CYP2B6 G516T polymorphisms and concomitant rifampicin use on the plasma efavirenz concentrations in HIV-infected Taiwanese.Plasma efavirenz concentrations in HIV-infected patients with tuberculosis (TB) may be affected by cytochrome P450 (CYP) 2B6 single-nucleotide polymorphisms and concurrent rifampicin use. We aimed to investigate the effects of CYP2B6 G516T polymorphism and plasma efavirenz concentrations with the use of polymerase-chain-reaction restriction fragment-length polymorphism and high-performance liquid chromatography, respectively.HIV-infected patients with or without TB who had received combination antiretroviral therapy containing efavirenz (600 mg daily) for two weeks or greater were enrolled for determinations of CYP2B6 G516G, 55 (32.2%) GT, and 3 (1.8%) TT genotype. Patients receiving rifampicin had a significantly lower median plasma efavirenz concentration than the control group ; however, all patients achieved target plasma concentration (>1 mg/L). Patients with GT or TT genotype had a significantly higher plasma concentration than those with GG genotype . Plasma efavirenz concentration >4 mg/L was noted in 38 (22.2%) patients, which was associated with a lower weight  and GT or TT genotype  in multivariate analysis.From October 2009 to August 2012, 171 HIV-infected patients, including 18 with TB, were enrolled 113 (66.1%) with CYP2B6 516 GT and TT genotypes and a lower weight are associated with higher plasma efavirenz concentrations.Despite combination with rifampicin, sufficient plasma efavirenz concentrations can be achieved in HIV-infected Taiwanese with TB who receive efavirenz 600 mg daily. Carriage of  Efavirenz is a potent non-nucleoside reverse-transcriptase inhibitor (NNRTI) and its combination with tenofovir and emtricitabine remains one of the preferred antiretroviral regimens for antiretroviral-naive patients without resistance-associated mutations to NNRTIs CYP2B6Efavirenz is metabolized primarily through hepatic cytochrome P450 (CYP) 2B6. The wide inter-patient variability of efavirenz concentrations has been reported, which may be related to sex, weight, ethnicity, drug-drug interactions, and single-nucleotide polymorphism (SNP) of CYP2B6 polymorphism, particularly G516T, is associated with high plasma concentrations of efavirenz and its drug-related toxicity CYP2B6 G516T polymorphisms may vary with different racial populations CYP2B6 G516T polymorphisms on the plasma efavirenz concentrations in HIV-infected Taiwanese patients.Efavirenz-containing antiretroviral therapy has been recommended for HIV-infected patients with tuberculosis (TB) when rifampicin-containing anti-tuberculous therapy is co-administered. Rifampicin may induce activity of CYP enzymes, which may lower the plasma concentrations of efavirenz. The early pharmacokinetic studies reported a reduction by 26% in the efavirenz concentration when co-administered with rifampicin Mycobacterium tuberculosis; or had hepatic transaminases greater than five times the upper limit of normal. The protocol was approved by the Research Ethics Committee of the National Taiwan University Hospital  and all patients provided written informed consent prior to enrollment.From October 2009 to August 2012, HIV-infected patients who were aged 18 years or greater and had been receiving an efavirenz-containing combination antiretroviral therapy at a daily dose of 600 mg for more than 14 days were enrolled when they sought routine HIV care at the National Taiwan University Hospital, the largest referral hospital for inpatient and outpatient HIV care in Taiwan. For patients who received a clinical or microbiologically confirmed diagnosis of TB, the daily dose of rifampicin was 450 mg for those with a weight less than 50 kg and 600 mg for those with a weight of 50 kg or greater. Patients were excluded from the study if they were pregnant; infected with HIV that was shown to harbor resistance-associated mutations to efavirenz or other NNRTIs; receiving anti-tuberculous regimens that did not contain rifampicin; infected with rifampicin-resistant CYP2B6 G516T genotyping and determination of efavirenz concentration. Plasma samples were stored at −20°C until analysis.We used a computerized case record form to collect data on the demographics, weight and height, and clinical characteristics including CD4 lymphocyte counts and plasma HIV RNA loads at baseline and during the follow-up, serostatus of hepatitis B virus and hepatitis C virus, concomitant medications , and the date and time of the last efavirenz dose that was usually taken at bedtime. Blood samples were collected 12±1 hours after the last dose of efavirenz into tubes containing EDTA as anticoagulant for Plasma HIV RNA load was quantified using the Cobas Amplicor HIV-1 Monitor test  with a lower detection limit of 40 copies/mL, and CD4 lymphocyte count was determined using FACFlow . The CD4 counts and plasma HIV RNA loads were monitored one month after initiation of combination antiretroviral therapy in antiretroviral-naive patients, or change of regimens in the presence of virological failure; and every three to six months thereafter according to the national HIV treatment guidelines.CYP2B6 G516T.High molecular weight genomic DNA was extracted from PBMC using the Wizard® Genomic DNA purification kit . The concentration of extracted DNA was determined by spectrophotometry and stored at −20°C before further analysis. Polymerase-chain-reaction restriction fragment-length polymorphism (PCR-RFLP) was performed to determine the SNPs of The plasma concentration of efavirenz was analyzed using high-performance liquid chromatography (HPLC) based on a validated method reported by Ramachandran et al. P values <0.10 in the univariate analyses were entered into a multivariate logistic regression model with the backward elimination method. Pearson’s correlations were used to evaluate the relationships between body weight and plasma efavirenz concentration. P values <0.05 were considered statistically significant.Statistical analysis was performed using SPSS software . Continuous variables were reported as medians and ranges, and were compared using the Mann-Whitney test. Categorical variables were expressed as numbers and percentages, and were compared using the Pearson χ2 test or Fisher’s exact test, as appropriate. Multivariate logistic regression analysis was used to evaluate the association between each independent variable and risk of high plasma efavirenz concentration (>4 mg/L). Variables with During the study period, 171 HIV-infected patients, all of whom were ethnic Chinese, were enrolled. The baseline characteristics of the patients are shown in CYP2B6 G516T polymorphism: 113 patients (66.1%) had carriage of GG genotype (wild-type), 55 (32.2%) GT genotype (heterozygous mutant), and 3 (1.8%) TT genotype (homozygous mutant). Compared with those who did not have TB, patients with TB and receiving rifampicin had a lower weight , though the body-mass index (BMI) between the two groups was similar (P = 0.041); and were more likely to have higher levels of total bilirubin levels in the serum . The demographics, HIV-related variables, and laboratory data were all similar among the patients with different genotypes of CYP2B6 G516T . Nevertheless, all patients receiving rifampicin achieved plasma efavirenz concentrations above the recommended target concentration (>1 mg/L), even in the eight patients who weighted 60 kg or more (the plasma concentrations ranging from 1.35 to 2.82 mg/L). CYP2B6 G516T genotypes. Patients with heterozygous or homozygous mutants had significantly higher plasma efavirenz concentrations compared to those with wild type .The median plasma concentration of efavirenz at 12 hours for all patients was 2.82 mg/L , and almost all of the patients achieved plasma efavirenz concentration of more than 1 mg/L except one with a level of 0.98 mg/L. CYP2B6 G516T genotypes were statistically significantly associated with elevated plasma efavirenz concentrations above 4 mg/L  had plasma efavirenz concentrations greater than 4 mg/L, a concentration that was reported to be associated with a higher risk for neuropsychiatric adverse effects e 4 mg/L . Figure P = 0.34). As for the 45 antiretroviral-naïve patients, 8 and 32 patients in each group continued efavirenz-containing combination antiretroviral therapy for at least 24 weeks, and the proportions of patients who achieved virological suppression was also similar .Fourteen of 18 patients (77.8%) receiving rifampicin and 143 of 153 patients (93.5%) not receiving rifampicin continued efavirenz-containing combination antiretroviral therapy for at least 24 weeks. Among the patients receiving rifampicin, three died of bacterial pneumonia complicated with respiratory failure and one was transferred to another hospital. On the other hand, among the patients not receiving rifampicin, one patient died of bacterial pneumonia complicated with respiratory failure; two discontinued efavirenz due to intolerable neuropsychiatric side effects with plasma efavirenz concentrations of 4.08 and >10.0 mg/L, respectively; three changed antiretroviral regimen because of genotypic resistance to efavirenz; and the other four were lost to follow-up. The proportion of patients with successful virological suppression (plasma HIV RNA load <200 copies/mL) at week 24 was similar between the patients receiving rifampicin and those not receiving rifampicin , which was statistically significantly associated with a lower weight and GT or TT genotypes of CYP2B6 G516T.This therapeutic drug monitoring and pharmacogenetic study that was performed in HIV-infected Taiwanese individuals showed that almost all patients could achieve therapeutic efavirenz concentrations (>1 mg/L) regardless of Efavirenz-containing combination antiretroviral therapy is the preferred antiretroviral therapy for HIV-related TB for which rifampicin-based anti-tuberculous therapy is co-administered CYP2B6 G516T polymorphism but not rifampicin co-administration significantly influenced the pharmacokinetics of efavirenz. The study by Kwara et al. found that patients receiving rifampicin had significantly higher mean efavirenz concentrations in those with the CYP2B6 516TT genotype P = 0.067), and the concentrations were significantly higher in blacks  In contrast, more recent and larger studies in HIV-infected patients with TB indicated either that there was no significant impact of rifampicin on efavirenz concentrations CYP2B6 516TT genotype, and none of them were taking rifampicin concomitantly in our study. Therefore, more studies are needed to determine if the same phenomenon of paradoxically elevated efavirenz concentrations in patients with CYP2B6 516TT genotype receiving rifampicin will be seen in Chinese population.In our study, patients taking rifampicin had significantly lower plasma efavirenz concentrations compared to the patients not receiving rifampicin, but all of them could achieve plasma efavirenz concentrations above the therapeutic target. Besides, most of the patients (88.9%) weighed greater than 50 kg. As a result, our findings suggest that the standard daily 600 mg efavirenz dose is adequate for patients on efavirenz-containing combination antiretroviral therapy who receive concurrent rifampicin for the treatment of TB. Of note, there were only three patients with the CYP2B6 gene is highly polymorphic. Many previous studies have shown CYP2B6 SNP, particularly G516T, to be associated with higher plasma efavirenz concentrations and its drug-related toxicity, which could result in early discontinuation of antiretroviral therapy CYP2B6 G516T varies among the different ethnicities, ranging from 14% to 21% in Asian populations CYP2B6 G516T was 17.8% (32.2% of GT genotype and 1.8% of TT genotype), which was similar to those in prior Asian studies Efavirenz is mainly metabolized by CYP2B6, and the Several studies have demonstrated that a higher weight were associated with lower efavirenz levels Our study has several limitations. First, it was performed at a single hospital in Taiwan, with most patients being male. Information on the efavirenz concentrations among HIV-infected female patients remains limited. Second, only patients who could tolerate efavirenz for two weeks or more were included in this study, and three quarters of the patients had been receiving efavirenz for more than 24 weeks. Therefore, the proportion of patients with toxic drug levels may be underestimated since patients with significantly higher drug concentrations may have interrupted or changed regimen earlier than two weeks because of severe psychiatric side effects and skin rashes. Third, this was a cross-sectional study, and we did not collect serial blood samples when the patients received and after they discontinued rifampicin-containing anti-tuberculous therapy, to avoid inter-patient variations. Last, we did not collect information on neuropsychiatric adverse effects to correlate with plasma efavirenz concentrations.CYP2B6 516 GT and TT genotypes were associated with higher plasma efavirenz concentrations that might be associated with an increased risk of neuropsychiatric adverse effects.In conclusion, with information obtained from therapeutic drug monitoring and pharmacogenetic study in HIV-infected Taiwanese, we have shown that most patients could achieve therapeutic efavirenz concentrations, with or without combination with rifampicin, when receiving a standard 600 mg of efavirenz daily; and lower weights and


Antiretroviral treatment (ART) has been introduced in Ethiopia a decade ago and continues to be scaled up. However, there is dearth of literature on the impact of ART on changes in CD4 lymphocyte count and weight among patients on treatment.To determine the predictors of change in CD4 lymphocyte count and weight among HIV/AIDS infected patients taking antiretroviral treatment in eastern Ethiopia.A retrospective cohort study was conducted among HIV/AIDS patients taking ART from 2005 to 2010. A sample of 1540 HIV infected adult patients who started antiretroviral therapy in hospitals located in eastern Ethiopia were included in the study. The primary outcomes of interest were changes in CD4 count and weight. Descriptive statistics and multivariable regression analyses were performed to examine the outcomes among the cohort.Both the median CD4 lymphocyte counts and weight showed improvements in the follow up periods. The multivariate analysis shows that the duration of ART was an important predictor of improvements in CD4 lymphocyte count  and weight . Advanced WHO clinical stage, lower baseline CD4 cell count, and baseline hemoglobin levels were factors associated with decline in weight. Actively working patients had higher CD4 lymphocyte count and weight compared to those that were ambulatory (p<0.05).We detected a substantial increment in weight and CD4 lymphocyte count among the patients who were taking ART in eastern Ethiopia. Patients who are of older age, with low initial CD4 lymphocyte count, late stage of the WHO clinical stages and lower hemoglobin level may need special attention. The reasons for the improved findings on CD4 count and weight throughout the five years of follow up merit further investigation. HIV/AIDS continues to be a global challenge. Sub-Sahara Africa is the most affected part of the world with an estimated 22.9 million people living with HIV and 1.2 million deaths from AIDS among children and adults in 2010 The introduction of antiretroviral treatment has altered the course of the HIV/AIDS epidemic Monitoring clinical and diagnostic progression of patients on anti-retroviral treatment (ART) is important to examine responses to the treatment and for clinical decision-making. Patient responses and clinical diagnostic measures to anti-retroviral treatment show differences based on individual and population characteristics, and the type of setting where treatment is delivered Ethical clearance was obtained from the Institutional Research Ethics Review Committee (IRERC) of Haramaya University. Further approval was obtained from the hospital management before accessing the patients' medical records. In Ethiopia, patients are not customarily requested for consent before their medical record is to be used for research. This includes at the beginning of the recording process and afterward. Initially we wanted to contact loss to follow up patients through nurses to check if they are alive or dead, however the IREC advised us against this citing interference and emotional burden to patients and their families. As a result, we abandoned this approach and collected only non-identifying data from patients' cards. This was done by anti-retroviral treatment nurses working in the respective hospitals in order to assure confidentiality. The IRERC has approved this procedure.The study was conducted in Hiwot Fana, Jugal and Dil Chora hospitals located in eastern Ethiopia. According to the 2007 ART guideline of Ethiopia, the eligibility criteria for initiation of ART are dependent on the availability of CD4 count instruments in the treating health facilities. When CD4 count is not available, ART can be initiated to patients in WHO clinical stages III and IV irrespective of total lymphocyte count (TLC), and WHO clinical stage II patients if TLC is less than 1,200/ml. Whereas in the presence of CD4 count measurement instruments in the health facilities, treatment is given to WHO clinical stage IV patients, irrespective of the CD4 count – to WHO clinical stage III, if the CD4 cell count is less than 350/mL; and all WHO clinical stages if the CD4 cell count is below 200/mL A retrospective cohort study was conducted among HIV patients on ART. Data was collected from September to November 2010. A random sample of 1540 patients who started treatment between September 11, 2005 and September 10, 2008 were selected and retrospectively followed until September 10, 2010. The patients' identification numbers were used to extract the necessary data from the database and cards of the HIV patients in the ART clinics.Socio-demographic characteristics, baseline and follow up clinical and laboratory data, and treatment outcomes were abstracted from patients' cards. The primary outcomes for this study were CD4 lymphocyte count and body weight measurements across time.3) and weight (kg) were measured every six months by the ART nurses, but in some instances such as dysfunction of CD4 counter machines measurements could be extended to longer intervals.A standard questionnaire was used to extract the data from patients' cards. This form is developed using the standardized ART entry and follow up form employed by the ART clinics. Four advanced ART nurses who were trained on comprehensive HIV care and treatment services and were involved in patient follow ups at the time of the study collected the data. Data collection was supervised by the researchers. All completed data collection forms were examined for clarity and consistency. Death was ascertained by reviewing cards and death certificates. Patients who died from unrelated diseases or accidents, as well as those alive at the end of the follow up period were considered censored. CD4 lymphocyte count  Descriptive statistics such as median, standard deviations (SDs) and tables were used to investigate the characteristics of the cohort. Descriptive statistics were analyzed using SPSS version 16, while regressions were fitted using STATA version 11. Linear regression was used to characterize and screen data for problems of multicollinearity. Mixed models regression was used to examine changes in CD4 cell count and weight after the baseline measurement. Models were fitted to both the raw and transformed data that approximate to a normal curve. Data transformations ranging from inverse to square roots of the CD4 counts and weight were attempted. Both the random intercept and slope-intercept models were examined to see the model fit. To check the reliability of the model based on the 60A total of 1540 patients were included in the study. The median (IQR) age of participants was 32 (28.0–40.0) years and 37.3% (574) were males. Three hundred forty eight (22.7%) and 890 (58.2%) patients were on the WHO clinical stages of II and III at baseline respectively. The median  of baseline CD4 count was 135 (76.0–198.3) per milliliter .From the registered patients in this cohort analysis, the outcome of patients as active, deceased, lost to follow up and transferred out were 1005 (67.2%), 86 (5.9%), 210 (14.0%) and 192 (12.8%) respectively.th and 60th months where the median CD4 cell count showed a slight decline . Advanced WHO clinical stages, lower baseline CD4 lymphocyte count, and baseline hemoglobin levels were associated with lower body weight. Higher age predicted improvement in weight . The model from the square root transformed data revealed similar findings with the one based on the raw data. We show both models in the tables. To examine sensitivity of the model to missing values we refitted it using values of observations up to the 48ollow up .This longitudinal study aimed to examine the progress of CD4 lymphocyte count and weight, and their predictors among a cohort of patients taking antiretroviral treatment in eastern Ethiopia. Our findings indicate that the duration of ART was an important predictor of improvements in CD4 lymphocyte count  and weight . Patient's functional status at the beginning of treatment also predicted improvements in both body weight and CD4 lymphocyte count. Furthermore, higher age, earlier WHO clinical stage, higher CD4 count and high hemoglobin levels were associated with improvements in weight.et al. classified patients being provided nutritional supplementation into CD4+ cell count strata, each 100-cell/mm3 decrease in CD4+ cell count was associated with an average of 1.9 kg reduction in weight The research literature indicates that declines in CD4 lymphocyte count and weight loss among patients on anti-retroviral treatment are associated with increased mortality and morbidity manifesting in the form of deterioration of clinical conditions and decreased functional status in both adults Studies have shown that longer duration on ART leads to an improvement of CD4 lymphocyte counts. Similarly, in this study, longer duration on treatment was significantly associated with improvements in CD4 count and weight. A study among 358 patients on ART from southern Ethiopia has documented similar findings In this study we found that older age is associated with improvement in weight. A similar finding has been reported by Mariz and colleagues where older age among patients on ART was associated not only with increased weight gain but also with becoming overweight among patients in Brazil We found that high baseline hemoglobin level predicts improvement in weight gain. Our finding is similar to previous literature in that BMI levels are predicted by not only CD4 counts but also by anemia and hemoglobin levels This study has some limitations. CD4 lymphocyte counts were not available for all patients due to lack of resources at the hospitals where the study was conducted. On top of this HIV RNA measurements were not collected since they are not part of the clinical monitoring of patients in Ethiopia because of their very prohibitive cost. Similarly, diagnostic tests that would confirm the presence of certain opportunistic infections were also limited. Due to this we were not able to include them in our analysis to examine and account for their potential contribution to CD4 count and weight changes. The retrospective study design limited our ability to gather data about factors that may influence morbidity, CD4 count and weight levels, such as food insecurity, social support and depression. However, our study has strengths in that we used a relatively large sample size, multiple referral hospitals, a robust analysis and a longer follow up period. It also has strengths in that it provides evidence about the implementation of ART and its impacts on clinical indicators in the context of treatment provided by lower level health workers in a resource poor country.We detected a substantial increment in weight and CD4 lymphocyte count among the patients who were taking ART in eastern Ethiopia. The findings indicate that the length of time patients stay on ART and patients' functional status improves both CD4 count and weight gain. Higher age groups, earlier WHO stage, higher CD4 count and high hemoglobin levels were associated with improvements in weight. Ambulatory patients during the initiation of ART should be monitored carefully for immunological response. Patients who are in old age, with low CD4 lymphocyte count, late stage of WHO and lower hemoglobin level may need special diet considerations such as food by prescription programs to help them gain weight over time.


Three decades into the HIV/AIDS epidemic there is a growing cohort of perinatally HIV-infected adolescents globally. Their survival into adolescence and beyond represent one of the major successes in the battle against the disease that has claimed the lives of millions of children. This population is diverse and there are unique issues related to antiretroviral treatment and management. Drawing from the literature and experience, this paper discusses several broad areas related to antiretroviral management, including: 1) diverse presentation of HIV, (2) use of combination antiretroviral therapy including in the setting of co-morbidities and rapid growth and development, (3) challenges of cART, including nonadherence, resistance, and management of the highly treatment-experienced adolescent patient, (4) additional unique concerns and management issues related to PHIV-infected adolescents, including the consequences of longterm inflammation, risk of transmission, and transitions to adult care. In each section, the experience in both resource-rich and limited settings are discussed with the aim of highlighting the differences and importantly the similarities, to share lessons learnt and provide insight into the multi-faceted approaches that may be needed to address the challenges faced by this unique and resilient population. With successful strategies for Prevention of Mother to Child Transmission (PMTCT), fewer infants are acquiring HIV perinatally or through breastfeeding, resulting in fewer children requiring HIV care. There are, however, approximately 2,000,000 children living with HIV globally, 90% of whom live in sub-Saharan Africa . The curThere is a wide spectrum in timing of diagnosis and entry into care for PHIV-infected adolescents. In the United States, Europe and other resource-rich settings, perinatal HIV infection has been contained by the implementation of maternal testing and PMTCT programmes since the 1990s, early testing of HIV-exposed infants, and close follow up of HIV-infected children through adolescence. In the United Kingdom and Ireland, for example, 62% of the current adolescent population presented to care at a year of age or less ,6. A few3 at presentation and are desperately in need of treatment  and 550 cells/mm3 [IQR 832–861], respectively   . Also, ap=0.31)] .3 should occur by six months after initiation, with the caveat that with significantly elevated viral loads and markedly suppressed CD4 counts, this may vary [In general, with appropriate cART, virologic suppression and a CD4 increase of 150 cells/mmmay vary . In settIn resource-rich settings, many PHIV-infected adolescents are significantly treatment-experienced, with over a decade of antiretroviral exposure on average, often with suboptimal single and dual ART regimens before transitioning to cART when it became available ,22. For et al. reported that in a UK and Ireland cohort, 52% of PHIV-adolescents had dual and 12% had triple-class ART resistance [In the setting of suboptimal ART drug levels there is resultant viral evolution and development of resistance . Certainsistance . Genotypsistance . Althougsistance –71. Datasistance . These dEmergence of drug resistance has been highly correlated with all-cause mortality, with resistance to particular classes of agents, NNRTI specifically, having a threefold higher correlation with mortality, likely due to virulence of these viral variants . This coIn resource-rich settings, resistance testing (genotypic and when necessary phenotypic assessment) is readily available and prudent in the setting of virologic failure in order to guide decisions about treatment. Particularly critical for adolescents who are highly treatment-experienced, is the cumulative genotype of all mutations previously documented as with decreased drug-selective pressure with poor adherence or switch to a different drug regimen, viral variants harbouring resistance may fade from the circulating plasma viral pool, but still be present in the latent reservoir, emerging when the drug-selective pressure is resumed. Also, all prior ART regimens and responses to those regimens should be reviewed to assess likelihood of residual activity and predicted presence of resistance ,18. In ret al., PHIV-infected youth on cART were assessed and their regimens characterized as optimal vs. sub-optimal based on cumulative genotypes and anticipated drug activity at the start of the regimen. More than half of the patients from each cohort had poor adherence. By 48 weeks, those in the optimal group had a greater median CD4 increase, +62 (+25 to +200) than those in the suboptimal group +8 (−93 to +54) cells/mm3 (p=0.04), and were four times more likely to have an increased CD4>50 cells/mm3, a difference that persisted throughout the study period. There were no differences in clinical events or accumulation of new resistance mutations between the two groups. The authors’ caution that the group of highly treatment-experienced adolescents with ongoing poor adherence could develop resistance to the optimal regimen and conclude that the choice of initiating a new regimen needs to consider adherence, adverse effects, pill burden and fear of accumulating resistance [Although the availability of newer agents, including integrase inhibitors and CCR5 antagonists in certain settings, has generated renewed hope for virologic suppression and immune recovery for heavily treatment-experienced PHIV-infected adolescents with extensive resistance, selecting an optimal background regimen to achieve virologic suppression is a challenge. As many PHIV-infected adolescents continue to struggle with adherence, timing of introducing the salvage regimen is critical to prevent failure on what is often the last available suppressive regimen. In a retrospective study by Wong sistance . In genesistance . The appsistance .et al. examined treatment interruptions in PHACS and reported that 23% of the cohort, significantly more in the earlier birth cohort (1991–1993) vs. younger cohorts, had discontinued ART for at least one period of ≥3 months after continuous ART for ≥6 months [Providers that care for PHIV-infected adolescents have been forced to be creative in managing treatment failure in this population. Possible strategies include: bridging strategies , and treatment de-intensification or even discontinuation –81. Once6 months . While iInflammation is increasingly being recognized as a significant consequence of HIV infection. Initially shown in adult studies, subsequent paediatric studies have also shown that PHIV-infected children have a high degree of inflammation related to uncontrolled HIV replication . The seqStudies have reported mixed findings regarding sexual activity in PHIV-infected adolescents with some studies reporting delayed penetrative sex in young HIV-infected adolescents ,85 with PHIV-infected adolescents often have complex psychosocial situations and clinical histories, including complicated resistance patterns ,10. ThesIn published studies of PHIV-infected adolescents, there is usually equal gender distribution between male and female PHIV-infected adolescents, a characteristic which distinguishes PHIV and non-PHIV-infected adolescents, where there tend to be varying gender distributions depending on the epidemic . Gender may significantly affect outcomes and clinical practice in PHIV-infected adolescents for a number of reasons. Contrasting findings regarding the impact of gender on adherence and virological suppression warrant further investigation. A French cohort demonstrated greater virological suppression rates in female adolescents in a multivariate analysis of the cohort , while tIn conclusion, the growing cohort of PHIV-infected children that are emerging into adolescence and young adulthood require cART treatment to control viral replication, prevent immune deterioration and avert secondary transmission. Successful treatment is complicated by developmental, cognitive and psychosocial challenges that can compromise adherence leading to the development of resistance and reduced treatment options, with resultant morbidity and mortality. While recent data in adults has estimated that the life expectancy for HIV-infected individuals has improved to near normal, with significant proportions of PHIV-infected adolescents emerging into adulthood with resistant virus, continued non-adherence, and the limited pipeline of new agents, there is concern that the survival seen in HIV-infected adults may not be duplicated for PHIV-infected adolescents. Resource-rich settings are over a decade ahead of resource-limited settings in their treatment of PHIV-infected adolescents, providing foreshadowing for some of the challenges ahead for resource-limited settings and insight into the multi-faceted approaches that may be needed to address these challenges. Lessons learnt from resource-rich settings and research about the unique barriers that may exist in resource-limited settings will be critical to assuring that PHIV-infected youth continue to benefit from treatment as they navigate the challenging period of adolescence.


To understand the relationship between HAART and preterm delivery (PTD).Analysis of prospectively collected data from all patients attending HIV antenatal services at a single UK centre 1995—2010. Fisher's exact test two tailed for categorical variables. T-test for continuous variables.Data are presented on all 324 deliveries up to 26/07/2010. 78% of the women were Black African, 10.3% Caucasian. Risk for HIV infection was heterosexual intercourse in 94%, injecting drug use in 1.2%. Median gestational age at first ante-natal clinic appointment was 13 weeks. 7 women took no therapy prior to delivery and 7 took dual NRTI treatment. Data on the remainder are summarised in Table 11. Of these 10 had a preterm delivery (30.3%).Delivery before 37 weeks (PTD) occurred in 14.4% pregnancies of which 62%  before 34 weeks. Difference not significant comparing PTD in patients starting new continuous HAART (17.2%) with pre-conception HAART (12.6%). PTD occurred in 17/142 (12.%) women treated with nevirapine-based HAART compared with 24/102 (23.5%) treated with protease inhibitor (PI)-based HAART (p 0.02). Of the 64 patients treated with a short-course of HAART (START) during pregnancy to prevent mother-to-child HIV transmission 33 were eligible, if willing to delivery by pre-labour caesarean section (PLCS) to receive zidovudine monotherapy (ZDVm) according to the 2008 BHIVA guidelinesThe role of HAART and PI-based HAART in PTD has been controversial. Even in this single-centre study, where all women were managed by one team in accordance with national guidelines, confounders abound. CD4 counts and viral loads differed significantly between patients starting HAART in pregnancy and those taking ZDVm or already on HAART at conception. We therefore compared PTD rates in women who were eligible for, and chose between, START  and ZDVm with PLCS. The rates of PTD were significantly higher (p 0.005)with START (30.3%) than with ZDVm (6.6%) in these ZDVm eligible mothers. These data suggest that PI-based HAART initiated during pregnancy is associated with a significantly increased rate of PTD and that this is influenced by maternal immune status. Further investigation to determine whether other regimens may have less impact on PTD are urgently required particularly with the increasing use of HAART in prevention of mother-to-child transmission.


In HIV-infected patients receiving rifampicin-based treatment for tuberculosis (TB), the dosage of lopinavir/ritonavir (LPV/r) is adjusted to prevent sub-therapeutic lopinavir concentrations. In this setting, South African clinicians were advised to administer super-boosted LPV/r (400 mg/400 mg) twice daily, instead of standard dosed LPV/r (400 mg/100 mg) twice daily. We sought to determine – in routine practice – the tolerability and HIV treatment outcomes associated with super-boosted LPV/r compared to unadjusted LPV/r in combination with rifampicin-based TB treatment.3 (IQR 21–159) and 39,457 copies/mL , respectively. According to physician preference, 15 (52%) of 29 patients received super-boosted LPV/r (400 mg/400 mg) every 12 hours during TB treatment and 14 (48%) of 29 patients received standard dose LPV/r (400 mg/100 mg) twice daily during TB treatment. Among patients who received super-boosted LPV/r there was a trend towards a higher rate of symptomatic transaminitis , gastrointestinal toxicity  and a significantly increased need for treatment discontinuation . The rate of virologic failure was not higher in patients with unadjusted LPV/r dosing.We conducted a retrospective review of HIV-infected patients who receiving second-line ART with a LPV/r-containing regimen who required concomitant TB treatment. We identified 29 patients; the median age was 36 years (IQR 29–40), 22 (76%) were female, the median CD4 cell count and viral load at first-line ART failure was 86 cells/mmWe observed a high rate of toxicity and need for treatment discontinuation among patients on standard rifampicin-based TB treatment who received super-boosted LPV/r. A significant proportion of HIV-infected patients in South Africa require second-line ritonavir-boosted protease inhibitor (PI)-based antiretroviral therapy (ART) as a result of virologic failure or intolerance of initial ART The reduction in PI concentrations associated with concomitant rifampicin can be attenuated with the use of higher doses of ritonavir Although widely used, there is very limited data from routine clinical settings in low and middle-income countries on the tolerability and antiviral efficacy of super-boosted lopinavir in HIV-infected adult patients receiving rifampicin for TB infection. We hypothesized that coinfected patients who received super-boosted lopinavir (400 mg twice daily) would experience a significantly higher rate of adverse events and tolerate a shorter duration of concomitant TB treatment compared to patients receiving standard lopinavir dosing.McCord Hospital, a state-aided hospital, provides treatment for HIV and tuberculosis to patients living in Durban and the province of KwaZulu-Natal. We conducted a retrospective review of HIV-infected patients who initiated second-line ART containing LPV/r at McCord Hospital between July 2004 and February 2007. Eligible for inclusion in the current study were adult patients who received both LPV/r-containing second-line ART and rifampicin for tuberculosis treatment for at least three months, and who underwent a viral load test during the overlap period. There was considerably clinician-to clinician variability regarding the decision to use – during treatment of tuberculosis in patients receiving LPV/r-based second-line ART – either super-boosted LPV/r (400 mg/400 mg) twice daily or standard boosted LPV/r (400 mg/100 mg) twice daily. In all instances, the dose of the lopinavir component was 400 mg twice daily and the nucleoside analogue dosing was standard. We excluded women who received LPV/r-containing ART only as part of a prevention of a maternal-to-child-transmission protocol.Rifampicin was administered as part of a 4-drug  fixed-dose combination (FDC) or as a 2-drug (rifampicin and isoniazid) FDC, depending upon whether the patient was receiving intensive phase or continuation phase TB treatment. The second-line ART agents available during the study included lopinavir/ritonavir ; ritonavir, lamivudine, didanosine (enteric-coated formulation); zidovudine; and stavudine.Patient monitoring followed South African Department of Health recommendations including HIV-1 RNA level (detection limit of <50 copies/ml) and CD4 cell count monitoring every 6 months. Liver function testing was not obtained routinely during coadministration of LPV/r and rifampicin-containing TB treatment but was available to clinic staff in the presence of symptoms or signs of hepatitis . Adverse events and abnormal laboratory results during coadministration were graded according to the Division of AIDS Regulatory Support Center guidance (7).Using a standardized instrument, we abstracted from the patient medical record clinical and demographic characteristics including HIV-1 RNA levels, CD4 cell count, tuberculosis treatment history and outcomes, adverse events detected by clinician and treatment modification.We compared baseline characteristics (at the time of first-line ART failure), rates of key adverse events, treatment discontinuation and virologic suppression among patients receiving second-line ART and concomitant tuberculosis therapy who received super-boosted lopinavir (400 mg twice daily) and patients who received standard boosted lopinavir (100 mg twice daily). Categorical variables were compared using chi-square and Fisher’s exact tests, and continuous variables were compared using Student’s t-test. We defined the event treatment discontinuation as the need to stop either rifampicin-based TB therapy or LPV/r-based ART prematurely resulting from toxicity. We compared event-free survival between patients who received super-boosted lopinavir and patients who received standard boosted lopinavir, by the Kaplan-Meier method.P<0.05 were considered significant.Analyses were performed using SPSS software, version 18.0. All tests of significance were two-sided; associations with The study was approved by the Research Ethics Committee at McCord Hospital in Durban, South Africa. The Committee waived the requirement for informed consent for this retrospective data analysis.A total of 3025 patients initiated first-line ART at the Sinikithemba clinic at McCord Hospital between July 2004 and February 2007 and over that period 189 (6%) subsequently required second-line LPV/r-containing ART as a result of virologic failure or an adverse effect to initial ART. During that period, we identified 29 patients who received concomitant rifampicin and LPV/r-containing ART, representing 15% of patients who initiated second-line ART. The median age for this subgroup was 36 years (IQR 29–40), 22 patients (76%) were female, the median CD4 cell count and viral load at first-line ART failure was 86 cells/µl (IQR 21–159) and 39,457 copies/mL , respectively. The most common nucleosides used with LPV/r in second-line ART were: AZT + DDI, 11 patients (38%); D4T +3TC, 8 patients (28%); AZT +3TC, 7 patients (24%); 3TC alone, 3 patients (10%).P = 0.03). There were no other significant differences in the baseline characteristics of the two groups at the time of first-line ART failure with respect to age, CD4 cell count, viral load, weight, and second-line nucleoside backbone.Lopinavir was super-boosted (LPV/r 400 mg/400 mg twice daily) in 15 (52%) of 29 patients and LPV/r dosing was standard in 14 (48%) of 29 patients (LPV/r 400 mg/100 mg twice daily). We compared the baseline characteristics of patients who received, during tuberculosis therapy, super-boosted LPV/r and the standard dose of LPV/r . Among pP = 0.3) and gastrointestinal toxicity .Among patients who received coadministered rifampicin-containing TB therapy and LPV/r -based ART, adverse reactions were common. Hepatitis and gastrointestinal toxicity were the most prominent adverse reactions . OverallP = 0.036; P = 0.035). The most common reason for treatment discontinuation was transaminitis in the presence of clinical symptoms or signs of hepatitis.Overall, the mean period of coadministration of rifampicin-containing TB therapy and LPV/r-based ART was 6.7 months [range 1–11 months]. Months of coadminstered TB and LPV/r-based ART treatment completed was evaluated as a function of lopinavir/ritonavir dosing strategy utilizing a Kaplan-Meir analysis. Patients who received the super-boosted lopinavir/ritonavir strategy had a significantly fewer months of dual therapy completed prior to treatment discontinuation compared who received standard lopinavir/ritonavir dosing  or rate of gastrointestinal toxicity (P = 0.965). A Kaplan-Meir analysis revealed no relationship between use of DDI or D4T in the backbone and treatment discontinuation .To explore if D4T or DDI may have been implicated in higher rates of toxicity among patients receiving co-administered LPV/r and rifampicin-containing TB treatment, we explored the relationship between receiving a nucleoside backbone containing DDI or D4T compared to a backbone without DDI or D4T on rate of symptomatic transaminitis, gastrointestinal toxicity, and rate of treatment discontinuation. There was no significant impact of use of DDI or D4T-containing backbone on rate of symptomatic transaminitis (P = 0.7).We evaluated the proportion of patients in the two ritonavir dose groups in whom virologic failure (HIV-1 RNA >200 c/mL) was observed . OverallP = 0.036). The most common reason for treatment discontinuation was transaminitis in the presence of clinical symptoms or signs of hepatitis. Early discontinuation of coadministered lopinavir/ritonavir and rifampicin-based TB treatment placed patients at risk for suboptimal treatment outcomes. As the number of patients requiring boosted protease inhibitor-containing therapy for HIV expands, the need for a more varied drug formulary – to respond to common situations such as TB co-infection – grows even more pressing.In South Africa, where the prevalence of HIV and tuberculosis are among the highest in the world, we found that, overall, during second-line ART with a LPV/r-containing regimen approximately 15% of patients required coadministration of rifampicin for tuberculosis therapy. In our sample, co-infected patients who received super-boosted LPV/r experienced a trend towards a higher rate of symptomatic transaminitis, gastrointestinal toxicity and the durability of coadministered treatment was significantly shorter resulting from adverse event-related treatment discontinuation  to prevent a reduction in lopinavir plasma concentrations We did not find a high rate of virologic failure associated with coadministration LPV/r-based ART and TB treatment, either among patients who received super-boosted LPV/r or standard dose LPV/r, despite the known effect of rifampicin on reducing lopinavir concentrations. However because of our relatively low sample size, we cannot exclude such an effect. It should also be considered that in the current study, patients were not homogenous with respect to time since initiation of second-line ART. Further, it has been previously reported that patients receiving long-term boosted-protease inhibitor-containing ART may tolerate more variation in lopinavir concentrations without loss of virologic control There are several limitations of this study. First, with regard to the adverse events, we implicated specific drugs (ritonavir and rifampicin) but cannot exclude a role for other drugs including the NRTIs and other anti-tuberculosis drugs in the standard TB treatment regimen including isoniazid. Second, patients who received less than three months of coadministered LPV/r-containing second-line ART and rifampicin-based TB treatment were not included, potentially excluding patients who experienced early adverse events. Third, adverse events were detected passively during coadministration and therefore we are likely to have underestimated the rate of these events. For example, a symptom-based monitoring approach was used to monitor for hepatic toxicity (transaminase levels were measured when symptoms or signs resulted in concern for drug-induced hepatitis), and therefore we were unable to detect subclinical elevations in transaminases. It is possible that the a more intensive lab monitoring strategy would have resulted in more adverse effects detected, but the relevance of subclinical laboratory abnormalities is not clear Managing the competing risks of coadministered LPV/r-based ART and rifampicin-containing TB treatment is complex, requires access to laboratory services and – with the increasing use of second-line ART – is likely to become a more common problem over time. In the absence of additional drug options, efforts to use existing agents to overcome the interaction between lopinavir and rifampicin have continued. An additional potential dosing strategy was illustrated by a recent small trial involving 18 co-infected patients in South Africa. Decloedt and colleagues used double-dose lopinavir/ritonavir in combination with rifampicin-based TB treatment and found no grade 3 or 4 level toxicity and no evidence of virologic breakthrough The replacement of rifampicin with rifabutin, in patients receiving boosted PIs requiring TB treatment, is recommended in high-income countries. Rifabutin can be substituted for rifampicin in the treatment of TB without loss of efficacy and rifabutin – when given at an adjusted dose of 150 mg every other day – does not substantially lower lopinavir concentrations More feasible than the wider introduction of rifabutin may be improving access to additional antiretroviral options in resource-poor settings with potentially fewer important interactions with rifampicin-based TB treatment. Raltegravir, an integrase inhibitor which does not require boosting, can be dose-adjusted during rifampicin-based TB treatment, is one promising alternative


Data on determinants of long-term disease progression in HIV-infected patients on antiretroviral therapy (ART) are limited in low and middle-income settings.Effects of current CD4 count, viral load and haemoglobin and diagnosis of AIDS-defining events (ADEs) after start of combination ART (cART) on death and new ADEs were assessed using Poisson regression, in patient aged ≥18 years within a multi-centre cohort in Thailand.Pneumocystis jiroveci pneumonia were the most commonly presented first new ADE . In multivariable analyses, low current CD4 count after starting cART was the strongest predictor of death and of new ADE. Even at CD4 above 200 cells/mm3, survival improved steadily with CD4, with mortality rare at ≥500 cells/mm3 . Haemoglobin had a strong independent effect, while viral load was weakly predictive with poorer prognosis only observed at ≥100,000 copies/ml. Mortality risk increased following diagnosis of ADEs during cART. The decline in mortality rate with duration on cART  was accounted for by current CD4 count.Among 1,572 patients, median follow-up from cART initiation was 4.4 (IQR 3.6–6.3) years. The analysis of death was based on 60 events during 6,573 person-years; 30/50 (60%) deaths with underlying cause ascertained were attributable to infections. Analysis of new ADE included 192 events during 5,865 person-years; TB and 3 minimizes mortality. However, patient monitoring could potentially be relaxed at high CD4 count if resources are limited. Optimal ART monitoring strategies in low-income settings remain a research priority. Better understanding of the aetiology of anaemia in patients on ART could guide prevention and treatment.Patients with low CD4 count or haemoglobin require more intensive diagnostic and treatment of underlying causes. Maintaining CD4≥500 cells/mm By the end of 2009, 5 of the 33 million HIV-infected patients in low- and middle-income countries were receiving antiretroviral therapy (ART) Differences between low and high-income settings such as patients’ characteristics at presentation to HIV medical services, prevalence of co-morbidities, distribution of AIDS-defining illnesses, spectrum of causes of death, and clinical management have generally been observed 3 combined, restricting scope to identify whether there is a target threshold above which CD4 level should be maintained to minimise mortality So far, analyses from low and middle-income settings on prognostic factors in treated patients have mostly considered baseline characteristics only and/or included relatively short-term follow-up Using data from a multi-centre cohort in Thailand, a middle-income country, we assessed the prognostic effects of CD4 count, viral load and haemoglobin at start of combination antiretroviral therapy (cART) and after treatment initiation on progression to death and to first new ADE. The effect of new ADE on death was also examined.The Program for HIV Prevention and Treatment (PHPT) cohort was approved by the Thai Ministry of Public Health and local ethics committees. Participants provided written informed consent at entry.www.clinicaltrials.gov). The study has been previously described 3http://www.who.int/classifications/icd/en/index.html).The PHPT adult cohort prospectively followed HIV-infected patients receiving ART in 40 public hospitals across Thailand , subsequently started cART with at least 3 antiretroviral drugs at age 18 years or older, and had at least one CD4 and one viral load evaluation after cART initiation.Outcomes evaluated were progression from cART initiation to (i) all-cause mortality and (ii) first new ADE (which included death in the absence of a new AIDS diagnosis after starting cART). In the analysis of new ADE, patients with AIDS diagnosis before starting cART were included and progression to the first clinically new ADE was considered, ignoring recurrence of ADE’s occurring before cART initiation. Follow-up was considered from date starting cART up to the date of the outcome of interest or date of last clinic visit for patients censored.At a given time point, we defined current CD4 count, viral load or haemoglobin as the most recent measurement taken after cART initiation which was within the last 9 months of that time point. The value at cART initiation was defined as the most recent measurement within 6 months prior to date of initiation. We classified ADEs as either mild or moderate/severe, based on prognostic categories proposed in a Antiretroviral Therapy Cohort Collaboration (ART-CC) study which showed different types of ADE diagnosed after cART initiation had varying impact on mortality First, we examined the predictive value of the following time-varying factors: current CD4 count; current viral load; current haemoglobin; time since cART initiation; and for progression to death only, diagnosis of new mild ADEs and of new moderate/severe ADEs after cART initiation. The effect of type of new ADEs on mortality was assessed based on patients without prior AIDS diagnosis at cART initiation, consistent with the ART-CC study We then assessed whether after accounting for current CD4 count, viral load and haemoglobin, there were any additional prognostic value in CD4 count, viral load, haemoglobin and AIDS diagnosis status at cART initiation.a priori confounders at initiation which were selected on the basis of findings from previous studies Effects of covariates were estimated using Poisson regression models, based on follow-up periods with both current CD4 count and viral load available. Clustering within hospitals was included as a random effect. Multivariable analyses were adjusted for time since cART initiation , current CD4 count, viral load and haemoglobin and, in addition, the following th, 50th and 90th centiles CD4 count was square root transformed to improve model fit. Current viral load was categorized as <400, 400 to 99,999 and ≥100,000 copies/ml; these categories were chosen since the detection limit of assays used had varied over time and a previous study showed high viral load at 6 months from starting treatment was associated with poorer survival but only at levels above 100,000 copies/ml Finally, we carried out sensitivity analyses with: (i) current CD4 count, viral load and haemoglobin defined assuming a given measurement was valid for a maximum of 6 and 12 months, rather than 9 months and (ii) including only patients who were ART-naïve at cART initiation (apart from ART use for PMTCT).All statistical analyses were undertaken in STATA, version 11.1740 patients were ART-naïve at PHPT cohort enrolment and subsequently started cART aged ≥18 years between 2001 and 2010. Of these, 1572 (90%) had follow-up period (s) after initiation with both current CD4 count and viral load measures and were included in analyses.3. Women had lower haemoglobin than men, with median 11.5 (IQR 10.5–12.4) versus 12.8 (11.4–14.2), respectively. A small proportion of patients  previously received ART for their HIV, all initially prescribed didanosine plus stavudine dual therapy at median 2.9 (IQR 2.2–3.3) years before switching to cART. Around a fifth of patients  were diagnosed with AIDS before cART initiation.Three-quarters (76%) of patients were female; Patients were followed up for a median 4.4 (IQR 3.6–6.3) years after starting cART. 145/1572 (9%) patients were lost to follow-up after cART initiation and 149 (9%) voluntarily withdrew. Median number of measurements per patient after initiation was 14 (IQR 11–17) for CD4 count, 14 (10–16) for viral load and 20 (16–27) for haemoglobin.3, increasing to 35% and 52% at 3 and 5 years, respectively. High CD4 count at cART initiation was associated with high absolute level at all subsequent times (data not shown). Haemoglobin increased following cART though mainly within the first year, with lower levels observed in women throughout follow-up (data not shown).The proportion of patients with HIV-1 RNA <400 copies/ml was 91.3% at 6 months from cART initiation, and around 95% from 3 years onwards . At 12 mThe analysis of progression to death was based on 60 deaths during 6573 person-years  when both current CD4 count and viral load were available; Pneumocystis jiroveci pneumonia (20%), generalized herpes simplex infection (10%) and recurrent pneumonia (5%).In the analysis of progression to first new ADE, 69 of the 1572 patients developed a new ADE after cART initiation but prior to any CD4 and viral load evaluation while on treatment, so were excluded; of these, 80% (n = 55) developed a new ADE within 6 months, 46% (n = 32) within 3 months. Among the remaining 1503 patients included, there were 192 events during 5865 person-years ; 153 pat3, decreasing substantially to 1.1 per 1000 person-years at ≥500 cells/mm3. In multivariable analyses, low level of current CD4 count (p<0.001) and haemoglobin (p<0.001) were the strongest predictors of mortality. Current viral load had a weak independent effect (p = 0.05), with poorer survival associated with levels ≥100,000 copies/ml. As expected, mortality rate declined steadily with time from cART initiation, from 21.3 per 1000 person-years during the first 6 months to 4.7 per 1000 person-years at ≥36 months; however, this trend disappeared after adjusting for current CD4 count (p = 0.82), but not when only either current viral load (p = 0.01) or haemoglobin (p = 0.006) were accounted for.The associations with mortality for factors after cART initiation are shown in Prior AIDS diagnosis before cART initiation was not predictive of mortality . HoweverCurrent CD4 count (p<0.001) and haemoglobin (p<0.001) were both strongly prognostic of new ADE, though the magnitude of these associations was weaker compared with that for death . Viral lAfter adjusting for current CD4 count, viral load and haemoglobin, higher viral load at cART initiation remained strongly associated with new ADE p<0.001, , with itResults remained the same when either (i) current CD4 count and viral load were defined by assuming a given measurement was valid for a maximum of 6 or 12 months or (ii) patients who initiated on dual therapy before switching to cART were excluded.3 was substantially high (124.8 per 100 person-years). Therefore, patients with low CD4 count, even if virologically suppressed, should be closely monitored, with underlying causes of immunosuppression  promptly investigated and addressed.This study was based on a well-run, long-term ART programme involving a wide range of public hospitals throughout Thailand, with good quality data collection including accurate ascertainment of AIDS diagnoses and cause of death for most patients. Consistent with other studies from low and high-income settings, CD4 count was found to be the strongest predictor of death and of new ADE 3, with mortality being rare at ≥500 cells/mm3 (rate 1.1 per 1000 person-years). A recent analysis of four large treatment cohorts from Sub-Saharan Africa reported a 1.7 fold increase in mortality risk at CD4 levels 350–499 cells/mm3 compared to ≥500 cells/mm3 , comparable to our estimate of 2.5 (95% CI 1.9–3.4) 3 had similar mortality rates as the general population 3 while on treatment. They also suggest that relaxing monitoring for patients on treatment with high stable CD4 count should be evaluated for settings with limited capacity for CD4 monitoring. In addition, early HIV diagnosis and early treatment initiation before onset of severe immunosuppression are indicated, given that high CD4 count at ART initiation is associated with improved long-term CD4 cell count recovery 3 compared to deferring until CD4 falls below 350 cells/mm3 is being investigated in the ongoing randomized START trial We observed improved survival with increasing CD4 count even at levels above 200 cells/mmPrevious studies across different settings already showed the independent association between low haemoglobin on cART and disease progression In our cohort, high current viral load was weakly associated with both mortality and new ADE but only at ≥100,000 copies/ml, consistent with a previous ART-CC study Substantial variation in the impact of different ADEs on mortality was previously observed in an ART-CC analysis including treated patients in Europe and US In our study, rate of loss-to follow-up was relatively low, with efforts made to trace patients missing clinic visits using telephone calls and home visits. Although voluntary patient withdrawal from the study was usually due to relocation or ART being accessed elsewhere through the national treatment programs, it is important to note that patients who were either lost to follow-up or voluntarily withdrew were less likely to be virologically suppressed after cART initiation compared to those still in follow-up, with loss to follow-up also associated with lower CD4 attained (data not shown). This could lead to under-estimation of the effects of CD4 count and viral load if patient drop-out was associated with mortality or progression to new ADE’s, conditional on the covariates in the models .A limitation to our analysis was that effects of covariates were not assessed separately for AIDS-related and non AIDS-related mortality due to limited number of deaths. An association between CD4 count and non-AIDS related mortality has been reported, albeit of weaker magnitude compared with that for AIDS-related mortality In conclusion, immunosuppression and anaemia may reflect the presence or development of conditions that lead to mortality. Half of the deaths in this Thai cohort were attributable to infections which were chronic and generally considered treatable, while the cause of death remained unknown in nearly a fifth despite regular follow-up. This suggests a significant proportion of patients died of conditions which were not diagnosed in time or appropriately treated, possibly due to constraints in this resource-limited setting and lack of experience in handling such complications at the beginning of the program when the priority was given to starting antiretroviral treatment for all those who urgently needed it. Patients with low CD4 count or haemoglobin should therefore receive more intensive case-management, while monitoring could potentially be relaxed for those with high CD4 if resources are limited. ART monitoring in low-income settings remains a research priority, and in particular, better understanding of predictors of morbidity and mortality due to non-AIDS disease is needed given patients are surviving longer on treatment.


We lack the understanding of why HIV-infected individuals in South Africaprogress to AIDS. We hypothesised that in end-stage disease there is a shiftingdynamic between T cell imposed immunity and viral immune escape, which, throughboth compensatory and reverting viral mutations, results in increased viralfitness, elevated plasma viral loads and disease progression. We explored how Tcell responses, viral adaptation and viral fitness inter-relate in South Africancohorts recruited from Bloemfontein, the Free State(n = 278) and Durban, KwaZulu-Natal(n = 775). Immune responses were measured byγ-interferon ELISPOT assays. HLA-associated viral polymorphisms weredetermined using phylogenetically corrected techniques, and viral replicationcapacity (VRC) was measured by comparing the growth rate of gag-proteaserecombinant viruses against recombinant NL4-3 viruses. We report that inadvanced disease (CD4 counts <100 cells/µl), T cell responses narrow,with a relative decline in Gag-directed responses (p<0.0001). This isassociated with preserved selection pressure at specific viral amino acids, but with reversion at other sites , most notably in Gag and suggestiveof “immune relaxation”. The median VRC from patients with CD4 counts<100 cells/µl was higher than from patients with CD4 counts ≥500cells/µl , potentially explaining the rise in viral loadassociated with disease progression. Mutations at HIV Gag T186S and T242Nreduced VRC, however, in advanced disease only the T242N mutants demonstratedincreasing VRC, and were associated with compensatory mutations(p = 0.013). These data provide novel insights into themechanisms of HIV disease progression in South Africa. Restoration of fitnesscorrelates with loss of viral control in late disease, with evidence for bothpreserved and relaxed selection pressure across the HIV genome. Interventionsthat maintain viral fitness costs could potentially slow progression. With few exceptions, untreated individuals infected with Human Immunodeficiency VirusType 1 (HIV-1) develop Acquired Immunodeficiency Syndrome (AIDS), associated withopportunistic infections, malignancies and, eventually, death. Some patientsprogress to AIDS quickly, whilst others maintain undetectable plasma viral loadswithout therapy and do not become unwell for many years. Deciphering the correlatesof this heterogeneous protection is important, as there are implications for thedesign of vaccines and other interventions.The pace of HIV disease progression is multifactorial - a mixture of host andpathogen genetics combined with factors such as the immune response and viraladaptation. In genome-wide association studies a limited number of SNPs and allelescorrelate with lower viral loads What determines this differential HLA Class I effect is unclear.‘Beneficial’ HLA Class I alleles may be associated with T cell cloneswith broader cross-reactivity to viral variants due to reduced thymic selectionEscape from an effective immune response is determined by the strength of the imposedselection pressure and may explain why the prevalence of HLA-associatedpolymorphisms is greater for HLA Class I alleles associated with protection These interactions between HLA Class I imposed selection, viral escape and viralfitness are together likely to influence clinical progression, however themechanisms that lead to progression to late disease and AIDS are poorly defined. Wehypothesised that the nature of these interactions can be understood better byinvestigating patients with late-stage HIV infection to determine if, or how,CD8+ve T cells are maintaining selection pressure, and whether the virus showsdifferent patterns of adaptation and fitness costs compared to patients with earlierinfection. There are limited reports that, despite the loss of CD4+ve cells,CD8+ve T cells may still be functional in AIDS, although with varied avidity,less polyfunctionality and less differentiation In this study we question whether progression to AIDS is associated with an increasein viral fitness, and whether this is related to changes in T cell imposed selectionpressure. We start by comparing T cell ELISPOT responses in patients acrossdifferent CD4 count strata, and then examine how the variation in these responsescorrelates with different patterns of selection across the HIV genome in patientswith very low CD4 cell counts compared with less progressed infections. Finally, wemeasure the viral fitness of these variants to correlate the replicative cost ofadaptation with outcome. We investigated 1053 untreated HIV-1 infected South Africanindividuals from Bloemfontein and Durban, and found that viral fitness was greaterin patients with advanced disease with examples of both viral compensatory mutationsand of reduction in escape mutations, the latter possibly due to ‘immunerelaxation’. Despite the complex interplay between host and pathogen, thesedata support a key role for the restoration of viral fitness in association withAIDS progression and help to explain the rise in viraemia in terminal stages.Full ethical approval was gained for the study of both cohorts. Written informedconsent was provided by all study participants. The study ethics for theBloemfontein cohort was approved by the University of the Free State (ETOVS10/04 and ETOVS 206/05) and for the Durban cohort by the University ofKwaZulu-Natal Review Board.gag,pol or nef sequences were included in theanalysis of HLA-linked polymorphisms, and comprised the ‘total’cohort. Subsequent analyses involved stratification of the patients according toHLA type, plasma viral load and CD4 T cell count. In the CD4 T cell countstratification, patients were assigned to either “High CD4” , or “LowCD4” .Participants from two South African cohorts were studied  - the ‘Bloemfontein’ cohort from theFree State (n = 278) and the ‘Durban’ cohortfrom KwaZulu-Natal (n = 775). Both are established cohorts,described elsewhere . InsummParticipants' HLA Class I type was determined to the oligo-allelic levelusing Dynal RELITM Reverse Sequence-Specific Oligonucleotide kits for the HLA-A,-B and –C loci . To obtain four-digit typing, Dynal BiotechSequence-Specific priming kits were used, in conjunction with theSequence-Specific Oligonucleotide type.Peptides, 18 amino acids in length (n = 410), overlapping by10 amino acids, and spanning the entire expressed HIV genome, were synthesizedbased on the consensus of available C-clade sequences in 2001. These peptideswere used in a ‘mega-matrix’ of 11–12 peptides per pool totest patient samples for HIV-specific T cell responses by interferon-gammaELISPOT assay, as previously described gag, pol and nef weresequenced using previously described methods and primers HIV 2-adjusted odds ratio.Amino acid polymorphisms that associated with host HLA class I alleles, wereidentified using previously described methods utilising ‘phylogeneticdependency networks’ http://www.hiv.lanl.gov/content/immunology/pdf/2008/optimal_ctl_article.pdf).The epitope flanking region was also included in the analysis, defined accordingto the five amino acids neighbouring to the defined epitope in both C and Nterminal directions.Identified polymorphisms were mapped to known cytotoxic T cell epitopes. Epitopemaps were defined from experimental data from the Durban cohort gag-protease-deleted pNL4-3 HIV plasmid(pNL4-3Δgag-protease) with autologousgag-protease amplified from virus extracted from patientplasma, as described elsewhere Chimeric viruses were constructed by recombining agag-protease viral RNAwas extracted from plasma, reverse transcribed and amplified using specificprimers in the first round of nested PCR using the Superscript III One-stepRT-PCR kit with high fidelity Platinum Taq polymerase(Invitrogen) . The second round of the nested PCR was conducted using a 100-merprimer pair  that completelymatched the pNL4-3 sequences using Platinum Taq polymerase(Invitrogen).In brief, to amplify the autologous HIV BstEII) sites at the 5′ end of thegag gene and 45 nucleotides downstream from the 3′end of the protease gene, as described elsewhere BstEII enzyme digest results in deletion of HIVgag-protease and self ligation of the remaining plasmid(pNL4-3Δgag-protease). For transfection, thepNL4-3Δgag-protease was linearised byBstEII digestion for two hours at 60°C (10 U/µlof enzyme per 1000 µg/mL of plasmid). For each sample, 10 µg of thelinearised pNL4-3Δgag-protease was co-transfected with 5µg of patient-derived gag-protease PCR product in3.9×106 tat-driven GFP reporter T cells (GXR cells of CEMorigin). After transfection by electroporation , 10 µl polybrene (4 µg/µl) and1×106 GXR cells were added and incubated at 37°C.Positive control samples were generated using co-transfection ofpNL4-3Δgag-protease with gag-proteaseamplified from pNL4-3, pHXB2 (subtype B), and pMJ4 (subtype C). After five days,GFP expression was measured every 1∼2 days using a FACSCalibur flowcytometer . Supernatant was harvestedwhen >15% of cells were positive for GFP, and stored at−80°C.The pNL4-3 HIV plasmid had been mutated to contain unique restriction enzyme(6 GXR cells to determinethe multiplicity of infection (MOI). The VRC was assayed by adding recombinantvirus supernatant to 1×106 GXR cells at an MOI of 0.003. TheVRC was measured, at least in duplicate, using gating strategies as previouslydescribed Viral titration was performed in 1×10Student t-tests (two tailed) and Mann-Whitney tests (two tailed) were used tocompare various clinical parameters and VRC measurements. The statisticalanalyses were carried out using Prism4 for Macintosh . Associations between HLA Class I alleles and viral polymorphisms weredetermined using phylogenetically-corrected algorithms, as described above.gag,pol and nef genes were available.Patients were recruited from the Bloemfontein and Durban cohorts 2 = 0.055) in contrast to theproportion of responses to other proteins such as Env  and Vif,which was greater in patients with lower CD4 cell counts. We hypothesized thatthe reduction in breadth of CD8+ve T cell responses might result in, andtherefore correlate with, a decline in selection pressure, particularly in Gagin light of the relative shift away from Gag targeting. As a result, this‘immune relaxation’ might allow the reversion of costly viral escapemutations back to a fitter wild type state. Accordingly, we next analysed HLAClass I-associated polymorphisms in the HIV-1 gag,pol and nef genes to identify anydifference in the strength of selection pressure in patients with high and lowCD4 cell counts.In order to measure the T cell-imposed selection pressure acting on HIV atdifferent stages of the infection, ELISPOT assays using overlapping peptides(OLPs) covering the entire HIV genome were analysed from 775 patients. Resultswere stratified according to CD4 cell count and are presented according to thenumber of OLPs seen per patient for each gene , and theTo identify evidence of differential HLA Class I imposed selection pressure inpatients with high and low CD4 cell counts, we carried out a study to identifyHLA Class I associations with particular amino acid polymorphisms in thiscohort. We accounted for potential confounding effects such as phylogeny,founder effect and multiple comparisons by using phylogenetic dependencynetworks gag, pol andnef sequences from the combined Durban and Bloemfonteincohorts and then we carried out sub-group analyses on patients with either‘high’  or‘low’  CD4cell counts. We then selected those HIV amino acid polymorphisms significantlyassociated with an HLA Class I allele in either, or both of, the‘high’ or ‘low’ CD4 count group of patients for furtheranalysis. The results for gag, pol andnef are presented in 2-adjusted odds ratios for the ‘high’and ‘low’ groups. In the final column, the p value is reported, whenthere is a significant difference between the two odds ratios.Initially, we analysed 916 HIV-1 gag,pol and nef genes, in either, or both of,the ‘high’ or ‘low’ CD4 count subgroups. Of these, 16,28 and 7 associations in HIV-1 gag, pol andnef, respectively, had significantly differentlog2-adjusted odds ratios in the two subgroups. These 51associations are shown in gag,pol and nef, respectively, there were 5/16(31%), 24/28 (86%) and 7/7 (100%) associations that weremore prevalent in the ‘low CD4’ group. These data show that, wherewe could detect a difference, associations were more prevalent at lower CD4counts in pol and nef, suggestive of continuedselection pressure, but that certain associations – particularly in HIV-1gag – were weaker. Why might HLA associations becomeweaker as disease progresses? One possible explanation is that as CTL pressureweakens due to immune exhaustion and loss of breadth, escape mutations withreplicative fitness costs are no longer required by the virus. These thereforerevert back to wild-type, resulting in a reduction in the observedlog2-adjusted odds ratio. Thus, low-cost mutations accumulateover the course of infection, leading to higher prevalence in Nef and Pol,whereas high-cost mutations begin reverting, leading to a lower prevalence inGag.We identified 40, 91 and 20 associations, respectively, between an HLA Class Iallele and a viral polymorphism in the HIV-1 gaggene was associated with the most potential examples of immune relaxation weimplemented a fitness assay targeting the gag gene and focussedon three beneficial HLA Class I alleles  and one allele associated with more rapid progression, HLAB*5802. In particular, we highlighted two associations: the T186S mutationin the TPQDLNTML (TL9) epitope, restricted by B*8101, with an observeddecrease in log2-adjusted odds ratio from 18.38 to 7.25 in patientswith high and low CD4 cell counts, respectively, , and theB*57/B*5801 restricted epitope TSTLQEQIAW (TW10), withlog2-adjusted odds ratios of 25.4 and 23.2 for high and low CD4 cellcount stratification analysis . We focussed onthese two associations as published data suggest that they are both associatedwith significant viral fitness costs 2-adjusted odds ratios.If changes in selection pressure were resulting in an increase or decrease in theprevalence of wild type virus during late-stage disease, we would expect to findchanges in viral replicative capacity in patients with low CD4 cell counts. Wetherefore undertook assays of viral fitness. As the HIV gag-protease mutations selected by differentHLA class I alleles at different clinical stages, we constructed 148 chimericHIV-1 NL4-3 viruses containing autologous gag-protease frompatients of the Bloemfontein cohort. Patients with specific HLA class I allelespreviously associated with control of HIV in this population(HLA-B*57/5801/8101) or lack of control (B*5802) gag-proteaseplasmid recombined with the wild type HIV-1 NL4-3 gag-proteaseinsert.To investigate further possible reasons for declining selection pressure withinGag epitopes with progression to AIDS, we considered the hypothesis that loss ofCD8+ T-cell responses as CD4 count declines results in reversion of escapemutants, with resulting increase in viral replicative capacity. To investigatethe impact of the Initially we determined whether CD4 cell count alone was associated with VRC.10 plasma viral load .The slope of the best-fit values (0.029±0.0079) indicated that a34.4% increase in VRC accounted for an increase of one log10in plasma viral load.There was also a weak but statistically significant positive correlation betweenVRC and logAs higher VRCs were associated with higher plasma VL and lower CD4 cell count, weexamined how this effect varied according to HLA Class I . The VRCTo determine whether the association between HLA Class I and viral fitness wasadditionally impacted by disease stage we stratified the VRCs according to highand low CD4 cell counts . This suThese analyses have shown that viral fitness is greater in viruses from patientswith advanced disease, and that certain protective HLA Class I alleles  are associated with greater fitness costs.The HLA-association analysis presented above ,2 and 3 For HLA-B*8101, we excluded from the analysis any viruses with key mutationsrestricted by other HLA Class I alleles  which wereknown to impact fitness. Viruses from patients with HLA B*8101 weresignificantly less fit than viruses from patients in the rest of the cohort Together these data suggest that advanced HIV disease is associated with anincrease in viral fitness, which may contribute to the rise in plasma viral loadfrequently seen in AIDS. However, this increase in fitness is multifactorial andwe report two processes specific to different HLA Class I alleles –reversion due to immune relaxation and compensatory mutations – topotentially explain these findings.In this study, we have explored the hypothesis that progression to AIDS is associatedwith a rise in viral replicative capacity, and the mechanisms associated with this.We proposed that a rise in viral fitness in AIDS could be due either to a relaxationof CTL-imposed selection pressure resulting in the reversion of costly mutations or,alternatively, if immune pressure is maintained, compensatory mutations mightrestore the fitness costs of persisting escape mutations.We initially tested the hypothesis by looking for evidence of changes in Tcell-imposed selection pressure measured by gamma interferon ELISPOT assays. Wefound that the breadth of the response narrowed as the CD4 count declined and that,specifically, the contribution of Gag responses to the overall response decreased.It has previously been reported that acute HIV infection is associated with narrow,high affinity T cell responses which broaden as patients progress into chronicinfection Although we identified a narrowing of the T cell responses in AIDS, the ELISPOT dataalone are not sufficient to infer changes in selection pressure. We therefore lookedfor evidence of differential selection in patients with high and low CD4 cell countsusing a statistical HLA Class I association study. By combining sequence and HLAdata from the Bloemfontein cohort and the Durban cohort, we were able to define twogroups of patients with CD4 counts less than 100 cells/µl(n = 196) and greater than 500 cells/µl(n = 299). These CD4 count strata were defined to representextremes of HIV-related disease without reducing the power of the analysis. The WHOhave defined that antiretroviral therapy should be commenced at a CD4 count of 350cells/µl. Below 200 cells/µl, opportunistic infections become morecommon. A count of 100 cells/µl or below, therefore, represents advanced HIVdisease and progression to AIDS. Above 500 CD4 cells/µl it would be highlyunusual for there to be opportunistic infections and the most recent NIH guidelinesdo not recommend treating above this level 2-adjusted odds ratios.To identify mutations in the HIV genome associated with individual HLA Class Ialleles we used an established technique using phylogenetic dependency networksgag, pol or nef that weresignificant in patients with either high or low CD4 cell counts. By comparinglog2 adjusted odds ratios in the CD4 strata, we found that there wasevidence for both immune relaxation and intensification in patients with low CD4counts compared with high CD4 counts. This suggests that for some HLA ClassI–restricted responses the selection pressure imposed by CTL is maintained atvery low CD4 cell counts but that for others there may be reversion of escapemutations associated with a reduction in selection pressure. Interestingly, themajority of events compatible with immune relaxation were in HIV Gag, which was alsothe protein for which there was a relative narrowing of T cell ELISPOT responses inAIDS patients.In our cohorts we identified 151 HLA-associated mutations in HIV-1Two key sites potentially reflecting immune ‘persistence’ and‘relaxation’ were the HLA B*57/B*5801 associated T242N mutationand the B*8101 associated T186S mutation, respectively. For the former, theassociation analysis provided evidence for on-going mutation associated withcompensatory mutations, whereas for the latter there was evidence for reversion. Alimitation of this study is that it is cross-sectional rather than longitudinal,potentially biasing our data interpretation. An alternative explanation for immunerelaxation is that the patients who progress are the ones who do not make an immuneresponse and so do not select mutants, therefore enriching patients with low CD4counts with wild-type viruses. This would be compatible with the finding thattargeting certain key epitopes may be associated with different speeds of clinicalprogression gag-protease was inserted into agag-protease-deleted pNL4-3 plasmid. The advantage of thisapproach is that it uses the full gag gene from the patientsequence as well as the associated protease that encodes the enzymefor cleavage of the Gag-Pol protein. The disadvantage of this assay is that it is ina parallel rather than competitive format, and therefore may lack sensitivity forsmall fitness differences. In addition, the construct is a recombinant of a subtypeB backbone with a subtype C insert. Although this approach has been publishedelsewhere gag-protease) into which werecombined the pNL4-3 gag-protease. This controlled for any changein fitness due to the recombination step.Viral fitness was measured using a recombinant assay in which a patient-derivedRT-PCR amplicon of HIV-1 ex vivo assayreflects viral replication in vivo. Analysing our data by HLA ClassI revealed that HLA Class I alleles that are associated with clinical advantage were associated with impaired viral fitness, ashad been inferred from previous analyses of the reversion of transmitted escapemutations in HLA mismatched hosts We found that VRC was significantly greater in patients with low CD4 cell counts andthere was a significant, although weak, correlation with plasma viral load. Thesedata, which are consistent with a population-based study of HIV subtype B-infectedindividuals The HLA-amino acid association analysis showed that for HLA B*8101 there was adecrease in mutations in the Gag TL9 epitope at low CD4 cell counts, whereas for HLAB*57/B*5801 there was persistence of the T242N escape mutation in TW10. Thefitness assays showed that the T186S mutation in TL9 was associated with a fitnesscost (P<0.0001) and it is therefore possible that in patients with B*8101 andlow CD4 counts, the decrease in prevalence of the T186S mutation reflects reversionto a fitter strain coincident with the weakening of T cell responses. ForB*57/B*5801 we saw a different pattern. Here, possession of HLAB*57/B*5801 was associated with less fit viruses in conjunction with theT242N mutation in the Gag TW10 epitope, but in patients with low CD4 counts fitnesswas restored even though T242N persisted. We therefore sought compensatory mutationsto explain this finding. For T242N there are four documented compensatory mutationsat Gag codons 219, 223, 228 and 248 These data show that viral fitness increases with progression to AIDS. Whether thisis cause or effect is difficult to determine, although the fact that we see evidencefor both reversion and compensatory mutations in the same cohort indicates thatprogression may be a mixture of the two. What is clear is that viral fitness is asignificant component of progression to AIDS and, as such, should be considered as atarget for intervention, for example through vaccines aimed at epitopes which escapewith high fitness costs. Trials such as DART have shown that antiretroviral therapycan be extremely effective in regions such as sub-Saharan Africa