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.

Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.

To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)-mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES-mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES-mediated translation.

Reflux episodes are similar in healthy African Americans and non-Hispanic whites.

Ambulatory esophageal pH monitoring is the current gold standard diagnostic exam for gastroesphageal reflux disease. Presently, no data are available for normal 24-hour esophageal pH monitoring among any US ethnic group. The aim of the present study was to obtain normal values of 24-hour esophageal pH monitoring in healthy adult African American (AA) volunteers and compare these with values obtained in healthy non-Hispanic white (nHw) volunteers to determine if ethnic variation exists in 24-hour esophageal pH testing. Twenty-four-hour dual esophageal pH monitoring was performed in healthy AA and nHw. Values for total number of reflux episodes, episodes longer than 5 min, total reflux time in minutes, and longest reflux episode in the proximal and distal esophagus were obtained for both ethnic groups. Differences between groups were considered significant if P < 0.05. Eighty subjects volunteered for the study and completed 24-hour pH testing. Forty-one were AAs and 39 were nHws, with males making up 49% of each group. The AAs were older and had higher body mass index than the nHws. No difference was observed between the AA and the nHw subjects for any measured pH parameter in either the proximal or distal esophagus. There is no difference in values obtained during esophageal pH monitoring in healthy African Americans and non-Hispanic whites. This indicates that the currently accepted normal values of ambulatory esophageal pH monitoring are readily applicable to African Americans and can be used without compromising diagnostic accuracy in this ethnic group.

Diagnosis of pancreatic cancer and prediction of unresectability using the tumor-associated antigen CA19-9.

Marked elevations of the tumor-associated antigen CA19-9 are relatively specific for pancreatic carcinoma and are associated with more advanced malignancies. We retrospectively reviewed 53 patients with CA19-9 values > 90 U/ml in whom the test had been done because of clinical suspicion of pancreatic malignancy. Pancreatic cancer was found in 45 patients (85%). If a cutoff value of CA19-9 > 200 U/ml is used, 36 of 37 (97%) patients had pancreatic cancer. Thirty patients with pancreatic cancer and no radiographic criteria of unresectability underwent attempted resection; five of these patients were judged to be potentially resectable and four of them underwent attempted resection. In only one patient with a CA19-9 value > 300 U/ml was resection possible; this patient had advanced carcinoma. Our results suggest that, in patients in whom the clinician suspects pancreatic carcinoma, CA19-9 > 90 U/ml is highly suggestive of pancreatic malignancy, while CA19-9 > 200 U/ml is virtually diagnostic of pancreatic malignancy. In similar patients with CA19-9 > 300 U/ml, resection is rarely possible and tumors are advanced.

Treatment of chronic hepatitis.

Chronic hepatitis is an etiologically diverse syndrome. The approach to treatment depends on the cause of the disease. The efficacy of immunosuppressive treatment of chronic autoimmune hepatitis has long been established, and most patients with this disease can be treated successfully with prednisone and azathioprine. Interferon therapy has revolutionized the treatment of chronic viral hepatitis. Although the response in chronic hepatitis delta is disappointing, hepatitis C is often controlled, and certain patients with chronic hepatitis B may actually be cured of the disease. Future studies will seek to optimize the therapeutic effects of interferon in these viral diseases. Certainly, studies with other antiviral agents and biologic response modifiers are forthcoming. We have entered a new era in the treatment of chronic liver disease. It is reasonable to hope and expect that progress will continue and that most forms of chronic viral hepatitis will become curable within the next several years.

Isolation of keratinophilic fungi from floors in Roman kindergarten and secondary schools.

We examined the dust collected from the floors of forty classrooms, twenty in kindergarten schools (children aged 2-5) and twenty in secondary schools (students aged 11-14) in order to determine the diffusion of keratinophilic fungi in respect to such different factors as human presence and children's age. In the kindergarten schools 268 colonies of keratinophilic fungi were isolated: 50 were Microsporum, 6 Trichophyton and 212 Chrysosporium species. Members of the Chrysosporium genus were found the widely diffused. It is interesting to note the isolation of M. gypseum in two schools. In the secondary schools 847 colonies of keratinophilic fungi were isolated: 727 were Chrysosporium, 81 Microsporum, 38 Trichophyton and 1 Epidermophyton species. Again the Chrysosporium species were the most widely diffused. It is remarkable to point out the isolation of pathogenic species such as Epidermophyton floccosum, Microsporum canis, Microsporum gypseum and the rather rare Microsporum vanbreuseghemii.

Isolation of keratinophilic fungi from floors in Roman primary schools.

Keratinophilic fungi are present in the environment with variable distribution patterns that depend on different factors, one of which, of fundamental importance, is human and or animal presence. The present study was conducted in the environment and classrooms of schools in order to evaluate the relationship between the human presence and the presence of keratinophilic fungi. In order to achieve this goal, a new isolation technique was used. From 20 samples, 253 colonies of keratinophilic fungi were isolated. The results showed that species of the genus Chrysosporium were present in 100% of the samples, while Microsporum and Trichophyton species were present in 40% and 65% of the samples respectively. The percentage of three pathogenic species, M. canis (25), T. mentagrophytes (10) and M. gypseum (10) was significant. The other species isolated were: T. terrestre (55%), Trichophyton sp. (35%), M. cookei (25%) and T. ajelloi (10%). A correlation between the amount of gathered dust and the number of colonies of keratinophilic fungi isolated was not found.