The impact of the emergence of drug-resistance mutations on mortality is not well characterized in antiretroviral-naïve patients first starting highly active antiretroviral therapy (HAART). Patients may be able to sustain immunologic function with resistant virus, and there is limited evidence that reduced sensitivity to antiretrovirals leads to rapid disease progression or death. We undertook the present analysis to characterize the determinants of mortality in a prospective cohort study with a median of nearly 5 y of follow-up. The objective of this study was to determine the impact of the emergence of drug-resistance mutations on survival among persons initiating HAART.Participants were antiretroviral therapy naïve at entry and initiated triple combination antiretroviral therapy between August 1, 1996, and September 30, 1999. Marginal structural modeling was used to address potential confounding between time-dependent variables in the Cox proportional hazard regression models. In this analysis resistance to any class of drug was considered as a binary time-dependent exposure to the risk of death, controlling for the effect of other time-dependent confounders. We also considered each separate class of mutation as a binary time-dependent exposure, while controlling for the presence/absence of other mutations. A total of 207 deaths were identified among 1,138 participants over the follow-up period, with an all cause mortality rate of 18.2%. Among the 679 patients with HIV-drug-resistance genotyping done before initiating HAART, HIV-drug resistance to any class was observed in 53 (7.8%) of the patients. During follow-up, HIV-drug resistance to any class was observed in 302 (26.5%) participants. Emergence of any resistance was associated with mortality . When we considered each class of resistance separately, persons who exhibited resistance to non-nucleoside reverse transcriptase inhibitors had the highest risk: mortality rates were 3.02 times higher  for these patients than for those who did not exhibit this type of resistance.We demonstrated that emergence of resistance to non-nucleoside reverse transcriptase inhibitors was associated with a greater risk of subsequent death than was emergence of protease inhibitor resistance. Future research is needed to identify the particular subpopulations of men and women at greatest risk and to elucidate the impact of resistance over a longer follow-up period. Emergence of resistance to both non-nucleoside reverse transcriptase inhibitors and protease inhibitors was associated with a higher risk of subsequent death, but the risk was greater in patients with NNRTI-resistant HIV. In the 1980s, infection with the human immunodeficiency virus (HIV) was effectively a death sentence. HIV causes AIDS (acquired immunodeficiency syndrome) by replicating inside immune system cells and destroying them, which leaves infected individuals unable to fight off other viruses and bacteria. The first antiretroviral drugs were developed quickly, but it soon became clear that single antiretrovirals only transiently suppress HIV infection. HIV mutates  very rapidly and, although most of these changes (or mutations) are bad for the virus, by chance some make it drug resistant. Highly active antiretroviral therapy (HAART), which was introduced in the mid-1990s, combines three or four antiretroviral drugs that act at different stages of the viral life cycle. For example, they inhibit the reverse transcriptase that the virus uses to replicate its genetic material, or the protease that is necessary to assemble new viruses. With HAART, the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. As a consequence, for many individuals with access to HAART, AIDS has become a chronic rather than a fatal disease. However, being on HAART requires patients to take several pills a day at specific times. In addition, the drugs in the HAART regimens often have side effects.Drug resistance still develops even with HAART, often because patients don't stick to the complicated regimens. The detection of resistance to one drug is usually the prompt to change a patient's drug regimen to head off possible treatment failure. Although most patients treated with HAART live for many years, some still die from AIDS. We don't know much about how the emergence of drug-resistance mutations affects mortality in patients who are starting antiretroviral therapy for the first time. In this study, the researchers looked at how the emergence of drug resistance affected survival in a group of HIV/AIDS patients in British Columbia, Canada. Here, everyone with HIV/AIDS has access to free medical attention, HAART, and laboratory monitoring, and full details of all HAART recipients are entered into a central reporting system.The researchers enrolled people who started antiretroviral therapy for the first time between August 1996 and September 1999 into the HAART Observational Medical Evaluation and Research (HOMER) cohort. They then excluded anyone who was infected with already drug-resistant HIV strains (based on the presence of drug-resistance mutations in viruses isolated from the patients) at the start of therapy. The remaining 1,138 patients were followed for an average of five years. All the patients received either two nucleoside reverse transcriptase inhibitors and a protease inhibitor, or two nucleoside and one non-nucleoside reverse transcriptase inhibitor (NNRTI). Nearly a fifth of the study participants died during the follow-up period. Most of these patients actually had drug-sensitive viruses, possibly because they had neglected taking their drugs to such an extent that there had been insufficient drug exposure to select for drug-resistant viruses. In a quarter of the patients, however, HIV strains resistant to one or more antiretroviral drugs emerged during the study (again judged by looking for mutations). Detailed statistical analyses indicated that the emergence of any drug resistance nearly doubled the risk of patients dying, and that people carrying viruses resistant to NNRTIs were three times as likely to die as those without resistance to this class of antiretroviral drug.These results provide new information about the emergence of drug-resistant HIV during HAART and possible effects on the long-term survival of patients. In particular, they suggest that clinicians should watch carefully for the emergence of resistance to NNRTIs in their patients. Because this type of resistance is often due to poor adherence to drug regimens, these results also suggest that increased efforts should be made to ensure that patients comply with the prescribed HAART regimens, especially those whose antiretroviral therapy includes NNRTIs. As with all studies in which a group of individuals who share a common characteristic are studied over time, it is possible that some other, unmeasured difference between the patients who died and those who didn't—rather than emerging drug resistance—is responsible for the observed differences in survival. Additional studies are needed to confirm the findings here, and to investigate whether specific subpopulations of patients are at particular risk of developing drug resistance and/or dying during HAART.http://dx.doi.org/10.1371/journal.pmed.0030356.Please access these Web sites via the online version of this summary at US National Institute of Allergy and Infectious Diseases fact sheet on HIV infection and AIDSUS Department of Health and Human Services information on AIDS, including details of approved drugs for the treatment of HIV infectionUS Centers for Disease Control and Prevention information on HIV/AIDSAidsmap, information on HIV and AIDS provided by the charity NAM, which includes details on antiretroviral drugs Post-surveillance cohort studies and long-term follow-up studies of clinical trials, like the Merck 035 study, continue to suggest the ongoing durability of highly active antiretroviral therapy (HAART) –3. MortaEmerging resistance poses a growing threat to the ongoing success and durability of HAART regimens. Nearly 80% of antiretroviral-treated patients with detectable viraemia in one large US-based clinic study had evidence of phenotypic drug resistance . The freThe current impact of the emergence of drug-resistance mutations on mortality, however, is not well characterized in antiretroviral-naïve patients first starting HAART. Patients may have sustained immunologic function with resistant virus , and theThe distribution and the population-based monitoring of antiretroviral therapy in British Columbia have been extensively described in the literature . Since 1Since October 1992, the distribution of antiretrovirals has been the responsibility of the HIV/AIDS Drug Treatment Program of the British Columbia Centre for Excellence in HIV/AIDS (the Centre). The Centre distributes antiretroviral drugs based on guidelines generated by the Therapeutic Guidelines Committee, which is made up of physicians, pharmacists, virologists, health service researchers, and economists . The CenAll antiretroviral treatment recipients in the province are entered into an Oracle-based monitoring and evaluation reporting system that uses standardized indicators to prospectively track the antiretroviral use and clinical and health status of HIV-1-positive individuals. Physicians enrolling an HIV-1-infected individual into the system must complete a drug request enrolment prescription form, which compiles information on the participant's address, past HIV-specific drug history, CD4 cell counts, plasma HIV-1 RNA, current drug requests, and enrolling physician data. Typically, persons receiving antiretroviral therapy are monitored by physicians at intervals no longer than 3 mo, at which time prescriptions are renewed or modified. At the time of the initial dispensation, participants are asked to provide informed consent for accessing medical electronic records  and to complete a participant survey, which elicits information on sociodemographic characteristics, clinical and health status, and alternative therapy use. Both the consent form and the participant survey are optional, and participant's refusal to do either does not limit his or her access to free antiretroviral therapy. At the same time, the treating physicians are asked to complete a clinical staging form using the World Health Organization clinical staging system.The Centre recommends that plasma HIV-1 RNA levels and CD4 cell counts be monitored at baseline, at 4 wk after starting antiretroviral therapy, and every 3 mo thereafter. Plasma HIV-1 RNA levels were determined using the Roche Amplicor Monitor assay  using either the standard method or the ultrasensitive adaptation. Plasma samples were stored chronologically as they were drawn and securely stored (frozen at −20 °C) for future use. CD4 cell counts were measured by flow cytometry, followed by fluorescent monoclonal antibody analysis .Resistance testing was also completed on stored plasma HIV-1 RNA samples –14. HIV All HIV-infected men and women in the current study were entered into the Centre's monitoring and evaluation system when they were first prescribed antiretroviral agents. Eligible study participants were persons who were antiretroviral naïve and were first dispensed triple combination therapy between August 1, 1996, and September 30, 1999. Participants must also have had a CD4 count and plasma HIV-1 RNA measurement within 6 mo of the first antiretroviral start date. Study data from eligible participants were extracted from the Centre's monitoring and evaluation system to form the HAART Observational Medical Evaluation and Research (HOMER) cohort.For all HOMER cohort participants, HIV drug resistance genotyping was attempted on all samples with HIV-1 RNA levels of 1,000 copies/ml or more collected in the first 30 mo following initiation of HAART. HIV isolates were assigned to one of four resistance categories based on a modification of the International AIDS Society–USA table . SamplesThe primary endpoint in this analysis was all cause mortality. Deaths occurring during the follow-up period were identified on a continuous basis from physician reports and through annual record linkages carried out with the British Columbia Division of Vital Statistics.The following explanatory variables were investigated: age, gender, CD4 cell count, plasma HIV-1 RNA levels, prior AIDS diagnosis, protease inhibitor use, current or past history of injection drug use, physician experience, adherence, and drug resistance. Physician experience was defined as the number of HIV-positive patients the physician had previously treated at the time the study participant was enrolled into the HIV/AIDS Drug Treatment Program. Estimates of adherence to antiretroviral therapy are based on medications actually dispensed, not prescribed. Patients receive new prescriptions at various time intervals ranging from monthly to, at most, every 3 mo. For this exercise we limited our measure of adherence to the first year of therapy and estimated it by dividing the number of months of medications dispensed by the number of months of follow-up.Cumulative mortality rates were estimated using Kaplan-Meier methods. Event-free participants were right censored as of June 30, 2003. Participants included in this analysis were not followed after this date, and those lost to follow-up were censored at the date of last known contact with the HIV/AIDS Drug Treatment Program.Cox proportional hazard regression was used to model the simultaneous effect of prognostic variables on survival . A forwaIn this analysis, a number of prognostic variables were treated as time-dependent variables including the following: protease inhibitor use, a diagnosis of AIDS, CD4 cell count, plasma HIV-1 RNA levels, and drug resistance. Protease inhibitor use (yes versus no), a diagnosis of AIDS (yes versus no), plasma HIV-1 RNA levels , emergence of resistance to antiretrovirals (yes versus no), and each class of resistance mutation (yes versus no) were treated as binary time-dependent variables. In our analysis we assumed that once resistance was detected, this variable was not allowed to change values until the end of the follow-up period. Other variables were measured at baseline and treated as either categorical or continuous. Gender  and history of injection drug use (yes versus no) were treated as fixed binary variables. Age (in years) and physician experience (per 100 patients followed) were treated as continuous variables.Marginal structural modeling was used to address potential confounding between time-dependent variables in the Cox proportional hazard regression models . This pop-value of less than 0.05 indicating that an association was statistically significant.Analyses were performed using SAS software version 9.1.3 service pack 3 . All tests of significance were two-sided, with a p < 0.001). No other significant associations were noted between the two groups.Between August 1, 1996, and September 30, 1999, a total of 1,312 antiretroviral-naïve participants aged 18 y and over initiated triple combination therapy consisting of two nucleosides plus a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor. Of these, 121 (9.2%) were excluded from this analysis for not having both baseline CD4 and plasma HIV-1 RNA level measures available within 6 mo prior to the start of antiretroviral therapy or for having initiated therapy as part of a clinical trial. Among the remaining 1,191 study participants, there were 679 patients with HIV-drug-resistance genotyping done on samples of plasma HIV-1 RNA before initiating HAART at the time of this analysis. HIV-drug resistance to any class was observed in 53 (7.8%) of the patients. Among these patients, 39 (73.6%) exhibited resistance to one class, ten (18.9%) to two classes, and four (7.6%) to three classes. A total of 14 (26.4%) exhibited mutations conferring resistance to lamivudine, 29 (54.7%) to other nucleoside reverse transcriptase inhibitors, eight (15.1%) to non-nucleoside reverse transcriptase inhibitors, and 19 (35.8%) to protease inhibitors. These patients exhibiting baseline resistance were excluded from the analysis, and the total study sample was based on the remaining 1,138 (86.7%) participants. Study participants had HIV-1 RNA levels higher than those of individuals excluded from the analysis , a total of 229 (20.1%) patients achieved durable viral suppression  during the entire follow-up period. There were 776 (68.2%) patients with at least one HIV-1 RNA level measurement at 1,000 copies/ml or above that showed no detectable resistance during the follow-up period, and we assumed these individuals had no drug resistance. Although we systematically genotyped all samples with HIV-1 RNA levels of 1,000 copies/ml or more during follow-up, there were three (0.3%) patients for whom the resistance test failed at least once for technical reasons, and for these particular tests we assumed that no mutation was detected.Among the remaining HOMER participants , plasma HIV-1 RNA level was 120,000 copies/ml , physician experience was 44 patients per physician . There were 317 (27.9%) participants who had a history of injection drug use and 149 (13.1%) study participants with a prior diagnosis of AIDS. Among all participants, 956 were males (84.0%).Study participants were first prescribed 26 different triple combination antiretroviral regimens. The overall median time of follow-up was 56.4 mo . The median number of CD4 count measurements was 3.59 per year , and the median number of plasma HIV-1 RNA measurements was 3.44 per year . A total of 17,632 CD4 cell counts and 17,358 HIV-1 RNA determinations were collected for participants over the study period. The median numbers of CD4 cell counts and HIV-1 RNA determinations per participant were 15  and 15 , respectively. In this study, a switch in drug regimen was recorded when any modification  in the original drug regimen occurred. Men and women switched one or more drugs in their regimen a median three times  over the follow-up period. Protease inhibitors were added to or removed from a regimen a median of one time , non-nucleoside reverse transcriptase inhibitors one time , and nucleoside reverse transcriptase inhibitors three times . The median time to first switch was 7.8 mo  in this study population.Drug-resistance testing was completed on a total 3,099 samples over the first 30 mo of follow-up. A median of three  samples were tested per participant. HIV-drug resistance to any class was observed in 302 (28.5%) participants. Of these, 19 (6.3%) exhibited resistance to all three classes, 137 (45.4%) to two classes, and 146 (48.3%) to one class. A total of 221 (73.2%) exhibited mutations conferring resistance to lamivudine, 98 (32.5%) to other nucleoside reverse transcriptase inhibitors, 157 (52.0%) to non-nucleoside reverse transcriptase inhibitors, and 66 (21.9%) to protease inhibitors. The median time to first emergence of resistance to any class of drug under study was 16.5 mo .p = 0.007), to have lower CD4 counts (p < 0.001), and to have higher plasma viral load (p < 0.001) than persons who did not develop any resistance over the first 30 mo.As of June 30, 2003, a total of 207 deaths were identified in the study population over the follow-up period, with an overall crude mortality rate of 18.2%. The product limit estimate of the cumulative mortality rate at 12 and 24 mo was 4.4% (±0.6%) and 8.7% (±0.8%), respectively. Of these deaths, 30 (14.5%) were to patients who exhibited resistance to one class, 26 (12.6%) to two classes, and one (0.5%) to three classes. A total of 41 (19.8%) deaths were to patients who exhibited resistance to lamivudine, 15 (7.3%) to other nucleosides, 32 (15.5%) to non-nucleoside reverse transcriptase inhibitors, and six (2.9%) to protease inhibitors. Nearly, three-quarters of the deaths involved no resistance to any class of drug .The univariable and multivariable analyses of the baseline and time-dependent factors associated with all cause mortality are presented in n = 160; 77.3%). Individuals dying from non-accidental deaths in the study had less experienced physicians , worse CD4 cell counts , and higher viral load levels  than the individuals included in the all cause mortality . These individuals had more experienced physicians , better CD4 cell counts , and lower viral load levels  than the individuals included in the all cause mortality . We believe that the differences among the results of these two studies and ours are due to differences between study populations  and differences in methodology used to assess the relationship between outcome and exposures.Two recent studies have reported on the association between drug resistance and risk of death ,25. Lucay et al. , based on = 229; 20.1%) were assumed to have no drug-resistance mutations. We therefore conducted a sensitivity analysis restricted to patients with plasma viral load levels of 1,000 copies/ml or more to assess the impact of this assumption on our results and the robustness of the marginal structural models in dealing with this possible source of bias. We observed that patients who showed the emergence of any resistance were 1.68 times  more likely to die than those who did not, which is consistent with the results found in our original analysis. Second, in the present study we did not exclude patients who achieved durable viral elevation  during the entire follow-up period . To assess whether this group drove our mortality trends, and not emergence of resistance per se, we conducted a second sensitivity analysis restricted to patients with no durable viral elevation. We observed that those patients that developed any type of resistance were 2.09 times  more likely to die than those who did not, which is also consistent with our previous results.Although we concluded that emergence of resistance was strongly associated with elevated risk of mortality, there are two important potential sources of bias that could explain our results. First, HOMER patients with samples of HIV-1 RNA of less than 1,000 copies/ml during the entire follow-up period  accession number for HIV HXB2 is K03455.The GenBank (


The study assessed the effect of some highly active antiretroviral therapies (HAART), used in the management of HIV/AIDS in Cameroon, on oxidative stress markers such as malondialdehyde (as TBARs), albumin, protein carbonyl content and protein sulfhydryls groups. 85 HIV positive patients (34.8 ± 9.3 years) were on three different highly active antiretroviral therapies (HAART patients). 65 HIV positive patients (32.2 ± 10.9 years) on no treatment (Pre-HAART patients), and 90 non-HIV infected patients (32.6 ± 9.3 years), were the control groups. Plasma TBARs as well as carbonyl levels were significantly higher in HIV patients on HAART compared to pre-HAART patients or non-HIV infected controls. On the other hand, the protein sulfhydryl group content was not different for patients on HAART compared to pre-HAART patients, but both were significantly lower than non-HIV infected controls . The combination treatment Therapy I [stavudin (80 mg) + Lamivudin (600 mg) + Nevirapin + (400 mg) zidovudin (600 mg)] brought about a significant (p < 0.05) reduction in the plasma concentration of protein sulfhydrl groups as well as TBARs compared to Therapy II [stavudin (80 mg) + Lamivudin (300 mg) + nevirapin (400 mg)] or with combination Therapy III of [zidovudine (600 mg) + lamivudin(300 mg) with efavirenz (600 mg)] (P < 0.05). The content of the antioxidant, Vitamin C was lower in the plasma of patients on Therapy I compared to those on Therapy II (P < 0.01) and Therapy III (P < 0.001).HIV infection therefore increases the oxidative stress process, while antiretroviral combination therapy increased protein oxidation as well as the level of oxidative stress already present in HIV infection. WHO currently recommends first-line therapy with two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitors (NNRTI) [The use of antiretroviral therapies (ART) is recommended worldwide for the management of HIV/AIDS. Different types of ART or combination therapies are available, and the prescription and use of a particular therapy depends on tolerabity, the cost, and the therapeutic objectives. The initiation of therapy is dependent on diagnosis as well as serological parameters, with therapy generally started when the CD4 count is less than 200 lymphocytes/mm (NNRTI) . BecauseFree radicals are compounds possessing an unpaired electron, which renders them highly reactive and capable of causing oxidative damage to all the major macro-molecules in cells, including lipids, proteins and nucleic acids. A major family of free radicals is the reactive oxygen species, derived metabolically from molecular oxygen via super oxide anions. Oxidative attack on proteins results in the formation of protein carbonyls , often w3) and viral load measurements. 65 positive patients were not on any form of antiviral therapy, while 85 were on one of the different therapies outlined below. These individuals showed no serological evidence for HBV and or HCV-infection. The control group comprised of 90 randomly selected age-matched healthy subjects who showed no serological evidence for HIV and/or HCV-infection and no abnormal laboratory finding .Patients were eligible if they had confirmed HIV infection, were older than 18 years, and had not taken antiretroviral drugs before (pre-HAART patients) or being on triple drugs therapy for least 3 to 6 months. HIV positive out patients (150) were recruited from the Yaounde Hospital. Patients were classified according to their sex, immune status such as the CD4 + count : Generic fixed-dose combination of stavudin + lamivudin + nevirapin associated with a generic fixed-dose combination of zidovudin +Lamivudin;Therapy II (n = 20): Generic fixed-dose combination of stavudin+ lamivudin + nevirapinTherapy III (25): Generic fixed dose combination of zidovudin + lamivudin associated with efavirenz.Over night (12 hour) fasting venous blood (10 ml) was collected from all subjects and divided between a heparinized tube and a tube containing Ethylene Diamine Tetra Acetate (EDTA). After centrifugation at 3000 g for 10 minutes at 4°C, a sample of the heparinized plasma was set aside for the analysis of protein sulphydryls and protein carbonyls. Samples of plasma containing EDTA were also set aside for the measurement of Vitamin C and MDA. All samples were stored at -80 °C and analysed within 2 weeks.Total protein was determined using a commercially available Biuret method . Lipid pp value of = 0.05 was considered significantValues are expressed as mean ± Standard error of mean (SEM). Data was analysed using the SPSS package, and normality of distribution of the data assessed using the normal plot method. The variables were normally distributed and comparisons made using ANOVA test. Differences between groups were assessed using the Bonferonni procedure.Multiple linear regression analysis was used to assess the influence of influence of BMI, age, sex, CD4 count and Viral load on oxidative stress markers in HAART patients. A The CD4 count was significantly p < 0.001) lower in the HIV positive subjects on HAART compared to pre-HAART patients  and lower levels of carbonyl  in HIV patients on antiviral therapy compared to those not following therapy or non HIV infected controls Table . On the 3 (Table Our results showed a non significant difference in the oxidative stress markers levels between patients with and without CD4 count less than 200 lymphocytes/mm3 Table .2). Among pre-HAART patients 33.9% had normal weight and 66.1% were overweight and only albumin level was significantly different between the two categories , normal (20–24.9 kg/m2), overweight or obesity  as indicator of malnutrition showed that 53.9% of pre-HAART and 74.1% of HAART patients had lower albumin level Table .Multiple linear regression analysis confirmed that BMI sex, age and CD4 count and viral load did not had a significant impact on TBARs and carbonyls concentrations but CD4 and Viral load significantly influenced the antioxidant capacity  in HAART patients Table .3. The consequence of this is patients being on antiviral therapy when the HIV infection is fairly advanced. When therapy is however initiated, it varies depending on the therapeutic objectives, the cost, and their availability on the market. Our results showed that, HIV infection increases the oxidative stress process, which is further increased by the use of ART. This was observed by the significantly higher TBARs (lipid peroxidation) concentrations  level (sulfhydryls group) and albumin, as well as Vitamin C with Therapy I. Abnormally high levels of free radicals as well as the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular organelles and enzymes as well as increased lipid peroxidation. Under normal conditions, numerous cellular antioxidant systems exist to defend against oxidant stress and maintain the redox balance of the cell. ROS are cleared from the cell by enzymatic systems including superoxide dismutases (SODs), catalase, and glutathione peroxidase, or the nonenzymatic system including alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), glutathione, and uric acid. Glutathione peroxidase plays an important role as defense mechanism in mammals, against oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. In addition to its role as a substrate in GSH redox cycle, glutathione, also act as a direct endogenous scavenger of hydroxyl radicals, involved in detoxification and metabolism of a number of substances in the liver . HAART mHIV infection increases the oxidative stress process, while anti retroviral combination therapy compounds this effect by increasing lipid oxidation. The decrease in antioxidants that accompanies HIV infection suggests a potentially important role of nutritional supplementation and good nutrition in general in the proper management of HIV/AIDS.


Pegylated interferon given for 24 or 48 weeks constitutes the most effective initial therapy for the treatment of chronic hepatitis C. It has been shown that viral load at week 2 appears the best time for predicting response to treatment. The objectives of this study were to assess whether the hepatitis C virus (HCV) RNA viral decline is predictive of sustained virological response (SVR) and to determine the best time for predicting complete response in our cohort of naïve patients treated with pegylated interferon alpha-2a  and ribavirin.Twenty patients treated with Peg-IFN alpha-2a and ribavirin for 48 weeks were studied. Six months after the end of treatment, a SVR (negative HCV RNA measured by PCR six months after the end of therapy) was obtained in 9 patients. Samples were obtained before and at week 2, 4, 8, and 12. At the end of week 2, viral load decreased more than 1.39 log in 8 out of the 9 patients with SVR and in 1 out of the 11 other patients. When we considered the viral load reduction from baseline to each week of treatment, week 2 appeared to be the best point time for predicting SVR, with a sensitivity of 91% , a specificity of 89% , a positive predictive value of 91%  and a negative predictive value of 89% .During treatment with Peg-IFN alpha-2a plus ribavirin in genotype 1 patients, when the main objective of the treatment is viral eradication, viral kinetics showed that week 2 appeared to be the best time point for predicting SVR. Our results must be further confirmed on a larger cohort. Interferon alpha plus ribavirin and more recently pegylated interferon (Peg-IFN) given for 24 or 48 weeks constitutes the most effective initial therapy for the treatment of chronic hepatitis C -4. In re12 virions/day [Understanding the kinetics and dynamics of human immunodeficiency virus (HIV) and of hepatitis B virus (HBV) has greatly improved the understanding of the life cycle of these viruses and their response to therapy . Studiesions/day . This raions/day ,9. Afterions/day . Infecteions/day .In the present study, viral kinetics during Peg-IFN alpha-2a plus ribavirin treatment were determined at week 2, 4, 8, and 12 of treatment in 20 patients after a first course of 48 weeks of Peg-IFN alpha-2a plus ribavirin. Our objectives were to assess: 1) whether the HCV RNA viral decline is predictive of a sustained virological response (SVR); and 2) the best time for predicting complete response in our cohort of patients.Epidemiological data for each patient are shown in Table 10) from baseline to each of the first weeks of treatment, week 2 point time tend to be better than other time points for predicting SVR, with an area under receiver operating characteristic (ROC) curve of 0.93 , a sensitivity of 91% , a specificity of 89% , a positive predictive value (PPV) of 91%  and a negative predictive value (NPV) of 89%  (P non-significant)  from baseline to week 2, 4, 8, and 12 of treatment, week 2 point time tend to be better than other time points for predicting SVR, with a PPV of 91% and a NPV of 89% and a very good sensitivity and specificity . Other studies have reported the same observation at week 2 [et al. reported that the absence of a 3 log10 viral decline after one month of therapy is associated with an absence of SVR [In the present study, when we considered the median reduction of the viral load  at week 12 (NPV of 100% and PPV of 50%). These results are in the same range as those of recent studies, which showed that failure to achieve EVR at week 12 of therapy is highly predictive of the absence of therapy , whereas the predictive value of achieving SVR after EVR at week 12 is not so strong (PPV = 65%) ,21,22.in vivo can be modified to give insights into the HCV dynamics, the mechanisms of action of interferon as well as the consequences of varying dosages of interferon [Methods introduced for analysing HIV dynamics terferon . The higterferon .When the main objective of the treatment is viral eradication, the study of viral kinetics during the first twelve weeks of Peg-IFN alpha-2a plus ribavirin treatment showed that week 2 tend to be the best time point for predicting sustained virological response. The early identification of patients with no SVR may lead for proposing an interruption of therapy for avoiding side effects and additional costs, or an early change of therapy . This obA total of 20 consecutive patients were prospectively enrolled after a first course of combination  and just before starting interferon and ribavirin therapy for chronic HCV infection, at the Hepatology and Gastroenterology Department of the Arnault Tzanck Institute . All patients tested positive for HCV RNA. All other causes of chronic hepatitis were excluded by appropriate serological testing and liver histology. All patients were treated with ribavirin 1 g once a week, orally, plus Peg-IFN alfa-2a (180 μg subcutaneous injection once a week) for 48 weeks. Written consent was obtained from each patient, and the study was approved by the local Ethics Committee in accordance with the 1975 declaration of Helsinki. Those patients were all genotype 1. Virological response was assessed by a qualitative HCV RNA assay with a lower sensitivity of 50 IU/ml . According to the qualitative HCV RNA results, patients were defined as virologic sustained responders (HCV RNA negative 6 months after the end of therapy) or as non-responders. SVR was obtained in 9 out of 20 patients. All patients had liver biopsy assessment according to Metavir histological staging.HCV RNA levels were measured at the same time on blood samples collected at baseline and at weeks 2, 4, 8, and 12 after the initiation of Peg-IFN alpha-2a treatment. Blood was collected in plasma preparation tubes (Becton Dickinson) that were centrifuged directly after collection in order to minimize RNA breakdown . Viral lThe ROC curve method was used to determine the best cut-off between week 2, week 4, week 8, and week 12 viral load measurements, by comparing the areas under the ROC curves. The Hanley-McNeil test was used for testing the statistical significance of the difference between the areas under ROC curves. The significant  level was set at 5%.The author(s) declare that they have no competing interests.DO was responsible for the conception, the design of the study, and the drafting of the paper. HJ has been involved in drafting and revising the paper HK has been responsible for testing the serums. GP has been responsible for the statistical analysis. PH has been involved in writing the paper. All authors have read and approved the content of this work.


Limited information exists on long-term prognosis of patients with sustained virologic response to antiretroviral therapy. We aimed to assess predictors of unfavorable clinical outcome in patients who maintain viral suppression with HAART.Using data collected from ten clinic-based cohorts in Spain, we selected all antiretroviral-naive adults who initiated HAART and maintained plasma HIV-1 RNA levels <500 copies/mL throughout follow-up. Factors associated with disease progression were determined by Cox proportional-hazards models.P = .002), and age at initiation of HAART . Initial antiretroviral regimen chosen was not associated with different risk of clinical progression.Of 2,613 patients who started HAART, 757 fulfilled the inclusion criteria. 61% of them initiated a protease inhibitor-based HAART regimen, 29.7% a nonnucleoside reverse-transcriptase inhibitor-based regimen, and 7.8% a triple-nucleoside regimen. During 2,556 person-years of follow-up, 22 (2.9%) patients died , and 40 (5.3%) died or developed a new AIDS-defining event. The most common causes of death were neoplasias and liver failure. Mortality was independently associated with a CD4-T cell response <50 cells/L after 12 months of HAART (adjusted hazard ratio [AHR], 4.26 ; Patients with sustained virologic response on HAART have a low mortality rate over time. Long-term outcome of these patients is driven by immunologic response at the end of the first year of therapy and age at the time of HAART initiation, but not by the initial antiretroviral regimen selected. In the majority of drug-naive patients, initiation of highly active antiretroviral therapy (HAART) results in a reduction in plasma virus load (pVL) to below the detection limit of currently available assays In the present study, we describe clinical outcomes and analyze predictors of disease progression in patients with sustained viral suppression from a large multicenter observational cohort constituted after HAART availability.This project is a joint activity of a recently created Research Network of Excellence  funded by the Spanish Research Council which incorporates basic scientists, immunologists, virologists, clinicians, epidemiologists and statisticians. A total of 10 HIV clinic-based cohorts from 7 of the 17 Autonomous Communities of the country (see appendix) participate in the project. Data were collected during the course of clinical care of HIV patients at cohort sites from January 1, 1997 through December 31, 2003. The cohort characteristics have been previously described in detail elsewhere For the purpose of these analyses, we selected all antiretroviral-naive patients who began HAART  since January 1, 1997. Patients were included in the analyses if they met all the following criteria: 1) pre-HAART CD4 cell count of <350 cells/µL and plasma virus load >1000 copies/mL, 2) ≥6 months of follow-up and at least one determination of CD4 cell count and pVL after HAART initiation, 3) a complete virologic response to therapy, defined as maintained virus suppression (pVL below 500 HIV-1 RNA copies/mL) throughout follow-up, and 4) a pVL confirming virologic suppression (<500 HIV-1 RNA copies/mL) within 6 months of the last database update. This value of 500 HIV-1 RNA copies/mL was chosen to overcome the heterogeneity of the assay detection limits used to quantify plasma HIV RNA during the study period in the different medical centres.The last available CD4 cell count obtained within the 2 months before the start of HAART was termed the pre-HAART CD4 cell count. CD4-T cell responses were defined according to changes in CD4 cell count from the pre-HAART value to 6 months (±2 months) and 12 months (±2 months) after HAART initiation. A “low” CD4-T cell response to HAART was defined as an increase in CD4 cell count <50 cells/µL from the pre-HAART assessment. This cut-off has been previously used in other studies Outcome measures were deaths and new ADE (i.e. CDC stage C) that occurred after the first 6 months of HAART. Patients who developed a new AIDS disease during follow-up were included only once; recurrences were not considered. The primary endpoint was all cause mortality. We also conducted sub-analyses of nonaccidental mortality (excluding deaths attributable to trauma), and of progression to a new ADE or nonaccidental death.P<0.1 in the univariate analysis were tried to enter simultaneously in a multivariate Cox model. Patients not known to be dead by December 31, 2003 were censored on the date they were last known to be alive. SPSS (version 11) was used for data management and statistical analysis. A two-tailed P value of 0.05 or less was considered significant.Univariate analyses were performed by using Kaplan-Meier estimates, and differences between curves were tested by using the log-rank tests. For multivariate analysis, we used Cox proportional-hazards models to determine the factors associated with outcome measures. age, gender, category of transmission, previous AIDS diagnosis, pre-therapy CD4 cell count, pre-therapy pVL, type of HAART combination and CD4-T cell responses to HAART, as defined above, were evaluated as potential predictors of clinical outcome. Age at HAART initiation was analysed as a continuous variable and dichotomized into two groups (<50 years and ≥50 years). This age cut-off is commonly used in other studies The cohort database contained data on 4643 patients with at least 6 months of follow-up. Of these, 73% were men and the median age was 35  years. Overall, 56.4% were intravenous drug users, 22.6% heterosexuals and 14.4% men who had sex with men. Twenty-one percent were diagnosed of AIDS prior to study entry. The incidence rate of AIDS during follow-up was 3.6 per 100 person-years. Overall, 310 (6.7%) patients died, being the death rate 1.7 per 100 person-years. Twenty-seven percent had started HAART prior to study entry. Of the 3421 treatment-naive subjects, 2613 started HAART during follow-up.A total of 757 patients fulfilled the inclusion criteria and they were, therefore, eligible for the present analyses. Baseline demographic and clinical characteristics of the patient sample are shown in Antiretroviral therapy administered is detailed in P<0.000), and were more likely to be intravenous drug users , but did not differ with respect to baseline pVL, baseline CD4 cell counts, prevalence of AIDS at baseline, or immunological responses at 6 and 12 months of HAART initiation.Median follow up period was 3.1 years . A total of 63 (8.3%) subjects were lost to follow-up. Lost to follow-up patients were younger . According to previous definition, 180 (24.9%) of the 722 patients with CD4 cell counts available at 6 months had a “low” CD4-T cell response. At 12 months, data were available for 650 (85.9%) patients, with 108 (16.6%) showing a “low” CD4-T cell responseCD4- T cells increased in the entire patient sample from a mean of 162  cells/µL before starting HAART, to 302  cells/µL and 346  cells/µL, at 6 and 12 months, respectively, after the initiation of HAART  patients died , 21 (95.4%) of them were nonaccidental deaths. A total of 21 (2.8%) subjects experienced a new ADE, and 40 (5.3%) developed an ADE or death throughout follow-up. Of the 21 nonaccidental deaths, analysis of cause-specific mortality indicated that 7 (33.3%) patients died of neoplasias ; 4 (19%) patients died of liver failure and/or cirrhosis; 4 (19%) of opportunistic or bacterial infections , 1 (4.8%) case from progressive end-organ disease , and 1 case committed suicide. Despite a thorough review, cause of death could not be ascertained for the remaining 4 (19%) individuals who died away from the hospital; in all of them the death was sudden and unexpected.P = 0.029), and low CD4-T cell response at 12 month  were independently associated with mortality , and low CD4-T cell response at 12 month  were independent predictors of new ADE or death .At Kaplan–Meier estimates, a significant difference was found in terms of proportion of patients experiencing death, and new AIDS defining event or death, during the follow-up period according to CD4-T cell response, and age at HAART initiation (<50 years versus ≥50 years) .The study has confirmed that patients with sustained virologic response to HAART have a very low mortality rate over time. The analyses have shown that low CD4-T cell responses at 12 months and age at the initiation of antiretroviral therapy are the main determinants of unfavourable long-term outcome. The initial antiretroviral regimen chosen, however, was not associated with different risk of clinical progression. Our results are based on a large population of patients with over 2500 patient-years of observation, which allowed us to examine clinical progression up to more than 3 years after starting HAART. The cohort includes many clinics treating HIV-1-infected patients from different regions of Spain, with a broad spectrum of patient characteristics and antiretroviral regimens. The results should therefore be applicable to many individuals with HIV-1 infection beginning HAART in Spain and elsewhere.Low immunological response to antiretroviral therapy was the strongest predictor of mortality. This finding is in agreement with those from previous studies assessing clinical outcomes in patients receiving HAART, that have suggested poorer prognosis in subjects with discordant immunologic and virologic responses In addition to suboptimal immunological response, older age at initiation of HAART was found to predict unfavorable outcome in our study. Most studies have shown that compared with younger patients, patients over 50 generally have a slower immunological response to HAART and experience more rapid clinical progression Several published randomized clinical trials have shown that different antiretroviral regimens have different capacities to decrease the pVL and raise the CD4 cell count thus leading to different clinical efficacy. In addition, it has been suggested that antiretroviral drugs might have effects on risk of AIDS and/or death, which are not mediated by their effect on HIV-RNA and CD4 cell count An expected finding from the study was the significant contribution of mortality from non-AIDS-defining illnesses, including cancer and end-stage liver disease. These results corroborate previous findings that indicate that the relative proportion of non-AIDS-related death has increased during the period of HAART A number of limitations in our study should be noted. First, to be included in the analysis, patients recorded in the database must have had at least a CD4 cell count and pVL assessment within 6 to 12 months after starting HAART. As a result, patients who died or were lost to follow-up before a second CD4 cell count assessment were not included, and consequently the risk for clinical progression may have been underestimated. The 8% losses to follow-up could have introduced a number of other different biases into our results. Lost to follow-up patients were younger and they were more likely to be intravenous drug users. However, considering the total number of losses and the lack of differences in other baseline parameters, including immunological responses to HAART, with respect to the other group, the effect of such biases would likely to be small. The analyses of the effect of different antiretroviral regimens is limited by the fact that it was a nonrandomized, observational cohort study, and the selection of therapy depended on the treating clinicians criteria and the availability of the antiretroviral drugs throughout the study. Finally, given the large number of participating sites, variations in CD4 cell counts and pVL might have occurred. A potential impact of such variations on the study results cannot be ruled out.Several confounding factors might have influenced the results. Unfortunately, hepatitis C virus (HCV) serostatus and adherence to therapy were not included among the set of pre-defined variables in the database, therefore the contribution of these factors to immunological response and clinical outcomes could not be ascertained in the study. The influence of HCV co-infection in immunological response to HAART remains controversial. Recent data from the British Columbia cohort suggest that HCV antibody-positive HIV-infected patients may have an altered immunologic response to HAART Despite the limitations, the study represents some of the first substantial body of data on long-term outcome of patients with sustained virologic response on HAART. The analyses were conduced in patients who started therapy with a broad spectrum of antiretroviral drugs and we selected the CD4 threshold at which antiretroviral therapy is currently recommended In summary, our results indicate that clinical outcome of patients who maintain a durable virologic suppression on HAART could be driven by the immunologic response at 12 months and the age at the time of HAART initiation, but not by the initial antiretroviral regimen chosen. The fact that the CD4-T cell response is strongly associated with long-term clinical outcome indicates that both immunologic and virologic responses are important when assessing the effectiveness of antiretroviral drugs. The study results suggest the need for alternative strategies to amend immune response in patients with low CD4-T cell increases at the end of the first year of therapy. Older age at initiation of HAART was also found to predict unfavorable outcome, indicating that age at which therapy is started should be carefully considered.


The risk of hepatotoxicity associated with different highly active antiretroviral therapy (HAART) regimens  in HIV-HCV co-infected patients has not been fully assessed.Retrospective analysis of a prospective cohort of 1,038 HIV-HCV co-infected patients who commenced a new HAART in the Italian MASTER database. Patients were stratified into naïve and experienced to antiretroviral therapy before starting the study regimens. Time to grade ≥III hepatotoxicity (as by ACTG classification) was the primary outcome. Secondary outcome was time to grade IV hepatotoxicity.Incidence of grade ≥III hepatotoxicity was 17.71 per 100 patient-years (p-yr) of follow up in naïve patient group and 8.22 per 100 p-yrs in experienced group . In the latter group, the only independent factors associated with shorter time to the event at proportional hazards regression model were: previous liver transaminase elevations to grade ≥III, higher baseline alanine amino-transferase values, and use of a non nucleoside reverse transcriptase inhibitor based regimen. In the naive group, baseline aspartate transaminase level was associated with the primary outcome.Use of a single or multiple protease inhibitor based regimen was not associated with risk of hepatotoxicity in either naïve or experienced patient groups to a statistically significant extent. A cautious approach with strict monitoring should be applied in HIV-HCV co-infected experienced patients with previous liver transaminase elevations, higher baseline alanine amino-transferase values and who receive regimens containing non nucleoside reverse transcriptase inhibitors. Highly active anti-retroviral therapy (HAART) is associated with a number of serious and potentially life-threatening adverse events, including drug-induced liver injury . Previous studies demonstrated the association of hepatotoxicity in HIV-infected patients treated with HAART, co-infected with hepatitis C virus (HCV) -10. HoweHepatotoxicity has been associated with any currently used anti-retroviral (ARV) drugs but existing studies fail to demonstrate a consistent association between a particular drug or drug class and the development of subsequent hepatotoxicity, although in a single cohort-study involving HCV positive and negative patients the recent use of nevirapine (within 12 weeks of initiating therapy) and the use of full-dose ritonavir (600 mg bid) have been implicated .It is a general belief that non nucleoside reverse transcriptase inhibitors (NNRTI), especially nevirapine, have a class effect in terms of abnormal liver enzyme levels, but an increased rate of serious clinical (symptomatic) hepatotoxicity has not been comparatively demonstrated in general patient populations yet. Moreover, risk of hepatotoxicity has been shown to be dependent on several concomitant conditions, such as viral co-infection, plasma drug levels, gender and degree of immune damage .Few data are available about the risk of hepatotoxicity during treatment including low-dose ritonavir co-administered with a protease inhibitor  in comparison with other kinds of regimens. A study, conducted in patients naïve to therapy , suggestBy contrast, data from a small population of Canadian HIV-positive subjects co-infected with HBV and/or HCV, demonstrated that concurrent use of lopinavir/ritonavir was an independent predictor of grade ≥III alanine amino-transferase (ALT) elevation . TherefoThe objective of this paper is to present incidence and risk factor estimates in one of the largest cohorts of HIV-HCV co-infected patients presented so far. As patients were selected for inclusion in the most recent years, it was possible to assess risk associated with modern antiretroviral treatment regimens .The study consists of an analysis of a prospective cohort of HIV-HCV co-infected patients in the Italian MASTER database. The study was conducted in 5 sites: Brescia, Bari, Pavia, Florence and Turin, in agreement with human experimentation guidelines of the declaration of Helsinki and after approval of the Ethic Committee in each participating centre. Patients were defined as HCV infected by the presence of a positive test for HCV antibodies.All sequential patients who started any HAART regimen, either PI-or NNRTI-based, from January 2001 to March 2004 have been included in this cohort, provided that a baseline (BL) determination of CD4+ cell count, HIV RNA, aspartate amino-transferase (AST) and ALT levels within 90 days of commencement of therapy were available, and no renal impairment (i.e. creatinine serum level >2 mg/dl), severe liver function test (LFT) elevation (either AST or ALT) or bilirubinaemia ≥5 times the upper limit of normality (ULN) were present. Date of "freezing" of the data-base for the present analysis was September 2004. Patients have been stratified into treatment experienced (E) and naïve (N) for antiretroviral (ARV) drugs before starting the study regimens. Patients were asked to return for clinical and laboratory follow-up (including liver transaminase levels) after 1 month from baseline and at least every 3 months thereafter. The ULN for transaminase was 45 IU/l for ALT levels and 50 IU/l for AST levels in all centres.Any increase by ≥5 × ULN in ALT or AST  during Finally, a clinical evaluation of patients who experienced grade ≥III or IV hepatotoxicity was performed to rule out that other concomitant causes of LFT elevation were present .On-treatment (OT) analyses were separately conducted in N and E groups with grade ≥III hepatotoxicity as primary outcome measure. Further analyses were conducted using grade IV hepatotoxicity as a secondary outcome. To ascribe hepatotoxicity to the current study regimens only, follow-up was censored at any modification or stopping of the treatment regimen, or achievement of the study outcomes, whichever came first.The following factors assessed at BL were tested at proportional hazards regression models for their possible associations with occurrence of the study end-points: gender, age, risk factor for HIV acquisition, nadir CD4+ T-cell count , chronic HBV infection (defined as surface antigen positivity), hepatitis delta virus (HDV) infection (defined as antibody positivity), ALT and AST serum levels, previous LFT elevation to ≥grade III, HIV-RNA, CD4+ T-cell count, previous use of interferon, use and length of exposure to dideoxynucleoside reverse transcriptase inhibitors (DDX) – i.e. stavudine, didanosine and zalcitabine. Type of HAART in the study regimens , and use of DDX, lamivudine (3TC) and tenofovir (TDF) in the current HAART were also considered. Moreover, to account for possible effect of immune reconstitution as risk factor for hepatotoxicity, the CD4+ T-cell count evolution (defined as slope of CD4+ T-cell count since BL throughout the entire follow-up) was imputed in the model as a time-dependent covariate .All variables with a P-value lower than 0.20 at univariate analysis were included in multiple proportional hazards regression model, if clinically meaningful. In all statistical analyses we stated a significance level P ≤ 0.05. Epi Info 3.2 package  and Egret 2.0.31 package  were used to perform the analyses.Table Among a total of 32 patients who were prescribed a single PI-based regimen, 27 (84.38%) received nelfinavir. All 50 patients who were taking multiple PIs were prescribed ritonavir at low dose (≤200 mg/twice daily), mainly at a dose of 100 mg/twice daily associated with lopinavir (84%). Among 73 patients taking NNRTI-based regimens, 42 (57.53%) received efavirenz.In Table A total of 20 cases of grade ≥III hepatotoxicity were observed , corresponding to an overall incidence of 17.71 per 100 p-yrs of follow-up. Incidence of grade ≥III hepatotoxicity was 20.32 per 100 p-yrs among patients receiving single PI-based regimen, 30.15 per 100 p-yrs among patients receiving multiple PIs and 10.26 per 100 p-yrs in those receiving NNRTI. Additional 90 patients were censored to the follow-up because treatment was changed or stopped for reasons different from hepatotoxicity . Forty-five patients were still on the initial HAART regimen at the time of "freezing" the study data base for the analysis. No patient deaths were recorded during the follow-up.Incidence of grade IV hepatotoxicity was 4.13 per 100 p-yrs , with 0 per 100 p-yrs in single PI group, 12.49 per 100 p-yrs in multiple PI group and 1.61 per 100 p-yrs in NNRTI group. No significant differences were reported comparing incidences of grade ≥III and grade IV hepatotoxicity in patients receiving nevirapine or efavirenz.Mean length of observation was 266 days (SD: 263). Mean number of measurements of transaminase levels per year was 5.35 .A total of 58 cases of grade ≥III hepatotoxicity were observed , corresponding to an overall incidence of grade ≥III hepatotoxicity of 8.22 per 100 p-yrs. Incidence of grade ≥III hepatotoxicity was 3.78 per 100 p-yrs in single PI group, 8.05 per 100 p-yrs in multiple PIs group and 10.82 per 100 p-yrs in NNRTI group. Additional 583 patients were censored to the follow-up because treatment was changed or stopped for reasons different from hepatotoxicity . No patient deaths were recorded during the follow-up. Two-hundred and forty-two patients were still on the initial HAART regimen at the time of "freezing" the study data base for the analysis.Incidence of grade IV hepatotoxicity was 1.08 per 100 p-yrs , with 0.62 per 100 p-yrs in single PI group, 0.74 per 100 p-yrs in multiple PI group and 1.64 per 100 p-yrs in NNRTI group. As in naïve patients, there was no significant differences in the incidences of grade ≥III or grade IV hepatotoxicity between patients who were prescribed nevirapine and those who received efavirenz.Mean length of observation was 292 days (SD: 278). Mean number of measurements of transaminase level per year was 4.68 .Figure When time-to-grade IV hepatotoxicity was considered as dependent variables, none of the factors resulted to be significantly associated with hepatotoxicity. Due to the small number of patients and relatively short follow up, a multivariable hazards regression model was not performed in patients who were antiretroviral therapy naïve, either for the ≥grade III or for the grade IV end-points.Clinical review did not reveal any concomitant causes for ≥grade III LFT elevations. One patient (an active IVDU), who was assuming a multiple PI-based regimen, had previously developed an acute HBV infection that resolved at the time of hepatotoxicity and another, who was taking a single PI-based regimen, admitted alcohol abuse although AST/ALT ratio did not support an acute alcohol hepatitis.Cumulative risk of time-to-developing a grade ≥III hepatotoxicity among experienced patients, stratified by treatment groups, is represented in Figure Figure At the univariate proportional hazards regression analyses performed using occurrence or time-to-grade IV as dependent variables, none of the factors that were tested showed significant associations with outcome.Among patients who had grade ≥III hepatotoxicity events, those who admitted alcohol abuse were equally distributed in the 3 treatment groups . No other concomitant causes for grade ≥III hepatotoxicity were found.This study has a number of strengths but also limitations. A large number of different drug combinations are used in the "real-life" setting but the attribution of an episode of hepatotoxicity to a single ARV agent can be arbitrary. In assigning a patient to a specific treatment arm, physicians may be influenced by many considerations. A subjective evaluation of a high risk of hepatotoxicity may have resulted in prescription of a regimen with an assumed better profile of liver tolerability. Notwithstanding these limitations, observational data reflect the real clinical setting and this is the widest observational cohort of HIV/HCV co-infected patients reported so far. In order to be able to ascribe hepatotoxicity events to ongoing regimen, only a OT approach may be used, discontinuing follow-up when any change to HAART occurs.Multiple PI-based regimens are not, in this analysis, associated with a significantly higher risk of developing grade ≥III hepatotoxicity when compared to single PI-based regimens, either among patients naïve or experienced to ARV drugs. Ritonavir, whose hepatotoxicity is the clearest defined among PIs, was used rarely at full dosage (1200 mg) in our cohort. When used for pharmacokinetic enhancement in association with another PI, its dosage was in most cases ≤400 mg/day. This low-dose of ritonavir did not appear to influence the liver-toxicity profile of the regimen, thus confirming the report by Sulkowski et al. in a small number of HIV-HCV co-infected patients treated with boosted PI regimens .By contrast, use of NNRTI rather than single PI-based regimens was independently associated, but only among treatment experienced patients, with a higher risk of grade ≥III hepatotoxicity. This finding suggests that careful management of these patients, with a strict follow-up should be performed, especially if other risk factors associated with hepatotoxicity are present.Among naïve patients, a higher incidence of severe hepatotoxicity and a different pattern of risk factors were reported than those in experienced patients (including the use of NNRTI).It is possible that more prominent immune-reconstitution occurring in naïve patients than in experienced ones may have increased the overall hepatotoxicity risk, diluting the impact exerted by NNRTI as a cofactor for severe hepatotoxicity. The CD4+ T-cell slope was more pronounced in patients receiving their first line treatment than in experienced subjects. Moreover, the lack of previous drug exposure in naïve patients may have led to prescription of drugs whose potential for hepatotoxicity was less than in the experienced patient cohort, where, due to HIV drug resistance, less choice is available.It is however important to recognize possible limitations of this study. The low number of patients in the naïve cohort, compared to the experienced cohort, may make the effect of specific drugs in determining hepatotoxicity less evident. Moreover, a different frequency in measuring LFTs, which resulted in more frequent monitoring among naïve patients taking PI-based regimens, may have led to an increased detection of toxicity in that group, although a statistically significant correlation between the frequency of LFT determinations and the risk of hepatotoxicity was not present (data not shown). The low incidence of grade IV hepatotoxicity events did not allow us to perform multivariable analyses. Further studies, with longer follow-up and conducted in a larger series of patients, are therefore necessary in this respect. Lastly, several variables which were not reliably detected in the whole cohort  should be further evaluated. However, the low prevalence of alcohol abusers in those who had severe hepatotoxicity, which did not cluster in a specific treatment group, may suggest that alcohol abuse was not a major confounder.The risk of severe hepatotoxicity was different among experienced and naïve patients, with patterns of risk factors associated with hepatotoxicity being different between these two groups. Amongst experienced patients, elevated ALT level at BL, a history of previous LFT elevation and use of NNRTI-based regimens were independently associated with the development of grade ≥III hepatotoxicity. The choice of multiple PI-rather than a single PI-based regimen did not influence the risk of hepatotoxicity to a significant extent. By contrast, a cautious approach and strict monitoring should be applied in experienced patients with previous hepatotoxicity events, high BL ALT values and prescribed NNRTI-containing regimens, who have the greatest risk of severe liver toxicity.3TC: lamivudineALT: Alanine Amino-TransferaseARV: Anti-RetroviralAST: Aspartate Amino-TransferaseBL: BaselineCI: confidence intervalDDX: dideoxynucleoside reverse transcriptase inhibitorsE: ExperiencedHAART: Highly Active Anti-Retroviral TherapyHBsAg: Hepatitis B Surface AntigenHBV: Hepatitis B VirusHCV: Hepatitis C VirusHDV: Hepatitis Delta VirusHIV: Human Immunodeficiency VirusHR: Hazard RatioIFN: InterferonIVDU: Intra-Venous Drug UserLFT: Liver Function TestN: NaïveNNRTI: Non Nucleoside Reverse Transcriptase InhibitorOT: On-Treatmentp-yr: patient-yearPI: Protease InhibitorSD: standard deviationTDF: TenofovirULN: Upper Limit of NormalityThe author(s) declare that they have no competing interests.CT conceived of the study, participated in its design and coordination and drafted the manuscript. GL participated in the design of the study, in the collection and in the interpretation of data, helped in the statistical analysis and helped in drafting the manuscript. SC performed the statistical analysis and revised the manuscript. MP participated in the design of the study and revised the manuscript. MN helped in the interpretation of data and critically revised the manuscript. EQR revised the manuscript. DB, GP, NL, LM, GS, FM, SLC, GDP and GF participated in the design of the study, in the acquisition of data and revised the manuscript. CT participated in the statistical analysis and revised the manuscript. GC participated in design and coordination of the study, revised the manuscript and collected funding for this study.The pre-publication history for this paper can be accessed here:


The association between baseline drug resistance mutations and subsequent increase in viral failure has not been established for HIV-infected children. We evaluated drug resistance mutations at 39 codon sites (21 protease inhibitor (PI) resistant codons and 18 nucleoside reverse transcriptase inhibitor (NRTI) resistant codons) for 92 clinically stable NRTI-experienced, PI-naive HIV-infected children 2 to 17 years of age who were initiating new therapy with ritonavir plus zidovudine (ZDV) and lamivudine or plus stavudine. The association between baseline drug resistance mutations and subsequent viral failure after 12 and 24 weeks of highly active antiretroviral therapy (HAART) was studied.There were few primary PI associated mutations in this PI-naïve population, but 84% had NRTI mutations – codons 215 (66%), 41 (42%), 67 (37%), 210 (33%) and 70 (32%). None of the specific baseline drug resistance mutations were associated with a higher rate of virologic failure after 12 or 24 weeks of HAART. Median week 12 viral load decreased as the total number of NRTI mutations at baseline increased (P = 0.006). Specifically, a higher level of baseline ZDV resistance mutation was associated with a decrease in viral failure after 12 weeks on a ZDV-containing HAART regimen (P = 0.017).No increase was seen in the rate of viral failure after HAART associated with the presence of resistance mutations at baseline. This paradoxical result may be due to adherence, replicative capacity, or ZDV hypersusceptibility to the new regimen. Nucleoside reverse transcriptase inhibitors (NRTI) were the first antiretroviral drugs available and continue to be a component of anti-retroviral therapy (ART), despite the emergence of drug resistance over time. Few studies have investigated the role of pre-existing drug resistance and response to therapy in children -4 comparThere were very few primary resistance mutations to PIs in this PI-naïve population, although 88% of the children had polymorphisms that included secondary minor resistance mutations. The most frequent secondary PI mutations were at codons 63 (78%), 77 (37%), 36 (17%) and 10 (12%) (data not shown). Only two children had a primary PI resistance mutation (V82A). Other PI mutations  were present in less than 10% of the study subjects. The most common NRTI mutations occurred at codons 215 (66%), 41 (42%), 67 (37%), 210 (33%), 70 (32%), 69 (22%), 118 (21%) and 219 (21%). The median numbers of baseline NRTI, thymidine analog mutations (TAM), PI and total mutations were 3, 3, 2 and 4.5, respectively .After 12 weeks on study, 51 (55%) subjects had viral loads suppressed below 400 copies/ml. The number of subjects with viral suppression dropped to 31 (34%) and 29 (32%) at weeks 24 and 48, respectively.The association between the presence of a specific baseline mutation and virologic failure after 12 weeks of HAART was studied (Table 10 RNA (copies/mL) associated with each additional NRTI resistance mutation (P = 0.006).When the association between the number of baseline resistance mutations and virologic failure after 12 weeks of HAART was investigated, the only significant relationship observed was in the ZDV/lamivudine (3TC)/RTV combination regimen. A higher median number of NRTI mutations at baseline was associated with virologic suppression . There was no apparent association between the number of baseline PI resistance mutations and the level of viral load after 12 weeks of HAART, although as the number of baseline NRTI mutations increased the level of viral load at week 12 decreased , and no suppression. According to this definition, 56 (60%) subjects achieved full viral suppression and an additional 13 (14%) achieved partial viral suppression during the first 24 weeks on HAART. There was no statistically significant (at the unadjusted 5% level) association between any baseline mutation and virologic suppression during the first 24 weeks on HAART.The association between the presence of a specific baseline mutation and virologic suppression during the first 24 weeks on HAART was explored. Viral response was categorized into three ordered levels: full suppression , partial suppression  had a viral rebound. In the three-drug arm children with mutation at the NRTI codon 67 seemed to be less likely to have a viral rebound after virologic suppression (unadjusted P = 0.002). There was also the suggestion of a potential association with virologic failure for the presence of any PI mutation (unadjusted P = 0.020) in the three-drug arm and for mutation at the NRTI codon 69 (unadjusted P = 0.028) in the two-drug arm, but these findings need to be treated cautiously due to the problem of multiple comparisons .For the 69 children with full or partial viral suppression as defined above, the association between the presence of a specific baseline mutation and viral rebound between the suppression and week 48 was evaluated. A rebound was declared if there was an increase in RNA of more than one logWe evaluated the association between resistance mutations and their potential effect on the ability of antiviral medications to reduce the viral burden in children with HIV disease. As expected, there was little PI resistance and considerable NRTI resistance in this PI-naïve, NRTI-experienced population. However, contrary to our expectations, we did not find an increase in the rate of viral failure after HAART associated with the presence of resistance mutations at baseline. The two specific associations identified at week 12 in our study  in combination therapy responded favorably and rapidly. We did not observe an increase in the rate of viral failure after HAART linked to the presence of resistance mutations at baseline. In fact, viral loads at the 12 week time point were inversely correlated with the number of baseline NRTI mutations. It is important to remember that resistance is not an all-or-none phenomenon, and treatment failure is not defined by a resistance test. In order to better understand both the virology of mutated viruses in vivo and their response to anti-retroviral drugs as well as the use of resistance testing, prospective randomized clinical trials linked with pathogenesis-related in vitro analysis should be performed in children.PACTG 338 was a multicenter, randomized clinical trial that enrolled 297 children aged 2–17 years. All subjects were HIV-infected, had been receiving continuous, unchanged antiretroviral therapy for the 16 weeks before study entry, and were naïve to protease inhibitors and lamivudine (3TC) or had received no more than 6 weeks of ZDV plus 3TC in the year prior to study entry and none in the 4 months prior to study entry. Children were randomly assigned to receive either ZDV/3TC (n = 100), stavudine (d4T)/RTV (n = 97) or ZDV/3TC/RTV (n = 100). Children initially assigned to the ZDV/3TC regimen were not included in this evaluation of drug resistance and subsequent viral failure as this regimen was demonstrated to result in a suboptimal virologic suppression compared to protease inhibitor-based regimens.3, proportion with CD4 cell count <500 cells/uL was 29%, proportion with CD4 percent <25% was 38%, median plasma HIV-1 RNA was 20,500 copies/mL, and the proportion with HIV-1 RNA copy number > 4 log10 was 75%. The majority of the children were African-American (66%) and 51% were male. Children had received prior treatment with ZDV monotherapy (46%), the combination of ZDV and ddI (37%) or other combination therapy (15%). Baseline characteristics of the children in this resistance analysis were very similar to the baseline characteristics for the overall PACTG 338 study of 297 children, except for minor differences in the CD4 cell count (648 cells/mm3), the proportion of African-American children (51%), and the proportion with HIV-1 RNA copy number > 4 log10 (64%). Children with viral loads <1,000 copies/mL were excluded from this resistance study. For the secondary analyses of viral suppression at weeks 12 or 24 and viral rebound after 48 weeks on HAART, two additional subjects were included, who did not have viral load measured at week 12 but did have information at week 24. The institutional review board at each institution approved the study and informed consent was obtained from all patients or their guardians.A total of 92 children assigned to a RTV-containing regimen had both baseline resistance information and viral load measurement at week 12, and they were included in any analysis involving the primary endpoint of viral failure at week 12. Of these 92 subjects, the median age was 7.3 years, median CD4 cell count was 602 cells/mmSequencing was determined in batch at the conclusion of the study in two laboratories that participated in the National Institute of Allergy and Infectious Diseases (NIAID) Virology Quality Assurance Program. For HIV-1 sequencing, plasma RNA was extracted using the QIAampViral RNA Mini Kit . Reverse transcriptase polymerase chain reaction (RT-PCR) and direct DNA sequencing of protease and reverse transcriptase genes were performed using the TruGene HIV-1 Genotyping Kit  according to the manufacturer's instructions. The International AIDS Society-USA recommended guidelines for resistance to protease inhibitor (PI) and nucleoside reverse transcriptase inhibitors (NRTI) were used ,19.HIV-1 RNA copy number was assessed using the NucliSens Assay  , which hFisher's exact test was used for associations between specific baseline resistance mutations and viral failure, the Kruskal-Wallis test was used to assess the association between the number of resistance mutations and viral failure, and the Mantel-Haenszel test for trend was used to evaluate the association between level of baseline ZDV resistance and viral failure defined as viral load > 400 cp/ml ,24. FurtThe author(s) declare that they have no competing interests.AAW, LMF, RY, KM, SIP, KES and SAN conceived, designed and implemented the primary study (PACTG 338), upon which this secondary resistance study was based. SF organized this secondary study and contributed to its design and implementation along with SAF, AK, LAP and SN. CH performed the statistical analysis. SF drafted the manuscript jointly with KES. All authors provided review comments and textual modifications during manuscript development. All authors read and approved the final manuscript.


Castro has advocated that adherence to HIV antiretroviral therapy should be understood within a patient's clinical and social context . Over 90In rural western Uganda, there is, for example, a 40-year-old man who is HIV-positive, has no education, and works as a farmer. He lives with his brother, sister-in-law, and three nieces in a three-room, mud-walled house without electricity. He owns a lantern, a bed, a sofa, a bike, and a radio, but does not own a watch. He was diagnosed with HIV in April 2005 and started generic D4T/3TC/NVP (Triomune) four months after developing disseminated herpes zoster and Kaposi sarcoma with a CD4 count of 151. His adherence was measured with an electronic medication monitor that records a date-time stamp in flash memory each time the pill container is opened. Over the 89 days of monitored treatment, he had 98.9% adherence by electronic monitor and took 90% of prescribed doses within ten minutes of 7:20 a.m. and within 17 minutes of 7:20 p.m. When asked how he knew when to take his dose, he said that he knows it is time to take his medications by “listening to Radio West's ‘News and Announcements’ every morning and evening.”While population levels of adherence will likely drop as treatment access expands and people begin to experience toxicities of long-term therapy, he is an example of how patients can have precise, if not perfect, adherence with creative solutions in a resource-limited setting.


HIV and AIDS are significant and growing public health concerns in southern Africa. The majority of countries in the region have national adult HIV prevalence estimates exceeding 10 percent. The increasing availability of highly active antiretroviral therapy (HAART) has potential to mitigate the situation. There is however concern that women may experience more barriers in accessing treatment programs than men.A systematic review of the literature was carried out to describe the gender distribution of patients accessing highly active antiretroviral therapy (HAART) in Southern Africa. Data on number of patients on treatment, their mean or median age and gender were obtained and compared across studies and reports.The median or mean age of patients in the studies ranged from 33 to 39 years. While female to male HIV infection prevalence ratios in the southern African countries ranged from 1.2:1 to 1.6:1, female to male ratios on HAART ranged from 0.8: 1 to 2.3: 1. The majority of the reports had female: male ratio in treatment exceeding 1.6. Overall, there were more females on HAART than there were males and this was not solely explained by the higher HIV prevalence among females compared to males.In most Southern African countries, proportionally more females are on HIV antiretroviral treatment than men, even when the higher HIV infection prevalence in females is accounted for. There is need to identify the factors that are facilitating women's accessibility to HIV treatment. As more patients access HAART in the region, it will be important to continue assessing the gender distribution of patients on HAART. There is increasing global interest to ensure that HIV infected persons have access to antiretroviral therapy. In the developed world, mortality from AIDS has significantly reduced, in part due to wide availability and accessibility to highly active antiretroviral therapy (HAART). In resource-limited countries however, although there has been steady progress in increasing accessibility to antiretroviral therapy (ART), most patients have no access to this life saving intervention . NatrassWhen patients in resource limited settings access HAART, treatment adherence has been reported to be comparable to industrialized countries . The benSouthern Africa is the region of the world that has been hardest hit by the HIV pandemic. For instance, Malawi's adult 15–49 years) HIV prevalence was estimated at 14.4% in the Malawi Demographic and Health Survey in 2004 –49 years. In NamiThe situation in southern Africa though has not been well described probably because comprehensive HIV care which incorporates HAART is a relatively new phenomenon. With the scaling up of HAART programs and comprehensive care going on in the continent, there is need to describe the distribution of patients obtaining HAART in terms of gender and age. Some of the questions that need to be addressed are: Is there gender balance in the distribution of patients obtaining HAART? What age groups are more likely to access HAART? Answers to these questions may help to guide researchers and policy makers to identify operational gaps, and suggest interventions that may improve accessibility of patients to health services. We aimed to obtain answers to these questions by conducting a systematic review of published literature following the suggestions of the Meta-analysis of Observations Studies in Epidemiology (MOOSE) Group published by Stroup et al .We searched for peer-reviewed articles and conference abstracts published in English language that reported on gender distribution of adult patients obtaining highly active antiretroviral therapy (HAART) in treatment programs in southern Africa. Different combinations of the following keywords: "antiretroviral", "anti-retroviral", "highly active antiretroviral therapy", "HAART", "AIDS treatment" and "HIV treatment" and a combination of these terms to each of the southern African countries were used. For the purposes of this paper, southern Africa was defined as all the 14 countries that make up the Southern African Development Community (SADC). These countries are: Angola, Botswana, Democratic Republic of Congo (DRC), Lesotho, Madagascar, Malawi, Mauritius, Mozambique, Namibia, Seychelles, South Africa, Swaziland, Tanzania, Zambia and Zimbabwe.Electronic searches of MEDLINE, PsycINFO, African Journals Online (AJOL), Ingenta and Ovid electronic databases were done for reports published from 2000 to August 15, 2006. We also searched electronically for conference abstracts on Gateway  and manuWe included a study or program document for review if it reported on the following within a treatment program: the gender distribution of patients on HAART, mean or median age of patients and time period of accumulation of patients on treatment of not less than 6 month. Journal articles and the other reports had to report on 'routine' program treatment and not randomized clinical trials or any aggregation of patients whose composition was determined by the authors or researchers i.e. we aimed to capture the natural flow of patients accessing HAART.The following information was extracted from the studies and relevant program documents: authors, setting of study, duration of accumulation of cases included in the study, male and female distribution of cases, whether program required patients to pay for medication or medications were provided for free and mean or median age of patients on treatment.We also contacted by email, 20 corresponding authors of the articles that we retrieved and HIV and AIDS national program officials asking them whether they were aware of any other reports on HAART provision in the southern African countries they were working.In order to determine whether there was gender balance or imbalance among patients accessing HAART care, we compared the estimated gender ratio of HIV infection prevalence in a specific country to the gender ratio on treatment. We used the UNAIDS 2005 data of national HIV prevalence estimates . This raWe did not attempt to assess whether any patients within a study area were included in only one study or report. As the purpose of the study was to assess the gender distribution of patients, patient serially recorded still served our purpose which was to estimate what was the gender distribution of patients at any particular time data were reported .We identified 21 reports that were eligible to be included in the review -35. We oTable The reported median or mean age of patients reported ranged from 33 to 39 years. In 3 studies, there was a male predominance in treatment programs, one each from Malawi, Swaziland and Zambia. The majority of the studies however reported a > 1.0 female to male ratio. Ten studies reported a female to male ratio exceeding 1.6  and Portuguese (Angola and Mozambique). We were however able to find articles in English from Mozambique, a Portuguese-speaking country, and these were included in the review. There was also likely to be overlapping of patients cohorts among the reports. For instance, in any country where both national treatment figures and localized patient numbers where obtained, many of the patients at the local setting would also be counted within the national figures. We do believe that such double counting in fact emphasizes the point that even when both larger and smaller patient cohorts were assessed, the gender distributions were maintained.The available published data suggest that females may not be disadvantaged in accessing highly active anti-retroviral therapy in Africa. From the limited available reports however, there was either gender balance within treatment programs or predominance of females within treatment programs. There is need to identify facilitating factors that enhance high uptake of women on HAART programs in order to sustain the current successes. There is also need to identify barriers against accessing care by males. This will ensure that both genders have fair access to treatment programs.ART: antiretroviral therapyARVs: antiretroviralsDRC: Democratic Republic of Congo (ex Zaire)HAART: highly active antiretroviral therapyHIV: Human immunodeficiency virusMACRO: Malawi AIDS Counseling and Testing OrganisationMOH: Ministry of HealthMOOSE: Meta-analysis of Observations Studies in EpidemiologySADC: Southern African Development CommunityThe author(s) declare that they have no competing interests.ASM conceived the idea, collected data, participated in analysis and drafting of manuscript.TJN collected data, participated in analysis and drafting of manuscript.SS collected data, participated in analysis and interpretation of findings and drafting of manuscript.EU participated in interpretation of data and the drafting of manuscriptCMM participated in interpretation of data and the drafting of manuscript.HWP collected data, contributed to drafting manuscript and interpretation of data.CSW collected data, participated in interpretation and writing of manuscript.RHM: collected data, participated in analysis and drafting of manuscript.The pre-publication history for this paper can be accessed here:


The impact of HIV drug resistance mutations in salvage therapy has been widely investigated in adults. By contrast, data available of predictive value of resistance mutations in pediatric population is scarce.A multicenter, retrospective, observational study was conducted in children who received rescue salvage antiretroviral therapy after virologic failure. CD4 counts and viral load were determined at baseline and 6 months after rescue intervention. Genotypic HIV-1 resistance test and virtual phenotype were assessed at baseline.vs. 5.4 (p = 0.115). Moreover, the mean number of susceptible drugs according to virtual phenotype clinical cut-off for maximal virologic response was 1.7 vs. 0.8 and mean number of susceptible drugs according to virtual phenotype cut-off for minimal virlologic response was 2.7 vs. 1.3 . Eighteen children were rescued with a regimen containing a boosted-PI and virological response was significantly higher in those subjects compared with the others .A total of 33 children met the inclusion criteria and were included in the analysis. The median viral load (VL) and median percentage of CD4+ at baseline was 4.0 HIV-RNA log copies/ml and 23.0% respectively. The median duration that children were taking the new rescue regimen was 24.3 weeks (23.8–30.6). Overall, 47% of the 33 children achieved virological response at 24 weeks. When we compared the group of children who achieved virological response with those who did not, we found out that mean number of PI related mutations among the group of responders was 3.8 Salvage treatment containing ritonavir boosted-PIs in children with virological failure was very efficient. The use of new tools as virtual phenotype could help to improve virologic success in pediatric population. Treatment with highly active antiretroviral therapy (HAART) has resulted in great reductions in mortality and progression of HIV-1 disease in both adults and children. Increasingly, these children are surviving through to adolescent into adult life ,3.Guidelines for antiretroviral treatment (ART) in children recommend an early and active approach, which usually includes one Protease Inhibitor (PI) or Non-Nucleoside Analogue Reverse Transcriptase Inhibitor (NNRTI) and two Nucleosides Analogue Reverse Transcriptase Inhibitors (NRTI) . AlthougThe use of co formulation of PIs with a fixed dose of ritonavir (rit) has shown a greater activity in both antiretroviral-naïve and treatment experienced HIV-1 infected children than previous PIs because of its pharmacokinetic advantages due to low-dose of rit which enhance the antiretroviral activity of the other PI. -9. HowevThe aim of this retrospective study was to analyze and determine predictive factors of virological success when PIs are used as a salvage therapy for HIV-1 infected children.A multicenter retrospective, observational study was conducted to evaluate the predictive factors of virological responses when PI-containing regimens were used as salvage therapy in vertically HIV-1 infected children. Patients were recruited from three large Spanish Hospitals , with a specialized HIV/AIDS unit in pediatrics. The inclusion criteria were: (1) older than one year of age, (2) been previously treated with antiretroviral therapy including PIs, (3) at least 6 months of follow-up with a salvage regimen and (4) having a resistance test (genotypic and phenotypic) at baseline.Plasma viremia was determined using the bDNA assay version 3.0 , which has a lower detection limit of 50 HIV-RNA copies/ml. A Virological Response (VR) was considered as significant when plasma HIV-RNA reductions were greater than 1 log and/or went to less than 50 HIV-RNA copies/ml. The CD4+ lymphocyte count was measured by flow cytometry . Patients were monitored every three months. Clinical examination and laboratory tests were performed at each visit. There was not a uniform approach regarding antirretroviral drugs included in the salvage regimen. Instead, each paediatrician administered the appropriate ART regimen and changed the drugs according to his/her interpretation of the children data. The adherence of antiretroviral drugs included in the salvage regimen, was measured by each paediatrician by pill count and through interviews with their parents or tutors.protease (PRO) and retrotranscriptase (RT) genes were obtained at baseline using and automatic sequencer . Interpretation of drug resistance mutations was performed following the latest IAS-USA guidelines  and percentages respectively. Univariate analysis was performed to compare baseline characteristics following virological response. Categorical and continuous variables, were compared with the chi-square test (Yates and Fisher correction when necessary) and non-parametric tests (Mann-Whitney U), respectively. Univariate and multivariate binary logistic regression analyses were used to identify baseline characteristics associated with VR. All statistical analyses were conducted using the SPSS package , and all differences were considered to be significant when p values were below 0.05.A total of 33 children were included in the study. Distribution according to sex was; 17 boys (51.5%) and 16 girls (48.5%). Baseline characteristics of the study population are recorded in Table All children had been exposed to at least one PI before the first determination. The proportion of children who have been treated with different PI were as follows: Nelfinavir (NFV) (78.8%), Lopinavir/ritonavir (LPV/rit) (36.4%), Ritonavir (RTV) (30.3%), Indinavir (IDV) (21.2%), Saquinavir (SQV) (21.2%) and Amprenavir (APV) (6.1%). The number of PI that the children have taken before the baseline determination was: 48.6% had taken 1, 24.2% had taken 2, 15.1% had taken 3, 9.1% had taken 4 and 3% had taken 5. At baseline all the children take a PI as part of the salvage regimen. At time of the analysis, 60.6% were using NFV, 30.3% LPV/rit, 6.1% IDV and 3.0% SQV/rit.The median CD4+ cell counts and percentage at baseline were 707.0 (537.5–941) and 23.0 % (19.5–32), and the CD8+ cell counts was 1309.0 (967.2–1996.5) and 45.0% (33.5–57.5). Median baseline viral load was 4.0 (3.4–4.6) HIV-RNA log copies/ml. At baseline, the median number of Nucleotide Analogue Mutations (NAMs) was 3.0 (1.5–4) and the median number of protease resistance mutations was 5.0 (2–6.5). M184V was found in 11 children (33%); 3 of them (30.3%) were resistant to Abacavir and 1 of them (15.2%) was resistant to Tenofovir according to the virtual phenotype. At baseline, 42.4% of children have NNRTI mutations; 30.3% have the K103N and 15.2% the Y181C.vs. -0.2 (p = 0.01). Furthermore, when we compared the variation of the percentage of CD4+ and CD8+ cell counts between the two groups, virological responders had a mean increase in the %CD4+ cell count of 5.3 vs. 0.6 (p = 0.14) and a mean decrease of %CD8+ cell count of -4.7 vs. 0.6 (p = 0.01). Finally, when we compared the group of children who achieved virological response with those who did not, we found the following differences: mean number of PI related mutations was 3.8 vs. 5.4 (p = 0.11), mean number of susceptible drugs according to cut-off for maximal virologic response (CCO1) 1.7 vs. 0.8 (p = 0.03) and mean number of susceptible drugs according to cut-off for minimal virlologic response (CCO2) 2.7 vs. 1.3 (p = 0.01), respectively.Virological response defined as plasma HIV-RNA reductions greater than 1 log and/or VL less than 50 HIV-RNA copies/ml was achieved in 46.9% of children in the first 24 weeks of follow-up. However, 66.6% of them reached undetectable viremia after 24 weeks of follow-up. When we analyze the decrease in VL among patients who achieved virologic response and those who did not, we found the following differences: the former group had a mean decrease of log VL of -1.8 vs. 28.6%, p = 0.09).PI combinations with low doses of ritonavir were used in 18 children; 15 received LPV/rit, 2 Fosamprenavir/rit and 1 SQV/rit. None of them received a double boosting of PIs. Virological response was significantly higher in those subjects rescued with boosting PIs combinations compared with the others  but it was not identify as a prognostic factor due to the short period of follow-up included in our study.In the multivariate analyses only the number of drugs used according with the CCO2 and a lower number of previous PI received were independently associated with virological response. Clinical cut-offs are based on virological response observations in treated patients giving an indication of how the response is affected by viral resistance ,14. Thisboosted-PIs are included in salvage regimens. New strategies as virtual phenotype could improve virological responses and could contribute to select the most appropriate regimen in treatment experienced children, when antiretroviral drugs are not so available.In conclusion, our results showed higher rates of virological success when The author(s) declare that they have no competing interests.BL had primary responsibility for protocol development, collecting and recording data, and outcome assessment and contributed to the writing of the manuscript. CM contributed to the writing of the manuscript and developed the preliminary data analysis. JM Bellón participated in analytic framework for the study. VS contributed to the writing of the manuscript. Paediatricians: MIJ, MJM and JTR were responsible for patient screening, and contributed to the writing of the manuscript. JTR supervised the design and execution of the study, the final data analyses, and the writing of the manuscript. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here: