Few studies have compared the impact of different antiretroviral regimens on residual immune activation and inflammation with discordant results. Aim of the study was to investigate the impact of various antiretroviral regimens on markers of immune activation and inflammation during the first two years of effective therapy.e-transformed values and multivariable linear regression models were used to study the effect of the treatment components, after adjusting for factors that might have influenced the choice of ART regimen or biomarker levels. Differences were expressed as the mean fold change percentage difference (Δ).We studied HIV-infected antiretroviral-naïve patients who began cART with either abacavir/lamivudine or tenofovir/emtricitabine, combined with ritonavir-boosted lopinavir (LPV/r), atazanavir (ATV/r) or efavirenz (EFV). All the patients had a virological response within 6 months, which was maintained for 2 years with no change in their ART regimen. C-reactive protein (hs-CRP), interleukin-6 (IL-6), soluble CD14 (sCD14), monokine induced by interferon-γ (MIG) and interferon-γ-inducible protein-10 (IP-10) were measured in stored plasma obtained at cART initiation and 24 months later. Mean changes from baseline were analyzed on log3 and HIV-1 RNA level 4.6 log10 copies/ml. During the 2-years study period, IL-6, IP-10 and MIG levels fell significantly, while hs-CRP and sCD14 levels remained stable. IP-10 and MIG levels declined significantly less strongly with ATV/r than with EFV , while no difference was noted between LPV/r and EFV. The decline in IL-6 did not differ significantly across the different treatment components.Seventy-eight patients  with a median age of 43 years met the inclusion criteria. Their median baseline CD4 cell count was 315/mmAfter the first 2 years of successful cART, IL-6, IP-10 and MIG fell markedly while hs-CRP and sCD14 levels remained stable. The only impact of ART regimen was a smaller fall in markers of immune activation with ATV/r than with EFV. Our results suggest that these markers could be worthwhile when evaluating new antiretroviral drugs. Antiretroviral therapy (ART) leads to a dramatic reduction in HIV-related morbidity and mortality  and currBy comparison with HIV-uninfected individuals, several studies have shown an excess of comorbidities in HIV-infected patients on virologically effective treatment, including metabolic disorders , decreasNow that ART is recommended for all HIV-infected patients, and life expectancy is greatly prolonged by suppressive ART despite the lack of viral eradication, it is important to compare the potential impact of different antiretroviral strategies on residual immune activation and inflammation. There are some arguments to suggest that different drugs from different classes may have different impact on immune activation and inflammation. In the Spiral study, a switch from PI-based therapy to a raltegravir-containing regimen in patients with suppressed viremia led to a decrease in biomarkers associated with inflammation, insulin resistance and hypercoagulability .Few studies have examined the impact of first-line ART on biomarkers of inflammation and immune activation, and they have given discordant results -31. ThesWe therefore explored the impact of different first-line ARV regimens on soluble markers of inflammation and immune activation during the first two years of effective therapy, while controlling for potential confounders such as HIV replication, and changes in the initial regimen.We compared the impact of commonly used first-line antiretroviral drugs on soluble markers of inflammation and immune activation, while controlling for potential confounders. In order to avoid the influence of previous ARV exposure, active viral replication and treatment switches, we restricted our analysis to a homogenous group of treatment-naive HIV-infected patients who had experienced a rapid and persistent virological response and remained on their initial regimen for 2 years. Because the biomarkers of interest were not part of the patients' routine biological monitoring, we restricted our analysis to patients for whom plasma samples stored at ART initiation and 2 years later were available. The analyses were also adjusted for baseline characteristics that might have influenced the choice of cART regimen or affected biomarker levels, such as the age, smoking status, CD4 cell count, prior AIDS-defining events, plasma HIV-1 viral load, and hepatitis virus coinfection.All HIV-infected patients receiving care in the Infectious Diseases department of Pitié-Salpêtrière Hospital  have their clinical, biological and therapeutic findings recorded prospectively in standardized electronic medical records (NADIS). Biological data obtained in the hospital, such as HIV RNA levels and immunological parameters, are directly imported from the laboratory computer system, thus minimizing collection bias. The quality of the database is ensured by automated checks during data capture, and by regular controls and annual assessments. Routine blood tests are performed at each hospital visit, and residual plasma is stored frozen, being identified by a serial number.All HIV-1-infected patients who started first-line cART between January 2006 and December 2009 were screened for eligibility, using the hospital database. Patients were included in this study if they received either tenofovir-emtricitabine (TDF/FTC) or abacavir-lamivudine (ABC/3TC), combined with efavirenz or with a ritonavir-boosted protease inhibitor (atazanavir (ATV/r) or lopinavir (LPV/r)). In order to control for potential causes of inflammation/activation due to persistent plasma viral replication, we only studied patients who had a rapid and persistent virological response, defined by a plasma HIV-1 viral load (VL) below 400 copies/mL at 6 months and below 50 copies/mL at 24 months, with no values above 1000 copies/ml between month 6 and month 24. Other eligibility criteria included no change in antiretroviral therapy throughout the 24 months of the study, and the availability of frozen plasma samples obtained at baseline (D0) and month 24 (M24). The study was approved by the Pitié-Salpêtrière institutional review board, and the patients were asked to give their written consent to the use of their medical information and plasma samples, as required by French law.We selected markers of inflammation and immune activation that can be reliably measured in frozen plasma. We evaluated IL-6 and hs-CRP as markers of inflammation, soluble CD14 (sCD14) as a marker of monocyte activation, and interferon-γ-inducible protein 10 (IP-10) and monokine induced by interferon-γ (MIG) as markers of T-lymphocyte and macrophage activation.Enzyme-linked immunosorbent assays (ELISA) were used according to manufacturer’s instructions to quantify IL-6, sCD14  and hs-CRP . IP-10 and MIG levels were determined on thawed diluted plasma with Cytometric Bead Array kits (BD™ CBA) on a BD FACS Canto I according to manufacturer’s instructions. Coefficients of variation were 6.9-7.8% for IL-6, 7.9–8.5% for hs-CRP, 4.8–7.4% for sCD14, 4% for IP-10 and 9–13% for MIG. Plasma samples were allowed to thaw for 50 minutes at room temperature before centrifugation for 5 minutes at 1000 rpm, followed by distribution into Eppendorf tubes in the amounts required for each assay kit. Plasma was diluted as recommended by the kit manufacturers. Standards provided with the kits were measured in duplicate and the mean value was used as reference. Samples with values higher than the highest standard value were further diluted and retested. D0 and M24 samples from each patient were tested in the same run.e-transformed for analysis. For each marker, changes between D0 and M24 (mean fold change) were expressed as the geometric mean of their ratio after loge transformation. A paired one-sample t test was used to identify significant differences in the overall changes in each marker.Two NRTI backbone combinations (TDF/FTC vs ABC/3TC) and three third agents  were compared in a factorial design. Baseline characteristics were compared between treatment groups by using the Wilcoxon and chi-squared tests in order to identify variables associated with the choice of treatment. Because the marker values were not normally distributed, they were logLinear regression models were used to investigate the impact of the different NRTI backbones and the different third agents on the biomarker changes. The results are expressed as the estimated percentage difference between the mean fold changes observed with a given drug, using TDF/FTC and EFV as the reference groups for the comparison. Relationships between baseline covariables and changes in each biomarker were examined in univariable linear regression models. These covariables were sex, age, body mass index, smoking status, hepatitis B or C virus (HBV or HCV) coinfection, prior AIDS-defining events, and the pre-ART CD4 cell count and viral load. Baseline covariables associated with changes in at least one biomarker (p < 0.10) and viral blips above 50 copies/ml between M6 and M24 were retained in all multivariable linear regression models in order to control for factors that might have influenced the choice of cART regimen or affected biomarker levels. Age and smoking status were included in the multivariable model since these variables are known to influence marker levels ,33. InteBetween January 2006 and December 2009, a total of 539 patients began first antiretroviral therapy and remained under care over two years in our department. Of those, 370 patients had a rapid and persistent virological response over the two years. Of them, 280 patients who began therapy with abacavir/lamivudine or tenofovir/emtricitabine plus efavirenz, atazanavir/r, lopinavir/r or fosamprenavir/r were eligible for the study. The remaining 90 patients were not considered since they either received no longer recommended antiretroviral therapy such as combivir and invirase (n = 64) or were included in protocols evaluating new drugs such as darunavir and rilpivirine. Frozen plasma samples at baseline and after two years were available for 149 patients. Characteristics of patients who had stored plasma were not different from those did not have it for age, CD4 cell count, HIV-RNA level, AIDS defining events and for prescribed antiretroviral therapy. Two patients who withheld their consent were excluded. Among the remaining 147 patients, 78 remained on the same antiretroviral regimen throughout the 2-year study period and comprised the study population. Modifications in first ART regimen were due to side effects, drug toxicity, or switch to newly available drugs.10 copies/mL, the median CD4 cell count was 315/mm3 and the median CD4/CD8 cell ratio was 0.30. Seven patients (9%) had an AIDS-defining event. Four patients were HCV-PCR-positive and two were HBsAg-positive.The NRTI backbone consisted of TDF/FTC in 61 patients (78%) and ABC/3TC in 17 patients. The third agent was EFV in 36 patients (46%), ATV/r in 27 patients (35%) and LPV/r in 15 patients. At baseline, the median viral load was 4.6 log3), a value significantly different from the median count in patients prescribed ATV/r (330 cells/mm3) or EFV (342 cells/mm3) (p = 0.020). Patients prescribed TDF/FTC had a lower median CD4 cell count than patients prescribed ABC/3TC . Four patients prescribed LPV/r had an AIDS-defining event, compared to only one patient prescribed ATV/r and two patients prescribed EFV (p = 0.030). All the patients with hepatitis virus coinfection were prescribed LPV/r (p < 0.001).Baseline characteristics were well balanced across the treatment groups, with the following exceptions. As shown in Table 3 and the CD4/CD8 ratio by 0.42. At M24, the median CD4 cell count was 530/mm3 (IQR 393 to 683) and the median CD4/CD8 ratio was 0.76 (IQR 0.47 to 1.1). No patient developed AIDS defining event during the two years of treatment, while 11 patients experienced a non-AIDS defining event, consisting of pneumonia (n = 5), nephropathy (n = 4), osteoporosis (n = 1) and idiopathic gout (n = 1). None of the 28 smoking individuals quitted smoking while on treatment.As requested, all the patients had plasma HIV viral loads below 400 copies/ml at M6 and below 50 copies/ml at M24. At M6, 72 patients (92%) had viral loads below 50 copies/ml. Between M6 and M24, twelve patients (15%) had isolated viral loads above 50 copies/ml . Among patients receiving TDF/FTC, 10 patients (16%) had isolated plasma viral loads above 50 copies/ml, compared to 2 patients (12%) receiving ABC/3TC (p = 0.64). Among patients receiving EFV, 3 patients (9%) had isolated viral loads above 50 copies/ml, compared to 4 patients (17%) receiving ATV/r and 5 patients (33%) receiving LPV/r (p = 0.08). During the 2-year study period, the CD4 cell count increased by 216/mmAt baseline, the only difference in plasma biomarkers levels across the treatment groups was a higher IL-6 level in patients prescribed LPV/r (median 2.17 pg/ml) than in patients prescribed ATV/r or EFV .At M24, IL-6, IP-10 and MIG levels were significantly lower than at baseline , while sCD14 levels were unchanged , no significant difference in the decline in the IL-6 level was found between the TDF/FTC and ABC/3TC groups   shows that the choice of the third antiretroviral agent influenced the degree of decline in markers of T-lymphocyte and macrophage activation. Plasma levels of IL-6, IP-10 and MIG fell by at least 40% in all the treatment groups, while the decline in hs-CRP levels failed to reach statistical significance and sCD14 levels were unchanged. The only observed difference between the treatment groups was that the T-lymphocyte and macrophage activation markers IP-10 and MIG fell less markedly in patients receiving ATV/r than in patients receiving EFV; no difference was found between LPV/r and EFV. Our patient population represents the naïve patients who initiated cART between January 2006 and December 2009 with a moderate immune deficit and a median viral load slightly below 100000 copies/ml and who experienced a rapid virological response in usual care in France.Several aspects of study design are important in studying the influence of different antiretroviral drugs on biomarker variations in patients starting first-line therapy. First, it is important to control for viral replication, which is a major cause of persistent immunological activation and inflammation. As a result, the approach used in randomized trials, which includes all patients independently of their virological response, may not be appropriate. Furthermore, removal of patients with uncontrolled viral load from a randomized trial will result in a simple observational study. Despite the lack of randomization in our study, baseline characteristics were well balanced across the treatment groups, and factors that might have influenced the choice of cART regimen or the time course of the markers of interest were systematically included in the analyses. In addition, all the patients under care in the Infectious Diseases department of Pitié-Salpêtrière Hospital were screened for eligibility criteria, thus avoiding any major selection bias. However, one cannot exclude that unrecognized confounding regarding treatment selection could be present. In our study, the strict inclusion criteria regarding the viral replication resulted in a limited sample size. However, even with this sample size, significant differences were detected for some of the comparisons. In addition, most p-values of non-significant tests were above 0.15 indicating that for most comparisons, power was not an issue. It is possible that the freezing of plasma sample could have modified the marker levels and diminished our ability to detect differences. However, all the groups of antiretroviral regimens were studied similarly and this should not have biased the comparison between groups. Moreover, as in most published studies, studying thawed plasma allowed to study all marker dosages simultaneously in the same experiment, limiting the inter-individual variability. The high variability of inflammatory markers between individuals could have precluded seeing differences between treatment groups.Elevated IL-6 and CRP levels before antiretroviral treatment initiation or after treatment interruption have been linked to a higher risk of AIDS-defining events and death ,34. In oElevated plasma sCD14 levels are an independent predictor of death among HIV-infected patients . SCD14, Persistent immune activation despite virologically effective therapy has been linked to immunological failure ,42. WhilThe differential effect of the studied antiretroviral drugs on changes of two immune activation markers such as IP-10 and MIG levels suggests that these markers could be worthwhile when evaluating new antiretroviral drugs. The presence of sCD14 despite two years of viral suppression reflects the persistence of immune activation, and may contribute to the maintenance of residual viremia and HIV DNA reservoirs . With thNo members of the study team have any financial or personal relationships with people or organizations that could inappropriately influence this work. Dominique Costagliola and Christine Katlama have received at some stage in the past travel grants, consultancy fees, honoraria and study grants from various pharmaceutical companies including Bristol-Myers-Squibb, Gilead Sciences, Janssen-Cilag, Merck-Sharp & Dohme-Chibret and ViiV Healthcare.Conception and design: SH, AG, MG, GC, A-GM, DC, CK. Plasma samples collection: SH, SF, A-GM. Biomarkers measurements: SH, AG, GC, BA. Collection and assembly of data: SH, AG, MG, SF, FC. Statistical analyses: SH, MG, DC. Critical revision of the article for scientific accuracy: all. Final approval of the article: all.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/122/prepub


Document progress in HIV-treatment in the Netherlands since 1996 by reviewing changing patterns of cART use and relating those to trends in patients' short-term clinical outcomes between 1996 and 2010.3 within 12 months of starting cART; ii) achieving viral load (VL) suppression ≤1,000 copies/ml within 12 months of starting cART; iii) switching from first-line to second-line regimen within three years of starting treatment; and iv) all-cause mortality rate per 100 person-years within three years of starting treatment.1996–2010 data from 10,278 patients in the Dutch ATHENA national observational cohort were analysed. The annual number of patients starting a type of regimen was quantified. Trends in the following outcomes were described: i) recovery of 150 CD4 cells/mmBetween 1996 and 2010, first-line regimens changed from lamivudine/zidovudine-based or lamivudine/stavudine-based regimens with unboosted-PIs to tenofovir with either emtricitabine or lamivudine with NNRTIs. Mortality rates did not change significantly over time. VL suppression and CD4 recovery improved over time, and the incidence of switching due to virological failure and toxicity more than halved between 1996 and 2010. These effects appear to be related to the use of new regimens rather than improvements in clinical care.The use of first-line cART in the Netherlands closely follows changes in guidelines, to the benefit of patients. While there was no significant improvement in mortality, newer drugs with better tolerability and simpler dosing resulted in improved immunological and virological recovery and reduced incidences of switching due to toxicity and virological failure. Combination antiretroviral therapy (cART) was first introduced more than 15 years ago. Since 1996, over twenty new antiretroviral drugs have been licenced for the treatment of HIV-infection A non-selective database that collects data from all HIV-infected patients in clinical care in the Netherlands provides a unique opportunity to record the progress of cART since 1996 across a variety of clinical and non-clinical markers. We aim to use this dataset to document progress in HIV-treatment by: i) reviewing the changing patterns of first-line cART regimens between 1996 and 2010 in the Netherlands; ii) to describe time trends in a variety of short-term clinical and non-clinical markers; and iii) to relate new regimens to trends in patients' short-term clinical outcomes as a measure of population-level effectiveness.www.hiv-monitoring.nl).ATHENA is a national observational cohort that includes all HIV-patients followed in 25 designated HIV treatment centres in the Netherlands since 1996. The design of this cohort has been described previously st January 1996. Patients were antiretroviral drug-naïve prior to entering the study and started cART during follow-up. Women known to have been pregnant during follow-up were excluded. Data was analyzed up to and including 31st December 2010. The number of patients in the analysis was 10 278 patients.Patients from the ATHENA cohort were included in this analysis if they were aged 18 or over, infected with HIV-1, and diagnosed with HIV from 1Combination therapy was defined as regimens containing three or more antiretroviral drugs. Regimen types were classified by nucleoside reverse transcriptase inhibitor (NRTI) backbone: i) lamivudine (3TC) and stavudine (d4T); ii) 3TC and zidovudine (AZT); and iii) tenofovir (TDF) with 3TC or emtricitabine (FTC). The analysis was further stratified into regimens combined with either: i) non-nucleoside reverse transcriptase inhibitors (NNRTI); ii) non-boosted protease inhibitors (PI); or iii) (ritonavir)-boosted-PIs. Regimens other than those described above were classified as ‘other’ in further analysis. The choice of regimen classification reflects the main regimen types used between 1996 and 2010 in the Netherlands.3 within 12 months of starting cART; ii) hazard rate of achieving viral load (VL) suppression ≤1,000 copies/ml within 12 months of starting cART; iii) the incidence of switching from first-line to second-line regimen within three years of starting treatment; and iv) all-cause mortality rate per 100 person-years within three years of starting treatment. A threshold of 1,000 copies/ml for VL suppression was chosen to allow for comparison across the years, due to the changing detection threshold for VL suppression, from 1,000 copies/ml in 1996 to 20 copies/ml in 2010.Clinical outcome was measured using: i) hazard rate of recovering 150 CD4 cells/mmA switch was defined as a change in regimen that included at least one new antiretroviral drug. Reasons for switching were classified as: i) toxicity, ii) simplification/new medication becoming available, iii) virological failure, and iv) other reasons st December 2010), or the outcome of interest, whichever occurred first. Follow-up was divided into three time periods, 1996–2000, 2001–2005, and 2006–2010, early cART, medium cART and late cART period. Mortality was treated as a censored event in analysis where mortality was not the outcome of interest. Wald statistics were used to determine significance of this analysis using a significance level of 0.05 throughout.Time-dependent Cox proportional hazard model and Kaplan-Meier curves were used as time-to-event analysis. Cox regression analyses were carried out on all four clinical outcomes defined above. When analysing switching, Cox analysis was limited to virological failure and toxicity. Follow-up time was defined as months from the start of treatment until the date of end of follow-up time, closure of the database , CD4 and VL at the start of treatment, route of HIV transmission (men-who-have-sex-with-men (MSM), heterosexual, injecting drug use (IDU), and vertical infection, blood infusion, or unknown), and region of origin. Categories of regions of origin were Netherlands, Europe (excluding the Netherlands), Sub-Saharan Africa, and other. CD4 counts categories were <200, 201–350, 351–500, and >501 cells/mmDemographic characteristics of the patients are summarized in The cART regimen types used between 1996 and 2010 are presented in Between 1996 and 2010, the number of patients starting treatment increased. In 1996, 156 patients started on therapy per year, increasing to around 1,000 patients per year after 2007. From 1996 to 2004 the majority of patients started on a backbone of 3TC/AZT . Its useThe Kaplan-Meier estimate of the percentage of deaths at three years was 6.4% (95% CI 5.9–6.9). The short-term all-cause mortality rate of patients on cART between 1996 and 2010 is presented in All-cause mortality was significantly increased in older patients , in patients who had acquired HIV via injecting drug use compared to MSM , in smokers , and with increasing cumulative AIDS-defining events . Patients on regimens of TDF/3TC or TDF/FTC with boosted-PIs may experience increased risk of mortality compared to patients on TDF/3TC or TDF/FTC with NNRTIs .3 within 12 months of treatment initiation. The CD4 recovery rate of 150 cells/mm3 following treatment initiation was significantly higher in 2006–2010 compared to 2001–2005  (Between 1996 and 2010 70.4% (95% CI 69.5–71.3) patients reached a CD4 increase of 150 cells/mm = 0.03) . The dif3 was improved in women , worse in older patients , patients from Sub-Saharan Africa compared to Dutch-born , and patients who had acquired HIV through heterosexual contact or injecting drug use compared to MSM . CD4 recovery was also worse in patients who had a CD4 count below 200 cell/mm3 or above 500 cells/mm3 and VL below 100 000 copies/ml at the start of treatment . Patients on regimens of 3TC/d4T or TDF/3TC or TDF/FTC with boosted-PIs may experience improved rates of immunological recovery, compared to patients on TDF/3TC or TDF/FTC with NNRTIs .CD4 recovery of 150 cells/mmThe Kaplan-Meier estimate of the percentage of patients achieving VL suppression <1,000 copies/ml by 12 months was 94.7% (95% CI 94.2–95.1). VL suppression following the start of cART was improved in recent years  . The difIn adjusted analysis, VL suppression was worse in patients non-Dutch, European patients , for those who had acquired HIV either via heterosexual contact or injecting drug use , and in patients with a VL above 100,000 copies/ml at the start of treatment . Regimens of 3TC/AZT with non-boosted or boosted-PIs, and TDF/3TC or TDF/FTC with boosted-PIs may be associated with worse rates of VL suppression .During follow-up, 4,481 patients (44%) switched to second-line. The short-term incidence of switching, due to virological failure or toxicity, from first-line regimens has declined between 1996 and 2010 , suggestIn models adjusted for cART regimen type, differences between time periods were not significant . The onl3 and VL above 100,000 copies/ml at the start of treatment. Patients on regimens of 3TC/d4T or 3TC/AZT with unboosted-PIs had an increased risk of virological failure, while patients on 3TC/AZT or TDF/3TC or TDF/FTC with boosted-PIs had a decreased risk of virological failure.Older patients and women had an increased risk of toxicity-driven switch, while patients who acquired HIV via heterosexual sex had a reduced risk of toxicity-driven switch compared to MSM. Patients on backbones of 3TC with either d4T or AZT had an increased risk of toxicity-driven switch compared to patients on TDF/3TC or TDF/FTC with boosted-PIs. The incidence of switching due to virological failure was higher in patients from Sub-Saharan Africa, and with a CD4 count below 200 cells/mmAmong patients who started treatment between 1996 and 2000, the three main adverse events leading to a switch in regimen were gastrointestinal- , hepatological- (15.2%), and hematological-related (10.1%) . In 2001Between 1996 and 2010, first-line regimens changed from 3TC/AZT-based or d4T/3TC-based regimens with PIs to TDF/3TC or TDF/FTC with NNRTIs. There was no significant reduction in short-term mortality between 1996 and 2010 when controlling for clinical and demographic factors, such as age and CD4 at start of cART, likely because the greatest impact on short-term mortality occurred before this, as a consequence of the introduction of combination therapy The pattern of changes in first-line cART regimens in the Netherlands between 1996 and 2010 parallels changes in Dutch national treatment guidelines and the publication of a number of clinical studies To our knowledge this is the first attempt to document progress in population-level effectiveness of HIV-treatment in the Netherlands since 1996 across a large number of clinical and non-clinical markers. By using a unique, non-selective dataset to review the changing patterns of cART use coupled with trends in patients' short-term clinical outcomes, it provides a valuable insight into how HIV treatment has changed and the impact this has had on treatment success. The analysis is limited factors typical of cohort data. Comparison of rates of VL suppression is limited by our use of the cut-off of 1,000 copies/ml in the definition of VL suppression. However, we used it in order to compare trends in VL suppression from 1996 to 2010. Analysis of CD4 recovery and VL suppression is restricted by the varying monitoring intervals amongst patients in clinical care. However, as these intervals have lengthened since 1996 and most patients will have a CD4 count and VL test by 12 months; this should not significantly affect the results. The analysis of mortality is limited by the cohort effect, and the fact that the risk of mortality may also depend on a number of factors, such as lifestyle, general health, and co-morbidities, which are not all routinely collected in the dataset. In the analysis we could not control for adherence as adherence data is not routinely collected in ATHENA. Adherence may have improved over time as drugs have become more tolerable The use of first-line cART in the Netherlands closely follows changes in guidelines, to the benefit of patients. While there was no significant improvement in mortality, newer drugs with better tolerability and simpler dosing resulted in improved immunological and virological recovery and reduced incidences of switching due to toxicity and virological failure.Table S1Adjusted hazard ratio  of 3-year mortality.(DOCX)Click here for additional data file.Table S2Adjusted hazard ratio  of reaching CD4 increase of 150 cells/mm3 by 12 months.(DOCX)Click here for additional data file.Table S3Adjusted hazard ratio  of virological suppression below 1,000 copies/ml by 12 months.(DOCX)Click here for additional data file.


We examined factors associated with late presentation of HIV using the new European consensus definition . The frequency of late presentation was highest in those aged 50 years or older and remained unchanged over time (64.3% in 1996-1998 and 65.4% in 2008-2010). In contrast, among those aged less than 50 years, the proportion with late presentation decreased over time (57.1% in 1996-1998 and 38.5% in 2008-2010). Other factors associated with late presentation were African ethnicity and being a male heterosexual.Of the 1536 included in the analysis, 86% were male and 10% were aged 50 years and older. Half the cohort (49%) had a CD4 cell count below 350 cells/mmThe mortality rate was 15.47/1000 person-years (pyrs) (95%-CI: 13.00-18.41). When compared with younger adults, older individuals had a higher mortality, after adjusting for confounders (rate ratio (RR) = 2.87; 95%-CI: 1.88-4.40).Older adults were more likely to present late and had a higher mortality. Initiatives to expand HIV testing in clinical and community setting should not neglect individuals aged over 50. Thcells/mm3,20. Howells/mm3[3 and the D4 count . Two recD4 count  while thD4 count .3), and mortality in a large UK cohort comprising predominantly of men who have sex with men (MSM) over a 14-year period.In this study, we examined factors associated with late presentation  patients with no documented baseline CD4 cell count for reasons stated in Figure For the analysis on mortality, 64 (3.5%) patients only attended once and did not contribute to follow-up time, while the dates of HIV diagnosis were missing for 9 patients leaving 1965 (96.4%) patients eligible for this analysis  estimated using logistic regression. The likelihood ratio test statistic was used to estimate the We also examined for the median time to initiation of HAART for the two age groups stratified by CD4 cell count at presentation using the Kaplan-Meier approach. For this analysis, patients were censored at the time started HAART for those on treatment and time last seen for those who remained untreated at the end of follow-up.To estimate mortality rates, participants were censored at the earliest of: death, date last seen, or end of follow-up (31 May 2010). Poisson regression modelling was used to estimate survival probability. Patients who attended at least once following the enrollment visit contributed person-years (pyr) for the analyses. Age was the primary exposure when fitting the multivariable Poisson regression model. This was adjusted for a priori confounders such as ethnicity and HIV risk group irrespective of whether they acted as confounders in this study or not. All analyses were carried out using STATA 11.0 .Of the 2038 patients enrolled with HIV infection between January 1996 and May 2010, 1536 (75.4%) patients had documented CD4 cell counts within 3 months of diagnosis and formed the basis of the analysis on late presentation. Of the 1536 patients, 1316 (86%) were male and 151 (9.8%) were aged 50 years and older when diagnosed with HIV infection. The median age at HIV diagnosis was 33.1 years (IQR 27.8-38.7) and 54.8 years (IQR 52.0-60.0) respectively, for those aged <50 and ≥50 years at diagnosis and 34.2 years (IQR 28.2-41.5) for the whole cohort.p = 0.74 and 0.41 respectively). Patients with missing information on CD4 count were significantly more often heterosexual and of African ethnicity (p = 0.007 and 0.008 respectively).The majority of the cohort were Caucasian (79.5%) and MSM (73.8%). Of the 239 participants of African-Caribbean ethnicity, 90% acquired their HIV through heterosexual intercourse while 87% of Caucasians acquired their HIV through homosexual intercourse. There was no difference in the age and sex distributions of patients with missing CD4 count at presentation (3 at HIV diagnosis and 23% (359/1536) presented with a CD4 count less than 200 cells/mm3. The median CD4 cell count for late and early presenters was 211 cells/mm3 (IQR 83-289) and 527 cells/mm3 (IQR 423-659) with median ages of 35.9 years (IQR 29.7- 43.6) and 32.9 years (IQR 27.5- 39.0) respectively.Approximately half (49%) the cohort presented late with a CD4 count less than 350 cells/mmOn univariable analyses, late presentation was associated with older age, being of non-Caucasian origin and being either a female or male heterosexual Table . On multp-trend = 0.0001). In contrast, among those over the age of 50 years, the proportion of patients presenting late was constant over time .The proportion presenting late decreased from 57.1% among those younger than 50 years in 1996-1998 to 38.5% in the 2008-2010 . The corresponding proportions among heterosexuals were 66.3%, 66.5% and 65.0% respectively (p-trend = 0.78).There was also evidence of a difference in trends of presenting late with sexual orientation. Among MSM, 51.3% presented late in 1996-2001 period, compared with 42.4% in 2002-2005 and 39.1% in the 2006-2010 (p (log-rank test) = 0.001 whilst amongst those presenting early it is 1041 days (IQR 467-1491) and 1236 day respectively (IQR 607-2290); p (log-rank test) = 0.0001  and 98.0 days respectively (IQR 39.0 -476.0); 3 respectively while 47 deaths occurred in individuals with unknown CD4 cell counts before death.Overall 127 deaths occurred in 1965 patients between Jan 1996 and May 2010; 26 and 54 deaths were in individuals presenting with CD4 cell count greater and less than 350 cells/mmpneumocystis jirovecii pneumonia (PCP) as well as AIDS-related malignancies in both age groups. 34/127(26.8%) of deaths were non-AIDS related death, predominantly due to malignancies. The causes for the remaining deaths were unspecified.64/127 (50.4%) of deaths were AIDS related death, and were predominantly due to AIDS- related bacterial pneumonia and Overall mortality rate in the cohort after 8210.30 person years (pyrs) of follow up was 15.47 per 1000 pyrs (95% CI: 13.00-18.41).3, 23/54(42.6%) in those with CD4 count less than 350 cells/mm3 and 29/47 (61.7%) in those with unknown CD4 count.53/127 (41.7%) of deaths occurred within the first year of follow up in cohort; 1/26(3.9%) in those presenting with CD4 count greater than 350 cells/mm3. 4/12(33.3%) and 8/12(66.7%) were in individuals with CD4 count less than 350 cells/mm3 and unknown CD4 count respectively.12/30 (40%) of deaths among individuals 50 years or older occurred in the first 12 months of presentation. None of these 12 deaths occurred in individuals with CD4 count greater than 350 cells/mm3. 19/41(46.3%) and 21/41(51.2%) of these deaths were in individuals with CD4 count less than 350 cells/mm3 and Unknown CD4 count respectively.41/97(42.3%) of deaths among individuals less than 50 years occurred in the first 12 months of presentation. Only 1 of these early deaths occurred in an individual with CD4 cell count greater than 350 cells/mmTable 3 was associated with a doubling of mortality while ART was associated with 37% reduction in mortality.On Univariable analysis, individuals who were 50 years or older at HIV presentation had a higher mortality compared to younger adults-HR 2.70 (95% CI: 1.80-4.08). Being diagnosed with a CD4 count <350 cells/mm3. Prior to this ART was initiated at CD4 <200 cells/mm3. We observed that there was no difference in the adjusted decrease in mortality in the 3-year period preceding the change in guidelines and the 2-year period following this, both periods showing an 80% decrease in mortality (full results not shown).On multivariable analysis, older age, earlier calendar period, and lower or unknown CD4 count were significantly associated with increased mortality. ART was also associated with a more marked reduction in mortality after adjustments. The reduction in mortality on ART was similar for the group who had been on it for less than 12 months with that of individuals who had been on it for longer, hence only the combined results for being on ART was shown. The decrease in mortality in the later calendar periods was further explored because of the change in UK guidelines in 2008 recommending the initiation of treatment when CD4 <350 cells/mmp-interaction = 0.67).No statistical interaction was detected between age at presentation and late presentation on mortality , with a likely associated increase in mortality.The association of late presentation with increased mortality has also been described in other studies both in the UK ,36,37 an3. Some deaths were coded as unspecified especially in those who died outside healthcare facilities as further information was lacking. This could have resulted in misclassification of some causes of deaths. Finally, the majority of the patients in this cohort were Caucasian MSM, hence the findings cannot be generalised to a cohort that is diverse in both ethnicity and sexual orientation; however this does not undermine the study validity.The strengths of this study lie in the utilization of the new European consensus definition for late presentation as this would facilitate regional comparisons within Europe and allow investigation of temporal trends after interventions , and theAs a result of the high mortality associated with late diagnosis, strategies must be put in place to encourage HIV testing. The CDC recommend routine testing in all healthcare settings for patients aged 13–64 years, unless the local HIV prevalence is known to be less than 0.1% .In addition to recommending routine testing in genitourinary medicine and antenatal clinics, 2008 UK guidelines for HIV testing also advocate that tests should be offered to all adults registering in general practice and to all general medical admissions patients in areas where diagnosed HIV prevalence is greater than 2 per 1,000 among 15 to 59 year olds . The UK HIV testing should take an ‘opt-out’ approach. Higher rates of HIV testing are achieved with such ‘opt-out’ approaches than opt-in approaches ,46. IdenThis paper showed that HIV late presentation is very common and does not appear to be improving in older adults. This impacts individual health as documented by increased mortality in this study. The consequent increase in hospitalizations also results in increased health care costs. Most HIV transmissions are from individuals unaware of their status.Prompt HIV diagnosis could be achieved through a policy that enables HIV test to be offered routinely alongside other investigations.All authors declare there are no competing interests that are relevant to this article.CI conceived the study and participated in the design, analysis and draft of the manuscript. DC made substantial contributions to the study design and draft of the manuscript. YG contributed to the design and draft of the manuscript. HW contributed to the statistical analysis and draft of the manuscript. MF participated in the design, analysis, coordination and draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/397/prepub


Highly effective combination anti-retroviral therapy (c-ART) has been applied in Bulgaria since 1999. The aim of the present study was to compare retrospectively the long-term immunological effect of c-ART in treatment-naïve and pre-treated HIV-1+ patients.The study included HIV-1+ patients (n=56) that have started c-ART between March 1999 and December 2001, have been on continuous treatment, with good adherence, death being the only reason for ART stop. Of them, 27 had a history of irregular pre-treatment with AZT or AZT/LMV for an average of 4.6 yrs (Group A), and 29 were ART-naïve (Group B). CD4 absolute counts (AC) were determined by single-platform flow cytometry (BD Biosciences). Viral load (VL) was measured by RT-PCR (Roche). Comparisons were performed by unpaired t-test (SPSS 17.0).The demographic characteristics of groups A and B did not differ significantly: mean age (yrs): 34 vs. 35; male to female ratio: 9 vs. 7, respectively. Baseline CD4 AC (cells/ml) and VL (log HIV RNA copies/ml) were comparable: mean 124 vs. 119, and 5.1 vs. 4.6, respectively, (p>0.05 for both comparisons). In the long term, suppression of viral replication was observed in both groups: mean VL at 5 yrs 3.7 vs. 3.1 for groups A and B, respectively, (p>0.05). However, treatment-naïve patients (group B) had a better immune recovery than group A, and the difference became significant in the long term: mean CD4 AC 177 vs. 252 after 6 months of c-ART, (p>0.05), 391 vs. 240 at 2 yrs , a more complete and lasting long-term immunologic response to c-ART was observed in treatment – naïve patients. According to us, a previous sub-optimal and irregularly applied ART regimen, may promote the selection of gradually outgrowing drug-resistant viral strains, compromising therapeutic efficacy in the long run.


To determine if tropism for CXCR4 or CCR5 correlates with cellular HIV DNA load, residual viraemia and CD4 count in 219 successfully treated naive subjects with HIV infection enrolled in five infectious diseases units in Northeastern Italy.A subset of subjects, achieving plasma HIV RNA level <50 copies/ml after initiation of first-line therapy and maintaining it until follow-up time points, was retrospectively selected from a prospective cohort. Blood samples were collected before the beginning of therapy (T0), at the first follow-up time (T1) and, when available, at a second (T2) follow-up time.= 0.0007). X4 tropism at T1 was negatively correlated with the possibility of achieving viraemia<2.5 copies/ml at T2 (p = 0.0076). T1-T2 tropism stability was significant (p <0.0001). T0 tropism correlated with T1 and T2 tropism (p < 0.001); therefore the stability of the tropism over the two follow-up periods was significant (p = 0.0003). An effective viremic suppression (viraemia<2.5 copies/ml) correlated with R5 coreceptor affinity (p= 0.047).HIV DNA, CD4 count and plasma viraemia were available from all 219 patients at T0 and T1, and in 86 subjects at T2, while tropism determinations were available from 109 subjects at T0, 219 at T1, and from 86 subjects at T2. Achieving residual viraemia <2.5 copies/ml at T1 correlated with having the same condition at T2 (p The tropism of archived virus was stable during an effective treatment, with 15-18% of subjects switching over time, despite a viraemia<50 copies/ml. R5 tropism and its stability were related to achieving and maintaining viraemia<2.5 copies/ml. Several chemokine receptors are coreceptors for HIV entry into cells. Non-syncytium-inducing HIV variants use the CC-chemokine receptor (CCR)-5 as a coreceptor (R5 viruses) -4, whereThe dynamics and the influence of viral tropism on the course of HIV-1 infection in subjects exposed to antiretroviral therapy (ARV) are not fully understood. Several studies have demonstrated a higher prevalence of X4 variants in patients exposed to antiretroviral drugs than in drug-naïve individuals ,11. The Studies aimed at assessing the influence and frequency of the switch of viral tropism during the course of therapy showed a low event rate in patients responding to HAART, and there have been conflicting results regarding the response to treatment, even if a direct correlation between the switch from an R5- to an X4-tropic virus and a low CD4 + nadir was found in treated patients -17.A chaUp to now, there are few data from longitudinal studies on viral tropism in patients fully responding to HAART, and in particular, there are insufficient data on the correlation between viral tropism and combined HIV-proviral DNA, residual viraemia and drugs used in treatment.To assess the impact of antiretroviral therapy on viral tropism evolution and the correlations between tropism and the response to therapy in terms of HIV DNA and residual viraemia values, we retrospectively examined a group of 219 HIV-1-infected naïve individuals who were successfully treated with ARV.The CAVeAT is a prospective cohort of HIV-infected patients enrolled starting from 2004 in five infectious diseases units in Northeastern Italy (Veneto region). We retrospectively selected from this cohort of patients a subset of subjects achieving virological suppression within 6 months after initiation of first-line therapy and maintaining plasma HIV RNA levels < 50 copies/ml, without virological failures, until evaluation at the follow-up time points. Patients interrupting or needing treatment modifications for failure, as well as patients who had more than one viral blip (plasma HIV RNA from 50 to 1.000 copies/ml) per year after achieving viral suppression, which was determined by frequent blood sampling (at least 4 times per year), were not included in the study. In the event of a change in the HAART, it was accepted if this change was due to intolerance or pill burden convenience. No patient was treated with CCR5 antagonists.Blood samples were collected at baseline (before the beginning of therapy), at the first follow-up time (T1) and, when available, at a second (T2) follow-up time.First follow up time (T1) was defined as the first available time point after achieving virological suppression within a range from 12 to 72 months, and T2 as a more recent time point available, collected a median time of 16 months after T1. All virological and immunological analyses were performed on all samples, with the exception of the tropism analysis, which was performed on all samples available at T1 and T2 but on only 109 out of 219 subjects at T0 due to depletion of the samples. This cohort was initially enrolled for another study on DNA load .The enrolled subjects gave informed consent for all procedures and the use of their blinded data for a scientific evaluation and publication. The local government, which was represented by the Veneto Regional Health Authority, approved the study and provided funding . This study was conducted in accordance with the Helsinki Declaration and local legislation .A primary HIV infection was defined by (i) a negative or indeterminate HIV antibody enzyme linked immunosorbent assay associated with HIV RNA positive plasma or (ii) an initially negative test for HIV antibodies followed by a positive serology within 18 months.6 PBMCs were stored at −80°C until use. Baseline and follow-up blood samples in EDTA were submitted within 6 hours from collection to the Laboratory of Virology at the University of Padova and stored until analysis.The blood samples were collected in tubes containing EDTA and separated into plasma and cells using Ficoll-Paque Plus density gradient centrifugation. Aliquots of plasma and dried pellets of 2 × 10The extraction and purification of DNA from cells were performed using the QIAmp Blood kit . The real-time TaqMan protocol was used to quantify the cellular HIV DNA copy number in PBMCs as previously described . The celA duplicate analysis within the same experiment was conducted for both HIV and beta-globin evaluation, and the mean of these values was calculated. When the delta values were more than 10%, the sample was analysed again. Baseline and follow-up samples from a patient were always evaluated in the same experiment; this procedure was possible because only complete sample sets  of the patients, retrospectively selected, were considered. A sample that had been previously evaluated was regularly added as an internal control.The plasma samples were obtained from the blood that was collected and were frozen at −80°C within 6 hours of collection and kept at this temperature until testing.Residual viraemia was quantified, using an ultra-ultrasensitive method based on a modified Amplicor HIV-1 Monitor test, version 1.5 , with a limit of detection limit of 2.5 copies/ml . ModificGenotypic analysis of viral tropism was performed on PBMCs as previously described . Brieflyhttp://coreceptor.bioinf.mpi-inf.mpg.de (accessed by June 2012).The generated V3 sequences were then interpreted using the bioinformatic tool Geno2pheno, with a false-positive rate of 20%. Geno2pheno is available at All PBMCs available from the subjects gave a useful amplification and sequencing of the V3 region, allowing us to analyse all subjects.All longitudinal discordant results were analysed twice, to confirm the tropism switch; similarly, all samples with an false positive rate from 10% to 30% were confirmed by a second analysis starting from the amplification of the sample. In summary, we determined and described a clear viral switch only with two concordant predictions.10 copies/106 PBMCs at T0 and follow-up. The HIV RNA log10 copies/ml was evaluated as continuous variable. HIV RNA at follow-up under the lowest threshold level (under 2.5 copies/ml), primary HIV infection at the start of therapy, stranger origin, male gender, risk categories  and tropism based on the presence of R5 or X4 variants in PBMCs at T0, T1 and T2 were evaluated as binary variables.The following variables were analysed: age, CD4 cell count at T0 and follow-up, and HIV DNA logAll variables were submitted to pairwise correlation analysis to outline the underlying link pattern in the evaluated population. The correlation was established by a linear regression analysis when at least one variable was continuous. When both paired variables were categorical (binary), the chi-square test by Pearson or the Fisher’s exact test (when applicable) were used.We performed a further statistical analysis based on X4 detection and R5 detection as binary variable and the patient as grouping variable (most patients had repeated observations). Unifying the time-points under a common time scale, a multilevel/mixed logistic regression was performed, using plasma RNA under low threshold as outcome, and the following covariates: log10DNA, baseline log10RNA, X4R5 (binary variable), and time. Conventional descriptive statistics were also applied when appropriate.The sequences from the PBMCs of 219 patients were genotyped at a median follow-up of 36 months (range 12–72) after the initiation of therapy. The demographic, virological and immunological data are reported in Table Of the 219 subjects, 146 (66.6%) had an R5 genotype in PBMCs. Eighty-three of 219 subjects (37.9%) had < 2.5 copies/ml of HIV RNA at T1. In particular, 62 of 146 patients (42.4%) harbouring a R5-tropic virus and 21 of 73 patients (28.7%) with an X4 virus reached fewer than < 2.5 copies/ml of HIV RNA (p=ns). The analysis of viro-immunological parameters before starting HAART showed a significantly higher CD4 cell count at baseline in R5 than in X4 patients .A tropism analysis of PBMCs at the baseline of therapy (T0) was available in 109 of 219 subjects from the study population.At the T0, 75 subjects (69%) had R5 tropism. The subjects harbouring an X4-tropic virus had a lower baseline CD4 absolute count than the patients with an R5-tropic virus p= 0.0012) and higher baseline plasma HIV RNA levels (p = 0.0431). With regard to the genotype stability, the results showed that at T1, after a median time of 24 months of follow up (range 12 – 48), most patients  maintained the same viral tropism. , and the patients with a stable R5 virus showed a decrease in HIV DNA from 896 to 127 copies/106 PBMCs.The patients who switched to an X4 virus showed a decrease in HIV DNA from 1432 to 159 copies/106 PBMCs, and the patients who switched to an R5 virus showed a decrease in HIV DNA from 419.5 to 111 copies/106 PBMCs.No differences in the follow-up duration were observed between the patients harbouring an R5- or an X4-tropic virus or between the switching or non-switching subjects (data not shown). The subjects with a stable X4 virus had a median CD4 cell gain of 235 cells (increased from 185 to 420 cells), and the patients with a stable R5 virus had a CD4 cell gain of 237 cells (from 305 to 542 cells). The patients who switched to an X4 virus presented a CD4 cell gain of 197 cells (from 228 to 425 cells), whereas the patients who switched to an R5 virus had a CD4 cell gain of 496 cells (from 104 to 600 cells). The patients with a stable X4 virus showed a decrease in HIV DNA from 516 to 58.5 copies/10At T1, 42.3% (11/26) of the patients with a stable X4 virus, 46% (29/63) of the patients with a stable R5 virus, 16.6% (2/12) of the patients who switched to an X4 virus and 43% (3/8) of the patients who switched to an R5 virus reached fewer than 2.5 copies/ml of HIV RNA (p = ns).A second evaluation of HIV tropism and of viro-immunological parameters was obtained in 86/219 subjects at T2 (median time of 16 months after T1). An R5 tropic strain was detected in 5 patients (67%) Table . In 73 oSix of the57 subjects harbouring an R5 virus at T1 switched to an X4 virus at T2, whereas seven of 22 patients with an X4 virus switched to an R5 virus at T2. No differences in the follow-up duration were observed among the four groups of patients. The ability to reach the lowest threshold of plasma HIV RNA at T1 correlated with having the same viral tropism at T2 (p = 0.0007). With respect to the correlation of residual viraemia with viral tropism, a condition of X4 tropism at T1 was negatively correlated with the possibility of reaching virological success with plasma RNA under the lowest threshold at the second time point (p= 0.0076). The relation among tropism evolution from T1 to T2 and the detection of plasma viremia lower than 2.5 copies/ml at T2 is reported in table The mean CD4 cell increase from T1 to T2 was significantly higher for R5 harbouring patients (p = 0.0497).Similarly, the persistence of HIV RNA less than 2.5 copies/ml at T1 and T2 was detected in non-switcher patients. In stable R5 patients, the persistence of an undetectable viral load was 76% (p = 0.002).In a subgroup of 51 patients, a viral tropism evaluation of the PBMCs at all 3 time points T0, T1 and T2 was available.The stability of the tropism over the long follow up, from T0 to T2, was significant (p = 0.0003).Six of the 37 R5 patients at T0 switched to X4 at T2. In the evaluation of the sequence of the coreceptor tropism status of the patients at the three time points, 5 of the 8 subjects with plasma RNA levels below 2.5 copies/ml at T2 were stable R5, one was R5-R5-X4, one was R5-X4-R5, and one was X4-R5-X4 ( p= ns).Twenty-four percent of patients that were stable R5 from T0 to T2 had less than 2.5 copies of HIV RNA, compared to none with a stable X4.A significant persistence (p = 0.047) of the virological effect, defined as reaching the <2.5 copies RNA level at T1 and maintenance at T2, was noted in non-switcher patients with R5 virus.Overall, 9 R5 patients had undetectable viremia at T1 and 5 confirmed virological success at T2.An RNA level below the lowest threshold at T1 and T2 was found in subjects with persistence of the R5 status at the transition from T0 to T1. The persistence of the acquired RNA level was 73% (p = 0.047).The baseline X4 condition was correlated with lower values of cellular HIV DNA, both at T1 (p= 0.0288) and at T2 (p= 0.0095). HIV DNA levels (p=0.002) and the condition of having a primary infection at the start of therapy (p= 0.008) were related to attaining RNA virological success, defined as having <2.5 copies/ml of HIV RNA.A total 47 patients had a FPR between 10-20%: 10 (9.2%) in the group of 109 patients evaluated at T0, 24 (11%) in the group of 219 patients with tropism available at T1 and 13 (15.1%) in the group of 86 patients studied at T2. Overall, 6 out of 21 (28.6%) patients with an FPR > 10% and 9/42 (21.4%) with an FPR < 10% switched to R5.The multilevel/mixed logistic regression confirmed that R5 is independently correlated to the achievement of plasma HIV RNA under 2.5 copies/ml.Three covariates appeared negatively correlated to the favorable virological outcome: log10DNA, baseline log10RNA, X4R5 in a significant manner. The coefficients of the regression are reported in Table This study is the first to analyse the relationship of tropism of archived virus with residual plasma viraemia below 2.5 copies/ml and HIV DNA cellular load in a cohort of naïve patients who underwent and mantained successful treatment, with a baseline and two follow-up time points. The observation time was prolonged, with a median of 36 months from the beginning of treatment and 16 months thereafter in a subgroup of patients.The stability of tropism in 109 subjects from the beginning of therapy to a median follow-up point of 24 months was significant (p< 0.001), with 89 subjects (82%) remaining in the same condition as that at the baseline, 8 subjects switching to R5 and 12 subjects switching to X4. A higher stability of viral tropism (88%) during ARV was previously reported  in a smaThe stability of tropism remained significant in the second period of observation (T0 to T2 p = 0.0003); in the second follow-up interval, 16% of the subjects switched, and a correlation between a switch to X4 and a decrease in the CD4 count was observed. No differences in relation to the therapy administered at the time of follow-up were observed in the different groups.The emergence of X4 viruses generally occurs in the later stages of disease -9, and iAmong 219 successfully treated subjects, 38% reached complete control of viral replication, defined as an HIV RNA copy number under the detection limit of 2.5 copies/ml, by the first follow-up. This rate is comparable with those of other studies reporting success rates of 36-40%, but it is lower than that of another cohort study reporting a success rate 60% ,23. A siThese results allow us to suggest that the total amount of time spent with the R5 condition appears to be relevant for reaching the best control of plasma viraemia, rather than the current tropism condition. To our knowledge, no data are available on this matter, making this observation the first demonstration of a correlation between an R5 genotype and a better virologic control during follow-up in treatment responder patients.In our cohort, 73 subjects (33.4%) had an X4 strain in their PBMCs at the first follow-up after treatment initiation; the absolute CD4 count and the CD4 percentage were higher in R5- than in X4-tropic patients at baseline and at the follow-up time points. The T0 absolute CD4 count was particularly high in R5-tropic patients, reaching statistical significance with respect to X4 subjects (p= 0.0216). On the contrary, no significant difference was found in the follow-up assessments at different time points in the recovery of CD4+ cells in patients harbouring an R5- or X4-tropic virus. This result confirms those obtained in previous studies showing no differences in the virological treatment response between patients harbouring an X4- or R5-tropic virus, although a poor recovery of CD4 cells has been shown in patients with an X4-tropic virus at baseline or after an R5 to X4 virus switch -13,24. NIn successfully treated naïve subjects, the tropism of archived virus was revealed to be stable, but with a substantial rate (15-18%) of subjects switching from one type to the other over time, despite a viraemia <50 copies/ml. An R5 tropism and its stability on follow-up were related to achieving and maintaining very low levels (less than 2.5 copies/ml of plasma) of viral replication in treatment responder patients, suggesting a relation among viral tropism and response to treatment in the long term.The authors declare that they have no competing interests.SGP designed and coordinated the study, supervised the laboratory experiments, collected the data, interpreted the findings, and wrote the paper; SA performed the laboratory experiments; CM interpreted the data and performed the statistical analysis; RS helped to design the study, managed the patients, and collected the samples; MC managed the patients, collected the samples and helped to interpret the findings and write the paper; RF managed the patients and collected the samples; VM managed the patients and collected the samples; SP managed the patients and collected the samples; MB helped to interpret the findings and write the paper; CB performed the laboratory experiments; SB performed the laboratory experiments; LS helped to interpret the findings and write the paper; MA helped to interpret the findings and write the paper; and GP designed the study and helped to interpret the findings and write the paper. All authors read and approved the final manuscript. SGP, RS, MC, RF, VM, SP, and GP are members of CAVeAT .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/314/prepub


Candidia esophagitis (CE) is an AIDS-defining condition, usually occurring in individuals with low CD4 counts of <200 cells/µL. Endoscopy is a valuable definitive diagnostic method for CE but may not be indicated for asymptomatic patients or for those with high CD4 counts or without oral candidiasis. This study assessed such patients to clarify the factors associated with CE and its severity on endoscopy in the highly active antiretroviral therapy (HAART) era.+ count, HIV-RNA viral load (VL), history of HAART, GI symptoms, GI diseases, and oral candidiasis were assessed. Endoscopic severity of CE was classified as mild (Kodsi's grade I/II) or severe (grade III/IV). Of the 733 subjects, 62 (8.46%) were diagnosed with CE . Of them, 56.5% (35/62) had no GI symptoms, 30.6% (19/62) had CD4 + ≥200 cells/μL, and 55.3% (21/38) had no oral candidiasis. Univariate analysis found lower CD4+ counts, higher HIV VL, and no history of HAART to be significantly associated with CE. With lower CD4+ counts and higher HIV VL, CE occurrence increased significantly (P<0.01 for trend in odds). Multivariate analysis showed low CD4+ counts and high HIV VL to be independently associated with CE. Of the severe CE patients, 55.2% (16/29) had no GI symptoms and 44.4% (8/18) had no oral candidiasis. Median CD4+ counts in severe cases were significantly lower than in mild cases .A total of 733 HIV-infected patients who underwent upper gastrointestinal (GI) endoscopy were analyzed. Sexual behavior, CD4Low CD4+ counts and high HIV VL were found to be factors associated with CE, and advanced immunosuppression was associated with the development of severity. Endoscopy is useful as it can detect CE, even severe CE, in patients without GI symptoms, those with high CD4 counts, and those without oral candidiasis. Candida esophagitis (CE) is the most common infectious disease of the esophagus + cell count >200, an HIV viral load <400, and the presence of gastric ulcers are considered protective factors for CE Only a limited number of studies have investigated the associated factors of CE, and no study has yet been conducted with a large population of patients. In the pre-HAART era, a low CD4+ cell count (<200 cells/μL) was identified as one such associated factor Endoscopy is a highly valuable definitive method for CE as it enables tissue biopsy and pathological and cultural examinations of specimens in addition to the evaluation of gross appearance We conducted a cross-sectional study to identify clinical factors associated with the diagnosis of CE. We also assessed the association between the severity of CE and clinical factors.Upper gastrointestinal endoscopy was performed in a cohort of 752 HIV-infected patients between 2003 and 2009 at the National Center for Global Health and Medicine (NCGM), a 900-bed hospital located in the Tokyo metropolitan area and the largest referral center for HIV/AIDS in Japan.Indications for endoscopy were: 1) gastrointestinal symptoms in symptomatic patients; and 2) fecal occult blood test results, abnormal findings on CT or annual health checkups, or laboratory abnormal findings of tumor markers or infections suspicious of colorectal cancer in asymptomatic patients. In Japan, screening endoscopy is frequently performed for the early detection of malignant or premalignant lesions, even as part of the examination for patients who are asymptomatic.We excluded patients who had received endoscopy for follow-up evaluation shortly after treatment for GI diseases. Written informed consent was obtained from all patients prior to endoscopy and biopsy. Study protocols were approved by the NCGM ethics committee.CE was suspected on endoscopic findings on the basis that candidal white plaques in the esophagus cannot be washed away Endoscopy was performed by gastroenterologists at NCGM with a high resolution gastroscope . Gross appearance on endoscopy was graded according to Kodsi's classification as grade I–IV, with grade I/II defined as mild and grade III/IV defined as severe  [14]. InGI symptoms were collected from medical records written by an attending doctor who interviewed each patient before endoscopy. Those without GI symptoms or descriptions of symptoms in their medical charts and those with a reason for receiving “screening” endoscopy were treated as asymptomatic cases.+ cell count values obtained within 2 weeks before or after endoscopy were collected from patient medical records and categorized into the following three groups: ≥200 cells/μL; 100–199 cells/μL; <100 cells/μL. HIV-RNA viral load (VL) as determined by real-time quantitative polymerase chain reaction (PCR) was also reviewed within 1 month of endoscopy. The minimum detection level was 40 copies/mL of plasma, and a positive result was defined as ≥40 copies/mL. We categorized HIV-RNA VL into four groups: VL ≤40 copies/ml ; 40<VL≤10,000 copies/ml; 10,000<VL≤100,000 copies/ml; and 100,000< VL copies/ml.GI symptoms comprised epigastralgia, nausea, heart burn, dysphagia, throat pain, appetite loss, hematemesis, and tarry stools. CD4Routes of infection were determined using face-to-face questionnaires conducted by the medical staff. HIV infection routes were classified into the following five categories: men who have sex with men (MSM); heterosexual; drug user; use of untreated blood products; and unknown. Sexual behavior was divided into two categories: MSM and heterosexual. Patients who were neither homosexual nor bisexual were regarded as heterosexual.Mycobacterium avium complex (MAC), malignant lymphoma (ML), and idiopathic ulcer, as well as other GI diseases such as esophageal varix, gastric adenoma, gastric adenocarcinoma, and reflux esophagitis were verified by examination of medical records and the endoscopic database. Information regarding history of HAART was collected from pre-endoscopy medical records.The presence of HIV-related GI diseases such as cytomegalovirus (CMV), herpes simplex virus (HSV), Kaposi's Sarcoma (KS), + cell count and HIV-RNA viral load were evaluated using the Chi-square test for linear trends. A multiple logistic regression model was used in multivariate analysis with factors showing values of P<0.2 on univariate analysis. A final model was then developed by backward selection of factors showing values of P<0.1. To identify the best performing combination of clinical factors, the area under the receiver operating characteristic curve (ROC-AUC) was calculated.The odds ratio (OR) and 95% confidence interval (CI) were used to determine factors associated with CE, and the relationships between CE and CD4The interobserver agreement of endoscopic severity (mild or severe) was measured with kappa statistics. Kappa values >0.80 were denoted excellent, 0.60–0.80 good, 0.40–0.60 moderate, 0.20–0.40 fair, and <0.20 poor.+ cell count, and HIV-RNA viral load. Fisher's exact test was used for sex, sexual behavior, history of HAART, and the presence of GI symptoms and oral candidiasis. Values of P<0.05 were considered significant, and all statistical analysis was performed using Stata version 10 software .Association between the severity of CE and clinical factors were evaluated. The Mann-Whitney U test was used for age, CD4Of the 752 potential study subjects we recruited who underwent endoscopy, 19 patients who underwent endoscopy for follow-up evaluation shortly after treatment for GI diseases were excluded. The remaining 733 patients were selected for data analysis.+ cell count was 234 cells/μL , and the median HIV-RNA viral load was <40 copies/mL . HAART had been administered to 545 patients (74.35%). The median CD4+ cell count was significantly higher in patients with HAART than in those without . The median HIV-RNA viral load was significantly lower in patients with HAART than in those without .Patient characteristics are summarized in GI symptoms were noted in 263 patients (35.9%) and included epigastalgia (n = 84), nausea (n = 36), tarry stool (n = 29), hematemesis (n = 25), heart burn (n = 22), dysphagia (n = 12), throat pain (n = 8), and appetite loss (n = 5). The endoscopic diagnosis of upper GI diseases is shown in + cell count, higher HIV-RNA viral load, and no history of HAART were significantly associated with CE  had no GI symptoms, 30.6% (19/62) had CD4 +≥200 cells/μL, and 55.3% (21/38) had no oral candidiasis . Univari with CE .+ count , the occurrence of CE increased significantly  and 29 in the severe group (grade III/IV: 24/5). A “white carpet” was observed in three grade-IV patients. The interobserver agreement for endoscopic severity was shown to be 0.77 (rated as “good”) by Kappa statistics.+ cell count in severe cases was significantly lower than in mild cases .CE is the most common esophageal disorder observed in HIV patients The presence of CE is an indication for systemic treatment with antifungals + cell count, and higher HIV-RNA viral load as factors associated with CE. Multivariate analysis identified a low CD4+ cell count and a higher HIV-RNA VL as independent associated factors. A low CD4+ cell count and high HIV-RNA viral load as associated factors are consistent with previous reports. López-Dupla and colleagues + cell counts were lower, albeit non-significantly, in CE patients. Their study included a large number of patients (n = 114) but, as a case series, did not compare the data with those obtained from control patients + cell count >200 cells/μl, and gastric ulcers as protective factors. However, their study did not consider the history of HAART or the presence of asymptomatic patients. Manfredi and colleagues In the present study, univariate analysis identified the absence of prior HAART, low CD4+ cell count as one such factor. Some patients with severe CE had characteristic endoscopic findings such as “white carpet” (CE is classified into mild and severe cases according to endoscopic findings, and severe cases are associated with complications such as hemorrhage, stenosis, and esophagotracheal fistula  carpet” , which h+ cell counts than other grades while there is no significant difference in CD4+ cell count between grade II–IV cases. Wilcox and colleagues + cell count <200, although their data are inadequate as their study included only a small number of HIV patients with CE. We also evaluated oral candidiasis, in addition to clinical factors, but found no association with the severity of CE.Werneck-Silva and colleagues There are several limitations in the present study. Although oral candidiasis is considered an important associated factor for the diagnosis of CE + cell counts and higher HIV-RNA VL were identified as clinical factors associated with CE, while higher endoscopic severity was shown to be associated with lower CD4+ cell counts. Endoscopy is useful as it can detect CE, even severe CE, in patients without GI symptoms, those with high CD4 counts, and those without oral candidiasis.Clinical factors should be considered when determining the endoscopic indication to diagnose CE. Low CD4


It remains controversial as to whether HIV-1 subtypes influence disease progression. Singapore offers a unique opportunity to address this issue due to the presence of co-circulating subtypes. We compared subtype CRF01_AE and non-CRF01_AE infected patients, with regards to estimated annual rate of CD4+ T-cell loss and time from estimated data of seroconversion (EDS) to antiretroviral therapy (ART).We recruited ART-naive patients with known dates of seroconversion between October 2002 and December 2007 at the Singapore Communicable Disease Centre, the national reference treatment centre. Multilevel mixed-effects models were used to analyse the rate of CD4+ T-cell decline. Time from EDS to ART was analyzed with the Kaplan-Meier survival method and compared with Cox proportional hazards models.10 viral load or haemoglobin concentration. The estimated annual rate of CD4+ T-cell loss was 58 cells/mm3/year  greater in subtype CRF01_AE infected patients compared to non-CRF01_AE patients, after adjusting for age, baseline CD4+ T-cell count and baseline log10 viral load. The median time from EDS to ART was 1.8 years faster comparing CRF01_AE to non-CRF01_AE infected patient with a 2.5 times  higher hazard for ART initiation, after controlling for age, baseline CD4+ T-cell count and baseline log10 viral load.54 patients with previously assigned HIV-1 subtypes  were observed for 89 patient-years. Subtype CRF01_AE and non-CRF01_AE infected patients did not differ in age, gender, risk factor, rate of symptomatic seroconversion, baseline CD4+ T-cell count, logInfecting subtype significantly impacted the rate of CD4+ T-cell loss and time to treatment in this cohort. Studies to understand the biological basis for this difference could further our understanding of HIV pathogenesis. The global HIV-1 pandemic is characterized by extensive genetic diversity Two recent studies from northern Thailand with cohorts of subtype CRF01_AE infected patients reported a 3-year shorter median survival compared to age-matched individuals for the Western CASCADE cohort, where subtype B predominates In Singapore, subtypes CRF01_AE and B exist in near equal proportions Details of the study participants and laboratory protocols have been previously published Clinical data was obtained from chart review. Only patients with 2 or more CD4+ T-cell counts measurements from at least 3 months following EDS were included in the current study. The 3-month interval was chosen to exclude results from the period of wide variability in CD4+ T-cell counts during recent seroconversion. Patients were followed until mid-May 2010 via chart review or censored either at ART initiation or loss to follow up (defined as more than 1 year without a clinic visit).Informed written consent was obtained from all participants. The National Healthcare Group ethics committee approved this study and its guidelines were followed in the conduct of this study.As published, HIV-1 pol gene sequences were previously determined using the Celera Diagnostics ViroSeq HIV-1 Genotyping System (version 2.0) . Phylogenetic analysis for sub-typing against reference sequences from the Los Alamos HIV Sequence Database has been published 10-transformed and those above the detectable limit were given the value equivalent to 750,000 copies/ml, the upper limit of the Roche assay. No result exceeded the detectable limit of the Abbott assay of 10 million copies/ml. The mode of transmission was divided into 5 categories . HIV-1 subtypes were dichotomised into subtype CRF01_AE and non-CRF01_AE. Baseline characteristics were compared between the subtype groups by Student's t-test and Wilcoxon Rank-Sum test for continuous variables and Fisher's exact test for categorical variables.The estimated date of seroconversion (EDS) was defined in order of priority as: (1) date of onset of clinical seroconversion syndrome, (2) date of indeterminate Western blot or (3) midpoint between date of last negative and first positive HIV-1 serology. Baseline CD4+ T-cell count was defined as the first result available at least 3 months from EDS. Viral loads in copies/ml were logMultilevel linear mixed-effects models with unstructured covariance were used to estimate the annual rate of CD4+ T-cell decline The Kaplan-Meier survival method was used to examine progression from EDS to ART initiation. Cox proportional hazards models were used to estimate crude and adjusted hazard ratios for this endpoint. All analyses were performed using Stata 11 , and all tests were conducted as 5% significance level.54 patients met the inclusion criteria. Subtype distribution was as follows: 24 CRF01_AE, 17 B, 8 B', 1 CRF33_01B, 3 CRF34_01B and 1 G. The median age was 30.6 years (range: 17.2 to 57.9 years). The majority of patients were men (98%) and of Chinese ethnicity (96%). Risk factor for HIV-1 acquisition was homosexual exposure in 27 patients (50%), heterosexual in 13 patients (24%), bisexual in 7 patients (13%), intravenous drug use (IDU) in 1 (2%) patient and 6 (11%) patients with no self-reported risk. 50% of the patients had a history of symptoms consistent with seroconversion illness.The mean baseline CD4+ T-cell count was 408 cells/mm3 (s. d.: 197 cells/mm3) with a median time from EDS to baseline count of 6.4 months (IQR: 4.8 to 7.9 months). Mean baseline log10-transformed viral load was 4.8 (s. d.: 0.76) obtained at median time from EDS of 5.8 months (IQR: 3.3 to 10 months).The total observation time was 89 patient years. The median follow-up time from baseline CD4+ T-cell count to last recorded pre-ART CD4+T-cell count was 1.8 years (IQR: 1 to 2.3 years). 6 patients (2 CRF01_AE and 4 non-CRF01_AE) defaulted follow up after a median duration of 1.1 years (IQR: 0.7 to 2.1 years). There were no statistically significant demographic or baseline laboratory test differences between the two subtype groups .3 per year (95%CI: 30 to 82 cells/mm3), controlling for age, baseline CD4+ T-cell count and log10 viral load. In the multilevel model including subtype, CRF01_AE infected patients had an average decline of 90 cells/mm3 per year (95%CI: 50 to 131 cells/mm3) compared to 32 cells/mm3 per year for non-CRF01_AE infected patients (95%CI: 1 to 64 cells/mm3), after controlling for age, baseline CD4+ T-cell count and log10 viral load. The difference in average yearly decline between the two subtype categories was statistically significant (P = 0.027) .The overall average rate of CD4+ T-cell decline without regard to subtype was 56 cells/mm= 0.027) . Square-10 viral load, the HR was unchanged at 2.5 .A greater proportion of CRF01_AE infected patients initiated ART  with similar CD4+ T-cell counts at ART initiation in the two groups . The med3/year greater adjusted loss of CD4+ T-cells compared to other infecting subtypes (mainly B) in our study. The resultant clinical outcomes were markedly different in that the median time to treatment initiation was 1.8 years faster in subtype CRF01_AE infected patients.In this cohort of seroconverters from Singapore, subtype status was strongly correlated with rate of CD4+ T-cell loss. Subtype CRF01_AE infected patients had a mean 58 cell/mm3 greater yearly loss in subtype D compared to subtype A infected patients Differences in subtype progression have been documented in Africa A study of injecting heroin users in Bangkok reported higher viral loads in subtype CRF01_AE compared to subtype B infected patients up to 9 months from seroconversion. However, no difference was documented in the rate of decline of CD4+ T-cell counts up to 2 years following seroconversion between CRF01_AE and B infected individuals A recent study from Canada demonstrated longer time to treatment initiation among non-subtype B  infected patients compared to subtype B In conclusion, we document a marked difference in CD4+ T-cell decline and time to treatment comparing CRF01_AE and non-CRF01_AE HIV-1 infection in Singapore. Larger studies to confirm this result are planned. If confirmed, studies to understand the biologic basis for these differences could bear fruit to further our understanding of HIV-1 pathogenesis.


The optimal treatment of early syphilis  in HIV-infected patients remains controversial. The Center for Diseases Control STD Treatment Guidelines recommended 1 dose of benzathine penicillin G (BPG) regardless of HIV infection. However, many providers modify the treatment for early syphilis.We performed a retrospective chart review of all cases of early syphilis with positive serologic test results in HIV-infected patients from May 2006 to May 2011 in 2 large, urban HIV clinics. Early syphilis includes primary, secondary, and early latent syphilis. Serological failure was defined as a lack of 4-fold decrease in rapid plasma reagent (RPR) titers 9 to 12 months after syphilis treatment. Patients whose RPR titers decreased after treatment and subsequently increased 4-fold at 9 to 12 months were excluded from the analysis of serological response because of possibility as “reinfection”. Baseline characteristics were tested as predictive factors of serological failure using a univariate and multivariate logistic regression model, respectively.Of 560 patients with confirmed cases of early syphilis, 51 (9.0%) experienced serological failure. Multivariate logistic regression modeling demonstrated that the predictive factors associated with serological failure after early syphilis treatment were baseline RPR titer ≤ 1:16 , a previous history of syphilis , and a CD4 T-cell count below 350 cells/ml . Of note, type of syphilis treatment (1 dose versus 3 doses of BPG) did not appear to affect the proportion of serological failure , however the power of this study to detect small differences was limited.HIV-infected patients with baseline RPR titer ≤1:16, syphilis history, and/or a CD4 T-cell count <350 cells/ml should be closely monitored for serologic failure after early syphilis treatment. This study did not detect a substantial difference between treatment with > 1 dose of BPG and decreased frequency of serological failure, supporting the current recommendation that one dose of BPG is adequate treatment for early syphilis in HIV-infected patients. Despite a significant decrease in syphilis cases in the United States during the 1990s, syphilis incidence has risen dramatically since 2000. A recenSeveral studies have demonstrated that certain risk factors are predictive of an increased risk for serological failure-8. For eAIDS Healthcare Foundation is one of the largest community-based HIV/AIDS medical providers in the nation, serving over 5000 patients in Los Angeles, California, USA. We performed a retrospective review of medical records to identify all cases of early syphilis with positive serologic test results in HIV-infected patients from May 2006 to May 2011 in our 2 large urban clinics in Los Angeles. The AIDS Healthcare Foundation human research protection approved the project. During the study period, RPR testing was performed on all patients every 3 months. Inclusion criteria are as follows: (1) serologic follow-up performed 9 to 12 months after syphilis treatment. (2) RPR titers are ≥ 1:4 at the time of diagnosis. (3) Clear documentation of the patient’s early syphilis stage as primary, secondary or early latent syphilis and type of syphilis therapy received within 2 weeks of diagnosis. (4) In patients with syphilis history, a more than 4 fold increase of RPR titer was required to diagnose a new infection. (5) Positive RPR results were confirmed using fluorescence treponemal antibody absorption test. Patients were excluded from the analysis for the following reasons: no follow-up testing within the period, no documentation of treatment, patients with diagnosis of late latent syphilis or syphilis of unknown duration, cases of primary syphilis with nonreactive serologic characteristics at the time of treatment. For those who satisfied inclusion criteria, medical record review was further performed to obtain information about patient characteristics, medical conditions, age, race, gender, syphilis stage , baseline RPR titer, CD4 T-cell count, plasma HIV viral load, ART use, syphilis history, current amphetamine use, syphilis treatment type such as 1 or more doses of BPG, and history of other sexually transmitted diseases. CD4 T-cell count and plasma HIV viral load were analyzed at the time of syphilis diagnosis. CD4 T-cell count, HIV viral load, and age were evaluated as a dichotomous variable . ART use was defined as the use of at least 3 antiretroviral drugs, from 2 different classes for a duration of > 6 months.Disease stage was classified on the bases of patient history and clinical examination. Patients were considered to have primary syphilis , secondary syphilis , or early latent syphilis .The primary outcome was response to syphilis treatment, determined on the basis of change in RPR titers after treatment. Serological failure was defined as a lack of a 4-fold decrease in RPR titers at 9 to 12 months after initial treatment or a titer pattern consistent with re-infection. Because we wanted to include only patients likely to have had true treatment failure, patients whose RPR titers decreased after treatment and subsequently increased 4-fold at 9 to 12 months were excluded from the analysis of serological response as “reinfection”.To identify potential predictors of developing serologic failure, we compared demographic and clinical characteristics of patients with serological failure to those with serological cure using univariate logistic regression. Demographic and clinical characteristics for serological failure were selected a priori based on previous literature, clinical plausibility, and ease of clinical collection. Combination of risk factors significant in univariate analyses were assessed and were selected in a stepwise algorithm, with variables entered and excluded for P > .025. Post-hoc power analysis tests were performed, with an alpha of .05, in order to assess the minimum level of effect that would be detected. Data were analyzed using SAS, version 9.3 (SAS Institute).During the study period, a total of 777 HIV-infected patients with early syphilis were identified. Among the 777 patients, 217 were excluded due to: lack of data (84 patients), loss of follow-up (48 patients), incomplete chart (37 patients), reinfection (28 patients), or because RPR titer were 1:4 (20 patients). “Reinfection” was defined as a RPR pattern of decreased RPR titers after treatment and subsequently 4-fold increase between 9 to 12 months.Table Table P value cutoff of .025, a model identifying serological failure was generated. This multivariate logistic regression modeling demonstrated that the predictive factors associated with serological failure after early syphilis treatment were a CD4 T-cell count below 350 cells/ml , syphilis history , and baseline RPR titer ≤ 1:16 , independent of other characteristics.Using a Our study was the large systematic evaluation assessing type of syphilis treatment associated with serological response in HIV-infected patients with early syphilis. Our population had a very high risk of past and current syphilis exposure as half had a history of syphilis infection. We found that overall rate of serological failure after therapy was low 9%) in our HIV-infected patients, and the duration of syphilis treatment (1 dose versus 3 doses of BPG) did not affect the proportion of serological failure (4% in a group of 1 dose of BPG versus 10% in a group of 3 doses of BPG) while our small number of serologic failure limited the power to detect a small difference in serological failure rates between individual who received one dose and three doses of BPG. However, based on our sample size and treatment distribution, we can exclude the difference greater than 13.5%. Our results have important clinical implications therefore it is important to assess whether there is a true lack of effect. HIV-infected patients with early syphilis often receive 3 doses of BPG. However, BPG may have adverse effects including allergic reaction, neurotoxicity, neutropenia, and local pain[% in our P > 0.05), and did not exclude patients who had been lost to follow-up and/or re-infected. Furthermore, the majority of previous studies suggested that HIV status is not associated with serological response to therapy[P = .995)[Limited data is available regarding a total dose of BPG for early syphilis in HIV-infected patients, possibly causing substantial variation in the management of early syphilis for HIV-infected patients among clinicians. In pre- therapy,7,15-19.Of interest is our finding that persons with CD4 T-cell count <350 cells/ml were more likely to develop serological failure, independent of type of treatment . This finding is consistent with previous studies that demonstrated HIV-co-infected patients with low CD4 T-cell count showed significantly slower treatment response[One of the strongest predictors of developing serological failure in our cohort was RPR titer ≤1:16 . This finding agrees with the observation that patients with low titers may be at risk of serological failure after treatment as alluded by Sena et al. in HIV negative populations; wherebyOur study has several limitations. First, a selection bias is possible because 28% of patients were excluded due to lack of data or loss of follow-up. Secondly, the number of patients treated with one dose of BPG was low , and thus a lack of statistical power cannot be excluded as the cause of the observed lack of effect of dosage on serological failure. However, the study did have adequate power to detect the risk difference of 13.5% or greater. Third, our study was based on medical record review and document may have been incomplete. Fourth, we excluded patients with primary syphilis whose serology was negative as well as those whose titer were <1:4. Therefore, our results may not be generalizable to those who have primary syphilis with negative serology or whose titers are <1:4. Finally, unmeasured potential confounding variables must be considered as limitations of all retrospective cohort studies such as inadvertent exposure to other efficacious antibiotics against syphilis.In conclusion, HIV-infected patients with baseline RPR titer ≤ 1:16, syphilis history, and/or a CD4 T-cell count <350 cells/ml should be closely monitored for serological failure after early syphilis treatment. Treatment with > 1 dose of BPG was not associated with a large difference in frequency of serological failure. Further research must be conducted to evaluate the current recommendation that one dose of BPG is adequate treatment for early syphilis in HIV-infected patients.They authors declare that they have no competing interests.SJ collected the clinical information of cohort data and participated in the design of the study, performed statistical analysis, and prepared manuscript. BA collected the clinical information of cohort data. PK prepared manuscript. CCB performed statistical analysis and prepared manuscript. JDK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/605/prepub