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Little is known about the effect of human immunodeficiency virus type 1 (HIV-1) resistance mutations present at time of regimen switch on the response to second-line antiretroviral therapy in Africa. In adults who switched to boosted protease inhibitor-based regimens after first-line failure, HIV-RNA and genotypic resistance testing was performed at switch and after 12 months. Factors associated with treatment failure were assessed using logistic regression. Of 243 participants, 53% were predicted to receive partially active second-line regimens due to drug resistance. The risk of treatment failure was, however, not increased in these participants. In this African cohort, boosted protease inhibitors successfully resuppressed drug-resistant HIV after first-line failure. [ABSTRACT FROM AUTHOR]

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r 2  > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r 2  = 0.94 over 10-10 4 cells/μL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites., (© 2023 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Rationale: We developed a standardized method, possible poor treatment response (PPTR), to help ascertain efficacy endpoints in Study S31/A5349 (NCT02410772), an open-label trial comparing two 4-month rifapentine-based regimens with a standard 6-month regimen for the treatment of pulmonary tuberculosis (TB). Objectives: We describe the use of the PPTR process and evaluate whether the goals of minimizing bias in efficacy endpoint assessment and attainment of relevant data to determine outcomes for all participants were achieved. Methods: A PPTR event was defined as the occurrence of one or more prespecified triggers. Each PPTR required initiation of a standardized evaluation process that included obtaining multiple sputum samples for microbiology. Measurements and Main Results: Among 2,343 participants with culture-confirmed drug-susceptible TB, 454 individuals (19.4%) had a total of 534 individual PPTR events, of which 76.6% were microbiological (positive smear or culture at or after 17 wk). At least one PPTR event was experienced by 92.4% (133 of 144) of participants with TB-related unfavorable outcome and between 13.8% and 14.7% of participants with favorable and not-assessable outcomes. A total of 75% of participants with TB-related unfavorable outcomes had microbiological confirmation of failure to achieve a disease-free cure. Conclusions: Standardized methodologies, such as our PPTR approach, could facilitate unbiased efficacy outcome determinations, improve discrimination between outcomes that are related and unrelated to regimen efficacy, and enhance the ability to conduct pooled analyses of contemporary trials.

Background: Treatment management after repeated failure of antiretroviral therapy (ART) is difficult due to resistance and adherence challenges. For people who have failed non-nucleoside reverse transcriptase inhibitor-(NNRTI-) and protease inhibitor-(PI-) based regimens with no or limited resistance, remaining on PI-based ART is an option. Using data from an ART strategy trial (A5288) in low/middle-income countries which included this option, we explored whether predictors can be identified distinguishing those who experienced further virologic failure from those who achieved and maintained virologic suppression., Methods: A5288 enrolled people with confirmed HIV-1 RNA ≥ 1000 copies/mL after ≥ 24 weeks of PI-based ART and prior failure on NNRTI-based ART. This analysis focused on the 278 participants with no resistance to the PI being taken and no or limited nucleoside reverse transcriptase inhibitor (NRTI) resistance, who continued their PI with flexibility to change NRTIs. Proportional hazards models were used to evaluate predictors of virologic failure during follow-up (VF: confirmed HIV-1 RNA ≥ 1000 copies/mL at ≥ 24 weeks of follow-up)., Results: 56% of participants were female. At study entry, median age was 40 years, time on ART 7.8 years, CD4 count 169 cells/mm 3 , HIV-1 RNA 20,444 copies/mL; and 37% had NRTI resistance. The estimated proportion experiencing VF increased from 39% at week 24 to 60% at week 96. In multivariable analysis, significant predictors at study entry of VF were higher HIV-1 RNA (adjusted hazard ratio: 2.20 for ≥ 10,000 versus < 10,000 copies/mL), lower age (1.96 for < 30 versus ≥ 30 years), NRTI resistance (1.74 for present versus absent), lower CD4 count (1.73 for < 200 versus ≥ 200 cells/mm 3 ), and shorter ART duration (1.62 for < 10 versus ≥ 10 years). There was a strong trend in proportion with VF at week 96 with the number of these five risk factors that a participant had, varying from 8% for zero, to 31%, 40%, 73%, and 100% for one, two, three, and four/five. Only 13% of participants developed new NRTI or PI resistance mutations., Conclusion: A simple count of five predictors might have value for identifying risk of continued VF. Novel antiretroviral and adherence support interventions are needed to improve virologic outcomes for higher risk individuals., (© 2023. The Author(s).)

We report a 23% asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) Omicron carriage rate in participants being enrolled into a clinical trial in South Africa, 15-fold higher than in trials before Omicron. We also found lower CD4 + T-cell counts in persons with human immunodeficiency virus (HIV) strongly correlated with increased odds of being SARS-CoV-2 polymerase chain reaction (PCR) positive., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

Introduction: ACTG A5288 was a strategy trial conducted in diverse populations from multiple continents of people living with HIV (PLWH) failing second-line protease inhibitor (PI)-based antiretroviral therapy (ART) from 10 low- and middle-income countries (LMICs). Participants resistant to lopinavir (LPV) and/or multiple nucleotide reverse transcriptase inhibitors started on third-line regimens that included raltegravir (RAL), darunavir/ritonavir (DRV/r) and/or etravirine (ETR) according to their resistance profiles. At 48 weeks, 87% of these participants achieved HIV-1 RNA ≤200 copies/ml. We report here long-term outcomes over 144 weeks., Methods: Study participants were enrolled from 2013 to 2015, prior to the availability of dolutegravir in LMICs. "Extended Follow-up" of the study started after the last participant enrolled had reached 48 weeks and included participants still on antiretroviral (ARV) regimens containing RAL, DRV/r and/or ETR at that time. RAL, DRV/r and ETR were provided for an additional 96 weeks (giving total follow-up of ≥144 weeks), with HIV-1 RNA measured at 48 and 96 weeks and CD4 count at 96 weeks after entry into Extended Follow-up. Proportion of participants with HIV-1 RNA ≤200 copies/ml was estimated every 24 weeks, using imputation if necessary to handle the different measurement schedule in Extended Follow-up; mean CD4 count changes were estimated using loess regression., Results and Discussion: Of 257 participants (38% females), at study entry, median CD4 count was 179 cells/mm 3 , and HIV-1 RNA was 4.6 log 10 copies/ml. Median follow-up was 168 weeks (IQR: 156-204); 15 (6%) participants were lost to follow-up and 9 (4%) died. 27/246 (11%), 26/246 (11%) and 13/92 (14%) of participants who started RAL, DRV/r and ETR, respectively, discontinued these drugs; only three due to adverse events. 87%, 86%, 83% and 80% of the participants had HIV-1 RNA ≤200 copies/ml at weeks 48, 96, 144 and 168 (95% CI at week 168: 74-85%), respectively. Mean increase from study entry in CD4 count at week 168 was 265 cells/mm 3 (95% CI 247-283)., Conclusions: Third-line regimens comprising of RAL, DRV/r and/or ETR were very well tolerated and had high rates of durable virologic suppression among PLWH in LMICs who were failing on second-line PI-based ART prior to the availability of dolutegravir., (© 2022 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

Evidence on the impact of human immunodeficiency virus (HIV) drug resistance on regimens following treatment failure is varied and inconclusive. Differential medication adherence may explain this variation. We aimed to test the association between drug resistance at first-line antiretroviral therapy (ART) switch and adherence to and virologic failure on subsequent ART. We conducted a secondary analysis of data from an open-labeled randomized trial of second-line ART (ACTG A5234). ART susceptibility was determined from study entry plasma using the Stanford Drug Resistance database version 8.7. Adherence was measured with microelectronic monitors. Three adherence variables and rates of virologic failure (HIV-1 RNA ≥1000 copies/mL) on second-line ART were compared between participants with and without resistance at first-line ART failure. Of 214 participants switching to second-line ART with baseline resistance results, 113 (53%) were men, mean age was 39 years (standard deviation 10.3), and 37 (17%) had susceptible virus at study entry. Cumulative genotypic susceptibility score (cGSS) was inversely associated with adherence, adjusted odds ratio (aOR) 0.15, 95% confidence interval (CI) (0.05-0.40), p  < 0.001. The aOR of virologic failure for a one-unit increase in cGSS was 1.72, 95% CI (1.22-2.41), p  < 0.001. Participants switched to second-line ART without resistance displayed inferior adherence and had higher rates of virologic failure. Therefore, these individuals warrant additional adherence interventions to help them achieve virologic success. Clinical Trial Registration number: NCT00608569.

Introduction: Cryptococcosis remains a leading cause of meningitis and mortality among people living with HIV (PLHIV) worldwide. We sought to evaluate laboratory-based cryptococcal antigen (CrAg) reflex testing and a clinic-based point-of-care (POC) CrAg screening intervention for preventing meningitis and mortality among PLHIV in South Africa., Methods: We conducted a prospective pre-post intervention study of adults presenting for HIV testing in Umlazi township, South Africa, over a 6-year period (2013-2019). Participants were enrolled during 3 phases of CrAg testing: CrAg testing ordered by a clinician (clinician-directed testing, 2013-2015); routine laboratory-based CrAg reflex testing for blood samples with CD4 ≤100 cells/mm3 (laboratory reflex testing, 2015-2017); and a clinic-based intervention with POC CD4 testing and POC CrAg testing for PLHIV with CD4 ≤200 cells/mm3 with continued standard-of-care routine laboratory reflex testing among those with CD4 ≤100 cells/mm3 (clinic-based testing, 2017-2019). The laboratory and clinical teams performed serum CrAg by enzyme immunoassay and lateral flow assay (Immy Diagnostics, Norman, OK). We followed up participants for up to 14 months to compare associations between baseline CrAg positivity, antiretroviral therapy and fluconazole treatment initiation, and outcomes of cryptococcal meningitis, hospitalization, and mortality., Results: Three thousand one hundred five (39.4%) of 7877 people screened were HIV-positive, of whom 908 had CD4 ≤200 cells/mm3 and were included in the analyses. Laboratory reflex and clinic-based testing increased CrAg screening (P < 0.001) and diagnosis of CrAg-positive PLHIV (P = 0.011). When compared with clinician-directed testing, clinic-based CrAg testing showed an increase in the number of PLHIV diagnosed with cryptococcal meningitis (4.5% vs. 1.5%; P = 0.059), initiation of fluconazole preemptive therapy (7.2% vs. 2.5%; P = 0.010), and initiation of antiretroviral therapy (96.8% vs. 91.3%; P = 0.012). Comparing clinic-based testing with laboratory reflex testing, there was no significant difference in the cumulative incidence of cryptococcal meningitis (4.5% vs. 4.1%; P = 0.836) or mortality (8.1% vs. 9.9%; P = 0.557)., Conclusions: Laboratory reflex and clinic-based CrAg testing facilitated the diagnosis of HIV-associated cryptococcosis and fluconazole initiation but did not reduce cryptococcal meningitis or mortality. In this nonrandomized cohort, clinical outcomes were similar between laboratory reflex testing and clinic-based POC CrAg testing., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To evaluate associations between hair antiretroviral hair concentrations as an objective, cumulative adherence metric, with self-reported adherence and virologic outcomes., Design: Analysis of cohort A of the ACTG-A5288 study. These patients in resource-limited settings were failing second-line protease inhibitor-based antiretroviral therapy (ART) but were susceptible to at least one nucleoside reverse transcriptase inhibitor (NRTI) and their protease inhibitor, and continued taking their protease inhibitor-based regimen., Methods: Antiretroviral hair concentrations in participants taking two NRTIs with boosted atazanavir (n = 69) or lopinavir (n = 112) were analyzed at weeks 12, 24, 36 and 48 using liquid-chromatography--tandem-mass-spectrometry assays. Participants' self-reported percentage of doses taken in the previous month; virologic failure was confirmed HIV-1 RNA at least 1000 copies/ml at week 24 or 48., Results: From 181 participants with hair samples (61% women, median age: 39 years; CD4+ cell count: 167 cells/μl; HIV-1 RNA: 18 648 copies/ml), 91 (50%) experienced virologic failure at either visit. At 24 weeks, median hair concentrations were 2.95 [interquartile range (IQR) 0.49-4.60] ng/mg for atazanavir, 2.64 (IQR 0.73--7.16) for lopinavir, and 0.44 (IQR 0.11--0.76) for ritonavir. Plasma HIV-1 RNA demonstrated inverse correlations with hair levels (rs -0.46 to -0.74) at weeks 24 and 48. Weaker associations were seen with self-reported adherence (rs -0.03 to -0.24). Decreasing hair concentrations were significantly associated with virologic failure, the hazard ratio (95% CI) for ATV, LPV, and RTV were 0.69 (0.56-0.86), 0.77 (0.68-0.87), and 0.12 (0.06-0.27), respectively., Conclusion: Protease inhibitor hair concentrations showed stronger associations with subsequent virologic outcomes than self-reported adherence in this cohort. Hair adherence measures could identify individuals at risk of second-line treatment failure in need of interventions., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The World Health Organization guidelines recommend that individuals living with HIV receive ≥ 6 months of isoniazid preventive therapy, including pregnant women. Yet, plasma isoniazid exposure during pregnancy, in the antiretroviral therapy era, has not been well-described. We investigated pregnancy-induced and pharmacogenetic-associated pharmacokinetic changes and drug-drug interactions between isoniazid and efavirenz in pregnant women. Eight hundred forty-seven women received isoniazid for 28 weeks, either during pregnancy or at 12 weeks postpartum, and 786 women received efavirenz. After adjusting for NAT2 and CYP2B6 genotype and weight, pregnancy increased isoniazid and efavirenz clearance by 26% and 15%, respectively. Isoniazid decreased efavirenz clearance by 7% in CYP2B6 normal metabolizers and 13% in slow and intermediate metabolizers. Overall, both isoniazid and efavirenz exposures were reduced during pregnancy, but the main determinants of drug concentration were NAT2 and CYP2B6 genotypes, which resulted in a five-fold difference for both drugs between rapid and slow metabolizers., (© 2020 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Competing Interests: The authors have declared that no competing interest exists.

Background: Human immunodeficiency virus (HIV) drug resistance profiles are needed to optimize individual patient management and to develop treatment guidelines. Resistance profiles are not well defined among individuals on failing second-line antiretroviral therapy (ART) in low- and middle-income countries (LMIC)., Methods: Resistance genotypes were performed during screening for enrollment into a trial of third-line ART (AIDS Clinical Trials Group protocol 5288). Prior exposure to both nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs and confirmed virologic failure on a protease inhibitor-containing regimen were required. Associations of drug resistance with sex, age, treatment history, plasma HIV RNA, nadir CD4+T-cell count, HIV subtype, and country were investigated., Results: Plasma HIV genotypes were analyzed for 653 screened candidates; most had resistance (508 of 653; 78%) to 1 or more drugs. Genotypes from 133 (20%) showed resistance to at least 1 drug in a drug class, from 206 (32%) showed resistance to at least 1 drug in 2 drug classes, and from 169 (26%) showed resistance to at least 1 drug in all 3 commonly available drug classes. Susceptibility to at least 1 second-line regimen was preserved in 59%, as were susceptibility to etravirine (78%) and darunavir/ritonavir (97%). Susceptibility to a second-line regimen was significantly higher among women, younger individuals, those with higher nadir CD4+ T-cell counts, and those who had received lopinavir/ritonavir, but was lower among prior nevirapine recipients., Conclusions: Highly divergent HIV drug resistance profiles were observed among candidates screened for third-line ART in LMIC, ranging from no resistance to resistance to 3 drug classes. These findings underscore the need for access to resistance testing and newer antiretrovirals for the optimal management of third-line ART in LMIC., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: The integrase strand transfer inhibitor dolutegravir (DTG) has a high genetic barrier to resistance. Only rare cases of resistance to DTG have been reported when it is used as a component of antiretroviral therapy regimens in treatment-experienced patients unless there was prior use of a first-generation integrase inhibitor., Patient Presentation: A 38-year-old woman diagnosed with tuberculosis was switched to a second-line antiretroviral regimen of zidovudine, lamivudine and dolutegravir 50 mg 12-hourly together with rifampicin-based TB treatment. Based on treatment history and a previous resistance test there was resistance to lamivudine but full susceptibility to zidovudine. The patient did not suppress her viral load on this regimen and later admitted to only taking dolutegravir 50 mg in the morning because of insomnia., Management and Outcome: A second resistance test was performed which showed intermediate level of resistance to dolutegravir. Her regimen was changed to tenofovir, emtricitabine and ritonavir-boosted atazanavir with rifabutin replacing rifampicin for the remainder of her TB treatment. She achieved viral suppression on this regimen., Conclusion: To our knowledge this is the first case report from South Africa of emergent dolutegravir resistance in a treatment-experienced, integrase inhibitor-naïve patient. Factors that may have contributed to resistance emergence in this patient were that there was only one fully active nucleoside reverse transcriptase inhibitor in the regimen and lower exposure to dolutegravir because of the reduced dosing frequency while on rifampicin., Competing Interests: The authors have declared that no competing interest exists., (© 2020. The Authors.)

Background: Sex differences in studies of antiretroviral (ART) drug exposure and treatment outcomes support the hypothesis that some ART combinations may not be well tolerated in women. We evaluated disparities in outcomes between men and women participating in ACTG A5288, an interventional strategy trial for individuals failing a protease inhibitor-based second-line ART regimen in low- and middle-income countries., Methods: Participants were assigned to one of 4 cohorts (A-D) based on resistance profiles and ART history. Cohort A had no lopinavir/ritonavir (LPV/r) resistance and stayed on their second-line regimen, and cohorts B, C, and D had increasing resistance and accessed novel ART regimens. In this secondary analysis, we evaluated sex differences in the primary endpoint, HIV-1 RNA ≤200 copies/mL at week 48; confirmed virologic failure ≥1000 copies/mL (VF); and clinical outcomes and adverse events (intent-to-treat)., Results: Women made up 258/545 (47%) of the study population. More women than men were assigned to cohort A. Median follow-up was 72 weeks. Fewer women than men had HIV-1 RNA ≤200 copies/mL at week 48: 39% vs. 49% in cohort A and 83% vs. 89% in cohorts B, C, and D combined. More women experienced VF, grade ≥3 signs and symptoms, but similar grade ≥3 diagnoses or laboratory abnormalities., Conclusions: More women than men entered the study with a resistance profile suggesting that their second-line regimen could have been effective in maintaining virologic suppression. The more frequent occurrence of grade ≥3 signs and symptoms in women suggests that tolerability issues were under recognized in women on protease inhibitor-based therapy.

Etravirine (ETR) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used in treatment-experienced individuals. Genotypic resistance test-interpretation systems can predict ETR resistance; however, genotype-based algorithms are derived primarily from HIV-1 subtype B and may not accurately predict resistance in non-B subtypes. The frequency of ETR resistance among recombinant subtype C HIV-1 and the accuracy of genotypic interpretation systems were investigated. HIV-1 LAI containing full-length RT from HIV-1 subtype C-positive individuals experiencing virologic failure (>10,000 copies/ml and >1 NNRTI resistance-associated mutation) were phenotyped for ETR susceptibility. Fold change (FC) was calculated against a composite 50% effective concentration (EC 50 ) from treatment-naive individuals and three classifications were assigned: (i) <2.9-FC, susceptible; (ii) ≥2.9- to 10-FC, partially resistant; and (iii) >10-FC, fully resistant. The Stanford HIVdb-v8.4 was used for genotype predictions merging the susceptible/potential low-level and low-level/intermediate groups for 3 × 3 comparison. Fifty-four of a hundred samples had reduced ETR susceptibility (≥2.9-FC). The FC correlated with HIVdb-v8.4 (Spearman's rho = 0.62; P < 0.0001); however, 44% of samples were partially (1 resistance classification difference) and 4% completely discordant (2 resistance classification differences). Of the 34 samples with an FC of >10, 26 were HIVdb-v8.4 classified as low-intermediate resistant. Mutations L100I, Y181C, or M230L were present in 27/34 (79%) of samples with an FC of >10 but only in 2/46 (4%) of samples with an FC of <2.9. No other mutations were associated with ETR resistance. Viruses containing the mutation K65R were associated with reduced ETR susceptibility, but 65R reversions did not increase ETR susceptibility. Therefore, genotypic interpretation systems were found to misclassify ETR susceptibility in HIV-1 subtype C samples. Modifications to genotypic algorithms are needed to improve the prediction of ETR resistance for the HIV-1 subtype C., (Copyright © 2020 American Society for Microbiology.)

Background: Cryptococcal antigen (CrAg) screening with fluconazole prophylaxis has been shown to prevent cryptococcal meningitis and mortality for people living with HIV (PLWH) with CD4 < 100 cells/mm 3 . While cryptococcal meningitis occurs in individuals with CD4 100-200 cells/mm 3 , there is limited evidence that CrAg screening predicts cryptococcal meningitis or mortality among this group with moderate immunosuppression. Current IDSA and WHO clinical guidelines recommend restricting CrAg screening to PLWH with CD4 < 100 cells/mm 3 ., Methods: We conducted a prospective cohort study of PLWH 18+ years who had not initiated ART in South Africa. We followed participants for 14 months to determine onset of cryptococcal meningitis or all-cause mortality. At study completion, we retrospectively tested stored serum samples for CrAg using an enzyme immunoassay (EIA). We calculated CD4-stratified incidence rates of outcomes and used Cox proportional hazards to measure associations between CrAg positivity and outcomes., Results: We enrolled 2383 PLWH, and 1309 participants had serum samples tested by CrAg EIA. The median CD4 was 317 cells/mm 3 (interquartile range: 173-491 cells/mm 3 ). By CD4 count at baseline, there were 209 individuals with a CD4 count of 100-200 cells/mm 3 and available CrAg test results. Of these, four (1.9%) tested positive. Two of four (IR: 58.8 per 100 person-years) CrAg+ participants and 11 of 205 (IR: 5.6 per 100 person-years) CrAg- participants developed cryptococcal meningitis or died for an overall rate of death or cryptococcal meningitis that was 10.0-times higher for those who were CrAg+ (95% confidence interval: 2.2-45.3). Among those with CD4 < 100 cell/mm 3 and CrAg EIA test results (N = 179), ten (5.6%) participants tested CrAg+. Among this group, seven of ten (IR: 137.6 per 100 person-years) CrAg+ participants and 26 of 169 (IR: 17.8 per 100 person-years) CrAg- participants developed cryptococcal meningitis or died, for a rate of death or cryptococcal meningitis that was 6.3-times higher for those who were CrAg+ (95% confidence interval: 2.7-14.6)., Conclusions: Although few PLWH with moderate immunosuppression screened CrAg positive, a positive CrAg test was predictive of increased risk of cryptococcal meningitis or death. Similar to those with a CD4 < 100 cell/mm 3 , systematic CrAg screening may reduce morbidity and mortality in PLWH with CD4 100-200 cells/mm 3 .

Since the ignition of the HIV-1 group M pandemic in the beginning of the 20th century, group M lineages have spread heterogeneously throughout the world. Subtype C spread rapidly through sub-Saharan Africa and is currently the dominant HIV lineage worldwide. Yet the epidemiological and evolutionary circumstances that contributed to its epidemiological expansion remain poorly understood. Here, we analyse 346 novel pol sequences from the DRC to compare the evolutionary dynamics of the main HIV-1 lineages, subtypes A1, C and D. Our results place the origins of subtype C in the 1950s in Mbuji-Mayi, the mining city of southern DRC, while subtypes A1 and D emerged in the capital city of Kinshasa, and subtypes H and J in the less accessible port city of Matadi. Following a 15-year period of local transmission in southern DRC, we find that subtype C spread at least three-fold faster than other subtypes circulating in Central and East Africa. In conclusion, our results shed light on the origins of HIV-1 main lineages and suggest that socio-historical rather than evolutionary factors may have determined the epidemiological fate of subtype C in sub-Saharan Africa., Competing Interests: Rebecca Rose is employed by a commercial company, Bioinfoexperts, LLC.

Background: Antiretroviral therapy (ART) management is challenging for individuals in resource-limited settings presenting for third-line treatment because of complex resistance patterns, partly due to reduced access to viral load monitoring. We aimed to evaluate use of newer antiretroviral drugs and contemporary management approaches, including population-based sequencing, to select appropriate antiretrovirals, plasma viral load monitoring, and interventions to improve adherence in individuals presenting with second-line viral failure., Methods: A5288 was a phase 4, third-line ART strategy study done at 19 urban sites in ten countries that enrolled adult participants with confirmed plasma HIV-1 RNA (viral load) of 1000 copies per mL or more after more than 24 weeks of protease inhibitor-based second-line ART. The primary objective was to use antiretrovirals (raltegravir, etravirine, and ritonavir-boosted darunavir) and diagnostic monitoring technologies, including viral load, genotyping, and adherence support to achieve viral load suppression (defined as ≤200 copies per mL) in 65% or more of participants. ART history and real-time drug resistance genotypes were used to assign participants to one of four cohorts: cohort A (no lopinavir resistance) stayed on second-line ART and cohorts B (B1, best available nucleoside reverse transcriptase inhibitors [NRTIs] plus ritonavir-boosted darunavir plus raltegravir; B2, ritonavir-boosted darunavir plus raltegravir plus etravirine; B3, ritonavir-boosted darunavir, raltegravir, and either tenofovir plus emtricitabine or tenofovir plus lamivudine), C (ritonavir-boosted darunavir plus raltegravir plus tenofovir-emtricitabine or tenofovir plus lamivudine), and D (best available NRTIs plus ritonavir-boosted darunavir plus raltegravir) were defined by increasing levels of resistance and received appropriate regimens, including new antiretrovirals. Participants in Cohort B without detectable hepatitis B surface antigen were assigned by blocked randomisation to cohorts B1 and B2, and those with detectable hepatitis B surface antigen were assigned to cohort B3. The trial is registered with ClinicalTrials.gov, number NCT01641367., Findings: From Jan 10, 2013, to Sept 10, 2015, 545 participants were enrolled. 287 (53%) were assigned to cohort A, 74 (14%) to B1, 72 (13%) to B2, eight (1%) to B3, 70 (13%) to C, and 34 (6%) to D. Overall, 349 (64%, 95% CI 60-68) participants achieved viral suppression at week 48, with proportions varying from 125 (44%) of 287 in cohort A to 65 (88%) of 74 in cohort B1, 63 (88%) of 72 in B2, eight (100%) of eight in B3, 63 (90%) of 70 in C, and 25 (74%) of 34 in D. Participants in cohort A remained on their second-line protease inhibitor, and had the most participants with grade 3 or higher adverse events (147 [51%])., Interpretation: Targeted real-time genotyping to select third-line ART can appropriately allocate more costly antiretrovirals to those with greater levels of HIV drug resistance., Funding: National Institutes of Health., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: To expand understanding of the virological potency of initial dolutegravir plus lamivudine dual therapy (dolutegravir/lamivudine), we compared the viral decay seen in the pilot ACTG A5353 study with the decay observed with dolutegravir plus two NRTIs in the SPRING-1 and SINGLE studies, while also exploring the impact of baseline viral load (VL)., Methods: Change in VL from baseline was calculated for timepoints shared by A5353 (n = 120, including 37 participants with pretreatment VL >100000 copies/mL), SPRING-1 (n = 51) and SINGLE (n = 417). The 95% CIs of change from baseline were determined for each observed week, using the mean log10-transformed VL, and compared between the dolutegravir/lamivudine and triple therapy groups using the Wilcoxon Rank Sum test for non-inferiority (δ = 0.5). To assess the impact of baseline VL on viral decay, we examined a bi-exponential non-linear mixed-effect model., Results: The mean VL change from baseline to week 24 was -2.9 log10 copies/mL for dolutegravir/lamivudine versus -3.0 log10 copies/mL for dolutegravir-based three-drug therapy (P < 0.001). In the decay model, baseline VL >100000 copies/mL was associated with a slower initial decay rate (d1). A faster initial decay rate was seen with dolutegravir/lamivudine, which was partially offset when baseline VL was >100000 copies/mL as indicated by a significant interaction between baseline VL and drug therapy group. The secondary decay rate (d2) was not significantly different from zero, with no significant associations., Conclusions: Viral decay with dolutegravir/lamivudine was comparable to viral decay with dolutegravir-based triple therapy, even in individuals with higher pretreatment VL (>100000 copies/mL)., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The AIDS Clinical Trials Group study A5353 demonstrated the efficacy and safety of dolutegravir and lamivudine for initial treatment of HIV-1 infection at week 24 in individuals with HIV-1 RNA 1000-500 000 copies/mL. Optimal ART for treatment-naive individuals must be durable., Objectives: The aim of this study was to estimate the efficacy and safety of dolutegravir plus lamivudine at week 48 and compare the efficacy in participants with baseline HIV-1 RNA ≤100 000 copies/mL versus >100 000 copies/mL., Methods: Virological success was defined as HIV-1 RNA <50 copies/mL by FDA Snapshot criteria. Definition of virological failure included confirmed HIV-1 RNA >200 copies/mL at week 24 or later. The proportion of participants with virological success was estimated using two-sided exact Clopper-Pearson 95% CI. Comparison between screening HIV-1 RNA (≤100 000 versus >100 000 copies/mL) strata was carried out by Fisher's exact test. The study was registered with ClinicalTrials.gov, number NCT02582684., Results: A total of 120 enrolled eligible participants were included in the analysis. At week 48, 102 of the 120 participants (85%; 95% CI 77%-91%) had virological success. Virological success was similar between screening HIV-1 RNA groups. Six (5%) participants had virological non-success and one additional participant experienced virological failure while on study but off study treatment. No new drug resistance mutations were observed. Six (5%) participants had study-related grade 3 or higher adverse events and none discontinued study treatment., Conclusions: These results add to the evidence that dolutegravir plus lamivudine is a safe and effective option for initial ART in individuals with HIV-1 RNA <500 000 copies/mL., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Antiretroviral therapy (ART) non-adherence causes HIV treatment failure. Past behaviour might predict future behaviour; failing second-line ART could indicate ongoing risk for subsequent non-adherence. We aimed to find out whether a two-way mobile phone-based communication intervention would increase HIV treatment success by improving medication adherence., Methods: We did a multinational, randomised controlled trial of patients at 17 sites in nine lower-income and middle-income countries in Africa, Asia, and the Americas. Patients aged 18 years and older, with HIV infection, and on second-line protease-inhibitor-based antiretroviral regimens, were randomly assigned (1:1) to either two-way mobile phone intervention plus standard of care (MPI + SOC) adherence support or standard-of-care alone (SOC). Our study was nested within a strategy study of ART after second-line ART failure (the main study, A5288). The main study had four cohorts, which were assigned regimens according to ART history and real-time genotype. Randomisation was stratified by the main study cohort with dynamic institutional balancing. Only the clinical management committee was masked, not the participants or site personnel. Text messages were sent over 48 weeks starting once a day and tapering down to once per week; participants were to respond once to each message if taking ART without issues. Repeated non-response to three messages over a 2-week period for the first 8 weeks, and then two messages over a 2-week period for the remainder of the study, triggered problem-solving counselling by staff. For this study, the primary endpoint was plasma HIV-1 RNA 200 copies per mL or less at 48 weeks and the secondary endpoint was virological failure (two consecutive HIV-1 RNA ≥1000 copies per mL) at 24 or more weeks. Prespecified intention-to- treat analyses were adjusted for cohort. Follow-up continued until the last participant had reached 48 weeks, with a median follow-up time of 72 weeks. The trial is registered with ClinicalTrials.gov, number ., Findings: Enrolment began on Feb 22, 2013, and ended on Dec 21, 2015, with the last participant completing follow-up on Feb 13, 2017. Of 545 participants in the main study, 521 (96%) were enrolled and randomly assigned to MPI + SOC (n=257) or SOC alone (n=264). 52% of patients were men and the median HIV-1 RNA 4-4 log 10 copies per mL (IQR 3.5 to 5-2). At week 48, HIV-1 RNA 200 copies per mL or less was reached in 169 (66%) of 257 patients in the MPI + SOC group and 164 (62%) of 264 patients in the SOC group (estimated difference 3-6% [95% CI -4-6% to 11-9%]; p=0-39). The adjusted odds ratio comparing MPI + SOC and SOC was 1-23 (0-82 to 1-84; p=0-32). Virological failure occurred in 66 (26%) patients in the MPI + SOC group and 89 (34%) patients in the SOC group during the median 72 weeks follow-up (adjusted p=0-027). Observed difference in virological failure favoured MPI + SOC in all cohorts. 23 (4%) participants died, 11 (4%) in the MPI + SOC group and 12 (5%) in the SOC group (p=0-89), with none of the deaths ascribed to ART, the MPI, or study procedures., Interpretation: Two-way MPI did not significantly improve week 48 suppression, but it did modestly affect virological failure. People failing second-line ART might not achieve benefits from phone-based triggers or enhanced adherence support (or both). More effective strategies are needed.

Since rapid cryptococcal antigen lateral flow assays (CrAg LFA) may expedite treatment of HIV-associated cryptococcal infections, we sought to validate clinic-based CrAg LFA testing. Among newly-diagnosed HIV-infected adults in South Africa, a trained nurse performed clinic-based testing of urine, fingerprick capillary and venous whole blood with rapid CrAg LFA (Immy Diagnostics, Norman, USA). We performed matched laboratory-based serum cryptococcal antigen testing with an enzyme immunoassay (EIA). We assessed diagnostic accuracy using EIA as the gold-standard, and performed additional validation testing on serum and among hospitalized adults with cryptococcal meningitis. Among 5,618 participants enrolled, 1,296 were HIV-infected and screened for cryptococcal antigenemia. Overall CrAg prevalence by serum EIA was 3.6% (95% CI 2.0-6.0%) for adults with CD4 < 200 cells/mm 3 , and 5.7% (95% CI 2.8-10.2%) for adults with CD4 < 100 cells/mm 3 . Using expanded screening guidelines (CD4 < 200 cells/mm 3 ), CrAg LFA testing of venous whole blood, fingerprick capillary blood, and urine had diagnostic sensitivities of 46% (95% CI 19-75%), 38% (95% CI 14-68%), and 54% (95% CI 25-81%), and specificities of 97%, 97%, and 86%, respectively. When tested on serum samples, CrAg LFA had sensitivity of 93% (95% CI 66-100%) and specificity of 100% (95% CI 88-100%). All venous and fingerprick whole blood CrAg LFA tests were positive among 30 hospitalized adults with cryptococcal meningitis. Two independent readers had strong agreement for all LFA results (p < 0.0001). When performed at the point-of-care by trained nurses, CrAg LFA testing was feasible, had the highest accuracy on serum specimens, and may accelerate treatment of HIV-associated cryptococcal infections.

Background: There is an urgent need for more accurate screening tests for tuberculosis(TB). We assessed the diagnostic accuracy of C-reactive protein (CRP) as a screening test for active TB in HIV-infected ambulatory adults., Methods: CRP levels were measured in blood collected at the time of HIV testing.Diagnostic accuracy of CRP for pulmonary TB was calculated (reference standard: TB culture), compared to the WHO 4-symptom screen, consisting of cough, fever, night sweats, and weight loss. Diagnostic accuracy was also calculated for CRP in a larger cohort of HIV-infected adults with a positive symptom screen (reference standard: clinical or microbiological TB)., Results: Among 425 HIV-infected outpatients systematically tested for pulmonary TB, TB culture was positive in 42 (10%), 279 (66%) had at least one TB-related symptom and 197 (46%) had a CRP more than 5 mg/l. The sensitivity of CRP and the TB symptom screen to detect TB was the same [90.5%; 95% confidence interval 77.4-97.3] but specificity of CRP was higher than for the TB symptom screen (58.5% vs. 37.1%, P < 0.001). Of persons with no symptoms and normal CRP, 99 (98%) had no TB. In another cohort of 749 patients presenting with at least one TB-related symptom and clinically evaluated, CRP had a sensitivity of 98.7% and specificity of 48.3%., Conclusion: In HIV-infected outpatients, CRP was as sensitive but substantially more specific than TB symptom screening. Use of CRP as a screening tool to exclude active TB could identify the same number of HIV-associated TB cases, but reduce the use of diagnostic sputum testing in TB-endemic regions.

Background: Limited data exist on initial human immunodeficiency virus type 1 (HIV-1) treatment with dolutegravir plus lamivudine., Methods: A5353 is a phase 2, single-arm, pilot study of once-daily dolutegravir (50 mg) plus lamivudine (300 mg) in treatment-naive participants with HIV-1 RNA ≥1000 and <500000 copies/mL. Exclusion criteria included active hepatitis B or major protease, reverse transcriptase, or integrase resistance. The primary efficacy measure was the proportion with HIV-1 RNA <50 copies/mL (FDA [US Food and Drug Administration] Snapshot) at week 24. Virologic failure (VF) was confirmed HIV-1 RNA >400 copies/mL at week 16/20 or >200 copies/mL at or after week 24. Dolutegravir levels and drug resistance testing were performed at VF., Results: One hundred and twenty participants (87% male, median age 30 years, 37 (31%) HIV-1 RNA >100000 copies/mL) initiated study treatment. Median entry HIV-1 RNA and CD4 count were 4.61 log10 copies/mL and 387 cells/mm3. Virologic efficacy at week 24 was 108/120 (90%, confidence interval [83%, 95%]), with comparable results in the >100000 copies/mL and ≤100000 copies/mL strata, that is, 89% (75%, 97%) and 90% (82%, 96%), respectively. Three participants with VF, had undetected plasma dolutegravir at ≥1 time points; the M184V and R263R/K mutations developed in 1 participant. Two participants experienced grade 3 possible/probable treatment-related adverse events; none discontinued treatment due to adverse events., Conclusions: Dolutegravir plus lamivudine demonstrated efficacy in individuals with pretreatment HIV-1 RNA up to 500000 copies/mL in this pilot trial, but a participant developed resistance mutations., Clinical Trials Registration: NCT02582684.

Background Rilpivirine (TMC278LA) is a promising drug for pre-exposure prophylaxis of HIV-1 because of its sub-nanomolar potency and long-acting formulation; however, increasing transmission of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 with potential cross-resistance to rilpivirine could reduce its preventive efficacy. This study investigated rilpivirine cross-resistance among recombinant subtype C HIV-1 derived from 100 individuals failing on first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy in South Africa whose samples were sent for routine HIV-1 drug resistance testing to Lancet Laboratories (Johannesburg, South Africa). Methods Plasma samples were selected from individuals with HIV-1 RNA > 10,000 copies/ml and ≥1 non-nucleoside reverse transcriptase inhibitor-resistance mutation in reverse transcriptase. Recombinant HIV-1 LAI -containing bulk-cloned full-length reverse transcriptase sequences from plasma were assayed for susceptibility to nevirapine (NVP), efavirenz (EFV) and rilpivirine in TZM-bl cells. Fold-change (FC) decreases in drug susceptibility were calculated against a mean IC 50 from 12 subtype C HIV-1 samples from treatment-naïve individuals in South Africa. Cross-resistance was evaluated based on biological cutoffs established for rilpivirine (2.5-FC) and the effect of mutation combinations on rilpivirine phenotype. Results Of the 100 samples from individuals on failing antiretroviral therapy, 69 had 2.5- to 75-fold decreased susceptibility to rilpivirine and 11 had >75-fold resistance. Rilpivirine resistance was strongly associated with K103N especially in combination with other rilpivirine-associated mutations. Conclusion The frequently observed cross-resistance of HIV-1 suggests that the preventive efficacy of TMC278LA pre-exposure prophylaxis could be compromised by transmission of HIV-1 from individuals with failure of first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy.

The emergence and spread of human immunodeficiency virus (HIV) drug resistance from antiretroviral roll-out programs remain a threat to long-term control of the HIV-AIDS epidemic in low- and middle-income countries (LMICs). The patterns of drug resistance and factors driving emergence of resistance are complex and multifactorial. The key drivers of drug resistance in LMICs are reviewed here, and recommendations are made to limit their influence on antiretroviral therapy efficacy., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: HIV-1 genotypic resistance test (GRT) interpretation systems (IS) require updates as new studies on HIV-1 drug resistance are published and as treatment guidelines evolve., Methods: An expert panel was created to provide recommendations for the update of the Stanford HIV Drug Resistance Database (HIVDB) GRT-IS. The panel was polled on the ARVs to be included in a GRT report, and the drug-resistance interpretations associated with 160 drug-resistance mutation (DRM) pattern-ARV combinations. The DRM pattern-ARV combinations included 52 nucleoside RT inhibitor (NRTI) DRM pattern-ARV combinations (13 patterns x 4 NRTIs), 27 nonnucleoside RT inhibitor (NNRTI) DRM pattern-ARV combinations (9 patterns x 3 NNRTIs), 39 protease inhibitor (PI) DRM pattern-ARV combinations (13 patterns x 3 PIs) and 42 integrase strand transfer inhibitor (INSTI) DRM pattern-ARV combinations (14 patterns x 3 INSTIs)., Results: There was universal agreement that a GRT report should include the NRTIs lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir disoproxil fumarate; the NNRTIs efavirenz, etravirine, nevirapine, and rilpivirine; the PIs atazanavir/r, darunavir/r, and lopinavir/r (with "/r" indicating pharmacological boosting with ritonavir or cobicistat); and the INSTIs dolutegravir, elvitegravir, and raltegravir. There was a range of opinion as to whether the NRTIs stavudine and didanosine and the PIs nelfinavir, indinavir/r, saquinavir/r, fosamprenavir/r, and tipranavir/r should be included. The expert panel members provided highly concordant DRM pattern-ARV interpretations with only 6% of NRTI, 6% of NNRTI, 5% of PI, and 3% of INSTI individual expert interpretations differing from the expert panel median by more than one resistance level. The expert panel median differed from the HIVDB 7.0 GRT-IS for 20 (12.5%) of the 160 DRM pattern-ARV combinations including 12 NRTI, two NNRTI, and six INSTI pattern-ARV combinations. Eighteen of these differences were updated in HIVDB 8.1 GRT-IS to reflect the expert panel median. Additionally, HIVDB users are now provided with the option to exclude those ARVs not considered to be universally required., Conclusions: The HIVDB GRT-IS was updated through a collaborative process to reflect changes in HIV drug resistance knowledge, treatment guidelines, and expert opinion. Such a process broadens consensus among experts and identifies areas requiring further study.

Background: HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings., Objectives: To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol., Study Design: Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol., Results: The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10 7 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10 7 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%., Conclusion: The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an alternative sample collection and transfer option in resource-limited settings and expands the utility of a viral load test to monitor HIV-1 ART treatment for infected patients., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: HIV-1 sequence variation is a major obstacle to developing molecular based assays for multiple subtypes. This study sought to independently assess performance characteristics of the ViroSeq™ HIV-1 Integrase RUO Genotyping Kit (Celera, US) for samples of multiple different HIV-1 subtypes., Methods: 264 samples were tested in the validation, 106 from integrase inhibitor naïve patients' sent for routine HIV-1 drug resistance testing after failing a 1st- or 2nd-line regimen, and 158 samples from an external virology quality assurance program (VQA). For the latter, 53 unique VQA samples were tested in two to five different laboratories to assess assay reproducibility. For all assays, viral RNA was extracted using the ViroSeq extraction module, reverse transcribed, and amplified in a one-step reaction. Four sequencing primers were used to span codons 1-288 of integrase. The Rega subtyping tool was used for subtype assignment. Integrase polymorphisms and mutations were determined as differences from the HXB2 sequence and by the Stanford database, respectively. Sequences obtained from the different laboratories were aligned and sequence homology determined., Results: HIV-1 RNA in the 264 samples ranged from 3.15 to 6.74 log copies/ml. Successful amplification was obtained for 97% of samples (n=256). The 8 samples that failed to amplify were subtype D (n=3), subtype C (n=1), CRF01&#95;AE (n=1), subtype A1 (n=2), and an unassigned subtype (n=1). Of the 256 that successfully amplified samples, 203 (79%) were successfully sequenced with bidirectional coverage. Of the 53 unsuccessful samples, 13 (5%) failed sequencing and 40 (16%) did not have full bidirectional sequence, as a result of failure of sequencing primers: Primer A (n=1); Primer B (n=18); Primer C (n=1); Primer D (n=7) or short sequences (n=16). For the 135 VQA samples (30 unique samples) that were assayed by different laboratories, homology of the sequences obtained ranged from 92.1% to 100%. However, Laboratory 2 detected more mixtures (74%) compared to the other four laboratories, whereas Laboratory 1 detected the least number of mixtures (35%), likely due to differences between the labs in the methods of sequence analysis. Mutations associated with integrase resistance were observed in seven of the 106 (7%) clinical samples [one sample: Q148K; E138K; G140A; two samples: T97A and four samples: L74I]. Of the four samples with L74I, 3 were subtype G., Conclusion: Of the total 264 samples tested, 243 (92%) of samples were able to be amplified and sequenced to generate an integrase genotype. Sequencing results were similar between the testing laboratories with the exception of mixture detection. Mutations associated with integrase inhibitor resistance were observed in only 7% of integrase inhibitor naive samples, and some of these mutations are likely to be due to subtype-specific polymorphisms rather than selection by an integrase inhibitor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1 LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC 50 ) from 12 recombinant subtype C HIV-1 LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC 50 s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC 50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring., (Copyright © 2017 American Society for Microbiology.)

Background.  The mechanism of virologic failure (VF) of lopinavir/ritonavir (LPV/r) monotherapy is not well understood. We assessed sequence changes in human immunodeficiency virus-1 reverse-transcriptase (RT) and protease (PR) regions. Methods.  Human immunodeficiency virus-1 pol sequences from 34 participants who failed second-line LPV/r monotherapy were obtained at study entry (SE) and VF. Sequence changes were evaluated using phylogenetic analysis and hamming distance. Results.  Human immunodeficiency virus-1 sequence change was higher over drug resistance mutation (DRM) sites (median genetic distance, 2.2%; Q1 to Q3, 2.1%-2.5%) from SE to VF compared with non-DRM sites (median genetic distance, 1.3%; Q1 to Q3, 1.0%-1.4%; P < .0001). Evolution over DRM sites was mainly driven by changes in the RT (median genetic distance, 2.7%; Q1 to Q3, 2.2%-3.2%) compared with PR (median genetic distance, 1.1%; Q1 to Q3, 0.0%-1.1%; P < .0001). Most RT DRMs present at SE were lost at VF. At VF, 19 (56%) and 26 (76%) were susceptible to efavirenz/nevirapine and etravirine (ETV)/rilpivirine (RPV), respectively, compared with 1 (3%) and 12 (35%) at SE. Participants who retained nonnucleoside reverse-transcriptase inhibitor (NNRTI) DRMs and those without evolution of LPV/r DRMs had significantly shorter time to VF. Conclusions.  The selection of LPV/r DRMs in participants with longer time to VF suggests better adherence and more selective pressure. Fading NNRTI mutations and an increase in genotypic susceptibility to ETV and RPV could allow for the reuse of NNRTI. Further studies are warranted to understand mechanisms of PR failure.

Background: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring., Methods: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories., Results: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude., Conclusions: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

Background: In resource-limited settings, where resistance testing is unavailable, confirmatory testing for patients with high viral loads (VL) delays antiretroviral therapy (ART) switches for persons with resistance. We developed a risk score algorithm to predict need for ART change by identifying resistance among persons with persistently elevated VL., Methods: We analyzed data from a Phase IV open-label trial. Using logistic regression, we identified demographic and clinical characteristics predictive of need for ART change among participants with VLs ≥1000 copies/ml, and assigned model-derived scores to predictors. We designed three models, including only variables accessible in resource-limited settings., Results: Among 290 participants with at least one VL ≥1000 copies/ml, 51 % (148/290) resuppressed and did not have resistance testing; among those who did not resuppress and had resistance testing, 47 % (67/142) did not have resistance and 53 % (75/142) had resistance (ART change needed for 25.9 % (75/290)). Need for ART change was directly associated with higher baseline VL and higher VL at time of elevated measure, and inversely associated with treatment duration. Other predictors included body mass index and adherence. Area under receiver operating characteristic curves ranged from 0.794 to 0.817. At a risk score ≥9, sensitivity was 14.7-28.0 % and specificity was 96.7-98.6 %., Conclusions: Our model performed reasonably well and may be a tool to quickly transition persons in need of ART change to more effective regimens when resistance testing is unavailable. Use of this algorithm may result in public health benefits and health system savings through reduced transmissions of resistant virus and costs on laboratory investigations.

Background: For second-line antiretroviral therapy, WHO recommends a boosted protease inhibitor plus nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs). However, concerns about toxicity and cross-resistance motivated a search for regimens that do not contain NRTIs. We aimed to assess whether boosted lopinavir plus raltegravir would be non-inferior to boosted lopinavir plus NRTIs for virological suppression in resource-limited settings., Methods: A5273 was a randomised, open-label, phase 3, non-inferiority study at 15 AIDS Clinical Trials Group (ACTG) research sites in nine resource-limited countries (three sites each in India and South Africa, two each in Malawi and Peru, and one each in Brazil, Kenya, Tanzania, Thailand, and Zimbabwe). Adults with plasma HIV-1 RNA concentrations of at least 1000 copies per mL after at least 24 weeks on a regimen based on a non-NRTI inhibitor were randomly assigned (1:1) to receive oral ritonavir-boosted lopinavir (100 mg ritonavir, 400 mg lopinavir) plus 400 mg raltegravir twice a day (raltegravir group) or to ritonavir-boosted lopinavir plus two or three NRTIs selected from an algorithm (eg, zidovudine after failure with tenofovir and vice versa; NRTI group). Randomised group assignment was done with a computer algorithm concealed to site personnel, and stratified by HIV-1 RNA viral load, CD4 cell count, and intention to use zidovudine, with the groups balanced by each site. The primary endpoint was time to confirmed virological failure (two measurements of HIV-1 RNA viral load >400 copies per mL) at or after week 24 in the intention-to-treat population. Non-inferiority (10% margin) was assessed by comparing the cumulative probability of virological failure by 48 weeks. This trial was registered with ClinicalTrials.gov, NCT01352715., Findings: Between March 13, 2012, and Oct 2, 2013, we randomly assigned 515 participants: 260 to the raltegravir group and 255 to the NRTI group; two participants in the raltegravir group and one in the NRTI group were excluded from analyses because of ineligibility. By the end of follow-up (October, 2014), 96 participants had virological failure (46 in the raltegravir group and 50 in the NRTI group). By 48 weeks, the cumulative probability of virological failure was 10·3% (95% CI 6·5-14·0) in the raltegravir group and 12·4% (8·3-16·5) in the NRTI group, with a weighted difference of -3·4% (-8·4 to 1·5), indicating that raltegravir was non-inferior, but not superior, to NRTIs. 62 (24%) participants in the raltegravir group and 81 (32%) in the NRTI group had grade 3 or higher adverse events; 19 (7%) and 29 (11%), respectively, had serious adverse events. Three participants in each group died, all from HIV-related causes., Interpretation: In settings with extensive NRTI resistance but no available resistance testing, our data support WHO's recommendation for ritonavir-boosted lopinavir plus NRTI for second-line antiretroviral therapy. Ritonavir-boosted lopinavir plus raltegravir is an appropriate alternative, especially if NRTI use is limited by toxicity., Funding: National Institutes of Health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Virologic failure in subtype C is characterized by high resistance to first-line antiretroviral (ARV) drugs, including efavirenz, nevirapine, and lamivudine, with nucleoside resistance including type 2 thymidine analog mutations, K65R, a T69del, and M184V. However, genotypic algorithms predicting resistance are mainly based on subtype B viruses and may under- or overestimate drug resistance in non-B subtypes. To explore potential treatment strategies after first-line failure, we compared genotypic and phenotypic susceptibility of subtype C human immunodeficiency virus 1 (HIV-1) following first-line ARV failure., Methods: AIDS Clinical Trials Group 5230 evaluated patients failing an initial nonnucleoside reverse-transcriptase inhibitor (NNRTI) regimen in Africa and Asia, comparing the genotypic drug resistance and phenotypic profile from the PhenoSense (Monogram). Site-directed mutagenesis studies of K65R and T69del assessed the phenotypic impact of these mutations., Results: Genotypic algorithms overestimated resistance to etravirine and rilpivirine, misclassifying 28% and 32%, respectively. Despite K65R with the T69del in 9 samples, tenofovir retained activity in >60%. Reversion of the K65R increased susceptibility to tenofovir and other nucleosides, while reversion of the T69del showed increased resistance to zidovudine, with little impact on other NRTI., Conclusions: Although genotype and phenotype were largely concordant for first-line drugs, estimates of genotypic resistance to etravirine and rilpivirine may misclassify subtype C isolates compared to phenotype., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

This guideline is an update of the post-exposure prophylaxis (PEP) guideline published by the Southern African HIV Clinicians Society in 2008. It updates the recommendations on the use of antiretroviral medications to prevent individuals who have been exposed to a potential HIV source, via either occupational or non-occupational exposure, from becoming infected with HIV. No distinction is made between occupational or non-occupational exposure, and the guideline promotes the provision of PEP with three antiretroviral drugs if the exposure confers a significant transmission risk. The present guideline aligns with the principles of the World Health Organization PEP guidelines (2014), promoting simplification and adherence support to individuals receiving PEP., Competing Interests: All expert panel members have completed and submitted conflict of interest disclosure forms. Disclosure information represents the previous 3 years (updated 10 June 2014) and includes relationships with pharmaceutical companies and medical aids: L.G.B. has received honoraria from Merck (MSD) for participating in consultative meetings. V.B. has received research support from Standard Diagnostics and served as a consultant to Novartis. F.C. has received support from Abbvie, Aspen and Mylan to attend conferences; research support from Janssen; honoraria for speaking engagements from Abbvie, GlaxoSmithKline, Janssen and MSD; and acted as a consultant to Sanofi Aventis. M.M. has received honoraria for speaking engagements from Aspen and MSD. F.V. has received support to attend conferences from Adcock Ingram, MSD and Aspen; received honoraria for speaking engagements from MSD, Aspen, Abbott, Novogen, Adcock Ingram and GlaxoSmithKline; served on advisory boards for Johnson and Johnson, Mylan and Tibotec; and received drug donations for research projects from Gilead and Mylan. C.L.W. has received support from Roche Molecular to attend conferences; honoraria for speaking engagements from Abbvie, MSD and Janssen; and acted as a consultant to Celera. B.H., P.H., G.M., N.S.P., K.R. and G.S. report no conflicts of interest.

Dried blood spots (DBS) are an easy to collect sample-type that can stabilize biological material at ambient temperature for transport and storage, making them ideal for use in resource-limited settings (RLS). We investigated the effect of storage temperature and duration on ability to detect mixed HIV-1 viral RNA populations, and subsequently viral RNA populations in a background of proviral DNA. Part one of the study used DBS samples of whole blood spiked with specific quantities of HIV-1 subtype-B and -C RNA to study mixed virus population detection. Part two used DBS comprising of HIV-1 subtype-B proviral DNA containing U1 cells combined with HIV-1 subtype-C RNA to mimic HIV-1 infected clinical samples as a model system to study the relative stability of HIV-1 RNA and DNA in DBS. Prepared DBS were stored at -20 °C and +30 °C for periods of one day, one, two, and four weeks. Samples were genotyped to determine changes in the detection of mixtures in the sample over time. From two weeks onwards, storage at +30 °C resulted in gradual, time-related reduction in the detection of mixed virus population at log10 VL 4.0 but not at log10 5.0. Proviral DNA and viral RNA were both stable for at least 52 weeks when stored at -20 °C, compared to progressive RNA decay over time at +30 °C. DBS storage conditions and duration had a significant effect on HIV-1 RNA amplification. Our results demonstrate that DBS storage at ambient temperature (+30 °C) should not exceed two weeks, with long-term storage at -20 °C or lower.

Background: Evaluation of pretreatment HIV genotyping is needed globally to guide treatment programs. We examined the association of pretreatment (baseline) drug resistance and subtype with virologic failure in a multinational, randomized clinical trial that evaluated 3 antiretroviral treatment (ART) regimens and included resource-limited setting sites., Methods: Pol genotyping was performed in a nested case-cohort study including 270 randomly sampled participants (subcohort), and 218 additional participants failing ART (case group). Failure was defined as confirmed viral load (VL) >1000 copies/mL. Cox proportional hazards models estimated resistance-failure association., Results: In the representative subcohort (261/270 participants with genotypes; 44% women; median age, 35 years; median CD4 cell count, 151 cells/µL; median VL, 5.0 log10 copies/mL; 58% non-B subtypes), baseline resistance occurred in 4.2%, evenly distributed among treatment arms and subtypes. In the subcohort and case groups combined (466/488 participants with genotypes), used to examine the association between resistance and treatment failure, baseline resistance occurred in 7.1% (9.4% with failure, 4.3% without). Baseline resistance was significantly associated with shorter time to virologic failure (hazard ratio [HR], 2.03; P = .035), and after adjusting for sex, treatment arm, sex-treatment arm interaction, pretreatment CD4 cell count, baseline VL, and subtype, was still independently associated (HR, 2.1; P = .05). Compared with subtype B, subtype C infection was associated with higher failure risk (HR, 1.57; 95% confidence interval [CI], 1.04-2.35), whereas non-B/C subtype infection was associated with longer time to failure (HR, 0.47; 95% CI, .22-.98)., Conclusions: In this global clinical trial, pretreatment resistance and HIV-1 subtype were independently associated with virologic failure. Pretreatment genotyping should be considered whenever feasible., Clinical Trials Registration: NCT00084136., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The AIDS Clinical Trials Group (ACTG) A5230 study evaluated lopinavir/ritonavir (LPV/r) monotherapy following virologic failure (VF) on first-line human immunodeficiency virus (HIV) regimens in Africa and Asia., Methods: Eligible subjects had received first-line regimens for at least 6 months and had plasma HIV-1 RNA levels 1000-200 000 copies/mL. All subjects received LPV/r 400/100 mg twice daily. VF was defined as failure to suppress to <400 copies/mL by week 24, or confirmed rebound to >400 copies/mL at or after week 16 following confirmed suppression. Subjects with VF added emtricitabine 200 mg/tenofovir 300 mg (FTC/TDF) once daily. The probability of continued HIV-1 RNA <400 copies/mL on LPV/r monotherapy through week 104 was estimated with a 95% confidence interval (CI); predictors of treatment success were evaluated with Cox proportional hazards models., Results: One hundred twenty-three subjects were enrolled. Four subjects died and 2 discontinued prematurely; 117 of 123 (95%) completed 104 weeks. Through week 104, 49 subjects met the primary endpoint; 47 had VF, and 2 intensified treatment without VF. Of the 47 subjects with VF, 41 (33%) intensified treatment, and 39 of 41 subsequently achieved levels <400 copies/mL. The probability of continued suppression <400 copies/mL over 104 weeks on LPV/r monotherapy was 60% (95% CI, 50%-68%); 80%-85% maintained levels <400 copies/mL with FTC/TDF intensification as needed. Ultrasensitive assays on specimens with HIV-1 RNA level <400 copies/mL at weeks 24, 48, and 104 revealed that 61%, 62%, and 65% were suppressed to <40 copies/mL, respectively., Conclusions: LPV/r monotherapy after first-line VF with FTC/TDF intensification when needed provides durable suppression of HIV-1 RNA over 104 weeks., Clinical Trials Registration: NCT00357552., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Adherence is key to the success of antiretroviral therapy. Enhanced partner support might benefit patients with previous treatment failure. We aimed to assess whether an enhanced partner-based support intervention with modified directly observed therapy would improve outcomes with second-line therapy in HIV-infected patients for whom first-line therapy had failed., Methods: We did a multicentre, international, randomised clinical trial at nine sites in Botswana, Brazil, Haiti, Peru, South Africa, Uganda, Zambia, and Zimbabwe. Participants aged 18 years or older for whom first-line therapy had failed, with HIV RNA concentrations greater than 1000 copies per mL and with a willing partner, were randomly assigned (1:1), via computer-generated randomisation, to receive partner-based modified directly observed therapy or standard of care. Randomisation was stratified by screening HIV RNA concentration (≤10 000 copies per mL vs >10 000 copies per mL). Participants and site investigators were not masked to group assignment. Primary outcome was confirmed virological failure (viral load >400 copies per mL) by week 48. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00608569., Findings: Between April 23, 2009, and Sept 29, 2011, we randomly assigned 259 participants to the modified directly observed therapy group (n=129) or the standard-of-care group (n=130). 34 (26%) participants in the modified directly observed therapy group achieved the primary endpoint of virological failure by week 48 compared with 23 (18%) participants in the standard-of-care group. The Kaplan-Meier estimated cumulative probability of virological failure by week 48 was 25·1% (95% CI 17·7-32·4) in the modified directly observed therapy group and 17·3% (10·8-23·7) in the standard-of-care group, for a weighted difference in standard of care versus modified directly observed therapy of -6·6% (95% CI -16·5% to 3·2%; p=0·19). 36 (14%) participants reported at least one grade 3 or higher adverse event or laboratory abnormality (n=21 in the modified directly observed therapy group and n=15 in the standard-of-care group)., Interpretation: Partner-based training with modified directly observed therapy had no effect on virological suppression. The intervention does not therefore seem to be a promising strategy to increase adherence. Intensive follow-up with clinic staff might be a viable approach in this setting., Funding: AIDS Clinical Trials Group and the National Institute of Allergy and Infectious Diseases, US National Institutes of Health., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania., Methods: CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall-Wallis tests., Results: Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site., Conclusions: The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV) has been co-formulated with emtricitabine and tenofovir disoproxil fumarate for initial therapy of HIV-1-infected individuals. RPV, formulated as a long-acting nanosuspension, will also be assessed for its ability to prevent HIV-1 infection in resource limited settings. In this study, we determined whether any pre-existing genetic differences occurred among different HIV-1 subtypes at residues in RT associated with decreased virologic response to RPV. We found that the E138A substitution occurs more frequently in subtype C (range: 5.9-7.5%) than B (range: 0-2.3%) sequences from both treatment-naïve and -experienced individuals (p<0.01) in 4 independent genotype databases. In one of the databases (Stanford University), E138K and E138Q were also more common in RTI-experienced subtype C sequences (1.0% and 1.1%, respectively) than in subtype B sequences (0.3% and 0.6%, respectively). E138A/K/Q in subtype C decreased RPV susceptibility 2.9-, 5.8-, and 5.4-fold, respectively. Taken together, these data suggest that E138A could impact treatment or prevention strategies that include RPV in geographic areas where subtype C infection is prevalent., (Copyright © 2014 Elsevier B.V. All rights reserved.)

There are many challenges to performing clinical research in resource-limited settings. Here, we discuss several of the most common laboratory issues that must be addressed. These include issues relating to organization and personnel, laboratory facilities and equipment, standard operating procedures, external quality assurance, shipping, laboratory capacity, and data management. Although much progress has been made, innovative ways of addressing some of these issues are still very much needed.