In a double-blind trial, Rajesh Gandhi and colleagues detect no significant reduction in viral load after people with low-level HIV viremia added an integrase inhibitor to their treatment regimen. Most HIV-1-infected patients on effective antiretroviral therapy (ART) with plasma HIV-1 RNA levels below the detection limits of commercial assays have residual viremia measurable by more sensitive methods. We assessed whether adding raltegravir lowered the level of residual viremia in such patients.n = 25) and the placebo (n = 24) groups , nor did the change in HIV-1 RNA level from baseline to week 10/12 . There was also no significant change in HIV-1 RNA level from weeks 10/12 to weeks 22/24 after patients crossed-over. There was a greater CD4 cell count increase from baseline to week 12 in the raltegravir-intensified group compared with the placebo group , which reversed after the cross-over. This CD4 cell count change was not associated with an effect of raltegravir intensification on markers of CD4 or CD8 cell activation in blood.Patients receiving ART who had plasma HIV-1 RNA levels below 50 copies/mL but detectable viremia by single copy assay (SCA) were randomized to add either raltegravir or placebo to their ART regimen for 12 weeks; patients then crossed-over to the other therapy for an additional 12 weeks while continuing pre-study ART. The primary endpoint was the plasma HIV-1 RNA by SCA averaged between weeks 10 and 12 (10/12) compared between treatment groups. Fifty-three patients were enrolled. The median screening HIV-1 RNA was 1.7 copies/mL. The HIV-1 RNA level at weeks 10/12 did not differ significantly between the raltegravir-intensified  has killed about 25 million people since 1981 and more than 30 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV is a retrovirus—its genetic blueprint is made of ribonucleic acid (RNA). HIV infects human immune system cells and destroys them, leaving infected individuals susceptible to other infections. Early during the AIDS epidemic, most HIV-positive people died within ten years of infection. Then, in 1996, effective antiretroviral therapy (ART) was developed. ART consists of combinations of drugs that prevent viral replication by inhibiting essential viral enzymes such as reverse transcriptase  and protease . Now, in industrialized countries, the life expectancy of HIV-infected patients treated with ART is similar to that of people with diabetes and other chronic conditions.Although ART can reduce the number of viral RNA copies in the plasma (the liquid portion of blood) of HIV-positive patients to less than 50 copies/mL , it is does not eradicate HIV. When very sensitive assays are used to detect viral RNA , most patients on ART have one copy or more of HIV RNA per mL of plasma. The origin of this low-level residual viremia (virus in the blood) is controversial. Residual viremia could arise from ongoing cycles of viral replication, in which case intensification of ART should reduce it. Alternatively, residual viremia could be due to HIV release from stable reservoirs such as latently infected resting immune system cells, in which case intensification of ART should have no effect on residual viremia. In this randomized, controlled trial (a study in which randomly selected groups of patients are given different treatments and the effects of these treatments compared), the researchers assess whether the addition of raltegravir (a drug that inhibits HIV integrase) to standard ART has any effect on residual viremia.The researchers enrolled 53 HIV-positive patients who had been receiving ART containing several reverse transcriptase inhibitors and, in some cases, a protease inhibitor for at least 12 months and who had a plasma HIV RNA level below 50 copies/mL but detectable viremia by SCA. The patients were randomly assigned to receive either raltegravir or a dummy drug (placebo) in addition to their normal ART for 12 weeks. They were then crossed-over (swapped) to the other therapy for a further 12 weeks. At baseline, the trial participants had an average plasma HIV RNA level of 1.7 copies/mL. The HIV RNA level at weeks 10/12 (the average of SCA results at 10 and 12 weeks) was similar in the raltegravir group and in the placebo group and did not differ significantly from this baseline level. There was also no significant change in plasma HIV RNA levels from weeks 10/12 to weeks 22/24 after the patients crossed-over between treatment groups.In this randomized, cross-over study, raltegravir intensification of ART for 12 weeks did not demonstrably reduce low-level residual viremia in HIV-positive patients receiving standard ART. It is possible that 12 weeks is too short a time to see an effect of raltegravir on residual viremia. Furthermore, although this is one of the biggest trials of this type done to date, it might be that insufficient patients were included in the trial to detect a subtle effect of raltegravir on residual viremia. Nevertheless, these findings argue against the hypothesis that residual viremia arises from ongoing cycles of viral replication and the infection of new cells. Instead, they suggest that residual viremia might be due to the release of HIV from stable reservoirs. If so, new therapeutic strategies designed to eliminate these reservoirs of latently infected cells will be required to cure HIV infection.http://dx.doi.org/10.1371/journal.pmed.1000321.Please access these Web sites via the online version of this summary at HIV infection and AIDS, and on the treatment of HIVInformation is available from the US National Institute of Allergy and infectious diseases on HIV InSite has comprehensive information on all aspects of HIV/AIDS, including information on antiretroviral therapiesAvert, an international AIDS charity on many aspects of HIV/AIDS, including the treatment of HIV and AIDS (in English and Spanish)Information is available from AIDS and on AIDS medicines (in English and Spanish)MedlinePlus has links to further resources on  Although currently recommended antiretroviral therapy (ART) lowers plasma HIV-1 RNA levels to below the detection limit of commercial assays , most patients have persistent low-level viremia when tested with more sensitive methods The causes and clinical implications of persistent low-level viremia in patients on ART are controversial .This study was conducted according to the principles expressed in the Declaration of Helsinki. The study  and S2 w3. Participants were also required to have documentation of a pre-ART HIV-1 RNA level of >100,000 copies/mL because detectable residual viremia is more likely in individuals with higher pre-treatment HIV-1 RNA levels The main inclusion criteria for the study  were:  on a scale of 1 to 4 that signifies severity ranging from mild to life-threatening.Eligible patients were randomized with equal probability to either immediate-intensification (group A) or delayed-intensification (group B) with raltegravir, using permuted blocks, stratified by whether they were on a PI- or NNRTI-containing entry regimen . Partici10 copies/mL values by SCA were averaged at pre-entry and entry (baseline [average of the pre-entry and entry measurements]), weeks 10 and 12 (10/12), and weeks 22 and 24 (22/24). CD4 and CD8 cell counts were assessed by flow cytometry at pre-entry and entry  and again at weeks 12 and 24. CD4 and CD8 cell activation in the blood was assessed on cryopreserved cells at pre-entry and entry  and at weeks 12 and 24. The percentage of CD4 and CD8 cells that expressed the activation markers CD38 and human leukocyte antigen (HLA)-DR was determined using phycoerythrin-conjugated anti-CD38, fluorescein isothiocyanate–conjugated anti-HLA–DR, allophycocyanin-conjugated anti-CD4, and peridinin chlorophyll protein-conjugated anti-CD8 monoclonal antibodies and analyzed by flow cytometry.SCA testing was performed at pre-entry, entry, and weeks 10, 12, 22, and 24. Samples from these time points were assayed in a batch for each patient . The plasma volume assayed was ≥7 mL in ≥90% of samples  and ≥10 mL in >83% of samples . The HIV-1 RNA log10 difference (1 standard deviation effect size) in the HIV-1 RNA level at weeks 10/12 between the two treatment groups using a Wilcoxon rank-sum two-sided test at a 0.05 level. We increased the target enrollment to 25 in each group with the expectation that some participants would discontinue study drug prior to week 12 and that treatment intensification might increase the proportion of participants who have HIV-1 RNA levels below the LLQ by SCA at weeks 10/12, which would affect statistical power. When averaging the week 10 and 12 measurements, if one or both measurements were below the SCA lower assay limits, the LLQ was used to compute the average and the primary endpoint was treated as below the averaged value. The primary endpoint was analyzed using the censored-data version of the Wilcoxon rank-sum test, which accommodates the fact that participants had different SCA lower limits due to varying plasma volumes. For analyses of secondary endpoints , treatment arms were compared using exact Wilcoxon rank sum tests for continuous endpoints or Fisher's exact tests for proportions. The within-participant effect of intensification was tested by Wilcoxon signed rank test. Confidence intervals (CIs) were estimated using Hodges and Lehmann method. Associations between responses and predictors were assessed using rank-based Spearman correlations. Reported p-values were two-sided without adjustment for multiple comparisons.The primary objective of the study was to compare the HIV-1 RNA level by SCA at weeks 10/12 in patients who added raltegravir to a PI- or NNRTI-containing regimen to the level in patients who added placebo to a PI- or NNRTI-containing regimen. We conducted an on-treatment analysis and excluded patients who had confirmed virologic rebound to >50 copies/mL of HIV-1 RNA. The target enrollment for the study was 50 participants (25 in each group). From prior data on assay and biologic variation in residual HIV-1 RNA levels One hundred twenty-five patients were screened for the study, of whom 75 (60%) had detectable plasma HIV-1 RNA by SCA . Fifty-t3, and the median time since the first undetectable HIV-1 RNA level on a commercial assay was 5.5 years. The median  screening HIV-1 RNA level by SCA was 1.7 copies/mL . Participants in the raltegravir-first and placebo-first groups were well-balanced with respect to these baseline characteristics. The median screening HIV-1 RNA by SCA was 1.2 copies/mL in the raltegravir-first group and 1.9 copies/mL in the placebo-first group; the interquartile ranges for the median HIV-1 RNA values in the two groups overlap  contributed data to the primary endpoint at weeks 10/12 and 46 (87%) completed all 24 weeks of the study treatment . In thosn = 25) and the placebo group (n = 24): median 1.2 (0.1 log10) versus 1.7 (0.2 log10) copies/mL  (n = 51). In the intent-to-treat analysis, the median HIV-1 RNA at week 10/12 in the raltegravir-first group was 1.2 (0.1 log10) copies/mL and in the placebo group was 1.7 (0.2 log10) copies/mL; there was no statistically significant difference between the two groups . The absence of a demonstrable difference in HIV-1 RNA level at week 10/12 between the two treatment groups was also seen in planned sensitivity analyses that adjusted for ART regimen at entry, baseline HIV-1 RNA by SCA and/or baseline CD4 cell count (using regression models for censored data) .The primary objective of the study was to compare the HIV-1 RNA level by SCA at weeks 10/12 in participants who added raltegravir to a PI- or NNRTI-containing regimen to the level in those who added placebo to a PI- or NNRTI-containing regimen. The average HIV-1 RNA level at week 10/12 did not differ significantly between the raltegravir-intensified (pies/mL) . A similp = 0.71, Wilcoxon rank sum test)  . In a protocol-specified secondary analysis, we also evaluated the change in HIV-1 RNA level from the pre-intensification level  to the post-intensification level . The median change in HIV-1 RNA level from pre-intensification to post-intensification was 0.0 (−0.07 log10) copies/mL (95% CI of −0.5 to 0.3 copies/mL or −0.1 to 0.1 log10 copies/mL). That is, we can exclude, with 95% confidence, a median decline from baseline HIV-1 RNA during intensification of more than 24% (>0.1 log10 copies/mL). In our study population, we can exclude a median reduction in HIV-1 RNA during intensification of more than 0.5 copies/mL. The proportion of participants who had either an increase or no change versus a decrease in HIV-1 RNA levels during the intensification period did not differ significantly between treatment groups (unpublished data).We also assessed the difference between the pre-intensification and post-intensification SCA values in the two treatment groups. The change in HIV-1 RNA from baseline to weeks 10/12 did not differ significantly between the two treatment groups  . The proIn addition, the cross-over design allowed us to evaluate whether there was an increase in HIV-1 RNA level after discontinuation of raltegravir by patients in the raltegravir-first group, or a decrease in HIV-1 RNA level after addition of raltegravir by those in the group that received placebo first. There was no significant change in HIV-1 RNA level after participants crossed-over from raltegravir to placebo or from placebo to raltegravir (unpublished data).3 vs. −44 cells/mm3), but this difference was not statistically significant . After the cross-over, the change in CD4 cell count reversed in the two groups: the CD4 count in the group that switched to placebo dropped by a median of 26 cells/mm3, whereas it increased by 54 cells/mm3 in the group that added raltegravir . When the treatment groups were pooled, the median CD4 count change was an increase of 42 cells/mm3 during raltegravir intensification (95% CI −2 to 57 cells/mm3). There was also no significant change in CD8 cell count during raltegravir intensification.We also examined the effect of raltegravir intensification on CD4 and CD8 cell counts, which were measured at baseline and at weeks 12 and 24 . There wr = 0.08 for the correlation between HIV-1 RNA and CD4+/CD38+/HLA-DR+ percentage; r = 0.01 for the correlation between HIV-1 RNA and CD8+/CD38+/HLA-DR+ percentage; both p-values>0.6, Spearman rank correlation).Because HIV-1 replication is associated with elevated T cell activation, we looked for a relationship between baseline activation and HIV-1 RNA level. CD4 and CD8 cell activation was determined by measuring the percentage of cells that expressed both CD38 and HLA-DR. There was no association between baseline HIV-1 RNA value by SCA and baseline T cell activation  or from week 12 to week 24 . We performed a similar analysis of the effect of raltegravir intensification on CD4 cell activation. At baseline, participants in both the raltegravir- and placebo-first groups had a median of 7% CD4+/CD38+/HLA-DR+ cells. We found no statistically significant difference in CD4+/CD38+/HLA-DR+ percentage between the treatment groups at week 12 or 24. There was also no significant difference between the two treatment groups in the change in CD4+/CD38+/HLA-DR+ percentage from baseline to week 12 or from week 12 to week 24.We then examined whether raltegravir intensification affected the level of CD8 and CD4 cell activation , as mighr = −0.31, p = 0.03, Spearman rank correlation). There was no association between age and CD8+/CD38+ percentage . The median change in CD8+/CD38+ from pre-intensification to post-intensification—when data from the two treatment groups were pooled—was −2.0% (95% CI −10% to 0%) . Although there was a marginally significant difference between the groups in the change in CD8+/CD38+ percent from baseline to week 12—there was a median of a 3% decline in the group that added raltegravir versus a 1% increase in the group that added placebo —the change in CD8+/CD38+ percentage from week 12 to 24 (after the cross-over) did not differ between the treatment groups . However, there was no significant difference between the groups in the change in CD4+/CD38+ percentage from baseline to week 12  or from week 12 to 24   .Finally, to determine whether the trend toward an increase in CD4 cell count during raltegravir intensification described above could be explained by an effect on T cell activation, we analyzed the relationship between change in activation markers and increase in CD4 cell count. We found no associations between the changes in CD4 or CD8 CD38+/HLA-DR+ percentage or CD38+ percentage and the trend towards a CD4 cell count increase during raltegravir intensification (unpublished data).In this randomized, double-blind, placebo-controlled cross-over study, 12 weeks of raltegravir intensification did not demonstrably reduce residual viremia in patients on PI- or NNRTI-containing ART. Although there was a trend toward an increased CD4 cell count during the period of raltegravir intensification, this change was not associated with an effect of raltegravir on T cell activation. CD4 and CD8 cell activation levels in blood, as determined by the percentage of CD38+/HLA-DR+ cells, were not associated with the baseline level of residual viremia, and we did not find evidence that raltegravir intensification reduced T cell activation by this measure.The findings of this study are consistent with the results of two smaller, single-arm trials. In a study of treatment intensification with 4–8 weeks of efavirenz, atazanavir/ritonavir or lopinavir/ritonavir in nine HIV-1 infected patients, there was no effect of adding the additional agent on HIV-1 RNA level measured by SCA + had received suppressive ART for a shorter time (mean 3.5 ± standard deviation (SD) of 2.5 years) than those who did not become 2-LTR+ (mean 5.2 ± SD 2.9 years) (p = 0.075). In our study, patients had been on suppressive ART for a mean of 5.6 ± SD 3 years (median of 5.5 years). The fact that increases in 2-LTR circles occurred mainly in patients who were on protease inhibitor-containing regimens suggests a mechanistic explanation independent of ongoing full cycles of viral replication, e.g., that the accumulation of 2-LTR circles is related to the stage at which different antiretroviral agents block HIV-1 rather than a reflection of complete cycles of viral replication. Finally, in the Buzon study, there was no effect of raltegravir intensification on plasma HIV-1 RNA levels, as measured by a single copy assay; this finding is the same as the result we obtained. The absence of an effect on plasma HIV-1 RNA implies that raltegravir intensification does not reduce ongoing viral replication, unless the virus is replicating in a compartment that does not communicate freely with the plasma. Studies to address this possibility by measuring the effect of raltegravir intensification on important anatomic compartments, such as gut-associated lymphoid tissue, are ongoing.A recently published study of raltegravir intensification has been interpreted as showing that ongoing viral replication occurs in a subset of patients on combination ART. Buzon et al. found that 13 of 45 (29%) patients on suppressive ART who were randomized to add raltegravir had a transient increase in 2-LTR circles—an episomal form of HIV DNA—which the authors interpret as reflecting persistent replication In addition to examining the effect of raltegravir intensification on residual viremia, we examined the immunologic effects of this intervention. Because residual viremia might affect immune recovery, perhaps by triggering abnormal T cell activation and increasing activation-induced cell death, we assessed whether raltegravir intensification affected the CD4 cell count. We observed an increase in CD4 cell count during the period of raltegravir intensification. A raltegravir-based regimen was also previously found to be associated with greater CD4 cell count gains than an efavirenz-based regimen in a randomized study conducted in treatment-naïve patients This study also allowed us to examine the relationship between residual viremia and T cell activation. Persistent viremia might explain the observation that T cell activation remains higher in patients who are receiving therapy and have HIV-1 RNA levels of <50 copies/mL than in uninfected individuals There are some potential limitations of our study. First, the duration of raltegravir intensification was 12 weeks, which may not have been long enough to see an effect of the agent on very long-lived reservoirs. However, if residual viremia arises from new cycles of infection, as opposed to release of virus from stable reservoirs, we would expect to detect an effect on HIV-1 RNA within the 12-week period of intensification. If there is ongoing HIV-1 replication, one would expect that both short- and long-lived cells would be infected—it seems implausible that new infection would only occur in long-lived cells—and, therefore, blocking infection of short-lived cells by adding raltegravir should reduce plasma viremia within a 12-week period. The absence of such an effect argues that residual plasma viremia does not primarily arise from complete cycles of HIV-1 replication and infection of new cells. A second limitation of our study is that the low HIV-1 RNA levels at study entry limit the ability to detect declines after intensification; as a result, our findings do not completely exclude the possibility that ongoing cycles of HIV-1 replication contribute to residual viremia. However, if adding raltegravir substantially reduced HIV-1 replication and residual viremia, we would expect that a higher proportion of patients would have HIV-1 RNA levels below the detection level of the single copy assay during intensification; we did not see such an effect. Finally, as noted above, we cannot exclude the possibility that there is ongoing viral replication in a compartment or compartments that do not contribute to plasma viremia and do not affect the level of T cell activation in blood. Studies of HIV-1 replication in other compartments, such as the gut-associated lymphoid tissue, are required to exclude this possibility.This study also has several strengths. First, unlike previous single-arm intensification studies, we performed a double-blind placebo-controlled randomized trial, which limits confounding. Second, this study has a longer period of intensification than a recently published trial that examined the effect of 4 weeks of raltegravir intensification In conclusion, in this randomized, double-blind cross-over study, 12 weeks of raltegravir intensification was not found to reduce low-level plasma viremia in patients on currently recommended ART. This finding argues against the hypothesis that ongoing, complete cycles of viral replication and integration are the main source of residual viremia. These results suggest that treatment intensification with raltegravir is unlikely to lead to eradication of HIV-1 infection, and that new therapeutic strategies to eliminate cellular or anatomic reservoirs will be required to cure HIV-1 infection Text S1Study protocol.(0.63 MB DOC)Click here for additional data file.Text S2CONSORT checklist.(0.19 MB DOC)Click here for additional data file.


The devastating impact of AIDS in the world especially in sub-Saharan Africa has led to an unprecedented global effort to ensure access to antiretroviral (ARV) drugs. Given that medication-taking behavior can immensely affect an individual's response; ART adherence is now widely recognized as an 'Achilles heel' for the successful outcome. The present study was undertaken to investigate the rate and predictors of adherence to antiretroviral therapy among HIV-infected persons in southwest Ethiopia.0) and third month (M3) follow up visits.The study was conducted in the antiretroviral therapy unit of Jimma University Specialized Hospital. A prospective study was undertaken on a total of 400 HIV infected person. Data were collected using a pre-tested interviewer-administered structured questionnaire at first month (M0) and at follow up visit (M3) respectively were interviewed. Self-reported dose adherence in the study area was 94.3%. The rate considering the combined indicator  was 75.7%. Within a three month follow up period, dose adherence decreased by 2% and overall adherence rate decreased by more than 3%. Adherence was common in those patients who have a social support . Patients who were not depressed were two times more likely to be adherent than those who were depressed . However, at the follow up visit, social support  and the use of memory aids  were found to be independent predictors of adherence. The principal reasons reported for skipping doses in this study were simply forgetting, feeling sick or ill, being busy and running out of medication in more than 75% of the cases.A total of 400 and 383 patients at baseline (MThe self reported adherence rate was high in the study area. The study showed that adherence is a dynamic process which changes overtime and cannot reliably be predicted by a few patient characteristics that are assumed to vary with time. Adherence is a process, not a single event, and adherence support should be integrated into regular clinical follow up. An estimated 33.2 million people worldwide were living with HIV, 2.5 million became newly infected and 2.1 million lost their lives to AIDS at the end of 2007 [Highly Active Antiretroviral Therapy (HAART) was a breakthrough in the industrialized world, leading to the reduction of mortality and the improvement of quality of life of people living with HIV and AIDS (PLWHA) ,4. It trEven though ART is the single most dramatic development yet in the treatment of HIV , many haAdherence is the second strongest predictor of progression to AIDS and death, after CD4 count . ConsistStudies report conflicting evidence about the association between socio-demographic factors and adherence behavior. Some literatures reported that certain socio-demographic variables have influence over adherence to HAART; however, others showed no association ,14,20,21et al showed that regimen complexity alone was not a significant predictor of patients' medication adherence [The complexity of the regimen, side effects and various demands around medication and food timing caused by it are also associated with non-adherence ,16,27. Hdherence . Variousdherence ,17,24. Fdherence ,28,29.As the world gears toward increasing access to antiviral treatment in the developing world it is critical to understand factors (motivators and barriers) that influence adherence to Antiretrovirals and apply the lessons learnt in improving existing and new programs. Available research in Ethiopia has shown that our understanding of factors associated with ART adherence is limited, and related literature in the study area is remarkably scarce. Understanding the predictors of adherence in the local context is a forefront agenda in Ethiopia, where little is known and scaling up of ART program is in progress. In view of this, and to assess whether the global experience works with the Ethiopian context, a prospective study was conducted in southwest Ethiopia to determine the rate, barriers, and factors associated with ART adherence. It is anticipated that the findings generated from this study will contribute to the knowledge and understanding of non-adherence to ARVs and be useful in developing evidence based interventions that are undertaken to address ARV adherence in Ethiopia.0), and was conducted from December 25/2006. In phase II the subjects were followed prospectively for three months and self reported adherence was measured at 3rd month (M3) starting from March 25/2007.The study was conducted in the antiretroviral therapy unit of Jimma University Specialized Hospital (JUSH) from December 25/2006 – April 30/2007 for a period of 4 months. The zone is one of 17 zones in Oromia Regional State; the capital Jimma, is located 335 KM Southwest of Addis Ababa. The Hospital and ART units launched their service as of July 2003 and till recently a total of 2538 PLWHAs were receiving care, of whom 1118 patients were on ART until the end of July 2007 [A prospective study was undertaken to investigate the possible factors for adherence. The sample size was calculated using Epi-Info 3.3.2 statistical software, assuming the following parameters. The proportion of adherence among non-depressed individuals was 83.6% with a relative risk of 2.8 . Other pThe dependent variable was adherence to antiretroviral therapy. Adherence in this study was measured by (1) Self-reported dose adherence, defined as patient's self-report of whether any antiretroviral medication had been skipped that day, the previous day, the previous three days or the previous seven days. A person was said to be adherent if he/she took ≥ 95% of the prescribed doses correctly. For the comparison assessment, we used adherence in the previous seven days. (2) Self-reported time adherence; where a person is said to be adherent when claiming to always follow scheduling instructions. (3) Self-reported food adherence; where a person is said to be adherent when always following dietary instructions agreed upon with the providers. Hence, for comparison purposes a combined indicator of adherence was made using the three adherence measures taking into account all questions pertaining adherence. Accordingly a person was said to be adherent when he/she took >= 95% of the prescribed doses correctly, always followed scheduling instructions and always followed dietary requirements. This type of measurement of adherence has been frequently used in a range of studies -33.Data were collected using a pre-tested questionnaire which consists of socio-demographic characteristics , psychosocial attributes , disease characteristics , regimen related variables , health care system and health care team related variables .Depression was measured using a 13 item scale widely used in the HIV/AIDS literature, after excluding items reflecting somatic symptoms. Accordingly, a cut-off point of 10 was used in this study to differentiate depressed from non-depressed individuals . Stigma 0) and repeat (M3) visits. Self-reported dose adherence to all antiretroviral agents was summarized as the ratio of the average daily number of antiretroviral medications adhered to correctly according to the standard instructions over the total number of antiretroviral medication prescribed. The analysis consisted of basic summaries of patient characteristics, and bivariate analysis of the relation between adherence and various factors. The reliability of scale items was evaluated using Cronbach's alpha; which measures how well each individual item in a scale correlates with sum of the remaining items. A cut-off value of 0.7 was used to indicate acceptable internal consistency [0 and M3 as the dependent variable to determine the constant predictors. In both cases, all explanatory variables that were associated with the outcome variable (adherence at M0 and M3) in the bivariate analyses with a P-value of 0.05 or less were included in the initial logistic models. The models were evaluated using a forward and backward stepwise elimination procedure. A P-value of 0.2 was used to select variables for entry into the model and 0.1 for removal from the model.The data collected from the respondents were cleaned, coded, entered and analyzed using SPSS 12.0.1 for Windows at baseline  of Jimma University.0). A total of 403 subjects gave informed consent and were interviewed, of which three had missing adherence data and were omitted from the analysis. At the follow up visit (M3) a total of 383 subjects completed the study with a median length of follow up and inter-quartile range of 90 (88–92) days. The remaining 17 subjects were not present at the third data collection phase and were not included in the follow up study.Patients who came to the ART unit during the first phase of the data collection were included in the baseline study . The majority 389 (97.3%) were from Jimma, were Oromo 161 (40.3%) by Ethnicity and Orthodox 231 (57.8%) by religion. One hundred eighty (45%) were married, 201 (50.3%) had attended secondary education, 143 (35.8%) had no job and 148 (37%) of the survey participants had no monthly income. Three hundred twelve (80.7%) of the study subjects were living with their husband/wife, family or their friends and only 77 (19.3%) lived alone  had CD4 count less than 200 cells/mm3; the range being 2 to 749 cells/mm3 with a median of 135 cells/mm3. The study subjects, at inclusion, were on HAART for a median duration of 8 months (3 to 67 month). Most 384 (96%) of them had monthly regular follow up visit for their drug refill  had started HAART while at WHO disease stage III. In addition for those patients in which the initial CD0) either from their family, friends/peers, HIV clubs or co-workers. The study subjects reporting social support decreased to 207 (54%) at M3 the difference being significant (P ≤ 0.05). Using the HIV/AIDS stigma scale  at M3 121 (32.2%) of the study subjects were stigmatized. Thirty eight (9.5%) of participants at M0 reported active substance use. However, 39 (10.2%) reported active substance use at M3 (P > 0.05). Based on the Beck's depression inventory (BDI) scale with a cut-off point of ten, 223 (55.8%) and 190 (49.6%) of the study subjects were depressed at baseline and follow up visit respectively (P > 0.05). Side effects were experienced by 209 (52.3%) of the patients at baseline. However, after 3-month follow up period only 78 (20.4%) reported having side effects (P > 0.05). Twenty eight (7%) of the study subjects at baseline were hospitalized after they started HAART. Of these, 24 (88.9%) were hospitalized at least once; the rate being 2 times higher among non adherent than adherent individuals. Hospitalization was further decreased at M3 in which only 8 (2.1%) had a history of admission (p > 0.05). Three hundred ninety five (98.8%) and 343 (89.6%) of the study participants reported using memory aids at M0 and M3 respectively  received social support at baseline (M0 and M3 respectively (P = 0.54). Three hundred eighty nine (97.2%) and 373 (97.4%) of the study subjects always followed the time restrictions (time adherence) agreed upon with their providers at M0 and M3 respectively (P = 0.77). Three hundred thirty eight (84.5%) and 319 (83.3%) subjects followed instructions related to food (food adherence) all the time at M0 and M3 respectively (P = 0.79). Hence, the rate of self reported adherence in the study area based on the combined indicator of the three adherence errors was 79.3% at baseline and 75.7% at follow up visit (P = 0.62) (table The three adherence errors were assessed in the study to get a combined adherence indicator Table . Accordiy P = 0.5. Three h0). Adherence was common in those patients who have a social support . Patients who were not depressed were two times more likely to be adherent than those who were depressed . However, at the follow up visit, social support  and the use of memory aids  were found to be independent predictors of adherence (Table In the multivariate logistic regression analysis; three variables were found to be predictors of adherence at baseline (MIn most studies, adherence refers solely to dose adherence, but successful treatment with ART also includes adhering to scheduling and adhering to dietary instructions that accompany many antiretroviral drugs ,33. In t3; which is higher that reported in Addis Ababa and Arbaminch, Ethiopia [3. Some studies in resource-rich settings have documented less than 50% of patients taking all their antiretroviral medications on time and according to dietary instructions [et al also found that low socio economic status was not a predictor of adherence for patients with fully subsidized therapy and concluded that adherence in developing countries has been found to be at least as good as adherence in developed countries [The rate of dose adherence in the study area was 96% at baseline and 94.3% at MEthiopia . ConsistEthiopia . The oveructions ,33. Thisountries .This relatively short prospective study underscores the dynamic nature of adherence. Though not significant, within three months, dose adherence decreased by 2%, food adherence by 1% and overall adherence rate reduced by more than 3%. The lack of statistical significance difference may be due to the short duration of follow up. Various studies have indicated the dynamic nature of adherence overtime ,33,40. TThe principal reasons reported for skipping doses were similar to other studies at both visits ,37,38. TResearches' have already shown the dynamic character of HAART-treated patients' adherence behaviors, which are influenced by multiple factors varying over time ,33. Our Our results also show that patients who claimed to use memory aids were three times more likely to be adherent than those who did not. This study shows that adherence interventions should include memory aids and other reminders to help patients take their drugs. Social support was a constant predictor of adherence identified in this study. Patients who reported social support were more likely to be adherent than those who did not. This is consistent with many comparable studies both in resource poor and resource rich settings ,18,24,38The findings of this study must be interpreted in the light of its limitations. Because it was conducted at a single site, the findings may not be generalizable to other clinical settings. There is no gold standard for measuring adherence and our measurement of adherence is only based on patients' reports of missed doses, scheduling instructions and dietary requirements. This may be subject to social desirability and recall biases and the literature suggests that patients tend to overestimate adherence . HoweverThe adherence rate found in this study seems to be encouraging. The findings emphasized the importance of multiple periodic assessments of adherence errors. Timely detection of non-adherence behaviors and appropriate monitoring of patients' difficulties with HAART could potentially help patients to maintain adherence and therefore improve the treatment outcome. Finally the results suggested that psychosocial and medical interventions aimed at increasing adherence of HAART-treated patients should integrate the dynamic dimensions of adherence behaviors. Adherence is a process, not a single event, and adherence support must, therefore, be integrated into regular clinical follow up. Investigation of factors related with long-term adherence would require longer follow-up than the present study.The authors declare that they have no competing interests.AA conceived and participated in the design, conduct, analysis and interpretation of the study. KW, SG and BG and KD involved in designing the survey and undertook preliminary analysis. All authors contributed to the final report and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Adequate antiretroviral drug potency is essential for obtaining therapeutic benefit, however, the behavioral aspects of proper adherence and readiness to medication, often determine therapeutic outcome. Therefore, this study aimed to assess the level and determinants of nonadherence and nonreadiness to highly active antiretroviral therapy (HAART) among people living with HIV/AIDS (PLWHA) at Gondar University Teaching Hospital and Felege Hiwot Hospital in Northwest Ethiopia.A cross-sectional study was conducted between July and September 2008 using structured interviewer-administered questionnaire. All consecutive adult outpatients who were receiving antiretroviral treatment for at least three months, seen at both hospitals during the study period and able to give informed consent were included in the study. Multivariate logistic regression was used to determine factors associated with nonadherence and nonreadiness.A total of 504 study subjects were included in this study. The prevalence rates of nonadherence and nonreadiness to HAART were 87 (17.3%) and 70 (13.9%) respectively. Multivariate logistic regression analysis revealed that medication adverse effects, nonreadiness to HAART, contact with psychiatric care service and having no goal had statistically significant association with nonadherence. Moreover, unwillingness to disclose HIV status was significantly associated with nonreadiness to HAART.In this study the level of nonadherence and nonreadiness to HAART seems to be encouraging. Several factors associated with nonadherance and nonreadiness to HAART were identified. Efforts to minimize nonadherence and nonreadiness to HAART should be integrated in to regular clinical follow up of patients. HIV/AIDS is the fourth most common cause of death in the world  and is eHighly Active Antiretroviral Treatment (HAART) has dramatically reduced mortality and morbidity due to HIV ,5. It isProlonged viral suppression is only achievable if the virus does not get the chance to replicate and develop drug-resistant HIV variants . The virDespite the high prevalence of HIV/AIDS in Africa including Ethiopia, the HAART coverage is extremely low due to limited resources, but in these days WHO as well as different countries are interested to intensify the HAART activities and expand the program as preventive strategy for HIV epidemic and AIDS patient care.Ethiopia has been started provision of HAART for the people living with HIV/AIDS since August 2003. However, by the end of June 2008, there were only 110,611 patients (75%) who were alive and on HAART out of the 150,136 patients who had been started on HAART since 2003 . This inOne of the main factors contributing to sub-optimal drug levels and resistance is non-adherence to treatment ,18. It hPatient's readiness to antiretroviral therapy means put the patient himself/herself feels ready to initiate, take responsibility for, and to maintain (including being adherent to) a prescribed treatment . ReadineAssessment of patient adherence and readiness to treatment are good opportunities to enhance patient understanding of medication regimen, to identify potential obstacles to taking medication and trusting relationship between patients and health care providers, and ultimately to prevent virologic break through . TherefoA cross-sectional study was conducted between July and September 2008 at Gondar University Teaching Hospital and Felege Hiwot Hospital in Northwest Ethiopia. These hospitals are tertiary level teaching hospitals that each hospital provides health service to over five million inhabitants in Northwest Ethiopia, located 727 Km and 540 Km away from the capital city, Addis Ababa respectively.All consecutive adult outpatients who were receiving antiretroviral treatment at least for three months, seen at both clinics during the study period and able to give informed consent were included in the study.Data was collected using structured interviewer-administered questionnaires which include the following variables: socio-demographic characteristics, knowledge of patients towards treatment and health care system, patient attitude towards health care provider and program and patients self-report to treatment adherence and readiness. Prior to data collection, training to nurses (interviewers) about the objectives of the study and methods of interviewing was given and the English version questionnaire was translated to the local language (Amharic). Institutional ethical clearance was obtained from the research and publication committee of Gondar University.Respondents were asked whether they had missed any doses the day prior to completing the questionnaire, and how often doses were missed in general (ranging from every day to never). Respondents, who were reported that they had not forgotten a dose the day prior to the completion of the questionnaire and those responded that they never forget doses were categorized as adherent. This strict definition of adherence was chosen, since the respondents providing us with these answers would theoretically reach an adherence level of at least 95% .Patients' readiness to HAART was determined based on the five indicators of readiness : These iData was coded, cleared, entered and analysed using SPSS statistical software version 13. Different variables were described and characterized by frequency distribution. Association between the dependent and independent variables was analyzed using chi- square test and multivariate logistic regression. In all cases p-value of less than 0.05 was taken to indicate level of statistical significance.A total of 504 study subjects  were included in this study. Of these, 310 (61.5%) respondents were females and 194 (38.5%) were males with the mean (SD) age of 35.3 (8.9) years. Most of the study subjects, 452 (89.7%) were urban resident, 488 (96.8%) were Amhara by ethnicity and 471 (93.5%) were Christian by religion. Large number of the respondents, 138 (27.4%) were illiterate, 107 (21.2%) were widowed, 114 (22.6%) were daily labourers and 254 (50.4%) had household income below 45.25 USD per month. Moreover, thirty six (7.1%) study subjects were reported active substance use   had disclosed their sero-status . Four hundred ninety five 98.2%) respondents thought that HAART had benefited them by improving their quality of life or improving their symptoms. One hundred ninety nine (39.5%) had an adverse reaction to HAART like skin rash, itching, nausea, and/or vomiting since starting HAART. More than half, 255 (50.6%) participants had discomfort when taking their drugs in front of others, and most of the respondents, 476 (94.4%) were satisfied with the health care providers service. Majority of respondents, 453 (89.9%) had access to pharmacy at any time and 108 (21.4%) patients were visiting their doctors monthly. Moreover, 59 (11.7%) respondents reported contact with psychiatric care services and 485 (96.2%) were having goals (Table % respondThe level of nonadherence and nonreadiness to HAART were 87 17.3%) and 70 (13.9%) respectively. The main reasons for nonadherence are drug side effects 27 (31.0%) and other health problems 19 (21.8%). On the other hand, the major reasons for nonreadiness to HAART are anxiety 31 (44.3%) and hopelessness 19 (27.1%). Of all study subjects, 419 (83.1%) were highly motivated to take HAART, 74 (14.7%) were partially motivated and 11 (2.2%) respondents were not at all motivated to take HAART , nonreadiness to HAART , contact with psychiatric care service  and having no goal  had statistically significant association with nonadherence  respondents had less than 95% adherence and 70 (13.9%) of the respondents had not been ready to HAART. The level of nonadherence in this study was comparable with those reported in Addis Ababa  where adherence rates were 81.2%  and 82.8The multivariate logistic regression analyses showed that medication adverse effects had statistically significant association with nonadherence to antiretroviral therapy. This is in agreement with the findings of other studies conducted in Brazil, Senegal and Addis Ababa ,26,27. EAlthough there are few published studies, and they have used different methods to assess readiness, a significant association between the level of readiness and the level of adherence has been observed . This obHaving long-term plans and goals, using time wisely and having a meaningful life are characteristics of patients who have fewer adherence difficulties ,35. ThisOur study has the following limitations. First, we measured adherence and readiness of patients to HAART by patient self - report, which may be subject to recall bias and overestimate adherence and readiness. Nevertheless, many other studies document that well collected self report data clearly correlates with virologic changes and is more practical in most settings ,38. SecoIn this study the level of nonadherence and nonreadiness to HAART seems to be encouraging. Medication adverse effects, nonreadiness to HAART, contact with psychiatric care service and having no goal were significant barriers to treatment adherence. Moreover, unwillingness to disclose HIV status was a significant factor for nonreadiness to HAART. Therefore, efforts to minimize nonadherence and nonreadiness to HAART should address these barriers among others, and should be integrated in to regular clinical follow up of patients. Furthermore, continuous measurement of patients' non adherence and nonreadiness, to identify when interventions are required, seems to be an approach worth further investigation.The authors declare that they have no competing interests.BT was the primary researcher, conceived the study, designed, conducted data analysis and drafted the manuscript for publication.FB, AM and AG assisted in data collection and reviewed the initial and final drafts of the manuscript.FE and US interpreted the results, and reviewed the initial and final drafts of the manuscript.All the authors read and approved the final manuscript for submission for publication.


Antiretroviral therapy (ART) has dramatically reduced morbidity and mortality among people with HIV infection; however, mortality after the start of ART is high in resource-limited settings. We investigated risk factors for mortality among adults starting ART in a multi-clinic community programme in South Africa.st March 2007. Kaplan-Meier survival estimates were used to describe overall mortality. Shared frailty Cox regression was used to identify baseline risk factors for early mortality.Cohort of adults starting ART at 27 clinics between February 2005 and June 2006, followed to 31Among 1350 participants ], median follow-up 13.4 months), there were 185 deaths, overall mortality rate 13/100 pyrs; for 0-3, 3-9 and >9 months from ART start mortality rates were 24, 13 and 6/100 pyrs respectively. 43% of the deaths were in the first 3 months of treatment. Risk factors for mortality in univariable analysis were baseline CD4 count, viral load, haemoglobin and body mass index, in multivariable analysis adjusting for age and gender, only CD4 count and haemoglobin remained independently associated with proportional hazards not being satisfied for haemoglobin. Adjusted hazard ratios (aHR) for participants with haemoglobin <8, 8.1-9.9, >11.9(f)/12.9 (m) g/mL were 4.99, 3.05 and 0.12 respectively comparing to 10-11.9 (f)/12.9 (m)g/mL in the first 3 months of ART. aHRs for CD4 counts were 0.40, 0.38 and 0.34 for 50-99, 100-200 and >200/μL comparing to <50/μL.The high mortality rate in the first 3 months underlines the need for earlier HIV diagnosis so that ART can be initiated earlier. Low haemoglobin and low CD4 count are both strong predictors of mortality, and could be used to identify individuals at high risk who might benefit from intensive case management. South Africa is estimated to have 5.7 million HIV-positive individuals out of a total population of 48.6 million  and has Although treatment programmes have been scaled up and increased in number they still face challenges. Studies in Sub-Saharan Africa show that one of these challenges is that mortality in these programmes is much higher in the first three months -9. One oThe data for this study were collected from a large community based programme set in clinics run by NGOs or private practitioners in five provinces of South Africa. The clinics provide standardised HIV care and treatment through a programme implemented by the Aurum Institute. We describe early mortality and loss to follow-up, and risk factors for mortality in this setting.This was a prospective cohort study examining routinely collected clinic data from an observational cohort of adult patients to examine risk factors for early mortality. The clinics contributing data to the study were in urban and peri-urban sites in five provinces of South Africa . The clinics were either run by a non-government organisation (NGO), or private practitioners. Training, clinical and financial support, drug procurement, and administration and monitoring support were provided by the Aurum Institute. This programme provided free antiretroviral treatment to patients who did not have medical insurance and were unable to pay for their treatment themselves. The organisation of the programme as it was originally developed in an industrial setting has been described elsewhere .The clinics followed the National South African HIV treatment guidelines for participants starting ART: either CD4 <200/μL or classified as in WHO stage 4. The first line regimen given to the majority of patients was stavudine, lamivudine, and either nevirapine or efavirenz. Patients starting ART attend monthly to collect medication; they have a clinical review and blood taken for full blood count and liver function tests at six weeks, three months and three monthly thereafter; and CD4 count and viral load at six weeks, six months and six monthly thereafter.st February 2005 and 1st June 2006, giving them an opportunity to have at least 10 months follow-up before the cohort was censored on the 31st March 2007.Patients were included in this analysis if they were 18 years or older when initiating ART treatment, if they were attending clinics that had over 50 participants in HIV care in the programme, and if they were ART-naïve (by self-report) when enrolled into the programme. The participants in this cohort all began their treatment between 1st March 2007, whichever came first. Deregistration forms provided information on participants leaving the programme alive and deaths whilst on treatment. Deaths were ascertained based on information from relatives or local hospital services, or by clinic staff attempting to contact patients who had not attended scheduled follow-up appointments. Records were checked to confirm that participants without a death or deregistration recorded were alive and attending the clinic as of 31st March 2007. Reasons for leaving the programme, documented on the deregistration form, were relocated out of the area, transferred out of the programme or patient stopped treatment. Patients were defined as lost to follow-up if either the deregistration form documented lost to follow-up with no supplementary data for the reason or the most recent clinic visit occurred more than 6 months before 31st March 2007.The data were collected through routine health care services. Forms, completed at both scheduled and unscheduled visits, included information on CD4 count, haemoglobin, viral load, height, weight, and previous treatments. Participants with less than 8 g/mL of haemoglobin were classified as severely anaemic, and those with < 12 (<13 for men) g/mL as anaemic. In thisData were analysed using STATA v. 10 . CD4 count, viral load, haemoglobin levels and body mass index (BMI) at baseline were defined as the results closest to the date of ART start, restricted to the time interval 90 days before and 15 days after ART start, with viral load restricted to within 4 days after ART start, and if more than one measurement was available, the viral load before ART start was used.The cohort was analysed using an intent-to-treat approach, assuming that once started on ART, patients remained on ART without interruption ,12. MortUnivariable analysis was carried out using the Cox proportional hazards model with shared frailty to calculate unadjusted hazard ratios for baseline variables. Shared frailty was used to take into account that individuals attending the same clinic may be more similar to each other with respect to outcomes and measures associated with outcomes than to those attending other clinics. Likelihood ratio tests were used to assess departures from linearity and for linear trend for ordinal variables. Proportional hazards were assessed by a lexis expansion at 3 months follow-up time and Schoenfeld residuals were also examined for departures from the proportional hazards assumption.a priori confounders and those that were associated in univariable analysis (p < 0.1 based on likelihood ratio tests). BMI as a baseline risk factor had more than 20% missing data so a model using multiple imputation was examined, with 10 imputed datasets derived [A multivariable model was constructed by retaining variables that were considered  derived  and esti derived . BaselinThis study was approved by the Research Ethics Committees of the University of KwaZulu-Natal, South Africa and the London School of Hygiene and Tropical Medicine, UK. Data were anonymised prior to analysis.st February 2005 and 1st June 2006. Patients were excluded if they had never collected their medication (n = 140), hence there were 1350 included in the analysis  and 8.7% were classified as severely anaemic (<8 g/mL) at baseline  and 59.8% were female  patients were defined as lost to follow-up and 80 (35.1%) patients left the programme early . Those who were censored alive before 31st March 2007 compared to those who were alive at the end of follow-up were more likely to be male  and their median haemoglobin level was 11.4 g/mL compared to 11.8 g/mL ; there was no evidence for differences between those who left the programme and those alive at the end of follow-up in terms of baseline CD4 count, BMI, and age at ART start (data not shown).The median follow-up time for the 1350 participants in this cohort was 13.4 months  with 937 participants alive and in the programme at the censoring date of 31There were 185 deaths during follow-up giving an overall mortality rate of 13 per 100 person years (pyrs), with 94% alive at 3 months and 88% at 9 months. The mortality rate in the first 3 months was 24.5 per 100 pyrs, decreasing to 13 and 6 per 100 pyrs for 3 - 9 months and greater than 9 months following the start of ART, respectively (p < 0.0001). Seventy nine of the deaths (42.7%) occurred in the first 3 months.In univariable analysis using Cox regression with shared frailty, lower CD4 count, lower haemoglobin, low BMI and higher viral load, were all found to be associated with higher mortality Table . The hazKaplan-Meier curves show the survival experience for the whole cohort stratified by baseline CD4 count and baseline haemoglobin in Figure In multivariable analysis CD4 count and haemoglobin were independent risk factors for mortality Table . After aThere was less than 10% change in the adjusted hazard ratios for BMI from multiple imputation compared to the adjusted hazard ratios from the reduced multivariable model with no imputation, after controlling for haemoglobin and CD4 count. Evidence for BMI being associated with mortality was weak (p = 0.11) which is consistent with the conclusions from the multivariable analysis with no imputation.This analysis, based on routinely collected data at clinics across South Africa, showed that there was high early mortality on ART with a large proportion (42.7%) of the deaths occurring in the first three months of treatment, consistent with other studies from sub-Saharan Africa . This isAt three months after ART start, 13% of those with a baseline CD4 count below 50 had died compared with 3% of those with CD4 count of 50 and above, highlighting the strong association between baseline CD4 count and early mortality, consistent with other studies ,12,18,19One in six participants in this cohort left the programme during follow-up, and for the majority of these there was none or only limited information on reasons for their departure, similar to other ART programmes in Africa ,15,23,24A strength of the data presented in this analysis is that individuals identified in this cohort had definitely started ART and collected their medication. This helps to reduce the bias that may occur if individuals who never started ART were contributing person-years to the study.There were missing data for BMI (36.7%) but it was evaluated in the univariable and multivariable analyses and conclusions from the multivariable model were supported by multiple imputation methods. There was also no information on the cause of death for this cohort which would have been useful, as what had caused the early deaths and information regarding the deaths of those with very low haemoglobin would help to inform an intensified case management system. However there is increasing evidence from other studies about the importance of tuberculosis as an early cause of death ,29The results from this analysis give evidence that CD4 count and haemoglobin predict early mortality for individuals on ART in sub-Saharan Africa. Individuals starting ART with either low CD4 count or low haemoglobin should be targeted for intensified case management, including investigation for and treatment of coexistent infections, particularly tuberculosis in settings of high tuberculosis prevalence. In clinics without access to CD4 counts, haemoglobin may be useful to identify those at highest risk.The authors declare that they have no competing interests.ECR, SC, ADG, KF designed the study; SC & LP collected the data; ECR, KF carried out data analysis and interpretation; ADG, SC and GJC carried out data interpretation; ECR, SC, ADG, KF drafted the manuscript; GJC & LP revised the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/433/prepub


To the Editor: Before 2007, two human polyomaviruses were known to infect humans: BK virus and JC virus  and were being treated with antiretroviral drugs. HIV-1 viral load determination, CD4+ counts, and HIV-1 genotyping were performed as part of the routine investigation. Plasma viremia levels ranged from <50 to 2,877,764 copies/mL, and CD4+ counts ranged from 150 to 1,218. Most patients (64.5%) were infected by HIV-1 subtype B. Other subtypes found were F, G, and C.T-Ag) and almost the entire small t antigen (t-Ag) genes. KIPyV was amplified as described (t-Ag fragments were sequenced as described (www.ncbi.nlm.nih.gov/BLAST). ClustalX software  was used to obtain alignment with a set of KIPyV and WUPyV isolates from Italy (www.mbio.ncsu.edu/BioEdit/bioedit.html) (http://paup.csit.fsu.edu/about.html). Maximum-likelihood (ML) trees were then inferred with the selected model and ML-estimated substitution parameters. The heuristic search for the ML tree was performed by using an NJ tree as starting tree and the tree–bisection-reconnection branch-swapping algorithm. NJ trees were also estimated by using pairwise distances inferred by ML with the best fitting nucleotide substitution model. Calculations were performed by using PAUP* 4.0b10 (http://paup.csit.fsu.edu/about.html)  and then stored at –80ºC until analysis. KIPyV and WUPyV PCR screening was carried out as described , respiratory tract (KIV-RM1 to KIV-RM4), and tonsils (KIV-RM12 to KIV-RM20), nor are they related to those previously identified  and WUPyV in 1 (1.6%). All 3 patients were infected by HIV-1 subtype B. Phylogenetic analysis of the ntified ,.To date, KIPyV and WUPyV have been detected in respiratory secretions and stool and serum specimens from pediatric patients with acute respiratory symptoms and have been found in respiratory tissue of adults and children (Molecular analysis of the KIPyV and WUPyV identified in plasma showed that these polyomaviruses were not closely related to strains identified previously in other countries nor to the KIPyVs and WUPyVs identified in Italy in stool, respiratory tract tissue, and tonsils. Whether this difference reflects a tropism of some strains for a particular tissue or organ remains to be established. Further studies are needed to clarify the possible pathogenic role of KIPyV and WUPyV in immunocompromised patients.


Coinfection with hepatitis B virus (HBV) or hepatitis C virus (HCV) in HIV-infected patients receiving a commonly used nevirapine-based antiretroviral therapy is a major concern for African clinicians owing to its high prevalence, the infrequent testing and treatment of viral hepatitis, and the impact of liver disease on the tolerability and effectiveness of anti-HIV treatment. We compared the hepatotoxicity and the immunological, virological and clinical effectiveness of a nevirapine-based antiretroviral therapy between patients infected with HIV only and patients coinfected with hepatitis B or C virus in Cameroon.A retrospective cohort study was conducted among HIV-1-infected patients. Plasma HBV DNA and HCV RNA were tested in positive or indeterminate samples for HBsAg or HCV antibodies, respectively. All patients received nevirapine and lamivudine plus stavudine or zidovudine.3 (interquartile range [IQR] 67-218), 21% were coinfected with HBV or HCV. In coinfected patients, the median viral load was 2.47 × 107 IU/mL for HBV (IQR 3680-1.59 × 108) and 928 000 IU/mL for HCV (IQR 178 400-2.06 × 106). Multivariate analyses showed that the risk of hepatotoxicity was 2-fold higher in coinfected patients (p < 0.01). The response to antiretroviral therapy was however comparable between monoinfected and coinfected patients in terms of CD4 cell count increase (p = 0.8), HIV-1 viral load below 400 copies/mL (p = 0.9), death (p = 0.3) and death or new AIDS-defining event (p = 0.1). Nevirapine was replaced by a protease inhibitor in 4 patients owing to hepatotoxicity.Of 169 HIV-1-infected patients with a median baseline CD4 count of 135 cells/mmThis study suggests that the nevirapine-based antiretroviral therapy could be used safely as first-line treatment in patients with low CD4 cell count in Africa despite frequent coinfections with HBV or HCV and infrequent testing of these infections. Although testing for HBV and HCV should be systematically performed before initiating antiretroviral therapy, transaminases elevations at baseline or during treatment should be a decisive argument for testing when hepatitis status is unknown. Coinfection with hepatitis B virus (HBV) or hepatitis C virus (HCV) in HIV-infected patients receiving a nevirapine-based antiretroviral therapy is a major concern for African clinicians owing to its high prevalence, the infrequent testing and treatment of viral hepatitis, and the impact of liver disease on the tolerability and effectiveness of anti-HIV treatment . While HNevirapine, the non nucleosidic reverse transcriptase inhibitor (NNRTI) most often used in first-line treatment in combination with two nucleosidic reverse transcriptase inhibitors, is associated with early hypersensitivity reactions  which can cause fulminant hepatitis leading to hepatic failure and death, and with later onset of direct drug-related hepatotoxicity leading to liver enzymes elevations -5. The WThe impact of HBV or HCV coinfection on the effectiveness of a nevirapine-based antiretroviral therapy remains unclear owing to the lack of specific studies especially in the African context. A recent South African study in HBV-coinfected patients receiving the less hepatotoxic efavirenz (the second NNRTI used in first-line treatment) found similar response to antiretroviral therapy between monoinfected and coinfected patients despite higher hepatotoxicity in the latter . RegardlWe therefore compared the hepatotoxicity and the immunological, virological and clinical effectiveness of a nevirapine-based antiretroviral therapy between patients infected with HIV only and patients coinfected with HIV and hepatitis B or C virus in Cameroon .3, a Karnofsky score over 50%, and no contraindications to antiretroviral treatment, including serum liver enzyme levels less than five times the upper limit of normal (ULN) in the first project or less than three times the ULN in the second project. All patients received nevirapine and lamivudine plus stavudine or zidovudine. Tenofovir was not available. Efavirenz was temporarily substituted for nevirapine in case of concomitant tuberculosis therapy. Care (visits and laboratory exams) and drugs  were provided free of charge.A retrospective cohort study was conducted in two major hospitals  in Yaoundé, the capital of Cameroon, among HIV-1-infected patients enrolled from 2001 to 2003 in two clinical research projects designed to assess antiretroviral treatments -25. The 8 IU/mL for HBV and 15 to 6.9 × 107 IU/mL for HCV).Hepatitis B and C markers were assessed on baseline blood samples frozen at -80°C. Enzyme immunoassays (EIA) were used to detect hepatitis B surface antigens  and antibodies to hepatitis C virus . Plasma HBV DNA and HCV RNA were tested in positive or indeterminate samples for HBsAg or anti-HCV, respectively, using the Cobas Ampliprep/Cobas TaqMan quantitative assay  classification.The CD4 cell counts were measured with a FACSCount device  at baseline and then every 6 months. The HIV-1 RNA load was determined using the Roche Amplicor HIV-1 Monitor assay  or the Bayer bDNA HIV-1 Quantiplex assay  at baseline, months 3 and 6, and then every 6 months. Serum liver enzymes  levels were assayed at baseline, weeks 2, 4 and 6, and then months 2, 3, 6, 9, 12, 18 and 24. Hepatotoxicity was graded with the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) toxicity scale in which an ALT or AST level of 1.25-2.5 times the ULN defines grade 1, >2.5-5 times the ULN defines grade 2, >5-10 times the ULN defines grade 3 and >10 times the ULN defines grade 4 2 test and, when sample sizes were too small, Fisher's exact test were used to compare the distribution of categorical variables between the infection groups (HBV or HCV-coinfected patients versus HIV-monoinfected patients). For continuous variables, comparisons were based on the non parametric Mann-Whitney two-sample test.Patients were classified as coinfected if they had positive HBV DNA or HCV RNA. The analyses were based on an intention-to-treat approach. Data were censored at the time of each patient's month 24 visit or, if follow-up was shorter, at the time of the last visit or death. The χHepatotoxicity was first assessed by using two end points: 1) occurrence of at least one episode of grade 2 or higher hepatotoxicity, and 2) occurrence of at least one episode of grade 3 or 4 hepatotoxicity. Kaplan-Meier curves were plotted and differences between monoinfected and coinfected patients were checked for significance by the log-rank test. Cox proportional hazard models were used to compare hepatotoxicity between groups adjusted on baseline covariates. The proportional hazards assumption was evaluated by both graphic and statistical  methods. A multivariate Poisson regression was then used to compare between groups the highest grade of hepatotoxicity reached by each patient during follow-up. The evolution of the CD4 cell count during follow-up was estimated using a mixed-effect linear regression model. Taking into account the biphasic evolution of the CD4 cell count during antiretroviral therapy , we alloversus men), age (≥ 42 versus <42 years), time since diagnosis of HIV infection (<24 versus ≥ 24 months), body mass index (≥ 21 versus <21 kg/m2), Karnofsky score (<100% versus 100%), CDC clinical stage, CD4 cell count (≥ 100 versus <100 cells/mm3), HIV-1 viral load (<5.0 versus ≥ 5.0 log10 copies/mL), hemoglobin level (≥ 10 versus < 10 g/dL), total lymphocytes count (≥ 1700 versus < 1700 cells/mm3), liver enzymes levels (≥ 1.25 versus < 1.25 × ULN), antiretroviral therapy and cotrimoxazole prophylaxis. Independent covariates associated with outcomes with a conservative p value of < 0.25 in univariate analysis were subsequently tested in multivariate analysis. A backward elimination procedure was used to determine the final model containing only the infection group, together with significant covariates and potential confounders. The incidence rates of hepatotoxicity, deaths, and deaths or new AIDS-defining events were expressed as the number of patients with at least one episode of the given event per 100 person-years of follow-up. For the incidence rates as for the survival analyses, data were censored at the time of the first episode of the given event. All analyses were performed using STATA 10.01 software .Multivariate analyses were adjusted on the following baseline covariates: gender (women 3 (interquartile range [IQR] 67-218). Fifteen women  had a CD4 count above 250 cells/mm3. No man had a CD4 count above 400 cells/mm3. Most characteristics were similar between HIV-monoinfected patients and those coinfected with an hepatitis virus. However, coinfected patients were older (p < 0.001), and had higher liver enzyme levels (p < 0.001). The median viral load was 2.47 × 107 IU/mL for HBV  and 928 000 IU/mL for HCV  in patients coinfected with the respective virus. The median time of follow-up was 23.9 months (IQR 17.2-24.0) in monoinfected patients and 24.0 months (IQR 20.0-24.0) in coinfected patients. Half the patients received zidovudine, lamivudine and nevirapine at baseline, and the other half received stavudine, lamivudine and nevirapine. Nevirapine was interrupted during follow-up in 16 monoinfected patients (12%) and 4 coinfected patients (11%) because of tuberculosis , adverse effects  and antiretroviral drug resistance .Of 169 HIV-1-infected patients enrolled between January 2001 and April 2003, 35 (21%) were coinfected with HBV (n = 14) or HCV (n = 21). The baseline patient's characteristics are shown in table p = 0.2). Two patients only (both were coinfected) had a grade 2 hepatotoxicity at baseline ; at week 2, hepatotoxicity dropped to grade 1 in one patient and remained at grade 2 in the other patient. No patient had a baseline grade 3 or 4 hepatotoxicity.A total of 1588 measures of liver enzymes were available. The number of measures per patient was similar in both infection groups , Karnofsky score  and liver enzymes levels . Of the 17 coinfected patients who experienced a grade ≥ 2 hepatotoxicity, 4 were positive for HBV  and 13 were positive for HCV .During follow-up, 39 patients experienced at least one episode of grade 2 or higher hepatotoxicity (22 monoinfected patients 16%] and 17 coinfected patients [49%]). The corresponding incidence rate was 12.4 per 100 person-years  in monoinfected patients and 54.0 per 100 person-years (CI 33.6-89.5) in coinfected patients. The Kaplan-Meier analysis showed the higher risk in coinfected patients  and liver enzymes levels . All 5 coinfected patients who experienced a grade 3 or 4 hepatotoxicity were HCV positive; their median viral load was 970 000 IU/mL (IQR 320 000-1.22 × 106).Only 12 patients (7%) experienced a grade 3 or 4 hepatotoxicity (7 monoinfected patients [5%] and 5 coinfected patients [14%]). The incidence rates were 3.6 (CI 1.7-7.6) and 9.9 per 100 person-years (CI 4.1-23.7) in monoinfected and coinfected groups, respectively. The risk tended to be higher in coinfected patients but the difference was not significant  and Karnofsky score .The higher risk for hepatotoxicity in coinfected patients was confirmed by the multivariate Poisson regression analysis . Only the male patient was coinfected . Nevirapine was replaced by a protease inhibitor in all three patients. The woman with a high CD4 cell count experienced simultaneously a grade 3 hepatotoxicity. Nevirapine was also replaced by a protease inhibitor in one other (monoinfected) patient with an isolated grade 3 hepatotoxicity. No patient had clinical hepatitis.Three patients developed a rash in the first four weeks (two women with baseline CD4 counts of 31 and 307 cells/mmp = 0.5). As shown in figure 3 in monoinfected patients and from 136 to 297 cells/mm3 in coinfected patients. The square root CD4 cell count increase was not significantly different between monoinfected and coinfected patients , age , HIV-1 viral load , hemoglobin level  and total lymphocyte count .The number of CD4 cell counts per patient was comparable between the monoinfected group  and the coinfected group . The proportion of patients with viral load below 400 copies/mL was 91% and 85% after 3 months and 91% and 78% after 24 months in monoinfected and coinfected patients, respectively , age , CD4 cell count  and hemoglobin level . In the monoinfected group, the deaths were related to advanced HIV disease (n = 5), poor general health (n = 5), multifocal tuberculosis, pulmonary infection, wasting, malaria, pancreatitis and hepatic carcinoma (n = 1 each). In the coinfected group, death was related to advanced HIV disease, poor general health and persistent fever of unknown origin (n = 1 each); the first two patients had taken traditional medicines.There were 16 deaths 12%) in monoinfected patients and 3 (9%) in those coinfected. The mortality rates were 7.2 (CI 4.4-11.7) and 4.8 (CI 1.4-15.0) per 100 person-years, respectively. The Kaplan-Meier curves did not differ significantly between the groups  and body mass index .Deaths or new AIDS-defining events occurred in 25 monoinfected patients (19%) and 5 coinfected patients (14%). The respective incidence rates were 10.6 (CI 7.0-16.1) and 6.7 per 100 person-years (CI 2.5-17.8). The Kaplan-Meier curves showed no significant difference between the groups . Indeed, transaminases elevations >5 times the ULN in patients receiving nevirapine has been associated with sex-dependant CD4 cell count [3 and in men with a CD4 count above 400 cells/mm3. In our study, only 13% of women and no man had a CD4 cell count above these respective cuttoffs. Most transaminases elevations in our patients were asymptomatic. Only 2% of patients had a rash following treatment initiation. In their analysis, Dieterich et al found a rash in 2.2% of patients [Overall, the hepatic tolerability of the nevirapine-based antiretroviral therapy was as expected. Seven percents of patients experienced liver enzymes elevations >5 times the ULN. In a comprehensive analysis of 17 randomized clinical trials of nevirapine, the rate was 10% after one year of treatment . A highe3 . Our lowpatients . On the The immunological response to the nevirapine-based antiretroviral therapy was comparable between coinfected and monoinfected patients, in accordance with several studies irrespective of the treatment prescribed ,14,18,20The virological response also was comparable between patients coinfected with an hepatitis virus and patients infected with HIV only. Increased risk for virological failure has been reported in HBV-coinfected patients in Taiwan . However3). Surprisingly, deaths or new AIDS-defining events were less common in coinfected patients. A possible explanation could be a close medical supervision of coinfected patients owing to frequent liver enzymes elevations. Higher rates of deaths  and/or new AIDS-defining events in patients coinfected with HBV or HCV has been reported by some [The clinical progression did not differ significantly between coinfected and monoinfected patients but treatment was initiated late  could have led to a selection bias  but this is concordant with the recommendations for nevirapine use. Classification biases for hepatitis status could also not be ruled out because seronegative blood samples were not tested for HBV DNA or HCV RNA. Higher rates of negative HBsAg or anti-HCV results in viraemic samples have been observed in immunocompromised HIV-infected patients ,4. In adThis study suggests that the nevirapine-based antiretroviral therapy could be used safely as first-line treatment in patients with low CD4 cell count in Africa despite frequent coinfections with HBV or HCV and infrequent testing of these infections. Although testing for HBV and HCV should be systematically performed before initiating antiretroviral therapy, transaminases elevations at baseline or during treatment should be a decisive argument for testing when hepatitis status is unknown.The authors declare that they have no competing interests.JBTM analysed the data and, with CL, wrote the first draft of the report. CL also designed the study and contributed to data collection, analysis and interpretation of data. HG and NM contributed to data analysis. CK, AB, LC, AC, SKS, JD, EMN and ED contributed to data collection, interpretation of data and drafting of manuscript. All authors read and approved the final draft of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/105/prepub


To investigate dynamic fluctuations of serum viral load and peripheral T-lymphocyte subpopulations of chronic hepatitis B patients and their correlation during entecavir therapy.Fifty-five patients received entecavir 0.5 mg/d therapy. Serum HBV DNA load was measured by Real-Time-PCR, and the levels of peripheral T-lymphocyte subpopulations by flow cytometry biweekly, every four weeks and every eight weeks during weeks 1–12, 13–24 and 24–48, respectively. Multilevel modelling was used to analyse the relationship between these variables.7 copies per milliliter. HBV viral load dropped sharply during the first two weeks. In 28 and 43 patients, the level became undetectable from week 24 and 48, respectively. Using pre-therapy level as the reference, a significant decrease in CD8+ T cells and increase in CD4+ T cells were found from week 12. Both parameters and CD4+/CD8+ ratio steadily improved throughout the 48 weeks. Multilevel analyses showed that the level of decrement of HBV DNA was associated with the increment of T-lymphocyte activities only in the later period (4–48 week). After 4 weeks of therapy, for each log10 scale decrement of HBV DNA, the percentage of CD4+ lymphocyte was increased by 0.49 and that of CD8+ decreased by 0.51.Of the 55 patients, all HBeAg positive and with detectable HBV DNA, the majority (81.8%) had serum levels of HBV DNA over 10T-lymphocyte subpopulations could be restored partially by entecavir treatment in patients with chronic hepatitis B concurrently with reduction of viremia. Patientnfection -5. The rnfection ,7.Most previous studies suggested that T cell failure is associated with a persistent high viral replication ,10. An eTwo major types of antiviral drugs are being used for the treatment of chronic HBV: drugs that directly interfere with virus replication and drugs that modulate the HBV-specific immune response. Nucleoside and nucleotide analogues, such as lamivudine, adefovir and entecavir, directly inhibit reverse transcriptase and thereby impair viral replication. Interferon (IFN) has marked immunomodulatory, but less pronounced direct antiviral, effects. Recently, novel antiviral agents have been evaluated for their therapeutic potential for treatment of chronic hepatitis B -18. Ente5 copies/mL, documented on at least two occasions, at least 3 months apart, during the 12 months before entry; aminotransferase levels higher than 2 times the upper normal limit for at least 12 months; and liver biopsy taken within 12 months before enrollment showing chronic hepatitis [Fifty-five consecutive CHB patients  who were recruited from the First Affiliated Hospital of Kunming Medical University, the Third Municipal People's Hospital of Kunming and the Yunnan General Hospital of The Chinese People's Armed Police Forces, between January 2006 and February 2007, were prospectively included into the study. All patients had presence of hepatitis B surface antigen (HBsAg) and HBeAg in the serum for at least 12 months; positive serum tests for HBV DNA with high viral load more than 10epatitis . PatientThis design used was single cohort with intervention study. The patients received entecavir at a daily dose of 0.5 mg for 48 weeks. Clinical, virological, biochemical and immunological parameters were assessed at baseline, then biweekly, every four weeks and every eight weeks during weeks 1–12, 13–24 and 24–48, respectively.Biochemical investigations including serum alanine aminotransferase (ALT), aspartate transaminase (AST) and total bilirubin (TBiL) were tested with routine automated techniques  . HBV markers  were measured at a virological laboratory with the use of ELISA (enzyme-linked immunosorbent assay) method . The experimental methods followed the guideline written in the reagent kit .Serum HBV DNA load was assessed with real-time fluorescent quantitative polymerase chain reaction method  using a Lightcycler PCR system  with a lowest limit of detection of approximately 1000 viral genome copies/mL. The handling procedures were performed in strict accordance with the reagent kit  package insert. The primer was provided in the kit, the reaction volume was 40 μL, and the reaction condition was 37°C for 5 min, 94°C for 1 min then 40 cycles as 95°C for 5 sec and 60°C for 30 sec.+, CD4+ and CD8+ T-cells found to be positive for the marker antigen in the total T cell population.The blood samples were analysed with a Multi-Q-Prep processor  and thereafter with Epics-XL flow cytometry (FCM) . Lymphocytes were analysed using a gate set on forward scatter versus side scatter, and a three-color flow cytometry to combination reagent of CD3, CD4, CD8. Anti-human monoclonal antibodies CD3-PE-CY5/CD4-FITC/CD8-PE were from Immunotech, Ltd, USA. For each sample, the detection was analyzed with the CELLQuest software . The results were expressed as the percentages of CD3t test or one-way ANOVA adjusted for multiple comparision, as appropriate. Categorical data were compared by a two-tailed chi-square test or Fisher's exact test. The aim in longitudinal analysis was to investigate the effects of covariates both on the overall level of the responses and on changes of the responses over time. Thus, multilevel regression with random effects among subjects was employed to assess the independent effects of variables on peripheral blood T-lymphocytes. The changes of peripheral blood T-lymphocyte subpopulations during treatment are dependent variables of the first level nested within the subject which is at the second level. On the other hand, the effects of age, sex, treatment time (week) and the changes of viral load and HBeAg expression were treated as fixed effects common to all subjects. Variables yielding a P value of ≤ 0.2 in the univariate analysis were included in the multivariate analysis, and the model refined by backward elimination guided by the change in log likelihood of successive models. A final P value of less than 0.05 was considered statistically significant. Computations were carried out with the aid of R softwere version 2.6.0, and glmmPQL function [Descriptive statistics were used to examine the age, sex, serum level of HBV viral load, T-lymphocyte subpopulation, HBeAg status, ALT, AST and total bilirubin. Continuous data were presented as means ± standard deviations and were compared by a two-tailed Student function . The lonfunction .7 copies per milliliter. Most patients  were moderate chronic hepatitis with moderate activity and mild or moderate fibrosis  and 15% (8/55) patients were severe chronic hepatitis with marked activity and marked fibrosis and developing cirrhosis . No patients were with previous hepatic decompensation. No patient dropped out, and all completed the 48-week therapy.Fifty-five consecutive patients were enrolled in this study. Characteristics of the patients are summarized in Table 10 copies/mL during the first two weeks, with a highly significant reduction (p < 0.0001) at week 2 and thereafter, as compared to those at baseline  and 9/55 (16%), and 4/55 (7.3%) and 9/55 (14.5%), respectively. Highly significantly decreasing serum aminotransferase and total bilirubin  (p < 0.0001) occurred during the first 2 weeks of the study  was observed in 11% and 15%, respectively. There was no evidence of drug resistance or adverse effect in CHB patients treated for up to 48 weeks.HBV DNA levels declined sharply by around 3 log10 copies/mL, at weeks 12–16, could significant increases in the CD3+ cells and in the CD4+/CD8+ ratio be observed . All T-lymphocyte parameters continued to improve and achieved highly significant difference from the baseline (p < 0.0001) by the 48th week. The increase in the CD4+/CD8+ ratio was a combined result of an increase in the number of CD4+ cells and a decrease in CD8+ cells. The magnitude of increase in CD3+, CD4+ cells and CD4+/CD8+ ratio and decrease in CD8+ cells was, respectively, 21.6%, 30.2%, 77.2% and 27.6% of pre-therapy levels at the end of study.The effect of the entecavir therapy on alterations of the T cell subpopulations are shown in Table +, CD4+ and CD8+ cells, which are the dependent variables, respectively. The fixed effects part consists of coefficient (β) and standard error (SE) of the effect of each predicting variable which act equally to all subjects, while the random effects reflect variation of the baseline level outcome variable among the subjects. Interaction terms between log HBV DNA and period are significant for all T-lymphocyte parameters, indicating that effect of HBV DNA on these parameters after the first 2 week of therapy was significantly different from that in the initial period. The main effect of HBV DNA was not significant indicating no relationship during the initial period. The signs of the interaction term were negative for CD3+ and CD4+, and positive for CD8+ indicating that in the later period, the level of decrement of HBV DNA was associated with the increment of T-lymphocyte activities. These effects have already been adjusted for time or "week" as a continuous variable in the models. After 4 weeks of therapy, for each log10 scale decrement of HBV DNA, the percentage of CD4+ lymphocyte was increased by 0.49 and that of CD8+ decreased by 0.51. The relationship before week 4 was not statistically significant.In Table This study demonstrated that entecavir results in an early rapid viral load reduction and continued virologic and biochemical improvement and HBeAg seroconversion through 48 weeks of treatment in CHB patients. Also a decreased viral load in serum (an inhibition of virus replication) during antiviral treatment was accompanied by a delayed restoration of peripheral T cell subpopulations. Although the relationship was not significant during the initial period (≤ 2 week), the level of decrement of HBV DNA was associated with an increment of T-lymphocyte activities in the later period (4–48 week). These responses were sustained up to the end of the observation period.10 copies/mL for entecavir and 4.84 log10 copies/mL for adefovir. PCR undetectability (HBV DNA < 300 copies/mL) was reached in 45% of entecavir treated patients vs 13% of those receiving adefovir [Our data showed that entecavir treatment led to a rapid drop of viremia in all patients and a slower HBeAg/HBeAb seroconversion. Most patients experienced improvements in serum ALT values after initiation of entecavir therapy. The efficacy of entecavir appears to result from its potent suppression of HBV replication, and this rapid viral suppression occurred especially during the first two weeks. The virologic and biochemical efficacy of entecavir continued through 48 weeks. The improvement of virologic and biochemical parameters found in this study was in agreement with recent studies of patients with chronic hepatitis B -22. Globadefovir . Signifiadefovir . A similadefovir -22. The adefovir .+/CD8+ ratio, owing to a relative decrease in the number of CD8+ cells and increase in the number of CD4+ lymphocytes, and an increase in the number of CD3+ cells, concomitantly with a quantitative reduction in viral replication. The benefit of entecavir on T cell profile was not immediately significant at the time of rapid reduction of viral load during the first 2 weeks. T cell subpopulations restoration had a tendency to increase or accumulate gradually after initiation of treatment. Although the relationship was not significant during the initial period (≤ 2 week), the level of decrement of HBV DNA was associated with the increment of T-lymphocyte activities in the later period (4–48 week). Our results also showed that the magnitude of restoration of these parameters was maintained up to the end of the study. The independent effect of viral load on peripheral T-lymphocyte subpopulations profile found in this study was partly in accordance with previous studies. Stoop et al [et al [+ T-cells, but no increase in HBV-specific CD8+ T-cells. Kondo et al [et al [+ and CD8+ cells can be restored by lamivudine treatment in CHB patients concurrently with reduction of viremia, but the restoration of HBV-specific T-cell reactivity is only transient. In their study, after an initial increase of frequency of functionally efficient T cells (first 5 months of therapy), peripheral blood CD4+ responses declined to pretreatment levels and this degree of reactivity was maintained after therapy discontinuation, throughout the follow-up period. In addition, this transient restoration of HBV-specific T-cell was also observed by Mizukoshi et al [To our knowledge, no data have been reported concerning the synchronous effects of entecavir on the serial measurement of peripheral blood lymphocyte subpopulations and serum HBV DNA concentrations in patients with chronic hepatitis B. During entecavir-therapy, we detected a shift in the CD4op et al  and Lau l [et al  reporteddo et al  suggestel [et al -13 that hi et al , who sughi et al -22.10 copies/mL and thereafter during entecavir-therapy. Furthermore, the decline of viremia viral load was clearly related with time (week). Therefore, these observations indicate that the restoration of impaired T cell profile may be actually directly related with the reduction of viral load resulting from inhibition of viral replication. Taken together with recent reports of previous studies [Our results reveal that T cell failure was significantly associated with viral replication level. The stronger independent predictive effect of time (week) on T cell improvement might be explained as the effect of treatment course. As a matter of fact, the restoration of peripheral T cell profile observed in our study is attributed to the effect of treatment with entecavir, not time (week) itself. However, entecavir reacts by directly inhibiting HBV DNA replication, not by modulating the HBV-specific immune response -22. Inde studies -13,28-31+ and CD4+ T cells, accompanied with a decrease of CD8+ lymphocytes and a restoration of CD4+/CD8+ ratio to a near normal profile are evident and also maintained for the whole study period. As the antiviral action of entecavir is directly on the virus, with no known direct effect on T cell subpopulations [This study suggests that entecavir can effectively reduce HBV viral load resulting from inhibition of virus replication and also maintain a low level for at least 48 weeks. As a consequence, an increase of CD3ulations -22, our The strength of this study lies in the large sample size and the measurements of T-lymphocyte subpopulations using modern advanced flow cytometry technology and viral load with quantitative Real-Time-PCR method. A limitation of this study is that the specific T-lymphocyte subpopulations and liver-derived T-lymphocytes as well as their functionality were not explored concurrently. Another one is that long-term effect of entecavir was not known from this data set. Although the strong correlation between viral load and T-lymphocyte subpopulations is illustrated, further studies are needed to confirm the T cell responsiveness during prolonged therapy and after withdrawal of entecavir, and to elucidate the complex interactions among them.Our results describe a dynamic relationship between T cell subpopulations and serum viral load that could potentially be used for a better design of HBV treatment in chronic hepatitis B patients. Inhibition of viral replication with agents such as novel nucleotide analogues may enhance the likelihood that therapeutic stimulation of the antiviral T cell reaponses will induce long-lasting viral suppression and HBV antigen seroconversion, ultimately leading to recovery from disease. Further clinical studies are needed to explore this possibility in persistent HBV-infected patients.In this study, profound effects on cellular distribution and viral replication were found during entecavir-therapy. The inhibition of virus replication induced by treatment with entecavir results in a partial restoration and improvement of the impaired cellular immune response. The restoration was clearly associated with the decreases in serum viral load, indicating a close correlation between viral load and T-lymphocyte subpopulations.ALT: alanine aminotransferase; AST: aspartate transaminase; HBV: hepatitis B virus; HBV DNA: hepatitis B virus DNA; HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B envelope antigen; CHB: chronic hepatitis B; ELISA: enzyme-linked immunosorbent assay; Real-Time-PCR: real-time fluorescent quantitative polymerase chain reaction.The authors declare that they have no competing interests.JY, HS, AG and VC conceptualized the study. JY and staff of the research group assisted with the data collection. JY was responsible for data management and data analysis. JY, HS, AG and VC were responsible for interpretation of data. HS, AG, VC and LZ provided advice and review. JY, HS, AG and VC collaboratively wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: