Hepatitis C Virus Core Antigen (HCVAg): an affordable assay to monitor the efficacy of treatment in DAAs era.

Hepatitis C virus (HCV) Core Antigen (HCVAg) and HCV-RNA were tested in 962 plasma/serum samples from 180 patients during Direct Antiviral Agents (DAAs) treatment and at follow-up. One hundred and eighty individuals were included: 71% carried advanced fibrosis and 43% were treatment-experienced. A Sustained Virological Response (SVR) was achieved in 166/180 (92%) individuals: 96/102 (94.1%) na ve and 70/78 (89.7%) treatment-experienced (p=0.20). The baseline median levels of HCV-RNA and HCVAg were not significantly different between individuals achieving SVR (5.92 x 105 IU/mL, IQR 5.4-6.4, and 3,417 fmol/L, 2,900-3,795) and those without SVR (6.06 x 105 IU/mL, 5.63-6.57, and 3,391 fmol/L, 2,828-4,077). The HCV-RNA vs. HCVAg assays results showed a fair correlation with an overall moderate qualitative agreement (kappa=0.52). Among treatment-failed individuals, at failure 100% of the assays results were positive for both techniques, with HCV-RNA median value 3.09 x 105 IU/mL (2.10-29.09) and HCVAg median value 1570.28 fmol/L (360.15-9317.67). Undetectable HCV-RNA at EOT showed sensitivity 54%, specificity 100%, negative predictive value (NPV) 93% and positive predictive value (PPV) 100%. Undetectable HCVAg at EOT showed sensitivity 74%, specificity 100%, NPV 97% and PPV 100%. The operative and economic advantages of the HCVAg support the alternative use of HCVAg to monitor DAAs treatment outcome.

Serum hepatitis B core-related antigen is an effective tool to categorize patients with HBeAg-negative chronic hepatitis B.

The discrimination between active chronic hepatitis B (CHB) and the clinically quiescent infection (CIB) is not always easy, as a significant portion of patients falls in a "grey" zone. Hepatitis B core-related antigen (HBcrAg) is a now quantifiable serological marker with potential applications in diagnosis and therapy monitoring. The aim of the present study was to evaluate the HBcrAg serum levels in HBeAg-negative HBV infection, and its ability in identifying the clinical profile, in comparison with HBsAg serum levels. HBcrAg was retrospectively assessed on serum samples from a population of treatment-naive HBeAg-negative patients by ChemiLuminescent Enzyme Immunoassay (CLEIA). HBsAg and HBV-DNA data were collected. Serological data were associated to clinical profile, defined in the subsequent follow-up of at least 1 year. In the overall population of 160 HBeAg-negative patients, HBcrAg results weakly correlated with qHBsAg levels (Spearman r = 0.471, P < 0.0001) and correlated closely with HBV-DNA (Spearman r = 0.746, P < 0.0001). HBcrAg levels were significantly higher in 85 CHB patients relative to 75 CIB carriers. A value of 2.5 logU/mL produced the optimal cut-off to identify CIB patients, with diagnostic accuracy comparable to HBsAg levels. In long-term clinical evaluation, a single measurement of HBcrAg at the established cut-off was optimally consistent with clinical outcome. Conversely, the HBsAg cut-off performed well in the true quiescent phase and less in more difficult-to-categorize patients. In conclusion, single-point use of HBcrAg serum levels provides an accurate identification of CIB and represents a useful tool for patient classification.

Syphilis in a high-density urban area in the North of Italy.

Although far less common now than in the past, syphilis continues to pose a danger to public health and should not be overlooked. In this study, we evaluated the presence and characteristics of syphilis in a group of patients attending an STI Clinic in the North of Italy. A retrospective study was carried out, analysing the data from the 5609 subjects who attended the STI Clinic of St. Orsola-Malpighi Hospital (Bologna) for syphilis screening from January 2016 to December 2017. Globally, 692 patients (12.3%) were found positive for treponemal tests, with a significant difference between males and females (16.6% vs 4.1%; P<0.0001). Moreover, positive women were more likely foreign (63.3%), in contrast to men, who were more likely Italian (86.1%; P<0.0001). A total of 306 patients (44.2%), mainly males (47% vs 25%; P=0.0003), received a diagnosis of early syphilis. These cases peaked among patients 35-44 years (31%) and 25-34 years (26.8%). Overall, 32.9% of the women found positive for treponemal tests were pregnant. Among them, 84.6% were foreign (mainly from Eastern Europe) and 38.4% received a diagnosis of early syphilis. No cases of mother-to-child syphilis were found. The presence of an HIV-syphilis co-infection was found in 21.5% of patients with early syphilis, with a significant association with the male sex (P<0.009). In-depth knowledge of the characteristics of syphilis could help set up effective strategies for its control.

Increase of ribavirin dose improves sustained virological response in HCV-genotype 1 patients with a partial response to peg-interferon and ribavirin.

BACKGROUND AND AIM: In patients with chronic hepatitis C receiving Peg interferon/ribavirin (PEG-IFN/RBV) who do not achieve ≥ 2 log-reduction in HCV-RNA at week 12 (null responders, NR) and in those with ≥ 2 log-decrease but detectable at week 24 (partial responders, PR) the probability to achieve the sustained virological response (SVR) is almost null. The aim of this study was to investigate the efficacy of individualized schedule of progressively increased RBV doses in the setting of PEG-IFN/RBV treatment. MATERIAL AND METHODS: PR or NR to PEG-IFN/RBV instead of discontinuing treatment were enrolled to receive increasing doses of RBV until a target theoretical concentration ([tRBV]) of ≥ 15 µmol/L (by pharmacokinetic formula based on glomerular filtration rate). HCV-RNA was assessed every 4 weeks and, if detectable, RBV dose was gradually increased until negativization. Twelve weeks later, patients with detectable HCV-RNA discontinued therapy while those with undetectable HCV-RNA continued for further 48 weeks. RESULTS: Twenty genotype-1 patients (8 NR and 12 PR) were enrolled. After 12 weeks 9 (45%) were still HCV-RNA positive and were discontinued, while remaining 11 had undetectable HCV-RNA. One stopped treatment for side effects. Ten completed treatment. Five (all PR) achieved SVR. Side effects incidence was similar to that observed during PEG-IFN/RBV. CONCLUSIONS: In conclusion, RBV high doses, according to individualized schedule, increase SVR in PR on a similar extent to that of triple therapy but without increase of side effects. Such treatment should be considered in PR with no access or intolerant to protease inhibitors (PI).

Recent and long-lasting infections: the need for avidity testing in HIV-1 infected subjects.

Standard serological tests have reached high levels of sensitivity and reproducibility but do not indicate whether infection is recent or long-standing. Among the 59960 sera analyzed for HIV positivity at the Retrovirus Laboratory, Operative Unit of Microbiology, Bologna, Italy, from January 2010 to July 2011, 134 samples showed an initial positive result. Application of the avidity test, able to distinguish between recent or long-standing HIV infection, classified 59 subjects as recently infected and 75 as chronically infected. Besides all the public health implications, the distinction between acute and chronic infection might serve to establish the time of infection and therefore reach any potential partners who might have been infected in a specific period of time. Although our results are limited to subjects referred to our laboratory and hence represent only a limited part of the problem, the routine application of methods able to distinguish recent from long-lasting infection could help monitor disease incidence, identify high-risk groups, and enhance epidemiological conclusions.

A randomized trial of induction doses of interferon alone or in combination with ribavirin or ribavirin plus amantadine for treatment of nonresponder patients with chronic hepatitis C.

BACKGROUND: Efficacy and safety of interferon induction therapy alone or in combination with ribavirin or ribavirin plus amantadine were evaluated in chronic hepatitis C patients who were nonresponders to primary antiviral treatment. METHODS: The study was designed to have 225 HCV nonresponder patients, but at an interim analysis the response rate difference between groups was lower than expected and the enrollment was stopped when 75 patients had been randomized to receive interferon-alpha2a (group A, n = 26), interferon-alpha2a plus 15 mg/kg per day of ribavirin (group B, n = 24), or interferon-alpha2a plus ribavirin plus 200 mg/day of amantadine hydrochloride (group C, n = 25). Treatment duration was 48 weeks. The dose of interferon was 6 MU/day for 4 weeks followed by 3 MU/day for the remaining 44 weeks. RESULTS: On intention-to-treat, the sustained virological response at 24 weeks of follow-up was 11.5% in group A, 12.5% in group B, and 12% in group C. Therapy was discontinued because of adverse effects in three patients in group A (11.5%), three in group B (12.5%), and two in group C (8%). CONCLUSIONS: Nonresponders with chronic hepatitis C may achieve a sustained virological response rate of approximately 12% if retreated with interferon induction treatment followed by administration of a daily dose. The addition of ribavirin or amantadine did not seem to improve the response rates.

Monitoring the treatment of hepatitis C with directly acting antivirals by serological and molecular methods.

AIM: To evaluate the potential value of using a serological assay to quantitate the hepatitis C virus core antigen (HCV-Ag) when monitoring patients with chronic hepatitis C being treated with direct-acting antivirals (DAAs). METHODS: Ninety-six patients treated with DAAs, either alone (91) or in combination with PEG interferon (5), were tested for HCV-RNA and for HCV-Ag at baseline and at weeks 2, 4, 8 and 12 during treatment and 12 weeks after completion. The concordance and correlation between the viral parameters as well as the respective kinetics during and after treatment were evaluated. RESULTS: A sustained viral response (SVR) was achieved in 82 patients (91%), whereas 11 relapsed (R) and 1 showed a virological breakthrough while receiving treatment. HCV-RNA and HCV-Ag showed good concordance (kappa = 0.62) and correlation. No significant differences between SVR and R was observed in either assay at 2 and 4 weeks after the start of treatment. At 8 weeks, HCV-Ag showed higher accuracy than HCV-RNA (AUC: 0.74 vs. 0.55) and there was a significantly greater decrease from baseline in SVR than in R (4.01 vs. 3.36 log10; p<0.05). CONCLUSIONS: Monitoring during treatment with DAAs by using either HCV-RNA or HCV-Ag has only a limited predictive value for SVR. Since those assays are equivalent for identifying a virological relapse, HCV-Ag may be preferred from an economical and organizational perspective.

Hepatitis B virus reactivation among hepatitis C patients treated with direct-acting antiviral therapies in routine clinical practice.

BACKGROUND: Hepatitis B (HBV) reactivation in chronic hepatitis C (CHC) patients treated with IFN-free direct acting antiviral (DAA) therapies has recently emerged as a potential risk. Given the potential burden of this issue, further data are needed to establish its actual clinical impact. OBJECTIVES: The aim of the present study was to analyze the occurrence of HBV reactivation in a cohort of CHC patient treated with DAAs in routine clinical practice. STUDY DESIGN: Consecutive CHC patients with different genotypes, treated with DAA between January 2015 and January 2016 were included in the study. Subjects had been tested for HBsAg and anti-HBc antibodies before antiviral therapy. HBV-DNA levels were examined in anti-HBc positive patients at baseline and 24 weeks after the end of treatment. Post-treatment HBsAg determination was performed in case of HBV-DNA positivity. Serum anti-HBs kinetics was analysed in anti-HBs and anti-HBc positive subjects. RESULTS: A cohort of 137 consecutive HCV patients treated with IFN-free regimens in routine clinical practice was evaluated. From this cohort, plasma samples of 44 subjects with positive serology for HBV (anti-HBc positive) were tested for HBV-DNA levels at baseline and 24 weeks after the end of treatment. Two of them were HBsAg-positive, while the others had signs of a past HBV exposure (HBsAg-negative±HBsAb-positive). No reactivation was found in HBcAb-positive and HBsAg-negative subjects. In the two HBsAg-positive, one experienced an increase in HBV-DNA levels of ≥2 log10 IU/mL during treatment. However, the reactivation was without clinical impact and, most important, was followed by HBsAg loss. CONCLUSIONS: Based on our experience, a past HBV infection seems not to be a condition predisposing to HBV reactivation. On the contrary, in HBsAg-positive subjects not in suppressive treatment with nucleos(t)ide analogs, regular monitoring of HBV-DNA during and after DAA treatment should be considered.

Patterns of HCV-RNA and HCV core antigen in the early monitoring of standard treatment for chronic hepatitis C.

BACKGROUND: An early drop of HCV-RNA levels is useful in assessing response to antiviral treatment in chronic hepatitis C; the first recommended time point is 4 weeks after the start of therapy. OBJECTIVES: We evaluated retrospectively HCV-RNA and HCVAg levels at different time points to assess the clinical value of an early monitoring. STUDY DESIGN: Thirty-five patients with chronic hepatitis C infected by genotype 1b and consecutively enrolled in an open-label study on PegIFN plus Ribavirin and/or ketoprofene were tested for HCV-RNA (real-time PCR) and HCVAg (ARCHITECT) at baseline and after 1 and 2 days and 1, 2, 4 and 12 weeks after the start of treatment. Treatment response was assessed according to the EASL consensus criteria. RESULTS: In the 17 sustained responders (SR) the median log decrease of HCV-RNA and HCVAg at the different time points was 0.40 and 0.37; 1.36 and 0.84; 1.47 and 0.97; 2.34 and 1.86; 2.51 and 2.32; 3.28 and 2.61, respectively. The best time point to predict SR was 2 weeks after the start of therapy, with a sensitivity, specificity and overall accuracy of 76.9%, 86.7% and 82.1% for HCV-RNA and 81.8%, 75.0% and 76.8% for HCVAg, respectively. DISCUSSION: An early monitoring is at least equally effective than standard monitoring in predicting response to hepatitis C therapy. The similarity of HCV-RNA and HCVAg kinetics suggests that HCVAg may be useful in the early phases as a trigger to evaluate HCV-RNA levels at earlier time points for a personalized approach to therapy monitoring.

Virus-specific immune response in HBeAg-negative chronic hepatitis B: relationship with clinical profile and HBsAg serum levels.

BACKGROUND AIMS: The immune impairment characterizing chronic hepatitis B (cHBV) infection is thought to be the consequence of persistent exposure to viral antigens. However, the immune correlates of different clinical stages of cHBV and their relation with different levels of HBsAg have not been investigated. The aim of the present study was to evaluate the relationship between HBV-specific T cells response and the degree of in vivo HBV control and HBsAg serum levels in HBeAg-HBeAb+ cHBV. METHODS: Peripheral blood mononuclear cells from 42 patients with different clinical profiles (treatment-suppressed, inactive carriers and active hepatitis) of cHBV, 6 patients with resolved HBV infection and 10 HBV-uninfected individuals were tested with overlapping peptides spanning the entire HBV proteome. The frequency and magnitude of HBV-specific T cell responses was assessed by IFNγ ELISPOT assay. Serum HBsAg was quantified with a chemiluminescent immunoassay. RESULTS: The total breadth and magnitude of HBV-specific T cell responses did not differ significantly between the four groups. However, inactive carriers targeted preferentially the core region. In untreated patients, the breadth of the anti-core specific T cell response was inversely correlated with serum HBsAg concentrations as well as HBV-DNA and ALT levels and was significantly different in patients with HBsAg levels either above or below 1000 IU/mL. The same inverse association between anti-core T cell response and HBsAg levels was found in treated patients. CONCLUSIONS: Different clinical outcomes of cHBV infection are associated with the magnitude, breadth and specificity of the HBV-specific T cell response. Especially, robust anti-core T cell responses were found in the presence of reduced HBsAg serum levels, suggesting that core-specific T cell responses can mediate a protective effect on HBV control.

Hepatitis C virus core antigen: analytical performances, correlation with viremia and potential applications of a quantitative, automated immunoassay.

BACKGROUND: Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE: To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN: Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS: The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS: HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.

Ketoprofen, peginterferon 2a and ribavirin for genotype 1 chronic hepatitis C: a phase II study.

AIM: To evaluate the safety of adding ketoprofen to pegylated-interferon (PEG-IFN) with or without ribavirin and the effect on viral kinetics, STAT1 activity and expression of 2'-5'-oligoadenylate synthetase (2'-5'OAS) in genotype 1 chronic hepatitis C in a phase II study. METHODS: Forty-five patients were studied: fifteen were randomized to PEG-IFN plus ribavirin (PR), 16 to PEG-IFN plus ketoprofen and 14 to PR and ketoprofen. The molecular study of IFN-dependent signal transduction was conducted in 9 patients from each group. RESULTS: The combination of ketoprofen and PEG-IFN with or without ribavirin was safe and well tolerated. An early activation of STAT1 was observed in ketoprofen-treated patients, but this activation was less sustained over time. Conversely, ketoprofen plus PEG-IFN and ribavirin induced an early and sustained increase of 2'-5'OAS transcription starting 24 h after the first dose until the 36th wk. These data are consistent with the clinical results, showing a better sustained virological response and a lower relapse rate in patients receiving ketoprofen plus PEG-IFN and ribavirin. CONCLUSION: The addition of ketoprofen to the standard therapy of chronic hepatitis C should be explored in larger randomized clinical studies.

Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples.

This paper describes the development of a SYBR Green-based multiplex real time RT-PCR for the simultaneous detection of HCV and HIV-1 genomes in plasma samples. Viral genomes were identified in the same sample by their distinctive melting temperature (Tm) which are 81.6 and 86.5 degrees C for HIV-1 gag 142 bp amplicon and HCV 5'-NCR region 226 bp amplicon, respectively. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of both HIV-1 and HCV. In addition, we also assayed HIV-1 and HCV viral load in 30 co-infected patients and in 15 blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HIV-1/HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors.

Slowly tapering off steroids protects the graft against hepatitis C recurrence after liver transplantation.

Chronic hepatitis C represents a major clinical problem after liver transplantation, but factors influencing the recurrent disease have not been well characterized. We analyzed the clinical records of all the patients transplanted for hepatitis C virus (HCV)-related liver disease in our Center between 1991 and 1997. Eighty consecutive HCV-positive (+) patients (60 men, ages 28 to 64) survived more than 1 month after transplantation and were followed for a median of 45 months. Diagnosis of recurrent chronic hepatitis C was made in 38 patients (47.5%), of whom 22 had moderate/severe chronic hepatitis. Decompensated cirrhosis occurred in six patients (7.5%). No difference in patient survival was found between patients with and without hepatitis C recurrence. No association was found between recurrent hepatitis C and presumed risk factors. The method of tapering off corticosteroids was significantly associated with both hepatitis C recurrence and the severity of hepatitis. In patients receiving a higher daily prednisone dose, 12 months after transplantation, the proportion of recurrent hepatitis C was 35.7% versus 66.6% (P = .02; odds ratio [OR], 3.6; 95% confidence interval (CI): 1.25 to 10.36), and among patients receiving a higher daily prednisone dose, 6 months after transplantation, the proportion of moderate/severe chronic hepatitis C was 40% versus 89% (P = .03; OR: 0.08, 95% CI: 0.008 to 0.84). Finally, prednisone dose at month six was significantly associated with disease-free survival of the liver graft. In conclusion, our results seem to indicate that in HCV-infected liver transplant recipients, a long-term treatment with corticosteroids, slowly tapered off over time, may prevent the more aggressive forms of recurrent liver disease.

Inter-laboratory comparison of HCV-RNA assay results: implications for multi-centre research.

To investigate whether it is appropriate to assume comparability of hepatitis virus C (HCV)-RNA results across laboratories in multi-centre studies, nine laboratories of the European Paediatric HCV Network participated in an international proficiency study of HCV-RNA assays. A panel of 12 samples of different dilutions and genotypes was sent to each laboratory and tested with qualitative and/or quantitative HCV-RNA assays according to local procedures. Commercial assays were used in seven laboratories and in-house assays in two. All six laboratories in which a commercial qualitative assay was used were proficient, as were four of six runs (in five laboratories) in which a commercial quantitative assay was used. The proficiency of the laboratories where in-house assays were used could not be assessed according to the VQC definition because of differences in the methods used. Overall, there were several false-negative results, but only one false-positive result with a quantitative assay and none with a qualitative assay. The false-negative results may have implications for the diagnosis of infection, and highlight the need for an antibody test to be performed at 18 months to confirm the absence of infection. The results of qualitative assays were generally consistent across laboratories but it was difficult to evaluate and compare the results of quantitative assays. Multivariate analysis of data collected in multi-centre studies should therefore allow for centre and/or assay used.