Progressive multifocal leukoencephalopathy (PML), a rare devastating demyelinating disease caused by the polyomavirus JC (JCV), occurs in severely immunocompromised patients, most of whom have advanced-stage HIV infection. Despite combination antiretroviral therapy (cART), 50% of patients die within 6 months of PML onset. We conducted a multicenter, open-label pilot trial evaluating the survival benefit of a five-drug cART designed to accelerate HIV replication decay and JCV-specific immune recovery.All the patients received an optimized cART with three or more drugs for 12 months, plus the fusion inhibitor enfuvirtide during the first 6 months. The main endpoint was the one-year survival rate. A total of 28 patients were enrolled. At entry, median CD4+ T-cell count was 53 per microliter and 86% of patients had detectable plasma HIV RNA and CSF JCV DNA levels. Seven patients died, all before month 4. The one-year survival estimate was 0.75 . At month 6, JCV DNA was undetectable in the CSF of 81% of survivors. At month 12, 81% of patients had undetectable plasma HIV RNA, and the median CD4+ T-cell increment was 105 per microliter. In univariate analysis, higher total and naive CD4+ T-cell counts and lower CSF JCV DNA level at baseline were associated with better survival. JCV-specific functional memory CD4+ T-cell responses, based on a proliferation assay, were detected in 4% of patients at baseline and 43% at M12 (P = 0.008).The early use of five-drug cART after PML diagnosis appears to improve survival. This is associated with recovery of anti-JCV T-cell responses and JCV clearance from CSF. A low CD4+ T-cell count (particularly naive subset) and high JCV DNA copies in CSF at PML diagnosis appear to be risk factors for death.NCT00120367ClinicalTrials.gov  Human polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease of the central nervous system (CNS) involving lytic infection of oligodendrocytes. PML occurs in severely immunocompromised individuals. The incidence of PML increased sharply during the HIV/AIDS pandemic, up until 1996 T-cell responses likely play a key role in controlling intracerebral JCV replication. Indeed, specific cytotoxic CD8+ T-lymphocyte responses to JCV were found to be undetectable in patients who subsequently died, in contrast to survivors To date, no specific anti-JCV treatment has shown significant clinical efficacy in patients with PML We conducted a prospective multicenter study evaluating the effect of five-drug antiretroviral treatment including enfuvirtide on PML outcome.The protocol for this trial and supporting CONSORT checklist are available as supporting information see .The ANRS 125 trial, sponsored by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS), was a multicenter open-label non comparative pilot study designed to assess the effect of an early use of five-drug cART including enfuvirtide on survival one year after PML diagnosis in HIV-1-infected patients. The study was designed to show an expected one-year survival rate of 70%, significantly higher than 45% which was the average of the one-year survival rate observed in previous reports All subjects had documented HIV type 1 (HIV-1) infection. All subjects were required to have a diagnosis of active PML, with less than 90 days of clinical progression All the participants received an effective cART for 12 months, plus enfuvirtide for the first 6 months. The choice of regimen was based on the individual ART history. Patients, who had never received ART when PML was diagnosed, received efavirenz, ritonavir-boosted lopinavir, and the fixed-dose emtricitabine-tenofovir combination. ART-experienced patients received drugs belonging to at least two different antiretroviral families, chosen on the basis of their treatment history and viral resistance genotyping results. Beyond 6 months, only patients with limited therapeutic options were maintained on enfurvirtide.Each participant was followed for one year. As the mean interval between the enrolment visit and the outset of enfuvirtide therapy was 1.4 days (range 0 to 11 days), the date of the first dose of enfuvirtide was chosen as the study baseline. The patients had physical examinations, including a standardized neurological examination and Karnosky performance-status measurement, at baseline, week 6 (W6), month 3 (M3) and month 6 (M6). Karnosky performance-status and the modified Rankin scale (MRS) We used a standardized neurological examination from the NIH Stroke Scale 5′ gga aag tct tta ggg tct tct acc ttt 3′) and PEP6 (5′ gaa gac cgt ttt tgc cat gaa ga 3′). PCR product was detected with the HuPo1 probe (5′ atc acg ggc aaa cat 3′). To control for the presence of PCR inhibitors, an internal DNA control  was simultaneously amplified in each PCR run. The detection limit was 100 copies per milliliter.JCV DNA was quantified in CSF by using a quantitative real-time PCR method, Flow cytometry used a FACsCalibur device , with monoclonal antibodies and reagents provided by the same manufacturer. T-cells subsets are defined by the following antibody expression patterns: Naive subset, CD45RA+CD62L+; Central memory subset, CD45RA−CD62L+; Effector memory subset, CD45RA−CD62L−; Effector subset, CD45RA+CD62L−.JCV-specific CD4+ T-cell responses were assessed in proliferation assays with purified JCV (MAD-4 strain) ex vivo interferon-gamma Elispot assay with PBMC and 14 pools of overlapping 15-amino-acid peptides covering the entire JCV VP-1 protein. In some patients, the effect of JCV peptide pools on T-cell responses was also examine by flow cytometry, the interferon-gamma responses mainly involved CD8 T-cells. The pools were used at 2 µg per milliliter. Positive controls used PMA (50 ng per milliliter) and ionomycin (500 ng per milliliter). The response to each pool was considered positive if the mean number of spot-forming cells (SFC) per 106 PBMC in activated wells (in triplicate) was more than 2-fold that of untreated wells (background) and also higher than the background plus 100 SFC. A patient was considered to be a responder, i.e. to have a detectable anti-JCV effector T-cell response, if he or she had a positive response to at least one peptide pool.To investigate anti-JCV effector T-cell responses, we used an The primary endpoint was the one-year survival rate. Secondary endpoints were the percentage of patients who survived at M12 with a MRS score below 3 ; the percentage change in neurological scores between baseline and M6; the change in Karnofsky performance-status between baseline and M12; the change in CSF JCV level and the proportion of patients with CSF JCV clearance at M6; and the changes in CD4+ and CD8+ T-cell subsets and in JCV-specific T-cell responses between baseline and W6, M3, M6 and M12. Non-parametric methods  were used to compare continuous and categorical variables. Survival rates were estimated with the Kaplan-Meier method from the first dose of enfuvirtide. Survival curves were compared with the log-rank test.Between April 2005 and December 2006, 29 patients were enrolled in six centers, including 21 patients in the main center . One patient subsequently withdrew consent, and the study population therefore consisted of 28 patients.Baseline clinical and immunovirological data are shown in All the patients received enfuvirtide until death or for at least 6 months. Five patients continued to receive enfuvirtide after 6 months, for an average total period of 11 months . In addition to enfuvirtide, 15 patients, including all 12 previously untreated patients, received efavirenz, ritonavir-boosted lopinavir and tenofovir/emtricitabine. The other 13 patients received other combinations consisting of 2 or 3 reverse transcriptase inhibitors  plus 1 or 2 ritonavir-boosted protease inhibitors . Percutaneous endoscopic gastrostomy tube for enteral feeding and drugs administration was inserted in 7 patients with swallowing disorders.Seven patients died during the study period, all from PML, and all before M4 including one with plasma HIV RNA below 40 copies/mL. The estimated one-year survival rate was 0.75  . No signAs shown in Treatment was rapidly and persistently effective on plasma HIV RNA level  panel C:See The naive CD4+ T-cell count  panel D Naive CD8+ T-cell numbers increased only from W6 to M3 (P = 0.05) (not shown). No significant change was observed in the numbers of central memory, effector memory or effector CD8+ T-cells.Significant restoration of anti-JCV CD4+ T-cell proliferation to purified JCV was observed, starting at M3  panel A.ex vivo interferon gamma Elispot assay and overlapping peptide pools covering the entire VP1 protein to test effector T-cell responses including mainly CD8+ T-cell responses, we found no significant changes in the percentage of patients responding to at least one peptide pool between baseline (19%) and M12  or any other time during the follow-up  of survivors and in none of the 7 patients who died , while specific effector T-cell responses were found in 76% (16/21) of survivors and in 29% (2/7) of patients who died (P = 0.06).Moreover no correlation was observed between JCV viral load in CSF and JCV-specific T-cell responses both at baseline and during follow-up (M3 and M6). Finally no relationship has been found between neurological outcome  and JCV-specific T-cell responses at M12 as well as JCV viral load during follow-up.A possible immune restoration inflammatory syndrome (IRIS) occurred in a treatment-naive patient with a baseline CD4+ T-cell count of 167 per microliter, whose neurological status markedly deteriorated  during the first 6 weeks of ART. At W6, the CD4+ T-cell count was 214 per microliter and cerebral MRI showed extension of demyelination to the white matter of both hemispheres, including the corpus callosum. A mass effect on the left ventricle and perilesional contrast enhancement were also observed. The patient improved rapidly on steroid therapy. Of note, this patient had no detectable specific CD4+ T-cell responses during the study period, and a positive anti-JCV effector T-cell response was observed only at M12.Six serious adverse events occurred in 6 different patients, consisting of one case each of bicytopenia, bilateral pulmonary embolism, partial epilepsy, acute respiratory distress syndrome, biochemical deterioration of hepatitis C, and hysterectomy for severe cervical dysplasia. With the exception of bicytopenia, these serious adverse events were assessed as not directly related to the antiretroviral therapy. All serious events resolved, except in the patient with severe hepatitis C, whose liver status continued to deteriorate despite interruption of ART and who died at week 6. All other patients were still alive at the end of the study. Enfuvirtide was never withdrawn. Urolithiasis occurred in a patient on indinavir, which was replaced by atazanavir. No significant variations in creatinine clearance, calcemia or phosphatemia occurred in the 24 patients receiving tenofovir.In this study the one-year cumulative probability of survival after PML diagnosis was 75% , a rate higher than previously reported in HIV-infected patients placed on conventional triple-drug ART after PML diagnosis (39% to 56%) There are few longitudinal studies of the clinical course of PML during cART. In a previous paper The low incidence of HIV-related PML led us to choose an open-label non comparative study design. To demonstrate in a randomized controlled study that a one-year survival rate of 70%, was significantly higher than 45% which was the average of the survival rate observed in previous published studies Use of triple-drug cART in patients with AIDS-related PML can cause IRIS In this study, survival of PML patients was associated with a high rate of JCV clearance from CSF, as previously reported in vitro for 10 to 14 days ex vivo procedure, without in vitro amplification, to assess circulating IFN-gamma-producing anti-JCV T-cell effectors including mainly CD8+ effectors. Survival was not associated with the detection of anti-JCV T-cell effectors at baseline, but most JCV-specific T-cell effectors might be located in brain tissue, where JCV replication occurs predominantly during active PML. This, in addition to the inconstancy of detection of anti-JCV T-cell effectors in a given patient during follow-up, would mean that the blood compartment does not accurately reflect anti-JCV T-cell responsiveness. This is consistent with recent studies based on the use of specific tetramers, that showed a very low frequency of anti-JCV specific CD8+ T-cells in peripheral blood, relative to infections by less anatomically restricted viruses such as HIV, EBV and CMV Better outcome of PML has been linked to the detection of anti-JCV peripheral blood CD8+ CTL after peptide stimulation By accelerating HIV decay, five-drug antiretroviral combination may lead to more rapid anti-JCV immune reconstitution. However, 25% of patients still die within 4 months of treatment initiation. Moreover, only 38% of survivors recovered adequate autonomy for activities of daily living. The size of the residual naive and central memory CD4+ T-cell pools may influence the time required for effective CD8-helping CD4+ T-cell responses to recover during cART. These pools could therefore represent targets for immunotherapy based on cytokines such as IL-7 In conclusion, our results support the use of five-drug cART early during the course of PML, for both antiretroviral-naive and -experienced patients.Table S1The PML Score is a clinical assessment tool adapted from the NIH Stroke Scale . To evaluate and document neurological status in PML patients, four items  have been added to the NIHSS. Moreover, two of the 15 original items  have been modified to take into account bilateral damage. PML Score ranges from 0 to 60 points. This score is not yet validated at present.(DOC)Click here for additional data file.Table S2Review of previous studies that reported effects of combination antiretroviral therapy on HIV-related PML: characteristics of patients at PML diagnosis. Results are expressed as median, (range) or [IQR]. EDSS, Expanded Disability Status Score; KPS, Karnofsky Performance Score; NA, not available; ud, undetectable.(DOC)Click here for additional data file.Checklist S1Consort Statement 2001 Checklist for the paper entitled “Improved Survival of HIV-1-Infected Patients with Progressive Multifocal Leukoencephalopathy Receiving Early 5-Drug Combination Antiretroviral Therapy”.(DOC)Click here for additional data file.Protocol S1Short version of the protocol of the ANRS 125 Trial “Early Intensification of Antiretroviral Therapy Including Enfuvirtide in HIV-1-Related Progressive Multifocal Leucoencephalopathy”.(DOC)Click here for additional data file.


Increasing rates of non-AIDS defining illnesses, and in particular liver diseases, have been found after the initiation of highly active antiretroviral therapy. However, there is little evidence concerning the risk factors for and clinical characteristics of liver disease in antiretroviral (ARV)-treated HIV infection, in the absence of hepatitis B or C viral co-infection.A nested case–control study of HIV infected volunteers, matched by starting date of anti-retroviral treatment, was conducted in a Thai cohort studied from Nov 2002 - July 2012. Cases were defined as those subjects with an elevated alanine aminotransferase (ALT ≥ 40 IU/L) at two consecutive visits six months apart, while controls were defined as individuals who never demonstrated two consecutive elevated ALT results and had a normal ALT result (< 40 IU/L) at their last visit. Both groups had normal ALT levels prior to ARV initiation. Clinical demographics and risk factors for chronic hepatitis including HIV-related illness, ARV treatment and metabolic diseases were collected and analyzed. Conditional logistic regression was used to determine risk factors for chronic hepatitis in HIV infection.10 copies/ml, metabolic syndrome at baseline visit, high BMI > 23 kg/m2, abnormal HDL cholesterol at time of ALT elevation and treatment experience with NNRTI plus boosted PI were selected  to the final model of multivariate analysis. Male sex had 3.1 times greater risk of chronic hepatitis than the females by multivariate analysis . High BMI ≥ 23 kg/m2 was also associated with 2.4 times greater risk of chronic hepatitis .A total of 124 matched pairs with HIV infection were followed over 3,195 person-years. The mean age (±SD) was 33.0 ± 7.3 years, with 41.1% of subjects being male. The incidence of chronic hepatitis was 5.4 per 100 person-years. The median time from initiation of ARV to chronic hepatitis was 1.3 years . From univariate analysis; male sex, plasma HIV-1 RNA level > 5 log 2 carry higher risks for developing chronic hepatitis in this study. Therefore, these patients should be closely monitored for long-term hepatotoxicity.Chronic hepatitis in ARV-treated HIV-infected patients is common and may lead to a major health care problem. Male sex and high BMI ≥ 23 kg/m Human immunodeficiency virus (HIV) infection continues to be a major global health problem. An estimated 33.4 million people are currently living with HIV worldwide . The eraThe etiologies of liver disease seen in HIV infection include hepatitis B or C virus co-infection, medication-induced hepatitis from antiretroviral (ARV) or non-ARV drugs (such as anti-tuberculous and anti-lipemic agents), metabolic syndrome, excessive alcohol consumption and HIV infection itself .2 and clinical parameters and laboratory assessments at an event visit .The main risk factors that were evaluated included clinical characteristics [age, gender, sexual risk behavior, HIV-related illness and disease severity, baseline CD4Clinical characteristics were described as frequency and percentage for categological data. Continuous data were reported as mean (x̄) and standard deviation (SD) if normally distributed or as median and inter-quartile range (IQR) if not normally distributed . For infA total of 1,680 HIV-infected individuals participated in the long term HIV-NAT cohort during the period of November 2002 to July 2012. We excluded individuals from the present study owing to inactive status  at the time of data analysis (n = 413), a confirmed positive result for chronic hepatitis B or C virus infection (n = 651), unavailable baseline ALT data (n = 120), baseline ALT ≥ 40 IU/L (n = 119) and loss to follow-up for at least 1 year (n = 9)  were slightly higher in the cases than the controls with borderline significance (p = 0.08) . There were only 12 patients who met the criteria for metabolic syndrome because of missing data for lipid profiles and waist circumference. There was no significant difference of metabolic syndrome between groups.The proportion of individuals with BMI greater than 23 kg/mThere was no difference in the prevalence of elevated blood pressure and hypertension, impaired fasting plasma glucose and lipid profiles between the groups.2 was significantly higher in the cases (44.4%) than the controls  .All patients were treated with ARV Table . The comEleven patients developed early acute adverse events from ARV treatment such as severe hepatitis, drug hypersensitivity reaction and severe fatigue. ARV was discontinued and the hepatitis completely resolved. The new regimens for these patients were used as the first ARV regimen for our analysis.The concomitant medications were similar between the 2 groups Table .10 copies/ml and metabolic syndrome at baseline visit, high BMI ≥ 23 kg/m2 and abnormal HDL cholesterol at time of ALT elevation and treatment experience with NNRTI plus boosted PI were selected to the final model of multivariate analysis  were the independent risk factors for chronic hepatitis.From the multivariate analysis, male sex  and high BMI ≥ 23 kg/mOur study examined clinical characteristics and the risk factors of chronic hepatitis by evaluating ALT level as a surrogate marker. We reported a case–control study of 124 matched pairs, ARV-treated HIV-infected individuals without HBV or HCV infection with a follow-up time of 3,195 person-years. Our incidence rate of hepatitis was slightly higher (5.4 cases per 100 person-years) than previously reported in Kovari et al. (3.9 cases per 100 person-years) [The ALT elevation in our study was mostly mild . There was no report of serious liver disease or hepatic encephalopathy, however, its long-term deleterious consequence is not well defined. The majority of previous studies mainly reported patients with severe elevation of ALT  who also tended to have symptomatic disease . In geneWe found that the median time from ARV initiation to chronic hepatitis was 1.3 years  which was similar to previous report of 1.2 year . As our Our study confirmed that males had 3.1 times  greater risk of chronic hepatitis than females. This finding supports the report by Guaraldi G et al. that non-alcoholic fatty liver disease (NAFLD) was associated with male sex and elevated ALT level . Previou2 was associated with 2.4 times  greater risk of chronic hepatitis. Previous studies showed that high BMI (>25 kg / m2) and overt obesity were associated with chronic hepatitis [Our study concurred that high BMI greater than 23 kg / mepatitis . Howeverigh BMI > kg / m2 epatitis .Our findings confirmed the study from the Swiss HIV cohort that HIV-related parameters such as baseline CD4+ cell count and HIV-1 RNA level are not associated with chronic hepatitis. Our finding disagrees with the study of Sterling et al. which reported that detectable HIV-1 RNA level was associated with chronic, rising ALT enzymes. However, that study was a cross-sectional study. Plasma HIV-1 RNA and ALT level were tested only one visit during the study period .We did not find exposure to ARV or concomitant medication to demonstrate an association with chronic hepatitis in contrast to previous reports ,10. The The strengths of this study include the availability of prospective longitudinal data collection of ALT that has been shown to accurately reflect liver steatosis. The determination of chronic hepatitis by using ALT level as a surrogate laboratory test increases the generalizability of the data to other resource-limited settings. This test is inexpensive, widely available and easy to perform. However, liver injury may exist in the absence of transaminases elevation. Limitations of this study include a lack of histological and clinical imaging data to confirm liver injury as well as missing data on alcohol use and parameters to diagnose metabolic syndrome. In addition, lipodystrophy may be under diagnosed or under reported. This study did not exclude other causes of chronic hepatitis including metabolic diseases such as hemochromatosis, Wilson’s disease and autoimmune hepatitis and infectious causes including cytomegalovirus or Epstein-Barr virus, chronic hepatitis E virus. Finally, this study also did not examine an association between duration of ARV treatment and chronic hepatitis because it had already matched for the date of ARV initiation.2 carry higher risks for developing chronic hepatitis in this study. Therefore, these patients should be closely monitored for long-term hepatotoxicity.In conclusion, chronic hepatitis in ARV-treated HIV infected patients is common and may lead to a major health care problem. Male sex and high BMI ≥ 23 kg/mAOR: Adjusted odds ratio; BMI: Body mass index; CDC: Centers for disease control and prevention; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDL cholesterol: High density lipoprotein cholesterol; HIV: Human immunodeficiency virus; IQR: Interquartile range; LDL cholesterol: Low density lipoprotein cholesterol; MSM: Men who have sex with men; NNRTI: Non-nucleoside reverse transcriptase inhibitor; NRTI: Nucleoside reverse transcriptase inhibitors; OR: Odds ratio; PI: Protease inhibitors; SD: Standard deviation; ULN: Upper limit of normal.The authors declare that they have no competing interests.NH, PT, AA, JA, TC and SP designed and conducted the study, interpreted the data, and prepared and edited the manuscript. All authors read and approved the final manuscript.


The visual-analogue scale (VAS), Likert item , pills identification test (PIT), and medication possession ratio (MPR) provide estimates of antiretroviral therapy (ART) adherence which correlate with HIV viral suppression. These simple adherence measures are inexpensive and easy to administer; however, require validation and adjustment prior to implementation. The objective of this study was to define the optimal adherence assessment measure in Namibia to identify patients at risk for sub-optimal adherence and poor virologic response 6 months after ART initiation. We conducted a cross-sectional survey in HIV-infected adults receiving ART for 6–12 months prior to the adherence assessment. Adherence measures included 30-day VAS, 30-day Likert item, self-reported treatment interruptions, PIT, and MPR. Association of adherence measures with 6-month HIV-1 RNA level was assessed using two thresholds (1000 copies/mL and 5000 copies/mL). Adherence was assessed in 236 patients, mean age 37.3 years, 54% female. Mean adherence was 98.1% by 30-day VAS, 84.7% by 30-day Likert item, 97.0% by self-reported treatment interruptions, 90.6% by PIT, and 98.8% by MPR. Agreement between adherence measures was poor using kappa statistic. 76% had HIV-1 RNA <1000 copies/ml, and 88% had HIV-1 RNA <5000 copies/ml. MPR (continuous) was associated with viral suppression <5000 copies/ml (p = 0.036). MPR <75% was associated with virologic failure at ≥5000 copies/ml with OR 3.89 , p = 0.013. Adherence was high with all measures. Only MPR, was associated with short-term virologic response, suggesting its cross-culturally utility for early identification of patients at high risk for virologic failure. As of December 2011, over 8 million people infected with HIV were receiving antiretroviral therapy (ART) in low- and middle-income countries which represents a 26-fold increase since 2003 Assessment of adherence by health care providers often results in an overestimation of patients’ medication adherence Namibia is a resource-limited country in sub-Saharan Africa that has been severely affected by the HIV epidemic. In Namibia, there are approximately 200,000 people living with HIV in a population of 2.1 million It is unclear which adherence measure would be most appropriate for use in Namibia’s ART program. Therefore, identifying the best tool to estimate patient adherence for Namibia would be valuable for quality-improvement of the ART program to minimize preventable HIV drug resistance and optimize patient care. The objective of this study was to define the optimal cross-cultural tool to provide ART care providers in Namibia with information to identify patients at risk for sub-optimal adherence and poor short-term virologic response.We conducted a cross-sectional survey in 236 HIV-infected adults in Namibia who had initiated ART 6–12 months prior to the adherence assessment. Adherence measures included a 30-day VAS, 30-day Likert item, self-reported treatment interruptions, and PIT. Pharmacy dispensing records were used to calculate MPR. A viral load was conducted 6 months after ART initiation on all ART patients and used to assess association with adherence measures. Viral load testing was conducted by the Namibian Institute of Pathology utilizing COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Hoffman-La Roche). The survey was performed between September 2010 and April 2011.Ethical approval was obtained from the institutional review board at Tufts University School of Medicine in Boston, USA and the Republic of Namibia Ministry of Health and Social Services Ethics and Research Committee in Windhoek, Namibia. Written informed consent was obtained from all participants.The study population consisted of patients diagnosed with HIV-1 who were being prescribed adult ART and being managed at a public ART delivery site in Windhoek, Namibia. Windhoek, the largest urban setting in the country, has an estimated population of 342,000 (2009) Patients were recruited from the three major public ART delivery sites in Windhoek: Katutura State Hospital, Katutura Health Centre, and Windhoek Central Hospital. Patients were eligible to be included in the survey if they were HIV positive, started an adult ART regimen for the first time 6–12 months prior to the date of the adherence assessment, were receiving an adult ART regimen at the time of the adherence assessment, and had a routine 6-month viral load available for analysis.Adherence was measured at the routine ART clinic visit with five different adherence measures: 1) VAS, 2) 5-choice Likert item, 3) self-reported treatment interruptions, 4) PIT, and 5) MPR. The VAS asked the patient to mark an “X” on a continuous scale describing his/her level of adherence to all their antiretroviral (ARV) medications prescribed over the previous 30 days on a scale of 0% to 100%. The position indicated by the patient was converted to a percentage. The 5-choice Likert item asked the patient to choose the word that most accurately described how well they took their ARV medication during the past 30 days: “excellent”, “very good”, “good”, “fair”, and “poor”. Self-reported treatment interruptions were assessed by asking, “How many times did you ever interrupt your ART for 2 days or more.” All self-reported adherence questions were preceded by a statement asking for truthful answers, as they would be kept strictly confidential and would in no way affect their future health care. The PIT measured whether a patient was able to identify all the medication that he/she was taking and state the number of pills that should be taken and the time. The PIT used in this study was adapted from the original PIT developed by Parienti et al The standardized questionnaire was administered via face-to-face interviews by a trained interviewer. The questionnaire was translated into Oshiwambo and Afrikaans and back-translated and pre-tested to ensure accuracy. The questionnaire was administered in English, Oshiwambo or Afrikaans depending on the participant’s preference. English is the official language in Namibia; and Oshiwambo and Afrikaans are spoken in 37% and 24% of households respectively Univariate statistics were obtained for each variable included in this analysis. Frequencies and distributions were carefully examined for unusual values. For the outcome, we chose to look at viral load level at two thresholds, <1000 copies/mL and <5000 copies/mL. These two thresholds were chosen to reflect the World Health Organization (WHO) criteria for prevention of HIV drug resistance (1000 copies/mL) Separate chi square analyses were conducted for each adherence measure to determine if associations existed between adherence and each viral load outcome. Bivariate associations were examined between other relevant demographic and potentially confounding variables and viral load using chi square tests for categorical variables and Wilcoxon Rank Sum tests for ordinal and continuous measures.To assess bivariate associations between outcome variables and each of the adherence measures and demographic variables of interest, three statistical tests were used: Spearman rank correlation was used to measure the association between two continuous variables; Wilcoxon Rank Sum tests for the association between binary and continuous variables; and Kruskal-Wallis tests for the association between categorical and continuous variables.The degree that adherence measures agreed with each other was assessed. Agreement among the dichotomized measures of adherence  was assessed using Cohen’s kappa statistic.For all statistical analyses, an alpha of 0.05 was used to evaluate statistically significant differences or associations. All analyses were performed using PASW version 18 .Adherence to ART was assessed in 236 patients , mean agMedian and mean adherence was 100% and 98.1% respectively by 30-day VAS. Ninety-six percent of respondents had a VAS percent of >95%. By the 30-day Likert item 85% reported “excellent” or “very good” adherence and 48% of respondents reported “excellent” adherence. Ninety-one percent of respondents had a satisfactory PIT score. Median and mean adherence by MPR was 100.0% and 98.8%. Ninety-three percent had ≥75% MPR. Only 3% of respondents reported having one or more treatment interruption of ≥48 hours. .Agreement between adherence measures was poor with low Kappa statistic values. The VAS had higher agreement with self-reported treatment interruptions  than other clinic-based measures. The second highest agreement was VAS with 30-day Likert .HIV-1 RNA was determined 6 months after ART initiation; 179/236 (76%) had HIV-1 RNA <1000 copies/mL, and 208/236 (88%) had HIV-1 RNA <5000 copies/mL. MPR (continuous variable) was significantly associated with viral suppression at <5000 copies/mL (p = 0.036). Having MPR <75% was significantly associated with virologic failure at ≥5000 copies/mL with OR 3.89 , p = 0.013. MPR <75% had borderline significant association virologic failure at ≥1000 copies/mL with OR 2.64 , p = 0.058. No other adherence measure had a significant association with viral load at ≥5000 copies/mL or ≥1000 copies/mL.This study is the first reported ART adherence assessment among HIV-infected patients in Namibia. The level of adherence observed in the three public ART delivery sites in Windhoek, Namibia was high as estimated by all five adherence measures. This finding is consistent with previously published levels of reported adherence in other African settings We used five different measures of adherence in this study to assess for a cross-cultural and simple measure of adherence associated with short-term virologic response. Viral load 6 months after ART initiation was used as a marker for short-term virologic response and was tested for associations with the different adherence measures. As shown previously in other settings No other measure of adherence in this study was found to be significantly associated with short-term virologic response. In addition, agreement between adherence measures was poor. These results could be explained by a variety of cultural-linguistic factors and/or limitations with the tools themselves such as recall and social desirability bias. In addition, some tools may be limited in their adherence assessment depending on the reasons for poor adherence and pattern of missed doses as detailed below.The level of adherence by 30-day Likert in our study was high, but was not associated with virologic suppression. Although, the 30-day Likert item has been validated in other settings, the overestimation of adherence by these types of self-reported adherence tools has been reported To our knowledge, this is the first study assessing PIT in Sub-Saharan Africa. As the PIT requires minimal use of words because it involves primarily identification of the pills or pictures of pills This study has several limitations. First, we conducted the adherence assessment in Windhoek, which is not representative of the country. However, because Windhoek has a mixture of the different socio-economic and ethnic populations present in Namibia, we would expect similar understanding and usefulness of these adherence tools. Results though may not be as generalizable to rural populations. Second, adherence assessments were conducted a mean two months after the 6-month viral load. This lag period leaves open the possibility that the lack of association between self-reported adherence and viral load was due to a change in viral load In conclusion, this study provides the first data from Namibia describing levels of ARV adherence comparing multiple adherence measures. The levels of adherence were high with all adherence measures, but self-reported adherence measures were not associated with virologic failure. Depending on the reasons and patterns of missed doses in particular populations, some tools may be more helpful than others. In resource-limited settings, self-reported adherence measures may be less useful tools because non-adherence and thus virologic failure may be due more to lack of access to medications rather than individual reasons for poor adherence. MPR was found to be associated with short-term virologic response, which suggests its utility in early identification of patients at high risk for virologic failure in resource-limited settings. Future research should focus on identifying optimal adherence measure tools for resource-limited settings where lack of access to medications may be the primary driver for treatment failure.


The recently observed association between the APOC3-related rs10892151 polymorphism and serum triglyceride levels has prompted us the possibility to explore whether this genetic variant may play a major role in human immunodeficiency virus (HIV)/antiretroviral therapy-induced dyslipidemia.n = 208) and in a group of age and sex-matched healthy volunteers (n = 200). Circulating lipid and lipoprotein levels were followed for 12 months after antiretroviral treatment initiation in the HIV-infected group.We determined the rs10892151 genotype distribution and serum apolipoprotein (apo) C-III concentration in a group of HIV-infected patients ; additionally, the A allele was not related to serum apo C-III concentration. However, among patients receiving protease inhibitor (PI) treatment, carriers of the A allele had significantly increased serum triglyceride (5.76 ± 2.54 mmol/L) and total cholesterol (6.63 ± 2.85 mmol/L) concentrations together with depressed levels of HDL-cholesterol (0.75 ± 0.3 mmol/L) when compared with patients not carrying the allele  at the end of the study. This effect was only evident for HDL-cholesterol concentration when patients were treated with non-nucleoside reverse transcriptase inhibitors (1.05 ± 0.4 vs. 1.28 ± 0.4 mmol/L).The A allelic variant of the rs10892151 polymorphism is not associated with serum apo C-III concentration, but predisposes HIV-infected patients to less favorable lipid profile, particularly in those patients treated with PIs. Of the 208 patients initially selected, 201, 198 and 192 of them remained on the same ART regimen at months 3, 6 and 12 of follow-up, respectively. 160 (76.9%) were treatment-The baseline clinically relevant characteristics of the patients included in the study are summarized in Table National Center for Biotechnology Information SNP public database; it was not significantly different to that predicted by the Hardy-Weinberg distribution.The genotype and allelic frequencies of the rs10892151 polymorphism did not show significant variations between healthy and HIV-infected participants (p = 0.7). A total of 18 subjects (8.6%) from the HIV-infected group and 15 (7.5%) from the healthy group were carriers of the A allele, while 190 (91.4%) HIV-infected patients and 185 (92.5%) healthy subjects were carriers of two G alleles (the wild-type genotype). No subjects were homozygous for the A allele. The A allelic frequency for these populations was similar to that reported in the When we examined serum apo C-III concentration based on genotypes, no statistically significant differences were observed in serum apo C-III levels among healthy participants  or among HIV-infected patients . We also failed to observe significant associations between the duration and nature of antiretroviral treatment or viral load and serum apo C-III concentration as well as between lipid profile and rs10892151 genotype distribution in healthy participants.Table Because hyperlipidemia is strongly associated with the PI treatment regimen, we focused on a subgroup of 95 patients receiving PIs as a component of their antiretroviral therapy, and we analyzed lipid profile changes over a 12-month follow-up period. As indicated, baseline values of metabolic parameters were comparable based on genotype. However, carriers of the A allele had consistently higher serum triglyceride concentration than non-carriers at three , six , and twelve  months, after adjustment of the data for age, sex, BMI, and HCV co-infection status Figure . ResultsWe separately evaluated a subgroup of 113 patients who were undergoing treatment with NNRTIs. Baseline values were also comparable between genotypes, and there were no differences between genotypes with respect to serum triglyceride Figure  or totalOur study represents the first report of a strong association between the rs10892151 A allelic variant and the expression, particularly during PI-treatment, of a dyslipidemic phenotype in HIV-infected patients, which includes hypertriglyceridemia and depressed HDL-cholesterol levels.APOE genotypes, are associated with the severity of antiretroviral treatment-induced dyslipidemia. For instance, the most severe dyslipidemic profiles after PI therapy were obtained in those patients carrying the three APOC3 variations  and a non-ε3/ε3 APOE genotype [et al. [APOA5 gene experienced marked increases in triglyceride levels (up to 80%) during a 3-year follow-up, while no change was recorded in patients carrying the normal -1131T allele. More recent studies [ARβ2) with the occurrence of lipodystrophy. In addition, tumor necrosis factor-α (TNFα) [The high prevalence of lipid disorders among treated HIV-infected patients requires the identification of specific polymorphisms in candidate genes that might predispose to these complications. However, the genetic basis of these metabolic abnormalities remains unclear. Some studies show that variants of apo A-V and C-III, interacting with genotype -21. Simi [et al.  indicate studies ,24 have α (TNFα) ,25,26 anα (TNFα) ,28 have et al. [APOC3 gene cluster, prompted us to test the presence of a genetic association with lipid outcomes in our patients. In their high-fat feeding intervention study in 809 Old Order Amish individuals, the authors observed that rs10892151 A carriers had lower fasting and post-prandrial serum triglycerides values than non-carriers, and they found a linkage disequilibrium with an APOC3 null mutation, which was likely the result of a founder effect. Conversely, we found that this polymorphism in our study has no effect on circulating apo C-III values in either HIV-infected patients or healthy subjects, indicating that the linkage disequilibrium between this two genetic regions could not be universal.The intriguing results obtained by Pollin et al.  in the pHigh serum triglyceride concentration and low HDL-cholesterol values were found in our population, irrespective of the treatment they were assigned , but these lipid disorders were not related to a differential or abnormal response to circulating apo C-III levels. In addition, we were not able to identify further associations between the polymorphism and other lipid and inflammatory parameters. However, being a carrier of the rs10892151 A variant was a primary determinant of the course of lipid alterations. During follow-up, these patients consistently showed higher serum triglyceride and cholesterol concentrations, as well as lower HDL-cholesterol levels than those carrying the G/G genotype. This effect was particularly evident in those under PI-treatment, although a residual effect in HDL-cholesterol levels was also evident in those under NNRTI-treatment indicating that the HIV-infection itself may have a considerable impact on lipid concentrations for patients with the G/A genotype. However, further studies in larger populations under other chronic inflammatory stimulus are required to confirm the direction of rs10892151 polymorphism effect and to explain the fact that the observed effects in our study were quite different than expected from HIV negative individuals as previously reported [Although the design of our study cannot address the mechanism by which antiretroviral treatment and HIV-infection interact with the polymorphism, it could be argued that antiretroviral treatment may interfere in the functioning of lipolytic enzymes, ,30 whichAnother possibility is that antiretroviral treatment may not affect just lipoprotein metabolism, but may exacerbate the chronic inflammatory state by the expression of pro-inflammatory molecules such as TNFα or MCP-1, which have a major role in lipid metabolism and, consequently, in cardiovascular disease ,32-34. HThe peculiar distribution of alleles seriously limits the reach of our conclusions but the differential response and pharmacogenetic implications among patients with different treatment strongly reinforces the need for replication. According to our data, this should be done in a sample with same ethnic origin and a sample size only achievable in multicenter-multicohort studies. The impact of population stratification in this study is unlikely because in our population sample this has been already assessed empirically by analyzing more than 25 unlinked single-nucleotide polymorphisms (SNPs) in several association studies spanning a range of disease states -39 and cWe also acknowledge that studies assessing only one polymorphism are less robust than those assessing several polymorphisms associated with a plausible role in the pathogenesis of dyslipidemia. Further studies are needed to assess the possibly coordinated association with other well-documented polymorphisms, particularly those involved in metabolism and in molecular targets of antiretroviral drugs in order to uncover a host-related predisposition towards developing metabolic complications. If successful this may aid for specific, individual design of antiretroviral regimens to diminish the rapid emergence of side effects and the consequent deleterious significance.These results suggest that the rs10892151 A allelic variant could be associated in HIV-infected patients with less favorable lipid profile, particularly those treated with PIs. The exact mechanism for this putative effect is unknown but, considering the therapeutic implications, clarification deserves further research.The authors declare that they have no competing interests.JJ and CA-V conceived and designed the study and reviewed critically the manuscript. GA and PP-R performed the statistical analysis and drafted the manuscript. AR, RB-D, ER-G and LF-S collated data or performed various measurements for the study and helped with the analysis and interpretation of the data. JC reviewed critically the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/120/prepub


HIV-1 subtype D infections, which are associated with a faster rate of progression and lymphocyte CD4 decline, cognitive deficit and higher mortality, have rarely been found in native Europeans. In Northwestern Poland, however, infections with this subtype had been identified. This study aimed to analyze the sequence and clinical data for patients with subtype D using molecular phylogeography and identify transmission clusters and ancestry, as well as drug resistance, baseline HIV tropism and antiretroviral treatment efficacy.Phylogenetic analyses of local HIV-1 subtype D sequences were performed, with time to the most recent common ancestor inferred using Bayesian modeling. Sequence and drug resistance data were linked with the clinical and epidemiological information.pol sequences clustered monophyletically with the clades of Ugandan origin and no evidence of transmission from other European countries was found. Time to the most common recent ancestor was 1989.24 (95% HPD: 1968.83–1994.46). Baseline drug resistance to nucleoside reverse transcriptase inhibitors was observed in 54.5% of cases  with evidence of clustering, no baseline integrase or protease resistance and infrequent non-R5 tropism (13.6%). Virologic failure was observed in 60% of cases and was associated with poor adherence (p<0.001) and subsequent development of drug resistance .Subtype D was found in 24 non-immigrant Caucasian, heterosexually infected patients . Partial Local subtype D represented an independently transmitted network with probably single index case, high frequency of primary drug resistance and evidence of transmission clusters. Phylogenetic analyses have been widely implemented in molecular epidemiology of HIV infection and allowed not only to understand genetic sequence variability, but also to trace the flow of the variants and drug resistant clades as well as to infer a rate of molecular evolution to date the ancestry Acquisition of non-B subtypes may be associated with high prevalence of minor drug resistance mutations in untreated individuals and changes in virus tropism In this study the sequences and clinical data from a cluster of locally diagnosed individuals infected with subtype D were analyzed. Firstly, we used molecular phylogeography to trace the import of subtype D into Poland, infer between-host phylogenies, indentify transmission clusters and determine ancestry with molecular clock methods. Secondly, baseline drug susceptibility  and V3 based predicted viral tropism were assessed. Finally, treatment efficacy among patients infected with this variant and secondary drug resistance were summarized.Of the total of 190 patient sequences sampled so far in at the Department of Infectious Diseases and Hepatology, Szczecin, Poland HIV-1 subtype D was found in 24 (12.63%) cases. The first case of the infection with this variant was confirmed in a year 1994 in a young female, who had never travelled outside Poland. All cases were of Caucasian origin with the predominance of females  and a median age at diagnosis of 49.5 years (IQR: 29–56 years). For only one patient presumed infection from a partner travelling overseas (sailor) was recorded, however the source sample was not available. Travel history for this contact is not specific, with frequent worldwide travels and no clear African connection. In the remaining cases no history of travel outside Europe was noted.For all cases heterosexual transmissions were recorded, and patients were HCV antibody negative at HIV diagnosis. In one case HCV infection with antibody production was observed in the third year of observation. Patients tended to be diagnosed at symptomatic stages of the HIV infection  with a low baseline and nadir lymphocyte CD4 counts  and a high baseline HIV-1 viraemia (median of 5.6 log HIV-1 copies/ml [IQR: 4.9–6 log HIV-1 copies/ml]).Pneumocystis jirovecii pneumonia and the fourth related to the cardiac event eight years after HIV diagnosis.Total observation period in the group was 116 person-years with a mean observation time of 57.82 months (95% CI: 35.37–80.27). In fourteen cases (58.3%) AIDS was diagnosed within the period of observation. Mortality rate was 3.44 per 100 person-years. Of the four deaths recorded, three were AIDS-related and occurred within the first year of observation; two cases of large B-cell lymphoma, one For the phylogeographic and dated phylogeny the earliest possible sample from every patient was selected and sequenced. The majority of samples were obtained from treatment-naive patients , the remaining two samples were from patients on ARV.In the maximum likelihood tree all sequences from Poland formed a monophyletic cluster nested within the Ugandan sequences  with pretreatment genotyping available. Primary drug resistance was the most common within the fourth, the youngest (tMRCA in the 2002 [95% HPD: 2005.36–1998.52]) sub-cluster , with nucleoside reverse transcriptase (NRTI) mutations observed. Ten patients within this sub-cluster were female and one male; epidemiological data from patients' records suggested possible sexual contacts between the man and at least three women. Median age in this subgroup was 56 years (IQR: 53–60 years); all patients were diagnosed with clinical features of immunodeficiency (category B and C according to the CDC 1993).In two patients non-nucleoside reverse transcriptase (NNRTI) drug resistant virus was observed in pretreatment samples (as shown in The group of 20 (83.3%) patients were commenced on stable antiretroviral therapy. Of the remaining one patient refused consistently, one was lost to follow-up within a month following the HIV diagnosis, one died before the treatment was introduced and one died within the first month of treatment. The median for the highest recorded lymphocyte CD4 count on cART was 369.5 (IQR: 294.25–596.5) cells/µl, with only five (25%) patients maintaining stable CD4 count >500 cells/µl for at least six months during the follow-up period. In 10 (50%) cases clinician recorded poor adherence, while pharmacy records confirmed <90% adherence in 9 (45%) patients. In 12 (60%) cases the primary therapy was virologically successful , and as Interestingly, the treated patients with the baseline drug resistance tended to present with higher rates of virologic success if compared to the ones with drug susceptible viruses , however this phenomenon was associated with higher adherence.In this study we have evaluated the subtype D infections occurring among Caucasian individuals from Northwestern Poland. Differently to the cases of infection with this subtype observed across Europe Observed infections with subtype D infections formed a network with probably one index case and subsequent local spread. This cluster of heterosexually acquired infections seemed independent from subtype B ones spreading among men who have sex with men (MSM),injection drug users (IDU) and heterosexual populations Additionally, the high frequency of primary drug resistance mutations (DRMs) observed in this study was associated to the observed clustering within the network of transmission and presence of M41L and T215 revertant mutations. Similar results were reported in various countries for different subtypes, with most common transmission of NRTI DRMs Immunologic and virologic antiretroviral therapy efficacy in the observed, stably treated group was poorer than expected for individuals on cART with no specific drug combination clearly superior in the group. Virologic failure was noted in 40% of cases, compared to the 24.7% in the European observational studies It has to be noted, that the limitations of this study can be related to the fact that the certain samples with subtype D might have remained undetected, as it was not possible to sequence all the samples from the patients followed-up in the centre. Neither index case, nor the certain source patient for the cluster 4 were found.To sum up, local spread of HIV-1 subtype D infections described here represented an independent cluster, detected among heterosexually exposed individuals in parallel to the widely distributed subtype B infections. In phylogenetic inference analyses Ugandan origin of the virus was identified with probable single transmission event. Frequency of drug resistance in this group was high, especially within sub-clustered sequences, while the treatment efficacy was poor and independent of the primary drug resistance. Late diagnoses and association with heterosexual mode of transmission might fuel spread of these infections, especially in the light of evidence for the epidemiological clustering which suggests that new transmissions of HIV subtype D have been under diagnosed.For the study participants followed up at the Department of Infectious Diseases and Hepatology, Szczecin, Poland were recruited. The centre provides care for HIV infected patients, mostly from the Northwest part of Poland from the early 90-ties, and so far 960 patients have been observed. Clinical, epidemiological and laboratory data have been collected as a part of a routine patient care. The study has been approved by the local ethical committee of Pomeranian Medical University, Szczecin, Poland. All patients for whom sequence data (n = 190) were available have been analyzed. Treatment adherence was assessed based on the patient records  as well as clinical assessment based on the patient's statement regarding missed doses at home or treatment interruptions. Antiretroviral therapy was commenced according to the clinical standard at the time of introduction, stable therapy was defined as >30 consecutive days of uninterrupted treatment. Virologic success of the therapy was defined as stable serum viral load <400 copies/ml in the first available analysis following 6 months of treatment, and as a consequence no evidence of secondary drug resistance.http://bioafrica.mrc.ac.za/rega-genotype/html/subtypinghiv.html) based on the partial pol sequence obtained by Viroseq methodology. Obtained protease/reverse transcriptase sequences were 1302 b.p. long; location from the start of HXB2 genome: positions 2253–3525. Subsequently, the subtype was confirmed in phylogenetic analysis with a selection of reference sequences listed in the HIV Sequence Compendium 2010 . For this purpose bootstrapped (1000 replicates) neighbor-joining tree with Kimura 2 parameter model was used . Obtained integrase sequences were 866 b.p. long, location from the start of HXB2 genome: positions 4230–5096. V3 loop sequencing was carried out following the protocol provided by the HIV Centre of Excellence , with the following amplification and sequencing primers: GAGCCAATTCCCATACATTATTGT (outer forward), GCCCATAGTGCTTCCTGCTGCTCCCAAGAACC (outer reverse), TGTGCCCCAGCTGGTTTTGCGAT (inner forward), TATAATTCACTTCTCCAATTGTCC (inner reverse), AATGTCAGYACAGTACAATGTACAC , GAAAAATTCCCTTCCACAATTAAA . Obtained integrase sequences were 107 b.p. long, location from the start of HXB2 genome: positions 7110–7217. Briefly, nested PCR was carried out following reverse transcription of extracted HIV-1 RNA as stated in the methodologies cited above. Amplicons were used for sequencing by standard techniques with BigDye technology using an ABI 3500 platform . V3 loop analyses were done in triplicate. Two overlapping sequencing reactions (forward and reverse) were performed for each sample. Reverse transcriptase and protease sequences were assembled with a software provided with a Viroseq 2.8 kit, while V3 and integrase sequences using the Recall online tool (http://pssm.cfenet.ubc.ca) www.geno2pheno.org) with two FPR thresholds: 10% (as suggested by the European Guidelines on HIV Tropism testing) and 5.75%  Plasma samples collected from patients between the years 1996 and 2011 were stored at −80 degrees C and analyzed retrospectively. HIV RNA extraction as well as reverse transcriptase and protease genotyping was performed using Viroseq 2.7 and 2.8 genotyping assays  according to manufacturer's protocol. Initial subtyping was performed using REGA genotyping 2.0 tool . This dataset of 741 sequences was aligned with our partial pol sequences using Clustal ×2.0.10 . Bootstrapped (1000 replicates) maximum likelihood phylogeny using a GTR+γ model with empirical nucleotide frequencies was inferred using PAUP* v. 4.0 (http://paup.csit.fsu.edu/order.html). Separate analyses were performed for the dataset of 24 local sequences with maximum-likelihood methodology (bootstrapped with 1000 replicates) using PHYML v. 3.0 software with subtree pruning and regrafting (SPR) algorithm under GTR substitution model To analyze the origin of our sequences, we have downloaded all HIV-1 group M subtype D Statistical comparisons were performed using the Fisher's exact t tests for nominal variables while continuous variables were analyzed using the U-Mann Whitney test using Statistica 8.0 PL software .HIV-1 subtype D sequences from this study sequences were submitted to the GenBank and may be accessed with the following IDs: GU906860, GU906864, GU906871, GU906872, GU906873, GU906874, JQ305750–JQ305792.


Sub-therapeutic and supra-therapeutic plasma concentrations of antriretrovirals are the significant causes of treatment failure and toxicity respectively among HIV-infected patients. We conducted this study to determine the pattern of efavirenz and nevirapine plasma drug concentrations among adult HIV-infected patients with immunological failure attending at a tertiary hospital in North-western Tanzania.A cross-sectional study was conducted among adult HIV-infected patients with immunological failure who have been on either efavirenz or nevirapine based antiretroviral regimen for more than 6 months. Patients were serially enrolled through routine Care and Treatment Clinic (CTC) activities. Plasma drug concentrations for efavirenz and nevirapine were determined by high performance liquid chromatography (HPLC) and Gas Chromatography (GC) respectively. Demographic, clinical and laboratory data such as viral load and CD4 counts were collected. Data analysis was done using STATA 12.p-value<0.001), NRTI backbone , HIV stage  and viral load  within sub-therapeutic, therapeutic and supra-therapeutic ARV plasma drug concentrations.Of the 152 patients with immunological failure enrolled, the sub-therapeutic, therapeutic and supra-therapeutic plasma antiretroviral drug concentrations were found in 43/152 (28.3%), 76/152 (50.0%) and 33/152 (21.7%) respectively. Half of the patients were outside therapeutic window with either sub-therapeutic or supra-therapeutic plasma ARV drug concentrations. There was a significant difference in distribution of ARV adherence  could assist identifying these patients early and making timely correction to avoid virological failure, poor immunological outcome and prevent associated drug toxicities. Nonetheless, ARV adherence should be strictly emphasized on HIV patients with immunological failure. The primary aim of antiretroviral therapy (ART) is to durably suppress the viral replication to undetectable levels to allow a satisfactory immune recovery Efavirenz and nevirapine are the core and first line non-nucleoside reverse transcriptase inhibitors (NNRTI) commonly used to treat HIV infection This was a hospital based cross-sectional study which was conducted between April 2011 and March 2012 at Bugando Medical Centre (BMC) at Care and Treatment Center (CTC) in Mwanza, Tanzania. BMC is a 1000-bed capacity, tertiary and teaching hospital for the North Western Tanzania. The hospital serves around 13 million people and CTC activities is one of the core part of outpatient activities, which started in 2004, and currently it serves more than 10,000 patients, of whom about 3,600 are active on ARTs. The permission to conduct this study was obtained from the Catholic University of Health and Allied Sciences (CUHAS)/BMC joint ethics review board. The written consent was obtained from all participants.The study population was adult HIV patients on either efavirenz or nevirapine based regimen for more than 6 months with immunological failure attending BMC CTC. All patients were treated with the standard dose of nevirapine 200 mg twicw daily and efavirenz 600 mg once daily. Inclusion criteria were adult HIV positive patients diagnosed as per WHO guidelines, age over 18 years with immunological failure as per WHO guideline and still on first line. Pregnant women and patients co-treated with anti-tuberculosis medications and other concomitant medications known to interact with NNRTI 3, ii) a drop of CD4 cell count below baseline pre-treatment level, or iii) a drop of CD4 cell count of 50% from peak on treatment value all in the absence of an ongoing co-infection and after a minimum of 6 months of ART. For criteria ii and iii, the CD4 cell count must also fall below 200 cells/mm3 to qualify as immunologic failure Immunological failure was diagnosed if the patient met one of the following criteria: i) persistent CD4 below 100 cells/mmPatients were instructed to have medication at night and come the following morning for blood sample collection before taking their next ART dose to determine their antiretroviral plasmatic trough concentrations for nevirapine and the mid concentrations for efavirenz. For each patient, 5 ml of whole blood was collected in plasma EDTA bottles for TDM, approximately 8 to 12 hours after the last dose of ART, just before the next dose was due. The samples were immediately centrifuged at 3000 rpm for 3 minutes to obtain plasma that was transferred into cryovials. The cryovials were stored at −20°C before shipment. The samples were packed and shipped to Germany in cold boxes with cooling packs maintaining a temperature of −30°C. The plasma concentrations of efavirenz (EFV) and nevirapine (NVP) were determined using High Performance Liquid Chromatography (HPLC) [HPLC Beckman Coulter System Gold] and Gas Chromatography (GC)  respectively as described previously Data were managed using Epi Data 3.1  and analysis was done using STATA version 12 . ARV drug concentrations were recorded as continuous variables. Based on the reference ARVs therapeutic ranges, defined as 1000–4000 ng/ml for EFV and 3400–8000 ng/ml for NVP A total of 152 HIV infected adult patients with immunological failure were enrolled in the study. Of these 79/152 (52.0%) were using nevirapine based regimen whereas 73/152 (48.0%) were on efavirenz based regimen. The ART regimens used were Zidovudine+Lamivudine+Nevirapine 46/152 (30.3%), Zidovudine+Lamivudine+Efavirenz 45/152 (29.6%), Stavudine+Lamivudine+Nevirapine 31/152 (20.4%), Tenofovir+Emtricitabine+Efavirenz 28/152 (18.4%) and Tenofovir+Emtricitabine+Nevirapine 2/152 (1.3%). The duration of use of these regimens ranged from 7 to 72 months. The mean age was 40.8±10.0 years with most patients 107/152 (70.4%) being females . Of the Of the 152 patients enrolled, the sub-therapeutic, therapeutic and supra-therapeutic plasma antiretroviral drug concentrations were observed in 43/152 (28.3%), 76/152 (50.0%) and 33/152 (21.7%) respectively. Half of the patients were outside therapeutic window with either sub-therapeutic or supra-therapeutic plasma ARV drug concentrations. Based on the ARV regimens, sub-therapeutic plasma concentrations were more common among patients using nevirapine than those using efavirenz, 35.4% (28/79) versus 20.5% (15/73). Supra-therapeutic plasma antiretroviral drug concentrations were slightly lower among patients using nevirapine than those using efavirenz, 20.3% 16/79 versus 23.3% (17/73). These differences were not statistically significant . Of the p-value<0.001), type of NRTI backobone , viral load  and WHO HIV stage  within the categories of ARV plasma drug concentrations . Sub-therapeutic drug concentrations were significantly more common (as supra-therapeutic was less common) among patients with poor ARV adherence, NRTI backbone comprising Stavudine+Lamivudine (d4T+3TC), advanced HIV stage and those with high viral loads than their counterparts. Generally, there was a significant difference in distribution of ARV adherence rate , increasing viral loads and advancing HIV stage. This finding is similar to that from previous studies done in Uganda and Italy . Since this study has demonstrated that a good adherence among patients with immunological failure is significantly associated with therapeutic ARV plasma level, strict emphasis on ARV adherence on this study population could be very helpful.Therapeutic drug concentrations are a key to successful ART There is a wide inter-individual variability of plasma ARV concentrations among HIV patients with immunological failure in routine clinical practice, with a large proportion of patients being outside therapeutic window. This variability is associated with ARV adherence, NRTI backbone, viral load and HIV stage. Routine therapeutic drug monitoring (TDM) could assist identifying these patients early and making timely correction to avoid immediate virological failure, long term poor immunological outcome and prevent associated drug toxicities. Good adherence is associated with therapeutic ARV plasma concentrations; therefore ARV adherence should be strictly emphasized on HIV patients with immunological failure.


Mycobacteria leprae(leprosy) and HIV coinfection are rare in Kenya. This is likely related to the low prevalence  of leprosy. Because leprosy is no longer a public health challenge there is generally a low index of suspicion amongst clinicians for its diagnosis. Management of a HIV-1-leprosy-coinfected individual in a resource-constrained setting is challenging. Some of these challenges include difficulties in establishing a diagnosis of leprosy; the high pill burden of cotreatment with both antileprosy and antiretroviral drugs (ARVs); medications' side effects; drug interactions; scarcity of drug choices for both diseases. This challenge is more profound when managing a patient who requires second-line antiretroviral therapy (ART). We present an adult male patient coinfected with HIV and leprosy, who failed first-line antiretroviral therapy (ART) and required second-line treatment. Due to limited choices in antileprosy drugs available, the patient received monthly rifampicin and daily lopinavir-/ritonavir-based antileprosy and ART regimens, respectively. Six months into his cotreatment, he seemed to have adequate virological control. This case report highlights the challenges of managing such a patient. It is a well-established fact that in TB/HIV coinfected patients, each disease contributes to the progression of the other. Active TB in HIV-1-infected patients is associated with increased immunodeficiency and mortality . It has Treatment of an HIV-1-leprosy coinfected patient requires a potent combination of ARVs as well as antileprosy agents. The ART must be taken with strict adherence to achieve maximal suppression of viral replication. This in turn limits the risk of developing drug resistance , 10. AchWe report the case of a patient coinfected with HIV-1 and leprosy who developed virological failure to first-line ART. He was subsequently switched to second-line ART. Six months later, antileprosy chemotherapy was initiated.A 66-year-old male was seen in a USAID, Academic Model Providing Access to Healthcare (AMPATH) Partnership clinic in western Kenya, complaining of general body itchiness and skin patches on the chest for 3 months prior to contact. He had tested HIV positive three months prior to enrollment at the clinic. He complained of poor appetite and had lost 3 kg. He did not report any allergies to food or drugs, did not smoke, but reported that he takes alcohol  2-3 times/week.On physical examination, he was in fair general condition with a BP of 100/60 mmHg, pulse rate of 92 beats per minute, temperature 36.1°C, and arterial oxygen saturation of 93% while breathing ambient air. He weighed 65 kgs. His skin had 3 hypopigmented and hypoaesthetic patches on the right chest wall and right upper arm. The largest lesion measured 5 by 6 cm and the smallest 2 by 3 cm. Other systems were essentially normal. Baseline investigations revealed a CD4 cell count of 145/mL, CD4 percentage 10%, white blood cell count of 2800/uL, Hemoglobin 12.3 g/dL, and platelets count of 178,000/uL. SGPT and creatinine were within normal ranges. His chest X-ray was without evidence of pathology.Tinea corporis was made and the patient assigned WHO clinical stage 2 due to weight loss of <10%. He was given cotrimoxazole prophylactic therapy and clotrimazole cream. One month later, the patient was started on an ART regimen composed of stavudine, lamivudine, and nevirapine after adherence counseling. The patient's progress  is summarized in An impression of The patient had poor adherence based upon missed clinic appointments and self report. There is documented history that the patient did not take his medications for a period of one month during the 3rd year of ART. He attributed this to social stressors. An outreach worker had to visit his home to remind him of clinic visits. After several adherence counseling sessions, his plasma viral load dropped from 1,084,798/mL at 29 months of treatment to 433,376/mL six months later. At this point, virological treatment failure was confirmed based on a plasma viral load of >5000 copies/mL. A decision was made to continue with first-line ART until perfect adherence is maintained for at least six months before initiating second-line ART. All this time the skin lesions persisted despite regular use of clotrimazole cream.After demonstrating good adherence, the patient was initiated on second-line ART regimen including abacavir, didanosine, and aluvia (Lopinavir/Ritonavir) 47 months after initiating first-line ART. The skin lesions persisted but did not increase in number or size. A decision to refer him to the regional center for leprosy was made where a skin smear was done and was positive for acid fast bacilli. According to Ridley-Jopling system, he was classified as having borderline tuberculoid leprosy based on having three lesions only that were well demarcated, asymmetrical, and unilateral. However, there was no enlargement of the nerve trunks and no neurological deficits other than the hypoaesthesia. According to the WHO classification, he was classified as having multibacillary leprosy based on having a positive skin smear on at least one site. Seven months after initiating second-line ART, he was put on rifampicin 600 mg monthly, clofazimine 300 mg once a month and 50 mg daily and dapsone 100 mg daily for 12 months which is the WHO recommended multidrug treatment (MDT) regimen for multibacillary leprosy. Six months later, the skin lesions had resolved, there was no neurological sequelea, and a check viral load was 188 copies/mL.Eighteen months since initiating MDT for leprosy, the patient remained stable without new lesions, nor neurological deficits. A follow-up skin biopsy showed mild chronic inflammation with noncaseating granulomas. Fite's Acid fast stain for leprosy was negative. Genotypic viral resistance testing performed revealed resistance mutations to nonnucleotide reverse transcriptase inhibitors (NNRTI's) and nucleoside reverse transcriptase inhibitors (NRTI's) but none to protease inhibitors (PI's).Globally, the new case detection rate of leprosy is generally declining. The registered prevalence rate as of 2011 was highest in the South East Asia  followed by the African region . In Africa, the highest number of new infections in 2010 was reported in the Democratic Republic of Congo followed by Ethiopia  .M.leprae-HIV true coinfection according to Talhari et al. classification [This is a case of fication . This clThe skin lesions of leprosy are classified morphologically according to the Ridley-Jopling classification system into five groups based on the immunity . PatientM. leprae are well preserved at the site of the disease [As noted earlier, HIV-1 does not seem to affect the clinical classification and progression of leprosy. In a study by AS Pereira et al.  the clin disease . In our The use of rifampicin in a patient on protease inhibitor-based ART is problematic because of the potential for drug interactions. Rifampicin is a potent inducer of the cytochrome P450-3A4 subenzyme, which is responsible for the metabolism of protease inhibitors (PI), amongst other drugs , 18. ThiIn our patient, the treatment of leprosy required administration of rifampicin 600 mg once a month. We presumed that monthly rifampicin would have minimal interactions with the lopinavir-/ritonavir-based ART. As such, we did not adjust the dose of lopinavir/ritonavir in the realization that such an adjustment would only serve to increase toxicity. Thus the patient received standard dose lopinavir/ritonavir together with monthly rifampicin. This combination was well tolerated and resulted in good outcome for both diseases. The skin lesions resolved within six months of treatment and the viral load revealed good virological control.M.leprae [ It is recommended that rifabutin should substitute rifampicin in the treatment of TB in TB/HIV-1-coinfected patients on PI-based ART , 25. HowM.leprae –30. HoweOur management of this patient was informed by the fact that there is no clinically validated substitute for rifampicin in the treatment of leprosy in HIV-1-coinfected patients. In addition to that, we did not have an alternative to lopinavir/ritonavir for the treatment of HIV. Though we were unable to monitor the drug concentrations in his blood due to resource limitation, surrogate markers such as the clinical, immunological, and virological parameters suggested a good response. Initiation of ART has been associated with Immune Reconstitution and Inflammatory Syndrome (IRIS) in various situations. IRIS in leprosy may trigger potential adverse effects, such as leprosy acute inflammatory episodes –34. ThisThe follow-up skin biopsy revealed mild chronic dermatitis, but Fite's Acid fast stain was negative, suggesting that the patient is cured of leprosy. The absence of PI resistance mutations suggests that the dosing schedule of rifampicin adopted in this patient did not adversely affect the efficacy of the PI-based regimen. This, however, must be interpreted with caution since it is known that PI's have a high genetic barrier to resistance.Standard dose lopinavir-/ritonavir-based ART coadministered with monthly rifampicin may still achieve virological suppression in HIV-1-leprosy coinfected patients.


The aim of this study was to examine the relationship between trends in CD4 counts (slope) and HIV viral load (VL) after initiation of combination antiretroviral treatment (cART) in Asian patients in The TREAT Asia HIV Observational Database (TAHOD).Treatment-naive HIV-infected patients who started cART with three or more and had three or more CD4 count and HIV VL tests were included. CD4 count slopes were expressed as changes of cells per microliter per year. Predictors of CD4 count slopes from 6 months after initiation were assessed by random-effects linear regression models.A total of 1676 patients  were included. The median time on cART was 4.2 years (IQR 2.5-5.8 years). In the final model, CD4 count slope was associated with age, concurrent HIV VL and CD4 count, disease stage, hepatitis B or C co-infection, and time since cART initiation. CD4 count continues to increase with HIV VL up to 20 000 copies/mL during 6-12 months after cART initiation. However, the HIV VL has to be controlled below 5 000, 4 000 and 500 copies/mL for the CD4 count slope to remain above 20 cells/microliter per year during 12-18, 18-24, and beyond 24 months after cART initiation.After cART initiation, CD4 counts continued to increase even when the concurrent HIV VL was detectable. However, HIV VL needed to be controlled at a lower level to maintain a positive CD4 count slope when cART continues. The effect on long-term outcomes through the possible development of HIV drug resistance remains uncertain. Studies show that latent infection of CD4 cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy .HIV-infected patients in most developing countries have limited second and third line antiretroviral treatment options . In manyThe aims of this study were to examine the relationship between trends in CD4 count and VL after initiation of combination antiretroviral treatment in HIV-infected Asian patients, using data from The TREAT Asia HIV Observational Database (TAHOD).Established in 2003, TAHOD is a collaborative observational cohort study involving 18 sites in the Asia-Pacific region (See acknowledgement). Detailed methods are published elsewhere . BrieflyThe following data were collected: patient demographics and baseline data, CD4 and CD8 count, HIV VL level, prior and new AIDS defining illness (ADI), date and cause of death, prior and current prescribed HAART, and reason for treatment change. Data are collected according to a common protocol. Upon recruitment, all available data prior to entry to TAHOD (considered as retrospective data) are extracted from patient case notes. Prospective data are updated six-monthly at each clinic and transferred to data management centre for aggregation and analyses. TAHOD sites are encouraged to contact patients who were not seen in the clinics in the previous 12 months.TAHOD patients were included in this analysis if they were treatment naïve and initiated with triple or more combination antiretroviral treatment, and had three or more concurrent CD4 and HIV VL test pairs (within 28 days if not tested on the same day) during follow-up. Both retrospective and prospective data were included in the analysis.Trends in CD4 count (slope) was calculated by linear regression with the values at time T, before T, and after T, and was expressed as changes of cells per microliter (μL) per year. The HIV VL was related to the CD4 count slope at time T. Previous studies reported a two-phase CD4 count response, demonstrated as a rapid increase (a high CD4 count slope) in the first several months after treatment initiation and followed by slower increase -14. PrelPredictors of the CD4 slope at time T was assessed by random-effect linear regression models which take account of within and between patient variability. Covariates included sex, age per 10 years), disease stage , hepatitis coinfection (hepatitis B or C antibody positive), haemoglobin level, time since cART initiation, initial cART regimen non-nucleoside/nucleotide reverse transcriptase inhibitor (NNRTI)-based, non-boosted PI, or boosted PI, and treatment containing abacavir. Disease stage, CD4 count and HIV VL were fitted in the model as time-dependent variable. We did not include CD4 count and HIV VL at baseline for the following three reasons: first, a large proportion of patients did not have the tests at treatment initiation , and Stata .There were 4699 patients with data collected in TAHOD as at September 2009. Approximately 75% of patients had a clinic visit in the 12 months before September 2009, and 214 patients died since entry to TAHOD . Among the 4699 TAHOD patients, 612 were not currently receiving antiretroviral treatment, 31 were receiving mono or dual therapy, and 4056 had initiated cART with three or more drugs. 1676 naïve patients initiated cART, and had three or more concurrent CD4 and HIV VL data pairs available beyond 6 months after cART initiation.Table At cART initiation, the median age of the patients included in the analysis was 36 years , median CD4 count 140 cells/μL (IQR 42-230), median HIV VL 5.00 log10 copies/mL (IQR 4.33-5.56), 12% had hepatitis B or C co-infection, and 36% were diagnosed with an AIDS defining illness (ADI). The median time on cART was 4.2 years (IQR 2.5 - 5.8). The median time between each CD4 and HIV VL tests was 165 days (IQR 106 - 223). The initial cART was predominantly an NNRTI-based regimen . Approximately 15% of the patients started a non-boosted PI  regimen with two NRTI drugs and 20% started with a ritonavir-boosted PI . The annual rate of a drug class change or change of at least two or more drugs was approximately 20%. After cART initiation, viral logical suppression (HIV VL < 400 copies/mL) was achieved in 83% of patients at 6 month and 82% in 12 months.Table In Figure The models shows that, after cART initiation, mean CD4 counts continued to increase even when the concurrent HIV VL was detectable. In addition, the model also shows that to maintain a positive CD4 count slope, the HIV VL needed to be suppressed to a lower level in later periods of cART. To illustrate, the estimated CD4 slopes from the model in two patients with specific baseline characteristics are shown in Table Hepatitis co-infection had a significant effect on the CD4 count slope. In one scenario, shown in Table The analyses were repeated in three subgroups as sensitivity analysis Table . The resIn a subset of TAHOD patients who were treatment naïve and initiated with three or more combination antiretroviral treatment and had concurrent CD4 count and HIV VL tests, the CD4 count slope was associated with age, concurrent CD4 count and HIV VL, disease stage, hepatitis coinfection and time since cART initiation. After cART initiation, CD4 counts continued to increase even when the concurrent HIV VL was detectable. However, HIV VL needed to be controlled at a lower level to maintain a positive CD4 count slope when cART continues at later stages, particularly from 6 months to more than 24 months after cART initiation.The inverse relationship between age and CD4 restoration has been reported in previous studies. In these studies younger age was associated with more rapid CD4 recovery and was associated with preserved thymic function ,13-15. TStudies have shown that neither HBV nor HCV coinfection influence virological response to cART -22. HoweThe PLATO study  reportedUsing data from EuroSIDA, Mocroft et al  reportedSimilar to other studies -13, our In many countries in Asia, second-line cART is not widely accessible -6. SeverSeveral limitations should be considered in interpreting the results in this paper. First, TAHOD participating sites are generally urban referral centres, and each site recruits 200-300 patients who are considered by local clinicians to have a reasonably good prospect of long-term follow-up. Hence TAHOD patients, and their treatment, are not representative of all HIV-infected patients in the Asia and Pacific region. Second, we do not have data on adherence and treatment against TB and other ADI. Finally, a more thorough analysis would include the survival outcome. However, because of the limited number and follow-up of patients who were failing virologically, this analysis is currently underpowered. Further analyses will be considered with longer duration of follow-up.The analyses suggest that after cART initiation, mean CD4 slope can continue to increase even when the concurrent HIV VL is detectable. HIV VL needed to be controlled at a lower level to maintain a positive CD4 slope beyond 2 years of cART. However, the effect on longer term outcomes through the possible development of HIV-drug resistance remains uncertain.The authors declare that they have no competing interests.JZ and ML originated the study concept and detailed the analysis plan. JZ performed the data manipulation, statistical analysis, interpretation of results and drafted the manuscript. TS, SK, YMC, NH, PLL, NK, JYC, TPT and EY commented on the study concept and analysis plan, helped interpretation of results and edited the manuscript. SO, AK, PP, CL, PL, VS reviewed the manuscript and provided clinical interpretations. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/361/prepub