Hepatitis E virus in domestic pigs and surface waters in Slovenia: prevalence and molecular characterization of a novel genotype 3 lineage.

A novel hepatitis E virus (HEV) genotype 3 lineage was identified in Slovenian pig herds. Stool samples from six Slovenian pig farms were collected and tested for the presence of HEV RNA. Of 85 individual samples 15 (20.3%) were positive for HEV RNA of which 2/38 (5.3%), 6/21 (28.6%) and 7/26 (26.9%) were from suckling, weanling and fattening pigs, respectively. Additionally, 51 pooled porcine stool samples were tested in one of the biggest pig farm and the estimated infection rate of individual pig was calculated, resulting in 7.8%, 10.6% and 24.2% for suckling, weanling and fattening pigs, respectively. The majority of HEV positive porcine samples were from the same pig farm. Out of 17 Slovenian patients with confirmed recent hepatitis E in the period 1999-2011, the serum samples of 10 patients were tested and 3 samples turned out to be HEV RNA positive. Furthermore, 60 surface water samples were tested throughout the country, of which 2 (3.3%) were positive for HEV RNA, one of them in the near vicinity of a pig farm. All HEV strains were analysed at 5' ORF1 and 5' ORF2 regions and both genome regions confirmed that Slovenian HEV strains represent a distinct genotype 3 lineage, diverse from all other genotype 3 lineages available in GenBank and described in the literature to date. All but one HEV strains detected in pigs in Slovenia represent a monophyletic branch in phylogenetic trees, with a high degree of sequence identity. One human HEV strain belonged to genotype 1 and two to genotype 3 but did not match the new genotype 3 lineage detected in Slovenian pig herds.

Twenty-four mini-pool HCV RNA screening in a routine clinical virology laboratory setting: a six-year prospective study.

The usefulness of combined anti-HCV and 24 mini-pool HCV RNA screening strategy was re-evaluated after a six-year continuous routine use in a clinical virology laboratory, at which more than half of newly diagnosed hepatitis C patients are intravenous drug users. Pools of 24 samples were prepared from 20,448 anti-HCV negative serum samples and tested using an automated commercial PCR assay with a lower limit of detection of 50 IU/ml. After detection of anti-HCV negative/HCV RNA positive patients, responsible physicians provided follow-up samples. Thirty-eight (0.19%) anti-HCV negative/HCV RNA positive samples from 30 patients (28 intravenous drug users) were detected. Follow-up samples were available for 27/30 patients. Twenty, six and one patient seroconverted in the second, third and fourth available samples, respectively. The interval between the first HCV RNA positive and the first available anti-HCV positive sample was 17-517 days. The costs of detecting a single anti-HCV negative/HCV RNA positive patient were 1227 Euros. Combined anti-HCV and 24 mini-pool HCV RNA screening is a useful and cost effective strategy, not only in blood-transfusion settings but also in a routine clinical virology laboratory, at which a significant proportion of the tested population belongs to a high-risk population.

Prevalence and clinical relevance of cagA, vacA, and iceA genotypes of Helicobacter pylori isolated from Slovenian children.

BACKGROUND: Although infection with Helicobacter pylori in children mostly induces asymptomatic chronic gastritis, the clinical outcome of H pylori infection is generally unpredictable. To identify the risk subgroup of infected children who can progress toward serious gastrointestinal disease, we assessed the prevalence of H pylori virulence genes cagA, vacA, and iceA in children from southeastern Europe and correlated their presence with the severity of histological changes in the stomach. MATERIALS AND METHODS: A total of 165 children (age range 4-18 years, mean 13 years) with H pylori infection were studied for a 6-year period. Virulence genes were determined by polymerase chain reaction from biopsy samples. RESULTS: The cagA gene was present in 61.2% of patients. The predominant vacA genotype was s1m1 (42%), followed by s1m2 (28%), and s2m2 (24%). IceA genotypes iceA1 and iceA2 were detected in 62% and 31% of the samples, respectively. Multiple genotypes were found in 11.5% of isolates. The H pylori density score, the degree of chronic and acute inflammation, correlated with a cagA-positive status (P < 0.01, P < 0.01, P = 0.01, respectively). Higher bacterial infiltration (P < 0.01) and degree of chronic inflammation (P = 0.03) were detected in vacA s1-positive samples. CONCLUSION: CagA, vacA s1m1, and iceA1 genotypes are the predominant genotypes of H pylori isolated from the southeastern European pediatric population. CagA and vacA s1 are important virulence determinants of H pylori in children, but were not found associated with an increased incidence of precancerous gastric lesions.

Hepatitis C virus genotypes in 1,504 patients in Slovenia, 1993-2007.

In order to identify the main routes of hepatitis C (HCV) transmission and to determine the HCV genotype distribution and its dynamics during a 15-year period in Slovenia, HCV genotypes were detected using the INNO-LiPA HCV II (Innogenetics) test for serum samples obtained from 1,504 patients representing 72.6% of all patients with chronic hepatitis C diagnosed from 1993 to 2007. HCV genotype 1 was predominant (56%), followed by genotypes 3, 2, and 4, with a prevalence of 37.8%, 5%, and 1.2%, respectively. HCV genotypes 5 and 6 were not detected in any patient. Patients infected with HCV genotype 3 were significantly younger (mean age 28.9 +/- 8.5 years) than those infected with genotype 1 (mean age 38.9 +/- 14.8 years; P < 0.0001) and those infected with HCV genotype 2 (mean age 50.3 +/- 18.2 years; P < 0.0001). Intravenous drug use was identified as the most frequent possible HCV transmission route (34.3%), followed by medical-related transmission such as transfusion of HCV-contaminated blood or blood products, and hemodialysis (12.5%). Being an intravenous drug user was found to be strongly associated with HCV genotype 3 (OR, 3.71 [95% CI, 2.97-4.65]; P < 0.0001) and reporting infection by transfusion of blood or blood products was found to be strongly associated with HCV genotype 1 (OR, 3.28 [95% CI, 2.18-4.95]; P < 0.0001). During the 15-year period, the proportion of genotype 3 increased substantially, reflecting the fact that the HCV epidemic in Slovenia is driven mostly by intravenous drug use.

GeneXpert enterovirus assay: one-year experience in a routine laboratory setting and evaluation on three proficiency panels.

A prospective unblinded comparative evaluation of three assays for the detection of enteroviral RNA performed on 83 positive and 79 negative cerebrospinal fluid samples showed initial and resolved sensitivities of 90.4% and 98.8%, respectively, for the Cepheid GeneXpert enterovirus assay; 94.0% and 97.6%, respectively, for the Argene enterovirus consensus kit; and 100% and 100%, respectively, for an in-house real-time PCR. The initial and resolved specificities were 100% for all three assays.

Genomic diversity of human papillomavirus genotype 53 in an ethnogeographically closed cohort of white European women.

Human papillomavirus (HPV) genotype 53 is classified taxonomically in alpha HPV genus-species 6, together with HPV-30, HPV-56, and HPV-66 and is considered to be one of three "probable high-risk" HPV genotypes. Recent worldwide comparison of 44 isolates of HPV-53 showed the existence of nine long control region (LCR) genomic variants, which formed a phylogenetic tree with two deep dichotomic branches. In order to investigate further the genomic diversity of HPV-53, a total of 94 isolates of HPV-53 obtained from an ethnogeographically closed cohort of 70 white European women was analyzed. The identification and characterization of HPV-53 genomic variants was based on analysis of three different HPV genomic regions: LCR, E6 and E7. A higher genomic diversity of HPV-53 was identified in the ethnogeographically closed cohort of white European women than has been reported previously on isolates collected worldwide. Altogether, 19 HPV-53 genomic variants, composed of 13 LCR, 13 E6, and 5 E7 genomic variants, were identified. Eleven out of 13 LCR, all E6, and four out of five E7 genomic variants were described for the first time. The present study confirmed dichotomic phylogeny of HPV-53 described previously and, in addition, showed for the first time that after a dichotomic split, both groups of HPV-53 genomic variants formed star-like phylogenetic clusters. In women with persistent HPV-53 infection, HPV-53 genomic variants remained unchanged for up to 51 months. In rare cases, infection with multiple HPV-53 genomic variants is possible. Taking into account the results of this and previous studies, at least 26 different HPV-53 genomic variants exist today.

Twenty-four mini-pool HCV RNA screening outside a blood transfusion setting: results of a 2-year prospective study.

The usefulness of 24 mini-pool hepatitis C virus (HCV) RNA screening was evaluated in a 2-year prospective study carried out on a total of 6432 consecutive anti-HCV negative specimens in a routine diagnostic laboratory setting. A total of 268 mini-pools were tested using an automated commercial PCR assay for qualitative detection of HCV RNA, with a lower limit of detection of 50 IU/ml. Eighteen (0.28%) anti-HCV negative/HCV RNA positive serum samples obtained from 12 patients (all intravenous drug users), were detected. Ten patients responded to an invitation for follow-up testing. Five, three and one patient seroconverted in the first, second and third follow-up sample, respectively. One patient had not seroconverted by the end of the study period. The interval between the first HCV RNA positive sample and the first anti-HCV positive samples was 24-192 days. The costs of detecting a single anti-HCV negative/HCV RNA positive sample and a single anti-HCV negative/HCV RNA positive patient using the 24 mini-pool HCV RNA screening strategy were estimated to be around euro 643 and 965, respectively. It was shown that screening for HCV infection using the 24 mini-pool HCV RNA screening strategy can also be both useful and cost effective outside a blood transfusion setting.

The role of core antigen detection in management of hepatitis C: a critical review.

Several assays in research format and two commercial assays for the detection of hepatitis C virus (HCV) core protein or HCV core antigen have been developed in recent years. In order to elucidate the role and significance of HCV core antigen detection in the diagnosis and management of hepatitis C, we reviewed 56 studies published in peer-reviewed journals until September 2004. Evaluations in transfusion settings showed that the HCV core antigen assay detects HCV infection, similarly as nucleic acid techniques (NAT), between 40 and 50 days earlier than the current third generation HCV antibody screening assays. HCV core antigen levels closely track HCV RNA dynamics, and allow clinical monitoring of a patient's therapy, independently of HCV genotype, however, mainly in the samples with HCV RNA levels above 20,000 IU/ml. Considering the lower sensitivity of HCV core antigen detection in comparison to NAT, the HCV core antigen assay is not practical for the determination of the end of treatment response and sustained viral response, but could be useful for the determination of early viral response in the pegylated interferon-alpha and ribavirin treated patients infected with HCV genotype 1. The HCV core antigen detection is a viable tool for study of hepatitis C pathogenesis. The HCV core antigen can be used as a marker of HCV replication in anti-HCV positive individuals in the areas of the world that cannot afford NAT and/or in the settings that are not equipped or competent to perform HCV RNA testing. Because the manufacturer of HCV core antigen assays recently stopped an active marketing of these assays in several countries, it will, unfortunately and probably, never be possible to determine the actual potential and usefulness of HCV core antigen testing in the management of hepatitis C.