Predominance of hepatitis C virus Q80K among NS3 baseline-resistance-associated amino acid variants in direct-antiviral-agent-naïve patients with chronic hepatitis: single-centre experience.

In the era of direct-acting antiviral agents (DAAs), hepatitis C virus (HCV) genotyping tests at baseline are controversial. The HCV NS3-Q80K polymorphism is associated with resistance to the recently approved NS3 inhibitor simeprevir (SMV) when combined with PEG-interferon and ribavirin (PEG-IFN/RBV) and alternative therapy should be considered for patients with baseline Q80K. The aim of this study was to provide an estimate of Q80K prevalence at baseline in a study group of 205 DAA-naïve patients (21% of them with HIV coinfection) using NS3 full-population direct sequencing to detect resistance-associated amino acid variants (RAVs). NS3 RAVs were identified in 56 patients (27.3%). Q80K was the most frequently reported one (41%), in both HIV/HCV-coinfected and HCV-monoinfected patients, but it was only detectable in cases of HCV-subtype 1a infection. Therefore, in clinical practice, an NS3-Q80K genotyping test prior to simeprevir plus PEG-IFN/RBV treatment is highly recommended.

Clinical impact of A/H1/N1/09 influenza in patients with cirrhosis: experience from a nosocomial cluster of infection.

A/H1N1/09 influenza is associated with a high risk of complications in patients with chronic diseases, but data on morbidity and mortality in patients with cirrhosis are limited. A cluster of A/H1N1/09 infection in 48 patients admitted to a Gastro-Hepatology Unit is reported. Nosocomial spread, clinical outcome, and viral characteristics of A/H1N1/09 strains from a study group of 48 inpatients (21 and 27 with and without cirrhosis, respectively) were compared with those from a control group of 44 outpatients with mild influenza-like illness and without cirrhosis. A/H1N1/09 infection was confirmed in 8/48 (17%) inpatients. A/H1N1/09 infection rate did not differ in patients with and without cirrhosis (4/21, 19%; 4/27, 15%), but three patients with cirrhosis died of pneumonia and acute respiratory distress syndrome, with fungal or bacterial superinfection in two cases, despite antiviral treatment. None of patients without cirrhosis died. Viral sequences showed the presence of hemagglutinin mutation D222G in two out of three fatal cases and S183P in seven out of eight infected patients. These mutants were not detected in the outpatients group. Even if A/H1N1/09 infection rate in hospitalized patients with and without cirrhosis was not significantly different, cirrhosis and D222G/S183P substitutions were significantly associated with severe disease and poor outcome, also suggesting fungal or bacterial superinfection and portal hypertension as risk factors for A/H1N1/09 disease severity in patients with cirrhosis. Vaccination, preventive and early treatment and a strict control of nosocomial spread should be activated carefully in patients with cirrhosis during epidemics influenza.

LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages.

The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS.

The RNA-binding protein KSRP promotes decay of beta-catenin mRNA and is inactivated by PI3K-AKT signaling.

Beta-catenin plays an essential role in several biological events including cell fate determination, cell proliferation, and transformation. Here we report that beta-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase-AKT signaling. AKT phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated level of control of beta-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of beta-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase-AKT signaling and beta-catenin accumulation.

Laboratory findings in Zika infection: The experience of a reference centre in North-West Italy.

BACKGROUND: Zika virus (ZIKV) remains a public health concern due to its association with fetal malformation and neurologic disease. OBJECTIVE: To report a reference centre experience on ZIKA virus (ZIKV) infection in travelers from epidemic countries from January 1 to September, 30, 2016 in Italy North-West (a geographic area covering 4.424 million inhabitants, corresponding to almost 73% of Italy North-West area). STUDY DESIGN: One hundred and twelve febrile travelers were studied to rule out a tropical fever [e.g. malaria, dengue (DENV), chikungunya (CHIKV), West Nile (WNV) and ZIKV]. Molecular tests for detecting ZIKV RNA were applied on serum or urine as well as IgG and IgM specific serology. RESULTS: ZIKV was the most frequent "tropical infection (11.6%) with 12 infected travelers and one sexual partner of an infected traveler. At the time of the diagnosis, ZIKV RNA was detected in the blood from 9 patients (69%) within 7 days from symptom onset; afterwards, the virus was detected only in urine (5 patients) and ZIKV IgM was reactive in 9 patients (69%). Travelers with ZIKV infection tested negative for DENV, CHIKV, WNV and malaria and completely recovered. Other infections identified in travelers were DENV (5 patients, 4.5%), CHIKV (1, 0.9%), malaria (Plasmodium vivax, 1, 0.9%), measles (1, 0.9%) and tuberculosis (1, 0.9%). CONCLUSIONS: The etiologic diagnosis of a febrile illness in travelers where ZIKV is endemic is highly desirable as they are sentinel of a challenging epidemiology including the risk of autochthonous transmission in non endemic countries where the competent or carrier vector is present.

Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling.

BACKGROUND: KSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile beta-catenin mRNA through an impairment of KSRP function. RESULTS: Aim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary alphaT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to beta-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation. CONCLUSION: Our study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway.

Genes regulated by hepatocyte growth factor as targets to sensitize ovarian cancer cells to cisplatin.

Advanced ovarian cancers are initially responsive to chemotherapy with platinum drugs but develop drug resistance in most cases. We showed recently that hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and that this effect is mediated by the p38 mitogen-activated protein kinase. In this work, we integrated genome-wide expression profiling, in silico data survey, and functional assays to identify transcripts regulated in SK-OV-3 ovarian cancer cells made more responsive to CDDP by HGF. Using oligonucleotide microarrays, we found that HGF pretreatment changes the transcriptional response to CDDP. Quantitative reverse transcription-PCR not only validated all the 15 most differentially expressed genes but also confirmed that they were primarily modulated by the combined treatment with HGF and CDDP and reversed by suppressing p38 mitogen-activated protein kinase activity. Among the differentially expressed genes, we focused functional analysis on two regulatory subunits of the protein phosphatase 2A, which were down-modulated by HGF plus CDDP. Decrease of each subunit by RNA interference made ovarian cancer cells more responsive to CDDP, mimicking the effect of HGF. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or revert chemotherapy resistance.

Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer.

Cancer is a genetic disease caused by mutations in somatic cells. Those that carry advantageous mutations are favoured by natural selection. In most cancers, genetic instability increases mutation rate and facilitates cancer cell evolution. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. As mutations in expressed genes are more likely to be functional, we developed a procedure for the systematic identification of mutant repeat-containing expressed sequences (amplification of repeat-containing transcribed sequences, ARTS). The entire cell mRNA was converted into short double-stranded cDNA fragments linked to an adapter at both ends. Repeat-containing cDNA fragments were PCR amplified using the adapter-specific primer in combination with different arbitrary primers including the repeat. ARTS yielded discrete PCR products with lengths that were directly correlated to the lengths of the endogenous repeats. Comparison between ARTS products obtained from control cells and cancer cells with microsatellite instability (MSI+) revealed mRNAs carrying insertions or deletions at repeats. The subsequent sequencing allowed the identification of a series of frameshift-mutated mRNAs in MSI+ cancer cells, including the already described mutant BAX transcript. These data show that ARTS provides an unbiased genome-wide approach to the discovery of functionally relevant genes that could be affected by MSI in cancer.

Deletion in a (T)8 microsatellite abrogates expression regulation by 3'-UTR.

A high level of genetic instability might cause mutations to accumulate in tumours. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. By scanning transcriptome databases, we found that microsatellites in the human genome are less numerous in coding DNA than in the 3'-untranslated region (UTR), known to mediate control of gene expression. By mutation analysis, we identified a 1 bp deletion in a (T)(8) microsatellite embedded in the 1801 nucleotide long 3'-UTR of CEACAM1 gene, thought to be involved in tumour onset and progression. By Lentiviral Vector- mediated gene transfer, we showed that the wild-type but not the mutated CEACAM1 3'-UTR greatly decreased transgene expression at both mRNA and protein level. Messenger RNA abundance was fully regulated by the most 3' region of CEACAM1 3'-UTR. This region includes the (T)(8) microsatellite but not any known classified regulatory element. These data show that CEACAM1 3'-UTR contains non-canonical elements contributing to mRNA regulation, among which a short repeat sequence could play a critical regulatory function. This suggests that, in cancer cells, a single mutation in a 3'-UTR short microsatellite might strongly affect gene expression.

Travelers With Chikungunya Virus Infection Returning to Northwest Italy From the Caribbean and Central America During June-November 2014.

Chikungunya virus (CHIKV) has recently emerged in the Caribbean. In Italy, CHIKV vector is documented in the Po river valley; therefore, a risk for autochthonous outbreaks is present. We report a case series of seven imported CHIKV infections in travelers returning from the Caribbean and Latin America occurring between June and November 2014, in the area of Turin, Northwest Italy, 3 years after the last imported cases were reported. These cases are a reminder of the need to always consider CHIKV infection in travelers from these epidemic areas as well as the importance of a prompt diagnosis.

Quantification of hepatitis B surface antigen with the novel DiaSorin LIAISON XL Murex HBsAg Quant: correlation with the ARCHITECT quantitative assays.

BACKGROUND: Recent technologic innovations allow for quantitative assessment of hepatitis B surface antigen (HBsAg) levels in serum; this has been used to monitor the course of chronic HBV hepatitis (CHB) and predict treatment response. LIAISON-XL Murex HBsAg Quant assay (DiaSorin, Saluggia, I) is the newest immunoassay CE approved to quantify HBsAg. OBJECTIVES: To compare LIAISON-XL performances with ARCHITECT-QT HBsAg (Abbott Diagnostics, IL, USA), as reference test. STUDY DESIGN: Sequential serum samples (n=152) from 14 HBe-negative patients with CHB, the majority of them infected by HBV genotype D undergoing antiviral treatment, were retrospectively tested with both assays. The 2nd WHO Standard 00/588 for HBsAg was used as reference. RESULTS: LIAISON-XL and ARCHITECT-QT correlated by r=0.95, p<0.0001; by Bland-Altman analysis agreement of mean difference was 0.21 ± 0.15 log 10 IU/mL, 95% CI: -0.07 to 0.5). Performance of LIAISON-XL against the 2nd WHO Standard was r=0.998, p<0.0001 (95% CI: 0.993-0.999) with results nearer to the expected WHO values compared to ARCHITECT-QT. Median baseline HBsAg level was similar with the two methods before antiviral treatment, throughout fluctuations of HBsAg level in treatment non-responders and during the decrease of HBsAg titer in treatment responders. Correlation between HBsAg levels and HBV DNA was statistically significant for both the two immunoassays (LIAISON-XL: r=0.4988, 95% CI: 0.3452-0.6264, p<0.0001; ARCHITECT-QT: r=0.480, 95% CI: 0.3233-0.6111, p<0.0001). CONCLUSIONS: Correlation between HBsAg measurement with LIAISON-XL and ARCHITECT-QT was high. LIAISON-XL accurately quantified HBsAg in clinical samples at baseline or during antiviral therapy; it can be applied for HBsAg quantification in clinical practice and decision making in CHB.

Combination of conventional blood cultures and the SeptiFast molecular test in patients with suspected sepsis for the identification of bloodstream pathogens.

We evaluated performances of the molecular test SeptiFast (SF) for the detection of agents of bloodstream infection (BSI) in patients with suspected sepsis, the majority of them under antibiotic treatment and at high prevalence of HIV-1 infection (10.5%). Matched SF and blood culture (BC) samples (n=1186) from 1024 patients were studied. Two hundred fifty-one episodes of BSI out of 1144 were identified with the combined methods (22%). SF identified more episodes of BSI than BC: 206 versus 176 (χ(2)=7.008, P=0.0081) and a significantly higher number of Gram-negative bacteria than BC (77 versus 53, χ(2)=9.12; P=0.0025), as well as of polymicrobial infections (χ(2)=4.50, P=0.0339). In conclusion, SF combined with BC improved the diagnosis of sepsis, especially in immunocompromised patients.

Evaluation of a rapid antigen and antibody combination test in acute HIV infection.

BACKGROUND: New strategies at implementing HIV testing including rapid HIV assays are highly recommended to avoid late diagnosis. To shorten the diagnostic window period, the first point-of-care HIV assay, Determine HIV ½ Ag/Ab Combo (D4G, Alere, I) for the combined detection of p24 and anti-HIV antibody has been recently marketed and mainly tested in high prevalence setting. OBJECTIVES: To establish D4G performances in acute HIV infection (AHI) in a setting at low HIV-1 prevalence. STUDY DESIGN: D4G performances were compared with HIV-1 RNA levels in a panel of well-characterized serum specimens from 17 patients with AHI. For specificity, 124 anti-HIV negative serum specimens from patients seeking HIV testing were studied. RESULTS: D4G detected HIV infection in 15/17 patients. D4G antigen was positive in only 5 patients (29.4%), 4 of them with a viral load >10 million copies/mL. D4G antibody was reactive in other 10 patients (sensitivity: 58.8%, viral load from 70,161 to 8,120,000 copies/mL). Combined D4G sensitivity for acute HIV-1 infection was 88.2%; no false positive or invalid result was recorded (100% specificity, positive and negative predictive values: 100% and 98.4%, respectively). CONCLUSION: In spite of a poor antigen sensitivity with optimal performances only for viral load >10 million copies/mL, D4G performances in acute HIV-1 infection were enhanced by the addition of p24 testing to the antibody. Improved HIV rapid testing to shorten the window period is important as rapid tests play a major role in expanding access to HIV testing and preventing HIV transmission.

Recent outbreak of aseptic meningitis in Italy due to Echovirus 30 and phylogenetic relationship with other European circulating strains.

BACKGROUND: Enteroviruses (EVs) are common human viral pathogens, causing a variety of diseases, including aseptic meningitis. Recently, EV aseptic meningitis outbreaks have been reported across Europe, but, in Italy, knowledge of recent EV molecular epidemiology is very limited. OBJECTIVES: We report an outbreak of EV aseptic meningitis in 10 adults in North-Western Italy, from October to November 2012. Patients were parents or close relatives of children <5 years old attending the same class of a nursery school, suffering from a mild febrile upper respiratory disease. Phylogenetic relationship with other European circulating strains was analyzed updating E30 circulation in Italy in recent years. STUDY DESIGN: EVs were detected from cerebrospinal fluid (CSF) specimens with a real-time reverse transcription polymerase chain reaction and virus isolation was achieved from rectal and pharyngeal swabs. For cluster definition and phylogenetic studies, viral VP1 region was directly amplified and sequenced from CSF. RESULTS: EVs were identified in CSF from all patients and from rectal and pharyngeal swabs in 7 of them. Direct sequencing of CSF revealed the presence of the same Echovirus 30 (E30) in all patients and phylogenetic analysis identified it as a diverging clade within E30 genotype VII, the most recent strain circulating in UK, Finland and Denmark since 2006. CONCLUSION: Molecular techniques allowed the rapid identification and typing of E30 from CSF. Phylogenetic analysis revealed that the cluster might be due to a new E30 variant within the genotype VII currently circulating in Europe, thus updating the epidemiology of EV circulation in Italy.

A(H1N1)pdm09 hemagglutinin D222G and D222N variants are frequently harbored by patients requiring extracorporeal membrane oxygenation and advanced respiratory assistance for severe A(H1N1)pdm09 infection.

BACKGROUND: In patients with A(H1N1)pdm09 infection, severe lung involvement requiring admission to intensive care units (ICU) has been reported. Mutations at the hemagglutinin (HA) receptor binding site (RBS) have been associated with increased virulence and disease severity, representing a potential marker of critical illness. OBJECTIVES: To assess the contribution of HA-RBS variability in critically ill patients, A(H1N1)pdm09 virus from adult patients with severe infection admitted to ICU for extracorporeal membrane oxygenation support (ECMO) during influenza season 2009-2011 in Piemonte (4·2 million inhabitants), northwestern Italy, was studied. PATIENTS AND METHODS: We retrospectively analyzed HA-RBS polymorphisms in ICU patients and compared with those from randomly selected inpatients with mild A(H1N1)pdm09 disease and outpatients with influenza from the local surveillance program. RESULTS: By HA-RBS direct sequencing of respiratory specimens, D222G and D222N viral variants were identified in a higher proportion in ICU patients (n=8/24, 33·3%) than in patients with mild disease (n=2/34, 6%) or in outpatients (n=0/44) (Fisher's exact test P<0·0001; OR 38·5; CI 95% 4·494-329·9). Eleven ICU patients died (42%), three of them carrying the D222G variant, which was associated with RBS mutation S183P in two. D222G and D222N mutants were identified in upper and lower respiratory samples. CONCLUSIONS: A(H1N1)pdm09 HA substitutions D222G and D222N were harbored in a significantly higher proportion by patients with acute respiratory distress for A(H1N1)pdm09 severe infection requiring ICU admission and ECMO. These data emphasize the importance of monitoring viral evolution for understanding virus-host adaptation aimed at the surveillance of strain circulation and the study of viral correlates of disease severity.