Persistence of plasma HIV-1 RNA during seemingly effective antiretroviral thereapy (ART) is incompletely understood. Using an ultrasensitive assay, this cross-sectional study investigated residual plasma HIV-1 RNA in subjects maintained on firstline ART with continuous viral load suppression <50 copies/mL for ≤15 years without recognized viral load blips or treatment interruptions and explored its relationship with the duration of suppressive ART, efavirenz concentrations in plasma, 2-LTR circular HIV-1 DNA (2-LTRc DNA) in peripheral blood mononuclear cells, and cellular  and soluble  markers of immune activation in peripheral blood. Residual plasma HIV-1 RNA, total HIV-1 DNA and 2-LTRc DNA were quantified by real-time and digital droplet PCR. Cellular  and soluble  markers of immune activation were measured by flow cytometry and ELISA.10 copies/mL higher with each log10 U/mL increase in sCD27 . No significant association was found between residual plasma HIV-1 RNA and other explored parameters. Residual plasma HIV-1 RNA and 2-LTRc DNA were detected in 52/104 (50%) and 24/104 (23%) subjects, respectively. Among subjects with detectable HIV-1 RNA, 50/52 showed levels ≤11 copies/mL. In adjusted analyses, HIV-1 RNA levels were 0.37 logThese findings point to an ongoing relationship between plasma HIV-1 RNA and selected markers of immune activation during continuously suppressive ART. The novel direct association with levels of sCD27 warrants further investigation. Maintaining plasma HIV-1 RNA suppression below the limit of detection of commercial viral load assays defines clinical success of antiretroviral therapy (ART) . UltraseIdentifying the determinants of HIV-1 RNA persistence during seemingly effective ART is central to our understanding of how the virus may be eradicated , 15. SucWe previously reported that treated patients with plasma HIV-1 RNA detected at levels ranging between approximately 10 and 49 copies/mL by a commercial viral load assay were more likely to experience viral load rebound above 50 copies/mL and above 400 copies/mL during 12 months of follow-up when compared with treated patients with undetectable HIV-1 RNA . The aimIn this cross-sectional study, eligible patients had started firstline ART with 2 nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) plus 1 non-nucleoside reverse transcriptase inhibitor (NNRTI), had achieved viral load suppression <50 copies/mL within 6 months of starting ART, and during subsequent follow-up had remained on the initial NNRTI, undergone ≥2 viral load measurements per year, and maintained continuous viral load suppression <50 copies/mL without any recorded viral load elevation >50 copies/mL or treatment interruption. To ensure balance in terms of duration of suppressive ART, recruitment was stratified to span from 1 year of treatment to 10 years or longer. Changes of the initial NNRTI were not allowed. Changes of the initial NRTIs were allowed to reflect evolving practice, provided they did not coincide with a viral load >50 copies/mL or a treatment interruption. The study took place at multiple centers across the United Kingdom. It was approved by the National Research Ethics Service (London-Dulwich) and included in the National Institute for Health Research Clinical Research Network Portfolio. Patients provided written informed consent.Plasma was separated from venous blood in EDTA within 2 hours of collection and stored at −80°. HIV-1 RNA was quantified using a modified version of the RealTime HIV-1 assay . Validation studies previously showed that the assay 50% and 95% detection rates were 1 and 3 HIV-1 RNA copies/mL, respectively, with high reproducibility across diverse HIV-1 subtypes , 20, 21.6 PBMC, respectively. 2-LTRc DNA was measured by droplet digital PCR as described [6 PBMC. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation and cryopreserved. Total HIV-1 DNA was quantified by real-time polymerase chain reaction (PCR) as described ; the assescribed ; the ass−6 µg/mL), soluble CD27 , soluble CD30 , and IL6 .Flow cytometry was performed at the Immunology Service of the Royal Free Hospital in London using freshly collected blood. Following centrifugation, cell pellets were lysed by 10-minute incubation at room temperature in 1× Hoffman’s lysis buffer and incubated with FITC-, Cy5- or PE-labeled antibodies targeting CD4 plus CD26, CD38, or CD69, and CD8 plus CD38 or HLA-DR/DP/DQ . Labeled cells were incubated in 1% paraformaldehyde for 30 minutes prior to acquisition with BD FACSCalibur and analysis by CellQuest. Soluble markers were measured in frozen plasma by commercial Elisa assays (EIAs) for soluble CD14  and nevirapine (NVP) concentrations were measured in untimed plasma samples using high-performance liquid chromatography–tandem mass spectrometry, as previously described . The timP ≤ .20) with the outcome of interest in the univariable analysis were included in the multivariable models, with the following modifications: (a) when modeling 2-LTRc DNA, total HIV-1 DNA was excluded due to high colinearity (P < .0001); (b) age and duration of ART were excluded in favor of duration of suppressive ART. Separate models explored: (a) the effect of forcing the nadir CD4 cell count and pre-ART viral load into the multivariable analyses and (b) the association between residual plasma HIV-1 RNA or 2-LTRc DNA and plasma concentrations of EFV (as a continuous variable) in EFV recipients, after adjustment for the time of sampling in relation to the last EFV dose. IBM SPSS v. 22.0  and SAS v9.4  were used for the analyses.Duration of suppressive ART was defined as the length of time following the first viral load measurement <50 copies/mL, which in all subjects was recorded within 6 months of starting ART. When analyzing residual plasma HIV-1 RNA and 2-LTRc DNA as continuous variables, undetectable values were replaced with the midpoint value between 0 and the lower limit of detection of the assays. EFV and NVP concentrations were categorized as above or below the recommended minimal effective concentration (MEC) for wild-type virus: 1000 ng/mL for EFV  and 3400The population comprised 104 patients that at the time of recruitment had experienced continuous viral load suppression <50 copies/mL for a median (IQR) of 5 (3–8) years while receiving firstline ART with 2 NRTIs plus either EFV  or NVP . Reflect6 PBMC that ranged between 4 and 119 copies/106 PBMC  patients . Levels 106 PBMC . Cellula106 PBMC  and solu106 PBMC  markers 106 PBMC . CD8 cel3/y) and lower expression of activation markers on CD4 cells (CD38) and CD8 cells . Levels of sCD14, sCD30, and IL-6 did not differ by duration of suppressive ART, whereas levels of sCD27 were significantly lower with longer duration of suppressive ART. A trend was observed for lower levels of 2-LTRc DNA with longer duration of suppressive ART. There was less evidence of a difference in residual plasma HIV-1 RNA, and no significant difference was seen in total HIV-1 DNA. A linear regression analysis confirmed an inverse association between 2-LTRc DNA and duration of suppressive ART, with an adjusted mean difference of 0.03 log10 copies/106 PBMC per year longer  .Differences in virologic and immunologic parameters according to the duration of suppressive ART, analyzed after exclusion of the 2 outliers, are shown in P = .02) . Forcing+CD38+ cells, and levels of sCD30 and IL-6 . The association similarly persisted in analyses including the 2 outliers (not shown).The univariable linear regression analysis indicated a significant direct association between residual plasma HIV-1 RNA and sCD27 among the 102 subjects with HIV-1 RNA ≤11 copies/mL . There wand IL-6 . There wP = .30) or 2-LTRc DNA (P = .47) and EFV concentrations (We first explored the association between plasma EFV concentrations and virologic parameters in 76 EFV recipients (including the 2 outliers) who had taken the last EFV dose a median (IQR) of 12 (10–13) hours prior to sampling, and were therefore in the range of the “mid-dose” interval, where targets have been previously defined . Althougtrations .This study demonstrated that half of subjects who had experienced viral load suppression <50 copies/mL continuously for up to 15 years while maintained on the initial NNRTI had detectable HIV-1 RNA in plasma when tested with an ultrasensitive assay, at levels that rarely exceeded 11 copies/mL. We previously reported that in treated patients, plasma HIV-1 RNA levels above approximately 10 copies/mL were associated with a risk of viral load rebound above 50 copies/mL and above 400 copies/mL over 12 months of follow-up . Taken t10 2-LTRc DNA copies/106 PBMC per year. This apparent, slow decay in 2-LTRc DNA is consistent with observations from Bushman [These data do not elucidate the debated source of residual plasma HIV-1 RNA , 31. The Bushman  and Sili Bushman , and pot Bushman .Previous studies did not detect an association between residual plasma HIV-1 RNA and markers of immune activation on CD4 or CD8 cells (including CD38 and HLA-DR) in patients treated for up to 4 years , 37, andThere are scarce data on the effect of ART on sCD30 . Levels trough. The patients analyzed in the models had taken the last EFV dose at a consistent interval relative to sampling, and time of sampling would be expected to have little impact overall. Finally, we recognize that both drug concentration and antiviral activity measured in peripheral blood do not necessarily reflect the situation in lymphoid tissue [Our study has limitations. First, findings obtained in a cross-sectional study should be confirmed with prospectively collected data, which is challenging when aiming to study long-term outcomes in populations maintained on the same regimen. To date, prospectively determined virologic indicators of HIV-1 persistence during long-term suppressive ART have been poorly documented. Compared with published studies addressing similar questions, one strength of our study is the relatively large population combined with stringent eligibility criteria and a consistent definition of virologic suppression. It could be argued that our study population was not selected based on the infection stage at the start of ART, which could have introduced a bias. We observed a lower nadir CD4 cell count and a higher pre-ART viral load in subjects with longer duration of suppressive ART; however, the analyses did not identify an effect of nadir CD4 cell count or pre-ART viral load on the measured parameters in our highly selected population. Of note, we excluded 2 outliers from the main analyses with residual plasma HIV-1 RNA levels >11 copies/mL. Whereas the main conclusions were not affected by their inclusion, it would have been of interest to determine any risk of viral load rebound with prospective follow-up in these 2 subjects. Further, we reported total HIV-1 DNA rather than integrated HIV-1 DNA as the indicator of the viral reservoir in this study. However, we previously described integrated HIV-1 DNA levels in a subset of the same population and demonstrated that they were highly correlated with the levels of total HIV-1 DNA, not influenced by the duration of suppressive ART, and not associated with residual HIV-1 RNA . We colld tissue , 9.In conclusion, this study provides evidence that residual plasma HIV-1 RNA and PBMC-associated 2-LTRc DNA can be detected in a subset of patients receiving long-term, continuously suppressive firstline ART, without evidence of a clear association between the two, and without evidence of an association of either parameter with plasma concentrations of EFV. While residual plasma HIV-1 RNA was not associated with commonly reported markers of immune activation, there was a novel, independent direct association with levels of sCD27. Further studies are warranted to confirm the findings, extend the immune activation profiling, and determine the underlying mechanism, including whether the relationship reflects ongoing virus replication in lymphoid tissue.Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ofy032_suppl_supplementary_tablesClick here for additional data file.ofy032_suppl_supplementary_figure1Click here for additional data file.ofy032_suppl_supplementary_figure2Click here for additional data file.ofy032_suppl_supplementary_figure_legendsClick here for additional data file.


These include individuals aged <30 years and/or with CD4/CD8 ratio of >0.8 and/or viral load of <5000. This is because these individuals are at very low risk of disease progression in the subsequent 2 to 3 years, the risk is minimally diminished by antiretroviral therapy and is virtually identical in the first 18 months of therapy regardless of treatment initiation. In addition, asymptomatic young individuals are at higher risk of loss‐to‐follow and of low adherence to treatment, and those with low viral loads are less likely to transmit the virus. In addition, lessons from START and Temprano can help design trials to investigate strategies to decrease losses‐to‐follow‐up, while minimizing risks to patients.Initiation of antiretroviral therapy is not a once in a lifetime opportunity. In some resource constrained settings financial limitations make it necessary to prioritize treatment initiation for some groups of patients. In developed countries, there are patients who are reluctant to initiate treatment. Subgroup analysis of the START trial can inform recommendations for which patients with CD4 counts >500 cells mm For less resource constrained settings that can afford to treat some but not all patients with CD4 counts of >500 cells/mm3, there are no specific recommendations In countries in resource limited settings (RLS) that cannot afford to treat all HIV‐infected individuals, the World Health Organization (WHO) recommends prioritizing the treatment of patients with CD4 counts of <350 cells/mm3 and for respecting patient readiness in developed and developing countries alike, here we attempt to discuss how data from the START Considering the need for prioritization in certain RLS for patients with CD4 counts >350 cells/mmThe optimal CD4 cell count at which ART should be initiated in asymptomatic individuals has been a hotly debated topic for three decades 3. Over 50% of the patients were from RLS. Participants were randomized to immediate initiation of ART or its deferral until the CD4 count declined to <350 cells/mm3. After a mean follow‐up of 3 years, the primary endpoint  had occurred in 1.8% and 4.1% of the participants in the immediate and in the deferred groups respectively. There was no evidence that the beneficial effect of immediate ART varied among various predefined subgroups, including region of the world. These results were interpreted as indicative that ART should be initiated in all asymptomatic HIV‐positive adults regardless of CD4+ count. Furthermore support for this interpretation was provided by the Temprano trial, which was conducted in Ivory Coast and included 2076 asymptomatic patients with CD4 counts of <800 cells/mm3. Patients were randomized to immediate ART or deferral until reaching contemporary WHO guidelines (CD4 < 200 and later <350 cells/mm3). When the analysis was restricted to participants with CD4 counts of >500 cells/mm3 at study entry (n = 868), the cumulative probability of death or severe HIV‐related illness (the primary endpoint) at 30 months declined from 12.4% to 6.9% in the deferred and immediate arms respectively.START was a large international trial that involved 4685 previously untreated asymptomatic adult HIV‐positive persons from 35 countries in five continents with CD4 counts of >500 cells/mm3 at study entry. For example, for those with a viral load of <3000 copies/mm3  the absolute risks of reaching an endpoint at 3 years of follow‐up were 2.4 and 1.9% in the immediate and deferred groups respectively, a difference of only 0.5%. In this subgroup, it would be necessary to treat 992 individuals to prevent one event. Moreover, the two curves in this subgroup were completely superimposable in the first 2 years of follow‐up Although in the START trial the relative risk reduction for the primary endpoint was large for all subgroups, absolute differences varied considerably. In some subgroups, the absolute risk of disease progression was substantial and thus the absolute impact of ART was numerically sizable. For example, for those over 50 years of age at study entry, 11.7% in the deferred arm had reached an endpoint by 36 months, compared to 2.9% in the immediate arm, a difference of 8.8%. In this subgroup, it would be necessary to treat 45 individuals to prevent one event. At the other extreme, three characteristics were associated with a low risk of progression during the follow up period, namely age of <30 years, CD4/CD8 ratio of >0.8, and viral load of <3000 (or 5000) copies/mmIn START participants were censored on the date an endpoint was reached or they were last known to be alive. In addition, over 98% of the participants who started ART had an undetectable viral load at 12 months of follow‐up. Thus, results in START should represent the best obtainable benefit with present‐day ART. Participants in clinical trials have greater adherence and health seeking behaviours than the general population and many programs report losses to follow‐up greater than 50% in the first 2 years after treatment initiation. Thus, it seems fair to predict that the actual effectiveness of ART for individuals with high CD4 counts might be significantly inferior to the efficacy reported in START. Furthermore, immediate ART might be even less effective in subgroups which have even higher rates of loss‐to‐follow up, such as healthy young individuals with high CD4 counts, which had the smallest absolute benefit in START. For these individuals, the future advent of long acting drugs, which might circumvent adherence concerns, is likely to affect the balance.The term “immediate” can be misleading. In the START trial, the reported median time since HIV diagnosis to being referred to the study was 1 year. The median time since the first study CD4 cell count and ART initiation in the immediate arm was 6 weeks. Hence, for most individuals in the immediate arm of START over 1 year had elapsed between diagnosis and ART initiation. On the other hand, “immediate” is often misinterpreted as referring to something that occurs suddenly. Thus, the concept of “immediate” utilized in START for treatment initiation is substantially different from its colloquial use or its meaning in the proposed strategy of same‐day HIV testing and ART initiation.3. The number of participants retained in care and with a 12‐month viral load of <50 copies/mm3 were 120 (42%) and 151 (54%) in the standard ART and same‐day groups respectively. Although same‐day initiation represented a statistically significant improvement, the result obtained at 12 months was far from the lifelong 90% UNAIDS target The strategy of whether same‐day HIV testing and ART initiation improves retention in care and virologic suppression was tested in a randomized trial conducted in Haiti involving 564 individuals with CD4 cell counts of >500 cells/mmThe probability of sexual transmission of HIV is determined by behaviour and biologic factors. Results of various studies have demonstrated that infected individuals with suppressed viremia are unlikely to transmit the virus, adding an important societal benefit to ART The effect of the universal test and treat strategy as recommended by WHO on HIV acquisition at the population level was tested in a cluster randomized trial involving 22 communities in South Africa. In this trial, the universal test and treat strategy failed to lower HIV incidence. Thus, when postponement of treatment is considered, it might be reasonable to weigh the societal risks and benefits of providing ART to individuals who are at a lower risk of transmission of the virus and of disease progression in the short term, but who are at high risk of low adherence. For example, asymptomatic young persons with high CD4 countsThe credibility of subgroup analysis to provide recommendations has been questioned 3 at study entry, IPT had an independent and statistically significant positive impact on severe morbidity, the main outcome of the study.The Temprano trial used a 2 × 2 factorial design in which participants were randomized to immediate or deferred ART according to contemporary WHO criteria, each with or without a 6‐month course of primary tuberculosis prophylaxis with isoniazid (IPT). Final results demonstrated that both in the entire study population and in the group with CD4 counts of >500 cells/mm3 with characteristics that predict low probability of disease progression but high probability of loss‐to‐follow‐up (e.g. young age and high CD4/CD8 ratio) Initiation of ART is not a once in a lifetime opportunity. As mentioned above, in some subgroups, including young individuals with high CD4 counts, low viral load, and preserved CD4/CD8 ratio, the risk of progression and the impact of ART over the ensuing one to 1 years are both low. Thus, a testable hypothesis to decrease losses‐to‐ follow‐up, while at the same time minimizing risks to patients, would be to combine lessons from START and Temprano to design studies of same‐day initiation, shown to improve outcomes as mentioned before. Inclusion could be restricted to individuals with CD4 counts >500 cells/mm3 temporary postponement of treatment initiation is safer. These include individuals aged <30 years and/or with CD4/CD8 ratio of >0.8 and/or viral load of <5000. This is because these individuals are at very low risk of progression in the subsequent 2 to 3 years, the risk is minimally diminished by ART and is virtually identical in the first 18 months of therapy regardless of treatment initiation. In addition, asymptomatic young individuals are at higher risk of loss‐to‐follow and of low adherence to treatment In summary, in some RLS financial constraints make it necessary to prioritize treatment initiation for some groups of patients. In developed countries, there are patients who are reluctant to initiate ART. Subgroup analysis of the START trial can inform recommendations for which patients with CD4 counts >500 cells mm3 with the greatest need for immediate antiretroviral therapy.Despite undisputable evidence of personal and societal benefits of ART, these benefits are small for some groups of individuals and little affected by ART in the short term. It has been demonstrated that biomarkers of inflammation and/or coagulation activation predict risk for serious cardiovascular disease, cancer, and all‐cause mortality Participation in advisory boards and/or honoraria for lectures – Gilead Sciences, GSK/ViiV, Janssen, Merck.


Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing.Buruli ulcer (BU) caused by M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms.Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4.Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable. Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain the M. ulcerans toxin, mycolactone, suggesting continuing infection after completion of antibiotic therapy. In the present study we have determined how soon M. ulcerans was killed during antibiotic treatment using the M. ulcerans 16S rRNA assay combined with qPCR for IS2404 to detect live bacilli in clinical samples and investigated its influence on healing. This assay is more sensitive than culture for the organism. Using samples collected from one hundred and twenty-nine BU patients prior to antibiotic treatment, viable organisms were detected by culture in 34% but the 16S rRNA assay was positive in 65%. The 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks, and 10% at week 16. Lesions with positive 16S rRNA after antibiotic treatment also contained a higher number of bacteria at baseline, had a lower rate of healing at week 4 and took a longer time to heal compared with those in which the organism was undetectable at baseline or by week 4. Positive 16S rRNA was less likely in ulcerative compared with nodular forms of disease 4 weeks after antibiotic treatment. It may be possible to shorten the treatment to 4 weeks in patients with low numbers of bacteria at baseline. Since persistent infection appears to contribute to slow healing, some patients with a high bacterial load at baseline may need antibiotic treatment for longer than 8 weeks.Buruli ulcer (BU) caused by  Mycobacterium ulcerans (Mu) which is common in rural parts of West African countries including Ghana [Buruli ulcer is a neglected tropical disease caused by infection with ng Ghana . It causng Ghana . The ining Ghana  4] 5]. [5].Myco. [5].Mycng Ghana but thereM ulcerans disease in Australia with antibiotic durations of less than 8 weeks suggesting that successful outcomes may be achieved in selected patients  [15 [1514] . With reM. ulcerans IS2404 dry reagent based (DRB) PCR presenting to treatment clinics at the Tepa Government Hospital, Nkawie-Toase Government Hospital, Dunkwa Government Hospital and Agogo Presbyterian Hospital were screened for inclusion. Patients who had already been under antimycobacterial treatment at the time of study initiation were excluded.In the period from June 2013 to June 2015, patients more than 5 years of age with suspected Buruli ulcer and subsequent confirmation by s samples were collected from skin lesions to confirm the diagnosis of Buruli ulcer by microscopy and conventional IS2404 DRB PCR. An additional sample for culture and another for the 16S rRNA reverse transcriptase/IS2404 qPCR assay (16S rRNA) were collected at baseline and during (week 4 and 8) or after treatment (week 12 and 16) from unhealed lesions, immediately placed in either 500μl PANTA media or 500μl RNA protect respectively on site. Human GAPDH mRNA assay was performed on the samples in the RNA protect to assess the stability of the RNA in the solution .Demographic data were collected using standard BU01 forms from the WHO together with a careful history to establish when lesions were first observed and their type. The dimensions of lesions were documented with Silhouette  a 3-dimensional imaging and documentation system together with digital photographs. The Silhouette camera captures an image of the wound, a tracing of the wound boundary is generated and the wound dimensions including the area, depth and volume are automatically calculated. For oedematous lesions, only digital photographs were obtained. Patients were reviewed at 2 weekly intervals during standard antibiotic treatment and monthly thereafter with further recordings of clinical data as routinely conducted for all BU patients until complete healing. These measurements enabled calculation of healing rate at week 4 and predicted healing time in relation to lesion size and type. Rate of healing in mm per week was calculated by subtracting the mean diameter of the lesion in millimeters determined at week 4 from that determined at week 0 and dividing this result by 4. Mean diameter was the mean of the maximum diameter and the largest diameter at right angles to that . Two finAll routine laboratory tests were conducted at Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) immediately upon arrival of samples. Prior to the study a human GAPDH mRNA reverse transcriptase qPCR was established and validated at the Department for Infectious Diseases and Tropical Medicine (DITM) of the University Hospital of the Ludwig-Maximilians-University (LMU) in Munich, Germany. During the study all molecular assays were conducted at the KCCR by trained laboratory staff supervised by Kwame Nkrumah University of Science and Technology (KNUST) staff.M. ulcerans 16S rRNA assay  15] [15]2404 [15]2404FNA and swab samples were transported from study site to the KCCR stabilized in 500 μl RNA protect . Whole transcriptome RNA and whole genome DNA were extracted from the same clinical sample. The RNA and DNA isolation was carried out within 5 hours of sample collection using the AllPrep DNA/RNA Micro kit  as previously described with minor modification. Here, hM. ulcerans whole transcriptome RNA extracted, 2 μl DNA wipe out buffer  was added to 12 μl of the total RNA extracts, incubated for 5 min at 42°C and the reaction was terminated by incubating at 95°C for 3 min. 2 μl gDNA free M. ulcerans whole transcriptome RNA extracted was included as a wipe out control. The remaining M. ulcerans whole transcriptome RNA was then reverse transcribed into cDNA using QuantiTect Reverse transcription kit  according to the manufacturer’s instructions as described elsewhere. The. The18].2404 qPCR to increase the specificity for M. ulcerans and quantification of the bacterial load as previously described  pro proM. ul margins . However margins . Individ margins  28]. Th. ThM. ululcerans . The pre healing . FurtherIn conclusion this study has demonstrated that current antibiotic therapy for BU disease is highly successful in most patients but it may be possible to abbreviate the treatment to 4 weeks in patients with a low initial bacterial load. On the other hand evidence has been presented that persistent infection contributes to slow healing in other patients, probably those with a high bacterial load, who may need antibiotics for longer than 8 weeks.S1 Protocol(DOCX)Click here for additional data file.S2 Protocol(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


Background: Antiretroviral therapy (ART) poses challenging drug-drug interactions with immunosuppressant agents in transplant recipients.  We aimed to determine the impact of specific antiretroviral regimens in clinical outcomes of HIV+ kidney transplant recipients. Methods: A single-center, retrospective cohort study was conducted at a large academic center. Subjects included 58 HIV- to HIV+ adult, first-time kidney transplant patients. The main intervention was ART regimen used after transplantation.  The main outcomes assessed at one- and three-years were: patient survival, death-censored graft survival, and biopsy-proven acute rejection; we also assessed serious infections within the first six months post-transplant. Results: Patient and graft survival at three years were both 90% for the entire cohort. Patients receiving protease inhibitor (PI)-containing regimens had lower patient survival at one and three years than patients receiving PI-sparing regimens: 85% vs. 100% (p=0.06) and 82% vs. 100% (p=0.03), respectively. Patients who received PI-containing regimens had twelve times higher odds of death at 3 years compared to patients who were not exposed to PIs .  Three-year death-censored graft survival was lower in patients receiving PI vs. patients on PI-sparing regimens . Patients receiving integrase strand transfer inhibitors-containing regimens had higher 3-year graft survival. There were no differences in the incidence of acute rejection by ART regimen. Individuals receiving PIs had a higher incidence of serious infections compared to those on PI-sparing regimens . Conclusions: PI-containing ART regimens are associated with adverse outcomes in HIV+ kidney transplant recipients. HIV infection is associated with a two- to three-fold increase in the risk of rejection3. Reduced exposure to immunosuppressive agents is considered the main mechanism for increased predisposition to rejection7.More than 500 kidney transplants in human immunodeficiency virus–infected  and calcineurin inhibitors (CNI), such as tacrolimus, pose a significant clinical challenge. Protease inhibitors (PI) and cobicistat increase the levels of CNI, whereas nonnucleoside reverse transcriptase inhibitors (NNRTI) reduce the levels of these agents. In contrast to PI and NNRTI, integrase strand transfer inhibitors (INSTI), which are not a substrate of CYP450, have become the preferred antiretroviral in many centers to overcome the problematic pharmacokinetic interactions10, it can cause renal tubular dysfunction in HIV+ individuals11 and tenofovir-related nephrotoxicity is always a concern in kidney transplant recipients.Although tenofovir disoproxil fumarate (TDF) has a good safety profile and is recommended as a first-line agent+ kidney transplant recipients is scarce. In the present study, we compared post-transplant outcomes by ART regimens in a group of 58 HIV+ kidney recipients transplanted at our institution over a 9-year period.Data on the impact of specific ART regimens on the clinical outcomes of HIV- to HIV+ adult, first-time kidney transplants performed in the Miami Transplant Institute affiliated to Jackson Memorial Hospital, a 1,550-bed academic medical center, between October 2006 and October 2015. All HIV+ recipients had an undetectable viral load, and all but one (a kidney-liver recipient) had a CD4 count > 200 cells/mm3 at the time of the transplant. The study was approved by the University of Miami institutional review board (#20150614). Written consent was waived by the institutional review board due to the retrospective observational nature of the study.A single-center, retrospective cohort study of 58 consecutive HIV5.Immunosuppression and antimicrobial prophylaxis protocols at our center have been previously described4.The one- and three-year outcomes assessed were: patient survival, death-censored graft survival, and biopsy-proven acute rejection; we also assessed serious infections within the first six months post-transplant, defined as infections requiring admission to the intensive care unit during initial transplant hospitalization or re-admission to the hospital after dischargeThe Fisher exact test and Wilcoxon Mann–Whitney U test were used where appropriate. Univariate analyses were performed using logistic regression with penalized likelihood estimation. Multivariable models were not pursued due to small number of events. Log-rank test was used to assess differences in time-to-event. Statistical analyses were performed using SAS University Edition .+ adult kidney allograft recipients were studied  plus PI, NRTI plus INSTI, and NRTI plus NNRTI (There were no ART restrictions in transplant eligibility for HIVs NNRTI .p<0.01) and 51% (p<0.0005) vs. 17%, respectively  patients underwent ART modifications after transplant; 22 (38%) of them prior to discharge, and an additional 8 (14%) during the first year post-transplant. Adjustments in ART were primarily done to avoid drug-drug interactions or added nephrotoxicity. There was a significant increase in the proportion of patients receiving INSTI at time of discharge and at 12 months post-transplant compared to pre-transplant period: 41% (ctively .p=0.06) and 82% vs. 100% (p=0.03), respectively (p=0.02). Hepatitis C and delayed graft function also increased the odds of death, but this finding did not reach statistical significance , thrombosis/hemorrhagic complications in two (33%), CNI toxicity in one (17%), and unidentified in another patient. The cumulative incidence of biopsy-proven acute rejection was 14 and 17% at one and three years post-transplant, respectively. There were no significant differences in rejection rates by ART ;Table 34. In total, 13 (87%) of these patients were on PI-containing regimens. Individuals receiving PI had a higher incidence of serious infections compared to those on PI-sparing regimens  patients. The etiology of such infections, mainly bacterial and fungal in nature, has been reported previouslyTacrolimus levels at 4, 12, 26 and 52 weeks post-transplant were within therapeutic range for most patient groups . AlthougRosa et al. Impact of ART in KT outcomes in HIV recipients: Raw dataClick here for additional data file.Copyright: © 2016 Rosa R et al.2016Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).5, we observed excellent transplant outcomes without evidence of HIV disease progression. The most important finding of the present study is the association between PI use and adverse outcomes, namely reduced three-year patient and graft survival, and increased risk of serious non-opportunistic infections. These observations remained true in analyses restricted to patients receiving NRTI “backbone”; thus, even after excluding the potential influence of other agents included in the ART regimen, PI continued to be associated with poor outcomes. The immunosuppression protocol at our institution remained constant during the study period, and the proportion of patients transplanted in the 2006–2010 (and consequently the 2011–2015) eras was similar between PI and non-PI groups, suggesting that this observation was also independent of variation in transplant practices over time that might have impacted outcomes.Consistent with previous studies of kidney transplantation in HIV+ individuals has been largely attributed to reduced exposure to immunosuppressive agents, due to drug-drug interactions with ART7. However, in this small cohort, we did not observe an association between ART regimens and the incidence of rejection. CNI levels at 4, 12, 26 and 52 weeks were comparable across ART groups. Other factors, such as infection of the allograft, previous alloimmunization and immune activation, might also play a role in predisposition to rejection6.Biopsy-proven acute rejection and CNI toxicity accounted for half of the cases of graft loss in patients taking PI in the present study. Increased risk of allograft rejection in HIV+ kidney recipients3, especially those with marginal pre-transplant CD4 counts4. Notably, the occurrence of serious infections in this cohort was almost five-fold higher in patients receiving PI.Non-opportunistic infections within six months post-transplant are common in HIVThis might be due to the effects of PI on tacrolimus levels, considering that the overwhelming majority of these patients were on PI-containing regimen and more-than-half had tacrolimus levels above target at the time of infection. PI could also influence the net state of immunosuppression by increasing the level or effect of other immunosuppressants, such as prednisone and mycophenolate.12. Whether there is added clinical benefit of TAF over TDF in kidney transplant recipients remains to be established.Contrary to our expectations, the use of NNRTI or TDF did not influence kidney allograft survival. Tenofovir alafenamide (TAF) is a new formulation of tenofovir associated with less kidney (and bone) toxicity9, patients receiving INSTI-containing regimens had excellent patient survival (96%) and graft survival (100%) at three years, and the lowest rejection rates in this cohort (8%). Current guidelines recommend the use of NRTI plus INSTI as a first-line therapy for HIV10. INSTI pose no interactions with CNI or mTOR inhibitors. In addition, INSTIs have no interactions with direct-acting antivirals, which is important in the setting of hepatitis C co-infection, as that has been associated with poor outcomes3. Thus, it has become our practice to preemptively switch HIV+ candidates pre-transplant or in the immediate post-transplant period to PI-sparing, preferably INSTI-based, ART regimens.Consistent with recent reports13. HIV+ recipients and their community HIV providers should be educated about what ART medications to avoid, and when not possible, how to adjust CNI doses and monitor levels accordingly.Although none of the patients studied here was on cobicistat, it is important to highlight that this pharmacokinetic enhancer, contained in several combination pills, can increase the levels of CNI+ kidney recipients seems to be the most prudent approach.Our study is limited by the small number of patients and retrospective design; serum levels for other immunosuppressants, such as mycophenolate were not available. The association found in the present study between PI-containing ART regimens and adverse outcomes needs to be confirmed in larger studies. Until more data becomes available, the use of PI-sparing regimens in HIVThe data referenced by this article are under copyright with the following copyright statement: Copyright: © 2016 Rosa R et al.Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).et al. Impact of ART in KT outcomes in HIV recipients: Raw data. doi,Dataset 1: Rosa10.5256/f1000research.10414.d14671714. This manuscript is a retrospective single- canter analysis of the outcomes of kidney transplantation in HIV infected recipients stratified by anti-retroviral therapy (ART) used. The authors found inferior 3 year graft and patient survivals in patients on protease inhibitor (PI) containing regimens. Six month opportunistic infections were also high in patients on PI-containing regimens. ART with integrase inhibitors was associated with better outcomes. Integrase inhibitors such as raltegravir has minimal drug-drug interactions and are being increasingly used in ART regimens. Overall, the manuscript is written well and conclusions are supported by the study findings.Was there any difference in outcomes based on induction agents used?Any postulations as to why the observed inferior graft and patient outcomes in the PI group other than drug-drug interactions? I have following comments:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et. al. is a well-done study with contributions from a multi-disciplinary group of medical providers . The findings add to a growing experience on the optimal management of HIV+ renal transplants including use of ART. Specifically, it provides statistical analysis demonstrating that PI-containing regimens are significantly associated with worse 3 year survival, 3 year graft function and severe infection rates at 6 months in a relatively large group of patients at a single transplant center.  The results add further weight to our own findings at our center1 about the preferential use of INSTI regimens in this population preferentially used pre-emotively before transplant.The paper entitled" Impact of antiretroviral therapy on clinical outcomes in HIV+kidney transplant recipients: Review of 58 cases" by Camargo The study design is well explained and tables and figures are clear.  The conclusions are balanced and justified on the basis of the data. My main question is that the outcomes are based on the ART regimen in the immediate post-transplant period (there were 33 patients on PI-containing regimen).  However, the authors state that at 12 months post-transplant, of their available data, only 21 were on PI-containing regimen, due to switches made, in part due to concerns for drug-drug interactions.  So the conclusions regarding 3 year patient and graft survival may not reflect an on-treatment analysis. In other words, at 3 years, how many patients were still actually taking PIs? A few other questions to clarify the study would be helpful: 1) There were 6 deaths at 3 years:  what were the causes of death?  Infections? How many were on PIs at time of death? 2) What were the specific antiretrovirals used? How was dosing altered post transplant? 3) Can the authors speculate on what other mechanisms might explain why PI use (aside from effect on CNI or mTOR levels) could affect patient and graft survival?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


In order to assess the level of transmitted and/or pre-treatment antiretroviral drug resistance to HIV-1, the World Health Organization (WHO) recommends that regular surveys are conducted. This study’s objective was to assess the frequency of HIV-1 antiretroviral drug resistance in patients initiating antiretroviral treatment (ART) in the public sector throughout South Africa.Pol sequences were obtained using RT-PCR and Sanger sequencing and submitted to the Stanford Calibrated Population Resistance tool v6.0.A prospective cross-sectional survey was conducted using probability proportional to size sampling. This method ensured that samples from each province were proportionally collected, based on the number of patients receiving ART in each region. Samples were collected between March 2013 and October 2014. 3 (IQR: 62–249) and, based on self-reporting, participants had been diagnosed as HIV-positive approximately 44 days prior to sample collection (IQR: 23–179). Subtyping revealed that 98.2% were infected with HIV-1 subtype C. Overall, 25 out of 277 patients presented with ≥1 surveillance drug resistance mutation . Non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations were the most numerous mutations detected (n = 23). Only two patients presented with a protease inhibitor (PI) mutation. In four patients ≥4 SDRMs were detected, which might indicate that these patients were not truly ART-naïve or were infected with a multi-resistant virus.A total of 277 sequences were available for analysis. Most participants were female (58.8%) and the median age was 34 years (IQR: 29–42). The median baseline CD4-count was 149 cells/mmThese results show that the level of antiretroviral drug resistance in ART-naïve South Africans has reached moderate levels, as per the WHO classification. Therefore, regular surveys of pre-treatment drug resistance levels in all regions of South Africa is highly recommended to monitor the changing levels of pre-treatment antiretroviral drug resistance. South Africa has the largest antiretroviral treatment (ART) program in the world with an estimated cumulative number of individuals on treatment of 3.1 million. The preThe definition of TDR can theoretically only be applied to recently-infected individuals as transmitted mutations may revert back to wild type, or only be present at levels below the sensitivity of standard genotyping assays . Since i3 were on treatment by the end of 2012, an increasing level of transmitted and/or pre-treatment drug resistance in the South African population can be expected.The WHO has classified levels of TDR into three categories: low (<5%), moderate (5–15%) and high levels (>15%) . The 201Since 2002, various surveys addressing the level of transmitted or pre-treatment drug resistance have been conducted in South Africa. For ease of reference no distinction was made between studies that targeted only recently-infected individuals versus any patient prior to ART initiation. Gauteng and KwaZulu-Natal were the most surveyed regions. The studies conducted in Gauteng, Free State and Mpumalanga consistently detected a low prevalence of TDR –14. The This study therefore aimed to conduct the very first national pre-treatment drug resistance survey to assess the current situation in the whole of South Africa.In estimating the proportion of ART naïve patients that had transmitted or pre-treatment resistance at a national level, we had two considerations: the size of the sample and how to select samples to represent South Africa. For the size of the sample, with 277 sequences we had sufficient statistical power to detect a 10% prevalence with a margin of error of +/- 3.5%, which we deemed sufficient to estimate this proportion. We selected the samples from the provinces proportional to the HIV population of the sample in order to achieve a proportional distribution of ART naïve individuals using probability proportional to size sampling (PPSS). South Africa has 9 provinces and 52 districts. Only tier two facilities with at least 500 patients in ART care were considered, to ensure adequate sampling options. The number of patients in care per facility was obtained from the South African District Health Information System. Any patient initiating ART or attending a pre-treatment follow-up visit, without prior ART exposure, who was at least 18 years of age and had signed informed consent, was eligible to be included in the study. Due to the lack of research staff at the health care facilities, a non-random sample of patients was used for this survey.pol sequences. The in-house genotyping assay used at Tygerberg hospital covers protease (PR) amino acid (aa) 1 to 99 and reverse transcriptase (RT) aa 1 to 262 and has a lower limit of detection of 400 copies/ml [Health care professionals at sentinel sites informed eligible patients of the HIV antiretroviral drug resistance survey. After obtaining written informed consent, a study questionnaire was completed. The study questionnaire included questions regarding basic demographics, ART history and CD4 counts. Subsequently, whole blood was collected in EDTA-vacutainers. The samples were then sent to one of three participating HIV genotyping laboratories: Tygerberg Hospital, Stellenbosch processed samples collected in the Western Cape; Universitas, Bloemfontein, processed samples collected in the Free State and the laboratory at Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg processed the samples collected in the remaining provinces. To ensure sample integrity, it was advised that the specimens should reach the relevant laboratories within 72 hours after sample collection. Upon receipt, samples were centrifuged and plasma was stored at -80°C until processing. Each laboratory used their own validated population-based in-house genotyping method to obtain opies/ml  The in-hopies/ml , 25.http://cpr.stanford.edu/cpr.cgi) was used to interpret the level of pre-treatment drug resistance in the ART naïve population. This tool is based on the 2009 WHO surveillance drug resistance mutation (SDRM) list [http://sierra2.stanford.edu/sierra/servlet/JSierra) was used to generate predicted resistance profiles. Predicted antiretroviral drug resistance profiles were categorised as either being susceptible , intermediate (including low-level resistance) or high-level resistance.All nucleotide sequences obtained were collated at Charlotte Maxeke Johannesburg Academic Hospital for analysis. In addition, the Stanford CPR v6.0  tool (RM) list . The Stahttp://hiv-web.lanl.gov), were generated using Clustal X (version 2.1)  to determine subtypes as well as confirm there was no cross-contamination amongst samples. The generated Clustal X alignment was used to construct a Neighbor-Joining phylogenetic tree in MEGA 7 (http://www.megasoftware.net) with the Kimura two-parameter model. The stability of the nodes was assessed by bootstrap analysis (1000 replicates), and bootstrap values greater than 70% were considered significant. An additional phylogenetic analysis was conducted for all sequences with at least one SDRM, to exclude clustering among resistant sequences.A multiple alignment of the 277 nucleotide sequences with the 2010 subtype reference sequences from HIV-1 subtypes A to K, and several CRFs (pol subtyping was confirmed by using the Rega HIV subtyping tool v2.0 (http://bioafrica.net/rega-genotype/html/subtypinghiv.html). Any sequence that was assigned to be a non-C subtype was also analysed using the NCBI genotyping tool (http://www.ncbi.nlm.nih.gov/projects/genotyping/) and the jumping profile HMM tool (http://jphmm.gobics.de/submission_hiv.html) to confirm the subtype.In addition Statistical analyses were performed using GraphPad Prism 6. For all prevalence calculations, 95% confidence intervals were calculated using the modified Wald Method.pol nucleotide sequences were submitted to GenBank using Sequin v13.70 (www.ncbi.nlm.nih.gov/Sequin) and are available under accession numbers KT892975-KT893251.The This study was conducted with ethical clearance by the Research on Human Subjects  Committee at the University of the Witwatersrand (Clearance Number M120254). Written informed consent was obtained from all participants prior to sample collection.3 (IQR: 62–249).Patient samples were collected between March 2013 and October 2014. Only 320 samples out of the originally targeted 336 samples were collected. Samples from North West and Western Cape provinces were underrepresented due to the inability to motivate health care workers and/or patients to participate in the survey. For 43 samples, sequences could not be included due to the inability to obtain a PCR product (n = 31), inadequate completion of the consent form (n = 5) or participants being younger than 18 years (n = 7). Finally, a total of 277 sequences were available across all nine provinces , S1 Fig.pol subtype was HIV-1 subtype C (98.6%). Five sequences belonged to non-C subtypes. One patient from the Eastern Cape was infected with HIV-1 subtype D; two patients from Gauteng were infected with HIV-1 subtype B and CRF02_AG, respectively. The last non-C subtype was detected in KwaZulu-Natal (recombinant between subtype C and A2).The most common Overall, 25 out of 277 surveyed patients presented with at least one antiretroviral drug resistance mutation. This finding translates into an overall moderate pre-treatment HIV-1 resistance prevalence of 9.0% , lamivudine/emtricitabine (3TC/FTC) and efavirenz (EFV) as per national guidelines . ResultsThis pre-treatment antiretroviral drug resistance survey was designed to assess the current prevalence of HIV-1 drug resistance among South African HIV-infected patients prior to ART initiation in all nine provinces for the first time. The obtained data is important to verify whether the currently recommended first-line ART regimens are still adequate to ensure a good treatment outcome in most HIV-infected patients initiating these regimens. This is the first survey which includes samples from every province in South Africa, based on the proportion of patients receiving ART in each region.The observed pre-treatment resistance prevalence is classified as a moderate level of 9.0% (95% CI: 6.1% to 13.0%) according to the WHO classification. This level of pre-treatment drug resistance was only detected in three South African studies prior to this survey: 9.3% in Limpopo  and 7.4 et al. demonstrated in a large multi-centre study that pre-treatment drug resistance does have an impact on the outcome of first-line treatment with an odds ratios for increased virological failure of 2.13 (95% CI: 1.44–3.14). In addition, patients with pre-treatment drug resistance are more likely to develop acquired drug resistance as compared to those patients without pre-treatment drug resistance  [Based on these findings, only 2.2% of the population initiating ART would likely be started on monotherapy, or a completely inactive regimen and 6.1% of the study population would be initiated on dual ART (active TDF and 3TC/FTC). Hamers It can be argued that the patients with multiple antiretroviral drug resistance mutations, without recorded treatment history, were either not truly drug-naïve or might have been infected with a multidrug resistant virus. Several studies, especially cluster transmission studies, have shown that resistance mutations with minimal impact on viral fitness may be transmitted serially without the need for intervening drug pressure , 30. HowDespite the adequate sample size and PPSS study design, the study had some limitations. Firstly, the enrolment of patients into the study was largely dependent on the willingness of clinicians and nurses to obtain consent from patients, complete study questionnaires and collect specimens, as there was no provision of study-specific staff to assist in the survey. These activities place a burden on clinics that are often already under-resourced and thus demonstrate the difficulties in conducting such surveys and the vast costs associated with their completion. These challenges resulted in a relatively long period to complete sample collection. Overall 82% of the planned sample size was collected and analysed. But there was under-sampling from North West, and Western Cape provinces.Secondly, patients were not systematically sampled at the clinic level. This approach might have introduced a selection bias as sample selection was dependent on willingness of health care workers and patients to comply with the added burden of the study. However, we don’t believe that it is possible for clinicians to choose treatment naïve patients based upon demographic or clinical characteristics that would bias the proportion of SDRM.3. It is known that TDR mutations can revert back to wild type in the bulk viral population, especially without the presence of drug pressure. It is therefore possible that archived mutations were missed in this analysis. However, these limitations are largely overcome by the large sample size of the study and the fact that samples were collected from all provinces, proportional to the number of patients on ART.Thirdly, data quality was dependant on the completeness and accuracy of the questionnaires. Moreover, no specific information regarding exposure to Prevention-of-Mother-to-Child-Transmission prophylaxis was obtained and may have potentially overestimated the level of pre-treatment drug resistance caused by the use of PMTCT regimens, especially since most patients were female. In addition, information regarding prior ART exposure could only be verified based on verbal confirmation from participants or notes in the patient’s hospital files, which might have led to the inclusion of patients with undisclosed ART exposure. Finally, resistance mutations were detected by population-based Sanger sequencing which might underestimate the prevalence of minority drug resistance mutations, due to sensitivity limitations of this approach, especially in patients who have been chronically infected before ART initiation. This study might have underestimated the level of pre-treatment resistance; since it is likely the sample included a substantial proportion of chronically infected patients, indicated by the low median CD4 count of 149 cells/mmOverall, this study has shown that the level of antiretroviral drug resistance in ART-naïve South Africans is on the rise. Regional analyses indicate that there might be a difference in the level of drug resistance between provinces, although this trend needs to be confirmed by repeating surveys in each province with larger sample sizes. Despite the availability of routine viral load testing in the public sector, there is often a delayed response to changing management based on virological failure which could have contributed to the transmission of drug resistance . These fS1 FigEach circle indicates a health care facility that contributed specimens to the survey. The size of the circles is proportional to the number of specimens collected at each health care facility. The grey scale indicates the number of patients on ART in each province.(DOCX)Click here for additional data file.S2 FigNo clustering is observed among these samples.(DOCX)Click here for additional data file.


Most patients  remained anemic during follow-up. Weight gained  and QOL  were lower for PLWHA with versus without IHS. Relative to anemia-free/normal ferritin, the risk of hospitalization/death was elevated for PLWHA with anemia , low or high ferritin  and those that developed new/persistent/progressive anemia . Among PLWHA, IHS predicted deficits in QOL, low weight gain and a high risk of hospitalization/death. Intervention to mitigate persistent IHS may be warranted among PLWHA on long-term highly active antiretroviral therapy (HAART) to improve health outcomes.Impaired hematologic status (IHS) was investigated as a determinant of immune function defined as cluster of differentiation 4 (CD4) T-helper cell count, quality of life (QOL) weight and hospitalization/mortality over 18-months among 398 adult persons living with HIV/AIDS (PLWHA) on anti-retroviral therapy. IHS was defined as having anemia at baseline (Hemoglobin: <12 g/dL for women and <13 g/dL for men), time-updated anemia or having low (<30 μg/L) or high (>200 μg/L for men and >150 μg/L for women) ferritin levels at baseline. Months-to-hospitalization/death or study-end (if no event) was calculated from enrollment. Multivariable linear-mixed models quantified associations between IHS and changes in CD4 cell-count, weight gain and QOL. Cox proportional hazards models calculated hazard ratios (HR) and corresponding 95% confidence intervals (CI) for IHS-related differences in time-to-hospitalization/death. The prevalences of anemia and high and low ferritin levels at baseline were 48.7% ( Impaired hematologic status (IHS)—including anemia and high or low ferritin—remains highly prevalent in chronic HIV despite highly active antiretroviral therapy (HAART) ,2,3. HIVFerritin is commonly measured in epidemiologic studies as biomarker of bio-available iron . High seAmong PLWHA on long-term HAART, the morbidity significance of IHS–particularly hypoferritinemia and anemia persistence, is poorly understood. This study informs existing knowledge gap by evaluating IHS as a determinant of adverse immunologic and clinical outcomes among adults with chronic HIV-infection stably connected to HIV-care in Kampala, Uganda. It specifically tests the hypothesis that impaired hematologic indices and/or their persistence over time will be associated with worse outcomes in treated HIV despite HAART.This longitudinal study uses data from multiple micronutrient trial  conducted among HIV-infected Ugandan adults between 2009 and 2012. All participants had World Health Organization (WHO) HIV disease stage 3 or higher, were initiating HAART upon study enrollment or had been on HAART for six months or less. Participants were followed-up every six months for a total of 18 months [Ferritin was measured at enrollment only in serum using enzyme-linked immunosorbent assays (ELISA) ,22. Per Six profiles of time-varying anemia were defined to classify major patterns of anemia evolution within PLWHA and allow explicit comparison of health outcomes among them. Patterns of anemia persistence expected to have a similar degree of impact on clinical outcomes were combined within profiles to enhance statistical efficiency for quantifying the association between this variable and respective outcomes.(i)Persistently anemia free: no anemia in any observation period.(ii)Completely Resolved Anemia: mild or moderate/severe anemia at enrollment only without evidence of anemia in any follow-up interval.(iii)Incompletely Resolved Anemia: baseline moderate/severe anemia that was down-modulated to mild in ≥1 follow-up interval.(iv)Incident moderate/severe or sustained mild anemia: baseline anemia that became moderate/severe in one of three follow-up intervals or mild baseline anemia that was sustained in ≥2 follow-up intervals.(v)Mild Anemia: one episode of mild anemia at baseline or developed mild anemia in one of the follow-up intervals.(vi)Progressive or Persistent moderate/severe Anemia: baseline moderate/severe anemia that was sustained in ≥2 assessment intervals or mild baseline anemia that progressed to moderate/severe in ≥1 follow-up intervals.Four outcome variables were each assessed at months 0, 6, 12 and 18.(i)Immune function: cluster of differentiation 4 (CD4) T-helper cell count: absolute T-cell lymphocyte count in cells/micro liter was measured using a FACS Calibur flow cytometer .(ii)Body mass index (BMI): was calculated as the ratio of weight (in kilograms) to squared height (in meters).(iii)QOL: assessed with the Medical Outcomes Study HIV Health Survey translated and culturally adapted for the study area . As desc(iv)Death/hospitalization: Occurrence of hospitalization or death event was, for each participant, a composite endpoint to enhance statistical efficiency and provide a more complete picture of serious adverse clinical outcomes among PLWHA, which should become rarer in the HAART era. Censoring was at date of first hospitalization or death. Participants that experienced both events were censored at hospitalization.Age was measured in whole years categorized by the approximate quartiles of age distribution in our sample as: 18–29, 30–35, 36–41 and 42+. Socioeconomic status was defined by years of education, employment status and household wealth or assets owned. Composite scores were categorized at quintiles . Historyα = 0.05. In addition, we estimated differences in outcomes according to high or low versus normal baseline ferritin, baseline anemia and baseline anemia-severity versus no anemia at months 0, 6, 12 and 18. Next, anemia persistence variable was used as an explanatory variable in the regression analysis of each outcome  at month 18. We constrained this analysis only to month 18 to ensure temporal sequence between evolution of anemia severity since enrollment and respective outcome measures. Lastly, we used Cox regression to test whether baseline ferritin level, baseline anemia (including severity) and anemia persistence were associated with time to hospitalization or death over 18 months of follow-up.In addition to baseline assessment, three repeated measures of CD4 + T-cell count, BMI and QOL  were analyzed using multivariable linear mixed effects models in relation to baseline ferritin status and baseline anemia (including anemia severity). An unstructured covariance matrix was assumed to accommodate non-independence of repeated outcome assessments within participants. A random intercept was specified to allow within-subject differences in outcome trajectory from enrollment. Empirical standard errors were used to derive robust measures of variance and to mitigate potential misspecification of the covariance matrix. We tested the null hypothesis of no hematologic status related differences in absolute CD4, BMI and QOL over 18 months at To address possible confounding by extraneous covariates, we adjusted the analyses for baseline measures of behavioral factors (smoking and drinking), socio-demographic characteristics , parent study trial arm, baseline HAART experience and vitamin D status. Final multivariable regression models included independent predictors of respective outcomes and candidate confounders as described in the literature. We adjusted for baseline CD4 and BMI in models where they are not primary outcomes. All analyses were implemented in Statistical Analyses Software (SAS) version 9.4 .At enrollment in the parent study, each participant provided written informed consent administered in the local language of Luganda. The study was approved by the Scientific Review Committee of the Infectious Diseases Institute at Makerere University College of Health Sciences and the Institutional Review Boards of Harvard School of Public Health (protocol number: 17361) and that of Makerere University School of Public Health (protocol number: HDREC 067). The study was further registered and approved by the Uganda National Council for Science and Technology .Low, normal and high ferritin groups were similar in terms of material wealth, prevalence of alcohol use, multivitamin use, mean CD4 cell count, vitamin D level, employment/work status, QOL score and education. Participants with high ferritin group tended to be older, have lower BMI, higher C-reactive protein (CRP) and slightly more anemia. Participants with low ferritin tended to be younger, include more females and were less likely to rate current health as good, very good or excellent. .n = 58) of baseline anemia was completely resolved and another 17.5% (n = 34) was only partially resolved. However, anemia developed after enrolment in 14.7% (n = 30) of PLWHA without baseline anemia. Anemia persisted at some level for most PLWHA with baseline anemia , including 19.6% (n = 38) for those whose anemia either progressed from mild to moderate/severe or was sustained at moderate/severe from enrollment  decreased from 48.5% at enrollment to 22.7% by month 18 . By studrollment .p = 0.78) . Specifically, patients with low or high ferritin status had no significant difference in weight gain compared to patients of normal ferritin status during months 0 and 6. By month 12 and 18 however, patients with low or high baseline ferritin gained between 0.2 and 0.5 kg/m2 more weight than those with normal baseline ferritin. This superior weight gain was only significant for high versus normal ferritin in the interval between 12 and 18 months. QOL scores over time was lower for persons with high or low versus normal baseline ferritin at three out of four follow-up intervals. Low versus normal baseline ferritin related deficits in QOL scores  was statistically significant at month 18 only.Baseline ferritin showed no association with absolute CD4 cell count over the study period  . Howeverp-value for anemia × time = 0.56) or its severity  at baseline. The change in weight gain and QOL over time differed by baseline anemia severity. Specifically, at enrolment, PLWHA with severe anemia  weighed significantly less than anemia-free PLWHA. The magnitude of baseline weight difference was lower and statistically insignificant for those with mild versus no anemia. During follow-up, all participants gained weight and magnitude of baseline anemia status dependent differences in weight gain declined and became statistically insignificant from study month six. The trend in QOL change by baseline anemia groups was similar to that for weight gain. Specifically, the evidence for anemia dependent differences in QOL existed at baseline only for persons with moderate/severe versus no anemia . Post-enrolment, anemia-dependent deficit in QOL was either reversed in direction or its magnitude was substantially smaller relative to baseline  and the amount of weight gained (p < 0.01) at month 18  and those whose anemia partially resolved gained 1.4 kg/m2  more weight. There was no association between anemia persistence and QOL at month 18.Anemia persistence was significantly and adversely associated with magnitude of CD4 cell count recovered  than those with no anemia.Among adult PLWHA from Uganda, IHS was highly prevalent at or near HAART initiation with 48.7 anemic, 17% with hypoferritinemia and 40.5% with hyperferritinemia at enrolment in this study. Anemia prevalence rate is slightly lower than the 69% and 70.5% rate respectively reported among adult HIV+ Congolese and South Africans at HAART initiation . On the In line with our study hypothesis, participants with baseline anemia had relative disadvantage with respect to weight gain and QOL at enrolment. Over the study period, as participants stabilized on HAART, these baseline anemia-related deficits in weight gain and QOL outcomes were strongly down-modulated. The contributory role of HAART for resolving pre-HAART anemia in PLWHA has been described ,3. IndeeOur data further suggests that PLWHA who have extreme ferritin levels experience more weight gain by study month 12 than those having normal ferritin levels. This association was statistically robust only among persons with baseline hyperferritinemia, an observation that we speculate is explained by relative changes in the etiology of anemia among persons on long-term HAART. This is the subject of ongoing investigation in our group. Briefly, our unpublished data shows that burden of ACD is greatest in this group 47%) at enrollment compared to participants with low (19.5%) and normal ferritin levels (40.2%). Equally relevant for explaining the differential weight gain among participants with high baseline ferritin, our unpublished data show that the resolution of ACD is highest among baseline hyper-ferritinemic participants by study month 18. Hence, HAART driven resolution of ACD will enable this group experience superior weight gains in the long term. We also noted that baseline low versus normal ferritin was associated with lower QOL by study end. This observation is consistent with the predominance of iron deficiency anemia, folate deficiency and other anemia of nutritional origin in this group. These kinds of anemias maybe beneficially impacted by iron and micronutrient supplementation but not necessarily HAART. Prior work has demonstrated that release of dietary iron from enterocytes is inhibited in the context of inflammation that is characteristic of HIV disease ,7. Hence% at enroRelationships between ferritin categories and most outcomes investigated demonstrated a U-shaped response where associations with morbidity/mortality were similar in direction for PLWHA with high or low compared to normal ferritin. Low ferritin and anemia is expected to increase fatigue, irregular heartbeat and depressed mood , whereasTime-varying anemia severity–particularly the development of moderate anemia, progressive, persistent and incomplete resolution of existing anemia, consistently predicted lower immune recovery by study end. Conversely, resolution of existing anemia–even when incomplete, was associated with substantial weight gain advantage in comparison with persistently anemia free patients. Our finding that non-hospitalized survival time was significantly higher and the risk of hospitalization or death was significantly elevated for persons with poor hematologic profile is internally consistent with associations noted herein with soft morbidity indicators–weight gain, CD4 cell count and QOL and with extant literature . Few priStrengths of this investigation include prospective design that permits temporal inference, a long follow-up duration with repeated assessment of hemoglobin, limited loss of participants to follow-up and control for potential confounders. The emphasis on hospitalization events and typical morbidities—weight loss, QOL and immune recovery appropriate for the post-HAART era of chronic HIV are additional strengths. However, our data does not allow for specific understanding of the predominant forms of anemia that develop or persist in HIV-positive persons on HAART. For example, is it possible that with extended time on treatment and expected down-modulation of severe inflammation that emergent anemia and/or anemia that persists in treated HIV is increasingly iron deficiency anemia and nutritional in etiology? Future prospective studies to understand variations in causes of anemia over time on HAART among PLWHA will crucially inform the appropriate clinical course of action for managing anemia in this population.In summary, we confirm previously reported observations of a high IHS burden among PLWHA on HAART and the spontaneous resolution of anemia over 18 months on HAART for some PLWHA. We extend current knowledge base by providing novel empirical data on the adverse impact of persistent anemia on immune recovery, hospitalization and mortality. Of equal importance, we show that resolution of baseline anemia was associated with comparable immune recovery, QOL and risk of hospitalization/death over 18 months relative to PLWHA anemia free throughout. We conclude that intentional management of anemia of all etiologies in chronic HIV-infection presents a key opportunity to enhance functional survival in the increasing number of PLWHA on long-term HAART. Our data shows that despite spontaneous resolution of anemia for some, anemia remains highly prevalent in PLWHA over 18 months on HAART. With antiretroviral therapy-related down-modulation of high level inflammation, the etiology of persistent anemia differs relative to pre- HAART. This possibility should be specifically investigated in future studies among PLWHA on HAART. Given the high prevalence of persistent anemia and the possibility that these could be nutritional or iron-deficiency in etiology, future iron supplementation interventions to clarify the safety, efficacy and proper targeting of iron supplements in PLWHA may be warranted. Such trials should ideally lag HAART initiation and begin when the level of inflammation in PLWHA has been down-modulated by HAART to maximize benefit derived from iron supplementation and minimize the risk of iron-overload.


Primary study outcome was absence of treatment failure  per protocol at week 48.Patients on 3-drug ART with stable HIV-1 RNA <50 copies/mL and CCR5-tropic virus were randomized 1:1 to maraviroc with darunavir/ritonavir qd (study arm) or continue current ART (continuation arm).In June 2015, 115 patients were evaluable for the primary outcome . The study was discontinued due to excess of VF in the study arm . The proportion free of treatment failure was 73.2% in the study and 59.3% in the continuation arm. Two participants in the study and 10 in the continuation arm discontinued therapy due to adverse events (p = 0.030). At VF, no emergent drug resistance was detected. Co-receptor tropism switched to non-R5 in one patient. Patients with VF reported lower adherence and had lower plasma drug levels. Femoral bone mineral density was significantly improved in the study arm.Switching to maraviroc with darunavir/ritonavir qd in virologically suppressed patients was associated with improved tolerability but was virologically inferior to 3-drug therapy. Antiretroviral therapy (ART) significantly reduces morbidity and mortality associated with HIV infection; this is best achieved by chronically maintaining undetectable levels of HIV-1 RNA in patients plasma –4.The long-term side effects of NRTIs are still of concern. Some agents are associated with renal and bone toxicity –7, otherThe efficacy of maraviroc as maintenance therapy in virologically controlled patients has been recently tested in the MARCH study . In thisThe aim of this study was to test non-inferiority of an NRTI-sparing dual therapy based on once daily maraviroc with darunavir/ritonavir as compared to continuing an ongoing, virologically successful 3-drug therapy. The experimental regimen tested here is not according to the label of maraviroc. A secondary aim was to perform a prospective validation of selecting patients with R5 virus based on single-sequence viral DNA genotyping .www.clinicaltrials.gov, number NCT01367210). The study protocol was authorized by the Italian Drug Regulatory Agency (AIFA) and approved by the local Ethics Committees at each study center; written informed consent was obtained from all patients before participation. The study was performed in accordance with the ethical guidelines of the Declaration of Helsinki (7th revision) and with the International Conference on Harmonization Good Clinical Practice guidelines. The authors confirm that all related trials for this intervention are registered. Study approval by the Ethics Committee of the Catholic University was obtained on November 18, 2010. Between May 23, 2011 and April 22, 2015, patients underwent screening for the study and the latest visit was on September 28, 2015. The complete list of Ethics Committes that approved study is: Ethics Committee of Umbria and Perugia; Ethics Committee of Azienda Ospedaliera Sacco, Milan; Ethics Committee of Modena; Ethics Committee Azienda Ospedaliero-Universitaria of Florence; Ethics Committee University of Study D’Annunzio and Chieti; Ethics Committee AUSL 9 of Grosseto; Ethics Committee Azienda Ospedaliera Universitaria S Martino, Genova; Ethics Committee AUSL 3 Pistoia; Ethics Committee Azienda Ospedaliera S. Salvatore of Pesaro; Ethics Committee AOU San Luigi Gonzaga of Orbassano; Ethics Committee Azienda Ospedaliero-Universitaria Policlinico S. Orsola-Malpighi of Bologna; Ethics Committee IRCC of Rome; Ethics Committee of Lecce; Ethics Committee Policlinico Umberto I of Rome; Ethics Committee Lazio 2; Ethics Committee IRCC Spallanzani of Rome; Ethics Committee San Paolo Hospital, Milano.GUSTA (GUided Simplification with Tropism Assay) was a multicenter, open-label, randomized study  were excluded.Main exclusion criteria were having a CD4 cell count at nadir <50 cell/mmprotocols.io repository with doi 10.17504/protocols.io.jmnck5e. Genotypic resistance to NRTIs, NNRTIs and PIs was determined using the Viroseq HIV-1 Genotyping System.All patients underwent genotypic testing for HIV-1 coreceptor tropism at screening by population sequencing on whole blood using a single DNA sequence of the gp120 V3 env region . DNA seqAt baseline, eligible patients were randomized 1:1 to receive maraviroc 300 mg qd  plus darunavir/ritonavir 800/100 mg  or to continue the previous antiretroviral regimen . Follow-up visits were scheduled at weeks 4, 12 and every 12 weeks thereafter. Routine physical examination and laboratory tests, including CD4 cell counts and HIV-1 RNA levels, were performed at baseline and at each follow-up visit.Treatment failure was defined as any of the following: virological failure, discontinuation or change of any current drug due to any cause, withdrawal of consent after treatment initiation, loss to follow-up, progression to AIDS or death. Virological failure was defined as the second of two consecutive HIV-1 RNA levels >50 copies/mL or a single HIV-1 RNA level >1,000 copies/mL. At the time of virological failure, an additional visit and blood sampling were performed. Clinical and laboratory adverse events occurring during the study were graded according to the latest available Division of AIDS tables. A Data Safety Monitoring Board was established to monitor patient safety and treatment efficacy data.The primary study endpoint was the proportion of patients free of treatment failure at 48 weeks on the per-protocol population. Based on the assumption that 90% of patients in the continuation arm would meet the primary outcome, enrolment of 330 patients distributed 1:1 in each study arm was planned in order to test non-inferiority of the study arm with a lower bound of 10%, a confidence interval of 95% and a power of 80%. Secondary endpoints were: the proportion of patients with treatment failure and virological failure at 48 weeks on the intention-to-treat population, modification of CD4 cell count and of blood metabolic parameters, changes in bone parameters, self-reported adherence and symptoms scores, and of health-related Quality of Life from baseline to week 48.At baseline and at week 48, patients from 7 study sites underwent dual-energy X-ray absorptiometry scans to analyze bone mineral density (BMD) . Total L2–L4 lumbar column and femoral neck BMD were recorded. Moreover, plasma alkaline phosphatase , 25(OH)vitamin D, parathyroid hormone (PTH) and osteocalcin were measured.trough were calculated accordingly using the formula C = C0 × e-kt ; e = base of natural logaritm). Liquid/liquid extraction was employed for purification and concentration of maraviroc, darunavir and ritonavir from plasma samples. Samples were injected into an AQUITY UPLC system employing an AQUITY UPLC BEH C18 column  for analysis. The UPLC was connected to a triple quadrupole tandem mass detector  for the mass spectrometric detection. Data were processed using MassLynx with a QuanLynx program version 4.1 . The detection limit for the drug determination was 30 ng/mL. We computed Ctrough when maraviroc levels were available 6–27 hours after intake and darunavir 10–27 hours after intake, i.e. during the linear phase of the elimination curve. All patients were instructed to take the study medications with food. In both arms, self-reported adherence , patient-reported symptoms and physical/mental Quality of Life (QoL) scores (weeks 0 and 48) were collected using validated questionnaires [Plasma drug levels were measured in arm S in blood collected before morning antiretroviral drug intake between week 4 and 48 by a validated tandem mass spectrometry (UPLC-MS/MS). The time of the last drug intake and of blood sampling were recorded for each patient and Connaires . Self-reDescriptive statistics were used to describe baseline patients characteristics, main study outcomes, laboratory toxicities, clinical adverse events and serious adverse events. Differences between categorical variables were tested by the Chi-square test, differences between continuous variables were tested using the t-test or the Mann-Whitney U test, as appropriate. Changes from baseline values at week 48 were assessed using Student’s t-test for paired samples. The primary efficacy endpoint, treatment failure, was investigated by per-protocol (PP) analysis at study week 48: this analysis excluded patients with major protocol violations. In addition, an intention-to-treat (ITT) analysis at study week 48 including all randomized patients who received at least one drug dose after randomization was also performed. In all the mentioned analyses on treatment failure, missing values and those who had changed or discontinued their ART regimen were counted as failures. In a further secondary analysis, time to virological failure was also analysed in all randomized patients divided by study arm, until last available follow-up at the time of study interruption, using the Kaplan-Meier method. Non-inferiority for the primary outcome was tested using the one-sided test procedure: noninferiority would be established at an alpha level of 0.05 if the dual therapy arm achieved the primary outcome in no more than 10% less patients than the current therapy arm.A two tailed P value <0.05 was considered to be statistically significant. All analyses were performed using the SPSS version 22 software package .Between May 23, 2011 and April 22, 2015, 259 patients underwent screening for the study. On June 2015, based on a pre-planned 48-week interim analysis, the Data Safety Monitoring Board recommended trial discontinuation due to an excess of virological failures in the study arm. At that time, screening failures were 89/259 (36%): 67 (26%) for non-R5 viral tropism, 11 (4%) for HIV-RNA >50 copies/mL, 6 (2%) for V3 region of HIV-1 gp120 not amplifiable, 5 (2%) due to other exclusion criteria. Five (2%) additional patients were excluded due to consent withdrawal before randomization. Therefore, 165 patients were randomized of whom 123 were evaluable for the 48-weeks ITT analysis (62 in arm S and 61 in arm C), and 115 were included in the main PP analysis (56 in arm S and 59 in arm C) .The main baseline characteristics of the PP study population, overall and divided by study arm, are summarized in In the PP population, 76/115 (66%) patients were free of treatment failure at 48 weeks: 41/56 (73.2%) in arm S and 35/59 (59.3%) arm C . The proIn the ITT population the proportion free of treatment failure at 48 weeks in arm S was noninferior to the proportion in arm C  . All 8 pWhen baseline characteristics of the 8 patients with virological failure in arm S (ITT population) were compared with the 54 without virological failure in the same arm, significant differences included a higher mean baseline FPR of the geno2pheno co-receptor assay , a shorter duration of previous virological suppression  in those with virological failure as compared to those without. Other characteristics  did not differ between patients with virological failure and those without . In a fuDuring the 48 weeks of the study 78 clinical adverse events were observed in 49 of the 123 patients  and 14 (23%) patients in arm S and 24 (41%) and 19 (31%) in the arm C experienced grade 3 and grade 4 laboratory toxicities, respectively, without significant differences between arms.2 in arm S, vs. -8 mL/min/1.73m2 in arm C, p = 0.12), although the study arm showed a better preservation of kidney function from baseline between week 12 and 36   and high-density lipoprotein (HDL) cholesterol did not show significant modifications . There w= 0.034) .Changes in BMD and bone metabolism markers were evaluated in 64 patients: 34 in the S arm and 30 in the C arm. At 48 weeks, patients in arm S showed significantly better gains over those in arm C in femoral neck BMD . Lumbar column BMD changes did not show significant differences between arms . Patients in arm S showed a more prominent reduction of total plasma alkaline phosphatase  and bone alkaline phosphatase  at 48 weeks, compatible with a reduced bone remodeling. No other significant changes were observed in the other bone metabolism biomarkers.trough of all drugs at virological failure were significantly lower as compared to those of patients without virological failure: darunavir median level 681 ng/mL  vs 1,368 ng/mL , p = 0.038, maraviroc 36 ng/mL (7–152) vs 121 ng/mL (100–127), p<0.001, and ritonavir 2 ng/mL (0–81) vs 33 ng/mL (17–49), p<0.001. Adherence, patient-reported symptoms, physical health or mental health-related QoL scores did not differ between arms at baseline and at week 48  had at least one assessment of self-reported adherence, patient-reported symptoms and QoL: baseline characteristics of these patients did not differ from those of the main stydy population (not shown). Detailed numbers are reported in  week 48 . Physica week 48 . Mean ad week 48 . Among pThis open-label randomized trial in virologically controlled patients on a 3-drug ART with R5 virus was designed to demonstrate the non-inferiority of switching therapy to once daily maraviroc plus ritonavir-boosted darunavir as compared to continuing the previous 3-drug regimen. Although the coadministration of once daily maraviroc 300 mg with darunavir/ritonavir 800/100 mg is not according to label, other studies demonstrated high tolerability and favourable pharmacokinetics when compared with 300 mg of maraviroc twice daily with 245 mg of tenofovir/200 mg of emtricitabine . The stuFor selecting candidate patients with R5-tropic virus we used a lower cut-off of the FPR of the geno2pheno interpretation system (10%) than suggested by a European panel (20%) . NotablyImportantly, in line with the lack of drug resistance development, all patients with virological rebound rapidly regained virological suppression with alternative therapies, showing that effective therapeutic options were not lost in these cases. Of note, patients enrolled in this study were more heavily pretreated and had lower nadir CD4 counts as patients enrolled in bPI monotherapy studies, in more recent trials of maintenance therapy with bPI plus lamivudine as well as in the MARCH study , 18, 28.As mentioned, study and continuation arm did not differ in the primary efficacy endpoint (proportion of patients free of treatment failure at 48 weeks on the per-protocol population). Indeed, more patients in the continuation arm interrupted their regimen because of toxicity. Most of these were renal or bone toxicities and were attributed to tenofovir disoproxil fumarate, a member of the NRTI class, which was discontinued in all cases. While the open-label study design could have facilitated treatment changes in the continuation arm, this did not happen in the study arm with maraviroc and boosted darunavir, showing a good tolerability of this regimen. In the subgroup of patients analyzed, BMD was improved in the study arm as compared to the continuation arm and, accordingly, markers of bone turnover were also improved. Altogether these findings suggest that the association of maraviroc 300 mg qd with boosted darunavir qd is well tolerated and prevents some NRTI-associated toxicity. Candidate patients should however be very carefully selected among those which guarantee the maximal adherence levels and those with prolonged viral suppression. Alternatively, it should be tested whether maintaining one of the less toxic NRTIs with this combination could be a virologically safer strategy .The main limitation of this study is represented by the open label design, the heterogeneity of the regimens in the continuation arm and the inclusion of less than half patients than planned in the study. The reduced number of patients due to premature study discontinuation did not allow us to formally conclude on the noninferiority of our experimental regimen on the primary study outcome, although the lower 95% confidence interval bound of the difference between experimental and control arm was within the pre-specified lower margin for defining noninferior efficacy. The heterogeneity of the comparator regimens allowed to compare the efficacy of a drug reduction strategy against a number of different standard 3-drug regimens which more closely reflect the reality of currently treated and virologically controlled patients in clinical practice.The inferior virologic efficacy of the dual regimen tested here contrasts with results obtained with other dual regimens showing non-inferior efficacy to 3-drug therapy, in particular ritonavir-boosted PI regimens combined with lamivudine and dolutegravir with rilpivirine –35. TherIn conclusion, switching a standard 3-drug therapy to maraviroc 300 mg qd plus boosted darunavir qd in virologically suppressed patients was associated with an improved tolerability but was virologically inferior as compared to continuing the previous therapy. Virological failures were associated with lower adherence, which caused suboptimal drug exposure. Genotypic tropism testing on viral DNA can be employed to select virologically suppressed patients to maraviroc but candidates to the dual therapy regimen tested here need to be carefully selected and instructed.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.S1 Text(DOC)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOC)Click here for additional data file.