Using patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, whom have been sampled longitudinally for more than 7 years, genetic change was modeled against to the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interventions, AIDS defining illnesses, and other factors. Phylogenetic methods based on the sequences of the LTR and tat exon 1 of the HIV-1 proviral DNA quasispecies were used to obtain an estimate of an average mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/year (yr) prior to initiation of antiretroviral therapy (ART). Following ART the baseline mutation rate was reduced to an average of 1.02 nt/kb/yr. The post-ART baseline rate of genetic change, however, appears to be unique for each patient. These studies represent our initial steps in quantifying rates of genetic change among HIV-1 quasispecies using longitudinally sampled sequences from patients at different stages of disease both before and after initiation of combination ART. Notably, while long-term ART reduced the estimated mutation rates in the vast majority of patients studied, there was still measurable HIV-1 mutation even in patients with no detectable virus by standard quantitative assays. Determining the factors that affect HIV-1 mutation rates in the peripheral blood may lead to elucidation of the mechanisms associated with changes in HIV-1 disease severity.As a result of antiretroviral therapeutic strategies, human immunodeficiency virus type 1 (HIV-1) infection has become a long-term clinically manageable chronic disease for many infected individuals. However, despite this progress in therapeutic control, including undetectable viral loads and CD4 The introduction of combination therapeutics have extended the life expectancy of those infected with the human immunodeficiency virus type 1 (HIV-1) for many years past what would have been observed even a decade ago. Due to the mutable nature of the HIV-1 genome, longer life expectancy and associated selective pressures has led to increased opportunities for the emergence of viral genetic variants that escape therapy , contribOver the past several years, a great deal of attention has focused on the fate of the infecting viral inoculum and the cells that are initially targeted in the genital tract, regional lymph nodes, and peripheral blood during the early acute stages of infection. The general conclusions from studies of heterosexual transmission have indicated that infection is typically acquired from a single viral genotype (80% of cases), and the transmitted virion typically utilizes the CCR5 coreceptor (designated an R5 virus), which replicates well in CD4+ T-cell cultures but not monocyte-derived macrophage cultures . AlthougViral genetic variation over time in a patient is likely the result of at least three processes. The first process involves the introduction of novel mutations between the entry and integration steps of the viral life cycle, due to errors in the proof-reading mechanism of HIV-1 reverse transcriptase . These eThe second major modulator of viral genetic variation within a host centers on how variant sequences are affected by selective pressures resulting from immune responses, therapeutic interventions, drug abuse, and other environmental factors, which all affect the frequency of preexisting viral variants. In most cases, this leads to a predominant sequence comprising between 20% to 80% of the total number of viral quasispecies sequences along with potentially dozens of lower frequency variant sequences . Viral qThe third general source of viral genetic variation in the peripheral blood is based on the periodic escape or release of HIV-1 quasispecies from cellular and anatomical reservoirs harboring virus. Subsequent or continued replication of such previously compartmentalized viruses after transition to the peripheral blood allows for the seemingly hidden evolution of new quasispecies in the peripheral blood. During the acute infectious process when the virus achieves very high viral loads it seeds several anatomic reservoirs including the brain , lung 22, gut, aThe Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort in Philadelphia, PA is a unique resource to examine the interplay between these three biologic processes that impact the course of viral genetic variation in individual infected patients. More that 550 HIV-1-infected patients within the CARES Cohort have been longitudinally sampled with return visits scheduled approximately every six months. PCR-based Sanger sequencing has been utilized to sample the viral sequence from multiple genomic regions of integrated HIV-1 provirus populations, and this has provided for a detailed inspection of the sequential variation of the dominant proviral quasispecies across many HIV-1-infected patients –32.Modern computational methods are able to integrate multiple time-stamped sequences into a phylogenetic reconstruction of current and past divergence histories. Bayesian Estimation of Ancestors by Sampling Trees (BEAST) is a well-established tool used to estimate the divergence time of historic sequences, as well as lineage-specific evolutionary rates using a relaxed molecular clock assumption . It has tat exon 1 regions of 36 patients in the CARES Cohort were sampled over a six-year period and BEAST phylogenetic analyses were performed to determine how the mutation rates changed over time, and how clinical factors may affect mutation rates.In this study, the LTR and Patients enrolled in the Drexel Medicine CARES Cohort were recruited from the Partnership Comprehensive Care Practice of the Division of Infectious Disease and HIV Medicine in the Department of Medicine at Drexel University College of Medicine  and the Center for Clinical and Translational Medicine in the Drexel Institute for Molecular Medicine and Infectious Disease. Patients from this Cohort encompass a broad range of clinical severities; however, in general, the CARES Cohort consists primarily of African American males that have been infected for a median of 15.9 [15.6–16.7] years and who have been on therapy for a median of 13.5 [12.9–14.3] years . For theWhole blood was collected from each of the 36 patients, processed, and assessed as previously described . This prtat exon 1 regions were amplified from genomic DNA isolated from PBMCs with three different methods: (1) The “PCR Read” method which consisted of direct Sanger sequencing of a PCR product generated from genomic DNA; approximately 90% of the patient samples have a PCR Read. (2) The “Clone Read” consisted of subcloning to isolate individual fragments from PCR products with subsequent sequencing of the clones; 31 patients had at least 10 clones and an average of 16 clones per sample  the PCR Read exactly matched at least one of the Clone Reads. Also of particular interest is whether the PCR Read matched the most predominant unique Clone Read. In 23/31 cases the PCR Read matched the predominant Clone Read . While ttat exon 1. Due to the current sequencing progress in the Drexel Medicine CARES Cohort and the exclusion criteria discussed in Materials and Methods, this resulted in the construction of 84 trees for the LTR, 12 trees for tat exon 1, and 6 trees for tat exon 2. BEAST trees that disagreed with standard phylogenetic trees generated using RAxML were then excluded. This resulted in a high confidence set of 36 LTR trees and 8 tat exon 1 trees , as indicated by narrower branches and a lower aggregated mutation rate.The BEAST trees shown in Interestingly, To quantify the effect of ART therapy on the HIV-1 mutation rate, 36 patients were further analyzed. This subset of patients was selected such that more than one sample had been collected from the patient prior to the initiation of combination ART (PreART) and at least one sample had been collected, in which they had been on combination ART with undetectable viral loads. The samples from each patient were subdivided into the two groups. Specifically; the PreART Group refers to samples collected before the introduction of ART. The Suppressed ART Group refers to samples collected after the virus has been reduced to undetectable levels until therapy was changed or the latest sampled time point. As expected, the 36 patients have spent different lengths of time on combination ART and have been well-suppressed for different lengths of time. In order to synchronize the time series across multiple patients, the time points were synchronized using clinical markers. The PreART Group time points were measured from the reported HIV infection date  while thtat exon 1). The random effects were assumed to be an independent intercept unique to each patient. This assumes that the virus in each patient has a unique basal mutation rate that is an aggregate of all unmeasured variables affecting viral mutation in that patient, thus accounting for repeated measure issues inherent with longitudinal data [To directly test the hypothesis that combination ART has a significant effect on the mutation rate, a mixed effects statistical model was built such that the mutation rate was due to three fixed effects: years seropositive, ART status, and genetic region sampled . Age showed a mild correlation with mutation rate (p = 0.074); however, this may be due to its covariance with Years Seropositive; more data is needed to resolve this difference. Current viral loads (those within the detectable range), CD4+ T-cell, and CD8+ T-cell counts also did not correlate with the mutation rate (p>0.1).Numerous other clinical parameters contained within the Drexel Medicine CARES Cohort database were examined with respect to their correlation with time-varying mutation rate but no associations with a statistically significant effect were identified. Peak viral load and nadir CD4The results, taken as a whole, indicate that the mutation rate was lowered by ART therapy in this dataset but that the overall effect is small and mutations remain ongoing even in the absence of detectable viral load for prolonged periods of time. Using this population of 36 patients sampled longitudinally for six years it is estimated that the effect of therapy on the mutation rate of HIV represents a 20% decrease. Notably, despite undetectable levels of viral loads the mutation rate has never been estimated to be zero. These results show that, even in the absence of detectable viral load, new genetic variation continues to arise in the proviral DNA population. Importantly, the analysis performed treated distinct lineages, or quasispecies, of provirus individually since lineage-specific mutation rates were estimated, therefore reactivation of latent reservoirs and patients infected by multiple distinct genotypes are not confounding the results.We have hypothesized that the genetic variation over time within the integrated HIV-1 proviral genome in the infected cells derived from the peripheral blood represents (1) a combination of sequential mutations within quasispecies, (2) changes in the relative frequencies of distinct quasispecies, and (3) the reemergence of compartmentalized genomes leaking from viral reservoirs including the brain, gastrointestinal tract, and perhaps other tissues that may include the bone marrow. Longitudinal analysis of HIV-1 proviral sequences derived from patients in the Drexel Medicine CARES Cohort is beginning to quantify the relative contribution of each of these forces. In the present analysis, we have demonstrated that it is possible to construct date-tipped phylogenies and estimate changes in mutation rates in response to clinical factors/events.Of the three potential sources of variation, this study focused on lineage-specific mutation rates and how these changed over time to ART. The sampling of Clone Reads performed on the 31 samples was insufficient to capture low frequency variants, usually defined as those below 1% . This maExamining the release of virus from a “leaky” viral reservoir will require additional experimental approaches in addition to a greater depth of sequence coverage. For example, fluorescence-activated cell sorting can be used to separate cells to specifically examine the CD4+ memory T-cell compartment, a likely cellular reservoir , 25. Exatat exon 1 has a higher observed mutation rate (0.636 nt/kb/yr) when compared to the LTR (0.527 nt/kb/yr). The LTR and tat differ in that tat is expressed as a protein where the LTR is not. Because of this Tat is processed as an antigen which is recognized by the immune system. This leads to an additional selective pressure on Tat with respect to immune evasion [The variable rate of sequence evolution between the differing regions of the HIV-1 genome is something that has been explored previously , 32. Thi evasion  that doeThe mutation rates calculated above conform to previous estimates of HIV-1 mutation rates as observed by Josefsson and colleagues . They fo+/CD8+ cell counts, current and peak viral loads, and age) did not have a significant impact on the result. The viral load was weakly correlated with the mutation rate but only when using a threshold at the level of detection and combined with ART status. The PreART Group was examined with respect to direct correlations between viral load and mutation rate but with no success. This could be due to numerous factors that include the fact that (1) increased viral loads may occur from a clonal expansion of a small number of infected cells leading to the development of a population of identical viral genomes leading to less overall variation [Attempts to account for these data using other clinical variables ariation ; (3) theAfter initiation of ART, the mutation rate converges to an apparently patient-specific rate that stays constant over numerous years, after therapy has reduced viral load to undetectable levels. This rate was estimated to be 3.69 +/- 2.88 mutations/kb/year. It is currently unclear what parameters affect this measure, but it is likely that a combination of the specific ART regimen of each patient, patient adherence to therapy, and viral factors such as the initial viral set point all play a role. In this dataset there is no correlation between the baseline rate of genetic change and typical clinical markers such as Nadir CD4, peak viral load, or years on therapy (p>0.1). It was not possible to test the effect of specific ART regimens due to the breadth of the therapeutic approaches used in this relatively small cohort. As such, a larger dataset will be required to associate the patient-specific baseline rate of genetic change with clinical parameters such as therapeutic protocol, viral set point, as well as others.This study is limited by the assumptions inherent in both the phylogenetic framework as well as the linear modeling. The BEAST analysis assumes that the tree is rooted at a specific time and that all observed variation results from mutations to that common founder virus. While this is likely to be true in most cases , 11, indAssumptions surrounding the linear modeling may also lead to potential misinterpretations of the results. Both ART suppression and increased length of infection are correlated with a decreased mutation rate. These results may lead one to hypothesize that after enough time on therapy the mutation rate would eventually reach zero. This mirrors previous hypotheses that sufficient therapeutic control would eventually lead to viral clearance , 48. HowWith the improvement of therapeutic interventions, HIV-1 patients are living longer. As such it is important to understand the factors that affect the viral mutation rate including (1) relative frequencies abundances of quasispecies, (2) reemergence of historical sequences, and (3) the nature of the reservoirs that harbor defective and fully functional proviruses as this will be critical to treating patients in the long-term. The results reported herein indicate that even under complete therapeutic elimination of detectable viral replication, mutations within the integrated proviral DNA still occur, albeit at a lower rate. It is likely that this is a combination of active replication in areas of poor therapeutic penetrance, such as the brain, gut, or other tissues, and undetectable levels of replication in the PBMC compartment. Deep sequencing experiments on longitudinally collected samples will allow researchers to sample all quasispecies at each time-point along with relative frequencies. When combined with rich clinical information, the factors influencing each mode of variation will be examined in even greater detail.The Drexel University College of Medicine Institutional Review Board (IRB) has approved this work under protocol 1201000748 . All patient samples were collected under the auspices of protocol 1201000748 through written consent.


Challenges to scaling up treatment as prevention (TasP) of HIV transmission are considerable in the developing-world context and include accessing at-risk populations, human resource shortages, adherence and retention in care, access to newer treatments, measurement of treatment effects, and long-term sustainable funding. Optimism about ending AIDS needs to be tempered by the realities of the logistic challenges of strengthening health systems in countries most affected and by balancing TasP with overall combination prevention approaches. Challenges to scaling up treatment as prevention (TasP) of HIV transmission are considerable in the developing-world context and include accessing at-risk populations, human resource shortages, adherence and retention in care, access to newer treatments, measurement of treatment effects, and long-term sustainable funding. Optimism about ending AIDS needs to be tempered by the realities of the logistic challenges of strengthening health systems in countries most affected and by balancing TasP with overall combination prevention approaches. The 2011 results of the HIV Prevention Trials Network 052 randomized clinical trial (RCT)There is broad consensus that prevention strategies need to involve a combination of proven prevention interventions.Mathematical models may be useful for raising policy implications, but they are highly susceptible to the assumptions that inform them. The heterogeneity of estimates of TasP benefits weakens their inferences severely.As early as 1991, mathematical projections highlighted the potential for TasP using only the antiretroviral drug zidovudine.The trial was conducted among serodiscordant couples (one partner had HIV infection and the other did not) in a well-monitored RCT environment. However, the randomized portion of the trial was stopped early due to treatment benefits,In light of this uncertain evidence, there are a number of important challenges to scaling up TasP, categorized into 6 distinct areas:Early provision of ART reduces transmission of HIV infection—by as much as 96% in randomized trials. But benefits in the real world are probably markedly lower.Prioritization of patient and population groupsHuman resources and health systemsAcceptance, adherence, and retention of patientsImproved access to more effective therapiesTools to measure the effect of TasPFinancial resources to cover new costsMany of these issues were put forward a decade ago as challenges to scaling up ART in under-resourced settings.The greatest public health benefits in targeted prevention come from stopping transmissions among those who are most likely to infect others. One approach to maximizing the benefits of TasP should be to address specific groups of individuals with HIV infection, such as sex workers, injecting drug users, men who have sex with men (MSM), and individuals with multiple sex partners. There is an expectation that targeting screening and treatment (test and treat) to specific groups of people who are at high risk of transmitting the virus—and thus reducing their viremia—may result in the greatest reduction in new infections, although this has yet to be evaluated in any research setting.While there is clearly a need to better engage these key affected populations in TasP programs, the decision to prioritize screening and treatment based on certain risk behaviors rather than on medical need continues to be a subject of debate.Other strategies to engage infected individuals early besides point-of-care testing include self-testing and home-based testing visits. A recent evaluation of house-to-house testing by health workers in Kenya found high levels of acceptance and that people accepting home testing had a median CD4 count of 323 cells/µL compared with a median CD4 count of 217 among those visiting voluntary testing centers.With increasing numbers of individuals screened and treated, health systems will need larger numbers of trained health care workers capable of testing, initiating treatment, monitoring adherence and retention, and engaging patients in long-term social support groups. In countries with HIV burden, health service constraints to expanding ART access are considerable. For example, in settings such as Swaziland  and Malawi , there are fewer than 0.05 physicians per 1,000 people and fewer than 0.2 nurses per 1,000 people.The simplification of screening, treatment, and monitoring of patients has allowed for task shifting from doctors to less specialized health staff. Despite numerous observational studies and several randomized trials validating this approach, task shifting for HIV treatment and care is still not universally accepted.Increasing numbers of patients engaged in treatment will also require that health systems adapt from facility-based care to community-based care and delivery. Several important examples of this model exist in Kenya, Mozambique, and Uganda.TasP provides an additional compelling argument for improving access to viral load monitoring as CD4 is a weak surrogate marker of viral suppression. Currently, due to costs, viral load monitoring is rarely provided in the lowest-resource settings. Advocates of viral load monitoring over CD4 monitoring argue that viremia is a better predictor of treatment failure than CD4 monitoring and that a renewed emphasis on decreasing costs of care should focus on making viral monitoring available in resource-limited settings.Viral load monitoring may be a better predictor of ART failure than CD4 monitoring.Linking patients with a positive HIV test to effective and uninterrupted ART represents an important challenge. Major attrition of patients occurs between each stage of the leaky treatment cascade—from diagnosis and assessment of ART readiness to receipt of initial ART and long-term retention in care .24Just because patients have been prescribed ART does not mean they are willing to begin lifelong treatment. A recent study from Kenya found that almost 40% of serodiscordant couples were unwilling to use early treatment for its preventive effects.Systematic reviews show that, on average, adherence among people with HIV infection that do start ART is below 80%,Reducing viremia effectively requires adequate and sustained adherence, but approaches to improve adherence remain poorly defined. Community-based organizations have used social groups, involved partners, and sent mobile phone text messages in efforts to retain patients along the cascade of care.Treating greater numbers of asymptomatic patients will require renewed efforts to improve adherence, including identifying adherence-support models for people who have never experienced an illness episode.Early treatment of HIV patients who may not have symptoms yet requires enhanced efforts to improve adherence.Although there have been recommendations to shift away from using drugs associated with severe adverse events, such as stavudine, toward better-tolerated tenofovir-based formulations, stavudine is still commonly prescribed in many resource-limited settings.Measuring whether TasP has a marked decrease on new infections in communities represents an important challenge to scale up.Incidence monitoring is challenging in many settings in Africa because few incidence cohorts exist. Also, many populations at high risk of infection, such as migrant workers, have high rates of mobility and may not be captured in incidence surveys.Additionally, determining the specific preventive contribution of treatment will be difficult in an environment where a range of positive prevention interventions, such as condom and clean-needle use, male circumcision, and decreasing the number of partners, are used by different populations to different extents. In 2 pragmatic, controlled cohorts of serodiscordant couples (in China and Uganda) where the index partner in the intervention group received ART, there were no differences in the rates of infection between groups.Implementing treatment as a prevention strategy will have financial costs associated with it in terms of drugs, human resources, laboratory monitoring, and evaluation. In the current global economic climate, where this money will come from is a mystery.In more developed settings, such as British Columbia, Canada, financial resources for TasP are obtained by moving resources from a previous allocation to the seek-and-treat program.Most organizations in Africa have a set allocation of patients who are permitted to begin treatment within defined funding periods, and they are not permitted to initiate ART for patients who do not meet their organization's initiation criteria. Engaging a large number of new patients with higher CD4 status than current eligibility criteria will require permission from funders. Yet neither PEPFAR nor the Global Fund has announced the allocation of significant new funding for TasP.Whether TasP is a cost-effective strategy compared with other preventive approaches in HIV-endemic settings has been controversial. A model evaluating the cost-effectiveness of TasP compared with medical male circumcision found that a focus on circumcision had greater cost-effectiveness than TasP alone.Important programmatic challenges have hindered scale up of ART for the purpose of treatment alone.While treatment as prevention holds promise, scale-up strategies need to be mindful of programmatic challenges.TasP has given much-needed impetus to the HIV-prevention and treatment agenda at a time when political support was waning. It has also focused attention on the health system challenges to enrolling and retaining more people with HIV infection, earlier in care.Other interventions, such as PrEP, prevention of mother-to-child transmission (PMTCT), condom and clean-needle use, and medical male circumcision, are also important strategies to prevent infections. All these interventions face challenges in terms of timely uptake by individuals at risk, health service capacity, and, in the case of PrEP and PMTCT, adherence to treatment.The appropriate strategic combination of these different biomedical prevention interventions will differ according to the epidemiologic, economic, and cultural realities of different settings.


The increasing number of people requiring HIV treatment in South Africa calls for efficient use of its human resources for health in order to ensure optimum treatment coverage and outcomes. This paper describes an innovative public-private partnership model which uses private sector doctors to treat public sector patients and ascertains the model’s ability to maintain treatment outcomes over time.The study used a retrospective design based on the electronic records of patients who were down-referred from government hospitals to selected private general medical practitioners (GPs) between November 2005 and October 2012. In total, 2535 unique patient records from 40 GPs were reviewed. The survival functions for mortality and attrition were calculated. Cumulative incidence of mortality for different time cohorts (defined by year of treatment initiation) was also established.3, this proportion reduced to 6.6% at 12 months and 4.1% at 48 months. Similarly, 88.4% of the patients had suppressed viral load (defined as HIV-1 RNA <400 copies/ml) at 48 months. The patients’ probability of survival at 12 and 48 months was 99.0% (95% CI: 98.4%–99.3%) and 89.0% (95% CI: 87.1%–90.0%) respectively. Patient retention at 48 months remained high at 94.3% (95% CI: 93.0%–95.7%).The median number of patients per GP was 143 (IQR: 66–246). At the time of down-referral to private GPs, 13.8% of the patients had CD4 count <200 cell/mmThe study findings demonstrate the ability of the GPs to effectively maintain patient treatment outcomes and potentially contribute to HIV treatment scale-up with the relevant support mechanism. The model demonstrates how an assisted private sector based programme can be effectively and efficiently used to either target specific health concerns, key populations or serve as a stop-gap measure to meet urgent health needs. With advances in antiretroviral treatment and improved survival of people living with HIV, the South African health system has to contend with an unprecedented demand for HIV/AIDS-related health care resources. Preliminary reports of the South African household HIV prevalence survey already suggest an almost 2% increase in prevalence between 2010 and 2012 In spite of the above, there has been an astronomical increase in the number of people living with HIV who are on treatment in South Africa Against this background, this paper describes the outcomes of an innovative public-private-partnership (PPP) model in an attempt to ascertain its ability to maintain patient treatment outcomes and the model’s success factors that can be harnessed for future programmes. The model – referred to as the private general medical practitioner (GP) model – assumes the treatment of stable patients who were originally initiated on treatment at public health facilities.The GP model was developed at the request of the North West Provincial Department of Health. The GP model is one of the three models used by BroadReach Healthcare (BRHC) – a private healthcare solutions company – to provide HIV treatment through selected private GPs. The GP model was designed to be sustainable with minimal external financial support. The costs supported by BroadReach Healthcare included the start-up costs of setting up a staffed office within selected public sector wellness clinics, patient data management, adherence support and a negotiated consultation fee for the private GPs. The Department of Health provided ART medication from their district pharmacy, and laboratory services were rendered by National Health Laboratory Services which is a government parastatal. Eligible patients were down-referred to a select group of GPs in the communities where the patients live. The GPs were selected based on their prior training and experience in HIV/AIDS treatment. Patients were considered eligible for down referral if they had been stable on ART for at least 3–6 months with a suppressed viral load, did not have private medical insurance, did not have concurrent illnesses (excluding TB) and voluntarily consented to participate in the programme. The patients were then cared for by the private GPs as an alternative to public sector health service outlets. The patients were initially referred back to the public sector for the management of acute co-morbidities and complications. This was changed after 2012 to allow the GPs to manage co-morbidities. The adherence and treatment support services provided by BroadReach Healthcare were in the form of patient education sessions and adherence/wellness training, which patients attended upon their down-referral and on an annual basis. Other adherence support services provided by BroadReach Healthcare included the use of clinic-based adherence supporters; home visits by adherence supporters and telephonic follow up.A key component of the model was its patient information management system, referred to as the Disease Management System (DMS). BroadReach Healthcare’s DMS is managed by Aid for AIDS (AfA) which is a private data company that is funded through donor funds. The DMS promotes efficient management of complex patient information; it is available both online and offline (for remote use); and provides real-time patient information such as treatment regimen and pick-up history, laboratory information, doctors’ consultation notes and adherence support data. The DMS is also automated to send important phone text messages, to remind patients, doctors, pharmacists and laboratory personnel of treatment activities. The reminders included scheduled treatment pick-up and laboratory test dates, details of defaulting patients, undesirable laboratory results and uncollected treatments. Some of these innovations were not part of the routine public sector treatment programme. This study was based on a retrospective design and used the electronic records of patients enrolled in the GP model. Patients included in this analysis are cared for by 40 GPs. The database used for the study was developed for patient management but contained the minimum scope of variables needed for this study’s objectives. The data elements taken from the database for this study were age, sex, treatment initiation dates, treatment regimen, results of routine clinical tests and respective test dates of the patients. Patients younger than 18 years were rarely recruited into the GP model and were thus excluded from the analysis. Outcome variables considered were viral load, CD4 cell counts and patient retention. The treatment outcomes were categorized, for example, viral load status was defined as either suppressed or unsuppressed using 400 copies/ml as the status threshold for suppression. Methods of CD4 count and viral load testing have been described The study is based on data that is collected and stored on DMS . The database used for this study had no individual patient identifier and the analysis was completely anonymous. Patients in the GP model also signed a consent for their information to be entered into the central database and used for routine decisions on patient and programme management and were made aware that any further analyses will exclude patient identifiers.3 (IQR 248–476). The database used for this analysis included 2535 longitudinal patient records from their respective time of down-referral to November 2012. The first group of patients were down-referred into the partnership model in November 2005. The patients’ duration in the care of the GPs ranged from 2–84 months with a 28 months median. The median number of patients each GP ever managed in the model was 143 (IQR: 66–246). The median age of the patients was 40 years old (IQR 34–46). About two-thirds (68.4%) of the patients were female. The median CD4 cell count at baseline was 342 cells/mmThe GP model showed similar treatment outcome when the probability of survival of cohorts initiated to treatment at different times for a minimum of 60 months (five years) using logrank test (p>0.05) was compared. The survival functions at six (6) and 48 months are 99.6% (95% CI: 99.3%–99.9%) and 89.0% (95% CI: 87.1%–90.7%) respectively. The retention function at six (6) and 48 months are 100% and 94.5% (95% CI: 93.0%–95.7%) respectively. Survival and retention rates at 6, 12, 24 and 48 months are also shown in The study findings demonstrate the ability of the private sector to successfully absorb and retain public sector patients over time. The GP model therefore presents an opportunity to significantly reduce the growing burden of HIV/AIDS care on the public sector. Leveraging more GPs for public sector patients or marginally decreasing doctor-patient ratio could further improve treatment coverage, decongest the public sector and obtain good patient retention and treatment outcomes 3 and 500 cells/mm3Unlike most HIV/AIDS programmes, this model only caters for patients who have been stabilized through public sector programmes. This makes it difficult to compare its outcomes with the outcomes of other programmes that enrol all patients irrespective of their viral load. Whilst a direct comparison may be spurious, patient progression noted after down-referral into the GP model is similar to the progression among patients initiated to treatment at CD4 cell counts of between 350 cells/mmThe successes reported in this study may also be attributed to the combination of established strengths of both public and private sector HIV/AIDS programmes. The GP model on the one hand harnesses public sector experience and regulations, and on the other leverages the private sector human resources and infrastructure to ensure patient retention and decongestion of public sector facilities. With this, the GP model proposes a complementary model that harnesses the arguments of both the proponents of universal state-based health care and the advocates of using the private sector to provide health services in areas where the public sector does not have capacity The private sector in general is often criticized for its high cost and inefficiencies in terms of unnecessary diagnostic tests and treatment options The limitation of this study was its exclusion of patients transferred-out of the GP programme for a variety of reasons. Following-up patients who have been transferred-out of a programme remains a major problem for many treatment programmes In conclusion, the GP model demonstrates the ability of an assisted private sector treatment programme to retain patients and maintain immunologic and virologic outcomes. This model offers lessons to guide strategic and operational decisions, specifically on how to use private sector resources to target defined health concerns or key populations or serve as a stop-gap measure to meet urgent health needs.


Tenofovir-based first-line antiretroviral therapy (ART) is recommended globally. To evaluate the impact of its incorporation into the World Health Organization (WHO) guidelines, we examined treatment failure and drug resistance among a cohort of patients on tenofovir-based first-line ART at the Academic Model Providing Access to Healthcare, a large HIV treatment programme in western Kenya.We determined viral load (VL), drug resistance and their correlates in patients on ≥six months of tenofovir-based first-line ART. Based on enrolled patients’ characteristics, we described these measures in those with (prior ART group) and without (tenofovir-only group) prior non-tenofovir-based first-line ART using Wilcoxon rank sum and Fisher's exact tests.Among 333 participants , detectable (>40 copies/mL) VL was found in 18%, and VL>1000 copies/mL (WHO threshold) in 10%. Virologic failure at both thresholds was significantly higher in 217 participants in the tenofovir-only group compared with 116 in the prior ART group using both cut-offs . Failure in the tenofovir-only group was associated with lower CD4 values and advanced WHO stage. In 35 available genotypes from 51 participants in the tenofovir-only group with VL>40 copies/mL (69% subtype A), any resistance was found in 89% and dual-class resistance in 83%. Tenofovir signature mutation K65R occurred in 71% (17/24) of the patients infected with subtype A. Patients with K65R had significantly lower CD4 values, higher WHO stage and more resistance mutations.In this Kenyan cohort, tenofovir-based first-line ART resulted in good (90%) virologic suppression including high suppression (99%) after switch from non-tenofovir-based ART. Lower virologic suppression (85%) and high observed resistance levels (89%) in the tenofovir-only group impact future treatment options, support recommendations for widespread VL monitoring in such resource limited settings to identify early treatment failure and suggest consideration of individualized resistance testing to design effective subsequent regimens. In 2014, 10.7 million people in sub-Saharan Africa received antiretroviral therapy (ART), an effort that reduced the number of AIDS-related deaths in the region by 48% .Current World Health Organization (WHO)-recommended first-line ART includes tenofovir (TDF), efavirenz (EFV) and XTC (lamivudine=3TC or emtricitabine=FTC), with zidovudine (AZT) and nevirapine (NVP) as alternatives for TDF and EFV, respectively . TDF hasIn Kenya, where adult HIV prevalence is 6.1%, 63% of medically-eligible patients receive ART and infection is diverse with predominant subtypes A (50–80%), D (10–20%) and C (5–15%) ,22. Few The Academic Model Providing Access to Healthcare (AMPATH) is a major HIV programme in western Kenya, treating >140,000 patients . As recoThis cross-sectional study was conducted at Moi Teaching and Referral Hospital (MTRH) clinic, AMPATH's largest, managing 26,791 adults . HIV-posCD4  and VL  testing were done at the Good Clinical Laboratory Practice (GCLP) compliant AMPATH reference laboratory, which participates in the United Kingdom National External Quality Assessment Service and Rush University Viral Quality Assurance programmes and is accredited for ISO 15189 by Kenya Accreditation Services. Confirmatory VL results after adherence counselling, as recommended by WHO guidelines, were not available.Genotyping was performed on all patients with detectable (>40 copies/mL) VL. Frozen plasma was shipped to the United States for genotyping. RNA was extracted from 200 µL of plasma via Biomerieux's MiniMAG . To increase assay sensitivity of samples with VL <1000 copies/mL, RNA was extracted from 400 uL plasma. An in-house genotyping assay was used to generate a 1300-bp fragment including the RT gene. Briefly, a nested RT-PCR was performed using SuperScript III One-Step RT-PCR , followed by second-round PCR using Platinum Taq DNA Polymerase High Fidelity (Life Technologies) per manufacturer's instructions. Cycling parameters for RT-PCR were 45°C for 45 minutes, 95°C for 2 minutes, followed by 40 cycles of 94°C for 15 seconds, 55°C for 20 seconds and 72°C for 2 minutes, followed by a 10-minute hold at 72°C. Cycling parameters for PCR were 35 cycles of 94°C for 15 seconds, 55°C for 20 seconds and 72°C for 2 minutes, followed by a 10-minute hold at 72°C. PCR products were Sanger sequenced at the Rhode Island Genomics and Sequencing Center and assembled with Sequencher v4.10.1 . SequencThe primary objective of the study was to evaluate virologic failure and drug resistance rates among patients on TDF-containing first-line ART. As described below, we observed during recruitment that participants meeting enrolment criteria fit into one of two distinct groups: those who began ART on a TDF-containing regimen (tenofovir-only group) and those who switched to TDF from a prior non-TDF first-line regimen . Analyses were therefore designed to (1) describe virologic failure and resistance in each group, (2) explore predictors of failure and resistance in the two groups as the data and sample sizes allow, and (3) examine HIV-1 subtype and associated potential genotypic pathways and mutation correlation for the development of resistance . We do not attempt a thorough comparison of the two groups because they differ substantially by key variables such as time on first-line ART and CD4 count at TDF initiation, and consequently standard adjustments for confounding are not feasible see .Two definitions for treatment failure were considered: VL>40 copies/mL (assay detection limit) and VL>1000 copies/mL (WHO threshold). Genotyping quality control and distance calculations were performed with SQUAT . ResistaTo evaluate potential impacts of subtype-specific codon usage on K65R selection, we compared nucleic acids at codons 64 to 66 in subtypes A, B, C and D sequences from ART-naïve or TDF-treated patients, derived from: (1) Stanford database (33) and (2) AMPATH (prior and current studies) .n=10), missing clinical data (n=6), poor adherence (n=2); and one declined participation.Inclusion criteria were met by 352 patients over the recruitment period who were approached for possible enrolment. Eighteen patients were excluded because of ineligible medication history (p=0.014), had lower median CD4 values , were on overall ART and TDF-based ART for substantially less time (p≤0.0001) and were less likely to be on NVP-based regimens . Most patients in the prior ART group had documented side effects upon switch to TDF-containing regimens, including lipodystrophy (70%), anaemia (9%), neuropathy (6%) and both anaemia and neuropathy (2%); (reason unavailable for 13%).Of 333 participants, 217 had no ART exposure before TDF-based regimens (TDF-only group), and 116 had prior non-TDF-based first-line ART exposure . Gender, adherence, WHO staging and TB history were similar between groups. Compared to the prior ART group, TDF-only group patients were younger  had detectable VLs and 34 (10%) had VL>1000 copies/mL . The medPatients in the TDF-only group failed TDF-based therapy at higher rates than the prior ART group by both VL thresholds . Treatment failure in either group was not significantly associated with age, gender, adherence, OIs, specific non-NRTI (NNRTI) exposure or time on ART, which could be due to small sample sizes.p=0.029) and lower CD4 values . With only eight failures in the prior ART group, ascertaining correlates of failure was difficult; however, failure was marginally associated with seven-day ART adherence .Among the TDF-only group, participants failing treatment were more likely to have advanced WHO stage , showed little to no overlap .Sequences were available from 35 of 51 patients with detectable VL from the TDF-only group and two of eight patients from the prior ART group. The 22 patients (16 from the TDF-only group and six from the prior ART group) whose genotypes were not available were treated for a median of 21 months (IQR 17–41) and had a median VL of 111 copies/mL . In univariate analysis, successful genotyping was significantly associated with higher VL. Due to the few sequences from the prior ART group, genotypic comparisons with the TDF-only group were not performed. Detailed resistance results for the TDF-only group are provided in RT resistance was detected in 89% (31/35) of patients, 89% (31/35) to NNRTIs, 83% (29/35) to NRTIs and 83% (29/35) to both. Of the 31 patients with any resistance mutation, 1/31 (3%) had one-class and 34/35 (97%) had dual-class resistance. Overall 155 mutations were detected , with a median of five mutations per patient (range 0–9), two NRTI (range 0–5) and two NNRTI-associated (range 0–4). Of five patients with VL<1000, two had no resistance, two had one NNRTI-associated mutation and one had multiple NRTI-associated mutations.p=0.223). Compared to those without K65R, those with this mutation had lower median CD4 values , more advanced WHO stage  and more RT mutations . There was no significant association between K65R development and specific NNRTIs , age, subtype, or time on treatment. Only 4/24 patients with K65R had any IAS-USA-defined thymidine analogue mutations (TAMs) [p=0.002); G190A/S ; and G190A/Y181C co-occurrence .K65R developed in 69% 24/35) of patients : 17/24 ( of patieOther common (>20%) RT mutations were NRTI-associated M184V/I  and NNRTI-associated Y181C/G/Y , G190A/S  and K103N/S . TDF-associated K70E was found in 3/35 (9%) patients, none with TAMs. Examination of specific mutations by NNRTI backbone revealed overall similarity . CompareIntermediate-high resistance to first-line ART drugs was seen in 89% of the patients (31/35), including 83% (29/35) to 3TC and FTC, 71% (25/35) to TDF, 86% (31/35) to EFV and 89% (31/35) to NVP . IntermeAvailable sequence data at RT positions 64–66 were compiled from 45,794 patients from the Stanford Database ; and 78 AMPATH patients  . The mosTo our knowledge, this is the largest report of HIV-positive Kenyans failing first-line TDF-based ART, highlighting three main findings. First, defining virologic failure as VL>1000 copies/mL, 99% of those switching to TDF and 85% of those starting on TDF-based first-line ART had virologic suppression after a median of 25 months, demonstrating good virologic response among 333 AMPATH adults infected mostly with HIV-1 subtypes A, C and D. Second, switch from non-TDF- to TDF-based first-line therapy was successful and associated with low treatment failure. Third, among failing patients, high levels of resistance were observed across subtypes, with high K65R rates posing particular concern. Our results demonstrate public health consequences of the implementation of first-line TDF-containing regimens in RLS and support switching to TDF-based regimens from other first-line medications. These data highlight the importance of virologic monitoring to avoid resistance accumulation upon treatment failure and suggest consideration of resistance testing for individual care in RLS.The proportions of study participants failing TDF-containing ART (18% detectable VL and 10% VL>1000) are within the range of published data, mostly from US and European clinical trials . The lowThis study provides new data on TDF use in diverse subtypes. The high overall 89%) and dual-class (83%) resistance is consistent with other reports from AMPATH ,26 and o% and duaK65R is a TDF signature mutation conferring resistance to all NRTIs but AZT, is rarely transmitted , is lessSeveral considerations are relevant to the demonstrated increased K65R levels. First, our data support the reported association between K65R, Y181C and G190A, suggesting synergistic fitness effects, and extend it to NVP (not only EFV) treatment . Second,Our results therefore support implementation of routine VL monitoring for earlier identification of treatment failure and consideration of individual resistance testing to optimize patient care. The latter may be particularly true for K65R detection because its presence would indicate the need to switch to AZT and its absence would indicate the possibility to recycle TDF in a subsequent first- or second-line regimen. In fact, in this study, 31% of the patients had no K65R, some after as many as 33 months of TDF-based first-line ART. Switching such patients to AZT would be premature. The one patient with enough accumulated resistance to both TDF and AZT is concerning and further justifies the need for VL and resistance testing. Such consequences are important for public health considerations, which should include improvement of treatment monitoring by increased VL testing resulting in less drug-resistance accumulation.The major study limitation was a small sample size, resulting in low power to examine some associations with failure and resistance, and inability to compare failure and resistance patterns between groups and subtypes. In addition, traditional Sanger sequencing may have underestimated K65R and other minority resistant variants; we were unable to follow the prior ART group from ART start or switch to TDF; VL failure was not confirmed; adherence was self-reported; time of failure was not quantified; and lastly, enrolled participants were on TDF for various time periods and those lost to follow-up were not included, potentially underestimating actual failure rates. Nevertheless, the study represents real-life clinical care circumstances and provides new needed data from RLS.In this study from a large HIV treatment programme in western Kenya, patients’ response to a TDF-based first-line ART was good and switch from non-TDF-based ART was successful. High prevalence of resistance, in particular K65R, was observed, impacting future treatment options. Results support recommendations for widespread VL monitoring in RLS to identify early treatment failure, and the need to consider individualized resistance testing to accurately design subsequent regimens . Even in


A key objective of combined antiretroviral therapy (cART) is to reach and maintain high CD4 cell counts to provide long-term protection against AIDS-defining opportunistic infections and malignancies, as well as other comorbidities. However, a high proportion of patients present late for care. Our objective was to assess CD4 cell count recovery up to seven years in naïve patients initiating cART with at least three drugs in usual clinical care.3 according to baseline CD4 cell count.From the French Hospital Database on HIV, we selected naïve individuals initiating cART from 2000 with at least two years of follow-up. Participants were further required to have achieved viral load suppression by six months after initiating cART and were censored in case of virological failure. We calculated the proportion of patients (Kaplan-Meier estimates) who achieved CD4 recovery to >500/mmA total of 15,025 patients were analyzed with a median follow-up on ART of 65.5 months (IQR: 42.3–96.0). At cART initiation, the median age was 38.6 years (IQR: 32.2–46.0), 9734 (64.8%) were men, median CD4 cell count was 239 (IQR: 130–336) and 2668 (17.8%) had a prior AIDS event. Results are presented in the This study shows that CD4 cell counts continue to increase seven years after cART initiation, whatever the baseline CD4 cell count. Failing to achieve CD4 recovery with continuous viral load suppression is rare for naïve patients initiating cART in routine clinical practice, but takes substantially longer in patients who initiate antiretroviral therapy at low CD4 cell counts.


Routine HIV viral load (VL) monitoring is the standard of care for persons receiving antiretroviral therapy (ART) in developed countries. Although the World Health Organization recommends annual VL monitoring of patients on ART, recognizing difficulties in conducting routine VL testing, the WHO continues to recommend targeted VL testing to confirm treatment failure for persons who meet selected immunologic and clinical criteria. Studies have measured positive predictive value (PPV), negative predictive value, sensitivity and specificity of these criteria among patients receiving first-line ART but not specifically among those on second-line or subsequent regimens. Between 2008 and 2011, adult ART patients in Nyanza, Kenya who met national clinical or immunologic criteria for treatment failure received targeted VL testing. We calculated PPV and 95% confidence intervals (CI) of these criteria to detect virologic treatment failure among patients receiving a) first-line ART, b) second/subsequent ART, and c) any regimen. Of 12,134 patient specimens tested, 2,874 (23.7%) were virologically confirmed as treatment failures. The PPV for 2,834 first-line ART patients who met either the clinical or immunologic criteria for treatment failure was 34.4% (95% CI 33.2–35.7), 33.1% (95% CI 24.7–42.3) for the 40 patients on second-line/subsequent regimens, and 33.4% (95% CI 33.1–35.6) for any ART. PPV, regardless of criteria, for first-line ART patients was lowest among patients over 44 years old and highest for patients aged 15 to 34 years. PPV of immunological and clinical criteria for correctly identifying treatment failure was similarly low for adult patients receiving either first-line or second-line/subsequent ART regimens. Our data confirm the inadequacy of clinical and immunologic criteria to correctly identify treatment failure and support the implementation of routine VL testing. In sub-Saharan Africa, there are an estimated 22.1 million adults aged 15 years and above living with HIV and 1.4 million of these reside in Kenya, the country with the fourth highest number of infected persons worldwide .A concern for persons on ART, particularly in resource-constrained areas, is the development of treatment failure followed by the change to more expensive regimens. In developed countries, identification of treatment failure is done through routine viral load (VL) testing. However, the costs, small number of laboratories with expertise in measuring VL, and difficulty with reliable transport of specimens, has greatly limited the use of routine VL testing in most resource-limited settings. Although the World Health Organization (WHO) recently adopted new recommendations for VL testing as the preferred routine method to monitor patients on ART, recognizing that this may not be feasible in all settings, the WHO continues to recommend the use of CD4 and clinical monitoring to diagnose treatment failure and VL testing to confirm failure in order avoid unnecessary changes in regimens –9. HowevIn 2008, targeted VL testing was initiated in Nyanza region for patients on first-line or second-line ART regimens suspected to be failing treatment and meeting national immunological or clinical criteria for treatment failure adopted from WHO. We measured the positive predictive value (PPV) of immunological and clinical criteria for treatment failure among adult patients on first-line, second-line or other ART regimens. In June 2012, the Kenya national HIV treatment guidelines were updated, adding routine VL testing at 6 and 12 months after ART initiation and thereafter one VL test per year, aligning with the 2013 WHO treatment guidelines , 10. HowTargeted VL testing was offered at 180 of the 600 health facilities in Nyanza that provided HIV care and treatment services as of the end of 2011. These facilities included dispensaries, health centers, sub-district hospitals, district hospitals, provincial, and referral hospitals. The study period was from September 2008 to December 2011.The Kenya National AIDS and Sexually Transmitted Disease Control Program established criteria for ART failure based on the WHO clinical and immunologic criteria . Adult pTo ensure adherence to VL testing guidelines, standardized laboratory request forms that included the indications for VL testing and job aids were developed in 2008 and revised in 2009. Health workers were trained to recognize the clinical and immunologic criteria for targeted VL testing, complete the laboratory requisition form, and interpret the test results. Additionally, the mechanism to transport the specimens and receive results were developed prior to implementation of targeted VL testing. The laboratory requisition form included patient demographic information, facility where patient was receiving care, the indications for VL testing, current and past ART regimens, and CD4 test dates and results. Note that only the clinician documented indication for VL testing was used regardless of the CD4 test results included on the laboratory requisition. Specimens collected from patients with reported poor adherence two weeks prior to VL testing, active infections, including newly diagnosed tuberculosis or fever, were not tested since these conditions may increase or cause transient VL increase.™ 1.5 [This analysis includes patients aged 15 years and above for whom the indication for testing was documented on the requisition form. Because VL testing was used to routinely monitor ART virologic response among pregnant women who did not have clinical or immunologic criteria, we excluded this sub-population from the analysis . We used™ 1.5 .diagt command. Positive predictive value was calculated as the number of persons who met the clinical or immunologic criteria for viral load testing divided by the total number of persons with virologic failure We calculated the PPV and corresponding 95% exact binomial confidence intervals of the clinical and immunologic criteria to identify virologic failure using Stata Ethical approval for the study was obtained from the Kenya Medical Research Institute and United States Centers for Disease Control and Prevention.A total of 12,134 adult patient records were included in the analysis. The majority of patients were receiving first-line ART; only 133, (1%) of patients were receiving second-line or subsequent regimens Of 12,134 patient specimens tested, 2,874 (23.7%) yielded an HIV RNA concentration > 1000 copies/mL, confirming virologic treatment failure. Among those with failure, the median RNA concentration was 41,150 copies/mL, interquartile range; 9,000–133,800. The PPV for virologic failure among all ART patients who met either the clinical or immunologic criteria for targeted VL testing was 34.4% (95% CI 33.1%-35.6%), 34.4% (95% CI 33.2%-35.7%) for first-line ART patients, and 33.1% Click here for additional data file.


Regimens sparing RATE  agents might have better long-term safety. We conducted a feasibility exercise to assess the potential for a randomised trial evaluating RATE-sparing regimens.Observational.We first calculated RATE-sparing options available to an average patient receiving RATE agents. We reviewed treatment history and all resistance assays from patients attending the St. Vincent’s Hospital (Sydney) clinic and receiving ≥2 RATE agents (n = 120). A viable RATE-sparing regimen with 2 or 3 fully-active agents was constructed from the following six ‘safer’ agents: rilpivirine or etravirine; atazanavir; raltegravir; maraviroc; and lamivudine. Activity for each drug was predicted as 1 (full-activity), 0.5 or 0 (no activity) using the Stanford mutation database. The utility of maraviroc was calculated assuming both maraviroc activity and inactivity where unknown. The analysis was restricted to regimens for which supporting evidence was identified in the literature or conference proceedings. Finally, we calculated the proportion of patients in the nationally representative Australian HIV Observational Database (AHOD) cohort receiving ≥2 RATE agents (n = 1473) to measure the potential population-level uptake of RATE-sparing agents.Assuming full maraviroc activity, 117(97.5%) and 107(89.2%) individuals had at least one option with 2 or 3 active RATE-sparing agents, respectively. Assuming no maraviroc activity this decreased to 113(94.2%) and 104(86.7%), respectively. In AHOD, 837(56.8%) patients were receiving ≥2 RATE agents.Feasible treatment switch options sparing RATE agents exist for the majority of patients. Understanding the pros and cons of switching stable patients onto new RATE-sparing regimens requires evidence derived from randomised controlled trials. Ritonavir (booster dose): long-term inhibition of CYP450, tolerance (diarrhoea) and dyslipidemia Abacavir: hypersensitivity Tenofovir: long-term concern for bone and renal disease Efavirenz: neuropsychiatric and cognitive effects While the development of antiretroviral therapy (ART) has seen tremendous success, there are ongoing concerns regarding the short- and long-term safety and tolerability of some of the common contemporary ART drugs. These agents and their associated concerns include: (i) Newer, apparently safer, more tolerable drugs continue to be approved, but the data derived from their clinical development is generally limited to their use in first-line or late salvage ART. There is a need for evidence on how agents can be innovatively combined and sequenced in order to optimally replace RATE agents and in turn achieve better long-term outcomes. A few recent small studies of novel, unconventional regimens have been reported and attest to the growing interest in more fully exploiting the opportunities offered by the growing HIV armamentarium. Before planning a full scale trial evaluating RATE-sparing agents, we conducted a preliminary feasibility study to identify key metrics needed for planning such a trial. We had two key objectives: (i) in an average patient receiving ≥2 RATE agents, how many feasible RATE-sparing regimen options containing 2 or 3 fully-active agents are available if they were to switch?; and (ii) what proportion of HIV+ individuals at the population level receiving successful ART have ≥2 RATE agents as components of their regimen and might be expected to benefit from RATE-sparing options?For objective(i), we conducted a detailed review of the individual records of the patients attending the St. Vincents Hospital’s ambulatory care clinic . This database was chosen as it provided access to detailed treatment history, tropism test, all archived resistance mutations and clinical history available in each patient. The retrospective study on the St. Vincent’s Hospital’s database was approved by the St. Vincent’s Hospital Human Research Ethics Committee, Sydney, Australia. Informed consent from participants was not obtained for this study. Requirement for informed consent for this study was waived by the above mentioned Ethics Committee. All of the data generated was made available in de-identified format to the study team.We analysed the data from all patients enrolled in clinics under the following investigators: MAB, ADK, GM and DAC. Patients had to be receiving ≥2 RATE agents, in active follow-up (last visit within one year of March 2013), virologically suppressed (<200 copies/mL measured on at least 2 occasions more than 7 days apart) and not known to be hepatitis-B infected. We reviewed patient demographics, treatment history and resistance and tropism assay reports. Given that the resistance assay has been standard of care in Australia for some years, most patients were expected to have had one performed at the time of virological failure.We defined a new regimen option as a RATE-sparing regimen constructed from a pool of the following six agents approved for use in Australia: rilpivirine or etravirine (cannot be used together); atazanavir (cannot be used with rilpivirine or etravirine because of possible unfavourable drug-drug interactions); raltegravir; maraviroc; and lamivudine. These agents were chosen for their proven efficacy and good safety profile. Also, from the PI class, only atazanavir is approved for use without a ritonavir booster. We limited consideration to raltegravir as the only integrase inhibitor available in Australia in the period we performed this analysis, but it could can be potentially be replaced by other integrase-inhibitors entering the market which will only increase the number of possible RATE sparing combinations available.To predict drug activity we entered all recorded patient mutations (including archived results) in the Stanford mutation database version-6.3.0 (June 2013) Finally, since most of the RATE-sparing regimens were unconventional, we conducted a review of the peer-reviewed literature (using MEDLINE database) and major conference presentations to identify regimens for which there was at least some empirical support . The search was conducted using key words for each of the agents in consideration (or their respective ART classes) using Boolean operators. We restricted the search to articles in English language published in the year 2006 or later. We included studies on regimens of interest regardless of study phase, number of patients or availability of a comparator arm.For objective(ii), we analysed the data from the Australian HIV Observational Database (AHOD) cohort. The AHOD is an observational cohort study of HIV-positive individuals attending specialised general practitioner sites, sexual health clinics and tertiary referral centres throughout Australia. This study has been ongoing since 1999, and currently has 27 sites throughout Australia . The AHOD study has been approved by the Human Research Ethics Committee of the University of New South Wales, Sydney, Australia, and all other relevant institutional review boards. Written informed consent was obtained from participating individuals in the AHOD study. The details of the AHOD study design have been published elsewhere We identified patients in the AHOD who were in active follow-up (documented visit within one year of March 2013 data transfer), receiving ART, virologically suppressed (HIV RNA <200 copies/mL) at their last visit, and not known to be co-infected with hepatitis B (HBsAg negative). Our aim was to identify the proportion of such patients receiving ≥2 of RATE agents at their last follow-up visit and describe their demographic and treatment-history characteristics.All analyses were performed using Ms Excel (Microsoft) and STATA ver. 12 .A total of 120 patients from the Hospital database matched the selection criteria and were included in analyses. Table-1 describes their characteristics. They had been receiving ART for a median of 8 years (IQR: 4.4–12.9) and about 17% had exposure to integrase inhibitors. An HIV tropism test for predicting maraviroc activity was unavailable or unsuccessful in 54.2% and 7.5% of patients, respectively and 19 (15.9%) patients had full expected activity of the maraviroc. All HIV tropism tests were available within the last year (earliest one was in March-2012). About 16% of patients had no expected activity of either unboosted atazanavir or lamivudine.Table-2 describes the number of available RATE-sparing regimen options under various scenarios. Assuming maraviroc activity = 1 where unknown, 117 (97.5%) and 107 (89.2%) individuals had at least one option with a score of 2 or 3, respectively. This decreased to 113 (94.2%) and 104 (86.7%), respectively, on assuming maraviroc inactivity where unknown.The literature review indicated that direct or indirect support was available for many novel regimens, mainly in the form of small pilot studies Table-2 also describes a number of available RATE-sparing regimen options for which we could identify supporting evidence. Although the median number of available regimen options decreased in all of the scenarios, there was no appreciable change in the proportion of patients without any option remaining.Of the 1473 eligible patients in AHOD, 837  were receiving ≥2 RATE agents. Table-3 provides the characteristics of patients in AHOD who were receiving ≥2 RATE agents. Patients with ≥2 RATE agents had been receiving ART for a median of 10.7 (interquartile range (IQR): 4.1–15) years, 38% with a history of mono/dual therapy exposure in the past; they had been exposed to a median of 3 classes of drugs; 13.7% had been exposed to integrase inhibitors. The most common RATE agent used was tenofovir (76.9%) followed by ritonavir (in boosting dose) (55.7%).Our analysis of patients in one hospital-based clinic suggested that most patients receiving ≥2 RATE agents would be expected to have at least one viable RATE-sparing regimen switch option containing 2 or 3 fully-active agents This remained the case when we restricted our assessment to only those regimen options which have shown promise in publicly presented studies. In our analysis of the AHOD we found that up to 57% of HIV-positive individuals under treatment in Australia currently have ≥2 RATE agents in their regimen and might benefit from a RATE-sparing option. These findings suggest that there exits the potential to perform randomised trials to evaluate RATE-sparing regimens. Such trials would provide valuable guidance on how best to combine and sequence ART agents to maximise patient safety and preserve future treatment options.Most of the regimens proposed and considered as ‘viable’ in this study have not been rigorously tested in clinical trials and might be regarded as unconventional, and perhaps as possessing a lower genetic barrier to the selection of resistance. However, there has been growing interest in testing novel combinations of ART agents, which exclude nucleoside(tide) and older non-nucleoside reverse transcriptase inhibitors (N(t)RTIs and NNRTIs, respectively) as well as ritonavir (booster dose) A few limited studies on unboosted atazanavir and raltegravir show encouraging results in terms of virological success and safety profile, although hyperbilirubinemia might be an issue with unboosted atazanavir containing regimens, especially if atazanavir is used at a dose of 300 mg twice-daily. In one small single-arm trial, ART-naïve patients achieving virological suppression with a four drug combination of tenofovir, emtricitabine, maraviroc and raltegravir, safely stopped the N(t)RTI component after 24 weeks without any failure 24 weeks after stopping the N(t)RTIs. Despite these observations there is a clear need for trials to rigorously evaluate RATE-sparing regimens, not only to demonstrate their virological success but also their expected greater tolerability and safety. Given the concern for possible resistance emergence in those with high viral loads, trials could initially focus on virologically suppressed patients with no or limited history of treatment failure and high expected adherence. The scheme of a proposed trial design is represented in The following additional/alternative trial strategies would be worth considering: (i) enrolling patients thought to be at a high risk of a chronic disease such as cardiovascular disease and therefore most likely to benefit from the trial. Our study has limitations. Firstly, it was a retrospective survey of patient data. It analysed data from patients attending a single large tertiary hospital centre in Sydney (for objective i). It is possible that more complicated or difficult to manage patients may have been selectively referred to this centre, resulting in lesser generalizability of our findings. However, this may have only resulted in underestimation of the actual number of options available to patients in general. Also, patient characteristics in the Hospital database were broadly similar to those in AHOD, suggesting minimal selection bias. Second, the Hospital database may not have accurately captured comorbidities and concomitant medication data which may impact the number of suitable options available to a patient. However, the agents considered for treatment options are known to have only few serious drug-drug interactions or contraindications. Our assessment of the recorded data on comorbidities and concomitant medications did not affect our conclusions. We therefore do not believe this limitation would seriously undermine the number of options available. We did not consider use of atazanavir and etravirine together as an option (due to drug-drug interactions), though a recent study suggests that the use of a higher dose of atazanavir in such a combination may not be necessary. In summary, our study suggests that most patients using RATE agents have viable RATE-sparing switch options which include 2 or 3 fully active agents in the switch regimen. The use of RATE drugs is common and a significant proportion of HIV-positive individuals might be expected to benefit from such options. There is a need for fully powered randomised trials to rigorously evaluate this strategy in order to optimise long-term patient outcomes.Table S1Summary of regimens supported by the literature.(DOCX)Click here for additional data file.


The HIV epidemic in the United States (US) disproportionately affects gay, bisexual, and other men who have sex with men (MSM). Pre-exposure prophylaxis (PrEP) using co-formulated tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) has demonstrated high efficacy in reducing HIV incidence among MSM. However, low adherence was reported in major efficacy trials and may present a substantial barrier to successful PrEP implementation. Rates of adherence to PrEP in “real-world” clinical settings in the US remain largely unknown.We reviewed demographic and clinical data for the first 50 patients to enroll in a clinical PrEP program in Providence, Rhode Island. We analyzed self-reported drug adherence as well as drug concentrations in dried blood spots (DBS) from patients who attended either a three- or six-month follow-up appointment. We further assessed drug concentrations and the resistance profile of a single patient who seroconverted while taking PrEP.r = 0.49, p = 0.02). One patient who had been prescribed PrEP seroconverted at his three-month follow-up visit. The patient’s drug concentrations were consistent with daily dosing. Population sequencing and ultrasensitive allele-specific PCR detected the M184V mutation, but no other TDF- or FTC-associated mutations, including those present as minor variants.Of the first 50 patients to be prescribed PrEP, 62% attended a follow-up appointment at three months and 38% at six months. Of those who attended an appointment at either time point , 92% and 95% reported taking ±4 doses/week at three and six months, respectively. Drug concentrations were performed on a random sample of 20 of the 35 patients who attended a follow-up appointment. TDF levels consistent with ±4 doses/week were found in 90% of these patients. There was a significant correlation between self-reported adherence and drug concentrations (In this clinical PrEP program, adherence was high, and self-reported drug adherence accurately reflected drug concentrations as measured by DBS. The HIV epidemic continues to be a significant public health concern in the United States (US), with over 1.2 million persons currently living with HIV and the number of newly diagnosed cases approaching 50,000 annually . AdherenAnother major finding from the initial efficacy studies was the significant discordance between self-reported adherence and serum drug levels. In the Pre-exposure Prophylaxis Trial for HIV Prevention among African Women (FEM-PrEP)  and VagiIn 2013, we implemented a clinical PrEP program in Providence, Rhode Island. We evaluated FTC and TDF concentrations by DBS and compared to self-reported adherence among patients. We also describe drug concentrations in a single patient who seroconverted while on PrEP, as well as the resistance profiles generated by commercial genotyping and minor variant assays. This is one of the first reports of adherence, using drug concentration levels, in which data were collected during routine clinical care.We reviewed medical records of the first 50 patients receiving PrEP care at an outpatient infectious diseases clinic in Providence, Rhode Island, between February 2013 and June 2014. Patients provided written informed consent to participate in this study. Consent procedure and study protocol were approved by The Miriam Hospital Institutional Review Board. Data collected included age, race and ethnicity, insurance status, referral source, indications for PrEP, attendance at follow-up appointments, HIV serostatus, and self-reported adherence. HIV serostatus was determined by third-generation antibody testing . Patients were scheduled for follow-up appointments every three months, in accordance with guidelines from the Centers for Disease Control and Prevention (CDC) for administering PrEP . MedicalFig 1). Blood for DBS analysis was drawn during the course of routine clinical venipuncture; DBS analysis was conducted as described elsewhere [Thirty-five of the first 50 PrEP patients attended three- or six-month follow-up appointments; of these, 20 patients were randomly selected for DBS samples to measure drug concentrations. One additional patient was included in the DBS study after seroconverting while taking PrEP .To compare patients who were and were not retained in care, and did and did not have DBS samples, we performed two-tailed t-tests for continuous variables and Fisher’s exact test for categorical variables across demographic and behavioral categories. Pearson’s correlation coefficient was calculated to measure the correlation between self-reported adherence and drug concentrations. Significance was determined for all tests at a two-tailed For the patient who seroconverted, commercial genotyping was performed using the Celera Diagnostics ViroSeq HIV-1 Genotyping System . Detection and quantification of drug resistance mutations M184V, K70E and K65R when present as minor variants was performed using allele-specific PCR as described elsewhere .Of the first 50 patients prescribed PrEP, 58% were non-Hispanic white, 26% were non-Hispanic black or African American, 26% were Hispanic or Latino, and 8% were another race. Eighty-eight percent were male, the majority (95%) of whom were MSM. The average age of the first 50 patients was 33.8 years (range: 18–58). All but one of the first 50 patients were insured. The mean annual income was $49,190, with a range of $0 to $350,000. Patients were referred to the PrEP program by the publicly-funded STD clinic (40%), the onsite HIV outpatient center (26%), outpatient physicians (20%), or other referral sources (14%), which included friends, community organizations, and the onsite post-exposure prophylaxis (PEP) program. A total of 88% (n = 38) and 82% (n = 31) of the first 50 patients reported taking PrEP at three and six months, respectively. Reasons for discontinuing PrEP included side effects (n = 3), moving (n = 3), and a reduction in behaviors associated with HIV acquisition (n = 1). A total of 70% (n = 35) of patients who were initially prescribed PrEP successfully attended a follow-up visit at either three or six months. Those who were retained in care three or six months after PrEP initiation did not differ significantly from those not retained in care on any measured demographic variables.Table 1). Patients in the DBS sample had higher mean self-reported drug adherence in the past 30 days (28.5 doses taken) compared to patients not included in the DBS sample .Thirty-one patients attended a three-month follow-up appointment and 19 attended a six-month follow-up appointment. Among those who attended visits at three or six months (n = 35), the mean self-reported drug adherence across follow-up visits was 6.2 and 26.8 doses taken in the previous seven and 30 days, respectively. Those included in the DBS sample (n = 21) did not differ significantly from those who were not included on the basis of age, race and ethnicity, gender, indications for PrEP, referral source, attendance of follow-up appointments, or self-reported drug adherence in the past seven days ; however, no significant correlation emerged between TFV-DP concentration and past-30 day adherence .DBS samples were collected an average of 4.4 months (range: 1.8–13.3) after PrEP initiation. Based on DBS analyses, patients had a mean TFV-DP concentration of 1493 fmol/punch (range: 31.9–4141.1 fmol/punch) and a mean FTC-TP concentration of 0.296 pmol/punch (range: 0.190–0.466 pmol/punch). FTC-TP concentrations for two patients fell below the level of quantification ; given that no measurements were obtained, these two patients were not included in calculating the mean. TFV-DP concentrations for the two patients with BLQ FTC-TP were 31.9 and 619.1fmol/punch. By TFV-DP concentration, 5% (1/21) of patients took fewer than two doses per week (BLQ-349 fmol/punch), 5% (1/21) of patients took two to three doses per week (350–699 fmol/punch), and 90% (19/21) of patients took four or more doses per week (±700 fmol/punch). TFV-DP concentration and self-reported adherence in the past seven days were significantly correlated  drug resistance were detected: D67N, M184V, T215S, and K219Q. The M184V mutation was also detected by AS-PCR, and the protease-associated mutation L10I was also noted.The present study is among the first to measure PrEP adherence by drug concentrations among early adopters in a clinical program and outside the controlled environment of a clinical trial in the US. Self-reported adherence was high and significantly correlated with drug concentrations obtained via DBS testing. PrEP adherence is critical for optimal intervention efficacy. In efficacy trials, the prevalence of seroconversions ranged from less than 1% in the immediate PrEP arm of the PROUD study to 6% in the oral TDF/FTC arm of the VOICE study, paralleling respective high and low adherence in these trials ,6. PharmDemonstration projects and open label studies have provided some insight into PrEP adherence in clinical settings. In the iPrEx OLE, TFV-DP concentrations consistent with taking no doses, fewer than two doses, two to three doses, and four to six, and seven doses per week were detected at 25%, 26%, 12%, 21%, and 12% of study visits, respectively ,21. HoweEarly results of the PrEP efficacy trials suggested that adherence would be a major obstacle during implementation ,6. HowevSuboptimal adherence to TDF/FTC for PrEP has contributed to reduced efficacy and, less commonly, the emergence of PrEP-selected mutations for drug resistance. Most drug resistance mutations in other PrEP studies were observed among those determined to have had acute HIV infection at study entry ,20. ConsThe current study presents a single case of seroconversion during PrEP clinical care. It is unclear whether the patient had acute HIV infection at baseline or seroconverted while on PrEP. Initiation of TDF/FTC as PrEP during acute HIV infection may facilitate development of drug resistance ,16,20. TThe seroconversion case underscores the importance of excluding acute HIV infection prior to PrEP initiation. Current CDC guidelines for PrEP recommend screening for acute HIV infection, which may include clinical screening for signs and symptoms or viral load testing . At our A limitation of this study is the small sample size. Future studies should evaluate PrEP adherence among a larger cohort and among different patient populations. The study site was an infectious disease clinic; the relationship between retention in care and adherence may differ in other settings  where patients receive other healthcare services in addition to PrEP. Further, the patient sample in this study was primarily non-Hispanic white or Hispanic; adherence, retention in care, and other outcomes of PrEP treatment may vary among other racial and ethnic groups.This study is among the first to demonstrate a strong correlation between PrEP self-reported adherence and drug concentrations in an exclusively clinical setting. Contrary to low adherence reported in early clinical trials, we report high adherence to PrEP among a sample of patients retained in care. PrEP patients who are successfully retained in care and who attend follow-up appointments are likely to be adherent to this treatment. Future research and interventions should consider retention in PrEP care as a target for improvement.