Treatment of hepatitis B with lamivudine and tenofovir in HIV/HBV-coinfected patients: factors associated with response.

As therapy for human immunodeficiency virus (HIV) infection evolves, optimizing hepatitis B virus (HBV) treatment and identifying factors that impact its response in the HIV/HBV-coinfected population is critical. We identified retrospectively 45 HBV/HIV-coinfected patients with detectable HBV DNA by the Bayer VERSANT HBV 3.0 bDNA assay (limit of quantification 2000 copies/mL) at baseline and/or year 1 of therapy. Patients were divided into three groups based on the active HBV agent in their antiretroviral regimen: group 1 (n = 15) received lamivudine; group 2 (n = 10), lamivudine plus tenofovir and group 3 (n = 20), lamivudine followed by lamivudine plus tenofovir. HBV genotypes and resistance profiles were determined by the Bayer Trugene HBV 1.0 assay. More patients in group 2 achieved HBV DNA suppression below 2000 copies/mL (80%), loss of HBe antigen (HBeAg) (40%) and loss of HBeAg and gain of anti-HBe (20%) than did patients in group 1 or 3. More patients with HBV genotype A, achieved HBV DNA suppression <2000 copies/mL than did patients with non-A genotypes [74% (26/35) vs 20% (2/10)], respectively (P = 0.003). Risk for virological nonresponse was significant in those with non-A genotypes [odds ratio (OR) 11.1; 95% CI: 2.0-50], previous HIV therapy (OR 6.5; 95% CI: 1.2-35) and <90% compliance (OR 3.7; 95% CI: 0.99-14.3). Simultaneous therapy with lamivudine/tenofovir suppresses HBV DNA more effectively than lamivudine or tenofovir added to lamivudine. More patients infected with HBV genotype A responded than the non-A patients, regardless of therapeutic regimen, compliance or prior HIV therapy.

Accurate model predicting sustained response at week 4 of therapy with pegylated interferon with ribavirin in patients with chronic hepatitis C.

Current models used to predict response to peginterferon plus ribavirin treatment, based on viral decline during the first 12 weeks of therapy, have focused on creating an early stopping rule to avoid unnecessary prolongation of therapy. We developed a multivariate model that predicted sustained virological response and nonresponse at baseline and during the first 12 weeks of therapy using collected data from 186 unselected patients with chronic hepatitis C treated with peginterferon plus ribavirin. This model employed ordinal regression with similarity least squares technology to assign the probability of a given outcome. Model variables include sex, age, prior treatment status, genotype, baseline serum alanine aminotransferase levels, histologic necroinflammation and fibrosis scores and serum hepatitis C virus RNA concentration at baseline and weeks, 4, 8, and 12. A multivariate model demonstrated high performance values at all time points. At baseline, the model demonstrated a negative predictive value (NPV) and a positive predictive value (PPV) of 91% and 95%, respectively. At week 4, these values improved to 97% and 100%, respectively, with 95% sensitivity, 89% specificity and 93% accuracy. At week 4, the model was equally efficient for naïve or previously treated patients. Internal validation demonstrated 90% PPV, 94% NPV, 95% sensitivity, 88% specificity and 92% accuracy. A week 4 stopping rule for patients with chronic hepatitis C treated with peginterferon with ribavirin might be proposed by using the model developed in our study.

Hepatitis B virus blood screening: unfinished agendas.

The risk of transfusion-transmitted hepatitis B virus (TTHBV) has been steadily reduced through the use of volunteer donors, enhanced donor questioning, and increasingly sensitive hepatitis B surface antigen (HBsAg) tests. In a few countries, screening by antibody to hepatitis B core antigen (anti-HBc) and/or hepatitis B virus (HBV) nucleic acid amplification tests (NAT) has also been introduced. Nevertheless, the risk of TTHBV in most countries is higher than the risk of either human immunodeficiency virus-1 (HIV-1) or hepatitis C virus (HCV). HBV screening practices vary both in terms of the markers screened and the tests employed, and are often dependent on a country's healthcare resources. Anti-HBc screening could be employed as an additional safety feature in HBV low-prevalence countries, but would lead to the rejection of a high percentage of otherwise acceptable donations in HBV moderate- and high-prevalence countries. It has been argued that only individual-donor HBV NAT testing would substantially reduce the risk of TTHBV beyond that achieved by the most sensitive HBsAg tests. However, studies from countries with low, moderate and high HBV prevalence have demonstrated NAT yield from window period- and late stage HBV-infected donors, even using minipool testing following the most sensitive HBsAg tests.

Role of nucleic acid testing in cadaver organ donor screening: detection of hepatitis C virus RNA in seropositive and seronegative donors.

Hepatitis C virus (HCV) transmission by both seropositive and seronegative cadaver organ donors has been documented, yet nucleic acid testing is not routinely used to identify active infection in these donors prior to transplantation. Between November 2001 and February 2004, we screened 1445 cadaver organ donors for anti-HCV antibodies with either HCV EIA-2.0 (Abbott Diagnostics, Chicago, IL, USA) and/or Ortho HCV Version 3.0 ELISA (Ortho-Clinical Diagnostics, Raritan, NJ, USA) and confirmed seropositive samples with Chiron RIBA3.0 SIA (Chiron Corporation, Emeryville, CA, USA). Samples with sufficient volume (n = 726) were tested by the VERSANT HCV [transcription-mediated amplification (TMA)] Qualitative assay (Bayer Healthcare LLC, Tarrytown, NY, USA) which can be performed in approximately 5 h. Those with detectable HCV RNA and sufficient volume were quantified by the VERSANT HCV 3.0 (bDNA) Assay (Bayer Healthcare LLC) and/or the HCV RNA TMA Quantitative Assay (n = 23) and genotyped (n = 57). Seventy-seven of 1445 (5.3%) donors were seropositive, reactive by either one or both anti-HCV assays. Fifty-two of 63 (82.5%) of the seropositive samples had detectable HCV RNA and were genotyped. Seventeen of these samples had quantifications ranging from 128,123 to >7,692,307 IU/mL. Six of 663 (0.9%) seronegative samples had detectable HCV RNA. Their quantifications ranged from <9.3 to 1,464,799 IU/mL, and five of these six were successfully genotyped. As HCV RNA was demonstrated in samples from both our seropositive and seronegative cadaver organ donors, we are now incorporating nucleic acid testing into our donor screening/diagnostic algorithm.

Transcription-mediated amplification is more sensitive than conventional PCR-based assays for detecting residual serum HCV RNA at end of treatment.

OBJECTIVE: In patients chronically infected with hepatitis C virus (HCV) undergoing antiviral therapy, sustained virologic response is suggested by viral clearance by end of treatment (EOT). Viral clearance is defined by nondetection of serum HCV RNA, usually by qualitative PCR-based assays with limits of detection ranging from 100 to 1000 copies/ml. However, some individuals relapse after achieving apparent viral clearance by EOT. These individuals may have low levels of viremia not detected by current PCR methods. The aim of this retrospective study was to determine whether the Bayer HCV RNA Qualitative Assay, which employs Transcription Mediated Amplification (TMA) and detects 50 HCV RNA copies/ml, could detect residual serum HCV RNA in patients who achieved apparent viral clearance by EOT and subsequently relapsed. METHODS: Samples were obtained at EOT (wk 24 or 48) and follow-up (wk 24-26 posttreatment) from 97 patients treated for HCV (78 relapsing patients, 19 sustained responders). All samples in which HCV RNA was not detected by PCR were tested in a blinded manner for HCV RNA by the TMA-based assay. RESULTS: HCV RNA was detected by the TMA-based assay in 27 (34.6%) EOT and 76 (97.4%) follow-up samples from relapsing patients, but not in any of the EOT or follow-up samples from sustained responders. CONCLUSION: Residual serum HCV RNA was detected by the TMA-based assay in EOT samples from 34.6% of patients that had achieved apparent viral clearance by PCR. The detection of HCV RNA by the TMA-based assay could help redefine EOT response and assist in the antiviral management of HCV infection.

Impact of chronic hepatitis B and interferon-alpha therapy on growth of children.

Interferon-alpha (IFN) has been approved as treatment for children with chronic hepatitis B (CHB). The aims of this study were to assess the impact on children's growth of the disease itself and of IFN treatment. The growth of 142 children with CHB (70 IFN-treated, 72 untreated) was monitored for a minimum of one year. Regression analysis models were used to determine which of the variables most affected children's growth. After adjusting for racial differences, the population of 142 children with CHB had a mean baseline height for age percentile of 39 and a mean baseline weight for age percentile of 38, which were significantly different (P < 0.0001) from the 50th percentiles of their respective reference populations. The height for age Z score of untreated children was inversely correlated with serum hepatitis B virus DNA and aspartate aminotransferase levels, and the weight for age Z score was inversely correlated with serum hepatitis B virus DNA levels. While undergoing IFN therapy, children displayed a "U-shaped" growth pattern, such that height for age and weight for age Z scores at 3 or 6 months were lower than scores at baseline or 12 months. In this study the average child with CHB showed compromised growth even in the absence of IFN therapy. During IFN therapy, children's growth was temporarily disrupted.

Statistical models for predicting response to interferon-alpha and spontaneous seroconversion in children with chronic hepatitis B.

To develop prognostic models for identifying children with hepatitis B who are likely to respond to interferon-alpha (IFN-alpha) or to spontaneously seroconvert, we evaluated results of a multinational controlled trial comprising 70 children with chronic hepatitis B who received IFN-alpha and 74 children who did not receive therapy. Prognostic models were developed using SMILES (similarity of least squares), which is a data analysis network that incorporates multidimensional relationships in the clinical data of complex diseases. Commonly collected clinical data included age, gender, serum aminotransferase (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) and hepatitis B virus (HBV) DNA levels, and IFN-alpha dose. Additional data included pretreatment directional information (e.g. increases or decreases in serum aminotransferase and HBV DNA levels), liver biopsy results, race and transmission mode. Using data available prior to initiation of treatment, the SMILES models achieved prospective predictions of 89% for responders, 96% for non-responders, 100% for seroconverters and 93% for non-seroconverters. Although not predictive by themselves, the variables that had the greatest impact on predictions for IFN-alpha response were HBV DNA pretreatment direction, baseline HBV DNA, IFN-alpha dose and gender. The variables that had the greatest impact on predictions for spontaneous seroconversion were ALT pretreatment direction, baseline HBV DNA level, age and AST pretreatment direction. Therefore, these models may be useful in determining, in children with hepatitis B, the likelihood of response to IFN-alpha and spontaneous seroconversion.

Assessment of hepatitis C virus RNA stability in serum by the Quantiplex branched DNA assay.

OBJECTIVES: Quantification of hepatitis C virus (HCV) RNA in serum is used to assess the probability of treatment response and to monitor antiviral therapy. Since serum specimens often are shipped to central sites for HCV RNA testing, it is important to define conditions that preserve HCV RNA integrity. METHODS: We evaluated the stability of HCV RNA in 25 previously frozen (PF) and 11 fresh, never previously frozen (NPF) specimens subjected to handling and short-term storage conditions that mimic those encountered during interlaboratory shipping. All sera were separated within 4 h of collection. PF samples covering a approximately 3 log10 HCV RNA dynamic range were thawed, divided into aliquots, incubated at 4, 23, and 37 degrees C (+/- 1.5 degrees C) for 24, 48, 72 and 96 h (+/- 2 h), and then refrozen at -70 degrees C prior to testing with the Quantiplex HCV RNA 2.0 assay. Eleven NPF samples were stored at -70, -20, and 4 degrees C (+/- 1.5 degrees C) for up to 1 month prior to testing. RESULTS: Linear regression analysis showed no HCV RNA degradation in PF specimens kept at 4 degrees C over 4 days. However, HCV RNA levels in PF specimens decreased over 4 days by 20 and 105% at 23 and 37 degrees C, respectively. Three independent statistical methods showed that the probability of specimen failure in PF specimens, defined as a loss of 20% or more of HCV RNA, was lowest at 4 degrees C and increased with increasing temperature. The HCV RNA quantification of the 11 NPF specimens stored at 4 degrees C was similar to their frozen controls. CONCLUSION: HCV RNA in separated serum specimens is stable for at least 4 days at 4 degrees C.

Effect of multiple freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA technology.

Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (P < 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable.

Multi-measurement method comparison of three commercial hepatitis B virus DNA quantification assays.

Serum specimens (327) from patients with chronic hepatitis B were evaluated for hepatitis B virus (HBV) DNA using three commercial assays--Chiron Quantiplex (CA), Digene Hybrid Capture (DA) and Abbott HBV DNA assay (AA). The HBV DNA values obtained following evaluation were used to compare the linearity, responsiveness and precision of each assay and to determine the conversion between the three different assay values. The comparison was accomplished using a new statistical approach termed the multi-measurement method (MMM). MMM is a minimal bias, non-linear regression technique that allows simultaneous multiassay performance evaluation as well as assay value interconversion. MMM analysis demonstrated that the CA was more sensitive and responsive than either the DA or the AA. Both the CA and DA were more precise than the AA. Validation of the MMM results was performed using two additional data sets of 551 and 100 specimens, respectively. MMM is a novel statistical tool that has a broad application for the generation of statistically normalized laboratory data and for interassay standardization.

Clinical evaluation of the branched DNA assay for hepatitis B virus DNA detection in patients with chronic hepatitis B lacking hepatitis B e antigen and treated with interferon-alpha.

The aim of this study was to evaluate the Chiron branched DNA (bDNA) assay for detection of serum hepatitis B virus (HBV) DNA in patients with chronic hepatitis B lacking hepatitis B e antigen (HBeAg) and undergoing interferon (IFN) therapy. Results obtained with the bDNA assay were compared with those obtained using the Abbott liquid hybridization (LH) assay and the polymerase chain reaction (PCR). Serial samples (274) from 34 patients were analysed. Analysis of variance results indicated that bDNA values were more significantly correlated than LH values with both PCR positive/negative results (probability of artifact (Prob > F) = 0.7 and 0.09 for LH and bDNA assays, respectively) and presence/absence of precore mutations (Prob > F = 0.21 and 0.001 for LH and bDNA assays, respectively). Both bDNA and LH results correlated highly with alanine aminotransferase (ALT) values (both had Prob > F values of 0.0) while PCR was not correlated with ALT (Prob > F = 0.05). In 26 evaluable patients, a model based on a generalized Knodell score was used to predict response to IFN therapy, as defined by normalization of ALT values during therapy. This model discriminated well between non-responders and responders. The bDNA results correlated well with the generalized Knodell score, while the LH results did not (Prob > F = 0.04 and 0.19 for the bDNA and LH assays, respectively). In conclusion, the bDNA assay appears to be useful for quantification of HBV DNA levels in HBeAg-negative chronic hepatitis as it correlates with biochemical and histological indications of disease severity as well as with response to IFN therapy.

Statistical models for predicting a beneficial response to interferon-alpha in patients with chronic hepatitis B.

Therapy with interferon-alpha has been reported to induce remissions in 35% of patients with chronic hepatitis B. The ability to identify patients likely to respond would be helpful in making recommendations for treatment. In this statistical analysis we included 82 patients with chronic hepatitis B who received interferon-alpha in clinical trials at the National Institutes of Health between 1984 and 1991. A response was defined as the loss of hepatitis B virus (HBV) DNA and hepatitis B e antigen (HBeAg) within 1 year of therapy. Multiple clinical parameters measured at pretreatment (month 0) and after the first month (month 1) of therapy were selected by stepwise regression to support the development of the prognostic models: the two-stage logistic regression model and a neural network that utilized higher-order non-linear interactions between variables. Among the 82 patients, 24 (29%) were responders. The two-stage logistic model using pretreatment variables: sex, hepatic fibrosis and alanine aminotransferase (ALT) levels correctly identified 61% of responders and 76% of non-responders. When HBV DNA at month 1 along with sex, initial ALT and fibrosis was included, the resultant model correctly identified 69% of responders and 77% of non-responders. The neural network, by incorporating interactions between variables, correctly identified 77% and 86% of responders, and 87% and 92% of non-responders, using pretreatment factors alone and the combination of pretreatment and month 1 factors respectively. Hence, the neural network was more accurate than the simple logistic regression model in predicting a response to interferon-alpha in chronic hepatitis B. The universality of these models needs to be further verified.

Assessment of hepatitis B virus DNA stability in serum by the Chiron Quantiplex branched-DNA assay.

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log10 HBV DNA dynamic range, were thawed and stored at -70, 4, 23, 37, and 45 degrees C (+/-1.5 degrees C) for 0, 24, 72, and 120 h (+/-2 h) and were refrozen at -70 degrees C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4 degrees C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at < or =4 degrees C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45 degrees C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at < or =4 degrees C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of > or =20% at 23 or 37 degrees C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at -70 or 4 degrees C.