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A series of 716 HIV-negative IDUs entering a large drug treatment centre in central Italy was studied to evaluate the pattern of T-lymphocyte subsets among IDUs and to determine the effects of individual variables such as age, gender, duration of drug use and infection with viral hepatitis B and C. To this end, white cell count, total lymphocyte count and absolute number of lymphocyte subsets (i.e. CD3+, CD4+ and CD8+), HBV markers and HCV serology, were determined. For each individual, information on age, gender and duration of drug use was collected. To evaluate the association between level of lymphocyte subsets and phases of HBV infection, participants were stratified into three groups: (A) negative for all markers; (B) HBsAg-positive, with or without HBeAg, and HBsAb-negative; and (C) HBsAg and HBeAg-negative, positive for any other serological marker. The median absolute number of CD3+, CD4+ and CD8+ was 1909, 1080 and 770 cells/mm3, respectively, and did not vary across age bands. The CD8+ count was higher in males than in females. There was a tendency for CD3+ lymphocyte counts (mainly due to CD8+ counts) to be higher in individuals with 6-8 years of drug use compared to those who started injecting 4 years before or less. There was no evidence of increasing counts for a duration of drug use of more than 8 years. With regard to HBV, there was a general tendency for individuals in group "C" to have higher CD3+ and CD8+ counts than those in group "A". Finally, there was no difference in absolute lymphocyte count subsets between HCV-positive and HCV-negative IDUs. In conclusion, demographic and behavioural factors such as long-term heroin use or infection with HCV do not seem to influence the level of lymphocyte subsets among HIV-negative IDUs, nor does the infection with HBV seem to determine dramatic changes in CD4+ counts.

Author(s): Grund, Birgit; Baker, Jason V; Deeks, Steven G; Wolfson, Julian; Wentworth, Deborah; Cozzi-Lepri, Alessandro; Cohen, Calvin J; Phillips, Andrew; Lundgren, Jens D; Neaton, James D; INSIGHT SMART/ESPRIT/SILCAAT Study Group | Abstract: BackgroundDespite effective antiretroviral treatment (ART), HIV-positive individuals are at increased risk of serious non-AIDS conditions (cardiovascular, liver and renal disease, and cancers), perhaps due in part to ongoing inflammation and/or coagulation. To estimate the potential risk reduction in serious non-AIDS conditions or death from any cause that might be achieved with treatments that reduce inflammation and/or coagulation, we examined associations of interleukin-6 (IL-6), D-dimer, and high-sensitivity C-reactive protein (hsCRP) levels with serious non-AIDS conditions or death in 3 large cohorts.MethodsIn HIV-positive adults on suppressive ART, associations of IL-6, D-dimer, and hsCRP levels at study entry with serious non-AIDS conditions or death were studied using Cox regression. Hazard ratios (HR) adjusted for age, gender, study, and regression dilution bias (due to within-person biomarker variability) were used to predict risk reductions in serious non-AIDS conditions or death associated with lower "usual" levels of IL-6 and D-dimer.ResultsOver 4.9 years of mean follow-up, 260 of the 3766 participants experienced serious non-AIDS conditions or death. IL-6, D-dimer and hsCRP were each individually associated with risk of serious non-AIDS conditions or death, HR = 1.45 (95% CI: 1.30 to 1.63), 1.28 (95% CI: 1.14 to 1.44), and 1.17 (95% CI: 1.09 to 1.26) per 2x higher biomarker levels, respectively. In joint models, IL-6 and D-dimer were independently associated with serious non-AIDS conditions or death, with consistent results across the 3 cohorts and across serious non-AIDS event types. The association of IL-6 and D-dimer with serious non-AIDS conditions or death was graded and persisted throughout follow-up. For 25% lower "usual" IL-6 and D-dimer levels, the joint biomarker model estimates a 37% reduction (95% CI: 28 to 46%) in the risk of serious non-AIDS conditions or death if the relationship is causal.ConclusionsBoth IL-6 and D-dimer are independently associated with serious non-AIDS conditions or death among HIV-positive adults with suppressed virus. This suggests that treatments that reduce IL-6 and D-dimer levels might substantially decrease morbidity and mortality in patients on suppressive ART. Clinical trials are needed to test this hypothesis.

Background. The impact of human immunodeficiency virus (HIV)‐associated tuberculosis on CD4 T-cell count response to combined antiretroviral therapy (cART) is poorly investigated. Methods. A collaborative analysis including HIV-infected patients prospectively enrolled in 4 Italian clinical cohorts was conducted. Patients were grouped according to Centers for Disease Control and Prevention stage at the start of cART as having tuberculosis, having AIDS but not tuberculosis (nontuberculosis AIDS), and not having AIDS (AIDS free). Time to CD4 T-cell count of at least 100, 200, and 300 cells/lL above pre-cART levels and to CD4 T-cell count of .500 cells/lL were major end points. Survival analysis with time-fixed and time-dependent covariates was used. Results. A total of 6528 patients were eligible; 125 patients (2%) had tuberculosis, 1062 (16%) had nontuberculosis AIDS, and 5341 (82%) were AIDS free. Patients with tuberculosis had a significantly reduced chance of CD4 T-cell count increase compared with AIDS-free patients as well as those with nontuberculosis AIDS, regardless of the primary outcome considered for a given value of confounders measured at baseline (eg, for .200 cells/lL above baseline; relative hazard, 0.71; P 5 .02), although it was no longer significant after further adjustment for current level of viral load suppression (relative hazard, 0.80; P 5 .11). There was a trend for reduced virological response in patients treated concomitantly for tuberculosis and HIV infection compared with those who were treated separately in time (P 5 .09). Conclusions. HIV-infected patients starting cART with a tuberculosis diagnosis showed an impaired immune recovery to cART compared with AIDS-free patients and those with nontuberculosis AIDS. It seems to be driven mainly by a delay in achieving viral suppression. Whether this may be due to interactions between antituberculosis drugs and antiretrovirals needs to be investigated.

Background: There is increasing interest in the role of immune activation and inflammation in HIV disease, but data on direct effects of HIV replication on immune cell activation are limited. Methods: High sensitivity multiplex bead array assays (MBAAs) were used to measure changes in plasma cytokines and chemokines [interleukin (IL)-1β, IL-2, IL-6, IL-7, IL-8, IL-12p70, IL-17, tumor necrosis factor-α (TNFα), interferon-γ, granulocyte macrophage colony-stimulating factor, IL-4, IL-5, IL-10, IL-13, CXCL10] from randomization (month 0) to month 2 in a random sample of 200 patients from both the drug conservation (DC) and viral suppression (VS) arms of the Strategies for Management of Antiretroviral Therapy (SMART) trial. IL-6 was also measured by ELISA. Data were evaluated using nonparametric correlation and censored parametric analysis of covariance and associations were declared as statistically significant when the Bonferroni-adjusted P-value was less than 0.003. Results: Compared with the VS arm, significant increases were seen in the DC arm for TNFα (+0.34 loge pg/ml, P = 0.0001), IL-10 (+0.33 loge pg/ml, P = 0.00001) and CXCL10 (+0.66 loge pg/ml, P = 0.00001). IL-6 ELISA poorly correlated with IL-6 MBAA (Spearman,s rho = 0.29, P = 0.0001). Conclusion: Resumption of HIV replication after ceasing antiretroviral therapy is associated predominantly with an increase of monocyte/macrophage-derived cytokines. Measurement of IL-6 levels may be affected by assay method and this should be considered in future studies of biomarkers.

To the Editor—We read with interest the comments of Manfredi. The aim of our study was to determine the seroprevalence of cytomegalovirus (CMV) antibodies among human immunodeficiency virus (HIV)–positive subjects in the ICONA cohort and to define the impact of CMV serostatus on the risk of AIDS and severe non-AIDS events/death. Although we agree that our analysis has several limitations, mentioned in the discussion section of the article [1], we believe that we have contributed useful new data to the debate about whether CMV infection might be associated with the risk of clinical progression in HIV-infected individuals. CMV is a herpes virus characterized by persistence in a latent state, resulting in lifelong infection [2]. Chronic CMV infection is diagnosed on the basis of detection of persistently positive CMV immunoglobulin G (IgG) antibodies, which can be detected in immunodeficient individuals and elderly individuals, except those with common variable immunodeficiency [3]. There is incontrovertible evidence that CMV infection has an important role in human immunity [4]. As shown by many studies, there is a significant correlation between the increase in CD57+ and CD28− T cells and CMV IgG antibody positivity [5–7]. These cells, characterized by short telomeres, loss of CD28 expression, and/or gain of CD57 expression, play a significant role in various conditions associated with chronic immune activation, such as cancer and autoimmune diseases [8, 9]. Because of these findings, CMV serostatus (and not, for example, T-cell–specific CMV response or level of CMV viremia) was included in the immune risk phenotype markers panel, a cluster of immune markers predictive of increased mortality in elderly individuals. For these reasons, we believe that it is appropriate to use CMV serostatus as a marker of CMV chronic infection, assessed by a standard test for anti-CMV IgG used in clinical practice. Manfredi also pointed out that a limitation of our study is the cumulated time elapsed from the CMV-positive serologic test until the occurrence of end-point events. Our analysis was, indeed, performed using the data from a large cohort of HIV-positive, disease-free Italian subjects monitored for almost 6 years, on average (interquartile range, 2–10 years), until they developed AIDS or a severe non–AIDS-defining event/death. It is also true that a single measurement of CMV antibodies was used in the analysis, which, in most cases, was performed at enrollment (baseline) and that, therefore, potential new incident CMV infections occurring after baseline were ignored. However, previous observations showed that the CMV seroconversion rate over time is low [10], so current exposure might have been potentially misclassified only for a minority of our population. Moreover, although it is true that clinical progression can be determined by many events over follow-up, it was originally hypothesized that would take several years for chronic CMV infection to influence the analyzed clinical outcomes. Indeed, CMV serostatus was evaluated at baseline with the aim of assessing whether it was a predictor of the risk of clinical progression independently of other classical factors (eg, nadir CD4+ T-cell count, Centers for Disease Control and Prevention HIV disease stage, and age). To reduce bias due to these potential confounding factors, in particular for the risk of cardiovascular and cerebrovascular disease, we conducted a multivariable analysis. Moreover, we also explored the potential confounding role of smoking status. In contrast, the interpretation of the association between CMV serostatus, fitted as a time-dependent covariate, and the risk of progression is not free of caveats and potentially requires sophisticated modeling able to correctly control for potential time-dependent confounding factors affected by CMV seroconversion, which was beyond the scope of our analysis. Finally, Manfredi was surprised by the absence of an association between positive anti-CMV serostatus and AIDS events/death. Most people included in the analysis achieved viral suppression during antiretroviral therapy (ART) and showed a good recovery in the CD4+ T-cell count. CMV reactivation with clinical manifestations are typical of severe immunodeficiency, which is uncommon among ART recipients. In fact, the natural history of CMV infection has changed in terms of viral replication and reactivation as a result of suppression of HIV replication and improved patient immunity. In conclusion, we think that our analysis contributes to existing knowledge regarding the role of CMV in HIV disease progression. Indeed, it is the first large cohort study in which CMV/HIV-coinfected individuals were compared to HIV-monoinfected individuals in terms of their risk of future morbidity and mortality. The analysis provided strong evidence that coinfected persons are at higher risk for severe non–AIDS-related events/death than those with HIV monoinfection. Additional studies are needed to investigate the possible mechanisms through which CMV might increase this risk and whether CMV coinfection should be considered for inclusion in scores for the prediction of severe non–AIDS-related events.

Background This study provides an estimate of the proportion of HIV-positive patients in Italian clinics showing an ‘adverse prognosis’ (defined as a CD4 count ≤200 cells/μL or an HIV RNA >50 HIV-1 RNA copies/mL) over time, and investigates whether this proportion varied according to patients' characteristics. Methods We estimated the annual proportion of patients with a CD4 count ≤200 cells/μL or HIV RNA >50 copies/mL out of the total number of patients in the Icona Foundation cohort seen in any given year, both overall and after stratifying by demographical and treatment status groups. Generalized estimating equation models for Poisson regression were applied. Results In 1998–2008, the prevalence of patients with a CD4 count ≤200 cells/μL decreased from 14 to 6% [adjusted relative risk (RR) 0.86/year; 95% confidence interval (CI) 0.84–0.88; P 50 copies/mL decreased from 66 to 40% (adjusted RR 0.95/year; 95% CI 0.95–0.96; P

Background Guidelines suggest that patients on continuous antiretroviral therapy for >4 months with current viral load (VL)>1,000 copies/ml should be tested for resistance. There are limited data showing the frequency of resistance testing in routine clinical practice following these recommendations. Methods In EuroSIDA, virological failure (VF) was defined as confirmed VL>1,000 copies/ml after ≥4 months continuous use of any antiretroviral in a ≥3-drug regimen started during or after 2002. We assessed whether a resistance test was performed around VF (from 4 months before to 1 year after VF) and used logistic regression analysis to assess factors associated with having a resistance test. Results A total of 1,090 patients experienced VF a median 8.1 months (range 4 months to 6.3 years) after starting their regimen. There were 395 (36.2%; 95% CI 33.4–39.1) patients with a resistance test around the time of VF. Predictors of having a resistance test following VF include availability of a resistance test earlier than 4 months before VF (OR 2.20, 95% CI 1.77–2.75 for yes versus no; PConclusions This analysis suggests a delay in genotypic testing after VF that seems longer than expected given current treatment guidelines. This delay is highly variable across Europe.

Background. Robust long-term estimates of the risk of development of drug resistance are needed for human immunodeficiency virus (HIV)-infected patients starting combination antiretroviral therapy (cART) regimens currently used in routine clinical practice.Methods. We followed a large cohort of patients seen in 1 of 11 HIV clinics in the United Kingdom after starting cART with nucleoside reverse-transcriptase inhibitors and either a nonnucleoside reverse-transcriptase inhibitor (NNRTI) or a ritonavir-boosted protease inhibitor (PI/r). Survival analysis was employed to estimate the incidence of virological failure and of detected drug resistance.Results. Seven thousand eight hundred ninety-one patients were included; 6448 (82%) started cART with an NNRTI and 1423 (17%) with a PI/r. The cumulative risk of virological failure by 8 years was 28%. The cumulative probabilities of detecting any mutation, >= 1 major nucleoside reverse-transcriptase inhibitor International AIDS Society-United States of America (IAS-USA) mutation, >= 1 major NNRTI IAS-USA mutation (in those starting an NNRTI), and >= 1 major PI IAS-USA mutation (in those starting a PI) were 17%, 14%, 15%, and 7%, respectively, by 8 years. The probability of detecting PI mutations in people who started PI/r-based regimens was lower than that of detecting NNRTI mutations in those starting NNRTI-based regimens (adjusted relative hazard, 0.36; 95% confidence interval, 0.26-0.50; P < .001). The risk of detecting nucleoside resistance did not vary according to whether an NNRTI or a PI/r was used in the regimen (adjusted relative hazard, 1.00; 95% confidence interval, 0.80-1.26; P = .98).Conclusions. In patients who started modern cART in clinical practice in the United Kingdom, virological failure by 8 years was relatively common and was paralleled by an appreciable risk of resistance detection, although the detection rate of class-specific resistance was lower for those who started a PI/r-based regimen.

Background The extent of short-term CD4+ T-cell recovery in patients tolerating first-line highly active antiretroviral therapy (HAART) and attaining undetectable HIV RNA levels is inadequately defined. Methods We retrospectively analysed patients in four Italian cohorts who started HAART between January 1996 and September 2006. All patients had known HCV coinfection status, did not modify the regimen for 6 months and had Results The analysis involved 1,488 patients (1,096 males, 73.7%) with a median age of 43 years (interquartile range [IQR] 39–49); 435 (29.2%) were positive for HCV, 71 (4.8%) were positive for hepatitis B surface antigen (HBsAg) and 76 (5.1%) had experienced a previous AIDS-defining event. At baseline, patient CD4+ T-cell counts were 226 cells/μl (IQR 99–332), CD4+ T-cell percentages were 14.7% (IQR 8.7–21.2) and HIV RNA levels were 4.91 log10 copies/ml (IQR 4.38–5.34). Overall, 24-week CD4+ T-cell recovery was 144 cells/μl (IQR 70–240). At multivariable analysis, T-cell recovery was positively related to the use of a boosted protease inhibitor ( P+ T-cells ( P+/CD8+ T-cell ratio and a history of AIDS-defining events had no independent effect on CD4+ T-cell recovery. Conclusions Among HIV-infected patients tolerating first-line HAART and with undetectable HIV RNA after 6 months, CD4+ T-cell recovery is significantly greater in those without HCV coinfection, with a high baseline viral load, a high baseline percentage of CD4+ T-cells and in those treated with a boosted protease inhibitor.

Objective: To assess the genotypic determinants of the virological response to didanosine (ddI) in HIV-infected patients.Methods: The Forum database on ddI was randomly divided into a derivation set (n=1000) and a validation set (n=453). Linear regression models and bootstrap sampling were used to select resistance mutations and to estimate their resistance scores. Linear regression models, accounted for the censoring of viral load measurements due to assay lower limits, of the week 8 reduction in viral load from baseline were adjusted for baseline viral load, the exact number of weeks between baseline and the week 8 viral load measurements, and the Stanford genotypic sensitivity score.Results: The ddI resistance mutations and their resistance scores based on the derivation set were as follows: M41L (score of 14), T69D (24), D123S (40), T139M (54), I180V (53), M184V (-12), V1891 (55), Q207K (37), L210W (25), and T215Y (eight). The total score is obtained by adding the individual scores. Viruses with scores of 19 or less, 20-59, and 60 or more are considered sensitive, intermediate, and resistant, respectively. In the validation set, respectively, 58.7, 36.9, and 4.4% of viruses were predicted to be sensitive, intermediate, and resistant to ddI. The observed viral load reductions at week 8 were, respectively, 1.51 log(10)copies/ml [interquartile range (IQR) 1.26-1.76] (P=0.0001 versus resistant), 1.11 (0.94-1.30) (P=0.0077 versus sensitive), and 0.46 (0.32-0.74) (P=0.0079 versus intermediate).Conclusion: We developed a genotypic resistance score for didanosine including four mutations never previously used. (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

OBJECTIVE AND DESIGN The present study aims at evaluating the influence of genetic polymorphisms on antiretroviral therapy (ART)-associated lipodystrophy. We included in the study 255 ICoNA. patients and we assessed the distribution of Fas -670 AG polymorphism, ApoC3 -455 CT and -482 CT polymorphisms, C161T silent substitution in the PPAR gamma gene, the Adrenergic beta3 Receptor (ARbeta3) codon 64 TC variant, and two polymorphisms in the Adrenergic beta2 Receptor (ARbeta2) codon 16 AG and codon 27 CG. Crude rates of lipoatrophy and fat accumulation and adjusted relative rates were calculated using Poisson regression. RESULTS In a multivariate model after adjusting for gender, HIV exposure, age, current viral load, hepatitis C virus (HCV) serology, nucleoside reverse-transcriptase inhibitor (NRTI) pair/'third drugs' currently used, months of pre-highly active antiretroviral therapy (HAART) exposures to NRTI, the following genotypes resulted protective against lipoatrophy: ApoC3 -455 CC genotype [adjusted relative risks (ARR) 0.2, 95% confidence interval (CI) 0.046-0.91 vs CT/TT, P = 0.037], ARbeta3 codon 64 TT genotype (ARR 0.39, 95% CI 0.14-1.06 vs TC/CC, P = 0.066), and Fas -670 GG genotype (ARR 0.51, 95% CI 0.26-1.01 vs AG/AA, P = 0.053). With regard to fat accumulation, in the multivariate model, the ARbeta2 codon 27 CC genotype resulted protective (ARR 0.21, 95% CI 0.08-0.51 vs CG/GG, P = 0.0006), whereas the ARbeta2 codon 16 AA genotype resulted associated with higher risk (ARR 3.72, 95% CI 1.58-8.76 vs AG/GG, P = 0.0026). CONCLUSION Our study suggests that genetic polymorphisms of genes involved in apoptosis and adipocyte metabolism are significantly related to ART associated lipodystrophy. Particularly, we evidenced a role for ApoC3 -455 in lipoatrophy and for the two variants of ARbeta2 in fat accumulation.

Background. This study was performed to investigate the concordance between commonly used human immunodeficiency virus type 1 (HIV-1) drug resistance interpretation systems for didanosine (ddI) and their ability to predict responses at weeks 8 and 24. Methods. The study included drug-experienced HIV-infected patients who had viral loads >500 copies/mL and who underwent a genotypic resistance test when beginning a ddI-containing therapy. The interpretations of the level of resistance to ddI were compared for the 6 interpretation systems. Linear and logistic regression were used to assess their ability to predict responses for weeks 8 and 24, respectively. Results. The 1453 patients had a median viral load of 4.3 logic copies/mL, and 31% were preexposed to ddl. Complete concordance was found for 19% of samples, partial discordance for 49%, and complete discordance for 32%. The median viral load reduction at week 8 was 1.36 logic 10 copies/mL, and 56% of patients had viral loads >400 copies/mL at week 24. At week 8, all systems correctly predicted a greater viral load reduction in patients with susceptible viruses than in those with resistant viruses, but only the Stanford system was able to discriminate between patients with resistant, intermediately resistant, and susceptible viruses. No systems predicted virological response correctly at week 24. Conclusions. Our results show the need for standardized methods to establish genotypic interpretation systems.

Objectives To investigate the concordance between any of the results of nine HIV-1 drug-resistance interpretation systems (ISs) and their ability to predict week 8 and week 24 virological responses to abacavir-containing combination therapy. Patients and Methods A total of 1306 HIV-infected patients with a viral load >500 HIV-1 RNA copies/mL and a baseline genotypic resistance test were included in the study. Predicted abacavir susceptibilities according to each rule-based IS were compared. Linear and logistic regressions were used to assess the prognostic value of each IS for week 8 and week 24 responses, respectively. Results A median of three (interquartile range 1–5) abacavir mutations were detected at baseline. Comparing the IS predictions for abacavir susceptibility, 9% to 45% of patients were predicted to have resistant (R) virus, 9% to 53% virus with intermediate (I) resistance, and 23% to 74% susceptible (S) virus. Overall, the median week 8 viral load reduction was 1.61 log10 copies/mL (95% confidence interval 1.52–1.71) and 50% of patients experienced virological failure at 24 weeks. Most ISs showed better virological responses with S and I viruses than with R viruses. Conclusions Despite some degree of variability in predicted abacavir susceptibility among ISs, most ISs are useful to predict virological response.

Recently, it was shown that cirrhotic patients without human immunodeficiency virus (HIV) infection had low CD4 cell counts and normal CD4 cell percentages, suggesting that, for HIV-infected persons, the CD4 cell percentage might be a more accurate marker of disease progression than the absolute CD4 cell count. In cirrhotic HIV-infected persons in the Italian Cohort of Antiretroviral-Naive Patients, the absolute CD4 cell count seemed to be better predictor of the risk of developing an acquired immunodeficiency syndrome-defining illness than the CD4 cell percentage.

BACKGROUND All-cause mortality and serious non-AIDS events (SNAEs) in individuals with HIV-1 infection receiving antiretroviral therapy are associated with increased production of interleukin-6 which appears to be driven by monocyte/macrophage activation. Plasma levels of other cytokines or chemokines associated with immune activation might also be biomarkers of an increased risk of mortality and/or SNAEs. METHODS Baseline plasma samples from 142 participants enrolled into the Strategies for Management of Antiretroviral Therapy study, who subsequently died, and 284 matched controls, were assayed for levels of 15 cytokines and chemokines. Cytokine and chemokine levels were analysed individually and when grouped according to function (innate/proinflammatory response, cell trafficking and cell activation/proliferation) for their association with the risk of subsequent death. RESULTS Higher plasma levels of proinflammatory cytokines (interleukin-6 and tumour necrosis factor-α) were associated with an increased risk of all-cause mortality but in analyses adjusted for potential confounders, only the association with interleukin-6 persisted. Increased plasma levels of the chemokine CXCL8 were also associated with all-cause mortality independently of hepatitis C virus status but not when analyses were adjusted for all confounders. In contrast, higher plasma levels of cytokines mediating cell activation/proliferation were not associated with a higher mortality risk and exhibited a weak protective effect when analysed as a group. CONCLUSION Whereas plasma levels of interleukin-6 are the most informative biomarker of cytokine dysregulation associated with all-cause mortality in individuals with HIV-1 infection, assessment of plasma levels of CXCL8 might provide information about causes of mortality and possibly SNAEs.

To assess the risk of clinical progression (CP) according to the duration of time spent without complete viral load (VL) suppression compared with that associated with periods of stably suppressed viremia in HIV-infected people who started highly active antiretroviral therapy (HAART) when previously naïve to antiretrovirals.A cohort study of patients having started HAART after enrollment in the Italian Cohort of Antiretroviral-Naive Patients (ICoNA) and being followed for at least 6 months.Person-years spent in different categories according to the VL level and the change in VL from the most recent value before the initiation of HAART were calculated. A multivariable Poisson regression model, including potential confounders, was constructed.A total of 3023 patients were studied. The overall rate of CP was 13.4 per 1000 person-years. Evidence for a higher risk of CP was observed for people with a current VL10,000 copies/mL. For each year longer spent on HAART with a VL100,000 copies/mL, a 5-fold increased risk was observed (relative risk [RR] = 5.34, 95% confidence interval [CI]: 2.83 to 1.08; P = 0.0001). An increased risk of CP in patients with current suppression1.5 log10 copies/mL (RR = 2.34, 95% CI: 1.16 to 4.74; P = 0.02) and in those with no suppression or a VL higher than their set point (RR = 2.39, 95% CI: 1.17 to 4.89; P = 0.02) was observed compared with those with suppression of3 log10 copies/mL, although it was not significant. Longer duration on HAART with a VL suppressed below set point seemed to confer protection against CP.Virologic failure to antiretroviral drugs is common. The risk of CP may remain low despite a low but detectable level of HIV viremia.

Hepatitis C virus (HCV) variability is mainly attributed to the ability of the virus to respond to host immune pressure, acting as a driving force for the evolution of quasispecies. This study was aimed at studying the changes in HVR-1 heterogeneity and the evolution of HCV quasispecies in HIV/HCV-coinfected patients according to the pattern of response to highly active antiretroviral therapy (HAART). Sixteen HIV/HCV-coinfected patients harbouring HCV genotype 1 and who had been on HAART for at least 1 year, 8 showing increasing CD4+T-cell counts (immunological responders) and 8 showing a stable or decreasing CD4+ T-cell counts (immunological non-responders), were selected from a prospective cohort study. After 1 year of HAART, 11 patients showed HIV viral load 10 cp/ml (virological responders), and 5 showed HIV viral load above this value (virological non-responders). Plasma samples, collected before starting therapy and after 1 year of HAART, underwent clonal sequence analysis for HVR-1 region of HCV. Non-synonymous/synonymous substitutions ratio (Ka/Ks), aminoacidic complexity (normalized Shannon entropy) and diversity (p-distance), were considered as parameters of quasispecies heterogeneity. After 1 year of HAART, heterogeneity of HVR-1 quasispecies significantly decreased in virological non-responders, whereas the heterogeneity tended to increase in virological responders. The differences in the evolution were less stringent, when considering immunological response. On the other hand, profound qualitative modifications of HVR-1 quasispecies were observed only in patients with both immunological and virological HAART response. On the whole, these findings suggest that, in patients undergoing HAART, the extent of HCV variability and the evolution of HVR-1 quasispecies is influenced by the pattern of response to antiretroviral therapy.

Background Although efavirenz (EFV) and lopinavir/ritonavir (LPV/r) are both recommended antiretroviral agents for combination therapy in drug-naive HIV-infected patients, no randomized comparison of their efficacy and tolerability is available yet. A multi-cohort prospective observational comparative study was performed. Methods Efficacy was examined comparing time to virological failure, CD4 recovery and clinical progression. Tolerability was examined comparing time to treatment discontinuation for any reason and for toxicity and time to liver enzymes or lipid alterations. Survival analysis was conducted by an intent-to-treat principle using the Kaplan–Meier method, and standard and weighted Cox regression models. Results A total of 674 antiretroviral-naive patients starting a two nucleoside reverse transcriptase inhibitor regimen plus either EFV ( n=481) or LPV/r ( n=193) were examined. At baseline, patients starting LPV/r had higher HIV RNA and lower CD4+ T-cell counts. There was no difference in the adjusted hazards of virological failure (LPV/r versus EFV relative hazard [RH] 1.16, 95% confidence intervals [CI]: 0.58–2.32, P=0.67), CD4 recovery (RH=0.93, 95% CI: 0.66–1.30, P=0.66), clinical progression (RH=1.64, 95% CI: 0.70–3.84, P=0.25), drug discontinuation for toxicity (RH=0.92, 95% CI: 0.51–1.64, P=0.76) and for any reason, and rates of liver enzyme and total/low density lipoprotein (LDL) cholesterol elevation. In contrast, the rate of triglycerides elevations (>1 NCEP Adult Treatment Panel III category increase) was higher in the LPV/r group (RH=1.69, 95% CI: 1.14–2.50; P=0.01). Models weighted for the inverse of conditional probability of receiving either drug applied to the efficacy endpoints yielded similar results. CD4 recovery with both drugs was also similar in the lowest CD4 strata. Conclusions Our analysis suggests similar efficacy and tolerability for EFV- or LPV/r-based first-line antiretroviral regimens. LPV/r was associated with higher rates of hypertriglyceridaemia.

Objective: To investigate the impact of 215 HIV-1 revertants on the risk of virological failure of the first thymidine analog-containing highly active antiretroviral therapy (HAART).Design: The study included 491 HIV-1 subjects of the Italian Cohort Naive for Anti retrovirals, 405 of whom received a genotypic assay before therapy and had a virological follow-up.Methods: Pre-treatment genotypic resistance was assessed by sequencing of the whole protease (PR) and reverse transcriptase (RT) region.Results: Three (3.2%) and 13 (3.3%) individuals with recent (n = 95) and chronic (n = 396) HIV-1 infection carried an HIV-1 strain with 215 revertants (215D/C/E/A/V), respectively. In contrast, nucleoside associated mutations were higher in the former (15.8%) compared with the latter group (6.8%) (P = 0.005). A multivariable regression model, considering pre-HAART viral load levels, use of saquinavir-hard gel as the only PI, use of zidovudine, number of other RT and PR mutations, indicated that patients carrying 215 revertants had an increased risk of virological failure compared with those not carrying such mutants (adjusted relative hazard = 2.97 95% confidence interval, 1.11-7.94, P = 0.03). Among patients with 215 revertants, who experienced virological failure, four out of seven showed the emergence of the 215Y resistant mutation. The probability of 215Y occurrence was different between patients carrying 215 revertants compared with those who did not carried these mutants (P = 0.006).Conclusions: HIV-1 215 revertants with an increased ability for selecting 215Y mutation are associated with a higher risk of virological failure and may lead to the appearance of virus carrying 215Y/F mutation in vivo. These findings suggest that 215 revertant viruses may compromise the efficacy of the first thymidine analog-containing regimen. (C) 2004 Lippincott Williams Wilkins.

Background: Subpopulations of HIV with mutations associated with resistance to antiretroviral drugs often have reduced replicative capacity, so virus with resistance mutations for all existing and new antiretroviral drugs is likely to be appreciably impaired. Issues of toxicity, quality of life and economics mean that the simultaneous use of all these drugs in combination is unrealistic. We aimed to explore the use of sequential monotherapy regimens using a mathematical model of quasi-species dynamics, to see if these could take advantage of the poor replicative capacity of highly resistant virus.Methods: We assume for each of seven drugs that a single mutation is associated with the ability to replicate (effective reproductive ratio, R>1) in the presence of that drug as monotherapy. Parameters included were drug efficacy, the cost of resistance mutations and the number of new target cells arising daily.Results: The use of seven drugs in a daily/weekly sequential monotherapy cycle led to substantial viral suppression (in the presence of all resistant viral subpopulations) for a wider range of parameter values than a continuous five-drug regimen. Although on any one day/week there is a viral subpopulation with R>1 (e.g. that with resistance only to the current drug), this subpopulation does not have time to grow sufficiently during the short period when that drug is being taken.Conclusion: These results provide a rationale for trials of sequential regimens, using as wide a number of drugs with different resistance-associated mutations as possible, as a potential 'resistance-proof' strategy for achieving significant viral load suppression. (C) 2003 Lippincott Williams Wilkins.

Objective To document the demographic changes in new HIV diagnoses at the Royal Free Hospital, London, UK, between 1994 and 2000. Design Retrospective case note review. Methods Data were extracted from the Royal Free HIV database identifying new diagnoses for 1994, 1997 and 2000. All case notes were reviewed and patients were included if they had their first positive HIV test at the Royal Free Hospital, or if they first tested positive elsewhere and attended the Royal Free HIV unit for their initial HIV care. Data extracted included sex, ethnicity, age, risk factor(s) for HIV, reason for test, clinical stage of disease, CD4 count and HIV RNA viral load at diagnosis. Results One hundred and forty-four patients were identified for 1994, 136 for 1997 and 110 for 2000. Over this time period the proportion of white patients dropped from 72% (n = 104) to 48% (n = 53), P= 0.0001, whilst the proportion of black Africans rose from 24% (n = 34) to 45% (n = 49), P= 0.0004. The median CD4 count at diagnosis of the white cohort was 475 cells/µL in 1994 and 286/µL in 2000, P= 0.005, whilst in the black African patients it was 240/µL and 230/µL for the same years. Conclusions There has been a reduction in new HIV diagnoses among the white population and a rise in the black Africans at this centre between 1994 and 2000. The clinical and immunological parameters of HIV disease have worsened over this time period for the white group, but have remained stable in the black Africans.

Objective: To assess the prevalence of mutations in the reverse-transcriptase (RT) and protease (PR) region in a cohort of chronically-infected HIV-positive patients requiring highly active antiretroviral therapy (HAART). Methods: The study included 347 patients enrolled in the Italian Cohort of Antiretroviral Naive patients (I.CO.NA) who had to initiate HAART. The whole PR-region, and aminoacids 1-320 of RT-region were sequenced from plasma samples at baseline. Results: Median CD4-lymphocytes and HIV-RNA at baseline were 231 × 10 6 cells/l and 4.89 log 10 copies/ml; 307 of 347 (88.5%) patients carried no mutations in the RT region, whereas 40 (11.5%) carried one or more mutations associated with resistance to nucleoside-RT inhibitor (NRTI) (7.8%), or non-nucleoside-RTI (NNRTI) (4.9%), with four patients carrying mutations to both classes. Among mutations associated with high-level resistance to RTI, T215Y was found in only two patients, M184V in two cases, T69D and T215C in other two cases (one each), and K103N in only one patient, for a total of six patients (one carrying both T215Y and M184V) (1.7%). Seventy-six patients (21.9%) carried no mutations in the PR region, whereas 271 (78.1%) had one or more mutations. Primary mutations associated with substantial resistance to protease inhibitors were found in only five of 347 patients (1.4%) (M46V/L, 154V, V82A/l); all the other patients carried only secondary mutations (L10F/I/V, M36I, L63P, A71T/V, V77I). Conclusions: Prevalence of mutations associated with high-level resistance to antiretroviral drugs is low in HIV-infected patients with long-term infection. This suggests no preclusion in principle to any antiretroviral drug at the time of decision of the first therapeutic regimen.

OBJECTIVE:: Use of antibiotics in critically ill patients may increase the risk of invasive Candida infection. The objective of this study was to determine whether increased exposure to antibiotics is associated with increased prevalence of invasive Candida infection.DESIGN:: Substudy using data from a randomized controlled trial, the Procalcitonin And Survival Study 2006-2010.SETTING:: Nine multidisciplinary ICUs across Denmark.PATIENTS:: A total of 1,200 critically ill patients.INTERVENTION:: Patients were randomly allocated to either a "high exposure" antibiotic therapy (intervention arm, n = 604) or a "standard exposure" guided by current guidelines (n = 596).MEASUREMENTS AND MAIN RESULTS:: Seventy-four patients met the endpoint, "invasive Candida infection," 40 in the high exposure arm and 34 in standard exposure arm (relative risk = 1.2; 95% CI, 0.7-1.8; p = 0.52). Among medical patients in the high exposure arm, the use of ciprofloxacin and piperacillin/tazobactam was 51% and 75% higher than in the standard exposure arm; no difference in antibiotic exposure was observed between the randomized arms in surgical patients. Among medical intensive care patients, invasive Candida infection was more frequent in the high exposure arm (6.2%; 27/437) than in standard exposure arm (3.3%; 14/424) (hazard ratio = 1.9; 95% CI, 1.0-3.6; p = 0.05). Ciprofloxacin used at study entry independently predicted invasive Candida infection (adjusted hazard ratio = 2.1 [1.1-4.1]); the risk gradually increased with duration of ciprofloxacin therapy: six of 384 in patients not exposed (1.6%), eight of 212 (3.8%) when used for 1-2 days (hazard ratio = 2.5; 95% CI, 0.9-7.3), and 31 of 493 (6.3%) when used for 3 days (hazard ratio = 3.8; 95% CI, 1.6-9.3; p = 0.002). Patients with any ciprofloxacin-containing antibiotic regimen the first 3 days in the trial had a higher risk of invasive Candida infection than did patients on any antibiotic regimen not containing ciprofloxacin (unadjusted hazard ratio = 3.7; 95% CI, 1.6-8.7; p = 0.003; adjusted hazard ratio, 3.4; 95% CI, 1.4-8.0; p = 0.006).CONCLUSIONS:: High exposure to antibiotics is associated to increased risk of invasive Candida infection in medical intensive care patients. Patients with ciprofloxacin-containing regimens had higher risk of invasive Candida infection. Other antibiotics, such as meropenem, piperacillin/tazobactam, and cefuroxime, were not associated with such a risk.

Introduction : Sexually transmitted diseases (STDs) data collected in HIV+ patients could be used as indicator of risky sexual behaviour possibly linked to HIV transmission. We described the STDs incidence over time and identified higher incidence factors. Methodology : All patients in the ICONA Foundation Study enrolled after 1998 were included. STDs considered: any-stage syphilis, human papilloma virus (HPV) diseases, gonococcal and non-gonococcal urethritis, herpes simplex virus (HSV) genital ulcers, vaginitis and acute hepatitis B virus (HBV), hepatitis A virus (HAV), and hepatitis C virus (HCV) infections (only for non-IVDU (intravenous drug user) patients). STDs incidence rate (IR): number of STDs divided by person years of follow-up (PYFU). Calendar periods: 1998 – 2002, 2003 – 2007 and 2008 – 2012. Predictors of STDs occurrence were identified using Poisson regression and sandwich estimates for the standard errors were used for multiple STD events. Results : Data of 9,168 patients were analyzed (median age 37.3 (SD=9.3), 74% male, 30% MSM). Over 46,736 PYFU, 996 episodes of STDs were observed (crude IR 17.3/1,000 PYFU). Median (IQR) CD4/mmc and HIV-RNA/mL at STD: 433 (251 – 600) and 10,900 (200 – 63,000). Highest crude IRs were observed for any-stage syphilis (3.95, 95% CI 3.59 – 4.35), HPV diseases (1.96, 1.71 – 2.24) and acute hepatitis (1.72, 1.49 – 1.99). At multivariable analysis (variables of adjustment shown in Figure 1), age (IRR 0.82 per 10 years younger, 95% CI 0.77 – 0.89), MSM contacts (IRR 3.03, 95% CI 2.52 – 3.64 vs heterosexual) and calendar period (IRR 1.67, 95% CI 1.42 – 1.96, comparing 2008 – 2012 with 1998 – 2002) significantly increased the risk of acquiring STDs. Moreover, having a HIV-RNA >50 c/mL (IRR 1.44, 95% CI 1.19 – 1.74 vs HIV-RNA 500) showed an increased risk of STDs. Being on ARV treatment significantly reduced the risk of developing an STD (IRR 0.37, 95% CI 0.32 – 0.43) compared to ART-naive people, even in the situation of temporary interruption of treatment (IRR 0.51, 95% CI 0.39 – 0.43) (see Figure 1). Conclusions : The overall incidence of STDs has been increasing in the recent years. Interventions to prevent STDs and potential further spread of HIV infection should target the recently HIV diagnosed, the young population and MSM. Being on ARV treatment (potentially an indicator of whether a person is regularly seen for care) seems to reduce the risk of acquiring STDs independently of its viro-immunological effect. (Published: 2 November 2014) Citation : Cingolani A et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19653 http://www.jiasociety.org/index.php/jias/article/view/19653 | http://dx.doi.org/10.7448/IAS.17.4.19653

Introduction : We aimed at evaluating the efficacy and durability of a lopinavir/ritonavir-based dual regimen (LPV/r-DR) in virologically controlled HIV-infected individuals in current clinical practice. Methods : Patients who have initiated for the first time a LPV/r-DR with HIV-RNA 50 copies/mL] and time to experience either a single VL>200 copies/mL or discontinuation/intensification (= treatment failure, TF). Individuals’ follow-up accrued from the date of starting the LPV/r-DR to event or last available VL. Kaplan-Meier curves and Cox regression analysis were used. Covariates included in the multivariable analysis were gender, age, route of transmission, hepatitis co-infection, calendar year of starting the DR, nadir CD4+ count, VL at initiation of first cART, previous failures to protease inhibitors (PIs), time with undetectable VL before starting the DR and the type of DR [nucleoside reverse transcriptase (NRTI), non-NRTI (NNRTI), raltegravir or maraviroc, with NRTI as reference group]. Results are presented as median (Q1, Q3) or frequency (%) as appropriate. Results : 108 individuals followed for 18 (7, 30) months were included; baseline (BL) characteristics are detailed in Table 1. LPV/r was associated with a NRTI in 51, with a NNRTI in 10, with raltegravir in 29, and with maraviroc in 18 individuals. By 36 months from switching to the LPV/r-DR, the proportion of individuals with VR and TF was 10% (95% CI 3–17%) and 36% (95% CI 22–50%), respectively. We did not find any factor independently associated with the risk of VR. Older age (ARH=0.49 (95% CI 0.30–0.78) per 10 years older; p=0.003) was found to be protective from TF. Mean (SE) CD4+ cells/µL increase from BL to month 36 resulted significant: 195 (40.1) cells/µL (p=0.0028). We did not observe significant changes in AST, ALT, eGFR (MDRD formula), triglycerides and both total and HDL-cholesterol. Conclusions : A LPV/r-DR can be considered a valuable option in patients with HIV-RNA

Introduction : PI/r monotherapy has been suggested as an attainable maintenance strategy in patients achieving stable HIV suppression in plasma. The objective of trial was to compare the virological outcome of two different PI/r QD monotherapy strategies (LPV/r or DRV/r) with maintaining a triple PI/r-based ARV regimen. Material and Methods : Phase III, open-label, non-inferiority (−12% margin), randomized trial of HIV adults with HIV-RNA 100 cell/mm 3 , without previous PIs virological failure. Eligible patients were randomized to continue PI/r+2NRTIs (Arm A), to switch to LPV/r 800/200 mg QD monotherapy (Arm B), or to switch to DRV/r 800/100 mg QD monotherapy (Arm C). Primary endpoint was proportion of patients with plasma HIV-1 RNA

Introduction : In clinical trials, toxicity leading to discontinuation of tenofovir (TDF) is a rare occurrence (3% by two years)[1,2]; however, in clinical practice it seems to be higher. Previous studies suggested that TDF toxicity is higher when it is co-administered with ritonavir-boosted protease inhibitors (PI/r)[3,4]. The aim of this study is to assess the rate of TDF discontinuations in clinical practice and to explore associated factors. Methods : All previously antiretroviral-naive patients initiating a TDF-containing regimen were selected from the ICONA cohort, unless they were positive for hepatitis B. The primary outcome was TDF discontinuation (>30 days) regardless of the reason, the secondary was TDF discontinuation due to toxicity. All analyses were repeated for the isolated stop of TDF (no stop of associated drugs). The main reason for discontinuation as reported by the treating physicians was used to classify stops. Kaplan–Meier (KM) analysis and Cox proportional hazards model were used. Results : A total of 3,303 naive patients were enrolled: 674 (20.4%) were female, the median age was 38 years (32–45), 55% were on PI/r-based regimen and 45% on NNRTI; 80% of calculated estimated glomerular filtration rates (eGFR) were >90 ml/min. The probability of discontinuation of TDF regardless of the reason was 10% (95% CI 8–11) at two years, 20% by eight years. The causes of discontinuation were: toxicity (33%), failure (10%), non-adherence (21%), simplification (16%) and other/unknown causes (20%). The five-year KM estimates in the PI/r vs. not PI/r groups were 23% vs. 10%, respectively (log-rank p=0.0001), for the outcome of stopping regardless of the reason, and 8% vs. 4% (p=0.18) for discontinuation due to toxicity. In a multivariable Cox model, PI/r use and lower body weight were associated with increased risk of discontinuing TDF regardless of the reason; lower eGFR at baseline was associated with TDF discontinuation for toxicity and PI/r use was associated with isolated stop of TDF (Figure). No differences in rates of TDF discontinuations between PIs were found. Conclusion : In our cohort, the observed frequency of TDF discontinuations was low although higher than estimated in clinical trials (10% by two years). Co-administration of TDF with PI/r was associated with an increased rate of TDF discontinuations. This finding should guide further investigations of the mechanism that may have led to discontinuation of TDF in patients using PI/r. (Published: 2 November 2014) Citation : Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Costarelli S et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19571 http://www.jiasociety.org/index.php/jias/article/view/19571 | http://dx.doi.org/10.7448/IAS.17.4.19571

Introduction : Serious non-AIDS events (SNAE) are frequent in HIV patients receiving cART. Current CD4 count was shown to be more strongly associated with infective compared to not-infective SNAE and unable to predict cardiovascular events. We investigated the relationship between baseline and current CD4 count and the risk of both infective and non-infective SNAE in HIV-positive patients according to current ART use. Methods : We included all HIV-positive persons enrolled in the ICONA Foundation Study cohort who had at least one follow-up visit. SNAE were grouped in infective (pneumonia, sepsis, endocarditis and meningitis) and non-infective (malignancies, chronic kidney disease, cardiovascular events, hepatic events and pancreatitis) aetiology. Incidence of these event groups were calculated overall and according to baseline and current CD4 count (grouped as 0–200, 201–350, 351–500, 501–750, and >750 cells/mm 3 ). Participants’ follow-up accrued from the date of enrolment (baseline) to a diagnosis of SNAE or their last visit. An event was defined the first time one of the considered SNAE occurred so that each person contributed a single event. A Poisson regression model for each of the two endpoints was used. Results : A total of 10,822 patients were included (25.3% females, 38.2% heterosexuals) and 26.6% had hepatitis co-infections. Median age was 36 (IQR 31–42) years. Overall, 423 not-infective and 385 infective SNAE were included. The most frequent non-infective SNAE were malignancies ( n =202) and the most frequent infective SNAE were pneumonia ( n =289). Crude rates of non-infective SNAE were 0.78, 1.08 and 0.80/100 PYFU, and those of infective SNAE were 1.00, 0.51 and 0.66/100 PYFU in ART naive, currently off and currently on ART patients, respectively. Higher current CD4 count was associated with reduced risk of both infective and non-infective SNAE in naives and in patients on ART (Table 1). The association was less strong in the group which suspended ART (for non-infective SNAE the p value for interaction between current log-CD4 and ART-status, p =0.004). Conversely, we found no association between baseline CD4 count and risk of non-infective SNAE in people treated with ART ( p value for interaction=0.0001). When CVD were considered separately, there was no association with CD4 count (not shown). Conclusions : Our findings show that, differently from ART naive, in ART-treated patients, non-infective SNAE are predicted by current but not by baseline CD4, suggesting that immune restoration is crucial to prevent these events. (Published: 2 November 2014) Citation : Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Madeddu G et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19509 http://www.jiasociety.org/index.php/jias/article/view/19509 | http://dx.doi.org/10.7448/IAS.17.4.19509

Objectives: To investigate differences in liver enzyme elevation (LEE) between HIV-infected patients with and without HCV coinfection who start a darunavir/ritonavir-containing regimen. Methods: HIV-infected patients enrolled in the Italian Cohort of Naïve to Antiretrovirals (ICONA) Foundation Study were included if they started darunavir/ritonavir for the first time. Patients were classified as not HCV coinfected, HCV active coinfected (HCV RNA positive), and HCV nonactive coinfected (HCV-Ab positive/HCV RNA negative). Time to LEE endpoint was defined using the ACTG toxicity scale, based on changes relative to baseline. Kaplan-Meier was used to estimate 1-year and 2-year probability of LEE. The incidence rate ratios (IRRs) of LEEs were estimated until the last follow-up (intention-to-treat analysis [ITT]) and up to darunavir/ritonavir discontinuation (on-treatment analysis [OT]). Results: Overall, 703 patients were included. Ninety-one were HCV-Ab positive; of those, 68 (9.7%) had active HCV coinfection. In 879 person-years of follow-up, 101 LEEs occurred (ITT). No severe hepatotoxicity event was registered in active HCV coinfected patients. HCV active coinfection was predictive of LEE in the overall population (OT: adjusted incidence rate ratio (IRR), 2.25; 95% CI, 0.70-7.24; P = .17; ITT: adjusted IRR, 3.62; 95% CI, 1.67-7.83; P < .001) and in naïve patients (OT: adjusted IRR, 6.29; 95% CI, 2.54-15.55; P = .00; ITT: adjusted IRR, 3.87; 95% CI, 0.99-15.16; P = .05). Conclusions: No grade 3-4 LEEs occurred in HCV active coinfected patients. HCV active coinfected patients experienced low grade LEEs more frequently than HCV-Ab negative patients. Darunavir/ritonavir seems to be safe whatever the HCV status, when liver enzymes are carefully monitored. © 2014 Thomas Land Publishers, Inc.

Introduction : The aim of this study was to analyze the likelihood and the predictors of discontinuation of first-line regimen in the late HAART era. Methodology : An observational multi-center analysis of HIV-positive patients enrolled in ICONA. Patients eligible were those starting a first-line HAART after 1 January 2008. Discontinuation was defined as stop and/or switch of at least one drug of the regimen. All causes of discontinuation, as reported by the treating physician, were evaluated and cumulative risk of stopping was investigated according to age, gender, co-morbidity, years since starting HAART, immuno-virological status, third drug and backbone of the first regimen. Kaplan Meier (KM) analysis and Cox proportional hazards model were used for the outcome discontinuation of ≥1 drug regardless of the reason. For the KM estimates a competing risk approach was used to estimate the contribution of each of the reasons over time to the cumulative risk of stopping over time. Results : Data of 1759 patients who started first HAART and had at least one month of clinical follow-up were analyzed. The overall discontinuation risk was 33% over a median follow-up of 12 months. The likelihood of discontinuation by KM was 27% by one year (95% CI 25–29) and 41% by two years (95% CI 38–44). Main reason for stopping at least one drug in regimen was simplification (10%), followed by intolerance (7%), toxicity (5%), failure (2%) and other causes (8%). Estimates of the cumulative risk of discontinuation of ≥1 drug over time and according to reason are shown in Figure 1. In a multivariable Cox model independent predictors of discontinuation regardless of the reason were: longer time from HIV diagnosis to date of starting HAART (hazard ratio [HR] 0.96; 95% CI 0.93–1.00; p=0.039), regimens containing ZDV/3TC (HR 2.86; 95% CI 1.42–5.76; p=0.003 vs TDF/FTC) and an NNRTI-based regimen (HR 2.47; 95% CI 0.91–6.72; p=0.07 vs regimens not NNRTI-based). Conclusions : In a previously reported analysis of the ICONA data [1], the overall risk of discontinuation of first-line HAART was 36% with 21% due to intolerance/toxicity. In this updated analysis, the main reason for stopping is simplification (accounting for 32% of stops), reflecting the recent changes in recommendations aimed to minimize drug toxicity, enhancing adherence and quality of life. (Published: 2 November 2014) Citation: Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Di Biagio A et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19825 http://www.jiasociety.org/index.php/jias/article/view/19825 | http://dx.doi.org/10.7448/IAS.17.4.19825

Background : Use of unboosted atazanavir (ATV 400 ) is approved in the US but not in Europe [1]. Due to pharmacokinetic interactions it should not be used with tenofovir but can be used with abacavir/lamivudine (ABC/3TC) [1, 2, 3]. Effectiveness data of ATV 400 +ABC/3TC as a switch strategy in clinical routine however are scant. Methods : We evaluated treatment outcomes of ATV 400 +ABC/3TC in pre-treated subjects in the EuroSIDA cohort with undetectable HIV-1 RNA, and previous ABC experience or assumed previous HLA B57*01 testing. We performed a time to loss of virologic response (TLOVR below 50 c/mL) and a snapshot analysis at 48, 96 and 144 weeks. Virological failure (VF) was defined as a confirmed plasma HIV-1 RNA >50 c/mL. Results : We included 258 subjects: 176 (68%) male, median age 46 (IQR 41, 53) y, 225 (87.2%) white, hepatitis virus co-infection 36%, median baseline CD4 at switch 540 cells (360, 700), time with VL≤ 50 c/mL 45 (24, 69) months. The median calendar year of switching was 2008 (2006, 2010). The 3rd drug in previous regimen was ATV/r in 70 (27.1%), other PI/r in 25 (9.7%), and other 163 (63.2%); 85 (32.9%) had previously failed with a PI. The virological response at 48/96/144 weeks was, respectively, 89.5 [95% CI 85.1, 92.9]/88 [83.4, 91.7]/86.3% [81.6, 90.4] (TLOVR, composite endpoint failure or stop for any reason) and the risk of VF was 8.3/7.6/7.6%. In the snapshot analysis HIV-RNA was below 50 c/mL in 72.5/65.9/51.6%, respectively, and >50 c/mL in 6.6/5.4/4.3%. Only 0.8/1.9/3.5% discontinued due to adverse events. There was a high rate of discontinuations due to other reasons or with VL missing in window. In a multivariate adjusted analysis, we observed an association between VF and nadir CD4 count (RH 0.60 [0.39, 0.93] per 100 cells higher), time with VL≤50 c/mL (RH 0.89 [0.81, 0.98] per 6 months longer) and previous failure with a PI (3.04 [1.36, 6.80]). There was no association with gender, age, hepatitis virus co-infection, CD4 count at time of switching or third drug used in the previous regimen. Conclusions : A switch to ATV 400 +ABC/3TC in selected subjects with HIV-RNA below 50 c/mL is associated with relatively low rates of VF and discontinuation due to adverse events. Use might be considered in those with long-term suppression and without prior PI failure. Larger cohorts are required to further define the appropriate selection criteria. (Published: 2 November 2014) Citation: Abstracts of the HIV Drug Therapy Glasgow Congress 2014 Llibre JM et al. Journal of the International AIDS Society 2014, 17(Suppl 3) :19810 http://www.jiasociety.org/index.php/jias/article/view/19810 | http://dx.doi.org/10.7448/IAS.17.4.19810

This study was designed to assess the influence of highly active antiretroviral therapy (HAART) on non-Hodgkin lymphoma (NHL) among patients infected with human immunodeficiency virus (HIV). Within EuroSIDA, a multicenter observational cohort of more than 8500 patients from across Europe, the incidences of NHL and subtypes (Burkitt, immunoblastic, primary brain lymphoma [PBL], and other/unknown histology) were determined according to calendar time of follow-up, and for those who initiated HAART (or =3 drugs) also time on HAART. Potential predictive factors of NHL were evaluated in Cox proportional hazard models. Over 26 764 person-years of prospective follow-up (PYF) from May 1994 to December 2000, the incidence of NHL decreased from 1.99 (95% confidence interval, 1.51-2.47) before September 1995 to 0.30 (0.19-0.42) cases/100 (PYF) after March 1999 (P.001). The incidence of all subtypes of NHL decreased significantly and most pronouncedly for PBL. Among patients who started HAART, the incidence of NHL decreased from 0.88 (0.60-1.16) within the first 12 months after starting HAART to 0.45 (0.31-0.60) cases/100 PYF after more than 24 months (P =.004). In an adjusted Cox model for patients on HAART, the latest CD4 cell count and plasma viral load were both significantly associated with diagnosis of NHL; the relative hazard was 1.39 (range, 1.14-1.69) per 50% lower CD4 cell count, and 1.51 (range, 1.21-1.88) per 1 log higher plasma viral load. In conclusion, the incidence of NHL among HIV-infected patients has decreased significantly after the introduction of HAART, and the decline was most pronounced for PBL. After starting HAART, patients with insufficient immunologic and virologic responses were at highest risk of NHL.

Background Relatively little is known about the long-term durability of viral suppression in individuals initially achieving a viral load of less than 50 copies/ml within 24 weeks of starting antiretroviral therapy, nor the extent to which therapy interruption accounts for the loss of suppression. Methods We intensely followed all 336 antiretroviral-naive patients attending the Goethe Universitat Clinic who began multi-drug combination regimens and in whom a viral load of less than 50 copies/ml was achieved within 24 weeks, in order to assess the risk of viral load rebound. Inspection of case notes allowed the distinction of viral rebound according to whether there was an associated complete interruption of therapy. Results A total of 61 patients experienced viral rebound during 543.1 person-years of follow-up, giving a 25.3% risk of rebound by 3.3 years from first achieving viral suppression. However, for 47 of the patients with viral rebound there was an associated documented complete interruption of antiretroviral therapy, mostly as a result of co-morbidities, leaving 14 who appear to represent a failure of the virological efficacy of therapy (viral breakthrough; 5.2% risk by 3.3 years). The risk of viral breakthrough declined with the increased duration of suppression (P = 0.01). Conclusion The intrinsic virological effectiveness of multi-drug antiretroviral therapy in previously drug-naive individuals appears to be such that viral suppression, once achieved, can be maintained for several years in patients not interrupting therapy. The major challenge is to develop regimens that can be taken consistently and safely for such long periods of time.

To describe the reasons for, and factors associated with, modification and discontinuation of highly active antiretroviral therapy (HAART) regimens at a single clinic.A total of 556 patients who started HAART at the Royal Free Hospital were included in analyses. Modification was defined as stopping or switching any antiretrovirals in the regimen, whereas discontinuation was defined as the simultaneous stopping of all antiretrovirals included in the initial regimen. Reasons were classified as immunological/virological failure (IVF) and toxicities and patient choice/poor compliance (TPC).The median CD4 count at starting HAART was 171 x 10(6) cells/l and viral load 5.07 log copies/ml. During a median follow-up of 14.2 months, 247 patients (44.4%) modified their HAART regimen, 72 due to IVF (29.1%) and 159 due to TPC (64.4%) and a total of 148 patients (26.6%) discontinued HAART. Older patients were less likely to modify HAART [relative hazard (RH), 0.73 per 10 years; P = 0.0008], as were previously treatment-naive patients (RH, 0.65; P = 0.0050), those in a clinical trial (RH, 0.64; P = 0.027) and those who started nelfinavir (RH, 0.57; P = 0.035). Patients who started with four or more drugs (RH, 2.21, P0.0001), who included ritonavir in the initial regimen (RH, 1.41; P = 0.035) or who had higher viral loads during follow-up (RH per log increase, 1.51; P0.0001) were more likely to modify HAART.There was a high rate of modification and discontinuation of HAART regimens in the first 12 months, particularly due to toxicities, patient choice or poor compliance.

Objectives: To describe the viral response to HAART by weeks 4 and 8 in previously antiretroviral-naive patients. To assess whether the weeks 4 or 8 viral loads are useful predictors of viral suppression by week 24.Design: A large clinical database including 453 antiretroviral-naive patients whose plasma viral load was monitored every 4 weeks.Methods: Observed probabilities of achieving a viral load less than or equal to 500 copies/ml by week 24 (days 84-168) from starting highly active antiretroviral therapy (HAART) were calculated according to viral loads at weeks 4 and 8.Results: A total of 42.4% of patients (153/361) reached less than or equal to 500 copies/ml viral load by week 4 and 70.4% (245/348) by week 8. Viral suppression below 500 copies/ml by 4-8 weeks was similar irrespective of the pre-HAART viral load. In patients with viral loads above 10 000 copies/ml at week 4, 60.6% (20/33) achieved less than or equal to 500 copies/ml by week 24. In patients with viral toads still above 10 000 copies/ml at week 8, only 42.3% (11/26) achieved less than or equal to 500 copies/ml by week 24, and only 33.3% (3/9) maintained viral suppression below 500 copies/ml to week 48.Conclusion: Viral loads at weeks 4 and 8 should be monitored to detect early signs of low subsequent viral suppression. For previously antiretroviral-naive patients whose viral loads after 8 weeks of HAART are still above 10 000, there is an urgent need to assess adherence to therapy, drug levels and resistance, so management can be modified accordingly to reduce the rate of week 24 virological failure. (C) 2001 Lippincott Williams & Wilkins.

Human immunodeficiency virus-1 (HIV-1) infection has been shown to result in up-regulation of the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes in vitro and in vivo. The objective of this study was to investigate whether this up-regulation is paralleled by higher serum levels of soluble uPAR (suPAR) in patients with advanced HIV-1 disease and whether the serum level of suPAR is predictive of clinical outcome. Using an enzyme-linked immunosorbent assay, the level of suPAR was measured retrospectively in serum samples from 314 patients with HIV-1 infection. By Kaplan-Meier and Cox regression analyses, the serum suPAR levels were correlated to survival with AIDS-related death as the end point. High levels of serum suPAR (greater than median) were associated with poor overall survival, and Kaplan-Meier analysis on patients stratified by suPAR level demonstrated a continuous increase in mortality rates with higher suPAR levels. After adjustment for accepted prognostic markers—including Centers for Disease Control and Prevention–defined clinical stages, CD4 counts, viral load, β2-microglobulin, and age—the prognostic strength of suPAR remained highly significant, indicating that the serum suPAR level is a novel, strong, and independent predictor of survival in HIV-1 infection. This report is the first to demonstrate an important association between the plasminogen activator system and disease progression in HIV-1 infection.

It is recommended that randomized controlled trials be analyzed on an intention-to-treat (ITT) basis whereby patients are analyzed in the group to which they were originally assigned, irrespective of the treatment actually received. However, in trials of antiretroviral therapy, it is quite common for patients to withdraw from the trial, and information on virological or immunological endpoints may not be available. The way in which this missing information is dealt with in the analysis can have a large effect on the results of a trial and, therefore, the principle of ITT may be adhered to more closely in some studies than in others. This article describes some simple approaches commonly taken to impute missing data values and discusses the possible effects of these approaches on the results of a trial.

Context: According to recent studies, women have lower plasma HIV RNA concentrations than men. However, these studies did not take into account the duration of HIV infection. Objectives: To analyze the relationship between viral load and gender among individuals with known date of seroconversion. Setting: Sixty infectious disease clinics in Italy. Design: Cross-sectional analysis of data collected at enrollment in a cohort study. Participants: Injecting drug users and heterosexual contacts naive to antiretroviral therapy at enrollment (245 men; 170 women). Main Outcome Measures: Plasma HIV RNA concentrations, measured using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) or signal amplification b-DNA assays before antiretroviral therapy. Results: Plasma HIV RNA concentrations were similar by age and exposure category (p = .80 and p = .39, respectively). Median viral load among women was roughly half that of men (p = .002). The association between viral load and gender remained significant after fitting a two-way analysis of variance (p = .03) and after adjusting for CD4 count, modality of HIV transmission, and age at enrollment in a regression model. Viral load was 0.27 log 10 copies/ml (95% confidence interval, 0.05-0.40; p = .01) lower in women (i.e., 50% lower in the raw scale). Conclusions: Plasma HIV RNA concentrations were found to be lower among women, even when considering the duration of HIV infection. Compared with men, it is possible women should be given highly aggressive antiretroviral therapy at lower HIV-RNA concentrations.

Summary Background We used data from Europe, North America, and Australia to assess the effect of exposure category on the AIDS incubation period and HIV-1 survival and whether the effect of age at seroconversion varies with exposure category and with time since seroconversion. Methods 38 studies of HIV-1-infected individuals whose dates of seroconversion could be reliably estimated were included in the analysis. Individual data on 13 030 HIV-1-infected individuals from 15 countries were collated, checked, and analysed centrally. We calculated estimates of mortality and AIDS incidence rates and estimated the proportions of individuals surviving and developing AIDS at each year after seroconversion from the numbers of observed deaths or cases of AIDS and the corresponding person-years at risk. Analyses were adjusted for age at seroconversion, time since seroconversion, and other factors as appropriate. Findings Mortality and AIDS incidence increased strongly with time since seroconversion and age at seroconversion. Median survival varied from 12·5 years (95% Cl 12·1–12·9) for those aged 15–24 years at seroconversion to 7·9 years (7·4–8·5) for those aged 45–54 years at seroconversion, whereas for development of AIDS the corresponding values were 11·0 years (10·7–11·7) and 7·7 years (7·1–8·6). There was no appreciable effect of exposure category on survival. For AIDS incidence, the exposure category effect that we noted was explained by the high incidence of Kaposi's sarcoma in those infected through sex between men. We estimated that among people aged 25–29 years at seroconversion 90% (89–91) and 60% (57–62) survived to 5 years and 10 years, respectively, after seroconversion, whereas 13% (12–15) and 46% (44–49), respectively, developed AIDS (excluding Kaposi's sarcoma). Interpretation Before widespread use of highly-active antiretroviral therapy (before 1996), time since seroconversion and age at seroconversion were the major determinants of survival and development of AIDS in Europe, North America, and Australia.

Objective: To evaluate the frequency of discontinuation of the first highly active antiretroviral regimen (HAART) and the factors predictive of discontinuing for toxicity and failure in a population-based cohort of HIV-positive individuals in Italy, naive from antiretrovirals at enrolment.Methods: The study population consisted of individuals who initiated HAART and had at least one follow-up visit. The primary end-points were discontinuation of any component of HAART for drug toxicity and discontinuation for failure. Survival analyses were performed to identify predictive factors for reaching the two end points.Results: Eight hundred and sixty-two individuals initialed HAART; in 727 of them (84.3%) this consisted of two nucleoside reverse transcriptase inhibitors (NRTI) and one protease inhibitor (PI). Over a median follow-up of 45 weeks, 312 patients (36.2%) discontinued therapy: 182 (21.1%) discontinued due to toxicity, 44 (5.1%) due to failure. The probability of discontinuing HAART at 1 year was 25.5% [95% confidence interval (Cl), 21.9-28.9] due to toxicity and 7.6% (95% Cl, 4.9-10.3) due to failure. Independent factors associated with discontinuation for toxicity were: gender [relative hazard (RH) = 0.51; 95% Cl, 0.32-0.80 for men versus women], type of treatment (indinavir-containing regimens, RH = 1.94; 95% Cl, 1.10-3.41 and ritonavir-containing regimens, RH = 3.83; 95% Cl, 2.09-7.03 versus hard-gell saquinavir) and time spent on treatment (RH = 0.89; 95% Cl, 0.80-0.98 for each additional month). Discontinuation due to failure was independently associated with the most recent HIV-RNA (RH = 3.20; 959/0 Cl, 1.74-5.88 for log(10) copies/ml higher), and with type of treatment (indinavir-containing regimens, RH = 0.21; 95% Cl, 0.06-0.78 and ritonavir-containing regimens, RH = 0.23; 95% Cl, 0.04-1.26 Versus hard-gell saquinavir).Conclusions: If the current HAART regimen caused no toxicity, less than 10% of naive patients discontinue their first HAART regimen because of failure after 1 year from starting therapy. (C) 2000 Lippincatt Williams & Wilkins.

The association between CD4 cell count and duration of virus load suppression was investigated in 558 patients in the Frankfurt HIV Clinic Cohort who had begun highly active antiretroviral therapy and who had virus load declines to/=500 copies/mL. The Kaplan-Meier method estimated viral rebound in 42.5% of patients by 24 weeks and in 64.3% by 84 weeks. Risk of viral rebound was independently associated with baseline and changes from baseline CD4 cell count. The achieved CD4 cell count was the most important factor for prediction of viral rebound (relative hazard, 5.4 for an updated CD4 cell count of20 vs.500 copies/mL; P=.0001). Baseline virus load was not associated with virus load rebound. Lower baseline CD4 cell counts were associated with increased risk of viral rebound; however, this risk was significantly reduced in persons with low baseline CD4 cell counts who experienced substantial increases in CD4 cell counts during follow-up.

OBJECTIVE To describe the changes over time in incidence of hospital admissions among patients with HIV, reasons for hospital admission, duration of stay, relationship with CD4 T-cell count and with antiretroviral treatment. METHODS The incidence of hospital admissions during each calendar year from 1988 to 1997 inclusive was calculated using a person-years analysis. In addition the proportion of patient follow-up spent in hospital and the impact of changing treatment regimens among all patients with HIV aged > or = 14 years and with at least one CD4 T-cell count seen at the Royal Free Hospital, London was also described. RESULTS A total of 1806 patients were investigated with median follow-up of 21.1 months. Among all patients, the proportion of follow-up time spent as an in-patient decreased from 3.9% in 1988 to 1.3% in 1997 (P = 0.0015; test for trend). Hospital admissions for any cause peaked during 1989 at 72.0 per 100 patient years of follow-up (PYFU) and was 28.5 per 100 PYFU during 1997 (P 30% before 1990 to < 5% during 1997 (P = 0.026; test for trend). Hospital admissions varied greatly according to treatment regimen; in 1996 and 1997 just 0.1% of follow-up time of patients on triple antiretroviral treatment regimens was spent as a hospital admission. CONCLUSIONS Admissions to hospital began falling before the introduction of combination therapy and declined strikingly during 1996 and 1997 following the introduction of highly active antiretroviral therapy. These results have important implications for future allocation of resources and for patient management.

Objectives: To illustrate a simple approach to adjusting for bias due to drop-outs (i.e., attrition bias) when evaluating the effect of a certain therapy in HIV clinical trials using the mean change in plasma viral load. To evaluate its validity and to compare its performance with that of another simple method for handling dropouts: the last observation carried forward (LOCF) method.Design: Data from a notional treated group of 100 patients followed up to 52 weeks were generated. Attrition bias was introduced by mimicking selective patient dropout (i.e., more likely in patients doing badly).Methods: The difference between the true mean change in HIV RNA levels at 52 weeks and the observed mean change because of drop-outs was calculated (attrition bias). The reduction in bias obtained by using the proposed approach was then calculated and compared with that obtained by using the LOCF method. To assess the performance of the methods over the entire follow-up, the mean areas under the curves were considered.Results: Our method reduced the bias by a clinically relevant amount in a variety of different settings. In most of our simulations, bias was reduced by a larger amount than that obtainable from using the LOCF method.Conclusions: The current situation is that results from trials in HIV infection are invariably presented with no associated attempt to quantify the attrition bias present. Attrition-adjusted plots of mean change in HIV RNA should, we believe, be presented alongside usual plots as a form of sensitivity analysis. (C) 1998 Lippincott-Raven Publishers.

Background: The role of sexual transmission of hepatitis C virus (HCV) infection is still not completely understood, partly because of the lack of longitudinal studies among cohorts of HCV-negative individuals who engage in at-risk sexual behavior. Goals: To evaluate the incidence of HCV infection in a population at risk for human immunodeficiency virus type 1 (HIV-1) infection and other sexually transmitted diseases (STD) and to identify factors associated with HCV seroconversion. Study Design: A retrospective longitudinal study was carried out on a cohort of consecutive attendees of a voluntary HIV-1 testing and counseling program in a large STD center in Rome. All individuals undergoing at least two consecutive tests for HCV antibodies were enrolled. Clinical data and information on individual behavior were collected for all study participants. Results: Between June, 1992 and December, 1994, a total of 709 individuals (12 intravenous drug users [IDU], 244 homosexuals, and 453 heterosexual non-IDUs), initially negative for HCV antibody, were tested more than once. Among these individuals, 15 HCV seroconversions occurred. The average follow-up time was 1.25 person/years (p/y) for an incidence rate of 1.69 per 100 p/y. The incidence rates by exposure category were 39.30 per 100 p/y among IDUs, 1.37 per 100 p/y among homosexual men, and 0.97 per 100 p/y among heterosexual non-IDUs. Excluding IDUs, of the 697 STD clinic attendees engaging in at-risk sexual behavior, HIV-1-positive status tended to be associated with HCV seroconversion (relative hazard = 5.48; 95% confidence interval = 0.85-35.40). The HCV crude incidence rates among HIV-1-infected patients at enrollment was 11.5%, 4.2%, and 2.4% in those with severe, moderate, and mild levels of immunosuppression, respectively (chi-square for trend = 2.38, P = 0.1). Conclusions: In this cohort, HCV infection was confirmed to be strongly associated with intravenous drug use. Nonetheless, the occurrence of two thirds of the total HCV seroconversions in non-IDU individuals engaging in at-risk behavior suggests a role of sexual practices in the transmission of the infection. Among non-IDU individuals, the risk for development of HCV infection tended to increase in those who were HIV-1 infected.

It has been suggested that the rate of CD4 cell decline accelerates in parallel with decreasing numbers of cells; however, the statistical literature suggests the opposite, CD4 cells were counted about every 6 months in a cohort of 1264 human immunodeficiency virus-infected subjects (the Italian Seroconversion Study cohort), Kaplan-Meier analysis was used to estimate the time for CD4 cells to decline by 100 cells/mm(3), conditional on reaching predefined levels, In addition, CD4 cell counts were modeled as a function of time since seroconversion in individuals with greater than or equal to 5 counts, Kaplan-Meier survival times for a 100 cell/mm(3) decrease in CD4 cells increased as lower counts were reached (log rank test, P