Performance characteristics of Allele-Specific PCR (ASPCR) in detecting drug resistance mutations among non-B HIV-1 Variants.

Allele-Specific Polymerase Chain Reaction (ASPCR) is an affordable point-mutation assay whose validation could improve the detection of HIV-1 drug resistance mutations (DRMs) in resource-limited settings (RLS). We assessed the performance of ASPCR onforty-four non-B HIV-1 plasma samples from patients who were ARV treated in failure in N'Djamena-Chad. Viral RNA was reverse-transcribed and amplified using LightCycler® FastStart DNA MasterPLUS SYBR Green I. Detection of six major DRMs (K70R, K103N, Y181C, M184V, T215F, T215Y) was evaluated on Roche LightCycler®480 automated system (with dilutions 0.01-100%). ASPCR-results were compared to Sanger-sequencing (gold-standard). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). ASPCR appears more efficient for detecting DRMs on diverse HIV-1 non-B circulating in RLS like Chad.

Determinants of Survival of HIV Patients Receiving Dolutegravir: A Prospective Cohort Study in Conflict-Affected Bunia, Democratic Republic of Congo.

This study aims to determine the factors influencing HIV-related mortality in settings experiencing continuous armed conflict atrocities. In such settings, people living with HIV (PLHIV), and the partners of those affected may encounter specific difficulties regarding adherence to antiretroviral therapy (ART), and retention in HIV prevention, treatment, and care programs. Between July 2019 and July 2021, we conducted an observational prospective cohort study of 468 PLHIV patients treated with Dolutegravir at all the ART facilities in Bunia. The probability of death being the primary outcome, as a function of time of inclusion in the cohort, was determined using Kaplan-Meier plots. We used the log-rank test to compare survival curves and Cox proportional hazard modeling to determine mortality predictors from the baseline to 31 July 2021 (endpoint). The total number of person-months (p-m) was 3435, with a death rate of 6.70 per 1000 p-m. Compared with the 35-year-old reference group, older patients had a higher mortality risk. ART-naïve participants at the time of enrollment had a higher mortality risk than those already using ART. Patients with a high baseline viral load (≥1000 copies/mL) had a higher mortality risk compared with the reference group (adjusted hazard ratio = 6.04; 95% CI: 1.78-20.43). One-fourth of deaths in the cohort were direct victims of armed conflict, with an estimated excess death of 35.6%. Improving baseline viral load monitoring, starting ART early in individuals with high baseline viral loads, the proper tailoring of ART regimens and optimizing long-term ART, and care to manage non-AIDS-related chronic complications are recommended actions to reduce mortality. Not least, fostering women's inclusion, justice, peace, and security in conflict zones is critical in preventing premature deaths in the general population as well as among PLHIV.

Incidence and Predictors of Loss to Follow Up among Patients Living with HIV under Dolutegravir in Bunia, Democratic Republic of Congo: A Prospective Cohort Study.

This study aimed to examine the incidence and predictors of loss to follow up (LTFU) in the context of ongoing atrocities caused by armed conflict, where HIV treatment programs and HIV-infected patients may face unique challenges in terms of ART adherence and retention in care. We conducted an observational prospective cohort study of 468 patients living with HIV (PLWHIV) under dolutegravir (DTG) in all health facilities in Bunia between July 2019 and July 2021. Kaplan-Meier plots were used to determine the probability of LTFU as a function of time as inclusive of the cohort. The main outcome variable was LTFU, defined as not taking an ART refill for a period of 3 months or longer from the last attendance for refill, and not yet classified as 'dead' or 'transferred-out.' The log-rank test was used to compare survival curves based on predictors. Cox proportional hazard modeling was used to measure predictors of LTFU from the baseline until 31 July 2021 (the endpoint). A total of 3435.22 person-months (p-m) were involved in follow up, with an overall incidence rate of 33.48 LTFU per 1000 p-m. Patients who had less experience with ART at enrolment and the ethnically Sudanese, had a higher hazard of being LTFU compared to their reference groups. This study reports a high LTFU rate in this conflict setting. An ART program in such a setting should pay more attention to naive patients and other particularly vulnerable patients such as Sudanese during the pre-ART phase. The study implies the implementation of innovative strategies to address this high risk of being LTFU, reducing either the cost or the distance to the health facility.

Predictors of Viral Non-Suppression among Patients Living with HIV under Dolutegravir in Bunia, Democratic Republic of Congo: A Prospective Cohort Study.

The Democratic Republic of the Congo adopted the integrase inhibitor dolutegravir (DTG) as part of its preferred first-line HIV treatment regimen in 2019. This study aimed to identify predictors of viral non-suppression among HIV-infected patients under a DTG-based regimen in the context of ongoing armed conflict since 2017 in the city of Bunia in the DRC. We conducted a cohort study of 468 patients living with HIV under DTG in all health facilities in Bunia. We calculated the proportion of participants with an HIV RNA of below 50 copies per milliliter. About three in four patients (72.8%) in this cohort had a viral load (VL) of <50 copies/mL after 6-12 months. After controlling for the effect of other covariates, the likelihood of having non-suppression remained significantly lower among the 25-34 age group and self-reported naïve patients with a baseline VL of ≥50 copies/mL. The likelihood of having non-suppression remained significantly higher among those who were at advanced stages of the disease, those with abnormal serum creatinine, those with high baseline HIV viremia over 1000 copies/mL, and the Sudanese ethnic group compared to the reference groups. This study suggests that we should better evaluate adherence, especially among adolescents and economically vulnerable populations, such as the Sudanese ethnic group in the city of Bunia. This suggests that an awareness of the potential effects of DTG and tenofovir is important for providers who take care of HIV-positive patients using antiretroviral therapy (ART), especially those with abnormal serum creatinine levels before starting treatment.

[Epidemiological, Clinical And Therapeutic Profile Of Hiv-Infected Patients On Antiretroviral Treatment In Kisangani, Democratic Republic Of Congo]. / Profil Epidemiologique, Clinique Et Therapeutique Des Patients Infectes Par Le VIH Sous Traitement Antiretroviral A Kisangani, Republique Democratique Du Congo.

MATERIAL AND METHOD: This was a cross-sectional and descriptive study with retrospective collection conducted from 1 January 2020 to 31 December 2021 in Kisangani on HIV-infected patients. Sociodemographic, clinical and therapeutic data of patients were recorded and analyzed. RESULTS: A total of 124 patients were identified, 71% of whom were females. The majority were aged 26-35, female and unemployed. Provider-initiated testing and counselling was the most common circumstance of discovery of HIV diagnosis (56.4%). Weight loss (48.4%), fever (40.3%) and cough (37.9%) were the main clinical manifestations found in patients. At the time of the discovery of HIV infection, the majority of patients were in category C at stage III of the disease according to the 1993 CDC classification. The most frequently encountered antecedents were sexually transmitted infections (22.6%) and tuberculosis (14.5%). HIV infection mainly affects young adults, females, married, unemployed, urban residents, secondary school and who consult health facilities at the advanced stage of the disease. CONCLUSION: Public awareness (targeting especially youth) and early use of screening could improve this situation.

MATÉRIEL ET MÉTHODE: Il s'est agi d'une étude une étude transversale et descriptive à collecte rétrospective menée du 1er janvier 2020 au 31 décembre 2021 à Kisangani portant sur les patients infectés par le VIH. Les données sociodémographiques, cliniques et thérapeutiques des patients ont été enregistrées et analysées. RÉSULTATS: Au total 124 patients ont été identifiés, dont 71% des sujets de sexe féminin. La majorité était âgée de 26-35 ans, de sexe féminin et sans emploi. Le dépistage et le conseil initié par le prestataire était la circonstance de découverte la plus fréquente du diagnostic de l'infection à VIH (56,4%). L'amaigrissement (48,4%), la fièvre (40,3%) et la toux (37,9%), étaient les principales manifestations cliniques retrouvées chez les patients. Lors la découverte de l'infection par le VIH, la majorité des patients était dans la catégorie C au stade III de la maladie selon la classification de CDC de 1993. Les antécédents les plus fréquemment rencontrés étaient les infections sexuellement transmissibles (22,6%) et la tuberculose (14,5%). L'infection par le VIH affecte principalement des adultes jeunes, de sexe féminin, mariés, sans profession, résidents en milieu urbain, de niveau d'étude secondaire et qui consultent les formations sanitaires au stade avancé de la maladie. CONCLUSION: La sensibilisation de la population (ciblant surtout les jeunes) et le recours précoce au dépistage pourraient améliorer cette situation.

Epidemiological, clinical and biological profile of neuromeningeal cryptococcosis among people living with HIV in Kinshasa, Democratic Republic of Congo.

Neuromeningeal cryptococcosis (NMC) is one of the most frequent opportunistic infections (OI) in Human Immunodeficiency Virus (HIV) infection. In Kinshasa, the latest data on cryptococcosis were published in 1996. The objective was to describe the epidemiological, clinical and biological profiles of NMC in HIV-infected people living in Kinshasa. This is a descriptive study based on the medical records of patients who attended three clinics in Kinshasa between January 1 s t 2011 and December 31st 2014. Only the medical records of HIV-infected people presenting the NMC were reviewed. During the 4 year-period of the study, 261 HIV-positive patients presented to the clinics for neuromeningeal syndrome, including 23 with NMC. The global prevalence of NMC was 8.8% for the three clinics. The mean age was 42.8 ± 9.5 years, with male predominance (65.2%). The main symptoms were headache (73.9%), neck stiffness (60.9%), fever (47.8%), and coma (47.8%). Biological records were as follows: median CD4 cell count was 79 cells/mm3; cerebrospinal fluid (CSF) was clear for 56.5% of the cases with predominance of neutrophils in 73.9%. The outcome was fatal in 34.8% of cases. The prevalence and therapeutic outcome of NMC show that it constitutes a non-negligible OI in Kinshasa, especially in HIV-infected people at the AIDS stage. As HIV-infected people with severe immunosuppression are the most affected by NMC, active preventive measures should benefit this vulnerable category of people.

Déterminants des parasitoses intestinales chez les enfants VIH-positifs à Kinshasa

Contexte et objectif. L'immunodépression induite par le VIH s'accompagne d'infections diverses et certaines parasitoses intestinales (PI) y sont fréquemment associées. Les données relatives à cette co-infection sont fragmentaires en Afrique subsaharienne. La présente étude a évalué l'ampleur des parasitoses intestinales chez les enfants seropositfs pour le VIH (VIH+) suivis dans les hôpitaux de référence de Kinshasa. Méthodes. Une étude transversale multicentrique a été menée dans huit hôpitaux de référence de Kinshasa, incluant 227 enfants séropositifs pour le VIH (VIH+), âgés de 18 mois à 15 ans. Les données sociodémographiques ont été enregistrées, et les échantillons de selles et de sang collectés chez chacun des participants. Les examens parasitologiques sur selles (selles directes et après concentration), ainsi que le Kinyoun (Ziehl modifié, ont été réalisés au laboratoire de parasitologie de la faculté de Médecine, et le taux de lymphocytes T CD4 sanguin déterminé au laboratoire de référence de l'Hôpital Général de Référence de Kinshasa. Résultats. Deux cent vingt sept enfants (sexe ratio H/F : 1,1/1) ont été examinés. Parmi eux, 56 (24,6%, IC 95% :19,0 -30,3%.) étaient infectés par au moins un des parasites intestinaux suivants: Ascaris lumbricoïdes (12,8%), Trichiuris trichiura (11,9%), Schistosoma mansoni (0,4%), Entamoeba coli (5,7%) et Giardia intestinalis (1,8%). Cryptosporidium sp et Isospora belli n'ont pas été détectés. Le niveau bas d'étude des parents a été significativement associé aux PI (p = 0,039). Conclusion. A Kinshasa, un enfant VIH-séropositif sur quatre, surtout issu d'un ménage où le niveau d'instruction des tuteurs était bas, présente une PI. L'éducation sanitaire et les mesures élémentaires d'hygiène sont à promouvoir comme moyen primordial de prévention et de lutte contre ces PI

Virological response, HIV-1 drug resistance mutations and genetic diversity among patients on first-line antiretroviral therapy in N'Djamena, Chad: findings from a cross-sectional study.

BACKGROUND: The national antiretroviral therapy in the Republic of Chad provides free of charge antiretroviral regimens and therapeutic monitoring for patients receiving antiretroviral therapy nationwide. For a successful programmatic uptake, these efforts merit to be supported by thorough assessments of antiretroviral therapy response and HIV-1 drug resistance surveillance, especially with risks of cross-resistance due to the gradual stavudine phasing out in such national settings. We therefore evaluated the virological response to antiretroviral therapy, HIV-1 drug resistance emergence and circulating HIV-1 clades in a Chad context. A cross-sectional and prospective study was conducted among 116 patients (41 [δ ± 6.87] years, 59% female) receiving first-line antiretroviral therapy for ≥ 6 months in Ndjamena, Chad, in 2011-2012, enrolled consecutively. To ensure accuracy, plasma viral load was concomitantly measured using Abbott Real-Time and Cobas AmpliPrep/TaqMan (v2.0), and virological failure defined as ≥ 1000 HIV-1 RNA copies/ml. Plasma from patients experiencing virological failure were processed for sequencing of HIV-1 protease-reverse transcriptase using the ANRS-AC.11 resistance testing protocol; drug resistant mutations were interpreted using the ANRS-AC11 algorithm; and phylogenetic analysis was performed using MEGA.v.6. RESULTS: Majority of patients was receiving zidovudine plus lamivudine plus nevirapine (46%), stavudine plus lamivudine plus nevirapine (41%) and tenofovir plus emtricitabine plus efavirenz (11%), for a median time-on-treatment of 5 [IQR 4-7] years. The rate of virological failure was 43% (50/116), with 86% (43/50) sequencing performance. Overall, 32% (37/116) patients presented ≥ one major drug resistant mutation(s), with 29% (34/116) to nucleos(t)ide reverse transcriptase inhibitors (67% [29/43] M184V/I, 30% [13/43] T215Y/F, 19% [8/43] V75A/F/I/L/M, 9% [4/43] K70P/R/W, 9% [4/43] K219E/N/Q and 5% [2/43] A62V); 86% (37/43) to non-nulceos(t)ide reverse transcriptase inhibitors (30% [13/43] K103N/S/E, 26% [11/43] Y181C/V/F/L, 2% [1/43] L100I, 2% [1/43] F227L, 2% [1/43] P225H); and 2% (1/43) to protease inhibitors (M46I, I54V, V82S). Six HIV-1 subtypes were found: 30% circulating recombinant form (CRF02_AG), 30% J, 16% G, 9% A, 9% D, 5% F. CONCLUSIONS: In Chad, almost half of patients are failing first-line antiretroviral therapy after 5 years, with considerable drug resistant mutations at failure. Absence of K65R supports the use of tenofovir-containing regimens as preferred first-line and as suitable drug for second-line combinations, in this setting with significant HIV-1 genetic diversity.