The goal of antiretroviral therapy (ART) is to suppress viral replication, reduce morbidity and mortality, and improve quality of life (QoL). For resource-limited settings, the World Health Organization recommends a first-line regimen of two-nucleoside reverse-transcriptase inhibitors and one non-nucleoside transcriptase inhibitor (nevirapine (NVP) or efavirenz (EFV)). There are few data comparing the QoL impact of NVP versus EFV. This study assessed the change in QoL and factors associated with QoL among HIV patients receiving ART regimens based on EFV or NVP.We enrolled 640 people with HIV eligible for ART who received regimens including either NVP or EFV. QoL was assessed at baseline, three months and six months using Physical Health Summary (PHS) and Mental Health Summary (MHS) scores and the Global Person Generated Index (GPGI). Data were analyzed using generalized estimating equations, with ART regimen as the primary exposure, to identify associations between patient and disease factors and QoL.QoL increased on ART. The mean QoL scores did not differ significantly for regimens based on NVP versus EFV during follow-up for MHS and GPGI regardless of CD4 stratum and for PHS among patients with a CD4 count >250 cells/μL. The PHS-adjusted β coefficients for ART regimens based on EFV versus NVP by CD4 count strata were as follows: −1.61  for CD4 count <100 cells/μL; 0.82  for CD4 count 101–250 cells/μL; and −1.33  for CD4 count >250 cells/μL. The corresponding MHS-adjusted β coefficients were as follows: −0.39  for CD4 < 100 cells/μL; 0.16  for CD4 count 101–250 cells/μL; and −0.75  for CD4 count >250 cells/μL. The GPGI-adjusted odds ratios for EFV versus NVP were 0.51  for CD4 count <100 cells/μL, 0.98  for CD4 count 101–250 cells/μL, 1.39  for CD4 > 250 cells/μL. QoL improved among patients on EFV over the 6-month follow-up period . Overall, patients with depression  had lower scores and women had lower MHS . Other factors associated with lower QoL included alcohol use, low education level and advanced HIV disease.ART improves QoL. The results support use of either NVP or EFV. Patients initiating ART should be assessed for depression and managed appropriately. Women may require extra support to improve their QoL. The World Health Organization (WHO) recommendation for combination antiretroviral therapy (ART) for HIV is two-nucleoside reverse-transcriptase inhibitors (zidovudine (AZT)/abacavir plus lamivudine 3TC) or nucleotide tenofovir (TDF) plus 3TC) and a non-nucleoside reverse-transcriptase inhibitor: efavirenz (EFV) or nevirapine (NVP) . Uganda TC or nucThe majority of clinical studies have focused on the immunological and virological outcomes associated with EFV and NVP. However, with the availability of ART, there is no doubt that survival has been improved. In Uganda, HIV/AIDS patients reportedly now have a life expectancy similar to that of the general population . For thiOther factors have been associated with PHS, including baseline viral load concentration ≥100,000 copies/ml and decline in plasma viral load. An increase in MHS has been associated with a decline in viral load. The presence of an adverse event during follow-up has also been associated with declines in both the MHS and PHS . AdherenIn summary, studies suggest similar virologic and clinical outcomes for regimens based on NVP or EFV, but findings for QoL outcomes have been inconsistent. With the increased access to ART in sub-Saharan Africa, we need to ensure both good clinical outcomes and optimal QoL. Research is needed to improve understanding of the impact on QoL of different treatment options. There is limited research on the differences in QoL between the recommended ART regimens and sparse data on the different factors associated with QoL in relation to ART regimens. The aim of this study was to assess the effect of either NVP or EFV based regimen on QoL the other factors associated with QoL and these ART regimens. Our findings will help to inform strategies to optimize patient outcomes.We enrolled 640 patients from an outpatient HIV clinic at the Ugandan National Referral Hospital between April 2012 and December 2013. Patients were eligible for the study if they were starting ART on the day of the interview, were prescribed either of the following regimens (TDF plus 3TC plus EFV/NVP or AZT plus 3TC plus EFV/NVP), had a CD4 count <350 and were 18 years or older. Owing to the variable nature of CD4 count with time of day, fatigue, stress or infections , patientThis was a prospective cohort study to assess QoL and factors associated with QoL among patients receiving ART regimens based on NVP or EFV. Patients were followed up for six months and were assessed at baseline, three months and six months.The study was approved by Makerere University School of Medicine, Research and Ethics Committee and the Uganda National Council for Science and Technology. All participants provided written, informed consent prior to participation in the study.Three patient-reported outcome measures were used. First, the Medical Outcomes Study (MOS-HIV) Health Survey was used to collect data on health-related QoL (HRQoL). It is a disease-specific measure that has been widely used . MOS-HIVWe collected data on sociodemographic characteristics including sex, age, religion, monthly income, employment status, education level, marital status and social behavioral characteristics . Information was documented on clinical characteristics such as WHO HIV stage, opportunistic infection, diagnosis of clinical depression, toxicity that warranted change of regimen, adherence to ART and CD4 + T lymphocyte counts. Blood was drawn at baseline and at the six-month visit for the CD4 cell count for all patients. All patients were screened for depression using CES-D at each of the three visits. We used a cutoff score of at least 16 to suggest probable depression as recommended by the author of CES-D .The MOS-HIV subscales were transformed into two summary scores, PHS and MHS , normaliTo identify determinants of QoL among patients on an EFV-based ART regimen and those receiving a NVP-based regimen, we analyzed the two groups separately. Descriptive statistics and frequencies were determined for all sociodemographic, clinical and outcome variables. For comparisons between the two groups we used chi-square tests for categorical variables, and student’s t-tests, Spearman’s correlation and Wilcoxon’s rank sum tests for continuous variables. We determined effect size as recommended by Cohen . We compFor the normally distributed outcome variables, we used univariate linear regression with generalized estimating equations (GEE) to identify factors associated with PHS and MHS. All variables determined to be significantly associated with PHS and MHS in univariate models at p < 0.20 and p < 0.10, respectively, were considered in the final multivariate models. Sex and age were considered as forced variables in the final multivariate models. Depression was not included in the MHS model to avoid collinearity since MOS-HIV also covered depression.We then ran two sets of models: one set with only the forced variables, and the other set with the significant p values (cutoff 0.20 for PHS and 0.10 for MHS). We observed the standard errors for the forced variables and found them to be approximately the same in the two models; these were the final models without multi-collinearity. To further test for collinearity, each of the other variables was dropped from the model and we observed changes in the standard errors for the forced variables. We used logistic regression with GEE to identify demographic, behavioral and clinical characteristics associated with GPGI score at univariate and multivariate analysis. All factors with p < 0.20 at univariate analysis were included in the final multivariate model. The same process was used to build the final multivariate model, as was done for MHS and PHS. The value p < 0.05 was used as a cutoff for the determinants of QoL at multivariable analysis. We tested for interaction between ART regimen and/or time and the significant factors at multivariate analysis. All statistical analyses were conducted using Stata 12.1 .Six hundred and forty patients were enrolled in the study, 481 (70 %) were initiated on an EFV-based ART regimen and 159 (30 %) on a NVP-based regimen. Of the total enrolled, 420 (66 %) were women. The mean CD4 change (cells/μL) from baseline to six months was 182 (SD 148.5). The patients on EFV had a higher mean CD4 change  than patients on a NVP regimen  p = 0.04  had drug reactions that warranted a change of regimen, 10 (2.1 %) changed from EFV, eight (1.7 %) had hypersensitivity skin reactions, and two (0.4 %) had severe dizziness. Of the eight patients (5 %) who changed from the NVP-based regimen, two  had Steven Johnson’s syndrome and the rest (3.8 %) had a hypersensitivity rash. Other clinical and sociodemographic characteristics are shown by ART regimen in Table Regardless of ART regimen, there was a trend towards an increase in the mean QoL scores from baseline to the six-month visit; PHS and MHS were normally distributed and presented as means and GPGI was negatively skewed presented as medians Table . The impDepression was the only factor significantly associated with PHS among patients receiving a NVP-based regimen at both univariate and multivariate analysis . People living with HIV/AIDS (PLHA) receiving a NVP-based ART regimen with probable depression (score ≥16) had on average 1.64 lower PHS than those without depression . There was no evidence of interaction between depression and ART regimen (p = 0.89).The following factors were associated with PHS among patients receiving an EFV-based ART regimen: study visit, level of education, WHO stage and probable depression. PHS improved from baseline to six months (p = 0.04). In multivariate analysis , there was a 0.50 mean increase in PHS at six months compared with baseline visit (95 % CI 0.06 to 0.93). Patents with higher education level had an overall increase in PHS (p = 0.005). For example, patients with tertiary education receiving an EFV-based regimen had a 2.16 mean increase in PHS compared with patients with a primary or no education (95 % CI 0.66 to 3.65). A patient with WHO stage 3&4 compared with those graded as WHO stage 1&2 on average had a 0.86 lower PHS (95 % CI −1.47 to −0.25). Depression was negatively associated with PHS among patients receiving EFV, and PLHA with probable depression had a 1.67 lower mean PHS than patients without depression (95 % CI −2.13 to −1.21). There was no evidence of interaction between WHO stage and ART regimen (p = 0.08), level of education and ART regimen (p = 0.80), study visit and ART regimen (p = 0.90) and depression and ART regimen (p = 0.88). There was evidence of interaction between WHO stage and time (p = 0.0002) and ART regimen and baseline CD4 count (p = 0.003).A woman on a NVP-based ART regimen compared with a man had on average a 1.57 point lower MHS score (95 % CI −2.40 to −0.73). Sex was the only factor significantly associated with MHS among patients receiving a NVP-based ART regimen. The factors adjusted for in the multivariate model were sex, age, study visit, WHO stage, marital status, social support, baseline CD4 count and level of education.Study visit was the only factor associated with MHS among patients on an EFV-based ART regimen. There was an improvement in MHS (p < 0.001) over time. Patients had a mean increase of 1.04 MHS at the three-month visit compared with the baseline visit (95 % CI 0.62 to 1.46), and a 0.7 point increase in MHS at the six-month visit compared with the baseline visit (95 % CI 0.33 to 1.17). There was no evidence of interaction between the study visit and ART regimen (p = 0.90). The factors adjusted for in the multivariate model were sex, age, study visit, WHO stage, marital status, social support, baseline CD4 count and level of education.For the GPGI score, depression was the only statistically significant predictor in multivariate analysis. A patient on a NVP regimen with probable depression (score ≥16) had 0.31 lower odds of having a high GPGI score (95 % CI 0.19 to 0.51) compared with a patient without depression. There was no evidence of interaction between ART regimen and depression (p = 0.48) and time and depression (p = 0.16). The other variables in the multivariate model included sex, age, study visit, WHO stage, education level, income per month, depression, employment status, alcohol use, social support and baseline CD4 count.Several factors were associated with the GPGI score among patients on an EFV-based regimen: study visit, alcohol consumption and depression. There was a significant overall association of odds of a high GPGI score over time from baseline visit to the six-month visit (p = 0.028). The odds of a high GPGI score at month six compared with baseline were 1.34 (95 % CI 1.01 to 1.77). In addition, in patients taking an EFV-based ART regimen, the odds of a high GPGI were 1.69 (95 % CI 1.18 to 2.42) for those who did not consume alcohol compared with those who did. Patients with probable depression had odds of 0.43 (95 % CI 0.33 to 0.55) for high GPGI score compared with patients without depression. There was no evidence of interaction between ART regimen and alcohol consumption (p = 0.56) and ART regimen and time (p = 0.61). The following variables were included in the multivariate model: sex, age, study visit, WHO stage, education level, income per month, depression, employment status, alcohol use, social support, opportunistic infection and baseline CD4 count.In summary, depression and sex were associated with QoL scores among patients on the NVP-based regimen and study visit, probable depression, level of education, WHO stage and alcohol use were associated with QoL scores among patients on the EFV-based regimen. Study visit was significant among patients receiving EFV for all the QoL measures, and depression was significant both for PHS and GPGI among patients receiving NVP. Table The objective of this study was to determine the factors associated with QoL among HIV patients receiving a first-line ART based on EFV or NVP, and to compare the pattern of results for people on the two medication regimens. There was a statistically significant improvement in QoL scores while on ART, with small to medium/large effect sizes observed. Effect sizes of this magnitude have been interpreted as “a real effect of ART on QoL scores that can be seen through a study to obvious effect” .The differences in QoL scores were small compared with what has been reported in other studies , 33. HowThe above studies reported greater differences in QoL than our study, possibly because of the longer time of follow-up. In addition, the majority of participants in these studies were WHO stage 3&4 and likely to have greater changes in QoL, whereas in our study the majority of patients were stage 1&2 and followed up for only six months. These changes would be more comparable to our findings if the effect sizes had been reported. The interaction between baseline CD4 count and time indicates that PHS may change with time while on ART in relation to baseline CD4 count. The borderline significant interaction term between CD4 count and GPGI and lack of evidence for interaction between CD4 count and MHS show that mental health and global QoL may not be dependent on CD4 changes with time. Further to this, there were no significant differences in change in MHS and GPGI QoL scores over time when comparing the EFV and NVP regimens. Patients with CD4 count <100 cells/μL and on EFV had a decrease in PHS. Those with a CD4 count between 101 and 250 cells/μL had an improvement in PHS; however, there was also no difference in PHS between EFV and NVP regimens for patients with CD4 > 250 cells/μL. Generally, there was some improvement in MHS and PHS from baseline to six months among patients on an EFV-based regimen. There was no evidence of interaction between regimen and time. EFV has been reported to improve QoL in other settings . AlthougThere were two severe reactions among patients on the NVP-based regimen (two cases of Steven Johnson’s syndrome). No severe neurological reactions to EFV were reported . A higher proportion of NVP patients than EFV patients required changing the regimen. Although we did not examine the relationship between adverse events and QoL, results from a qualitative sub study of this population revealed minimal interference with QoL related to side effects . FurtherTo our knowledge, this study is the first to assess QoL and factors associated with QoL among HIV patients receiving ART regimens based on EFV and NVP in sub-Saharan Africa. The majority of studies have assessed virological outcome and not QoL. For instance, EFV has shown advantages over NVP in some studies, including a lower risk of virological failure , greaterIn general, data are still limited regarding changes in QoL related to ART regimens based on NVP or EFV. Previous studies from developed countries have studied QoL and ART regimens by comparing either NVP with protease inhibitor or EFV with a protease inhibitor. Among ART-experienced patients who were switched to either EFV or protease inhibitor, patients who received EFV reported a better QoL . Another Adherence to ART has been reported in other studies to be associated with QoL , 48. A mAmong patients receiving a NVP-based ART regimen, depression score was the only variable associated with lower PHS and GPGI. Being female was associated with lower MHS. WHO stage 3&4, low education level and probable depression were associated with poor QoL in this study among patients on EFV. Depression and alcohol use had a significant negative association with global QoL among patients receiving an EFV-based regimen. Depression was independent of time during the follow-up period. Depression has been associated with poor QoL in PLHA in other studies in both resource-limited settings and developed countries –54. In t This was not a randomized study but future studies may build on these results. We recommend a randomized study with women with a CD4 count of <250 cells/μL and men with a CD4 count of <400/μL cells randomized to receive either NVP or EFV, with assessment of QoL as the main outcome measure. Follow-up of six months is short for a chronic illness and does not allow for assessment of regimen efficacy or the proportion of patients who fail to respond to therapy or switch to another regimen. We recommend studies with long follow-up and a large sample of patients initiating ART to confirm the continuous improvement in QoL that occur when ART is initiated at relatively high CD4 counts.There was a trend towards an increase in mean and median QoL scores among patients on ART in this study, especially with the EFV-based regimen. The changes in MHS and GPGI did not differ between patients using EFV or NVP as the first-line treatment from baseline to six months, and no difference was found in PHS for patients with a CD4 count >250. Patients with a very low CD4 count (<100 cells/μL) have better PHS while on a NVP regimen than an EFV regimen. By contrast, patients with a CD4 count between 101 and 250 cells/μL have a better PHS with an EFV regimen than a NVP regimen. Initiating an ART regimen containing EFV at a higher CD4 count led to improvement in QoL with time. NVP caused more severe toxicity and a relatively higher incidence of toxicities that warranted regimen change than EFV. This study supports the WHO guidelines that were in effect at the time of the study for resource-limited settings to initiate ART at a higher CD4 count and to use either a regimen based on NVP or EFV as a first-line therapy, albeit with a preference to EFV.Regardless of ART regimen, women initiating ART may require more psychosocial support to improve their mental health. All PLHA initiating ART should be screened for depression and alcohol use, and appropriate support should be given in order to improve or maintain a high QoL.


The efficacy of first-generation protease inhibitor based triple-therapy against hepatitis C virus (HCV) infection is limited in HIV/HCV-coinfected patients with advanced liver fibrosis and non-response to previous peginterferon-ribavirin. These patients have a low chance of achieving a sustained virologic response (SVR) using first generation triple-therapy, with a success rate of only 20%. We investigated the efficacy and safety of lead-in therapy with intravenous silibinin followed by triple-therapy in this difficult-to-treat patient group.Inclusion criteria were HIV/HCV coinfection with advanced liver fibrosis and documented previous treatment failure on peginterferon-ribavirin. The intervention was a lead-in therapy with intravenous silibinin 20 mg/kg/day for 14 days, followed by triple-therapy (peginterferon-ribavirin and telaprevir) for 12 weeks, and peginterferon-ribavirin alone for 36 weeks. Outcome measurements were HCV-RNA after silibinin lead-in and during triple-therapy, SVR data at week 12, and safety and tolerability of silibinin.We examined sixteen HIV/HCV-coinfected patients with previous peginterferon-ribavirin failure, of whom 14 had a fibrosis grade METAVIR ≥F3. All were on successful antiretroviral therapy. Median (IQR) HCV-RNA decline after silibinin therapy was 2.65 (2.1–2.8) log10 copies/mL. Fifteen of sixteen patients (94%) had undetectable HCV RNA at weeks 4 and 12, eleven patients (69%) showed end-of-treatment response , and ten patients (63%) reached SVR at week 12 (SVR 12). Six of the sixteen patients (37%) did not reach SVR 12: One patient had rapid virologic response (RVR)  but stopped treatment at week 8 due to major depression. Five patients had RVR, but experienced viral breakthroughs at week 21, 22, 25, or 32, or a relapse at week 52. The HIV RNA remained below the limit of detection in all patients during the complete treatment period. No serious adverse events and no significant drug-drug interactions were associated with silibinin.A lead-in with silibinin before triple-therapy was safe and highly effective in difficult-to-treat HIV/HCV coinfected patients, with a pronounced HCV-RNA decline during the lead-in phase, which translates into 63% SVR. An add-on of intravenous silibinin to standard of care HCV treatment is worth further exploration in selected difficult-to-treat patients.NCT01816490ClinicalTrials.gov  Treatment of hepatitis C virus (HCV) infection has currently entered a new era ,2. The asilybum marinum and commercially available as Legalon SIL—on HCV-RNA decline and treatment outcome in HIV/HCV coinfected patients with advanced liver fibrosis/cirrhosis and previous failure of peginterferon-ribavirin therapy. These patients are most in need of immediate therapy as they are at highest risk for HCV-related morbidity and mortality [In 2011, we investigated as a proof of concept the effect of intravenous silibinin—an extract of the milk thistle ortality . Here, wWith this proof of concept study we aimed to assess the virologic outcomes of the lead-in with intravenous silibinin and the subsequent triple-therapy , as well as the safety and tolerability of silibinin in HIV/HCV-coinfected patients with advanced liver fibrosis who failed on previous treatment with peginterferon-ribavirin.http://clinicaltrials.gov, ID NCT01816490) , (ii) partial response  or (iii) viral breakthrough . Advanced liver fibrosis was defined as a METAVIR fibrosis score ≥2 in at least one documented liver biopsy since diagnosis of HCV infection. SVR at week 12 is defined as an undetectable HCV RNA level 12 weeks after the end of treatment. Of note, a SVR 12 correlates strongly with a permanent clearance of the virus and predicts a cure in 99 to 100% of patients. [We treated all 16 patients (14 men and two women) with an intravenous silibinin lead-in and subsequently initiated triple-therapy. Patient selection was based on documented advanced liver fibrosis and a documented history of previous failure of standard-of-care peginterferon-ribavirin, including three different scenarios: (i) non-response  for 30 years for the treatment of hepatic intoxication by Amanita phalloides. In Austria, Legalon SIL was approved in December 2010 for treatment of chronically HCV-infected patients with non-response or partial response to peginterferon-ribavirin. In Switzerland, Legalon SIL is accepted for compassionate use for the same indication. All patients received lead-in therapy with intravenous silibinin at 20 mg/kg/day once daily for 14 days. Silibinin was administrated over one hour. After the 14-day intravenous silibinin lead-in treatment, patients started standard triple-therapy on day 15 (i.e. week 0), including telaprevir 375mg TID, pegylated interferon alfa-2a 180μg once a week, and weight-based daily ribavirin 1000mg (body weight <65kg) or 1200mg (body weight ≥65kg). Triple-therapy was continued for 12 weeks, followed by 36 weeks of peginterferon-ribavirin dual therapy.Laboratory assessments to ensure drug safety were made on days 1, 8, 12, and 15 of intravenous silibinin lead-in, and during subsequent triple therapy in weeks 1, 2, 4, 12, 24, 36, and 48, and additionally according to patient’s clinical state. These assessments included measurements of full blood count, electrolytes , aspartate aminotransferase, alanine aminotransferase, total and fractionated bilirubin, gamma-glutamyl transferase, total protein, alkaline phosphatase, creatinine, albumin, and international normalized ratio (INR). Serum HCV RNA levels were determined by the TaqMan PCR assay . HCV RNA was measured on days 1, 8, and 15 of silibinin treatment. During triple-therapy, HCV viral load was monitored at weeks 2, 4, and 12. The tolerability of intravenous silibinin was determined by daily clinical assessment during the silibinin treatment period.Plasma samples obtained from HIV-HCV infected individuals were isolated by immediate centrifugation at 1850 X g (3000 rpm) and stored at -80°C. For the plasma quantification of antiretroviral drugs, samples were inactivated for virus at 60°C for 60 minutes. Plasma levels were determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after protein precipitation with acetonitrile according to our previously reported validated analytical method . The labTo measure silibinin plasma concentration, the plasma concentrations of silibinin isomers (A and B) and silibinin dihydrogen succinate isomers (A and B) were determined by LC/MS-MS. Of note, the range of plasma concentrations of the LC-MS/MS assay was selected according to the levels reported in the study from Sala et al . BrieflyChromatographic separations were performed using a gradient program with 10 mM ammonium formate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes were quantified by electrospray ionization-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the negative mode. Importantly, the selected gradient program allows adequate separation to quantify isomers A and B individually. The method has a mean inter-day coefficient of variation (CV) % of <10% and an inter-day deviation from nominal values of <7%.We used an intention-to-continue treatment approach to calculate virologic responses as percentage of patients with undetectable HCV-RNA, using all patients who started treatment as denominator. Plasma concentrations are presented as median (range). Two-tailed paired t-tests were conducted to detect difference in trough levels of antiretroviral drugs between pre- and post-administration of silibinin. GraphPad Prism 6  was used with an alpha of 0.05.+ T-cell count of 586 cells/μL (range 175–2529). Fourteen patients (88%) were infected with HCV genotype 1a, and one each with genotypes 1b and 1e. Nine patients (57%) had a METAVIR fibrosis score F4, five (31%) F3, and two (12%) F2. Previous peginterferon-ribavirin therapy in the 16 patients elicited 11 non-responses, 4 partial responses, and one viral breakthrough. The ART regimens of the patients included ATV, DRV, RAL, EFV, tenofovir (TDF), emtricitabine (FTC), abacavir (ABC), and lamivudine (3TC).The 16 patients had a median age of 50 years (range 38–60) . The mosmax and days 2, 4, 8, 12, and 15 for Cmin. Median (range) trough levels for silibinin A, B isomers and silibinin dihydrogen succinate A, B isomers are reported in min of isomer A of both silibinin dihydrogen succinate and silibinin were 2- and 7-fold higher than the corresponding values of isomer B [Plasma concentrations for silibinin A, B isomers, silibinin dihydrogen succinate A, B isomers and antiretroviral drug levels were measured at several occasions at days 1 and 12 for Cisomer B .Most patients received RAL (n = 13) in combination with ATV/r (n = 5), EFV (n = 1), or DRV/r (n = 1). ETV was prescribed in 2 patients, combined with DRV/r in one participant. Among the 16 patients who completed the study, ART plasma levels are reported for 15 patients. One patient was excluded, because there was no plasma sample for drug level measurement available. As depicted in 10 IU/mL (n = 1), 3–5 (n = 2), 2–3 log10 IU/mL (n = 12), and 1–2 log10 IU/mL (n = 1); the median interquartile range (IQR) of HCV RNA decline was 2.65 (2.1–2.8) log10 IU/mL  did not reach SVR 12: One patient had a rapid virologic response (RVR) but stopped treatment at week 8 due to major depression. Five patients had a RVR, but experienced a viral breakthroughs at week 21, 22, 25, or 32, or a relapse at week 52. The HIV RNA remained suppressed in all patients during the complete treatment period, and at SVR 12.Intravenous silibinin treatment was well tolerated in all patients without any serious adverse events . Mild orWe demonstrated as a proof of concept that an intravenous silibinin lead-in was safe, well tolerated and highly effective in a difficult-to-treat patient population and resulted in a pronounced HCV RNA decline during the lead-in phase. The subsequent initiation of telaprevir-based triple-therapy led to a SVR 12 in ten of sixteen (63%) HIV/HCV-coinfected patients with advanced liver fibrosis/cirrhosis and previous failure of peginterferon-ribavirin.We recently published data on the first six patients treated with a silibinin lead-in followed by triple-therapy and reported an SVR rate of 80% . Meanwhimax and Cmin observed in this population and indicate the wide variability between individuals. The finding that silibinin does not influence the pharmacokinetics of antiretroviral drugs is in line with a previous report from Molto et al. [We acknowledge that in the light of the extremely potent and well tolerated next-generation DAAs the current value of silibinin for treatment of HCV infection is limited . Howevero et al. .We would like to share our data since a silibinin add-on could have some benefit in special clinical settings for selected patients: First, in HIV/HCV coinfected patients on salvage ART in whom significant drug-drug interactions may limit the use of some newer DAAs. For example, HIV protease inhibitors cannot be administrated together with the HCV protease inhibitor simeprevir or with the new compounds MK-5172 and MK 8742 , 25, 27.in vitro and therefore might serve as an investigational antiviral agent in the setting of life-threatening, to date untreatable viral diseases  [Apart from its anti-HCV effect, silibinin shows a broad-spectrum antiviral activity atients) , 30. In atients) , and as atients) , 33. TheIn conclusion, a lead-in course with intravenous silibinin prior to the initiation of triple-therapy resulted in a 63% SVR 12 rate in ten of sixteen HIV/HCV coinfected patients with advanced liver fibrosis and previous failure to peginterferon-ribavirin. Tolerability and safety of silibinin was favorable and no significant drug-drug interactions with ART occurred. Due to its anti-viral and anti-inflammatory properties, silibinin may serve in the future as part of the cure for hepatitis C and other viral infections and liver diseases.S1 CONSORT Checklist(DOC)Click here for additional data file.S1 ProtocolA phase II, multi-center, open-label, interventional study to evaluate the safety of intravenous silibinin and its effect on the hepatitis C virus load in treatment-experienced HCV-HIV co-infected individuals with advanced liver fibrosis in the Swiss HIV Cohort Study (SHCS).(PDF)Click here for additional data file.


We analysed the impact of different parameters on genotypic tropism testing related to clinical outcome prediction in 108 patients on maraviroc (MVC) treatment.[coreceptor] and T-CUP tools with FPR cut-offs ranging from 1%-20%. Additionally, 27 RNA and 28 DNA samples were analysed in triplicate, 43 samples with the ESTA assay and 45 with next-generation sequencing. The influence of the genotypic susceptibility score (GSS) and 16 MVC-resistance mutations on clinical outcome was also studied.87 RNA and 60 DNA samples were used. The viral tropism was predicted using the geno2phenoConcordance between single-amplification testing compared to ESTA and to NGS was in the order of 80%. Concordance with NGS was higher at lower FPR cut-offs. Detection of baseline R5 viruses in RNA and DNA samples by all methods significantly correlated with treatment success, even with FPR cut-offs of 3.75%-7.5%. Triple amplification did not improve the prediction value but reduced the number of patients eligible for MVC. No influence of the GSS or MVC-resistance mutations but adherence to treatment, on the clinical outcome was detected.Proviral DNA is valid to select candidates for MVC treatment. FPR cut-offs of 5%-7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this drug. In addition, the new prediction system T-CUP produced reliable results. Since MVC binds exclusively to the CCR5 molecule, its administration must be preceded by a coreceptor usage (or tropism) phenotypic or genotypic analysis. Among the phenotypic assays, the most-widely used is the Enhanced Trofile (ESTA) test, whose validity has been shown in the MOTIVATE, MERIT and A4001029 trials , althougidelines ,15. For [coreceptor], whose predictions are mainly based on geometric distances of amino acid pairs within the structure of V3   and T-CUP tools, using different FPR cut-offs: 1%, 3.75%, 5%, 7.5%, 10%, 15% and 20%. All samples with enough remaining material were additionally analysed with the ESTA assay, NSG [[454] tool and classified as X4 when more than 2% of the sequences displayed a FPR value above the considered cut-off.The viral tropism was predicted with the geno2phenosay, NSG , and/or http://www.genafor.org; for protease-, reverse transcriptase-inhibitors, and raltegravir) and HIV-GRADE  for all the patients where PR/RT and IN sequences were available. The output of the algorithms was mapped to numerical values: susceptible = 1 (or 0.5 for NRTIs), intermediate = 0.5 (0.25 for NRTIs) and resistant = 0.CD4 counts, VL, and therapies were collected. Therapy success was defined as a decrease in VL≥2 logs with respect of baseline or VL<50 copies/mL at two consecutive sampling times. Positive Predictive Value (PPV) was calculated as the percentage of patients carrying baseline R5 viruses who succeeded under MVC treatment. The number of active drugs in the concomitant optimised background therapy  was calculated with the prediction tools geno2pheno  .162 patients were initially enrolled in this study, whereof 108 were finally included in the analysis as at least one baseline V3 sequence and clinical follow-up on MVC treatment were available. 88/108 were male. The median age was 44 years (range 18–81), patients were in median 17 (2–24) years infected with HIV, 9 patients were therapy-naive and 99 patients had undergone a median of 8 (1–25) previous antiretroviral therapies; the 99 therapy-experienced patients had experienced a median of 2.5 (0–25) virological failures. The median VL was 320 copies/mL (40–1265000), whereof 31 patients started the MVC-therapy with a VL≤50, and 77 with detectable viremia [median 829 copies/mL (52–1265000)] . The med87 RNA and 60 DNA baseline sequences were obtained and their FPR calculated , panel B97/108 (89.8%) patients achieved therapy success under MVC treatment (responders). The earliest sampling point for success was in median 13 weeks under MVC (range 4–28). Baseline VL was lower for the 97 responders  than for the eleven non-responders , although no correlation between baseline VL and response to treatment was found . At weekThe univariate analysis of baseline parameters showed that baseline VL≤66 copies/mL (p = 0.005), baseline CD4>120 cells/μL (p = 0.008), and nadir>25 cells/μL were predictors of therapy success; GSS, and the number of previous therapeutic regimens and virological failures were not predictors of treatment outcome. The multivariate analysis of baseline parameters showed no significant associations, except for viral load and nadir CD4 count . However, these associations were rather weak (-9.204e-07 and 7.786e-04). These results are also reflected in the prediction performance in a leave-one-out cross-validation, where the logistic regression model was not able to accurately predict MVC response (AUC = 0.611). Thus, the results from the multivariate analysis are in agreement with those from the univariate analyses.[coreceptor] interpretation was in average 80.2% (range 71.0%-87.1%), and between ESTA and T-CUP analysis 81.6% (80.6%-87.1%). Concordance was very similar for all the FPR cut-offs tested.We analysed the concordance in tropism prediction between population-based sequencing, ESTA and NGS . The con[coreceptor] was in average 76.7% (71.7%-81.1%), and with T-CUP 66.3% (60.0%-70.0%). Predictions by NGS and bulk sequencing from RNA samples accorded more [average 78.2 (geno2pheno) and 70.5 (T-CUP)] than those from DNA samples [median 72.9 (geno2pheno) and 59.5 (T-CUP)] did. Concordance with NGS was also higher at lower FPR cut-offs.For NGS, concordance with bulk sequencing with geno2pheno[coreceptor] from both RNA and DNA samples associated with therapy success. For the 87 RNA samples, statistical correlations between baseline R5 tropism and successful treatment were detected with the geno2pheno-FPR cut-off = 5%(p≤0.039), and T-CUP-FPRs>5% (p≤0.041). For the 60 DNA samples, significant correlations were found with the geno2pheno-FPR cut-offs = 5% and 20% (p≤0.023), and all tested T-CUP-FPRs (p≤0.012). Classification of baseline sample as R5 by ESTA and NGS (<2% of the sequences with FPR≥10%) also correlated with therapy success . Baseline R5 detection using the FPR the cut-off = 0%  for bulk and NGS analyses did not correlate with therapy success.Baseline R5 tropism prediction by geno2pheno[coreceptor] and 80.5% (69.2%-100%) for T-CUP. Three patients displayed high discordances between the FPR values from the concomitant RNA and DNA. Patients #96 [RNA: 75.9% (geno2pheno[coreceptor]), 68.3% (T-CUP); DNA: 6.6% (geno2pheno[coreceptor]), 3.2% (T-CUP)] and #105 [RNA: 41.2% (geno2pheno[coreceptor]), 17.1% (T-CUP); DNA: 7.9% (geno2pheno[coreceptor]), 2.0% (T-CUP)] were non responders. Patient #145 [RNA: 7.0% (geno2pheno[coreceptor]), 2.2% (T-CUP); DNA: 47.1% (geno2pheno[coreceptor]), 58.7% (T-CUP)] achieved undetectable VL at week 18 and remained so until week 43, the last VL value available to us.The PPV for all patients with predicted baseline R5 viruses was around 90% , withoutThe number of patients carrying baseline R5 viruses decreased as the FPR cut-off increased. The FPR cut-off = 5% classified an average of 6.6% more patients as R5 carriers compared to FPR cut-off = 10%, and 17.3% more patients when compared to FPR cut-off = 20% .Triplicate amplifications of 27/31 RNA and 28/33 DNA samples were achieved and their tropism correlated with the clinical outcome. Viruses were considered R5 when all three sequences produced a FPR value above the specific cut-off. For concordance comparison with single amplification (singleton), the first amplification of each sample was used as control.Detection of baseline R5 tropism associated with therapy success in control and triple amplifications. For the controls, significant correlations between baseline R5 tropism and therapy success were found for RNA samples . For DNA samples, correlations were found with T-CUP-FPR cut-offs>7.5% (p≤0.022). These results are concordant with those obtained with the whole dataset of 87 RNA and 60 DNA sequences previously described. For the triplicates, significant correlations were found for RNA samples , and for DNA samples with T-CUP-FPR cut-off = 7.5% (p = 0.035).The PPVs of triple amplifications were close to 90% and not significantly superior to the corresponding singleton control, independently of the type of sample or FPR cut-off used . The use[coreceptor] and T-CUP, respectively) of the sequences from failing patients . Increases of FPR were detected in 3/24 (12.5%)- 5/24 (25.0%) sequences . X4 sequences were found in 2/24 (8.3%)- 9/24 (37.5%)  or 4/24 (16.7%)– 9/24 (37.5%)  of the sequences. Additionally, 19 samples from 16 patients with a successful MVC treatment were available. 2/19 (10.5%) (geno2pheno and T-CUP) of the samples displayed a FPR<3.75%, 3/19 (15.8%)- 1/19 (5.3%)  showed FPRs between 5%-20%, and 14/19 (73.7%) -16/19 (84.2%) FPRs≥20%. Based on these data, therapy failure did not correlate with the detection of X4 viruses after virological rebound, at any of the FPR cut-offs tested.From 19 patients failing their MVC therapy, 24 samples after virological rebound could be sequenced . Marked Changes in the GSS after failure were detected in 7 out of the 17 (41.2%) of the failing patients whose GSS could be calculated . In 6/17No correlation between virological rebound and the GSS on MVC therapy or the duration of the treatment was detected.The mutations 4L, 11SR, 13HS, 18G, 19ST, 20F, 21I, 22T, 25D, 26V, INS 15, INS24, DEL18, and the combinations 11S+26V, 18G+22T, 19ST+26V, 20F+25D+26V, and 20F+21I have been related to MVC resistance independent of CXCR4 tropism  tool for tropism prediction in clinical settings has been extensively demonstrated [[coreceptor], ESTA and NGS for tropism prediction in clinical settings and presents the first genotypic results of the T-CUP tool on clinical samples.The usefulness of population-based sequencing followed by use of the geno2phenonstrated –7,23–25.[coreceptor]) or >5% (T-CUP), which correlated with therapy success. Besides, our study showed that predictions from RNA samples were concordant to the predictions from concomitant DNA and that the same FPR cut-offs may be applied [[coreceptor] have been developed to identify X4 viruses with highest sensitivity, so the use of too high FPR cut-offs should greatly overestimate X4 variants. Supporting our results, increasing evidence published in the latest years have also suggested the use of FPR cut-offs ranging 5.75%-10% in order to avoid exclusion of patients who would succeeded in their MVC treatment, while still guaranteeing a save administration of the drug  tool for tropism prediction for patients currently attending HIV day units. Our data suggest that genotypic testing with single amplification from RNA or DNA FPR and use of cut-off = 5.0% would assure a safe administration of MVC without excluding many patients who could benefit from this potent antiretroviral drug that also seems to reduce the immune activation. Promising results have also been obtained for T-CUP that should be confirmed in future works. In addition, we also showed that treatment adherence may be a critical factor for therapy success in patients on MVC-containing therapies with R5 tropism at baseline.In conclusion, our work analyses the usefulness of population-based sequencing followed by use of the geno2pheno


In the UK, patients undergo HIV viral load and genotype testing before they are prescribed antiretroviral therapy. The genotype test guides clinicians in prescribing antiretroviral therapy with maximum efficacy against the patient’s specific viral strain. HIV viral load escape under antiretroviral drug therapy, to which the virus was thought to be genotypically susceptible, is commonly observed in patients with poor adherence. We observed early viral escapes in two-newly diagnosed patients, during antiretroviral treatment, with different sequences compared to their original viral resistance test and who reported excellent adherence to and tolerance of their therapy. HIV superinfection with a new viral strain was identified in a patient with multiple risk factors and co-infections with sexually transmitted infections. The second patient was a case of the emergence of primary resistant virus under drug pressure. Both suppressed their virus promptly after treatment switch. Acute HIV sero-conversion syndrome was diagnosed. Sequencing of protease (PR) and first 234 amino acids of the reverse transcriptase (RT) genes indicated a fully antiretroviral-susceptible HIV-1 subtype B strain with minor PR and RT differences to the HIV reference strain HXB2 . HCV RNA was undetectable, suggesting spontaneous clearance of the HCV. Sixteen weeks later, despite initial recovery, the CD4 had declined to 185 × 106/L (12%). The HIV-1 VL was 167,200 copies/mL (Log10 5.2). Abacavir, lamivudine and nevirapine were commenced and the VL suppressed to 270 copies /mL (Log10 2.4) at 12 weeks. Four weeks later, the patient presented feeling unwell with night sweats. On examination, he had right-sided inguinal lymphadenopathy and enlarged liver and a palpable spleen. A pale macular rash was visible on the trunk and extremities. He was diagnosed with rectal Neisseria gonorrhoeae and urethral Chlamydia trachomatis, his HCV RNA remained negative. However, the HIV VL had risen to 14,900 copies/ml (Log10 4.2) despite good reported adherence and tolerance. HIV genotyping revealed HIV-1 subtype B with multiple new mutations: PR: K14R, R83K, RT: A98S, K101E, M184I, G190A, T215D, I135T, D177E, I178L, V179I, T200A, I202V, Q207K, R211K. This virus, showed high-level resistance to lamivudine and nevirapine and low-level resistance to abacavir. The sequences were highly divergent exhibiting 45 nucleotide differences of which 20 were non-synonymous including the four resistance-associated mutations (10 1.6) within three weeks. Fifteen months later, routine tests revealed newly raised AST (73IU/L) and bilirubin (89 µmol/L) levels and HCV RNA levels of 2.6 × 106 copies/mL. The two HCV strains were identical in the 135 base region of the 5’ NCR. Considering the previous HCV RNA negative tests and ongoing unprotected sex, HCV reinfection was thought to be more likely than reactivation.A 41-year-old, White British, man who has sex with men (MSM) presented complaining of drenching night sweats and fatigue of recent onset. He reported unprotected oral and anal intercourse with casual partners and had recently been diagnosed with hepatitis C virus (HCV). He gave no travel history of note. He was diagnosed with and treated for early, secondary syphilis. His HCV genotype was 1a (HCV RNA: 3.87 × 104 copies/mL) and his HIV serology testing was negative. Four weeks later, he presented with night sweats, anorexia and dysphagia. Oesophageal candidiasis was suspected and treated with fluconazole 50 mg once daily for seven days. HIV-1 antibodies were positive . He was commenced on tenofovir, emtricitabine and efavirenz. He improved clinically, but the CD4 count plateaued at 11 × 106/L, 2.6%) and the VL only decreased to 88,000 copies/mL (log10 4.9) at 4 weeks and stalled at 56,000 copies/mL (log10 4.7) at 12 weeks. He reported no side-effects, no new sexual contacts and 100% adherence to HAART. He gave no travel history of note. Resistance testing revealed K65KR, M184V, K101E, Y181CY and G190CS. Comparison of the two sequences revealed only four out of 1007 sequences nucleotide changes between baseline and the second test, excluding residues exhibiting mixed bases, of which three resulted in amino acid changes within known resistance foci at RT residues 101, 184 and 190. The fourth was a synonymous nucleotide change. Based on the fact that the patient denied lack of exposure, reported good adherence and the commonly under drug pressure observed mutations, this lack of sufficient VL response was thought to be due to an unmasking of a primary minority species virus under drug pressure, rather than HIV superinfection. The patient's treatment was switched to zidovudine, lamivudine, raltegravir and boosted darunavir. Twenty-four weeks later he was feeling well and his VL was 41 copies/mL (log10 1.6) and his CD4 count 155 × 106/L, 17%.A 41-year-old, White British MSM receiving treatment for We report viral escape as a consequence of potential transmitted drug resistance due to HIV-1 superinfection and the unmasking of a primary minority species resistant virus under drug pressure. In both case, viral drug resistance was discovered following an insufficient immunological and virological response. Both patients had received information on transmission and prevention of sexually transmitted infections as well as HIV superinfection and adherence to and side-effects of antiretroviral medication. We routinely provide free condoms and advise to refrain from sex during the sero-conversion period.1 The latter two are rare but should be considered in patients with good adherence to HAART. A detailed history and HIV VL measurement, genotype and phylogenetic analysis, especially ultradeep pyrosequencing,2 are useful in distinguishing the cause of viral escape. HIV superinfection with a different virus can be acquired after primary seroconversion.4 It is associated with early HIV infection6 and estimated to occur at a rate of around 5 per 100 person-years.2 It may lead to the acquisition of drug resistance,7 viral escape, may necessitate treatment switch and be associated with disease progression.8 In our case, repeat genotyping detected the distinctly different new virus and guided a successful treatment switch. We believe that the archived resistance in case 2 was 194V and 101E as these were not present in mixtures and that 65R and 181C then rapidly emerged as indicated by these being mixed with wild type. The residue at 190 was also mixed but not with wild type, and we cannot be sure whether one or both of these were archived or emerged in different quasi-species. We were not able to conduct ultradeep sequencing, since it is not yet validated at our routine virology service. However, the high divergence in the first case and the lack of divergence in the second case make superinfection in the first case and the unmasking of a previous resistant variant in the second case, the most likely explanation  over time and a more rapid loss of NNRTI and PI mutations compared to TAMs.12 Using these data, in our second case, the M184V and Y181C may have been lost quicker than the remaining three; however, they re-emerged under drug pressure.Random samples of treatment-naïve HIV-infected subjects harboured 5%, 3% and 3% primary resistant HIV strains to NNRTIs, NRTIs and PIs, respectively.13 advising patients to avoid unprotected sex, measuring VL at baseline at 4, 8, 12 and 24 weeks, tailoring treatment to the viral genotype, repeating genotype in viral escape and switching treatment in response to the new genotype.We support the current practice:Both patients have consented for the cases to be published.


Access to antiretroviral treatment (ART) becomes more and more effective in resource-limited settings (RLS). However, this global effort would be even more profitable if the access to laboratory services especially in decentralized settings was strengthened. We report the virological outcome and HIV-1 drug resistance in three West African countries using dried blood spots (DBS) samples.pol gene was performed using in-house protocol.We included HIV-1-infected adults on ART ≥6 months and followed up in capital cities and decentralized sites in Senegal, Mali and Guinea-Conakry. Patients were consecutively enrolled and DBS were collected in field conditions and kept at ambient temperature before transfer to the reference laboratory. Viral load (VL) was quantified using the NucliSENS EasyQ HIV-1 v1.2. Genotyping of HIV-1 10 copies/mL) was observed in 26% , 18–35; n=31), 11%  and 24%  of patients in Senegal, Mali and Guinea-Conakry, respectively (p=0.001). Of samples presenting VL≥3log10 copies/mL (n=80), 70 were successfully genotyped. At least one drug resistance mutation (DRM) was detected in the following proportions: 70% , 93%  and 68%  in Senegal, Mali and Guinea-Conakry, respectively (p=0.22). Twenty-six per cent  of patients in VF harboured wild-type viruses, which is likely indicative of weak adherence. Phylogenetic analysis showed the predominance of CRF02_AG subtype .Of the 407 participants, 119, 152 and 136 were from Senegal, Mali and Guinea-Conakry, respectively. The median treatment duration was 36 months [IQR: 6–136]. Virological failure (VF) (VL≥3logWe describe the ART outcome in capital and rural settings of Senegal, Mali and Guinea-Conakry. Our results in all of the three countries highlight the need to reinforce the ART adherence in order to minimize the occurrence of drug resistance. In addition, these findings provide additional evidence that the use of DBS as a sampling support could assist virological monitoring of patients on ART in remote areas. In the latest UNAIDS report, the number of people on antiretroviral therapy (ART) has dramatically increased in the past few years . HoweverWe conducted a multi-site study on patients on ART in capital cities and decentralized medical centres of three West African countries . We included patients who were at least 18 years old, under ART treatment for at least six months and consenting to participate in the study. Women who received the protocol for the prevention of mother-to-child transmission (PMTCT) as well as patients positive for HIV-2 or co-infected (HIV-1 and HIV-2) were not included. Patients were consecutively enrolled between February 2010 and December 2011 in 17 sites distributed as follows: Senegal (n=7), Mali (n=6) and Guinea-Conakry (n=4). In Mali and Guinea-Conakry, recruitment sites were chosen depending on the active file of patients. In Senegal, all samples received for routine virological monitoring, from patients who met the inclusion criteria were considered.National Ethics Committees of participating countries approved this study. Patients were recruited on a voluntary basis. To keep confidential data of the participants, a unique identifier was assigned to each sample and used throughout the study, ascertaining anonymity. Only the attending physician could establish the correspondence between this identifier and the patient.For each patient, two DBS cards  were spotted: 50 µL/spot, five spots/card from whole blood EDTA tube. Cards were left to dry overnight at ambient temperature (range 22–37°C) before being packed in an individual sealed bag with desiccants and a humidity indicator card. These individual bags were in turn packed in zip-lock plastic bag and sent at ambient temperature to the reference laboratory in Dakar, Senegal, for testing. On site, collected DBS specimens were checked frequently for humidity and sent within one month after sampling. At the reference laboratory, upon reception, the conformity  was checked and DBS were stored at −80°C until testing. The samples were discarded if they did not meet the criteria mentioned above.Total HIV-1 nucleic acids were extracted from two spots with NucliSENS miniMAG  with magnetic silica as previously described . Briefly10 (1000) copies/mL as recently recommended by WHO . Of the 407 patients enrolled at 17 collection sites in three West African countries, 93%  were on first-line ART (2NRTI+1NNRTI) with zidovudine (AZT), lamivudine (3TC), stavudine (D4T), nevirapine (NVP) and efavirenz (EFV) the most commonly used . Patients on second-line ART (2 NRTI+1 boosted-PI)  were distributed as follows: Senegal (n=5), Mali (n=16) and Guinea-Conakry (n=7). The ART median follow-up duration was 36 months [IQR: 6–136], with a higher proportion of females , , Thiès (n=18), Diourbel (n=13), Fatick (n=8), St-Louis (n=2), Ziguinchor (n=2)] and one in the suburb of Dakar [Roi Baudouin (n=49)]. The median age was 42 years [IQR: 18–65] and 79%  of patients were female. Among the 119 patients, 96%  were on first-line ART mainly with AZT+3TC+NVP/EFV combination 96%  . The med10 copies/mL) and the median VL was 3.63log10 copies/mL [IQR: 3–5.48]. According to the treatment duration, patients in VF were distributed as follows: 19.2% , 39.1%  and 24.2% , respectively, at 6–12, 13–24 and >24 months (p=0.24). Furthermore, data from 10 copies/mL and were distributed as follows, according to treatment duration: 60% , 44%  and 41% , respectively, at 6–12, 13–24 and >24 months. Among the patients in the group >24 months (n=17), three were on second-line ART. Based on medical centres (n=7), no difference was found in the occurrence of VF between patients followed in Dakar, the capital city , and those of the regional centres , (p=0.91).A total of 26%  of the patients had detectable VL (≥800 copies/mL). All of them were in VF . The median VL of genotyped samples was 3.6log10 copies/mL [IQR: 3–5.4] and those not amplified (n=4) presented VL of: 3, 3.1, 3 and 3.4log10 copies/mL. Resistance analysis among 27 amplified samples showed that at least one drug resistance mutation (DRM) was found in 19 cases. Global resistance rate was 16% . Of the samples with VL≥3logAmong the 27 genotyped samples, 30%  presented no DRM  with thrPhylogenetic analysis  shows thIn Mali, 152 patients were enrolled at six sites  distributed in four regional medical centres . The median age was 41 years [IQR: 18–66] and 67%  were female . Most ofFor patients on first-line ART regimen, AZT+3TC+NVP/EFV  and d4T+3TC+NVP/EFV  combinations were most frequently observed . For som10 copies/mL [IQR: 2.9–6.1]. The VF (VL≥3log10 copies/mL) was observed in 11% . According to the treatment duration, 5% , 11%  and 12%  of patients in VF were, respectively, at 6–12, 13–24 and >24 months (p=0.61) (10 copies/mL (p=0.47).Seventeen patients  had detectable VL (≥800 copies/mL). The median of detectable VL was 3.9log(p=0.61) . Among topies/mL . In a gr10 copies/mL, resistance testing was successfully performed in 94% . The only one unamplified sample had a VL equal to 3.9log10 copies/mL. All amplified samples (n=15), except one, harboured at least one DRM representing global resistance rate of 9%   . Resista5; n=14) .Phylogenetic analysis of 15 viral sequences showed a relative predominance of CRF02_AG 47% . Other subtypes were also found: D (n=2), CRF09_cpx (n=1), CRF06_cpx (n=1), C (n=1) and three URFs  .10 copies/mL) was observed in 24%  and median VL was 3.6 [IQR: 3–6.7] log10 copies/mL. The VL distribution showed that 48%  of patients in VF had VL greater than 3.7log10 copies/mL. Stratified by treatment duration, 31% , 23%  and 24%  of patients in VF were, respectively, at 6–12, 13–24 and >24 months (p=0.22) (p=0.15).In Guinea-Conakry, 136 patients were enrolled in this survey. Recruitment took place in Conakry, the capital city  and three regional medical centres . The median age of patients was 38 years [IQR: 18–61] and 61%  were female. Ninety-five per cent of patients  were on first-line ART (2NRTI+1NNRTI) and 5%  on second-line ART (2NRTI+1boosted-PI). The ART median time was 35 months [IQR: 6–108]. For patients undergoing first-line ART regimen, AZT+3TC+NVP/EFV  and d4T+3TC+NVP/EFV  combinations were most frequently observed . The tre(p=0.22) . In the 10 copies/mL, genotypic tests were successfully performed in 85% . The median VL of genotyped samples (n=28) was 4 [IQR: 3–6.7] log10 copies/mL and those not amplified (n=5) was 3.1 [IQR: 3–3.6] log10 copies/mL (p=0.02). Nineteen of the 28 patients with available genotypic results harboured at least one DRM giving a global resistance rate of 14% . Resistance to both NRTIs and NNRTIs was observed in 79% , and four patients had only NNRTI-associated DRMs. Otherwise, no PI-associated DRM has been observed among the patients on first-line or among those on second-line ART. A total of 52 NRTI-associated DRMs were detected and the M184V/I mutation was found in 25% . TAMs were found in 60% . Five patients had T69 insertion complex conferring resistance to all NRTIs currently approved by the US FDA. All of them harboured CRF02_AG virus and were at >24 months of treatment. Their treatment regimens were: AZT+3TC+NVP/EFV (n=3), d4T+3TC+NVP (n=1) and AZT/3TC/LPVr (n=1). The most common NNRTI mutation was K103N  followed by Y181C/I, V90I/V and V108I. Each of these three mutations mentioned was found four times  of patients were infected by CRF02_AG subtype. Subtypes D and CRF06_cpx have been found in the respective proportions of 7%  and 11%   .The aim of this work was to document the virological outcome and HIV-1 drug resistance in adult patients followed in capital cities and decentralized settings in Senegal, Mali and Guinea-Conakry using DBS samples. Most of the patients in this study were on first-line ART regimen , with a wide predominance of AZT+3TC+NVP/EFV combination followed by d4T+3TC+NVP/EFV according to WHO 2006 recommendations for resource-limited settings (RLS) . Similarp=0.13). However, our findings show differences between the VF (VL≥3log10 copies/mL) rate noted in Senegal , Mali  and Guinea-Conakry , (p=0.01). This difference could be due partly to the size of the study populations between the three countries. In Senegal, all samples received routine virological monitoring and those who met the inclusion criteria were considered. Therefore, the number of patients failing treatment is not representative of the actual situation in the sub-population of persons on ART. Recently, a study outlining virological outcome in patients receiving HAART, and monitored with the World Health Organization (WHO) Public Health Approach, showed a VF of 10.7% among patients at 24 months’ treatment duration and recruited consecutively [et al.[Despite the difference in VL thresholds (800 copies/mL for “DBS” vs. 50 copies/mL for “Plasma”), the rate of viral suppression in our study  is comparable to those reported in multicentre studies in Burkina Faso and Mali  , , (p=0.74) [In Senegal, the study showed a similar VF rate  as previously reported by Diouara (p=0.74) . The dif(p=0.74) .For Guinea-Conakry, to our knowledge, this is the first data of VL and antiretroviral drug resistance in HIV-1-treated patients. So, our results cannot be compared to the national level. However, VF is comparable to what was previously obtained in the neighbouring countries, namely Senegal and Mali ,19. Moreet al. observed a lower PCR amplification rate  [10 copies/mL [p=0.03). It has also been reported in several studies that many factors, mainly the conditions and storage time, transportation, temperature, humidity level and genotyping methods could influence the efficiency of DBS samples PCR amplification[PR and RT (at least codon 41–236) as described above seem to be most suitable in addition to the prerequisites for the conservation of sample integrity throughout the pre-analytical process [et al. in 2012 and recently by Parry et al. (2014). Their reports have also mentioned that the international shipment at an ambient temperature did not affect amplification success rates [To the best of our knowledge, this is the first work in West Africa using DBS samples in VL and drug resistance testing. Overall, we had a good rate of PCR amplification , which is comparable to that obtained previously in Senegal  and thosn=18/55) . Also, ln=18/55) . In addiopies/mL . This isfication–14,31,32ss rates ,33.p=0.15). The DRM profiles in these three countries were similar and show that TAMs (p=0.81), M184V/I (p=0.37) and K103N (p=0.45) mutations were by far the most frequently encountered. Their strong representation seems to be associated with the wide use of AZT/D4T+3TC+EFV/NVP in first-line protocol. Our results are in line with previous studies [Of a total of 407 patients on ART according to WHO 2006 revised recommendations for RLS, 13%  had at least one DRM. Depending on the country, no significant difference was observed between the DRM rate: 70%  in Senegal, 93%  in Mali and 68%  in Guinea-Conakry,  of genotyped samples were distributed as follows: Senegal (n=8), Mali (n=1) and Guinea-Conakry (n=9) had wild-type viruses . This ev viruses . It is kPhylogenetic analysis shows the predominance of CRF02_AG for each of the three participating countries. And, HIV-1 genotypes found in our study are concordant with those previously observed in Senegal , in MaliOur study has some limitations. First, to assess treatment adherence, a questionnaire was not provided to patients during the enrolment process. Second, the CD4 cell count at ART initiation was not available and sometime data relating to the treatment history (combination and substitution) too. Other limitations were the small number of patients recruited in some sites, the heterogeneous treatment duration of patients (ranging from 6 months to 11 years) and the recruitment process. For example, in Senegal, all samples received for routine virological monitoring and who met the inclusion criteria were considered. It was not the case in Mali and Guinea-Conakry, where the selection of recruitment sites was made based on the active file of patients. So, the results are not representative nationwide.Our data show a relatively high rate of patients in VF while among them an important proportion harboured wild-type viruses. This highlights a real need to reinforce treatment adherence and expand VL and resistance testing in remote areas. Regarding virological monitoring of patients followed in remote areas, these results provide additional evidence that the use of DBS samples could facilitate access to viral load and resistance testing for better care of patients in a context of limited resources.


A total of 1,321 documents of patients reviewed revealed timely immunological monitoring were inadequate. There was adequate immunological response, with pediatric patients, females, those with less advanced illness (baseline WHO Stage I or II) and those with higher baseline CD4 cell count found to have better immunological recovery. Thirty-nine patients (3%) were not evaluated for immunological failure because they had frequent treatment interruption. Despite overall adequate immunological response at group level, the prevalence of those who ever experienced immunological failure was 17.6% (n=226), while after subsequent re-evaluation it dropped to 11.5% (n=147). Having WHO Stage III/IV of the disease or a higher CD4 cell count at baseline was identified as a risk for immunological failure. Few patients with confirmed failure were switched to second line therapy. These findings highlight the magnitude of the problem of immunological failure and the gap in management. Prioritizing care for high risk patients may help in effective utilization of meager resources.Immunological monitoring is part of the standard of care for patients on antiretroviral treatment. Yet, little is known about the routine implementation of immunological laboratory monitoring and utilization in clinical care in Ethiopia. This study assessed the pattern of immunological monitoring, immunological response, level of immunological treatment failure and factors related to it among patients on antiretroviral therapy in selected hospitals in southern Ethiopia. A retrospective longitudinal analytic study was conducted using documents of patients started on antiretroviral therapy. Adequacy of timely immunological monitoring was assessed every six months the first year and every one year thereafter. Immunological response was assessed every six months at cohort level. Immunological failure was based on the criteria: fall of follow-up CD4 cell count to baseline (or below), or CD4 levels persisting below 100 cells/mm In Ethiopia, access to highly active antiretroviral therapy (HAART) started on massive scale in 2005 and by 2013 it has reached to some 317,443 people [As important as it is to get people who need the treatment be on HAART, it is even more important to ensure that patients adhere to the treatment so that they benefit from it in the long run as well. This is because poor adherence to treatment is associated with increased risk of drug resistance and thus increased risk to treatment failure , 6. Achith and 24th month follow-up CD4 tests (n = 1163) [Literature is scanty on the issue of immunological monitoring in Ethiopia, but the available data reveals a suboptimal CD4 monitoring among pediatric patients on HAART in southern Ethiopia with only 68 and 37% of children having 6 = 1163) . As for  = 1163) , 20. Whi = 1163) , 22 and  = 1163) . Another = 1163) , 16, 24.There is no published study for adult patients in Ethiopia indicating the level of adherence to immunological monitoring guidelines, which by far accounts for more than 90% of patients on HAART. Furthermore, there is no study indicating the prevalence and determinants of patients with immunological failure that may benefit from targeted viral load monitoring in the Ethiopian care setting. This study, therefore, investigated the level of immunological monitoring, the level of immunological response, both in the form of CD4 cell counts, as well as the prevalence of treatment failure and its determinants in selected areas in Ethiopia with a focus on immunological criteria.Approval for this study was obtained from the National Research Ethics Review Committee, and the Southern Nations Nationalities and People’s regional health bureau of Ethiopia. Ethical approval was also sought from the Department of Higher Degrees of the Department of Health Studies at the University of South Africa (UNISA). Permission was also obtained from the directors of each of the hospitals from whose HIV clinics data was used for the purpose of this study. Informed consent was not obtained since there was no interaction with human subjects. In order to keep anonymity, data was de-identified (de-linked) before analysis.This is a retrospective, longitudinal study conducted in SNNP region, which is located to south of the Ethiopian capital. It has a population of around 18 million people . AccordiImmunological monitoring (CD4 cell count test) is expected at baseline just before starting HAART. After starting treatment CD4 cell count should be measured every six months for the first year, then once yearly in line with the health management information system (HMIS) guidance for cohort reporting .fall of follow-up CD4 count to baseline (or below), or3, orCD4 levels persisting below 100 cells/mm50% fall from on-treatment peak value.These same criteria were used in this study to identify patients with immunological failure according to the WHO 2010 guidance [Although the WHO recommends three types of criteria to define antiretroviral treatment failure, namely clinical, immunological, and virologic, the focus of this study was on immunological criteria which the WHO defines as follows:guidance . A patiest 2005 until December 31st 2012 formed the study population. Transfer in cases, patients with fewer than two CD4 tests and patients with follow-up for less than six months on HAART were excluded. The total sample size required was estimated for the combined accessible patients’ documents on HAART treatment in the two hospitals. Sample size calculations were done assuming random sampling for a finite population based on estimation of a 50% proportion, at a 5% significance level, 2% precision, and 15% estimated missing records. This put the sample size to be 1,304.PLHIV five years and older started on HAART in the selected facilities from September 1The primary outcome variable was immunological failure as defined earlier. Predictor variables included adherence as measured by appointment for medication refill, baseline WHO clinical stage, baseline CD4 cell count, age, gender, and current tuberculosis. A document checklist was used to collect data from medical records and registers. The data collection checklist and data cleaning program codes were pretested prior to data collection and cleaning. This was done by a statistician among transferred-in patients found in one of the study facilities.Epi Info version 3.5.1 statistical software (United States Centers for Disease Control and Prevention 2007) was used to clean the data while Stata version 12.0 was used to analyze it. Pattern of immunological monitoring was assessed using the national monitoring standard for determination of follow-up CD4 cell count for patients on antiretroviral therapy. The timing for evaluation of patients was every six months the first year and yearly then after . A CD4 cThe values of adherence and tuberculosis status varied with time. The variable ‘adherence’ was assigned a value of ‘0’ as long as patients were adherent to treatment with respect to their appointment to HAART medication prescription refill. If they failed to show up, this variable was assigned a value of ‘1’ for the time of follow-up after the time of interruption. This enabled the investigator to compare if the risk of immunological treatment failure varied between times after treatment interruption as compared to that before treatment interruption. In practical terms, this required splitting the observation time of patients into that up to the point at which treatment was discontinued, where the value to adherence was ‘0’, and the time period after that, where the value given to adherence was ‘1’. So, the time an individual spent being adherent to appointments was taken into account. This is justified for this study since patients who failed to show up for their medication refill in effect discontinued treatment for at least more than two weeks and thus fell beneath the 95% adherence limit, thus putting them at risk of experiencing treatment failure . Tubercu3 . After the initiation of HAART, tuberculosis was diagnosed in 17.79% (n = 235) of patients. Eighteen per cent (n = 241) of patients showed poor adherence as they could not show up to their appointments for medication refill. These patients were followed up for a median time of 30 months (interquartile range: 15 to 48 months) after restarting treatment to censoring or diagnosis of failure. By the end of the follow-up, 73% (n = 960) of patients were still on HAART in the same facility where treatment was initiated. There was no difference with respect to being active on follow-up with regard to baseline covariates .  recovery was observed among patients on antiretroviral therapy. The magnitude of follow-up CD4 cell count increase was rapid in the first few years of follow-up after starting antiretroviral therapy and reached a plateau thereafter . ChildreOf the 1,321 patients’ medical records reviewed, 2.95% (n = 39) discontinued their treatment for considerable time. Such patients would have needed additional follow-up before treatment failure diagnosis could be made, for which reason they were not evaluated for treatment failure. After excluding these patients, the prevalence of patients who experienced immunological failure at least once was 17.6% (n = 226) . But, with further evaluation and follow-up CD4 cell count determination this percentage dropped to 11.5% (n = 147)  . DiagnosPatients not evaluated for treatment failure were excluded (n = 39) from survival analysis. This left 97%  of patients. The median duration of follow-up from HAART initiation to diagnosis of immunological failure was 1.8 years (range: 0.51–7.92). For those who never experienced treatment failure, the median follow-up time was 3.73 years (0.51–8.36). The patient data scrutinized amounted to a total of 4,649 person-years at censoring or at diagnosis of immunological failure . Table 1(0.2840678) = 1.33). While an increase in baseline CD4 cell count by 100 cells/mm3 increased the hazard of immunological failure by 34% (adjusted hazard rate ratio of 2.718(100x0.0029061) = 1.34). None of the other covariates  predicted immunological treatment failure. . However with further evaluation and follow-up CD4 cell count determination, it dropped to 11.5%. Since a significant number of patients who were re-evaluated after immunological failure didn’t fulfil immunological failure criteria, it could be possible that the actual percentage of patients with immunological failure would have been even lower than this, had there been proper follow-up immunological evaluation.This is the first paper that assessed the level of immunological failure for both adult and paediatric patients on HAART according to the WHO 2010 criteria for diagnosing immunological treatment failure  and the It should be noted that the gold standard for determining antiretroviral treatment failure is viral load testing, which is not routinely available in Ethiopia. For this reason, interpretation and utilization of immunological failure criteria should be based on its strength and limitations. This is provided by the predictive values of immunological failure on virologic failure. Several studies have made that assessment and found out that the benefit of screening patients with the WHO’s immunological failure criteria is in ruling out virologic failure among those who don’t have immunological failure according to those criteria. This is because the WHO’s immunological failure criteria has very high negative predictive value (87% to 99% depending on the study) but low positive predictive value (9% to 37% depending on the study) –36. For The overall prevalence of patients on second line HAART, 1.67% , is comparable to what was reported by Assefa et al , which iThe fact that the majority with failure (58%) were diagnosed with the criteria ‘50% drop from peak follow-up CD4 cell count value’ implies that they experienced an initial increase in CD4 cell count before it slumped down later on. This is consistent with the finding that the majority in follow-up experience considerable CD4 cell count recovery especially in the first year of follow-up. This trend of adequate initial recovery has been reported in other studies as well , 20, 37.The suboptimal level of immunological monitoring, which could be one contributing cause for some patients to remain in treatment failure, has been documented in another study in southern Ethiopia . This is3 at one year’ after HAART initiation. All these criteria are different from that issued by the WHO [Two factors were important in predicting immunological treatment failure. Baseline WHO stage and baseline CD4 cell count. Those with higher baseline WHO stages (III or IV) or higher baseline CD4 cell count were found to be at high risk for immunological treatment failure. Other variables like age, gender, adherence to medication refill, and tuberculosis were not associated with it. There are studies that assessed predictors of immunological failure but comparison was not possible with most because different immunological failure criteria were used. For instance, Kassa et al used the following criteria to define immunological failure: increase of CD4 cell count by fewer than 50 cells/μl at month six, and the criteria of less than 100 cells/μl at months 18 and 24 of HAART; Anude et al used the criteria ‘fall of follow-up CD4 cell count to baseline or below’, or ‘an increase in the CD4 cell count of less than 50 cells/mm the WHO , 30, 42  the WHO . The fac the WHO . Therefo the WHO –47.This study may have important inputs both for clinical practice and policy design on the issue of immunological monitoring and management of those with immunological failure while on HAART. Firstly, the cut off CD4 cell count for initiating HAART for adults and adolescents in Ethiopia has been raised in Ethiopia to be 500 since 2013. In addition to that mothers are initiating lifelong HAART at any CD4 level for PMTCT purposes. The same is true for HIV infected children and adolescents below 14 years of age who are all eligible for HAART in spite of WHO staging or baseline CD4 cell count . This meDespite the overall positive contributions of this study for clinical practice and program implementation, it has important limitations. Since this was a retrospective study based on existing medical records, the effect of possibly erroneous entries as well as incomplete entries of data should not be underestimated. For instance, these reasons have led us to drop the variable weight. In addition, the study could not assess the effect of clinical disease progression on immunological failure except for tuberculosis as follow-up staging (treatment staging) was not well documented. Not all patients were active on follow-up at last observation because of treatment interruption or transfer to other facility for follow-up. For this reason, immunological failure could only be measured for as long as patients were on follow-up. This effect was, however, accounted for during survival analysis, which is specifically, designed to analyze such data. Moreover, there was no association between baseline covariates and being active on follow-up at last observation making the effect of incomplete observations minimal. Also, since only two hospitals were selected for convenience, the overall prevalence of immunological failure couldn’t be generalized for the whole region.This study merely identified the problem of poor immunological determination. Therefore, further studies need be made to address barriers to proper CD4 cell count determination. In the face of confirmed immunological failure, very few patients were managed by shifting antiretroviral treatment regimen to second line according to the guideline. Hence, providers’ knowledge, attitude, and practice to managing patients with immunological failure should be evaluated in order to design an intervention to address gaps in care being observed. Also, the proposed tool to monitor and manage immunological assessment and management for those with failure needs to be validated and piloted before implementation.Immunological treatment failure was prevalent in the study setting. There is suboptimal level of immunological monitoring which if addressed may contribute to reduction in the number of patients with immunologic failure diagnosis, and thus unnecessary switching to second line therapy where targeted viral load testing is not yet possible. Patients with confirmed immunological failure diagnosis were not managed as per the guideline which may contribute for further drug resistance. Further studies need to be made to identify root causes of poor monitoring and management of patients with failure.S1 Dataset(DTA)Click here for additional data file.S2 Dataset(DTA)Click here for additional data file.S3 Dataset(DTA)Click here for additional data file.S1 Dofile(DO)Click here for additional data file.S2 Dofile(DO)Click here for additional data file.S3 Dofile(DO)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Worldwide circulating HIV-1 genomes show extensive variation represented by different subtypes, polymorphisms and drug-resistant strains. Reports on the impact of sequence variation on antiretroviral therapy (ART) outcomes are mixed. In this review, we summarize relevant published data from both resource-rich and resource-limited countries in the last 10 years on the impact of HIV-1 sequence diversity on treatment outcomes. The prevalence of transmission of drug resistant mutations (DRMs) varies considerably, ranging from 0% to 27% worldwide. Factors such as geographic location, access and availability to ART, duration since inception of treatment programs, quality of care, risk-taking behaviors, mode of transmission, and viral subtype all dictate the prevalence in a particular geographical region. Although HIV-1 subtype may not be a good predictor of treatment outcome, review of emerging evidence supports the fact that HIV-1 genome sequence-resulting from natural polymorphisms or drug-associated mutations-matters when it comes to treatment outcomes. Therefore, continued surveillance of drug resistant variants in both treatment-naïve and treatment-experienced populations is needed to reduce the transmission of DRMs and to optimize the efficacy of the current ART armamentarium. At any given time, an HIV-infected individual harbors a heterogeneous population of HIV-1 referred to as quasispecies ,3,4. Mor (TDRMs) .Four distinct genetic groups of HIV-1 exist worldwide: M (major), O (outlier), N , and P (new group) ,6. The min vitro and clinical data indicate that HIV-1 with primary mutations at codon 155 or 143, and the T66I and E92Q mutants remain susceptible to dolutegravir, whereas mutations at codon 148 in the presence of other secondary mutations  leads to decreased dolutegravir efficacy [Sequence variations that result from drug selection pressure are referred to as drug resistance mutations (DRMs). Therefore, DRMs are antiretroviral drug class dependent. Resistance to nucleoside analogs could result from either a single mutation or combinations of mutations in HIV-1 RT gene. A high level of resistance to zidovudine (AZT) or stavudine (d4T) results from accumulation of mutations in the RT  . These mefficacy .The primary goal of antiretroviral therapy is to suppress viral replication to undetectable levels as assessed by standard HIV RNA quantification assays. Viral suppression serves two main functions: immune restoration and the reduction of AIDS-related morbidity and mortality ,24. TherThe majority of HIV-1 infections world-wide are due to non-subtype B virus. Fifty percent of all HIV-1 infections are actually attributed to subtype C, followed by A at 12%, and B at 10% . Subtypeversus non-B group, there were no differences in virologic response [versus 98 in subtype C, p < 0.001), no such difference existed in viral loads or resistance rates. Interestingly, in a 2012 Italian study patients were matched across subtypes with the same resistance mutations; no difference in viral suppression was found by week 12 [et al. reported a faster immunologic decline in subtype CRF01_AE compared with subtype B, followed by a comparable slower and less robust CD4 count recovery upon initiation of antiretroviral therapy [Although differences in drug susceptibility exist between subtypes, recent large international studies assessing effectiveness of various ART regimens have not demonstrated increased treatment failures in specific subtypes, although there have been instances of delayed CD4 cell count recovery ,34,35,36response . In thei week 12 . CD4 cel therapy .These findings corroborate ours and that of other investigators in sub-Saharan Africa who have demonstrated the effectiveness of ART in HIV-infected children and adults with predominantly non-B subtypes ,41,42,43versus another might impact the probability of developing a particular drug resistance, at the population level, no difference in treatment outcomes has been demonstrated among the subtypes. Thus, the best method for determining drug susceptibility still appears to be genome sequencing to look for specific drug resistance mutations or polymorphisms in individual patients; subtype alone is not predictive of drug treatment success or failure.The variation in intra-subtype drug susceptibility seems to stem from polymorphisms that have a higher prevalence within specific subtypes, such as non-B subtypes . A well-versus ≥ 1 minor polymorphisms in the protease gene [et al. showed that 57% of HIV infected patients in their London cohort had at least one polymorphism in the reverse transcriptase gene, and that those with ≥ 2 polymorphisms had significantly higher rates of virologic failure [There are conflicting reports at the population level as to the effect of polymorphisms on treatment outcomes. For instance, the Swiss Cohort Study did not show any difference in time to virological failure or time to virological suppression between HIV infected patients with 0 ase gene . In cont failure . Thus, iPolymorphisms are of interest when they directly impact either viral fitness or antiretroviral susceptibility. Interestingly, most of the major drug-resistance mutations (DRMs), as outlined by the Stanford HIV Resistance Database are not considered polymorphisms; they are only rarely, if ever, identified in drug-naïve populations of HIV-infected individuals . The Stasame amino acid may increase the probability of a virus developing DRMs by altering the way in which reverse transcriptase moves along the reading frame [Naturally occurring HIV-1 variants, polymorphisms, confer resistance or improve viral replicative fitness through multiple mechanisms. For example, DRMs can reduce the replication capacity of the virus in addition to rendering it more resistant to a particular drug class . Howeverng frame . Polymorng frame ,49. We win vitro [et al. showed that mutations at position 190 led to the production of virions with incompletely processed Gag-Pol protein, leading to downstream inefficiency of both protease and reverse transcriptase. It was observed that the presence of a L74V polymorphism increased replicative fitness in those viruses containing a G190V/E mutation (conferring resistance to nevirapine and/or efavirenz) by potentially improving the processing of Gag-Pol and increasing the stability of reverse transcriptase [Some polymorphisms only confer resistance in the presence of other DRMs. For example, M50I often appears in infected cell cultures exposed to integrase inhibitors following the emergence of the R263K mutation. This combination confers resistance to dolutegravir, a second-generation integrase inhibitor, and has been observed in patients failing treatment with first generation-integrase inhibitors . Alone, in vitro . One meccriptase .in vitro drug resistance studies using subtype B isolates are natural polymorphisms in non-B subtypes [Polymorphisms at drug target sites may affect drug susceptibility. There is wide variability in the susceptibility of HIV-1 viral isolates to entry inhibitors; up to a 1000-fold difference in 50% inhibitory concentrations have been demonstrated . Much ofsubtypes . M426L, subtypes .et al. reported that polymorphisms found in the nucleotides encoding the Gag-Pol protein cleavage sites (the targets of protease) affected susceptibility to protease inhibitors (PIs) [gag polymorphisms, especially in the p2/NC cleavage site. Furthermore, non-B subtypes were more likely to have more than two polymorphisms at this site; these changes were associated with virologic failure while on a protease inhibitor regimen.Polymorphisms at the active site may influence the catalytic efficiency of the enzyme. In some circumstances, increased enzyme activity or expression is enough to overcome the drug effect. Ghosn rs (PIs) . Intereset al. demonstrated that the higher rate of the K65R mutation in subtype C is due to polymorphisms at codons 64 and 65 that change the more common AAG-AAA sequence (subtype B) to an AAA-AAG (subtype C) [Polymorphisms do not always confer antiretroviral resistance directly. Occasionally, the effect of a polymorphism on a viral enzyme simply increases the risk of developing broad antiretroviral resistance. For instance, HIV-1 subtype C has higher rates of the K65R major mutation which confers multidrug resistance to reverse transcriptase inhibitors. Coutsinos btype C) . All thrbtype C) .i.e., reducing the genetic barrier. For example, “AAG” (lysine) to “AGG” (arginine) requires one nucleotide change. However for “AAA” (lysine) to become “AGG” (arginine), requires two nucleotide substitutions. Thus, “AAA” has a higher genetic barrier to the development of the K65R mutation than “AAG” [et al. examined the mutational pathway to protease inhibitor resistance in non-B subtypes and found that M89L (ATG→ACG) is present in >95% of HIV-1 subtypes A, CRF01_AE, CRF02_AG, and G in addition to > 80% of subtype C worldwide [Moreover, polymorphisms sometimes simply reduce the number of mutations it takes to for an individual virus to develop a major resistance mutation, an “AAG” . Martineorldwide . When coet al. recently reported significant variability in the overall prevalence of TDRMs across African study populations with an increasing prevalence over time (<5% to >15%) [TDRMs are the current menace in HIV therapeutics. TDRMs could reverse the gains of ART, particularly during the unprecedented rollout in resource-limited countries. TDRMs can persist for several years with debatable effects of treatment outcomes ,60,61,62to >15%) . The repto >15%) ,72.et al. reported that using the WHO consensus guidelines, DRMs were not observed over a 5-year period in an HIV treatment-naïve cohort in Kuala Lumpur [TDRMs are of public health concern particularly in resource-limited countries where most HIV-infected individuals are initiated on ART when CD4 counts fall below 350 cells/μL. Thus, at the time of initiation of treatment, TDRMs might have become minority variants. Minority variants constitute less than 15% of the viral population and, therefore, allele-specific sequencing assays are more sensitive for detecting the variants than bulk or population sequencing assays . Furthera Lumpur . Howeveret al. found no association between the presence of minor PI DRMs at baseline and treatment outcome  in the Swiss HIV Cohort [et al. recently used a mathematical model to estimate the potential long-term impact of TDRMs on mortality [Although Scherrer V Cohort . HoweverV Cohort ,89,90. IV Cohort . TDRMs hV Cohort ,93,94. IV Cohort . In a stV Cohort . This fiV Cohort . Cambainortality . This moortality .i.e., HIV drug resistance “Early Warning Indicators” (EWI) [Appreciation of HIV-1 sequence diversity is a critical element for drug discovery and for optimization of ART, especially in drug-naïve patients. Although HIV-1 subtype may not be a predictor of treatment outcome, review of emerging evidence supports the fact that natural polymorphisms or drug-associated mutations matter when it comes to treatment outcomes. HIV-1 continues to evolve rapidly, thus introducing ever more complex sequence diversity. Therefore, continued surveillance of DRMs in both treatment-naïve and treatment-experienced populations is needed to reduce the transmission of DRMs and to optimize the efficacy of the current antiretroviral armamentarium. An ideal treatment algorithm would include the assessment of a patient’s HIV-1 genotype prior to ART initiation. However, this is not technically and economically feasible at the present time, particularly in resource-limited countries. ART programs in resource-limited countries have adopted the WHO HIV drug resistance guidelines: a population-based survey of transmitted HIV drug resistance in recently infected individuals . The WHOs” (EWI) ; (2) surs” (EWI) ; and (3)s” (EWI)  and relas” (EWI) . The pres” (EWI) , there as” (EWI) . As the


In a previous interim 24-week virological safety analysis of the PROTEST study , initiatPROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910) carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL) <50 c/mL on stable ART during the previous 6 months, requiring an ART change due to toxicity, with no antiretroviral resistance to the ART started, and R5 HIV by proviral DNA genotypic tropism testing (defined as a G2P FPR >10% in a singleton), initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week)*100.3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62%) subjects, NNRTIs in 27 (36%) and integrase inhibitors (INIs) in 1 (2%). The main reasons for treatment change were dyslipidemia (42%), gastrointestinal symptoms (22%), and liver toxicity (15%). MVC was given alongside TDF/FTC in 40 (54%) subjects, ABC/3TC in 30 (40%), AZT/3TC in 2 (3%) and ABC/TDF in 2 (3%). Sixty-two (84%) subjects maintained VL<50 c/mL through week 48, whereas 12 (16%) discontinued treatment: two (3%) withdrew informed consent, one (1%) had a R5→X4 shift in HIV tropism between the screening and baseline visits, one (1%) was lost to follow-up, one (1%) developed an ART-related adverse event (rash), two (3%) died due to non-study-related causes , and five (7%) developed protocol-defined virological failure, although two of them regained VL<50 c/mL with the same MVC regimen (Tropism results were available from 141/175 (80.6%) subjects screened: 87/141 (60%) were R5 and 74/87 (85%) were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm regimen . Initiation of MVC plus 2 NRTIs in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA is associated with low rates of virological failure up to one year.


Chronic low-grade inflammation and immune activation may persist in HIV patients despite effective antiretroviral therapy (ART). These abnormalities are associated with increased oxidative stress (OS). Bilirubin (BR) may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UGT1A1, thus increasing unconjugated BR levels, a distinctive feature of this drug. We compared changes in OS markers in HIV patients on ATV/r versus efavirenz (EFV)-based first-line therapies.Cohort of the Spanish Research Network (CoRIS) is a multicentre, open, prospective cohort of HIV-infected patients naïve to ART at entry and linked to a biobank. We identified hepatitis C virus/hepatitis B virus (HCV/HBV) negative patients who started first-line ART with either ATV/r or EFV, had a baseline biobank sample and a follow-up sample after at least nine months of ART while maintaining initial regimen and being virologically suppressed. Lipoprotein-associated Phospholipase A2 (Lp-PLA2), Myeloperoxidase (MPO) and Oxidized LDL (OxLDL) were measured in paired samples. Marker values at one year were interpolated from available data. Multiple imputations using chained equations were used to deal with missing values. Change in the OS markers was modelled using multiple linear regressions adjusting for baseline marker values and baseline confounders. Correlations between continuous variables were explored using Pearson's correlation tests.145 patients  were studied. Mean (SD) baseline values for OS markers in EFV and ATV/r groups were: Lp-PLA2 [142.2 (72.8) and 150.1 (92.8) ng/mL], MPO [74.3 (48.2) and 93.9 (64.3) µg/L] and OxLDL [76.3 (52.3) and 82.2 (54.4) µg/L]. After adjustment for baseline variables patients on ATV/r had a significant decrease in Lp-PLA2  and a significantly lower increase in OxLDL . Changes in BR and changes in OS markers were significantly correlated.In virologically suppressed patients on stable ART, OS was lower in ATV/r-based regimens compared to EFV. We hypothesize these changes could be in part attributable to increased BR plasma levels.