A new targeted CFTR mutation panel based on next-generation sequencing technology

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling

A rapid and sensitive method to measure the functional activity of shiga toxins in human serum

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins)

Group B streptococcal meningitis in an adult: A possible complication of olecranon bursitis

Background: We report a man with septic olecranon bursitis who had an early development of meningitis. Case Summary: A 74-year-old man presented to the emergency room with malaise, headache, mental confusion, a fever unsuccessfully treated with oral NSAIDs and ice, and with a 10-day history of pain and swelling in his right elbow. Clinical and laboratory evaluation excluded other causes and microbiological evaluation documented a S. agalactiae infection. Antibiotic treatment induced a rapid improvement, without the development of side effects. Conclusion: This is the first report on olecranon bursitis and concomitant meningitis related to S. agalactiae infection

Cost analysis of residual viremia detected by two real-time PCR assays for response-guided (dual or triple) therapy of HCV genotype 1 infection

Cost analysis of residual viremia detected by two real-time PCR assays for response-guided (dual or triple) therapy of HCV genotype 1 infectionIntroductionThe duration of interferon-based dual and triple therapies for HCV-G1 is determined by assessment of early viral kinetics. We conducted a cost analysis to determine the mean cost of dual or triple therapy treatment for a patient with HCV-G1, where the therapy duration (24 or 48 weeks) is guided by HCV-RNA assay.MethodsHCV-RNA was assessed by two widely used real-time PCR-based assays, Cobas Ampliprep/Cobas TaqMan (CAP-CTM) and Real-Time HCV (ART). As far as the dual therapy (PegINFα-2b and RBV) is concerned, at week 12 of treatment 16.0% of patients (27/169) were eligible to receive a shorter duration of therapy (24 weeks) according to CAP-CTM and 8.9% (15/169) according to ART: 26 patients achieved SVR (15.4%) with CAP-CTM and 15 with ART (8.9%). With regard to triple therapy (TPV, PegINFα-2a and RBV), at week 12 of treatment 59.6% of patients (31/52) were eligible to receive a shorter duration of therapy (24 weeks) according to CAP-CTM and 28.8% (15/52) according to ART: 30 patients achieved SVR (57.7%) with CAP-CTM and 13 with ART (25.0%). The cost analysis was conducted from the perspective of the Italian National Health Service (NHS). Only drug (TPV, PegINFα-2a, PegINFα-2b and RBV) and test (CAP-CTM and ART) costs were taken into account. Ex-factory prices (included all discounts) and national tariffs were used to calculate drug consumptions and tests, respectively. Costs were assessed in Euros 2015.ResultsWith regard to dual therapy, the overall mean treatment cost per patient with CAP-CTM (€9,572.77) was lower than with ART (€9,876.19). With regard to triple therapy, the overall mean treatment cost per patient was lower with CAP-CTM (€31,354.40) than with ART (€33,155.26).ConclusionsCAP-CTM HCV-RNA assay was cost-saving from the Italian NHS perspective compared with ART HCV-RNA assay in the dual (-€303.42 per patient) and triple (-€1,800.96 per patient) HCV therapy

Evaluation of reactivation of HSV1, HHV6, CMV and EBV in a population of patients undergoing allogeneic bone marrow transplantation

Herpes viruses are recognized as important pathogens as a result of viral reactivation in immunocompromised hosts, especially in patients undergoing bone marrow transplantation. Objectives of this study were the assessment of the reactivation of herpes virus HSV1, HHV6, CMV and EBV and the correlation between viral reactivation and progression of transplantation in a population of patients undergoing allogeneic bone marrow transplantation at Fondazione IRCCS Ospedale Maggiore Ca’ Granda Policlinico, Milan. Viral DNA was detected and quantified by Real Time PCR in a population of 35 patients undergoing allogeneic bone marrow transplantation. The viral reactivation was observed in 7 patients for HSV1 (20%), 6 patients for HHV6 (17.1%), 11 patients for CMV (31.4%) and 4 patients for EBV (11.4%). Difference in the incidence of aGVDH between patients with viral reactivation versus those for which there was no reactivation was statistically significant. These data confirm the importance of monitoring viral load for the management of antiviral therapy in order to prevent CMV disease and complications related to herpes viruses reactivation

Enterovirus-D68 in the Cerebrospinal Fluid of Two Children with Aseptic Meningitis

This case report describes two previously healthy children with aseptic meningitis whose cerebrospinal fluid was positive for enterovirus-D68, which indicates direct involvement of this infectious agent in the development of this neurologic disease

Shigella flexneri-induced vaginitis in a prepubertal children: description of a case

In prepuberal girls vulvo-vaginitis are caused by germs of intestinal origin,mycetes, Gardnerella vaginalis, protozoa. Shigella is an uncommon agent able to induce valvovaginitis in children. We report the case of a 7-year-old girl with chronic vulvo-vaginitis caused by S. flexneri. Antibiotic Susceptibility Testing revealed that S. flexnery was sensible to cefotaxime, amoxicillin, imipenem, ciprofloxacin, but resistant to amikacin, cefazolin, gentamycin, ampicillin and tetracycline. A treatment with ciprofloxacin brought to a rapid resolution of all symptoms. At the follows up at 3 and 6 months the patient did not report symptoms of infection or articular cartilage abnormality; microbiological evaluations were also negative. Even if it is a single case report and other clinical trial may be performed in order to validate this hypothesis,we speculate that in patient with vulvo-vaginal infection living in environment with low hygiene care, a carefully microbiological evaluation of uncommon agents may be performed