Universal access to first-line antiretroviral therapy (ART) for HIV infection is becoming more of a reality in most low and middle income countries in Asia. However, second-line therapies are relatively scarce.We developed a mathematical model of an HIV epidemic in a Southeast Asian setting and used it to forecast the impact of treatment plans, without second-line options, on the potential degree of acquisition and transmission of drug resistant HIV strains. We show that after 10 years of universal treatment access, up to 20% of treatment-naïve individuals with HIV may have drug-resistant strains but it depends on the relative fitness of viral strains.If viral load testing of people on ART is carried out on a yearly basis and virological failure leads to effective second-line therapy, then transmitted drug resistance could be reduced by 80%. Greater efforts are required for minimizing first-line failure, to detect virological failure earlier, and to procure access to second-line therapies. HIV/AIDS arose in Asia in the early-to-mid 1980s. By the 1990s HIV epidemics had established in numerous countries; among the worst affected were Thailand and Cambodia with HIV prevalence levels of 1–2%. Currently Thailand, Cambodia, and Myanmar have been experiencing declines in HIV prevalence Effective antiretroviral therapy (ART) is currently being scaled up in most countries in the region. In principle, anyone who is treatment eligible, according to country-specific guidelines but generally similar to the WHO treatment guidelines for resource limited settings Transmitted drug resistance is a problem around the world, including the Southeast Asia region. Documented rates of transmitted drug resistance include 4% in 2003–2004 in Japan The primary means to detect transmitted drug resistance is to perform blood tests on newly infected treatment-naïve individuals. Resistance strains can be divided up into two broad categories, namely, majority-resistant and minority-resistant variants. Majority resistant strains are detected through conventional nucleotide sequencing methods after polymerase chain reaction (PCR) amplification, however, these methods are not sensitive enough to detect minority-resistant strains that comprise less than ∼25% of the viral population We sought to estimate the potential levels of acquired and transmitted (majority and minority) drug resistant strains of HIV that could emerge in a typical Southeast Asian population. We do this through the development of a biologically realistic mathematical transmission model. We use the situation in Thailand as a representation for a general Asian epidemic and thus calibrated the model to reflect the epidemic in Thailand. Thailand is a leading example of treatment scale-up with the introduction of ART through the National Access to Antiretroviral Program for People who have AIDS by the Ministry of Public Health Access to Care program Our model describes the unique nature of Asian HIV epidemics whereby epidemics typically emerge and are initially driven by injecting drug use and sex work. Waves of infection occurred in these population groups, followed by infection among clients of sex workers and their regular sexual partners which led to generalized epidemics. In recent years HIV epidemics have emerged among men who have sex with men. This epidemic pattern has been observed in numerous Southeast Asian countries The level of adherence to ART is associated with clinical success The model was then used to assess the impact of regular viral load testing in a setting where second line treatment is available and commenced once virological failure is detected. We assumed that viral load tests could be performed at regular intervals on all those who are receiving treatment. We simulated different scenarios of frequency of viral load testing: once every 2 years, every year, twice yearly, or quarterly. We also assumed that a period of one week was required between the time of the test and receiving the test results and starting the patient on effective second-line treatment. Full technical detail of the model structure, assumptions and parameter values can be found in the supporting information.After 10 years of universal ART without access to any second line therapies, moderately high levels of drug resistance can be expected in the HIV-infected population. People on ART will start to acquire drug resistant strains of virus. If second and subsequent lines of therapy are not widely available and failed regimens continue to be used then the emergent drug-resistant strains can be transmitted to susceptible individuals. Subsequently, the proportion of newly-infected treatment-naïve HIV cases that have drug-resistant strains could be substantial. Our model estimates that after 10 years of universal ART without monitoring of treatment failure and optimizing therapy ∼24% of new infections could include drug-resistant mutations . ApproxiIn the absence of the pressure of ART, majority-resistant strains of HIV tend to revert to become minority-resistant variants that appear to be exclusively wild-type and not detected by standard sequencing methods. According to our model, after 10 years of universal access to ART without second-line options ∼20% of treatment-naïve cases in asymptomatic stage would have some drug-resistant strains and ∼17% of cases at treatment-eligible stage of infection would have some drug-resistant strains . HoweverKey factors giving rise to the prevalence of drug resistance differ between populations of treatment-naïve and treatment-experienced individuals. Multivariate sensitivity analyses revealed that the average time for resistant strains to appear to revert to wild-type virus and the relative fitness of drug-resistant strains were the most important parameters for determining the prevalence of majority-resistant variants in treatment-naïve cases . The relIn many Southeast Asian countries, treatment failure is often realized due to clinical symptoms rather than the presence of mutations or virological or immunological failure. Frequent viral load testing is generally infeasible due to financial constraints. However, viral load testing for monitoring patients' responses to ART is available in some settings and it could be expected that it will become more common across the region in the future. Therefore, we used our model to estimate the expected proportion of newly acquired HIV infections to have drug-resistant strains versus the frequency of viral load testing of individuals on ART . In Effective treatment with antiretroviral drugs reduces viral load which improves the health of treated individuals and also decreases infectiousness and the potential to transmit the virus to others We demonstrated that if treatment options are limited for those who fail first-line therapy then the prevalence of acquired and transmitted drug resistant strains of HIV could be relatively large. The prevalence of transmitted drug resistance could be ∼24% after ten years of universal treatment access if there is no viral load monitoring and access to second-line therapy. However, most (99%) of the drug resistance could remain ‘hidden’ as minority-resistant variants that are not detectable by standard sequencing methods. Majority-resistant variants are likely to emerge at significantly faster rates than expected once treatment is initiated To reduce the prevalence of drug resistance among treatment-naïve individuals it is recommended that treated cases are regularly monitored and that second-line and subsequent lines of therapy are made available for those who have failed first-line regimens. We investigated the expected impact on transmitted drug resistance of different frequencies of viral load monitoring and access to second-line therapy when required. Even with a modest testing frequency of once every two years for patients on ART, the model demonstrates a large reduction in the amount of transmitted drug resistance would be achieved. Testing as frequently as quarterly could reduce the prevalence of transmitted drug resistance by ∼90%. In Thailand, since 2008 second-line therapy with TDF/3TC/LPV/r has been widely available as well as once yearly viral load monitoring and genotyping . However, there are limited treatment options in Thailand and patients with TDF resistance will have difficulties in finding effective second line treatment options. Wide availability of third line treatments for patients in this region will be unlikely in the near future. Therefore, it is highly important to minimise drug resistance. Based on our model, yearly testing can reduce transmitted drug resistance to below 5%. It is important for countries in Southeast Asia to procure access to second-line therapies and determine ways of implementing regular viral load monitoring. It will then be important to procure third-line and salvage therapies for patients in this region, however, this is unlikely to be feasible in the near future. Viral load testing is not widely available in many Asian countries and the emergence of drug resistant HIV is not typically assessed during patient consultation Limited combinations of antiretrovirals are available for first-line treatment in most Southeast Asian countries. In Thailand, first-line therapy is based on NNRTIs and usually consists of a fixed dose combination of d4T/3TC/NVP, with a newer regimen of ZDV/3TC/NVP recently rolled out While first-line therapy continues to scale-up around Southeast Asia it is important to plan for, and control, the emergence of drug-resistant HIV, particularly as most drug-resistant cases in the future could be ‘hidden’ as minority-resistant variants. Current surveillance programs, which are based around testing newly diagnosed subjects aged less than 25 years rather than genuinely acute infections, will not detect the scale of the problem. Hidden transmitted drug resistance has the potential to drive relatively high levels of drug resistance over the next 5–10 years unless treated cases are monitored regularly and initiate second-line therapies soon after the failure of first-line options. Data from TAHOD suggest that around half of patients beginning ART will require second-line therapies 3 years after beginning treatment While our model is specifically constructed and calibrated to reflect the unique epidemiology of HIV transmission in Southeast Asia, the conclusions drawn from our study can also be applied to other settings. Most countries in Southeast Asia still use d4T-based first-line therapy, which is similar to Sub-Saharan Africa. Access to antiretrovirals is similarly limited in both regions. Our results are generally applicable to non resource-rich settings in which suboptimal regimens are used and there are limited therapeutic options. Our conclusions concerning the dangers of continued use of failed treatment regimens and important value of regular viral load monitoring coupled with access to second-line therapies may assist countries in their scale-up of antiretroviral treatment.Mathematical Details S1Detailed description of mathematical model.(0.54 MB DOC)Click here for additional data file.Figure S1The seven population subgroups contained within the model. Lines between groups indicate interactions for sexual mixing.(0.82 MB TIF)Click here for additional data file.Figure S2Response surface plot from sensitivity analysis. This plot shows the proportion of cases on ART that have majority-resistant variants (colored contours) versus the rate at which people infected with wild-type acquire drug resistant virus (x-axis) and the average time for majority-resistant variants to emerge for people infected with minority-resistant variants (y-axis) after 20 years of universal treatment access.(0.32 MB TIF)Click here for additional data file.


Several decision support systems have been developed to interpret HIV-1 drug resistance genotyping results. This study compares the ability of the most commonly used systems  to predict virological outcome at 12, 24, and 48 weeks.Included were 3763 treatment-change episodes (TCEs) for which a HIV-1 genotype was available at the time of changing treatment with at least one follow-up viral load measurement. Genotypic susceptibility scores for the active regimens were calculated using scores defined by each interpretation system. Using logistic regression, we determined the association between the genotypic susceptibility score and proportion of TCEs having an undetectable viral load (<50 copies/ml) at 12 (8–16) weeks (2152 TCEs), 24 (16–32) weeks (2570 TCEs), and 48 (44–52) weeks (1083 TCEs). The Area under the ROC curve was calculated using a 10-fold cross-validation to compare the different interpretation systems regarding the sensitivity and specificity for predicting undetectable viral load. The mean genotypic susceptibility score of the systems was slightly smaller for HIVdb, with 1.92±1.17, compared to Rega and ANRS, with 2.22±1.09 and 2.23±1.05, respectively. However, similar odds ratio's were found for the association between each-unit increase in genotypic susceptibility score and undetectable viral load at week 12; 1.6 [95% confidence interval 1.5–1.7] for HIVdb, 1.7 [1.5–1.8] for ANRS, and 1.7 [1.9–1.6] for Rega. Odds ratio's increased over time, but remained comparable (odds ratio's ranging between 1.9–2.1 at 24 weeks and 1.9–2.2 at 48 weeks). The Area under the curve of the ROC did not differ between the systems at all time points; p = 0.60 at week 12, p = 0.71 at week 24, and p = 0.97 at week 48.Three commonly used HIV drug resistance interpretation systems ANRS, Rega and HIVdb predict virological response at 12, 24, and 48 weeks, after change of treatment to the same extent. The effectiveness of antiretroviral therapy has been limited by the development of HIV-1 drug resistance. Resistance occurs frequently in patients and may decrease both the magnitude and the duration of the response to treatment Several prospective studies have shown that the use of genotypic resistance analysis to guide the new treatment choice for patients failing their current HAART improves virologic outcome ANRS, Stanford HIVdb, and Rega are the three most commonly used and publicly available drug resistance interpretation systems, which are all regularly updated. The systems are rule based algorithms, providing scores for specific (combinations of) mutations. The scores are then translated into different levels of susceptibility. The rules for these scores are based on literature and expert's opinion. The Rega system was the first to be validated in drug experienced patients A good way to compare systems is by using virological response data in correlation with the prediction of interpretation systems. However, some systems may be better for short-term virological outcomes, and others may be better for longer-term outcomes. The results of a comparison between systems may therefore depend on the virological outcome time point that is used. In this study, a large data set of HIV-1 patient's sequences was collected together with virological data to compare the three most commonly used interpretation systems in genotypic susceptibility score and in the prediction of virological response. We used 3 different virological outcome time points to analyze the effect of therapy duration on the prediction of systems.http://www.hivarca.net/), Germany (AREVIR database); Sweden , and Luxembourg . The time-periods of available therapies in the ViroLab and EuResist database ranged between 1996 and 2008.Data was made available through the EU-sponsored ViroLab and EuResist projects These databases were used to extract treatment change episodes (TCEs). TCEs were defined, in patients aged ≥18, as follows : (1) a bThe genotypic results were interpreted using three commonly used rule-base interpretation systems: Agence Nationale de recherches sur le SIDA (ANRS) version 17; Stanford HIVdb, version 5.1.2; and Rega Institute version 8.0.1. The ANRS and Rega both report 3 levels of resistance: susceptible, intermediate, and resistant. For ANRS, we translated the definitions ‘susceptible’, ‘intermediate’, and ‘resistant’ into susceptibility scores of 1, 0.5, and 0, respectively. For the Rega scores, we used the weighted score suggested by Rega, which uses the following changes: NNRTI were scored 0.25 (with the exception of etravirine with a score of 0.5) for intermediate resistance, and ritonavir-boosted PI were scored 0.75 and 1.5 for intermediate resistance and susceptible, respectively. The Stanford algorithm uses 5 levels of resistance. We assigned the following scores to these 5 levels of Stanford: 0, 0.25, 0.50, 0.75, and 1 for respectively the high-level resistance, intermediate resistance, low-level resistance, potential low-level resistance, and susceptible. In a separate analysis we used the unweighted scores for Rega. We assigned the scores 0, 0.5, and 1 to the ‘resistant’, ‘intermediate’, and ‘susceptible’ groups for all drugs, respectively. The three systems did not include a score for ritonavir. We therefore excluded eleven TCEs that used ritonavir as only protease inhibitor, as we could not calculate a GSS of their treatment regimens.The arithmetic sum of the individual score for the specific drugs provided the total GSS of that treatment. For brevity, we classified the total GSS score in the following categories: 0 to <1, 1 to <2, 2 to <3, 3 to <4, and ≥4. The 0 to <1 group contains viral sequences almost entirely resistant to the drugs in their regimen, and the ≥4 group contains viral sequences susceptible to more than 3 drugs given in their regimen.To calculate the prevalence of drug resistance we used the mutation list published by the International AIDS Society USA (IAS-USA) Kaplan-Meier curves were estimated to determine the association between GSSs and the proportion of TCEs having an undetectable viral load (<50 copies/ml). The association between GSS scores and undetectable viral load was analyzed with a logistic regression. In the multivariate analyses we adjusted for real time to viral load measurement  and log viral load at start of therapy. Furthermore, we used logistic regression, to calculate Odds Ratios for each GSS group compared to the GSS group of 0 to <1. The receiver operating characteristic (ROC) curves were calculated to analyze the trade-off between the proportion of true-positive (correct virologic response prediction) and false-positive (incorrect virologic response prediction) results across the range of possible prediction cutoffs. The AUC (Area Under the Curve) is a value between 0 and 1 that corresponds to the probability that a randomly selected virologic success receives a higher score than a randomly selected virologic failure. We used the AUCs to calculate how well the systems separate the GSS groups into those with and without undetectable viral load (<50 copies/ml). Robust extra-sample error estimation was obtained by 10-fold cross-validation 10 copies/ml , and the median CD4+ cell count was 233 cells/µL . The most commonly given treatments were lamivudine (59%), tenofovir (37%), and lopinavir (35%). A combination of lamivudine, zidovudine, and lopinavir/r was the most frequently given therapy combination, with a percentage of 8%, followed by 6% for the therapy combination lamivudine, tenofovir, and lopinavir/r.The baseline characteristics are shown in The percentage of sequences having a drug resistance mutation is shown in The genotypic susceptibility scores for a TCE was calculated as the total score of genotypic susceptibility scores for all drugs in one regimen as explained in the ‘The GSS of TCEs with longer follow-up were slightly higher compared to TCEs with a short follow-up time (data not shown), with baseline GSS means ranging between 1.93 and 2.23 at 12 weeks, 1.98 and 2.29 at 24 weeks, and 1.98 and 2.32 for TCEs with viral load measurement available at 48 weeks.The virologic responses of all TCEs are described in Adjusted odds ratios for reaching a viral load below 50 copies/mL for each unit increase in GSS are reported in The ROC curves in In In this study, data from treated HIV-1 patients were modeled to predict virological outcome comparing genotypic drug resistance with the most commonly used interpretation systems. We used logistic regression and AUC calculations and showed in 3,763 treatment change episodes that ANRS, HIVdb, and Rega, do not differ in predicting virological outcomes.Comparisons of interpretation systems have been previously reported An explanation for the findings in this study is that the systems all make use of the same literature available on correlations between genotypic and phenotypic analyses as well as correlations with treatment history and clinical response.Several studies showed small changes in genotypic susceptibility scores between different systems. For example Ravela et al. A possible limitation of studies comparing different interpretation systems lies in the translation of the indications from the interpretation systems into numeric values, which are taken arbitrarily. However, we have used the same principles used by authors of HIV drug-resistance algorithms for calculating the genotypic susceptibility score. Therefore we were able to compare the three systems in the way they are used in practice. We also used the Rega scores without the suggestions about weighting of scores for boosted PI drugs and NNRTI. Using these unadjusted scores did not change in GSS distributions and virological outcome to a great extent.Some novel drugs  were not frequently used in our study population. Similarly, drugs belonging to the newly approved classes, such as raltegravir and maraviroc, were not included. Therefore, the predictive value we found is not a validation for all individual rules in the system and we did not attempt to validate individual rules. Continuous validations in large dataset with recent drug data will therefore remain needed.No restriction on therapies was performed; therefore suboptimal regimens (fewer than three full-dose drugs) were included. However, the group of patients receiving suboptimal regimens was small and the same for all three interpretation systems. Furthermore, it was previously demonstrated that removal of suboptimal treatment reduces the accuracy of the models th week after start of therapy, will do better on therapy and will have better virological responses than patients who switch to another therapy at earlier stages. In accordance, we found stronger associations between interpretation systems and virological outcomes at later time points compared to earlier time points in the logistic regression analyses. However, in the logistic regression that compared the different GSS groups to the GSS group of 0 to <1, the Odds Ratios were similar between week 24 and week 48. Therefore, week 24 may be a well suitable time point to measure long term responses. However, confidence intervals in week 48 were large, because of low numbers of included TCEs, therefore creating a bias at this time-point.Much discussion has been going on about which follow-up period is most suitable to validate a system. Short term responses might be more directly attributable to the antiviral drug activity whereas longer term outcomes might be more clinically relevant but more easily confounded by other issues such as loss in adherence, drug discontinuations and switches In conclusion, we found that the three most common used interpretation systems do not differ in their ability to predict virological response. Also, when looking into different time points, the prediction abilities between the systems were similar. Since the overall performance is comparable, these systems might evolve towards a more consistent scoring in the future. New breakthroughs might be needed for further improvement in genotypic resistance test interpretation.


The recent origin and great evolutionary potential of HIV imply that the virulence of the virus might still be changing, which could greatly affect the future of the pandemic. However, previous studies of time trends of HIV virulence have yielded conflicting results. Here we used an established methodology to assess time trends in the severity (virulence) of untreated HIV infections in a large Italian cohort. We characterized clinical virulence by the decline slope of the CD4 count (n = 1423 patients) and the viral setpoint (n = 785 patients) in untreated patients with sufficient data points. We used linear regression models to detect correlations between the date of diagnosis (ranging 1984–2006) and the virulence markers, controlling for gender, exposure category, age, and CD4 count at entry. The decline slope of the CD4 count and the viral setpoint displayed highly significant correlation with the date of diagnosis pointing in the direction of increasing virulence. A detailed analysis of riskgroups revealed that the epidemics of intravenous drug users started with an apparently less virulent virus, but experienced the strongest trend towards steeper CD4 decline among the major exposure categories. While our study did not allow us to exclude the effect of potential time trends in host factors, our findings are consistent with the hypothesis of increasing HIV virulence. Importantly, the use of an established methodology allowed for a comparison with earlier results, which confirmed that genuine differences exist in the time trends of HIV virulence between different epidemics. We thus conclude that there is not a single global trend of HIV virulence, and results obtained in one epidemic cannot be extrapolated to others. Comparison of discordant patterns between riskgroups and epidemics hints at a converging trend, which might indicate that an optimal level of virulence might exist for the virus. The AIDS epidemic claims more lives per year than any other infectious disease, even though its cause, the Human Immunodeficiency Virus (HIV), is the youngest of all major human pathogens. The recent origin and great evolutionary potential of the virus raise the possibility that the virus might still be adapting to humans. Of primary interest is whether the virulence of the virus, i.e. its ability to cause disease, has been changing over time. Unfortunately, previous results have yielded conflicting results. We investigated time trends of virulence in the Italian HIV epidemic and found increasing virulence. The use of an established methodology allowed, for the first time, direct comparison with results obtained in other epidemics. The comparisons revealed that genuine differences exist in the trends of HIV virulence between different epidemics. Thus, there is no single time trend of HIV virulence worldwide. Our results are consistent with the hypothesis of increasing HIV virulence; however, clinical virulence combines viral and host factors, and the effect of host factors could not be excluded in our analysis. Human immunodeficiency virus type 1 (HIV-1) is a recent pathogen of humans, estimated to have jumped to the human species about 80 years ago The most direct measure of virulence available in untreated HIV infections is the rate of decline of the CD4 cell count in the blood. After a data selection procedure see , the CD4The analysis of the point estimates of the CD4 slope per patient cannot account for the misspecification of the slope of any one individual. To ensure reliable estimation, we required five CD4 measurements for the estimation of the slope; however, this requirement introduced a selection bias against fast progressors, who cannot accumulate sufficient data points before starting therapy. We therefore assessed time trends in the CD4 slope also with a mixed-effect model fitted to the original CD4 count data. The model included the same fixed effects as the general linear model, plus random intercept and slope to account for the correlation of repeated measurements within each individual. We included patients with at least three CD4 counts; this more relaxed selection criterion increased sample size to 1889 patients  except among IDUs . Female gender was associated with lower virus load  and higher age correlated with lower virus load in the IDU exposure category . All other factors (including riskgroup) and interaction terms were dropped from the regression design due to lack of significance during model simplification. To further explore potential differences between riskgroups we carried out separate regression analyses restricted to each category. The effect of date proved strongest among MSM , weaker among HET  and weakest among IDU , where it failed to reach significance. Factors for adjustment retained after model simplification included the baseline CD4 count for HETs and MSM, and gender in the HET exposure category . Because the distribution of setpoints displayed considerable negative skew due to a long tail of low setpoints, we repeated all setpoint analyses after discarding low setpoint values to ensure normality. The results (including the effect of the date of confirmed infection) were consistent with those of the analyses performed on the complete dataset.The magnitude of virus load in the blood after the resolution of primary infection  has been shown to correlate with the rate of disease progression We thus observed consistent trends of increasing virulence towards both faster CD4 decline and increasing setpoints during the time span of the cohort. Because higher setpoints have been associated with faster disease progression Finally, to visualize short-term trends in the virulence markers, we plotted a moving average of both markers . Each poWe found robust time trends both in the rate of CD4 decline and in the viral setpoint towards increasing virulence in untreated patients of the MASTER cohort. Over two decades of observations, the rate of CD4 decline in newly diagnosed infections has almost doubled, while the viral setpoint has been increasing at a rate of about one log per 16 years since RNA measurements had become available. The finding of increasing virulence is consistent with some We note that our “empirical” measures of virulence reflect the combined effect of both viral and non-viral  factors, and our findings are therefore not direct evidence for virus evolution. In addition to the factors incorporated in our analyses , the progression of HIV infections has been shown to be influenced by host genetic factors Transmitted drug resistance has been associated with faster CD4 decline in the first year of infection We also found differences in the time trends of virulence between riskgroups, which raises the possibility that the epidemics of the main exposure categories might have been founded by independent introductions of the virus. The strongest effect was the finding of a faster trend towards steeper CD4 slopes among IDUs, who initially had the slowest CD4 decline among the riskgroups. This suggests a plausible scenario that the IDU epidemic was by chance founded by a virus strain of reduced virulence, and occasional contact between the riskgroups later transmitted more virulent (and apparently fitter) viruses into this group. This hypothesis could be tested in the future by phylogenetic analysis. Alternatively, the increase in virulence might have reflected viral evolution within the IDU epidemic. It has been observed that explosive epidemics (characteristic of early spread in IDU populations) are associated with slow HIV evolution Finally, we note that the overall median of the rate of CD4 decline was faster in the Swiss HIV Cohort Study (55 cells/µL/year) that had stable virulence than in the Italian MASTER cohort (50 cells/µL/year) that showed increasing virulence. This observation and the finding of converging levels of virulence among the riskgroups of the MASTER cohort are consistent with, but do not provide direct evidence for, the hypothesis that HIV has evolved close to the level of virulence that is optimal for its transmission We conclude that trends and patterns of HIV virulence may differ between geographical regions and even between different riskgroups within the same region. The results of individual studies can therefore not be extrapolated to predict the global patterns of the evolution of HIV virulence.The Italian MASTER  Cohort is a prospective longitudinal multicenter cohort comprising the general HIV patient population in referral centres throughout Italy. The analyses were restricted to patients of Italian nationality and white ethnicity for best representation of the local epidemics, and to the three major exposure categories: heterosexually infected patients (HET), intravenous drug users (IDU) and men having sex with men (MSM). Patients under age 15 were excluded from the analysis.Markers of virulence were defined as in We estimated the effect of the date of confirmed infection on the virulence markers by analysis of covariance . Categorical factors included gender and exposure category (riskgroup); reference levels were chosen to reflect the majority in the study groups. Continuous predictors included the earliest date of confirmed infection (converted to Julian date), age at the date of confirmed infection and the baseline CD4+ cell count, defined as the first CD4+ count obtained at least 200 days after the earliest date of confirmed infection. The starting models included the interaction between gender and riskgroup, and two-way interactions between all pairs of a continuous and a categorical predictor. Significant effects were assessed by stepwise elimination of non-significant interaction terms and non-significant factors, and finally by merging non-significant levels of categorical predictors The time trend of the CD4 slopes was also investigated with mixed-effect models fitted to the CD4+ T cell counts (relative to baseline) from all eligible patients over time; the models accounted for the correlation of repeated measurements within each individual by random intercept and time slope. The primary explanatory variable was time since the earliest date of confirmed infection (estimating the CD4 slope), and the models also included the interactions of the time with gender, riskgroup, the earliest date of confirmed infection, age at the date of confirmed infection, the baseline CD4+ cell count and  the viral setpoint, to account for the effect of these factors on the CD4 slope; and the 3-way interaction between time, date and riskgroup. Stepwise elimination of non-significant factors was performed based on likelihood ratio tests.www.r-project.org).All statistical analyses were performed with the R software package (st in the list, etc. Each average was dated on the horizontal time axis to the mean date of confirmed infection among the respective 50 patients.The datasets of both virulence markers were sorted into ascending order according to the date of confirmed infection of each patient. CD4 slope values below the 5% and above the 95% quantiles were excluded from the analysis. The average of 50 patients was calculated at a time, with the window of averaging moving along the lists of patients. The first point in each graph represents the average of the first 50 patients in the respective list; the second point represents the average of the patients second through 51


The aim was to identify predictive markers of CD4+ cell count declines to < 350/μL in CD4-guided antiretroviral treatment interruptions.The common response to stopping anti-HIV treatment is an increase of HIV-RNA load and decrease in CD4+ count > 600/μL, and HIV-RNA < 50 copies/ml for at least 6 months were offered the option to discontinue antiretroviral therapy. The main outcome was a decline in CD4+ cell count to < 350/μL.27 HIV-infected patients participated in a prospective multicenter study in with a 24 month follow-up. Patients on stable highly active antiretroviral therapy (HAART), with CD4+ cell count to < 350/μL. Patients with a CD4+ nadir of < 200 cells/μL had a greater risk of restarting therapy during the follow-up (RR (CI95%): 3.37 ). Interestingly, lymphoproliferative responses to Mycobacterium tuberculosis purified protein derivative (PPD) below 10000 c.p.m. at baseline ), IL-4 production above 100 pg/mL at baseline ) in PBMC cultured with PPD, and increased IL-4 production of PBMC with p24 antigen at baseline ) were associated to declines in CD4+ cell count to < 350/μL.After 24 months of follow-up, 16 of 27 (59%) patients (who discontinued therapy) experienced declines in CD4+ nadir) and the functional activity of CD4+ (lymphoproliferative response to PPD) predict the CD4+ decrease associated with discontinuation of ART in patients with controlled viremia.Both the number (CD4 After more than ten years of use, combination antiretroviral therapy (ART) continues to have a significant impact on the morbidity and mortality of HIV infection ,2. Despi+ counts fall below 200 cells/mm3. These studies have uniformly identified the nadir CD4+ count or the CD4+ count at the time of initiation of ART as the single, most important predictive marker of the duration of treatment interruption [+ T-cells in the decrease of CD4+ counts in persons who discontinue successful ART. The aim of this study was to identify predictive markers for declines in CD4+ cell count to < 350/μL and to evaluate the long-term effects of CD4-guided antiretroviral treatment interruptions (TI).Recently, large clinical trials have concluded that treatment interruptions can no longer be recommended due to the risk of clinical progression in some circumstances . Howeverrruption . No othe+ count decrease after a follow-up of 24 months, in these 27 patients.A prospective study of the immune, viral, and clinical outcomes of ART interruptions in 27 patients with chronically suppressed HIV-1 infection was carried out in four Spanish hospitals to evaluate the long-term effects of ART interruptions. In addition to the main results of the study, we report herein the factors that have been identified as predictors of the likelihood of a CD4+ > 600/μL, and HIV-RNA load < 50 copies/ml for at least 6 months before discontinuing therapy. Patients were excluded if they previously had AIDS, ART failure, or poor adherence to HAART (< 90%), or if they were pregnant or had been splenectomized. Patients were evaluated monthly during the first three months and every two months thereafter. The criteria for reintroducing HAART, if needed, were: (i) patient's choice, at any CD4+ count level; (ii) appearance of symptoms that could be manifestations of HIV infection (CDC category B events and/or AIDS defining events); and (iii) a decreasing CD4+ count before falling below 350 cells/μL. We checked clinical parameters and measured T-cell subsets, HIV-RNA load, and lymphoproliferative responses (LPR) to different stimuli in vitro.The decision to interrupt ART was made jointly by the physician and the patient, during two weeks, while an offer to be included in this study was made to all the HIV patient from several different hospitals. 27 patients responded affirmatively and a written informed consent form was obtained from all patients. The Ethics Committee of the participating hospitals approved the study. Inclusion criteria for HIV-infected patients on highly active antiretroviral therapy (HAART) interruption were age > 18 years, stable HAART, CD4T lymphocyte CD4 cell counts were determined in whole blood by flow cytometry . Plasma HIV-1 RNA was quantified using the Amplicor Ultransensitive Assay  with a limit of detection of 50 copies/mL.5/200μl per well) resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum. The cells were stimulated with several stimuli (p24 HIV-1 antigen (p24-Ag), streptokinase (SK), tetanus toxoid (TT), Mycobacterium tuberculosis purified protein derivative (PPD), and pokeweed). Cultures were made as reported previously [Peripheral blood mononuclear cells (PBMC) were seeded in 96-well flat-bottom microtiter plates  to identify protein expression in culture supernatants according to the manufacturers' specifications. Multiplex kit  was used to specifically evaluate IL-4 and INF-γ. A minimum of 100 events (beads) was collected for each of the analyte protein and median fluorescence intensities (MFI) were obtained. Analyte protein concentrations were automatically calculated based on standard curve data using MasterPlex™ QT Analysis version 2 . A five-parameter regression formula was used to calculate the sample concentrations from the standard curves.All p-values were 2-tailed, and the threshold of significance was set at 0.05. All statistical analyses were performed with SPSS 12.0 software .+  and an increase of VL .The outcome variables examined were analyzed by the Kaplan-Meier method. This was considered as the duration between the baseline timepoint (HAART interruption) and the appearance of a decrease in CD4+ cell counts to < 350/μL in HIV-infected patients after treatment interruption was analyzed by Cox regression Immunologic characteristics (predictive markers) as a continuous and dichotomic variable. We did not make a multivariate analysis due to the low number of patients included in this study.The value of immunological characteristics to predict declines in CD43) for which they had to restart HAART (with CD4+ > 350 cell/μL). The rest of patients remained completely asymptomatic and did not show signs of clinical progression.Table + 24 months after treatment interruption. A decrease in the CD4+ count was observed in all the patients, 23 (85%) patients experienced declines in CD4+ cell count to < 500/μL, 19 (70%) in CD4+ to < 400/μL, and 16 (59%) in CD4+ to < 350/μL. The patients who experienced declines in CD4+ cell count < 350/μL restarted HAART. Figure Figure + nadir, the CD4+ count at of initiation of HAART, and the CD4+ at study entry (at baseline) were all predictive of declines in CD4+ cell count to < 350/μL during ART interruption in the period of study. We looked for functional characteristics of CD4+ T cells that could be associated with the decline of CD4+ count after ART discontinuation. Interestingly, lymphoproliferative responses to PPD at baseline was inversely related to a decline in CD4+ cell count to < 350/μL during follow-up after ART interruption  did not reveal any significant association.The analysis of characteristics shown in Table on Table . Patient+ cell count to < 350/μL during ART interruption  but also the function (lymphoproliferative response to some antigens) of the immune system seem to be important predictors of the CD4+ decrease associated to discontinuation of ART in well controlled subjects.This study confirms the importance of the CD4n of ART ,9. Not sAs to be expected from previous reports, patients in this study experienced rebounds in HIV-RNA load to pre-HAART levels during TI . Moreove+ T cells, including the decrease of LPR to mitogens and antigens [+ and increase of more than 4 log10 of HIV-RNA load during follow-up.HIV-1 infection is characterized by persistent viremia and a progressive decline in both the number and function of CD4antigens  and the antigens . It is aantigens ,13. In oin vitro is a good test to measure the quality of immune response. LPR to PPD is a marker of cellular immune damage, and its loss is associated with immunodeficiency [+ falling below 350/μL. This may be a consequence of prior severe immune damage leading to the loss of antigen-specific memory T cells [+ count in HIV-infected patients during ART interruption. Our results indicate that LPR to PPD appears to be a useful marker to measure the quality of immune response along with the quantification of CD4+ (quantitative immune response) for the selection of patients to discontinue HAART. However, further study is required to confirm this data.LPR ficiency . The pat T cells . So, LPR+ falling below 350/μL. A previous report showed that there are two types of lymphocytes according to the cytokine secretion profile, and these profiles can be affected by HIV-infection [+ T cells, it is now well known that some of these cytokines are also secreted by CD8+ and non-T cells. It is documented that the type-1 cellular immune response is impaired during the course of HIV infection [We also found that IL-4 production in supernatant of PBMC cultured with PPD and p24-Ag were strong predictive markers of CD4nfection . Type-1 nfection . In addinfection . In our nfection . Thus, tnfection .+ nadirs are also those with poor CD4+ T cell function and these assays simply identify the same individuals. Further studies to look at this as an independent predictor should be carried out with a larger number of patients. In spite of this, we consider our data to be very interesting as well as never having been previously described in non-structured treatment interruption.The main limitation in our study is the small number of patients. Thus, while LPR to PPD may have been predictive of a certain outcome in this small number of patients it is premature to conclude that it is clinically useful for the selection of patients to discontinue HAART. We have not shown it to be predictive in a multivariate analysis. It is, in fact, quite possible that those individuals with lower CD4+ nadir and a good LPR response. Most significantly, we have identified immunological markers that may help explain the dynamics of CD4+ count decline after discontinuing ART and may help identify patients that are most likely to benefit from a potential interruption of therapy.The conclusions of the study may be limited by the small number of patients evaluated. However, the results suggest that ART interruptions may be useful in a specific subset of patients with a minimal CD4The author(s) declare that they have no competing interests.ES carried out the immunoassays and participated in the design of the study and performed the statistical analysis. SR participated in the design of the study, performed the statistical analysis, and contributed to the writing of the manuscript. SM, JCL, AM, RR, JGG, JRA, and FP carried out patient screening, collecting and recording data, and contributed to the writing of the manuscript. MAMF conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Long-term outcomes of antiretroviral therapy (ART) in children remain poorly documented in resource-limited settings. The objective of this study was to assess two-and three-year survival, CD4 evolution and virological response among children on ART in a programmatic setting in Cambodia.Children treated with first-line ART for at least 24 months were assessed with viral load testing and genotyping. We used Kaplan-Meier analysis for survival and Cox regression to identify risk factors associated with treatment failure.3 at 24 months and 737 at 36 months. Median CD4 percentage gain for children under five years old was 15.2% at 24 months and 15% at 36 months. One hundred and thirty children completed at least 24 months of ART, and 138 completed 36 months: 128 out of 268 (48%) were female. Median age at ART initiation was six years.Of 1168 registered HIV-positive children, 670 (57%) started ART between January 2003 and December 2007. Survival probability was 0.93 (95% CI: 0.91-0.95) and 0.91 (95% CI: 0.88-0.93) at 24 and 36 months after ART initiation, respectively. Median CD4 gain for children aged over five years was 704 cells/mmOverall, 22 children had viral loads of >1000 copies/ml  and 21 of 21 presented mutations conferring resistance mostly to lamivudine and non-nucleoside reverse transcriptase inhibitors. Risk factors for failure after 24 and 36 months were CD4 counts below the threshold for severe immunosupression at those months respectively. Only two out of 22 children with viral loads of >1000 copies/ml met the World Health Organization immunological criteria for failure (sensitivity = 0.1).Good survival, immunological restoration and viral suppression can be sustained after two to three years of ART among children in resource-constrained settings. Increased access to routine virological measurements is needed for timely diagnosis of treatment failure. Pediatric HIV/AIDS care programmes in resource-limited settings have been reporting good antiretroviral treatment (ART) outcomes, comparable to those in high-income countries -15. HoweWith an estimated HIV prevalence of 0.9% in the adult population (15-49 years) by the end of 2008 (estimated at 2.2% in 1997) and an estimated 4400 children living with HIV, Cambodia is one of the worst-affected countries in south-east Asia ,17. MédeMore than 1100 HIV-positive children had been enrolled and more than 650 had been started on ART by the end of 2007. These two large-scale pediatric programmes represented 25% of the national pediatric HIV cohort and more than 25% of the total children on ART in the country. We previously described excellent outcomes, with more than 80% virological success in this pediatric cohort, after 12 months of treatment ,5.The aim of the current study was to assess whether the effectiveness of ART was sustained after 24 to 36 months of follow up. We report on survival, CD4 count evolution, virological response, and antiretroviral drug resistance patterns among HIV-positive children receiving first-line ART in a programmatic setting in Cambodia.3 for children older than five years.In collaboration with NCHADS, MSF supported pediatric HIV programmes in two hospitals in Cambodia: the Angkor Hospital for Children, a charity hospital located in Siem Reap province , and Donkeo Referral Hospital, a public health facility in Takeo Province . Since 2003 and 2004, respectively, ART was offered free of charge to HIV-positive children, confirmed with serology or reverse transcriptase polymerase chain reaction (RT-PCR) for children under 18 months, and CD4 count below 20% for children younger than five years of age, or below 200 cells/mmThe standard first-line regimen was stavudine, lamivudine and nevirapine, as recommended by the World Health Organization (WHO) . ZidovudPreparation for ART initiation included a minimum of three counselling sessions for the caregiver and patient, depending on the age of the child. Follow-up visits were monthly throughout the study period.CD4 count and/or CD4 percentage were monitored every six months. Viral load monitoring and genotyping were not routinely performed, but only used to confirm clinical and/or immunological failure by treating physicians. Adherence support was provided to caregivers and children at each visit by specially trained counsellors through individual and age-specific support group sessions. An active tracing system for patients who failed to attend a clinic appointment was set up, and included home visits, additional counselling sessions and psychosocial support.All HIV-positive children aged 15 years or younger who had registered in the two pediatric HIV/AIDS programmes and who had completed at least 24 months of first-line ART by December 2007 were eligible for this study. All children ever started on ART, regardless of time on treatment, were included in the overall survival analysis of the total cohort and the cohort CD4 evolution analysis.Between December 2007 and October 2008, eligible patients had a VL measurement done together with their routine CD4 test. CD4 measurements were performed at a Cambodian Ministry of Health Reference Laboratory  and at the National Institute of Public Health Laboratory in Phnom Penh. HIV-1 RNA VLs were measured at the Pasteur Institute of Cambodia by real-time RT-PCR, using the ANRS G2 LTR-based real-time RT-PCR assay  . For pathttp://www.hiv.lanl.gov/content/sequence/HIV/refer.html as well as for phylogenetic analysis using a nucleotide-distance matrix and the bootstrap neighbor-joining method. The resistant mutations were defined according to the WHO HIV Drug Resistance Database http://hivdb.stanford.edu/pages/WHOResistanceList.html and the drug susceptibility was predicted according to the French National Agency for AIDS Research (ANRS) algorithm http://www.hivfrenchresistance.org/.Chromatograms, sent back electronically to Pasteur Institute of Cambodia, were verified, analyzed and interpreted. Clustal X 1.81 software was used for alignment with subtype reference sequences set from the Los Alamos HIV Sequence Database .© monitoring software .Medical follow-up data were routinely collected at each consultation and entered prospectively into FUCHIAThe Kaplan-Meier method was used to estimate survival for all children on an intention-to-treat basis. Patients were censored on the date of their last visit, or date of transfer, or date of death, before 31 December 2007. CD4 gains were calculated on a six-monthly basis after ART initiation and weight-for-age z-score increases were recorded annually. Success ratio of the entire ART cohort was calculated on an intention-to-treat basis.Treatment failures were defined as patients with VLs of >1000 copies/ml (before and after 24 months of ART), and patients who died, were lost to follow up, or were not assessed. Patients who did not return within three months of their scheduled follow-up visit were considered lost to follow up. Rate ratios were calculated to identify factors associated with virological failure after 24 months of ART. The Cox proportional hazards model was fitted to identify determinants of virological failure. Statistical analyses were performed using Stata 8.2 software .The study protocol was approved by the National Ethics Committee for Health Research of Cambodia, the Ethics Review Board of Médecins Sans Frontières, and the Institutional Review Board of Angkor Hospital for Children, Cambodia. All participants' caregivers gave their written informed consent.A total of 1168 HIV-positive children were registered in the two HIV clinics between March 2002 and December 2007, of whom 670 (57%) started ART between January 2003 and December 2007. Among the 670 children who started ART, 37 (5.5%) had died, 13 (2.0%) were lost to follow up and 47 (7.0%) were transferred out by the end of December 2007. Median time of follow up since ART initiation for all children ever started ART was 24 months (IQR: 12-34). Estimates of survival after admission were 0.93 at 24 months (95% CI: 0.91-0.95) and 0.91 at 36 months (95% CI: 0.88-0.93) when death and loss-to-follow-up events were combined , and median CD4 cell percentage for children under five years was 11.0% (IQR: 6.0-15.8) (Table One hundred and twenty-eight out of 268 (48%) were female and the median age was six years (IQR: 4-8). Only one child was aged less than 18 months at ART initiation. A high proportion of children were already at an advanced stage of HIV disease when starting treatment: 103 (38%) and 53 (20%) were at CDC stages B and C, respectively. Median CD4 count was 190 cells/mm3 (IQR: 469-1034) at 24 months (n = 158) and 737 (IQR: 546-924) at 36 months (n = 115) , six had a VL between 250 and 1000 copies/ml and 22 had a VL above 1000 copies/ml, of whom 15 were over 10,000 copies/ml. Among 130 and 138 patients on ART for at least 24 and 36 months, 112 (86.1%) and 134 (97.1%) patients, respectively, had undetectable VL. The overall treatment success ratio for the cohort on ART for at least 24 months was 86% in an intention-to-treat analysis. Among 22 children with a VL above 1000 copies after 24 months of ART, only two were in immunological failure according to the WHO criteria .HIV-1 reverse transcriptase genotyping was performed for 21 patients with VLs above 1000 copies/ml after 24 months of ART. Genetic analysis revealed that all these children were infected with CRF01_AE viruses. According to the WHO HIV epidemiological survey for scoring resistance-associated mutations list, the most frequent mutations were M184I (n = 18), Y181C (n = 15), K103N (n = 7) and G190A (n = 6)  drugs were available, common to most HIV clinics in the developing world. Despite excellent short- and medium-term programme outcomes in these settings, an expanded drug formulary will eventually be needed as pediatric cohorts mature and ARV resistance becomes inevitable.Unfortunately, drugs used for standardized second-line regimens, such as abacavir, didanosine and lopinavir/ritonavir, are still discouragingly expensive . In addiIn the multivariate model, the only significant risk factor for treatment failure after 24 and 36 months of ART was a CD4 count or CD4 percentage below the threshold for severe immunosupression at those months. A recent study in Thailand has found that children who received nevirapine were 3.7 times more likely to develop virologic failure than those receiving efavirenz, but in our setting we have only few children receiving efavirenz .We calculated the sensitivity, specificity and positive and negative predictive value  of the WHO immunological criteria for failure among this cohort of pediatric HIV patients. While these criteria showed a high specificity, PPV and NPV , the sensitivity was unacceptably low at only 10%. Thus, relying exclusively on WHO immunological criteria would have led to a misclassification of 20 out of 22 treatment failures among the children in our cohort. This finding supports the importance of viral load measurement as a routine monitoring tool for ART.In our previous 12-month study, being an orphan was found to be a greater risk for virological failure, but this factor was not seen here . At thatFinally, regular training sessions were provided to staff in orphanages to improve their care of the children. The current study design could not establish a causal relationship between increased support and improved adherence, but it underlined the importance of regular and systematic analyses of programme outcomes that can result in concrete action.A very low loss-to-follow-up rate was observed in our cohort. Even though we were not able to systematically record treatment adherence as expressed by actual pill intake or evaluation of plasma ARV concentrations, we systematically recorded clinic attendance as a proxy for treatment adherence. Home-based care and early systematic tracing of defaulters was established and followed up with intensive counselling and psychosocial support for both children and their caregivers. Caretakers often reported that the clinical benefits of the treatment were rapidly apparent and that this motivated them to adhere to treatment.These factors might explain not only the very low lost-to-follow-up rate, but also the good overall programme outcomes. Studies from similar settings suggest that higher rates of ART adherence are observed in developing countries and this holds true even for pediatric patients . Meta-anThere are several limitations to this study. First, due to the unavailability of diagnostic tools for infants in the early years of the programme, only a few were started on ART. This probably biased the results towards a better overall outcome. Second, previous ART experience, including exposure to prevention of mother to child transmission regimens, was only self-reported in our clinic (by the caregiver) and we could not verify this. Third, we were not able to systematically record antiretroviral drug toxicities and adverse events in this programme. Fourth, the small number of treatment failures limited the power of the multivariate analysis for identifying risk factors. However, the routine system for monitoring patients, using patient files and electronic records, was robust and regularly checked by supervising teams, and we believe that the results are reliable and representative.Despite these limitations, our study provides reassuring evidence on the medium-term effectiveness of ART in a non-urban, resource-constrained setting.Good survival, immunological restoration and viral suppression were sustained after two to three years of ART among children in resource-constrained settings. Increased access to routine virological measurements is needed for timely diagnosis of treatment failure. Improved access to tolerable, affordable pediatric formulations of second-line ART is also critical so that children in whom virologic failure is diagnosed will have treatment options available.The authors declare that they have no competing interests.PI and MER conceived and designed the study protocol. PI was the study coordinator. TV, CST, KA, EA and VK carried out the study and collected data in the field. SN and EN did the virological evaluation. MER did the statistical analysis. PI and RZ participated in the statistical analysis and data interpretation. PI led the writing of the manuscript; all investigators participated in its final writing and editing.


Pneumocystis jirovecii pneumonia (PcP) using clinical features, induced sputum for immunofluorescent staining, real-time PCR, and posttreatment follow-up. PcP incidence was highest in patients with the lowest CD4 counts but uncommon compared with incidences of pulmonary tuberculosis and bacterial pneumonia.In a prospective study of 660 HIV-positive Malawian adults, we diagnosed  Pneumocystis jirovecii pneumonia (PcP) in HIV-infected adults in the sub-Saharan African region remains uncertain. That PcP is common in African children <1 year of age is well documented  in combination with immunofluorescent (IF) staining for HIV-infected adults (>15 years of age), who sought treatment at a government health center in the township of Ndirande, Blantyre, Malawi, were enrolled in a prospective, community-based study to determine the incidence of infections that were preventable by trimethoprim-sulfamethoxazole prophylaxis  was performed on DNA extracted from the stored induced sputum slides  based on Poisson distribution. First and subsequent episodes in the same person were counted separately, except for PcP, because patients with PcP received secondary prophylaxis and exited the study. The CD4 count at the time of the episode or within the previous 6 months was used for analysis.2, Mann-Whitney, and Student t tests for analysis of age, sex, and CD4 counts among diagnoses, respectively, using SPSS version 12 software . The study was approved by the Institutional Review Boards of the University of Malawi College of Medicine, the University of Maryland, and Michigan State University.We used χBeginning in September 2002, 660 adults were enrolled in the study and followed up through August 2004. Baseline CD4 and World Health Organization stage data are shown in Pneumocystis colonization were recorded, with a mean follow up of 26 weeks (range 4–48 weeks). Ninety-five episodes of suspected PcP occurred in 75 persons. Outcomes of these episodes are given in 3, range 1–103) was not significantly lower than in those with Pneumocystis colonization , but was significantly lower than in those with other diagnoses . Mean age and sex distribution of confirmed PcP, Pneumocystis colonization, and other diagnoses were not significantly different. The case-fatality rate of confirmed PcP was 50%.With full diagnostic workup including posttreatment follow up as the gold standard for the diagnosis of PcP, the sensitivity of PCR alone was 100%, the specificity 88%, and the positive predictive value 31%. Among episodes in which PcP was suspected, the mean CD4 count in patients with confirmed PcP cases (42.5 cells/mm3 (2.5/100 person-years) was clearly lower than in AIDS patients in the United States before the introduction of routine trimethoprim-sulfamethoxazole prophylaxis and highly active ART (10/100 person-years [3), PcP was common, although the incidence was low compared with that of bacterial pneumonia and pulmonary tuberculosis.This is the first community-based prospective study of PcP in a developing country. We found an incidence of PcP in Malawian HIV-infected adults of 1.0/100 person-years, similar to the rates observed in studies that used less comprehensive diagnostic approaches in South African miners (0.5/100 person-years) (We believe it is unlikely that we missed many PcP cases among other diagnoses or losses to follow-up because of the intensive active and passive follow-up and because our facility provided expeditious, high-quality care free of charge. Allowing for reduced sensitivity of induced sputum compared to BAL (Pneumocystis colonization and confirmed PcP were equally common among patients with suspected PcP. More sensitive molecular detection methods would possibly have detected higher rates of colonization. It remains uncertain why certain HIV-infected persons clear Pneumocystis colonization while others develop PcP. The level of immune suppression as indicated by the CD4 count is a possible explanation, although our data do not support this. Genetic differences between P. jirovecii strains may be relevant (We found that 3. Among the most immunocompromised patients, PcP is an important diagnostic consideration.The incidence of PcP in HIV-infected Malawian adults, diagnosed clinically and confirmed with molecular analysis, was low compared with the incidence of bacterial pneumonia and pulmonary tuberculosis at all levels of immunosuppression. PcP rarely occurred with CD4 cell counts >100 mm


The purpose of this study was to evaluate the efficacy and safety of three nevirapine-based antiretroviral treatments for adult antiretroviral-naïve Chinese patients with HIV-1 infection.This was a prospective, multicenter study. 198 antiretroviral-naïve HIV-1 positive subjects with CD4 lymphocyte counts between 100/ul and 350/ul and plasma HIV-1 RNA levels more than 500 copies/ml were randomized to start three NVP-based antiretroviral treatments: group A, NVP+AZT+ddI; group B, NVP+3TC+d4T; group C, NVP+AZT+3TC. Viral responses, immunologic responses, adverse events and drug resistence were monitored at baseline and the end of week 4, 12, 24, 36, 52. Viralogical response and immunological response were also comparaed in different strata of baseline CD4 T lymphocyte counts and plasma HIV-1 RNA concentrations. At baseline, the plasma HIV-1 RNA was 4.44±0.68, 4.52±0.71 and 4.41±0.63 lg copies/ml in group A, B and C respectively (p = 0.628). At the end of the study, the plasma viral load reached 2.54±1.11, 1.89±0.46 and 1.92±0.58 lg copies/ml in group A, B and C respectively (p<0.001). At week 52, suppression of plasma HIV-1 RNA to less than 50 copies/ml was achieved in more patients in group B and C than in group A . In planned subgroup analyses, the decrease of viral response rate was seen in group A when CD4 cell count >200/ul (subgroup H). But in subgroup L, viral response rate of three groups has no significant statistic difference. There were no statistically significant differences among three groups in immunological response wthin any of the CD4 or pVL strata. 3 out of 193 patients with available genotype at baseline showed primary drug resistant. Of 26 patients with virologic failure, 17 patients showed secondary drug resistant, 16 subjects in group A and 1 subject in group B. Logistic regression analysis indicated that presence of hepatotoxicity was associated with HCV-Ab positive  and higher CD4 baseline (CD4 count >250/ul).Our findings strongly support the use of 3TC+d4T and 3TC+AZT as the nucleoside analogue combination in NVP-based antiretroviral therapy. The regimen of AZT+ddI+NVP produced poor virological response especially in the stratum of CD4 count more than 200/ul. More patients showed secondary drug resistant in this arm too. Patients with HCV-Ab+ and CD4 count >250/ul appear to have significantly high risk of hepatoxicity.NCT00618176ClinicalTrials.gov  Acquired immunodeficiency syndrome (AIDS) is a life-threating disease caused by human immunodeficiency virus (HIV). The advent of highly active antiretroviral therapy (HAART) has resulted in profound suppression of HIV replication, substantial increase of CD4+ T cells and partial reconstitution of the immune system.The protocol for this trial and supporting consort checklist are available as supporting information; see This study was approved by the ethics committee of Peking Union Medical College Hospital. Enrollment began in January 2005. Patient Information and Consent to Medical Treatment and sign a written consent form. Consenting individuals 18 years or older were recruited from thirteen research centers of China. They were found to be HIV-seropositive by standard serum enzyme-linked immunosorbent assay (ELISA) tests and also by Western blot analysis. Patients were considered for inclusion in this study if they were antiretroviral drug-naïve. The eligibility criteria for participants were CD4+ T-cell count from 100 to 350 cells/mm3; plasma viral load over 500copies/ml. Main exclusive criteria were pregnancy or breastfeeding, anticipated nonadherence, AIDS-defining illness within 2 weeks of entry, white blood cell count less than 2.0×109/L, absolute neutrophil count less than 1.0×109/L, hemoglobin level less than 90g/l, platelet count less than 0.75×1012/L, transaminase and alkaline phosphatase level more than 3 times the upper limit of the normal range, bilirubin level more than 2.5times the upper limit of the normal range, serum creatinine level more than 1.5 times the upper limit of the normal range.Patients were randomly allocated to three treatment groups: group A, AZT(300mg twice daily [bd])+ddI (200mg bd or 125mg bd when less than 60kg)+NVP(200mg once daily for 2 weeks and 200mg bd thereafter); group B, 3TC(150mg bd)+d4T(30mg bd or 20mg bd when less than 60kg)+NVP; group C, AZT+3TC+NVP.  For farther analysis, patients were stratified by baseline CD4+T cell count: Subgroup L was defined as the baseline CD4+T cell ≤200/ul; subgroup H was defined as the baseline CD4+T cell >200/ul, and baseline plasma viral load (pVL): subgroup A, the pVL ≤10,000copies/ml; subgroup B, the pVL >10,000 and ≤100,000copies/ml; subgroup C, the pVL>100,000copies/ml. Patients were also stratified by baseline CD4+ T cell≤250/ul or CD4 + T cell>250/ul in the analysis of adverse effects.The present study was divided into a screening period of 1–4 weeks and a follow-up period of one year. At the end of the screening period, the eligible patients were randomly allocated to one of the three treatment groups. The randomization was performed by drawing of envelopes. The follow-up period then started, with five visits at 4, 12, 24, 36, 52 weeks. During all of the visits the clinical assessment was recorded in the CRF and the subjects had samples taken for laboratory assessment and further detection including T cell subset, pVL and analysis of genotype resistance. The adverse events were classified and graded according to division of AIDS table for grading the severity of adult and pediatric adverse events.Viral load:Plasma was separated from whole blood by centrifugation within 4h of collection and was stored frozen at −80°C until tested. The QUANTIPLEX HIV-1 RNA assay, version 3.0 (bDNA 3.0 assay), was performed according to the manufacturer's instructions that were provided with the assay kit. The limits of detection of the assay, indicated by the manufacturer, was 50∼500,000 HIV-1 RNA copies/ml.Immunofluorescent surface staining and flow cytometric analysis:Peripheral blood mononuclear cells were obtained by separation from the centrifugation gradient. Subpopulations of CD3+, CD4+, and CD8+ cells were determined by three-color flow cytometry  at baseline and at the end of week 4, 12, 24, 36, 52. The following groups of monoclonal antibodies were used: PEcy5-CD4/PE-CD8/FITC -CD3 (CD4+/CD8+T cell counts). All monoclonal antibodies were purchased from Beckman-Coulter and Immunotech, USA.Blood routines, liver functions, sero-amylase, HCV-Ab, and serum lipids were administered by the clinical laboratory department of each research centers.http://hivdb.stanford.edu) to detect drug-resistance mutations. The protocol of nest PCR and DNA sequencing were described elsewhere.Genotype resistance: Viral RNA was extracted from the isolated viruses using a QIAamp Viral RNA Mini Kit  according to the manufacturer's protocol, and stored at −70°C until use. Reverse transcript reaction was run to synthesis of cDNA. The 1100-bp pol gene fragment encompassing the complete protease gene and the first 220 codons of the RT gene of HIV-1 RNA were amplified by nested PCR strategy and purified using a QIAquick Gel Extraction Kit (Qiagen Inc.). The product was sequenced and the results were compared to the consensus B reference sequence using HIVdb software  we need about 60 patients in each treatment group. Allowing for 20% loss to follow up, a total sample of 220 was required.All statistical analysis was performed using the SPSS 11.5 statistical package. In present study, we performed two separate and complementary analyses of these data: the analysis according to the actual treatment and the intention-to-treat analysis. For the analysis according to actual treatment, we included only patients for whom outcomes were measured at specified time points. The intention-to-treat analysis involved patients who underwent randomization, received at least one dose of study medication, and had at least one plasma HIV-1 RNA measurement after treatment began. Missing values were imputed for the analysis with the use of a last-observation-carried-forward method. When a continuous outcome variable is considered, we use analysis of covariance to control for baseline variables. The change between the baseline value and the follow-up value of a particular outcome is evaluated by Kruskal-Wallis test or Mann-Whitney U test. Using a chi-square test, we compared the rates of response among the treatment groups in terms of the percentage of patients with suppression of plasma HIV-1 RNA levels to less than 400 copies per milliliter as well as the percentage for whom it was less than 50 copies per milliliter. Multivariate logistic regression analyses were used to identify factors associated with the presence of certain outcome. For all tests, P<0.05 was considered to be statistically significant.Of the 362 subjects screened, 198 HIV-positive antiretroviral-naive patients were recruited into this study. Demographic characteristics of the groups are summarized in In an intent-to-treat (ITT) analysis, the changes of plasma viral load during 52-week HAART in group A, B and C, are shown in 110 patients (58.8%) achieved a plasma VL of <50 copies/ml, 155 patients (82.9%) achieved a plasma VL of <400copies/ml. The percentage of patients who achieved a plasma VL of <50 copies/ml was 39.7% in group A, 68.2% in group B and 69% in group C (P<0.001). The percentage of patients who achieved a plasma VL of <400 copies/ml was 63.5% in group A, 92.4% in group B and 93.1% in group C (P<0.001).The viral response rate was analyzed when the subjects stratified by baseline CD4+T cell count. The baseline pVLs were 4.67±0.63 lg copies/ml in subgroup L and 4.29±0.66 lg copies/ml in subgroup H (P<0.001). At the end of 52 weeks, the pVL reached 2.07±0.80 lg copies/ml in subgroup L and 2.09±0.77 lg copies/ml in subgroup H (P = 0.744). The viral response rate of patients who divided into three therapy groups and stratified by baseline CD4+T cell count are showed in The increase in CD4+T cell count was divided into two phases: a rapid increase in the first 12 weeks constituted the first phase and a slow increase after week 12 constituted the second phase. The increased magnitudes of CD4+T cell count were 72/ul at week 4 and 105/ul at week 12. At the end of the study, the increased magnitudes of CD4+T cell count reached 149/ul. There was no significant difference among the mean CD4+T cell count of three groups at weeks 4, 12, 24, 36, 52. When the subjects stratified by baseline CD4+T cell count, the CD4+T cell count reached 289±128/ul at subgroup L and 432±144/ul at subgroup H (P = 0.581) at week 52. The increase trends of CD4+T cell count in two subgroups were parallel to each other. In each subgroup, there is no statistic difference of mean CD4+T cell count and the increased magnitute of CD4+T cell count in three treatment groups.When the subjects stratified by baseline pVL, the baseline CD4+T cell counts were 250±68/ul in subgroup A, 216±74/ul in subgroup B, 195±73/ul in subgroup C (P<0.001). At the end of this study, CD4+T cell count reached 366±139/ul, 368±158/ul, 338±163/ul in subgroupA,B, C (P = 0.172).There is no statistical difference of CD8+T cell count and CD4/CD8 ratio of three therapy groups at the baseline and at weeks 4, 12, 24, 36, 52.After 52 weeks of HAART, totally 14 patients (7.1%) lost to follow-up. In remain 184 patients, common complaints, such as fatigue, malaise and weight loss, were released at the end of this study. No one developed new AIDS-defining illness and died.285 cases of adverse events occurred in 198 subjects. The details can be seen in 37 patients changed the corresponding drug because the adverse events were intolerable and could not be controlled by symptomatic treatment.  Bone marIn patients in all three groups, the most common treatment-related adverse events were hepatotoxicity. It occupied 47.1% severe adverse events (grade 3–4). The ratio of severe hepatotoxicity was 12/64(18.8%), 19/69(27.5%), 10/65(15.4%) respectively in treatment group A, B, C. Multivariate logistic regression analyses were used to identify factors associated with the presence of hepatotoxicity (grade 2–4). Patients have higher CD4 baseline (CD4 count >250/ul) and HCV-Ab positive were more likely to have hepatotoxicity after controlling for treatment group and the gender. If the subjects divided by the gender, logistic regression analyses showed that higher CD4 baseline (CD4 count >250/ul) increased the odds of developing hepatotoxocity in women  but not in men(P = 0.511). The bone morrow suppression occupied 29.9% severe adverse events (grade 3–4).Genotypic testing was carried out on baseline specimens and on specimens with pVL more than 1000copies/ml after 12 weeks. At baseline, among the 193 patients for whom HIV-1 RNA could be amplified, 3 subjects had isolates showing resistance mutations.At virologic failure after 12-week therapy, of 26 patients without preexisting resistance, 17 patients had genotypic results available. Of the 17 with genotypic information available, 16 patients were in group A, and the other one was in group B. The mutations mostly occurred at 24-week therapy. Mutations related to NNRTI  were detected in 17 patients. Mutations related to NRTI  were detected in 8 out of 17 patients.175 patients had HCV-Ab test at baseline. 58 subjects (29.3%) were HCV-Ab positive. The baseline HIV pVL of HCV(+) subjects and HCV(−) subjects were 4.43±0.72 copies/ml, 4.47±0.64 copies/ml (p = 0.75). The baseline CD4+T cell count of HCV(+) subjects and HCV(−) subjects were 231.1±75.6/ul, 218.0±75.3/ul (p = 0.281). At the end of this study, 57.4% HCV(+) patients and 57.9% HCV(−) patients achieved a pVL of less than 50copies/ml (p = 0.952). The increased magnitute of CD4+T cell count was 169.3±123/ul in HCV(+), 131.7±121/ul in HCV(−) (p = 0.162).As we metioned before, logistic regression analysis indicated that presence of hepatotoxicity was independently associated with HCV-Ab positive .NVP-based antiretroviral treatments are most accessible regimens in China. This was a prospective, multicentre study to evaluate three NVP-based regimens in terms of antiviral efficacy and tolerability in Chinese HIV-1 infected patients.To date, information on the direct comparison among NVP-containing antiretroviral therapy has been scarce. The dissimilar criteria used of inclusion and exclusion in the studies makes it difficult to compare the efficacy of the regimens with different nucleoside backbones described in the different trial. In this study, we found significant difference over 52 weeks among three NVP-based regimens in the initial treatment of HIV-1 infection with regard to virologic response and resistance mutation. The regimen of AZT+ddI+NVP produced poor virological response especially in the stratum of CD4 count more than 200/ul. In the INCAS trail, the maximal effect in the AZT+ddI+NVP group was observed at week 16, with 68% of patients demonstrating a pVL<20 copies/ml, with 51% maintaining this level of suppression at week 52.In this study, two of the NVP-containing regimens: NVP+d4T+3TC, NVP+AZT+3TC, are effective in ART-naïve patients, producing a durable virological and immunological response. There are no significant differences over 52 week between these two regimens with regard to virological response, immunological response, adverse events or resistance mutations at virologic failure. In a comparison with an Australian trial of the same NRTIs with NVP, the 3TC+d4T and 3TC+AZT arm of the current study had similar proportion of patients at the end of the study with plasma HIV RNA <50copies/ml. There were no statistically significant differences of virological response in any pVL or CD4 count strata. After 4-week therapy, there were no statistically significant differences of pVLs in any strata at all. And this trend lasted until the end of the study. Lower CD4 cell counts and higher viral loads at baseline were not associated with poorer virological outcome of HAART. A report from Andrew Phillips,Primary resistance is common among adults recently infected with HIV-1 in North America and Europe. The safety profiles of the three therapeutic arms were also assessed in present study. Hepatotoxicity was the major adverse events of NVP-based therapy. Stern JO and his colleagues reported the increased risk of severe hepatotoxicity in women with CD4 count >250/ul and men with CD4 count >400/ul.The incidence of hyperlipidemia in treatment group A and C is accordant with the result of 2NN.All of the three therapeutic arms contain at lest one of AZT and 3TC. As the papers reported The adverse events such as rash, gastrointestinal disorders, peripheralneuropathy are always mild and can be controlled by symptomatic therapy. But it is important to well handle these adverse events because it may affect the adherence of HAART.In conclusion, as virological response and incidence of viral resistance has been concerned, 3TC+d4T and 3TC+AZT as the nucleoside analogue combination of NVP-based HAART is recommended. The diversity of immunological response of three NVP-based arm do not reach the statistic significant. We confirmed that incidence of hepatotoxicity is significantly higher in subgroup of CD4 count >250/ul and subjects with HCV-Ab positive in Chinese.Checklist S1CONSORT Checklist(0.05 MB PDF)Click here for additional data file.Protocol S1Trial Protocol(0.07 MB PDF)Click here for additional data file.


We report 4 chinese patients with hepatitis B e antigen-positive chronic hepatitis B achieving clearance of HBsAg by using pegylated interferon alpha-2a for less than 1 year, to provide one clinical clue for the treatment of chronic hepatitis B. Chronic infection with Hepatitis B virus(HBV) is a major global health problem, affecting 350–400 million people worldwide ,2. Patie6 copies/mL to 5.00 × 107 copies/mL. They came to our hospital for pegylated interferon alpha-2a(Pegasys) treatment from March 2006 to Ten 2007. All of them had neither complications nor other accompanying diseases, also with no history of anti-HBV drug use.All 4 patients aged from 14 to 49 yrs-old, including 3 males and 1 female, had suffered from CHB for 1 to 3 years with the sera profile of HBeAg, HBsAg positive, and HBV DNA leveled from 2.71 × 10Pegasys monotherapy Case 1 was treated with pegasys at a dose of 135 ug intracutaneously one time per week(for her age was below 18). Case 2 was treated with pegasys at a dose of 180 ug intracutaneously one time per week.Sequential therapy As both leucopenia and thrombocytopenia occurred in case 3 and case 4 during the treatment of pegasys, we adopted sequential therapy with pegasys and entecavir(Baraclude). Pegasys was administered at a dose of 180 ug intracutaneously one time per week. Baraclude was administered at a dose of 0.5 mg orally one time per day.Leukocyte and platelet counts, serum HBV DNA and ALT were detected once every month. HBV markers were detected once every three months. Among them, serum HBV DNA was arrayed by fluorescent quantitative PCR and HBV markers were arrayed by ELISA.Within less than 1 year, serum HBV DNA loss, ALT normalization, HBeAg seroconversion, HBsAg clearance occurred in the 4 patients treated with either pegasys monotherapy  or sequential therapy with pegasys and baraclude . Among them, the latest time when HBsAg clearance occurred (in case 4) was at week 44, and the earliest time when HBsAg clearance occurred (in case 1) was at week 17. Furthermore, HBsAg seroconversation occurred in case 1 at week 20, which is sustained positive in 48 weeks follow-up, while HBsAg negative also kept steady in case 2, case 3, case 4 in 60 weeks follow-up, 60 weeks follow-up, 72 weeks follow-up, respectively. All of them are kept under observation until now. The Variations of sera HBV DNA, ALT and HBV markers in the four patients are outlined in Table During the course of pegasys treatment, apparent reduction of leukocyte and platelet counts had occurred in 2 patients (case 3 and case 4) at week 4 and week 10 of therapy respectively, but returned to baseline levels one month after sequential therapy was adopted . There were no other remarkable adverse events in all the four cases during the total course of anti-viral therapy.The aims of treatment for CHB are to achieve sustained suppression of HBV replication, thereby inducing remission of liver diseases and interrupting progression to liver cirrhosis or hepatocellular carcinoma. PegylatBy analyzing above materials, we had observed one phenomenon that the clearance of HBsAg occurred in the 4 patients not very long after they had achieved HBeAg seroconversion. This may imply that pegylated interferon alpha-2a probably plays an important role in helping clear HBsAg by means of activating the host immune reaction. Additionally, ALT levels in the 4 patients were elevated during treatment but gradually normalized at the end of therapy, they also had no clinical symptoms. Considering that pegylated interferon alpha-2a takes effect by improving the host immune function, we suppose the occurrence of transient elevated ALT might be a reflection of the course during which HBV infectious hepatocytes are being eradicated.Although limited case numbers are reported in this article and more patients need to be included for further study, one certain clue, which can be provided to clinical doctors, is that some patients with HBeAg-positive CHB could probably be expected to achieve the ideal therapeutic goal of HBeAg seroconversion, HBsAg clearance and even HBsAg seroconversion if they are treated with effective anti-viral drugs and appropriate therapeutic schemes.Based on our clinical observation, either pegasys monotherapy or sequential therapy with pegasys and baraclude is efficacious and relatively safe for treating patients with CHB.The authors declare that they have no competing interests.YJ conceived the study and made substantial contributions to its design, acquisition, analysis and interpretation of data. ZL participated in the design and revised the manuscript critically for important intellectual content.