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.

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.

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.

Patterns of cytodistribution of prosomal antigens on the vimentin and cytokeratin networks of monkey kidney cells.

Prosomes were found as mRNA-associated ribonucleoprotein particles (RNP) and cofactors of untranslated (ribosome-) free mRNP. Previous data have shown the presence of prosomal networks in the cytoplasm of PtK1 and HeLa cells and their superposition onto the intermediate filaments (IF) of cytokeratin type but little if any of vimentin type. Here it is shown that in LLC-MK2 cells various prosomal antigens are present on both, vimentin and cytokeratin networks, individual prosomal antigens superposing to variable degrees onto the IF subnetworks. Some prosomal antigens in variable relative concentrations were also observed in the nuclei of these cells. We suggest the existence of prosomal subnetworks specific for each prosomal antigen superposing to a variable extent onto the IF of both types.

Specific types of prosomes distribute differentially between intermediate and actin filaments in epithelial, fibroblastic and muscle cells.

First observed as components of non-translated mRNP complexes, prosomes harbour RNase and several proteinase activities; they are also the central constituent of the "Multicatalytic Proteinase (MCP) complexes" or "26S-proteasomes". In two recent publications (Arcangeletti et al., 1997b; De Conto et al., 1997) we have shown, by applying a new fixation technique, that these particles distribute differentially between the cytoskeletal networks of intermediate filament (IF) and actin types; previously they had been observed exclusively on the intermediate filaments. Here we further investigate the distribution of prosomes of several types, distinct by their subunit composition, between the IF of vimentin type and the actin network, as well as in the 3D space of the cell. It is shown that subtypes of prosomes occupy specific networks of the cytoskeleton, and that this pattern is specific for a given cell type. Confocal microscopy shows that prosome cytodistribution is not homogeneous in the 3D space: in the perinuclear area they colocalize most strongly with the IF, and more peripherally with the microfilament/stress fiber system; connections may exist between the two networks. Furthermore, new data indicate that the prosome-actin interaction may participate in the molecular structure of the stress fibers.

Rhodococcus equi cavitary pneumonia in HIV-infected patients: an unsuspected opportunistic pathogen.

Two patients seropositive for human immunodeficiency virus (HIV) and with no previous acquired immunodeficiency syndrome-defining conditions developed cavitary pneumonia and pleural disease caused by Rhodococcus equi. R. equi was isolated from these patients' sputum and lung biopsy specimens, respectively, but the microorganism was initially considered to be a contaminant (patient 1) or misidentified as a nontuberculous mycobacterium (patient 2). The R. equi infection was fatal in one patient, who died after 4 months without specific antimicrobial therapy; the second patient was unresponsive to combination therapy with various antimicrobial agents. R. equi may cause life-threatening infections in HIV-infected patients. Microbiology laboratories should be cognizant of the need to exclude R. equi as a cause of infection in highly immunosuppressed patients.

Detection by immunofluorescent anti-idiotypic antibodies of yeast killer toxin cell wall receptors of Candida albicans.

Yeast killer toxin cell wall receptors of Candida albicans were observed by indirect immunofluorescence using an affinity purified rabbit anti-idiotypic antiserum. The antiserum had been raised against a monoclonal antibody neutralizing the in vitro activity of a killer toxin produced by a selected strain of Hansenula anomala UCSC 25F. This simple procedure permitted the location of killer toxin cell wall receptors in various morphological phases of the yeast cells. The use of the indirect immunofluorescence technique with anti-idiotypic antibodies may have potential value in determining the occurrence of killer toxin receptors in other microbial systems.

Abortive replication of influenza A viruses in HeLa 229 cells.

Several experimental data support the idea that certain mammalian cells are unable to replicate influenza viruses type A, although these viruses can efficiently penetrate the cells. This cannot be attributed to a lack of specific receptors on the cell surface, but depends upon the failure of specific step(s) to occur during viral growth. Here we report a study of abortiveness of human and avian type A influenza viruses in HeLa 229 cells. Viral polypeptide synthesis was monitored by [35S]methionine pulse labelling at several time points after infection, showing that normal amounts of virus-induced components were synthesized. Cellular fractionation of HeLa 229 cells infected by influenza viruses showed that the distribution of viral proteins into nuclear and cytoplasmic compartments was comparable to that seen in the permissive host, chick embryo fibroblasts. Viral HA glycoprotein, produced during the infectious cycle, was entirely found in the cytoplasm of infected HeLa 229 cells. The polypeptide was able to agglutinate red blood cells but did not show positive haemadsorption even at late times of infection. Therefore it seems that during the maturation of viral particles there is a failure of the haemagglutinin to perform a correct insertion into the plasma membrane of infected HeLa 229 cells.

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.

Modification of cytoskeleton and prosome networks in relation to protein synthesis in influenza A virus-infected LLC-MK2 cells.

Modifications of the cytoskeleton and protein synthesis were investigated in LLC-MK2 cells during infection by FPV/Ulster 73, an avian strain of influenza A virus. During infection, the cytoskeleton and the prosome networks undergo a dramatic reorganization, which seems to be at least temporally differentiated for each cytoskeletal system, i.e. microfilaments (MFs), microtubules (MTs), intermediate filaments (IFs). In order to evaluate the role of the three different cytoskeletal networks during FPV/Ulster infection, studies were carried out on cellular and virus-specific protein synthesis and viral production, using drugs which selectively affect individual cytoskeletal systems. Our data show that the perturbation of the IF system, but not that of the MFs or MTs, seems to have a strong inhibitory effect on virus production and cellular and viral protein synthesis. Furthermore, the dynamics of IFs and prosomes were investigated during viral infection and, at no time, dissociation of the prosome and IF networks was observed. Taken together, these results strongly support the idea that the interactions between the protein synthesis machinery, the cytoskeleton, and the prosomes are all affected by viral infection in a partially coordinated manner.

Value of cerebrospinal fluid leukocyte aggregation in distinguishing the causes of meningitis in children.

BACKGROUND: Current laboratory tests often cannot distinguish between bacterial and aseptic meningitis rapidly and accurately. The ability to make a prompt diagnosis has important implications for the management and outcome of children with meningitis. The observation that leukocytes aggregate in the cerebrospinal fluid (CSF) has been previously reported, and it has been advocated as a reliable method to distinguish the causes of meningitis in children. OBJECTIVE: To investigate the utility of CSF leukocyte aggregation as a screening test to distinguish between bacterial and aseptic meningitis. METHODS: We compared the clinical and laboratory indices of 109 prospectively enrolled patients with meningitis (67 bacterial, 23 viral, 19 undefined etiology) and evaluated the validity of the CSF leukocyte aggregation test. The predefined leukocyte aggregation scores (LAS) were compared among the types of meningitis, and correlations with other markers of inflammation were calculated. RESULTS: The median LAS was significantly higher (P < 0.001) in the bacterial (32.1%; range, 0 to 84.1%) than in the viral (0%; range, 0 to 16.6%) or undefined (0%; range, 0 to 20.7%) groups. The optimal sensitivity of the leukocyte aggregation test, 98.5 to 92.5%, was demonstrated with LAS values of 0 to 3%. The corresponding specificity was 64.3 to 88.1%. The peripheral white blood cell (WBC) count, serum C-reactive protein, CSF WBC count, blood culture, CSF Gram stain and CSF culture were inferior to the LAS as screening tests when compared individually. The LAS was as effective as CSF protein, TNF-alpha, IL-1-beta, IL-6 and IL-8 to predict bacterial meningitis. In a logistic regression model that included routine laboratory tests, the best predictor of bacterial meningitis was the LAS (odds ratio, 1.6 to 3.7). Significant correlations were demonstrated between the LAS and CSF protein, CSF WBC count, IL-1-beta, IL-6 and IL-8. Duration of symptoms before diagnosis, pretreatment with antibiotics, HIV-1 infection status and CSF red blood cell count did not significantly alter the LAS. CONCLUSIONS: There is no single test to diagnose the etiology of meningitis in children promptly and accurately. The finding of leukocyte aggregation in CSF might be of value as a sensitive adjunctive screening tool for the timely diagnosis of bacterial meningitis, recognizing that it has low specificity and potential practical limitations.

Differentiation between vaccine-related and wild-type polioviruses using a heteroduplex mobility assay.

A heteroduplex mobility assay (HMA) was developed for intratypic differentiation between poliovirus isolates. The assay is based on polymerase chain reaction (PCR) amplification of a 480 base pair fragment which encodes a variable segment of VP1, followed by denaturation and reannealing of the resulting single strands with those from reference Sabin targets. Mismatches between wild-type and Sabin vaccine templates result in the formation of detectable heteroduplexes of reduced electrophoretic mobility. Poliovirus strains confirmed previously as wild-type or vaccine-like by PCR and sequencing were all correctly identified using the HMA. Mixtures of both wild-type and vaccine-like strains in a single isolate could also be detected using this technique. The results of this study demonstrate that heteroduplex analysis is a simple, rapid, and sensitive means for differentiating between vaccine-like and wild-type poliovirus isolates.

Natural and 'in vitro' selected antigenic variants of influenza A virus (H2N2).

We provide data on the prevalence of SRH antibody to influenza A/Singapore/1/57 (H2N2). Approximately 10.3% of sera had antibody to the influenza A (H2N2) subtype virus in comparison to the 36.9% of positive sera to a representative influenza A (H3N2) and 31.5% to influenza A (H1N1) viruses. The percentage of subjects with antibody constantly decreased from the older to the younger age groups. Persons born after 1968 were essentially seronegative, whereas subjects born before 1900, and in the decade 1950-1959, showed the highest antibody levels to influenza A (H2N2) viruses. These age groups also appeared to have 'protective' levels of anti-HA antibody to influenza A (H2N2) virus. An antigenic variant of A/Singapore/1/57 virus was selected in the laboratory using a monoclonal antibody to HA. Serological comparison of the new in vitro variant with the parental virus and two naturally occurring viruses, namely A/England/12/64 and Tokyo/3/67, showed that certain human sera were able to distinguish the variant, indicating a restricted antibody repertoire in these adult and children's sera, providing an explanation of how such variants could actually arise in nature.

A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy.

Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.

Radio immune Western blotting: a possible solution to indeterminate conventional Western blotting results for the serodiagnosis of HIV infections.

Western blot is used worldwide as a confirmatory assay after a positive or doubtful ELISA result in the serodiagnosis of HIV infections. Despite the use of this test some results may be not definitive due to the presence in Western blot of only few or a single band to HIV protein. This result has been defined as indeterminate. To resolve indeterminate results obtained with conventional Western blotting (WB) we used radio immune Western blotting (RIWB), a method capable of detecting antibodies to HIV-1 proteins with greatly increased sensitivity with respect to conventional WB. We used RIWB to perform a retrospective survey on 20 sera belonging to individuals undergoing HIV serodiagnosis (not blood donors) with positive or borderline EIA results, and indeterminate WB results who only later fully seroconverted. Fifteen of the sera tested by RIWB showed a positive result which clarified any doubtful indeterminate WB result; all bands present in conventional WB were enhanced in intensity using RIWB and new bands, absent in the WB assay, were highlighted using RIWB. RIWB may resolve most indeterminate WB results.

Comparative growth of pure cultures of human intestinal spirochaetes on six selective media.

The growth of pure cultures of 24 human intestinal spirochaetes (HIS) was analysed comparatively in six selective media with antibiotics and in a control medium without antibiotics. The selective media SR and SP were the only two media among the six tested which allowed the growth of all the strains studied. These media contained spectinomycin (400 micrograms/ml) and rifampin (30 micrograms/ml) (SR), and spectinomycin (400 micrograms/ml) and polymyxin B (5 micrograms/ml) (SP), respectively. Moreover, most of the strains tested showed in these two media a number of CFU/ml equal to, or, for a few strains, not more than ten-fold lower than that observed in the control medium. The other four selective media tested were: SRVC (spectinomycin 200 micrograms/ml; rifampin 12.5 micrograms/ml; vancomycin 6.25 micrograms/ml; colistin 6.25 micrograms/ml), CSp (colistin 6.25 micrograms/ml; spiramycin 25 micrograms/ml), SRSp (spectinomycin 200 micrograms/ml; rifampin 12.5 micrograms/ml; spiramycin 25 micrograms/ml) and SRVCSp (spectinomycin 200 micrograms/ml; rifampin 12.5 micrograms/ml; vancomycin 6.25 micrograms/ml; colistin 6.25 micrograms/ml; spiramycin 25 micrograms/ml). The growth of most of the spirochaetes was strongly reduced in the media SRVC, CSp, SRSp and SRVCSp. Furthermore, several of the 24 HIS examined did not grow in the medium SRVC (3 spirochaetes, 11%), CSp (15 spirochaetes, 62%), SRSp (17 spirochaetes, 70%), SRVCSp (19 spirochaetes, 79%). The results reported in this paper indicate that the media SR and SP, of the six selective media tested, may profitably be used in the isolation of HIS as they did not significantly affect the growth of the HIS tested.

The use of radioimmune western blotting to evaluate indeterminate western blotting results in blood donor HIV serodiagnosis.

The serodiagnosis of HIV infection is sometimes puzzling due to questionable "indeterminate" results obtained by western blotting (wb), the confirmatory test used world-wide after a positive or borderline ELISA result. These situations cause anxiety in the person being tested and determine additional laboratory costs. We showed that radioimmune western blottting (riwb) an improved and sensitive modification of conventional wb, was able to resolve most "indeterminate" results in individuals who later fully seroconverted. We report in this paper the use of riwb in sera of 20 uninfected blood donors with an indeterminate wb continuing for at least 6 years, to verify the serological status in indeterminate not at risk individuals. Thirteen out of the 20 were indeterminate with conventional wb due to the presence of antibodies to p 24, two to p 17, two to p 55, two to p 51 and p 55 and one to p 24 and p 51 antibodies. The presence of all these bands was confirmed using riwb; moreover, in comparison with wb, the intensity of all bands was clearly enhanced. Despite the use of this highly sensitive method no new bands were found with riwb in indeterminate sera; negative results were obtained when sera borderline or positive with ELISA but negative with wb, were subjected to riwb.