Antiretroviral therapy (ART) has been successfully introduced in low-middle income countries. However an increasing rate of ART failure with resistant virus is reported. We therefore described the pattern of drug resistance mutations at antiretroviral treatment (ART) failure in a real-life Tanzanian setting using the remote genotyping procedure and thereafter predicted future treatment options using rule-based algorithm and the EuResist bioinformatics predictive engine. According to national guidelines, the default first-line regimen is tenofovir + lamivudine + efavirenz, but variations including nevirapine, stavudine or emtricitabine can be considered. If failure on first-line ART occurs, a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and boosted lopinavir or atazanavir is recommended.pol region was performed locally and the amplified DNA fragment was sent to Sweden for sequencing (split genotyping procedure). The therapeutic options after failure were assessed by the genotypic sensitivity score and the EuResist predictive engine. Viral load was quantified in a subset of subjects with second-line failure (n = 52).Plasma was obtained from subjects with first (n = 174) or second-line (n = 99) treatment failure, as defined by clinical or immunological criteria, as well as from a control group of ART naïve subjects (n = 17) in Dar es Salaam, Tanzania. Amplification of the pol region was successfully amplified from 55/174 (32%) and 28/99 (28%) subjects with first- or second-line failure, respectively, and 14/17 (82%) ART-naïve individuals. HIV-1 pol sequence was obtained in 82 of these 97 cases (84.5%). Undetectable or very low (<2.6 log10 copies/10−3 L) viral load explained 19 out of 25 (76%) amplification failures in subjects at second-line ART failure. At first and second line failure, extensive accumulation of NRTI  and NNRTI  DRMs but a limited number of PI DRMs (11% at second line failure) was observed. First line failure subjects displayed a high degree of cross-resistance to second-generation NNRTIs etravirine  and rilpivirine , and to abacavir  which is reserved for second line therapy in Tanzania. The predicted probability of success with the best salvage regimen at second-line failure decreased from 93.9% to 78.7% when restricting access to the NRTIs, NNRTIs and PIs currently available in Tanzania compared to when including all approved drugs.The HIV-1 The split genotyping procedure is potential tool to analyse drug resistance in Tanzania but the sensitivity should be evaluated further. The lack of viral load monitoring likely results in a high false positive rate of treatment failures, unnecessary therapy switches and massive accumulation of NRTI and NNRTI mutations. The introduction of regular virological monitoring should be prioritized and integrated with drug resistance studies in resource limited settings. In 2015, more than 500 000 human immunodeficiency virus (HIV) infected subjects in low-middle income countries received second-line antiretroviral therapy (ART). A recent modelling study estimated that the number of subjects on second-line ART will increase substantially, and by 2020 between 0.5 million and 3 million people in sub-Saharan Africa (SSA) will need second line therapy  subjects−6 L) is tenofovir (TDF) + lamivudine (3TC) + efavirenz (EFV) although the NRTIs may be changed to other available options such as zidovudine (AZT) or emtricitabine (FTC) and EFV may be replaced by nevirapine (NVP) under certain conditions. Treatment failure is identified using clinical and immunological parameters while virological monitoring, although recommended, is not part of routine clinical practice. If failure on first-line ART occurs, a combination of two NRTIs  and boosted lopinavir (LPV/r) or atazanavir (ATV/r) is recommended. However, in case of second-line ART failure no third-line regimen is described in the national guidelines [In Tanzania, ART has been freely available since 2004. In 2015, the prevalence of HIV-1 infection was 4.7% among adults and the ART coverage was estimated to be 53% . AccordiThis cross-sectional study aimed to describe the viral genotypic pattern at treatment failure of first-line ART as well as second-line PI/r-based ART in a real-life Tanzanian setting using the established clinical and immunological failure (CIF) criteria. Since drug genotypic resistance testing (GRT) is not readily available in SSA, we used the “split genotyping procedure” , where PEthical approval for carrying out the study was obtained from the Senate Research and Publications Committee at the Muhimbili University of Health and Allied Sciences (Ref. No. MU/01/1022/0122/10). Written informed consent was obtained from the subjects included in the study. All participants were given a copy of the signed consent form, which contained information about the study and contact details of the principal investigator and the ethics review board, both in English and Kiswahili.3 while on treatment [Subjects with suspected first or second line treatment failure were approached consecutively at two clinics; Muhimbili National Hospital and Amtulabhai Karimjee Treatment failure Clinic in Dar es Salaam, Tanzania, between June 2009 and September 2014. As viral load testing is not part of clinical practice in Tanzania, identification of subjects with ART failure was based on CIF only . WHO guireatment . One-hunCD4 cells were determined by routine flow cytometry at the Muhimbili National Hospital. Plasma from a subgroup of 52 subjects with CIF on second line PI/r-based therapy, of which 27 were PCR positive and 25 were PCR negative, were tested for HIV-RNA using COBAS AmpliPrep/Cobas TaqMan HIV-1 test v 2.0 (CAP/CTM 48)  at the National Health Laboratory Quality and Training Centre, Dar es Salaam.pol gene amplification was carried out according to EuResist guidelines, as described elsewhere [GRT was achieved using the previously described split genotyping procedure  in whichlsewhere  and visuSequences were manually scrutinized and edited using Geneious R6.1.6 . Sequences with full length protease gene  and partial reverse transcriptase gene  were used for further analysis. For positions where a second peak was observed with peak height ≥50% of the main peak, a wobble nucleotide was assigned. The sequences were deposited to GenBank with accession numbers KX775225-KX775306.www.hiv.lanl.gov). Multiple sequence analysis was performed in ClustalW algorithm incorporated in Geneious R6.1.6. The alignment file was used for phylogenetic analysis by maximum likelihood method in PhyML with the general time reversible plus gamma substitution model (GTR+G) as best fitted model identified by the FindModel tool (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). The approximate Likelihood Ratio Test (aLRT) was applied for branch support and the tree consisting of only Tanzanian sequences was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). For clustering analysis, 5–7 sequences per cluster plus 3 outliers were submitted to the HIV-1 BLAST tool. The top three hits per sequence were downloaded and aligned to generate a new phylogenetic tree as described above. The presence of drug resistance mutations was assessed by using the Stanford HIVdB online tool [HIV-1 subtyping was performed with REGA version 3.02  followedine tool .http://engine.euresist.org/) was used for prediction of the optimal salvage treatment regimen, based on the genotype at failure.For assessment of GSS for current and future drug options, Rega version 9.1.0  was emplData were analysed using SPSS Statistics 22  and graphs were generated with the aid of GraphPad Prism 5 . Descriptive analysis was performed: mean or median and interquartile range (IQR) was used for summarising continuous variables, and frequencies and percentages for categorical variables. A p-value < 0.05 was considered significant. For analyses examining the correlation between viral load and CD4, and between viral load and treatment duration, nonparametric correlation analysis  was used in SPSS.pol gene sequences suitable for further analysis. For first-line and second-line failures respectively, as defined by CIF criteria, only 55 of 174 (32%) and 28 of 99 (28%) samples were successfully amplified, with corresponding 43 and 26 sequences generated. Clinical information about the subjects for whom sequencing succeeded is given in Using the split genotyping procedure, 14 of 17 (82%) samples from ART-naïve individuals were successfully amplified, of which 13 yielded pol region while for the remaining 25 PCR amplification had been unsuccessful despite decreasing CD4 counts indicating treatment failure. The results revealed that for the majority of second-line samples for which PCR amplification was unsuccessful (n = 25), HIV-RNA levels were below the limit of quantification  or very low  or low . Only three samples (12%) were > 3 log10 copies/10−3 L . Consequently, among the samples tested for viral load, PCR amplification was successful in 27/30 (90%) of the samples with a viral load of >3 log10 copies/10−3 L. Among the PCR-positive second-line failure samples (n = 27), the viral load ranged between 3.06 and 5.74 log10 copies/10−3 L. For the 26 samples which generated usable sequences and were included in the genotypic analysis, the median viral load was 4.8 log10 copies/10−3 L . There was no significant correlation between viral load and CD4 at time of failure. Neither was there a correlation between viral load and treatment duration .To determine whether the low rate of amplification success was due to failure of the methodology or due to misclassification of treatment failure by the CIF criteria, viral load testing was performed in 52 subjects having second-line CIF for whom residual plasma was available, of which 27 had successfully amplified HIV-1 pol sequence was obtained, almost all  had ≥1 NNRTI mutation as well as ≥1 NRTI mutation. The proportion of subjects with NRTI and NNRTI mutations increased with the duration of ART. For those treated <36 months (n = 9 out of 38 subjects with data on ART duration), six subjects (67%) had NRTI mutations and seven subjects (78%) had NNRTI mutations (one patient had NNRTI mutations only and six had both NRTI and NNRTI mutations). For those treated 36–60 months (n = 24), 23 of 24 subjects (96%) had NRTI mutations and similarly 23 of 24 subjects (96%) had NNRTI mutations (100% had either NRTI and/or NNRTI mutations: NRTI only: 1 (4%); NNRTI only: 1 (4%); both NRTI and NNRTI: 22 (95%)). For the five subjects treated >60 months, all subjects had both NRTI and NNRTI mutations (Among 43 first-line failing subjects from whom a utations . For comOnly three out of 26 (11.5%) subjects failing PI/r-based second-line ART with detectable viral load and a readable sequence had major PI mutations . From a public health point of view in a setting without access to viral load testing, the rate of major PI mutation was only 3% of the 99 subjects considered to have second-line ART failure, according to CIF criteria. Among the 26 subjects failing second-line therapy, 17 (65%) had both NRTI and NNRTI mutations and 4 (15%) had either NRTI or NNRTI mutations.Among first-line ART failures, there was a widespread resistance to the first generation NNRTI, efavirenz and nevirapine (93% resistant for both drugs), and a high degree of cross-resistance to second-generation NNRTIs etravirine  and rilpivirine . Forty-nine percent (49%) of first-line failures also displayed cross-resistance to ABC, which in general is reserved for second line therapy in Tanzania. Second line failure subjects displayed somewhat lower resistance to EFV and NVP (69% resistance for both drugs) and lower cross-resistance to ETR (15% intermediate and 0% resistant) and RPV (8% intermediate and 31% resistant), while resistance to FTC and 3TC was high (58% resistant for both drugs). .https://rega.kuleuven.be/cev/avd/files/software/rega_algorithm/Rega_HIV1_Rules_v9.1.0.pdf).Future therapy options were assessed by the GSS approach  and by tThe success probability of the predicted optimal therapy was also investigated using the EuResist prediction engine for all first-line and second-line failure subjects, given their individual DRM profile. Firstly, when considering the TDF + FTC + LPV/r combination , the median success probability was 87.7% (range 64.2–94.2%) for 39 out of the 43 first-line failure subjects. For the remaining four subjects, this combination was not among the top ten recommended ART regimens (and hence no estimated success probability was given). Two of these subjects had DRM patterns which confer resistance to FTC according to the REGA criteria, and one of them additionally displayed intermediate resistance to TDF . Secondlpol sequences primarily clustered with other sequences from Tanzania, Kenya and Uganda (data not shown). Subtype D pol sequences clustered with other sequences from Tanzania and Uganda. Subtype C pol sequences clustered together with pol sequences from Tanzania, South Africa, Zambia and Botswana (9 sequences) or with pol sequences from Ethiopia (7 sequences).The subtype analysis identified 41% subtype A1, 27% subtype C, 9% subtype D, 5% CRF10_CD, 17% recombinant , and 1% unclassified. Subtype A1 Despite the increasing use of PI-based second-line ART in SSA, little is known about the real-life treatment outcomes, DRM patterns at therapy failure, and further treatment options thereafter. As viral load monitoring is not yet part of routine clinical practice in Tanzania, treatment failure is defined by clinico-immunological criteria. The study therefore evaluated the consequences of using these criteria for the definition of ART failure and the assessment of drug resistance. Also, since access to point of care (POC) diagnostics is lacking also for GRT all over the world , the splIn pilot evaluations, the “split genotyping procedure” has shown that laboratory technicians without or with limited background in molecular diagnostics can be successfully trained to generate HIV DNA fragments to be shipped and sequenced remotely with satisfactory results , althougAlthough we did not perform any formal analysis of the sensitivity of the approach, the split design is likely to be a valid alternative to e.g. GRT on dried blood spots or other approaches of POC diagnostics for GRT . We recopol gene amplification success rate at MUHAS was however very low when we tested a large number of plasma samples from subjects who had been defined as first-line ART failures (success rate 38%) or second-line PI/r-based ART failures (success rate 28%) by clinico-immunological assessment in real-life settings by Tanzanian physicians. When we determined the viral load in a subset of the second-line failures  by quantitative PCR it was found that almost all subjects for whom the HIV-1 pol amplification had failed, had undetectable or very low viral load. In contrast, most  of the subjects with a viral load >3 log10 copies/10−3 L had amplification success. It can be argued that the local PCR failed in three subjects with a viral load of 2.6–3 log10 copies/10−3 L, which in a high-income country would be a potential problem. However, it is important to emphasize that WHO recommends switch to a new regimen when the viral load is > 1000 copies/ml (3 log10 copies/10−3 L) since the risk of HIV transmission is very low below this threshold[10 copies/10−3 L can be handled through increased adherence support[et al. showed that 46.9% of subjects who were defined as treatment failures based on the clinico-immunological criteria were found to have an HIV RNA level <1000 copies/10−3 L (<3 log10 copies/10−3 L) [The HIV-1 e support, avoidine support, 19, Kene support as well e support demonstr/10−3 L) . In our Furthermore, the subjects with the longest duration on a failing first line regimen in our study had accumulated the highest number of DRMs. Other studies have demonstrated that absence of virological monitoring and a delayed switch of therapy lead to a high degree of accumulation of DRMs  and an iIn our cohort of 26 HIV-1 infected subjects with virologically confirmed failure on second-line therapy, we detected major PI mutations in 11.5% , which is the first report on emergence of major PI mutations in Tanzania. This data mirrors a larger study including 490 subjects receiving LPV/r in South Africa where the prevalence was 11% [In order to evaluate the remaining treatment possibilities for the failing subjects, we analysed the GSS as well as predicted the remaining options by the EuResist predictive engine . NotablyThe probability of success based on a predicted optimal therapy was also investigated using the EuResist prediction engine. Despite the high burden of NRTI mutations, when considering the TDF + FTC + LPV/r regimen the median success probability was as high as 87.7% range 64.2–94.2%) for 39 of the 43 first-line failure subjects. These results are in line with the finding of the EARNEST trial, in which NRTIs retained substantial virological activity when given with a PI in second-line therapy [–94.2% foIn summary, our study underscores the importance of using viral load for monitoring ART in resource-limited settings since the clinico-immunological monitoring in our study resulted in a very high frequency of unnecessary ART switches but also to an accumulation of extensive NRTI and NNRTI resistance in subjects failing with undetected viremia. When viral load is introduced for monitoring ART in these settings, our split genotyping procedure could be a valid and simple method for monitoring drug resistance to present and future antiretroviral agents although further validation is required to address potential PCR amplification issues. If a viral sequence can be obtained, bioinformatics methods can help the physicians to optimize the ARV regimens, especially for third-line ART in settings with limited therapeutic options.S1 Filest line ART failure subjects, 2nd line ART failure subjects and treatment naïve subjects.Age, gender, HIV-1 subtype, years since diagnosis, WHO stage, CD4 count, treatment regimen(s), treatment duration, optimal regimen and success probability predicted by EuResist prediction engine, genotypic sensitivity scores and HIV-1 drug resistance mutations are shown for 1(XLSX)Click here for additional data file.


Eculizumab is approved for atypical hemolytic uremic syndrome (aHUS). Guidelines discuss the importance of prompt treatment. We report a post hoc analysis investigating the effect of baseline factors, including patient characteristics and time from the latest aHUS manifestation to eculizumab initiation, on change from baseline in estimated glomerular filtration rate (eGFR) and other outcomes.2 for ≥28 days) and platelet count normalization were evaluated 1 year post-treatment. Baseline characteristics and eGFR outcomes were summarized by time to treatment from last aHUS manifestation [≤7 days (n = 21) versus >7 days (n = 76)].Data were pooled from four phase 2, open-label, single-arm, prospective clinical studies of eculizumab for patients with aHUS. Multivariate regressions identified predictors of eGFR change from baseline. The proportion of patients achieving sustained eGFR increase . After 1 year, 17/21 and 36/76 patients in the ≤7 and >7 day groups, respectively, achieved a sustained increase in eGFR. Mean time to platelet count normalization was similar between groups.Baseline eGFR were similar between groups. Multivariate regression analysis demonstrated time from aHUS manifestation to eculizumab treatment, age, baseline lactate dehydrogenase (LDH) and baseline hemoglobin were independently predictive of eGFR change from baseline. Mean eGFR change from baseline at 1 year was significantly higher in patients treated in ≤7 days than >7 days  contains supplementary material, which is available to authorized users. Atypical hemolytic uremic syndrome (aHUS) is a life-threatening condition  caused bThe rapid progression of TMA, associated with potentially irreversible damage to organs in patients with aHUS, indicates a need for urgent treatment. TMA may lead to acute renal dysfunction in early disease, and evidence suggests acute kidney injury (AKI) and chronic kidney disease (CKD) are closely interrelated, with each being a risk factor for the other . Plasma 9/l [95 % confidence interval (CI) 67–116] in the first trial; TMA event–free status for ≥12 weeks was achieved in 85 % of patients in the second trial. Both trials achieved normalization of platelet count and lactate dehydrogenase (LDH) levels (88 and 90 % respectively) [Eculizumab is a recombinant humanized monoclonal antibody that blocks the cleavage of C5, preventing formation of the proinflammatory peptide C5a and the cytotoxic membrane attack complex C5b-9 . Multiplctively) . A recenGuidelines recommend children and adults with a clinically definitive aHUS diagnosis be treated with eculizumab ideally within 24 h , 12. It While the timing of therapy initiation for aHUS has been investigated in small patient cohorts, it remains to be studied in a substantive population . We ther2. All patients were receiving eculizumab for the first time. In this pooled analysis, three major clinical outcomes were considered: (1) eGFR change from baseline at 1 week and monthly over 1 year, (2) proportion of patients achieving a sustained eGFR increase of ≥15 ml/min/1.73 m2 (improvement maintained for at least 28 days) from baseline to 1 year of follow-up, and (3) time to platelet count normalization (≥150 × 109/l). Baseline was defined as: at enrollment prior to first eculizumab dose. It should be noted that when patients were on dialysis, their eGFR was imputed to 10 ml/min/1.73 m2.Data were pooled from the four previously described phase 2, open-label, single-arm, prospective clinical studies which enrolled patients aged 1 month to 80 years . Inclusit test. To test the difference between the two groups (≤7 and >7 day) on the proportion of patients achieving a sustained eGFR increase of ≥15 ml/min/1.73 m2, Fisher’s exact test was performed for each visit.In order to explore in a clinically relevant and dichotomous way the effect of time from the latest aHUS manifestation to initiation of eculizumab treatment, results were stratified according to whether patients had started eculizumab treatment ≤7 or >7 days after the onset of the latest aHUS manifestation (herein referred to as time to treatment). Baseline characteristics were compared between the ≤7 and >7 day groups using the Wilcoxon rank-sum test for continuous variables and the Fisher exact test for categorical variables. The difference between the ≤7 and >7 day groups in time to platelet count normalization was tested using the two-group Multivariate regressions using repeated measures analysis were performed to identify predictors of change in eGFR from baseline through 1 year. Baseline eGFR and trial visit (scheduled post-dose visits in months) were always kept in the model as the dependent variable was change from baseline in eGFR and the analysis was a repeated measures analysis over post-dose visits up to 12 months. Ten other baseline variables or patient characteristics were tested, including six categorical variables: age group (<18 vs. ≥18 years), mutation , history of TMA manifestation (single vs. multiple), transplant history (yes vs. no), dialysis at baseline (yes vs. no), PE/PI at baseline (yes vs. no), as well as four continuous variables: time to treatment, baseline platelet count, baseline LDH, and baseline hemoglobin. Selection of variables was performed using a forward stepwise procedure to identify the main effects and their interaction terms for inclusion in the final model. In order to test the effect of time to treatment as both a continuous and a categorical variable, after the final model was identified (including time to treatment), it was repeated replacing continuous time to treatment with categorical time to treatment (≤7 vs. >7 days).The raw mean changes in eGFR from baseline over time up to 12 months are presented for the two dichotomized time to treatment groups (≤7 and >7 days). To evaluate the differences in eGFR change from baseline between the two groups not controlling for other factors, a two-group t test was performed at each visit.2. Sixty-eight (70 %) patients had data at 12 months follow-up on eculizumab.Of the original 100 patients in the intent-to-treat population, 97 were eligible for the pooled analysis. Three patients were not eligible because they either lacked a recorded date for onset of signs of aHUS or had a baseline eGFR > 90 ml/min/1.73 m2, in the ≤7 day vs. >7 day groups, respectively (p = 0.30).Stratification resulted in 21 patients in the group starting eculizumab at ≤7 days of presentation of TMA, and 76 patients starting at >7 days of presentation. Baseline demographics and disease characteristics are shown in Table The final model obtained through stepwise forward variable selection contained significant main effects: time to treatment, age group, baseline LDH, and hemoglobin. Significant interaction terms that remained in the final model were visit (post-dose visits in months) by time to treatment, visit by age group, visit by baseline LDH, visit by baseline hemoglobin, and age group by baseline hemoglobin Table . The finThe final multivariate analysis model showed that time to treatment as a continuous variable was a significant factor in determining eGFR change from baseline when controlling for other factors such as age group, baseline LDH, hemoglobin, eGFR and post-dose visits. Time to initiation of eculizumab treatment when dichotomized into groups for ≤7 and >7 days was not significant when controlling for these other factors.2 in the ≤7 day group compared with 23 ml/min/1.73 m2 in the >7 day group  greater mean improvement in eGFR from month 1 onwards. At 1 year, the eGFR increase from baseline was 57 ml/min/1.73 mTime to treatment and age group both remained in the final model as significant factors. A scatter plot of eGFR change from baseline to 6 months versus time to treatment showed a similar trend in the relationship between eGFR change and time to treatment in both children and adults Fig. a. LDH an2 for at least 28 days) at 3 months follow-up. This proportion remained stable through 1 year. In contrast, 26 (34 %) and 36 (47 %) patients in the >7 days group achieved a sustained eGFR at 3 months and at 1 year, respectively  was significantly higher (p < 0.05) for the ≤7 day group compared to the >7 day group at all visits based on Fisher’s exact test.Seventeen patients 81 %) in the ≤7 day group had achieved a sustained eGFR response  in the ≤7 day group and 42 (55 %) in the >7 day group. Median time to platelet count normalization was similar in the two groups (7 vs. 7.5 days), although the range of values in the ≤7 day (1–80 days) group was narrower than in the >7 days group (1–189 days). Corresponding mean (SD) times to platelet normalization were 14.3 ± 20.3 and 27.8 ± 38.7 days, respectively (p = 0.083).aHUS is a severe disease with poor outcomes in which progression can be rapid; prior to the availability of eculizumab over 50 % of patients with aHUS died, required dialysis or developed permanent kidney damage in the first year after diagnosis . This in2 was achieved more rapidly in the ≤7 day group while improvement progressed more slowly in the >7 day group, indicating the possibility to also continue eGFR improvement after 6 months of eculizumab treatment.This post hoc multivariate analysis of the time from last aHUS manifestation to treatment suggests that a shorter time to treatment with eculizumab results in a significantly better renal outcome for patients with aHUS. This is the largest group of patients with aHUS in whom the clinical impact of the timing of eculizumab initiation relative to presentation with disease has been explored. The analysis presents data up to 1-year because this was considered sufficient follow-up to detect differences in outcomes without excessive loss of patients available for evaluation over time. Renal outcomes were significantly better in the group of patients initiating eculizumab treatment in ≤7 days of presentation of TMA than in patients initiating treatment after 7 days. Of interest, the sustained improvement of >15 ml/min/1.73 mFactors other than early treatment initiation with eculizumab that were independently associated with eGFR outcome were younger age, higher LDH, and lower hemoglobin. Clinically it can be hypothesized that early treatment and younger age are suggestive of less renal damage at treatment initiation and, therefore, possibly greater potential of recovery of renal function.Overall, these data support the importance of prompt treatment of aHUS to optimize recovery of renal function and to avoid dialysis or transplantation in both adults and pediatric patients, as reported elsewhere , 12, 13.Recent guidelines recommend treating children and adults with a diagnosis of aHUS with eculizumab within 24 h. In patients where further investigations are required, and where no significant improvement of both renal and hematological parameters is observed, eculizumab should be initiated after no more than 5 plasma exchanges when aHUS diagnosis has been confirmed , 12. TheNo significant difference in time to platelet count normalization was shown between patients receiving treatment in ≤7 or >7 days, although a trend favoring earlier treatment was evident from mean numbers of days to normalization and ranges of times around median values. In the original source studies, platelet counts normalized rapidly: for example, in the two published trials which enrolled patients aged ≥12 years, 53 % of those with abnormal platelet counts at baseline had normal counts by day 7 . Of noteLimitations of this study include the non-randomized study design and the retrospective nature of the analysis, although the source data were collected prospectively. Further, it is impossible to know how patients treated in ≤7 days would have responded if treatment had been delayed until >7 days. Also, patients who may have already been receiving dialysis for an extended period before starting eculizumab therapy may not have been expected to show major improvements in eGFR, which might have resulted in bias favoring the ≤7 day group. The median time on dialysis was, however, fairly short (12 days) in the >7 day group with only two patients on dialysis for >90 days. It is also not possible to exclude a tendency for the initiation of eculizumab more rapidly in patients presenting with more severe symptoms.In summary, the results of this large pooled analysis indicate overall that patients with aHUS derive greater and more sustained eGFR recovery benefit from treatment with eculizumab when therapy is started earlier. Our data concur with previous observations and treatment guidelines that recommend prompt treatment with eculizumab in patients diagnosed with aHUS.Supplementary material 1 (DOCX 15 kb)Supplementary material 2 (DOCX 14 kb)Below is the link to the electronic supplementary material.


Despite successful virological suppression, HIV transcription frequently persists intracellularly. In this study, we hypothesize that HIV persistent transcription(HIVpt) may affect to a different extent patients on stable efavirenz(EFV) versus atazanavir(ATV)-based regimens. The role of the expression of drug efflux transporters in HIVpt was also investigated.+ T-cells and in the CD4+CD45RO+ T-cell subpopulation. The differential mRNA expression of P-glycoprotein(P-gp/ABCB1) and multidrug resistance-associated protein-1(MRP1/ABCC1) was also evaluated. Univariate logistic regression models were used to evaluate predictors of HIVpt.We prospectively enrolled 51 virologically suppressed patients on first-line treatment for one year with EFV or ATV combined with emtricitabine and tenofovir and followed them up for one year. Simultaneous ultrasensitive subpopulation staining/hybridization in situ(SUSHI) was performed to identify HIVpt in CD4+ T-cell population, HIVpt affected 13/30 of patients on EFV versus 10/21 on ATV. In the CD4+CD45RO+ T-cell population, HIVpt was present in 14/30 of patients on EFV versus 15/21 on ATV. A trend for association was observed between the risk of HIVpt and ATV treatment in the CD4+CD45RO+ T-cell population . HIVpt status was not associated with loss of virological suppression or CD4 evolution. We found no evidence of differential expression of the drug efflux transporters P-gp and MRP1.In the CD4Further study is required to evaluate whether the HIVpt profile in specific cell populations may differ across different antiretroviral regimens and to elucidate the potential clinical impact. The estanfection. Several nfection.–6 The imnfection.There is limited evidence derived from raltegravir intensification studies that residual viral replication as evidenced by increases in 2-long terminal repeat circles may affect to a lesser extent patients on non-nucleoside transcriptase inhibitors(NNRTIs) versus protease inhibitors(PIs).,4 For drIn our prospective study, we used a method which combines immunophenotyping with ultrasensitive-FISH to detect unspliced HIV-1 gag-pol in relevant cell populations in order to compare HIVpt in virologically suppressed patients on efavirenz(EFV) or atazanavir/ritonavir(ATV/r) and a backbone of emtricitabine-tenofovir(FTC-TDF).,10,11 WeWe conducted an observational prospective cohort study enrolling HIV-1 infected patients followed in the HIV clinic of the AHEPA University Hospital in Greece. The study was approved by the University Bioethics Committee and all patients provided written informed consent prior to study inclusion. Eligible patients were aviremic HIV-1 infected adults without hepatitis B or C co-infection on stable treatment for 12 months with either efavirenz or atazanavir/ritonavir and a backbone of emtricitabine—tenofovir disoproxil fumarate. Patients should be virologically suppressed (<50 copies/ml) as determined with the standard of care method  at sampling and for the preceding 6 months. Age, risk factor for HIV diagnosis, country of origin, pretreatment viral load, nadir CD4 count, CD4 count within 3 months before enrollment (baseline CD4 count), CDC stage as well as time from HIV diagnosis to treatment initiation and time from treatment initiation to study enrollment were recorded. Patients were followed up according to guideline recommendations for clinical progression and virological suppression for a period of one year after sampling. The CD4 count one year after sampling was recorded to evaluate the impact of HIVpt on CD4 evolution. In a post hoc calculation, a sample of 22 patients per treatment group would have 80% power to detect a 40% difference in the prevalence of HIVpt between the two treatment groups (α = 0.05).We isolated PBMCs from 6ml of whole blood using Histopaque, within 4 hours after collection. Viability and cell count were performed by using 7-AAD  staining. An average number of 3.96 million cells from each individual were stored in -20 °C for the total RNA extraction.+ as well as in the CD4+CD45RO+ memory T-cell subpopulation. Cell samples were incubated with appropriate volumes of CD4-PE and CD45RO-PE-Cy5 monoclonal antibodies  and the expression of intracellular HIV-1 unspliced gag-pol HIV RNA was evaluated with SUSHI  according to manufacturer’s instructions. [In our study, we focused on HIVpt in the CD4uctions.  Six HIV uctions. ,13 Measu2 First Strand Kit(Qiagen), according to the manufacturer’s instructions.Total RNA was extracted from PBMCs using the RNeasy Mini Kit for application in QIAcube automated system, according to the manufacturer’s instructions(Qiagen). RNA concentration was determined with the NanoDrop 1000(Thermo Scientific); in order to evaluate the integrity and quality of the isolated RNAs, random samples were checked using an Agilent 2100 Bioanalyzer. Extracted RNA was stored in -80 °C. Total RNA (500 ng) was reverse-transcribed using RT2 Profiler PCR Array(Qiagen) to compare the mRNA expression levels of P-gp(ABCB1) and MRP1(ABCC1) in patients with and without HIVpt on EFV-based versus ATV-based regimens. cDNA was combined with 2x RT2 SYBR Green MasterMix(Qiagen), and loaded onto an optically clear 96-well plate in duplicate where gene specific primers for target genes  and endogenous control gene  were preloaded. At the same plate, genomic DNA contamination, efficiency of the reverse transcription reaction and efficiency of the polymerase chain reaction were tested by amplification of the genomic DNA control , the reverse-transcription control  and positive PCR control  respectively. The cycling conditions were as follows: 95°C for 10 min,  × 40 cycles on the 7500 Fast Real-Time PCR System (Applied Biosystems).We performed qPCR using Custom RTContinuous variables were summarized as mean and standard deviation(SD) for normally distributed data and as median and interquartile(IQR) otherwise. Discrete variables were expressed as percentages and 95% exact binomial confidence intervals(CI) were calculated. The independent samples t-test was used to compare normally distributed continuous variables and the Mann–Whitney for non-normally distributed variables. The chi-square test and the Fisher’s exact test were used for discrete variables as appropriate. The association of various predictors with persistent HIV transcription status was evaluated with univariate logistic regression models. All statistical tests were two-sided. P-values less than 0.05 were considered statistically significant.-ΔΔCq. The expression levels were analyzed with Student’s test and a sensitivity analysis was conducted with one-way ANOVA.For gene expression analysis, raw Cq values of the expression of control and target genes were exported. The relative expression levels were calculated with the ΔΔCq method and fold-changes as 2Statistical analyses were performed with IBM SPSS Statistics version 23 except for the sample size and the binomial confidence intervals calculations (R version 3.3.1 –stats(power.prop.test) and binom packages).,15 Data During the period from October 2012 to July 2014, a total of 51 participants were enrolled in the study (30 on EFV and 21 on ATV). The two groups were similar in baseline cohort characteristics with the exception of time on HAART, which was longer in the ATV group  .+ cell populations in 23/51 patients : 13/30 patients in the EFV group versus 10/21 in the ATV group. In the CD4+CD45RO+ cell population, persistent HIV transcription was present in 29/51 patients  with 14/30 in the EFV group versus 15/21 in the ATV group . The percentage of cells with persistent HIV transcription for each patient sample is depicted in + cells were not different between the two treatment groups (p = 0.693 for CD4+ cells and p = 0.747 for CD4+CD45RO+ cells).Persistent unspliced HIV RNA transcription as captured by SUSHI was identified in the CD4+CD45RO+ cell population and ATV treatment   .+CD45RO+ cell reservoir (3) was observed with no evidence of a difference between the two treatment groups(p = 0.240). HIVpt status in CD4+and CD4+CD45RO+ cells was not associated with the evolution of the CD4 cell count .After study enrollment and sampling, one patient from the ATV group was lost to follow-up and one patient from the EFV group had a treatment switch due to renal toxicity to tenofovir. No patient presented clinical progression. Viral loads were below the limit of detection throughout the observation period in all patients, except for two patients in the EFV group who presented transient viral load elevations. Viral load was below the level of detection in subsequent evaluations. Of note, both patients were classified as negative with regard to HIVpt in the CD4+ T cell population and one was classified as positive in the CD4eservoir . A mean We did not observe differences in the levels of mRNA expression of P-gp and MRP1 between patients in the two treatment groups . Furthermore, there was no evidence for differential expression of P-gp and MRP1 in patients with HIVpt versus those without HIVpt overall as well as in the two treatment subgroups.+ cells. However, a trend for an association between an ATV regimen and the risk of HIVpt was observed in the CD4+CD45RO+ cell population. Furthermore, we did not observe an association between HIVpt status and CD4+ count evolution and the two patients with transient elevations of viral load that were observed did not map consistently into the HIVpt positive population. Finally, our data do not support a differential expression of the drug efflux transporters P-gp and MRP1 in PBMCs of patients with and without HIVpt.In our prospective study, we compared the prevalence of HIVpt as evaluated by SUSHI among virologically suppressed patients on two popular first-line regimens at the time the study was designed (EFV-based versus an ATV-based). HIVpt status was confirmed in approximately half of the patients. There was no evidence of a difference in the prevalence of HIVpt between the EFV-group and the ATV-group in CD4+ cell counts and plasma viral load.[The shift of the HIV research agenda towards strategies for the quantification and eradication of the viral reservoir has renewed interest in biomarkers beyond CD4ral load. Cell-assral load. and suggral load. Chronic ral load.–22+CD45RO+ cells. Despite the fact that patients in the ATV group were on HAART for a longer period compared to the EFV group , the decay dynamics of cells with persistent HIV transcription suggest that such a difference should not impact the results meaningfully. [We used a conceptually different method which identifies individual cells with low levels of HIVpt, instead of relying on average values that may be skewed importantly by a minority of cells with high levels of CA-RNA. SUSHI has been used previously to evaluate the “active” reservoir of HIV which was linked to persistent HIV-1 specific immune response.,12 The pngfully. Previous studies have suggested a role for CA-RNA as a predictive biomarker of virological failure(VF). Our studP-glycoprotein and MRP1 are among the most studied drug transporters that affect the disposition of antiretroviral drugs. We hypothesized that the interactions of antiretroviral drugs with these molecules may reduce the intracellular drug levels and be associated with HIVpt in PBMCs. Similarly, increases in CA-RNA levels in the absence of virological failure have been described in patients with suboptimal adherence to cART.  The vari+ cells, we examined the CD4+CD45RO+ memory cell population which includes cells that contribute significantly to the latent HIV reservoir such as the central memory and effector memory cells.[Several limitations apply to our study. The identification of HIVpt with SUSHI may not reflect concomitant HIV protein production as transcripts may be defective. Nevertheless, such transcripts may still encode chimeric proteins with implications for HIV pathogenesis. Except fry cells.,29 Howevry cells. SimilarlLarge prospective studies with representative patient samples are required to validate the use of new biomarkers against clinical outcomes. Further study is required to evaluate whether persistence of HIV transcription may not affect to the same extent different cell populations across treatment regimens.


MaxART Early Access to ART for All (EAAA) implementation study was designed to determine the feasibility, acceptability, clinical outcomes, affordability, and scalability of offering early antiretroviral treatment to all HIV-positive individuals in Swaziland’s public sector health system.There is robust clinical evidence to support offering early access to antiretroviral treatment (ART) to all HIV-positive individuals, irrespective of disease stage, to both improve patient health outcomes and reduce HIV incidence. However, as the global treatment guidelines shift to meet this evidence, it is still largely unknown if early access to ART for all  is a feasible intervention in the resource-limited countries where this approach could have the biggest impact on the course of the HIV epidemics. The This is a three-year stepped-wedge randomized design with open enrollment for all adults aged 18 years and older across 14 government-managed health facilities in Swaziland’s Hhohho Region. Primary endpoints are retention and viral suppression. Secondary endpoints include ART initiation, adherence, drug resistance, tuberculosis, HIV disease progression, patient satisfaction, and cost per patient per year.Sites are grouped to transition two at a time from the control (standard of care) to intervention (EAAA) stage at each four-month step. This design will result in approximately one half of the total observation time to accrue in the intervention arm and the other half in the control arm. Our estimated enrolment number, which is supported by conservative power calculations, is 4501 patients over the course of the 36-month study period.A multidisciplinary, mixed-methods approach will be adopted to supplement the randomized controlled trial and meet the study aims. Additional study components include implementation science, social science, economic evaluation, and predictive HIV incidence modeling.A stepped-wedge randomized design is a causally strong and robust approach to determine if providing antiretroviral treatment for all HIV-positive individuals is a feasible intervention in a resource-limited, public sector health system. We expect our study results to contribute to health policy decisions related to the HIV response in Swaziland and other countries in sub-Saharan Africa.NCT02909218. Registered on 10 July 2016.ClinicalTrials.gov, The online version of this article (doi:10.1186/s13063-017-2128-8) contains supplementary material, which is available to authorized users. The global community has made extraordinary strides in scaling up treatment for people living with HIV/AIDS over the past two decades. Since 2000, there has been a 30-fold increase in people accessing antiretroviral therapy (ART) globally from 250,000 people to more than 17 million people in 2015 [However, despite the significant progress, the global community is still far from ensuring that all people living with HIV/AIDS (PLHIV) eligible for treatment are receiving it. Moreover, without a significant reduction in new infections, the number of individuals in need of treatment will continue to expand, as will the costs of HIV treatment programs , 5. IdenOver the past several years, HIV research has demonstrated that earlier and expanded access to ART could have a significant impact on HIV incidence. Results from the HIV Prevention Trials Network 052 (HPTN 052) trial showed that early access to ART prevents onward transmission of the virus to the uninfected partner in heterosexual HIV-discordant couples. In 2011, the trial reported not only a 96% decrease in HIV transmission, but also a 41% decrease in HIV-related morbidity from early initiation of ART . In respWhile the clinical impact of Early Access to ART for All (EAAA) is evident, critical implementation questions remain. Recently, results from the Treatment as Prevention (TasP) Trial in rural KwaZulu-Natal in South Africa did not demonstrate a difference in HIV incidence between communities randomized to either immediate offer of ART for all HIV-positive individuals compared to the standard of care . The stuIt remains unknown, however, what the impact of EAAA policies will be on public sector health systems in sub-Saharan Africa. The overall HIV treatment program effectiveness in successfully treating HIV patients could change, as could patient satisfaction, patients’ economic welfare, and provider satisfaction. Policymakers in high HIV prevalence countries and stakeholders in the international community require empirical evidence to assess the feasibility and acceptability of EAAA policies, as well as the clinical outcomes, affordability, and scalability of national implementation of this intervention through routine, public sector health systems.This study was designed in response to questions posed by Swaziland’s policymakers about the impact of an EAAA policy on their national HIV/AIDS care and treatment program. The study aims to generate the evidence needed to more fully understand what is required to successfully implement an EAAA strategy in a public sector health system in sub-Saharan Africa.quantify the causal impact of early access to ART for all HIV-infected adults, irrespective of CD4 count or clinical staging, on ART retention and viral suppression in a public sector health system;quantify the causal impacts of EAAA on average health care expenditures and resources use;quantify the causal impacts of EAAA on patient satisfaction, patients’ welfare, and provider satisfaction;to establish the feasibility and acceptability of EAAA;establish the role of PLHIV, traditional leaders, civil society, and communities for implementing EAAA;determine the social and institutional processes throughout EAAA implementation to help interpret differences in health service delivery and patient experiences of ART initiation, adherence, and retention before and after the intervention;estimate the potential change in HIV incidence and ART program size if EAAA were to be implemented nationwide in Swaziland; andpredict the long-term cost-effectiveness of EAAA.This trial aims to:This is a randomized seven-stepped-wedge design on seven pairs  in Hhohho Region  that are already providing a comprehensive package of HIV care and treatment services per Swaziland’s national adult HIV treatment guidelines .Sites are grouped to transition two at a time from the control  to the intervention (EAAA) stage every four months. As illustrated in Fig. Individuals who arrive at one of the study facilities during the control (“C”) stage will be offered ART according to the current national guidelines. Sites will follow the current national standard of care for HIV care and treatment while in the control stage.During the transition period (“T”) and the intervention (“I”) stage, all HIV-positive patients will be offered ART per the EAAA intervention (an offer of immediate ART initiation irrespective of CD4). Study participants who were already in pre-ART at the start of the study or who were enrolled into pre-ART during the control stage according to national standard of care will be offered ART at their first follow-up appointment at their enrollment site following the site’s transition to the intervention stage. This way, everyone who is enrolled into the study population will eventually be offered the EAAA intervention Fig. .Fig. 3TrMaxART EAAA Trial, indicating which manuscript page contains each element of the study protocol).An additional file provides the protocol checklist  and estimates of need [The Swaziland National AIDS Program (SNAP) has been in existence since 2004 and has scaled up its program each year, resulting in a significant increase in the number of people on treatment. The country has made great strides in their HIV response, including a robust national ART program and expanding the immunological treatment eligibility criteria in their national HIV treatment guidelines since 2010 to individuals with CD4 < 350 cells/mmAll HIV-positive individuals who are aged 18 years or older and are ART naïve—excluding pregnant or breastfeeding women—who attend the health facilities included in the study will be asked for their verbal consent to enroll in the study. Those individuals whose CD4 is greater than the current national guidelines for ART initiation and whose WHO stage is not 3 or 4 are the primary study population. Written informed consent will be secured for participation in social science research and the economic evaluation.Participants must be able and willing to give verbal consent for trial participation. Individuals considered unable to provide and participate in informed consent include those with uncontrolled psychiatric disorders or severe neurological impairment.3 will be enrolled to form a total study population of 4501. With recent changes in Swaziland’s national guidelines, the number of patients enrolled within the primary population is expected to be lower than projected due to the change in ART initiation threshold.The primary study population will be 2008 newly diagnosed or returning pre-ART patients as defined by the current national guidelines who are enrolled during the control or the intervention stage over the 36-month study period. In addition, 2493 patients with CD4 ≤ 350 cells/mmThe trial statisticians performed randomization of the study sites before the start of the study. The 14 sites have been grouped into seven pairs: four pairs were grouped according to geographic proximity; two pairs were grouped to coordinate the timing with operational or logistical issues; and one pair was grouped and excluded from the randomization so that it can transition to the intervention first to accommodate the social science research timeline. The six remaining pairs will be randomized into steps 2 to 7 when each site transitions to the intervention stage, but the health workers, study participants, and study teams are blinded to the timing to minimize bias.Power calculations were undertaken to ensure that the minimal detectable difference in the two primary endpoint rates, given that the projected sample size was well within the expected given prior literature . For oneUsing the standard method for power calculations for binary endpoints in stepped-wedge designs, with a 5% two-tailed Type I error rate, an 80% power or more was found to detect a 6–8 percentage point increase in the one-year retention rate over a range of scenarios considered for the one-year retention rate in SOC from 40–80% and an intra-cluster correlation coefficient (ICC) of 0–0.5, with no prior data available in Swaziland to allow ICC estimation. It is well-known that the stepped-wedge design is insensitive to the assumed value of the ICC . To be cFor the six-month viral load suppression after ART initiation endpoint, 1869 patients were projected to enroll in the EAAA (intervention) phase and 1728 in the control phase over the first 30 months to ascertain a six-month viral load after ART initiation. Following the methods as above, we found that there was 80% power or more to detect a 7–8 percentage point increase in the six-month viral suppression rate over a range of scenarios considered for the six-month viral suppression rate in standard of care of 20–70% and an intra-cluster correlation coefficient of 0.10–0.20.Community mobilization is used to sensitize communities on early ART and create demand for uptake of services. A comprehensive messaging strategy was designed to address the complex personal, cultural, social, and sexual considerations that influence decisions around HIV testing and ART initiation. Messages are delivered through a variety of methods and activities, including sensitization meeting with key community actors, including traditional leaders to gain their support. Community dialogues targeting community members are conducted using edutainment providing HIV testing services for active case finding at the community level.Door-to-door visits where information, education, and communication materials are delivered by trained community-based volunteers. Community-based HTC is provided during the community events and linkage to care is ensured through working with expert patients and HIV testing partners. Support groups are equipped with skills and tools to provide treatment literacy to PLHIV. These activities are conducted within a 20–30-km radius around each study site.The study aims to promote a continuum of care and to evaluate feasibility of an EAAA strategy within a public sector health system setting. Therefore, the study will be implemented in a public sector health system where there is already an essential package of services available through the current national ART program. The main differences in the package of services delivered in the standard of care vs. the intervention stage of the study are outlined in Table Additional blood investigations will be conducted to inform the measurement of the study endpoint, viral load suppression, but these lab tests will be taken in both the standard of care and intervention groups. Aside from the eligibility criteria and the messaging used during pre-ART counseling sessions, there will be no differences in the package of follow-up services provided to patients between the control and intervention study groups.Per national guidelines, ART initiation takes place within four weeks of HIV testing or two weeks of enrollment at the trial clinic, unless delayed per clinician’s guidance. All ART drugs used in the trial follow the Swaziland adult HIV management guidelines. All individuals who are eligible for treatment will be initiated on Swaziland’s recommended first-line ART regimen (TDF + 3TC + EFV), unless contraindicated when recommended alternate regimens will be used per national guidelines. These alternatives are: TDF + 3TC + NVP or AZT + 3TC + NVP (when EFV cannot be used); ABC + 3TC + EFV or AZT + 3TC + EFV (when TDF cannot be used); ABC + 3TC + EFV or d4T + 3TC + EFV (when AZT cannot be used).All patients in the control and intervention phase will follow the nationally recommended follow-up schedule for individuals who are on ART or enrolled in pre-ART.Routine viral load and drug resistance testing are important to understand the implications and impact of the interventions. Patients in both study groups will receive routine viral load and drug resistance testing in addition to the routine standard of care laboratory tests per the recommendation of and as dictated by Swaziland’s National Comprehensive HIV Package of Care, including: HIV test; CD4 test, full blood count; AST/ALT; creatinine; and Hepatitis B surface antigen.Each patient will provide samples every six months for viral load testing. For drug resistance testing, a pre-treatment sample will be collected from each patient upon study enrollment and at ART initiation. Upon initial signs of ART failure , a post-treatment sample will be collected for drug resistance testing.The data for the clinical endpoints will be collected at the facility on an ongoing basis as individuals are enrolled into the study and return for follow-up visits at the facility. Health workers will collect data from individual study participants at each site using the national chronic care file paper-based data collection forms.Where possible, the study will collect data from existing data sources  to inform the research. Data will also be collected from individual study participants at each visit. For data collected on-site outside of existing Swaziland Ministry of Health procedures, paper-based data collection forms will be used. Two copies of this form are kept: one copy in the patients’ clinical file and the second copy as part of the study records.An electronic database will also be developed to organize all study-related data. Data clerks will be responsible for the daily entry of data from paper forms into the electronic database. All data will be encrypted to ensure patient confidentially before, during, and after the trial. The principal investigator, study statistician, and data manager will have access to the final trial dataset.Individuals enrolled in this study and initiated on ART will continue to receive care and treatment through Swaziland’s public sector health system for the rest of their lives.A multidisciplinary, mixed-methods approach will be adopted to meet the study aim, including implementation science research, social science research, economic evaluations and cost-effectiveness modeling, and incidence modeling.This is one of the first studies in the treatment-as-prevention field that has been designed to answer critical implementation questions (i.e. acceptance and retention among patients initiated on ART at higher CD4 thresholds), and determine the “real world” potential of this new prevention intervention , 16–19. Social science research methods are used to triangulate the study’s findings on ART initiation, adherence, and retention; to help identify effect moderators by analyzing throughout the implementation social and structural factors and differences in health service delivery that may differently affect the uptake of services before and after facilities have transitioned to EAAA; and to contribute to a deeper understanding of reasons for delayed initiation, non-adherence, and non-retention. These objectives will be met by conducting mixed-methods research in a sample of nine study sites including a random quantitative survey, semi-structured interviews with health providers and patients, and participant observation.The quantitative survey will be conducted with a random sample of patients tested HIV-positive during the control phase and during the intervention phase at six and 12 months . The baseline survey will be conducted in 12 months and will include a random sample of HIV-positive patients who have initiated ART less than 12 months prior to the interview. A follow-up survey will be conducted six months after the site’s last day of its first transition month with patients who were initiated within six months of the site’s transition to the EAAA strategy (intervention stage). The second follow-up survey at 12 months after the site last day of its first transition months will allow for the comparison of the experiences of those being initiated on ART and adherence before and after the intervention, at six months and at 12 months of being on ART. Changes across time of distal/proximate factors will be studied, including sex, age, family context, socioeconomic status, employment status, health-seeking history, gendered decision-making patterns, and illness experience. Outcomes will include ART initiation, adherence and retention, patients’ experience of immediate ART initiation process .To assess health providers’ experiences and attitudes about EAAA, semi-structured interviews will be conducted with a limited number at two points in time: the second month into the transition and then again four months after the last transition month (information-rich case sampling of those most involved in EAAA). To understand reasons for delayed initiation, non-adherence, and non-retention, qualitative in-depth interviews will be conducted among a diverse sample of 50 HIV-positive individuals  who have initiated ART. These interviews will take place at random points of time during the intervention stage at different stages of each individual’s cascade of care and treatment. The categories include EAAA patients who delayed or refused ART initiation and, identified through the EAAA database, patients who are not virally suppressed after six months on treatment, patients who are virally suppressed after six months on treatment, patients who have not been retained in care/on ART at six months, and patients who have been retained in care/on ART at six months.Finally, at each of the sampled facilities, three weeks of observation will be conducted of pre- and post-test counselling, ART initiation, and adherence counselling: at baseline before the site transitions to the intervention stage, in the second month during transition, and seven months after the first transition month. These observations will take place simultaneously with the baseline patient semi-structured survey interview, health provider, and/or qualitative patient interviews to limit the time spent at each site.MaxART study aims to quantify empirically the causal impacts of EAAA on a range of important economic outcomes, including healthcare expenditure, resources use, and patients’ welfare. In addition, the cost-effectiveness of the EAAA approach will be estimated through a predictive economic evaluation. To this end, a comprehensive costing study will be conducted, which includes systematic assessments of both the government and private activities for EAAA and the unit costs for these activities. As part of the costing study, the national health sector budgets and expenditure reports will be regularly reviewed and the data relevant to EAAA will be recorded, such as provider salaries and facility, supply chain, drug, and laboratory costs. A time-and-motion study will also be conducted to determine how much time healthcare providers spend on the different HIV treatment-related activities, including consultation time, physical examination, counseling, and interpreting laboratory results. Finally, patient exit interviews will be used to measure private healthcare expenditure and the patient and provider time required under a EAAA protocol.The To inform the national policymaker’s interpretation of the stepped-wedge trial, as well as long-term planning of a national EAAA program, a mathematical model has been developed to simulate the heterosexual transmission of HIV and the provision of ART in the catchment area of the study sites and for Swaziland as a whole .The objectives of the HIV incidence modeling component of this study are twofold. First, a narrow, data-driven simulation study will be conducted to estimate the impact of the EAAA stepped-wedge trial on HIV incidence during the study period in the catchment area of the study sites. Second, a larger, projection study will be conducted to estimate the impact of a national EAAA program on HIV incidence over three time horizons: (1) five years, (2) ten years, and (3) 15 years (Oct 2016–Sept 2031) for the entire country of Swaziland.Outcome indicators for both analyses are the incidence rate ratio, number of HIV infections averted, the number of HIV-negative life-years gained, the number of life-years gained, and the number of additional person-years of ART provided . Outcome indicators for the first analysis will be calculated using model output from both the control and EAAA phases of the clinics, weighted for the population size of the clinic catchment area and the amount of time spent in each phase. Besides population-average outcome indicators, the outcome indicators stratified by age group and gender will also be calculated.For the estimation of the impact of a national EAAA program on HIV incidence, two scenarios will be contrasted against one another. In the control scenario, the country will maintain its current national treatment guidelines for the entire simulation period. In the EAAA scenario, a shift to universal treatment will be simulated from October 2016 onwards. The rates of HIV diagnosis, ART initiation, viral suppression, and program retention among HIV-positive patients with CD4 cell counts > 500/mL in the intervention scenario will be equal to those inferred from the narrow, data-driven simulation study.The epidemiological model will be calibrated to historic data on key demographics , historic HIV prevalence data (stratified by age group and gender), historic ART program size data , and recent HIV incidence data, by age group and gender . As paraOnce the “model world” is deemed sufficiently resembling of the real world, the “model trial” will be calibrated to the real trial by adjusting the hazard functions for initiating ART and dropping out of the ART program in order to match key summary statistics: the size of the ART patient population by clinic, age group, gender, baseline viral load, and baseline CD4 cell count. In addition, the percentage of patients that is virally suppressed six or more months after ART initiation will be matched across all sites, during the period that the sites were offering EAAA, as well as the percentage of patients that are virally suppressed across all sites, during the period that the sites were in the control phase.The primary analysis will follow the intent to treat approach, with each patient’s intervention status assigned as the one in place at their facility at the time of study enrollment. The log rank test, stratified by step time, will be used to assess the statistical significance of any differences observed in one-year retention and six-month suppression rates between the standard of care and early access groups. Kaplan–Meier curves will be produced and used to obtain point and interval estimates of the cumulative incidence of the two primary endpoints at the landmark time points. In the retention analysis, censoring will occur only by administrative end of follow-up or one year from enrollment, whichever happens first. In the viral suppression analysis, only patients initiated to ART who have at least six months of follow-up at the time of administrative end of follow-up and at least one viral load measured after that time will be included. Thus, this endpoint will be interpreted as viral suppression given comprehensive clinical retention.p values for the intervention effects using the Cox proportional hazards model, adjusting for potential measured confounders, including facility, sex, age, BMI, disclosure to partner, occupation, education, baseline CD4, baseline viral load, date of first HIV-positive test, baseline opportunistic infections, baseline WHO stage, and baseline tuberculosis history. Additional analysis will adjust for those variables which are repeatedly updated over time as time-varying covariates, and will, in addition, consider patient’s intervention status as time-varying, allowing for the transition of facilities over time, as well as a time-varying eligibility status, allowing for changes in national guidelines and patient preferences.Censoring for this endpoint occurs at the minimum of the date of the last viral load six months after ART initiation, the administrative end of follow-up, and date of death, whichever happens first. Secondary analysis of the primary endpoints will include estimation of relative risks, their 95% confidence intervals, and Marginal structural models will be explored to adjust for possible bias due to dependent censoring –24. ModiSince one of our primary outcomes is retention, where attrition can be considered the opposite of retention, the intent to treat analysis is ideally situated to assess the impact of the intervention on retention/attrition and cannot be biased by it. However, six-month viral suppression can be biased by differential non-retention. Therefore, in secondary analysis, as recommended by Little et al. , we willMaxART trial is designed to better understand the “real world” complexities of implementing this treatment for all approach. The quantitative and qualitative social science research produced from this study will be essential to understand the social and behavioral impact of offering ART to HIV-positive individuals soon after diagnosis. Community perception of the intervention and research on how to best engage communities to support this intervention will have an important influence on uptake, linkages to care, retention, and adherence. Further, a comprehensive economic evaluation to assess how expanding access to treatment will improve livelihoods and productivity and, ultimately, decrease the cost to the health system as a whole. Modeling will be used to also understand the cost-effectiveness of the intervention, which is key evidence for the Government of Swaziland’s national financial planning. This study has been designed to answer the critical implementation questions posed by the Government of Swaziland as the country works towards rolling out a treatment for all approach across the country to turn the tide on their epidemic.The This trial is underway. Clients will be enrolled into the study through August 2017.


A community based, second-line treatment cohort of 102 subjects was followed monthly in community clinics with a total of 178 patient-years of follow-up; second-line virological failure was frequent in this setting. Second-line antiretroviral therapy (ART) based on ritonavir-boosted protease inhibitors (bPIs) represents the only available option after first-line failure for the majority of individuals living with human immunodeficiency virus (HIV) worldwide. Maximizing their effectiveness is imperative.This cohort study was nested within the French National Agency for AIDS and Viral Hepatitis Research (ANRS) 12249 Treatment as Prevention (TasP) cluster-randomized trial in rural KwaZulu-Natal, South Africa. We prospectively investigated risk factors for virological failure (VF) of bPI-based ART in the combined study arms. VF was defined by a plasma viral load >1000 copies/mL ≥6 months after initiating bPI-based ART. Cumulative incidence of VF was estimated and competing risk regression was used to derive the subdistribution hazard ratio (SHR) of the associations between VF and patient clinical and demographic factors, taking into account death and loss to follow-up.P < .001).One hundred one participants contributed 178.7 person-years of follow-up. Sixty-five percent were female; the median age was 37.4 years. Second-line ART regimens were based on ritonavir-boosted lopinavir, combined with zidovudine or tenofovir plus lamivudine or emtricitabine. The incidence of VF on second-line ART was 12.9 per 100 person-years (n = 23), and prevalence of VF at censoring was 17.8%. Thirteen of these 23 (56.5%) virologic failures resuppressed after a median of 8.0 months  in this setting where viral load monitoring was available. Tuberculosis treatment was associated with VF (SHR, 11.50 ; Second-line VF was frequent in this setting. Resuppression occurred in more than half of failures, highlighting the value of viral load monitoring of second-line ART. Tuberculosis was associated with VF; therefore, novel approaches to optimize the effectiveness of PI-based ART in high-tuberculosis-burden settings are needed.NCT01509508. Despite clinical and public health gains following antiretroviral therapy (ART) rollout, treatment failure of first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)–based ART is common , with upData from observational studies of second-line bPI-based ART treatment outcomes in sub-Saharan Africa suggest a 14%–32% prevalence of virological failure (VF) , with raPreviously published cohort studies have used a Kaplan-Meier survival analysis and standard Cox regression models that can result in inflated estimates due to competing events. Competing risk regression analysis overcomes this limitation by accounting for events, such as death, that preclude the subject from experiencing the study outcome. This is particularly important in sub-Saharan Africa where mortality remains significant following ART initiation due to advanced disease stage as well as loss to follow-up . Here weThis cohort study was nested within the French National Agency for AIDS and Viral Hepatitis Research (ANRS) 12249 Treatment as Prevention (TasP) trial , an ongoing cluster-randomized trial evaluating the impact of immediate vs deferred ART initiation (according to South African guidelines) on HIV incidence  , follow-up was censored as the latest of either the date of the last laboratory test or last clinic visit.http://hivdb.stanford.edu/) [At presentation to the trial clinics, all consenting HIV-infected participants were asked to complete the study questionnaires and underwent clinical examination by trial nurses. CD4 count was measured in TasP clinics using a commercial point-of-care CD4 test . VL was measured using The Abbott m2000 RealTime System with HIV type 1 (HIV-1) VL determination from human plasma of HIV-1–infected individuals in the range of 40–10000000 copies/mL . The viral load assay was CE (Conformité Européene) marked and performed at the Africa Centre laboratory; the laboratory participated in Quality Control for Molecular Diagnostics (QCMD) for VL quality assurance testing. Both CD4 and viral load were measured at baseline, and at months 3 and 6 after ART initiation and every 6 months thereafter. Full-genome deep sequencing was performed after 2 consecutive VL measurements >1000 copies/mL at least 6 months apart following second-line VF, as per adapted protocol from Gall et al . In brierd.edu/) . We repoParticipant characteristics were reported using frequency and percentage for categorical variables and median and interquartile range (IQR) for continuous variables. The incidence of second-line failure per 100 person-years was estimated with its 95% confidence interval (CI).P value of <.1. Although age and sex were not significantly associated with VF in the univariable analysis, they were kept in the final model as they were a priori specified confounders. Analysis was done using Stata software version 13.We estimated the cumulative incidence function of VF on second-line treatment, taking into account death and loss to follow-up as competing risks. Competing risk regression was used to estimate the subdistribution hazard ratio (SHR) of the associations between VF and participant clinical and demographic factors, accounting for the competing risks of death and loss to follow-up, according to the model of Fine and Gray . In the Ethics approval was granted by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (BFC 104/11) and the Medicines Control Council of South Africa. The study was also authorized by the KwaZulu-Natal Department of Health in South Africa. Written informed consent was obtained from all participants.One hundred one participants were included in this analysis. Sixteen (15.8%) individuals were already on second-line treatment at enrollment into the trial for a median of 2.7 years . Three of these participants had VF at the baseline clinic visit. Seven (6.9%) participants had not initiated ART at enrollment into the trial. The remaining 78 (77.2%) were on first-line NNRTI-based ART for a median duration of 4.9 years  at the time of enrollment; 41 (52.6%) of these had VF at the baseline clinic visit in the TasP trial, necessitating a switch to bPI along with 2 nucleoside reverse transcriptase inhibitors (NRTIs). Median duration on bPI for the 85 patients was 0.6 years .The majority of participants were female (65.4%) and the median age at initiation of second-line treatment was 37.4 years . There was a high level of unemployment (91.9%) in this cohort of participants residing in a rural setting . ThirteeThe 101 participants contributed 178.7 person-years of follow-up to the analysis. The overall incidence of VF on second-line ART was 12.9 per 100 person-years. At administrative censoring, 76 participants were alive and in care, 1 had died, and 1 was lost to follow-up before any VF was documented, and 23 participants had VF at least 6 months after initiating bPI second-line ART . Following intensification of adherence counseling, 13 of the 23 participants (56.5%), including the 3 patients with bPI VF at the baseline clinic visit, resuppressed with a VL <1000 copies/mL after a median of 8.0 months  . Eight of these 13 participants subsequently rebounded with a VL >1000 copies/mL. The prevalence of VF in patients alive and on bPI ART at the time of administrative censoring was 17.8% (18/101).P < .0001) and a lower level of adherence (median pill count <97%) . In the multivariable analysis, the association of TB treatment with VF on bPI second-line treatment remained , whereas the association with median pill count was no longer present  participants had acquired major PI mutations: M46I, I54V, L76V, and V82A. This individual received rifampicin containing TB treatment started at the same time as double-dose bPI.We determined the incidence rate for VF on second-line ART and associated risk factors in rural KwaZulu-Natal within the TasP trial. The incidence rate of VF was 12.9 per 100 person-years , and prevalence was 17.8% at the end of the observation period. A meta-analysis of studies conducted in resource-limited settings reported a pooled prevalence of VF of 23.1%  after 12 months of treatment with bPI ART , 27. HowThere are considerable differences between studies in the definition of VF (from VL >50 to >1000 copies/mL), and guidelines are not clear on a definition of second-line failure that should trigger a switch of treatment regimen. We therefore chose a pragmatic definition of a single VL >1000 copies/mL, particularly as the follow-up time was relatively short for patients in TasP who switched to second-line ART within the trial. We found that many patients with VF on bPI ART resuppressed to VL <1000 copies/mL following a period of intense adherence counseling. This phenomenon has been reported in patients treated with first-line ART in both South Africa  and otheThe association between TB treatment and VF of NNRTI-based first-line ART has previously been reported . Here wePoor adherence has been shown to be associated with VF in other studies , 17, 18;pol sequencing ignore the influence of mutations in other genes such as gag [env [We found multiple major NRTI and NNRTI mutations at first-line failure in the participants who had genotype testing (9 participants), consistent with data from resource-limited settings , 38. Thoh as gag  and env gag [env  on PI regag [env , 50, furThe main methodological strength of this study is the application of regression methods, which account for the presence of competing risks to estimate the rate of VF on second-line treatment and the association between covariates of interest and VF. Other cohort studies reporting outcomes on second-line treatment and factors associated with VF have used standard Kaplan-Meier survival analysis and Cox regression models, which can lead to biased or inflated estimates of association. As this analysis was done on prospectively collected data within a trial context with preset procedures rather than routine data, we also minimized the common problem of missing data and information bias.There are some limitations of the study. First, although the sample size is small, the prevalence of VF on bPI we found is in line with other published studies. The inclusion of whole-genome sequencing data, albeit from 9 patients, is unique to this cohort. Second, the study was nested in a randomized community trial and as such the patients may not be truly reflective of the general HIV population, given the intense monitoring and provision of counseling and adherence interventions that may impact their attitude toward health and promote more successful treatment outcomes. We did not measure drug levels to assess pharmacokinetic drug–drug interactions or adherence before a clinical visit. Finally, 16 (15.8%) patients were included who were already on second-line ART at enrollment, for a median of 2.7 years, representing a potential bias.There was also heterogeneity in the patient population. We included all participants from the parent study irrespective of study arm, though study arm had no effect on outcome, largely due to poor linkage to care in this setting . We included newly diagnosed as well as ART-experienced patients, as both present for first-line therapy under “real-world” programmatic conditions, sometimes with evidence of drug resistance to thymidine analogues arising from prior ART  or with In conclusion, this study found that second-line PI-based VF was common in this population accessing ART in rural South Africa under trial circumstances but recruited at the population level. Further research is needed to understand the mechanisms behind VF of bPI ART in TB-coinfected patients. Novel approaches to optimize second-line ART in resource-limited settings are still urgently needed as this population is likely to grow rapidly, owing to the WHO 2015 guidelines to test and treat all people living with HIV .Supplementary DataClick here for additional data file.


This retrospective cohort study is aimed to assess reasons and predictors of regimen change from initial highly active antiretroviral therapy among 1533 Human Immunodeficiency virus-infected adult patients at the Jimma University Tertiary Hospital.One in two (47.7%) adults changed their antiretroviral therapy regimen. Patients who were above the primary level of education [Hazard ratio (HR) 1.241 (95% CI 1.070–1.440)] and with human immunodeficiency virus/tuberculosis co-infection [HR 1.405 (95% CI 1.156–1.708)] had the higher risk of regimen change than their comparator. Individuals on Efavirenz [HR 0.675 (95% CI 0.553–0.825)] and non-stavudine [HR 0.494 (95% CI 0.406–0.601)] based regimens had lower risk of regimen change. Globally, the number of people living with human immunodeficiency virus (HIV) reached 38.8 million in 2015  for ART regimens changed. Among documented toxicities: 82 (61.19%) were fat changes, 30 (22.38%) were peripheral neuropathies, 14 (10.45%) were anemic, 5 (3.73%) were central nervous system (CNS) toxicities and one hepatotoxicity.A new TB treatment [n = 120 (16.42%)], planning pregnancy or being pregnant [n = 29 (3.96%)] and treatment failure [n = 24 (3.28%)] were the other reasons for regimens changed. Others (n = 127) had no documented reason for regimen changed.The results from the multivariable Cox-proportional hazards regression analysis found patients who were above the primary level of education [HR 1.241 (1.070–1.440)] and with HIV/TB co-infection [HR 1.405 (1.156–1.708)] had a higher risk of regimen change than their comparator as shown in Table Regimen change affects the success of the treatments to achieve United Nations Program on HIV and AIDS (UNAIDs) goals . Near toToxicities were the predominant reasons for ART regimens changed. This is consistent with other studies , 32–34. New TB drug treatment was also a major reason for the initial ART regimen change, especially for NVP-based regimens. This was due to an interaction of NVP with the anti-TB drugs such as Rifampin , 39. In In the current study, the hazard of regimen change was higher in Tb/HIV co-infected patients than in patients with HIV infections alone. This is consistent with other studies , 41, 42.EFV-based regimens had the lower hazard of change than NVP-based regimens. This is similar to other studies , 17, 45.Patients who were above the primary level of education had the higher hazard of regimen change than those below primary level education. Although we did not assess their knowledge, this might be due to better awareness of ART-associated toxicities and reasons for regimen change among the educated groups.One in two patients changed their ART, mainly due to drug toxicity. New TB drug treatment, being pregnant and treatment failure were other reasons for the regimen changed. Regimen changed patients were more likely to be HIV/TB co-infected, above primary level education, and on D4T or NVP based regimens. We recommended strengthening adherence level and adopting benchmarking programs such as a linkage-case-management to enhance ART linkage and retention, which have demonstrated to be effective in similar settings , 47.Reasons for regimen change were documented only if they were incidental to regimen change, therefore, their frequency cannot be used to estimate their actual occurrence.The outcome status of regimen changed patients was not known.Some variables such as mental illness and stigma were not assessed as our data source lacked this information.We acknowledge the limitations of the current study including: