Pierlanfranco D'Agaro

HBV, HCV, and TTV detection by in situ polymerase chain reaction could reveal occult infection in hepatocellular carcinoma: comparison with blood markers.

Objective: To report a retrospective analysis on the presence of hepatitis B virus (HBV), hepatitis C virus (HCV), and transfusion transmitted virus (TTV) sequences in formalin fixed, paraffin embedded liver biopsies from eight patients with hepatocellular carcinoma, in comparison with blood markers. Methods: A direct in situ polymerase chain reaction (PCR) technique was developed for the detection and localisation of genomic signals in the liver tissue. Conventional serological and molecular methods were used for blood evaluation. Results: In situ PCR showed the presence of one of the three viruses (four HCV, two HBV, and one TTV) in seven of the eight patients. In addition, a co-infection with HBV and HCV was detected in one patient. HCV and HBV sequences were located in the cytoplasm and the nucleus, respectively. When compared with blood markers, these findings were compatible with one occult HBV and two occult HCV infections. Conclusions: These findings provide further evidence for occult HBV and HCV infections in cancerous tissues from patients with hepatocellular carcinomas. In situ PCR could be an additional tool for evaluating the viral aetiology of hepatocellular carcinoma alongside conventional diagnostic procedures.

Hemorrhagic cystitis in children undergoing bone marrow transplantation: a putative role for simian virus 40.

Background. Late-onset hemorrhagic cystitis (HC) is a well-known severe complication of bone marrow transplantation (BMT), both in adults and in children. Protracted postengraftment HC is associated with graft-versus-host disease and viral infections, mainly caused by BK virus (BKV) or adenovirus (AV). This study investigated whether simian virus 40 (SV40) DNA sequences can be detected in specimens from pediatric patients affected by severe postengraftment HC. Methods. The clinical diagnosis of HC was made in 7 of 28 BMT children. DNA from peripheral blood mononuclear cells (PBMC) and urine sediment cells and supernatants was analyzed by polymerase chain reaction (PCR) for human cytomegalovirus (HCMV), AV, BKV, JC virus (JCV), and SV40. DNA filter hybridization and sequencing was carried out in SV40-positive samples. Results. SV40 footprints were detected in two of seven cases of HC. Specific SV40 DNA sequences were detected by PCR and by filter hybridization both in urine and in PBMC samples at the HC onset and during the follow-up. The DNA sequencing proved that the amplicons belonged to the SV40 wild-type. Urine samples of the two HC cases tested negative by cell cultures, PCR, or both for HCMV, BKV, JCV, and AV. Conclusions. The detection of SV40 DNA sequences suggest that this simian polyomavirus could be involved, at least in some cases, in the HC occurring in children after BMT.

Group B streptococcus prevalence in pregnant women from North‐Eastern Italy: advantages of a screening strategy based on direct plating plus broth enrichment

Aims: To assess the sensitivity of a combined selective broth enrichment technique plus selective plating for the detection of group B streptococcus (GBS) colonisation in a large cohort of pregnant women from North-Eastern Italy. Methods: During 2002–2005, 5020 pregnant women were screened between the 35th and the 37th week of gestation. A lower vaginal sample and a rectal sample were collected and inoculated onto LIM broth and a selective colistin aztreonam blood agar plate (CAP). Direct agar plates were examined after 18–24 hours and, if negative, after 48 hours. LIM broth was subcultured after 18–24 hours onto a Columbia blood agar plate. All colonies suggestive for GBS were submitted to phenotypic identification. Results: 901 Women (17.9%) were positive for GBS. On 728 positive samples, corresponding to patients enrolled between 2003 and 2005, the results of selective direct plating and selective broth enrichment were compared. A total of 561 (77.1% of positive samples, corresponding to 13.9% of patients) were positive on direct selective agar; an additional 167 isolates (22.9% of samples, 4.1% of patients) were recovered from the LIM broth subculture. Conclusions: The prevalence of GBS carriage in this population-based study is a reliable estimate considering the sensitivity of the microbiological methods used, the rate of attendance of pregnant women to clinical and laboratory settings and the compliance to the protocol. Results confirm that the combination of selective enrichment broth and selective direct plating is a time-saving and sensitive method.

Prevalence of Tick-borne encephalitis virus in Ixodes ricinus from a novel endemic area of North Eastern Italy.

In Alpine area of extreme North Eastern Italy the first autochthonous case of TBE was reported in 1998 and was followed by 45 cases during the period 2001–2007, thus defining this area as definitely endemic. An ecological survey evaluated the tick density and the Tick‐borne encephalitis virus (TBEV) infection prevalence in tick collected in selected sites. In addition, TBE strains were characterized by sequencing and phylogenetic analysis. Overall, 2,361 ticks (2,198 nymphs and 163 adults) of the Ixodes ricinus L. species collected during 2005 and 2006 were examined. Five samples were positive for TBEV, corresponding to an overall prevalence rate of 0.21%. When analyzed by place, TBEV was discovered in three sites where the highest tick density was found. The difference of prevalence between high and low density areas tested to be statistically significant (P = 0.028). Phylogenetic analysis showed that four sequences clustered with the Neudoerfl prototype, while the other clustered with the Isosaari 17 strain and with a number of Slovenian isolates. In addition, a sequence detected in archival samples from one human case segregated with another variant, namely the Swedish Torö strain. J. Med. Virol. 81:309–316, 2009.

Ticks and Lyme borreliosis in an alpine area in northeast Italy.

A 2‐year study was conducted in a mountainous area of northeast Italy to evaluate the occurrence and distribution of ticks, as well as to assess the prevalence of the spirochaete Borrelia burgdorferi sensu lato. All ticks collected were Ixodes ricinus L. (Parasitiformes: Ixodidae). In general, most nymphs and adult ticks were collected from April to July. Tick density was highly variable among sites; however, two areas with different infestation levels were recognized. Prevalences of B. burgdorferi s.l. in nymphal stages were rather variable between sites; overall the prevalence of infected nymphs in the whole area was slightly higher than 20%. The prevalence of B. burgdorferi s.l. in nymphs does not seem to be correlated with nymph density. The correlation between the incidence of Lyme borreliosis (reported human cases/1000 inhabitants/year) and Borrelia prevalence in nymphs was not significant, although a significant correlation was found between borreliosis incidence and nymph density.

Formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling

Interferons are key mediators of the innate antiviral response of the cell against viral infections. Viruses on the other hand have evolved various strategies to delay innate immunity in order to establish a productive infection. In this work we analyzed the pathway of interferon induction by the tick-borne encephalitis virus. We initially observed a consistent delay of interferon induction following virus replication. RIG-I, but not MDA5, and nuclear translocation of IRF3 were eventually required for interferon activation pointing to a defect in pattern recognition receptors signaling. However, viral proteins could not directly inhibit the pathway suggesting an indirect mechanism. We found that dsRNA replication intermediates and replicated viral RNA localized to membrane-defined perinuclear compartments that resisted RNAse treatment. Thus, initial escape from innate immunity involved the formation of replication vesicles that may function as a barrier to pattern recognition receptors.

A Molecular Case―Control Study of the Merkel Cell Polyomavirus in Colon Cancer

To explore the putative role of the Merkel cell polyomavirus in human colon cancer, a prospective molecular case–control study was undertaken in patients and their relatives enrolled during a screening program. Fresh tissue samples from 64 cases of colon cancer (mean age 69.9 ± 11.0 years; 40 males) and fresh biopsies from 80 relatives (mean age 53.7 ± 8.6 years; 43 male; 55 son/daughter, 23 brother/sister, 2 parents) were analyzed by PCR and sequencing. Pre‐cancerous lesions, namely adenomas and polyps, were detected in 15 (18.8%) and 9 (11.2%) of the controls, respectively. In addition, 144 blood samples were examined. Merkel cell polyomavirus DNA was detected in 6.3% of cases and 8.8% of controls. This difference was not statistically significant in the logistic regression analysis, after adjustment for age. Whereas blood samples from both cases and controls tested negative, the DNA Merkel cell polyomavirus was identified in 12.5% of adenoma/polyp tissues. No statistically significant difference was found when prevalence rates of Merkel cell polyomavirus in normal, pre‐cancerous and cancer tissues were compared. Sequence analysis of the viral LT3 and VP1 regions showed high homology (>99%) with those of strains circulating worldwide, especially with genotypes detected in France. The findings of this survey are consistent with the hypothesis that the Merkel cell polyomavirus, in addition to other human polyomaviruses, can be recovered frequently from the gastrointestinal tract, because it is transmitted throughout the fecal‐oral route. Moreover, the study does not indicate a role for Merkel cell polyomavirus in the genesis of colon cancer. J. Med. Virol. 83:721–724, 2011.

Latent viral infections in young patients with inflammatory diseases treated with biological agents: Prevalence of JC virus genotype 2

Treatment with biological drugs is associated with increased susceptibility to viral infections. Reactivation of JC virus (JCV) and human cytomegalovirus (HCMV) in adults after therapy has been documented. The long‐term effects of biological and conventional therapy on human herpesviruses and polyomaviruses infections in young patients were assessed. One hundred eighty‐six samples [urine, serum, and blood cells (PBMCs)] from 62 patients (15.8 ± 6.2 years old) with Crohns disease, ulcerative rectocolitis or juvenile rheumatoid arthritis treated with immunotherapy or conventional therapy for over 12 months were tested by real time PCR. One hundred twenty‐four samples (urine and blood) from 62 matched healthy volunteers (13.8 ± 8.6 years old) were included as controls. Sequencing of the JCV viral protein 1 (VP1) and transcriptional control region (TCR) was performed. Herpes simplex virus 1/2 and varicella zoster virus genomes were not detected in any patients, whereas Epstein–Barr virus, HCMV, and human herpesvirus‐6 genomes were detected in 4.8%, 3.2%, and 1.6% of the patients, respectively. JCV was detected in 22.6% (14/62) of urine samples from patients and in 8% (5/62) from controls, in 50% (7/14) of sera from patients shedding JCV, and in 71.4% (5/7) of matched PBMCs. There was a significant association between infliximab treatment and excretion of JCV genotype 2. Subclinical infection/reactivation of JCV genotype 2 in young patients during infliximab therapy was demonstrated. Conversely, increased susceptibility to herpesviruses infection was not shown. Future studies are warranted to investigate the effects of JCV reactivation on the health of young patients treated with infliximab. J. Med. Virol. 85:716–722, 2013.

Human herpes virus 6 in archival cardiac tissues from children with idiopathic dilated cardiomyopathy or congenital heart disease

Objective: To explore the possible role of human herpes virus 6 (HHV-6) in cardiac disorders in childhood in a retrospective study on archival specimens of explanted hearts. Methods: 16 children (median age at transplantation 11.0 years) with idiopathic dilated cardiomyopathy (DCM) and 19 children (median age at transplantation 1.0 year) with congenital heart disease (CHD), previously found to be negative for other cardiotropic viruses such as enteroviruses, adenovirus, parvovirus B19, cytomegalovirus and Epstein–Barr virus, were tested for HHV-6 by quantitative real-time PCR and by genotyping. In addition, HHV-7/8 infection was investigated by qualitative PCR. Results: HHV-6 B variant was detected in 11 of 35 samples (31.4%) with a mean viral load of 3.1×102 copies/μg of DNA. When assessed by heart disorder, the prevalence was different in the two groups (43.7% in DCM and 21% in CHD) while the mean viral loads were similar. In a logistic multivariate analysis HHV-6 was independently associated with DCM, taking CHD as reference and adjusting for age (best estimate: OR = 6.94; 95% CI 1.00 to 49.85; p = 0.05). Conclusions: Although the clinical significance of the results is unknown, HHV-6 B genome is frequently detected in explanted hearts from children with DCM and to a lesser extent with CHD, thus adding evidence for HHV-6 cardiac involvement.

Secondary lymphoid tissue as an important site for WU polyomavirus infection in immunocompetent children

The polyomaviruses KI and WU (KIPyV and WUPyV) have been identified in respiratory specimens from children with acute respiratory infections, which suggests the respiratory tract as a possible site of infection. However, the persistence of infection in the lymphoid system is unknown. Fresh samples (n = 211) of tonsils, adenoids, and peripheral blood mononuclear cells (PBMCs) from 83 immunocompetent children (mean age 4.8 years) were tested for amplification of the KIPyV VP1 and WUPyV VP2 genes. The known BK and JC polyomaviruses and the lymphotropic human herpesvirus (HHV)‐6 were also investigated by quantitative real‐time PCR and direct sequencing. In addition, 98 nasopharyngeal swabs collected from children (mean age 6.2 years) affected by seasonal influenza‐like illness were tested. Of the lymphoid tissues, 34.9% were positive for WUPyV, 4.8% for BK virus, and 33.8% for HHV‐6. KIPyV and JC virus were not detected in these specimens. None of the polyomaviruses were detected in PBMCs. Among the nasopharyngeal samples, the prevalence of WUPyV was 27.5%, although 70% of the positive samples were co‐infected with at least one of the following respiratory viruses: influenza virus, adenovirus, and respiratory syncytial virus. Phylogenetic analysis revealed high sequence homology (99%) between lymphoid‐ and nasopharynx‐derived WUPyV strains. These results suggest that the tonsils and adenoids of immunocompetent children are a reservoir for WUPyV infection; probably due to the respiratory route of transmission. In addition, the prevalence of WUPyV was high among the children, and the virus was identified more frequently in older children than during the first years of life. J. Med. Virol. 83:1446–1450, 2011.