T values, and clinical characteristics in patients with suspected TB who provided expectorated (n = 438) or induced sputum (n = 128), tracheal aspirates (n = 71), bronchoalveolar lavage fluid (n = 152), pleural fluid (n = 76), cerebral spinal fluid , pericardial fluid (n = 131), or urine (n = 173) specimens. Median bacterial load (TTP in days) was the strongest associate of MTB/RIF positivity in each fluid. TTP correlated with CT values in pulmonary specimens but not extrapulmonary specimens . Inhibition affected a greater proportion of pulmonary specimens than extrapulmonary specimens (IPC CT > 34: 6% (47/731) versus 1% . Pulmonary specimens had greater load than extrapulmonary specimens . HIV-infection was associated with a decreased likelihood of MTB/RIF-positivity in pulmonary specimens but an increased likelihood in extrapulmonary specimens. Mycobacterial load, which displays significant variation across different body compartments, is the main determinant of MTB/RIF-positivity rather than PCR inhibition. MTB/RIF CT is a poor surrogate of load in extrapulmonary specimens.The determinants of Xpert MTB/RIF sensitivity, a widely used PCR test for the diagnosis of tuberculosis (TB) are poorly understood. We compared culture time-to-positivity , MTB/RIF TB-specific and internal positive control (IPC)-specific C Tuberculosis (TB) is a leading cause of morbidity and mortality, and accurate and rapid diagnostic tests for TB are key to limiting the spread of the epidemic14Bacillus globigii spores and a primer pair specific for the DNA in these sporesXpert MTB/RIF  is an automated real-time PCR system that simultaneously detects TB and resistance to rifampicin. The test has excellent accuracy when performed on sputum8Information regarding Xpert MTB/RIF's performance on non-sputum specimens is emerging513141516171819202122232425While the relationship between sputum bacillary load  has been previously characterised2728293032T) values] in these fluids. We evaluated the degree of MTB/RIF PCR inhibition in each fluid, and how this modified the relationship between MTB/RIF and culture results.In this study, we first compared mycobacterial load in different fluids from different cohorts of patients with TB recruited from similar settings in South Africa . We identified clinical factors, including HIV co-infection and CD4 count, and specimen characteristics that may modulate liquid culture time-to-positivity (TTP) and MTB/RIF quantitative information  ; Table 2Scatter plots of TTP in MTB/RIF-positive and –negative culture specimens of different types are shown in Median TTPs (IQR) amongst culture-positive patients were shorter for expectorated sputum and induced sputum in HIV-uninfected compared to -infected patients . When data were pooled, patients that were HIV-uninfected had a similar TTP to those that were HIV-infected .HIV-infected patients with a CD4 count ≤200 cells/μl had a longer median TTP versus those with a CD4 count >200 cells/μl for expectorated sputum [14  vs. 8  days (p = 0.0027)] and CSF [26  vs. 18.5  days (p = 0.0110)], but not for induced sputum [24  vs. 16.5  days (p = 0.1071)] or pericardial fluid [21  vs. 24  days (p = 0.4755)]. When data from HIV-infected patients were pooled, patients with a CD4 count ≤200 cells/μl had a longer median TTP compared to those with a CD4 count >200 cells/μl for pulmonary specimens [15  vs. 9.5  days (p = 0.0052)], but not for extrapulmonary specimens [22  vs. 20  days (p = 0.2241)].Multivariable linear regression analyses of culture-positive patients showed the following clinical and demographic factors to be associated with increased TTP: younger age (p = 0.035) and HIV-infection (p = 0.047) for induced sputum , previouT values; a smaller CT value indicates greater load) (IQR) were compared across fluids, those from pleural fluid, CSF, pericardial fluid, and urine were greater than expectrated sputum  vs. 29.4 (26.4–32.2); p < 0.0001].When median MTB/RIF-generated cycle threshold values  amongst MTB/RIF-positive patients were similar in HIV-uninfected patients versus -infected patients for expectorated sputum [21.37 (17.71–26.71) vs. 25.09 ] or induced sputum [24.41 (21.87–29.11) vs. 23.34 ], and no differences were detected when pooled pulmonary or extrapulmonary data were used .Median CT values versus those with a CD4 count >200 cells/μl for expectorated sputum [29.81  vs. 20.60 ], but not for pericardial fluid [29.15  vs. 28.10 ] or urine [29.53  vs. 31.01 ]. Patients with a CD4 count ≤200 cells/μl had higher median CT values compared to those with a CD4 count >200 cells/μl for pulmonary specimens [28.68  vs. 20.70 ] but not for extrapulmonary specimens [29.47  vs. 29.51 ].HIV-infected patients with a CD4 count ≤200 cells/μl had higher CMultivariable logistic regression analyses showed MTB/RIF-positivity to be associated (p ≤ 0.100) with TTP for expectorated sputum  are shown in T values for expectorated sputum differed to those for induced sputum, tracheal aspirates, BALF, pleural fluid, and CSF, and were similar for pulmonary specimens and extrapulmonary specimens. The proportion of MTB/RIF results with an IPC CT value >34, which have been shown to be due to inhibition in sputumT value >34 for pulmonary specimens and extrapulmonary specimens was 6% (47/731) and 1% , respectively. Median (IQR) IPC CT values were similar for comparisons between MTB/RIF-positive and –negative culture-positive specimens of each type, except for CSF . MTB/RIF-negative, culture-positive pulmonary specimens and extrapulmonary specimens had median IPC CT values of 28.45  and 27.30 , respectively (p = 0.0048).Scatter plots of IPC CT values for expectorated sputum , female gender was associated with decreased internal control Csted see , howeversted see  for eachT values, which may be modulated by PCR inhibition, were compared, similar Spearman correlation coefficients  of 0.501, 0.623 (p = 0.5252), 0.100 (p = 0.5287), −0.051 (p = 0.5287), and 0.199 (p = 0.1211) for expectorated sputum, induced sputum, pleural fluid, CSF, and pericardial fluid, respectively were obtained. When data for pulmonary specimens were pooled , however, there was no significant correlation amongst extrapulmonary specimens , and the correlation observed amongst pulmonary specimens was stronger (p = 0.030). CT values correlated less strongly with low or high levels of bacterial load, rather than due to any intrinsic properties of extrapulmonary specimens. For example, although CT values and TTP exhibited a significant correlation overall for pulmonary specimens, this was not present amongst specimens with a TTP in the bottom  or top tertile , but was amongst those in the middle tertile .When the strength of the correlation between culture TTPs and Ce pooled , TTPs anT values, and MTB/RIF inhibition in specimens from different body compartments. Briefly, our key findings are: (i) compared to expectorated sputum, MTB/RIF is inhibited more in induced sputum, tracheal aspirates, and BALF, but less in pleural fluid; (ii) “false-negative” MTB/RIF results  from CSF displayed a greater inhibition compared to “true-positive” results, and pulmonary specimens inhibited MTB/RIF more than extrapulmonary specimens; (iii) CT values correlate with TTP in pulmonary specimens but not in extrapulmonary specimens, suggesting the assay to be unsuitable for estimation of mycobacterial load amongst patients with extrapulmonary TB; (iv) TTP is the strongest correlate of MTB/RIF-positivity in both pulmonary specimens and extrapulmonary specimens, even after adjusting for inhibition; and (v) extrapulmonary specimens are more paucibacillary than pulmonary specimens and, of the pulmonary specimens, expectorated sputum had the highest bacillary load.This study is the first to compare mycobacterial load using culture TTP, MTB/RIF-generated CT value >34We found pulmonary specimens to have a greater proportion of MTB/RIF results with evidence of inhibition [IPC CIn our study, we found “false-negative” MTB/RIF results to display more inhibition on CSF than those that are “true-positive”, suggesting that this fluid contains material that interferes significantly with the PCR and thus may be a cause of false-negative results. This is the first description of MTB/RIF inhibition in extrapulmonary specimens. Interestingly, we have shown in a separate studySeveral studies have detailed the performance of MTB/RIF on extrapulmonary specimens and pulmonary specimens other than sputum51314151617181920212223242526In pulmonary specimens, HIV-infection was associated with a decreased likelihood of a positive MTB/RIF result, however, in extrapulmonary specimens, HIV-infection was associated with an increased likelihood of MTB/RIF-posivity. This is reflective of the lower bacillary load seen in the lungs of HIV-coninfected patients with pulmonary TB (due to the lower frequency of caviation in these patients) compared to those who are HIV-uninfected. In contrast, patients who are HIV-infected displayed a higher TB bacillary load in specimens from extrapulmonary sites than those who were HIV-uninfected, and thus those who are HIV-infected are more likely to be MTB/RIF-positive for EPTB. Although EPTB is more frequent in HIV-infected patients, their extrapulmonary bacillary load is lower than HIV-infected patients with pulmonary TB. This means that EPTB specimens with a concetration of bacilli below the limit of detection of MTB/RIF will occur more frequently, and that patients with suspected TB who have a negative MTB/RIF result should still be investigated further. A further rammification of the low load seen in extrapulmonary specimens is that in fluids such as pleural fluid or pericardial fluid a biomarker-based approach using a molecule such as interferon-γ might be superiorT values to correlate significantly with culture time-to-positivity in pulmonary specimens. Such a conclusion is important because, for pulmonary TB, sputum bacillary load at diagnosis is one of the strongest baseline predictors of long-term outcome464748T values-TTP correlation deteriorating at low levels of bacterial load. While a direct association between baseline MTB/RIF CT values and clinic outcome has not yet been demonstrated, it appears that MTB/RIF would not be useful for such a purpose amongst patients with extrapulmonary TB, or for a meaningful estimation of disease severity as approximated by bacterial load.As has been documented by others2328303444This study has limitations. Although this is the first study to report on MTB/RIF inhibition in fluids other than sputum, we did not capture data on specimen-specific factors such as viscosity, appearance, or salt concentration, which may interefere with MTB/RIF. Although shown to be useful by us and other2541287In summary, this study has demonstrated that low mycobacillary load in extrapulmonary specimens is, rather than inhibition, primarily responsible for the diminished sensitivity of MTB/RIF in these specimens compared to those from the pulmonary system. While “false-negative” CSF displayed more inhibition than “true-positive” specimens, pulmonary specimens displayed the most inhibition overall, suggesting that MTB/RIF quantitative information might not be useful in a significant minority of patients with suspected pulmonary TB. Furthermore, the quantitative information generated by MTB/RIF from extrapulmonary specimens does not correlate with bacterial load, and is unlikely to be useful. Future studies on the exact clinical and specimen-specific determinants of MTB/RIF inhibition are important, as well additional specimen preparation steps that may reduce inhibition, especially if MTB/RIF quantitative information will used for patient management.Online supplement


The primary objective of this study was to estimate the incidence of treatment failure (TF) to protease inhibitor monotherapies (PI/r-MT) with lopinavir/ritonavir (LPV/r) or darunavir/ritonavir (DRV/r).A multicenter cohort of HIV-infected patients with viral load (VL) ≤50 copies/mL, who underwent a switch from any triple combination therapy to PI/r-MT with either LPV/r or DRV/r.VL was assessed in each center according to local procedures. Residual viremia was defined by any HIV-RNA value detectable below 50 copies/mL by a Real-Time PCR method. Standard survival analysis was used to estimate the rate of TF . A multivariable Cox regression analysis with automatic stepwise procedures was used to identify factors independently associated with TF among nadir and baseline CD4+ counts, residual viremia, time spent with <50 HIV-RNA copies/mL before switch, history of virological failure, HCV co-infection, being on a PI/r and hemoglobin concentrations at baseline.Six hundred ninety patients fulfilled the inclusion criteria and were included in this analysis. Their median follow-up was 20 (10–37) months. By month 36, TF occurred in 176  patients. Only CD4+ nadir counts  and residual viremia (aHR = 1.48 [95% CI: 1.01–2.17] vs. undetectable VL) were independently associated to TF.Residual viremia and nadir CD4+ counts <100 cells/μL should be regarded as the main factors to be taken into account before considering switching to a PI/r-MT. Ritonavir-boosted-PI based monotherapy (PI/r-MT) is considered by Italian guidelines a possible alternative switch strategy to standard combination antiretroviral therapy (cART) in case of drug toxicity . Indeed,PI/r-MT has been tested in different randomized studies, showing that this switching strategy is safe in the large majority of subjects with undetectable viral load. These studies have also demonstrated that in case of failure, no PI-related resistance mutations were selected and re-introduction of triple therapy was successful, without loss of subsequent drug options –25.The largest study conducted on PI/r-MT (PIVOT) showed that this strategy, with regular viral load monitoring and prompt reintroduction of combination treatment in case of viral rebound, preserved future treatment options and did not change overall clinical outcomes or frequency of toxic effects .Different studies were able to identify a number of factors associated with failure to PI/r-MT, including nadir and baseline CD4+ count, duration of viral suppression, previous failure to ART, HCV co-infection, PI in the baseline cART, residual viremia levels at time of switch, hemoglobin levels, age, VL at cART initiation, gender, mode of HIV transmission –37. In aThe primary objective of the current analysis was to estimate the incidence of virological and treatment failure of PI/r-based monotherapies with LPV/r or DRV/r in an unselected population with undetectable viral load achieved using a previous triple cART. Other objectives were to identify predictors of treatment failure in virologically suppressed patients undergoing simplification of cART with MT with PI/r and, based on the identified single predictors, to develop and refine a prediction score able to reliably anticipate failure to PI/r-MT.This is a prospective study of a cohort of people who was followed-up prospectively at each of the clinical sites. The database for the analysis has been put together retrospectively using some specific criteria by including only patients who underwent a switch from any triple combination therapy to PI/r-based MT with either LPV/r or DRV/r and with a viral load suppressed to a level ≤50 copies/mLwww.icona.org]. Sites participating to the Icona Foundation Study could contribute by including additional patients (who formed the hereafter named “Mono PI/r database”) not enrolled in ICONA who satisfied the inclusion criteria for this protocol. Once included in the protocol, these additional participants have been followed-up using the same standardized monitoring procedures and data collection as that used for patients in the ICONA Foundation Study, until the date in which the database has been frozen for the analysis .Most of the included patients are currently under follow-up in the ICONA Foundation Study cohort (case-cohort nested within ICONA). ICONA Foundation Study (ICONA) is a multi-centre prospective observational study of HIV-1-infected patients, which was set up in 1997. Eligible patients are those starting cART when they are naive to antiretrovirals, regardless of the reason for which they had never been previously treated and of the stage of their disease. The ICONA Foundation study has been approved by IRB of all the participating centers; sensitive data from patients are seen only in aggregate form. All patients sign a consent form to participate in ICONA, in accordance with the ethical standards of the committee on human experimentation and the Helsinki Declaration (1983 revision). Demographic, clinical and laboratory data and information on therapy are collected for all participants and recorded using electronic data collection [The main inclusion criteria were: i) having achieved a viral load ≤50 copies/mL while receiving triple combination therapy (cART) over follow-up with at least 2 consecutive measures below this threshold. The date of the second viral load was defined as baseline; the second condition in order to be included in this analysis was to have experienced a switch to monotherapy with LPV/r or DRV/r after baseline while current viral load was still ≤50 copies/mL. If a person had more than one episode of switch to one of the considered PI/r only the first ever occurring episode was included for this analysis. The date of switching after the first of two consecutive viral loads ≤50 copies/mL was defined as the baseline for this analysis.Viral load was assessed in each center according to local procedures. A subset of individuals was tested by a Real-Time PCR method: in these cases, undetectable viremia could be defined in the presence of a “target not detected” result and residual viremia by any HIV-RNA value detectable below 50 copies/mL by a Real-Time PCR method. The reason why the remaining individuals were not tested by a Real-Time PCR method is that this assay was not available at the time of PI/r-MT start.Standard survival analysis was used to estimate the rate of virological failure . We used 50 copies/mL  and 200 copies/mL  as thresholds. A composite endpoint to define treatment failure (TF) was also used to study the durability of the monotherapy: in this analysis an event was defined by virological failure (defined as above), or by modification of monotherapy. TF was the primary outcome of this analysis.Kaplan-Meier curves have been derived to estimate the time to event, with corresponding 95% confidence interval (CI), and thus to evaluate the durability of PI/r-MT. Unadjusted and adjusted relative hazards (RH) of the different endpoints have been calculated from fitting Cox regression models and tabulated.Analyses were repeated after stratifying according to whether patients had been switched to a DRV/r- or LPV/r- based mono-therapy. A similar analysis has been performed to test for heterogeneity between the two sources of enrolment (“Icona patients” vs. other patients enrolled at Icona sites but not in Icona). Results of these additional analyses are available as supplemental online material only.Besides the investigation of the predictive values of individual factors, we also aimed at constructing a score vector linear combination of these variables for prediction purposes. Before running the analyses, all factors previously identified as a predictor of mono PI/r-based therapy even in a single study (based on literature data) have been considered for inclusion in the score. These include the eight main candidates listed below:CD4 count at the date of switch (binary variable: ≤200 cells/μL vs. >200 cells/ μL)CD4 count nadir (binary variable: ≤100 cells/ μL vs. >100 cells/ μL)Duration of time with a VL ≤50 copies/mL before switching to monotherapy -Evidence of previous virological failure on ART Co-infection with HCV (binary yes/no)Being on a PI/r-including regimen (binary yes/no) at the date of switch10 higher)Haemoglobin  or residual viremia .HCV-RNA was available only in a subset of individuals and was not used to classify people for HCV status.Other important factors  could not be included as predictors, because they were not collected or were available only for a small subset of patients. We did however describe resistance (at baseline and/or at failure) in a subset of patients with available genotypic resistance testing (GRT) results. Baseline resistance data are result of historical tests performed at the time of previous virological failures using population sequencing in plasma. Patients with HIV-RNA >50 copies/mL confirmed in two consecutive samples or with HIV-RNA >200 copies/mL during follow-up were eligible for studying PI resistance by GRT.The construction of the score followed the steps described below using a Cox regression analysis framework:Best subset of the predictors out of the 8 a priori identified was chosen using automatic stepwise procedures .The model which was consistently chosen by all three procedures was our final chosen best model. In addition, internal 5-fold cross validation in Icona (training set) and external validation  set was independently used to develop the score. This approach identified the same predictors selected by the steps described above (data not shown).The Results of the full model including all eight factors have also been reported.Cox regression prediction estimates of treatment failure for exact values of CD4+ count nadir were also calculated.All analyses were performed using SAS version 9.4 .Six hundred ninety patients fulfilled inclusion criteria and were included in this analysis. Their median  age, nadir CD4+ count, current CD4+ count and duration of virological suppression below 50 copies/mL were 44  years, 359  cells/μL, 636  cells/μL and 44  months, respectively. Two hundred eight (30%) were female, 63 (9%) had a previous AIDS diagnosis, 95 (14%) previously failed to a PI, 537 (78%) were receiving a PI/r, 168 (24%) were co-infected with HCV and 323 (59%) out of 543 evaluable for residual viremia had undetectable viral load; further baseline characteristics are detailed in The median follow-up was 20  months. By month 36, treatment failure occurred in 176  patients  with theThe number of participants who experienced virological failure with viral load >50 and >200 copies/mL by 36 months was 71  and 31 , respectively  but not nadir CD4+ cell count and residual viremia. This bivariable model was the best choice also when using the “best subset” selection, with adjusted relative hazards (RH) of 2.09  comparing CD4+ nadir counts of ≤100 and >100 cells/μL and 1.75  comparing residual viremia vs. undetectable viral load.Results were similar when the analysis was repeated using the only Icona or the Mono PI/r database (data shown in supplementary tables only).In the present study the number of participants who experienced virological failure by 36 months was between 6.5% and 13.6%, depending on the threshold used to define this event; by the same time interval from baseline, 30% experienced treatment failure, thus highlighting that in a relevant number of patients the PI/r-MT was interrupted because of toxicity or convenience. Our data do not allow us to analyze in depth the causes of MT discontinuation and thus to speculate further on this issue.We identified in a large number of patients a nadir CD4+ count of ≤100 cells/μL and the detection of residual viremia  at the start of MT as the only predictors of failure to PI/r-MT. The results of this study suggest that these two predictors contribute both a fair amount of risk of failure although not in a synergistic manner .Although we were unable to define a true score for the outcome of PI/r-MT (as we identified only two predictors of response), we were able to provide robust independent estimates of failure for different nadir CD4+ counts, in the presence or in the absence of residual viremia at the time of switch to this strategy. Thus, our results are relevant as they document the prognosis of individuals treated with a strategy commonly used in clinical practice but with a limited support from available data. In addition, as estimates of failure are calculated using a large data set from a group of unselected individuals treated in every-day clinical practice, these estimates reflect more faithfully than those obtained from clinical trials the trends in the average HIV-infected individual in care in Italy. Finally, our results confirm those from previous studies, showing that PI-resistant HIV variants are almost never selected in patients experiencing virological failure.In patients treated with LPV/r-MT, duration of previous viral suppression was a main predictor for long-term success of this strategy , 30, 38;Beyond CD4+ nadir counts, recognized risk factors for failure in patients who received DRV/r-MT are the baseline presence of residual viremia, higher HIV-DNA load, shorter time of antiretroviral treatment before MT, as well as an adherence <100% during MT –34, 36. HIV-DNA has been associated to a higher risk of failing MT , 33. In One limitation of our study is that we could not investigate baseline HIV-DNA load as a potential predictor of failure to a PI/r-MT, because this information was available for very few patients. Nevertheless, our finding that nadir CD4+ and the presence of baseline residual viremia predicts failure to PI/r-MT is in keeping with similar findings , 32, 37 The main limitation of this analysis is the observational context, as we cannot exclude that individuals undergoing MT have been selected as those with a better immune-virological response and a good tolerability to ART. Although, we tried to correct for all measurable confounders, we cannot exclude also that some unknown or unmeasured confounding still remained. A further limit is the absence of data on adherence; however, we believe that, under a clinical perspective, information on the role of nadir CD4+ cell count and residual viremia on the risk of failure to a PI/r-MT are truly important in selecting patients for this strategy, independent of adherence to therapy. As CSF samples were not prospectively collected we were not able to identify failures that may occur only in the CNS compartment. Finally, the follow-up of patients in the present study might have been too short to observe failures, in particular when virological failure occur at CNS, as recently highlighted by Kahlert and coll. .In conclusion, in our large clinical setting, a PI/r-MT simplification strategy showed a risk of treatment failure consistent with that observed in clinical trials. Residual viremia and a CD4+ count nadir <100 cells/μL were the only predictors of failure to this strategy and should be thus considered the main factors to be taken into account before considering switching a virologically suppressed patient to a PI/r-MT.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.


The prevalence of HIV-1 resistance to antiretroviral therapies (ART) has declined in high-income countries over recent years, but drug resistance remains a substantial concern in many low and middle-income countries. The Q151M and T69 insertion (T69i) resistance mutations in the viral reverse transcriptase gene can reduce susceptibility to all nucleoside/tide analogue reverse transcriptase inhibitors, motivating the present study to investigate the risk factors and outcomes associated with these mutations.We considered all data in the UK HIV Drug Resistance Database for blood samples obtained in the period 1997–2014. Where available, treatment history and patient outcomes were obtained through linkage to the UK Collaborative HIV Cohort study. A matched case–control approach was used to assess risk factors associated with the appearance of each of the mutations in ART-experienced patients, and survival analysis was used to investigate factors associated with viral suppression. A further analysis using matched controls was performed to investigate the impact of each mutation on survival.A total of 180 patients with Q151M mutation and 85 with T69i mutation were identified, almost entirely from before 2006. Occurrence of both the Q151M and T69i mutations was strongly associated with cumulative period of virological failure while on ART, and for Q151M there was a particular positive association with use of stavudine and negative association with use of boosted-protease inhibitors. Subsequent viral suppression was negatively associated with viral load at sequencing for both mutations, and for Q151M we found a negative association with didanosine use but a positive association with boosted-protease inhibitor use. The results obtained in these analyses were also consistent with potentially large associations with other drugs. Analyses were inconclusive regarding associations between the mutations and mortality, but mortality was high for patients with low CD4 at detection.The Q151M and T69i resistance mutations are now very rare in the UK. Our results suggest that good outcomes are possible for people with these mutations. However, in this historic sample, viral load and CD4 at detection were important factors in determining prognosis.The online version of this article (10.1186/s12981-018-0198-7) contains supplementary material, which is available to authorized users. Highly potent and effective antiretroviral therapies (ART) to treat HIV-1 infection are now available and although drug resistance was a considerable problem in the early years of ART use, its impact has now declined in high-income countries. In the UK HIV Drug Resistance Database (UK-HDRD) only about 30% of treated patients receiving resistance tests following virological failure in 2014 showed any drug resistance, compared with 72% in 2002 –7. This HIV mutations that confer resistance to multiple drugs, and hence have the potential to severely limit treatment options, are of particular concern. The T69 insertion (T69i) and associated mutations in reverse transcriptase confer high level resistance to all nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) . LikewisThe T69i mutation has been linked to DDI use  whilst fThis paper reports changes in the prevalence of multi-drug resistance mutations Q151M and T69i in the UK-HDRD over time and evaluates outcomes in affected patients. Risk factors for the occurrence of the mutations and predictors of subsequent viral suppression are investigated, with the aim of helping to inform clinical management in regions in which the development of drug resistance is an increasing concern.We considered all available data in the UK-HDRD for blood samples obtained in the period 1997–2014. Few data are available before this period, as resistance testing was not widely available, with no recorded observations of either Q151M or T69i mutations. The prevalence of each resistance mutation studied was assessed in relation to calendar time stratified by whether patients were recorded as ART-naïve or -experienced in the UK-HDRD. Where possible, clinical data were obtained through linkage to the UK Collaborative HIV Cohort (UK CHIC) study . PatientThe identification of drug resistance mutations was based on output from the Stanford University HIV Drug Resistance Database program. Following established convention (and software output) , the terA Bayesian approach to statistical analysis was used throughout, with models implemented in the Stan probabilistic programming language  using thMatched case–control analyses were conducted in order to investigate factors associated with the occurrence of each of the mutations studied. These analyses only included patients for whom resistance test data could be matched to clinical records in the UK CHIC study. ART-experienced patients were defined as ‘cases’ at time of blood sampling for the first observation of the relevant mutation and were matched in a 1:10 ratio to ‘controls’, who were randomly sampled (without replacement) from a subset of patients with at least one resistance test available for whom the relevant mutation had never been detected. Matching in each instance was conditional on the control patient having first initiated ART within 6 months  of the case patient doing so, and current and historic treatment variables for both case and control patients were defined with respect to the time of blood sampling for the resistance test in the case patient. Multivariable conditional logistic regression analyses were conducted accounting for the matched case–control groups. This form of analysis was chosen so that key factors associated with the development of the resistance mutations could be investigated whilst controlling for the evolution of ART drug combinations and treatment strategies over time. The complexity of ART histories for each patient and the limited numbers of cases available for analysis precluded more complex modelling of the probability of the development of each resistance mutation conditional on full details of ART history.Details of variable selection for these models are provided in Additional file Multivariable Cox regression was used to investigate the factors associated with successful viral suppression following detection of either the Q151M or T69i mutation. Change to ART regimen following detection of a resistance mutation was taken as the zero time point for these analyses, and the survival outcome was defined as the time to the first of two consecutive undetectable VL observations (considering observations < 200 copies/mL to be ‘undetectable’). Patients in whom there was no change to ART regimen recorded were not included in these analyses. Baseline CD4 cell counts and VL were defined as the most recent observation recorded within 6 months prior to the start of the new ART regimen, and outcomes were censored at any subsequent change to ART regimen.10(VL) were included as predictors, alongside treatment-naïve or experienced status, sex, patient age and variables representing individual drugs received by ≥ 10% patients. Protease inhibitors (PIs) were grouped into the presence or absence of un-boosted or ritonavir-boosted PI (ubPI/rbPI) and non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs) were also grouped into a single variable. A dummy variable was included to indicate patients receiving an ART regimen containing drugs from only a single class. Presence or absence of the K65R mutation was also included as a predictor, and analyses were also conducted including the respective accessory mutations for Q151M and T69i. Further analyses were conducted to investigate the durability of viral suppression in ART-experienced patients, based on the proposal of McKinnon et al. [In the Cox regression analyses, baseline square-root(CD4) and logn et al. ; brieflyTo investigate the association between Q151M and 69i and mortality, we carried out a matched cohort analysis of comparable patients in whom ART data were available. Patients were matched from a subset of patients with at least one resistance test available for whom the relevant mutation had never been detected. Matching was conditional on the observation of a resistance test within 12 months of that in the case patient (effectively matching on treatment failure or interruption in ART-experienced patients), date of ART initiation within 12 months of the case patient in those who were ART-experienced at the time of detection (or ART-naïve status in ART-naïve cases), and patient sex and mode of infection. Matching was carried out with up to 10 ‘controls’ for each ‘case’ (dependent on availability). Survival analyses (for the outcome of any-cause death from date of resistance test) were then conducted using Cox regression including a normally distributed shared frailty term for each matched group and adjusted for CD4 count at time of resistance test, age at resistance test, prior diagnosis of an AIDS-defining illness at resistance test and number of other reverse transcriptase resistance mutations detected at the index resistance test (grouping ≥ 9 mutations).A total of 180 patients with at least one observation of the Q151M mutation were recorded in the UK-HDRD, out of a total of 80,281 patients with at least one resistance test. At first observation of the Q151M mutation, 18 (10%) patients were recorded as being ART-naïve, 139 (77%) as experienced and 23 (13%) as ‘not classified’. Figure Table Of the patients in whom the Q151M mutation was detected, 96 could be linked to clinical data. Of these patients, 82 (85%) were recorded as ART-experienced and one as ‘not classified’, with 13 being ART-naïve. Information regarding the ART regimen prior to the resistance test was available in 74/82 (90.2%) of the ART-experienced patients, and these 74 patients were included in the matched case–control analysis to investigate factors associated with the occurrence of the mutation  as did use of DDI , whilst the use of a rbPI showed a positive association   patients, a confirmed undetectable VL was observed (at any point) following the detection of the Q151M mutation at a median of 1.0 (IQR 0.5–2.3) years from the date of the resistance test sample. For a total of 62 patients there were data on change to ART regimen after detection of the Q151M mutation and before viral re-suppression (for patients in whom this occurred), but six of these patients were missing a baseline CD4 cell count  and so were dropped from the Cox analysis. Hence, 56 patients were included in the Cox analysis, with a total of 22 events  observed before censoring. Higher baseline VL (prior to new ART regimen) showed a substantial negative association with the probability of viral suppression (standardised hazard ratio (sHR) 0.32, 0.14–0.64; HR of 0.30 on original scale of logA model was fitted to assess the durability of viral suppression that included parameters for baseline VL, rbPI and DDI use. This analysis indicated that the durability of viral suppression was good for patients with low baseline VL, but that the overall probability of suppression was worse for patients with higher baseline VL values Fig. .Fig. 3MoFor the matched cohort analysis of mortality, 72 patients with and 665 patients without the Q151M mutation were included. Higher age and lower CD4 count were strongly associated with mortality, but no clear association with the number of reverse transcriptase resistance mutations was observed. There was no evidence that presence of the Q151M mutation was associated with increased mortality . In patients with Q151M, most deaths occurred amongst those with CD4 count < 100 cells/µL  patients were recorded as being ART-naïve, 69 81.2%) as experienced and nine (10.6%) as ‘not classified’. Figure .2% as exTable Of the patients with T69i mutation detected, 45 could be linked to clinical data. Of these patients, 39 (86.7%) were recorded as ART-experienced and six as naïve. Full information regarding the ART regimen prior to the resistance test was available in 36/39 (92.3%) of the ART-experienced patients, and these 36 patients were included in the matched case–control analysis to investigate factors associated with the occurrence of the mutation. For the final fitted model, only ‘total years of virological failure’ showed a substantial positive association with the occurrence of the T69i mutation (standardised odds ratio (sOR), 95% CrI 2.16, 1.34–3.60; OR of 3.48 on original scale (years)), although there was also some evidence for a positive association with ‘DDI use ever’   showed a substantial negative association with the probability of viral suppression , as did the use of D4T . 3TC  also showed some evidence of a negative association  patients, at least one confirmed undetectable VL was observed (at any point) following the detection of the T69i mutation at a median of 1.5 (IQR 0.5–2.2) years from the date of the resistance test blood sample. For a total of 31 patients there were data on change to ART regimen after detection of the T69i mutation and before viral re-suppression, but five of these patients were missing both baseline CD4 cell count and VL (prior to new ART regimen) and so were dropped from the Cox analysis. Hence, 26 patients were included, with a total of 12 events  observed before censoring. Higher baseline logA model was fitted to assess the durability of viral suppression that included parameters for baseline VL and D4T use. This analysis indicated that the durability of viral suppression was good for patients with low baseline VL, but that the overall probability of suppression was worse for patients with higher baseline VL values Fig. . The widFor the matched cohort analysis of mortality, 35 patients with and 294 patients without the T69i mutation were included. Lower CD4 count was strongly associated with mortality, but no clear association with the number of reverse transcriptase resistance mutations was observed. There was no evidence that presence of the T69i mutation was associated with increased mortality . In affected patients, the hazard of death was notably higher amongst those with CD4 count < 100 cells/µL  and Q151M was even rarer in ART-naïve patients (0–0.5%) whilst T69i was not observed. The levels amongst treatment-experienced patients are similar to those in subtype-B patients in the Swiss HIV Cohort Study, for which Scherrer et al. reported an overall prevalence up to February 2010 of 0.8 and 0.5% for Q151M and T69i, respectively . In the Patients who received ART in the earlier years of the HIV epidemic in the UK were treated using a wide variety of drug combinations, with changes often implemented as new drugs became available. The very high level of complexity of treatment histories for HIV hampers epidemiological investigation of risk factors for the occurrence of specific resistance mutations, particularly for the analysis of rare mutations such as Q151M and T69i. There are also equivalent issues to consider when analysing response to treatment conditional on patient characteristics and ART regimen. We were concerned that the analysis of a large number of potential risk (or predictive) factors with a small number of cases would lead to a high risk of false-positive results, and so we chose to carry out our analyses using a Bayesian framework that would penalise estimates of effect sizes without substantial evidence in the data; this approach also has the advantage that results regarding estimation of effect sizes can be interpreted in a directly probabilistic manner rather than purely dichotomised into present or absent.We found strong associations between D4T exposure and emergence of the Q151M mutation, a link previously identified by Nouhin et al.  drawn laD4T has been subject to a global phase-out over recent years, with a drop in total market share for NRTI use in adults from 29% in 2011 to just 0.2% in 2016 , and as The overall level of confirmed viral suppression (62/96:65%) was similar to that reported by Scherrer et al. 14/25:56%) [/25:56%  trials oWe should note that owing to the historic nature of the cases described, no patients were switched to an integrase inhibitor regimen directly following detection of the mutation and so we have not provided any information on the relative efficacy of this drug class. Our models indicate that virological outcomes were substantially better for patients that had lower baseline VL at detection of the mutation, but it may now be possible to achieve better outcomes for patients with high VL using modern ART regimens.We did not find evidence that presence of the Q151M mutation is associated with increased risk of mortality relative to comparable patients matched on ART initiation and resistance testing dates, but it is not possible to draw strong conclusions from the limited data available. Mortality outcomes will also be dependent on the availability of effective ART options, and rbPI regimens may not always be available in LMICs. Scherrer et al.  assessedRecent data from LMICs has indicated that T69i remains a rare mutation, for example Tang et al. identified only two cases (0.1%) in a review of 1825 patients failing D4T-containing first-line ART  (this stThe baseline VL prior to treatment switch (or initiation in ART-naïve patients) and continued D4T use were the strongest (negative) predictors of subsequent virological suppression, but the small sample size and large number of potentially important factors made it impossible to rule out important associations with other variables including drug choices and associated mutations. The overall level of confirmed viral suppression (33/45:73%) was very similar to that reported by Scherrer et al. (9/13:69%) . As for Our data confirm that modern ART and laboratory monitoring have greatly reduced the occurrence of multi-NRTI resistance due to T69i and Q151M in the UK, a change that is very likely to have also occurred in other developed countries. It remains to be seen whether similar progress can be achieved in LMICs. In addition this report demonstrates that ART regimen changes can be successful despite these mutations, although whether outcomes in patients with the poorest prognosis can be improved with more modern ART regimens also remains to be determined.Additional file 1. Appendix containing further details of methods and results.


The emergence, accumulation and spread of HIV-1 drug resistance strains in Africa could compromise the effectiveness of HIV treatment programs. This study was aimed at determining the incidence of virological failure and acquired drug resistance mutations overtime and identifying the most common mutational pathways of resistance in a well characterized HIV-1C infected Ethiopian cohort.A total of 320 patients (220 ART naïve and 100 on first lines ART) were included and followed. ART initiation and patients’ monitoring was based on the WHO clinical and immunological parameters. HIV viral load measurement and genotypic drug resistance testing were done at baseline (T0-2008) and after on average at a median time of 30 months on ART at three time points .The incidence of virological failure has increased overtime from 11 at T1 to 17 at T2 and then to 30% at T3. At all time point’s almost all of the patients with virological failure and accumulated drug resistance mutations had not met the WHO clinical and immunologic failure criteria and continued the failing regimen. A steep increase in the incidence and accumulation of major acquired NRTI and NNRTI drug resistance mutations have been observed (from 40% at T1 to 64% at T2 and then to 66% at T3). The most frequent NRTIs drug resistance associated mutations are mainly the lamivudine-induced mutation M184V which was detected in 4 patients at T1 and showed a 2 fold increase in the following time points (T2: n = 8) and at (T3: n = 12) and the thymidine analogue mutations  which were not-detected at baseline T0 and T1 but were increased progressively to 10 at T2 and to 17 at T3. The most frequent NNRTIs associated mutations were K103N, V106M and Y188C.An upward trend in the incidence of virological failure and accumulation of NRTI and NNRTI associated acquired antiretroviral drug resistance mutations are observed. The data suggest the need for virological monitoring, resistance testing for early detection of failure and access for TDF and PI containing drugs. Population-level and patient targeted interventions to prevent the spread of mutant variants is warranted. In Africa, over the past decade a remarkable increase in access to combination antiretroviral therapy (cART) has been continued and has substantially contributed to the dramatic reduction in morbidity and mortality. It has also improved quality of life and life expectancy and altered perceptions on HIV/AIDS from an epidemic to a manageable chronic illness , 2. ThesAlong with the expanded coverage of ART, prevalence of transmitted drug resistance (TDR) is rising overtime –7 predomIn Ethiopia, according to the recent report from the Federal HIV Prevention and Control Office, there are about 740,000 people living with HIV in Ethiopia, and only 400,000 are receiving antiretroviral treatment. Previous studies in early years of HIV and recent studies including our previous works show the presences of HIV-1C clade homogeneity in 99% of Ethiopian isolates . ART in https://aidsfree.usaid.gov/]. Although D4T has been omitted from ART recommendations because of its side effects in 2008, it is still used extensively in Ethiopia. ART initiation and monitoring is based on the WHO clinico-immunological approach. However, as this approach lacks viral load determination and drug resistance testing HIV infected individuals may be at risk for “unrecognized” virologic failure and the subsequent development of antiretroviral drug resistance.one NNRTI with first line regimens of lamivudine (3TC) combined with stavudine (d4T) or zidovudine (AZT), and either nevirapine (NVP) or efavirenz (EFV) permitting four alternative treatment regimens: D4T+3TC+NVP; D4T+3TC+EFV; ZDV+3TC+NVP; or ZDV+3TC+EFV. For second line therapy, the nucleoside backbone is changed to ABC, TDF, ddI or ZDV (if not used in first-line therapy); and, in addition, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) NVP or EFV was replaced by one of the boosted protease inhibitors (PIs) lopinavir (LPV/r), saquinavir (SQV/r) or indinavir (IND/r) . But with time like NRTIs, the frequency and pattern of NNRTIs associated mutations increased significantly. Briefly, by the time point T1, 8 NNRTIs associated mutations were observed which then increased substantially to 18 by the time point T2 and further increased to 26 by the time point T3 . The mosDual-class resistance to NRTIs and NNRTIs were not detected at baseline T0 . But eveMajor drug resistance mutations on PR region were not detected at all time points. However, in the majority of the patients at all time points, several secondary mutations that may facilitate the development of PI resistance were found at higher frequency which could be naturally occurring minor mutations/polymorphic changes  but their clinical significance is uncertain. Seven out of eleven at T1 and 9/15 at T2 who had treatment switch to second line ART (PI/r + 2NRTIs regimen) were found to be virologically suppressed.10 copies /ml.At baseline, most of the viral isolates were fully susceptible for the available NRTIs and NNRTIs. However, the mean GSS for both all NRTIs and NNRTIs decreased overtime and the number of treatment option decreased overtime with viremia. This study describes a decade virological outcomes and measures the incidence of virological failure and acquired drug resistance mutations in Ethiopian HIV-1C infected adults at 4 time points from the Horn of Africa. The study has the following main findings in relation to the incidence of virological failure, accumulation of acquired drug resistance mutations and the pattern of mutational pathways.First, a relative upward trend in the incidence of both virological failure and acquired drug resistance mutation driven by NRTIs and NNRTIs is observed over the years supporting the notion that prolonged exposure to ART drives the emergence of mutant variants , 16 and The higher rate of NRTI resistance may reflect the expected pattern of mutations with increasing use of first line therapy . MoreoveSecond, the absence of major protease inhibitors drug resistant mutations in this cohort could explain the relation between drug access and emergence of drug resistant mutations. This could reflect the presence of limited access of the drug class in the country as less than 2% of patients are taking drug regimens containing this drug class. The occurrence of naturally occurring polymorphic changes on PR region may have clinical relevance as these changes may result in the evolution of drug resistance along distinct mutational pathways, or in the incidence of different pathways when considering long-term treatment strategies of HIV-1C infected patients with PI based regimen . A rapidLastly, the study in one side identified an increasing common mutational pathways overtime with two thymidine non-analog (M184V and K65R) and three thymidine analog  major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs and four major NNRTI-associated DRMs  which is consistent with previous studies , 29. ButIn summary, an upward trend of both virological failure and NRTI and NNRTI associated drug resistance acquired drug resistance mutations has been observed overtime. The findings indicate that ART programs in east Africa could not achieved WHO suggested targets of long term virological suppression overtime. Thus, regional focused strategies to decrease the rate of virological failure and acquired drug resistance, to prevent the spread of mutant variants, to increase access for TDF, PI drugs, HIV viral load and resistance testing are needed if the 90-90-90 targets are to be attained. As a result treatment success of current first-line ART could be improved, the efficacy of second-and third-line therapies could be preserved and transmission of resistant variants which could be the future challenge of the region unless controlled early could be prevented.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Real-life data on access and response to direct antiviral agents (DAA) in HIV-HCV coinfected individuals are lacking.HCV viremic, HIV-positive patients from Icona and Hepaicona cohorts naïve to DAA by January 2013 were included. Access and predictors of starting DAA were evaluated. Switches of antiretroviral drugs at starting DAA were described. We calculated sustained virological response (SVR12) in those reaching 12 weeks after end-of-treatment (EOT), and defined treatment failure (TF) as discontinuation of DAA before EOT or non-SVR12. Statistical analyses included Kaplan-Meier curves, univariable and multivariable analyses evaluating predictors of access to DAA and of treatment outcome (non-SVR and TF).2,607 patients included. During a median follow-up of 38 (IQR:30–41) months, 920 (35.3%) patients started DAA. Eligibility for reimbursement was the strongest predictor to access to treatment: 761/1,090 (69.8%) eligible and 159/1,517 (10.5%) non-eligible to DAA reimbursement. Older age, HIV-RNA≤50 copies/mL were associated to faster DAA initiation, higher CD4 count and HCV-genotype 3 with delayed DAA initiation in those eligible to DAA reimbursement. Up to 28% of patients  underwent antiretroviral (ART) modification at DAA initiation. 545/595 (91.6%) patients reaching EOT achieved SVR12. Overall, TF occurred in 61/606 patients (10.1%), with 11 discontinuing DAA before EOT. Suboptimal DAA was the only independent predictor of both non-SVR12  and TF .Only 35.3% had access to HCV treatment. Despite excellent rates of SVR12 rates (91.6%), only 21%  of our HIV-HCV co-infected patients are cured. The natural history of chronic Hepatitis C virus (HCV) infection has dramatically changed upon the introduction of Direct Antiviral Agents (DAA) regimens. In clinical trials settings these regimens have led to sustained virological response at 12 weeks (SVR12) after end of treatment (EOT) in up to 80–96% of cases. DAA response in HIV-HCV coinfected individuals are similar to HCV mono-infected ones [Nevertheless, universal access to anti-HCV treatment is still unreachable. Despite International recommendations that all HCV-infected persons should receive treatment –11, payeIn addition, introduction of reimbursement has varied in many countries by specific DAA: in Italy sofosbuvir (SOF) was reimbursed by the Italian Agency of the National Health System (AIFA) from December 2014, simeprevir (SIM) from February 2015, daclatasvir (DCL) from April 2015 and sofosbuvir+ledipasvir (SOF/LDV), ombitasvir+paritaprevir+ritonavir (2D) and dasabuvir (DSB) from May 2015. This may lead to use of suboptimal treatment schedules.Concerning Human Immunodeficiency Virus (HIV)-HCV co-infected individuals, interactions with antiretroviral agents (ART) could occur, with possible toxicities or inadequate drugs levels . Thus, pThe main aim of this analysis was to evaluate the rate of access and response to DAA treatment in a HIV-HCV co-infected population seen for care in Italy. Issues related to the management of their HIV disease in relation to DAA treatment were also examined.Patients from HepaICONA and Icona Italian cohorts are included. HepaIcona is a cohort of HIV-HCV co-infected patients started on January 2013 to address issues related to access and response to DAA. The main inclusion criteria are to be ART-experienced, HIV-HCV coinfected and currently DAA-naïve with detectable HCV-RNA. This analysis includes also the subset of HIV-HCV co-infected patients enrolled in Icona  under active follow-up on January 1, 2013, with detectable HCV-RNA and DAA-naive. Details of Icona cohort have been described elsewhere . All patst, 2013, whichever was the latest. Socio-demographic factors were collected at baseline, HIV- and HCV-related factors have been collected at baseline and during follow-up: biochemistry, HCV-RNA, HCV-genotype, hepatic stiffness by transient elastography, anti-hepatitis drugs received before and after enrolment , ART regimens and reasons for discontinuation, all severe clinical events, including liver-related . Suboptimal DAA (sDAA) was defined by rating recommendations for currently available options described in the 2016 EASL (European Association for the Study of the Liver) guidelines [Baseline for this analysis was the date of enrolment in the cohorts or January 1idelines  i.e. theCharacteristics of the study population at baseline were described after stratification by the AIFA eligibility criteria. Differences in categorical factors were tested using the chi-square test and median values for continuous variables compared using the Wilcoxon test.In people displaying AIFA criteria for DAA reimbursement, we estimated the median time to access to DAA from date of enrolment by Kaplan-Meier method. Factors independently associated with the probability of starting DAA were identified by log-rank test and proportional hazards Cox regression analysis. When data on stiffness by transient elastography were not reported we used the Fibrosis-4 (FIB-4) index to evaluate fibrosis and considered equivalent to F4 cases with FIB-4 >3.25 . A sensiIn those who started DAA, we described the changes in ART regimen occurring in the previous 3 months before DAA. We calculated the median duration of DAA treatment in subgroups stratified by HCV-genotype, RBV use and decompensated cirrhosis and durations between groups was compared using the Wilcoxon signed rank test.We included all those who started DAA and had 12 weeks data after EOT available to study the binary response (SVR12 yes/no) by logistic regression. We evaluated the rate of treatment discontinuation before EOT.SVR12 was defined as a HCV-RNA result below the limit of detection at 12-weeks follow-up (SVR12) or thereafter. We defined treatment failure as a combined endpoint including treatment discontinuation or lack of SVR12 by intention-to-treat (ITT) switch = failure analysis.Multivariable models have been constructed by including potential predictors in the models.In those who started DAA and completed the treatment course we performed univariable and multivariable analyses of covariance (ANCOVA) on the absolute CD4 count  and HIV-RNA  to evaluate the potential effect of the inclusion of RBV in the DAA on these biomarkers.A total of 2,607 HIV-HCV co-infected patients have been analysed using data frozen at December 15, 2016; 1,090 (41%) displayed AIFA criteria for DAA reimbursement. In addition to the expected difference in terms of stage of fibrosis, previous failure to HCV therapy, and comorbidities, eligible patients showed different demographic features: they were older, less frequently females, more frequently infected through intravenous drug addiction, less frequently non-Italian, less frequently unemployed. Further, they had a longer history of HIV infection as documented by year of diagnosis, year of starting ART, CD4-positive lymphocyte (CD4) nadir .During a median follow-up of 38 (30–41) months, 761 (69.8%) patients eligible to reimbursement started DAA, and a further 159 (10.5%) not eligible, with F1-F2, started DAA within compassionate use programs.Median calendar year of starting DAA was 2015 (2015–2016); 94 (12.4%) eligible and 17 10.7%) non-eligible started suboptimal DAA regimens according to EASL [.7% non-eIn the 1,090 patients eligible to DAA reimbursement, the median time to DAA initiation was 12.8 (95% CI:10.8–15.0) months and by 42 months the probability of starting DAA was of 89% (95% CI: 86–92%) . NonetheA number of factors were independently associated with the probability of starting DAA among patients eligible for reimbursement in the multivariable model . Older aA total of 920 patients started DAA. A total of 79/142 (55.6%) patients with decompensated cirrhosis or HCC initiated DAA, with median Model For End Stage Liver Disease (MELD) score of 10.4 (IQR: 8.6–13.3).DAA duration according to different characteristics is shown in CD4 cell counts and HIV-RNA copy levels variations from DAA initiation to EOT and 12 weeks after EOT are summarized in In 829 (90%) of the 920 who started DAA, we had information on whether there has been a change in ART at DAA initiation as compared to 3 months prior to starting. A total of 230/829 (28%) underwent a modification of the third drug before DAA: atazanavir-ritonavir was replaced in 36/145 (25%) of cases, darunavir-ritonavir (DRV/r) in 40/117 (34%), rilpivirine (RPV) in 10/86 (12%), efavirenz in 36/75 (48%), raltegravir (RAL) in 6/221 (3%), elvitegravir in 8/17 (47%), 0/34 in dolutegravir (DTG).A total of 118/328 (36.0%) participants receiving a ritonavir-boosted protease inhibitor (PI/r)-based regimen 3 months prior to DAA initiation were switched to an integrase inhibitor (INI) (n = 113) or RPV (n = 5); 36/175 (21%) patients on a non nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen were switched to an INI-based regimen (n = 30) or to other classes (n = 6). Only 5/272 (2%) patients receiving an INI-including regimen changed the third drug class .A total of 595 patients (65.9%) of the 920 who started DAA reached the SVR follow-up time while still on DAA and week 12 HCV-RNA value was recorded; 545 (91.6%) experienced SVR and 50 (8.4%) virological failure (non-SVR12). SVR12 was reached in 106/120 (88.3%) of genotype 3 (92% if treated with SOF+DCL+RBV for 24 weeks) and 439/475 (92.4%) of patients with genotype other than 3 (p = 0.15); in 297/324 (91.7%) patients treated with RBV and 248/271 (91.5%) starting a RBV-free regimen (p = 0.95); in 51/59 (86.4%) patients with decompensated cirrhosis and in 494/536 (92.2%) without  treatment failures: 50 with non-SVR12 like in the previous analysis plus the 11 who had suspended treatment prematurely  were eligible to DAA reimbursement, and only 35.3%  were actually treated. In 595 , i.e. those reaching 12-week follow-up after EOT, we observed a success rate of 90%. So National Health System reimbursement was the main driver of treatment access in HIV-HCV as expected.When restricting to people eligible for reimbursement of treatment, by 2 years from enrolment approximately 70% of patients had access to DAA. Older age and HIV-RNA≤50 copies/mL were associated to faster DAA initiation, higher CD4 count with delayed DAA initiation. These findings might be explained by the fact that longer HIV and HCV infections occur in older people and with lower CD4 counts; among these individuals DAA was reserved to those with virologically controlled HIV disease supposed to be more compliant also with DAA. Individuals harbouring HCV-genotype 3 had a delayed access to DAA consequent to the only recent availability of DAA regimens effective on this genotype.There are differences across Europe regarding the prioritization of DAA initiation; for example in the Netherlands, there is no limitation to access to DAA and higher percentages of men sex with men (MSM) with less advanced liver disease have been treated compared to Italy; however, the rate of HCV cure in this population has been only slightly greater (80%) than in those with advanced disease (70%) . Of inteOverall, in the 595 patients who started DAA and received a full course of therapy the rate of SVR12 was very high, at 92%. This high percentage of success, very close to that observed in clinical trials, could not be predicted at the outset because our population was unselected and enriched with people with advanced liver disease .The only factors associated with the risk of treatment and virological failure were use of suboptimal DAA. Patients with decompensated cirrhosis showed a nearly double risk of failure compared to patients with advanced fibrosis, consistent with the findings of clinical trials ; 22, 23.It is interesting to note that, in our analysis, genotype 3-infected individuals, if treated with optimal DAA regimen, showed the same rate of success than that of individuals infected by other genotypes, suggesting that also individuals with difficult-to-treat genotypes might achieve eradication with DAA in real-life using a schedule such as SOF+DCL+RBV for 24 weeks that has never been verified in a controlled study.Our data confirm in a larger setting the observations of a French cohort , where tMore than 60% of the patients have been treated for more than 12 weeks even in combination with RBV. Treatment duration longer than 12 weeks was more frequent in HCV-genotype 3 (87.3%) and in decompensated cirrhosis (82.8%). This suggests that presumed treatment efficacy rather than schedule simplicity could be the main drivers of the choice of treatment schedule. Our data confirm also the transient lymphopenic effect of RBV resulting in a transient lower CD4 counts at EOT returning to normal after RBV completion .ART was switched before DAA in 28% of the patients, mainly for concerns on interactions; actually 36% of patients on PI-r regimens switched to other regimens. The availability of INI as RAL or DTG allowed the formulation of effective anti-HIV regimens even in patients with long history of anti-HIV therapy resulting in the possibility to effectively treat HCV without interactions with ART , 16.Our study has some limitations; first of all it is observational and therefore unmeasured confounding cannot be ruled out; we do not routinely collect ART adherence data in our cohorts; finally not for all patients included we had week 12 virological data.In conclusion: despite high SVR12 rates, only 21%  of our HIV-HCV co-infected patients are currently cured. These data document the gap between universal access and effective treatment in Italy, caused by economic limitations, a low capability of treatment centres and limited availability of optimal treatments. Some of these are unlikely to be unique to the Italian setting. In our unselected population, HIV-HCV co-infected patients show a very high success-rate on DAA even those with advanced liver disease and difficult to treat genotypes. The possible impact of prioritization reimbursement policies in different countries needs to be assessed using simulations and stochastic modelling.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The GEE analysis indicated that of the different factors, the duration of ETV therapy had a strong impact on the achievement of virological suppression, HBeAg seroconversion and SRR in adolescents. Each month after the initiation of therapy, the odds of loss of HBV DNA increased by approximately 5% , on average. Additionally, the GEE analysis revealed that adolescents with an age at infection of ≥10 years had 3 times higher odds of achieving undetectable HBV DNA than patients with a younger infection age . None of the ETV-treated patients reported significant adverse effects. ETV is an effective and safe treatment option for adolescents with CHB. Undetectable HBV DNA in the 6th and/or 12th month of ETV treatment and older age at infection could predict maintained virological suppression.This study evaluated the long-term (up to 4 years) efficacy and safety of entecavir ETV treatment and analysed the significance of baseline and on-treatment factors in long-term ETV outcomes in adolescents with chronic hepatitis B (CHB). We determined the cumulative virological and serological outcomes of 44 adolescents with CHB receiving ETV for up to 4 years. To investigate the dynamics of HBV DNA, ALT activity and hepatitis B e antigen (HBeAg) seroconversion over time and their associations with the considered factors, generalized estimating equation (GEE) models were used. The cumulative rates of undetectable HBV DNA (<20 IU/ml) and HBeAg seroconversion after 4 years were 89.7% and 55.4%, respectively. In the study group, we showed that having undetectable HBV DNA at the 6 Hepatitis B virus (HBV) infection is still one of the most important infectious diseases, with 2 billion people infected worldwide and more than 600,000 deaths each year . DespiteAmong children, HBV infection concerns those who have not been vaccinated at all or have not gone through the full cycle of vaccinations, those who were exposed to HBV prior to vaccination or were born to HBV-infected mothers and did not receive immuno-prophylaxis. However, Haber et al. observed the occurrence of vertical HBV infection in 5% of infants despite appropriate immuno-prophylaxis and vaccination . HBV infThe goal of hepatitis B treatment is to reduce viral replication and to minimize hepatic injury. To date, five medications for the treatment of children/adolescents with chronic hepatitis B (CHB) have been approved. These are interferon-alpha, lamivudine, adefovir, entecavir and tenofovir .The treatment of CHB in children and adolescents is controversial due to its unsatisfactory results in immune-tolerant states and the risk of resistance , 5. SomeThe aim of this study was to evaluate the long-term efficiency and safety of entecavir (ETV) treatment in children and adolescents infected with HBV. This is a unique analysis of the long-term 4-year treatment of adolescents with ETV in a real-life setting. The results of this study may be useful in enhancing the importance of ETV treatment in clinical practice as one of the available therapeutic options for adolescents with CHB.In our study, 57 patients were evaluated. We excluded 13 patients who were treated for less than 12 months or who didn’t have the necessary medical documentation. Patients with histological evidence of HCC or a chronic liver disease other than CHB and those who were co-infected with HCV or HIV were excluded. There were no pregnant women in the study group. Our study did not include patients in the immunotolerant phase. Overall, we collected data from 44 consecutive patients who, in most cases, were infected with HBV in childhood and had begun treatment with ETV at the Department of Paediatric Infectious Diseases and Hepatology. All examined patients were treated with ETV monotherapy at dosages of 0.5 mg/day or 1 mg/day for at least 48 weeks. The eligibility criteria were having detectable serum HBsAg for 6 months and the presence of two of the following criteria: detectable HBV DNA in the serum, increased ALT activity and histopathological changes in the liver. In all patients, a liver biopsy and liver ultrasound were performed at baseline. The liver biopsy specimens were assessed according to the modified Scheuer scale (grading from 0–4 and staging from 0–4) [th year of age, and in 6, at ≥10 years of age. Information about the occurrence of infection at ≥10 years of age was based on medical records, which negated the presence of HBsAg and anti-HBc in these patients before they were 10 years old. The median age at the start of ETV treatment was 16 years. We did not use any specific age to begin treatment. Although ETV therapy was only recommended in children older than 16 years (FDA and ESPGHAN recommendation), this study included patients younger than 16 years for ETV therapy. Every six months of treatment, the patients were required to return to the clinic to receive a physical examination and laboratory tests for serum HBV DNA levels, presence of serum HBeAg and HBsAg and ALT activity and to report adverse events.In the analysed group of patients, the age at HBV infection ranged from 4 months to 14 years (median 3 years). In 38 patients, HBV infection was recognized before the 10The effectiveness of the treatment was evaluated on the basis of the patient’s virological, serological and biochemical response, which was assessed by analysing HBV DNA levels, HBeAg status and ALT activity before and during the treatment . Furthermore, the combined response was assessed. The effectiveness of the treatment was evaluated based on the sustained response rate (SRR), defined as the suppression of viral replication to undetectable HBV DNA levels (<20 IU/mL), HBeAg seroconversion to anti-HBe (HBeAb), and normalization of ALT activity (<40 IU/L) in HBeAg-positive (HBeAg(+)) patients [th month were unavailable. Thus, the number of observations at the considered time points decreased from 44 at baseline to 12 months to 41 at the 24th month, 31 at the 36th month, and 23 at the 48th month.Observations up to month 48 were not available for all patients. For patients who started ETV therapy later, the period of therapy was shorter, and data up to the 48The protocol was approved by the NCU Bioethics Committee at the Collegium Medicum NCU. All procedures conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All patients older than 16 years and all legal guardians signed a written informed consent.P-value was less than 0.05. The statistical analysis was performed with the use of R-software, version 3.0.3. (package gee) [The summary statistics are presented as medians with interquartile ranges or absolute and relative frequencies, n (%), as appropriate. Differences between continuous variables were analysed by Mann-Whitney U test, and differences in categorical variables were tested using chi-square or Fisher’s exact test. The concentration of HBV DNA was analysed as a binary variable (undetectable and ≥20 IU/mL). The ALT activity was also considered a categorical variable  vs elevated ALT activity (≥40 IU/L)). The long-term treatment effects during the 4-year period of ETV therapy were analysed using generalized estimating equations (GEE), which account for the correlation between repeated observations from the same individual at multiple time points. To model the progression in virological suppression to HBV DNA levels <20 IU/mL, normalization of ALT activity, HBeAg seroconversion and SRR during ETV therapy, a GEE logistic regression model with logit link and an autoregressive working correlation structure was fitted to the data. Initially, the time of ETV therapy, HBeAg status at baseline, HBV DNA viral load, ALT activity at baseline, gender, previous NA-therapy, grading, staging, age at infection and age at treatment start were included in the model as covariates. In the analysis, all patients’ HBV DNA levels at baseline were determined and described by the percentage of patients in established categories: </≥2,000 or </≥ 20,000 IU/ml. On the basis of the Wald statistic, forward elimination was performed, and finally, the model with significant covariates was fitted to the data. The cumulative rates of virological suppression, HBeAg seroconversion and SRR achievement were estimated using the Kaplan-Meier method, and differences between treatment subgroups were calculated by the log-rank test. The results were considered statistically significant when the age gee) .This study recruited HBeAg(+) (47.7%) and HBeAg(-) (52.3%) CHB patients. The characteristics of the examined patients in the whole group and stratified by HBeAg status at baseline are presented in The percentages of baseline HBeAg(-)  and HBeAP = 0.027). The final cumulative rates of HBV <20 IU/mL were 95.2% for HBeAg(-) patients at year 1 and 82.6% for HBeAg(+) patients at year 4.For all patients, the cumulative rates of virological suppression to HBV DNA <20 IU/mL were 79.5%, 82.1%, 87.2% and 89.7% for years 1, 2, 3 and 4, respectively. The cumulative rates of HBV <20 IU/mL in patients stratified according to HBeAg status were shown in th month, elevated ALT activity was only present in 2/44 patients (4.6%) (In 29/44 (65.9%) patients, increased ALT activity was observed (73.9% in HBeAg(+) patients at baseline and in 57.1% patients who were HBeAg(-) at baseline). The highest increase in ALT <40 IU/L was observed in the first 6 months of therapy, and in the 12s (4.6%) . Then, tIn the HBeAg(+) group (23 patients), HBeAg/anti-HBe seroconversion occurred in 12 patients (52.2%): in the first year of ETV therapy for 7 patients, in the second year of therapy for 3 patients and in the third and fourth year, single cases of seroconversion occurred. The cumulative rate of HBeAg seroconversion up to year 4 was 55.4% . None ofP = 0.029).Finally, the dynamics of the changes over time in SRR achievement were studied. The percentages of patients with SRR during ETV treatment are shown in vs ≥20 IU/mL) during ETV therapy. Based on the Wald statistic, the final model with the covariates time of ETV therapy, HBeAg status at baseline, HBV DNA expressed as a binary variable  and age at infection was fitted to the data  subgroup .P = 0.014, OR = 0.25 (0.08–0.76)), age at infection ≥10 years ) and time of therapy ). As we have shown previously, these variables were significant also for predicting the loss of HBV DNA for the whole group of patients. In the next step for this model, we analyzed prediction of SRR for HBeAg(-) and HBeAg(+) subgroups. We showed that just like in the model containing HBeAg as a covariate, only the time of therapy was significantly associated with SRR in the HBeAg(+) subgroup.The obtained estimates indicate that each month of therapy increased the odds of the loss of HBeAg by approximately 3% (OR = 1.03 (1.01–1.05)). According to the presented model, it was revealed that HBV DNA ≥20,000 IU/mL decreased the probability of HBeAg loss (OR = 0.19 (0.05–0.67)). Similarly, modelling for SRR showed that the passage of a month increased the odds of SRR achievement by approximately 3% (OR = 1.03 (1.01–1.06)). According to the definition of SRR  patients and HBV DNA <20 IU/mL and normalization of ALT for HBeAg(-) patients), quite understandably, the analyses of all patients showed that being HBeAg(+) may decrease the probability of the loss of SRR. However, in the HBeAg(+) subgroup, only time of therapy had a significant impact on the achievement of SRR. Analyses of the HBeAg(-) subgroup showed no covariates that affected the achievement of SRR. In order to better compare the SSR difference between HBeAg positive and negative patients, we excluded the HBeAg as a covariate from the GEE analysis. Resulting data showed that SRR in the whole group of patients was predicted by HBV DNA >20,000 IU/mL  of SRR achievement in the 24th month of therapy than patients with HBV DNA ≥20 IU/mL in the 6th month of therapy. Undetectable HBV DNA at month 12 was also associated with undetectable HBV DNA and achievement of SRR up to year 3. Patients with undetectable HBV DNA in their 12th month of therapy had much higher odds of undetectable HBV DNA in their 36th month of therapy than patients with detectable HBV DNA in the 12th month of therapy . Finally, undetectable HBV DNA in month 12 seemed to be a good predictor of achieving SRR in the 36th month of therapy .In the next step, we analysed the significance of the association of HBV DNA levels in months 6 and 12 with virological suppression and SRR in years 2, 3 and 4 . Of the Overall, in your study there were 5 patients who remained viremic at the end of observation. From this group, 4 patients (9.1%) developed virological breakthrough between month 30 and 48, 1 of whom had a coexisting biochemical breakthrough  (40 IU/L) in patients who had normalized ALT and as ALT > 2 times the nadir in those who had never had normal ALT). Resistance profiling was determined among patients with detectable viremia during ETV therapy. ETV-resistant mutations observed as substitutions at positions T184, S202, M204 and L180 were present in 5 (11.4%) patients. Of this group, 3 (6.8%) patients developed virological breakthrough after 30 months of ETV therapy. The remaining 2 patients with ETV-resistant mutations did not develop virological breakthrough and were thus continued on therapy. In 1 of the 4 patients who developed virological breakthrough, resistant variants were not found. In the 4 patients with virological breakthrough, there were 3 patients previously exposed to LAM. One patient with virological breakthrough was naïve.The patients in whom viremia  was detected during the observation period were switched to therapy with tenofovir. If the viral load increased during the therapy—which was not qualified as a virological breakthrough—the patient continued therapy for 3–6 months, until the next inspection. In case of the one patient, the viral load at the end of the observation period (18 months) is still detectable, however, during to the constant drug administration, viremia was decreased to a level of 800 IU/mL whereby the patient continued therapy for 3–6 months, until the next inspection.The treatment was safe and well tolerated. During antiviral treatment, there were no serious adverse events associated with the administered drug . The mosIn our study we analyzed patient’s height, weight and BMI before the beginning of ETV therapy and at the end of the observation. Height, weight, and BMI were assessed using centile charts according to patient’s sex and age. By comparing the centile intervals distribution of height, weight and BMI from baseline to end of ETV treatment, we observed no impact of ETV therapy on expected growth .Despite the enhanced treatments for HBV infection in children and adolescents, improved by the optimization of existing treatment programs and the introduction of new drugs for therapy, currently we can speak at most of the half success of currently available therapies aiming at stop of progression of the underlying liver injury , 10.To the best of our knowledge, this is a unique longitudinal study that specifically examines the long-term efficacy of ETV treatment in adolescents in real life. Until now, no data regarding the long-term efficacy of ETV monotherapy for the treatment of CHB in children and adolescents have been presented. In fact, there is limited information regarding the results of ETV monotherapy in children and adolescents infected with CHB, restricted by the small size of the test groups and the limited duration of ETV therapy. A study conducted by Saadah et al. reported retrospectively the efficacy of ETV treatment in 8 HBeAg(+) CHB children, including both those who were previously treated with lamivudine and those who were treatment-naive. The mean duration of ETV treatment was 24 months. In those patients, HBV DNA levels were undetectable in 37.5%, with ALT normalization in 87.5% and HBeAg seroconversion in 37.5% . In our et al. assessing ETV therapy in adults, at year 5, 94% of patients had HBV DNA levels <300 copies/mL, and in 80%, ALT activity was normal [et al. study among 474 patients with CHB, by the fourth year of treatment, increases in the rates of undetectable HBV DNA and ALT activity normalization up to 96% and 93% of patients, respectively, were observed [Our data concerning the virological, biochemical and serological suppression during long-term ETV treatment in adolescents are in accordance with the numerous studies in adults showing high rates of outcomes. The results of a study among 533 CHB patients treated with ETV demonstrated that the rate of undetectable HBV DNA increased steadily from year 1 to 3, reaching 91.2% . In a sts normal . Luo et s normal . Yan et s normal . In an Oobserved .th month of treatment was selected as the best predictor of assessed virological suppression; however, the results of our study demonstrated that undetectable HBV DNA in the 6th month of treatment can also be considered an early predictive factor of virological outcomes up to year 3 of ETV therapy. In our study, undetectable levels of HBV DNA in the 36th month of therapy were identified in 21/21 (100%) and 23/24 (95.8%) patients who had undetectable HBV DNA at months 6 and 12 of therapy, respectively. These data are also consistent with previous findings in adults. In a multivariate analysis, HBV DNA level at month 12 was identified as an independent predictor of undetectable HBV DNA in the third year of therapy [th or 12th month does not necessarily indicate unsuccessful treatment, although it can. Zoutendijk et al. revealed that NA-naïve patients with a partial response to ETV may benefit from prolonged therapy without the development of resistance [th or 12th month of therapy are also more likely to achieve SRR in years 2 and 3 of therapy than patients with HBV DNA ≥20 IU/mL. These results are consistent with those presented by Wong at al. for NA-naïve adults, who found that an undetectable HBV DNA level at month 12 of ETV therapy was an independent predictor of virological and serological suppression during 3 years of treatment [HBV DNA suppression is a primary goal of therapy for CHB. The relationship between baseline and on-treatment factors and ETV treatment outcomes has not been widely studied in paediatric patients. Regarding the most predictive factor, undetectable HBV DNA in the 12 therapy . Howeverreatment .Information regarding the long-term HBV DNA kinetics during treatment with NAs in adolescent is limited. The analysis performed in this study revealed that the probability of the loss of HBV DNA is associated with the length of the treatment period and that the passage of each month during therapy increases the odds of the loss of HBV DNA by approximately 5%. The passage of one year of therapy more than doubles the odds of the loss of HBV DNA. Patients with HBeAg(+) CHB at baseline have, on average, approximately 80% lower odds of the loss of HBV DNA than patients who are HBeAg(-) at baseline, at any time point.P = 0.031). We can suppose that the lack of a link between previous LAM treatment and the therapy results determined by GEE analysis, is due to the more significant impact of HBV DNA ≥20,000 IU/mL variable on the final therapeutic effect. Moreover, in our study the proportion of mutations resulting from the administration of ETV was relatively low, was not always associated with previous LAM treatment, did not always have an impact on the virological supression, and therefore the previous NA-therapy variable was not relevant in predicting the effectiveness of treatment. In our study, 4 of the 5 patients with entecavir resistance had prior LAM exposure, but only 2 of them developed virological breakthrough.In our retrospective study, the patients were referred for ETV treatment only if there were no mutations predisposing them for acquiring entecavir resistance before treatment. GEE analyses include a variable indicating the presence of previous LAM treatment, which proved to be irrelevant. We can assume that this is related to the fact that patients with mutations resulting from the administration of LAM did not take part in the study. However, this is currently the therapeutic standard, developed on the basis of previous clinical trials and observational studies conducted in groups of patients previously treated with LAM. Univariate analysis showed no association of previous NA-therapy with baseline factors like: gender, age, ALT activity and HBV DNA viral load with cut off 2,000 IU/mL. Only for HBV DNA ≥20,000 IU/mL we noted significant association  and the loss of HBV DNA; higher infection ages increased the probability of the loss of HBV DNA. These results seemed to confirm the opinion that it is not appropriate to treat younger children in the immune-tolerant phase. Age at the start of treatment appeared to be non-significant in the model; therefore, no differences in the evolution of HBV DNA loss during treatment in adolescents and in adults has been detected, and it seems that the treatment of adolescents is as effective as it is in adults. Despite the frequent inability to determine the age of infection in clinical practice, the significance of the higher infection age in ETV therapy needs to be assessed in large studies.The results of the available studies and clinical trials suggest that entecavir was well tolerated , 14, 15.There are several limitations to this study, including the relatively small sample size of the treatment subgroups. Another limitation is the heterogeneous population of adolescents, including patients with different previous treatment histories, HBeAg status and baseline ALT activity. Most of the currently available studies on NA therapy efficacy in children and adolescents have this limitation. However, by stratifying according to HBeAg status and using GEE analyses for longitudinal studies, we could evaluate the importance of individual differences at baseline on the long-term outcomes. Multicentre clinical trials with ETV therapy in children and adolescents are ongoing , 24, andThese results support ETV as an effective and safe treatment option for children and adolescents with CHB. Extended therapy with ETV leads to high rates of HBV DNA suppression, HBeAg seroconversion and ALT activity normalization. Patients who maintained virological suppression could be predicted by having undetectable levels of HBV DNA in the 6th and/or 12th month of ETV therapy. This study revealed the role of age at infection in predicting virological suppression after ETV therapy in children and adolescents chronically infected with HBV. Thus, knowledge of the age at infection may be useful in clinical practice in enhancing the prediction of long-term outcomes in ETV-treated children and adolescents.