Genetic diversity of Streptococcus suis clinical isolates from pigs and humans in Italy (2003-2007).

Streptococcus suis, a major porcine pathogen, is emerging as a zoonotic agent capable of causing severe invasive disease in humans exposed to pigs or pork products. S. suis infection is rare in industrialised countries and usually arises as sporadic cases, with meningitis the most common clinical presentation in humans. Recent reports of two cases of meningitis in Sardinia and northeastern Italy prompted this first characterisation of Italian S. suis isolates. Fifty-nine S. suis strains, the two recent human strains and 57 swine clinical isolates collected between 2003 and 2007 from different Italian herds and regions, were tested for antimicrobial susceptibility, PCR-screened for virulence and antibiotic resistance genes, and subjected to molecular typing. Phenotypic and genotypic analysis demonstrated an overall high genetic diversity among isolates, the majority of which were resistant to macrolides (78%) and tetracyclines (90%). The erm(B), tet(O), mosaic tet(O/W/32/O), tet(W), and tet(M) genes were detected. The tet(O/W/32/O) gene, the most frequent tet gene after tet(O), had never been described in the genus Streptococcus before. In addition, a virulent cps2, erm(B) tet(O) clone, belonging to sequence type 1 (ST1) of the ST1 complex, was found to be prevalent and persistent in Italian swine herds. Finally, the two human isolates (both ST1) carrying cps2, erm(B) and tet(W) were seen to be closely related to each other.

Magnetization switching in high-density magnetic nanodots by a fine-tune sputtering process on a large-area diblock copolymer mask.

Ordered magnetic nanodot arrays with extremely high density provide unique properties to the growing field of nanotechnology. To overcome the size limitations of conventional lithography, a fine-tuned sputtering deposition process on mesoporous polymeric template fabricated by diblock copolymer self-assembly is herein proposed to fabricate uniform and densely spaced nanometer-scale magnetic dot arrays. This process was successfully exploited to pattern, over a large area, sputtered Ni80Fe20 and Co thin films with thicknesses of 10 and 13 nm, respectively. Carefully tuned sputter-etching at a suitable glancing angle was performed to selectively remove the magnetic material deposited on top of the polymeric template, producing nanodot arrays (dot diameter about 17 nm). A detailed study of magnetization reversal at room temperature as a function of sputter-etching time, together with morphology investigations, was performed to confirm the synthesis of long-range ordered arrays displaying functional magnetic properties. Magnetic hysteresis loops of the obtained nanodot arrays were measured at different temperatures and interpreted via micromagnetic simulations to explore the role of dipole-dipole magnetostatic interactions between dots and the effect of magnetocrystalline anisotropy. The agreement between measurements and numerical modelling results indicates the use of the proposed synthesis technique as an innovative process in the design of large-area nanoscale arrays of functional magnetic elements.

Sexual transmission of the hepatitis C virus and efficacy of prophylaxis with intramuscular immune serum globulin. A randomized controlled trial.

OBJECTIVE: To estimate the risk of sexual transmission of hepatitis C and to assess the value of prophylaxis with periodic intramuscular immune serum globulin administration. METHODS: Of 1102 steady heterosexual partners of patients with antibodies to the hepatitis C virus (HCV), 899 were enrolled in a single-blind, randomized, controlled trial. All the partners tested negative for antibodies to HCV and had normal baseline serum aminotransferase concentrations. The partners were assigned to receive 4 mL of 16% polyvalent immune serum globulin prepared from unscreened donors every 2 months (n = 450) or a placebo (n = 449). Tests for HCV infection were performed every 4 months. RESULTS: Eight hundred eighty-four partners completed the study. Seven partners became infected with HCV: 6 in the control group (incidence density, 12.00 per 1000 person-years; 95% confidence interval, 3.0 21.61) and 1 in the immune serum globulin group (incidence density, 1.98 per 1000 person-years; 95% confidence interval, 0-5.86). The risk of infection was significantly higher for partners in the control group (P = .03): for each year approximately 1% of the partners became infected. Sequence homology studies strongly suggest the sexual transmission of HCV. All immune serum globulin lots used had high enzyme-linked immunosorbent assay titers of neutralizing antibodies to HCV envelope glycoproteins and high neutralization titers in the neutralization of binding assay. CONCLUSIONS: Hepatitis C can be sexually transmitted. Immune serum globulin prepared from unscreened donors significantly reduced the risk. The treatment was safe and well tolerated. Because only immune serum globulin from unscreened donors (and not from those screened for HCV) contain anti-HCV neutralizing antibodies, hyperimmune anti-HCV immune serum globulin should be prepared from blood testing positive for antibodies to HCV, which is currently discarded.

Detection of hepatitis B virus DNA in serum using synthetic non-radioactive oligonucleotides.

A rapid and simplified technique for detecting hepatitis B virus (HBV) DNA by spot hybridisation in the sera of patients with different clinical forms of HBV infection was investigated using enzyme conjugated synthetic oligodeoxyribonucleotides as probes. These are able to hybridize to the S and C regions of the HBV L(-) DNA strand. When compared with a complete 32P-labelled HBV DNA probe, the synthetic oligonucleotides provided a sensitive and quick method for the routine survey of HBV infection. Moreover, the DNA extraction procedure used allowed the spot hybridisation technique to be applied and read easily and the results obtained within a few hours. It is concluded that synthetic cold probes can be used in hybridisation assays HBV DNA detection as part of current clinical laboratory procedures.

Chronic liver disease and active hepatitis C virus infection in patients with antibodies to this virus.

AIMS: To assess the association between active hepatitis C virus (HCV) infection and liver damage in randomly selected patients with antibodies to the virus. METHODS: Thirty three consecutive subjects with serologically confirmed positivity for antibodies to HCV were studied for the presence of liver and circulating viral sequences by using the reverse transcription polymerase chain reaction (RT-PCR) and specific primers for the 5'-untranslated region (5'-UTR) of the HCV genome. Parallel clinical, biochemical, and histological investigations were carried out in all cases. RESULTS: A comparative virological and histological investigation showed the presence of molecular signs of active viral replication and different degrees of liver damage in all cases. Baseline values of liver and plasma samples from all the patients showed (with one exception) the presence of detectable HCV RNA sequences, despite alanine amino transferase activities being within normal values or within 1.5 times the upper limit of normal in 13 of them. Examination of percutaneous liver biopsy specimens showed the presence of confirmed liver damage (ranging from chronic persistent hepatitis to cirrhosis) in all 33 patients. CONCLUSIONS: Circulating HCV RNA sequences (a direct sign of active HCV infection) are associated with liver damage, even in the absence of clinical or biochemical signs of overt liver disease. Parallel molecular, histological, and clinical follow up of these patients is needed to understand precisely the natural history of HCV infection and for correct clinical management.

PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.

Adenoma-carcinoma sequence of colorectum. Prevalence of K-ras gene mutation in adenomas with increasing degree of dysplasia and aneuploidy.

One hundred and fifty colorectal adenomas were investigated in order to detect the presence of K-ras gene mutation. The adenomas were classified according to the severity of the histological lesion (mild, moderate, or severe dysplasia and carcinomatous transformation) and to the degree of aneuploidy. K-ras mutation was found in 30.8% of cases, mostly consisting of a point mutation of codon 12. K-ras mutation was more frequently found in adenomas > 1 cm and in the villous type. No correlation was otherwise demonstrable with the ploidy pattern of the lesion.

A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction.

A rapid single-step procedure for the isolation of low molecular weight DNA using guanidinium thiocyanate and phenol as protein denaturants is described and applied for the detection of specific hepatitis B virus (HBV) DNA sequences from serum samples by the polymerase chain reaction (PCR). The novel technique is efficient and, when compared to the standard proteinase K/phenol/chloroform method has the advantage of being faster and easily adaptable to the routine processing of a high number of clinical samples by PCR and spot hybridization techniques.

Detection of human immunodeficiency virus type 1 transcripts in peripheral blood lymphocytes by the polymerase chain reaction.

A simplified application of the polymerase chain reaction (PCR) to the routine detection of human immunodeficiency virus type 1 (HIV-1) transcripts from peripheral lymphocytes of infected subjects is described. This technique is simpler than previously described assays and was shown to be highly sensitive after ethidium bromide staining of polyacrylamide gel electrophoresis of amplified material. The method can be used for the virologic evaluation of HIV-1-infected subjects, thus allowing early identification of seropositive patients with signs of active infection.

Prophylaxis of hepatitis C with intramuscular immunoglobulin: clinical and economic appraisal.

Hepatitis C virus (HCV) affects millions of individuals worldwide. In most cases, HCV infection progresses to chronic liver disease and, subsequently, to liver cirrhosis and hepatocellular carcinoma. HCV is transmitted by the parenteral route, for example by transfusion of blood or blood products, injection during drug abuse, etc., and by the inapparent parenteral route (penetration of the virus through difficult-to-identify microlesions present on the skin or mucosae), for example, sexual exposure or household exposure to infected contacts, etc. The cost of chronic hepatitis C and its sequelae is high in both financial and human terms. At present, only anti-HCV screening of blood/organ/tissue donors and universal precautions for the prevention of blood-borne infections are recommended for HCV prevention. Before the discovery of the main aetiological agent of non-A, non-B hepatitis (HCV), several randomised controlled clinical trials demonstrated that standard intramuscular immunoglobulin exerted a preventive effect on post-transfusional and sexual and /or horizontal transmission of non-A, non-B hepatitis. When serological tests for HCV infection became available, bimonthly inoculation of standard unscreened intramuscular immunoglobulin (prepared from plasma pools containing about 2% of anti-HCV-positive units) was demonstrated to significantly prevent sexually transmitted HCV infection. The immunoglobulin used contained high titres of anti-HCV neutralising antibodies (anti-E2 neutralisation of binding assay), whereas currently available commercial screened immunoglobulin (prepared from anti-HCV-negative blood units) did not. This finding suggested that anti-HCV neutralising antibodies are concentrated only in anti-HCV-positive units (which are currently discarded). Thus, anti-HCV hyperimmune globulin (HCIg) can be produced only from anti-HCV-positive units. The neutralising titre can be increased by the exclusive use of units with higher titres of neutralising antibodies. Unlike other hyperimmune globulins, which are produced from a limited number of selected donors, HCIg should be produced from a large number of units so as to contain neutralising antibodies to the different HCV strains. HCIg will have a number of advantages: (i) it is easy to produce and inexpensive; (ii) it has a long half-life, allowing infrequent administration; (iii) new additional viral inactivation procedures have been introduced to eradicate transmission of infection, and (iv) it may be possible to neutralise all the emerging HCV strains. HCIg could be used in all individuals at risk of HCV infection (sexual partners, haemodialysis patients, etc), in preventing reinfection of transplanted livers, and perhaps also in the treatment of chronic hepatitis C, alone or associated with other drugs.

Humoral immune response and natural killer activity in patients with mixed cryoglobulinemia.

OBJECTIVE: Based on serological and molecular evidence of hepatitis C virus (HCV) infection in a significant proportion of patients with mixed cryoglobulinemia (MC), a direct association between HCV and MC has been suggested. The goal of the present study was to investigate the role played by HCV and by the immune response to the virus in the pathogenesis of mixed cryoglobulinemia. METHODS: A competitive reverse transcription polymerase chain reaction was employed to evaluate the concentrations of specific HCV RNA sequences in different clinical specimens (plasma, sera, cryoprecipitates, bone marrow and peripheral blood cells). Using recombinant and synthetic peptides covering the HCV core, envelope 1 (E1) and nonstructural regions 4 (NS4) and 5 (NS5), the humoral immune response in a group of MC patients was assessed with an enzyme-linked immunosorbent assay. Natural killer (NK) cell activity was estimated using a 4 hr 51 Cr release assay. RESULTS: Quantitation of the RNA molecules in the biological samples confirmed an increased virion concentration in cryoprecipitates from 13/15 patients with mixed cryoglobulinemia. Analysis of the humoral immune response against the synthetic peptides suggested a distinct response to HCV antigens in MC patients when compared to patients with HCV infection but without serological evidence of cryoglobulinemia. Unstimulated NK cell functioning was below the normal range in all patients tested. However, peripheral blood mononuclear cells showed no enhancement of NK activity by the interferon inducer polyinosinic acid:polycytidilic acid. Enhancement by interferon-alpha was normal, suggesting an impairment in interferon production. CONCLUSION: The quantitative data are in line with the hypothesis of a direct or indirect role of HCV in mixed cryoglobulinemia. The abnormal immune response could be involved in the onset and persistence of HCV infection, and possibly in the appearance of cryoglobulinemia.

Sexual transmission of hepatitis C virus and prevention with intramuscular immunoglobulin.

The sexual transmission of hepatitis C virus (HCV) has long been debated. The prevalence of infected at-risk partners varies from 0% to 30%. In a prospective study, the risk of infection was quantified in steady heterosexual partners and the prophylactic effect of normal human polyvalent immune serum globulin (ISG) was evaluated. A total of 899 at-risk partners of HCV-infected patients were enrolled in a single-blind randomized controlled trial and assigned to receive every 2 month 4 mL of intramuscular ISG from unscreened donors (450 partners) or placebo (499 partners). Seven partners developed acute HCV infection (increased aminotransferase levels and appearance of HCV-RNA): six of the placebo group (incidence density [ID] 12.00/1,000 person year; 95% confidence interval [CI] 3.0 to 21.61), and only one of the ISG-treated group (ID 1.98/1,000 person year; 95% CI 0 to 5.86). The risk of infection was significantly higher in controls versus treated individuals (p = 0.03). Six couples had genotype 1b (85%), and one couple had genotype 1a; HCV sequence homology strongly supported sexual transmission. Our trial demonstrates that HCV infection can be sexually transmitted and quantifies the risk of sexual transmission: for every year of at-risk sexual relationship, almost 1% of the partners became infected. Intramuscular ISG is safe and well tolerated. Unlike ISG from screened donors, ISG from donors unscreened for anti-HCV contains high titers of anti-gpE1/gpE2 neutralizing antibodies and high neutralizing activity. Anti-HCV hyperimmune globulin could be prepared from anti-HCV-positive blood units and could be used to protect sexual partners and in other at-risk situations of exposure to HCV infection.

Umbilical blood flow and placental pathology.

The aim of the study was to establish whether or not placental morphostructural damage correlates with umbilical artery Doppler waveform and neonatal condition. To this end, seriated ultrasonographic monitoring, flowmeter tests on the cord artery and computerized cardiotocography were carried out in a population of 93 pregnant women in the second half of pregnancy. After birth placentas were subjected to macroscopic and microscopic examination. The Resistance Index showed a good correlation with placental vascular lesions, characterized by a distinct reduction in terminal villi and muscular wall arterioles. Two types of intrauterine growth retardation were discernible, the first of genetic origin with a low-profile growth curve and therefore not amenable to treatment, but with a positive fet l-neonatal prognosis, and the second with a pathologic placental component, presenting a late flattening growth curve with evolution towards fetal distress and a negative fetal-neonatal prognosis.

Characterization of hepatitis C virus genotypes in an hemodialysis unit in Paysandú, Uruguay.

Hepatitis C virus types were investigated by using samples from eight sero-reactive and PCR positive patients attending our Hemodialysis Unit en Paysandú, Uruguay. After HCV RNA detection by reverse transcription and polymerase chain reaction, HCV genotyping was carried out by a nested PCR amplification, using type specific primers of HCV core region. These results were confirmed using a method based upon reverse hybridation of amplified products by enzyme-labeled type-specific probes to portions of the 5' UTR region. HCV genotypes were assigned according to Simmonds' classification. Type 1b was found in five patients, type 3a was found in one and one patient was not classified. There was a patient who became PCR negative at the moment the genotyping was carried out.

Usefulness of capillary electrophoresis-based multiplex PCR assay for species-specific identification of Candida spp.

The study evaluated the performances of a commercial multiplex PCR assay, the Seegene Seeplex STI Master Panel 3, for Candida spp. identification. Eighty clinical strains of Candida spp. were identified with this system and a homemade multiplex PCR assay. The results were also compared with those obtained with two phenotypic methods. The study provided a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection of amplified products. The Seeplex assay was found to be a rapid and useful method for identifying large numbers of yeast isolates in the clinical laboratory context.

Dynamics of viral quasispecies in hepatitis C virus infection.

The genomic heterogeneity of hepatitis C virus (HCV) was addressed in the different phases of HCV infection. Viral sequences of the HVR-1 and NS5a regions were obtained by reverse transcription polymerase chain reaction from plasma samples of two patients with acute type-C hepatitis and two patients with chronic infection treated with interferon. The data indicate that in primary infection different degrees of genomic heterogeneity in biologically important viral regions might be associated with different clinical outcomes.

Quantitative molecular methods in virology.

During the past few years, significant technical effort was made to develop molecular methods for the absolute quantitation of nucleic acids in biological samples. In virology, semi-quantitative and quantitative techniques of different principle, complexity, and reliability were designed, optimized, and applied in basic and clinical researches. The principal data obtained in successful pilot applications in vivo are reported in this paper and show the real usefulness of these methods to understand more details of the natural history of viral diseases and to monitor specific anti-viral treatments in real time. Theoretical considerations and practical applications indicate that the competitive polymerase chain reaction (cPCR) and competitive reverse-transcription PCR (cRT-PCR) assay systems share several advantages over other quantitative molecular methodologies, thus suggesting that these techniques are the methods of choice for the absolute quantitation of viral nucleic acids present in low amounts in biological samples. Although minor obstacles to a wide use of these quantitative methods in clinical virology still remain, further technical evolution is possible, thus making the quantitative procedures easier and apt to routine applications.

Competitive polymerase chain reaction and analysis of viral activity at the molecular level.

Due to the high sensitivity level (which can be pushed to the limit of one molecule) and its extraordinary flexibility, the polymerase chain reaction (PCR) is the method of choice for the detection of nucleic acids present in very low concentration in biological samples. Since the qualitative features of PCR amplification have limited its use, several PCR-based approaches for the quantitation of low-abundance nucleic acid species have been planned and proposed in the last few years. Recently, different lines of evidence have indicated that competitive PCR and competitive reverse-transcription-PCR share several advantages over other quantitative approaches. This evidence opens up unexpected possibilities in many biological fields, including virology; in fact, availability of reliable techniques for the absolute quantitation of DNA and RNA species may be the key to a better understanding of the pathogenic steps of most viral diseases and for a more precise monitoring of patients treated with specific antiviral compounds. In this review article, we summarize the procedures adopted for this quantitative molecular approach; additionally, several important technical aspects to plan novel competitive PCR-based applications are analyzed, and early results obtained using cPCR for the direct evaluation of viral activity in vivo are discussed.

Characterization of growth parameters of a human hepatoma cell line cultured under serum-free conditions.

The PLC/PRF/5 human hepatoma cell line has at least four complete series of hepatitis B virus (HBV)-DNA sequences integrated into the host DNA and produces hepatitis B surface antigen (HBsAg) and several serum proteins in the culture medium. In order to study serum proteins and HBsAg released by these cells under controlled conditions, the serum-free growth of several human hepatoma-derived cell lines was recently investigated. In this paper the growth of PLC/PRF/5 human hepatoma cell line in a serum- and hormone-free medium was investigated. The results represent a tool which might be used in pharmacological research, studies on hormone-binding and virus gene expression.

Growth-factor independence of a new differentiated hepatitis B virus DNA-negative human hepatoma cell line.

The establishment of a new, differentiated, hepatitis B virus DNA-negative, human hepatoma cell line (named PLC/AN/2) is described. Neoplastic liver tissue was obtained during hepatectomy in an HBsAg-negative man. The established cell line is negative for alpha-fetoprotein and carcinoembryonic antigen; it has retained in vitro some of the differentiated functions of normal hepatocytes. Additionally, it presents a distinctive rearrangement (translocation) at the long arm of chromosome 4. The high degree of independence from serum growth factor requirements appears to be a major in vitro characteristic of PLC/AN/2 cells, making them a suitable model system for the more precise definition of the human hepatocellular carcinoma phenotype, including mechanisms of growth control.