The profile of immune activation markers in tuberculosis and HIV-infected patients is already known. The impact of simultaneous infections on the immune parameters is still not fully explored.3) and group 2 (CD4>200 cells/mm3). These parameters were measured at the time of HAART initiation (started about 30 days after the onset of TB treatment) and at the follow-up visits after 30, 60, 90 and 180 days. Trajectories were estimated using least squares estimates of the coefficients of a restricted cubic spline function in time after adjusting for subject effects, bootstrapping it 500 times.We conducted a prospective study to estimate trajectories of activated T cell subsets and the profile of anti- and pro-inflammatory cytokines in a group of HIV-TB individuals, previously naïve for HAART, recruited from a randomized clinical trial during TB treatment and first antiretroviral therapy with efavirenz. Patients were evaluated according to the immunosuppression levels at baseline as group 1 . Except for TNF-α and IL-2 discrete variations were observed for the other cytokines. Differences in the trajectories of these parameters were observed for groups 1 and 2. Higher values of IFN-γ, IL-2, IL-6 and IL-10 were observed for group 1 from the baseline to two months after treatment initiation, whereas reduced levels of TNF-α were observed for this group between 60 and 120 days of HAART.Increase of CD4 T cell counts and suppression of HIV viral load were observed for all patients under HAART and TB treatment. Descendent trajectories were observed for the activated CD8+ T cell counts, and control viral replication and immune activation parameters over time.Independent of the immunosuppression profile at baseline, HIV-TB patients under HAART were able to recover the CD4 TNF also plays a pivotal role in HIV-1 pathogenesis, being found in increased levels in acute and chronic HIV-1 infections 3) for the trends of such parameters.Although the cytokines are activated as regulatory networks, the secretion of individual cytokines may provide limited information. In the present study, we conducted a prospective analysis to estimate trajectories of activated T cell subsets and the profile of anti- and pro-inflammatory cytokines produced in a group of TB-HIV individuals, previously naïve for HAART, in TB-HIV patients during treatment for both diseases and the impact of the CD4 immunosuppression levels at baseline . The exclusion criteria for the trial included: resistance to rifampicin, baseline (grade 3) hepatic enzymes elevation or CD4 counts above 350 cells/mm3 for patients with pulmonary tuberculosis. Therapy was adjusted to replace isoniazid or other antimycobacterial drug, if resistance was detected.We conducted a prospective study in a cohort of HIV positive patients with tuberculosis (TB) at the Tuberculosis Clinic of the Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, from April 2006 to July 2011. Patients were recruited from a randomized clinical trial “Randomized Clinical Trial to Assess the Efficacy and Safety of Concomitant Use of Rifampicin and Efavirenz 600×800 mg” registered at clincaltrials.gov (NCT00533390). For this trial patients were eligible if they had 18 years or more, were naïve for HAART, had TB diagnosis based on clinical signs and symptoms suggestive of tuberculosis, the radiological aspects were compatible, and other opportunistic diseases were excluded, even if specimen baciloscopy was negative. Eligibility was confirmed if the Patients were selected to the immune reconstitution study if they performed at least 3 of the scheduled visits.HAART was initiated 30±10 days after initiation of tuberculosis therapy and included two reverse transcriptase nucleoside analogs plus efavirenz (600 or 800 mg). Follow-up visits were performed at 30, 60, 90 and 180 (30 days after the end of TB treatment) days after initiation of HAART. Blood samples were collected at baseline (15 days before HAART initiation) and at all follow-up visits to study the plasmatic immune profile. Tuberculosis and HIV therapies were prescribed according to the Brazilian Ministry of Health recommendations + T cell counts at baseline, as follows: group 1 (<200 cells/mm3) and group 2 (>200 cells/mm3). The CD4<200/mm3 threshold was chosen to meet the CDC laboratory criteria for AIDS (1994).In order to verify the association of the immune activation markers with the immune suppression profile, the patients were further distributed in two groups, according to CD4Patients were also assessed for the onset of immune reconstitution inflammatory syndrome (IRIS) defined as a documented worsening of signs or symptoms of TB during appropriate anti-tuberculosis treatment and after the initiation of anti-retroviral (ARV) therapy, not explained by any other disease or by an adverse effect of drug therapy This protocol was approved by the Instituto de Pesquisa Clínica Evandro Chagas Ethical Board (CAE: 0052.0.009.000-10), affiliated to the Brazilian National Ethics Council (CONEP) and all patients who accepted to participate in the study signed an informed consent form.+ T cell counts and viral load determinations were performed according to the Brazilian guidelines as part of the routine exams for HIV positive patients. BD Multitest monoclonal antibodies specific for CD45+, CD3+, CD4+ and CD8+, respectively conjugated to PerCP, FITC, APC and PE were used to determine the absolute counts of CD4 and CD8 T cell subsets according to the manufacturer . Samples were evaluated using a FACSCalibur and analyzed with Multiset software . Plasma samples were obtained by centrifugation within 4 hours of blood collection and aliquots were stored at −70 C freezer until use for viral load and cytokine measurents. Quantification of the plasma viral load was determined for all HIV/−TB patients at each visit , assuming the lower detection limit (LDL) of 80 copies/ml.CD4+ FITC/CD38+ PE or CD3 FITC+/HLA-DR+ PE. After 30 min incubation, the red blood cells were lysed (Becton Dickinson lysing solution), the lymphocytes were washed once with PBS pH 7,2 and fixed with (300 ul) of 1% paraformaldehyde in PBS; samples were evaluated in the FACSCalibur flow cytometer (BD Biosciences). In general, 10.000 events were collected in the lymphocyte gate by forward scatter (FSC) and side scatter (SSC) using CellQuest™ Software (BD Biosciences). CD3 or CD8 T cell populations were determined in the lymphocyte population based on the forward scatter vs fluorescence dot plot. HLA-DR and CD38 percentages were defined in CD3 and CD8high T cell populations, respectively.Whole blood EDTA (50 ul) was incubated with a combination of antibodies included in the BD Simultest  as follows: anti-CD8Plasma level of cytokines  were measured using the Cytometric Beads Array kit  using a FACSCalibur cytometer. The reagents and samples were prepared according to the manufacturer’s instructions.3 (group 1) and >200 cells/mm3 (group 2). Mann whitney test, as well as Fisher’s exact and Chi-square tests were employed for this study and a P value <0.05 was considered significant.Viral load and immunological parameters  were assessed for the HIV-TB patients along the follow-up treatment. The patients were further distributed in two groups according to CD4 T cell counts at study entrance: in <200 cells/mmTrajectories of markers were estimated as follow: least squares estimates of the coefficients of a restricted cubic spline function in time after adjusting for subject effects through the use of subject dummy variables. Then the fit was bootstrapped 500 times, by treating time and subject as random variables. Samples are taken jointly from the time, subject, and response vectors to obtain unconditional distributions.The bootstrap sampling was based on samples with replacement from subjects rather than from individual data points. In other words, either none or all of a given subject’s data will appear in a bootstrap sample. This cluster sampling takes into account any correlation structure that might exist within subjects, so that confidence limits are corrected for within-subject correlation. Assuming that ordinary least squares estimates, which ignore the correlation structure, are consistent  and efficient (which would not be true for certain correlation structures or for datasets in which the number of observation times vary greatly from subject to subject), the resulting analysis will be a robust, efficient repeated measures analysis for the one-sample problem. The method of Tibshirani was used to obtain simultaneous confidence sets for the set of coefficients of the spline function as well as the average intercept parameter (over subjects).Statistical analysis was done using the SPSS Software version 17.0 and for the graphics R-project 2.15.3 software with package Hmisc.+ T cell counts) of the remaining 54 HIV-TB patients at baseline are shown in + T cell count and viral load were 112 cells/mm3 and 5.02 log10copies/ml, respectively, with predominance of heavily immunosuppressed patients (61%). In spite of that, few cases of IRIS were observed in our cohort (11%). Moreover, the HIV-TB patients included in this cohort were treated with two distinct doses of efavirenz (600 mg or 800 mg/day). As no differences were observed for the virological and immunological parameters here analyzed, they were considered as a single group (data not shown).In total, seventy HIV-TB patients agreed to participate in the study. Sixteen patients were thereafter excluded due to non-adherence to the HIV treatment (n = 7) or for attending less than 3 follow-up visits along treatment (n = 9). Demographic, clinical and laboratory data  or levels of immunosuppression of the HIV-TB patients. Moreover, Bal et al., evaluating in vitro stimulated T cell response to mitogens, found lower IFN-γ, TNF-α and IL-2 cytokine levels when comparing TB positive patients, HIV positive or HIV-TB co-infected ones, suggesting that HIV could be responsible for negatively regulating both cytokines Cytokines are important immunomodulating agents of the immune system. IFN-γ and TNF-α have key roles in HIV-TB infection and are associated with inflammatory granuloma organization, immune protection against intracellular mycobacteria and cell mediated immunity IL-6 plays a role in the development of the inflammatory response during immune restoration and can act as a marker for persistent immune activation + T cells counts as <200 (group 1) and >200 cells/mm3 (group 2).We further hypothesized that the immunosuppression level of HIV-TB patients at baseline could influence the immune activation profile and the cytokine production in response to the tuberculosis treatment and HAART. For that reason, we evaluated those parameters in HIV-TB patients along both treatments with emphasis in their immunosuppression status according to the CD4+/HLA-DR+ and CD38+/CD8+ T cell activation profiles were observed for group 1 after HAART initiation, followed by a slight reduction of these immune activation markers along the HIV-TB treatment for group 1. This finding suggests that patients with severe immunosuppression under HAART initially have a cellular activation more intense than those included in group 2, independent of viral control. If the presence of Mycobacterium tuberculosis antigens is also playing a role to support this immune activation is still an open question. Two recent studies from a prospective HIV-tuberculosis cohort treated for tuberculosis, conducted in Uganda showed that TB therapy strongly reduced the expression of CD38 and/or HLA-DR expression on CD8 T while for CD4 cells the reduction in the expression of these markers was less pronounced. Similar results were observed for HIV-tuberculosis patients receiving antiretroviral therapy concurrent with tuberculosis treatment, highlighting the relationship among M. tuberculosis, immune activation and circulating T cell CD8+/CD38+ cells Although no differences in the T cell activation markers were observed at baseline for the patients included in groups 1 and 2, higher CD3Leishmania antigens can increase the degree of immune activation in individuals coinfected with HIV + T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART.In fact, in a previous study from our group we observed that et al.In general differences on the IFN-γ, IL-2, IL-6 and IL-10 levels between the two groups were more evident at baseline and just after the beginning of HAART. Although IL-10 and IL-4 are anti-inflammatory cytokines related to immune suppression et al.3.Distinct profiles of IFN-γ and TNF-α pro-inflammatory cytokines were observed according to the immunosuppression status of the HIV-TB patients included in our study. Indeed, higher IFN-γ levels were observed for the heavily immunosuppressed group from baseline to 60 days after HAART, whereas no variation was observed for the TNF-α levels overtime. Higher levels of plasma IFN-γ were also observed by Subramanyam + T cell median counts observed for that study group (25 cells/mm3 IQR 10–55). The low incidence of paradoxical tuberculosis-associated IRIS observed in our study group was probably due to the high CD4+ counts at baseline (112 cells/mm3 IQR 46–236) and the time point to introduce HAART which was longer than in the Cambodia study. Moreover, genetic background of the two populations could also be implicated in the risk of developing IRIS. No deaths were observed among IRIS patients in the present study. The low number of cases could explain this finding. Previous study of our group and other authors had also shown a low incidence of IRIS deaths Some limitations of the current study should be noted mainly in relation to the low incidence of paradoxical tuberculosis-associated IRIS cases. Indeed, a recent study from Cambodia comparing early versus later start of HAART in adult HIV-TB patients showed a high frequency of IRIS cases, with differences according to the time of HAART introduction, showing significant increase of IRIS in the earlier-HAART group + T cell counts and control of viral replication and immune activation over time. No specific pro and anti-inflammatory cytokine patterns could be observed in our study, although higher IFN-γ levels have been observed in the heavily immunosuppressed group associated to the increase of the CD4+ T cell counts and immune activated T cell subsets early upon HAART initiation.In conclusion, our results indicate that independent of the immunosuppression profile, HIV-TB patients under tuberculosis treatment and HAART are able to recover the CD4


Magnitude and frequency of HIV viral load blips in resource-limited settings, has not previously been assessed. This study was undertaken in a cohort from a high income country  known as AHOD  and another cohort from a mixture of Asian countries of varying national income per capita, TAHOD .Blips were defined as detectable VL (≥ 50 copies/mL) preceded and followed by undetectable VL (<50 copies/mL). Virological failure (VF) was defined as two consecutive VL ≥50 copies/ml. Cox proportional hazard models of time to first VF after entry, were developed.5040 patients (AHOD n = 2597 and TAHOD n = 2521) were included; 910 (18%) of patients experienced blips. 744 (21%) and 166 (11%) of high- and middle/low-income participants, respectively, experienced blips ever. 711 (14%) experienced blips prior to virological failure. 559 (16%) and 152 (10%) of high- and middle/low-income participants, respectively, experienced blips prior to virological failure. VL testing occurred at a median frequency of 175 and 91 days in middle/low- and high-income sites, respectively. Longer time to VF occurred in middle/low income sites, compared with high-income sites (adjusted hazards ratio (AHR) 0.41; p<0.001), adjusted for year of first cART, Hepatitis C co-infection, cART regimen, and prior blips. Prior blips were not a significant predictor of VF in univariate analysis . Differing magnitudes of blips were not significant in univariate analyses as predictors of virological failure . 209 of 866 (24%) patients were switched to an alternate regimen in the setting of a blip.Despite a lower proportion of blips occurring in low/middle-income settings, no significant difference was found between settings. Nonetheless, a substantial number of participants were switched to alternative regimens in the setting of blips. One or a combination of causal factors have been suggested including random biological fluctuation and statistical variation In addition it is possible that blips in the setting of drugs with a low genetic barrier to resistance, such as NNRTI, may have different impact on subsequent risk of VF compared with other classes of drug such as protease inhibitors or integrase inhibitors. Few studies have formally assessed the role of blips on virological outcomes between classes of antiretrovirals Blips could have different significance in settings where HIV virological monitoring occurs less frequently, and because of resource limitations blips may be managed differently in resource-limited, compared with resource-rich, settings. In addition, less frequent virological monitoring in resource-poor settings may result in different interpretations of blips when they are detected. To our knowledge there have been no previous studies examining the significance of blips in resource poor settings.The primary objective of this study was to compare the significance of blips in resource-poor and resource-replete settings. Secondary objectives were to assess the significance of differing magnitudes and frequency of blips in a given year, and the significance of blips with differing definitions of virological failure.25. Sites are stratified into low-, middle- and high-income based on gross national income per capita This was an analysis of patients from the Treat Asia HIV Observational Database (TAHOD) and Australian HIV Observational Database (AHOD). TAHOD is an observational cohort of 17 low- middle- and high-income clinical sites in the Asia and Pacific region, specifically Cambodia, China, Hong Kong, India, Indonesia, Japan, Malaysia, the Philippines, Singapore, South Korea, Taiwan, and Thailand Patients were commenced on cART, with a minimum of 3 antiretrovirals, in accordance with national guidelines and clinician discretion. Core data variables recorded in TAHOD and AHOD include: gender; date of birth; date of most recent visit; HIV exposure; CD4 and CD8 cell counts; HIV viral load; antiretroviral treatment data; AIDS-defining illnesses; hepatitis B virus (HBV) surface antigen status; hepatitis C virus (HCV) antibody status; and date and cause of death.Patient selection and extraction of data occur at data centres of the participating sites. Written informed consent is obtained from all patients at the time of enrollment. TAHOD and AHOD data is aggregated at The Kirby Institute, University of New South Wales. Ethical approval for the study was obtained from the University of New South Wales Ethics Committee. Each site also obtained approval from their local ethics committee. As part of the approved AHOD protocol, all data is formally owned by the contributing clinical sites, and is collated on their behalf at The Kirby Institute. Data is transferred to the Kirby Institute in a de-identified format. Data is stored in a combined format on The Kirby Institute local area network, a password protected system, with access restricted to the study’s Research Coordinators and Statisticians. Data may be released to researchers on application in a de-identified format following local ethics approval.Blips were defined as detectable viral load (≥50 copies/ml) preceded and followed by undetectable viral load (<50 copies/mL). Episodes of persistently detectable, defined as two or more consecutive viral loads ≥50 copies/mL, were not considered blips. “Real” blips were those blips that did not involve a change in therapy as a result of a VL>50 copies/ml. “Switched blips” were defined as undetectable viral load followed by a single elevated VL followed by a change in therapy. Virological failure was defined as two consecutive viral load ≥50 copies/ml after viral suppression post commencement of cART. Only data available from the time of each site having a “sensitive” viral load assay which could read down to 50 copies was included in the study so biases due to different types of assay reading down to 400 copies rather than 50 were reduced. Instances of subsequent intermittent tests at thresholds of less sensitive assays were excluded from the analysis.Based on survival models, the covariate ‘Year of first cART’ was dichotomized to before or after 2004, with equal numbers commencing cART either before or after this date. Subsequent inclusion into the model was based at time of virological control. Cox models of time to first virological failure after entry, following control of viral load after commencement of cART, were developed. Patients were censored at death, or the earlier of lost to follow up, 180 days after last viral measure, or 31 March 2011. Periods off treatment of greater than 14 days were excluded from models. Intervals of blip duration were excluded. Durations of blip, were excluded from analysis follow-up time. Multivariate models were developed using forward stepwise selection from significant univariate predictors (p<0.05) with forced inclusion of income level and prior blips. Low- and Middle-income site data was amalgamated because of insufficient observations in the low-income category. Sensitivity analysis of differing definitions of virological failure were conducted, specifically virological failure defined as 2 consecutive viral loads of ≥200 copies/mL, ≥400 copies/mL, and ≥1000 copies/mL. Sensitivity analyses were performed to assess the significance of varying magnitudes of blips, specifically to viral load thresholds of 200, 400 and 1000 copies/mL. A sensitivity analysis of ‘switched blips’ as determinants of virological failure was conducted.3 in high- and middle/low-income settings, respectively. Viral load testing occurred at a median of 175 and 91 days in low/medium-, and high-income sites respectively. The median duration of follow-up between viral control after first cART to censor was 3.20 years [IQR 1.36–6.13]. Other patient demographic and clinical characteristics are outlined in A total of 5040 patients, 2597 from AHOD and 2521 from TAHOD were included. 4167 (82.7%) participants were male, mean CD4 at cART initiation was 294.4 and 152.5 cells/mmVirological failure  occurred in 1037 patients (20.6%) at a median of 1.63 years (IQR: 0.87–3.23) and 1.71 years (IQR 0.74–3.12) post commencement of cART in high- and low/middle- income sites respectively.Hepatitis C antibody positivity was associated with a shorter time to virological failure. cART containing NRTI+PI), NNRTI+PI±NRTI, 3+NRTI/no PI/no NNRTI HR, and other regimens were associated with significantly shorter time to virological failure, compared to the most frequently used first-line regimen (3+NRTI+NNRTI/no PI). Commencement of cART after 2004, and heterosexual exposure compared with MSM, was associated with significantly longer time to virological failure. Income was associated with significantly reduced time to virological failure in multivariate analysis adjusted for all covariates as outlined in Sensitivity analysis assessing differing definitions of virological failure showed that income remained a significant predictor of time to virological failure for VL ≥200 copies/mL (HR 0.35 (95% CI 0.28–0.43) p<0.001) comparing low/middle-income sites to high-income sites, for VL ≥400 copies/mL (HR 0.37 (95% CI 0.29–0.46) p<0.001)and for VF ≥1000 copies/mL (HR 0.0.36 (95% CI 0.28–0.47) p = 0.07). Prior blips were not significant with these differing definitions of virological failure; VF ≥200 copies/mL HR 0.92 (95% CI 0.69–1.23) p = 0.57, VF ≥400 copies/mL HR 0.94 (95% CI 0.69–1.27) p = 0.67, VF ≥1000 copies/mL HR 0.1.00 (95% CI 0.72–1.39), p = 0.98.Nine hundred and ten (18%) of patients experienced blips ever . 744 (21%) and 166 (11%) of high- and middle/low-income participants, respectively, experienced blips ever. Seven hundred and eleven (14%) of all patients experienced blips prior to the earlier of censoring or virological failure. 559 (16%) and 152 (10%) of high- and middle/low-income participants, respectively, experienced blips. The overall rate of blips was 50.3 (95% CI: 46.7–54.1), and 31.7 (95% CI: 27.4–36.6) per 1000 person years of follow-up in high- and middle/low-income sites, respectively. However, the ratio of blips to number of viral load tests performed was similar across sites; 1.47% and 1.64% in high- and low/middle-income sites respectively.Testing frequency was not adjusted for in the primary analysis. A sensitivity analysis adjusting for testing frequency showed no qualitative difference in results. A sensitivity analysis to assess the significance of blips allowing for different testing intervals was conducted by using every second test result from HIC. This corresponded to a biannual testing frequency equivalent to the testing frequency of L/MIC. A separate sensitivity analysis using entry after more than one VL<50 copies/mL after first cART is qualitatively the same as primary analysis.572 (63%) of all blips were between 50–199 copies/mL, 114 (13%) were between 200–399 copies/mL, 98 (11%) were between 400–1000 copies/mL and 126 (14%) were greater than 1000 copies/ml. Differing magnitudes of blips were not significant in univariate analyses as predictors of virological failure . Differing frequencies of blips, compared to no blips, were not significant predictors of virological failure in univariate analysis; single blip HR 0.96 (95% CI 0.75–1.22); multiple blips HR 1.19 (95% CI 0.59–2.41).209 of 866 (24%) patients were switched to an alternate regimen in the setting of a single low viral load reading, thus meeting our definition of a ‘switched blip’. The median viral loads of patients that triggered this switch were 285.5 copies/mL (IQR 89–1000), and 257 copies (IQR 72–757) for high-, and low/middle-income sites respectively. There was no significant increase in time to virological failure with ‘switched’ blips HR = 1.10 (95% CI 0.91–1.33), p = 0.33). 63 (30%) of ‘switched blips’ occurred in middle/low-income sites. Most ‘switched blips’ occurred in high-income settings and before 2006 (68%). Most were switched from NRTI+PI (48%) regimens; of patients with ‘switched blips’, the majority was switched to NRTI+PI (46%) or NRTI+NNRTI (45%) regimens.Our study demonstrates that the presence of blips, irrespective of magnitude or frequency, is not predictive of virological failure. This finding also holds true in low/middle- income countries, where viral load testing is much less frequent than in high-income countries. Our study, additionally, demonstrates that a large proportion of patients undergo switching of antiretroviral regimen in the setting of blips.Our study findings are in keeping with previous studies, conducted only in resource-rich settings, that found no association between blips and virological failure A major strength of our study is that, to our knowledge, it is the first to assess the significance of blips in resource-limited settings. It reflects ‘real-world’ decision-making and outcomes, and has identified a sizeable proportion of patients who have potentially undergone unnecessary switching of cART in the setting of blips. While it does not obviate the need for viral load testing in resource-poor settings, it provides reassuring outcomes regarding infrequent viral load testing in this very specific setting of blips. Our study is also the first to address the significance of blips with differing definitions of virological failure – relevant in resource-limited settings, which often have higher thresholds for defining virological failure.Our study has several limitations. Analysis of duration of specific antiretroviral regimens, and switch to therapy prior to the development of virological failure were not included in this analysis. Regimens varied widely between sites and have changed significantly over the treatment duration included in this analysis, including triple NRTI regimens and un-boosted protease inhibitors. This analysis did not include details of the different viral load assays used at various sites at different times. Viral load assays are known to have different sensitivities This study demonstrates that frequency of viral load testing varies enormously between sites. Despite this, no significant increased risk of virological failure was demonstrated in the setting of blips. Questions remain about the significance of more prolonged low-level viremia in settings with infrequent viral load testing that warrant further investigation.


Limited data exist for the efficacy of second-line antiretroviral therapy among children in resource limited settings. We assessed the virologic response to protease inhibitor-based ART after failing first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens.A retrospective chart review was conducted at 8 Thai sites of children who switched to PI –based regimens due to failure of NNRTI –based regimens. Primary endpoints were HIV RNA < 400 copies/ml and CD4 change over 48 weeks.10 copies/ml for HIV RNA were included; 104 (41%) received a single ritonavir-boosted PI (sbPI) with 2 NRTIs and 137 (59%) received double-boosted PI (dbPI) with/without NRTIs based on physician discretion. SbPI children had higher baseline CD4 , lower HIV RNA , and less frequent high grade multi-NRTI resistance  than the dbPI children. At week 48, 81% had HIV RNA < 400 copies/ml  with a median CD4 rise of 9% . However, only 63% had HIV RNA < 50 copies/ml, with better viral suppression seen in sbPI .Data from 241 children with median baseline values before starting PI-based regimens of 9.1 years for age, 10% for CD4%, and 4.8 logSecond-line PI therapy was effective for children failing first line NNRTI in a resource-limited setting. DbPI were used in patients with extensive drug resistance due to limited treatment options. Better access to antiretroviral drugs is needed. The most commonly used first-line antiretroviral therapy (ART) in HIV-infected children in resource-limited settings (RLS) is a non nucleoside reverse transcriptase inhibitor (NNRTI)-based treatment ,2. Data However, in RLS without routine virologic monitoring, the diagnosis of treatment failure is usually late, of which brings about concerns for multi-NRTI resistance . ReporteThe primary objective of this study is to describe the immunologic and virologic efficacy of second-line ARV regimens containing either single- or double-boosted PI over a 48-week period in children with NNRTI-based treatment failure in Thailand.We formed a network of 8 large pediatric HIV centers in Thailand to retrospectively collect treatment outcome data of all children who failed NNRTI-based therapy and received ritonavir-boosted PI regimen as a second-line drug regimen. Immunologic treatment failure was defined according to the Thai guideline either CD4 percentage decline > 5 percentage point in a patient with CD4% less than 15, or CD4 cell count drop > 30% of baseline within 6 months [2/dose) combined with one other PI drug.Cases selected were HIV-infected children aged < 18 years, with a documented history of immunologic or virologic failure on NNRTI-based ART who received ritonavir-boosted PIs-based regimen for at least 24 weeks. They were excluded if they had previously received PIs treatment prior to the ritonavir-boosted PIs for longer than 30 days, or received second-line drugs not belonging to the NRTI, NNRTI, and PI classes. Standardized forms were used for retrospective hospital chart extraction: include demographics, CDC HIV clinical classification, history of ART, CD4 cell count and percentage, and genotypic resistance test result before switching to PI-based HAART. Follow up CD4, plasma HIV RNA and adverse events after switch to PI regimen were extracted and censored at last patient visit. After switching to second-line therapy, CD4 was uniformly monitored every 6 months and HIV RNA every 6–12 months. Single-boosted PI was defined as low dose ritonavir combined with one other PI drug. Double-boosted PI was defined as low dose ritonavir combined with another two PI drugs, or therapeutic dose ritonavir  at 5 sites, ViroSeq HIV-1 Genotyping System  at 1 site and with an in house method using Stanford and International AIDS society (IAS) database  at 2 sitPrimary endpoints were the proportion of children with plasma HIV RNA < 400 copies/ml at week 48 and the CD4 changes at 48 weeks after switching to second-line PI therapy. Secondary endpoints were the proportion of children with plasma HIV RNA < 50 copies/ml and the prevalence of adverse events. The analysis was performed using available data.The cumulative probability of virologic treatment failure defined as HIV RNA > 400 copies/ml after at least 6 months of second-line boosted PI regimen was calculated using the Kaplan Meier estimates. The data included patients who had HIV RNA result at least once after 24 weeks of treatment and censored at 24 months or earlier in cases of discontinuation of the follow-up. The difference between single- and double -boosted PI was tested using the log rank test.3), plasma HIV RNA at time of switch to boosted PI regimen , ART regimen as single-boosted PI or double-boosted PI, use of lopinavir/ritonavir (LPV/r) –containing regimen, and grade of resistance mutations. Definition of high grade multi-NRTI resistance was defined as ≥ 4 thymidine analog mutations (TAMs) or the presence of Q151M or 69 insertion. Low grade multi-NRTI resistance was defined as 1 to 3 TAMs. Factors with p-value < 0.2 in the univariate analysis were tested in the multivariate analysis. Analyses were performed using SAS version 9.1.The predictors for treatment failure defined as HIV RNA < 400 copies/ml at week 48 were explored in a logistic regression model. Factors included CD4 cell count at time of switch to boosted PI regimen , and a lower proportion of genotypes with multi-NRTI resistance mutation  compared with children who received double-boosted PI regimen.Between September 2002 and June 2007, 241 children were enrolled, including 1, 11, 62, 63, 60, and 44 children per calendar year. There were 104 children who switched to single-boosted PI and 137 children who switched to double-boosted PI (137 children) at 8 HIV pediatric clinics. The baseline characteristics of the children are shown in Table The single-boosted PI regimens included 55 (53%) indinavir/r (IDV/r) and 49 (47%) lopinavir/r (LPV/r). The NRTI backbones used were 50 (48%) zidovudine (AZT) plus lamivudine (3TC), 29 (28%) AZT plus didanosine (ddI), 17 (16%) ddI plus 3TC and 8 (8%) miscellaneous. Among children in the double -boosted PI group, 77 (56%) did not receive any NRTI, 37 (27%) received 3TC as the only NRTI, and 23 (17%) received other NRTIs. The double-boosted PI regimens were LPV/r + IDV in 46 (34%) children, IDV/r + Nelfinavir (NFV) in 24 (18%), LPV/r + Saquinavir (SQV) in 20 (15%), LPV/r + NFV in 17(12%), IDV + RTV in 16(12%), IDV/r + SQV in 8 (6%) and other PIs in 6 (4%) children.At week 24, 223 had CD4 information and 190 had plasma HIV RNA information. The overall median (IQR) CD4% change was 5 (1–8) % with more CD4 rise in children who received double- compared to single-boosted PI . Overall 78% achieved HIV RNA < 400 copies/ml. The proportion with HIV RNA < 400 copies/ml was not different between groups; 80.9% (72/89) in single- vs. 76.2% (77/101) in double-boosted PI regimens. The proportion with HIV RNA below 50 copies/ml was 59% overall with a higher proportion in single- compared to double-boosted PI groups: 66.7% (58/87) vs. 51.2% (44/86) p = 0.038).The outcomes at week 48 after initiation of boosted PI regimens are shown in Table Subgroup analysis was performed for the outcome HIV RNA < 400 copies/ml at week 48. Among children in single-boosted PI group, 30/36 (83.3%) of children who received indinavir/r and 24/29 (82.8%) of children who received lopinavir/r had undetectable viral load . Among children in double boosted PI group, 47/57 (82.5%) of children who did not receive any NRTI and 16/22 (72.7%) of children who received NRTIs had undetectable viral load .During the follow-up period, there were 31 adverse events that led to discontinuation or substitution of PI drugs. The median time of discontinuation or substitution were 13.3 months IQR 7.1-26.0) after start PI-based regimen. There were 20 events for IDV-related toxicity (13% of children who used IDV)due to nephrotoxicity (n = 11), hyperbilirubinemia (n = 4), nausea vomiting (n = 2), intolerance (n = 1), blurred vision (n = 1), hypertriglyceridemia (n = 1); 5 events for full dose RTV (31% of children who used full dose of RTV) due to intolerance (n = 3), hyperbilirubinemia (n = 1) and hypertriglyceride (n = 1); 4 events for NFV (10% of children who used NFV) due to intolerance (n = 2), rash (n = 1) and diarrhea (n = 1); 1 event for SQV (4% of children who used SQV) due to intolerance and 1 event for LPV/r (0.8% of children who used LPV/r) due to dyslipidemia. Proportion of children with elevated low density lipoprotein was significantly more in the double-boosted compared to single-boosted PI groups .The predictors for virologic suppression at week 48 were explored by multivariate logistic regression analysis as shown in Table Our study provides information on outcomes of second-line ART in NNRTI-failing children as part of a large multicenter observational study in a RLS. This study showed good virologic efficacy of second-line boosted PI regimens with 81% achieving HIV RNA < 400 copies/ml at 48 weeks of treatment. Children with more advanced HIV disease were preferentially treated with double-boosted PI versus single-boosted PI regimens. Both regimen types performed equally well in suppressing HIV below 400 copies/ml but further suppression to less than 50 copies/ml was significantly less with double-boosted PI.This study supports recycling of 2NRTIs in combination with a potent boosted PI as second-line therapy in children. The 2010 WHO guideline recommend replacing AZT/3TC or d4T/3TC in the first line regimen with abacavir (ABC)/3TC or ABC/ddI ; however10 copies/ml) than our children. The Spanish multicenter retrospective observational study showed 71.5% of PI-experienced children on LPV/r-based having HIV RNA < 400 copies/ml [The virologic response to second-line single boosted PI regimen in our study compares favorably to reports from other countries -17. In aopies/ml  In anothopies/ml . We alsoopies/ml .Double-boosted PI is an acceptable alternative second-line therapy option in Thailand for children with late treatment failure who have few or no fully active drugs aside from PIs . The ped3 or HIV RNA is > 5,000 copies/ml [3 or HIV RNA was < 10,000 copies/ml. Delayed in switching to second-line therapy may lead to resistance mutations accumulation which would be problematic in RLS where ARV options are limited. The PENPACT-1 study showed that children failing first-line NNRTI who were randomized to a delayed switch at HIV RNA > 30,000 copies/ml had more TAMs compared to those who switched at HIV RNA > 1,000 copies/ml [There is a lack of evidence to inform the optimum time to switch to second line regimens in RLS. The WHO guideline, relying heavily on a public health approach to care, recommends switching to second-line regimen when CD4 is < 100 cells/mmopies/ml . We demoopies/ml  This howThis study has several limitations. First is incomplete data, which is an inherent limitation of a retrospective study design. The information of adherence to treatment is not captured. Second, the lack of randomization of single- versus double-boosted PI regimens hinders the ability to directly compare the outcome between these two regimens. Third, the duration of NNRTI-based first line regimen in our study was relatively short, around 2 years. Furthermore, we have access to laboratory monitoring including CD4, plasma HIV RNA and genotypic resistance testing to guide the design of the new regimens which may limit the applicability of our results to other resource-limtted settings where treatment failure is detected later and genotyping is unavailable. However, the constraint in available ARV choices in our population prohibited us to fully utilize genotyping information. Therefore, we believe that our data represent typical clinical care settings in most resource-limited countries.In conclusion, this study provides security to clinicians in RLS to give children second-line treatment despite drug choice and laboratory monitoring constraints. Although we saw a good success rate in our study, one-fifth did not achieve viral suppression. Such children in RLS currently have no treatment option beyond second-line regimens. This emphasizes the urgent need to derive strategies to study, plan and procure new drugs and drug classes for children with treatment failure worldwide.The HIV Netherlands Australia Thailand Research Collaboration (HIV-NAT), The Thai Red Cross AIDS Research Centre, Bangkok: T. Bunupuradah, C. Phasomsap, P. Kaew-on.Institut de Recherche pour le Développement (IRD), UMI 174, Program for HIV Prevention and Treatment, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand, The Global Fund to Fight AIDS, Malaria and Tuberculosis supported drug and laboratory monitoring for some children: S Kanjanavanit, Nakornping Hospital, Somdej Pranangchao Sirikit Hospital, Chonburi, Somdej Pranangchao Sirikit Hospital, Chonburi: T Hinjiranandana, Bhumibol Adulyadej Hospital, Bangkok: P Layangool, Somdej Prapinklao Hospital, Bangkok: N Kamonpakorn, Mae Chan Hospital, Chiang Rai: S Buranabanjasatean, Prapokklao Provincial Hospital, Chantaburi: C Ngampiyaskul.Queen Sirikit National Institute of Child Health, Bangkok: T. Chotpitayasunondh, S. Chanpradub, P. Leawsrisuk. Department of Pediatrics, Faculty of Medicine, Siriraj Hospital, Mahidol University: S. Chearskul, N. Vanprapar, W. Phongsamart, K. Lapphra, P. Chearskul, O. Wittawatmongkol, W. Prasitsuebsai, K. Intalapaporn, N. Kongstan, N. Pannin, A. Maleesatharn, B. Khumcha.Research Institute for Health Sciences (RIHES), Chiang Mai University, Chiang Mai: L. Aurpibul, N. Wongnum, R. Nadsasarn.Department of Pediatrics, Faculty of Medicine, Khon Kaen University: P. Lumbiganon, P. Tharnprisan, T. Udompanich. Petchburi Hospital, Petchburi: M. Yentang.Chiang Rai Regional Hospital, Chiang Rai: A. Khonponoi, N. Maneerat, S. Denjunta, S. Watanaporn, C. Yodsuwan, W. Srisuk, S. Somsri, K. SurapanichadulAll authors declare no conflict of interest and that member of their immediate families do not have a financial interest in or arrangement with any commercial organization that may have a direct interest in the subject matter of this article.TP, GJ, KC, and JA designed the study, collected data, wrote the first draft, reviewed manuscript before submission. PS, TS,WP,VS, PK,WP,RH, NW collected data, reviewed and commented of manuscript before submission. US analyzed, reviewed and commented draft of manuscript before submission. All authors have read and approved the final manuscript.


To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects.In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%.7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir.In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects. The rate of virological failure of the 3 original drug classes is low, but not negligible, and does not appear to diminish over time from starting antiretroviral therapy.Studies have shown that pre-existing minority drug-resistant mutants increase the risk of virological failure to first-line antiretroviral therapy with non-nucleoside reverse transcriptase inhibitors (NNRTIs) It is particularly uncertain if ultrasensitive genotypic tests could provide clinically relevant information in heavily pre-treated HIV-1-infected subjects beyond that provided by standard population sequencing genotypic tests. On one hand, detection of additional minority drug-resistant mutants could improve the assessment of viral susceptibility to drugs with intermediate or high drug genetic barrier. On the other hand, the presence of extensive drug resistance could compromise the ability of deep sequencing to add relevant genotypic information to that already obtained with population sequencing, particularly when alternative treatment options are severely limited. Moreover, mutant fixation during virological failure in the presence of ART pressure could potentially complicate the detection of additional low-frequency variants We therefore sought to explore the potential of deep sequencing to provide additional, clinically relevant genotypic information that could be used to improve treatment decisions in heavily pre-treated HIV-1-infected subjects, relative to population sequencing.This proof-of-concept observational study included HIV-1-infected adults with previous virological failure (VF) to protease inhibitors (PIs), nucleos(t)ide (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), who developed virological failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Virological failure was defined as the presence of HIV-1 RNA levels >200 copies/mL 24 weeks after salvage therapy initiation or beyond. Adherence was self-reported by the patient and collected from medical charts. Adequate adherence was defined as intake of all medication doses. Partial adherence was defined as the presence of missed doses during treatment. Treatment interruption meant the complete interruption of therapy during follow-up. The Institutional Review Board of the Hospital Universitari Germans Trias i Pujol, Badalona, Spain, approved the study; participants provided written informed consent for retrospective sample testing.TM, QIAGEN, Valencia, CA). Three One-Step RT-PCRs  were performed in parallel per each sample. Primers used were 1571-L23 (HXB2: 1417→1440) 5′-ATT TCT CCT ACT GGG ATA GGT GG-3′ and 5464-L27 (HXB2: 5464→5438) 5′-CCT TGT TAT GTC CTG CTT GAT ATT CAC-3′. Cycling conditions were: 30 min. at 52°C, 2 min. at 94°C; 30 sec. at 94°C, 30 sec. at 56°C, 4 min. at 68°C, for 20 cycles; followed by 5 min. at 68°C. Triplicate RT-PCR products were pooled, column-purified  and used for both viral population and deep HIV-1 sequencing.HIV-1 RNA was extracted from 1 mL of plasma within 4 weeks before initiation of deep salvage therapy (baseline) and at virological failure  with primers 2084-U26 (HXB2: 2084→2109) 5′-ATT TTT TAG GGA AGA TCT GGC CTT CC-3′ and 5456-L26 (HXB2: 5456→5431) 5′-TGT CCT GCT TGA TAT TCA CAC CTA GG-3′. Cycling conditions were: 2 min. at 94°C; 30 sec. at 94°C, 30 sec. at 56°C, 4 min. at 68°C, for 20 cycles; followed by 5 min. at 68°C. Nested PCR products were pooled and column-purified. Protease (PR), reverse transcriptase (RT) and integrase (IN) were sequenced in-house  and resolved by capillary electrophoresis .Pooled purified RT-PCR products were used as template to generate eight overlapping amplicons covering PR and RT and 3 amplicons covering codons 51 to 215 in IN. Each codon was interrogated by at least 2 independent amplicons, which were generated in triplicate during 20 cycles of PCR amplification  followed by pooling and purification of triplicate PCR products . PCR reactions were pooled and purified using the Agencourt AMPure Kit  to eliminate primer-dimers. The number of molecules was quantified by fluorometry . When amplicon concentrations were below 5 ng/mL, amplicon size and primer-dimer content were analysed by spectrometry using BioAnalyzer . Emulsion PCR was carried out as in -5, within the range of the expected PCR error. The mean (SD) error rate for any aminoacid mismatch was 0.14% (0.19%), but the mean (SD) error rate for actual drug resistance mutations was 0.03% (0.07%). The 99th percentile of mismatches would establish the threshold for nucleotide errors in 0.61% and the limit for identifying true drug resistance mutations in 0.20%.Using a conservative approach, only mutations present in ≥ 1% of the virus population where considered in this study. An in-house analysis of 992 pNL43 clonal sequences obtained by deep sequencing under the same PCR conditions used to generate patient samples showed a mean (SD) nucleotide mismatch rate of 0.07% (0.13%), which is almost identical to previous reports Genotypes were interpreted with the HIVdb v6.0.8 3 at baseline; and 36,700  copies/mL and 150  cells/mm3 at virological failure. Median (IQR) nadir CD4 counts were 14  cells/mm3. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively.  age was 44  years. The median (IQR) year of HIV diagnosis was 1988 . Median (IQR) HIV-1 RNA levels and CD4 counts were, respectively, 110,600  copies/mL and 50  cells/mmctively.  Treatmenctively.  and partctively.  interrupThe median (IQR) deep sequencing coverage per base was 3938 (3130 – 6945) sequences at baseline and 3965 (3688 – 7091) sequences at virological failure. Deep sequencing detected all mutations found by population sequencing in all subjects, and found additional mutations in 6 out of 7 individuals.  AdditionDeep sequencing provided limited additional genotypic information at baseline relative to PS. Baseline DS changed the ANRS predicted susceptibility of lopinavir/r and saquinavir/r from sensitive to intermediate in subject 1 and the ANRS susceptibility of darunavir/r from intermediate to resistant in subject 2 . In subjMost additional resistance mutations leading to changes in susceptibility predictions were found after virological failure to deep salvage therapy . ConsistMost baseline resistance mutations were lost in subject 7 during treatment interruption becoming undetectable even by deep sequencing at the time of virological failure. No changes in predicted susceptibility were observed for tipranavir or raltegravir, although detection of minority G140S in subject 3 suggested improved fitness of the Q148R mutants detected by population sequencing at virological failure .While being technically non-inferior to population genotyping, deep sequencing enabled the detection of additional resistance mutations with potential clinical significance in 6 out of 7 individuals included in this study. Additional mutations, however, only modified about 5% of antiretroviral susceptibility predictions, including decreased etravirine efficacy in the 2 subjects developing virological failure to this drug, and decreased NRTI, efavirenz and indinavir/r efficacy in 2, 1, and 1 subject, respectively. Changes in darunavir susceptibility observed in 2 individuals were not consistent across algorithms. Deep sequencing had no impact on susceptibility predictions for tipranavir or raltegravir.Deep sequencing could modify patient clinical management by avoiding drugs whose resistance may have been underestimated by population sequencing. Indeed, baseline low-frequency PI-resistant mutants were selected during treatment exposure in two subjects: I54T in subject 1, and V32I, Q58E and L89V in subject 2. I54T is a PI-related mutation that appears to be associated with decreased susceptibility to each of the PIs, although its effect has not been well studied. Q58E is a non-polymorphic PI-related mutation associated with reduced susceptibility tipranavir Most additional mutants in our study, however, were detected at virological failure. Varghese et al. also detected additional minority variants with major etravirine mutations only in patients failing an NNRTI-containing regimen The increased sensitivity of deep sequencing could also reassure clinicians about the absence of additional genotypic resistance when making clinical management decisions. This is particularly important for the management of subjects with suboptimal adherence. In our study, deep sequencing provided additional genotypic information in all adherence strata.  Deep seqDetection of low-frequency resistance mutations associated with hypersusceptibility could also improve predicted susceptibility to certain antiretrovirals. For example, the incorporation of a minority T215F mutation in subject 2 changed the HIVdb predicted susceptibility to tenofovir from “resistant” to “intermediate” at virological failure. Therefore, ultrasensitive genotyping also has the potential to refine genotype interpretation rules towards increased virus susceptibility.Our findings extend those of Le et al. The main limitations of our study are its small sample size, its retrospective observational nature and the fact that adherence was self-reported. This study makes two arguable assumptions: First, that a mutant detected above 1% is clinically relevant. This threshold is clearly above the PCR and 454 sequencing error found in our own and other studies In conclusion, in this subset of heavily pre-treated individuals, deep sequencing showed technical non-inferiority to population sequencing. However, although additional mutations improved the assessment of genotypic resistance to etravirine, deep sequencing did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility relative to population sequencing. Further studies should extend our findings and address the clinical impact of ultrasensitive genotyping in HIV-1 infected individuals experiencing virological failure to their first or second-line antiretroviral therapy. Proper throughput escalation, sample multiplexing and adequate sequence coverage may potentially turn ultrasensitive genotyping into a feasible strategy for HIV drug resistance management in the clinical setting.Table S1a, b, c. a Only mutations with a score ≥ 5 in the Stanford HIV Drug Resistance Database were included in the analysis. b ART: antiretroviral therapy; DS: deep sequencing; PI: protease inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor; InSTI: Integrase Strand Transfer Inhibitor; ZDV: zidovudine; ABC: abacavir; d4T: stavudine; ddI: didanosine; TDF: tenofovir; EFV: efavirenz; NVP: nevirapine; ETR: etravirine; ATV: atazanavir; IDVr: indinavir/ritonavir; FAPVr: fosamprenavir/ritonavir; SQVr: saquinavir/ritonavir; LPVr: lopinavir/ritonavir; DRVr: darunavir/ritonavir; TPVr: tipranavir/ritonavir; ANRS: algorithm of the French ANRS  AC11 Resistance group, version 18, July 2009, France; HIVdb: HIV db program version 6.0.8, implemented at the Stanford HIV Drug Resistance Database, Stanford University, USA; REGA: Algorithm of the Rega Institute version 8.0.2, University of Leuven, Belgium. c Predicted antiretroviral susceptibility: S: susceptible; I: intermediate; R: Resistant.Additional genotypic information provided by deep HIV-1 sequencing and changes in predicted phenotypic susceptibility, relative to population sequencing (DOC)Click here for additional data file.Table S2Predicted antiretroviral susceptibility according to Sanger or quantitative deep 454 sequencing. Color code: Green =  Sensitive; Yellow = Intermediate; Red = Resistant. Changes in predicted phenotypic susceptibility between Sanger and 454 sequencing sequencing are highlighted in black boxes. DLV: delavirdine; EFV: efavirenz; ETR: etravirine; NVP: neviapine; 3TC: lamivudine; ABC: abacavir; AZT: zidovudine; d4T: stavudine; ddI: didanosine; FTC: emtricitabine; TDF: tenofovir dippivoxil fumarate; ATV: atazanavir; ATVr: ritonavir-boosted atazanavir; DRVr: ritonavir-boosted darunavir; FPVr: ritonavir-boosted fosamprenavir; IDVr: ritonavir-boosted indinavir; LPVr: ritonavir-boosted lopinavir; NFV: nelfinavir; SQVr: ritonavir-boosted saquinavir; TPVr: ritonavir-boosted tipranavir; RA: ralegravirL; ELV: elvitegravir.(XLS)Click here for additional data file.


Raltegravir(RAL) is a the first compound of a novel class: integrase inhibitors. Aim of our study was to evaluate efficacy and safety of RAL based regimen.We proceeded to retrospective analysis from patients(pts) referring to our service. We searched our databases for epidemiological, clinical, immunological and virological issues. Moreover we recorded HCV and HBV status. We focused our attention on the reason of ARV switching , duration of ARV before RAL introduction, immunological gain, virological suppression and tolerability of the prescribed regimen. We followed side effects in order to define safety of prescribed regimen.We enrolled 70 pts. , according to CDC 1993 they were classified as A(12); B(32);C(26). Median age was 49 years, 42 pts were HCV-positive and only 4 presented an active HBV coinfection. Risk factors for HIV infection resulted: injective drug abuse(33) homosexual(20) and heterosexual(17)exposition. They were switched to RAL therapy for drug resistance(39)(DRR); regimen simplification(7) or drug toxicity (24). Most of patients presented multiple drug failures to ARV therapy . Median follow up was with RAL is 9 months and median extent of ARV before RAL was 9 years. Among DRR. 29 were coming from a PI failure whereas 9 from NNRTI based regimen and one from 3TC monotherapy. The most represented drug associated in new ARV regimen was DRV/rtv (42) followed by etravirine(12) and maraviroc (10).We collected data of immune and virological response after 3 and 6 months, so at 6 months 59 pts.  presented undetectable(<20) viral load and a medium CD4 gain of 89 cc/ml(90 cc/ml in DRR). No relevant side effects were observed.Pts. treated with RAL based therapy presented a good outcome, most of them achieve (after 6 months) virological suppression and immunological recovery. In our case list, darunavir/rtv and RAL obtained the best results with no relevant side effects. So more studies are needed to confirm that RAL based ARV could be very efficacious and safe even in long term follow up period.


To evaluate the reasons and risk factors for antiretroviral discontinuation in a cohort of HIV-infected patients in the first year after starting combined antiretroviral therapy (cART) for the first time.A cohort of naïve HIV-infected patients who started cART in the context of a national reimbursement program from January 2007 onwards is being prospectively followed. For each individual, the date and reason of the first discontinuation of any drug in the initial regimen was identified. Changes to same drug formulations were not counted as discontinuations.3 (range 2-833). Viral load (VL) >100000 copies/mL in 55.6%. The cART regimen was based in NNRTIs /PIs in respectively 67.3% and 31.9% of the patients.399 patients were enrolled, with a mean follow-up of 82±48 weeks. This study concerns the 248 (62.2%) patients who have a follow-up of ≥1 year. Mean age 42.2±12.3 years (range 78-20), 71.8% were males. 98.8% were HIV-1-infected. HIV was sexually transmitted in 77.8% of the patients, 21.0% were IVDUs; in 1,2% risk was undetermined. HCV/HBV co-infection respectively in 24.6% and 4.4%. At baseline: 28.6% of the patients had AIDS. Mean CD4 cell count 185 cells/mmA total of 109 (44.0%) patients discontinued at least one drug in their cART regimen. The main reasons for discontinuation were: drug related adverse effects (39/35.8%), lack of adherence/lost to follow up (20/18.3%), virological failure (18/16.5%), regimen simplification (17/15.6%), increased CV risk (7/6.4%), pregnancy (4/3.7%). Four patients died. Low median CD4 count (p=0.021) and high median viral load (p=0.075) were found to be associated with virological failure.Drug related adverse effects were the main cause of antiretroviral discontinuations in this cohort, with a rate higher than the combined rates for lack of adherence/lost to follow-up and virological failure. Continued attempts to improve the tolerability of cART regimens and patient adherence may help to minimize drug discontinuation rates over the longer term.


Children with viral respiratory infections who undergo general anesthesia are at increased risk of respiratory complications. We investigated the impact of RSV and influenza infection on perioperative outcomes in children undergoing general anesthesia.We performed a retrospective case-control study. All patients under the age of 18 years who underwent general anesthesia at our institution with confirmed RSV or influenza infection diagnosed within 24 hours following induction between October 2002 and September 2008 were identified. Controls were randomly selected and were matched by surgical procedure, age, and time of year in a ratio of three controls per case. The primary outcome was postoperative length of stay (LOS).Twenty-four patients with laboratory-confirmed RSV or influenza who underwent general anesthesia prior to diagnosis of viral infection were identified and matched to 72 controls. Thirteen cases had RSV and 11 had influenza. The median postoperative LOS was three days (intra-quartile range 1 to 8 days) for cases and two days (intra-quartile range 1 to 5 days) for controls. Patients with influenza had a longer postoperative LOS (p < 0.001) and patients with RSV or influenza were at increased risk of unplanned admission to the PICU (p = 0.04) than matched controls.Our results suggest that children with evidence of influenza infection undergoing general anesthesia, even in the absence of symptoms previously thought to be associated with a high risk of complications, may have a longer postoperative hospital LOS when compared to matched controls. RSV and influenza infection was associated with an increased risk of unplanned PICU admission. Screening for symptomatic respiratory illness is a key element of the pediatric preoperative anesthesia assessment. Children with upper respiratory illnesses (URI) who undergo general anesthesia are at increased risk of respiratory complications  includinRespiratory syncytial virus (RSV) is an enveloped RNA paramyxovirus that causes annual epidemics during winter and early spring in temperate climates . RSV is The goal of the present study is to determine whether perioperative outcomes are negatively impacted and the incidence of complications is higher for patients who have occult RSV or influenza virus infection at the time of anesthetic induction than for matched controls.The Institutional Review Board at the Johns Hopkins Hospital reviewed and approved this study. We performed a retrospective case-control study identifying all patients under the age of 18 years who underwent induction of general anesthesia at the Johns Hopkins Hospital between October 2002 and September 2008 and who were subsequently diagnosed with laboratory-confirmed RSV or influenza. Viral infection was defined as identification of RSV or influenza virus by immunochromotography, direct fluorescent antibody, hemeadsorption, tube viral culture or shell vial culture from a nasopharyngeal or endotracheal specimen obtained at the time of, or within 24 hours of, induction of general anesthesia. Patients known to have RSV or influenza virus infection prior to induction were excluded from the study.Review of the patient chart and clinical databases using a standardized data collection tool was conducted to establish postoperative length of stay, prolonged positive pressure ventilation, need for extracorporeal life support (ECLS), and mortality. The anesthesia record was reviewed to determine pre-anesthesia history including assessment of the presence or absence of fever, symptoms of upper respiratory infections such as cough and rhinorrhea, and passive smoke exposure. We defined perioperative and postoperative events as bronchospasm, laryngospasm or unplanned re-intubation in the operating room or postanesthesia care unit (PACU). Unplanned PICU admission was defined as patients that were preoperatively scheduled for recovery in the PACU but required PICU admission. Prolonged postoperative positive pressure ventilation (PPV) was defined as the requirement of PPV for more than six hours following admission to the PICU. The primary outcome of interest was postoperative length of stay.Controls were randomly selected from a hospital clinical database for the years 1999 to 2010, and were individually matched with cases by surgical procedure, age, and time of year in a ratio of three controls per case. Previous research at our institution has established the predominant time frame for annual seasonal RSV and influenza epidemics extends from the months of November to March . For theWe identified patients with chronic medical conditions associated with an increased risk of complications from viral illness -11. ThesContinuous variables were compared using Wilcoxon matched pairs signed rank testing. Categorical variables were compared using McNemar's testing for matched pairs or McNemar's Exact testing as appropriate. All statistical calculations were performed using Stata/IC 10.1 .There were 24 patients with laboratory confirmed RSV or influenza virus infection who underwent general anesthesia prior to diagnosis of viral infection during the period of study. Thirteen patients had RSV and 11 patients had influenza (91% with influenza A). Fifty-four percent of the patients were female and the median age was 10 months (intra-quartile range (IQR) 5 to 28 months). Eighty-three percent of the surgical procedures were conducted during the months of November to March.A total of 72 controls were randomly selected, matched at a ratio of three controls to each case. Forty-two percent of the controls were female and the median age was 10 months (IQR 6 to 27 months). Baseline characteristics of the cases and their matched controls analyzed by virus are shown in Table The median postoperative length of stay was three days (IQR 1 to 8 days) for the cases and two days (IQR 1 to 5 days) for the controls. Fifty-four percent of cases were admitted to the PICU following surgery compared to 35% of the controls. Of those patients admitted to the PICU following surgery, 31% of cases were unplanned PICU admissions compared to 17% of the controls. No patients in either group required ECLS or died prior to discharge. Outcomes for cases and matched controls analyzed by virus are shown in Table Patients with influenza had longer postoperative lengths of stay (p < 0.001) than matched controls. There was no difference in postoperative lengths of stay between cases and matched controls in patients with RSV. There was an increased risk of unplanned postoperative PICU admission (p = 0.04) in patients with RSV or influenza as compared to matched controls. There was no difference in the incidence of perioperative or postoperative events, or requirement of prolonged postoperative positive pressure ventilation between cases and controls.Our results suggest that children with occult influenza undergoing general anesthesia have a longer postoperative hospital length of stay as compared to matched controls. Children undergoing general anesthesia with occult RSV or influenza infection have an increased risk of unplanned PICU admission. We observed no difference between cases and matched controls related to the risk of perioperative complications, requirements for prolonged positive pressure ventilation, or mortality.It is not uncommon for the pediatric anesthesiologist to encounter a child with URI symptoms preoperatively. Such patients may experience increased airway reactivity in the perioperative period often presenting as coughing, bronchospasm, laryngospasm or hypoxemia. This study quantifies the impact of these complications on our young patients with RSV or influenza infections receiving general anesthesia for surgical procedures. Interestingly, our findings are significant despite a lack of URI symptoms in 50% of cases. This supports the concept that even trained pediatric anesthesiologists are unable to accurately detect infection with these agents, and raises further questions regarding the sensitivity of preoperative assessment of symptoms.The issue of pre-operative screening of children undergoing surgery for viral respiratory illness is complex. In children with signs or symptoms of serious infection , it should be an easy decision for the anesthesiologist or surgeon to postpone surgery if appropriate. It is the children with mild symptoms, or no symptoms at all, that can prove to be difficult.While rapid viral testing may provide helpful data for these decisions, there are important testing limitations. The current method for rapid screening of viral respiratory antigens at most institutions is immunochromotography. Despite the high specificity (> 90%) that most commercially available immunochromotography tests possess, sensitivity tends to be low (e.g. approximately 60% sensitive for RSV) . Direct Our study has several limitations. Initiation of viral testing at our institution during the period of study, whether in the operating room, PACU or inpatient ward, was performed on a case-by-case basis by the responsible anesthesiologist, pediatrician or surgeon. It is reasonable to postulate that some patients with viral infection underwent general anesthesia and were not included in our case sample due to a lack of testing. Not all of our controls have documented negative tests. However, because our routine involved both viral testing for symptomatic patients and procedure postponement for patients with systemic or lower respiratory disease, it is unlikely that patients with occult RSV or influenza infection in perioperative period would have been misclassified as controls. More importantly, if such misclassification did occur, it should only serve to decrease the likelihood that the present study would find a significant difference between groups; thus it may be that our results would show an even greater difference between groups if negative testing results were available for all controls.Our investigation was limited to the types of viruses for which testing was readily available for the time period of study at our institution. Other viral causes of acute respiratory infections in children include the parainfluenza viruses, rhinoviruses, adenovirus, human metapneumovirus and enteroviruses. With polymerase chain reaction testing becoming increasingly available for these viruses, it will be important to assess the impact these viruses have on perioperative outcomes in children undergoing general anesthesia.We have no information as to the vaccination status of the children in our study. It is reasonable to consider that children with chronic medical conditions were more likely to have received vaccination for seasonal influenza and as such, may be underrepresented in our sample. Our study was comprised of a relatively small number of cases at a single institution. Further research, including a multicenter trial, would be beneficial for confirmation of our outcomes.The current study provides a framework for evaluating the symptomatic child, and predicting the escalation in perioperative care associated with concurrent RSV or influenza infection in children who are undergoing elective surgery. For elective cases during the late fall and winter months, we believe it may be reasonable to test symptomatic children preoperatively for the presence of RSV or influenza virus; future studies are required to evaluate the cost-effectiveness of this practice. Our results indicate that children with RSV and influenza virus infections who undergo elective surgical procedures may be at increased risk for postoperative ICU admission and an increased length of hospital stay. These are discrete end points that should be considered in the discussion of risk and benefits associated with proceeding with elective surgery in the child with URI symptoms.The authors declare that they have no competing interests.MS participated in the study design, data collection and analysis, manuscript preparation and coordinated the day-to-day activities of the project. JL participated in the study design, data collection and analysis, manuscript preparation and assisted in the coordination of the day-to-day activities of the project. RG participated in the study design and manuscript preparation. JF participated in the study design and manuscript preparation. JS participated in the study design, data analysis, and manuscript preparation and oversaw the conduct of the study as senior author. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/11/16/prepub


The design of an optimal highly active antiretroviral therapy (HAART) customised on patient's background and viral genotyping, is still a challenge. EuResist has been the first data-driven system to be implemented as a free web-service for customised HAART optimisation, and was proven to be superior to all existing genotypic interpretation systems (GIS) since it takes into account not only genotype but multiple other variables.The EuResist database stores and updates periodically demographic, clinical, and genomic information of HIV+ patients from several countries in Western Europe. The EuResist system is trained on treatment change episodes (TCE) drawn from the EuResist data base, composed of a new drug regimen with a baseline HIV-1 RNA load and a CD4+ count, a baseline viral pol genotype, demographic and previous treatment information. Each TCE is associated to an HIV-1 RNA measurement after 8 weeks, which is used for the definition of virological success (below 500 copies/ml or >2 Log10 reduction from baseline HIV-1 RNA). The system is a combination of three independent machine learning models . The 2010 update has been trained on >5,000 TCE, composed of 20 FDA/EMEA approved nucleoside/tide, non-nucleoside, and protease inhibitors, including the recently approved compounds (RAC) tipranavir, etravirine and darunavir.The EuResist combined system performance in predicting the correct virological success of a TCE after 8-weeks  exhibited an area under the receiver operating characteristic (AUROC) of 0.8 . The inclusion of therapy history, clinical, and demographic covariates was shown to increase significantly prediction performance. In the subset of regimens containing RAC (n=151), the EuResist AUROC was 0.7 and Stanford HIVdb AUROC was 0.63, not allowing to assess a significant difference owing to the small sample size. See Figure Based on patient’s information and virus genotype, the EuResist web-service ranks the most effective HAART regimens by the probability to achieve an undetectable HIV-1 RNA load after 8 weeks. Thus, it might be useful in clinical practice. The 2010 update includes also RAC and achieved fair performance, which is expected to increase with expanding training data.


After the advent of highly active antiretroviral therapy, HIV infection has turned into a chronic disease and the patient’s profile has changed dramatically. We are now treating patients that are active both socially and professionally, in whom virological response has already been achieved and quality of life has become of utmost importance, directly correlated with the management of drug-associated adverse reactions. To describe this new patient profile, we present the management of a young family, living with HIV for more than 7 years.A family was referred to our clinic in the summer of 2012, by the Italian HIV Clinic where they had been under medical evaluation for over 7 years. They had tested positive at the routine pregnancy test 7 years back, when the mother was started on lopinavir/r, lamivudine+zidovudine. She gave birth to a healthy HIV-negative girl, who received postpartum prophylaxis for 3 months . After delivery the mother was switched to efavirenz plus lamivudine+zidovudine, the same therapy as her husband’s, a regimen they would both take for 7 years.In 2012 they moved to Romania and started attending our clinic. Both had undetectable viral loads and high CD4 counts (>700 cells/cmm), consistent with their medical history. However, both complained of lipodystrophy. The female also complained of peripheral neuropathy, sleep disturbance, vivid nightmares while the male presented increased serum cholesterol and triglycerides, irritability and sleep disturbance.Because their quality of life had started to decrease, after psychological evaluation and support, therapy was changed to raltegravir, abacavir+lamivudine (both had negative HLA-B*5701). This regimen was well tolerated, the symptoms improved, the CD4 count remained high, the male’s lipid profile returned to normal, and dual-energy X-ray absorptiometry (DEXA) evaluation at 3 months showed an almost normal lipid distribution for both patients, with a slight tendency toward android abdominal lipid distribution for the female patient. She remained undetectable (data available for 1 year follow-up), however, in the case of the male patient HIV-RNA rose to 320 copies/mL at 3 months, possibly associated with the impact of the drug regimen on viral reservoirs. At 6 months, the viral load became undetectable and remained that way to date.Correct and prompt management of adverse reactions is an important aspect in patients on antiretroviral therapy, since this may lead to improved quality of life and better social and professional integration of patients.


The clinical relevance of residual low level viremia (LLV) in patients on steady HAART is debated. Similarly the clinical usefulness of HIV-RNA cut-offs lower than 50 copies/ml is questioned. Aim of this study was to analyze the dynamics of LLV in patients on HAART by means of a high resolution test for HIV-RNA.This is a prospective, single-center, cohort study in patients on stable HAART . All patients with a confirmed viremia <50 copies/ml were enrolled. Patients were monitored prospectively with determinations of HIV-RNA every 4 months performed with an enhanced PCR test with a lower limit of detection of 3 copies/ml. ITT analysis is reported.A total of 505 patients  with a mean age of 45.6 years (SD 7.6) were enrolled. At baseline the mean CD4 count was 667 cells/µl (SD 268) and VL was < 3 copies/ml in 73.9% and between 3 and 50 copies/ml in the remaining 26.1% of cases. Over the following 8 months period, patients with a baseline VL < 3 copies/ml presented a stable HIV-RNA below this threshold in 72.8% of cases, a level between 3 and 50 copies/ml in 27.2% of cases while no patient steadily rebounded above the 50 copies/ml threshold. On the contrary, patients with a baseline HIV-RNA between 3 and 50 copies/ml, in the follow-up, presented a stable VL < 3 copies/ml in 43.8% of cases, a VL between 3 and 50 copies/ml in 53.1% of cases, while 3.1% of patients steadily rebounded above the 50 copies/ml threshold (P < 0.0001). In the multivariate analysis the only variable significantly associated with viral dynamics was the third drug in the HAART regimen. A steady VL < 3 copies/ml, a VL between 3 and 50 copies/ml or a steady VL > 50 copies/ml was detected, respectively, in 71.8%; 27.7% and 0.6% of NNRTI-treated patients, while the same figures for PI-treated subjects were 56.2%; 42.9% and 1.0% (P = 0.029).The presence of a LLV is associated to a low risk of virological failure; however, in selected patients it may be indicative of effective virus replication leading to virological rebound. Patients treated with a NNRTI-based HAART compared to those receiving a PI-based regimen show a statistically significant more pronounced and steady control of viral replication. Further, prolonged studies are needed to assess the clinical relevance of residual LLV and eventually define new cut-off values predictive of a better control of virus replication.


Isospora belli causes diarrhoea in patients with AIDS. Most respond to targeted therapy and recommendations are that secondary prophylaxis can be stopped following immune reconstitution with ART. We report eight cases of chronic isosporiasis that persisted despite standard antimicrobial therapy, secondary prophylaxis, and good immunological and virological response to ART. Median CD4 nadir was 175.5 cells/mm3 and median highest CD4 while symptomatic was 373 cells/mm3. Overall 34% of stool samples and 63% of duodenal biopsy specimens were positive for oocytes. Four patients died, two remain symptomatic and two recovered. Possible explanations for persistence of symptoms include host factors such as antigen specific immune deficiency or generalised reduction in gut immunity. Parasite factors may include accumulating resistance to co-trimoxazole. Research is required to determine the optimum dose and duration of co-trimoxazole therapy and whether dual therapy may be necessary. Mortality was high and pending more data we recommend extended treatment with high-dose co-trimoxazole in similar cases. Isospora belli is among the most frequently identified causes of chronic diarrhoea in patients with AIDS 33 for greater than 6 months on ART although this has not been validated in clinical trials Isospora belli following immune reconstitution and chronic isosporiasis has only rarely been reported in immunocompetent hosts Isospora belli in South Africa is co-trimoxazole 960 mg 6 hourly for 10 days followed by co-timoxazole 960 mg daily until CD4 count >200 cells/mm3. We report 8 cases of recurrent, chronic isosporiasis in HIV-infected adults on ART in whom HIV replication was suppressed to undetectable levels and in whom immunological evidence of immune reconstitution was demonstrated.The coccidian parasite Isospora belli oocytes, persistent symptoms despite appropriate targeted treatment and virological suppression and immune reconstitution on ART.Local databases of patients referred to the infectious diseases services at GF Jooste and Groote Schuur hospitals in Cape Town were searched to identify patients with HIV infection and chronic diarrhoea. Paper and electronic clinical records were then examined to identify those with >1 stool or biopsy sample positive for Local research ethics committee approval was granted to analyse data held on a departmental database. Information reflecting the identity of the patients was removed.Salmonella and Shigella species and negative Clostridium difficile toxin testing. In addition 7 patients underwent duodenal biopsy, often on multiple occasions, with no alternative cause for chronic diarrhoea being identified. All patients achieved an undetectable HIV viral load (<50 copies/ml) while remaining symptomatic. Four patients never had an HIV viral load >50 copies/ml despite regular measurement. Three patients had only low level intermittent viraemia (maximum 2780 copies/ml) and one experienced high level virological failure (306671 copies/ml) but remained symptomatic despite subsequently suppressing HIV viral load to <50 copies/ml. Median CD4 nadir was 175.5 cells/mm3 and all patients were symptomatic despite sustained improvement in CD4 count (median increase 227 cells/mm3). The highest CD4 count at which a patient was symptomatic was 1013 cells/mm3 and the median of the highest CD4 count at which patients were symptomatic was 373 cells/mm3. Four patients died of complications of chronic diarrhoea and 2 remain symptomatic. Two patients eventually recovered but had been symptomatic for 2 years despite adequate targeted treatment, virological suppression and immune recovery.From January 2005 to January 2010 eight patients meeting the search criteria were identified. Five were female and median age at presentation was 34 years; their clinical course is summarised in 3 weighing 52 kg, presented with 3 months of watery diarrhoea. No cause was found despite investigations including multiple stool samples examined with modified acid-fast staining. Stavudine, lamivudine and efavirenz were started 2 months later, with efavirenz being switched to lopinavir/ritonavir following a hepatitis B flare at 3 months. Despite ART, profuse watery diarrhoea continued and after 4 months she was admitted to hospital for rehydration, weighing 44 kg. Isospora belli oocytes were observed in stool and treatment with co-trimoxazole at 1920 mg b.i.d was successful. She gained weight to 48 kg although her symptoms returned 3 months later, when oocytes were again identified in stool, despite secondary prophylaxis with co-trimoxazole 960 mg daily. At this time, her CD4 count had risen to 174 cells/mm3 and HIV viral load was 1000 copies/ml. Repeat episodes of severe diarrhoea with electrolyte disturbance continued despite secondary prophylaxis; 1 year after starting ART she weighed 38 kg. She required inpatient treatment for diarrhoea and dehydration on 18 occasions over 4 years. Each time, she improved on treatment, which included the use of single drug therapy or combinations of oral or intravenous co-trimoxazole, ciprofloxacin, albendazole and nitazoxanide. She relapsed on each occasion, despite secondary prophylaxis with co-trimoxazole 960 mg b.i.d with or without ciprofloxacin 500 mg b.i.d. She was supported with intravenous fluids, electrolyte supplementation and required total parenteral nutrition on several occasions. Diarrhoea was unlikely to be a side-effect of lopinavir/ritonavir as she remained asymptomatic between acute episodes while continuing this medication. Her CD4 was consistently >150 cells/mm3, peaking at 237 cells/mm3 after 20 months of ART. HIV viral load was consistently <50 copies/ml from 12 months of ART. She died from complications of chronic diarrhoea 4 years after starting treatment for isosporiasis.A 33 year old HIV-infected woman with a CD4 count of 32 cells/mm3 was treated with co-trimoxazole for microbiologically-proven Isospora belli in stool and started on secondary prophylaxis. Stavudine, lamivudine and nevirapine was started, but her gastro-intestinal symptoms never completely settled despite secondary prophylaxis with co-timoxazole at doses between 960 mg o.d. and 1920 mg b.i.d. CD4 count rose to 729 cells/mm3 and HIV viral replication was suppressed after 6 months of ART. After 2 years of intermittent symptoms, when her weight was 43 kg she was referred for gastroscopy and Isospora belli was observed in duodenal biopsy. She deteriorated and diarrhoea became more continuous; over the following 30 months she was admitted to hospital with increasing frequency. Isospora belli was observed in duodenal biopsies on 2 further occasions, but was absent from multiple stool samples taken at the same time. Co-trimoxazole, ciprofloxacin, paromomycin, pyrimethamine and nitazoxanide were all tried for relapses, with good initial response, but subsequent relapse on a combination of high dose co-trimoxazole and ciprofloxacin 500 mg b.i.d. secondary prophylaxis. Her weight dropped to 32 kg and she required 6 admissions in a 4 month period. Her clinical course was complicated by a deep vein thrombosis, major depressive episode, and hypocalcaemia-induced tetany requiring intubation and ICU admission. ART was continued throughout. HIV viral load recordings were consistently <50 copies/ml and her CD4 count peaked at 1013 cells/mm3, 5 years after starting ART. Five years after her first presentation, she died at home having declined hospital admission for a further episode of severe diarrhoea.A 33 year old HIV-infected woman with a CD4 count of 210 cell/mmTo our knowledge, this case series represents the first description of chronic, relapsing isosporiasis in HIV-infected adults despite evidence of reconstitution of CD4 T-cell count and suppression of HIV replication on ART. We think that Immune Reconstitution Inflammatory Syndrome (IRIS) is an unlikely explanation for our observations given the absence of a disproportionate inflammatory reaction when initiating ART. Our experience was of considerable morbidity necessitating multiple hospital admissions and high mortality of up to 50%. Depression was a common consequence.Isospora belli DNA in stool has been developed recently with promising results although prospective studies on clinical specimens are lacking There is no accepted gold standard method for diagnosing isosporiasis and the sensitivity of diagnostic tests is therefore unknown. However, we believe observing oocytes in stool or duodenal biopsy has a specificity approaching 100%. Our experience was of multiple negative stool examinations in patients even during acute episodes and when duodenal biopsy was positive; in total only 34% of stool samples were positive. This suggests that stool microscopy is insensitive and of limited use in excluding isosporiasis. Duodenal biopsies were more likely to be positive in our patients, in total 63% were positive, but duodenal biopsy is not routinely available in low resource settings where most patients with HIV live. A molecular test for identifying 3, but our observation was that whilst a low CD4 nadir may be necessary for infection to become established, this case series represents a sub-group of patients who failed to clear the infection following reconstitution of T cell numbers. We hypothesize that several factors may contribute to this phenomenon. There may be a failure of recovery of the antigen-specific immune response to Isospora belli alone, as despite recurrent clinical episodes related to this specific infection, our patients did not manifest other opportunistic infections which would indicate a broader functional defect in T cell immunity. ART is known to lead to substantially delayed, and only partial restoration of gastrointestinal CD4 T cells Isospora belli is a possible mechanism. Drug malabsorption is another possible factor although each patient responded adequately to ART and initial oral therapy in hospital suggesting otherwise. However, specific malabsorption of co-trimoxazole cannot be ruled out. Poor compliance with secondary prophylaxis was not judged to be a contributor, due to pill count monitoring and adherence counselling. Therapeutic drug monitoring was not performed, hence anomalies in drug pharmacokinetics cannot be ruled out.Isosporiasis is typically associated with CD4 counts <200 copies/mmIsospora belli have been described in the lymphoid tissue of a patient with AIDS Drug resistance to co-trimoxazole may have played a role in relapse. Acute episodes of diarrhoea generally responded to standard co-trimoxazole doses, arguing against resistance, although intermediate susceptibility of strains in patients who relapse may explain our observations. Unizoic cysts of Pneumocystis jirovecii and Cryptococcus neoformans in patients with sustained improvements in CD4 counts Isospora belli infection are lacking and our data suggest that similar guidelines may not be safe, at least in a sub-group of patients who remain symptomatic.Prospective studies have shown the safety of discontinuing secondary prophylaxis against The research priority in chronic isosporiasis is to determine the optimal dose and duration of the co-trimoxazole, the most efficacious drug. The efficacy of alternative treatments including pyrimethamine and nitazoxanide must be determined and combination therapy, possibly for extended periods, should be assessed in non-responders. In similar patients we currently recommend co-trimoxazole 1920 mg b.i.d. continued for up to 2 years while monitoring for myelotoxicity.Isospora belli. Chronic isosporiasis was likely in these cases, so clinical diagnosis may be necessary in areas where duodenal biopsy is not routinely available.We have managed several similar cases of chronic diarrhoea in HIV patients with immune restoration and virological suppression in rural primary care facilities, who despite negative stool samples (endoscopy and biopsy unavailable) have responded to empiric treatment for There are several limitations to our study. Detailed analysis of strains from these patients was not undertaken, so we are unable to differentiate between reactivation of clinical disease with the same strain or re-infection with another strain. There are no accurate data for the incidence of isospora diarrhoea in our setting so we are unable to directly compare our patients with a group who were successfully treated. However, a survey of laboratory reports over 2 ½ years at GF Jooste hospital found 36 individuals with at least 1 stool sample positive for isospora oocytes. Three of these are included in our case series but the outcome of the others is unknown (unpublished data). The ability to exclude all other causes of diarrhoea was limited by resources. However multiple stool cultures in all patients and small bowel biopsies in most patients failed to reveal an alternative cause.We have described a case series of chronic isosporiasis due either to relapse or re-infection, in patients with good immunological and virological response to ART. Stool microscopy was often negative and although duodenal biopsy seemed to perform better, this investigation is rarely available outside of the major tertiary hospitals in Africa. The mortality rate in our case series was high, suggesting that new approaches to treatment such as long term intravenous therapy need to be assessed in this sub-set of patients.