Temporal dynamics of hepatitis C genotypes in a five-year hospital-based surveillance in Northern Italy.

Hepatitis C virus (HCV) genotypes have became important epidemiological markers in the management of HCV-infected subjects and infection treatment. The dynamics of HCV genotypes are changing in Europe. During a five-year (2009-2013) hospital-based surveillance in the area of Parma, Northern Italy, serum/plasma samples from 1,265 HCV RNA-positive subjects were genotyped. Subtypes 1b, 3a, and 1a were predominant (32.6 %, 19.1 %, and 17.8 %, respectively), with a correlation between viral load and gender. Subtypes 1a and 3a were more frequent in adults and males with a significant difference with the over-50 age group and females (P > 0.0001). Subtype 1b, as well as 2a/2c and G2 not-subtypeable (15.7 % and 7.2 %, respectively), were more common in females and in the over-50 age group compared to males (P < 0.0001, P < 0.0001, and P < 0.05, respectively) and the under-50 age group (P < 0.0001). While subtype 1b showed a nearly constant trend, subtype 1a peaked in 2012, when a consistent decrease in G2 was observed. The increasing detection of G4, mainly in adults, and subtypes 1a and 3a suggests their epidemiological relevance in the population. The detection of more than one HCV genotype in the same sample (0.2 %) and different genotypes in distant samples (5.1 %) from the same subject reinforces the opinion that re-infection and super-infection with different genotypes are not negligible events, especially in HIV-infected subjects. The dynamics of HCV genotypes could have significant implications for infection control.

Epidemiological and molecular features of norovirus infections in Italian children affected with acute gastroenteritis.

During a 5-year (2007-2011) surveillance period a total of 435 (15·34%) of 2834 stool specimens from children aged <14 years with acute gastroenteritis tested positive for norovirus and 217 strains were characterized upon partial sequence analysis of the polymerase gene as either genogroup (G)I or GII. Of the noroviruses, 99·2% were GII with the GII.P4 genotype being predominant (80%). GII.P4 variants (Yerseke 2006a, Den Haag 2006b, Apeldoorn 2008, New Orleans 2009) emerged sequentially during the study period. Sequence analysis of the capsid gene of 57 noroviruses revealed that 7·8% were recombinant (ORF1/ORF2) viruses including GII.P7_GII.6, GII.P16_GII.3, GII.P16_GII.13, GII.Pe_GII.2, and GII.Pe_GII.4, never identified before in Italy. GII.P1_GII.1, GII.P2_GII.1, GII.P3_GII.3 and GII.P6_GII.6 strains were also detected. Starting in 2011 a novel GII.4 norovirus with 3-4% nucleotide difference in the polymerase and capsid genes from variant GII.4 New Orleans 2009 was monitored in the local population. Since the epidemiology of norovirus changes rapidly, continuous surveillance is necessary to promptly identify the onset of novel types/variants.

Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

Multicentric evaluation of new commercial enzyme immunoassays for the detection of immunoglobulin M and total antibodies against hepatitis A virus.

A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively).

Multicenter evaluation of a transcription-reverse transcription concerted assay for rapid detection of mycobacterium tuberculosis complex in clinical specimens.

A European multicenter study was performed to evaluate the performance of a new method, based on the transcription-reverse transcription concerted reaction (TRC-2), which enabled one-step amplification and real-time detection of the Mycobacterium tuberculosis 16S rRNA target directly in clinical specimens. A total of 633 respiratory and nonrespiratory specimens were tested, and the results were compared with those from smears and cultures. A total of 129 patients (Paris center) were followed up in order to evaluate the clinical performance of TRC-2. By using M. tuberculosis complex strains to inoculate sterile sputa, the detection limit of TRC-2 was found to be 30 to 50 CFU/ml. A total of 548 respiratory specimens and 59 extrapulmonary specimens were assessable. For pulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 86.8% and 50.4%, respectively (P = 0.002). The specificities were 97.5% and 100%, respectively. For extrapulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 83.3% and 8.3% (P < 0.0001), and the specificities were 95.8% and 100%, respectively. Fifteen of 129 patients were diagnosed with pulmonary tuberculosis (TB). The sensitivities of culture and TRC-2 were 80% (12/15) and 86.7% (13/15) (P = 0.16), and the specificities were 100% and 93.9%, respectively. Based on an 11.6% incidence of TB in our population, the positive predictive values of TRC-2 and culture were 81.3% and 100%, respectively, and the negative predictive values were 98.2% and 97.4%, respectively. These results demonstrated that detection of M. tuberculosis complex in clinical specimens by TRC-2 with ready-to-use reagents was an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB.

An outbreak of norovirus infection in an Italian residential-care facility for the elderly.

On December 2006, an outbreak of gastroenteritis occurred at a residential-care facility for the elderly in northern Italy. Thirty-five of 61 individuals interviewed (attack rate, 57.4%) fell ill. In 94.3% of cases, the onset of illness was within 48 h of a Christmas party at the facility. Norovirus (NoV) was detected by RT-PCR in 24 of 31 individuals examined, including three asymptomatic food-handlers, in whom there was evidence of long-lasting excretion of viral particles. The identification of a sequence referring to the '2006a GII.4 NoV variant' in all examined strains supported the hypothesis of a common point source. This retrospective cohort study is the first report on an outbreak of NoV gastroenteritis in an Italian residential-care facility for the elderly.

Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase.

Genetic polymorphisms influence Plasmodium ovale PCR detection accuracy.

Detection of Plasmodium ovale by use of a nested PCR assay with a novel Plasmodium ovale primer set was superior to detection of Plasmodium ovale by real-time PCR assays. Nested PCR was also better at detecting P. malariae. The detection of P. ovale in many patients first admitted >2 months following their return to Italy indicated that P. ovale relapses are common.

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs.

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.

Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs.

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.

Evaluation of a new plate hybridization assay for the laboratory diagnosis of imported malaria in Italy.

A new molecular diagnostic method "Malaria-IBRIDOGEN" (Amplimedical S.p.A.--Bioline Division, Turin, Italy) based on a plate-hybridization assay for the simultaneous detection and identification of human malaria parasites was evaluated in this study. A target DNA sequence of the plasmodial 18S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and hybridized in microtiter wells with five biotinylated probes each specific for Plasmodium falciparum, P. vivax, P. malariae, P. ovale and the beta-globine human gene, respectively. Compared to the nested-PCR actually used in our laboratory for the molecular diagnosis of malaria, "Malaria-IBRIDOGEN" revealed an overall sensitivity of 100% (51/51) for the four human Plasmodium species testing 100 whole blood samples from people with malaria-like symptoms and fever. Specificity was 92% (45/49) considering four discordant samples as "false positive" by "Malaria-IBRIDOGEN". The assay showed a threshold of parasite density (detection limit) of 0.07 P. falciparum parasites/microliter, 0.15-1.5 P. vivax parasites/microliter, 0.3 P. malariae parasites/microliter and 0.4 P. ovale parasites/microliter of whole blood, respectively. This assay could be successfully applied to the laboratory diagnosis of malaria as a useful aid to microscopy.

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis.

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts.

The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.

Evaluation of susceptibility of Mycobacterium bovis to antituberculous drugs by radiometric BACTEC 460TB system.

Susceptibility of Mycobacterium bovis strains to antituberculous drugs (isoniazid and rifampin) was detected by radiometric BACTEC 460TB system. M.bovis strains were isolated from tissue samples showing tuberculous lesions collected at an abbattoir from cattle belonging to 47 tuberculosis outbreaks occurring in Northern Italy in 1995-1999. Forty-six out of 61 strains (75.4%) resulted susceptible to both isoniazid and rifampin. Thirteen strains (21.3%) were resistant to isoniazid only. No strains showed resistance to rifampin only. Two strains (3.3%) resulted resistant to both drugs, showing antituberculous multidrug-resistance. Given the compulsory eradication program of bovine tuberculosis by elimination of infected animals and the ban on antituberculous drug treatments in animals, detection of resistant M. bovis strains appears of great interest.

Identification of Plasmodium falciparum, P. vivax, P. ovale and P. malariae and detection of mixed infection in patients with imported malaria in Italy.

The species-specific nested-PCR previously described by Snounou and others for detecting the four parasite species that cause human malaria is evaluated in the current study testing 230 blood samples. The results are compared with those obtained by microscopy and, for 101 samples out of 230, with those previously obtained by a genus-specific PCR based method (pg-PCR) followed by species-specific Southern-blot hybridization. All blood specimens were obtained from patients (127 foreigners and 103 Italians) with a suspect clinical diagnosis of imported malaria in Italy: 76 were positive by microscopy and 83 were positive by nested-PCR. The last method also revealed 10 double infections (8 foreigners and 2 Italians) which were not identified by microscopy or by pg-PCR with species-specific Southern-blot hybridization. Fifty-four out of 83 positive samples tested by nested-PCR were submitted to genomic sequence analysis, which confirmed the presence of DNA region portion encoding the 18S rRNA corresponding to the Plasmodium species identified by nested-PCR. These results demonstrate that the nested-PCR assay surpasses microscopy and pg-PCR with species-specific Southern-blot hybridization, both in sensitivity and in diagnostic accuracy. Moreover, it is quicker because it requires no further blotting or hybridization of PCR amplification products. This method also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment but also for the study of malaria epidemiology. Finally, our study also highlights the value of genomic sequence analysis for validating PCR results.

Brachyspira (Serpulina) pilosicoli of human origin interfere with the growth of Clostridium perfringens alpha-toxin producer.

Brachyspira (Serpulina) pilosicoli of human origin interfere with the growth of Clostridium perfringens alpha-toxin producer reducing the clostridial growth area and colonies number when bacteria were cultivated together in sheep blood agar plates. The growth inhibition of C. perfringens was only observed when B. (S.) pilosicoli grew 72-96 hours sooner than C. perfringens and after the inoculum of this latter the plates were anaerobically incubated for additional 48 hours. The phenomenon was observed at concentrations of B. (S.) pilosicoli ranging from 10(7) to 10(4) CFU/ml and at concentrations of C. perfringens ranging from 10(7) to 10(1) CFU/ml when the bacteria were 0-10 mm away from each other. When B. (S.) pilosicoli and C. perfringens were inoculated at the same time and when B. (S.) pilosicoli grew 24-48 hours sooner than C. perfringens, the clostridial growth inhibition was not appreciated and only a cooperative haemolysis was observed between the bacteria.

Brachyspira (Serpulina) pilosicoli of human origin interfere with the haemolytic activity and the growth of Staphylococcus aureus beta-toxin producer.

Brachyspira (Serpulina) pilosicoli related to intestinal spirochaetosis were found to interfere in vitro with the haemolytic activity and the growth of Staphylococcus aureus beta-toxin producer. This interference was clearly appreciated because a reduction of the zone of the staphylococcal beta-toxin activity, the reduction and/or absence of cooperative haemolysis between bacteria, and the growth reduction of S. aureus were observed when B. (S.) pilosicoli were grown 72-96 hours sooner than S. aureus and after the inoculum of the latter the plates were anaerobically incubated for additional 48-72 hours. The phenomenon was more clearly observed when B. (S.) pilosicoli had a concentration of 8x10(6)-8x10(7) CFU/ml and S. aureus at a concentration ranging from 10(7) to 10(1) CFU/ml was inoculated at a distance from the streaks of B. (S.) pilosicoli ranging from 0-10 mm. When B. (S.) pilosicoli and S. aureus were inoculated at the same time and when B. (S.) pilosicoli grew 24-48 hours sooner than S. aureus only a cooperative haemolysis was observed.

Weakly beta-haemolytic human intestinal spirochaetes antagonize the haemolytic activity of Clostridium perfringens alpha-toxin producer.

The production of haemolytic antagonism between weakly beta-haemolytic human intestinal spirochaetes (wbetaHIS) related to human intestinal spirochaetosis and Clostridium perfringens alpha-toxin producer was investigated. A reduction of the clostridial haemolytic activity and a distortion of the haemolytic halo of clostridial alpha-toxin surrounded by a small zone of poorly cooperative haemolysis was clearly observed on the level of the spirochaetal growth area when 40 out of 41 wbetaHIS were cultivated in sheep blood agar plates together with Clostridium perfringens alpha-toxin producer. This phenomenon of haemolytic antagonism was observed only when wbetaHIS grew 72-96 hours sooner than C. perfringens and after the inoculum of the latter at a distance of 0 to 10 mm from wbetaHIS the plates were anaerobically incubated for an additional 48 hours and the bacteria were used at concentrations ranging from 10(7) to 10(4) CFU/ml. These results were also observed between C. perfringens and weakly beta-haemolytic intestinal spirochaetes related to animal intestinal spirochaetosis including avian strains and Brachyspira (Serpulina) pilosicoli of porcine origin.

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect.

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.