To investigate the potential of nevirapine 200 mg once-daily regimen and evaluate the influence of patient characteristics on nevirapine concentrations.This was a prospective, multicentre cohort study with 532 HIV-infected patients receiving nevirapine as a part of their initial antiretroviral therapy. Plasma samples were collected at trough or peak time at the end of week 2 (lead-in period) and week 4, 12, 24, 36, and 48 (steady-state period), and nevirapine concentrations were determined using a validated HPLC method. Potential influencing factors associated with nevirapine concentrations were evaluated using univariate and multivariate logistic regression.v.s. 4.86 µg/mL, for ALT <1.5×ULN and ≥1.5×ULN, respectively, P = 0.045). No significant difference was observed in median nevirapine trough concentration between lead-in and steady-state periods in patients with baseline ALT and AST level ≥1.5×ULN , which was different from the patients with ALT/AST level <1.5ULN. The median trough concentrations were significantly higher in HIV/HCV co-infected patients than those without HCV at week 48 .A total of 2348 nevirapine plasma concentrations were collected, including 1510 trough and 838 peak values. The median nevirapine trough and peak concentration during the lead-in period were 4.26 µg/mL (IQR 3.05–5.61) and 5.07 µg/mL (IQR 3.92–6.44) respectively, which both exceeded the recommended thresholds of nevirapine plasma concentrations. Baseline hepatic function had a moderate effect on median nevirapine trough concentrations at week 2 (4.25 µg/mL The 200 mg once-daily regimen of nevirapine might be comparable to twice-daily in plasma pharmacokinetics in Chinese population. Hepatic function prior to nevirapine treatment and HIV/HCV coinfection were significantly associated with nevirapine concentrations.NCT00872417Clinicaltrial.gov ID:  Nevirapine is a human immunodeficiency virus type 1 (HIV-1) specific non-nucleoside reverse transcriptase inhibitor that binds directly to the viral reverse transcriptase of HIV-1 to block polymerase activity by causing disruption of the enzymes catalytic site Ctrough) and higher peak concentrations (Cmax). Because high nevirapine concentrations are associated with increased adverse events The substantial benefits conferred by cART, however, require strict patient adherence to the prescribed medication because poor adherence will lead to virologic failure. Adherence might be improved with more convenient dosing regimen. A possible measure for simplifying antiretroviral therapy is the use of once-daily dosing regimen. The long plasma elimination half-life of nevirapine (∼25 to 30 h) after multiple dosing may justify once daily dosing throughout the therapy with equivalent efficacy to that of twice daily regimen i.e. 200 mg twice daily, had excessively high plasma concentrations, which may contribute to the higher prevalence of hepatotoxicity in these patients. Another prospective study Ctrough and liver toxicity among Chinese HIV-infected patients, especially in males. To date, Food and Drug Administration approves one-pill, once-daily nevirapine extended-release tablets for use in combination with other antiretroviral agents for treatment of HIV-1 infection in adults. In the context of attempts to simplify treatment regimen while securing efficacy, reducing toxicity and enhancing adherence, there is great interest in nevirapine 200 mg once daily dosing for the treatment of Chinese HIV-infected patients in a long run. The present study aims to investigate the potential of nevirapine 200 mg once daily regimen in Chinese HIV-infected patients by comparing nevirapine plasma concentrations during the lead-in period with the steady-state concentrations. The influence of patient characteristics was evaluated on nevirapine concentrations.Our group has found that the pharmacokinetic profiles of nevirapine in Chinese patients are different with those in Caucasians and blacks Five hundred and thirty-two (n = 532) eligible HIV-infected patients were included and completed this study . DemograCtrough were available during the 48-week period, of which 290 at week 2, 270 at week 4, 247 at week 12, 244 at week 24, 233 at week 36 and 226 at week 48. Ctrough was 4.26 µg/mL (IQR 3.05–5.61) at the end of week 2 when the patients were administered with nevirapine 200 mg once daily, significantly lower than those in the later weeks when patients were receiving twice-daily dosing  (P<0.0001). The steady-state nevirapine Ctrough at week 4, 12, 24, 36 and 48 were 5.90 µg/mL (IQR 4.54–8.14), 6.21 µg/mL (IQR 4.68–8.15), 6.23 µg/mL (IQR 4.56–8.47), 6.15 µg/mL (IQR 4.51–8.07) and 6.37 µg/mL (IQR 4.72–8.64), respectively  shows that the mean nevirapine Ctrough (±SD) at the end of week 2 (4.59±1.81 µg/mL) was significantly lower than the levels at week 4 (6.40±2.59 µg/mL), week 12 (6.59±2.16 µg/mL), week 24 (6.85±2.83 µg/mL), week 36 (6.58±2.35 µg/mL), and week 48 (6.76±2.68 µg/mL) (One hundred and five HIV-infected adults (n = 105) had 6 consecutive 8 µg/mL) .Cmax of nevirapine were obtained, of which 167 at week 2, 159 at week 4, 137 at week 12, 129 at week 24, 126 at week 36 and 120 at week 48. It was demonstrated in Cmax of 5.07 µg/mL (IQR 3.92–6.44) during the lead-in period was significantly lower than the steady-state levels  (P<0.0001). The observed Cmax of nevirapine at week 4, 12, 24, 36 and 48 were 6.89 µg/mL (IQR 5.23–8.84), 6.34 µg/mL (IQR 4.72–8.03), 6.32 µg/mL (IQR 5.40–7.85), 6.48 µg/mL (IQR 5.19–8.35) and 6.91 µg/mL (IQR 5.49–8.56), respectively   . There wCtrough of nevirapine between lead-in and steady-state periods in patients with baseline ALT level ≥1.5×ULN  and baseline AST level ≥1.5×ULN , whereas the median Ctrough during lead-in period was significantly lower than the steady-state levels in patients with ALT/AST <1.5 ULN prior to initial treatment . The median Ctrough of nevirapine at week 48 in patients with HCV coinfection was significant higher than those without HCV coinfection . The difference was not observed in the patients with HBV coinfection  and higher (6.69 versus 5.74 µg/mL) compared with the 200 mg twice daily. The 2NN sub-study confirmed this findings and showed that Cmin (3.26 versus 4.44 µg/mL) was lower and Cmax (7.88 versus 6.55 µg/mL) was higher in the 400 mg once-daily dosing Cmax might result in a higher incidence of toxicity in patients with nevirapine once-daily dosing than those assigned twice-daily administration The efficacy and safety of nevirapine 400 mg once daily in treatment of HIV-infected patients had been assessed in several studies Gonzalez de Requena et al.Wang J et al.Ctrough and liver toxicity among Chinese HIV-infected patients, especially in males (P = 0.015).The liver toxicity is one of the most common hypersensitivity reactions to nevirapine and may be associated with its plasma concentrations. 1/2 (30.94 h), AUC0–12 h (92.82 µg·h/mL), Cl/F (0.71 L/h), Cmax (10.09 µg/mL) and Ctrough (7.88 µg/mL), respectively Ctrough in Chinese males and females (8.95 µg/mL and 6.59 µg/mL) were significantly higher than the levels in Caucasians and blacks The steady-state pharmacokinetic study in 15 Chinese patients with HIV infection Ctrough of nevirapine 200 mg once daily at the end of week 2 was significantly lower than the twice-daily dosing in later weeks . Similarly, the median Cmax of nevirapine during the lead-in period was also lower than the steady-state levels . The decreased nevirapine concentrations with 200 mg once daily dosing may suggest a lower incidence of liver toxicity than twice daily. To test this assumption, an small trial in seven treatment-naïve Chinese HIV-infected patients was carried out by our team. The seven HIV+ adults patients were administered with nevirapine 200 mg once daily as a part of initiating antiretroviral therapy. Till now, they maintained a high level of efficacy and comparable tolerability during 2-month follow up compared with the twice-daily dosing regimen.The present study showed that the median Vries-Sluijs et al.Ctrough of 3.0 µg/mL Ctrough at 3.9 µg/mL for Chinese patients with HIV infection, higher than the commonly recommended 3.0 µg/mL. The current study demonstrated that median Ctrough  and Cmax  of nevirapine in Chinese HIV-infected patients receiving 200 mg once daily were above the recommended thresholds of nevirapine concentrations, suggesting that nevirapine 200 mg once daily regimen may produce adequate viral inhibition in Chinese HIV-infected patients. The ongoing pilot trial in Chinese patients confirmed this assumption. Certainly, we cannot rule out the possibility that some patients will have nevirapine levels lower than the target thresholds due to inter-individual variability and unpredictable features, which may result in virologic treatment failure and even drug resistance. So routine therapeutic drug monitoring should be carried out.On the other hand, there are some concerns about virologic failure which was related to low nevirapine plasma levels in clinical practice. The relationship between nevirapine concentration and virologic response has been explored in previous studies. The INCAS trial v.s. 4.25 µg/mL, for ALT level≥v.s.<1.5×ULN, P = 0.045) and might exert an influence on the metabolism and clearance of nevirapine at steady state, because no significant difference was observed in median Ctrough of nevirapine between lead-in and steady-state periods in patients with baseline ALT and AST level ≥1.5×ULN , which was different from the patients with ALT/AST level <1.5×ULN. Gender, weight, HBV or HCV coinfection, baseline CD4 cell counts and viral load appeared to have no significant influence on nevirapine plasma concentrations. Consistently, the univariate and multivariate logistic regression models showed that none of the examined factors was found to predict nevirapine trough concentration at week 2 lower than 3.0 µg/mL or 3.9 µg/mL.Previous studies have found ethnicity, gender, weight and underlying hepatic disease to be predictive of nevirapine plasma concentrations v.s. 6.15 µg/mL, P = 0.004). We assumed that a high incidence of liver toxicity in Chinese patients and particularly a great risk of severe hepatotoxicity in HIV/HCV coinfected patients were both significantly associated with plasma nevirapine exposure. This finding was consistent with our previous study The nevirapine trough concentration increased gradually in HIV/HCV coinfected patients during the follow-up periods and finally was significantly higher than those patients without HCV coinfection at week 48  and serum creatinine level >1.5×ULN. Another important exclusion criterion was non-adherence to the study treatment regimen which was defined as less than 95% adherence.Patients were recruited from January 2009 to December 2010. Male and female antiretroviral-naïve patients with documented HIV-1 infection were eligible for inclusion if they were between the age of 18 and 65 years with CD4+T cell count <350 cells/mmThe cohort study was approved by institutional review boards and carried out in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. Written informed consent was obtained from each patient and the study protocol was approved by the ethics committee of Peking Union Medical College Hospital. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see All the patients received a standard ART regimen based on nevirapine together with two nucleoside reverse transcriptase inhibitors including stavudine plus lamivudine or zidovudine plus lamivudine. Nevirapine  was administered at 200 mg once daily for 2 weeks and 200 mg twice daily thereafter. The bioequivalence of nevirapine in reference to Viramune® from Boehringer Ingelheim was demonstrated in the previous study During the treatment period, patients were monitored at baseline, week 2, 4, 12, 24, 36, and 48 for clinical features (particularly severe adverse events), plasma nevirapine concentrations and laboratory values including blood routine examination, hepatic and renal function, and hepatitis B (HBV) or hepatitis C (HCV) serological state.Ctrough and/or 2 h post-ingestion for Cmax. All samples were collected in spray dry powdered EDTA tubes and centrifuged in the real time to obtain plasma, which were stored at −80°C and thawed at the day of analysis. The exact time of nevirapine dose and blood sampling was recorded.Blood samples were drawn prior to the next drug administration for The nevirapine concentration in plasma was determined by a validated HPLC assay modified from a previous study Plasma HIV-1 RNA viral load was measured by either bDNA Analyzer System 340  or the COBAS Ampliprep/TaqMan 48  according to the manufacturer's instructions. The measurement ranges were 50–500,000 copies/mL and 40–1,000,000 copies/mL, respectively.PBMCs were stained with combinations of immunofluorescent monoclonal antibodies FITC-CD3 and PEcy5-CD4  followed by flow cytometer analysis  to determine the number of CD4+T cells (CD4+CD3+).Statistical analyses were performed with Statistical Product and Service Solutions for Windows . Mean , median  and frequencies (%) were used to describe characteristics of patients as appropriate. Normal distribution of values was examined by Kolmogorov-Smirnov methods. Categorical variables were tested with Chi-Square or Fisher's exact test, and continuous variables were tested with Kruskal-Wallis or student's t test or one-way ANOVA.trough at the 6 follow-up visits in the 105 patients. An independent t-test or Mann-Whitney U test was used when two groups or two variables were compared.The differences between lead-in and steady-state periods for nevirapine plasma concentrations were assessed by the Kruskal-Wallis H test. ANOVA was used to determine whether there were significant differences in nevirapine CFactors affecting nevirapine plasma exposure were estimated using univariate stratification analysis. Risk factors predicting nevirapine trough concentration at week 2 lower than the recommended cut-off thresholds were evaluated using univariate and multivariate logistic regression. Odd ratios (OR) and 95% confidence intervals (95% CI) were also obtained.For all tests, P<0.05 was considered to be statistically significant.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


In attempt to clarify this aspect, we analyzed clinical samples of HIV-1 infected patients failing PR Inhibitor (PI) and RT Inhibitor (RTI) treatment among a cohort of five infectious diseases units located in Veneto in northeastern Italy.The Human Immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) are key enzymes in viral replication and major target of Antiretroviral (ARV) therapy. The mechanisms of resistance mainly involve mutations altering the interaction of viral enzymes and inhibitors. Recent studies reveal that, besides the enzymes-encoding ones, other regions might contribute to the development of resistance. In particular, some specific cleavage and non-cleavage site mutations in Gag increase the replication ability of mutant viruses. The effect of amino acid substitutions in different domains of Gag selected gag and pol gene PCR amplification and sequencing. Selected patient PCR-derived gag products were adopted to reconstitute recombinant HIV-1 viruses in an otherwise wildtype background (HIV-1 LAI). Gag processing experiments, single-cycle infectivity assay and replication kinetics were performed.Plasma and PBMCs samples from ARV treatment patients were used for gag and pol sequences derived from clinical samples of HIV-1 infected patients failing PR Inhibitors (PIs) and RT Inhibitors (RTIs). Besides polymorphisms and multiple amino acid substitutions associated with inhibitor resistance, genotypic analyses have identified insertions within the Gag at the level of the matrix domain and the p6 domains. The effect of these mutations have been examined in terms of: (i) Gag and Gag-Pol processing and particle release; (ii) virus infectivity as well as iii) viral replication capacity.We analyzed the Our results contribute to better characterizing the role of Gag and the relationship to PR and RT in resistance development, their relevance in viral replication and evolution in the presence or in the absence of drugs.


Objective. To determine the association between Yang-Deficient Constitution and the clinical outcomes of HIV/AIDS patients who have initiated highly active antiretroviral therapy (HAART). Method. A total of 197 antiretroviral-naive adults who initiated HAART between 2009 and 2011 were recruited. The participants were asked to complete a questionnaire twice to assess their Yang-Deficient Constitution status before HAART. During the study, signs and symptoms and CD4 or CD8 T cell counts were recorded. Routine blood and biochemical tests were conducted. For the patients who were found to have infections, pathologic examination was performed. Statistical test of association of clinical attributes and demographic factors with Yang-Deficient Constitution was conducted. Result. Good test-retest reliability was observed for Yang-Deficient Constitution scoring. The median Yang-Deficient Constitution score of 142 eligible participants was 25. Female , hepatotoxicity , nephrotoxicity , total number of adverse events (P < 0.1), and mortality  were associated with Yang-Deficient Consitution, while annual changes or nadir values of CD4 or CD8 T lymphocytes, and newly acquired infections after starting HAART were not. Mortality was also associated with total number of adverse events (P < 0.05), hepatotoxicity (P < 0.05), and nephrotoxicity (P < 0.05). Conclusion. Yang-Deficient Constitution score has a potential to be developed as a predictor for early HIV-related mortality and side effects. The interrelation and underlying mechanisms should be further investigated for evidence-based design of a more appropriate treatment strategy. Many of the newly admitted cases of HIV infections are indeed in the end stage of AIDS. Poor immunological response and adverse events are also common during initial highly active antiretroviral therapy (HAART). These partially account for early death in the first year of HAART [After China's National Free Antiretroviral Treatment Program has been initiated since 2002, the rate of receiving antiretroviral therapy cases among the eligible HIV infection reached 84% nowadays. Although the mortality rate has decreased to 50% n = 7740 for Individual differences are known to have important impact on the progression of diseases. In particular, differences in the composition of genome account for many of the variations observed and more evidence is emerging that much of the wisdom of traditional medicine can in fact be explained at genomics level  was shown to prevent cold-stress-induced hypothermia and immunosuppression in mice . HigenamTreatment-naive HIV-1 infected patients aged 18 years or older at the Guangzhou 8th People's Hospital between January 1, 2010, and May 1, 2012, who joined the National Free Antiretroviral Treatment Program with a combination of at least three drugs, including NRTIs, PIs, and NNRTIs, were enrolled. Subjects were excluded if they were pregnant or unable to understand or finish the questionnaire due to linguistic or mental problems. All subjects received a full explanation of the study and provided written informed consent. The study was approved by ethics committees of Guangzhou 8th People's Hospital. The recruited patients were asked to complete the constitution questionnaire before the initiation of HAART. They were then scheduled follow-up visits once every 3 months. This study period was one year and the dropouts were also excluded.http://www.cdc.gov/mmwr/preview/mmwrhtml/00018179.htm). People without an AIDS defining disease but with a CD4 cell count below 200 cells/L were classified as having AIDS.We adopted the definitions published by Centers for Disease Control and Prevention revision of the AIDS in 1993 case definition , CD4 and CD8 count at baseline and 3, 6, 9, and 12 months after HAART, infections acquired before and after HAART , immune reconstitution inflammatory syndrome, the number of days after HAART initiation for an infection to occur, adverse events including fatigue, allergy, alopecia, gastrointestinal reaction, mouth epithelium ulcer and pharyngitis, cerebral symptom, arthralgia, diabetes, hematologic toxicity, hepatotoxicity, abnormal lipid metabolism, pancreatitis, nephrotoxicity, sensory nerve dysfunction, and hyperlactacidemia, and total number of adverse events. The routine blood and biochemical tests conducted and their normal range are shown in Reliability of duplicate tests was evaluated. To assure quality, patients were requested to complete the same questionnaire twice in two visits (separated by one week) and the absolute differences of the scores between the two visits were calculated. Local outlier factor (LOF), an algorithm for identifying density-based local outliers , was useWe recruited 197 HIV/AIDS patients but only 158 of whom completed the questionnaire twice. There was no difference between test-retest scores for two-third of the cases and about 90% with 1 or fewer  absolute score difference . Nine ofThe other 149 patients were assigned scores by taking average of two visits, along with 39 patients that only completed once. As a result, there were 188 HIV/AIDS subjects at the beginning. Some of them dropped out during followups and finally there were 142 HIV positive individuals for in-depth analysis, with 57 (40%), 16 (11%), and 69 (49%) of whom were classified as types A, B, and C, respectively, according to CDC 1993 Revised Classification System (1993 Revised Classification System for HIV Infection and Expanded Surveillance Case Definition for AIDS Among Adolescents and Adults). The details of demographical information are summarized in P > 0.1) with Yang deficiency were also excluded.HAART drugs 3TC, KALETRA, TDF, encephalitis and meningitis, previous diagnosis of sexually transmitted disease, newly acquired hepatitis B, hepatitis C, pulmonary and extrapulmonary TB, pneumonia, superficial and visceral fungal infections, alopecia, mouth epithelium ulcer and pharyngitis, arthralgia, diabetes, and pancreatitis were excluded for further analyses due to their low variance. Other attributes that were not significantly associated (P = 0.54) nor CD8  reconstitution pattern  with Yang deficiency. Yang deficiency was associated with both HIV disease staging and mortality.Compared to males females showed significantly more serious Yang deficiency , but ageThere were 5 deaths in this study, due to pneumonia, infectious diarrhea, extrapulmonary tuberculosis, meningitis, encephalitis, and multiple organ dysfunction syndrome. The mortality rate was 3.5% (95% CI: 1.4%–8.6%). Indeed, the total number of newly acquired infections after starting HAART, previous pneumonia, intestinal infections, and IRIS and total number of the adverse events, particularly syndromes of hepatotoxicity and nephrotoxicity, were associated with mortality .Since the introduction of HAART in 2002 AIDS mortality (with and without receiving HAART) has begun to decrease from 39.2 to 14.2 per 100 person-years in 2009 in China . In thisIn resource-limited settings, prioritization allows better allocation of resources for diagnostic tests and appropriate and timely health services. AIDS patients with low CD4+ cell counts incur higher expenditures in the first year of antiretroviral treatment, due to high incidence of adverse drug events and opportunistic infections. Annual costs of AIDS patients with opportunistic infections were 10 times higher than those of HIV infection alone , hardly Constitution is largely inborn and Yang deficiency is one of the manifestations. Independent of age, female patients had higher Yang-Deficient Constitution score, which alludes to possible genetic effects. Our result of the association of female with Yang-Deficient Consitution was consistent with the Yin-Yang theory in TCM, which says male is characterized by Yang while female Yin. With regard to age, according to Yellow Emperor's Canon of Traditional Chinese Medicine, Yang-qi changes along with age, but not in a monotonic manner. Most of the people recruited in our study were between 20 and 55 years old, change of Yang-qi during this age group may follow a bell-shaped pattern, thus Yang-Deficient Consitution was proved to have no linear correlation with age in this study. Whether the correlation between Yang-Deficient Consitution and gender was due to sex hormonal level needs further elucidation , 28. MalHepatotoxicity and nephrotoxicity might be the indirect causes of death of the AIDS patients, which are generally caused by overlapping anti-infection treatment during opportunistic infections. Liver injury can be resulted from antiretroviral treatment , includiTo the best of our knowledge, this is the first report on the correlation between Yang-Deficient Constitution and hepatotoxicity and nephrotoxicity. Mitochondrial toxicity, commonly observed in HAART, might be responsible for hepatotoxicity and nephrotoxicity . Mitocho


The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence.http://clinicaltrials.gov/, NCT01038999). In more than 88% of treated patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. ROS (reactive oxygen species) production and ΔΨm  were measured by flow cytometry in blood lymphocytes and monocytes . Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins  were analysed by western blotting.49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres  or morphological  parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria.In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes), or from first line ART (monocytes). Together with other tissue impairments, these changes may contribute to global aging.NCT01038999ClinicalTrials.gov NCT01038999  Since 1996, antiretroviral therapy (ART) has increased life expectancy in HIV-infected patients who exhibit aging-related diseases This paper is focused on mitochondria, the main energy-producing factories in the cell, which are known to be involved in ROS production The production of ATP by the respiratory chain involves multiheteromeric enzymatic complexes located in the inner mitochondrial membrane (IMM). Protons are pumped from the mitochondrial matrix to the intermembrane space to establish an electrochemical gradient that results in the IMM potential (ΔΨm) required for ATP synthesis. More than 90% of the oxygen in tissues is consumed by mitochondria, and between 1 and 2–5% of the oxygen is transformed into reactive oxygen species (ROS) as respiratory chain by-products Mitochondrial network dynamics, cell apoptosis and autophagy exhibit close reciprocal relationships with innate antiviral signaling and mitochondrial morphological or functional  parameters. These events are coordinated by common mitochondrial or cytosolic partner proteins that are regulated by post-translational modifications Mitochondria form a dynamic reticulum that is continuously remodeled by balanced fission and fusion (or “kiss and run”) events controlled by two sets of outer (OMM) and inner (IMM) mitochondrial membrane specific proteins Molecular partners that link ROS overproduction, ΔΨm decrease, mitochondrial fission and mitophagy through the sequential recruitment and interaction of cytosolic proteins, OMM GTPases, IMM GTPases and oxidative phosphorylation (OXPHOS) complexes, have been implicated in the pathogenesis of Parkinson’s disease ROS HIV-encoded proteins or miRNAs trigger mitochondrial-mediated apoptosis, which may explain the progressive decline in CD4+ T cells in infected patients ART mainly targets two steps of the HIV lifecycle. Nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) block reverse transcription, whereas ritonavir-boosted protease inhibitors (PI/r) prevent the cleavage of HIV-encoded gag-pol proteins. A combination of molecules from these two groups currently represents the most common HIV infection treatment. However, ART with primarily first generation NRTIs can cause mitochondrial toxicity through mitochondrial DNA polymerase γ inhibition We used PBMCs as an easily available cell type targeted by both HIV-1 and ART. As a direct or indirect response of HIV-1 infection, flow cytometry demonstrated that ROS production and mitochondrial ΔΨm were altered in lymphocytes but not in monocytes from ART naive patients. ART partially rescued these lymphocyte parameters but induced slight mitochondrial changes in monocytes. Mitochondrial network morphology parameters  observed by confocal microscopy in these two cell types confirmed the functional mitochondrial results. Network fragmentation was stable regardless of subject status, and an increase in network volume and branching argued against mitophagy or mitochondria-linked apoptosis related to ROS and ΔΨm changes. No modifications in the amounts of mitochondrial proteins were observed in PBMCs. Even in patients considered to be clinically stable, the changes in PBMCs reported here, together with alteration of mitochondrial functional and morphological parameters in other tissues, may contribute to aging in HIV-1 infected patients.The ANRS EP45 “Aging” study is a cross-sectional and longitudinal (3 years) multicentre study. Patients were recruited by three specialised AIDS centres in France at Marseilles (the main coordination centre), Nice and Montpellier, and control subjects were recruited by CIC-UPCET  in Marseilles.Forty-nine HIV-1 patients infected for at least 24 months but who had not received ART (ART naive), and eighty-one HIV-1 infected patients in the first line of ART for at least 12 months, were enrolled. According to their ART combination, patients were distributed into three subgroups: patients treated with 2NRTIs and PI/r; patients treated with 2NRTIs and 1NNRTI; and patients treated with 3NRTIs. Forty-nine seronegative control subjects were age- and sex-matched with the ART naive patients.et al., in preparation). All experiments described below were performed blinded with respect to subject status . We report here the mitochondrial data measured at baseline.The basic demographic, clinical and biological parameters of the study participants, together with their ART combinations, are detailed in The protocol was approved by the French Health Products Safety Agency Regulatory Authority  and Marseille’s Ethical Committee (Comité de Protection des Personnes Sud Méditerranée I). The study was registered on the ClinicalTrials.gov web site  and performed in accordance with the Declaration of Helsinki. All subjects provided written informed consent before participation.The processing of blood samples is described in detail in the Methods S1. Briefly, PBMCs were isolated by Ficoll® gradient centrifugation  according to the manufacturer’s instructions. Leucocyte formulae were evaluated by May-Grünwald-Giemsa staining of cytospin samples. Cell viability was >97%  for 20 minutes was used as a positive control for mitochondrial membrane depolarization. Cells incubated without JC-1 probe were used as auto-fluorescence negative control cells. Monocytes were labelled using CD14-PC5 antibody (Beckman Coulter Inc) according to the manufacturer’s instructions. Residual red blood cells, if any, were hypotonically lysed using a solution of 100 µM EDTA, 150 mM NH4Cl, 1 mM KHCO3, pH 7.4. HIV-1 was inactivated by fixation for 30 minutes in ice-cooled 0.5% paraformaldehyde solution. As previously described ΔΨm was determined using the red/green fluorescence intensity ratio of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide probe . PBMCs were stained with 0.5 µM probe for 15 minutes at 37°C, 5% CO2DCFDA, Life Technologies). PBMCs were incubated with 5 µM probe for 30 minutes at 37°C, 5% CO2. Co-incubation of CM-H2DCFDA with 50 ng/ml of phorbol 12-myristate 13-acetate  was used as a positive control. Cells incubated without CM-H2DCFDA probe were used as auto-fluorescence negative control cells. Staining of CD14-monocytes, lysis of residual red blood cells (if any), fixation and flow cytometry analysis were performed as described above.ROS production was detected using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate acetyl ester probe (CM-H2. After fixation for 15 minutes at room temperature (RT) with 4% paraformaldehyde solution, cytospin samples of PBMCs were prepared at 23 g for 5 minutes and nuclei were labelled with DAPI  for 10 minutes at RT. Slides were mounted using FluorSave™ reagent (Merck). A single confocal slide  was recorded at 100×magnification from 30 lymphocytes and 30 monocytes identified according to their nuclear size and shape after DAPI staining. Three individuals from each of the 5 groups  were selected for analysis. The individuals were chosen on the basis of possessing maximal (44 to 71), minimal (15 to 22) and medium (31 to 37) ROS values in high-ROS producing lymphocytes from flow cytometry data. ΔΨm was confirmed as being inversely correlated with ROS values in the total lymphocyte population (http://rsbweb.nih.gov/ij/index.html). Three mitochondrial network stereological parameters  were calculated for lymphocytes and monocytes using a specific macro designed by Léon Espinosa (available on request). The mitochondrial network was stained using Mitotracker® Red CMXRos probe (Life Technologies). PBMCs were stained with 25 nM probe for 15 minutes at 37°C, 5% COpulation . Image aTotal PBMC proteins from 160 cohort participants were extracted and analysed by a standard western blotting procedure, as detailed in Methods S1.Statistical analysis was conducted using SAS software, version 9.2 (SAS Institute Inc). ΔΨm, ROS, mitochondrial network and quantitative protein parameters were compared between groups by analysis of variance or Kruskall-Wallis test. Pairwise comparisons were conducted using the Bonferroni method for multiple comparisons. Relationships between parameters were assessed by Pearson’s correlation coefficient. XLSTAT® software (AddinSoft) was used for box plot drawings and discriminant analysis, whereas comparison between groups used Wilks’ Lambda (Rao’s approximation) or Roy’s greatest root tests 3; viral load <40 copies/ml), out of the ANRS EP45 cohort. Regardless of HIV-1 status, four lymphocytes subtypes, CD3+, CD3+ CD4+, CD3+ CD8+ and CD3− CD19+, exhibited similar patterns of ROS production within the total lymphocyte population. This indicated that these cells were subdivided into both high- and low-ROS cells, whereas CD3− CD19− CD16+ CD56+ NK cells were always low-ROS producing cells , or in 72–77% of patients according to HIV-1 and ART status (lymphocytes) is shown in representative zebra plots and histograms of CD14+ monocytes  or lymphng cells . HIV-1 iMeasurements of ΔΨm by flow cytometry after incubation with the ΔΨm inhibitor, CCCP, clearly identified two subpopulations of lymphocytes . These tBecause the emission spectra of the probes overlapped, simultaneous measurement of ROS and ΔΨm was not performed in lymphocytes or monocytes. Therefore, the relationships, if any, between high- or low-ROS and ΔΨm remain unknown. A negative correlation (p<0.001) was highlighted between ΔΨm and ROS production within the total lymphocyte population , but notBecause changes in low-ROS cells between infected and uninfected subjects were not observed, we focused our investigations on high-ROS cells.Lymphocyte mitochondria ROS- and ΔΨm-related parameters in ART naive patients were statistically different to those in control subjects  and those from each ART group . However, no statistical difference was observed by comparison of ART naive patients with each ART group, or by comparison of the three ART groups, as shown by the close superposition of circles representing each patient group .In the ART naive patients, changes were mainly observed in volume density  and branIn contrast, the same parameters either in isolation , or in cThe amounts of porin , CoxIV-2  and CoxIV-4 (encoded by the nuclear genome) purified from PBMCs were analysed relative to GAPDH in all cohort participants . No statA 29 kDa band corresponding to CoxIV-4 was detected in 21% of the ART naive patients, 30% of the 2NRTI+1NNRTI patients, and 36% of the 3NRTI patients. However, this band was detected in only 1 of 38 PBMC homogenates from control subjects, and in 1 of 28 2NRTI+1PI/r patients . Co-labeThe goal of the ANRS EP45 “Aging” study is to analyse, in a large cohort of HIV-1 infected and uninfected subjects, the impact of HIV-1 infection and of antiretroviral regimen on mitochondrial parameters.PBMC mitochondrial parameters from ART naive patients compared to control subjects were used to delineate the consequences of HIV-infection, including the effects of viral proteins Western blot analysis showed a minor 29 kDa CoxIV-4 band in 21% of ART naive patients, which may have represented mono-ubiquitined CoxIV-4. Confirmation by co-immunoprecipitation experiments could not be performed because of the large amount of protein required. Mitochondrial protein mono- or poly-ubiquitinylation No variations in the amounts of the mitochondrial proteins, porin, CoxIV-2 and CoxIV-4, relative to GAPDH, were observed in PBMCs from ART naive patients compared to control subjects. Because protein assays were performed on total PBMC lysates, no distinction could be made between lymphocytes or monocytes, or between cell subgroups defined by their ROS production and ΔΨm. Thus, the variations between the different cell types may be masked. Nonetheless, proteome analysis in a cohort with similar clinical characteristics to ours showed that, compared to healthy donors, PBMCs from ART naive patients exhibited decreased levels of some Complex I (3), IV (5a) and ATP synthase  subunits but not of CoxIV-2 or CoxIV-4 Mitochondrial functional investigations were performed on lymphocytes and moncytes separately. Lymphocyte FSC/SSC gate included various lymphocyte subpopulations such as T lymphocytes , B lymphocytes (CD3− CD19+ ≈ 4%) and NK cells (CD3− CD19−CD16+ CD56+ ≈ 3%) as shown by a ancillary study we performed. Our lymphocyte FSC/SSC gate may also contain dendritic cells present in blood (0.3 - 0.5% of PBMC) Flow cytometry identified two lymphocyte and CD14+ monocyte subgroups characterized by either high- or low-ROS production. While high- and low-ROS producing monocytes have been described previously IMM permeability, which is measured by ΔΨm, and ROS production are two related parameters Discriminant analysis combining ROS and ΔΨm measurements better emphasized the mitochondrial differential effects of HIV-1 infection on the two peripheral blood cell types than comparison of single parameters. While lymphocyte mitochondria were statistically different in ART naive patients compared to control subjects, monocyte mitochondria showed no differences. HIV infection has previously been shown to induce general oxidant stress associated with a decrease in both lymphocyte and blood serum oxidant defence systems Significant correlations between functional parameters and viral load further underlined the effect of HIV-1 infection on lymphocyte mitochondria. Numerous events may result in mitochondrial impairment in both infected cells and uninfected bystander cells through various signaling events. Indeed, ROS production by NADPH oxidase has been observed in uninfected bystander CD4+ lymphocytes after binding of the HIV-1 protein gp41 to a plasma membrane receptor Decreased ATP supply due to increased ROS production and lowered ΔΨm have been reported to result in mitochondrial network fragmentation, mitophagy, or cell apoptosis In summary, HIV-1 infection in ART naive patients with a controlled CD4+ cell count and viral load resulted in significant alterations in four of the lymphocyte mitochondrial parameters , without irreversible damage that may lead to mitophagy and/or apoptosis.in vitro with HIV-1 and cultured for 7 days In contrast to lymphocytes and previously published ROS data Our study of ART naive patients who were considered stable according to their clinical parameters, highlighted that HIV-1 infection did not affect monocyte mitochondria as much as it directly or indirectly impaired lymphocyte mitochondria, leading to minor oxidant stress known to favour viral replication, immune dysfunction and disease progression in vitro studies Three different ART combinations did not modify the amounts of porin, CoxIV-2 and CoxIV-4, or the CoxIV-4/CoxIV-2 ratio, as previously reported Discriminant analyses integrating functional and morphological mitochondrial parameters revealed another discrepancy between lymphocytes and monocytes in response to ART. While ART regimens partially improved lymphocyte mitochondrial parameters, none of the ART combinations fully restored mitochondrial changes induced by HIV-1 infection. In monocytes, the mitochondrial parameters remained unchanged in ART naive patients whereas the three ART regimens resulted in slight  or significant  modifications. Previous studies have shown either ART-induced ΔΨm recovery None of the three first line ART regimens appeared to be more toxic towards PBMC mitochondria than the others. Moreover, the majority of 2NRTI+1PI/r and 2NRTI+1NNRTI patients shared the same NRTI backbone. Because no statistical difference was found between these two populations using discriminant analysis, our data suggested that boosted Lopinavir (68% of PI) and Efavirenz (72% of NNRTI) did not elicit major mitochondrial effects. The ART regimens in our patient cohort mainly included drugs of the second generation, whose mitochondrial toxicity is reduced in comparison with first generation drugs In contrast to lymphocytes, HIV-1 infects very few blood monocytes (less than 1%) as shown by the lower proviral DNA copy number in monocytes than in CD4+ T cells. Moreover, blood monocytes supported only a very low viral replication rate, as shown by the detection of 2 LTR circles, HIV mRNAs, or changes with time in viral nucleotide sequences. Monocyte migration out of the blood into tissues, followed by differentiation into macrophages or dendritic cells, increased the inhibition of viral replication  on mitochondria could be balanced by the positive effects of ART, i.e. decreased viral load and replication Taken together, these data suggest that mitochondria from blood lymphocytes and monocytes exhibit differential responses to either HIV-1 infection or ART. Whereas mitochondrial ROS and ΔΨm functional parameters exhibited changes, mitochondrial morphological modifications argued against apoptosis Figure S1Cell viability analysis. Representative density plots of peripheral blood lymphocytes (A) and CD14+ monocytes (B) from an HIV-1 infected patient. Blood collected in EDTA Vacutainer tubes was rotated overnight prior to PBMC isolation. Cell viability was determined after PBMC incubation for 3 h at 37°C, 5% CO2 in RPMI medium supplemented with 10% fœtal bovine serum and 2 mM L-glutamine. Dead cells were double stained with 7AAD and annexin V, and early apoptotic cells were identified by annexin V staining only. Viability remained over 97%. Apoptotic cell numbers were negligible. Control cells incubated overnight with 1 µM staurosporin were used as a positive control for apoptosis .(TIF)Click here for additional data file.Figure S2Correlation between ROS production and ΔΨm in lymphocyte mitochondria. Negative correlation between basal ROS production and ΔΨm . Total lymphocyte population from all cohort participants (n = 157).(TIF)Click here for additional data file.Figure S3Morphological analysis of the monocyte mitochondrial network. The mitochondrial network was imaged using confocal microscopy . One single slice was recorded for each monocyte identified using DAPI nuclear stain. 30 pictures were concatenated and analysed using a specifically designed Image J macro (available on request). The cell surface area and the mitochondrial network area were measured after automatic thresholding and binarization. Mitochondrial network images were then skeletonised, and skeleton connectivity was determined as follows: isolated pixels and pixels with only one neighbour were numbered (network ends); pixels with two neighbours were used for network length and fragmentation measurement; and pixels with three or more neighbours were used for the estimation of network branching. Data were stored in Excel files. The volume density of the mitochondrial network was calculated as the ratio of mitochondrial network surface area to monocyte surface area and expressed as a percentage. Fragmentation was estimated as the ratio of the number of skeleton fragments of the mitochondrial network to the total length of the skeleton. Branching was determined as the ratio of the number of pixels having three or more neighbours to the total skeleton length. The flow cytometric FSC parameter showed that the mean volume of both lymphocytes and monocytes was not statistically different among the 5 patient groups. This allowed for comparison between the stereological parameters related to lymphocyte or monocyte mean volume. Image processing of 4 monocytes exhibiting different representative morphological characteristics. From top to bottom: (A), (D), (G), (J) Original Mitotracker confocal slide pictures (B), (E), (H), (K) Binary pictures of the mitochondrial network after thresholding (C), (F), (I), (L) Mitochondrial network skeleton (white line) Isolated pixels are highlighted in blue, skeleton end pixels are highlighted in red, yellow points indicate pixels with three or more neighbours and correspond to branching points. (Bar = 2 µm)(TIF)Click here for additional data file.Figure S4ROS production by lymphocyte subtypes. Representative graphs for HIV-1 infected patients. Lymphocyte subtypes were stained using specific CD antibodies (Beckman Coulter Inc) after treatment with ROS according to a standard procedure. 20,000 FSC/SSC-gated lymphocytes were analysed using a Navios flow cytometer (Beckman Coulter Inc). Two panels were used to identify lymphocyte subpopulations, one for T subtypes and a second for B and NK lymphocytes. Overlays of dot plots  and histograms  of ROS production by the total lymphocyte population (gray) and lymphocyte subtypes (red) were reported. ROS MFI ± SD are shown for each population. The percentages were related to the total lymphocyte population (FSC/SSC).  CD3+ T lymphocytes  CD3+ CD4+ T lymphocytes  CD3+ CD8+ T lymphocytes  CD3− CD19+ B lymphocytes  CD3− CD19− CD16+ CD56+ NK lymphocytes CD3− CD19+ B, CD3+ CD4+ and CD3+ CD8+ T lymphocytes, representing the large majority of lymphocytes, exhibited the same pattern as the total lymphocyte population with both low- and high-ROS producing cells, whereas CD3− CD19− CD16+ CD56+ NK lymphocytes were defined as low-ROS producing cells only.(TIF)Click here for additional data file.Figure S5Western blot analysis of PBMC mitochondrial proteins. (A) Representative western blot: porin , CoxIV-2  and CoxIV-4 . GAPDH was used as a total protein loading control (37 kDa). Participants were age- and sex-matched. No variation in protein amounts was observed between the participant groups. Human alveolar macrophages were used as a control for antibodies. (B) Discriminant analysis using three PBMC mitochondrial proteins. The five cohort populations are delineated by their 95% confidence circles around the means. The two perpendicular axes describe the combined variance (97.45%) of the parameters analysed. The inset shows the contribution of the amounts of each protein to the variance associated with X and Y axes. No statistical differences are observed between the groups. (C to E) Box plot of PBMC mitochondrial protein comparison from the five cohort groups: porin (C), CoxIV-2 (D) and CoxIV-4 (E). No statistical differences are observed between the groups . (F) Western blot showing a faint 29 kDa CoxIV-4 positive band in some patients. (G) Western blot showing an absence of bands corresponding to a defect in the cleavage of CoxIV-4 mitochondrial targeting signal in patients treated with 2NRTI+1PI/r.(TIF)Click here for additional data file.Table S1Demographic, clinical and biological parameters of the subjects/patients in the ANRS EP45 “Aging” cohort. No differences with respect to age and sex were present in the five groups. CD4+ cell count, CD4+/CD8+ and viral load indicated that the HIV-1 infected patients could be considered clinically stable.(DOC)Click here for additional data file.Table S2Details of the ART treatments. Three ART combinations  were used in accordance with World Health Organisation and French Health Ministry current recommendations. 70% of the 2NRTI+1PI/r and 2NRTI+1NNRTI patients shared the same Tenofovir/Emtricitabine NRTI backbone, and 93% of the 3NRTI patients shared a Lamivudine/Zidovudine NRTI backbone. Lopinavir/r and Efavirenz were the main PI/r (68%) and NNRTI (72%) used, respectively.(DOC)Click here for additional data file.Protocol S1he ANRS EP45 “Aging” Study.Thttp://clinicaltrials.gov/, NCT01038999.(PDF)Click here for additional data file.Methods S1Cell viability and western blot procedures.(DOC)Click here for additional data file.


Results of plasma viral load  and CD4 count of all patients obtained every 3 or 6 months were abstracted. The distribution of 5-year CD4 count was described using medians and compared between classes of clinical and demographic variables using Wilcoxon's or Kruskal-Wallis' test. Variation in CD4 count over 5 years was described using means and compared between classes using Student's t test or ANOVA. In order to quantify the associations between independent variables and CD4 count variation, linear regression coefficients and respective 95% confidence intervals (95% CI) were calculated, and adjusted for sex, naïve status, transmission mode, baseline viral load and age at viral load suppression.3) was significantly higher in women , in younger patients (<30 years-old: 656 vs. 526 in those over 49), in non-AIDS patients  and in those with the highest baseline CD4 count . Mean CD4 variation was greater in women , in naïve patients , in those with the highest baseline viral load (>750000 copies: 490.3 vs. 271.0 in those under 10000 copies) and correspondingly in patients with the lowest baseline CD4 count (<100: 469.7 vs. 359.5 in those over 350). Patients on NNRTI had larger improvement than those on PI (436.2 vs. 403.0) but no significant differences in CD4 count were found after 5 years. In multivariate analysis, mean CD4 count improvement remained significantly higher in women and lower in older patients. Improvement was also directly associated with baseline viral load.In crude analysis, 5-year CD4 count  widened over the follow-up period between women and men and between younger and older patients.


The debate regarding ‘When to Start’ antiretroviral therapy has raged since the introduction of zidovudine in 1987. Based on the entry criteria for the original Burroughs Wellcome 002 study, the field has been anchored to CD4 cell counts as the prime metric to indicate treatment initiation for asymptomatic individuals infected with Human Immunodeficiency Virus. The pendulum has swung back and forth based mostly on the relative efficacy, toxicity and convenience of available regimens.In today’s world, several factors have converged that compel us to initiate therapy as soon as possible: 1) The biology of viral replication (1 to 10 billion viruses per day) strongly suggests that we should be starting early. 2) Resultant inflammation from unchecked replication is associated with earlier onset of multiple co-morbid conditions. 3) The medications available today are more efficacious and less toxic than years past. 4) Clinical trials have demonstrated benefits for all but the highest CD4 strata (>500 cells/μl). 5) Some cohort studies have demonstrated the clear benefit of antiretroviral therapy at any CD4 count and no cohort studies have demonstrated that early therapy is more detrimental than late therapy at the population level. 6) In addition to the demonstrated and inferred benefits to the individual patient, we now have evidence of a Public Health benefit from earlier intervention: treatment is prevention.From a practical, common sense perspective we are talking about life-long therapy. Whether we start at a CD4 count of 732 cells/μl or 493 cells/μl, the patient will be on therapy for over 40 to 50 years. There does not seem to be much benefit in waiting and there likely is significant long-term harm. Do not wait. Treat early.http://www.biomedcentral.com/1741-7015/11/148.The counter-argument to this debate topic can be freely accessed here:  All Scientific work is incomplete - whether it be observational or experimental. All scientific work is liable to be upset or modified by advancing knowledge. That does not confer upon us a freedom to ignore the knowledge we already have, or to postpone the action that it appears to demand at a given time.”“- Sir Austin Bradford Hill[ford Hill.Pneumocystis jirovecii pneumonia. After 24 weeks of treatment, 19 placebo recipients and 1 AZT recipient died (P <0.001). This remarkable benefit of AZT led to early discontinuation of the placebo-based, first successful HIV treatment trial [10 copies/ml and their individual use was followed by breakthrough HIV viremia with resistant virus [In 1986 zidovudine (AZT) had a striking efficacy in decreasing mortality among patients with Human Immunodeficiency Virus (HIV) infection and advanced Acquired Immunodeficiency Syndrome (AIDS). Those patients had very low CD4 T cell counts, profound immunodeficiency and a very high risk of developing opportunistic infections (OIs), especially nt trial . Howevernt trial -5. The rnt virus .10 copies/ml and certain non-nucleoside blockers of the reverse transcriptase, such as nevirapine, exhibited inhibitory effect of 1.0 to 1.5 log10 copies/ml [10 copies/ml in vivo[Over subsequent years substantial progress was made in developing more potent antiretroviral agents and regimens. Novel inhibitors of the HIV protease, such as ritonavir and indinavir, were able to lower plasma viremia by 2.0 logopies/ml ,9. Combil in vivo. The advl in vivo,12. The l in vivo,14.BMC Medicine[The debate regarding optimal time to initiate antiretroviral (ARV) therapy has continued since the early years of HAART ,16 and r Medicine.10 copies/ml) was noted as soon as one week after initiation of therapy followed by a fast, nearly symmetric return to baseline levels within one week after treatment discontinuation [de novo virus infection occur, making 1 to 10 billion new viral copies per day [During AZT monotherapy, an 80% reduction in viral load  clearly demonstrated that the adjusted mortality rates were statistically higher among the 6,935 patients who deferred therapy until their CD4 counts fell to <500 cells/μl than in the 2,200 patients who started therapy with CD4 counts >500 cells/μl . However, the absolute risk of death was low in both groups: 5.1% in the deferred therapy and 2.9% in the early therapy group . AlthougDue to these intrinsic limitations of cohort studies in analyzing rare outcomes, investigators have tried different methods and designs in evaluating the evidence behind early treatment. A recent clinical trial (Setpoint Study) randomly assigned patients, who were within six months of HIV seroconversion, to receive either immediate treatment for 36 weeks or deferred treatment (when CD4 counts were <350 cells/μl). More than 57% of the study participants had CD4 counts >500 cells/μl. The deferred treatment group had a statistically higher risk of meeting treatment initiation criteria  resulting in earlier discontinuation of the study. While this study was not a clinical endpoint study, these results illustrated that the time from diagnosis of early infection to the need for initiation of therapy was shorter than anticipated .Other data support earlier initiation of treatment. Chronically infected patients delaying therapy until CD4 T cells ≤350 cells/μl have suboptimal CD4 T cell count recovery. After six years of ARV therapy, those who delayed therapy reached a CD4 count plateau below 500 cells/μl, which was significantly lower than patients starting therapy earlier .The benefits of early treatment go beyond hard immunologic parameters and translate into a higher likelihood of overall treatment success. The implementation of universal treatment of all HIV-infected persons in a large, publicly-funded clinic in San Francisco in 2010 led to a six-fold increase in the probability of viral suppression. In 534 patients entering the clinic with CD4 counts >500 cells/μl, the one-year incidence of viral suppression increased from 14% to >52% after adopting the approach . These rAs we start to adopt the “test and treat” approach, data from the Johns Hopkins HIV clinic have demonstrated that starting therapy earlier is a cost-effective strategy by the generally accepted benchmark in the US .Many of the observational cohort studies have supported the earlier initiation of HAART, resulting in a renewed confidence among many guideline committees to recommend initiation of therapy for those with higher CD4 counts in resource rich countries . These results conveyed the message that early therapy is more effective in preventing transmission of HIV than all other behavioral and biomedical prevention interventions studied to date, including condom use, male circumcision, vaginal microbicides, HIV vaccination and pre-exposure prophylaxis.In addition to potential gains in viral load suppression for the individual, in terms of improved outcomes and resistance mitigation, the reduction in viral load substantially reduces new HIV infections at the community level and, therefore, is extremely important from the standpoint of public health. The results of the HIV Prevention Trials Network (HPTN 052) proved this benefit unequivocally. This was a multi-continental trial that enrolled 1,763 HIV-serodiscordant couples comparing immediate treatment versus delayed therapy for the HIV-infected partner . At studOther observational studies and modeling analyses have provided similar conclusions as it relates to a decreased rate of HIV transmission in serodiscordant heterosexual couples following the introduction of ART -57. In tDespite the high-quality, cohort-derived evidence and the balance in favor of early treatment, some investigators remain skeptical and reluctant to adopt this strategy due to the lack of clinical trials of early versus delayed therapy for those with CD4 cell counts >500 cells/μl. The Strategic Timing of Anti-Retroviral Treatment (START) trial has been in enrollment phase and is expected to provide the first randomized trial evidence of whether immediate initiation of treatment in patients with CD4 cell counts greater than 500 cells/μl is superior to delaying initiation of HAART until the CD4 cell count falls below 350 cells/μl . AlthougSecond, although clinical trials have the advantage of randomization, there still are inherent limitations in study eligibility criteria and study referral patterns that can limit the generalizability of the findings. In particular, the patients referred to this study will be only those deemed to have clinical equipoise regarding when to start treatment. Those who the clinicians either do not want to treat now (poor treatment candidates or those who are not willing to start treatment) or who they do want to treat now  will not be referred to the study. This could represent up to three quarters of potentially eligible patients. So, in essence, some of the same ‘channeling biases’ present in clinical practice and, therefore, cohort studies, are at play among those individuals referred (or not referred) to a clinical trial. If the question was whether to ever treat HIV, a clinical trial seems like a great approach to answer the question. In the case of defining subtle differences within a narrow window of time, the results do not seem worth the efforts and/or the costs.The decision to start early treatment is an event that occurs in a relatively small window of time in the life span of a person with HIV infection. While some patients have stable CD4 counts over time , the majority experience drops in CD4 counts of 40 to 80 cells/μl/year. As an example, over as few as two years, and on average five years, CD4 cell counts can drop from 500 cells/μl to 350 cells/μl. Five extra years of therapy out of a total of 40 to 50 years on treatment for those living a near-normal life span  represents relatively minor differences in long-term exposure to treatment. However, those five extra years of continued exposure to unchecked viral replication represent potential substantial harm as demonstrated by the known biology of the infection. Rather than the ‘feared’ unnecessary exposure to drugs for only a fraction of a person’s lifetime, the likely harm comes from relentless replication of HIV, inflammation, destruction of lymphoid tissue, likely increased cardiovascular events, higher rates of certain malignancies and accelerated cognitive decline.In conclusion, the balance of available data strongly supports starting treatment in nearly all individuals regardless of CD4 T cell counts. Early treatment recommendations are based on our understanding of HIV biology, HIV pathogenesis, the availability of better drugs, the evidence from cohort studies, and the public health implications of viral load suppression and decreased transmission. Exceptions might be among the very small population of individuals who are ‘elite controllers,’ defined as those who have undetectable virus in the absence of antiretroviral therapy. For everyone else, to wait on randomized clinical trial data could well be doing harm. The time spent waiting is time that the patients cannot get back and the long-term damage associated with waiting could well be irreversible.Just prior to the quote referenced in the Background section above, Sir Austin Bradford Hill had stated:In asking for very strong evidence I would, however, repeat emphatically that this does not imply crossing every ‘t’, and swords with every critic, before we act”[.“e we act”.Until proven otherwise, we should heed Sir Austin Bradford Hill’s admonition and act upon the evidence we have in hand, which overwhelmingly tells us to treat early. What are we waiting for?ART-CC: Antiretroviral Therapy-Cohort Collaborative; ARV: Antiretroviral; AZT: Zidovudine; CASCADE: Concerted Action on SeroConversion to AIDS and Death in Europe; CD4 count: A measure of the number of helper T cells per cubic millimeter of blood, used to analyze the prognosis of patients infected with HIV; CI: Confidence interval; EACS: European AIDS Clinical Society; ESRD: End-stage renal disease; HAART: Highly active antiretroviral therapy; HPTN: HIV Prevention Trials Network; HR: hazard ratio; MI: Myocardial infarction; NA-ACCORD: North America-AIDS Cohort Collaboration on Research and Design; OIs: Opportunistic infections; RR: Relative risk; START: Strategic Timing of Anti-Retroviral Treatment; VACS: Veterans Aging Cohort Study; WHO: World Health Organization.RF has no competing interests. MSS is a consultant for BMS, Gilead, Merck, ViiV and Janssen. He has received research support from Abbvie, BMS, BI, Gilead, GSK, Merck, ViiV and Janssen.Dr. Franco is an Associate in Infectious Diseases at UAB. Dr. Saag is Professor of Medicine and Director of the UAB Center for AIDS Research. This work was presented, in part, at the “HIV 11” meeting in Glasgow, Scotland, November 2012.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/147/prepub


To evaluate the efficacy and safety of Entecavir (ETV) plus adefovir (ADV) for chronic hepatitis B (CHB) patients after multiple nucleos(t)ide analogue (NAs) failure treatment.Hepatitis B e antigen (HBeAg)-positive patients who had a suboptimal response or developed resistance to two or more previous NAs treatments were included, and all subjects were treated with ETV in combination with ADV for ≥ 24 months. Complete virologic response (CVR) was defined as an undetectability of serum hepatitis B virus (HBV) DNA level during treatment. Safety assessment was based on the increasing of serum creatinine and creatine kinase levels.A total of 45 eligible patients were included. Twenty-five patients had been treated with lamivudine (LAM) or telbivudine (LdT) and developed genotypic resistance. Resistance to ADV was present in 18 patients and 4 patients had a suboptimal response to ETV. Two patients had a resistance to both LAM and ADV. The cumulative probabilities of CVR at 12 and 24 months of ETV + ADV treatment were 88.9% (40/45) and 97.8% (44/45), respectively. Although one patient failed to achieve CVR, its serum HBV DNA level decreased by 3.3 log copies/mL after 24 months of combination therapy. The cumulative probability of HBeAg seroconversion was 15.6% (7/45) and 26.7% (12/45) at 12 and 24 months of treatment, respectively. History of prior exposure to specific NAs did not make a difference to ETV + ADV treatment outcome. There were no significant adverse events related to ETV + ADV therapy observed in the study subjects.ETV + ADV can be used as an effective and safe rescue therapy in patients after multiple NA therapy failures, especially in the areas where tenofovir is not yet available. About a quarter of the world’s population have been infected with hepatitis B virus (HBV), including 350 million patients with chronic hepatitis B (CHB); and about 15% to 25% of CHB patients would progress to life-threatening liver disease including cirrhosis and hepatocellular carcinoma , and an In past decade in China, many CHB patients have undergone sequential treatment with lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LdT) and/or entecavir (ETV) at 1 mg to manage antiviral resistance or insufficient suppression of HBV DNA. In fact, majority of multidrug failure were developed in patients who switched to ADV monotherapy for LAM refractory, and the virological and biochemical outcomes of single ETV salvage therapy were not satisfactory . And theCurrently, more and more evidences had showed that the development of drug resistance was associated with viral relapse, biochemical breakthrough, clinical deterioration, and even losing of favorable effects obtained by previous NAs treatment. Thus, to achieve sustained suppression of HBV DNA replication and remission of liver disease, successful management of CHB patients who developed treatment failure due to antiviral resistance or incomplete inhibition of viral replication is critical ,10.Recently, the increasing evidence suggests that combination therapy could effectively suppress viral replication and significantly delay or prevent the emergent of drug resistance of HBV strains, and combination therapy has become a potentially attractive therapeutic option in management of refractory CHB patients -16. HoweIn this study, we evaluated the efficacy and safety of the combination of ETV plus ADV in CHB patients after the failure of multiple NA therapies, and the data generated in this study were anticipated to provide researchers and practitioners with more information regarding the management of CHB patients after multiple failures.CHB patients with failures of two or more previous NAs therapies were treated with 0.5 mg of ETV (Bristol-Myers Squibb) plus 10 mg of ADV (GlaxoSmithKline) daily for at least 24 months. Failures of previous NAs therapies included suboptimal viral suppression (serum HBV DNA level > 10 000 copies/mL despite continued therapy for more than 1 year) or the development of resistance. Exclusion criteria were coinfection with human immunodeficiency virus or hepatitis C virus, and history of underlying renal problems. The study protocol was approved by the ethics committee of Chengdu Military General Hospital, and was conducted in accordance with the principles of the Declaration of Helsinki.Serum HBV DNA, hepatitis B e antigen (HBeAg), anti-hepatitis B e antibody (Anti-HBe), alanine aminotransferase, creatinine and creatine kinase levels were detected every 3 or 6 months. Serum HBV DNA level was determined by real-time polymerase chain reaction assay, which has a lower limit of detection at 500 copies/mL. Serum HBeAg and Anti-HBe statues were measured using Enzyme-Linked Immunosorbent Assay . To identify mutations associated with resistance in the gene encoding HBV polymerase, PCR amplification and direct sequencing was performed.The mean reduction of serum HBV DNA level was assessed during treatment. Complete virological response (CVR) was defined as a decrease of serum HBV DNA ≤500 copies/mL. The primary non-response was defined as a decrease of serum HBV DNA of less than 2 log copies/mL at 24 weeks of therapy. Viral breakthrough was defined as an increase of HBV DNA > 1 log copies/mL from nadir during ETV + ADV treatment. HBeAg seroconversion was defined as loss of HBeAg and appearance of anti-HBe on two occasions at least.P values less than 0.05 were considered statistically significant. All data were analyzed using SPSS 15.0 .To describe continuous variables with normal distributions, the mean ± standard deviation was used. Continuous variables without normal distributions were expressed as the median with range. Cumulative probability of CVR during the treatment period was calculated using the Kaplan-Meier method. A total of 52 patients with failures of two or more previous NAs therapies were screened, of which 7 patients were excluded by underlying renal problems, and the remaining 45 patients were included in the study. Detailed demographics of those 45 included patients are presented in Table The probability of CVR was 88.9% (40/45) at 12 months and 97.8% (44/45) after 24 months of treatment . Genotypic resistance to ADV (rtA181V/T or rtN236T) and resistance to LAM/LdT did not affect CVR rates .To define whether there is any difference in the rates of CVR according to prior exposures to different antiviral agents and genotypic resistance profile, the CVR rates were compared according to these variables using a log-rank test. There were no significant differences between patients with prior exposure to LAM and ADV Cumulative probability of ALT normalization and HBeAg clearance was calculated with the Kaplan-Meier method. After 24 months of ETV + ADV combination therapy, all patients obtained ALT normalization; and the rate of HBeAg/Anti-HBe seroconversion was 15.6% (7/45) at 12 months and 26.7% (12/45) at 24 months of treatment ide analogues; ALT: Alanine aminotransferase; HBeAg: Hepatitis B e antigen; CVR: Complete virological response.All authors declare that they have no competing interests.LGL conceived the study and revised the manuscript critically for important intellectual content. XXH drafted the manuscript. XXH, QY, LQ, HFQ and LYJ made substantial contributions to its design and acquisition of data. PQR and DJY participated in the analysis and interpretation of data. All authors read and approved the final manuscript.


Life expectancy has increased for newly diagnosed HIV patients since the inception of combination antiretroviral treatment (cART), but there remains a need to better understand the characteristics of long-term survival in HIV-positive patients. We examined long-term survival in HIV-positive patients receiving cART in the Australian HIV Observational Database (AHOD), to describe changes in mortality compared to the general population and to develop longer-term survival models.Data were examined from 2,675 HIV-positive participants in AHOD who started cART. Standardised mortality ratios (SMR) were calculated by age, sex and calendar year across prognostic characteristics using Australian Bureau of Statistics national data as reference. SMRs were examined by years of duration of cART by CD4 and similarly by viral load. Survival was analysed using Cox-proportional hazards and parametric survival models.The overall SMR for all-cause mortality was 3.5 (95% CI: 3.0–4.0). SMRs by CD4 count were 8.6 (95% CI: 7.2–10.2) for CD4<350 cells/µl; 2.1 (95% CI: 1.5–2.9) for CD4 = 350–499 cells/µl; and 1.5 (95% CI: 1.1–2.0) for CD4≥500 cells/µl. SMRs for patients with CD4 counts <350 cells/µL were much higher than for patients with higher CD4 counts across all durations of cART. SMRs for patients with viral loads greater than 400 copies/ml were much higher across all durations of cART. Multivariate models demonstrated improved survival associated with increased recent CD4, reduced recent viral load, younger patients, absence of HBVsAg-positive ever, year of HIV diagnosis and incidence of ADI. Parametric models showed a fairly constant mortality risk by year of cART up to 15 years of treatment.Observed mortality remained fairly constant by duration of cART and was modelled accurately by accepted prognostic factors. These rates did not vary much by duration of treatment. Changes in mortality with age were similar to those in the Australian general population. Mortality has decreased for newly diagnosed HIV-positive patients since the inception of combination antiretroviral therapy (cART) Long-term survival in HIV-positive populations with access to effective treatment appears to be approaching that of the general population Further, there is strong evidence associating immunologic resilience with age, and age at cART initiation has been associated with the rate and extent of immunologic recovery The primary objective of this analysis is to measure all-cause mortality in adult HIV-positive patients receiving cART in Australia. Specifically we want to compare mortality rates in these patients with those of the general population, over the long-term, and examine how these rates are affected by duration of treatment when adjusted for prognostic factors. A secondary objective of this analysis is to examine the effects of ageing on mortality in HIV-positive populations relative to the general population.The Australian HIV Observational Database (AHOD) is an observational clinical cohort study of patients with HIV infection seen at 27 clinical sites throughout Australia. AHOD utilises methodology which has been described in detail elsewhere Ethics approval for the study was granted by the University of New South Wales Human Research Ethics Committee, and all other relevant institutional review boards. Written informed consent was obtained from participating individuals. All study procedures were developed in accordance with the revised 1975 Helsinki Declaration.This analysis included all patients who had been recruited to AHOD prior to 31 March 2011 and who had at least 1 subsequent clinical visit after the date of first recorded cART .www.cphiv.dk/CoDe). These are reviewed by an independent HIV specialist clinician at the Kirby Institute who determines the primary and secondary causes of death or recommends additional information be provided by the study site as required.The study endpoint was mortality. The method of collection of mortality in AHOD has been described in detail elsewhere The covariates considered were: sex; age; mode of HIV exposure (men who have sex with men (MSM), heterosexual, injecting drug user (IDU), other/unknown); time updated instance of first AIDS defining illness (ADI); mono or dual antiretroviral treatment prior to first cART; HCV antibody ; HBV surface antigen (HBVsAg) ; time updated CD4 cell count ; time updated viral load ; year of first cART ; and time updated number of regimens received .Follow up was calculated from the start of cART and censored at the earlier of death, lost to follow up or 31 March 2011 (cohort censoring date). Lost to follow up date was defined as the most recent clinic visit if no clinic visit had been recorded in the 12 months prior to 31 March 2011. We used an intent-to-continue treatment approach and ignored any changes to treatment after baseline including periods off treatment. This approach avoids problems of modelling causal effects of patients stopping cART.Standardised mortality ratios (SMR) across prognostic characteristics were calculated referent to the Australian Bureau of Statistics (ABS) general population mortality rates by age group, gender and calendar year Cox proportional hazards models were initially developed to identify important survival covariates. Initially univariate models were developed and all significant univariate predictors were considered for inclusion in multivariate models. Final multivariate models were developed using a backwards stepwise approach to reduce to a parsimonious set of statistically significant (2p<0.05) covariates. A sensitivity analysis was conducted using Cox proportional hazards models of survival in patients who had commenced cART after 1 January 1999.Parametric survival-time Weibull models using clustered variance estimators were developed based on these covariates, and used to investigate survival trends by age. Probability of 10 year survival was calculated for a range of representative prognostic covariate values. Results were compared with 10 year survival rates for general population Australian males 2007–2009 using ABS data Data were analysed using Stata version 12 .Of 3,173 people in AHOD, 2,675 had commenced cART and had at least 1 subsequent clinical visit or result recorded post therapy. During 15,936 patient years of follow-up 206 deaths were observed. The rate of loss to follow-up of patients was 40.4 per 1000 person years of follow-up (95% CI: 37.3–43.6). The number of patients at first cART by selected characteristics is shown in Observed mortality by patient characteristics is shown in A large difference was observed in mortality rates for patients with CD4 cell counts of <350 cells/µl  32.0; 95% CI 27.0–38.0) compared to patients with CD4 cell counts of 350–499 cells/µl ; and CD4 cell counts of ≥500 cells/µl . Similarly the SMR for patients with CD4 cell counts of <350 cells/µl  was much higher than for patients with CD4 cell counts of 350–499 cells/µl ; and for patients with CD4 cell counts of ≥500 cells/µl  which was slightly higher than population level mortality. An increased SMR was observed for patients with viral loads of >400 copies/ml  compared to patients with viral loads of ≤400 copies/ml .SMRs by time updated CD4 cell count category by years that a patient has been receiving cART are shown in SMRs by time updated viral load category by years that a patient has been receiving cART are shown in Univariate and multivariate Weibull model predictors of survival are shown in th or greater regimen with no HBVsAg ever and mode of exposure other than IDU because these were the most representative observed states based on duration of analysis time. Scenarios are presented variously controlling for incidence of prior ADI and viral load. Survival probabilities for patients with CD4 cell counts of ≥350 cells/µL did not differ significantly from those of the general population for all age groups when controlling for other model covariates. Survival probabilities for patients maintaining CD4 cell counts of ≥500 cells/µL and controlling for other model covariates were similar to general population levels for all age groups: the 10 year survival probability for a 20 year old HIV-positive person controlling for other model covariates was 0.98 (95% CI 0.96–1.00) compared to 0.99 for 20 year olds in the male general population Weibull model 10 year survival probabilities for specified ages by CD4 cell count are shown in In a sensitivity analysis, 10 year survival probabilities for a multivariate model of survival in patients who had commenced cART after 1 January 1999 were similar to those developed in the primary analysis (results not shown).In this analysis from AHOD, overall mortality in adult HIV-positive patients receiving cART was found to be higher than general population level mortality, but mortality rates were close to general population levels in patients with high CD4 cell counts . There was no evidence of increased mortality by duration of cART when controlling for CD4 cell count level. There was no observed evidence of changes in mortality associated with age relative to the general population after controlling for time updated incidence of prior AIDS, mode of HIV exposure, time updated CD4 cell count, time updated viral load and time updated regimen number.Prognostic indicators in our study are consistent with other studies There was no observed association between duration of treatment and survival when adjusted for CD4 cell count level. At CD4 cell counts less than: 350 cells/µL there was a mild decrease in SMRs by duration of cART but rates remained much higher than population level; while above this level, average rates remained fairly constant, and just above population level. Although we observed a decrease in SMRs associated with recent cART commencement and year of cART commencement was a significant univariate predictor of survival, it was not a significant covariate in multivariate models. We also observed slightly decreased SMRs for patients with less than 3 years of cART experience compared to those with 3–6 years cART experience when stratified by CD4 cell count level, and these rates are weighted heavily by more recent cART starters. However these differences were not significant. These findings can be contrasted with a recent study by the Antiretroviral Therapy Cohort Collaboration of decreasing mortality by period of cART initiation  This analysis provides a robust assessment of the association between age and survival for this population. Decreasing SMRs were observed with age, which adjust for different distributions of age, gender and temporal structure of AHOD compared to the general Australian population. However these ratios do not adjust for additional covariate factors, such as changes in CD4 cell count and viral load, because of limited sample size. Instead, survival models of the data demonstrated that values of, and decreases in 10 year survival probabilities over the range of ages included in these analyses (up to 70 years old) were similar to those of the Australian general population when adjusting for other factors . We found no evidence of increasing mortality compared to general population rates associated with age.Some caution should be used when extrapolating such trends given the relatively limited history of HIV infection. To compare, type 1 childhood-onset diabetes patients have relatively low and stable mortality rates at earlier durations (up to 15 years) but increased rates at extended durations (up to 45 years) The findings of this study apply generally to the AHOD population and are subject to the real life prospect that patients do not always adhere to therapy. In AHOD, while time off treatment is well reported, there are limited measures of the extent to which this is associated with adherence. However, the use of intent-to-continue treatment principles in this report reflects the applied utility of cART and results can be reasonably simply interpreted as describing the mortality risk in all patients who start cART.Generally AHOD adherence is expected to be high for several reasons including low rates of loss-to-follow-up, cohort effect, and resourcing.There are some limitations to our analyses. First, our data had insufficient observations to robustly assess survival at ages over 70. Some studies have accounted for insufficient data at these ages by applying assumed relative rates of mortality Second, the generalisation of these findings may be limited by likely survivor bias in our analysis associated especially with patients who commenced cART prior to 1999. AHOD commenced in 1999 nearly 5 years after the inception of cART and does not include mortality from before this time in patients . However, a sensitivity analysis of patients who had commenced cART after 1999 was qualitatively similar to the model used in the primary analysis suggesting that the effects of survivor bias are limited. Also, the year of cART commencement and duration of treatment were not important as covariates in survival models for the study population especially when adjusting for recent CD4 cell count.Third, the omission of the effects of prognostic lifestyle factors may reduce the accuracy of analyses, especially where the distribution of these factors differs from that of the general population. In particular, smoking is not widely recorded in AHOD. In a subsample of the cohort for which these rates were collected, 25% of patients had reported as a smoker at most recent visit, and 40% as ex-smokers, compared to 22% and 29% of males in the general Australian population 2007–2008 A strength of our analyses is the long-term follow-up compared to other studies, as few cohorts have data on patients who have received cART for 15+ years. Also, while randomised clinical trials have been used to investigate mortality among adults infected with HIV receiving cART In conclusion, mortality rates in this large cohort of Australian HIV-positive individuals were close to those of the Australian population, particularly for patients with CD4 counts above 500 cells/µL. These rates did not vary much by duration of treatment. Changes in mortality with age were similar to those in the Australian general population. Further long-term follow-up is needed to characterize survival and other outcomes in HIV-positive patients on cART at older ages.


We retrospectively studied virus-transmission patterns of these subtypes in comparison to the longer-established subtype B, evaluating in particular risk-group related differences. We also examined to what extent distinct drug-resistance patterns in subtypes A/AE Reverse-transcriptase (RT) and protease sequences were retrospectively analyzed along with clinical and epidemiological data. MEGA, ClusalX, and Beast programs were used in a phylogenetic analysis to identify transmission networks.318 drug-naive individuals with A/AE or patients failing combination antiretroviral therapy (cART) were identified. 61% were IVDU. Compared to infected homosexuals, IVDU transmitted HIV infrequently and, typically, only to a single partner. 6.8% of drug-naive patients had drug resistance. Treatment-failing, regimen-stratified subtype-A/AE- and B-patients differed from each other significantly in the frequencies of the major resistance-conferring mutations T215FY, K219QE and several secondary mutations. Notably, failing boosted protease-inhibitors (PI) treatment was not significantly associated with protease or RT mutations in either subtype.versus newer regimens. The phenomenon of treatment failure in boosted-PI-including regimens in the apparent absence of drug-resistance to any of the drugs, and its relation to adherence, require further investigation.While sizable transmission networks occur in infected homosexuals, continued HIV transmission among IVDU in Israel is largely sporadic and the rate is relatively modest, as is that of drug-resistance transmission. Deviation of drug-naive A/AE sequences from subtype-B consensus sequence, documented here, may subtly affect drug-resistance pathways. Conspicuous differences in overall drug-resistance that are manifest before regimen stratification can be largely explained in terms of treatment history, by the different efficacy/adherence limitations of older  Several interrelated factors detrimentally influence the efficacy of measures to control the HIV epidemic at the individual and community levels, including risk-behaviors, sub-optimal treatment regimens, incomplete patient adherence and drug-resistance development and transmission. It is difficult to assess the relative roles of these factors: the constitution of the infected population and of the population at risk is heterogeneous and variable; drug-resistance mechanisms in non-B subtypes are incompletely understood Since every HIV-infected Israeli citizen has free access to cART and the collection of clinical, epidemiological and laboratory data is centralized, such comprehensive analyses have been facilitated. They enable comparisons of drug-resistance patterns in conjunction with other parameters among patients infected with different subtypes and/or belonging to different risk-groups. Recently, we were able to infer from the evolution of such patterns over time, and from the extent and character of phylogenetic clustering of HIV sequences, a striking increase in the frequency of unprotected and multi-partner sex in the gay community in Israel Subtypes A, A1, the recombinant virus CRF01_AE and related variants  are widespread in the far East and Former Soviet Union (FSU), two major epicenters of the HIV pandemic today versus B, and inferring risk-behavior trends in IDVU versus men who have sex with men (MSM) from the different patterns of HIV transmission within these groups.Our aim in this study was two-fold: discerning the impact of antiretroviral treatment on subtypes A/AE We analyzed genotypic information from 318 individuals carrying A/AE viruses along with clinical and demographic data. We compared to earlier-reported data from B-infected patients Demographic and clinical data, including detailed antiretroviral-treatment history, are provided on standardized forms when samples are submitted for genotyping. These data are cross-checked with the national HIV, tuberculosis (TB) and sexually transmitted disease (STD) registries and are stored in an anonymous database.“Recent” HIV infection at diagnosis was identified retrospectively either by documented evidence that sero-conversion occurred in the preceding 12 months or when acute retroviral syndrome was documented, based on a compatible pattern of viral load, CD4 count and clinical history Hepatitis: HIV infected patients in Israel are routinely screened biannually for co-infection with hepatitis B and C.Tuberculosis: HIV and TB registries are cross-matched annually.Syphilis: Infectious syphilis  was defined as previously described www.hivdb.stanford.edu/hiv/) et al. Blood samples from HIV-infected patients were sent for drug resistance evaluation as part of patients’ routine follow-up, as previously described Phylogenetic and molecular-evolution analyses of protease and reverse-transcriptase (RT) sequences were performed using MEGA, version 5.05 2 test for the categorical independent variables and Student’s t-test for continuous variables. P<0.05 was considered significant. Analyses were conducted using SPSS version 19.0.Clinical data and mutation frequencies were compared across patient groups using ChiThe retrospective analysis of clinical and laboratory data, which were obtained from the medical charts of HIV-1 patients attending the Sourasky and Sheba Medical Centers, was approved by the respective ethical committees. Specifically, permission was granted by the Sourasky Ethical Committee to analyze such data without the need of a signed informed consent by the patients. The samples obtained at the Sheba Medical Center that were used in this study belonged to patients who had signed an informed consent agreeing to participate in a range of studies.Seventy-six percent of more than 300 infected immigrants from FSU whose HIV virus was genotyped carried A/AE viruses. Most of them were IVDU . A/AE viFor a summary of clinical parameters at diagnosis, see One hundred seventy six of the 318 A/AE-HIV carriers (55.3%) were co-infected with hepatitis B and C viruses (HBV and HCV). Co-infection among IVDU and/or FSU-born individuals was significantly higher than in other transmission groups or among Israeli-born, respectively p<0.005;  and 3. Cp<0.001). Thirteen (92.8% of the TB infected) immigrated from FSU and one from Kenya; eleven (78.5%) were IDVU and 12 were co-infected with HCV. Ten of the 318 (3.1%) had Syphilis (Fourteen of the 318 (4.4%) had tuberculosis, a much higher co-infection rate than in B-subtype patients (Syphilis .p = 0.07).Evidence for “recent infection” was found at the time of diagnosis in three of 44 MSM (6.8%) and in four of 198 IVDU  was constructed . Figure Typically, we could link with high posterior probability of having a recent common ancestor only single A/AE-infected IVDU to each other or to single heterosexuals. Large networks, of more than four infected individuals, did conspicuously occur only within A/AE-infected MSM ((EFV) as the third drug. These were also the most common regimens for treatment-failing patients, but the treatment-failure frequencies were significantly disproportionate, ∼44% for regimens including ZDV+3TC, and only ∼23% for those including TDF+FTC . Resistance-conferring mutations were found in 16 individuals (6.8% of the drug-naive group), one heterosexual , 7 IVDU , and 8 MSM . Two carried the protease mutation M46I; three had NRTI-related mutations; 11 non-nucleoside reverse transcriptase inhibitors (NNRTI); and one was resistant to NRTI and NNRTI. The most frequent mutation was K103N, in 7 individuals. Mutation frequencies that differed significantly between drug-naive and drug-treated patients (see below) and/or between A/AE and B are shown in Thirty-four drug-naive patients (14.5%) carried the RT mutation A62V. The frequency of this mutation (6%) decreased significantly under treatment  and therAmong the 78 treatment-failing patients seven (9.1%) carried major PI-related mutations , nine 1.Of the 78 treatment-failing patients, 55 (70.5%) were exposed to PIs and 34 43.6%) were genotyped while failing PI treatment. Notably, only 7 (20.6%) of the 34 had any major PI mutation. Six of those had been treated previously with non-boosted PIs. All the 15 patients, who failed treatment while receiving LPV/r as first-line therapy, did not have any major mutations, either protease or RT , 6, 7. T% were geTo gain more insights into the factors shaping the above-described pattern, we compared A/AE viruses to B-viruses from all treatment-failing patients genotyped at NHRL Although A/AE are among the most rapidly-spreading HIV variants, in particular in the Far East, FSU, and recently also in the Western hemisphere, relatively little has been documented about treatment outcomes in this group relatively to other variants. Here we summarize more than 10 years of well-documented treatment of A/AE viruses in Israel, including clinical, epidemiological and laboratory surveillance data.As shown here, A/AE variants spread in Israel in the wake of a wave of accelerated immigration from FSU in the mid-1990’s A major concern has been that the use and/or sharing of contaminated needles, indicated by this clinical association and partially accounting for the rapid spread of HIV in other parts of the world, may be commonly practiced by IVDU also in Israel. Several lines of evidence reported here suggest that certain measures taken by health authorities to limit the spread of HIV via intravenous drug injection The average fractional rate of 5% does not tell us whether infection events occur sporadically and uniformly or rather are the consequence of highly-risky behaviors of a relatively few. Behavioral factors that distinguish risk-groups from each other can be revealed through phylogenetic analyses of virus sequences. We earlier identified large clusters of B-infected MSM Both demographics and behavior explain the lack of A/AE clustering. A large proportion of the A/AE-IDVU patients were infected in the FSU, as indicated also by the fact that only a few were diagnosed in Israel near sero-conversion (data not shown), in sharp contrast to subtype-B MSM In B-infected MSM, we had interpreted frequent evidence for “recent infection” at the time of diagnosis, including sero-conversion, as an indication of risk-awareness, and of engagement in risk-behavior despite such awareness e.g. the protease mutations M36I, L89M or the RT mutation R211S, and others (et al. a-priori account for the observation of different pathways (e.g., Comparing the overall pattern of resistance-conferring mutations in A/AE-infected patients with such mutations in subtype B revealed large differences, mainly in the frequencies of PI-associated mutations and TAMs. The frequency of certain mutations in these categories in the treatment-failing B-population reached more than 30% s et al. . There ays e.g., . The infMost B-patient genotypic data in our database pertain to patients treated before 2001, who received non-boosted PIs, while only twelve A/AE-infected individuals started treatment before 2001. To better understand resistance-pattern differences, we next restricted the B-subtype data used for comparison to patients diagnosed between 2001 and 2011. The differences in drug-resistance mutation frequencies between the two subtypes diminished drastically  and 6. BRecently, analyses of in-vitro pharmacokinetic and pharmacodynamic data Not surprisingly, we found almost no PI-resistance mutations in A/AE viruses from drug-naive individuals . While KOur dataset necessarily involves a degree of idiosyncrasy, as would any region-specific data, limiting generalization. An example is the discrepancy between our failure to find the mutation G190AS among drug-naive individuals, in both subtypes, and the transmission of this mutation to subtype-B infected individuals reported elsewhere versus B does not exclude differences related to polymorphism, and understanding of such effects may be required to optimize treatment of non-B HIV infection, as indicated in other subtypes Our comparative analysis of the patterns of drug-resistance mutations in treated patients infected with A/AE-HIV In summary, this study provides evidence that the practice of needle sharing among IVDU in Israel is not wide-spread as it might have been. In all classes of patients, strict adherence to treatment should be maintained to minimize both the emergence of drug-resistance and wild-type virus replication in the presence of drugs. Structural differences between subtypes A/AE and B may subtly affect drug-resistance pathways. Our study underscores the difficulties in discerning inherent viral effects from clinical, demographic, epidemiologic and behavioral factors and the need for comprehensive multidimensional analyses of consolidated data in order to weigh these factors relative to each other for the benefit of clinicians and health-policy makers.