In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens . Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth.gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated .HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints .In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol region prior to ART initiation were associated with better treatment outcomes.In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV  In adults, high HIV diversity in the gag and pol regions were associated with older age and decreased 5-year survival gag, pol, and env regions) and older age in a separate cohort of 76 Ugandan children aged 0.6–12.4 years We developed an assay based on high resolution melting (HRM) technology that can be used to quantify the genetic diversity of HIV populations without sequencing gag, pol, and env and response to ARV treatment (ART) among 139 African children in the P1060 multi-national, randomized, clinical trial (NCT00307151) In this report, we used the HRM diversity assay to evaluate the relationship between HIV diversity in Written informed consent was obtained from the parents or legal guardians of all children enrolled in the study. The P1060 trial was approved by the Ministries of Health and the ethics review committees at the local study sites: University of Kwazulu-Natal Biomedical Research Ethics Administration; University of the Witwatersrand Ethics Committee; Stellenbosch University; Medical Research Council of Zimbabwe; University of Zambia Research Ethics Committee; Malawi National Health Sciences Research Committee; Uganda National Council for Science and Technology HIV/AIDS Research Committee; Kilimanjaro Christian Medical College Institutional Ethics Committee; and the Medicines Control Council. The P1060 trial was also approved by the institutional review boards of the participating institutions in the United States: University of Alabama at Birmingham; Johns Hopkins University School of Medicine; University of North Carolina at Chapel Hill; and Duke University Health System Cohort I of the P1060 trial enrolled 164 children (ages 6–36 months) at nine sites in six African countries  gag , one in pol (POL), and three in env . The HRM diversity assay was performed as previously described CD4 cell count, CD4%, and HIV viral load were measured in real time during the P1060 trial Fisher's exact and Wilcoxon rank-sum tests were used to compare the characteristics of participants with and without HRM results for categorical and continuous variables, respectively. Spearman correlations for continuous variables and Kruskal-Wallis tests for categorical variables were used to determine whether HRM scores were associated with characteristics at study enrollment. Median regression was used in multivariable analyses because of outliers in the HRM scores, which could over-influence results using linear regression techniques. Multivariable analysis was performed using backwards stepwise median regression starting with any variables that were significant in the univariate analysis at the P<0.10 level. The least significant variable was sequentially dropped from the model until any remaining variables were significant at the P<0.05 level.Cox proportional hazards regression was used to assess whether HRM scores were associated with time to virologic suppression, virologic failure, virologic failure or death, and virologic failure or discontinuation of the randomized NVP or LPV/r component of the study regimen. Virologic failure was defined previously as either not achieving virologic suppression within 24 weeks or rebounding after 24 weeks We also analyzed the relationship between age and treatment adherence, which was documented in the P1060 trial as missed doses at the 2, 4, 8, 12, 16, and 24 week visits. Perfect adherence at each study visit was defined as the caregiver reporting no missed doses of study treatment in the three days prior to the visit. Wilcoxon rank sum tests were used to compare the age distribution of the participants with and without perfect adherence at each study visit.The HRM diversity assay was performed using samples collected prior to initiation of ART (pre-treatment samples). Samples were available for analysis from 139 (84.8%) of the 164 children enrolled in Cohort I of the P1060 trial , Table 1We evaluated the association between pre-treatment HRM scores and demographic and clinical characteristics of the study participants. Results obtained for continuous and categorical variables are shown in Ninety-four (68%) of the 139 children analyzed in this report achieved virologic suppression on ART, defined as two consecutive HIV viral load values <400 copies/mL after initiation of ART. In these analyses, statistical models were adjusted for study treatment regimen, age (years), CD4%, and HIV viral load see . Higher We next assessed the association of HRM scores with time to study endpoints . Among tThe models reported in pol diversity and better treatment outcomes may have reflected better adherence to treatment regimens among older children . In the P1060 trial, treatment adherence was assessed by maternal report of missed doses at study visits 2 to 24 weeks after treatment initiation. Of the 139 children included in this study, over 92% did not have any report of missed doses. The percentage of participants with no missed doses ranged from 93.0% at week 8 (n = 114) to 98.4% at week 4 (n = 128). Furthermore, there was no indication that adherence was better among older children.We considered whether the observed associations between higher pol region prior to ART initiation. This association remained statistically significant in multivariate models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and ART regimen. The association of higher pol diversity with improved clinical outcomes was seen with a variety of outcome measures including: shorter time to virologic suppression after ART initiation, longer time to virologic failure, and longer time to the composite outcomes of virologic failure with either death or discontinuation of the study ART regimen. In this cohort, the definition of virologic failure took into account both initial viral suppression on ART and viral rebound  diversity measures were used. Similar associations were seen for some of these comparisons when the analysis was performed using composite diversity measures .In this study, we identified a novel factor associated with clinical outcome on ART: a higher level of HIV diversity in the ound see . The assenv or gag regions and study outcomes, but those findings were less consistent. For example, higher diversity in the ENV2 region was associated with shorter time to virologic failure (a worse study outcome). However, this association held for only one of the outcome measures. Higher diversity in the GAG1 region was associated with shorter time to virologic suppression and with longer time to the composite endpoint of viral failure or treatment discontinuation (better study outcomes). However, other regions of env (ENV1 and ENV3) and gag (GAG2) were not associated with any study outcome.We also observed associations between HIV diversity measures in individual env and gag diversity were associated with ART failure, while pol diversity was not. There are many differences between that study and this report that may account for the differences in our findings. This report included 139 children (vs. 76), children from four countries (vs. one country), children who were mostly infected with HIV subtype C (vs. A and D), children ages 6 to 36 months (vs. ages 6 months to 12.4 years), children who discontinued the ART regimen if they experienced viral failure , a variety of study endpoints , and was performed in the context of a randomized, clinical trial . Therefore, while the results of both studies are valid, we feel that the results from this study may be of more general relevance.Results in this study are different from those of an observational cohort of Ugandan children on ART pol region. An association between higher levels of HIV diversity and older age was also observed in our previous studies of Ugandan infants and children In this study of children ages 6–36 months, older age was significantly associated with higher diversity in the pol diversity would be associated with more rapid virologic suppression or other improved clinical outcomes on ART. Because older age was also associated with higher pol diversity, we considered that older children might have been more adherent to their study regimens and that this may have explained the better treatment outcomes in children with higher pol diversity. However, there was no significant difference in treatment adherence among older vs. younger children. Therefore, it was unlikely that the association between higher pol diversity and better treatment outcomes was the result of better adherence among older children. We also note that the multivariate models used for the analysis were adjusted for age, in addition to other factors.It is not clear why a higher level of env diversity and higher viral load gag diversity and higher viral load env diversity and viral load gag or pol and a borderline association in env (p = 0.05). This is consistent with our studies of Ugandan infants where we found no association between viral load and gag or pol diversity pol diversity and treatment outcomes in this study was independent of HIV viral load.We also considered whether HIV viral load, which may be associated with viral diversity, could have confounded our results. Previous studies examining the relationship between HIV diversity and viral load found conflicting results. In adults, one study found an association between higher pol region. While antibodies that target products of the HIV pol gene have been described In theory, higher viral diversity might reflect a more vigorous immune response to HIV infection, resulting in faster or greater viral diversification and evolution. The strength of the immune response in HIV-infected infants might be influenced by the viral adaptation that appears to occur in vertical HIV transmission pol diversity prior to ART initiation may reflect properties of the viral strain, such as faster replication. The presence of ARV drug resistance mutations is often associated with reduced viral fitness pol diversity was still associated with worse outcomes after controlling for treatment arm and other measures of HIV disease. Therefore, prior exposure to NVP for PMTCT was not likely to be responsible for the lower pol diversity observed in children with worse treatment outcomes.Alternatively, higher levels of pol diversity was associated with better treatment outcomes. Higher replication capacity is associated with more rapid viral turnover, which could result in faster viral suppression on ART pol diversity with better treatment outcomes in this cohort.Even though we did not see an association between ARV drug resistance and HIV diversity, differences in viral replication capacity could still explain why higher in utero and during breastfeeding Another finding in this study was the association of increased GAG2 diversity among infants whose mothers did not have a history of ARV drug use (other than sdNVP for PMTCT). Maternally-administered ARV drugs are known to be transferred to infants pol region diversity were associated with better treatment outcomes among African children on ART. Understanding the relationship between HIV diversity and the response to ART in HIV-infected children and adults may provide new insights into factors that impact clinical responses to ART.Previous studies on HIV diversity in children have relied on sequence-based methods for diversity analysis. Those methods are more costly and labor intensive than the HRM diversity assay and also produce more complex data sets that may be more difficult to analyze than single numeric HRM scores. In contrast, the HRM diversity assay is relatively simple to perform and is well-suited for analysis of multiple genomic regions. This makes it feasible to perform larger studies such as this one, which evaluated six genomic regions in 139 children (>800 measures). The major finding in this study was that higher levels of


To the Editor: Genotypic drug resistance testing before initiation of first-line antiretroviral therapy (ART) is recommended to detect drug-resistant viruses and to avoid treatment failure caused by preexisting drug-resistant viruses  and high HIV-1 viral load of 980,000 copies/mL plasma, we started directly observed ART with tenofovir and emtricitabine plus nevirapine. Genotypic resistance testing showed no evidence of resistance. Within the first 4 weeks of ART, the viral load decreased to 540; however, 4 weeks later it increased to 15,000, and then 12 days later to 71,000 HIV-1 RNA copies/mL . ResistaWe hypothesized that preexisting drug-resistant HIV-1 minority variants might have caused this early treatment failure. Thus, we performed clonal analysis of the RT gene before and during ART. At baseline, 17/222 clones (7.7%) carried the K65R mutation, synonymous to ≈51,000 HIV-1 RNA copies. Later, the K65R mutation was comprised in all clones. Further preexisting drug-resistance mutations have been detected in single, K65 wild-type, separate clones: K70R, V106A, and V108I. However, neither the K103N nor the M184V mutation was detected in any of those 222 clones, but both mutations were rapidly selected during early treatment failure.Thus, the presence of the K65R mutation in a substantial fraction of the virus population and the rapid acquisition of the K103N and M184V mutations led eventually to early treatment failure. We cannot formally rule out that K103N mutants were present before ART at a very low frequency <0.5% and that K65R/K103N double mutants were potentially selected. The M184V mutation was acquired later because only 12.5% of K65R/K103N viruses carried the M184V mutation at the first time point during treatment failure.Because of the considerable absolute number of less replication-competent K65R-harboring viruses in the patient, we assume that this variant has been transmitted. The additional presence of isolated, nonnucleoside RT inhibitor (NNRTI)/nucleoside RT inhibitor (NRTI) resistance mutations is another indication for the transmission of drug-resistant viruses, although they and the K65R mutation were not found in the same viral genomes. Presumably, the index patient was treated with nevirapine, stavudine, and lamivudine, the commonly prescribed first-line ART in resource-limited settings (The prevalence of K65R-harboring drug-resistant HIV-1 minority variants is not negligible in treatment-naive patients. We have shown that 2.7% of HIV-1–infected patients, mainly those infected with HIV-1 subtype B, harbor the K65R mutation as a minority variant (Immigration from resource-limited settings is increasing, and data suggest that minority variants harboring the K65R mutation are quite prevalent in those infected with HIV-1 subtype C, who are treatment naive (


This observational analysis examined the clinical outcomes of patients receiving etravirine-(ETR-) based therapy, particularly with protease inhibitors (PIs) other than darunavir (DRV) and with raltegravir (RAL). Data included treatment-experienced adults in the HIV Research Network who began ETR-containing antiretroviral regimens in 2008–2010. The primary objective was to assess 6-month outcomes . The cohort included 587 patients receiving ETR; 42% of ETR use was in patients not on DRV/ritonavir (r). Patients receiving ETR plus DRV/r had longer durability versus those on ETR plus a PI other than DRV/r at months 6 (91.2% versus 85.5%) and 12 (77.4% versus 65.2%), respectively. Patients on regimens with a PI other than DRV/r were the least likely to be receiving ETR at month 6 (85.5%) versus patients on other ETR-based regimens. Patients on regimens without a PI and without RAL had lower virologic suppression  versus patients on other ETR-based regimens. In a clinical care, nontrial setting, ETR was used in regimens without DRV/r. In this population, the 6-month response rates were similar and durable across all regimens, except when ETR was used without RAL and without a PI. Aside from registrational trial data, there is limited information on the utilization and clinical outcomes of patients treated with etravirine-(ETR-) based therapy. To date, ETR has demonstrated high efficacy rates, as well as good tolerability and safety profiles , 2. HoweDespite encouraging data on the use of ETR plus DRV/r, limited data exist on the use of ETR with protease inhibitors (PIs) other than DRV/r, and with novel agents like raltegravir (RAL) , 4–6. ThWe evaluated outcomes with the use of ETR in a consortium of high-volume HIV care sites to describe the current utilization of antiretroviral therapy. We analyzed antiretroviral medication utilization among HIV-infected adults enrolled in the HIV Research Network (HIVRN), a consortium of clinics that provide primary and subspecialty care to HIV patients. Fifteen sites treat adult patients. Data from 12 sites, located in the northeastern (5), midwestern (1), southern (3), and western (3) United States, were included in this analysis. The remaining three sites discontinued participation during the study period and did not provide complete data. Nine sites have academic affiliations. Adult patients (≥18 years of age) who enrolled at an HIVRN site between 2000 and 2010 and who had at least one outpatient visit and a CD4 count in any calendar year between 2000 and 2010 were eligible for inclusion. Data encompassing the period from January 1, 2008, through December 31, 2010, were gathered from medical records at each site and sent to a data coordinating center after personal identifying information was removed. Problematic data elements were identified, reviewed with the site, and corrected. After quality control and verification, data were combined across sites to produce a uniform database. The study was approved by institutional review boards at the Johns Hopkins University School of Medicine and at each participating site.Over 20,000 patients are represented at 12 sites. Of these, 29% are women, 51% are black, 22% are Hispanic, 51% are receiving Medicare and/or Medicaid, 18% are receiving commercial insurance, and 24% are receiving Ryan White services . Here, we present data collected from the HIVRN on the outcomes of patients receiving a variety of ETR-based therapies over 6 and 12 months.Demographic and clinical data were collected from treatment-experienced adult (≥18 years of age) patients in the HIVRN who began an ETR-containing antiretroviral regimen in 2008 or 2009. Demographic data for treatment-experienced adult patients in the HIVRN who began an antiretroviral regimen not containing ETR in 2008, 2009, or 2010 are also included for comparison. To be included, ETR had to be prescribed for at least 60 days and patients had to have at least 24 weeks of follow-up after beginning the regimen. The primary objective was to assess 6-month outcomes . Outcomes are reported by regimen up to 12 months. As not all HIV-1 RNA tests performed in routine care had a lower limit of quantification of <50 copies/mL, HIV-1 RNA <400 copies/mL was chosen as the primary end point and considered suppression for this study.Sample size was based on convenience and represented all available patients who met inclusion criteria in the cohort. As the sample was based on convenience and the study was not powered for between-group comparisons, only descriptive statistics are presented. Analyses were performed using SAS 9.1.3 . In total, 587 patients on ETR-containing regimens met inclusion criteria and were included in the cohort. Compared with the 15,084 patients from the HIVRN who were treatment experienced and did not receive ETR, the patients from this cohort were older, had lower median baseline CD4+ cell counts and CD4+ nadir counts, and had been in care for a longer period .n = 69), 27 (39.1%) received lopinavir/r, 17 (24.6%) received atazanavir, and 4 (5.8%) received fosamprenavir. In this cohort, 42% of the ETR use was by patients not on DRV/r, and 46% of ETR-based regimens included both a PI and RAL . Of the At baseline, the group of patients treated with ETR without a PI and those treated with ETR without a PI and without RAL had a lower proportion of male and white patients, a higher proportion of Hispanic patients, a shorter median time in care, and a high median nadir CD4 count compared with patients on other regimens . PatientPatients treated with ETR and DRV/r were more frequently male and had lower median nadir CD4+ cell counts and a lower incidence of injection drug use compared with those treated with ETR and other PIs or those treated with ETR and no PI . Patients treated with ETR plus DRV/r had higher durability  than those treated with ETR plus a PI other than DRV/r at month 6 and at month 12 . PatientRates of viral suppression (<400 copies/mL) were similar for patients receiving ETR plus DRV/r compared with those receiving ETR plus a PI other than DRV/r through month 6, whereas rates of viral suppression at 12 months were higher in patients receiving ETR plus DRV/r compared with those receiving ETR plus a PI other than DRV/r . PatientPatients on regimens without a PI and without RAL had lower rates of HIV-1 RNA suppression compared with patients on other ETR-based regimens at month 6 and at month 12 . In contThe registrational studies for ETR all included DRV/r as part of the treatment regimen . HoweverSuppression rates at month 6 and month 12 were lower when ETR was combined with neither a PI nor RAL. Suppression at month 6 was similar between patients treated with ETR plus RAL (80.0%) and those treated with ETR plus RAL and a PI (80.6%). These results suggest that, when deemed appropriate based on patient history and resistance testing, an ETR plus RAL-based regimen may be a viable treatment option. Although still high, the suppression rates in patients receiving ETR with a PI and RAL  in this trial were slightly lower than those seen in the TRIO study (88%) [Several limitations to this analysis should be noted. First, there was only a small population of patients receiving ETR in the HIVRN. Second, the differences in baseline characteristics of patients between the different treatment regimens were likely due to providers prescribing the different regimens based on perceived needs of the patients that included some of the observed characteristics as well as other characteristics not noted in this study . Adjustment for these variations in baseline characteristics by PI and RAL use was not performed in this analysis. Finally, the potential antiviral activity of ETR and other antiretrovirals was unknown before treatment initiation, as resistance testing results before starting ETR were not available.Results from this observational analysis demonstrated that, in a real-world setting, ETR was used in regimens not containing DRV/r and that in a highly treatment-experienced patient population, the response rates at 6 months were similar and durable across all combinations studied—except when ETR was used without RAL or a boosted PI. Future studies will need to explore whether certain populations may be more likely to benefit from one regimen compared with another.


It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects.To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen.Patients experiencing VF on a Tenofovir/Emtricitabine+PI/r regimen were evaluated by ultra deep sequencing (UDS) for mutations classified/weighted by Stanford HIVdb. Samples were evaluated for variants to 0.4% levels. 36 VF subjects were evaluated by UDS; 24 had UDS for PI and RT DRMs. Of these 24, 19 (79.2%) had any DRM by UDS. The most common UDS-detected DRM were NRTI in 18 subjects: M184V/I (11), TAMs(7) & K65R(4); PI DRMs were detected in 9 subjects: M46I/V(5), F53L(2), I50V(1), D30N(1), and N88S(1). The remaining 12 subjects, all with VLs<10,000, had protease gene UDS, and 4 had low-level PI DRMs: F53L(2), L76V(1), I54S(1), G73S(1). Overall, 3/36(8.3%) subjects had DRMs identified with Stanford-HIVdb weights >12 for ATV or LPV: N88S  in 1 subject on ATV; I50V  and L76V  in 1 subject each, both on LPV. All VF samples remained phenotypically susceptible to the treatment PI/r.Among persons experiencing VF without PI DRMs with standard genotyping on an initial PI/r regimen, low-level variants possessing major PI DRMs were present in a minority of cases, occurred in isolation, and did not result in phenotypic resistance. NRTI DRMs were detected in a high proportion of subjects. These data suggest that PIs may remain effective in subjects experiencing VF on a PI/r-based regimen when PI DRMs are not detected by standard or UDS genotyping. HIV treatment guidelines recommend combination antiretroviral (ARV) regimens for treatment naive patients consisting of a nucleoside/nucleotide reverse transcriptase inhibitor (N(t)RTI)-based backbone along with either a non-nucleoside reverse transcriptase inhibitor (NNRTI), an integrase inhibitor (INI) or a ritonavir (RTV)-boosted protease inhibitor (PI) Emtricitabine (TDF/FTC) plus a ritonavir boosted protease inhibitor (PI/r) is a solitary reverse transcriptase (RT) M184V mutation or no resistance mutations The most common drug resistance mutation pattern by standard HIV genotyping in patients experiencing virologic failure on an initial regimen of Tenofovir/The objective of this study was to determine if patients with virologic failure on an initial PI/r-based regimen without resistance by standard genotyping possess low-level drug resistant variants that could affect the use of a subsequent PI/r containing regimen. To investigate this question, ultra deep sequencing was performed on the virologic failure specimens from the CASTLE study All available Week 48 and 96 CASTLE virologic failure specimens from patients without PI resistance mutations by standard genotype were evaluated by ultra deep sequencing  for N(t)RTI+PI/r mutations see  and clasAll IRB approvals for this study were already obtained under the parent CASTLE study. All subjects had given informed consent. The parent CASTLE study is registered with ClinicalTrials.gov, number NCT00272779.Of 78 patients with virologic failure at Weeks 48 and/or 96 from the CASTLE study without PI resistance mutations by standard genotype, 57 were available for ultra deep sequencing . Of the N88S  and 2 patients on LPV: one with I50V (0.44%-mutation load 110) and the remaining patient with L76V (0.52%-mutation load 20). The mutations identified by UDS in each specimen and the estimated mutational loads with the corresponding phenotypes are listed in Overall, 3/36 (8.3%) patients had PI mutations identified with Stanford-HIVdb weights >12 for ATV or LPV: 1 patient on ATV with The results from this study demonstrated that among patients on an initial PI/r based regimen experiencing virologic failure without PI resistance mutations by standard genotyping, low-level variants possessing major PI mutations were present in a minority of cases, occurred in isolation, and did not result in phenotypic PI resistance. Only 3 patients (8.3%) had a PI mutation with a Stanford algorithm weight >12 for the PIs used in the study (ATV or LPV). However, they were at very low levels with low mutational loads. It should be noted that none of the low-level variants with a major PI mutation were at levels ≥1% of the viral population, a level that has been shown to be clinically significant for NNRTIs based regimens These findings underscore the limitation of stand-alone phenotypic susceptibility results and emphasize the importance of complementary and/or more sensitive genotyping techniques when evaluating for resistance As previously reported by our group, low-level variants possessing PI mutations are infrequently identified in treatment-naïve persons and did not significantly affect the efficacy of PI/r based regimens The results of this study may have implications for first line and subsequent ARV sequencing strategies. According to current guidelines, second line ARV regimens for subjects with resistance to NNRTI or PI/r based regimen should consist of at least 2, preferably 3 new and fully susceptible ARVs Our study had several limitations. Given the requirement for other predetermined study analyses we were only able to recover specimens for 57 of 78 Week 48 and/or 96 CASTLE virologic failures. Further, we were unable to perform ultra deep sequencing on 21 virologic failure specimens that had an HIV viral load ≤1,000 c/mL. Full length reverse transcriptase ultra deep sequencing for samples with HIV VL>10,000 c/mL was limited to 24 samples. Given stochastic effects of RNA sampling for the samples with HIV VL<10,000 c/mL, the reported variants may or may not represent the proportion of variants in plasma. Thus, we are unable to confidently analyze all specimens to low levels which may have lead to an undetected selection bias.In conclusion, among patients on an initial PI/r based regimen experiencing virologic failure without PI mutations by standard genotyping, low-level variants possessing major PI mutations by ultra deep sequencing were infrequently identified and if PI mutations were present they were found in isolation and did not result in phenotypic PI resistance. Only 3 patients (8.3%) had a PI mutation with a Stanford algorithm weight >12 for ATV or LPV, however they were at very low levels (<1%) with low mutational loads. NRTI mutations were detected in a high proportion of patients with virologic failure and with greater frequency than PI mutations. These data suggest that PIs may remain effective in patients experiencing virologic failure on a PI/r-based regimen, and may be re-used in subsequent ARV regimens with new optimized background therapy when PI resistance are not detected by standard or ultra deep sequencing. As the HIV population grows older, clinicians will need to conserve agents and use potent and relatively safer ARVs for as long as possible.These data were presented in part at the 2010 International HIV & Hepatitis Virus Drug Resistance Conference. Dubrovnik, Croatia, Abstract #102.Table S1UDS data for RT and PR and phenotype for 24/36 VF subjects. Ultra deep sequencing, mutational load, and phenotypic susceptibility results for 24 samples from subjects experiencing virologic failure on TDF+FTC+PI/r. # UDS for two virologic failure (VF) timepoints – both VF specimens had a M184V mutation. NRTI DRMs: M184V and K65R are bolded in black. PI DRMs with a Stanford HIVdb weight >5 are bolded in black with an asterix *. Please note that if a sample had different HIV variants with the same mutation they are listed separately e.g. Subject #2 had different HIV variants with a M184V at 3.92% and M184V at 94.94%.(DOCX)Click here for additional data file.Table S2UDS data for PR only and phenotype for 12/36 VF subjects. Ultra deep sequencing and mutational load for PIs only, and phenotypic susceptibility results for 12 samples from subjects experiencing virologic failure on TDF+FTC+PI/r. PI DRMs with a Stanford HIVdb weight >5 are bolded.(DOCX)Click here for additional data file.


In Mozambique, the evaluation of retention in HIV care and ART programmes is limited. To assess rate and predictors of attrition (no retention in care) and HAART effectiveness in HIV-1 infected patients who pay for medication and laboratory testing in Mozambique, we conducted a multicenter survey of HIV-1-infected patients who started HAART during 2002–2006. Cox proportional hazard models were used to assess risk of attrition and of therapy failure. Overall, 142 patients from 16 healthcare centers located in the capital city Maputo were followed-up for 22.2 months (12.1–46.7). The retention rate was 75%, 48% and 37% after one, two and three years, respectively. Risk of attrition was lower in patients with higher baseline CD4 count (P = 0.022) and attending healthcare center 1 (HCC1) (P = 0.013). The proportion of individuals with CD4 count ≤200 cells/µL was 55% (78/142) at baseline and decreased to 6% (3/52) at 36 months. Among the patients with available VL, 86% (64/74) achieved undetectable VL levels. The rate of immunologic failure was 17.2% (95% CI: 12.6–22.9) per 100 person-years. Risk of failure was associated to higher baseline CD4 count (P = 0.002), likely reflecting low adherence levels, and decreased with baseline VL ≥10,000 copies/mL (P = 0.033). These results suggest that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate. Mozambique suffers from one of the highest HIV/AIDS burdens in the world, with HIV-1 prevalence of 21% in the southern regions where the capital city Maputo is located The study was conducted according to the Declaration of Helsinki with the approval of the ethics committee of the Faculty of Medicine . Written informed consent was obtained from all participants.The Maputo HIV-1 Cohort was established to better describe the epidemiology of HIV-1 infection and care patterns and treatment among patients with medium-high socioeconomic status who self-paid laboratory analyses and ART. The Cohort prospectively enrolled antiretroviral-naïve HIV-1-infected patients recruited in 16 healthcare centers throughout the Maputo province. All patients aged ≥18 years at ART initiation, with documented date of start of highly active antiretroviral therapy (HAART) comprised of at least 3 antiretrovirals, with baseline CD4 T-cell count (for the six months before HAART initiation) and with follow-up of ≥3 months were eligible for this retrospective survey. The HIV-1 infection was determined on serum or plasma samples by Uni-Gold Recombigen  and Determine HIV-1/2  assays. Immune cell counts were determined by flow cytometry  and plasma HIV-1 RNA  by COBAS Amplicor test v1.5 , which has a detection limit of 400 copies/mL. Resistance genotyping was performed by ViroSeq HIV-1 Genotyping System .2 were used to compare categorical variables. The median of HIV-1 markers and biochemical parameters was calculated for each patient per semester. A window period of 6 months was allowed for measurements at each time-point. A median for the study population was then calculated to evaluate the evolution of each marker (Wilcoxon tests). Rates of change (slopes) in total lymphocyte count (TLC), CD4 and CD8 counts were calculated from 3 consecutive measurements (Deming regression). Differences in slopes among groups according to different baseline CD4 count were analyzed  and correlation between slopes was assessed (Spearman Rank test). The risk of attrition (no retention in care) after HAART initiation and the risk of immunologic failure over time were estimated by survival analyses using Kaplan-Meier curves and Cox proportional hazards models. Time was calculated from the date of HAART initiation to the outcome event. Data were censored on the last visit date for individuals not retained in care. Information on vital status  for those not retained in care was not available, thus the definition of outcome measure of attrition from care combined both patients who died and those lost to follow-up, who had failed to return for treatment for 3 months after their scheduled visit for reasons unrelated to death. Immunologic failure was defined as: fall of CD4 count to baseline (or below) or 50% fall from on-treatment peak value or persistent CD4 levels below 100 cells/µL 10; a reduction of VL >2 log10. SPSS  and GraphPad Prism  were used. Statistical significance was defined as P<0.05 (2-tailed).Baseline characteristics were reported with medians and interquartile range (IQR) for continuous variables and percentages for categorical variables. Kolmogorov-Smirnoff test was used to evaluate the distribution of variables. Fisher exact test or χ10 copies/mL, and was inversely correlated to CD4 counts . Median CD4 count and age were similar between patients with available and unavailable VL. Median CD4 count, VL and age were similar between patients attending HCC1 and HCC2. Other baseline characteristics and ART use are listed in Overall, 142 patients from 16 healthcare centers (HCC) who started HAART between 2002 and 2006 were eligible. Most patients  were from a centrally located clinic (HCC1); the remaining patients were from 15 small private clinics located around Maputo. For convenience of analysis these clinics were grouped into one and named HCC2. The median follow-up was 22.2 months (12.1–46.7). The median baseline CD4 count was 185 cells/µL (126–290). Baseline VL was available in 54% (76/142) of patients, who had a median of 5.3 (4.8–5.9) logThe retention rate was 75%, 48% and 37% after one, two and three years, respectively . Risk ofMedian CD4 count increased with time on HAART with a rapid average increase [113 cells/µL (38–187)] during the first semester (P<0.001) . ConsistAmong the 60 patients with available data at month 24, median CD4 slopes were uniform across CD4 strata (gain of 6.9 cells/µL/month [3.2–10.6]), with the exception of individuals with baseline >500 cells/µL that lost a median of 7 CD4 cells/µL/month . Median 10 occurred in 88% and of >2 log10 in 69% participants  experienced a decrease to undetectable VL levels after a median of 6.8 months (5.3–8.4). A VL reduction of >1 logicipants . ResistaThis is the first study evaluating the predictors of attrition after initiation of HAART as well as HAART effectiveness in a population paying for antiretrovirals and laboratory testing in Mozambique. The retention rate  was 75%, 48% and 37% after one, two and three years, respectively. Similar high rates of attrition have been previously reported in ART programs in several African countries A sustained immunologic response was observed, with most patients experiencing CD4 recovery above 200 cells/µL, a clinically meaningful threshold for clinical progression and opportunistic infections (OI) As previously found in the EuroSIDA survey, patients with higher baseline CD4 counts were at higher risk of immunological failure than patients with lower CD4. One reason for this finding could lie in the lower rates of ART adherence that individuals with higher baseline CD4 may have had because at lower risk of OI The main shortcoming of this study was the limited size of the study population and the difficulty in retaining patients in care might have led to a survivor effect. Notwithstanding these limitations and despite the fact that improved strategies are clearly required to increase in-care retention levels, this study suggests that HAART can be effective in HIV-1 infected patients from Mozambique that pay for their medication and laboratory testing. Further studies are required to identify the causes for low retention rates in patients with low CD4 counts and to better understand the association between healthcare setting and attrition rate.Figure S1Kaplan-Meier estimate of time from antiretroviral therapy initiation to attrition during follow-up according to healthcare centers.(TIF)Click here for additional data file.Figure S2Evolution of immunologic and biochemical parameters during follow-up in HIV-1-infected patients. Legend: Dots represent median values over the study period; bars represent interquartile range.(TIFF)Click here for additional data file.Figure S3Median changes in total lymphocytes, CD4 and CD8 counts per month during follow-up. Legend: Slopes were computed by Deming regression analysis; correlation between slopes was assessed by Spearman Rank test. TLC, total lymphocyte count.(TIFF)Click here for additional data file.Table S1Comparison of baseline characteristics and antiretroviral therapy use among HIV-1 infected patients retained in the cohort and those not retained.(DOC)Click here for additional data file.Table S2Evolution of CD4 T-cell count at different time points during follow-up in the study population.(DOC)Click here for additional data file.Table S3Analyses of risk of immunologic failure in the study population.(DOC)Click here for additional data file.Table S4Type of immunological failure according to baseline CD4 count strata in the study population.(DOC)Click here for additional data file.Table S5Slopes of CD4 count during the first two years of antiretroviral therapy in the study population.(DOC)Click here for additional data file.


Long term efficacy of raltegravir (RAL)-including regimens in highly pre-treated HIV-1-infected patients has been demonstrated in registration trials. However, few studies have assessed durability in routine clinical settings.Antiretroviral treatment-experienced patients initiating a RAL-containing salvage regimen were enrolled. Routine clinical and laboratory follow-up was performed at baseline, week 4, 12, and every 12 weeks thereafter. Data were censored at week 96.3; 273 out of 300 patients (91%), who were still receiving RAL at week 96, achieved viral suppression (HIV-1 RNA <50 copies/mL). When analyzing the immuno-virologic outcome according to the number of drugs used in the regimen, 2 (n = 45), 3 (n = 111), 4 (n = 124), or >4 (n = 40), CD4+ T-cell gain was similar across strata: +270, +214, +216, and +240 cells/mm3, respectively, as was the proportion of subjects with undetectable viral load. Laboratory abnormalities  were rare, ranging from 0.9 to 3.1%. The mean 96-week total cholesterol increase was 23.6 mg/dL.Out of 320 patients enrolled, 292 (91.25%) subjects maintained their initial regimen for 96 weeks; 28 discontinued prematurely for various reasons: death (11), viral failure (8), adverse events (5), loss to follow-up (3), consent withdrawal (1). Eight among these 28 subjects maintained RAL but changed the accompanying drugs. The mean CD4+ T-cell increase at week 96 was 227/mmIn a routine clinical setting, a RAL-based regimen allowed most patients in salvage therapy to achieve optimal viral suppression for at least 96 weeks, with relevant immunologic gain and very few adverse events. The efficacy of raltegravir (RAL) in combination with optimized background therapy (OBT) in HIV-1 infected, treatment-experienced patients, was assessed in a multicenter, dose-ranging, randomized, placebo-controlled phase II study 3. Engsig et al. Apart from these sponsored studies, the amount of observational cohort data is relatively small. In the ANRS CO3 Aquitaine Cohort As well as efficacy, also tolerability data are mainly obtained from the few official trials and describe a very well tolerated drug Treatment-experienced patients starting a raltegravir-including antiretroviral regimen between March, 2007 and June, 2009, were consecutively enrolled in a collaborative Italian study, called SALIR , having signed informed consent within the Expanded Access Program (EAP), approved by the local Ethics’ Committees, and followed beyond the time limit of the EAP, as convened among the authors, according to the Good Clinical Practice (GCP) Guidelines.List of the Ethics’ Committees:Comitato Etico Azienda Ospedaliera Luigi Sacco – Polo Universitario.Comitato Etico delle Aziende Sanitarie dell’Umbria.Comitato Etico Arnas Garibaldi.Comitato Etico Irccs Ospedale San Martino di Genova.Comitato Etico Provinciale di Modena.Comitato Etico Sperimentazione Clinica Azienda Ospedaliera-Universitaria Careggi.Comitato Etico Istituto Nazionale delle Malattie Infettive “Lazzaro Spallanzani”.San Gerardo Hospital Ethics Committee.Comitato Etico Ospedale di Circolo Di Busto Arsizio.Comitato Etico per la Sperimentazione dei Farmaci della Ausl 9 di Grosseto.Comitato Etico per la Sperimentazione Clinica dei Medicinali dell̀Azienda Sanitaria di Firenze.Comitato Etico Scientifico A.O.U. Policlinico "G. Martino" – Messina.Comitato Etico per la Regolamentazione della Sperimentazione Clinica dei Farmaci dell’Azienda U.S.L. di Pescara.Comitato Etico Provinciale per la Sperimentazione Clinica dell̀Azienda ULSS 18 di Rovigo.Companion drugs were chosen by each investigator on the basis of the genotypic sensitivity of the strains and of the patient’s history of treatment, intolerance, toxicity or allergy to antiretrovirals. All patients had blood drawn and visits at week 4 and 12 after the introduction of the new regimen, and approximately every 12 weeks thereafter. For the toxicity analysis we considered only those parameters that were routinely collected in all centers  and considered the Common Terminology Criteria for Adverse Events  Baseline patients’ characteristics were described. Continuous variables were reported as means and standard deviation (SD) if they were normally distributed and as medians and interquartile range (IQR) if not. Ordinal and categorical variables were reported as frequency and percentage.Patients who had missing data before week 96 were considered as failures. The proportion of subjects reaching <50 copies HIV-1 RNA/mL was analyzed as on-treatment  and intention-to-treat: missing  =  failure . Variables associated to treatment failure were compared using logistic regression, according to the Cox model. Results were given as hazard ratio (HR), and 95% confidence interval (CI). Odds ratio (OR) and 95% confidence intervals (CI) was used to evaluate the association between metabolic alterations at study end  and baseline patients’ characteristics. As appropriate, we included selected variables to adjust simultaneously for their potentially confounding effects, using a backward selection method and stating the significance cut-off P at 0.15.3  were still receiving their baseline therapy at week 96. Demographic and immuno-virological characteristics of all the subjects are shown in 3 .By the week 96, 27 patients (8.4%) had detectable viremia, mainly due to adherence problems, as detected by pill count. Overall, 29 patients (8.9%) experienced early or late virologic failure.Genotypic analysis was obtained for 14 out of 29 failing patients, and RAMS are shown in Nine (3.1%) and 7 (2.1%) patients reached AST and ALT serum levels over grade 2 toxicity respectively, while one and 28 (8.8%) had over grade 2 toxicity of TC and TG serum levels, respectively. Only TC showed an overall increasing trend over time, with a mean increase of +23.6 mg/dL at week 96 . Duration of HIV infection (P = 0.02), hepatitis B or C coinfection  and baseline elevated TC (P<0.0001) predicted week 96 TC elevation over the National Cholesterol Education Program guidelines P = 0.018), and baseline elevated TG (P<0.0001). Other adverse events with their CTCAE grading are reported in The multivariable analysis took into account gender, age, route of HIV transmission, GSS of the backbone therapy, CDC stage C, duration of HIV infection, number of regimen changes, HBV/HCV coinfection, baseline CD4+ T cell counts, HIV-1 RNA, and baseline TG, TC and ALT levels. Only baseline altered ALT  were a significant risk factor of having any level of ALT elevation by week 96 , while the baseline viremia did not influence such trend . The absolute CD4+ T-cell gain was comparable between arms , as was the proportion of subjects maintaining HIV-1 RNA <50 copies/mL at week 96 , 3 , 4 , or >4 . The clinicians’ decision to combine more or less drugs was oriented by the GSS  and the baseline CD4+ T-cell count  introduced at least a new drug class together with RAL, while groups B, C and D this occurred to 33 , 29  and 10 (25%), respectively.The main accompanying drugs were darunavir , lamivudine or emtricitabine , tenofovir , etravirine  and maraviroc . Overall, the accompanying drugs were generally recycled as the least damaged in long treatment histories.In arm A, 10/19 subjects receiving boosted darunavir had RAMs conferring significant loss of sensitivity towards this drug at baseline, in arm B the proportion was 35/58, in arm C 40/83 and in arm D 31/36.Lamivudine/emtricitabine RAMs were present in 42/54 3TC/FTC recipients in arm B, 92/113 in arm C and 35/39 in arm D, tenofovir RAMs in tenofovir-treated patients were 2/12 in arm B, 55/95 in arm C and 25/33 in arm D, and finally etravirine RAMs in etravirine-treated subjects were in 0/1 in arm A, 12/28 in arm B, 7/21 in arm C and 16/30 in arm D.Maraviroc sensitivity was based upon prediction by Enhanced Trofile and the drug could not be administered without this result, so it is considered to be active at baseline in all cases.3 with 7,3% virologic failures, while in darunavir-sparing regimens (n = 117) CD4+ T-cells increased by +236/mm3 on average with a 10% virologic failure rate.Finally, we analyzed the contribution of darunavir to virologic suppression and CD4+ T-cell gain. Darunavir-containing regimens (n = 203) achieved a mean CD4 increase of +223/mmTo our knowledge, this is the largest observational cohort of salvage therapy with raltegravir and the longest follow-up reported to date.The main comparison that can be made is with the BENCHMRK study data at 96 weeks 3 vs + 227/mm3).The ANRS 139 TRIO study showed very good results in a completely different setting, where a brand new regimen composed of etravirine, boosted darunavir and raltegravir acted in the setting of an optimal GSS ( = 3) The main results of the present study are indeed the immunologic gain and the protection from progression to AIDS after 96 weeks of salvage therapy in a consistent cohort (n = 320), representative of numerous centers from Italy. Also, the liver and metabolic toxicity that we observed in our cohort is quite mild and liver enzymes’ elevations were mainly seen in HCV-infected subjects, as reported by Vispo et al The progressive increase in total cholesterol is confirmed in the pilot SHIELD trial (n = 35) Furthermore, confirming a previous smaller experience In treatment-experienced subjects, a raltegravir-including salvage ART achieved and maintained high levels of virologic suppression, with few virologic failures, mostly accompanied by the emergence of resistance-associated mutations. The impact on metabolic and liver function was mild, also because of the presence of relatively low-impact backbone regimens, mostly containing darunavir and/or etravirine and/or tenofovir/emtricitabine. The proportion of deaths could be explained with the advanced stage of HIV-1 infection of the involved subjects.Considering the highly advanced HIV-infection of our cohort, we observed few serious adverse events and deaths; tumors were rare; no other new AIDS-defining events were recorded. The SALIR study provides a consistent glance on safety and efficacy of a raltegravir-based salvage therapy in the long run in a “real world” clinical setting.


The HIV epidemic continues unabated, with no highly effective vaccine and no cure. Each new infection has significant economic, social and human costs and prevention efforts are now as great a priority as global antiretroviral therapy (ART) scale up. Reverse transcriptase inhibitors, the first licensed class of ART, have been at the forefront of treatment and prevention of mother to child transmission over the past two decades. Now, their use in adult prevention is being extensively investigated. We describe two approaches: treatment as prevention (TasP) - the use of combination ART (2NRTI and 1NNRTI) following HIV diagnosis to limit transmission and pre-exposure prophylaxis (PrEP) –the use of single or dual oral agents prior to sexual exposure. Prevention of mother-to-child transmission using NRTI has been highly successful, though does not involve sustained use of NRTI to limit transmission. Despite theoretical and preliminary support for TasP and PrEP, data thus far indicate that adherence, retention in care and late diagnosis are the major barriers to their successful, sustained implementation. Future advances in drug technologies will be needed to overcome the issue of drug adherence, through development of drugs that involve both less frequent dosing as well as reduced toxicity, possibly through specific targeting of infected cells. The HIV epidemic has been devastating in its magnitude and devastation , despiteBy contrast, in the absence of an effective vaccine and/or cure, transmission has continued largely unabated over the last two decades, particularly in sub Saharan Africa, where 67% . CAPRISA3[Clearly, more effective prevention tools are needed. Prevention of mother to child transmission has proved highly effective when implemented efficiently, and serves as a model for prevention using antiretroviral drugs. Antiretrovirals, the cornerstone of HIV treatment, are now being assessed as tools for limiting transmission in two ways: treatment as prevention (TasP) and pre-exposure prophylaxis (PrEP). The potential for TasP to curb the epidemic is being explored following a report showing that transmission amongst discordant couples was reduced by 96% when the HIV infected partner initiated immediate antiretroviral therapy as compared to delaying treatment until a CD4 <250 cells/mm3. Modelin3.In this review, we highlight the achievements of ART in reducing morbidity and mortality, with particular emphasis on reverse transcriptase inhibitors (RTI). We review RTI use in prevention strategies and its anticipated impact on the HIV epidemic. We highlight the potential for drug resistance and the challenge that this presents to implementation of such prevention strategies.NRTI was the first class of antiretrovirals, with the thymidine analogue zidovudine (ZDV) introduced in 1987 . SubsequThymidine analogue resistance usually develops in a stepwise fashion, involving mutations , which increase the drug excision activity of RT ,24. ThesImportant mutations in the RT active site include L74V, K65R and M184V/I. The latter is selected by and confers resistance to 3TC  but incrConnection domain resistance mutations such as G335C/D, N348I, A360I/V, V365I, and A376S have been identified and N348I, in particular, emerges following nevirapine (NVP) exposure [Second-generation drugs in various classes with activity in the face of mutations associated with drug resistance to first generation drugs have been developed . As ABC and TFV are compromised by mutations selected by older NRTI, these drugs are potentially vulnerable in regard to future use in prevention in areas in which ART scale up has occurred.Two currently licensed NNRTI are efavirenz (EFV) and NVP, and both are highly effective when combined with 2NRTI . Both biThe long plasma half-life of NNRTI predisposes them to development of resistance, particularly when a fixed dose tablet is stopped . This maWide scale availability of ART outside industrialized countries has been largely possible through generic production of fixed dose combinations (FDC) and accreditation/quality control by the World Health Organization. Thymidine analogues have featured in the most widely used regimens along with 3TC/NVP with good outcomes ,49.However, NVP interacts with rifampicin-containing tuberculosis therapies and is also associated with potentially serious skin reactions and liver toxicity . Data suRates of viral suppression vary widely across sub Saharan Africa , but alsThe HPTN052 study showed that immediate treatment of HIV-infected individuals results in a 96% reduction of transmission to an uninfected sexual partner in the context of a discordant couple analysis . It is nThe first practical constraint of TasP is that adherence to ART initiated at high CD4 counts in the absence of any clinical illness may not reflect that achieved in HPTN052. A number of studies are underway to address this issue.3[The second constraint is that immediate treatment is frequently not possible as a substantial proportion of patients are diagnosed relatively late during their infection. For instance, epidemiological studies in resource-rich settings reported that 50% of patients are diagnosed with a CD4 < 350 cells/mm3-62. As a3,64.Third, in sub-Saharan Africa there is a problem with retention of patients in clinical care -68. In oAlthough TasP may not completely eliminate the epidemic, modeling studies suggest that ART could be effective in reducing transmission on a population level ,69-71. OThere are legitimate concerns regarding these of agents for co-temporaneous treatment for clinical indications and public health guided prevention efforts. Both efforts would potentially be compromised by rising drug resistance , especiaUse of boosted PI instead of NNRTI as first line could potentially avoid this problem given that viral failure after modern boosted PI is rarely accompanied by the occurrence of resistance mutations ,44. HoweSeveral modeling papers predicted the future prevalence of transmission of drug resistant HIV-1 in sub-Saharan Africa ,72. PhilIn resource-rich settings, implementation of TasP is not expected to result in a substantial increase of drug resistance. In these settings it is standard practice to perform a genotypic resistance before start of treatment, to estimate the plasma HIV-1 RNA load during treatment , and a gOne mathematical model predicted that over a ten-year time span, TasP can reduce the number of new infections by 34% in Los Angeles County and that at the same time multi-drug resistance can almost double from 4.8% to 9.1% . It shouWe do not know how good adherence will be where individuals are treated as part of a TasP strategy. Greater risk taking behavior is possible, with individuals not knowing whether their viral load is suppressed in the absence of point of care viral load testing. Under such a scenario, transmission of drug resistant variants could occur. Although further studies are underway to better evaluate TasP, effects on transmitted drug resistance will be difficult to measure given the long follow-up times that will be required to obtain definitive results.Due to favourable tolerability/toxicity and efficacy profiles, tenofovir will be the most widely used NRTI in first-line therapy ,80, and Multiple clinical trials have been undertaken in resource limited settings using TDF/FTC or TDF alone as PrEP for predominantly heterosexual transmission. Clinical trials reported that use of PrEP reduced the risk of infection by 44-75% -86. Two There is a concern that use of PrEP could result in emergence and transmission of HIV drug resistance . StudiesResistance was not common in the trials and was detected in only nine individuals, of who most had an unrecognized acute infection -86. ImpoAssessing the impact of PrEP on drug resistance will require large-scale epidemiological follow-up studies that are expensive and time-consuming. Mathematical models have therefore been developed which predict the impact of PrEP on the future HIV epidemic and the impact of PrEP on HIV drug resistance . An inteBehavioral issues regarding TasP would also apply to wide scale PrEP, where, in the absence of early detection of incident HIV infection, risk behavior combined with intermittent PrEP could contribute to emergence and transmission of drug resistant variants. Multiple studies are underway to better understand risk behavior in the context of PrEP, though there is likely to be considerable heterogeneity of results, based on geographical location, risk group and calendar time ,101.RT inhibitors have a long and distinguished history in PMTCT. A few years after introduction of ZDV, the landmark ACTG 076 study showed that a three component ZDV regimen reduced MTCT from 25% to 8% in the absence of breastfeeding . TransmiUnfortunately, important challenges have ensued. Firstly, the inherently low genetic barrier to resistance of NVP, in addition to its long half-life, increases the likelihood for selection of drug resistant virus. ZDV/3TC for a week following sd NVP has been shown to reduce the emergence of NVP resistance . A high Secondly, in RLS the disastrous impact of unsafe replacement feeding on child survival on the one hand  and breaThere has been a move from sdNVP to HAART in pregnant and breastfeeding women to limit vertical transmission, with prolonged ART following weaning. However, in a South African study, pregnant women were substantially more likely to be lost to follow-up than non-pregnant women for both pre-ART care and while on ART ,137, andData suggest that treatment as prevention and PrEP may be used to prevent transmission of HIV, and these could be powerful tools in curbing and reversing the global epidemic. The experience of PMTCT provides evidence for the ability of governments and health systems to target and treat a risk group with single dose ART with the aim of preventing new HIV infections. WHO option B + (life-long ART for pregnant HIV infected women as opposed to limited duration therapy) is now recommended, and future studies will address its success. These data will inform the feasibility of TasP. Adherence has been identified as a key barrier to successful implementation of both strategies, and sustained public health messaging will likely be crucial for success. This is a major research priority.It should be borne in mind that there are long-term toxicities associated with current and probably future ART . EFV is The field is also in need of new classes for treatment that do not overlap with prevention strategies. Basic science can help identify new targets, for example capsid destabilisation/stabilisation agents, maturation inhibitors and antagonists of viral accessory genes. As new agents would need to be cheap in order to be widely available, dose optimization should be a priority for future clinical trials of new antiretrovirals. Critically, basic science can also assist in the development of long acting agents to address the adherence issues that pervade both therapeutic and preventive HIV strategies. One promising approach is nanoparticle technology that might incorporate RTI as well as other agents ,143.The authors declare that they have no competing interests.RG, SM, MWDvdV, MW wrote the manuscript. All authors read and approved the final manuscript.


Citizenship of patients was as follows: 72.6% Italians, 9.3% South Americans and 7.3% Rumanians. Heterosexuals, Homo-bisexuals, Intravenous Drug Users accounted for 58.1%, 24.0% and 8.8% of patients, respectively. Phylogenetic analysis indicated that 70% of sequences clustered in 27 transmission networks. Two distinct groups were identified; the first clade, encompassing 56 sequences, included all Rumanian patients. The second group involved the remaining clusters and included 10 South American Homo-bisexuals in 9 distinct clusters. Heterosexual modality of infection was significantly associated with the probability to be detected in transmission networks. Heterosexuals were prevalent either among Italians (67.2%) or Rumanians (50%); by contrast, Homo-bisexuals accounted for 71.4% of South Americans. Among patients with resistant strains the proportion of clustering sequences was 57.1%, involving 14 clusters (51.8%). Resistance in clusters tended to be higher in South Americans (28.6%) compared to Italian (17.7%) and Rumanian patients (14.3%). A striking proportion of epidemiological networks could be identified in heterosexuals carrying F1 subtype residing in Italy. Italian Heterosexual males predominated within epidemiological clusters while foreign patients were mainly Heterosexual Rumanians, both males and females, and South American Homo-bisexuals. Tree topology suggested that F1 variant from South America gave rise to the Italian F1 epidemic through multiple introduction events. The contact tracing also revealed an unexpected burden of resistance in epidemiological clusters underlying the need of public interventions to limit the spread of non-B subtypes and transmitted drug resistance.About 40% of the Italian HIV-1 epidemic due to non-B variants is sustained by F1 clade, which circulates at high prevalence in South America and Eastern Europe. Aim of this study was to define clade F1 origin, population dynamics and epidemiological networks through phylogenetic approaches. We analyzed  HIV-1 is characterized by a high evolutionary rate that led to the establishment of different viral lineages. Group M is the most widespread group representing the main source of the HIV/AIDS pandemic. Based upon latest classification, it is further divided into nine subtypes , six sub-subtypes , an increasing number of Circulating Recombinant Forms (CRFs) (of whom 52 are known at present) and an undetermined number of Unique Recombinant Forms (URFs) As a consequence of migratory waves from low-middle income areas, non-B variants entered and circulates in almost all previously B-restricted European countries HIV-1 F1 subtype was firstly isolated in the Democratic Republic of Congo, where it accounts for a small percentage (<5%) of diagnosed cases at present In Europe, subtype F1 has a massive prevalence in Rumania (>70%), where it spread among adults few years before its striking emergence among institutionalized children around 1989 The origin of HIV-1 non-B infections in Italy has been not thoroughly investigated by specific population studies Since HIV-1 gene sequencing for drug-resistance monitoring has been widely adopted, new phylogenetic methods have been developed which allow to infer evolutionary dynamic from sequence data www.istast.it), indicates that Rumanians with about 1 million of persons, account for the main foreign community, while no information are available for specific countries of South America that contribute to immigration with about 350,000 presences. The aim of this study was to reconstruct the epidemiological features of F1 subtype in Italy by analyzing data from a nation-wide collaboration. Furthermore, we identified the epidemiological networks and established the relationships among the main demographic variables such as citizenship, route of infection and gender.The burden of immigration in Italy, reported by the National Institute for Statistics (ISTAT)  and in a dedicated database in Milan. They provided an informed written consent to have their anonymized data stored on a central server and used for academic studies. Informed consent statements are available at each clinical Centre. Demographic data  were collected by physicians from patient interviews and recorded in the databases together with virological, immunological and treatment information. The authors were exempted from approval by Ethic Committee of ‘L. Sacco’ Hospital because no human experimentation and no investigation of host genetics were conducted. The study was conducted in accordance with the 1964 Declaration of Helsinki and the ethical standards of the Italian Ministry of Health.Patients received a genotypic resistance assays at diagnosis or prior the start of therapy or at treatment failure. Tests were performed at local Services of participating clinical Centers. For each patient included in the analysis, the earliest available HIV-1 genotype was evaluated. All patients are included in the Antiretroviral Resistance Cohort Analysis  model of nucleotide substitution, a proportion of invariant sites, and gamma distributed rates among sites http://beast.bio.ed.ac.uk) implementing the GTR+Invariant+Gamma model 6 generations, and sampled every 30,000 steps. Convergence was assessed on the basis of the effective sampling size (ESS) after a 10% burn-in using Tracer software version 1.5 (http://tree.bio.ed.ac.uk/software/tracer/). Only parameter estimates with ESS’s >200 were accepted. Uncertainty in the estimates was indicated by 95% highest posterior density (95% HPD) intervals. Trees were summarized in a target tree by Tree Annotator program included in the Beast package choosing the tree with the maximum product of posterior probabilities (maximum clade credibility) after a 50% burn-in.The dated trees, evolutionary rates and population growth were co-estimated by using a Bayesian MCMC approach .Categorical variables were summarized as frequencies and comparisons between groups were performed using the χWe studied 343 HIV-1 F1 infected patients with sequence data obtained from 1998 to 2009. According to country of origin, 72.6% (n = 249), 9.3% (n = 32), 7.3% (n = 25) and 10.8% (n = 37) of patients were Italians, South Americans, Rumanians and other/unknown, respectively. Patients from South America had Brazil (n = 25), Paraguay (n = 3), Argentina (n = 2) and Columbia (n = 2) as country of origin.p<0.001).Among patients with known modality of infection (n = 217), heterosexuals (HETs), homo-bisexuals (HO-BISEXs), intravenous drug users (IDUs) and others accounted for 58.1% (n = 126), 24.0% (n = 52), 8.8% (n = 19) and 9.2% (n = 20) of patients, respectively. The heterosexual contacts were significantly more frequent among Italians with known risk factor  and Rumanians , than among South Americans, who were mainly HO-BISEXs  .Overall, three out of four subjects were males ; the male to female ratio was about 4∶1, 7∶1 and 1∶1.5 among Italians, South Americans and Rumanians, respectively. Males were predominant among Italians and South Americans but not among Rumanians (87.5% and 79.8% The median age was 38 years (min-max 10–73 years); HIV-1 viral load levels and CD4 cells ranged from 2 to 6 log copies/ml and from 3 to 1.990 cells/µL (medians 4.5 log copies/ml and 331 cells/µL), respectively.The Bayesian tree of HIV-1 F1 sequences is shown in The epidemiological characteristics of all the patients and of the individuals included in the epidemiological networks are reported in p = 0.04). No association was detected between gender and clustering probability.The heterosexual modality of infection was significantly associated with the probability to be detected in networks . As a consequence, a significant difference could be detected for gender distribution among distinct citizenships in networks; while males were prevalent among Italians (79.2%) and South Americans (80%), females accounted for 60% of Rumanians (p<0.001).Within the epidemiological networks, heterosexual risk factor was predominant among Italian  and Rumanian patients , whereas homo-bisexual contacts were prevalent among South Americans and accounted for 5/7 (71.4%) of these patients. Therefore, a different distribution of modality of infection was present in clusters according to citizenship  for the HIV-1 F1 pol sequences. On the basis of this value we estimated the Time of Most Recent Common Ancestor (TMRCA) of the root and every internal nodes of the tree.In order to obtain a time scaled phylogeny, we estimated the evolutionary rate based on known sampling date of our sequences. The comparison of the different models by BF test, shown in The dated tree is shown in Nine smaller clades , including a mean of 5 patients (range 3–12 isolates), involved a total of 34 and 10 patients from Italy and South America, respectively. Of note, 8 of 10 South American subjects were from Brazil. Each clade included one patient from the latter geographical area with the exception of clade #25 that encompassed two Italians and two South Americans. HO-BISEXs were 40% of clustering subjects in this group, including 100% of South American patients .The remaining 17 clusters  included only Italian isolates. HETs and HO-BISEXs represented 75.4% and 24.6% of these clustering patients, respectively, of which males were largely prevalent (81.3%).p<.0001). Median TMRCA estimates of different clusters are reported in The mean TMRCA estimation for the tree root was 46.4 years ago (95% HPD 34–106 years ago) implying an origin of the HIV-1 F1 subtype back to the year 1963. Most of the clades originated more than 19 years ago (85.2%), being the cluster with Rumanians the eldest  could be identified in our study. A relevant role of sexual networks was highlighted as about 1 of 3 heterosexuals of which 67.7% were man, and about 2 of 3 homosexuals/bisexuals were involved in epidemiological chains. These data extend previous results that demonstrated a high connectivity of contact networks when analyzing transmission events occurring by sexual contacts Different modalities of infection in clusters were linked to country of origin. Italian HET males predominated within epidemiological clusters while foreign patients were mainly HET Rumanians, both males and females, and South American HO-BISEXs. The high frequency of Italian HET males and South American HO-BISEXs in epidemiological networks indicated that homosexual or bisexual intercourses have lead to a relevant circulation of F1 subtype in Italy. Despite some reports indicated a partial control of HIV-1 transmission in male to female transsexuals living in Italy, due to condom use Phylogenetic analyses are sharp tools to trace potential contact chains, analyze their structure, date their occurrence and correlate them with citizenship risk behaviors and gender. The tree topology indicated that all Rumanian patients segregated in a single clade, including only few Italian individuals, thus suggesting a limited role for Rumanian F1 variant spread among Italians. In contrast, F1 variant from South America had given rise to the Italian F1 epidemic through multiple penetration events, as several independent entry occurrences involved South American and Italian individuals over time.Some limitations could have affected our work. Firstly, the ARCA database, despite its national coverage and representativeness, like other any other observational database, do not guarantee full records of demographic, clinical and immuno-virological parameters. The relative lack or citizenship and risk factor information for some patients could have weakened the strength of the detected associations. Furthermore, it is well established in the phylogenetic field that the sample density warrants sharp data leading to reliable reporting of clusters. Nevertheless, the frequency of contact networks was exceedingly high in our national cohort despite the probable underestimation related to incomplete data due to intervening individuals not sampled.As the time-dependent phylogeny is calibrated in calendar years, we were able to estimate when most of the transmissions in each clusters and sub-clusters occurred. Information from dated phylogeny indicated that the entry of F1 clade from Rumania occurred within a long space of time (from 11.8 to 68 years ago) and an internal order of transmission could be defined with high confidence.A more recent entry could be established for the South American variant, as it occurred likely around 1988 (range: 1974–1992).We lately established that the spread of F1 subtype among non-B clades was apparently favoured and accompanied with a high frequency of detection of BF genetic mosaic forms in Italy Overall, our findings provide new insights to strengthen the surveillance system for HIV-1 and the planning of prevention programs. This study provides sharp information about the association of F1 subtype with geographic origin as well as the sexual routes of transmission among patients living in Italy and carrying such variant. Furthermore, previous data regarding resistance to antiretrovirals in F1 strains circulating in Italy indicate a remarkable frequency of transmitted drug resistance in F1 subtype infected population Figure S1Starting tree obtained with MrBayes program showing 27 identified clusters (grey colour).(TIF)Click here for additional data file.Table S1Bayes factor comparison of the parametric and Bayesian skyline plot (BSP) models.(DOC)Click here for additional data file.


Among 1207 (80.1%) first-line participants with viral load measured, HIV RNA was <400 copies/ml in 963 (79.8%), 400–999 copies/ml in 37 (3.1%), 1,000–9,999 copies/ml in 110 (9.1%), and ≥10,000 copies/ml in 97 (8.0%). The proportion with HIV RNA <400 copies/ml was slightly lower  in CDM (76.3%) than in LCM (83.4%). Among 252 (64.8%) second-line participants with viral load measured (median 2.3 years after switch), HIV RNA was <400 copies/ml in 226 (89.7%), with no difference between monitoring strategies. Low switch rates and high, sustained levels of viral suppression are achievable without viral load or CD4 count monitoring in the context of high-quality clinical care.In contrast to resource-rich countries, most HIV-infected patients in resource-limited countries receive treatment without virological monitoring. There are few long-term data, in this setting, on rates of viral suppression or switch to second-line antiretroviral therapy. The DART trial compared clinically driven monitoring (CDM) versus routine laboratory (CD4/haematology/biochemistry) and clinical monitoring (LCM) in HIV-infected adults initiating therapy. There was no virological monitoring in either study group during follow-up, but viral load was measured in Ugandan participants at trial closure. Two thousand three hundred and seventeen (2317) participants from this country initiated antiretroviral therapy with zidovudine/lamivudine plus tenofovir (n = 1717), abacavir (n = 300), or nevirapine (n = 300). Of 1896 (81.8%) participants who were alive and in follow-up at trial closure (median 5.1 years after therapy initiation), 1507 (79.5%) were on first-line and 389 (20.5%) on second-line antiretroviral therapy. The overall switch rate after the first year was 5.6 per 100 person-years; the rate was substantially higher in participants with low baseline CD4 counts (<50 cells/mmISRCTN13968779  Most resource-limited countries have adopted a public health approach to anti-retroviral therapy (ART) for the treatment of HIV infection in which the public sector provides a single first-line regimen, with alternative substitute drugs as required, and a standard second-line therapy for those who fail first-line When viral load is not routinely monitored some patients may experience periods of prolonged undetected viraemia, which has several potential negative consequences. First, long delays in switching therapy may place the patient at increased risk of opportunistic infections although regular CD4 monitoring should mitigate against this However, these concerns clearly need to be balanced against the critical point that in any financially-constrained healthcare system facing static or diminishing funds for HIV/AIDS programmes, resources directed towards laboratory testing mean that fewer patients in need of treatment are able to receive it The debate on viral load monitoring in resource-limited settings has been conducted with remarkably few relevant data to inform it. Here we report cross-sectional viral load results after five years on ART among Ugandan patients in the DART trial, where clinical management  was based on clinical symptoms with or without access to CD4 counts in the absence of real-time viral load monitoring 3, enrolled from three clinical centres in Uganda and one in Zimbabwe between January 2003 and October 2004 DART  was an open randomised trial in ART-naive, symptomatic HIV-infected adults with a CD4 count ≤200 cells/mmAlthough there was no real-time viral load monitoring in DART, Ugandan participants were eligible for a viral load test under the national programme shortly after trial closure. However, participants who were enrolled in either OHFS or SARA were ineligible since viral load was measured (retrospectively) at specific time points as part of the protocol of these sub-studies. To increase the number of available measurements and to decrease potential bias, we included viral load results from these two sub-studies that coincided with the testing done within the national programme (January 2009 to April 2009). All viral load assays were done in two centres: Joint Clinical Research Centre, Kampala  and the Infectious Diseases Institute, Mulago . Viral suppression was defined as HIV RNA<400 copies/ml (i.e. the higher of the two LLDs).3 in the LCM group only First-line ART regimens comprised co-formulated zidovudine (ZDV)-lamivudine (3TC) plus either tenofovir (TDF), abacavir (ABC), or nevirapine (NVP) All second-line ART regimens included the boosted protease inhibitor (PI), lopinavir/ritonavir (LPV/r). The nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and/or non-nucleoside reverse transcriptase inhibitors (NNRTI) prescribed in combination with LPV/r were allocated according to the OFHS randomisation schedule if the individual participated in this sub-study, or otherwise at the discretion of the patient's clinician The probability of switching to second-line ART by trial closure was examined by multivariate logistic regression analysis, including a priori selected baseline predictors . A simplified version of this model, combining the two triple NRTI regimens  and excluding non-significant covariates (P>0.05), was used to estimate absolute probabilities (by converting from odds) of switching for combinations of variables. Logistic regression analysis was used to examine if the absence of a viral load measurement was related to any of the baseline variables or last available CD4 count. Logistic regression analysis, applied separately to participants on first-line and second-line ART, was similarly used to examine predictors of viral suppression at trial closure, adjusting for duration of the specific line of ART. Estimates of the overall prevalence of viral suppression (i.e. combining first-line and second-line regimens) were derived using weighted averages to account for the relative under-representation of participants on second-line ART. All analyses were performed using STATA (version 12.0).The study was approved by the Uganda Virus Research Institute, Entebbe.A total of 2317 Ugandan patients were randomised in DART, of whom 275 died  and 146 were lost to follow-up before the trial closed . The fol3) compared with the highest group (150–199 cells/mm3).The switch rate to second-line ART was low (5.6 per 100 person-years after the first year) and by trial closure only 389  patients had switched to second-line therapy . From mu3, these probabilities were remarkably low, with a minimum value of 3.1% .The predicted probability of switching to second-line ART by five years was calculated for all combinations of significant baseline predictors; estimates are shown separately by initial ART regimen (triple NRTI or NNRTI-based) . Apart fThe source and number of viral load measurements are shown in A viral load measurement at trial closure was available on 1207 (80.1%) patients on first-line ART. HIV RNA was <400 copies/ml in 963 (79.8%) patients, 400–999 copies/ml in 37 (3.1%), 1,000–9,999 copies/ml in 110 (9.1%), and ≥10,000 copies/ml in 97 (8.0%). The frequency of viral suppression (HIV RNA<400 copies/ml) was slightly lower in CDM (76.3%) than in LCM (83.4%), a difference of 7.1% (95% CI 2.5–11.5%). This was accompanied by a shift towards a higher proportion of patients with HIV RNA ≥10,000 copies/ml in CDM  . MultivaA viral load measurement at trial closure was available on 252 (64.8%) patients on second-line ART, at a median  of 2.3  years after switching. HIV RNA suppression was even higher than among patients on first-line ART: <400 copies/ml in 226 (89.7%) patients, 400–999 copies/ml in seven (2.8%), 1,000–9,999 copies/ml in 11 (4.4%), and ≥10,000 copies/ml in eight (3.2%) . The disCombining results on patients on first-line and second-line ART, an estimated 81.8% (95% CI 79.8–83.7%) of patients were virologically suppressed . StratifIn this cross-sectional analysis of patients who did not receive real-time viral load monitoring, an estimated 82% had HIV RNA<400 copies/ml at an average of five years after ART initiation. An impressive 79% of patients were still on first-line ART at trial closure – thus, the high prevalence of viral suppression was not explained by frequent switching to second-line ART, which included a highly potent boosted PI. These findings are an important contribution to the debate on laboratory monitoring strategies in resource-limited settings.We identified several factors associated with the rate of switching to second-line ART and/or the prevalence of viral suppression among patients who remained on first-line ART. The factors with the strongest effects were monitoring strategy, ART regimen, and baseline CD4 count.LCM was associated with a significantly higher rate of switch to second-line therapy as a change in regimen could be triggered by a clinical event or a low CD4 count compared with clinical events only in the CDM group. Consequently, among participants on first-line ART, episodes of viraemia are likely to have been more prolonged in CDM, as evidenced by a lower prevalence of viral suppression at trial closure in this group.A distinctive feature of DART was the use of first-line triple NRTI regimens . The outcomes for the two NRTI regimens, ZDV/3TC/TDF and ZDV/3TC/ABC, were broadly similar. However, this is not a randomised comparison, and the large change in the odds ratio (from 0.83 to 1.29) in the multivariate analysis of switch to second-line ART is suggestive of confounding, underling the need for cautious interpretation. The findings on patients who initiated ART with ZDV/3TC/NVP are likely to be of most interest as this has been one of the most frequently used drug combinations worldwide. Patients on this regimen had a significantly lower switch rate to second-line ART and, consistent with week 48 data, better virological outcomes 3 remained on their first-line regimen at the end of follow-up , it is likely that the overall proportion of patients who switched to second-line ART would have been substantially lower.The effect of baseline CD4 count on the durability of first-line ART was remarkably strong, and up to 97% of patients with a value between 150–199 cells/mmollow-up . The mucMost information on viral load outcomes in resource-limited settings have been reported from treatment programmes that utilised real-time viral load monitoring. In a large meta-analysis , McMahon and colleagues estimated a pooled prevalence of viral suppression of 84%  after one year of ART DART also compares favourably with other published studies that examined response to second-line ART. A recent inclusive meta-analysis of randomised and observational studies in resource-limited settings estimated that the cumulative pooled proportion of patients with virological failure after two years of second-line ART was 27% (based on five studies) and 38% after three years of second-line ART (three studies) One plausible explanation for the impressive clinical and virological outcomes in DART is that the clinical care received by DART participants was generally superior to that received in routine treatment programmes, enabling high levels of adherence achievable, and that widespread viraemia is not an inevitable consequence of not using viral load monitoring, as has been predicted Our study has several limitations. First, as discussed above, the high quality clinical care received by DART participants implies that the results may not be widely generalizable In principle, the best evidence on the value of viral load monitoring should come from randomised trials of monitoring strategies that included a viral load monitoring arm . Of the three published studies with such a study design to date, none discerned any effect of viral load monitoring on clinical outcomes, but the relatively small study sizes and short follow-up  limits their relevance to the debate on the role of viral load monitoring The cost-effectiveness of viral load monitoring has mainly been studied in several computer simulation models New WHO guidelines issued in July 2013 include a number of important changes to previous guidelines, including a recommendation that viral load is the preferred monitoring approach to diagnose and confirm ART failure


We screened 735 HIV-infected patients in Switzerland with unexplained alanine aminotransferase elevation for hepatitis E virus (HEV) immunoglobulin G. Although HEV seroprevalence in this population is low (2.6%), HEV RNA can persist in patients with low CD4 cell counts. Findings suggest chronic HEV infection should be considered as a cause of persistent alanine aminotransferase elevation. Unexplained liver enzyme elevation is frequently encountered in HIV-infected persons  history of >2 consecutively elevated ALT values (>60 IU/L); and 3) negative for both hepatitis B antigen and hepatitis C virus antibody. Serum HEV immunoglobulin (Ig) G was detected by an enzyme immunoassay (EIA), the MP Diagnostics HEV ELISA Kit . Positive samples were retested, and only those with a repeated positive result were considered positive. HEV RNA was determined in plasma by real-time PCR of a 189-bp product located in the open reading frame 2 region. Strains were sequenced and compared with reference HEV strains (GenBank) as described earlier  levels. Patients were eligible if they fulfilled the following criteria: 1) For all patients meeting inclusion criteria, HEV serology was performed on the most recently stored blood sample. When available, for patients with positive serologic test results, HEV serology and blood HEV real-time PCR were performed on plasma samples stored 3 months before the first documented elevated ALT value. In these patients, HEV PCR was also performed on samples stored at the time of, and 3 months after, the first elevated ALT level.3 at the time of initial ALT elevation. To characterize more fully the patients with positive PCR, additional serologic testing was performed by using EIAgen HEV IgG and EIAgen HEV IgM kits .To exclude occult infection, HEV PCR analysis was also performed on all available samples from IgG-negative patients with <150 CD4 cells/mm3 were 4.7 times more likely to be HEV positive compared with those with lowest CD4 counts <100 cells/mm3  before the first ALT elevation. Seroconversion from a prior negative to a positive result was found in 5 of these 16 patients (31.3%). There were no significant differences in ALT values , CD4 count, or HIV viral load of those who exhibited HEV seroconversion compared to those who did not.3 (5%) and a normal ALT level. ALT level increased 1 month later, but HAART was continued because the patient was asymptomatic. The patient refused a liver biopsy. Real-time PCR for HEV RNA was performed between August 2001 and December 2004. HEV PCR became negative for the virus when the CD4 count reached 83 cells/mm3 (17.5%), and ALT levels became normal 6 months later, when the CD4 count exceeded 100 cells/mm3. Real-time PCR for HEV RNA was positive in 1 of the 19 HEV IgG–positive patients (genotype 3b), with presence of HEV RNA over a 24-month period, while HEV IgG remained negative for the first 12 months. This 46-year-old white man, who had sex with men (MSM) and received a diagnosis of HIV infection in June 2001, started highly active antiretroviral therapy (HAART) with a CD4 count of 34 cells/mm3) at the time of initial ALT elevation. Real-time PCR for HEV was positive in 1 HEV IgG–negative patient over a 5-month period  were identified between October 2005 and March 2006. Additional serologic analysis revealed the presence of IgM over a 7-month period following first positive real-time PCR but absence of IgG seroconversion.Given the prolonged viremia and late seroconversion (IgG) observed in patients with low CD4 counts, we performed real-time PCR for HEV RNA on 135 samples obtained from 54 HEV IgG–negative patients with low CD4 counts (<150 cells/mmh period . This 59This study of participants of the Swiss HIV Cohort Study shows a 2.6% (19/735) seroprevalence of HEV in HIV-infected patients without HBV or HCV infection with a history of persistently elevated ALT level. We did not find higher HEV prevalence in subgroups previously considered at higher risk of HEV infection such as MSM, injection drug users, and prisoners. The prevalence in Switzerland is lower than in other European countries and the United States .We observed anti-HEV seroconversion in 5 patients, including one with prolonged HEV RNA (>24 months). In this single patient, who had a very low CD4 count, seroconversion (IgG) was delayed until immune reconstitution occurred. As the CD4 count exceeded 100 cells/mme normal . AccordiThis study faced 2 common limitations regarding HEV diagnosis and comparability of seroprevalence: lack of an approved algorithm and variability of serologic tests in terms of sensitivity and specificity. According to a recent comparative study, the test used in our study may underestimate seroprevalence because of its inherent lower sensitivity  may be delayed or not occur. When investigating unexplained, elevated ALT level in HIV-infected patients, we propose that HEV infection should be considered.