Real-life 3D therapy failure: Analysis of NS5A 93H RAS plus 108 K polymorphism in complex with ombitasvir by molecular modeling.

We report a real-life 3D therapy failure in a patient treated with ombitasvir (OMV)/paritaprevir/ritonavir and dasabuvir without ribavirin (3D-R). He had therapy failure at week 12 after the end of treatment. We detected resistance-associated substitutions (RASs) plus polymorphisms on NS3, NS5A, and NS5B target regions by population sequencing (15% cut-off) at baseline, at relapse and during follow-up. About this, NS5A RASs generally persist longer than resistances in the other target genes and may impact treatment outcome. Therefore, to evaluate OMV drug-resistance mechanism, we studied the acquired RAS plus polymorphisms on NS5A phosphoprotein by computational studies. OMV showed a higher affinity towards baseline and 93H/108 K mutant structure (follow-up) with respect to 93H/R108 mutant structure (relapse) on phosphoprotein. By Molecular Dynamics simulations (MDs), structural information about the protein stability in presence of OMV were observed. According to our data, molecular modeling approach has proved to be a powerful method to evaluate the impact of these RASs plus specific amino acid (AA) changes on phosphoprotein.

Mumps Outbreak Among a Highly Vaccinated University Community-New York City, January-April 2014.

Background: On 14 January 2014, a vaccinated student presented with parotitis. Mumps immunoglobulin M (IgM) testing was negative and reverse-transcription polymerase chain reaction (RT-PCR) testing was not performed, resulting in a missed diagnosis and the start of an outbreak at a New York City (NYC) university. Methods: Mumps case investigations included patient interviews, medical records review, and laboratory testing including mumps serology and RT-PCR. Case patients were considered linked to the outbreak if they attended or had epidemiologic linkage to the university. Epidemiologic, clinical, and laboratory data for outbreak cases residing in NYC were analyzed. Results: Fifty-six NYC residents with mumps were identified with onset between 12 January and 30 April 2014. Fifty-three cases (95%) were university students, 1 (2%) was a staff member, and 2 (4%) had epidemiologic links to the university. The median age was 20 years (range 18-37 years). All cases had parotitis. Three cases were hospitalized, including 1 of 2 cases with orchitis. Fifty-four (96%) cases had received ≥1 mumps-containing vaccine, 1 (2%) was unvaccinated due to religious exemption, and 1 (2%) had unknown vaccination status. Two of the 44 (5%) cases tested by serology were mumps IgM positive, and 27 of the 40 (68%) tested by RT-PCR were positive. Conclusions: Mumps outbreaks can occur in highly vaccinated populations. Mumps should be considered in patients with parotitis regardless of vaccination status. RT-PCR is the preferred testing method; providers should not rely on IgM testing alone. High vaccination coverage and control measures likely limited the extent of the outbreak.

Present, old and future strategies for anti-HCV treatment in patients infected by genotype-1: estimation of the drug costs in the Calabria Region in the era of the directly acting antivirals.

BACKGROUND: In Italy, anti-HCV drugs are provided free of charge by the National Health System. Since 2011, three drug regimens including a directly acting antiviral (DAA) are considered the gold standard for HCV treatment. However, these drugs add a significant cost (roughly €26,000) to the combination of pegylated-interferon-α/ribavirin (PEG-IFN/RBV), which before DAA represented the unique treatment. To provide the National Health System potential useful information, we estimated costs to provide anti-HCV drugs to treat a population experienced for PEG-INF/RBV. METHODS: Genotype 1 HCV mono-infected or HIV/HCV co-infected individuals who were treated with PEG-IFN/RBV between 2008 and 2013 were included. The cost to treat these patients with PEG-IFN/RBV was calculated (cost 1). We also estimated costs if we had to treat these patients with a lead-in period of PEG-INF/RBV followed by PEG-IFN/RBV and a DAA in naïves (cost 2), in addition to cost 1 plus the estimated cost to re-treat with PEG-IFN/RBV and a DAA patients who had a relapse or a non response (cost 3). Moreover, all costs were normalized by SVR. Rates of foreseen response with DAA were obtained from literature data. RESULTS: The overall study population consisted of 104 patients. The rate of sustained virological response (SVR) was 55%, while it was estimated that SVR would be obtained in 75% of patients with a lead-in period with PEG-IFN/RBV followed by a DAA combination, and in 78% if this treatment is used to re-treat experienced patients with a DAA. Drug costs associated with these treatments were: €1,214,283 for cost 1, €3,474,977 for cost 2 and €3,002,095 for cost 3. Costs per SVR achieved were: €22,284 for cost 1, €44,643 for cost 2 and €38,322 for cost 3. CONCLUSIONS: Treatments including DAAs achieve a SVR in more patients than PEG-IFN/RBV but they cost around three times more than PEG-IFN/RBV alone regimens. Also, cost per SVR is almost twofold greater than PEG-IFN/RBV regimens. Therefore, it is mandatory to implement use of DAA in clinical practice, but the National Health System should allocate adequate resources to provide drugs, which challenges sustainability. Cost reduction for anti-HCV drugs should be pursued.

Update on epidemiology of HCV in Italy: focus on the Calabria Region.

The epidemiological profile of HCV infection is evolving in Europe, as well as in Italy. We have previously showed genotype distributions and their dynamics in 2,153 HCV RNA positive patients living in Calabria, Southern Italy, over 11 years. In this study, we extend and update this information by evaluating a hospital-based cohort of 945 HCV RNA positive patients attending five hospitals in the Calabria Region from January 2011 to August 2013. We assessed rates of HCV genotypes according to age and gender and the dynamics of HCV genotype distribution over the 3-year period studied. Data showed that genotype 1b is the most prevalent, followed by subtypes 2a/2c and genotype 3. Genotype 4 exhibited an increase between 2011 and 2013. Also, we found a significant decrease in the median age of subjects infected with HCV genotype 3 and 4 during the period studied. Since HCV genotypes are important in epidemiology, pathogenesis and response to antiviral therapy, a continuous epidemiological surveillance is needed.

Public Health Consequences of a 2013 Measles Outbreak in New York City.

Importance: Internationally imported cases of measles into the United States can lead to outbreaks requiring extensive and rapid control measures. Importation of measles from an unvaccinated adolescent in 2013 led to what has been the largest outbreak of measles in New York City, New York, since 1992. Objective: To describe the epidemiology and public health burden in terms of resources and cost of the 2013 measles outbreak in New York City. Design, Setting, and Participants: This epidemiologic assessment and cost analysis conducted between August 15, 2013, and August 5, 2014, examined all outbreak-associated cases of measles among persons residing in New York City in 2013. Exposures: Measles virus. Main Outcomes and Measures: Numbers of measles cases and contacts. Total personnel time and total direct cost to the New York City Department of Health and Mental Hygiene (DOHMH), calculated as the sum of inputs (supplies and materials, equipment, and logistics) and personnel time (salary and fringe benefits). Results: Between March 13, 2013, and June 9, 2013, 58 persons in New York City with a median age of 3 years (range, 0-32 years) were identified as having measles. Among these individuals, 45 (78%) were at least 12 months old and were unvaccinated owing to parental refusal or intentional delay. Only 28 individuals (48%) visited a medical health care professional who suspected measles and reported the case to the DOHMH at the initial clinical suspicion. Many case patients were not immediately placed into airborne isolation, resulting in exposures in 11 health care facilities. In total, 3351 exposed contacts were identified. Total direct costs to the New York City DOHMH were $394 448, and a total of 10 054 hours were consumed responding to and controlling the outbreak. Conclusions and Relevance: Vaccine refusals and delays appeared to have propagated a large outbreak following importation of measles into the United States. Prompt recognition of measles along with rapid implementation of airborne isolation of individuals suspected of measles infection in health care facilities and timely reporting to public health agencies may avoid large numbers of exposures. The response and containment of measles outbreaks are resource intensive.

[Familial intrahepatic cholestasis. Novel advances]. / Sulla colestasi intraepatica familiare. Recenti acquisizioni.

In the last years hepatology has known remarkable improvement to understanding the mechanisms involved in familial intrahepatic cholestasis. The role of genetic anomalies is very important. Identification of genes involved in familial intrahepatic cholestasis has proven to be an important strategy to unravel the processes of bile acid synthesis and bile salt transport. The complexity of the mechanisms of bile flow clearly suggests that many more genetic abnormalities have yet to be identified.

Rubella Surveillance and Diagnostic Testing among a Low-Prevalence Population, New York City, 2012-2013.

The New York City Department of Health and Mental Hygiene (DOHMH) receives clinical and laboratory reports for rubella. Because rubella immunoglobulin M (IgM) assays may produce false-positive results and rubella infections may be asymptomatic, interpretation of positive IgM results can be challenging. Rubella reports received by DOHMH in 2012 to 2013 were reviewed. The rubella IgM testing purpose was determined through case investigation. Results of IgM testing by indirect enzyme-linked immunosorbent assay (ELISA) and capture enzyme immunoassay (EIA) were compared to determine positive predictive value (PPV) and specificity. DOHMH received 199 rubella reports; 2 were true cases. Of all reports, 77.9% were tested for rubella IgM erroneously, 19.6% were tested for diagnostic purposes, 2.0% had unknown test purpose, and 0.5% were not tested. PPV of indirect ELISA was 6% overall, 14% for diagnostic tests, and 0% for tests ordered erroneously. PPV of capture EIA was 29% overall, 50% for diagnostic tests, and 0% for tests ordered erroneously. Overall, specificity was 52% for indirect ELISA and 85% for capture EIA. Limiting rubella IgM testing to patients for whom rubella diagnosis is suspected and using a more specific IgM assay have the potential to reduce false-positive rubella IgM results.

Is neutrophil gelatinase associated lipocalin useful in hepatitis C virus infection?

AIM: To evaluate neutrophil gelatinase associated lipocalin (NGAL) in patients infected by hepatitis C virus (HCV) before and during treatment with directly acting antivirals (DAAs). METHODS: NGAL was measured in a group of patients with chronic HCV infection ranked, at baseline, by age, gender, anti-hypertensive therapy, HCV viral load, liver fibrosis stage and, either at baseline or after 1 year, estimated glomerular filtration rate (eGFR). Then, NGAL and eGFR evolutions were monitored in a subgroup of patients who started antiviral therapy with DAAs. Differences of median NGAL levels were evaluated through Wilcoxon-Mann-Whitney test for non-parametric data. Differences in dichotomous variables were evaluated through χ (2) test. At baseline, a univariate regression analysis was conducted to verify if NGAL values correlated with other quantitative variables [age, fibrosis four (FIB-4), AST to platelet ratio index (APRI), and eGFR]. RESULTS: Overall, 48 patients were enrolled, 8 of them starting HCV treatment. At baseline, statistically significant differences were found in median NGAL values only between patients with eGFR < 60 mL/min vs patients with eGFR ≥ 90 mL/min. Differences in NGAL were not significant among patients ranked by HCV viral load, FIB-4 score and APRI, when patients with NGAL > 118.11 ng/dL were compared with those of NGAL ≤ 118.11 ng/dL, not statistically significant differences were present for age, gender, chronic kidney disease classification and liver fibrosis (P > 0.05). Linear correlation was found between NGAL and both age (P = 0.0475) and eGFR (P = 0.0282) values. Not statistically significant predictions of NGAL at baseline were demonstrated for eGFR evolution 1 year later. Interestingly, in the 8 patients treated with DAAs, median NGAL significantly increased at week 12 compared to baseline (P = 0.0239). CONCLUSION: Our results suggest that NGAL should be further evaluated as an adjunct marker of kidney function in these patients.

Boceprevir or telaprevir in hepatitis C virus chronic infection: The Italian real life experience.

AIM: To check the safety and efficacy of boceprevir/telaprevir with peginterferon/ribavirin for hepatitis C virus (HCV) genotype 1 in the real-world settings. METHODS: This study was a non-randomized, observational, prospective, multicenter. This study involved 47 centers in Italy. A database was prepared for the homogenous collection of the data, was used by all of the centers for data collection, and was updated continuously. All of the patients enrolled in this study were older than 18 years of age and were diagnosed with chronic infection due to HCV genotype 1. The HCV RNA testing was performed using COBAS-TaqMan2.0 (Roche, LLQ 25 IU/mL). RESULTS: All consecutively treated patients were included. Forty-seven centers enrolled 834 patients as follows: Male 64%; median age 57 (range 18-78), of whom 18.3% were over 65; mean body mass index 25.6 (range 16-39); genotype 1b (79.4%); diagnosis of cirrhosis (38.2%); and fibrosis F3/4 (71.2%). The following drugs were used: Telaprevir (66.2%) and PEG-IFN-alpha2a (67.6%). Patients were naïve (24.4%), relapsers (30.5%), partial responders (14.8%) and null responders (30.3%). Overall, adverse events (AEs) occurred in 617 patients (73.9%) during the treatment. Anemia was the most frequent AE (52.9% of cases), especially in cirrhotic. The therapy was stopped for 14.6% of the patients because of adverse events or virological failure (15%). Sustained virological response was achieved in 62.7% of the cases, but was 43.8% in cirrhotic patients over 65 years of age. CONCLUSION: In everyday practice, triple therapy is safe but has moderate efficacy, especially for patients over 65 years of age, with advanced fibrosis, non-responders to peginterferon + ribavirin.

FAS and FAS-L expression by tumor cells and lymphocytes in breast carcinomas and their lymph node metastases.

FAS receptor (FAS, CD95) and FAS ligand (FAS-L, CD95-L) are complementary members of a particular apoptotic pathway that plays a major role in immune regulation. The activation of FAS-L may trigger cytotoxic mechanisms leading to the death of FAS-expressing cells. Tumor cells and tumor-infiltrating lymphocytes (TIL) may express FAS and FAS-L in various proportions, and their interplay may affect tumor behavior. In the present study, we explored the expression of FAS and FAS-L in 28 mammary carcinomas (19 ductal and 9 lobular) and in their lymph node metastases. The expression of these mediators in immunostained sections was graded and evaluated comparatively between normal and neoplastic mammary epithelium, between tumor cells and TILs, and between mammary carcinoma cells and their lymph node metastases. We demonstrated the coexpression of FAS and FAS-L by breast carcinoma cells and TIL, with FAS expressed more strongly by normal epithelial cells and TIL than tumor cells. FAS-L was better stained on tumor cells than on TIL. There was equal or greater expression of FAS and FAS-L in the primary tumors and their TIL than in the metastatic counterparts. Comparing the expression of FAS with that of FAS-L, we recorded FAS equal or stronger than FAS-L in the primary mammary tumors and the reversal of their expression, FAS-L greater than FAS in the lymph node metastases. These results are consistent with reports of studies with other tumors, suggesting that the upregulated FAS-L indicates an increased ability of tumor cells to induce apoptosis in TIL and in the normal tissues invaded. However, it is understood that the FAS/FAS-L system, although essential for apoptosis, is only a contributing factor to the complex process of tumor invasion and antitumor defense.

The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States.

BACKGROUND: A new HIV diagnostic algorithm has been proposed which replaces the use of the HIV-1 Western blot and HIV-1 immunofluorescence assays (IFA) as the supplemental test with an HIV-1/HIV-2 antibody differentiation assay. OBJECTIVES: To compare an FDA-approved HIV-1/HIV-2 antibody differentiation test (Multispot) as a confirmatory test with the HIV-1 Western blot and IFA. STUDY DESIGN: Participants were screened with an HIV-1/HIV-2 combination Antigen/Antibody (Ag/Ab) screening assay. Specimens with repeatedly reactive results were tested with Multispot and either Western blot or IFA. Specimens with discordant screening and confirmatory results were resolved with HIV-1 RNA testing. RESULTS: Individuals (37,876) were screened for HIV infection and 654 (1.7%) had a repeatedly reactive Ag/Ab assay result. On Multispot, 554 (84.7%) were HIV-1 reactive, 0 (0%) were HIV-2 reactive, 1 (0.2%) was reactive for both HIV-1 and HIV-2 (undifferentiated), 9 (1.4%) were HIV-1 indeterminate, and 90 (13.8%) were non-reactive. HIV-1 RNA was detected in 47/90 Multispot non-reactive (52.2%) specimens. Among specimens confirmed to have HIV infection (true positives), Multispot and Western blot detected HIV-1 antibody in a similar proportion of cases (93.7% vs. 94.4% respectively) while Multispot and IFA also detected HIV-1 antibody in a similar proportion of cases (84.5% vs. 83.4% respectively). CONCLUSIONS: In this study, Multispot confirmed HIV infections at a similar proportion to Western blot and IFA. Multispot, Western blot, and IFA, however, did not confirm all of the reactive Ag/Ab assay results and underscores the importance of HIV NAT testing to resolve discordant screening and confirmatory results.

Fine-needle aspiration in the evaluation of nucleated red blood cells in the human placenta.

OBJECTIVE: The purpose of this study was to evaluate the correlation between placental and umbilical cord nucleated red blood cell counts. STUDY DESIGN: Eighty placentas and their matched umbilical cord blood samples were collected prospectively immediately after delivery. In vitro fine-needle aspiration biopsy specimens were used to obtain placental tissue samples. Nucleated red blood cells were counted by both manual microscopy and flow cytometry. Statistical analysis included Wilcoxon signed rank test and Spearman correlation. RESULTS: The median nucleated red blood cell counts/100 white blood cell counts for manual microscopy in umbilical cord blood; placental samples were 7.5 and 3.0, respectively (P <.0001). The median nucleated red blood cell counts for flow cytometric determination in umbilical cord blood and placental samples were 11.3 and 8.6, respectively (P <.0001). The Spearman correlation between manually counted umbilical cord blood samples and the placental tissue specimens was 0.66 (P <.0001). The Spearman correlation between flow cytometrically counted umbilical cord blood nucleated red blood cell and nucleated red blood cell counts that were obtained from the placenta was statistically significant (r = 0.74, P <.0001). The Spearman correlation between manual microscopy and flow cytometry for umbilical cord samples and their matched placental tissue specimens were 0.80 and 0.58, respectively, with all probability values at <.0001. CONCLUSION: Previous studies have reported an association between acute and chronic hypoxia and elevated nucleated red blood cells. Our results indicate that in vitro placental nucleated red blood cell counts correlate with umbilical cord nucleated red blood cell counts and suggest that antenatal evaluation of fetal nucleated red blood cells could be achieved by placental fine-needle aspiration biopsy.