We used demographic and clinical data to design practical classification models for prediction of neurocognitive impairment (NCI) in people with HIV infection.The study population comprised 331 HIV-infected patients with available demographic, clinical, and neurocognitive data collected using a comprehensive battery of neuropsychological tests. Classification and regression trees (CART) were developed to obtain detailed and reliable models to predict NCI. Following a practical clinical approach, NCI was considered the main variable for study outcomes, and analyses were performed separately in treatment-naïve and treatment-experienced patients.The study sample comprised 52 treatment-naïve and 279 experienced patients. In the first group, the variables identified as better predictors of NCI were CD4 cell count and age . In treatment-experienced patients, the variables most closely related to NCI were years of education, nadir CD4 cell count, central nervous system penetration-effectiveness score, age, employment status, and confounding comorbidities . In patients with an undetectable viral load and no comorbidities, we obtained a fairly accurate model in which the main variables were nadir CD4 cell count, current CD4 cell count, time on current treatment, and past highest viral load .Practical classification models to predict NCI in HIV infection can be obtained using demographic and clinical variables. An approach based on CART analyses may facilitate screening for HIV-associated neurocognitive disorders and complement clinical information about risk and protective factors for NCI in HIV-infected patients. Neurocognitive impairment (NCI) is a concern in HIV infection. Between one-third and two-thirds of HIV-infected individuals experience this complication Neuropsychological test batteries are widely recommended for assessment of neurocognitive status in HIV-infected persons Demographic and clinical data are easily collected and could help to identify NCI during the routine care of HIV-infected patients, since they provide crucial information not only about physical status, but also about central nervous system (CNS) functioning The study was based on data from 331 HIV-infected outpatients attended at 7 HIV units in Barcelona, Catalonia, Spain. Individuals were selected because they had voluntarily undergone a comprehensive neuropsychological test battery, which was offered routinely in the daily clinical practice of the participating centres. Only participants with available demographic and clinical data were included. The remaining inclusion criteria were age ≥18 years and positive results for HIV infection in ELISA and Western-blot. Participants whose data were incomplete for any of the study variables were excluded. All participants provided their written informed consent to undergo neurocognitive assessment, and the Ethics Committee of Germans Trias i Pujol University Hospital  approved the study. Data were collected from September 2009 to March 2013.Demographic variables included age, gender, route of transmission, employment status, and years of education. Data were self-reported.The clinical variables collected were current CD4 cell count, nadir CD4 cell count, plasma viral load, past highest viral load, previous or no experience of antiretroviral therapy, current antiretroviral therapy, CNS penetration-effectiveness (CPE) score, previous treatment interruptions, time since HIV diagnosis, time since initiation of the first regimen, time on the current regimen, coinfection with hepatitis C virus (HCV), AIDS condition, and existence of comorbidities with the potential to affect neurocognitive performance. Data were retrieved from medical reports and local clinical databases at the study sites. The CPE score was calculated using the proposal by Letendre and collaborators Neurocognitive functioning was evaluated using a neuropsychological battery composed of 14 tests covering 7 domains that are recommended for assessment in HIV infection NCI was determined based on a neuropsychological criterion, which was defined as performing at least 1 standard deviation below the normative mean in at least 2 neurocognitive areas, as extensively reported elsewhere The classification of HAND was also used to describe the study sample and was obtained by applying the Frascati criteria We used a tree-structured approach to provide useful classification models for predicting NCI in HIV-infected individuals from a clinical practice perspective. Specifically, we used CART analyses, a methodology that generates rules for decisions involving categorical outcomes CART analyses are based on the successive selection of a variable and its value to subdivide the individuals comprising a sample so as to better classify them. Consequently, the criteria for subdivision are variables that make it possible to reduce the difference in scores. This approach has several advantages. First, CART analysis generates structured and visual classification rules, which facilitate understanding for the study findings. Second, the resulting outcomes depend on the different levels of accuracy. Third, as for quantitative variables, CART provides specific cut-off points that inform the user about key values that can affect the final outcome. Fourth, as a non-parametric method, CART analyses are not affected by the data distribution and thus enable rigorous assumptions to be established without the undesirable effects produced by outliers, collinearity, or heteroscedasticity. Therefore these characteristics make it possible to apply classification variables with different origins and a specific typology .In our study, the presence of NCI was the main study variable and was therefore considered a final endpoint in the classification models, namely, presence or absence of a disorder. The cross-validation method was used to determine the optimal size of the tree as a function of misclassification and of the cost-complexity parameter, depending on which the initial tree was pruned to obtain a simpler classification rule.Because we followed a practical clinical approach, models were elaborated separately for antiretroviral treatment-naïve and treatment-experienced patients, and we finally selected those with the greatest accuracy and simplicity according to the accuracy of classification found and the number of terminal nodes displayed.http://www.r-project.org).All study analyses were performed using SPSS for Windows, version 15.0  and R, version 2.11 , middle-aged . The simplest tree had 3 final nodes and included only CD4 cell count and age as predictive variables Figure 1A total of 279 patients had previously received antiretroviral therapy and were all currently on treatment. Most were men (83%) and middle-aged , with an undetectable viral load (86%), a median current CD4 count of 539 cells/µL (383–737), and a nadir CD4 count of 189 cells/µL (87–313). NCI was present in 140 individuals (50%), of whom 71 (53%) reported complaints. The remaining characteristics are displayed in The highest correct classification in the trees obtained was 83.5% in a model with 12 final nodes, including a total of 8 variables: years of education, CPE score, nadir CD4 cell count, age, employment status, years since HIV diagnosis, years on antiretroviral therapy, and presence of potential confounding comorbidities.Pruning the initial model provided a simple tree that did not substantially increase the misclassification rate. In a model with 7 final nodes and 6 variables, a correct classification of 82.1% was reached. The variables included in this tree were education, CPE score, nadir CD4 cell count, age, employment status, and existence of comorbidities Figure 2In our search for a homogeneous subset of patients, we also studied individuals with an undetectable viral load (n = 240). The simplest model from the trees comprised 3 nodes and 2 variables and had a 73.4% correct classification. Months on current antiretroviral treatment and years since HIV diagnosis were the variables included. NCI was not predicted when patients had been on current therapy for ≥5 months. In patients with <5 months on treatment, the number of years since HIV diagnosis determined the presence of NCI. Impairment was found in people diagnosed with HIV infection ≥13 years age, but not in those diagnosed <13 years ago Figure 3Analysis of an even more homogenous subgroup—individuals with an undetectable viral load and no presence of potential confounding comorbidities (n = 148)—revealed remarkable accuracy with a correct classification of 88%, included 6 final nodes, and comprised 4 main predicting variables: nadir CD4 cell count, current CD4 cell count, months on current treatment, and past highest viral load Figure 4We used a practical approach to analyze demographic and clinical data that could predict NCI in patients with HIV infection. Our data complement current knowledge on HIV-associated NCI, a major concern in the therapeutic management of HIV-infected patients. Different classification models were obtained, and we provide those that are potentially more useful in clinical practice, specifically in terms of accuracy and simplicity. Accuracy was based on the level of correct classification, and simplicity on the number of variables included in the models and the number of final nodes.In our sample of antiretroviral-naïve patients, the variables most significantly identified as predictors of impairment were CD4 cell count and age. CD4 cell count is widely acknowledged as an optimal marker of progression of HIV infection and our observations confirm this fact. As our approach was clinical, we applied graphical statistical methodology, which provided clear and specific visual study outcomes. Accordingly, classification trees obtained for naïve patients revealed cut-offs of 123 cells/µL and age 45 years. The value for the CD4 cut-off is consistent with previous findings indicating comparable links between worse immunological status and existence of neurocognitive disruption, particularly in advanced immunosuppression In patients on antiretroviral therapy, relevant parameters were years of education, nadir CD4 cell count, and CPE score. Education appeared at the first level of prediction. In fact, even in more complex models , level of education proved consistently relevant for determination of NCI. Greater education has been shown to act as a protective factor in the development of neurocognitive complications We performed separate analyses of patients with undetectable viral load and patients with an undetectable viral load and no confounding comorbidities in order to investigate further the mechanisms of HIV-associated NCI. Significant differences in time variables were found in patients with an undetectable viral load: time on current antiretroviral treatment and time since HIV diagnosis were the main predictors. Both variables have previously been shown to play a key role in HIV-related cognitive decline, with theoretical advantages for longer time on therapy and disadvantages for a more extended time since HIV diagnosis Our study has several limitations. First, although we aimed to obtain a large sample, our sample size was reduced by performing separate analyses of 2 groups (treatment-naïve and treatment-experienced patients) and because of the number of subjects studied for each subgroup in the final nodes. As mentioned above, the statistical methodology applied was based on CART; consequently, the sizes for the category groups were further reduced. A second limitation is the clinical variables analyzed. Because we used available medical data from the participating centres, valuable supplementary clinical information  was ignored for the purposes of the study. Examples include cardiovascular risk factors and hormonal markers, which are known to be associated with cognitive dysfunction in both the general population and HIV-infected individuals. Another limitation is the use of NCI as a primary outcome measure, instead of a specific HAND. This approach was adopted because of the clinical utility of the dichotomous character of NCI, and also due to the controversy surrounding the distinction between asymptomatic NCI (ANI) and mild neurocognitive disorder (MND). Nevertheless, the HAND classification would have made it possible to integrate valuable information about the severity of the impairment.Despite these limitations, and considering that our sample was from a single geographic area, the current work presents significant strengths. First, the models provided visual, easy-to-read outcomes that can be used immediately in clinical practice. Second, specific numeric cut-off points are offered for quantitative variables, also a major advantage in daily clinical practice. And third, different models are provided according to the level of accuracy and simplicity. Hence, clinicians and care providers are able to decide which models fit best in a specific setting. In fact, the degree of accuracy of the classifications was high. If we compare this feature with the accuracy provided by other screening tools for HIV-related NCI, it is clearly significant. Therefore, although the methodology presented should be seen as a novel approach when screening for HAND, the results generated can substantially help in the management and monitoring of neurocognitive complications in HIV-infected persons, particularly in those scenarios where clinical resources are limited. We fully recommend the application of neuropsychological test batteries for the rigorous assessment of neurocognitive functioning in HIV-infected individuals, but now we offer a potential screening method to identify the existence of risk and protective factors for NCI, considered as a supplementary resource for the therapeutic management of HAND.


Human immunodeficiency virus (HIV) infection is usually complicated by high rates of tuberculosis (TB) co-infection. Impaired immune response has been reported during HIV/TB co-infection and may have significant effect on anti-retroviral therapy (ART). TB/HIV co - infection is a major public health problem in Ethiopia. Therefore, the aim of the study was to assess the effect of TB incidence on immunological response of HIV patients during ART.A retrospective follow-up study was conducted among adult HIV patients who started ART at the University of Gondar Hospital. Changes in CD4+ T - lymphocyte count and incident TB episodes occurring during 42 months of follow up on ART were assessed. Life table was used to estimate the cumulative immunologic failure. Kaplan-Meier curve was used to compare survival curves between the different categories. Cox-proportional hazard model was employed to examine predictors of immunological failure.3 (adjusted hazard ratio (AHR) 1.8; 95%CI: 1.10 - 2.92, p = 0.023) and being male sex  were found to be significant predictors of immunological failure. There was borderline significant association with incident TB . The risk of immunological failure was significantly higher (38.5%) among those with incident TB compared with TB - free (21.1%) (Log rank p = 0.036).Among 400 HIV patients, 89(22.2%) were found to have immunological failure with a rate of 8.5 per 100 person-years (PY) of follow-up. Incident TB developed in 26(6.5%) of patients, with an incidence rate of 2.2 cases per 100 PY. The immunological failure rate was high (20.1/100PY) at the first year of treatment. At multivariate analysis, Cox regression analysis showed that baseline CD4+ T - cell count <100 cells/mm3 and being male sex were significant predictors of immunological failure. The result highlighted the beneficial effects of earlier initiation of ART on CD4+ T - cell count recovery.High incidence of immunological failure occurred within the first year of initiating ART. The proportions of patients with impaired immune restoration were higher among patients with incident TB. Lower baseline CD4+ T - cells count of <100 cells/mmThe online version of this article (doi:10.1186/1471-2334-14-468) contains supplementary material, which is available to authorized users. In 2012, about 8.6 million incident TB and 1.3 million deaths due to TB were reported globally. The majority of TB cases occurred in Asian (58%) and African (27%) countries [Despite recent advances in anti-infection rate was 20 - 50% [In developing countries, TB remains a major public health threat among HIV-infected individuals , 4. HIV 20 - 50% . Accordi20 - 50% .- cells and the functional restoration of patents’ immune response and decline in HIV viral load as well. However, the requirement of regular and lifelong medication of HIV patient is challenged with emergencies of treatment failure [- RNA which results ARV drug resistance [-retroviral treatment failure can be assessed clinically, immunologically and virologically. However, on the basis of clinical criteria treatment failure can’t be concluded. Despite viral load testing is preferred approach for monitoring ART response, in resource limited settings, immunological failure criteria, trends in CD4+ T - cells counts over time, remains the strongest predictor of treatment failure [With the advent of ARV drugs, HIV/AIDS has become a treatable chronic disease. Effective anti-retroviral therapy (ARV) therapy is usually convoyed by an increase in the number of CD4+ T  failure –10. Impasistance . HIV tre failure .- cells count during ART in HIV/TB co-infected patients is less clear. However, studies assessing immune responses to ART have found poor CD4+ T - cells recovery to occur in patients who develop incident TB after initiating ART [-lymphocytopenia has been observed in TB patients [M. tuberculosis-induced apoptosis of T - cells [- infected individuals. Although TB/HIV co-infection is a major public health problem in Ethiopia, no studies have reported the effect of TB on immunological responses of HIV patients during ART. Hence, assessing the effect of TB on immunological responses of HIV patients will provide information for clinicians for appropriate management of TB/HIV co-infected patients. Moreover, policy makers and health professionals can use the findings to design ART related programs. Therefore, the aim of this study was to assess the effect of incident TB on immunological response of HIV/AIDS patients during ART at the University of Gondar Hospital.The recovery of CD4+ T ting ART –16. Morepatients . An in v - cells . Therefo- cells counts, follow-up and supportive care of HIV-infected individuals. ART is provided free of cost to all HIV - infected individuals in need of treatment. The study populations were those patients who are 15 years old and above and ever started ART at the University of Gondar Hospital. Patients start ART based on WHO criteria for the initiation of ART in adults and adolescents; patients with CD4+ T - cell count <200 cell/mm3 or WHO stage 4 irrespective of CD4+ T - cell count or WHO stage 3 with CD4+ T - cell counts <350 cells/mm3. Routine monitoring of CD4+ T - cell counts is performed every six months, or more frequently if clinically indicated [-up visit through clinical evaluation and microbiological tests and chest-radiography based on WHO national guidelines. Patients diagnosed with incident TB were treated with a standard eight months regimen with two months intensive phase, combination of 4 drugs , and a subsequent 6 months continuation phase, 2 drugs  [- lymphocyte count measurements were performed by FACS count (Becton Dickinson) every 6 months or more frequently if clinically indicated after initiating ART.The study was conducted at the University of Gondar Hospital, North-west Ethiopia, in June 2013. University of Gondar Hospital is a referral hospital with more than 400 beds serving a population of about 5 million people in North-west Ethiopia. The hospital offers a wide range of services including voluntary counseling and testing, treatment, referral services, monitoring of treatment response with CD4+ T ndicated . All HIV-up time. Adult HIV patients whose charts were available and who had at least 6 months of follow - up (having at least two CD4+ T-cell measurements) and started on first line ART during the study period were eligible for the study. HIV/AIDS patients who had active TB  at the initiation of ART and missing charts or incomplete baseline and follow-up data were excluded from the study. Changes in CD4+ T - cells count and incident TB episodes occurring during the 42 months of follow-up on ART were assessed. Socio - demographic information of the patients and clinical characteristics such as time of ART start, baseline and follow-up CD4+ T - cell count, and WHO clinical stage, co-infection with TB, time of TB development and functional status were extracted from the hospital’s ART register. Baseline measurements of CD4+ T - cell count was included if performed within six months prior to ART initiation and once on treatment follow-up measurements was included if performed within ±2 months of the 6 month time points. Data extraction format was prepared and pretested on 20 charts. The data were extracted from patients’ charts by three nurses who had ART training and experience in HIV care.A retrospective follow-up study was conducted on patients initiating ART from the 1st of September 2007 and 30th of August 2008 at the University of Gondar Hospital. This period was selected to collect patient’s information from sufficient follow- cell count to pre -ART level or below, decrease in CD4+ T - cell count from on-treatment peak value by more than 50% or persistent CD4+ T - cell count <100 cells/mm3 after six months of therapy [- to - follow-up or didn’t show the event until the last visit was considered as censored.Prevalent TB was defined as patients taking anti-TB treatment at the time of starting ART. Incident TB was defined as a new active TB developed after initiation of ART. Immunological failure was defined based on WHO criteria: decrease in CD4+ T  therapy . Patient-Info version.3.5.3 then exported to Statistical Package for the Social Sciences (SPSS) version 20. Descriptive analyses were used to determine baseline socio-demographic and clinical characteristics of the patients. Life table was used to estimate the cumulative probabilities of immunologic failure. Kaplan-Meier survival curve was used to estimate the median survival time from initiation of ART to immunologic failure. Bivariate and multivariate Cox-proportional hazard model was employed to identify predictors of immunological failure. Hazard Ratio (HR) with 95% confidence intervals were computed and p-value < 0.05 was considered statistically significant for all cases.Immunologic failure rates were calculated per 100 person years at risk. Data were entered and cleaned by using Epi-identified prior to analysis. Individual records were coded and accessed only by research staff.The study was reviewed and approved by the Institutional Review Board (IRB) of the University of Gondar. Official permission was obtained from University of Gondar Hospital management. The patient records were anonymized and de-up data. Among 484 HIV patients who had at least 6 months of follow-up, 84(17.4%) had active TB at the initiation of ART and the analysis was restricted to those who did not have active TB at ART start. Therefore, a total of 400 adult HIV/AIDS patients record were analyzed. Of the 400 adult HIV/AIDS patients who met the inclusion criteria for the study, 315 (78.8%) remained within the programme at study censorship, 4 (1%) died, 67 (16.8%) transferred out and 14(3.5%) were lost to follow - up from the programme. Of 400 adult HIV/AIDS patients, the majority (64.5%) were females. The median age of patients at ART start was 33 years (Inter Quartile Range (IQR) = 27-40 years) and almost half of the patients were married (50.5%). One hundred seventeen (29.3%) of the study participants had no formal education and only 62 (15%) patients were employed . One hundred and twenty seven (31.8%) of patients had baseline CD4+ T - cell counts <100 cells/mm3. Majority of the patients started treatment with zidovudine (AZT)/lamivudine (3TC)/nevirapine (NVP) or efavirenz (EFV) (47.3%) followed by tenofobir (TDF)/3TC/NVP or EFV (40.5%) and stavudine (D4T)/3TC/NVP or EFV (12.3%) based combination first line ARV drug regimens. Three hundred and forty (85%) patients were working by their functional status  of patients were at WHO clinical stage III. At the time of ART initiation, patients had advanced immunodeficiency with the median baseline CD4+ T % of pati- cell counts performed per patient during the follow -up period was 7 (IQR = 6 - 8). Of the 400 HIV/AIDS patients who had at least 6 months of follow -up, 85(21.3%) patients were lost to follow - up before the study censorship. Eighty nine (22.2%) of the patients were found to have immunological failure. Based on WHO criteria, 49(55%) immunological failure were defined by decrease in CD4+ T - cell count to pre -ART level or below, 12(13.5%) decrease in CD4+ T - cell count from on-treatment peak value by more than 50% and 28 (31.5%) persistent CD4+ T - cell count below 100 cells/mm3. The median time for occurrence of immunological failure was 6 months (IQR = 6 - 12 months). The median time for immunological failure was 6 months for both TB and TB-free cohorts. Among all 89 immunological failures, 60(67.4%) patients were failed at 6 month of follow-up on ART. The overall immunological failure rate of our cohort was 30 per 100PY at the end of 6 months, 20.1 per 100 PY at the end of one year, 16.2 per 100 PY at the end of 18 months, 13.0 per 100 PY at the end of two years, 11.1 per 100 PY at the end of 30 months, 10.8 per 100 PY at the end of three years and 8.5 per 100 PY at the end of 42 months of follow-up. The cumulative probability of survival of patients from immunological failure at the end of 6 months was 85% while at the end of one year was 79%, at the end of three years 78% and at the end of 42 months 75% . The median number of CD4+ T -up. The median time for development of incident TB during the follow-up was 9.5 months . Among those who developed TB during the follow-up time, 17(57.7%) of the incident TB occurred within the first year of initiating ART. After 42 months on ART, the risk of immunologic failure was 38.5% versus 21.1% among patients with and without incident TB respectively (log rank p = 0.036)  patients within 42 months on ART, with an incidence rate of 2.2 cases per 100 PY of follow- cell count <100 cells/mm3 when compared to those CD4+ T - cell count of 100 cells/mm3 and above   1.7, 95% CI: 1.11 - 2.64, p = 0.015) and being male sex  were found to be significant predictors of immunological failure. Even though the survival from immunological failure was significantly lower among patients with incident TB (p = 0.036), Cox regression analysis showed borderline significant association between incident TB and immunological failure   which is concordant with findings reported in other African countries . This mi0 PY whicresponse , 21–23.-free for the same follow-up time on ART. However, in the multivariate Cox regression analysis we observed borderline significant association between incident TB and immunological failure. This may be due to relatively small number of cases with incident TB. Different studies reported that despite TB treatment patients who developed TB after ART initiation were more likely to have lower immune recovery. Studies on South African [- cell responses. Furthermore, a study from Senegal indicated that independent of HIV status development of incident TB causes severe CD4+ T-lymphocytopenia [- cell counts at enrolment to an ART programme is associated with increased risks of TB and of mortality during the first year of ART [- cell counts may expose patients to an increased risk of developing incident TB which can in turn contribute to immunological failure. Another explanation for the lower CD4+ T - cell recovery in patients with incident TB during ART may be due to decreased adherence to ART during TB treatment because of high pill burden and side-effects [In this study, the log rank test showed that the immunological failure free survival proportion was significantly lower among patients with incident TB when compared to those patients remaining TB African  and Ugan African  cohorts ytopenia . These fr of ART , 7. In t-effects .- cell counts were independently associated with immunological failure. This is supported by other studies conducted in Debremarkos, Ethiopia [- cell counts. Moreover, this finding is concordant with other reports where immune restoration is largely dependent on baseline CD4+ T - cell count and thus the timing of ART initiation is important in order to optimize the CD4+ T - cell response to therapy [- cells count at baseline have poor long term CD4+ T - cells responses. Therefore, our finding supports the new WHO 2013 recommendations of expanding eligibility for ART initiation to CD4+ T - cell counts ≤500 cells/mm3 for all adults and children above 5 years [The most important finding of our study was that in multivariate Cox regression analysis lower baseline CD4+ T Ethiopia  and ThaiEthiopia , 22 in w therapy . These r 5 years .Although the causal relationship is not known, keeping the report from Uganda  immunolo- cell count recovery of HIV patients during ART prospective cohort study should be considered.As a retrospective design our study had limitations. We excluded a large number of patient charts that could not be verified by chart review due to unavailability of full information which may have introduced bias. Moreover, because of the nature of the study design, it was difficult to control all possible confounders like body mass index and opportunistic infections other than TB. The sample size was also too small to include representative incident TB cases and with large sample size the effect of TB on immunological failure could be more elaborated. Given that virologic response is the preferred standard for monitoring ART, this study only assessed immunological responses of the patients and as a result that immunological failure could be related to viral resistance or non adherence. Therefore, for better understanding of the association between incident TB and CD4+ T - cell count of <100 cells/mm3 and being male sex were found to be significant predictors of immunological failure. The timing of initiation of ART was major determinants of the change in CD4+ T - cells count recovery. The result highlighted the beneficial effects of earlier initiation of ART on CD4+ T - cells recovery and strengthening early detection of TB through active screening and appropriate management of TB patients.High immunological failure rate in our cohort was occurred at six months of ART enrolment. The proportions of patients with impaired immune restoration were significantly higher among patients who developed incident TB during ART. Lower baseline CD4+ T


Access to antiretroviral treatment (ART) in resource-limited countries has increased significantly but scaling-up ART into semi-rural and rural areas is more recent. Information on treatment outcome in such areas is still very limited notably due to additional difficulties to manage ART in these areas.387 HIV-1 infected adults (≥18 years) were consecutively enrolled when attending healthcare services for their routine medical visit at 12 or 24 months on first-line ART in five HIV care centers . Among them, 102 patients were on first-line ART for 12 ± 2 months (M12) and 285 for 24 ± 2 months (M24). Virological failure was observed in 70 (18.1 %) patients ranging from 13.9 to 31.6 % at M12 and from 8.1 to 22.4 % at M24 across the different sites. For 67/70 patients, sequencing was successful and drug resistance mutations were observed in 65 (97 %). The global prevalence of drug resistance in the study population was thus at least 16.8 % (65/387). Moreover, 32 (8.3 %) and 27 (6.9 %) patients were either on a completely ineffective ART regime or with only a single drug active. Several patients accumulated high numbers of mutations and developed also cross-resistance to abacavir, didanosine or the new NNRTI drugs like etravirine and rilpivirine.The observations on ART treatment outcome from ART clinics in semi-rural areas are close to previous observations in Lomé, the capital city suggesting that national ART-programme management plays a role in treatment outcome. Scale-up of antiretroviral treatment (ART) programs in resource-limited countries was possible because standardized first and second line antiretroviral (ARV) combinations and clinical and/or immunological criteria to start and monitor ART were used as recommended by WHO . HoweverIn Togo, a country of six million inhabitants in West Africa, scaling-up of ART started in 2007 in Lomé, the capital city, and has expanded to semi-rural areas in 2008. In 2013, almost 50 % of patients who were in need for ART according to WHO guidelines from 2010 (CD4 count <350) were receiving ART . Given tA cross-sectional study was conducted in 2012 between January and July in five HIV care centers that administer ARV drugs and monitor treatment. They were located in four semi-rural cities: Aného (AN), Kpalimé (KP), Atakpamé (AT) and Kara (KA) at respectively 60, 120, 160 and 410 km from Lomé, the capital city, and in one rural city, Kouvé (KO) at 70 km distance from Lomé  was determined with EasyQ HIV assay  or RealTime m2000rt  in Lomé (BIOLIM/FSS-UL). According to WHO recommendations, genotypic drug resistance testing was done in a WHO accredited laboratory  on patients with VL ≥1000 copies/ml. Protease and partial Reverse Transcriptase (RT) were amplified with the in-house protocol from the Agence Nationale de Recherche sur le Sida et les Hépatites en France (ANRS) . Drug re3 was 176 (IQR 86–261) and 152 (IQR 88–219) for the patients who were on ART for 12 ± 2 and 24 ± 2 months, respectively. Overall, 153/373 (41.1 %) and 220/373 (58.9 %) of the patients were in WHO stages 1 or 2 and WHO stages 3 or 4, respectively at ART start. However, CD4 counts and WHO stages at ART initiation could vary across the different sites  or 24 ± 2  months on ART. Table Seventy patients  had VL >1000 copies/ml; 20/102  at M12 and 50/285  at M24. Virological failure ranged from 13.9 to 31.6 % at M12 and from 8.1 to 22.4 % at M24. For 67 (95.7 %) patients, sequencing was successful and DRM were observed in 65 (97 %) of them; i.e. 20/20 at M12 and 45/47 at M24. Among the 65 drug resistant HIV strains, 59 were resistant to NRTIs and NNRTIs, two to NRTIs only and four to NNRTIs only. The global prevalence of drug resistance in the study population was thus at least 16.8 %  (65/387), but 27 patients  were infected with HIV strains resistant to two of the three first-line ARVs and 32  to all three first line ARVs.As expected, the observed DRM were associated with the drugs used in first-line regimens Table . M184 V Overall, we showed that 18 % of patients on ART for 12 or 24 months in semi-rural and rural ART clinics were on virological failure and almost all of them (97 %) were infected with drug resistant HIV-1 strains. These observations are high and close to what has been noticed in previous studies from Lomé, the capital city; for example in a survey conducted about 1 year earlier in 2010/2011 using the same cross-sectional approach, 19 % (124/642) of patients for 12 or 24 months on ART were infected with HIV drug resistant strains [Today, only very few studies reported observations on ART outcome from semi-rural or rural areas in resource limited settings, especially from Africa , 8, 9. IOverall it seems that treatment outcome varies among countries, but within countries treatment outcome in semi-rural settings seem to be similar to those in urban settings except in extreme conditions. These observations are in line with the fact that national ART-programme management plays a role in treatment outcome in resource-limited countries . It is iLike in other reports on treatment outcome, several patients in our survey accumulated high numbers of mutations and developed also cross-resistance to potential second and/or third line drugs –18. In aThe observations on ART treatment outcome in semi-rural areas show high failure rate but are close to those in Lomé, the capital city. Lowering the rates of drug resistance represents a challenge for the country. The first goals will be to identify factors associated with drug resistance.


We assessed the virological response of DRV/r-based dual therapy in drug-experienced patients included in the Italian antiretroviral resistance database (ARCA).Patients included in the study were treated with DRV/r in association with raltegravir (RAL), etravirine (ETV) or maraviroc (MAR) following treatment failure(s) and with a resistance test and at least one follow-up visit available. Observation was censored at last visit under dual therapy and survival analysis and proportional hazard models were used, taking virological failure (confirmed >50 c/mL HIV-RNA) as the end-point.Of the total 221 patients included, 149 (67.4%) started DRV/r with RAL, 45 (20.4%) with ETV, 27 (12.2%) with MAR. Patients characteristics at the start of dual regimen were as follows: mean number of previous regimens, nine (IQR: 5–13); non-B subtype, 17 (7.7%); median CD4 count, 347 (IQR: 246–544); undetectable viral load, 74 (33.5%). Full DRV/r resistance was detected in one , 13  and 17 patients . 69 virological failures (31.2%) were observed during follow-up. At survival analysis, the overall proportion of failure was 29.2% at one year and 33.8% at two years. The proportion of failure was lower in patients starting with undetectable versus detectable viral load  and in patients treated with DRV 600 BID versus 800 QD . By regimen, patients treated with DRV/r-RAL showed a non-significant lower proportion of failure  if compared with DRV/r-MAR  and DRV/r-ETV . In the adjusted proportional model, no significant difference among the three regimens was detected. A significant lower risk of failure was associated with higher overall GSS , while a higher risk of failure was associated with detectable HIV-RNA .Among experienced patients, the best candidates to dual-therapy regimens including DRV/r are those with undetectable viral load and higher GSS. The association with RAL is the most commonly used but no clear advantage with respect to ETV or MAR was observed in our dataset, possibly due to the limited sample size.


In resource-limited countries, NNRTI-based regimen may result in emergence of more HIV drug resistance because of a low genetic barrier. We compare the efficacy and tolerance of LPV/r and NVP-based regimens and 2 WHO nucleoside backbones in naive HIV infected patients (p.).Naive p. from 5 clinics in Lubumbashi (Congo-DRC) were randomized to receive LPV/r versus NVP combined with TDF/FTC or ZDV/3TC. VL and CD4 were performed at baseline (BL) and every 24 weeks (W). The primary endpoint was the % of p. with therapeutic failure defined as clinical and virologic failures (VL>1000 c/ml)(missing data=failure), assessed at W48 and 96. We present here the results of 144 W of follow-up.425 Black African p.  age 38 years, md CD4 165/µL; md VL 5.2 log c/ml) were randomized . BL characteristics were comparable. In the ITT analysis, previous results showed no difference between LPV/r and NVP treatment arms at W96 except a higher proportion of virologic failure (VF) in p. on NVP-based regimens. W144 ITT analysis showed a significant difference on endpoints between LPV/r (94/216) and NVP (111/209)(p=0.0479) and persistence of a significant difference in VF rate (p= 0.015). BL genotypes showed NNRTI mutations (mt) in 3/31 NVP-failing p. and no PI mt in LPV/r-failing patients. At time of failure, NNRTI mt were seen in 23/26 NVP-failing p. and 0/13 primary PI mt in LPV/r failing patients. NRTI mt were seen in 19/26 p. in NVP arm (including K65R in 7p. and M184V in 18p.) vs 3/13p. in LPV/r arm (M184V in 3p.).Md CD4 change from BL was significant higher in LPV/r arm (251 cells/µL ) compared with NVP arm (174 cells/µL [IQR 102-330])(p= 0.0093). Percentage of p. with adherence >95% was similar (73.6 vs 74.4 for LPV/r vs NVP).In a resource-limited setting after 144 weeks of follow-up NNRTI-NRTI first-line regimen is associated with more virologic failure, more drug resistance mutations and a lower immunologic response than a PI-based regimen.


High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI.Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the  Highly Active Antiretroviral Therapy (HAART) has been a significant advance for the treatment of HIV infection; however, problems that emerge during its administration, such as chronic toxicity, poor adherence and costs, are factors contributing to treatment failure and limiting its long-term success. Recently, evaluation of Structured Treatment Interruptions (STI) has been proposed as an alternative therapeutic strategy to reduce complications associated with continuous HAART administration . Based oTwo HIV-1 infected children with a viral load (VL) undetectable by HAART (<400 copies/ml), without immunosuppression according to the 1994 CDC classification  and HIV 9 molecules/μL and a working dilution of 1 x 107 molecules/μL was prepared. For the emPCR, a 0.66:1 ratio of library molecules was mixed with DNA capture beads following the emPCR Lib-A protocol from Roche . The enriched DNA-capture beads were sequenced in GS Junior Titanium Series equipment (Roche) following manufacturer's instructions.Viral RNA was isolated from plasma samples collected at the end of each HAART interruption period  using the MagMax Viral RNA Isolation kit . Reverse transcription was performed with Super Script III First-Strand Synthesis System  and random hexamer primers according to manufacturer’s instructions. Using the HXB2 reference sequence (GenBank K03455.1), a set of five pairs of primers was designed to amplify overlapping regions of the protease and the first 1,348 nucleotides of the reverse transcriptase. Each one consisted of the Universal Sequence 454, A or B, at its 5' end required for labeling the amplicons of each sample prior to the emulsion-PCR reaction (emPCR) and the complementary region to the HIV-1 sequence at the 3' end . The ampThe sequencing analysis was made using the GS-Amplicon Variant Analyzer (AVA) software (v 2.8.0). Sequences were demultiplexed using either 5’ or 3’ MID barcodes. AVA was then used to generate alignments based on error corrected consensus reads for each sample. An in-house perl code was used to call variants at the codon level. A minimal coverage of 500 reads per position was required for further analysis to ensure maximal probability of detecting low-abundance DRM’s. In addition, according to sequencing strand-dependent homopolymer related error patterns and routine negative control results, only those variants showing frequency values on forward and reverse reads within a 1 Log ratio and an overall frequency greater than 1.0% were considered true variants and used for downstream analysis. A variant list containing all drug resistant mutations was reported in the Stanford HIV Drug Resistance Database algorithm  through the HIVdb Sierra interface.For the purpose of discussion in this paper, we used the term “low-abundance” DRM’s to define mutation that can be detected at <20% of viral quasispecies and “high-abundance” DRM’s as mutations detected at ≥20% of viral quasispecies through ultra-deep sequencing .Two children were included in the study. Patient No. 1 was a 13.9 year-old female with immune category B3. Patient No. 2 was a 15 year-old male with immune category B2. Both patients acquired HIV-1 infection perinatally and they were receiving zidovudine (AZT) + lamivudine (3TC) plus ritonavir (RTV) as a HAART regimen at the recommended pediatric dosages. Full-dose RTV as the only protease inhibitor (PI) was still recommended for the initial HAART regimen in children in Mexico at the time this study began .There were no symptoms related to HIV infection and no adverse events related to HAART were observed during the follow-up of the patients. In both patients, the third viral rebound was significantly lower than first and second. VL always fell under detection limits after the therapy was restarted. According to the absolute CD4+ T-Cell counts, Patient No. 1 experienced moderated immunosuppression at the second interruption period (361 cells/ml); however, subsequently counts of these cells tended to increase reaching 791 at the end of follow up. Patient No. 2 experienced severe immunosuppression at the first interruption period (147 cells/ml), but it was transitory reaching afterwards CD4+ T-Cell counts similar than baseline .pol gene, including the complete protease-coding region (residues 1–99) and part of the reverse transcriptase-coding region (residues 1–448) were analyzed in the viral rebounds generated in patients during interruption periods of the STI program. The dataset have been deposited in the NIH/SRA repository with the accession number PRJNA284433. Genotypes for resistance prediction were obtained using the HIVdb of the Stanford University [Amplicons covering part of the nucleotide sequences of the iversity . No mutaAntiretroviral drugs represent a significant progress in the treatment of HIV infection; however, problems that emerge during their administration limit their long-term benefits . In pediIn a previous study by our research group , four chThe L63P, V77I, and I93L mutations in the protease region were found in patient No. 1 and L63P and I64V in patient No 2. The HIVdb genotypic resistance interpretation algorithm classifies these mutations as “other mutations” and refers them as accessory mutations. Such mutations are not associated with antiretroviral resistance. The L63P mutation is a common polymorphism in patients receiving PI; V77I is a common polymorphism associated with treatment with Nelfinavir (NFV) and the I93L mutation is also a common polymorphism associated with PI therapy in subtype B viruses. The HIVdb does not show comment for the I64V mutation. However, resistance mutations in the protease gene are classified as “major” or “minor” in the 2014 Update of the Drug Resistance Mutation in HIV-1 from the International Antiviral Society-USA (IAS-USA) . Accordiin vitro and/or in vivo and is associated with minimal or no reduction in the susceptibility to ETR. In the first rebound of this patient, the V106I mutation was detected, which is a polymorphic mutation selected by the EFV therapy and which, in combination with V179D, can reduce susceptibility to NVP and EFV. The A62V mutation, found in the third rebound of patient No. 2, is the only one associated with NRTI resistance but it requires the presence of the changes K65R and Q151M or the T69 insertion to reduce susceptibility to NRTI. Alone, A62V has no effect on the susceptibility to antiretroviral drugs, and although it is indicated as a resistance mutation to NRTI, the viruses that present it are susceptible to all antiretroviral drugs.On the other hand, several mutations associated with low-level or intermediate-level resistance to NNRTI were found in the viral rebounds of both patients. In two viral rebounds of patient No. 1, the K101E mutation was found, which causes intermediate resistance to Nevirapine (NVP) and low-level resistance to Efavirenz (EFV) and Etravirine (ETR) and, in combination with M184I, causes intermediate resistance to Rilpivirine (RPV). The mutation was maintained at low-abundance and it never surpassed 2% of the reads, despite the cycles of treatment interruption/restart in this patient. In the third rebound of the same patient, the V108I mutation was detected, which is selected by NVP, EFV and ETR and may slightly reduce the susceptibility to NVP and EFV. The E138A mutation detected in the three viral rebounds of patient No. 2 is a common polymorphic change, which can be selected for the treatment with ETR and RPV. This mutation may slightly decrease susceptibility to ETR and RPV. The V90I substitution, found in the first and third viral rebounds of patient No. 2 is a common accessory polymorphism that may be slightly selected for the treatment with NNRTI Since mutations K101E in patient No. 1 and E138A and V90I in patient No. 2 were present from the first period of interruption. It is thus possible that these mutations were not generated during the interruption/restart periods of HAART, but they were already present before patients be included in the study; however, no genotyping study was carried out prior to initiating the STI program to ensure this. In addition, V108I in patient No. 1 and A62V in patient No. 2 emerged as low-abundance DRM’s at the third viral rebound of the STI program, so subsequent evaluations are needed to monitoring the behavior of these mutations. Importantly, the mutations related with resistance to NNRTI detected are generally selected by the treatment with the same NNRTI and both children in our study never received and were not receiving such antiretroviral drugs.In the present study, not any HIV-infection related symptom or HAART related side event was identified, such as we observed in our previous study that included four pediatric patients submitted to the same STI program . SimilarIn summary, it could be concluded that the STI program did not lead to the development of any meaningful DRM’s which suggests that the detected mutations do not represent a risk for virologic failure during subsequent therapy in patients. Thus, this study highlight the need to continue studies about the impact of STI in pediatric patients with a strict and careful planning and with a greater number of interruption/restart cycles to evaluate the behavior of DRM’s that can emerge.This study was approved by the institutional research and ethics review board of the Hospital de Especialidades No. 25, Instituto Mexicano del Seguro Social, Monterrey, Mexico.


In Tanzania, HIV-1 RNA testing is rarely available and not standard of care. Determining virologic failure is challenging and resistance mutations accumulate, thereby compromising second-line therapy. We evaluated durability of antiretroviral therapy (ART) and predictors of virologic failure among a pediatric cohort at four-year follow-up.This was a prospective cross-sectional study with retrospective chart review evaluating a perinatally HIV-infected Tanzanian cohort enrolled in 2008-09 with repeat HIV-1 RNA in 2012-13. Demographic, clinical, and laboratory data were extracted from charts, resistance mutations from 2008-9 were analyzed, and prospective HIV RNA was obtained.161 (78%) participants of the original cohort consented to repeat HIV RNA. The average age was 12.2 years (55% adolescents ≥12 years). Average time on ART was 6.4 years with 41% receiving second-line (protease inhibitor based) therapy. Among those originally suppressed on a first-line (non-nucleoside reverse transcriptase based regimen) 76% remained suppressed. Of those originally failing first-line, 88% were switched to second-line and 72% have suppressed virus. Increased level of viremia and duration of ART trended with an increased number of thymidine analogue mutations (TAMs). Increased TAMs increased the odds of virologic failure (p = 0.18), as did adolescent age (p < 0.01).After viral load testing in 2008-09 many participants switched to second-line therapy. The majority achieved virologic suppression despite multiple resistance mutations. Though virologic testing would likely hasten the switch to second-line among those failing, methods to improve adherence is critical to maximize durability of ART and improve virologic outcomes among youth in resource-limited settings.The online version of this article (doi:10.1186/s12879-014-0567-3) contains supplementary material, which is available to authorized users. An estimated 3.4 million children between 0-14 years of age are living with HIV, of whom 91% live in sub-Saharan Africa ,[3]. DesTherapeutic monitoring poses an additional challenge as HIV-1 RNA  testing is frequently unavailable in resource-limited settings. Unsuppressed viremia leads to accumulation of resistance mutations and patients monitored by clinical or immunologic criteria tend to have increased durations of unsuppressed viremia compared to those monitored with viral load . DespiteIn Tanzania, an estimated 230,000 children between 0 - 14 years of age are living with HIV, and according to UNICEF only approximately 26% receive ART . PatientIn 2008-9, our research team enrolled 206 Tanzanian children in a cross-sectional study to evaluate the prevalence of virologic failure and associated risk factors . Among tThis study re-enrolled participants who previously enrolled in a cross-sectional study to evaluate the prevalence of virologic failure among perinatally HIV-infected Tanzanian children receiving ART and to document associations with virologic outcomes. Methods of enrollment in the initial study have been described elsewhere . BrieflyBetween 2012-13, we re-enrolled the same cohort and obtained a repeat HIV-1 RNA to determine long-term durability of ART, including efficacy and predictors of failure. Didanosine was phased out in 2012-13 such that the majority of those on second-line currently receive ABC, 3TC and LPV/r.Participant sociodemographic data, ART regimen, CD4+ cell count, opportunistic infections, World Health Organization (WHO) staging, and physician-reported adherence were retrospectively collected from patient charts and research case report forms. Physician-reported adherence was recorded from progress notes and the standardized HIV form used in care and treatment centers (CTC) across Tanzania. This form has a dedicated column to assess adherence in which physicians document "G" for good and "P" for poor adherence. Poor is defined as missing two or more doses of medication by self-report during the previous month and is reassessed monthly. Adherence was coded as poor for this study if the physician documented two or more "P's" in their progress notes or on the standard CTC form in the year preceding the participant's HIV-1 RNA measure.All participants underwent plasma HIV-1 RNA quantification. HIV RNA testing was performed at the Kilimanjaro Christian Research Institute (KCRI) Biotechnology Lab using the Abbott m2000  with 40 copies/mL as the lower limit of detection. Due to a mechanical failure of the instrument at the KCRI laboratory, specimens collected between January to March 2013 were sent for HIV-1 RNA quantification to the Bio Analytical Research Corporation (BARC) laboratory. BARC is KCRI's U.S. Division of AIDS approved back-up laboratory in Cape Town, South Africa. Both laboratories participate fully in international external quality assurance programs including Viral Quality Assurance. CD4+ cell counts were tested in the clinical laboratory at KCMC per the standard of care.Plasma samples from the original 2008-9 enrollment with HIV-1 RNA >1,000 copies/mL were sent to Duke University Medical Center  for genotypic ART resistance testing and HIV subtyping. These tests were performed in 2009 by directly sequencing the PCR products. All HIV-1 RNA levels obtained as part of the study, both in 2008-9 and again in 2012-13, were made immediately available to the treating provider, but the resistance data were utilized strictly for research purposes. Interim HIV-1 RNA testing was sporadically available by private donation and/or support from Elizabeth Glaser Pediatric AIDS Foundation for patients felt to be at high risk for virologic failure. The decision to switch from a first-line to a second-line regimen was at the discretion of the treating physician. NRTIs were chosen according to Tanzanian guidelines.Descriptive statistics were used to describe the age , gender, time on ART, ART regimen, adherence, WHO stage, and CD4+ cell count  according to WHO cutoffs. Logistic regression was used to determine univariate associations between virologic failure (defined as HIV-1 RNA >400 copies/mL) and key demographic and clinical covariates as itemized above. Any covariates with p < 0.2 on univariate analyses were incorporated into a multiregression model. Continuous variables were preferentially used in the multivariate model if no significant difference was demonstrated between models with continuous versus categorical variables using the likelihood ratio test. Stata 13.1  was used for statistical analyses.HIV-1 subtypes were described and resistance data analyzed using the Stanford University drug resistance database to predict low, intermediate, or high level resistance to ART . The numWritten informed consent was required from a parent or guardian of children aged <18 years of age with assent from children aged 13 years or older. Those 18 years and older were able to consent for themselves. The Duke University Institutional Review Board, the KCMC Research Ethics Committee, and the Tanzanian National Institute Medical Review approved the study protocol.Of the 206 participants originally enrolled, 161 (78.2%) consented for this follow-up study , 37/39 (95%) who continued to receive first-line were suppressed and 63/72 (88%) were recently suppressed with any regimen. Of the children who transitioned into adolescence between the 2008-9 and 2012-13 assessments, 28/34 (82%) remained suppressed on first-line, with 44/65 (68%) suppressed on any regimen. Finally, those who were 12 years or older for the duration of the study, 9/15 (60%) remain suppressed on first-line therapy, with 11/24 (46%) suppressed on any regimen. Compared to those who remained under 12 years of age, there was a 3.3 fold increased odds of virologic failure on any regimen during transition to adolescence (p < 0.01), and an 8.3 fold increased odds of failure for those receiving any ART regimen who remained in adolescence (p < 0.01).Adherence to ART was also evaluated by age. Compared to those who remained less than 12 years old, those who transitioned into adolescents during this study had a 2.3 fold increased odds of poor adherence (p = 0.05). Those who were 12 years of age or older throughout the study (since 2008-09) demonstrated a 4 fold increased odds of poor adherence (p < 0.01).Resistance data were available for 35/54 (65%) originally failing first-line therapy in 2008-9  participants, of whom 4/10 (40%) fully suppressed on a second-line regimen. The other six were failing second-line, two with reported good adherence. Two patients had high predictive resistance (6 TAMs and 3 TAMs respectively) and both were fully suppressed on second-line with good physician-reported adherence.10 copies/mL) to the presence of any TAMs demonstrated a significant 4.85 increased odds of having TAMs for every log increase in viremia (p < 0.01).Among participants who changed to second-line therapy and who had no TAMs, 83% suppressed virus, whereas 60% with one TAM and 57% with two or more TAMs suppressed virus , physician-reported poor adherence (OR 5.0 p < 0.01), and lower CD4  to be significantly associated with virologic failure  of the original cohort over a four-year period. Of the 161 participants, 73.3% were virologically suppressed at four-year follow-up. These numbers are similar to data from a prospective study in Uganda evaluating children initiating ART with regular viral load monitoring which demonstrated an average of 74% virologic suppression (HIV RNA <400 copies/mL) over 3 years. While only 11/108 (10%) switched to second-line therapy, all suppressed in that study . In our Though viral load testing is the "gold standard" to monitor ART response in high income countries, resource-limited settings experience difficulty implementing this preferred option due to cost, logistical concerns, and technical challenges. Tanzanian guidelines continue to rely on immunologic and clinical criteria, which have a low sensitivity to predict virologic failure, especially in children . There iThis study supports increasing attention toward the growing number of adolescents living with HIV. It demonstrated that adolescents were more likely than younger children to have both poor adherence (p = 0.01) and virologic failure (p < 0.01). Reasons for poor adherence among youth 12 years and older are often multifactorial, including logistical complications in day-to-day routines (simply forgetting), adverse side effects, and emotional difficulties such as depression, traumatic stress, and anxiety -18]. Ad. Ad18]. Nearly all participants with virologic failure and resistance testing harbored dual NRTI and NNRTI resistance (89%). These data demonstrate slightly higher levels of dual-class resistance than found in a small study in rural Tanzania (58%) , and verAdherence may ultimately be the most important predictor of success of second-line therapy. Despite a high number of TAMs, both this study and similar Ugandan studies found a majority of children were still able to suppress on second-line therapy ,24]..24].A high level of viremia in 2008-09 did increase the odds of having one or more TAMs (p < 0.01), but did not predict the ability to suppress at four year follow up (p = 0.11) Table . ResultsConsideration of optimal drugs for first and second-line therapy is vital to maximizing the durability of ART. While M184V is a common resistance mutation that develops early in the majority of patients failing regimens with 3TC or FTC, therapeutic options remain given its association with hyper-susceptibility to other NRTIs. TAMs, on the other hand, tend to accumulate over a longer period of time with a failing AZT regimen. They are thought to convey much higher risk of regimen failure given their association with reduced HIV susceptibility to a number of NRTIs with no enhanced efficacy. K65R, L74V, T215Y slowly accumulate with ABC or TDF and do not affect susceptibility to AZT . For theThis study has several limitations. The cross-sectional design gives a snap shot of two time points. The original viral load does not provide information on the duration of virologic failure prior to testing; however, by using the accumulation of TAMs as a proxy, presumably many individuals had a prolonged period of viral replication on therapy. Only two-thirds of the original genotype samples were able to amplify for resistance testing. This is likely due to recurrent heating and cooling cycles in the storage facility that may have compromised sample quality.After switching to second-line, participants may have initially suppressed, but then rebounded. Information on this time sequence is incomplete. We do not have resistance data on the recent viral load to better understand whether maladherence versus further accumulation of resistance, or both, were at play in those with recent virologic failure.Though this study was not powered to show statistically significant differences among resistance mutations and their ability to predict suppression on second-line, it does provide a real world example of the durability of ART as perinatally HIV-infected children age into adolescence. While ultimately no factors remained significantly associated with virologic failure in multivariate analysis, colinearity of factors such as adolescence and poor adherence may have contributed to this finding and the results of the univariate analysis should bear weight in considering future policies targeted towards improving adherence and recognizing risk for failure.Finally, our data reflect an "aging" population of HIV-infected children who were largely infected before PMTCT programs were available. While the number of new HIV infections among HIV-exposed children is declining, the complexity of choosing and managing ART for those who are infected, most of whom are now nevirapine-exposed, was not addressed in this cohort. As WHO recommendations now call for initiation of PI-based therapy in all HIV-infected infants, the availability of future options for ART will become increasingly critical.This study provides both optimism for retention in care and durability of virologic suppression among children in resource-limited settings initiated on NNRTI-based regimens. Resistance data among children in resource-limited settings are scarce, as are the overall outcomes of standard PI regimens as second-line in settings where clinical and immune-based monitoring strategies are the norm. Our data suggest that virologic monitoring may lead to faster switch to second-line therapy resulting in fewer resistance mutations and potentially preserve treatment options. Nonetheless, despite accumulation of TAMs, most patients were able to suppress on second-line therapy when adherence was excellent. Validated methods to improve adherence among adolescents living with HIV are urgently needed, as is improved drug sequencing in settings where options are limited. Improved understanding of second-line ART is critical to maximize durability of second-line as currently most resource-limited settings have no access to third-line therapy.DED participated in project oversight, data entry, statistical analysis, and manuscript preparation. AMS participated in data collection and manuscript preparation. CKC participated in project management and manuscript preparation. EAR participated in study conception and design and participated in manuscript preparation. All authors read and approved the final manuscript.


Background: Boosted protease inhibitors (PIs), including ritonavir-boosted atazanavir (ATV/r), are a recommended option for the initial treatment of HIV-1 infection based upon clinical trial data; however, long-term real-life clinical data are limited.Objective: We evaluated the long-term use of ATV/r as a component of antiretroviral combination therapy in the real-life setting in the REMAIN study.Methods: This was an observational cohort study conducted at sites across Germany, Portugal, and Spain. Retrospective historical and prospective longitudinal follow-up data were extracted every six months from medical records of HIV-infected treatment-naïve patients aged ≥ 18 years initiating a first-line ATV/r-containing regimen.Results: Eligible patients (n = 517) were followed up for a median of 3.4 years. The proportion remaining on ATV/r at 5 years was 51.5% with an estimated Kaplan-Meier median time to treatment discontinuation of 4.9 years. Principal reasons for discontinuation were adverse events  and virologic failure (6.8%). The Kaplan-Meier probability of not having virologic failure (HIV-1 RNA < 50 copies/mL) was 0.79  at five years. No treatment-emergent major PI resistance occurred. ATV/r was generally well tolerated during long-term treatment with no significant changes in estimated glomerular filtration rate over five years.Conclusions: In a real-life clinical setting over five years, treatment-naïve patients with HIV-1 infection initiating an ATV/r-based regimen showed sustained virologic suppression, an overall treatment persistence rate of 51.5%, an absence of treatment-emergent major PI resistance mutations at virologic failure, a long-term safety profile consistent with that observed in clinical trials, and no significant decline in renal function. Following the dramatic improvements in the survival of individuals with HIV-1 infection in the era of combined antiretroviral (ARV) therapy,Boosted protease inhibitors (PIs) possess a high genetic barrier to the development of viral resistance.The aim of the REMAIN observational cohort study was to evaluate long-term outcomes in ARV-naïve patients initiating ATV/r-containing regimens in a real-life clinical setting over a maximum follow-up period of five years.The REMAIN study was a non-comparative, observational study conducted at a total of 51 sites across Germany, Portugal, and Spain . This study was conducted in accordance with the International Society for Pharmacoepidemiology Guidelines for Good Epidemiology Practices, the ethical principles originating from the Declaration of Helsinki, and local regulatory requirements. Local ethical committees approved the protocol, amendments, and the patient informed consent form. Written informed consent from all participants was obtained.Consecutive patients attending the investigating center(s) with HIV-1 infection aged ≥ 18 years who were ARV treatment-naïve when an ATV/r-based regimen was initiated were selected for this study. The ATV/r-based regimen, which included at least two nucleos(t)ide reverse transcriptase inhibitors, must have been initiated between 1 February 2008 and 31 July 2010, and patients must have been seen in routine outpatient consultations between 1 January 2011 and 31 March 2012. Retrospective historical and prospective longitudinal follow-up data were extracted every six months from the medical records with follow-up continuing until 31 July 2013 . Patients with more than four weeks of prior ARV exposure or who were participating in an atazanavir (ATV) clinical trial were excluded.The primary endpoint was the proportion of patients remaining on an ATV/r-based regimen over time. Other endpoints examining treatment persistence included: (1) the probability of remaining on treatment and time to treatment discontinuation assessed using the Kaplan-Meier method; (2) baseline factors associated with treatment discontinuation, assessed using univariate and multivariate Cox proportional hazards models; and (3) the overall proportion of patients discontinuing treatment and reasons given by the investigator for treatment discontinuation.Virologic and immunologic outcomes included: (1) time to virologic failure, defined as two consecutive HIV-1 RNA values of ≥ 50 copies/mL, or as one HIV-1 RNA value of ≥ 50 copies/mL followed by discontinuation, regardless of the reason, more than six months from ATV/r initiation ; (2) the proportion of patients with virologic failure (as defined above) and its inverse, virologic response; (3) treatment-emergent new major or minor PI resistance mutations, defined using the International Antiviral Society–USA classification system,The long-term safety and tolerability profile of ATV/r was assessed by the following parameters: (1) the frequency of adverse events (AEs) and AEs leading to discontinuation; (2) laboratory abnormalities graded according to the Division of AIDS system;For the analysis of time-to-event data, the Kaplan-Meier method was considered to be the most appropriate technique. Patients with missing data or who were lost to follow-up were censored. Patients still participating in the study as of December 2012, but who did not provide their consent to continue participation in the one-year extension to the follow-up period during 2013, were considered as having discontinued ATV/r.Baseline factors potentially associated with time to treatment discontinuation were investigated using Cox proportional hazards models. Gender, viral load at baseline , and co-infection with hepatitis B or C virus were entered into all models. Other factors, including country, age at initiation, disease duration, CD4 cell count, mode of HIV acquisition, and history of opportunistic infections, were preselected on the basis of a significance level of 0.2 in univariate Cox models provided excessive collinearity was not present. Pairwise interactions were tested in the final model and further selected for retention or exclusion by backward elimination.For the statistical analysis of changes in HIV-1 RNA, CD4 cell counts, and eGFR over time, mixed-models repeated measures (MMRM) analysis was considered to be a more appropriate method for imputing missing data than the last observation carried forward (LOCF) method because MMRM generates estimates that are valid for most types of missing data, whereas LOCF does not.The long-term safety profile of ATV/r treatment was evaluated by collecting data on reported AEs (as listed by study investigators without predefinition) and laboratory abnormalities. AE data were collected and analyzed using descriptive statistics  for all patients up to the point of ATV/r treatment discontinuation or, for those patients who remained on treatment at the end of the study, until the end of the follow-up period.3 in 38.2% and < 50 cells/mm3 in 10.4%, and hepatitis C virus co-infection in 18.6%. The majority of patients were receiving a fixed-dose combination of either tenofovir disoproxil fumarate/emtricitabine  or abacavir/lamivudine  as backbone ARV therapy. Patients were followed-up for a median period of 2.0 years (interquartile range [IQR]: 1.3 to 2.7) during the retrospective period and a median period of 1.5 years (IQR: 1.3 to 2.1) during the prospective period, giving a total median duration of exposure to ATV/r of 3.4 years (IQR: 1.8 to 4.3).Of 590 patients enrolled, 517 patients were eligible for inclusion in the study analyses. Details of patient disposition from enrollment to study analysis eligibility are summarized in FigureAt the end of the follow-up period, after a mean follow-up duration of 3.1 (standard deviation: 1.5) years, a total of 264 patients  remained on an ATV/r-based regimen. Accounting for censored observations by survival analysis techniques (i.e. patients lost to follow-up and missing data), the probabilities of remaining on an ATV/r-based regimen were 0.85 , 0.74 , 0.67 , 0.57 , and 0.47  for the first, second, third, fourth, and fifth years, respectively .On univariate Cox proportional hazards analysis, country of origin and a history of opportunistic infection at baseline were associated with an increased risk of treatment discontinuation Table . On multOverall, 223/517 patients (43.1%) had discontinued ATV/r by the end of the follow-up period for up to five years, with a lower rate in Germany than in Portugal or Spain , 0.85 , 0.80 , 0.79 , and 0.79  for the first, second, third, fourth, and fifth years, respectively  had treatment-emergent minor PI resistance mutations, but no patient had treatment-emergent major PI resistance mutations.p < 0.0001). In parallel, immunologic recovery was observed; the median CD4 cell count at the last available assessment was 549 cells/mm3 (IQR: 376 to 730), representing a median increase from baseline of 331 cells/mm3 (IQR: 158 to 466). Using a statistical method robust to data missing at random, MMRM analysis of CD4 cell counts showed progressive and significant increases over time (p < 0.0001), reaching 610 cells/mm3 at five years   over the first six months after ATV/r initiation (the associated proportion with HIV-1 RNA < 50 copies/mL at six months was 82.7%), with little change thereafter , which did not result in any change to the ATV/r-based regimen.Treatment-emergent clinical AEs (of any grade) and laboratory AEs (of grade 3–4 severity), regardless of causality, are shown in Table Within the group of patients discontinuing because of AEs, few patients <1.5%) discontinued for gastrointestinal or renal reasons with the most common reason being hyperbilirubinemia-related Table . Hyperbi% discontOverall, renal/urinary disorders occurred in 17 patients (3.3%). Grade 3–4 creatinine elevations were rare (<1%). The incidence of nephrolithiasis in the overall population was 0.6% per patient-year of exposure. Renal failure, which also led to treatment discontinuation in all cases, occurred in five patients , one of whom was also receiving concomitant TDF/FTC. At the last available patient assessment, median eGFR for the overall patient population was 95.3 mL/min (IQR: 81.4 to 113.8), representing a median change from baseline of −4.7 mL/min (IQR: –21.3 to 0.0). Using a statistical method robust to data missing at random, MMRM analysis of eGFR showed no significant change over time with treatment  for up to 5 years was high at 80%, confirming previous findings from ATV/r clinical trials.n = 21,801), treatment modification and interruption rates were high (51% after 2.3 years); however, patients taking ATV had lower rates of changing drug class compared with patients on efavirenz, whereas patients on lopinavir or nevirapine had higher rates of interruption.n = 1,294) who switched to an ATV/r-containing regimen, a treatment discontinuation rate of 21.2% over the first year was reported, with a probability of remaining on treatment after three years of 0.56  for patients with baseline virologic suppression (<500 copies/mL).Persistence with ATV/r-based regimens over time was good, with 51.5% of patients remaining on treatment up to five years in the current study; this compares favorably with the results of other cohort studies examining discontinuation rates with several different first-line ARV-regimensAEs were reported as reasons for treatment discontinuation in approximately 16% of patients in the current study, with the majority of individual events occurring in <5% of patients. Consistent with ATV inhibition of uridine 5’-diphospho-glucuronosyltransferase (UGT),Given that the current multivariate Cox proportional hazards analysis indicated that the overall risk of treatment discontinuation was lowest in Germany, factors other than treatment discontinuation due to hyperbilirubinemia-related reasons need to be considered. Of note, the proportion of patients at baseline with intravenous drug use was about threefold less in Germany than in Portugal or Spain Table , which mIn common with all observational studies, this study has acknowledged limitations. While the study sought to recruit patients from a wide variety of participating sites and consecutive clinic attendees were enrolled, it is not possible to state that the population studied here is representative of the general population of patients with HIV-1 infection commencing ARV therapy. Factors such as motivation to participate, reliability and completeness of historical and ongoing data collection, and ability to enroll sufficient numbers of patients within a set recruitment period were key issues contributing to the site selection process. It is possible, therefore, that patients in this study received a higher standard of clinical care leading to superior outcomes than that associated with non-participating centers. The observational and mixed retrospective–prospective study design meant that only historical data were collected for some patients, whereas for others the data collected were primarily prospective, and for both types of data collection, the length of follow-up varied. As a consequence, events may have been underreported during the retrospective phase. In common with all observational studies, bias from missing data or patients having been lost to follow-up cannot be excluded. In contrast to randomized trials where all AEs are systematically reported and assessed for causality, AEs could have been underreported. In addition, establishing whether there were causal relationships with ATV/r regimens was difficult owing to the lack of a comparator regimen in the current study.Despite these methodological limitations, this study followed a large number of patients from different European countries over an extended period beyond that normally associated with randomized trials. In addition, the lack of narrow selection criteria, as commonly occurs in randomized trials, enabled the long-term outcomes of ATV/r-based regimens in a real-life clinical setting to be evaluated in patients who may have been ineligible for participation in randomized controlled trials.To conclude, in a real-life clinical setting over five years, ATV/r-based regimens in treatment-naïve patients with HIV-1 infection showed sustained virologic suppression, an absence of treatment-emergent major PI resistance mutations at virologic failure, a lower overall rate of treatment discontinuation compared with other similar cohort studies examining alternative regimens, a long-term safety profile consistent with that previously observed in clinical trials, and no evidence of a significant decline in renal function for up to five years. ATV/r is an effective and well-tolerated therapeutic option for treatment-naïve patients with HIV-1 infection.Eugénio Teófilo has received honoraria for attendance at advisory boards and investigator grants for clinical trials from Abbvie, Bristol-Myers Squibb, Gilead Sciences, Janssen-Cilag, Merck Sharp & Dohme, and ViiV Healthcare. Nuno Rocha-Pereira has received honoraria to attend meetings and congresses both in general infectious diseases medicine and in HIV medicine from Abbvie, Bristol-Myers Squibb, Gilead Sciences, Janssen-Cilag, Merck Sharp & Dohme, and ViiV Healthcare. Birger Kuhlmann declares no conflicts of interests. Antonio Antela has received investigator grants for clinical trials, and honoraria for attendance at advisory boards or for speaker engagements from Abbvie, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Janssen-Cilag, Merck Sharp & Dohme, and ViiV Healthcare. Heribert Knechten has received investigator grants from Abbott and Octapharma, and has received investigator grants and personal fees from AbbVie, Biotest, Bristol-Myers Squibb, Boehringer Ingelheim, Gilead Sciences, Janssen-Cilag, Merck Sharp & Dohme, and ViiV Healthcare. Jesús Santos has carried out consultancy work for Bristol-Myers Squibb, Gilead Sciences, and Janssen-Cilag; he has received monetary payments for giving talks from Bristol-Myers Squibb, Gilead Sciences, Janssen-Cilag, and Merck Sharp & Dohme, and has received payments for the development of educational presentations for Bristol-Myers Squibb, Gilead Sciences, Janssen-Cilag, and Merck Sharp & Dohme. Maria Jesús Jiménez-Expósito is an employee of Bristol-Myers Squibb.This study was conducted in accordance with the International Society for Pharmacoepidemiology (ISPE) Guidelines for Good Epidemiology Practices, the ethical principles originating from the Declaration of Helsinki, and local regulatory requirements.Registered at clinicaltrials.gov (NCT01236235).


Estimates of CD4 response to antiretroviral therapy (ART) obtained by averaging data from patients in care, overestimate population CD4 response and treatment program effectiveness because they do not consider data from patients who are deceased or not in care. We use mathematical methods to assess and adjust for this bias based on patient characteristics.We examined data from 25,261 HIV-positive patients from the East Africa IeDEA Consortium.We used inverse probability of censoring weighting (IPCW) to represent patients not in care by patients in care with similar characteristics. We address two questions: What would the median CD4 be “had everyone starting ART remained on observation?” and “were everyone starting ART maintained on treatment?”Routine CD4 count estimates were higher than adjusted estimates even under the best-case scenario of maintaining all patients on treatment. Two years after starting ART, differences between estimates diverged from 30 cells/µL, assuming similar mortality and treatment access among dropouts as patients in care, to over 100 cells/µL assuming 20% lower survival and 50% lower treatment access among dropouts. When considering only patients in care, the proportion of patients with CD4 above 350 cells/µL was 50% adjusted to below 30% when accounting for patients not in care. One-year mortality diverged 6–14% from the naïve estimates depending on assumptions about access to care among lost patients.Ignoring mortality and loss to care results in over-estimation of ART response for patients starting treatment and exaggerates the efficacy of treatment programs administering it. CD4+T-cell (CD4) count is a marker for disease progression in HIV-positive patients , 2. For Previous studies have reported robust immunological response among patients initiating ART in low- and middle-income settings –7. HowevAnother analysis, which would be helpful for decision makers, involves questions of the response to therapy under some hypothetical intervention such as, for example, if it were possible to maintain on therapy, a higher percentage of patients starting ART. Such hypothetical scenarios have been routinely addressed through mathematical modelling but these procedures and results are frequently not transparent or well understood and are heavily influenced by unverified assumptions or hypotheses based on historical data external to the population under study. As we show in this paper, all these analyses can be approached through adjustments calculated from available data on actual rather than potential patients as well as strong but plausible assumptions on the outcome of patients who discontinue participation in care.Obviously, a major obstacle in obtaining reasonable adjustments to address the bias generated by the anticipated informative dropout of the sickest among HIV-positive patients is the fact that it is very difficult to physically measure CD4 counts among patients who have discontinued care and impossible to do so among those who have died. To overcome this obvious limitation we can use methods such as inverse probability of censoring weighting (IPCW) . The cruOne of the advantages of the IPCW approach is that it can be customized to address specific questions, some contrary to fact or “counterfactual,” which may not be directly measurable. Two examples of such questions relevant to our problem in the resource-limited setting are the following: “What would the long-term median CD4 count be if all patients starting therapy had remained under observation (but not necessarily continued to receive treatment)?” and “What would be the long-term median CD4 count had all patients starting therapy remained both in care and on treatment?”In this article we address these two questions in the context of a large HIV care and treatment program in sub-Saharan Africa. The former question is relevant because it measures patient outcomes on all patients starting therapy . The latter question measures the benefit of policies which increase patient retention in care and on treatment. The answers to these questions are therefore of substantial clinical and public health interest. If, in the process of addressing the first question, it is established that CD4 response to treatment is overestimated when not considering patients who dropped out or died, the estimated median CD4 counts after ART has been initiated will be adjusted downward and so will the effectiveness of programs with higher dropout and death rates among their clients. Similarly if, when addressing the second question, the adjustment of estimated median CD4 counts after ART is positive, the implication would be that there is a significant benefit resulting from interventions increasing patient retention in care and treatment. Such a conclusion  would inform decision-making about, for example, the comparative effectiveness of efforts aimed at increasing patient retention in care and treatment versus focusing all program resources in initiating therapy on a higher number of patients.We included adult HIV-positive patients aged ≥15 years, receiving routine HIV care and initiating ART after 1st February 2004 in the United States Agency for International Development and the Academic Model Providing Access to Healthcare (USAID-AMPATH) partnership in Eldoret, Kenya  32–44), the median CD4 cell count at the start of treatment was 116 cells/µL (53–180) . Within Rates of discontinuation from follow-up (loss to follow-up), treatment discontinuation and observed mortality (among patients on observation) are presented in and continued treatment. The IPCW analysis showed that the estimated median CD4 count under Scenario 2 was slightly higher than the estimated median CD4 count under Scenario 1 during later periods, but this difference was not particularly pronounced  and one assumed that survival rates among patients who have discontinued from care at the original clinic were 20% lower than those continuing in care.We also analyzed longitudinal CD4 counts without adjusting for death . An additional summary measure involving longitudinal CD4 counts estimated under Supplementary Figure 2 shows the routine cumulative mortality estimates produced by considering only observed deaths versus the adjusted estimates which consider both the characteristics of lost patients as well as an estimate of the mortality rate among patients no longer on observation. Assuming 20% lower survival among patients no longer in follow-up, the adjusted cumulative mortality rate is twice as high as the observed mortality rate at one year and more than three times at two years.A final by-product of this analysis is a revised estimate of the mortality rates adjusting for differential dropout and higher mortality and lower treatment access rates among patients no longer in follow-up. This study addressed two questions related to the estimation of CD4 counts over time after initiation of ART: “What would the median CD4 count be had all patients remained in care and possibly on treatment?”  and “Whaoriginal population of patients starting ART is alive with CD4 count above that threshold. Thus, in addition to measuring the effectiveness of a program in maximizing patient CD4 count response to treatment, program evaluation takes into account the program's success in keeping its clients alive, the ultimate criterion of program effectiveness.An additional feature of this method is that it incorporates patient survival in the estimation of longitudinal CD4 counts. The interpretation of the median CD4 count over time, in the present analysis, is the CD4 count threshold in each interval where 50% of the Regardless of the approach, this study found a substantial positive bias in the estimation of CD4 counts when observations are obtained by simply averaging data from patients under observation. In fact, the difference between the routinely reported CD4 count and the adjusted estimate, which accounts for selective dropout and mortality by the sicker patients, diverged over time. This suggests that the subgroup of patients who remain in care are increasingly unrepresentative of the cohort initiating ART.The answer to the counterfactual question in given that patients remain alive and in care?” This question is irrelevant from the patient-level perspective because it is not known at the start of treatment which patients will remain in care and/or survive in the future. It is also of dubious usefulness at the population level as it is obtained from a cohort which bears decreasing resemblance with the one starting therapy, particularly as the time from ART initiation increases.It is notable that, despite the robust benefit associated with maintaining patients on treatment, these estimates, produced under the best-case scenario of maintaining the entire patient cohort on treatment once ART has been initiated, were still lower than the unadjusted estimates, which are based on averaging data from patients in care . This shWe found that, considering data solely on patients under observation, about 50% of patients are estimated to have CD4 counts > 350 cells/µL two years after ART starts. This is potentially a remarkable achievement of treatment with ART given that, at the start of therapy, the median CD4 count in the cohort initiating therapy was barely above 100 cells/µL . HoweverSupplementary Figure 1). CD4 count estimates in patients who were alive at each interval of estimation show that a similar, albeit attenuated, downward adjustment of the routine estimates results when taking into account differential dropout rates among patients.We have also carried out an analysis without incorporating death in the estimation of CD4 count  among patients who are lost to clinic, results in almost double one-year and two-year mortality rates as compared to the usual estimates based on observed data. Differences are even more pronounced if higher mortality is assumed among dropouts. Under the best-case scenario where all patients are maintained in care and on treatment  The IPCW methodology produces adjusted mortality estimates which incorporate differential patterns of dropout and higher rates of mortality among patients who discontinue care. As shown in reatment , mortalie cohort  correspoA major limitation of this study is that, due to lack of additional data on CD4 counts past discontinuation from care, we modelled the CD4 trajectories of patients out of care and treatment, by CD4 counts of patients not on treatment but still in care. It is questionable whether this is appropriate. Remaining in care may be advantageous, even if treatment has been interrupted, because of access to medical care and prophylaxis treatment (e.g. access to co-trimoxazole prophylaxis) as well as other ancillary services. Thus, remaining in care will be likely associated with a less steep decline in CD4 count and less sharp increases in morbidity and mortality rates compared to patients who are out of care. For this reason, even the significantly downwardly adjusted estimates of the median CD4 count after ART initiation should be considered as an upper bound of the true median CD4 count .An additional limitation of this analysis is that we have no information on connection to care and survival status among patients no longer on observation. That is, we do not know how many of these individuals continue receiving care and treatment services at other facilities. Recent work has shown that, particularly in regions with robust scale-up of care and treatment services, an increasing proportion of patients who are lost to follow-up have simply moved to another clinic providing ART , 27. We This study indicates that there is significant overestimation of the CD4 response to treatment for a cohort starting ART when available data are simply averaged to estimate long-term CD4 response to therapy. At the same time, retention of patients to treatment results in a salutary effect which manifests in much higher median CD4 counts over time. Policies, programs and ART regimens which improve patient retention into care and treatment are desirable and will significantly improve patient outcomes. IPCW is an elegant and intuitive methodology, which can be generalized to other settings and longitudinal measures  and can be implemented using existing statistical software to address a number of critical clinical and public-health questions. With these methods rational decisions of the likely outcome of a number of interventions can be made based on more accurate quantification of the likely impact of patient behaviour, access to treatment and patient retention efforts.