Cancer causes significant morbidity and mortality among HIV patients in the US due to extended life expectancy with access to effective antiretroviral therapy. Low, detectable HIV RNA has been studied as a risk factor for adverse health outcomes, but its clinical impact on cancer risk remains unclear. The objective of this study was to determine whether HIV RNA <1,000 copies/mL six months after starting therapy was associated with 10-year first cancer risk.We followed 7,515 HIV therapy initiators from a US-based multicenter clinical cohort from 1998 to 2014. We used nonparametric multiple imputation to account for viral loads that fell below assay detection limits, and categorized viral loads six months after therapy initiation into four groups: <20, 20–199, 200–999, and >999 copies/mL. We calculated estimates of the cumulative incidence of cancer diagnosis, accounting for death as a competing event. Inverse probability of exposure and censoring weights were used to control for confounding and differential loss to follow up, respectively.Crude 10-year first cancer risk in the study sample was 7.03% , with the highest risk observed among patients with viral loads between 200 and 999 copies/mL six months after ART initiation (10.7%). After controlling for baseline confounders, 10-year first cancer risk was 6.90% , and was similar across viral load categories.Overall risk of first cancer was not associated with incomplete viral suppression; however, cancer remains a significant threat to HIV patients after treatment initiation. As more HIV patients gain access to treatment in the current “treat all” era, occurrences of incomplete viral suppression will be observed more frequently in clinical practice, which supports continued study of the role of low-level HIV RNA on cancer development. Effective antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) levels to below the detection limits of assays used in clinical practice in the United States. Uptake of ART has resulted in lower incidence of acquired immune deficiency syndrome (AIDS)-defining illnesses, prolonged survival, and rising incidence of non-AIDS-defining cancers and chronic diseases among people living and aging with HIV . Cancer Not all HIV patients on treatment achieve and maintain undetectable viral loads, and the impact of low levels of detectable HIV ribonucleic acid (RNA) under 1,000 copies/mL on the risk of comorbid disease, such as cancer, remains unclear. Incomplete viral suppression appears to increase the risk of certain cancers –9, but tWe used data from the Center for AIDS Research (CFAR) Network of Integrated Clinical Systems (CNICS), a multicenter clinical cohort of over 30,000 HIV patients in the United States. CNICS maintains a clinical data repository from electronic medical record systems to support HIV research . The CNIUpon entry into CNICS, demographic and historical information, including prior diagnoses and antiretroviral treatment, was collected. After enrollment, patient data were prospectively captured at clinic visits and include prescribed medications, laboratory test results, and conditions diagnosed by providers. CNICS participants were typically seen in clinical care every three to four months, though frequency of follow-up was patient specific. The research of CNICS was approved by the Office of the Institutional Review Board for Human Use at the University of Alabama at Birmingham. All participating study sites received consent from their local institutional review boards to conduct the research activities of CNICS. The study conducted here, a secondary analysis of extant, de-identified CNICS data, was reviewed by the Office of Human Research Ethics at the University of North Carolina at Chapel Hill, which determined that it did not constitute human subjects research.A total of 27,865 patients entered the CNICS cohort between 1 January 1998 and 31 December 2013. We excluded patients who initiated monotherapy or dual therapy prior to or with no history of starting combination ART (defined as three or more ART drugs prescribed concurrently) , initiated ART prior to entering CNICS , initiated ART after 31 December 2013 (n = 282), or had no history of initiating ART . We excluded patients who were diagnosed with cancer prior to ART initiation (n = 570), were diagnosed with cancer within six months of starting ART (n = 151), died within six months of starting ART (n = 99), or did not have at least one viral load measurement six months (-30/+90 days) after ART initiation . Patients with missing covariate data  or pre-ART viral load measurements that suggested unrecorded prior exposure to treatment   were also excluded. The final study sample comprised 7,515 patients.The exposure of interest was HIV RNA six months (-30/+90 days) after ART initiation. For patients who had more than one eligible viral load measurement during the specified 120-day window, we used the measurement that was closest to six months after date of ART initiation. Viral load measurements were determined by quantitative amplification assays and expressed as the number of HIV copies per milliliter of blood plasma (copies/mL). Viral load assay sensitivity varied over time and by CNICS site; the lower limits of detection for the most commonly used assays were 20, 30, 40, 48, 50, 75, and 400 copies/mL. Observed viral load measurements six months after ART initiation ranged from 6 to over 4 million copies/mL.The outcome of interest was time from six months after ART initiation to diagnosis of first invasive cancer, excluding nonmelanoma skin cancer. All cancer cases diagnosed through 31 December 2014 and recorded at CNICS sites were verified by medical record review . Cancer Death from any cause was considered a competing risk in the analysis. National Death Index and state death certificate records were queried regularly by all CNICS sites to confirm recorded dates of deaths.The start of follow-up for each patient was six months after date of ART initiation. Patients were followed until the earliest of the following: first cancer diagnosis, death, or loss to follow-up . Death from any cause without a cancer diagnosis was considered a competing event. Data were administratively censored after 10 years or on 31 December 2014.We used a proportional subdistribution hazards model  to computh, 35th, 65th, and 95th percentiles [The majority of viral load observations included in our analyses were reported to be below specified lower limits of detection . For the original study population and each bootstrap sample, we used a nonparametric imputation approach with a censoring score model to account for left-censored viral load data . For eaccentiles . We compcentiles , 23 of tPatients were assigned to the following exposure categories based on their observed or imputed viral load at baseline (six months after ART initiation): <20, 20 to 199, 200 to 999, and >999 copies/mL. Because the US Department of Health and Human Services and the AIDS Clinical Trials Group currently define virologic failure as one confirmed viral load measurement at or above 200 copies/mL , we diviWe used inverse probability of exposure weights , 25 to cAdditionally, we estimated inverse probability of censoring weights to account for potentially informative loss to follow-up by viral load category. The inverse probability of exposure and inverse probability of censoring weights were stabilized, and the product of the stabilized weights had a mean of 1.0 in the imputed datasets of the original study sample, with a minimum of 0.12 and maximum of 13. We used SAS version 9.4  for analyses, and R version 3.3.1  for figures.3. At baseline, 26% of study patients had been diagnosed with AIDS, 40% had been prescribed a protease inhibitor (PI)-based regimen, and 48% were prescribed a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. Patients were followed for a median of 4.9  years, for a total of 40,110 person-years. We recorded 290 cancer diagnoses during the study period, and 1,731 (23%) patients were lost to follow up.We identified 7,515 CNICS patients who met our study inclusion criteria . Median Of the 7,515 patients included in the study, 68% had viral loads six months after ART initiation that were below assay detection limits. After imputation, 56% of viral loads were under 20 copies/mL, 23% were between 20 and 199 copies/mL, 5% were between 200 and 999 copies/mL, and 15% were over 999 copies/mL. Plots of the distribution of viral load comparing our nonparametric multiple imputation approach to ad hoc substitution  are shown in Patients differed across viral load categories at baseline . PatientThe most common cancers observed in our study population were non-Hodgkin lymphoma , Kaposi sarcoma , lung cancer , Hodgkin lymphoma , prostate cancer , anal cancer , breast cancer , and liver cancer  . Crude c3 six months after starting therapy (vs. 36%). Crude and standardized risk curves for 10-year cumulative cancer incidence are shown in After controlling for baseline characteristics, overall 10-year cancer risk was 6.90% , with little variation in cancer risk by viral load category (range: 6.76% to 7.44%). The cumulative cancer incidence estimate for patients with viral loads between 200 and 999 copies/mL was markedly reduced after adjustment (crude risk of 10.7% vs. standardized risk of 6.82%); race/ethnicity, year of ART initiation, ART regimen, baseline CD4 count, and study site accounted for 75% of the change in estimate in this viral load category. Among patients with viral loads between 200 and 999 copies/mL six months after ART initiation who were diagnosed with cancer, 62% were black , 53% started ART between 1998 and 2000 (vs. 21%), 63% had been prescribed a PI-based regimen (vs. 46%), and 48% had a CD4 count of less than 200 cells/mmThe overall standardized risk of death without a cancer diagnosis, which was considered a competing risk in the analysis, was 12.2%  , Fig 2. Our objective was to evaluate 10-year cancer risk among HIV patients on antiretroviral therapy with low viral load under 1,000 copies/mL, while accounting for death from any cause as a competing event. Crude 10-year risk of first cancer diagnosis was highest for patients with viral loads between 200 and 999 copies/mL after six months of therapy, though this was largely due to imbalances in race, year of ART initiation, ART regimen, CD4 count, and study site; after controlling for these factors and other baseline confounders, there was no association between HIV RNA six months after ART initiation and first cancer risk.Nearly 70% of all viral load observations used in the analysis fell below assay detection limits, which was expected given that the study population had been on ART for six months at baseline. Prior studies have typically replaced viral load observations below assay detection limits with a constant value, which can result in substantial bias, particularly when the proportion of censoring is high , 27. In Studies that characterize cancer risk often measure incidence rates, typically expressed as the number of cancer events per 100,000 person-years, which assume that incident cancers occur at a constant rate over time. Here we estimated the probability of developing cancer over a specific 10-year period, which may provide a more intuitive measure of cumulative cancer risk. Additionally, a number of previous studies evaluating cancer trends among people with HIV have censored deaths in their analyses rather than using a competing risks approach. Treating deaths as censored observations  ignores the fact that HIV patients may die before being diagnosed with cancer, and will thereby overestimate cancer risk. While censoring competing events may not lead to significant bias when the risk of the competing event is rare, we observed nearly twice as many deaths as first cancer diagnoses (560 deaths vs. 290 cancer diagnoses) in our study sample of HIV patients on treatment. Moreover, censoring competing events may lead to additional bias when the risk of the competing event is differential by exposure, as was the case in this study. We expect that we arrived at less biased risk estimates by modeling the cumulative incidence function of cancer, while explicitly accounting for death from any cause without a cancer diagnosis as a competing risk.We did not account for adherence, switching, or cessation of ART regimen in the analyses. For each treatment-naïve patient, we considered the first recorded date of concurrent prescription of three or more ART drugs as an indicator of starting a combination ART regimen, and ignored changes in treatment. We assumed that variables included in the analyses were measured without error, which is unlikely for self-reported behaviors such as tobacco, alcohol, and illicit drug use; however, we do not expect measurement error of confounders to be differential by exposure or outcome. Outcome misclassification was minimized in this study as cancer cases were confirmed through medical record review, and deaths verified using national and state death records.Nearly a quarter of our study sample was lost to follow-up over the study period, and patients were followed for a median of five years. Given that the outcome of interest was 10-year cancer risk, it would be worthwhile to reassess our risk estimates after additional person-time has accumulated in the CNICS cohort. Due to the relatively small number of cancer cases observed in our study population, our estimates had limited precision and we were unable to make inferences about the potential relationship between incomplete viral suppression and specific cancer types. Therefore, pooling data from other clinical cohorts to verify and expand upon our results is warranted. Nevertheless, we expect that the results of this study are generalizable to HIV patients receiving care and treatment at academic medical centers in the US, and we observed clinically meaningful trends that highlight potential avenues for increased and/or early cancer screening and prevention among people living and aging with HIV. In this study, non-Hodgkin lymphoma, Kaposi sarcoma, lung cancer, Hodgkin lymphoma, prostate cancer, anal cancer, breast cancer, and liver cancer were the most commonly observed cancer types, consistent with prior studies of cancer in HIV patients after the introduction of ART , 28. ThiWe observed a 10-year standardized first cancer risk of 6.9% in our sample of HIV patients after starting therapy, which provides support for existing evidence that cancer poses a significant threat to HIV patients after ART initiation, even among patients who have rapidly achieved viral suppression. Exposure status was based on a single HIV RNA measurement collected approximately six months after ART initiation, as we considered viral suppression at this time point a relevant marker of early treatment success. However, a single detectable viral load measurement could represent either a transient increase in viral load or sustained low-level viremia. While we did not observe an association between early virologic response and subsequent cancer risk in this study, using longitudinal viral load measurements to assess exposure is warranted to better understand the dynamic nature of HIV RNA suppression. Continued research on the role of low-level circulating HIV RNA on cancer development is of particular importance, as occurrences of incomplete viral suppression will be observed more frequently in clinical practice with more people with HIV gaining access to effective ART in the current era of universal treatment.S1 FigDotted line indicates 20 copies/mL. (a) After nonparametric multiple imputation of left-censored viral load observations, averaged over 30 imputations of original dataset; (b) After substitution of left-censored viral load observations with half of detection limit of viral load assay.(TIF)Click here for additional data file.


Switch from first line antiretroviral therapy (ART) to second-line ART is common in clinical practice. However, there is limited knowledge of to which extent different reason for therapy switch are associated with differences in long-term consequences and sustainability of the second line ART.th, 20th, 30th and 40th percentile of time to viral failure (VF).Data from 869 patients with 14601 clinical visits between 1999–2014 were derived from the national cohort database. Reason for therapy switch and viral load (VL) levels at first-line ART failure were compared with regard to outcome of second line ART. Using the Laplace regression model we analyzed the median, 10Most patients  switched from first-line to second-line ART without VF. Patients switching due to detectable VL with  or without drug resistance mutations (DRM)  experienced VF to their second line regimen sooner  and 3.20 (95% 2.65–3.75), respectively) compared with those who switched without VF (4.53 years). Furthermore level of VL at first-line ART failure had a significant impact on failure of second-line ART starting after 2.5 years of second-line ART.In the context of life-long therapy, a median time on second line ART of 4.53 years for these patients is short. To prolong time on second-line ART, further studies are needed on the reasons for therapy changes. Additionally patients with a high VL at first-line VF should be more frequently monitored the period after the therapy switch. Antiretroviral therapy (ART) has substantially reduced mortality and morbidity in individuals with human immunodeficiency virus type 1 (HIV-1) infection –4, but aTo date, cohort studies have reported partly conflicting results on the consequences for disease progression by the various patterns of drug resistance at treatment failure –13. The The aim of the study was to analyze the consequences of different reason for therapy switch from first line ART on second-line ART outcome, using the Swedish cohort which represents a highly diversified HIV epidemic in a real-life setting . This wa+ T-cell counts, HIV-1 subtypes, type of ART, and presence and type of any HIV DRM (if detectable) were collected. ART was classified as: non-nucleoside reverse transcriptase inhibitor (NNRTI) based, ritonavir-boosted protease inhibitor (PI/r) based, protease inhibitor without boosting (PI), and other. A total of 869 patients and 14601 clinical visits were included in the study see Our study used observational data from the Swedish InfCare HIV database, which collects data through a clinical decision support tool and includes >99% of living HIV infected patients and the majority of patients diagnosed between 1983–2014 at 30 infectious disease clinics from all regions of the country . As of JPrimary outcome was time to viral failure (VF) after second-line ART initiation. Therapy switch from first to second-line ART was defined as any change between (1) NNRTI and PI or (2) change of any NRTI, PI or NNRTI while remaining on PI or NNRTI based ART regimen. Viral failure was defined as one VL> = 200 copies/mL after at least six months of a new ART line initiation (first-line or second-line ART). Sensitivity analysis were done using different definitions of VF: 1) one VL> = 50 copies/mL; 2) two consecutive VL> = 50 copies/mL after six months and nine months, respectively, of second-line ART initiation, with no differences in the estimates (data not shown). We controlled the frequency of VL measurements in our study population and all patients had at least one VL measurement per year. On average, each patient had 5 VL measurements per year.Secondary outcomes were: (1) median CD4+ T-cell counts at 12 and 24 months of second-line ART; (2) time to death and (3) time to AIDS.Switch to second-line ART was categorized into three different categories: (1) switch without VF and therefore GRT was not performed; (2) switch due to VF and no detectable DRM at standard GRT; (3) switch due to VF and detectable DRM.Real time PCR RNA quantification tests (COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test) were used for VL monitoring. VL at second-line ART initiation was categorized in six categories: (1) 0–200; (2) 200–500; (3) 501–1000; (4) 1,001–10,000; (5) 10000–100 000 and (6) 100001+ HIV RNA copies/mL.Potential confounders for time to second-line VF and changes in CD4+ T-cell counts were chosen as: VL , and CD4+ T-cell count  at baseline and at second-line ART initiation; age  at first-line ART initiation; type of ART regimen  at first and second-line ART; country of birth (Sweden vs Non-Sweden); gender ; route of transmission , people who inject drugs (PWID), other); and follow-up time on first-line ART as a continuous variable, and time on second-line ART as a continuous variable.Any DRM detected appearing at first line ART failure and before switch were described. In case of several tests, the closest one before switch was used. Thus, this test showed all DRM which had appeared from start of virological treatment failure. Of the total number of patients included, 479 (56%) had a DRM test at viral failure.Viral nucleotide sequence data was submitted to the Stanford University HIV Drug Resistance Database (HIVdb)  using Sith, 20th, 30th and 40th percentiles of time to VF of second-line ART, death and AIDS were modeled by using a Laplace regression model [+ T-cell count at 12 and 24 months second-line ART was modeled by using a quantile regression model [+ T-cell count in the interval of 11 to 13 months or in the interval of 23 to 25 months of second-line ART, so there was a high rate of missing values. To take that into account, we conducted a weighted analysis [+ T-cell count at 12 (11–13 month) and 24 months (23–25 month) with no changes on the estimates (data not shown). Weights were estimated modeling the probability of having a missing CD4+ T-cell count at 12 (11–13) or 24 (23–25) months separately and taking its inverse.Laplace regression is a statistical method which estimate multivariable survival percentiles and evaluate the effects of exposures on them. Survival percentiles are important summary measures of a time-to-event outcome of interest. For instance, in this study, the 50th survival percentile is that value of time to VF of second-line ART for which 50% of the study individuals have a value below it and 50% above it. Using Laplace regression we were able to estimate the effect of switch to second-line ART on different percentiles of time to VF of second-line ART adjusting for potential confounders. More specifically, assume that a significative effect of switch to second-line ART on median time to VF of second-line ART is found. This means that the median time to VF of second-line ART across categories of switching to second-line ART varies significatively, i.e. switching has an effect on median time to VF. Similar interpretation can be done with all other survival percentiles. The median, 10on model  adjustinanalysis  on mediaBaseline demographic and laboratory characteristics of the study population (n = 869) are described in Out of the 869 patients, 495 (57.0%) switched to second-line ART without a virologic failure, 250 (28.8%) switched with a VF without DRM, and 124 (14.2%) switched with a virological failure and PI, NNRTI and/or NRTI DRM.A total of 207 patients on NNRTI based first-line ART switched to second-line ART of whom 99 (47.8%) switched without a VF, 64 (30.9%) switched with VF without any DRM, and 44 (21.3%) with VF and DRM. For the 394 patients on a boosted PI based first-line ART, the number of individuals in the same categories of switches were: 265 (67.3%), 88 (22.3%), and 41 (10.4%), respectively. For the 178 patients on an unboosted PI based first-line ART, the number of individuals in the same categories of switches were: 73 (41.0%), 70 (30.9%), and 35 (19.7%), respectively. Eighty-six patients started other ART regimens .An NNRTI-based regimen was given as second-line in 364 patients, 292 were given PI/r based regimen, 43 an unboosted PI based regimen and 170 other treatments. At treatment failure, 287 DRM occurred among 85 patients and the most common were: M184I/V , K103N , V108I , D67N , K70R , M41L , K101E , T215F , Y181C .Patients switching from first-line to second-line ART due to virological failure with (n = 124) or without (n = 250) any DRM experienced VF to their second line regimen sooner compared to second-line VF in all studied survival percentiles compared with patients switching without failure (n = 495). For example, the first 50% (median) of virologic failures occurred within 4.53 years of second-line ART among patients who switched first line ART without failure (Reference group) and within 3.43 years (1.1 year before) among patients who switched due to virological failure and at least one DRM. th, 40th and median survival percentile compared with patients who initiated it with a VL between 0 and 200 copies/mL. Nonetheless, no clear difference was seen among the failures that occurred in the lower percentile (10th and 20th). For example, the first 50% (median) of second-line ART failures occurred within 4.68 years for the patients with a VL 0–200 copies/ml at first-line treatment failure (reference group). For patients switching with a VL of 201–500 copies/ml it occurred within 3.86 years ). Patients initiating second-line ART with VL 201–500, 501–1.000, 1.001–10.000, 10.001–100.000 and >100.000 copies/mL, respectively, experienced VF to their second line regimen sooner in the 30Type of switch and VL at second-line ART initiation did not show any significant effect on median CD4+ T-cell counts at 12 and 24 months, respectively  Tables. In this study we analyzed the effect of different causes to first line ART switch, and the level of VL at switch, on the long-term outcome of second-line treatment using the InfCare cohort, which represents >99% of all diagnosed HIV infected patients in Sweden. The majority of the patients switched therapy due to other reasons than VF with no difference over the calendar years (data not shown). This consistent pattern of reason for therapy switch could be due to that although the virological treatment failure was more common in the past, switches due to side effects of the older drugs also occurred to a higher extent. Our data thus confirm that toxicity and/or convenience are by far the most common reason for therapy switch of first-line ART in a real-life situation in a high-income country.+ T-cell counts at 12 and 24 months of second-line ART and also no effect on the few AIDS and death cases.Among the patients who had a detectable VL at treatment failure, the vast majority did not have any DRM and in total only 65 (8%) of the patients switching from first to second-line ART had one or more DRM. The low rate of DRM among patients with detectable VL could be due to either a poor adherence or use of drugs with a high genetic barrier, the latter illustrated by the low proportion (10.4%) of DRM in patients with PI/r based ART as compared to NNRTI-based ART (21.3%) and unboosted PI (19.7%). However, once VF occurred, it had a significantly negative effect on time to second-line VF regardless of DRM status. The significant differences started to appear around 2.5 years. The median time to failure was 4.3 years of second-line treatment for patients with no detectable VL at switch compared to 3.2 (95% 2.65–3.75) years for patients with VF only and 3.43 (95% CI 2.9–3.96) years for patients with at least one DRM at failure. Several factors can explain this finding. For example at virological failure the reservoirs are reseeded rapidly resulting in a high viral burden to contain with the new treatment. Also, there may be drug resistant quasispecies present that are not detected by routine resistance testing. In addiPartly in contrast to our results a study from Italy with GRT performed between 1998–2004, showed that three classes DRM was associated with clinical progression only when the model was not adjusted for calendar years, whereas another Italian study performed between 1999–2003 could establish the relationship . The FIRST study showed that NNRTI-DRM is the strongest predictor of poor clinical outcome . FurtherIn summary since our study includes patients from 1999 until 2014 and adjusts for time in follow up, it reflects on the current clinical practice and state of the art antiretroviral regimens, although no patients with integrase inhibitors were included. Treatment modifications were commonly done due to other reasons than viral rebound. The different reasons for therapy switch studied are not related to poor CD4+ T-cell gain on second-line treatment. In the context of life-long therapy, the median time on second line ART of 4.53 years is short. To improve time on second-line therapy further evaluations are needed of the reasons to therapy switch if patients have an undetectable VL. Furthermore the patients with a high viral load at first-line treatment failure should be more frequently monitored the time period after therapy switch.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


We assessed the validity of a self-report measure of undetectable viral load (VL) among women with HIV in British Columbia (BC), Canada. Questionnaire data from the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study was linked with population-based clinical data from the BC Centre for Excellence in HIV/AIDS. Self-reported undetectable VL was assessed by the question: “What was your most recent VL, undetectable (i.e. <50 copies/mL) or detectable (i.e. ≥50 copies/mL)?” Laboratory measurements of VL <50 copies/mL (closest to/before study visit) were the criterion for validity analyses. We measured positive and negative predictive values  and likelihood ratios .Of 356 participants, 99% were linked to clinical data. Those unlinked (n = 1), missing self-report VL (n = 18), or missing self-report and laboratory VL (n = 1) were excluded. Among the remaining 336: median age was 44 (IQR 37–51); 96% identified as cis-gender; 84% identified as heterosexual; and 45% identified as Indigenous, 40% White, 8% African, Caribbean, or Black, and 8% other/multiple ethnicities. Overall, 85% self-reported having an undetectable VL while 82% had clinical data indicating viral suppression. The PPV was 93.7 (95% CI 90.2–96.2) indicating that 94% of women who self-reported being undetectable truly were. The NPV was 80.4 (95% CI 66.9–90.2). LR+ was 3.2 (2.1–4.6) and LR− was 0.05 (0.03–0.10).Our self-report measure assessing undetectable VL strongly predicted true viral suppression among Canadian women with HIV. This measure can be used in research settings without laboratory data in regions with high rates of VL testing and suppression. Combination antiretroviral therapy (cART) is now recommended for all people living with HIV regardless of CD4 cell count . The cliThe efficacy of using treatment for individual health and for population-level prevention has been demonstrated in jurisdictions around the world including British Columbia (BC), Canada, where “Treatment as Prevention” (TasP) was formally adopted into policy in 2010 .To monitor and evaluate progress on 90–90–90 goals, accurate assessment of viral suppression is essential. Laboratory technologies of plasma samples are the gold standard for measuring VL in clinical practice . Howeverby, with, and for women living with HIV, in collaboration with researchers, clinicians, service providers, policy-makers and other stakeholders . In totParticipants completed structured questionnaires at baseline, administered by Peer Research Associates (women living with HIV who have received training in community-based research) . Study v“don’t know” or “prefer not to answer”) to the survey question assessing VL or self-reported never receiving HIV medical care or VL results (n = 18), or who were missing both self-reported and laboratory VL (n = 1) were excluded from the analysis, leading to a final analytic sample of 336.For this analysis, we used baseline questionnaire data from participants enrolled in CHIWOS in BC between August 27, 2013 and May 1, 2015, and linked clinical data from the BC Centre for Excellence in HIV/AIDS. Of 356 participants, 99% were linked to the DTP clinical database (n = 354). Those who remained unlinked (n = 1), who provided invalid responses  or detectable (i.e. 50 copies/mL or more)?” Criteria for validity analyses were corresponding laboratory measurements of VL <50 copies/mL, with tests closest to and before the study visit selected for analysis. For those included in analyses (N = 336), median time between study visit and most recent VL lab result was 50.5 days (IQR 20.5–87).In the CHIWOS questionnaire, self-reported VL was assessed by the following survey question: vs. White vs. African, Caribbean, or Black vs. other/multiple ethnicities), education (<High school vs. ≥High school), and illicit drug use within the past 3 months (Yes vs. No). PPV is the probability that participants who self-report being “undetectable ” truly have laboratory measurements of VL <50 copies/mL, while NPV is the probability that participants who self-report being “detectable ” truly have laboratory measurements of VL ≥50 copies/mL , one-third of whom were interviewed onsite [n = 41 (31%)], versus women who did not access this clinic. The PPVs for these two groups were similar [Oak Tree Clinic: 95.4 (95% CI 89.6–98.5) vs. Non-Oak Tree Clinic: 92.6 (85% CI 87.6–96.0)]. However, the NPVs were much higher for Oak Tree Clinic patients [91.3 (95% CI 72.0–98.9)] compared with non-Oak Tree Clinic patients [71.4 (95% CI 51.3–86.8)]. Thus, while prompting participants for clinical information does not appear to have altered the proportion of those undetectable that are able to correctly identify as such, likely given the high VL suppression rates to begin with, these data do suggest this screening procedure may have impacted accurate reporting of a detectable VL.“don’t know”, “prefer not to answer”, or “never received care/VL results”) and thus excluded from this analysis were more, less, or equally likely to be undetectable than those who were included. Among these participants, 56% (n = 10) were detectable. This compares to only 18% (n = 59) of those were included in this analysis. It is possible that these women did not feel comfortable sharing this personal information or were simply unaware owing to competing life demands preventing access to and engagement in care. All of these limitations are important to consider in research assessing VL levels.Finally, we also conducted a sub-analysis to determine whether the 18 participants who were unable to self-report their VL (either because they reported Engagement in treatment, care, and support is critical to sustained viral suppression, and, in turn, improved individual health and HIV prevention –9. Futur


HIV-1 drug resistance to older thymidine analogue nucleoside reverse transcriptase inhibitor drugs has been identified in sub-Saharan Africa in patients with virological failure of first-line combination antiretroviral therapy (ART) containing the modern nucleoside reverse transcriptase inhibitor tenofovir. We aimed to investigate the prevalence and correlates of thymidine analogue mutations (TAM) in patients with virological failure of first-line tenofovir-containing ART.We retrospectively analysed patients from 20 studies within the TenoRes collaboration who had locally defined viral failure on first-line therapy with tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleoside reverse transcriptase inhibitor  in sub-Saharan Africa. Baseline visits in these studies occurred between 2005 and 2013. To assess between-study and within-study associations, we used meta-regression and meta-analyses to compare patients with and without TAMs for the presence of resistance to tenofovir, cytosine analogue, or NNRTIs.vs 95·0 cells per μL [37·0–177·0] in patients without TAMs; p=0·007) and were more likely to have tenofovir resistance , NNRTI resistance , and cytosine analogue resistance . We detected associations between TAMs and drug resistance mutations both between and within studies; the correlation between the study-level proportion of patients with tenofovir resistance and TAMs was 0·64 (p<0·0001), and the odds ratio for tenofovir resistance comparing patients with and without TAMs was 1·29 Of 712 individuals with failure of first-line tenofovir-containing regimens, 115 (16%) had at least one TAM. In crude comparisons, patients with TAMs had lower CD4 counts at treatment initiation than did patients without TAMs (60·5 cells per μL [IQR 21·0–128·0] in patients with TAMS TAMs are common in patients who have failure of first-line tenofovir-containing regimens in sub-Saharan Africa, and are associated with multidrug resistant HIV-1. Effective viral load monitoring and point-of-care resistance tests could help to mitigate the emergence and spread of such strains. Given the potential substantial prevalence of unrecognised virological failure and drug resistance in this setting,11 programmatic single-drug substitutions risk more rapid acquisition of high-level drug resistance not only to NNRTIs and cytosine analogues, but also to tenofovir.12 Importantly, NNRTI resistance and thymidine analogue resistance mutations (TAMs) can be transmitted to uninfected individuals who are subsequently at increased risk of ART failure themselves.13WHO has recommended first-line tenofovir disoproxil fumarate (tenofovir) instead of thymidine analogues since 2012.We did a systematic review using PubMed and Embase, searching from Jan 1, 2000, up to Aug 15, 2016, without language limitations. Manuscripts of interest were also identified from the reference lists of selected papers, clinical trials registries, and abstracts from the Conference on Retroviruses and Opportunistic Infections (CROI) and International AIDS Society (IAS). We used the search terms “HIV” AND “Tenofovir” AND “thymidine analogue” OR “stavudine” OR “zidovudine” OR “AZT” OR “d4T”. We found no studies reporting the implications of previous thymidine analogue use on outcomes following tenofovir-based antiretroviral therapy (ART). One study investigated the implications of transition from thymidine analogue to tenofovir by use of a cross sectional survey in Myanmar before the introduction of tenofovir. The investigators tested viral loads in more than 4000 patients after 12 months of thymidine analogue-based ART to avoid substitutions in viraemic patients. They noted that a substantial proportion of patients were having treatment failure , in whom direct tenofovir substitution for the thymidine analogue would not be appropriate.Our results show that tenofovir-based first-line regimens are failing in a substantial proportion of patients who have evidence of previous exposure and drug resistance to older nucleoside (thymidine) analogues such as zidovudine and stavudine in sub-Saharan Africa. These individuals are likely to have developed drug resistance to the non-nucleoside reverse transcriptase inhibitor as well as the cytosine analogue, and therefore have high-level resistance to at least two of the three drugs present in tenofovir-based first line ART. Our data show that these individuals with thymidine analogue mutations have lower CD4 counts and therefore are at greater risk of clinical complications than are those without previous ART exposure.Cheap and effective viral load monitoring, resistance testing, or both could prevent the transition of patients with virological failure onto tenofovir-based first-line ART and also identify individuals with pre-existing drug resistance to first line agents arising from undisclosed prior ART. These individuals could then be treated with second-line regimens.14 Accordingly, we have previously reported unexplained TAMs in patients after viral failure of tenofovir-containing first-line regimens.15 In this Article, we characterise the prevalence, determinants, and implications of TAMs in patients after virological failure of tenofovir-containing first-line regimens in sub-Saharan Africa.A further complication to the introduction of tenofovir in sub-Saharan Africa is shown by data suggesting that individuals presenting as treatment naive often do not disclose previous ART exposure, which is most likely with thymidine analogue-based ART.15 Briefl y, we collected data from cohorts with documented virological failure after first-line ART consisting only of tenofovir plus either lamivudine or emtricitabine plus either efavirenz or nevirapine, with no previously known exposure to additional nucleoside reverse transcriptase inhibitors such as zidovudine or stavudine  per mL. Patient characteristics were broadly similar between patients with and without TAMs, with the exception of baseline CD4 count, which was roughly 30 cells per μL lower in patients with TAMs in all regions (p=0·007). We noted that usage of emtricitabine was 10% lower in patients with TAM compared to those without. 33 (16%) of 209 women with available data on single-dose nevirapine had known previous exposure to single-dose nevirapine. Prevalence of NNRTI resistance was 88% (29 of 33 patients) in patients with single-dose nevirapine exposure and 82%  in those without single-dose nevirapine exposure (p=0·38). For many patients, it was not known whether or not they had received single-dose nevirapine, including men, for whom single dose nevirapine use was always answered as no.We assessed 34 studies and excluded 14 because they contained fewer than ten patients (56 patients excluded). We identified 712 patients who had viral failure with WHO-recommended, tenofovir-based first-line regimens in 20 studies across sub-Saharan Africa . Most 4. Median TAMs were detected in 115 (16%) of 712 patients . The prevs 378 [63%] of patients without TAMs; p=0·0002) and nevirapine or efavirenz , with consistent findings across all regions , as well as resistance to cytosine analogues , or baseline CD4 count . Under this scenario antagonism with Lys65Arg would be active and we would therefore not expect to see Lys65Arg and TAMs together in the same individuals.There are three possible sources of TAMs in patients on first-line tenofovir. The first is transmitted drug resistance, which is unlikely to account for the majority of cases in this study because transmitted drug resistance of TAMs is rare (<1% of TAMs in untreated patients result from being transmitted).20 and would trigger adherence counselling and then a possible switch to a second-line protease inhibitor-based regimen instead of continuation of a failing first-line regimen with the substitution of a thymidine analogue for tenofovir. A large study in Myanmar (where tenofovir substitution is planned) has monitored viral loads in more than 4000 patients after 12 months of thymidine analogue-based ART, with the aim of avoiding substitutions in viraemic patients. The investigators found that 13% of patients had viral loads greater than 250 copies per mL, which was halved after adherence counselling was done, reinforcing the need for viral load monitoring before drug substitution.21The second possibility is programmatic substitution, wherein tenofovir was used to replace a thymidine analogue at a time when the patient had occult treatment failure. Under this scenario the most likely sequence of events would be, first, acquisition of cytosine analogue resistance, TAM, and NNRTI mutations during prolonged viral failure, followed by a switch to tenofovir and subsequent emergence of Lys65Arg that confers tenofovir resistance. Therefore, prevention of the develop ment of Lys65Arg mutation could only be achieved by viral load suppression confirmation before the switch in treatments. Effective viral load monitoring has been identified as a priority area23 The third possibility, which we believe could account for most of the TAMs in the present study is previous undisclosed ART use with undocumented viral failure and drug resistance. This hypothesis is supported by the lower CD4 counts detected in patients with TAMs. Moreover, significant variation has been reported in viral load monitoring practices between rural and urban settings in South Africa,24 possibly explaining how unrecognised viral failure and drug resistance during tenofovir substitution could occur in settings where viral load monitoring is centrally funded and part of national guidelines.However, the second scenario cannot account for many of the TAMs identified in the present study because we detected TAMs in cohorts in which no programmatic substitution had occurred and tenofovir-based ART was used at the outset in apparently untreated patients .22,23 Th25 could be used to assist in the identification of patients with resistance to the components of first-line ART. We have previously identified key mutations that could be used in such assays, including Lys65Arg, Lys103Asn, Val106Met, Tyr181Cys, Gly190Ala, and Met184Val,26 and on the basis of the present study, Asp67Asn and Lys219Gln/Glu could be added to this list. If HIV-1 drug resistance is detected with such assays, second-line ART could be initiated, taking into account the mutations identified. If they become sufficiently cheap and reliable, drug resistance assays could be used in place of viral load monitoring at treatment initiation or switches.To prevent drug resistance due to undisclosed previous ART use, accessible point-of-care baseline resistance screening23 we calculate that if 15% of people initiating ART have failure at 1 year , then our data represent about 4750 patients initiating tenofovir-based first-line ART.Our study has some limitations. The sampling was not systematic and therefore prevalence estimates might not be fully representative of countries and regions. Our drug resistance prevalence estimates represent prevalence for participants with documented virological failure. We can only estimate the overall number initiating treatment, because it was not systematically assessed. Using data from WHO and Uganda on the prevalence of virological failure,27 Additionally, we did not assess thymidine analogue resistance conferred by mutations in the connection domain between HIV-1 reverse transcriptase and RNAseH that are known to be selected by zidovudine,28 leading to further underestimation of drug resistance.Although none of the studies overtly used targeted viral load testing in individuals suspected of having treatment failure, such targeting might have occurred at the clinical level, potentially biasing our estimates of TAM resistance upwards. Conversely, Sanger sequencing can miss drug resistance mutations in 30% or more of patients.31 However, given that TAM and Lys65Arg are not selected together by a single stavudine-based regimen,32 exposure to stavudine would probably not explain the genotypes with both TAM and Lys65Arg that were seen in our study.Notably, stavudine selects not only for TAMs, but also for Lys65Arg in up to 20% of patients who have failure of stavudine.8 and pre-exposure prophylaxis.33 First, a single point-of-care viral load test could be implemented to prevent substitution of first line zidovudine for tenofovir in patients with virological failure. Regular viral load monitoring has been advocated in the past for treatment monitoring and could identify early virological failure in patients with previously undisclosed ART and drug resistance. However, this regular monitoring might be less cost effective than targeted viral load measurement. Second, simple resistance test kits could both assist in screening for drug resistance before ART initiation and also contribute to population level surveillance of HIV-1 drug resistance25 in both treated and untreated populations—a priority in sub Saharan Africa given the substantial mortality now recognised to be associated with HIV-1 drug resistance.5 These proposals should be part of a multipronged approach and subjected to cost effectiveness assessment in the wider context of other interventions that aim to limit burden of the HIV epidemic.This study has important policy implications for the limitation of drug resistance as tenofovir becomes more widely used both as treatmentSupplementary data


The viral load (VL) in patients receiving antiretroviral therapy (ART) is the best predictor of treatment outcome. The anticipated benefits of VL monitoring depend on the actual uptake of VL test results for clinical decisions. The objective of this study was to assess the uptake and utilization of VL test results for clinical decisions on HIV treatment in Cameroon, from 2013 to 2017.This was a retrospective cohort analysis of data from files of patients receiving ART at Buea, Limbe, Bamenda and Bafoussam regional hospital HIV treatment centers. A simple random pick of six file blocks was performed in each shelf that corresponded to a year of initiation, and the contents of all selected files were reviewed and the information needed for the study entered a structured questionnaire. The data collected was recorded in Epi Info (version 7.1.5.2), and analyzed using SATA .3). Approximately 54% of the patients with virologic failure were switched to a second-line regimen.Eight hundred and thirty files were reviewed. The mean duration on ART was 39.4±12 months. Viral load testing uptake was 24.33% and only one VL test had been done by all patients. Approximately 65% of the patients did the first VL after more than 24 months on ART. The median turnaround (TAT) time for VL testing was 6 days (Interquartile range (IQR) 3-7days). Among 201 patients who did a VL test, 94.55% had VL suppression (≤1000copies/mmThe uptake of viral load testing is low in North West, South West and West Regions of Cameroon. The current TAT for VL testing is plausible. The rate of switch to second line regimen is low. It is time to strengthen the scale up of VL testing and improve the rate of switch to second-line regimen in Cameroon. In December 2013, the Joint United Nation’s Program on HIV/AIDS put into action a ‘90-90-90’ strategy with the goal of bringing an end to the AIDS epidemic . The uptake of VL testing significantly improved with time [p(x2 trend) = 0.012]. The median turnaround (TAT) time for VL testing was 6 days (IQR: 3-7days). Viral load suppression was observed in 94.53% of patients who did a VL after starting ART.This is demonstrated in Approximately 89% of the participants with VL suppression were continued on same ART regimen while 5/11 of ART patients with virologic failure were continued on same first-line ART regimen. Five of the 11 participants with virologic failure were switched to a second-line ART regimen while one person had a substitution in the initial ART regimen. Details of these and other criteria for decision making are shown in The results of this study show that the current viral load testing uptake is very low in frequency and in the proportion of patients accessing a VL test at any given time during ART. The median turnaround time, from request of VL testing specimen to return of results, is 6 days. Viral load suppression is high in this study (94.5%) while only 5.5% of the patients had a virologic failure (VF). The proportion of treatment switch in the small number of patients with VF was inadequate. However, in the absence of clear national guidelines on VL monitoring during the time of the study, these findings should be interpreted with caution.We learn from the study results that HIV infected individuals in these regions were beginning ART soon after diagnosis (IQR (time from diagnosis to start of ART): 1.5–5 months), which is a good thing. However, one would have expected a wider IQR because the immunologic criteria for ART enrolment have been in use during the time when most of the cohorts included in this study started ART. This finding has several implications. Firstly, some patients were initiated on ART at a high CD4 count. Secondly, it could be suggestive that some patients were often HIV-diagnosed clinically (contrary to voluntary testing) at low immunologic status and therefore enrolled into treatment and care earlier-this is possible considering the commitment of the government of Cameroon into campaigns against stigmatisation during the period from 2013 and beyond.st century. There are several possible explanations to the low VL testing uptake in these centres. An obvious explanation would be the lack of laboratory infrastructure for VL testing at the treatment centres. It is also possible as anecdotal reports suggest that laboratory equipment is available but the laboratory lacks human capacity and the reagents to conduct VL testing. As VL remains the key predictor of treatment success or failure in ART, low VL testing rates provide limited information about success in the ART programs. In Cameroon, VL testing is still centralised for most of the treatment centres involved in this study and many other treatment centres within the primary health care structure. To raise hopes towards achieving the United Nation’s AIDS (UNAIDS) program’s target of 90% viral load suppression in the population, political commitment and other leverages are required to strengthen large scale VL testing.The overall and timely uptake of VL testing is very poor in this study. This was, however, improving with year of enrolment and duration on ART. It did not, however, fit into the recommendation of VL monitoring as none of the patients had done consecutive VL assessments for the duration on treatment. This finding of low access to VL testing is consistent with a global call for governments in resource-limited settings to commit politically and ensure scale up of viral load testing that align with the new guidelines for ART monitoring in the 21A turnaround time (TAT) of 6 days (IQR: 3-7days) is plausible when compared with reports from Malawi, where only the pre-analytic period of VL testing was found to be up to 39 days in 2015 . The preThe anticipated benefit of a VL test result lies on the readiness for a clinical decision to switch the treatment regimen or maintain similar regimen with enhanced adherence counselling. It is unfair to judge a clinical decision to switch or substitute a treatment regimen without considering information on adherence by the patient. It is possible that a patient who poorly adheres to the first line ART regimen would equally not adhere to the second line even if they are switched based on VF without enhanced adherence counselling. The criteria that were used for substitutions of first line regimens like features of drug toxicity, as in previous studies , and worThe ministry of public health in Cameroon is presently making efforts with the support of many global health partnerships to ensure improved access to VL testing across all regions of Cameroon. The available conventional laboratories underway for full functionality will greatly support the achievement of the third ‘90’of the UNAIDS targets  in CamerThis is the first study from Cameroon that demonstrates the uptake and utilization of viral load test results for HIV patient management at a time when VL testing is the preferred laboratory monitoring strategy. The strength of this study lies in its large sample size, multi-site nature which to an extent attempts to paint a national picture about VL testing in Cameroon. We were limited in our findings of low VL testing rate which made it inappropriate to infer the information about switch of regimen, since only a small number of patients were failing treatment. This study did not explore the availability of second-line ARVs, which could give more clarity on factors limiting the switch to second-line regimen. We cannot also account for the quality of the data collected as the study design dictates. However, the large sample size improves the precision of our findings.The uptake of viral load monitoring of ART in North West, South West and West Regions of Cameroon is inadequate. The turnaround time for VL testing is plausible. Adequacy of switch to second line regimen is low, but can be best exploited with larger number of patients testing for VL. Political action and other leverages is needed to strengthen the scale up of VL testing in Cameroon.S1 Dataset(ZIP)Click here for additional data file.


Genome-wide association studies (GWAS) have considerably advanced our understanding of human traits and diseases. With the increasing availability of whole genome sequences (WGS) for pathogens, it is important to establish whether GWAS of viral genomes could reveal important biological insights. Here we perform the first proof of concept viral GWAS examining drug resistance (DR), a phenotype with well understood genetics.st line antiretroviral treatment in rural KwaZulu-Natal, South Africa. The majority and minority variants within each sequence were called using PILON, and GWAS was performed within PLINK. HIV WGS from patients failing on different antiretroviral treatments were compared to sequences derived from individuals naïve to the respective treatment.We performed a GWAS of DR in a sample of 343 HIV subtype C patients failing 1GWAS methodology was validated by identifying five associations on a genetic level that led to amino acid changes known to cause DR. Further, we highlighted the ability of GWAS to identify epistatic effects, identifying two replicable variants within amino acid 68 of the reverse transcriptase protein previously described as potential fitness compensatory mutations. A possible additional DR variant within amino acid 91 of the matrix region of the Gag protein was associated with tenofovir failure, highlighting GWAS’s ability to identify variants outside classical candidate genes. Our results also suggest a polygenic component to DR.These results validate the applicability of GWAS to HIV WGS data even in relative small samples, and emphasise how high throughput sequencing can provide novel and clinically relevant insights. Further they suggested that for viruses like HIV, population structure was only minor concern compared to that seen in bacteria or parasite GWAS. Given the small genome length and reduced burden for multiple testing, this makes HIV an ideal candidate for GWAS. For this reason, genetic research is increasingly moving towards whole genome sequencing approaches to capture the full range of genetic variants in a population.Genome-wide association studies (GWAS) have led to significant advances in the understanding of complex human traits and diseases. They involve the analysis of hundreds of thousands or millions of common genetic variants, usually single nucleotide polymorphisms (SNPs), testing for an association between each variant and a phenotype (see ). This aIn this respect, the field of pathogen genomics is quickly catching up with human genomics, with international collaborations currently generating thousands of whole genome sequences (WGS) for pathogens such as HIV and malaria  resistance in HIV. The provision of ART to over 6.2 million people living with HIV in sub-Saharan Africa has been one of the most successful public health interventions ever undertaken , improvi343 samples with phenotype and genotype data remained after variant calling and quality control . A totalGWAS was performed to identify variants associated with drug resistance. The drug resistance phenotype was binary for each drug and defined as any history, or not, of failure while treated with the given drug. Failure was defined as at least one measure of viral load >1000 copies/ml after 12 months of treatment. GWAS identified eight independent associations at permutation adjusted genome-wide significance. Five of the associations were known loci involved in DR and all but one were in the reverse transcriptase region (RT), the functional target of these drugs see . Failure2 = 22%). Looking at known DR mutations ).The study sampled 319 HIV-infected adults and 24 children on ART with virological failure in the Hlabisa HIV Treatment and Care Programme in South Africa for which a whole genome of HIV-1 was produced. The inclusion criteria were: ART regimen for at least 12 months followed by virological failure, defined as one viral load >1000 copies/ml. Exclusion criteria were: prior use of nucleoside reverse transcriptase inhibitor (NRTI) monotherapy or dual therapy (not including regimens for the prevention of mother-to-child transmission (pMTCT)). All individuals were seen by a physician, who performed a clinical evaluation and obtained written informed consent for the study. A 5 ml EDTA whole blood sample for HIV DR genotyping was collected during the clinical evaluation. Basic clinical and demographic data, including GPS data on household location, were collected on a clinical form and clinical and treatment information was compared with the records in the Africa Centre’s ART Evaluation and Monitoring System (ARTemis), an operational database holding treatment and laboratory monitoring information from the national ART programme in South Africa. The clinical information was entered in anonymised form into a relational sequence database, the SATuRN REGA database . FurtherThe study was approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (ref. BF052/10) and the Health Research Committee of the KwaZulu-Natal Department of Health (ref. HRKM 176/10). South African legal guidelines define a person able to give informed from consent from age 17. Written informed consent was obtained from all the study participants and their parent or legal guardian in the case of paediatric patients (≤16 years).The median duration of ART among patients in this cohort was 42 months (IQR 32–53). The most common first line ART regimens were: tenofovir/stavudine/zidovudine +Lamivudine +efavirenz/nevirapine. The most common second line ART regimen were: Lopinavir (+ Ritonavir), Lamivudine, zidovudine/tenofovir. The median duration of antiretroviral failure was 27 months (IQR 17–40 months). Details on drug exposure data and DR results have been described previously , 29. DruRNA was extracted from samples using the manual QIAamp Viral RNA Mini Kit (Qiagen). The near complete HIV-1 genome was amplified by a previously described RT-PCR strategy with primers modified to be more subtype C specific . The amplification involved the production of four overlapping genetic fragments of lengths of 1.9kb, 3.6kb, 3.0kb and 3.5kb. This included all nine open reading frames and partial regions of the 5’- and 3’-LTR. The DNA concentration of individual amplicons was quantified using the Qubit sdDNA HS Assay Kit (Thermo Fischer Scientific-Life Technologies). Pooled amplicons were prepared for sequencing using the Nextera XT DNA Sample Preparation kit (Illumina) and the Nextera XT DNA Sample Preparation Index Kit (Illumina), following the manufacture’s protocol. The runs comprised pools of 96 samples that included three controls . All processes to generate WGS were undertaken locally at the Africa Centre laboratory, Nelson R Mandela Medical School, University of KwaZulu-Natal, South Africa.http://www.geneious.com) [Fastq quality control was performed using FASTQC(0.11.3) and QUASR(3.1) software applications. Reads of less than 100bp in length and a quality score lower than 30 were excluded. In addition, the reads were trimmed up to 10bp from 5’ and 30bp at the 3’ to exclude poor quality sequence at the beginning and end of reads. We noticed that the second pair read of the Illumina Nextera XT was of lower quality and that excluding the last 30bp increased quality score to > 33. We imposed these exclusion criteria in order to decrease the probability of ambiguous read mapping, which occurs when shorter reads of lower accuracy are included in assemblies [ous.com) . After aIn order to determine if there was clustering of sequences (i.e. sequences that were very similar with low genetic diversity), we aligned all of the whole genomes with a reference dataset for HIV-1 subtype C. The tree was constructed with HKY+Gamma site rate variation in a MPI version of RaxML. Reliability of internal nodes was evaluated by 100 bootstrap replicates. Phylogenies were analysed using Phylotype software application  in orderThe processing of WGS data to the performing of GWAS is outlined in As GWAS software was originally designed for diploid organisms (i.e. those with two chromosomes and so two copies of any given loci), each sample can be called either as homozygous for an allele (e.g. AA or TT) or as heterozygous (e.g. AT). While heterozygosity is incorrect in the sense that HIV is haploid, it captures an important reality of viral infection: genetic differences within the host’s viral population. We wanted to retain the feature of diploidy to account for samples with diversity at a given DR loci. We expected heterozygous samples to have an intermediate effect size compared to samples where the DR variant was either entirely non-existent or fixed. The downside of this approach was that given numerous sequence reads for each loci, some variation is expected due to sequencing errors. To account for this, we allowed for diploid calling in the following manner. If the reference allele frequency was present in >85% of reads at a loci, the loci was called as homozygous for the reference allele. A heterozygous call with one copy of the reference variant and one of the non-reference variant was made if the reference allele frequency was between 85% and 15% of reads. Finally, a homozygous non-reference call was made if the reference allele frequency was found in less than 15% of reads. While these cut-offs are simply defaults of the software, this worked as a crude calling approach for whether an individual sample’s HIV population was fixed or mixed for any given loci.VCFs were then merged in GATK , then thA logistic regression was performed in PLINK1.09  with druhttp://www.hiv.lanl.gov/). This was done to ensure our variant frequencies were in agreement with those observed elsewhere.Genome-wide significant SNPs within the Pol region were able to be taken forward for replication in a publically available independent sample. This was the Stanford University HIV Drug Resistance Database , 20, whehttps://figshare.com/articles/PLOSONE_DR_GWAS_HIV/3569766). Access to the full genomes of HIV can be done by application of a proposal to PANGEA_HIV (http://www.pangea-hiv.org/).Summary statistics for all SNPs of each GWAS are available online Click here for additional data file.


China’s National Free Antiretroviral Treatment Program (NFATP) has substantially increased the survival rate since 2002. However, the emergence of HIV drug resistance (HIVDR) limits the durability and effectiveness of antiretroviral treatment (ART) in at risk patients.A cross-sectional survey was conducted among patients having received a median of 13.9 months of ART in eight provinces in China. Demographic and clinical information was collected, and venous blood was sampled for CD4 cell counts, measurement of the HIV viral load (VL), and HIV drug resistance (HIVDR) genotyping. Possible risk factors for HIVDR were analyzed by the logistic regression model.The study included 765 patients. Among them, 65 patients (8.5%) had virological failure (VLF) defined as ≥1,000 copies/ml. Among the individuals with VLF, 64 were successful genotyped, and of these, 33 had one or more HIVDR mutations. The prevalence of HIVDR mutations among patients receiving first-line ART was 4.3% (33/765). All of the patients with HIVDR mutations were resistant to non-nucleoside transcriptase inhibitors, 81.8% were resistant to nucleoside reverse transcriptase inhibitors, and only 3% had mutations that caused resistance to protease inhibitors. Having lower ratios of drug intake in the past month and dwelling in two southwestern provinces were factors independently associated with the emergence of HIVDR.Most patients receiving first-line ART treatment achieved sound virological and immunological outcomes. However, poor adherence is still a key problem, which has led to the high rate of HIVDR. It was notable that the proportion of drug resistance widely varied among the provinces. More studies are needed to focus on adherence. Since its development in 1996, combination antiretroviral therapy (cART) has significantly improved the quality of life of HIV-infected persons and dramatically lowered their morbidity and mortality . It has In China, the National Free Antiretroviral Treatment Program (NFATP) was begun in 2002, after a pilot study . As a puWe conducted a cross-sectional survey in eight provinces or cities of China: Beijing, Jilin, Hunan, Guangxi, Sichuan, Guizhou, Yunnan and Xinjiang Province. The survey protocol was taken from the WHO recommended cross-sectional survey on acquired HIVDR in adult patients receiving ART. Patients included were 18 years or older, had begun free ART treatment in 2013, and had received first-line ART for 9–18 months at enrollment. Eligible patients were enrolled at routine clinic visits in 2014. All participants provided written informed consent.Institutional review board approval was granted by National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention (China CDC).An interview-administered questionnaire  was usedhttp://hivdb.stanford.edu/pages/algs/sierra_sequence.html). HIV drug resistance mutations were defined as those conferring low-, intermediate, or high- level resistance [Blood specimens were collected after the interview. CD4+ T cells were quantified using flow cytometry at local CDCs within 12 hours. Plasma was isolated and sent under cold chain to the laboratory at NCAIDS, China CDC where the HIV viral load was measured. Viral suppression was defined as an HIV RNA level <1000 copies/ml. In samples with a viral load ≥1000 copies/ml, HIV drug resistance genotyping was performed at the NCAIDS laboratory by using an in-house method , 16. A dsistance , 18.All questionnaire data were double-entered using Epidata 3.1 . Statistical Analyses  were perThis survey included 765 patients having received ART for 9–18 months . The meaAmong the patients, the proportion of a CD4 count of 0–199, 200–349, and ≥350 cells/ul before ART were 45.4%, 37.4%, and 17.2%, respectively. After 13.9 months of treatment, the proportion of a CD4 count of ≥350 cells/ul was increased to 37.4%. The median CD4 count before ART increased from 222  cells/ul to 303  cells/ul at the time of the survey. The great majority of patients  had a plasma HIV viral load <1000 copies/ml. Among the individuals with VLF, 64 were successful genotyped, and of these, 33 had one or more HIVDR mutations. However, the proportion of viral load failure among patients from Sichuan and Guizhou were 27% and 24%, respectively, which was higher than patients from other provinces.Among the 33 patients identified with HIVDR mutations, all patients harbored HIV-1 strains resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs) . 81.8% oThe risk factors for HIVDR that were significant in the univariate logistic analysis were included in the multivariate logistic regression . AccordiIn this study, we analyzed HIVDR data in order to evaluate the prevalence and risk factors among 765 patients who were undergoing treatment. Our findings showed that 91.5% (700/765) achieved virological suppression (VL<1000 copies/ml) after 13.9 months of ART, which is better than the outcomes of previous surveillance studies in China –21. 33 pAlthough our study showed that China has met the WHO target for 90% of patients having their viral load suppressed, as HIV/AIDS patients live longer and are on ART for life, the number of patients with drug resistance is likely to increase. A combination of strategies is required to combat drug resistance. New medicines that can more robustly cope with drug resistance mutants are needed, especially for those with common mutations such as K103N and M184I/ V , 26, 27.Factors independently associated with the incidence of HIVDR were: the ratio of drug intake in the past month; and place of residence. The first factor reflected that adherence was a direct factor causing HIVDR, with 21(2.7%) patients having reported to have lower than 90% of drug intake in the past month. Similar findings had been reported in our previous studies in China , 27, 30,The reasons why patients from Sichuan and Guizhou had worse outcomes compared to patients from other provinces was unclear. We found, however, that the composition of patients from this population differed compared to patients from other provinces. Their differential risk for drug resistance may have been mediated through factors influencing adherence such as education, economic level, healthcare providers support, and adherence to ART. 78.2% of the Sichuan and Guizhou patients had received middle school education or lower compared with 69.5% in other provinces, 32.7% of patients in Sichuan and Guizhou were poor versus 22.3% s in other provinces, 59.1% of patients in Sichuan and Guizhou get support from healthcare providers compared with 89.9% in other provinces, and 14.5% in Sichuan and Guizhou had a poor adherence to ART compared with 7.8% in other provinces. There may, however, have been unmeasured confounding variables that led to this observation. Observations about drug resistance found on the population level, may not apply to individual patients. Future studies should explore health systems and individual level differences to better elucidate why resistance was greater in Sichuan and Guizhou provinces than other provinces.We also found that regimens with or without TDF showed the same results in causing HIVDR, which needs further investigation. TDF is preferred to its predecessors AZT and d4T in the ART program because of its better safety profile  which haThis study has some limitations. First, this was a cross-sectional study and patients who terminated treatment  would not have been sampled, which may have led to overestimated treatment effectiveness. Second, adherence was assessed by self-reporting of having missed doses in the past month, which may not reflect the true adherence. Finally, we found the drug resistance difference between Guizhou, Sichuan, and other provinces. Explaining the differences focused on the socioeconomic status of the inhabitants, but in fact transmitted drug resistance and possible presence of transmission clusters with HIV drug resistant variants may differ in different provinces which we do not know.In conclusion, a representative national sample of HIVDR surveillance across China demonstrated excellent virological and immunological outcomes at 9–18 months among patients receiving first-line ART treatment. However, poor adherence to treatment is still a key problem regardless of the efforts on the regimens, which has led to the high rate of HIVDR. Drug resistance widely varies among provinces. More research needs to focus on the adherence of patients and long-term studies monitoring drug resistance should be completed in some select cases.S1 File(DOC)Click here for additional data file.S1 Table(SAS7BDAT)Click here for additional data file.


T97A is an HIV-1 integrase polymorphism associated with integrase strand transfer inhibitor (INSTI) resistance. Using pooled data from 16 clinical studies, we investigated the prevalence of T97A (pre-existing and emergent) and its impact on INSTI susceptibility and treatment response in INSTI-naive patients who enrolled on elvitegravir (EVG)- or raltegravir (RAL)-based regimens. Prior to INSTI-based therapy, primary INSTI resistance-associated mutations (RAMs) were absent and T97A pre-existed infrequently ; most often among non-B (5.3%) than B (0.9%) HIV-1 subtypes. During INSTI-based therapy, few patients experienced virologic failure with emergent INSTI RAMs , among whom T97A emerged infrequently in the presence (n = 6) or absence (n = 8) of primary INSTI RAMs. A comparison between pre-existing and emergent T97A patient populations  showed no significant differences in EVG or RAL susceptibility in vitro. Furthermore, among all T97A-containing viruses tested, only 38–44% exhibited reduced susceptibility to EVG and/or RAL , while all maintained susceptibility to dolutegravir. Of the patients with pre-existing T97A, 17 had available clinical follow-up: 16 achieved virologic suppression and 1 maintained T97A and INSTI sensitivity without further resistance development. Overall, T97A is an infrequent integrase polymorphism that is enriched among non-B HIV-1 subtypes and can confer low-level reduced susceptibility to EVG and/or RAL. However, detection of T97A does not affect response to INSTI-based therapy with EVG or RAL. These results suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing T97A. Combination antiretroviral therapy has revolutionized HIV/AIDS management, reducing viral burden and HIV-related morbidity and mortality rates . However®)  or INSTI-treated patients were antiretroviral treatment-naive (TN) and enrolled on cobicistat-boosted (/COBI) EVG once a day as part of a fixed-dose combination with FTC/TDF (Stribild®) [Studies 236-0102/0103/0104/0118/0128 and 292-0104/0111] or FTC/TAF (Genvoya®) [Studies 292-0102/0104/0106/0111].Sequences of the entire integrase gene derived from HIV-infected patients screened and/or enrolled across multiple clinical studies  were ana™ Integrase or GenoSure™ Integrase (IN) and Phenosense™ Integrase assays, respectively. Primary INSTI RAMs against EVG and/or RAL were based on IAS-USA Guidelines of RAMs  that experienced confirmed viral rebound  at a first protocol-defined virologic failure visit (Week 40) on EVG/r+TDF+DRV maintained T97A and sensitivity to EVG without further development of antiretroviral resistance. This patient had pre-existing NNRTI (V90I) resistance, was noncompliant , and discontinued study drug 21 days later (HIV-1 RNA = 330 copies/mL). Overall, pre-existence of T97A at screening or baseline (with or without reduced INSTI susceptibility) does not appear to affect treatment response to INSTI-based therapy with two other fully active antiretrovirals.Among the 17 patients with pre-existing T97A and available clinical follow-up (17 of 18), 16 patients [15 TN patients on EVG/COBI/FTC/TDF and 1 TE patient on EVG/r+TDF+DRV] achieved and maintained high rates of virologic suppression  and 1 patient (described below) experienced virologic rebound . One patient [1 TE patient on EVG/r+TDF+DRV] did not achieve virologic suppression prior to early study drug discontinuation at a non-protocol-defined virologic failure visit (Week 8) and was excluded from treatment outcome analysis, consistent with previously reported study-specific efficacy analyses see  Fig 2; . The proAmong the 7 patients with emergent T97A alone  at virologic failure and available clinical follow-up (7 of 8), 1 patient [1 TN patient on EVG/COBI/FTC/TDF] maintained M184V in reverse transcriptase and T97A in integrase before regaining virologic suppression with no change in therapy  and 6 TE patients remained viremic  , 89. OneThe introduction of INSTI-based regimens for the treatment of HIV-1 infection has proven highly effective and well-tolerated in INSTI-naive patients with or without prior antiretroviral experience. Nevertheless, major/primary INSTI RAMs may develop at highly conserved integrase positions in patients experiencing virologic failure on INSTI-based therapy. Although all major/primary INSTI RAMs that significantly reduce INSTI susceptibility are treatment-selected mutations, the converse is not necessarily true . Many miTo date, most reports show that primary INSTI RAMs are not present prior to the initiation of INSTI therapy , 107–109In agreement with previous studies , 64, 110Because genetic barrier is an important determinant of drug resistance, genetic variability among patients with different HIV-1 subtypes may influence the ease with which an integrase mutation can occur before or during INSTI treatment –70, 113.Because HIV-1 explores sequence space under drug pressure by selecting resistance and compensatory mutations, adaptive evolution in treatment-experienced patients could also shape integrase variability before or during INSTI treatment. Indeed, prior antiretroviral exposure has been associated with greater integrase divergence, leading to the suggestion that treatment-induced changes in other parts of the HIV-1 genome may be linked to the prevalence of specific integrase polymorphisms , 81, 114An important tool in the prognosis of HIV-1 drug resistance and impact on treatment-outcome is the prediction of phenotypic INSTI sensitivity based on genotypic information using virtual software-based algorithms. However, there exist varying degrees of discordance in these interpretative algorithms of the level of INSTI sensitivity conferred by integrase mutation(s). While ANRS  and REGA™ Integrase assay, ranging from low-level to no effect at all. In contrast, a site-directed T97A mutant virus was previously shown to exhibit low-level reduced EVG susceptibility and no effect on RAL susceptibility [Our study provides useful information concerning the phenotypic INSTI sensitivity of viral isolates harboring T97A relative to treatment outcome. Differences in susceptibilities to EVG and/or RAL were noted among T97A-containing isolates relative to their biological cutoffs using the PhenoSensetibility . Becausetibility , 118, thtibility –88, howeIn the current study, the T97A+L74M combination was detected in 2 TN patients with pre-existing T97A+L74M and 2 TE patients with emergent T97A+L74M  Tables. Since T97A has been associated with INSTI resistance in patients experiencing virologic failure on EVG or RAL , 85–89, Overall, natural or transmitted INSTI resistance in INSTI-naive HIV-1 infected patients was extremely rare, confined to only a restricted few integrase polymorphisms. The T97A integrase mutation, infrequently associated with emergent EVG and RAL resistance at virologic failure, was identified as a low frequency naturally occurring integrase polymorphism of greater prevalence in HIV-1 non-B than B subtypes. While T97A may confer low-level reductions in INSTI susceptibility in the context of other integrase polymorphisms, mutational pathways of INSTI resistance at virologic failure were not driven by the persistence of T97A. More importantly, pre-treatment detection of T97A did not increase the risk of virologic failure on EVG or RAL-based therapies. Taken together, these analyses support the current clinical paradigm that INSTI-naive patients are likely to benefit from INSTI-based therapy without a concern of inherent reduced susceptibility or compromised virologic response based on natural integrase polymorphisms.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Pediatric uptake and outcomes in antiretroviral treatment (ART) programmes have lagged behind adult programmes. We describe outcomes from a population-based pediatric ART cohort in rural southern Malawi.Data were analyzed on children who initiated ART from October/2003 –September/2011. Demographics and diagnoses were described and survival analyses conducted to assess the impact of age, presenting features at enrolment, and drug selection.The cohort consisted of 2203 children <15 years of age. Age at entry was <1 year for 219 (10%), 1–1.9 years for 343 (16%), 2–4.9 years for 584 (27%), and 5–15 years for 1057 (48%) patients. Initial clinical diagnoses of tuberculosis and wasting were documented for 409 (19%) and 523 (24%) patients, respectively. Median follow-up time was 1.5 years (range 0–8 years), with 3900 patient-years of follow-up. Over the period of observation, 134 patients (6%) died, 1324 (60%) remained in the cohort, 345 (16%) transferred out, and 387 (18%) defaulted. Infants <1 year of age accounted for 19% of deaths, with a 2.7-fold adjusted mortality hazard ratio relative to 5–15 year olds; median time to death was also shorter for infants (60 days) than older children (108 days). Survival analysis demonstrated younger age at ART initiation, more advanced HIV stage, and presence of tuberculosis to each be associated with shorter survival time. Among children <5 years, severe wasting (weight-for-height z-score </ = -3.0) was also associated with reduced survival.Cumulative incidence of mortality was 5.2%, 7.1% and 7.7% after 1, 3, and 5 years, respectively, with disproportionate mortality in infants <1 year of age and those presenting with tuberculosis. These findings reinforce the urgent need for early diagnosis and treatment in this population, but also demonstrate that provision of pediatric care in a rural setting can yield outcomes comparable to more resourced urban settings of poor countries. Pediatric HIV remains a major challenge to the health of children globally. Many advances in pediatric HIV treatment and programming have been realized in recent years–better antiretroviral (ARV) formulations for children , universDignitas International has been present in Zomba District in southern Malawi since 2004, working collaboratively with the Ministry of Health (MOH) to expand capacity for ART program delivery. In an attempt to ensure sustainable strengthening of local health systems, ART provision has adhered to the successive iterations of Malawi’s national HIV treatment guidelines. ART was initially provided exclusively at Zomba Central Hospital, a tertiary level referral centre in Zomba municipality  ; d4T/3TC/efavirenz (EFV) [n = 55 (2.5%)]; or AZT/3TC/EFV [n = 8 (0.36%)]. Alternate regimens were used in fewer than 1% of cases.The median (range) duration of follow-up was 1.5 (0–8) years, with a cumulative total of 3900 patient-years of follow-up. Over the period of observation, 134 (6.1%) patients died, 1324 (60%) remained actively followed in the cohort, 345 (16%) transferred out of the program and were alive at the last point of contact, and 387 (18%) defaulted; outcome data was missing for 13 patients. The overall mortality rate was 3.4 deaths per 100 patient years (PYs) (95%CI 2.9–4.0). Of the 134 deaths, 105 (78%) occurred in the first year after initiation of ART, with a 12-month mortality rate of 6.6 deaths per 100 PYs (95%CI 5.5–7.9). The median time to death was shorter for infants <1 year at 60 days compared to 108 days for older children.Survival analysis (Kaplan-Meier) showed that younger age at ART initiation , p<0.001Results of the multivariable Cox proportional hazard analysis of the time to death are presented in Several alternative multivariable models were explored to assess the robustness of model estimates in a sensitivity analysis . The priCompeting risk modeling of the risks of death, transfer out of cohort, and defaulting was performed, and estimated cumulative incidence of these competing events are shown in This study presents outcomes from a large population-based cohort of children receiving care in resource-limited southern Malawi over an 8-year period. The use of routinely collected surveillance data from a national ART program should be considered a strength with respect to providing highly relevant results that can inform pediatric HIV policy in countries highly endemic for HIV infection. In our cohort, the cumulative probability of death was estimated to be 5.3% at 1 year after enrolment; similarly, the 12-month mortality rate was 6.6 deaths per 100 PYs of follow-up. These results are similar to other large pediatric cohort studies in SubSaharan Africa –24, but Our mortality incidence rate of 3.4 deaths per 100 PYs however was somewhat lower than Peacock-Villada’s overall rate of 8.0 and African country rate of 7.5 deaths per 100 PYs. This contrasts with other large cohort reports from east  and soutThe combination of these findings with the trend towards higher mortality we noted in children 2 to <5 years old provides soft support for the 2013 WHO recommendations for universal treatment of children <5 years. While firm evidence suggesting better health outcomes with empiric treatment in this older group compared with immunologic or clinical indications is currently lacking, operational advantages may favour this approach. Only 10% (250/2203) of our cohort had baseline CD4 testing prior to initiation of ART, reflecting the reality of attempting to provide ART in resource-limited settings and the need for pediatric assessment skill in determining clinical disease staging for children. While availability of flow cytometers for CD4 count and percentage measurement has improved steadily, particularly with the roll-out of point-of-care technology in recent years , the lagTuberculosis diagnosis at baseline was associated with lower survival in our cohort, similar to a previous study in Uganda , but difIt was interesting to note that there was no impact of year of entry into the cohort, as seen in other studies , as one Rates of patients defaulting, or being “lost to follow up” (LTFU) are increasingly recognized as important outcomes to understand in HIV cohort surveillance. Amongst patients categorized as defaultted or LTFU, a number of actual outcomes may be contributing, including death, ART discontinuation, registration at another ART site, or alternate acquisition of ART (eg. private purchase). We found that 7.1%, 18% and 31% of children in our cohort defaulted at 1, 3 and 5 years, respectively. These rates are higher than those seen in most other paediatric cohort studies from Africa) , 27, 33.This study has several limitations. First is the issue of incomplete data, such as only 10% of patients having CD4 measured at baseline and no documentation of CD4%. However, clinical outcome data was available for >99% of patients, which was the primary outcome of interest. Despite scale up of CD4 and viral load measurement capacity in HIV endemic countries, many programs will none the less still have these same limitations, and relying on clinical assessment parameters will remain a reality in such limited resource settings. We believe our results will reflect those realities and provide insights for such programs. The high rate of loss to follow up also leaves us with uncertainty regarding the true mortality rate in the cohort. As discussed above, this is a common problem that needs to be systematically addressed in scale up of pediatric ART programs in order to gain better understanding of the true outcomes of such programs.Another limitation is that despite the opportunity to comment on the impact of decentralization of pediatric care on HIV outcomes, we were unable to obtain details of decentralization with respect to pediatric HIV outcomes in our cohort due to limitations within our programmatic surveillance data. Decentralization may have impacted health outcomes, though it is not clear if this may have been positive or negative. Our group’s previous examination of outcomes of decentralized patients  showed lFinally, we verified the robustness of conclusions from our statistical models in numerous ways Click here for additional data file.