HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

A cluster of fulminant Clostridium difficile colitis in an intensive care unit in Italy.

We describe, for the first time, a cluster of lethal fulminant health-care associated Clostridium difficile (CD) colitis in Italy, observed in the intensive care unit (ICU) of an Italian tertiary care hospital in Rome. For all cases the cause of ICU admission was CD-related septic shock. Three out of seven patients were residents in a long-term care facility in Rome, and the others had been transferred to the ICU from different medical wards of the same hospital. Five patients died within 96 h of ICU admission. Because of a clinical deterioration after 4 days of adequate antibiotic therapy, two patients underwent subtotal colectomy: both of them died within 30 days of surgical intervention. In four cases, ribotyping assay was performed and ribotype 027 was recognized. This high mortality rate could be attributable to three findings: the extent of disease severity induced by the strain 027, the delay in antimicrobial therapy administration, and the lack of efficacy of the standard antibiotic treatment for fulminant CD colitis compared to an earlier surgical approach. In order to contain a CD infection epidemic, control and surveillance measures should be implemented, and empirical therapy should be administered. Because of potential 027 ribotype CD spread in Italy, CDI should be regarded with a high index of suspicion in all patients presenting with shock and signs or symptoms suggesting abdominal disease, and an early surgical approach should be considered.

Recommendations for the management of acute hepatitis B: position paper of the Italian Society for the Study of Infectious and Tropical Diseases (SIMIT).

PURPOSE: To develop recommendations for the management of acute hepatitis B by the Italian Society for the Study of Infectious and Tropical Diseases. METHODS: Development of the recommendations divided into three levels of evidence according to the GRADE system: A (high), B (medium) and C (low experts opinion), together with three recommendation levels: 1 (strong), 2 (medium), 3 (weak). RESULTS: The treatment with antivirals is in selected cases the mainstay of management of severe acute hepatitis, and should be started as a matter of urgency in order to prevent death. CONCLUSIONS: These recommendations are meant to provide the rationale and practical indications for the management of acute hepatitis B (AHB).

Sex disparity and drug-induced liver injury.

Drug-induced liver injury (DILI) is a potentially serious clinical condition that remains a major problem for patients, physicians and those involved in the development of new drugs. Population and hospital-based studies have reported incidences of DILI varying from 1.4 to 19.1/100.000. Overall, females have a 1.5- to 1.7-fold greater risk of developing adverse drug reactions and the female/male ratio increases after the age of 49 years, suggesting a clear susceptibility of DILI after menopause. Sex differences in pharmacokinetics and pharmacodynamic, sex-specific hormonal effects or interaction with signalling molecules that can influence drug efficacy and safety and differences in abnormal immune response following drug exposure are the main probable causes of the higher vulnerability observed among female patients. A novel phenotype of autoimmune-mediated DILI following the use of check-point inhibitors in oncology and haematology has been recently described. Finally, there have been increasing reports of DILI associated with use of herbal and dietary supplements that is more frequently reported in women.

Recommendations for screening, monitoring, prevention, prophylaxis and therapy of hepatitis B virus reactivation in patients with haematologic malignancies and patients who underwent haematologic stem cell transplantation-a position paper.

SCOPE: Hepatitis B virus (HBV) infection reactivation is associated with high morbidity and mortality in patients with haematologic malignancy and/or haematopoietic stem cell transplantation (HSCT). However, information on this issue is limited. The scope of this position paper is to provide recommendations on HBV screening, monitoring, prophylaxis, treatment and vaccination in the patients described above. METHODS: These recommendations were developed from one meeting of experts attended by different Italian scientific societies as well as from a systematic literature review (of articles published through December 31, 2016) on HBV infection in haematologic patients and in patients who underwent haematopoietic stem cell transplantation published in the same issue of the journal. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess each recommendation's quality. QUESTIONS ADDRESSED: These recommendations provide the answers to the following questions: (a) HBV screening and monitoring: Who should be screened before chemotherapy? Which screening tests should be used? Should HBV-DNA detection be used to monitor HBV reactivation before starting antivirals? What is the best timeline to monitor HBV reactivation? (b) Prophylaxis in HBsAg-positive patients: Which antiviral drugs should be used to treat HBsAg-positive patients? How long should antiviral prophylaxis be provided to HBsAg-positive patients? (c) Prophylaxis in patients with resolved HBV infection: Which patients with resolved HBV infection should receive antiviral prophylaxis? Which antiviral drug should be used? How long should antiviral prophylaxis be provided? (d) HBV infection management strategy in autologous (auto-HSCT) and allogeneic HSCT (allo-HSCT): Which HSCT recipients should receive antiviral prophylaxis? Which antiviral drug should be used? How long should antiviral prophylaxis be provided? (e) Choice of antiviral drugs in the treatment of HBV reactivation: Should third-generation anti-HBV drugs be preferred to first- or second-generation antiviral drugs in the treatment of HBV reactivation with or without hepatitis flare in haematologic patients? (f) Immunization against HBV in patients with haematologic malignancies and/or patients who underwent HSCT: Should these patients be vaccinated? Which HBV vaccination schedule should be adopted? RECOMMENDATIONS: Haematologic patients should be screened for hepatitis B surface antigen (HBsAg) plus anti-hepatitis B core protein (HBc), and HBV DNA before chemotherapy. HBV DNA levels should be monitored monthly in all HBV-positive patients who do not receive prophylaxis. HBsAg-positive haematologic patients and those undergoing HSCT should receive third-generation antiviral therapy as prophylaxis. Anti-HBc-positive lymphoma patients and those receiving HSCT should receive antiviral prophylaxis. All HBV-negative haematologic patients should be vaccinated for HBV. The acquisition of data from well-designed studies is desirable in the near future.

Fatal acute hepatitis C virus infection in patients with hematological malignancies.

Patients in hematology units are at risk of hepatitis C virus infection. In these patients acute infection is reportedly mild, presents only moderately increased ALT levels, is characterized by a significant delay in anti-HCV seroconversion and does not influence the course of the underlying disease. We describe two fatal cases of acute HCV infection occurring in patients with hematologic malignancies and we hypothesize that, in a subset of immunocompromised patients, acute HCV infection may play a still unrecognized but not marginal role in contributing to death. Prospective studies are needed to define the frequency of fatal acute HCV infection among hematologic patients undergoing chemotherapy.

Validity and reliability of the Italian version of the Chronic Liver Disease Questionnaire (CLDQ-I) for the assessment of health-related quality of life.

BACKGROUND: The Chronic Liver Disease Questionnaire is a specific health-related quality of life assessment designed for patients with liver diseases. AIM: The aim of this paper is to report on the validity, reliability and sensitivity to change of the Italian version (Chronic Liver Disease Questionnaire-I) in subjects with HCV infection. SUBJECTS: The Chronic Liver Disease Questionnaire-I was administered to 350 subjects with HCV infection together with the World Health Organization Quality of Life Assessment, abbreviated version, a generic quality of life assessment. METHODS: The instrument was translated from English, backtranslated and reviewed in focus groups in the framework of a large multicentre study. Exploratory factor analysis identified five factors accounting for 65% of the variance of Chronic Liver Disease Questionnaire-I items and only partially overlapping with those found in the original version. RESULTS: The Chronic Liver Disease Questionnaire-I proved to discriminate between subjects with and without comorbid diseases at baseline (t-test = 3.59, p < 0.001). Test-retest reliability was moderate (ICC = 0.60). The Chronic Liver Disease Questionnaire-I was sensitive to change in patients who deteriorated after one month of treatment. Change in the overall Chronic Liver Disease Questionnaire-I score in deteriorated patients was correlated with changes in World Health Organization Quality of Life Assessment, abbreviated version scores in the physical, psychological and environment, but not in the social area. CONCLUSIONS: The Italian version of Chronic Liver Disease Questionnaire is a valid and reliable instrument to be used in cross-sectional and longitudinal studies.

Reversible alopecia universalis during treatment with PEG-interferon and ribavirin for chronic hepatitis C.

The combination of PEG-interferon and ribavirin is currently recommended for the treatment of chronic hepatitis C, which is a common cause of morbidity and mortality worldwide. Hair disorders have often been described during interferon therapy, which include reversible hair discoloration, hypertricosis and alopecia. Ribavirin is reported to cause photoallergic reactions. We report two cases of alopecia universalis, with complete hair loss extended to the whole body, secondary to PEG-interferon and ribavirin combination therapy for chronic hepatitis C virus infection. Both female patients were infected by genotype 1 and presented alopecia during the second half of a 48-week therapy, concurrently with low levels of ferritin and thyroid dysfunction (patient 1) or depression (patient 2). Patient 1 withdrew from the therapy on week 26 and, due to the occurrence of maculo-erythematous cutaneous eczema, underwent corticosteroid therapy with complete hair regrowth. Patient 2 completed the scheduled therapy and showed a spontaneous complete hair regrowth. It should be noted that in spite of an early (within 4 weeks of therapy) virological response, patient 1 had a disease relapse after therapy withdrawal and corticosteroid therapy, while patient 2 maintained a sustained virological response. In conclusion, interferon therapy may trigger reversible alopecia universalis in susceptible patients. However, given the benign and reversible nature of this side effect, patients who achieve a virological response should be strongly advised to complete the treatment in order to prevent disease relapse.

PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.

Isolate antibody to hepatitis C virus core antigen (C22) by RIBA-2: correlation with HCV-RNA and anti-NS5.

The presence of circulating hepatitis C virus genome (HCV-RNA), elevated ALT levels and antibodies to an NS5-derived synthetic peptide have been examined in 13 subjects with isolate positivity for antibodies to the HCV core antigen (C22) on RIBA-2 testing. All subjects were followed up for 8-18 months (mean 12.4 months). In seven subjects (54%), intermittent or persistent viremia was associated with abnormal ALT levels (6 subjects) and with positivity for antibodies to NS5-peptide (6 subjects). On the other hand, in 6 out of 13 subjects (46%) no viral replication, no liver cytonecrosis and no antibodies to NS5 were found. It is concluded that isolate reactivity to C22 by RIBA-2 is a heterogeneous condition that corresponds to two distinct categories of subjects: those with active HCV infection and those without evidence of virus replication. Although HCV-RNA determination is the most reliable means of identifying HCV carriers, antibodies to NS5 can be a useful marker of virus activity. In fact, antibodies to NS5 were detected in 6 out of 7 viremic patients, compared to 0 out of 6 non-viremic patients (P = 0.004). It remains to be elucidated whether the isolate reactivity to core antigen found in non-viremic subjects represents a specific, HCV-induced antibody response, or is an unrelated crossreactivity.

Multiple viral infections in HIV-infected children with chronically-evolving hepatitis.

Hepatic involvement was investigated in 31 children with perinatal HIV-1 infection, who were followed for 2-82 months (mean 30.5). Liver disease, as revealed by increased aminotransferase levels, liver biopsy or necroscopy, was diagnosed in 18 children (58%), of which 7 (22.5%) had acute hepatitis and 11 (35.5%) showed chronic liver disease. Overall, 40 persistently active or recurrent viral infections, as demonstrated by positive culture and/or detection of serum DNA, specific IgM, IgA and high levels of IgG, were revealed in the children with liver disease, while 12 similar infections were detected in 13 children without liver disease (p < 0.001). In particular, the children with liver disease showed a significantly (p < 0.002) higher incidence of cytomegalovirus (CMV) infections than children without liver disease (13 versus 3). Moreover, hepatitis C and B virus infections were revealed only in children with liver disease (5 and 1 patients, respectively). Clinical outcome showed a significantly (p < 0.001) higher mean survival in the children without liver disease than those with liver disease (47.5 versus 18.2 months). In fact, nine of the children with liver disease (50%) died, as opposed to only one of the children without liver disease (7.7%; p = 0.01). Based on these findings, liver disease is indicative of a poor prognosis in children with HIV infection, being related to the presence of multiple active viral infections.

Hepatitis in children with thalassemia major.

Since thalassemia major patients are transfusion dependent, they are at a particularly high risk of contracting post-transfusion hepatitis. In this study, 36 transfusion-dependent children were followed up for evidence of viral hepatitis. Of 23 with increased ALT levels, 17 were anti-CMV and 12 were anti-HCV positive, 9 were positive for both CMV and HCV. Of 13 children with normal transaminase levels, 5 were CMV positive and 3 were HCV positive. These results show that CMV may be a very common cause of non-A, non-B hepatitis in transfusion dependent thalassemic children.

Recombinant immunoblot assay for hepatitis C virus antibody in chronic hepatitis.

Testing for hepatitis C virus by ELISA requires confirmation by recombinant immunoblot assay (RIBA). The first-generation RIBA uses the same antigen as used in the ELISA and one further antigen. A second-generation RIBA in which two further antigens are present, detects positivity that is not found by either the ELISA or the original RIBA. Consequently, although it is adequate to test ELISA positive sera with the first-generation RIBA, the second-generation assay is recommended for confirming negativity.

One course versus two courses of recombinant alpha interferon in chronic C hepatitis.

Fifty-five patients with antibodies to HCV and chronic liver disease have been enrolled in the study. Thirty-four patients were treated with recombinant alpha interferon (IFN, 3 MU daily for 10 days followed by 3 MU twice/week for 3 months), and were compared to 21 untreated controls. Alanine aminotransferase (ALT) normalization was observed in a significant proportion of treated patients (52.9%), but 66.6% of them experienced a relapse after discontinuation of the therapy. The evaluation of the early ALT behavior after the 10 days priming with daily IFN administration was useful in predicting the response. The administration of a second IFN course with the same schedule and duration as the first course did not increase the efficacy of the treatment. Increased dosage and/or prolonged administration are probably required.

Hepatitis C virus infection in chronic liver disease in Somalia.

To assess the role of hepatitis C virus (HCV) in liver disease in Somalia, antibody to HCV (anti-HCV) was studied by enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) in 110 patients with chronic liver diseases, in 309 healthy adults, in 179 institutionalized subjects with a high prevalence of intestinal parasites and Schistosoma haematobium, and in 287 children with diseases other than hepatitis. According to the RIBA test, anti-HCV was present in three healthy adults (0.97%), in four institutionalized individuals (2.2%), but in none of the children. The prevalence of anti-HCV was 4.8% in patients with hepatitis B surface antigen (HBsAg)-positive chronic liver diseases and 20.6% in patients with HBsAg-negative chronic liver diseases. Thus, HCV infection appears to play a minor role in HBsAg-positive liver disease in Somalia but may be an important factor in HBsAg-negative chronic liver disease. The low anti-HCV prevalence in individuals with no hepatic disorders is consistent with the fact that HCV does not spread by nonpercutaneous transfer. We found also a large proportion of both patients with hepatic disease and institutionalized individuals who tested positive by ELISA but not confirmed by RIBA. However, the likelihood of a true positive result increases proportionally with the ELISA value; thus, in most cases a low ELISA value probably represents a false-positive reaction, while a high ELISA value probably represents a true positive reaction.

Lumbar pain in a married couple who likes cheese: brucella strikes again!

A 69-year-old man living in Florence reported fever and acute lumbar pain one month after transurethral resection of a superficial transitional cell carcinoma of the bladder. The radionuclide bone scan suggested metastatic lesions of the L3-L4 vertebrae. However cobalt treatment was ineffective. A bone biopsy of L4 showed an inflammatory pattern and antibiotic therapy was started which did not produce any clinical improvement. Six months after the onset of the back pain brucellar spondylitis was serologically diagnosed and treatment with doxycycline and streptomycin produced a significant clinical and radiological improvement. After 2 months the patient's wife presented with fever and lumbar pain, and brucellar spondylitis was diagnosed as well. An extensive epidemiological examination revealed that 8 months earlier the family had eaten unpasteurized goat cheese and serological examination of the entire family showed that 3 out of 4 members had significant titres of brucellar antibodies. Finally it was discovered that 4 months after consuming the cheese the third infected subject experienced an episode of epidydimoorchitis for which no diagnosis and effective treatment was found. This family cluster of brucellar infection indicates that a high degree of suspicion in the diagnosis of brucellosis is necessary even in non-endemic areas, to reduce the delay in the diagnosis and treatment of the disease and to prevent the occurrence of complications that may prove difficult to treat.

Risk management of HBsAg or anti-HCV positive healthcare workers in hospital.

Recommendations are made for controlling the transmission of the hepatitis B and hepatitis C viruses from healthcare workers to patients. These recommendations were based both on the literature and on experts' opinions, obtained during a Consensus Conference. The quality of the published information and of the experts' opinions was classified into 6 levels, based on the source of the information. The recommendations can be summarised as follows: all healthcare workers must undergo hepatitis B virus vaccination and adopt the standard measures for infection control in hospitals; healthcare workers who directly perform invasive procedures must undergo serological testing and the evaluation of markers of viral infection. Those found to be positive for: 1) HBsAg and HBeAg, 2) HBsAg and hepatitis B virus DNA, or 3) anti-hepatitis C virus and hepatitis C virus RNA must abstain from directly performing invasive procedures; no other limitations in their activities are necessary. Infected healthcare workers are urged to inform their patients of their infectious status, although this is left to the discretion of the healthcare worker; whose privacy is guaranteed by law. If exposure to hepatitis B virus occurs, the healthcare worker must undergo prophylaxis with specific immunoglobulins, in addition to vaccination.

Surgical procedures as a major risk factor for chronic hepatitis C virus infection in Italy: evidence from a case-control study.

OBJECTIVES: The study was carried out to evaluate the risk factors associated with chronic hepatitis C virus (HCV) infection. METHODS: This case-control study used multiple logistic regression analysis to determine risk factors associated with HCV infection. Study participants were followed at 10 liver or gastroenterologic units and included 294 subjects with chronic HCV infection and 295 age and sex matched anti-HCV-negative controls. RESULTS: The use of glass syringes and surgical procedures was reported by as many as 77.6% and 73.8% of cases, respectively; blood transfusion was recorded in nearly a quarter of cases; 10.2% of cases, but none of the controls, reported past or current intravenous drug use. Multiple logistic regression analysis showed that blood transfusion, being the sexual partner of an intravenous drug user, and surgery all were independent predictors of the likelihood of HCV infection. CONCLUSIONS: These findings indicate that, besides the well-known sources of infection, such as blood transfusion and intravenous drug use, surgical procedures may play an important role in the spread of HCV infection in Italy. Given that a large proportion of the general population undergoes surgery, a rational and relatively inexpensive policy for the prevention of HCV infection must focus on implementing efficient procedures for the sterilization of instruments and the use of disposable materials in surgical units.

Polyomavirus BK infection in end-stage renal disease: analysis of viral replication in patients on hemodialysis or peritoneal dialysis.

Patients in end-stage renal disease undergoing renal replacement treatment (ESRD-RRT) are considered immunocompromised. The hemodialysis (HD) or peritoneal dialysis (PD) procedures seem to produce alterations of the immune status. Interest in immunosuppression has increased due to the poliomavirus BK (BKV) infection. Our study evaluated the prevalence of BKV infection in ESRD-RRT patients and viral replication on HD or PD. From 2006 to 2011 we selected 58 patients (34 males) in ESRD-RRT for inclusion in our study. BKV replication was evaluated by qualitative real-time polymerase chain reaction. In ESRD-RRT patients, the prevalence of BKV replication on plasma was 21%. We identified two groups of patients according to the dialysis procedure: 36 patients on HD (HD group) and 22 on PD (PD group). BKV replication in the HD group was 33% (12 of 36) versus 0% (0 of 22) in the PD group. Different age, number of months on RRT, and preserved diuresis was observed in the HD versus PD groups. With our results we can speculate that BKV infection in ESRD-RRT patients is linked to factors involved in the uremia-related immune dysfunction but also to specific mechanisms related to the different RRTs. PD is an option that could be associated with a better transplant outcome for patients undergoing kidney transplantation.

Polyomavirus BK infection before liver transplantation in patients with chronic kidney disease.

End-stage liver disease (ESLD) and chronic kidney disease (CKD) patients are both immunocompromised populations but polyomavirus BK (BKV) replication before liver transplantation is rare. We evaluated BKV prevalence among liver transplant recipients with renal dysfunction and the possible role of CKD as a risk factor for BKV replication in ESLD. From 2010 to 2011 we selected 31 ESLD patients awaiting liver transplantation to identify, the presence of CKD: No CKD (n = 22; 18 males) and CKD group (n = 9; 5 males). BKV infection was defined on the basis of viremia evaluated using quantitative real-time polymerase chain reactions. The prevalence of viremia among the No CKD group was 14% versus 56% in the CKD group (Fisher test; P = .027). We hypothesized that the presence of CKD may represent an additional condition of immunologic dysfunction regarding antiviral surveillances other than the antibacterial one that characterizes ESLD immunodysfunction, which could have promoted BKV replication. The specific immunologic mechanisms involved in pretransplantation diseases may have a role in BKV reactivation that could become responsible for nephropathy after transplantation.