to 92.7% were aware of Mpox, with social media (58.7%) and radio/television (49.2%) as the main sources. The mean knowledge score was 14.0 ± 3.0 (4 to 20), with only 12.9% having excellent knowledge (≥80%) and 42.1% having good knowledge of Mpox. Younger age (26-30 years old) was associated with good knowledge, while workplace type was associated with excellent knowledge of Mpox (aOR [95% CI]: 4.01 [1.43-11.24]). Knowledge of treatment/management of Mpox was generally poor across the different professional categories. Conclusion: Knowledge of Mpox among HCWs is substandard across different professionals. Thus, for optimal preparedness and immediate interventions for Mpox and similar emerging pathogens, capacity-strengthening programs should be organized for HCWs while encouraging scientific literature and organizational social media websites.

Performance characteristics of INDICAID antigen rapid diagnostic test on SARS-CoV-2 samples during the omicron wave in Cameroon.

Background: WHO recommends the use of COVID-19 antigen rapid diagnostic tests (Ag-RDT) with at least 80 % sensitivity and 97 % specificity. In the era of Omicron variants, we sought to ascertain the performance of the INDICAID™ Ag-RDT compared to real-time PCR (RT-PCR) as the gold standard. Methods: A laboratory-based study was conducted among consenting individuals tested for COVID-19 at the virology laboratory of the Chantal BIYA International Reference Centre, Yaoundé-Cameron. The samples were processed by INDICAID™ Ag-RDT and DaAn Gene real-time PCR according to the manufacturer's instructions, and PCR-results were interpreted as per cycle thresholds (CT). The sensitivity, specificity, positive and negative predictive values (PPV and NVP) of INDICAID™ Ag-RDT were evaluated according to PCR CT-values. Results: A total of 565 nasopharyngeal swabs were collected from participants (median age [IQR]: 40 [31-75]; M/F sex-ratio was 1.2 and 380 were vaccinated). Following PCR, overall COVID-19 positivity was 5.66 %. For CT < 37, INDICAID™ Ag-RDT sensitivity was 21.9 % (95%CI: [8.3-39.9]), specificity 100 % (95%CI: [99.3-100]); PPV 100 % (95%CI: [59.0-100]), NPV 95.5 % (95%CI: [93.4-97.1]) and kappa = 0.34 (95%CI: [0.19-0.35]). For CT < 25, sensitivity was 100 % (95%CI: [47.8-100.0]), specificity 99.6 % (95%CI: [98.7-99.9]); PPV 94.4 % (95%CI: [51.7-100]), NPV 100 % (95%CI: [99.3-100]) and kappa = 0.83 (95%CI: [0.6-1.0]). COVID-19 sequences generated were all Omicron BA.1 subvariants. Conclusion: For patients infected with high viral loads (CT < 25), INDICAID™ Ag-RDT has high intrinsic (sensitivity and specificity) and extrinsic (predictive values) performances for COVID-19 diagnosis. Due to its simplicity and short turnaround time, INDICAID™ Ag-RDT is, therefore a reliable tool to prevent the spread of COVID-19 at community level in the current era of Omicron subvariants.

XBB.1, BQ1.1 and atypical BA.4.6/XBB.1 recombinants predominate current SARS-CoV-2 Wavelets with flu-like symptoms in Cameroon: A snapshot from genomic surveillance.

As of December 2022, Cameroon had observed a slight resurgence of COVID-19, raising concerns on genomic surveillance of related-SARS-CoV-2 variants under circulation. Following a laboratory-based survey, positive SARS-CoV-2 samples detected from December-2022 through March-2023 were processed for targeted sequencing at the Chantal BIYA International Reference Centre (CIRCB) in Yaoundé-Cameroon. From all positive cases detected, 13 were successfully sequenced (mean age 34 years, 70% female); the majority of the cases were unvaccinated (70%, 9/13) and symptomatic (92%, 12/13); all with flu-like symptoms (100%, 12/12). Following RT-PCR, the median cycle threshold was 22.23 [18-24] for the N gene; and 24.09 [20-26] for the ORF gene, underscoring high viral loads. Phylogenetic analysis of nucleotide sequences identified four major sub-variants in circulation, of which BA.5 (3/13), the recombinants BQ.1.1 (4/13), XBB.1 (4/13) and novel atypical variant of BA.4.6/XBB.1 (2/13). This snapshot surveillance indicates the introduction/emergence and circulation of new Omicron sub-variants, all accompanied by minor/mild symptoms. However, these new sub-variants and recombinants call for continuous genomic surveillance to prevent further resurgence of Covid-19 epidemiological wave.

SARS-CoV-2 genomic surveillance and reliability of PCR single point mutation assay (SNPsig® SARS-CoV-2 EscapePLEX CE) for the rapid detection of variants of concern in Cameroon.

Background: Surveillance of SARS-CoV-2 variants of concern (VOCs) and lineages is crucial for decision-making. Our objective was to study the SARS-CoV-2 clade dynamics across epidemiological waves and evaluate the reliability of SNPsig® SARS-CoV-2 EscapePLEX CE in detecting VOCs in Cameroon. Material and methods: A laboratory-based study was conducted on SARS-CoV-2 positive nasopharyngeal specimens cycle threshold (Ct)≤30 at the Chantal BIYA International Reference Centre in Yaoundé-Cameroon, between April-2020 to August-2022. Samples were analyzed in parallel with Sanger sequencing and (SNPsig® SARS-CoV-2 EscapePLEX CE), and performance characteristics were evaluated by Cohen's coefficient and McNemar test. Results: Of the 130 sequences generated, SARS-CoV-2 clades during wave-1 (April-November 2020) showed 97 % (30/31) wild-type lineages and 3 % (1/31) Gamma-variant; wave-2 (December-2020 to May-2021), 25 % (4/16) Alpha-variant, 25 % (4/16) Beta-variant, 44 % (7/16) wild-type and 6 % (1/16) mu; wave-3 (June-October 2021), 94 % (27/29) Delta-variant, 3 % (1/29) Alpha-variant, 3 % (1/29) wild-type; wave-4 (November-2021 to August-2022), 98 % (53/54) Omicron-variant and 2 % (1/54) Delta-variant. Omicron sub-variants were BA.1 (47 %), BA.5 (34 %), BA.2 (13 %) and BA.4 (6 %). Globally, the two genotyping methods accurately identified the SARS-CoV-2 VOCs (P = 0.17, McNemar test; Ka = 0.67). Conclusion: Genomic surveillance reveals a rapid dynamic in SARS-CoV-2 strains between epidemiological waves in Cameroon. For wide-spread variant surveillance in resource-limited settings, SNPsig® SARS-CoV-2 EscapePLEX CEkit represents a suitable tool, pending upgrading for distinguishing Omicron sub-lineages.

Challenges faced by the HIV testing system in low- and middle-income countries.

Introduction: Determining the HIV status of some individuals remains challenging due to multidimensional factors such as flaws in diagnostic systems, technological challenges, and viral diversity. This report pinpoints challenges faced by the HIV testing system in Cameroon. Case presentation: A 53-year-old male received a positive HIV result by a rapid testing algorithm in July 2016. Not convinced of his HIV status, he requested additional tests. In February 2017, he received a positive result using ImmunoComb® II HIV 1 & 2 BiSpot and Roche cobas electrochemiluminescence assays. A sample sent to France in April 2017 was positive on the Bio-Rad GenScreen™ HIV 1/2, but serotyping was indeterminate, and viral load was < 20 copies/mL. The Roche electrochemiluminescence immunoassay and INNO-LIA HIV I/II Score were negative for samples collected in 2018. A sample collected in July 2019 and tested with VIDAS® HIV Duo Ultra enzyme-linked fluorescent assay and Geenius™ HIV 1/2 Confirmatory Assay was positive, but negative with Western blot; CD4 count was 1380 cells/mm3 and HIV proviral DNA tested in France was 'target-not-detected'. Some rapid tests were still positive in 2020 and 2021. Serotyping remained indeterminate, and viral load was 'target-not-detected'. There were no self-reported exposure to HIV risk factors, and his wife was HIV-seronegative. Management and outcome: Given that the patient remained asymptomatic with no evidence of viral replication, no antiretroviral therapy was initiated. Conclusion: This case highlights the struggles faced by some individuals in confirming their HIV status and the need to update existing technologies and develop an algorithm for managing exceptional cases.

Laboratory Based Surveillance of HIV-1 Acquired Drug Resistance in Cameroon: Implications for Use of Tenofovir-Lamivudine-Dolutegravir (TLD) as Second- or Third-Line Regimens.

Increased HIV drug resistance (HIVDR) with antiretroviral therapy (ART) rollout may jeopardize therapeutic options, especially in this era of transition to fixed-dose tenofovir-lamivudine-dolutegravir (TLD). We studied acquired HIVDR (ADR) patterns and describe potentially active drugs after first- and second-line failure in resource-limited settings (RLS) like Cameroon. A laboratory-based study with 759 patients (≥15 years) experiencing virological failure was carried out at the Chantal Biya International Reference Centre (CIRCB), Yaoundé, Cameroon. Socio-demographic, therapeutic and immunovirological data from patient records were analysed according to HIV-1 genotypic profiles. Median (IQR) ART-duration was 63 (50-308) months. Median CD4 and viremia were 153 (IQR:50-308) cells/mm3 and 138,666 (IQR:28,979-533,066) copies/mL, respectively. Overall ADR was high (93.4% first-line; 92.9%-second-line). TDF, potentially active in 35.7% of participants after first-line and 45.1% after second-line, suggested sub-optimal TLD-efficacy in second-line (64.3%) and third-line (54.9%). All PI/r preserved high efficacy after first-line failure while only DRV/r preserved high-level efficacy (87.9%) after second-line failure. In this resource-limited setting (RLS), ADR is high in ART-failing patients. PI/r strategies remain potent backbones for second-line ART, while only DRV/r remains very potent despite second-line failure. Though TLD use would be preferable, blind use for second- and third-line regimens may be sub-optimal (functional monotherapy with dolutegravir) with high risk of further failure, thus suggesting strategies for selective ART switch to TLD in failing patients in RLS.

HIV-1 genotypic profiling ensures effective response to third-line antiretroviral therapy in Cameroon.

In order to limit the emergence of human immunodeficiency virus (HIV) drug resistance in a context of limited antiretroviral options, we sought to evaluate the efficacy of third-line (3L) regimens considering HIV genotypic resistance profile at initiation of 3L in Cameroon. A cohort-study was conducted from January-September 2020 among patients initiating a 3L antiretroviral therapy regimen at the Yaoundé Central Hospital. HIV-1 protease-reverse transcriptase was sequenced at the Chantal Biya international reference center for research on HIV/AIDS prevention and management and results were interpreted using Stanford HIVdbv8.3. Good virological response (viral load < 390 copies/mL) was assessed after 12 months using OPP-ERA platform. Statistical analyses were performed using Epi Info v7.2.2.6, with P < .05 considered statistically significant. Of the 38 patients initiating 3L with an available genotyping (42% female; median age, 49 [39-57] years), median cluster of differentiation type 4 count and viral load were 173 [34-374] cells/µL and 169,322 [30,382-551,826] copies/mL, respectively. At enrollment, all patients harbored resistance to reverse transcriptase inhibitors and 66% (25/38) to protease-inhibitors, although 63% (24/38) were still susceptible to darunavir/ritonavir. Preferred 3L regimen was dolutegravir + darunavir/r + tenofovir + lamivudine (51%) and median duration on 3L was 21 [17-32] months. Interestingly, 82% (31/38) of the participants achieved good virological response on 3L, regardless of genotypic profile at recruitment, variations in 3L regimens (P = .9) and baseline cluster of differentiation type 4 count (P = .3). Despite the high burden of reverse transcriptase inhibitor - and protease inhibitor boosted by ritonavir drug resistance, genotyping-guided 3L regimens is accompanied by virological success in most patients. This high efficacy, most likely due to use of high genetic barrier antiretrovirals, requires continuous adherence support alongside close monitoring for long-term effectiveness in similar programmatic settings.