To date, data on the durability of a regimen of stavudine, lamivudine and nevirapine are very limited, particularly from the resource-limited settings.A prospective cohort study was conducted among 140 antiretroviral-naïve patients who were enrolled to initiate d4T, 3TC and NVP between November 2004 and March 2005. The objectives were to determine immunological and virological responses after 144 weeks of antiretroviral therapy. Seventy patients with tuberculosis also received rifampicin during the early period of antiviral treatment (TB group).3; median (IQR) baseline HIV-1 RNA was 433,500  copies/mL. The average body weight was 55 kilograms. By intention-to-treat analysis at 144 weeks, the overall percentage of patients who achieved plasma HIV-1 RNA <50 copies/mL was 59.3% (83/140). In subgroup analysis, 61.4% (43/70) patients in TB group and 57.1% (40/70) patients in control group achieved plasma HIV-1 RNA <50 copies/mL . Eight (5.8%) patients discontinued d4T due to neuropathy and/or symptomatic lactic acidosis.Of all, median (IQR) baseline CD4 cell count was 31 (14–79) cells/mmThe overall durability and efficacy of antiviral response of d4T, 3TC and NVP are satisfied and they are not different between HIV-1 infected patients with and without co-administration of rifampicin due to tuberculosis. However, stavudine-related adverse effects are concerns.ClinicalTrials.gov Identifier NCT00703898 Currently, the preferred first-line antiretroviral regimens use a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a ritonavir-boosted protease inhibitor . AlthougIn addition, HIV-1 infection has contributed to a significant increase in the worldwide incidence of tuberculosis and this has substantially affected the mortality ,4. RifamMycobacterium tuberculosis, (3) receiving rifampicin-containing anti-TB regimen ≥1 month prior to enrollment, (4) CD4 cell count <350 cells/mm3 and (5) willing to participate and give consent form. Inclusion criteria for the control group were: (1) HIV-infected individuals ≥15 years of age (2) not receiving RFP within 1 month prior to enrollment, (3) CD4 cell count <350 cells/mm3 and (4) willing to participate and give consent form. Exclusion criteria for both two study groups was: (1) previous antiretroviral therapy, (2) pregnancy, (3) receiving a medication that has drug-drug interactions with NVP or RFP and (4) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) >5 times of upper limit of normal range. The administered antiretroviral drugs were stavudine, lamivudine and nevirapine. All patients received NVP 200-mg once-daily lead-in dose for 14 days, prior to escalation to 200 mg twice daily. In the present study, the patients in both groups were followed up through 144 weeks in which period of time they were assessed clinically and evaluated for adverse events. CD4 cell counts and plasma HIV-1 RNA were assessed every 12 weeks until 96 weeks of ART and then every 24 weeks through 144 weeks. The patients in TB group were repeatedly tested for chest X-ray and clinically evaluated for tuberculosis at 144 weeks of ART. The institutional ethics committees of Bamrasnaradura Infectious Diseases Institute and Ministry of Public Health approved the study. All patients signed the inform consent.The study design was a prospective cohort study involving 140 HIV-infected Thai patients in the Bamrasnaradura Infectious Diseases Institute, Ministry of Public Health, Nonthaburi, Thailand. There were equally 70 patients in TB group and control group. Initial enrollment was from November 2004 to March 2005 as previously described . Inclusi® version 1.5 ; lower limit of HIV-1 RNA detection, 50 copies/ml. The virological failure was defined as either a rebound plasma HIV-1 RNA of >1,000 copies/mL after having previously undetectable value or lack of achievement to <50 copies/mL at 24 weeks of ART. Genotypic resistance testing  was performed after the patient was documented virological failure.The dosage of stavudine was adjusted by body weight . The general characteristics , previous opportunistic infections and site of tuberculosis infection) were recorded. Blood samples were obtained to study CD4 cell counts by flow cytometry and HIV-1 RNA by polymerase chain reaction using Roche AmplicorAnti-tuberculosis regimen included isoniazid, rifampicin, ethambutol and pyrazinamide were administered in the first two months followed by isoniazid and rifampicin for the subsequent four months. Tuberculosis treatment outcomes  were evaluated by using definitions from WHO and the European Region of the International Union Against Tuberculosis and Lung Disease (IUATLD) .P value < 0.05 was considered as statistically significant. All analyses were performed using SPSS version 11.5.Power and Sample Size version 1.01  was usedP = 0.731). Mean CD4 cell count response is shown in figure A total of 140 patients were eligible and initially enrolled to the study. Seventy patients were equally included in each group. The patients' general characteristics, baseline CD4 counts and plasma HIV-1 RNA of each group are shown in table P = 0.688). The reasons were as follow: lost to follow-up , HIV drug resistance , NVP-related skin rashes grade II-III , d4T-related neuropathy and/or symptomatic lactic acidosis , deceased , transferred care  and drug interaction due to receiving itraconazole , respectively. Of 70 patients in each group, 10% (7 of 70) and 9% (6 of 70) patients in the TB group and control group developed HIV-1 RNA >1,000 copies/mL, respectively (P = 1.000). Among 13 patients with drug resistance, 5 (38%) had mutations contributed to only NRTI resistance; 1 (8%) had mutations contributed to only NNRTI resistance; and 7 (54%) patients had mutations contributed to both NRTI and NNRTI resistance. For NRTI-resistance mutations, M184V/I was the most common . Thymidine analogue associated mutations (TAMs) were found in 1 (8%) patients. K65R was observed in 2 of 13 (15%) patients. For NNRTI-resistance mutations, there were Y181C/I  and K103N  as shown in figure P > 0.05).Of all, 57 patients needed to discontinue ART after 144 weeks of ART, 27 patients were in TB group and 30 patients were in control group. There was no difference in term of ART discontinuation between the two groups  patients were cured or completed treatment; 7 (10%) lost to follow-up; 5 (7%) deceased ; 2 (3%) transferred care; and 1 (1%) had relapse.P = 0.048) [The present study has demonstrated that an antiretroviral regimen of stavudine, lamivudine and nevirapine showed acceptable long-term antiviral effectiveness in advanced HIV-1 infected patients. By intention-to-treat analysis, approximately 60% patients still achieve undetectable plasma HIV-1 RNA through 144 weeks. Consequently, CD4 cell count continues to increase significantly over the study period. This is considered favorable outcome when compared to the other NNRTI-based regimen -17. In s= 0.048) . AlthougLong-term acceptability and safety may be a concerned issue for this regimen. It is well established that long-term administration of NRTI, particularly stavudine, can cause mitochondrial toxicity. The clinical manifestations of this adverse effect present as hyperlactatemia and polyneuropathy . In the The initial sample size of the present study is calculated from the difference of minimum plasma concentration of nevirapine between the two groups of patients . Thus, iIn conclusion, a regimen of stavudine, lamivudine and nevirapine provides the satisfied durability and immunological response in very advanced HIV-infected patients. There is no difference of the 144-week efficacy between HIV-1 and tuberculosis co-infected patients receiving rifampicin and HIV-1 mono-infection not receiving rifampicin. However, long-term safety of stavudine is a concern. Relapse rate after 3 years of initiation of tuberculosis treatment in the patients who are receiving ART is low. In resource-limited settings, a regimen of stavudine, lamivudine and nevirapine is still an important first-line option for advanced HIV-1 infected patients. Strategy to minimize long-term toxicity of stavudine, such as switching to other drugs at an optimal timing, should be evaluated further.The authors declare that they have no competing interests.WM participated in the design of the study, draft the manuscript and performed the statistical analysis. PT participated in its design and coordination. SL carried out the laboratory testing. SS participated in draft the manuscript and statistical analysis.The pre-publication history for this paper can be accessed here:


Immune Reconstitution syndrome following antiretroviral therapy is common in HIV/AIDS patients due to boosting of immunity. A case is reported here wherein AIDS-related Non-Hodgkin‘s lymphoma patient received CHOP regimen and antiretroviral therapy. Patient developed tubercular lymphadenopathy paradoxically as a manifestation of IRIS. Immunodeficiency Virus (HIV) patients are on Highly Active Anti-Retroviral Therapy, and there is significant improvement in the CD4+ T cell count, reduction in viral load, and significant clinical improvement. This paradoxical response is seen mainly in advanced HIV infection (CD4+ T cell count < 50 cells/l). IRIS may manifest as the reactivation of dormant infections, such as, tuberculosis, cryptococcosis or leprosy.[4Acquired Immune Deficiency Syndrome AIDS-related lymphoma following chemotherapy and Highly Active Antiretroviral Therapy (HAART).Immune Reconstitution Inflammatory Syndrome (IRIS) is defined as a paradoxical worsening of the pre-existing opportunistic infection(s) when Human  leprosy. Sometime leprosy.3 Their i leprosy.45 There A case of AIDS-related Non-Hodgkin Lymphoma (ARL), which was treated with the cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone / prednisolone (CHOP) regime and Efavirenz-based Anti-Retroviral Therapy, developed IRIS with tubercular infection within nine months of therapy, and is discussed here.The 53-year-old male patient presented in April 2003, with fever, right axillary lymphadenopathy, weight loss, and oral candidiasis for two months. Routine investigations confirmed diffuse large B cell lymphoma and HIV-1 infection. Profound immune deficiency was observed. His CD4+ T cell count was 43 cells/L, CD8+ T cell count 339 cells/l and plasma HIV RNA level was 110,174 copies/ml (by PCR ultrasensitive). The physical examination revealed oral thrush, axillary lymphadenopathy, and hepatosplenomegaly. Routine investigations revealed hemoglobin 11.6 Gm/dl, with normal WBC, platelet counts and indices. The bone marrow examination was normal. Liver function tests and renal function tests were normal. S. LDH was raised, (900 IU/L). He was treated with six courses of chemotherapy with CHOP regime. Zidovudine, Lamivudine, and Efavirenz were started for control of HIV infection. He showed significant improvement with chemotherapy and tolerated HAART. He received cotrimaxazole, antifungal, and tuberculosis prophylaxis as well. He was in good condition during the next six months of observation. His CD4+ T lymphocyte count increased significantly and reached 156 cells/L, CD8+ T cell count was 875 cells/l and plasma HIV RNA was less than the level of assay quantification by PCR ultrasensitive (50 copies/ml).  There weThree months later the patient was admitted with painful right axillary lymphadenopathy, fever and sweating on follow-up. An axillary lymph node biopsy demonstrated acid fast bacilli but no evidence of Non-Hodgkin Lymphoma. IRIS with tuberculosis was diagnosed. Routine blood counts, urine analysis, liver function tests, renal function tests and serum electrolytes were normal. There was no evidence of active tuberculosis on chest X-ray. Ultrasonic study of the abdomen and pelvis showed hepatosplenomegaly. The antituberculosis regimen and corticosteroids along with HAART were started for tubercular lymphadenopathy. He developed hyperlipidemia following anti-Retroviral therapy. Serum cholesterol was 355 mg per deciliter. He was prescribed Atorvastatin 20-mg daily along with anti-Retroviral, antitubercular agents. After six years, the patient is receiving regular HAART and is in good health.de novo lymphoma.[Real incidence of AIDS-related lymphoma in India is not known. Non-Hodgkin Lymphoma (NHL) is the most common AIDS-defining cancer in Indian patients. In an Indian study of 250 cases of AIDS-associated cancers, 30.8% had Non-Hodgkin Lymphoma, while inProtease-inhibitor based Highly Active Anti-Retroviral Therapy is expensive and is associated with profound neutropenia when concurrent chemotherapy is administered and alterations in anticancer drug levels, due to drug–drug interactions. When HAA11Corticosteroids such as methylprednisone along with other specific treatments, are required for the control of HIV-associated IRIS. AIDS-ass


Aripiprazole is the first medication approved by the FDA as an add-on treatment for MDD. The impact of aripiprazole on the response to HIV is unknown. The patient we report on was diagnosed HIV-positive in 1997 and has been treated with antiretroviral therapy since then. In 2008, we diagnosed resistant major depression, hypochondria, and panic disorder. On that occasion, blood tests showed a significantly reduced CD4 count and a positive viral load. We treated this patient with aripiprazole and citalopram. Mood, somatic symptoms, and occupational functioning progressively improved. The last blood examination showed an increase in the CD4 count and a negative viral load. On the basis of the present case study and the review of the literature concerning the effects of psychotropic agents on viral replication, we suggest that the use of aripiprazole in HIV-infected subjects warrants further research. In November 2007 the Food and Drug Administration (FDA) approved the use of aripiprazole as an adjunctive, or add-on, treatment to antidepressant therapy in adults with major depressive disorder (MDD). Aripiprazole is the first medication approved by the FDA as an add-on treatment for MDD , modulatDrugs active on the dopaminergic system, such as methamphetamine, have been claimed to exert an effect on HIV infectivity , 4. LentWe present a case of resistant depression, somatoform disorder, and panic disorder in an HIV-infected subject, treated with aripiprazole as augmentation therapy. In this case aripiprazole led, not only to significant improvement in depressive symptomatology, but also to an improvement in CD4 cell count and viral load.μL and a viral load of 3000 copies/mL. All the other diagnostic tests were negative. In the meantime, he was prescribed different therapies to control anxiety, somatisation, and mood instability: prokinetics (domperidone 30 mg/die), omeprazole (20 mg/die), levosulpiride (50 mg/die), lorazepam (3 mg/die), trimipramine (100 mg/die) for 8 weeks, paroxetine (20 mg/die) for 10 weeks, and mirtazapine (30 mg/die) for 12 weeks. None of these drugs led to substantial clinical improvement. In September 2008 he came to our outpatient clinic owing to persistent symptomatology. We diagnosed resistant major depression, somatoform disorder (hypochondria), and panic disorder. Resistant depression was defined as a depressive episode with at least two previous failed antidepressant trials, each lasting at least 6 weeks (stage II of antidepressant resistance according to Thase and Rush)  in microglia [As regards aripiprazole's immunological impact, we know that several antidepressants  show up such an effect in that they exert an immunomodulatory effect, reducing proinflammatory cytokines and increasing anti-inflammatory cytokines . Antidepγ(IFN-γ) . In vitricroglia . We suppAnother possible reason for the improvement in the patient's immune status is an indirect effect of aripiprazole on cortisol levels brought about by improvement in depression. The dysregulation of the HPA axis function  has been associated with stress and depression in humans, and such dysregulation may negatively impact the immune response. There are data supporting the fact that glucocorticoids may affect HIV pathogenesis directly, through increased viral replication, or more indirectly by inhibiting the immune response to other pathogens. In fact, in 1997 Christeff et al.  demonstrAs a final point, we cannot exclude the possibility that the improvement in depression symptomology may result in an increased adherence to antiretroviral treatment.On the basis of the present case study and the review of the literature about the effects of psychotropic agents on viral replication, we suggest that the use of aripiprazole in HIV-infected subjects warrants further research.


Little is known about immunovirological treatment outcomes and adherence in HIV/AIDS patients on antiretroviral therapy (ART) treated using a simplified management approach in rural areas of developing countries, or about the main factors influencing those outcomes in clinical practice.Cross-sectional immunovirological, pharmacological, and adherence outcomes were evaluated in all patients alive and on fixed-dose ART combinations for 24 months, and in a random sample of those treated for 12 months. Risk factors for virological failure  and subtherapeutic antiretroviral (ARV) concentrations were investigated with multiple logistic regression.At 12 and 24 months of ART, 72% (n = 701) and 70% (n = 369) of patients, respectively, were alive and in care. About 8% and 38% of patients, respectively, were diagnosed with immunological failure; and 75% and 72% of patients, respectively, had undetectable HIV RNA (<400 copies/ml). Risk factors for virological failure  were poor adherence, tuberculosis diagnosed after ART initiation, subtherapeutic NNRTI concentrations, general clinical symptoms, and lower weight than at baseline. About 14% of patients had low ARV plasma concentrations. Digestive symptoms and poor adherence to ART were risk factors for low ARV plasma concentrations.Efforts to improve both access to care and patient management to achieve better immunological and virological outcomes on ART are necessary to maximize the duration of first-line therapy. In 2007, approximately 810,000 adults were living with HIV/AIDS in Uganda, and 25–49% of those requiring antiretroviral therapy (ART) were receiving treatment  patients when the 30-day VAS was used). In the M24 cohort, good adherence to ART in the last 4 days was reported by 258 (93.1%) patients (250 [90.3%] using VAS)  and correlation was stronger for M12 than for M24 measurements . Twenty-seven (6.0%) patients considered as having good adherence by VAS reported to have forgotten taking one or more pills in the last 4 days; while 37 (8.1%) of those considered as having good adherence through 4-day recall reported poor adherence with VAS. A total of 85 (16.8%) patients were categorized as poorly adherent to ART by any of the two methods.3, immunological failure was diagnosed in 7.9% of patients, and 40% had <200 cells/mm3  had HIV viral suppression, and 81.9% (227/277) had <1,000 HIV RNA copies/ml.At M24, median CD4 cell count was 238 cells/mmWe performed genotypic tests for 62 (82.7%) of patients with VL ≥ 1,000 copies/ml (PCR amplification failed for 13 samples) . One virus was resistant to 3TC, d4T, zidovudine (AZT), and NNRTIs (EFV and NVP), and 1 harbored K65R, K103N, Y181C, and G190A mutations conferring resistance to tenofovir (TDF), EFV, NVP, and possibly didanosine (ddI).In the M24 cohort, only 1 virus was resistant to NVP and EFV, and 22 to both 3TC and NNRTIs. Six viruses with resistance mutations to both 3TC and NVP had additional resistance to d4T and/or AZT. Three viruses harbored a K65R mutation conferring resistance to TDF and possibly to ddI. One virus had a multidrug resistance profile , conferring resistance to all the NRTIs and to EFV and NVP.Thymidine analogue mutations (TAMs) conferring resistance to AZT and d4T were observed in 5% and 14% patients at M24, respectively. Resistance to both TDF and ddI were described in 5% (1/20) at M12 and 7% (3/42) at M24. The NRTI multidrug resistance complex (Q151M) was found in 1 patient at M24. None of the viruses had mutations conferring resistance to the new NNRTIs (etravirine). Seven patients were resistant to AZT, d4T, 3TC, NVP, and EFV (6 in M24).Factors significantly associated with virological failure were longer time on therapy , ≥ 35 years of age at ART start , TB diagnosis during ART , weight less than baseline weight at time of ART initiation , poor adherence , low NNRTI plasma concentration , and presentation of general signs or symptoms   and 4.3% (1/23) of patients, respectively, and high in 24.5% (50/204) and 39.1% (9/23), respectively. NFV plasmatic concentrations were within normal therapeutic ranges (n = 2). In the M24 cohort, low plasma NVP concentrations were observed in 12.4% (32/258) of patients and high NVP and EFV concentrations in 20.9% (54/258) and 38.9% (7/18), respectively.P = 0.02). This association was observed in both cohorts  and reported digestive disorders  were significantly associated with the presence of low plasma NNRTI or NFV concentrations (Table 2). Estimates of retention in care appear to be more favorable than those reported during the early years of the Drug Access Initiative (DAI) program among individuals treated in urban hospitals of Kampala, where patients were requested to pay the care fees  and more than 30% were ARV-experienced [In the HIV care and treatment program of Arua, in rural Uganda, more than 70% of the patients remained in care after 1 or 2 years of treatment, but over 35% of patients remained at risk of developing opportunistic infections  laboratory-based toxicity were diagnosed. Self-reported adherence at 12 and 24 months of ART was high and similar to that reported in other studies, both in resource-limited countries  and NortThe observed immune and virological responses at 1 and 2 years of ART were similar to those reported in other African settings ,22,35,36Overall, 7% of patients with virological failure at M12 and 13% at M24 had resistant virus, which is similar to estimates reported for patients treated with 2 NRTI/1 NNRTI-based therapy in east Africa or South-east Asia after more than 9 months of treatment (9–18%) ,35,38. TEstimates of resistance and virological and immunological failure might also have been underestimated if some of the deaths or patients who were lost to follow-up died as a result of treatment failure and resistance. However, most deaths and losses to follow-up occurred within the first months of therapy when the likelihood of treatment failure was lower. Conversely, because our study is based on outcome data collected at a single time point and no confirmation of laboratory data was obtained in the following weeks, we might have slightly overestimated rates of virological and immunological failure due to measurement errors or to individual temporal variability in biological markers . As in previous studies conducted in patients using primarily d4T/3TC/NVP, the most commonly observed resistance mutations were those associated with the use of NNRTIs K103N) and 3TC (M184V) ,10,38, a3N and 3TPatients on ART for 2 years were twice as likely as those on 1 year treatment to develop virological failure. Patients diagnosed with TB after ART initiation were also more likely to fail therapy, suggesting that patients' virological response could be impaired when rifampicin-based anti-TB treatment is initiated while a patient is under ART. The potential interaction between NVP and rifampicin in HIV-infected patients treated for TB has been reported in previous studies -43. ThisEarly identification of patients with virological failure and switch to second-line therapy is important to prevent the accumulation of resistance that might compromise the effectiveness of subsequent lines of treatment. In this program where virological monitoring of patients is not routinely performed, patients presenting general clinical symptoms, those with a weight loss below the value observed at therapy initiation, or those reporting poor adherence were more likely to have virological failure. The association between high VL levels and subtherapeutic NNRTI concentrations has been previously reported in Malawi  and CambThe pharmacological assessment showed increased plasmatic levels of NNRTI in 23% of patients on NVP-based therapy and 39% of those on EFV. These figures are higher than what has been described in Western countries, and are likely to be related to the lower weight of our patients at ART initiation  comparedIn conclusion, in this HIV/AIDS care program implemented in a rural African setting using a simplified scale-up approach and free generic ART, survival and retention in care were satisfactory at up to 2 years of therapy, but efforts to increase earlier access to treatment and to improve patient management and support to achieve better immunological and virological outcomes on ART remain necessary to prolong patient survival. Early identification of poor adherence during follow-up, especially in the presence of intolerance to ARV and when TB treatment is provided, should be considered to maximize the duration of first-line therapy.The authors declare that they have no competing interests.LA designed and implemented the study, analyzed and managed data, interpreted results, and wrote the manuscript. GG and CO set up and implemented the study in the field and contributed to the interpretations of results. LP performed statistical analyses. PA contributed to the design, set-up, and implementation of the study. M-LC and CLT performed the virological and pharmacological tests and contributed to the interpretation of the results. SB and DO helped to design the study, interpret the results, and draft the manuscript. MP-R performed statistical analysis, interpreted results, and co-wrote 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/1471-2334/9/81/prepub


We tested the hypothesis of changing HIV-1 virulence by examining trends in prognostic clinical markers of disease progression from 1984 to 2005 among nearly 400 antiretroviral-naïve participants in the United States Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV infection in men who have sex with men (MSM).Several long-term cohort studies and 10 copies/mL/year, p = 0.76; at ∼15 months: slope = −0.005 log10 copies/mL/year, p = 0.71), CD4 cell count after seroconversion , or rate of CD4 cell decline over the first three years after seroconversion .Because clinical AIDS endpoints could not be used , three prognostic markers of disease progression were used as proxies for HIV-1 virulence: plasma viral RNA load and CD4+ T cell count at “set point” (between ∼9 and ∼15 months after seroconversion), and rate of CD4 cell decline within three years after seroconversion. We performed multivariate analyses of the association between these markers and seroconversion year, with covariates including MACS site, race/ethnic group, seroconversion age, and CCR5Δ32 status. No statistically significant association was found between year of seroconversion and “set point” plasma viral load (at ∼9 months after seroconversion: slope = −0.004 logThe lack of significant trends from 1984 to 2005 in these prognostic markers of HIV disease progression suggests no major change in HIV-1 virulence over the AIDS pandemic in MSM in the US. Arien et al. et al.in-vitro methods in an Amsterdam cohort (n = 25), found that HIV replication capacity was greater in recent (late 1990s) than in earlier isolates (mid-1980s), suggesting the opposite conclusion. In a recent large study of 22 cohorts from Australia, Canada, and Europe, Dorrucci et al. (2007) found trends of decreasing CD4+ T-cell counts (n = 3687) and increasing viral loads (n = 1584) in measurements at ∼4 months after seroconversion between 1985 and 2002, suggestive of increasing HIV-1 virulence over time Whether HIV-1 has become more or less virulent since its introduction into the human population has significant implications for the course of the AIDS pandemic. Over the last two decades, several studies of long-term cohorts have resulted in conflicting data on this question, including reports of increasing Studies of host-pathogen interactions suggest that hosts and pathogens can co-adapt to each other, with pathogens becoming less virulent over time Pathogen virulence is most commonly measured in host mortality 10 copies/mL (n = 357); at the third seropositive visit (∼15 months) this figure was 4.399 log10 copies/mL (n = 318) (The demographic characteristics of the study population are shown in n = 318) . The medn = 318) .10 copies/mL/year,  , we found no significant temporal trend in viral load      . These tAll of the above analyses were also done using calendar years in bins comprised of data from 5 year intervals, and yielded results consistent with the above analyses of individual calendar years. We also tested for a trend over time in the lag between estimated date of seroconversion and the second and third seropositive visits; no trend was found.We tested for associations between calendar year of seroconversion and three prognostic markers of disease progression in the MACS cohort between 1984 and 2005. Our results showed no significant trends in set point plasma viral RNA load, CD4 cell count after seroconversion, or the rate of CD4 cell decline in the first three years after seroconversion. Moreover, estimates of change in these markers over time were very close to zero. Thus, the results of this study do not support the hypothesis that there has been any important change in the virulence of HIV-1 over this time period in this cohort. This is consistent with recent findings that revealed no long-term changes in virulence over 20 years in a Swiss cohort et al. et al.'s results, we estimated trends from 1984 to 2005 in the plasma viral load and CD4 cell count at the first seropositive visit in the MACS cohort, corresponding to ∼3 months after seroconversion. We again found no significant association between viral load and seroconversion year , but did observe a decreasing trend in square-root transformed CD4 cell count . We suspect this result, and the discrepancy between our analyses at ∼9 and ∼15 months after seroconversion and those of Dorrucci et al., may be due to the significant CD4 cell fluctuations that occur during acute and early infections et al. cohorts.Our results are contrary to those from studies of long-term cohorts have found decreasing in-vitro competitive fitness data between early (1986–1989) and recent isolates (2002–2003) from Belgium et al. in-vitro fitness are not directly or easily compared to clinical prognostic markers or virulence in humans, and that larger sample sizes may be required in these assays.Our results are also contrary to the recent inference of HIV-1 attenuation based on e.g. CCR5Δ32, CCL3L1) have changed significantly in humans between 1984–2005. HIV-1 subtypes can vary in disease progression rates independently from viral load as well Host factors  can affect rates of disease progression independently of viral load e.g., prophylaxis and treatment of opportunistic infections, and development of more potent antiretroviral therapies). Thus, we had to rely on prognostic markers to study trends in HIV-1 disease progression rates. Third, because we excluded men who started antiretroviral therapy, there is a possible selection bias; men who experienced disease progression would not contribute to our analysis, and any decrease of the mean time from seroconversion to first therapy (into the second or third seropositive visit) over the study period would not be detected. However, the number of men excluded is small , and this likely did not impact our analyses. Fourth, we could not correct for the changes in laboratory methods for CD4 or viral load measurements over this time period, as the these changes correlate completely with year (and we are looking for changes in estimates of these markers by year). This issue is one that plagues all similar studies, including Dorrucci et al. (2007). Lastly, although we found no significant changes in set point viral load over the 20 years of observation, the 95% confidence intervals encompass a range of ∼−0.6 to +0.4 log10 copies/mL over this period  is an ongoing prospective study of the natural and treated histories of HIV-1 infection in homosexual and bisexual men conducted by sites located in Baltimore, Chicago, Pittsburgh and Los Angeles At the time of the present analysis, there were 2884 men who had entered the study as HIV-seroprevalent, and 596 men with a known date of seroconversion. Estimated date of seroconversion was the median time between the last seronegative visit and the first seropositive visit. For each participant, we included only data from visits prior to the start of any antiretroviral treatment ). We further restricted the analysis to those 483 seroconverters who had less than one year between their last seronegative and first seropositive visits. Of these, 357 had plasma viral load measurements at ∼9 months after estimated date of seroconversion, and 318 had viral load measurements at ∼15 months after seroconversion date. The corresponding numbers for CD4 cell counts were 375 and 348, respectively . To incrThe 596 men with known dates of seroconversion included 59 men from Pittsburgh who enrolled in the MACS already seroprevalent but with a known seroconversion date; 49 of these men had less than one year between their last seronegative and first seropositive visits, and 11 of these had viral load and CD4 measurements within 6 to 18 months of seroconversion. For our tests of seroconversion year versus rate (slope) of CD4 cell decline, 412 men contributed at least two measurements within three years following seroconversion and had no missing information on race, age, and CCR5Δ32 genotype, and thus contributed to the multivariate analysis.At each semiannual MACS visit, absolute CD4+ T lymphocyte counts were obtained by flow cytometry 10 copies/mL) at the second (∼9 months) and third (∼15 months) seropositive visits; 2) the CD4 cell count  at the second and third seropositive visits; and 3) slope of the CD4 cell decline per year within three years after seroconversion. Covariates included MACS center location , race , age at seroconversion (centered at 30 years old), and CCR5 gene status (homozygous wild type vs. heterozygous Δ32). Statistical associations were performed using SAS 9.1 . Graphs for figures were generated using S-Plus 6.2 .We performed univariate and multivariate linear regression of the association between date of seroconversion (independent) and prognostic markers of HIV infection (dependent): 1) plasma viral load (log


Concern about costs and antiretroviral therapy (ART)-associated toxicities led to the consideration of CD4 driven strategies for the management of HIV. That approach was evaluated in the SMART trial that reported an unexpected increase of cardiovascular events after treatment interruption (TI). Our goal was to evaluate fasting metabolic changes associated with interruption of antiretroviral therapy and relate them to changes of immune activation markers and cardiovascular risk.ACTG 5102 enrolled 47 HIV-1-infected subjects on stable ART, with <200 HIV RNA copies/mL and CD4 cell count ≥500 cells/µL. Subjects were randomly assigned to continue ART for 18 weeks with or without 3 cycles of interleukin-2 (IL-2) (cycle = 4.5 million IU sc BID x 5 days every 8 weeks). After 18 weeks ART was discontinued in all subjects until the CD4 cell count dropped below 350 cells/µL. Glucose and lipid parameters were evaluated every 8 weeks initially and at weeks 2, 4, 8 and every 8 weeks after TI. Immune activation was evaluated by flow-cytometry and soluble TNFR2 levels.By week 8 of TI, levels of total cholesterol (TC)   mmol/L, p<0.0001), LDL, HDL cholesterol mmol/L, p = 0.0007 and −0.05, p = 0.0033, respectively) and triglycerides decreased  mmol/L, p = 0.005). However the TC/HDL ratio remained unchanged , p = 0.2). Glucose and insulin levels did not change . After TI there was marked increase in immune activation , p<0.0001) and soluble TNFR2 , p = 0.0008) coinciding with the rebound of HIV viremia.Our data suggests that interrupting antiretroviral therapy does not reduce cardiovascular disease (CVD) risk, as the improvements in lipid parameters are modest and overshadowed by the decreased HDL levels. Increased immune cell activation and systemic inflammatory responses associated with recrudescent HIV viremia may provide a more cogent explanation for the increased cardiovascular risk associated with treatment interruption and HIV infection.NCT00015704ClinicalTrials.gov  The continuous use of antiretroviral therapy (ART) has been associated with a series of metabolic complications An unexpected result of the recently discontinued SMART trial, the largest study that evaluated CD4 driven therapy, was an increase in frequency of cardiovascular events in the drug conservation arm. Although hyperlipidemia is clearly been linked to the development of atherosclerosis, only recently has the critical role of inflammatory mechanisms in the initiation, progression and acute decompensation of the atheromatous plaque been appreciated. Antiretroviral treatment interruption (TI) in chronically suppressed individuals represents a unique situation because it links the acute metabolic changes associated with the discontinuation of ART with acute changes in the systemic degree of inflammation associated with reinitiating HIV replication. Individuals who discontinue ART will predictably go from a state of relatively low systemic inflammation  to a state of high inflammation, coinciding with the increase in HIV viremia that tends to occur approximately 2 weeks after discontinuation of therapy. The goal of this analysis was to evaluate the metabolic changes associated with the discontinuation of ART and the changes in immune activation markers to identify changes in cardiovascular risk in this situation. We used the setting of a prospective randomized trial, ACTG 5102, 3) for at least three months at the time of enrollment and had a CD4 cell count ≥500 cells/mm3. The study was approved by the IRB of all participant institutions and all subjects provided written informed consent.This study is a sub-study of ACTG study 5102. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see 47 subjects were then randomly assigned to continue ART for 18 weeks (Step 1) with (ARM A) or without (ARM B) 3 cycles of IL-2 (cycle = 4.5 million IU sc BID x 5 days every 8 weeks). After 18 weeks ART was discontinued in all subjects (Step 2). Subjects were followed monthly with monitoring of their CD4+ T-cell counts and plasma HIV-1 RNA levels. ART was restarted when the CD4+ cells reached a confirmed level of <350 cells/µL. At that point ART was restarted for 6 months and if the CD4+ cells again reached ≥500 cells/µL, the cycle was repeated. In this analysis we report only the results of the first treatment interruption. Recombinant human interleukin-2 (Proleukin®) was provided by Chiron Corporation. ART were provided by prescription through the subject's physician. No changes of antiretroviral regimen were allowed during Step 1. There were protocol defined recommendations for the management of IL-2 toxicities.16. Serum TNF Receptor alpha 2 was measured before TI and 24 weeks after discontinuation of ART. The metabolic effects of IL-2 had not been described. If there were no changes in the metabolic parameters during the first phase of the study, we planned to combine both arms for the purpose of this analysis.CD4+ and CD8+ T-cell counts were measured using standard flow cytometry techniques at the same local ACTG-certified flow laboratory throughout the study . HIV viral load was determined at weeks 0, 8 and 16 of Step 1 and weeks 2, 4, 6, 8, 16 and every 8 weeks thereafter in Step 2 using the UltraSensitive Roche Amplicor HIV-1 Monitor™ assay in a central laboratory. Fasting metabolic testing  were analyzed at the same time points  at each participating site laboratory. The baseline for Step 1 analysis were the values at randomization into the study. Baseline for changes after treatment interruption were the values obtained immediately before treatment interruption (week 0 of step 2). The number and percentage of CD8+ cells expressing activation markers (CD38+/HLA-DR+) were measured at entry and at week 16 of Step 1 while the participant was receiving ART and at weeks 2, 4, and 8 of Step 2 (TI) using 3 color flow cytometry following ACTG consensus methodologySummary statistics for continuous variables were presented as median . Comparison between treatment arms was carried out using a Wilcoxon Rank Sum Test for ordinal valued outcomes, and a Fisher's Exact Test or Pearson's Chi-square Test for categorical factors. Within each arm, a Wilcoxon Signed Rank Test was used to assess differences over time. Spearman's rank correlation was used to measure the relationship between immunologic and metabolic markers. The level of significance used was 0.05. These results are not adjusted for multiple testing and are therefore exploratory. Laboratory results from participants were censored after reinitiating antiretroviral treatment.The results of the primary analysis of the study have been recently published. Baseline and week 16 changes from baseline in fasting total cholesterol, triglycerides, HDL cholesterol and non-HDL cholesterol, fasting glucose and insulin were comparable between the study arms . AlthougThe baseline values for subsequent analysis are the ones immediately before treatment interruption. After the discontinuation of ART there was a rapid decrease of the concentrations of total cholesterol. By week 8 of TI, levels of total cholesterol (TC) decreased  = −0.73  mmol/L, p<0.0001) as well as triglycerides  mmol/L, p = 0.005). LDL and HDL cholesterol also decreased mmol/L, p = 0.0007 and −0.05, p = 0.0033, respectively) . These dThe total cholesterol to HDL cholesterol ratio decreased slightly during the first 4 weeks  after TI but returned to baseline by week 8 , p = 0.2) and remained unchanged afterwards due to the marked decreases in HDL cholesterol .After interruption the percentage of individuals with HDL cholesterol less than 40 mg/mL (1.04 mmol/L) went from 53% before discontinuation to 76% after 48 weeks. After the discontinuation of ART there were no significant changes in either the fasting glucose or insulin levels.3 in the no-IL2 arm, at the end of step 1 they were 267 and 231 per mm3 respectively, p = 0.55). There was no significant difference within the arms in the change from baseline in the percentage of CD8+ T-cells expressing activation markers (HLA-DR and CD38). Nevertheless, during Step 1 subjects in the IL-2 group  tended to experience a decrease in the percent of activated CD8+ T-cells while those not receiving IL-2 tended to experience an increase .During Step 1 (continuous ART), there was no significant difference within the arms in the change from baseline in the percentage of CD8+ T-cells expressing activation markers (HLA-DR and CD38). The median baseline absolute number of CD8+/HLA DR+ cells were 220 in the IL-2 arm and 204 cells per mmAs expected, after the discontinuation of treatment there was a significant increase in cellular and soluble activation markers in all participants. The increase in CD8+ T-cell activation was faster among individuals that received ART alone during Step 1. These patients experienced a significant increase in activation at week 2 of Step 2 , while those not receiving IL-2 did not experience a significant increase in activation until week 4 . By eight weeks after ART discontinuation the median increase in activated CD8+ cells was similar between groups . After 8 weeks of TI the percentage of activated CD8 cells in the whole group (HLA-DR+ and CD38+ positive) had increased a median of 34% . The levels of TNF receptor alfa II increased approximately by 30% after 24 weeks after the discontinuation of ART , p = 0.0008) .There were no significant correlations between the changes in lipid or glucose parameters and the changes in immune activation markers or viral markers during the first weeks of follow up (data not shown).Our study shows the metabolic effects of discontinuing ART. They generally appear to be the reverse pattern of the sequence of changes observed after the initiation of ART. Over the long term, although the decrease in total and LDL cholesterol might be associated with a decreased cardiovascular risk, those benefits may be offset by the marked decrease in HDL cholesterol observed. Total/HDL cholesterol ratio may more accurately capture the overall cardiac risk as compared to other individual lipoprotein measurements Following treatment interruption there is a prompt and obvious increase in cellular activation, which could negate any potential cardiovascular benefits of TI. The simultaneous combination of these two phenomena (modest decreases in lipids with no net cardiovascular benefit and significant pro-inflammatory changes) could partially explain the increase in cardiovascular events observed in the SMART trial and other studies after the discontinuation of ART. Our study may also provide some insight explaining two apparently discordant large data sets regarding cardiovascular risk and HIV infection: the one from the Veterans Administration study conducted by Bozzete et al. Our study has limitations that are important to point out; first it is a pilot study has a relatively small sample size, especially when compared to the more than 5,000 participants in the SMART trial. However our sample has enough power to identify the immediate effects of treatment discontinuation, both in lipid parameters and immune activation, our stated goals. Second the evaluation of immune activation is relatively crude and completed in a number of limited time points; however the results are consistent with what is known about ongoing HIV viremia. The lost to follow up rate was comparable to antiretroviral treatment trials of similar duration, with only 10 participants dropping out of the study (21%).Our results suggest that TI would not reduce CVD risk, as any improvements in lipid parameters are modest and overshadowed by the decreased HDL levels and the increased immune cell activation due to systemic inflammatory responses associated with returning HIV viremia. These effects could put the patients at a greater cardiovascular risk than when they were receiving antiretroviral treatment. Decreasing immune activation after TI and potentially among patients with ongoing viral replication could mitigate the cardiovascular morbidity and mortality associated with HIV viremia. Prospective trials of anti-inflammatory interventions to mitigate the risk of acute cardiovascular events in cases where treatment must be interrupted are warranted. Treatment interruptions provide a setting in which the relationship between viremia, immune activation and humoral inflammatory markers can be accurately assessed.Checklist S1CONSORT Checklist(0.12 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(0.99 MB DOC)Click here for additional data file.


Knowledge of the predictors of sustained viral response (SVR) to pegylated interferon (PEG-INF) alfa-2a and ribavirin (RBV) therapy in patients with hepatitis C genotype-4 (HCV-4) is crucial for selecting patients who would benefit most from therapy. We assessed the predictors of SVR to this combination therapy in Saudi patients with chronic HCV-4 infection.This retrospective study included 148 patients with HCV-4 infection who underwent clinical, biochemical and virological assessments before treatment and at 12, 24, 48 and 72 weeks post-treatment.2. Seventy-nine of 148 (60.1%) patients were treatment naïve and 110 (74.3%) underwent pre-treatment liver biopsy. Eighteen (12.2%) patients did not complete therapy because of side effects or they were lost to follow up. Early virological response was achieved in 84 of 91 (92.3%) patients. In the 130 (87.8%) patients who completed therapy, 34 (26.2%) were non-responders and 96 (63.8%) achieved end-of-treatment virological response (ETVR). SVR and virological relapse (24 weeks after ETVR) occurred in 66/130 (50.7%) and 30/130 (31.2%) patients, respectively. Compared to relapsers, sustained responders were significantly younger (P=.005), non-diabetic (P=.005), had higher serum albumin (P=.028), lower alpha-fetoprotein level (P=.026), lower aspartate aminotransferase (AST) (P=.04) levels, and were treatment-naivve (P=.008). In a multivariate regression analysis, the independent predictors of SVR were younger age (P=.016), lower serum AST (P=.012), and being treatment naivve (P=.021).Of the 148 patients, 90 (60.8%) were males. Mean (SD) for age was 48.5 (12.7) years and BMI was 27.9 (7.5) kg/mApproximately half of HCV-4 patients who complete the course of combination therapy achieve an SVR, especially if they are young, treatment naivve and have lower AST levels. Chronic hepatitis C virus (HCV) infects approximately 170 million people worldwide, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma and represents the most frequent cause for liver transplantation in the US and Europe.2Pegylated interferon (PEG-INF) plus ribavirin (RBV) therapy given for 48 weeks is now established as the standard therapy for patients with chronic HCV infection with genotypes 1 and 4.6610HCV-4 is known to be endemic in Central Africa and in the Middle East.1116This retrospective study included 148 consecutive patients with chronic HCV-4 infection referred to King Faisal Specialist Hospital and Research Centre (KFSHRC), Riyadh, Saudi Arabia between February 2003 and November 2005. Baseline characteristics of subjects are shown in 37Patients were then treated with PEG-INF alfa-2a  plus RBV  for 48 weeks. Clinical, biochemical and viral parameters were collected both pre-treatment and at weeks 12, 24, 48 and 72 of follow-up.Serum HCV RNA was extracted using an automated extraction system. HCV detection and quantification were performed using an Abbott Real-Time M2000rt PCR assay, which utilized two sets of primers and probes and targeted a conserved region of the 5′ untranslated region of the genome and an internal control. This assay detects and quantifies HCV genotypes (1-6) with a detection limit that ranges from 30 to 100 000 000 IU/mL, where 1 IU/mL=4 copies/mL. Prior to treatment, HCV genotype was performed in all patients  using INNO-LiPA HCV II .Before 2006, viral load testing was performed using the Bayer Quantiplex bDNA System . The lower quantification detection limit was 3200 copies/mL. The highest detection limit was 40 000 000 copies/mL. The average period between the quantitative PCR test and the start of therapy was 2.7 months. The National Institute of Health guidelines state a drop of ≥2 log10 in serum HCV viral load is indicative of response. An early viral response (EVR) was defined as ≥2 log10 drop in serum HCV viral load at 12 weeks after start of treatment. An end-of-treatment virological response (ETVR) was defined as an undetectable serum HCV RNA at 48 weeks. A sustained viral response (SVR) was defined as a persistently undetectable HCV RNA at 72 weeks (6 months after the end of course of treatment). Nonresponse (NR) was defined as a persistent positive HCV (PCR) after 48 weeks of treatment.t tests or non-parametric tests (Wilcoxon and Mann Whitney), as appropriate. The chi-square or Fisher exact tests were used to compare frequencies and proportions. Multivariate stepwise logistic regression analysis was performed to determine the independent predictors of sustained response. An intention-to-treat analysis was used. Patients who discontinued treatment and those who did not complete their course of treatment either due to adverse effects or loss to follow-up were not included in the analysis for ETVR or SVR.Data were collected initially in a specialized data collection form, then introduced into a Microsoft Excel worksheet and finally transferred to the Statistical Package for Social Sciences (SPSS) version 15.0 for Windows  for analysis. Means of continuous variables were compared using 2. Diabetes mellitus, prior interferon-based therapy, and concomitant HBV or HIV infection were present in 45 (30.4%), 59 (39.9%) and 29 (19.6%) patients, respectively . Steatosis in the liver biopsy was similar in the diabetic group (9 of 33) versus non-diabetics (16 of 78) (P=.33). Sustained responders and viral relapsers had similar serum ALT  at time points apart from week 72, in which those who relapsed after ETVR showed significantly higher serum ALT (P=.001)  were males. Mean and standard deviation for age was 48.5 (12.7) years and BMI was 27.9 (7.5) kg/mectively . Pre-tre(P=.001) .P=.016) (P=.157).PCR at 12 weeks post-treatment was done in 131 (91.9%) patients from the whole cohort (n=148) and in 91 (94.8%) of those who completed the full course of treatment (n=96). EVR was achieved in 96 patients (64.9%) in the whole cohort (n=148) and in 84 (92.3%) of those who achieved ETVR. EVR was significantly more common in patients who received treatment for the first time compared to those who previously received treatment  . HoweverP=.008) and lower relapse rate after ETVR (P<.008) compared to those who were previously treated with interferon-based therapy. However, the difference in EVR rate between treatment-naïve patients and those who were previously treated by interferon-based therapies did not reach statistical significance (P=.083) (Eighteen patients (12.2%) failed to complete the 48-week therapy due to side effects or loss to follow up. The full course of therapy was given to 130 (87.2%) patients; 96 (72.9%) achieved ETVR and 34 (26.2%) were non-responders. SVR was achieved in 66 of 130 (50.7%), while the remaining 30 (31.2%) developed virological relapse after ETVR . Virolog(P=.083) . BecauseP=.005), had a lower rate of diabetes mellitus (P=.005), higher serum albumin (P=.028), lower pre-treatment serum aspartate aminotransferase (AST) levels (P=.04), lower serum alpha-fetoprotein (AFP) levels (P=.026), and were more treatment-naïve (P=.008) than patients who developed virological relapse after ETVR   .A total of 158 side effects were encountered in 66 patients (44.6%) during follow-up . EffectsThe present study involved the largest cohort of patients infected with HCV-4 to be reported in the literature after treatment with the combination of PEG-INF alfa-2a and RBV for 48 weeks, and shows that this group of patients can no longer be considered “difficult to treat”. Indeed, with the use of this regimen, SVR was achieved in 44.6% of the whole cohort, in 50.8% of those who completed treatment and in 68.8% of those who achieved ETVR. These results are similar to the responses achieved in previous studies that involved cohorts with predominantly genotype 1 and are less than the responses in patients infected with genotype 2 or 3.363940Only 18 (12.2%) did not complete their course due to either side effects (n=5) or loss to follow up (n=13) and a total of 34 (26.2%) patients were classified as non-responders. This rate of non-response can be accepted if we put into consideration the tertiary nature of our hospital and the inclusion of many complicated cases such as those who failed previous interferon therapy, cases with organ transplantation, and cases copinfected with HIV and/or HBV. The impression that patients infected with HCV-4 respond poorly to interferon-based therapy and are generally “difficult to treat” came from many earlier studies where conventional interferon-alfa was used alone or in combination with RBV .242830–324282325Neither the fibrosis stage nor the inflammation grade in the pre-treatment liver biopsy was found to be statistically different between sustained responders and those who developed virological relapse after ETVR. This is contrary to what was previously reported by other studies in patients infected with genotype 1Contrary to other reports,40The safety profile of the combination therapy of PEG-INF alfa-2a and RBV used in the present study is comparable to what was previously described in the literature.2529The significantly lower SVR in our patients who previously received interferon therapy (28.8%) compared to those who were treatment-naïve (55.1%) is consistent with the results of many studies in both genotype 1 and genotype 4.27294446Better identification of the pre-treatment host or viral factors that can identify which patients respond better to therapy is currently attracting more attention. For instance, HCV-4 subtyping has been proposed to affect the response to PEG-INF alfa-2a plus RBV combination therapy.51This study demonstrates for the first time that lower baseline serum AST and not ALT is an independent predictor of SVR to PEG-INF alfa-2a and RBV in patients with chronic HCV-4. We believe that these lower AST levels reflect less severe histological parameters in the sustained responders. A study by Zechini et al showed a statistically significant positive correlation of baseline aminotransferase values with the hepatitis activity index and fibrosis score.Unlike the situation in most randomized controlled trials, the patients included in this study were heterogeneous: 10.1% had previous organ transplantation, 8.1% were positive for liver autoantibodies (classified as overlap syndrome), 19.6% were positive for HBV or HIV serology, 39.9% were non-responders to previous interferon-based therapy and 6.1% had renal impairment. These factors are known to affect the natural history of chronic HCV infection. However, this sample of patients represents what we usually face in real life. If we exclude cases with all the above co-morbidities, our results will only be applicable to patients with isolated HCV infection. As shown in The potential limitations of the current study include the fact that post-treatment biopsy was not done as our main objective was to assess pre-treatment predictors of SVR. There is a solid evidence that SVR is associated with improved outcomes,In conclusion, combination therapy with PEG-INF alfa-2a and RBV, if tolerated and completed, is effective in treating chronic HCV-4 patients especially if they are younger than 40 years of age, have no previous interferon therapy and have lower pre-treatment AST levels. Attempts to improve adherence to therapy and the early detection together with treatment of complications are needed to achieve better response to therapy. Further studies addressing other potential predictors of SVR in chronic HCV-4 patients such as the IP10, insulin resistance, HCV-4 subtype heterogeneity are warranted.


Human immunodeficiency virus (HIV) infection that persists despite antiretroviral therapy (ART) is a daunting problem. Given the limited evidence that resting CD4+ T cell infection (RCI) is affected by the histone deacetylase (HDAC) inhibitor valproic acid (VPA), we measured the stability of RCI and residual viremia in patients who added VPA with or without raltegravir (RAL), or enfuvirtide (ENF) with or without VPA, to standard ART.Patients with plasma HIV RNA<50 c/mL added sustained-release VPA (Depakote ER®) twice daily, RAL 400 mg twice daily, or ENF 90 mcg twice daily. Change in RCI was measured by outgrowth assays. Low-level viremia was quantitated by single-copy plasma HIV RNA assay (SCA).In three patients on standard ART a depletion of RCI was observed after 16 weeks of VPA, but this effect waned over up to 96 weeks of further VPA. In two patients ENF added to stable ART had no effect on RCI. Simultaneous intensification with ENF and addition of VPA had no effect on RCI frequency in one patient, and resulted in a 46% decline in a second. No significant depletion of RCI (>50%) was seen in six volunteers after the addition of RAL and VPA. In 4 of the 6 patients this lack of effect might be attributed to intermittent viremia, low VPA levels, or intermittent study therapy adherence. Overall, there was no effect of the addition of RAL or ENF on low-level viremia measured by SCA.The prospective addition of VPA and RAL, VPA and ENF, or ENF failed to progressively reduce the frequency of RCI, or ablate intermittent and low-level viremia. New approaches such as more potent HDAC inhibition, alone or in combination with intensified ART or other agents that may disrupt proviral latency must be pursued. Given the costs and difficulties in maintaining suppression of viremia in the HIV-infected patients for decades, and the challenges that face effective prevention of HIV infection, interest has reawakened in interventions that might eradicate HIV infection Histone deacetylase (HDAC) is a critical regulator of HIV latency In this prospective trial HIV-infected volunteers receiving stable ART with plasma HIV-1 RNA<50 copies/ml and a CD4 count of >300/µl for at least 6 months were studied. 3 HIV-infected males stably suppressed on ART and previously treated with VPA  in whom a significant decline in RCI was measured after 16 weeks of VPA agreed to further study on treatment with VPA. As reported 2 HIV-infected males stably suppressed on ART in whom no effect of VPA was measured  agreed to intensify baseline ART with ENF. When RCI was unchanged after ENF, patients 6 agreed to later intensify baseline ART with ENF and VPA simultaneously. One newly enrolled HIV-infected male stably suppressed on ART without previously VPA exposure (patient 12) simultaneously added VPA and ENF to baseline ART. As previously reported, patients 6 and 9 stopped VPA when no decline of RCI was measured, but continued baseline ART In a parallel study, seven HIV-infected males, stably suppressed on ART (patients 13–18) enrolled to study the effect of the receipt of VPA and RAL. Baseline ART included nucleoside RT inhibitors and protease inhibitors (7 patients), or non-nucleoside RT inhibitors (5 patients). CD4 cell counts at entry ranged from 320 to 1242 cells/µl (17–42%). Study therapy for this parallel protocol included RAL 400 mg twice daily and VPA (Depakote ER®) 500–750 mg bid for patients 13–18.Enrollment criteria for both protocols have been described Lymphocytes were obtained by continuous-flow leukapheresis. Isolation of resting CD4+ T cells, recovery and quantification of replication competent virus was performed as described, with inter-assay variance of 0.3 log Plasma HIV-1 RNA concentrations were measured by Roche Amplicor  with an assay detection limit of 50 copies/ml. To accurately measure the extent of low-level viremia, an ultra-sensitive, quantitative real-time, RT-PCR single copy assay (SCA) capable of detecting and quantifying plasma HIV-1 RNA to a limit of detection of 1 copy per ml was performed Overall, VPA therapy was generally well tolerated, with only minor or transient adverse events not clearly related to VPA. VPA levels were infrequently above 60 mcg/ml. RAL was well tolerated without adverse events. ENF was well tolerated, with minor, transient injection site reaction in one patient. No significant adverse effects (greater than ACTG Grade II) were noted in any patients.Patients 1–3 were found to have a >50% depletion of RCI on two resting cell assays at week 12 and 16 of VPA therapy in a study reported previously Patients 6 and 9 in the same prior study Four months later patient 6, in whom RCI had been unaffected by the addition of either VPA or ENF, agreed to undergo simultaneous intensification with ENF and the reinitiation of VPA. After 16 weeks of ART, VPA, and ENF, RCI remained low but stable. VPA levels were adequate, and SCA stably <1 copy/ml.The use of the sustained-release formulation of VPA (Depakote ER®) in these patients and those previously described Patients 13–18, stably suppressed on ART, intensified ART with the addition of 400 mg RAL twice daily and simultaneously added VPA. Surprisingly, despite RAL intensification, patients 13, 14, and 15 had episodic viremia detected by Amplicor. Patients 13 and 15 had four episodes of detectable viremia by Amplicor assay, while patient 14 was >50 copies on one occasion. VPA dose was increased to 1500 mg/day in patients 15 and 17 for trough levels consistently below 40 mcg/ml; levels of >50 mcg/ml were then measured in patient 17, but not in patient 15. In patients 13, 14, and 15, VPA levels were suboptimal, and patients 13 and 14 were unwilling to escalate the dose of drug. Patient 14 missed at least a week of study drug while traveling. None of patients 13–18 had significant declines (>50%) in RCI. Overall, the addition of RAL or ENF had no effect on viremia measured by SCA.increase of RCI of more than 50% was not seen in any subjects. As assay variation is expected to be bimodal, this suggests the declines observed were biological. Further, we have not observed a significant (>50%) upward fluctuation of the RCI assay in any of the 22 patients we have intensively studied In an initial study of the effect of 16 weeks of VPA prospectively added to stable ART In this follow-up study, depletion of RCI was not sustained in three patients studied over 48–96 weeks. It is therefore likely that the initial decline of RCI and its eventual return to baseline represents a biological event, rather than an assay artifact. A mechanistic hypothesis to explain the transient decline in RCI observed is that a population of latently HIV-infected cells are affected by VPA exposure, but that homeostatic forces later restore the size of the infected resting cell pool, as recently suggested by Chomont and colleagues Low-level viral expression as measured by SCA was not significantly diminished by VPA therapy in combination with either ENF or RAL, or by ENF alone. ENF intensification has recently been reported to have no effect on RCI, measured by similar outgrowth Anecdotally, it is of interest to note that the level of residual viremia appears to be related to the frequency of RCI. Given the small sample size this can only be a preliminary observation, but is consistent with the hypothesis that residual viremia originates from persistently infected, long-lived T cells.These findings provide several important insights for future study of persistent HIV infection, and approaches to attack it. Despite a sound scientific rationale for the testing of HDAC inhibitors, only a single weak inhibitor, VPA, has been tested, and only in a handful of patients. Furthermore, since 2005 VPA donated for research use has been in a sustained-release formulation, designed to blunt the exposure of patients to higher peak serum levels of VPA. Future studies of HDAC inhibitors may seek to maximize peak drug exposure levels and measure biological markers such as acetylation of nucleosomal histone via chromatin immunoprecipitation assays. One of the patients reported herein (patient 12) was treated with generic VPA, with a modest depletion of RCI that did not achieve the pre-determined standard for significance.Further, many patients had persistent or intermittent viremia detected by standard Amplicor assay or by SCA. Such viremia may blunt the ability to deplete RCI or to measure a depletion of RCI. The persistence of this viremia despite novel ART highlights a second significant obstacle to the eradication of HIV infection.Depletion of RCI in the majority of HIV-infected patients with stable suppression of viremia is an unrealized goal. Vorinostat, a more potent HDAC inhibitor, can induce expression of latent HIV


Whereas many patients respond quickly to treatment with tumour necrosis factor (TNF) inhibitors, some patients may experience significant but delayed responses.To evaluate the clinical response between 12 and 24 weeks in subjects with rheumatoid arthritis from the Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes.Clinical response was assessed at 24 weeks in 12-week non-responders, according to American College of Rheumatology (ACR) response criteria. The proportion of subjects who successfully maintained response to 52 weeks was analysed, as were radiographic outcomes.Data from 682 subjects were included in the analysis. Non and partial responders in all three groups  at week 12 showed an improvement in responses at week 24. Over 80% of the week 24 ACR20/50/70 responders in the etanercept plus methotrexate arm sustained their response to 52 weeks. In the etanercept arms, a delayed clinical response was not associated with increased radiographic progression at week 52.A significant proportion of non and partial responders to etanercept with or without methotrexate therapy at week 12 achieved a good clinical response or improved their overall clinical response at week 24. Discontinuing TNF inhibitor therapy at 12 weeks may be premature in some rheumatoid arthritis patients. Tumour necrosis factor (TNF) inhibitors, particularly in combination with methotrexate, have demonstrated excellent symptomatic and radiographic control in rheumatoid arthritis.6In this analysis, we used data from the Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes (TEMPO) to evaluate the extent to which subjects not responding to TNF inhibitor therapy at 12 weeks might respond at 24 weeks. The long-term sustainability of the improvement in response and radiographic outcome at week 52 was also assessed.Data from subjects treated with etanercept, methotrexate and etanercept plus methotrexate from the TEMPO study were used in this analysis.American College of Rheumatology (ACR)20/50/70 responses and Disease Activity Score using 28 joints (DAS28) were assessed at weeks 12, 24 and 52. Based on their clinical response at week 12, subjects were categorised into ACR20 non-responders (no response at week 12), ACR50 non-responders  and ACR70 non-responders .Among the subjects who showed an improvement in clinical response at week 24, the proportion of subjects who successfully maintained the improvement to at least 52 weeks was assessed. The radiographic outcome at week 52 was evaluated using the mean change in total Sharp score (TSS) and the percentage of non-progressors (TSS change ≤0).Frequencies and percentages were provided for improvement and decrease in ACR response and disease activity scores. One-way analysis of variance was used to test the difference in DAS28 scores among groups . The paired-sample t test was used to examine the difference in DAS28 scores between week 12 and week 24 and for week 12 European League Against Rheumatism (EULAR) non-responders who responded at week 24. Fisher’s exact test was used to compare the difference in radiographic progression between the week 12 responders and week 12 non-responders who became responders at week 24 within each treatment group. Bonferroni adjustment was used for multiple comparisons. Logistic regression models were used to identify predictors of response at 12 weeks. Results were considered significant at p⩽0.05 two-sided. Non-responder imputation was used to handle missing clinical and radiographic data. All analyses were performed using the SAS STAT system version 9.1.The TEMPO trial included 682 subjects . Baseline demographic characteristics for TEMPO subjects have been described previously.Results from the analysis of ACR20/50/70 responses at week 24 in week 12 non and partial responders are shown in Of 21 ACR20, 37 ACR50 and 23 ACR70 responders at week 24 in the etanercept plus methotrexate arm, 17 (81.0%), 31 (83.8%) and 19 (82.6%) subjects, respectively, showed a sustained response at week 52 . The susA decrease in ACR20/50/70 response was observed in some subjects between weeks 12 and 24. In the etanercept plus methotrexate arm, 18 out of 175 ACR20 responders (10.3%), five out of 96 ACR50 responders (5.2%) and five out of 51 ACR70 responders (9.8%) decreased their response at week 24 (data not shown). Similarly, 11.5% of ACR20, 23.3% of ACR50 and 4.8% of ACR70 responders in the etanercept monotherapy arm and 16.5% of ACR20, 10.2% of ACR50 and 23.1% of ACR70 responders in the methotrexate monotherapy arm decreased their response between weeks 12 and 24.In subjects from all three treatment arms who were EULAR (moderate or good) non-responders at week 12, but became responders at week 24, their mean DAS28 scores showed a significant decrease from baseline to week 24 and from week 12 to week 24 (p<0.05) . In the To determine if there was a slowing of radiographic progression in subjects who were ACR20 non-responders at week 12, but became responders at week 24 compared with week 12 responders, the mean change in TSS and the percentage of non-progressors were evaluated at week 52. Results are shown in No baseline characteristics or week 12 clinical variables  were significantly associated with a delayed response at 24 weeks.In this retrospective analysis, a substantial improvement in clinical response was observed between 12 and 24 weeks in subjects with rheumatoid arthritis in all three treatment arms from the TEMPO trial, including those who were non or partial responders at 12 weeks. The improvement in response at 24 weeks was most apparent in the etanercept plus methotrexate arm, in which 37.5% of ACR20, 46.8% of ACR50 and 51.1% of ACR70 non-responders became ACR20, ACR50 and ACR70 responders, respectively, at week 24. Similar improvements were observed for DAS28 scores in all three treatment arms.The delayed response observed at 24 weeks was largely sustained in 80% of subjects to 52 weeks and was not associated with an inferior outcome with regard to the inhibition of radiographic progression at 52 weeks in the etanercept and etanercept plus methotrexate arms.–12There are no clear guidelines on the appropriate treatment trial duration for TNF inhibitors. Recommendations from international guidelines vary, with most indicating 12 weeks as a time to assess response after TNF inhibitor therapy. Whereas some recommend discontinuing therapy at 12 weeks for non-responders, others allow for doctor’s discretion on whether or not to continue therapy.7Importantly, an analysis of disease variables and patient characteristics failed to identify any predictive factors.–The implications of this assessment are relevant with regard to the subsequent treatment of patients “failing” TNF inhibitor therapy at 12 weeks. It has been shown that patients who have “failed” therapy with a TNF inhibitor have somewhat lesser ACR responses to any second-line biological therapy.13Limitations of the current study include the retrospective nature of the analysis. Moreover, these findings are from patients enrolled in a randomised clinical trial who met the specific inclusion criteria and had high disease activity. Therefore, these observations may not reflect patients in a real-world setting.In conclusion, findings from this retrospective analysis suggest that a significant proportion of patients who do not achieve a clinical response 12 weeks after the initiation of etanercept with or without methotrexate therapy may benefit from continuing therapy for up to 24 weeks. Additional benefits of continuing therapy for up to 24 weeks include the sustainability of the 24-week gain in response through at least one year while maintaining radiographic protection.


In a routine service delivery setting in Uganda, we assessed the ability of the WHO clinical stage to accurately identify HIV-infected patients in whom antiretroviral therapy should be started.6/l were 52%  and 68%  respectively. Plasma viral load was tested in a subset of 1453 subjects in whom ART was initiated. Among 938 subjects with plasma viral load of 100,000 copies or more, 391  were at WHO stage I or II.Among 4302 subjects screened for ART, the sensitivity and specificity (95% CI) of WHO stage III, IV against a CD4 count < 200 × 10In this setting, a large number of individuals could have been denied access to antiretroviral therapy if eligibility to ART was assessed on the basis of WHO clinical stage. There is an urgent need for greater CD4 count testing and evaluation of the utility of plasma viral load prior to initiation of ART to accompany the roll-out of ART. In developed countries, CD4 count and plasma viral load testing are essential for monitoring HIV-infected subjects and for assessing when antiretroviral therapy (ART) should be initiated . ART is There are limited data on how well the WHO HIV clinical stage criteria identifies people correctly for ART. The WHO clinical stage was associated with survival and biological outcomes, done in the pre-ART era -6. A hosWe have screened and initiated ART in a large number of subjects in a normal health service delivery setting, and here we compare the different criteria for determining ART eligibility.The study was based at The AIDS Support Organisation (TASO) clinic in Jinja, SE Uganda. The setting has been described previously ,10. In b6 cells/l or advanced WHO stage III  or stage IV. From February 2005, all TASO clients starting ART were invited to join a pragmatic effectiveness trial evaluating two different strategies of ART delivery (home-based versus facility-based). The trial was integrated into routine health service delivery [In September, 2004, TASO began providing ART as part of HIV care. Registered clients were asked to come forward for screening. Eligibility for ART was assessed over 3 visits, usually scheduled over 4–6 weeks. At the first visit, patients were examined clinically by a physician and CD4 count was tested. Most physicians were medical officers with 1–2 years experience post-qualification. Clinical criteria for receiving ART were CD4 count below 200 × 10delivery ,10. Clindelivery . The TASBlood samples for CD4 counting were delivered to the laboratory about 100 km southwest of the clinic within 36 hours of collection. Testing was done using the BD FACS Calibur, Multitest™ method . Samples for plasma viral load testing were processed and stored at the trial site within 2 hours of collection. They were transported to the laboratory and tested using the HIV-1 RNA 3.0 Assay (bDNA) (Bayer AG).The study was approved by the ethics committees of Uganda Virus Research Institute and the London School of Hygiene & Tropical Medicine and the US Centers for Disease Control and Prevention.6/l of whom 1091  were classified at WHO stage I or II. The sensitivity and specificity (95% CI) of WHO stage III or IV against a CD4 count of <200 × 106/l were 52%  and 68%  respectively  was 37 (18–90) years and 74% were women. Overall 4302 were examined and tested for CD4 count (the remainder 19 subjects did not return); 2254 (52%) had CD4 count < 200 × 106/l and 163200  copies per ml respectively. There was a weak association between CD4 count and plasma viral load  subjects who had plasma viral load tested within 30 days of CD4 testing; p < 0.0001 for both, Spearman's correlation).Recruitment into the trial began in February 2005. Eleven subjects did not meet the eligibility criteria and a remaining 1477 were invited to join; 24 refused and 1453 were recruited. One thousand and thirty-one (71%) were women. The median age was 38 (range 18–83) years, 1050 (72%) had primary school or no education, 516 (36%) were married or living with someone, and 396 (27%) were unemployed. Subjects had taken a median 1 hour (range 0.25–6) to travel to clinic. Twenty (1%) were WHO stage 1, 642 (44%) at stage II, 672 (46%) at stage III, and 119 (8%) at stage IV. Most subjects (78%) at WHO stage I or II had presented with prurigo or upper respiratory infections. Of those at WHO stage III, 160 (24%) had either chronic fever of unknown origin, chronic diarrhoea of unknown origin, oral hairy leukoplakia, or pyomyositis, 437 (65%) had pulmonary tuberculosis in the last 5 years or oral candidiasis and the remainder 75 (11%) had of variety of conditions. Plasma viral load was tested within a median 32  days of the CD4 count and clinical examination. The median (IQR) CD4 counts and plasma viral loads were 109  × 106/l, 613  were classified as WHO stage I, II, 760  were classified as WHO stage I, II and early stage III and 1219  were classified as WHO stage I, II or III. Furthermore, of 938 subjects with plasma viral load of 100,000 copies or more, 391  were at WHO stage I or II, 507  were classified as WHO stage I, II and early stage III and 869  were classified as WHO stage I, II or III. Sixty of 132  of subjects with CD4 ≥ 200 × 106/l had plasma viral load of 100,000 copies per ml or more .Of 1321 subjects with CD4 count <200 × 106/l. Furthermore, over 40% of subjects at this clinical stage had high plasma viral load in excess of 100,000 copies per ml. These findings confirm those reported from Rakai, Uganda, earlier this year [6/l have high rates of death and infections and a greater risk of serious complications when they eventually start ART [6 cells/l or higher and when plasma virus load is below 100,000 copies per ml [6/l, following recent research findings suggesting significant risk to patients with CD4 counts above 200 × 106/l [This study shows that about a half of subjects with WHO stage I or II, as assessed in routine health service delivery in a typical African setting, had CD4 count <200 cells × 10his year . In the tart ART . In deve × 106/l .6/l, or earlier, will be a major challenge as the vast majority do not know their HIV status, and the number of new HIV infections far exceeds the number being placed on treatment [Our study highlights the urgent need for greater CD4 count testing prior to the initiation of ART to accompany the roll-out of antiretroviral therapy so that patients can start treatment in a timely manner. Besides the obvious survival benefit, the costs of baseline CD4 count testing would probably be far less than the health services costs incurred when ART is deferred to the time when the patient has a low CD4 count. Treating patients in Africa at around the CD4 count 200 cells × 10reatment .6/l were at WHO stage III or IV. Some of these subjects may have been incorrectly classified and may not have needed antiretroviral therapy, while others may have had AIDS defining conditions despite a high CD4 count. This requires research as unnecessary early treatment may be difficult to sustain over the long term for some patients without the necessary adherence support.Our study showed more than half of the subjects with CD4 count ≥ 200 × 106/l had plasma viral load of 100,000 copies or more in whom treatment should be considered. This is probably an underestimate since we measured this only in subjects in whom ART was initiated. This group of subjects will be at increased risk of progressing to disease and transmitting the virus [Our study also showed that 45% of subjects with CD4 count ≥ 200 cells × 10he virus  and testThe author(s) declare that they have no competing interests.SJ wrote the first draft of the paper. All authors reviewed drafts critically and contributed to ideas and writing on multiple occasions.


Viral load (VL) monitoring is an essential component of the care of HIV positive individuals. Rates of VL monitoring have been shown to vary by HIV risk factor and clinical characteristics. The objective of this study was to determine whether there are differences among regions in Canada in the rates of VL testing of HIV-positive individuals on combination antiretroviral therapy (cART), where the testing is available without financial barriers under the coverage of provincial health insurance programs.The Canadian Observational Cohort (CANOC) is a collaboration of nine Canadian cohorts of HIV-positive individuals who initiated cART after January 1, 2000. The study included participants with at least one year of follow-up. Generalized Estimating Equation (GEE) regression models were used to determine the effect of geographic region on (1) the occurrence of an interval of 9 months or more between two consecutive recorded VL tests and (2) the number of days between VL tests, after adjusting for demographic and clinical covariates. Overall and regional annual rates of VL testing were also reported.3,648 individuals were included in the analysis with a median follow-up of 42.9 months and a median of 15 VL tests. In multivariable GEE logistic regression models, gaps in VL testing >9 months were more likely in Quebec (Odds Ratio (OR) = 1.72, p < 0.0001) and Ontario  than in British Columbia and among injection drug users  and were less likely among older individuals , among men having sex with men , within the first year of cART , among individuals on cART at the time of the blood draw  and among individuals with VL < 50 copies/ml at the previous visit .Significant variation in rates of VL testing and the probability of a significant gap in testing were related to geographic region, HIV risk factor, age, year of cART initiation, type of cART regimen, being in the first year of cART, AIDS-defining illness and whether or not the previous VL was below the limit of detection. Viral load (VL) testing is an essential component of the care of HIV-positive individuals, both with regard to timing of initiation of antiretroviral therapy (ART) and to monitoring of virologic response to combination ART (cART) . The goaAccess to VL testing has been studied previously. In an Ontario cohort, injection drug use, younger age and residence in Toronto were associated with lower VL testing rates . In anotIn this study, we examine whether there are regional differences in patterns of VL testing among individuals who initiated cART therapy since January 1, 2000 in Canada, where VL testing is available without charge to all HIV-positive residents as part of the provincial universal health insurance plans. Furthermore, we identified demographic and clinical factors associated with suboptimal frequency of VL testing.st 2000. The study was established in March 2008 with funding from the Canadian Institutes of Health Research (grant# 711098) and the CIHR Canadian HIV Trials Network (CTN242) and includes cohorts and investigators from across the country (listed at the end of the manuscript). The collaboration is open to all Canadian HIV treatment cohorts with more than 100 eligible patients.The Canadian Observational Cohort (CANOC) collaboration is a Canadian cohort study of antiretroviral naïve HIV-positive patients initiating cART since January 1Data used in this analysis were from nine cohorts of HIV-positive individuals in British Columbia (BC), Ontario, and Quebec, including the BC Centre for Excellence in HIV/AIDS Drug Treatment Program, Montreal Chest Institute Immunodeficiency Cohort, The Electronic Antiretroviral Therapy, Clinique Médicale l'Actuel, The Canadian HIV/HCV Co-infection Cohort, Ontario Cohort Study, Maple Leaf Medical Clinic, Toronto General Hospital and Ottawa Hospital HIV/HCV Cohort .Patient selection and data extraction were performed at the data centres of the participating cohort sites. In provinces with multiple cohorts, VL data were entered from each cohort site and not from a provincial data source. Non-nominal data from each cohort on a predefined set of demographic, laboratory, and clinical variables were then pooled and analyzed at the Project Data Centre in Vancouver. All participating cohorts have received approval from their institutional ethics boards and governance committees to contribute non-nominal patient-specific data to CANOC. Ownership of individual cohort data remains with the contributing cohort and cohort data can only be used for studies approved by the CANOC Steering Committee.Eligibility criteria for inclusion in CANOC were documented HIV infection, residence in Canada, aged 18 years and over, initiation of three or more antiretroviral drugs for the first time (i.e. ART-naïve cART start) after January 1, 2000, and a viral load measurement and CD4 cell count within 6 months of the start of therapy. To be included in this analysis, individuals had to have at least one year of follow-up. Viral load measurements were available both before and after starting cART.Three measures were used to assess the frequency of VL testing. First, the primary outcome of interest was defined as a gap between VL tests in excess of 9 months, corresponding to at least three missed or delayed tests assuming the optimal frequency between tests is three months. This was felt to be a clinically important gap in VL testing. Second, the annual rate of VL testing was calculated by dividing the total number of tests for an individual by the duration of follow-up for that individual in years. Third, the time interval, defined as the number of days between two successive VL tests for a subject, was examined.Demographic and clinical characteristics such as gender, race, HIV risk factors, age, geographic region, CD4 count and type of cART regimen were compared among regions with chi square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.The proportion of individuals with at least one suboptimal interval and the annual rate of VL testing experienced were compared among regions and by demographic and clinical characteristics with the chi square test and Wilcoxon rank sum test respectively. Generalized Estimating Equation (GEE) logistic regression models, which account for correlation among multiple observations within subjects, were used to determine factors associated with the occurrence of a suboptimal testing interval. An exchThe relationships between the length of the VL inter-test interval and individual characteristics were examined using GEE linear regression models. Plasma VL levels, CD4 counts, characteristics of the antiretroviral regimen and the calendar year of VL tests were treated as time-varying covariates in all regression models. Covariates with a p < 0.10 in the univariate regression models were considered as candidates for inclusion in the multivariable model.10 copies/mL were considered to be duplicate measurements. In such cases, the first VL date was kept and the average of the VL measurements was assigned as the VL value.When there was a gap between two consecutive VL tests in excess of nine months, the accompanying CD4 test dates were examined to validate the VL test dates. Viral load tests within 8 days of each other and with results within 0.1 log3,648 subjects met cohort inclusion criteria. Demographic characteristics are described by region in Table 5 copies/mL, an HIV risk category of "men who have sex with men" (MSM), history of an AIDS-defining illness, boosted-protease inhibitor (PI)-based cART regimen, not being co-infected with Hepatitis C and not being an injection drug user (IDU)  Table .Of the 3,648 patients eligible for the analysis, 26% and 51% of the population had experienced at least one nine-month and one six-month gap during their follow-up, respectively. Proportions of patients with at least one nine-month gap and with at least one six-month gap are shown by demographic and clinical characteristics in Table In univariate GEE linear regression models, covariates which were associated with a decrease in the number of days between VL tests included age, male gender, later year of initiating cART, being within the first year of cART, receiving PI-boosted cART regimen, higher baseline VL and having been diagnosed with an AIDS-defining illness Table . CovariaThere was considerable regional variation in annual rates of VL measurement documented in this Canada-wide study. This variation remained significant even after adjusting for demographic variables such as age and HIV risk factors and clinical variables such as year of cART initiation, type of cART regimen, being in the first year of cART, AIDS-defining illness and whether or not the previous VL was below the limit of detection. In Ontario and Quebec, VL was measured quarterly on average. In BC, rates of VL measurement were even more frequent than guidelines suggest, with an average measurement frequency of almost five times annually.There are a number of possible explanations for the regional differences in rates of viral load measurement. Some of the difference in measurement rate was due to regional differences in demographic factors such as the proportions of IDUs, who typically have less frequent viral load testing, or pregnant women, in whom viral load is monitored more closely. Further differences could be due to variation in rates of VL blips among regions, after which a repeat VL measurement is typically ordered. Recent data has documented differing rates of blips by VL assay ,11 and t10 copies/mL) decrease in plasma VL in the first month and suppression to below 50 copies/mL by 24 weeks, depending on the pretreatment VL level [Effective therapy should result in at least a 90% or 10-fold (1.0 logIn terms of the demographic findings, our findings are similar to those of the study conducted in Ontario several years ago and could likely be generalizable to other settings with universal health care programs. In settings with user-pay programs for virologic monitoring, it seems likely that socioeconomic factors will have a considerably greater influence on rates of testing. In resource limited settings, availability and travel distances are likely to remain significant barriers to regular monitoring of VL levels.Our results have important implications for guidelines and for research into the monitoring of VL. For patients for whom VL is measured less frequently, virologic rebound will be detected later on average and patients will remain on failing regimens longer than is necessary. In some circumstances, however, it is safe to measure VL less frequently . A reducA strength of our study is that VL monitoring is provided free of charge to all HIV-positive individuals in Canada, so that we were able to examine correlates of rates of VL testing in the absence of financial barriers to testing. Further, by combining data from nine sites across Canada, we achieved a large sample size as well as a good representation by region, gender, race, and other demographic characteristics. CANOC represents about a quarter of all HIV-positive people in the country who are currently on ART and approximately half of those who have initiated on more modern regimens since 2000 . This isFuture plans for investigation include further examination of the reasons for the regional differences in rates of VL testing. Also, an economic analysis to determine if the less frequent testing found in Ontario and Quebec is adequate in terms of clinical outcomes and could result in financial savings would be informative. Particularly, an analysis of VL testing frequency in patients with full virologic suppression where less testing may be adequate would be of interest and its impact on cost savings could be important.In our setting, with universal health care and similar regional guidelines for viral load testing, significant variation in rates of VL measurement and the probability of a significant gap in testing were related to geographic region, age, HIV risk factors and clinical variables such as year of cART initiation, type of cART regimen, being in the first year of cART, AIDS-defining illness and whether or not the previous VL was below the limit of detection.AIDS: acquired immune deficiency syndrome; ART: antiretroviral therapy; BC: British Columbia; CANOC: Canadian Observational Cohort; cART: combination antiretroviral therapy; CI: confidence interval; GEE: generalized estimating equation; HCV: hepatitis C virus; HIV: human immunodeficiency virus; IDU: injection drug user; IQR: interquartile range; MSM: men who have sex with men; NNRTI: non nucleoside reverse transcriptase inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; OR: odds ratio; PI: protease inhibitor; VL: viral load.The authors declare that they have no competing interests.DS conducted the statistical analyses under the direction of JMR, MRL and AMB. JMR and MRL drafted the manuscript. RSH, CC, MBK, MRL, NM, JSGM, JMR, SBR, CT are principal investigators of CANOC and contributed cohort data to CANOC. All authors reviewed the manuscript during preparation, provided critical feedback and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/40/prepub