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To assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (<20% of individual's viral quasispecies) affects antiretroviral treatment (ART)-suppression at term. A case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls. HIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared. PDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases' median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (p<0.0001), a shorter duration of ART prior to delivery (p<0.0001), and randomization to efavirenz- (versus raltegravir-) based ART (p = 0.0085). We observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted.

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01_AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01_AE genotyped by PacBio (n = 15) or Sanger (n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection.

Background: HIV drug resistance (DR) mutations can compromise antiretroviral therapy (ART) success among children living with HIV (CLHIV). We conducted a secondary analysis using data from a randomized control trial for ART monitoring among CLHIV in Kisumu County, Kenya from 2019 to 2023, to assess clinical, psychosocial, and structural factors associated with HIV DR. Methods: 704 CLHIV were followed for 12+ months, with characteristics captured at enrollment and follow-up visits in the “parent” randomized-controlled-trial (of point-of-care plasma viral load testing and for viremias ≥ 1000 copies/mL HIV genotyping for DR vs. standard-of-care) and an observational “extension” substudy (of participants on a dolutegravir-containing ART with genotyping performed on viremic specimens ≥ 200 copies/mL). A multivariate modified Poisson regression model was used to analyze factors associated with sequences yielding a Stanford HIVDR database DR penalty score (DR-PS) ≥ 30 to a nucleos(t)ides and/or non-nucleoside reverse transcriptase inhibitor, protease inhibitor (PI), and/or integrase inhibitor (INSTI). Results: Among 113 (16.1%) participants who underwent genotyping, 93 (82.3%) had a DR-PS ≥ 30. DR-PS ≥ 30 were associated with age 1–5 years (adjusted risk ratio (ARR) = 1.84; 95% confidence interval (CI): 1.07, 3.14), history of viremia ≥ 1000 copies/mL (ARR = 4.18; 95% CI: 2.77, 6.31), prescription of a PI- (ARR = 6.05; 95% CI: 3.43, 10.68) or INSTI-containing regimen (ARR = 1.83; 95% CI: 1.08, 3.11), poor adherence to ART (ARR = 1.91; 95% CI: 1.32, 2.76), lack of caregiver confidence in ART administration (ARR = 1.89; 95% CI: 1.11, 3.22), and mid-sized clinic populations (ARR = 0.55; 95% CI: 0.33, 0.92). Conclusion: Addressing social factors associated with DR-PS ≥ 30 may improve ART success among CLHIV.

Objective To assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (<20% of individual’s viral quasispecies) affects antiretroviral treatment (ART)-suppression at term. Design A case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls. Methods HIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared. Results PDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases’ median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (p<0.0001), a shorter duration of ART prior to delivery (p<0.0001), and randomization to efavirenz- (versus raltegravir-) based ART (p = 0.0085). Conclusions We observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted.

Background and AimsThe prevalence of naturally occurring HCV-NS5A resistance-associated substitutions (RAS) to DAA drugs might affect the response to treatment in HCV/HIV coinfected subjects. There are limited data on the frequency of HCV-NS5A naturally occurring drug-RAS at baseline in HCV/HIV coinfected patients when ultra-deep sequencing methodologies are applied.MethodsHCV-NS5A-RAS were evaluated among 25 subjects in each group. Patients were matched by age, gender, and hepatic fibrosis stage category to control for selection bias.ResultsWithin subtype 1a, RAS were observed in 28% of HCV monoinfected and 48% of HCV/HIV coinfected subjects. More patients in the HCV/HIV coinfected group had clinically relevant mutations to DAA directed at NS5A.ConclusionWhile the clinical significance of this observation may be limited in highly drug adherent populations, some HCV/HIV coinfected persons may be at greater risk of viral resistance if suboptimal dosing occurs.

Hepatitis B virus (HBV) exhibits a high degree of heterogeneity with at least 10 genotypes (A–J) identified to date. Intergenotypic recombination is relatively common. Previously, we investigated HBV drug resistance in HIV/HBV co-infected individuals in Ghana. After identifying multiple circulating genotypes and a novel D/E recombinant, we sought to determine if additional individuals were also infected with recombinant HBV. Partial genome sequences from three individuals were initially identified as genotype A4. Full-length HBV genomes were obtained using rolling circle amplification followed by PCR and shown to cluster with known A/E recombinant viruses. Similar recombination breakpoints were observed in these three individuals suggesting local spread of this novel recombinant HBV in Ghana.

Globally, there are approximately 240 million people chronically infected with hepatitis B virus (HBV)—a major cause of hepatocellular carcinoma. Ten different HBV genotypes (A–J) have been identified with distinct geographic distributions. Novel variants generated by recombination between different HBV genotypes have been documented worldwide and represent an important element of genetic variability with possible clinical implications. Here, the complete genome sequence of an HBV genotype D/E recombinant from Ghana is reported. The full-length sequence was obtained using rolling circle amplification followed by PCR and sequenced using next-generation sequencing (NGS). A consensus sequence was extracted from the NGS data and underwent phylogenetic analysis to determine genotype, as well as the recombination pattern. Subsequently, the sequence was compared to recombinants described previously in Africa. Based on MCMC phylogenetic analysis, SimPlot recombination analyses, and intragroup genetic distance, the isolate 007N full-length genome is unique compared to other reported D/E recombinants in Africa.

HIV pretreatment drug resistance – defined as resistance detected prior to the initiation of antiretroviral therapy (ART) – has increased in prevalence over time in parallel with the increased global utilization of ART. The prevalence of pretreatment drug resistance has surpassed 10% to non-nucleoside reverse transcriptase inhibitors (NNRTI) in multiple countries, potentially threatening the efficacy of NNRTI-containing regimens which have been recommended for first-line ART in resource-limited settings for nearly two decades. In 2019, the WHO updated the recommended first-line regimen to integrase inhibitor-based ART to address the high levels of NNRTI pretreatment resistance; however, NNRTIs are still recommended in the alternative first-line regimen and for postnatal prophylaxis in infants to prevent vertical HIV transmission. The work presented in this dissertation sought to better understand the impact of HIV drug resistance on maternal treatment outcome and vertical transmission. We performed a series of clinical trial sub-studies that investigated genotypic HIV drug resistance – using Sanger and/or next-generation sequencing assays – among women and their infants living with HIV in resource-limited settings. Salient observations from the three studies include that (1) NNRTI resistance was prevalent among women prior to efavirenz-based ART initiation, but NNRTI resistance alone was not associated with virologic failure by 12 months of treatment; (2) maternal drug resistance was a risk factor for vertical transmission during breastfeeding, and drug resistance was prevalent among infants and accumulated during breastfeeding; and (3) pretreatment drug resistance among pregnant women living with HIV did not significantly contribute to virologic non-suppression at term; however, in women, higher pretreatment HIV RNA load, randomization to efavirenz- versus integrase inhibitor-based ART, and shorter duration of ART were associated with non-suppression. Taken together, these results support WHO recommendations of early prenatal care engagement and initiation of integrase inhibitor-based ART for pregnant women living with HIV for optimal maternal outcomes and to prevent vertical transmission. However, for individuals who are unable to take integrase inhibitor-based ART, our data and others suggest the utility of TLE could be extended despite the high prevalence of NNRTI pretreatment resistance in resource-limited settings. Lastly, our findings suggest that NNRTI infant prophylaxis may be insufficient against drug-resistant variants in maternal breast milk and support the recommendation to replace NNRTI infant prophylaxis with regimens that have a greater barrier to drug resistance to prevent vertical transmission and retain NNRTI susceptibility in infected infants.

Abstract Background Two large studies suggest that resistance mutations to only nonnucleoside reverse transcriptase inhibitors (NNRTI) did not increase the risk of virologic failure during antiretroviral therapy (ART) with efavirenz/tenofovir disoproxil fumarate/lamivudine (or emtricitabine). We retrospectively evaluated a third cohort to determine the impact of NNRTI resistance on the efficacy of efavirenz-based ART. Methods Postpartum women living with human immunodeficiency virus (HIV) were studied if they initiated efavirenz-based ART because of the World Health Organization’s recommendation for universal ART. Resistance was detected by Sanger genotyping plasma prior to efavirenz-based ART and at virologic failure (HIV RNA >400 copies/mL). Logistic regression examined relationships between pre-efavirenz genotypes and virologic failure. Results Pre-efavirenz resistance was detected in 169 of 1223 (13.8%) participants. By month 12 of efavirenz-based ART, 189 of 1233 (15.3%) participants had virologic failure. Rates of virologic failure did not differ by pre-efavirenz NNRTI resistance. However, while pre-efavirenz nucleos(t)ide reverse transcriptase inhibitors (NRTI) and NNRTI resistance was rare (8/1223 [0.7%]) this genotype increased the odds (adjusted odds ratio, 11.2 [95% confidence interval, 2.21–72.2]) of virologic failure during efavirenz-based ART. Age, time interval between last viremic visit and efavirenz initiation, clinical site, viremia at delivery, hepatitis B virus coinfection, and antepartum regimen were also associated with virologic failure. Conclusions Resistance to NNRTI alone was prevalent and dual-class (NRTI and NNRTI) resistance was rare in this cohort, with only the latter associated with virologic failure. This confirms others’ findings that, if needed, efavirenz-based ART offers most people an effective alternative to dolutegravir-based ART.

Human pegivirus (HPgV) is a positive single-stranded RNA virus in the Flaviviridae family. Phylogenetic analysis reveals the presence of multiple HPgV genotypes with distinct geographic locations. HPgV is of interest because of its potential beneficial impact on HIV disease progression. Despite this, the effects of HPgV in the context of other viral infections, such as hepatitis B virus (HBV), are poorly understood, and data from resource-limited settings are scarce. Therefore, we conducted a cross-sectional analysis of HPgV in HIV/HBV co-infected patients in Ghana. Sera from 100 HIV/HBV co-infected individuals were evaluated for HPgV RNA, and the genotype determined by sequencing the 5′ untranslated region. HPgV RNA was detected in 27 samples (27%). Of these, 26 were genotyped successfully with 23 belonging to HPgV genotype 1 and 3 belonging to HPgV genotype 2. The presence of HPgV RNA had no statistically significant impact on CD4 cell count or HBV DNA titers in the HIV/HBV co-infected patients. However, there was a trend towards decreased HBV DNA levels in HPgV RNA-positive patients with CD4 cell count < 200 (p = 0.0626). HPgV co-infection is common in Ghana. The effect of HPgV on HIV or HBV disease among HIV/HBV co-infected patients was minimal. However, decreased HBV DNA levels in HPgV RNA-positive patients with low CD4 cell counts highlight the need for prospective studies of HPgV in HIV and hepatitis co-infected patients, especially in those with advanced HIV disease, to study further the effects of HPgV on liver disease.