Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013.

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak

Evaluation of the Uro4 HB&L™ system for the rapid diagnosis of lower respiratory tract infections in intensive care units

Respiratory tract infection is a common and important problem in the intensive care unit (ICU) setting. It has been demonstrated that an appropriate initial regimen or its early modification (within 6-12h from diagnosis) based on microbiological results leads to a higher survival rate. Here we evaluated the Uro4 HB&L automated system for the rapid diagnosis of respiratory tract infections in ICU patients. A total of 644 lower respiratory tract specimens collected from 400 inpatients from nine ICUs at the Padova University hospital were collected during a 12-month period. All samples were processed both with the Uro4 HB&L system and with the reference culture method. Out of 322 samples, 312 were concordant positive, 276 out of 276 were concordant negative, 66 samples were declared uncertain and discarded because of an excess in turbidity. The diagnostic accuracy was good, compared with standard cultures from BAL specimens, in terms of sensitivity (0.972), specificity (1.00), likelihood ratios and diagnostic odds ratio. Ten discordant samples, resulted positive with the reference culture and not detectable with the Uro4 HB&L, were confirmed positive by Gram-stain smear analysis performed after incubation. The Uro4 HB&L system, compared to the standard culture method, revealed a very high sensitivity and a full specificity in identifying clinically relevant microorganisms from lower respiratory tract samples after merely 6h. Overall our results indicate that Uro4 HB&L is a reliable system for the surveillance of the respiratory tract infections in ICUs; it could speed up the laboratory procedures and provide fast, reliable results for clinicians

Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer.

Dear Sir, Studies in literature suggest that some human viruses, including JC polyomavirus (JCV),1\u20133 human cytomegalovirus (HCMV),4 human papillomaviruses (HPVs),5\u20138 and Epstein-Barr virus (EBV),9, 10 have a pathogenic role in starting or promoting colorectal cancer. However, a revision of the literature indicates that this association between colorectal tumors and viral infection is still matter of debate11\u201315 and further studies using robust methods to detect viral infection are required. In fact, the prevalence of viral infection in colorectal cancer samples varies widely among series, and this variability appears to be mainly related to technical aspects, such as in the case of JCV, for which the prevalence in colorectal tumors has been reported to range from 011 to 90%1\u20133, 16 in different studies. In our study, we used sensitive quantitative real-time PCR analysis and PCR and sequencing to investigate the prevalence of genome sequences not only of JCV, but also of other polyomaviruses which have been involved in human infection, i.e., BK virus (BKV), Simian virus 40 (SV40), and the newly discovered Washington University polyomavirus (WUV), Karolinska Institute polyomavirus (KIV) and Merkel cell carcinoma polyomavirus (MCV),17 in a large series of colorectal adenocarcinomas, adenomas and normal mucosa samples. Moreover, in the same samples, we investigated the presence DNA sequences of the herpesviruses HCMV and EBV and HPVs, which have been also suggested to be associated with colorectal tumorigenesis. A detailed description of methods is reported in the supporting information table. Patients gave informed consent to the study, which was approved by the local ethics committee and performed according to Declaration of Helsinki guidelines. Originally, the aim of our study was to investigate the prevalence of JCV infection in colorectal cancers from different age groups. To this aim, we first analyzed 144 archival samples of formalin-fixed paraffin-embedded tissues (group 1, Table I), collected at the Department of Medical Diagnostic Sciences and Special Therapies of the University of Padova in the period ranging from 1998 to 2007. Group 1 included colorectal adenocarcinomas and adjacent mucosa, from 72 patients, who were selected on the basis of age (36 patients were 6445 years, age range 32\u201345 years; 36 patients were 6575 years, age range 75\u201389 years) and matched for gender (14 males and 22 females in both subgroups), tumor site (28 in sigmoid, 30 in ascendent, 11 in descendent, and 3 in rectal colon in both subgroups), and stage (11 stage I-II, 17 stage III, 8 stage 4 in both subgroups). Quite unexpectedly, all samples of group 1 tested negative for JCV DNA at real-time PCR analysis. So, we tried other 3 validated primers and probe sets to detect JCV DNA and investigated whether genome sequences of other polyomaviruses, including the newly discovered viruses, were detectable in colorectal samples. But, no BKV, SV40, KIV, WUV and MCV DNA was detectable in any sample (Table I). Regarding the study of herpesviruses, 3 cancers and 1 cancer-adjacent mucosa from group 1 were positive for EBV DNA; likewise, 2 cancers and 2 cancer-adjacent mucosa samples were HCMV DNA-positive, without significant differences between age groups and among tumor sites and stages (Table I). Analysis of HPV infection demonstrated that 2 sigmoidal cancers from a male and a female patient <45 years of age were positive for HPV-16 DNA (Table I). The low frequency of viral DNA detection in this group of archival tumor samples prompted us to subsequently investigate fresh-frozen tissue samples to obtain better quality DNA. In particular, we investigated colorectal biopsies and tissue samples, which were consecutively collected during colonoscopy or surgery performed at the Department of Surgical and Gastroenterological Sciences of the University of Padova in the period ranging from January 2007 to July 2007 (group 2, Table II). Group 2 samples were obtained from 22 patients with colorectal adenomas (2 hyperplastic, 15 tubular, 5 tubulovillous) and 28 patients with colorectal adenocarcinoma (1 stage 0, 5 stage I, 8 stage II, 10 stage III and 4 stage IV). Mean age was 71.6 years (range, 41\u201392 years), 24 were males and 26 females. Overall, 5 tumors were located in caecum, 8 in ascendent, 1 in transvers, 4 in descendent, 17 in sigmoid colon and 15 in rectum. From each patient, pathologic tissues and tissues at about 3 and 10-cm distance from the lesions were collected. In addition, we analyzed biopsies of rectal, ascending and transverse colonic mucosa, obtained from 15 control subjects without medical history of colorectal pathology and without detection of lesions at colonoscopy (mean age, 58 years; age range, 36\u201382 years; 10 males, 5 females) (group 3). Like in group 1, no polyomavirus DNA sequences were detected in any sample of groups 2 and 3 (Table II). Regarding herpesviruses, EBV-DNA was detected in 11 colorectal cancers (including 2 with positive adjacent mucosa), in 5 adenomas (including 4 with positive adjacent mucosa), in 2 samples of 3-cm-adjacent mucosa, and in 1 sample of 10-cm-adjacent mucosa (with negative associated cancer or adenoma samples); HCMV-DNA was detected in 3 carcinomas (including 1 with positive adjacent mucosa) and in 1 sample of 10-cm-adjacent mucosa, but not in adenoma samples (Table II). EBV and HCMV-DNA load in tissue samples, calculated as the number of viral genome equivalents per cell, was very low in both tumor samples and adjacent mucosa (median EBV DNA load, 50 copies/106 cells; range, 10\u201310,000 copies/106 cells; median HCMV DNA load, 70 copies/106 cells; range, 10\u201330000 copies/106 cells) and consistent with lymphocyte/macrophage infiltration, rather than infection of tumor cells. The rate of EBV-DNA detection in carcinoma and adenoma samples was significantly higher than in adjacent mucosa (\u3c72-test, p < 0.05) and was explained by a greater inflammatory cell infiltration in neoplastic tissues than in normal mucosa. In the control group (group 3), EBV-DNA was detected in 5 out of 15 subjects (in the rectal mucosa in 2 cases, in the ascending colon in 2, and in all 3 colon samples in one subject); HCMV-DNA was detected in the rectal mucosa from a patient who was also EBV-positive. High-risk HPV-16 DNA was detected by both nested PCR/sequencing and real-time PCR in 2 rectal carcinomas and in a sigmoid adenoma of group 2, but not in control group 3 samples (Table III). In the 3 positive cases, the adjacent mucosa was also HPV-16 DNA-positive. In the literature, JCV genome sequences were detected by PCR amplification and Southern blot hybridization in 90% of colorectal cancers and in the adjacent normal colonic epithelium, with a 10-fold higher viral load in cancers than in adjacent mucosa,1, 2 as well as in 82% of sporadic adenomatous polyps.18 In colorectal mucosa, JCV was suggested to promote tumorigenesis through a direct interaction of its large T antigen (Tag) with \u3b2-catenin and p53.2, 3 Since a variant of the JCV Mad-1 strain was preferentially detected in colon cancers, it was hypothesized that this strain might be selectively activated in colonic epithelial cells.19 It cannot however be excluded that detection of a unique JCV strain represented a laboratory contamination, since the same Mad-1 strain was used as positive control in the laboratory.11 In our study, we used real-time PCR to detect viral sequences. This method is very sensitive and specific, but much less susceptible to amplicon contamination than PCR followed by Southern-blot, which was employed in the other studies. A study on large series of colorectal cancer/normal tissue pairs,11 which also employed real-time PCR, failed to demonstrate the presence JCV-DNA sequences in all samples, in agreement with our results. Immunostaining for SV40 TAg has been reported to give problems of false positive results,20 so it cannot be excluded that reported JCV TAg detection in colorectal tissues is also a technical artefact, since the same antibodies against SV40 TAg were employed to investigate JCV expression, exploiting their cross-reactivity with JCV Tag.2, 18 Reports regarding detection of BKV and SV40 in colorectal cancers are conflicting, with negative results in 1 study,21 in agreement with our data, but positive findings in a recent Italian report which demonstrated BKV and SV40 DNA in 6 and 10 out of 66 colorectal carcinomas, respectively.22 Problems of PCR contamination cannot be excluded even in this study, since the primers employed could amplify SV40 DNA sequences which are present in some laboratory plasmids.22 Our data seem also to exclude WUV, KIV and MCV polyomavirus have any role in colorectal tumorigenesis. HCMV has also been implicated in colorectal tumorigenesis because of its detection in about 80% of tumors, but not in adjacent non-neoplastic mucosa,4, 23 and because of the demonstration that HCMV infection of colorectal cancer cell lines in vitro resulted in induction Bcl2 and cycloxygenase-2 proteins, both of which are involved in important pathways of colorectal cancer progression.4 In contrast, other studies failed to detect HCMV DNA sequences in tumour samples.12\u201314 Regarding EBV, it has been associated with gastric adenocarcinoma and gastric lymphoepithelioma, whereas its involvement in colorectal tumorigenesis seems to be excluded by several studies which, generally, did not detect EBV-positive cancer cells, even though some EBV-positive tumor infiltrating B lymphocytes were frequently found.9, 24\u201326 However, we do not rule out the possibility that the occasional presence of EBV and HCMV in infiltrating lymphocytes/monocytes in colorectal mucosa may promote the production and release of cytokines and growth factors or deregulate tumor suppressor genes and thus cooperate with cancer development. Among candidate oncogenic viruses for colorectal tumorigenesis, HPV is the most reliable, as indicated by the epidemiological evidence of high-risk HPV infection in a relatively high percentage of colorectal tissues from patients with cancer, but not in control subjects without cancer.5\u20138 Accordingly, we also identified HPV-16 DNA in some cases of distally localized colorectal cancers. Persistent high-risk HPV infection has been definitely demonstrated to be a necessary cause for development of cervical cancer and other anogenital cancers27, 28 and might also be responsible for some cases of colorectal cancer. While waiting for further epidemiological data and for experimental studies to confirm this hypothesis, we can predict anti-HPV vaccination might have an impact on colorectal cancer prevention. In conclusion, our study, which for the first time systematically investigated the presence of genomic sequences of several putative oncogenic viruses in a large series of colorectal neoplasms, seems to exclude JCV, BKV, SV40, WUV, KIV, MCV, HCMV and EBV have a prominent role in the pathogenesis of colorectal cancer, whereas it suggests high-risk HPV could be involved in some cases of distally localized colorectal carcinoma. Yours sincerely, Valentina MILITELLO, Marta TREVISAN, Laura SQUARZON, Maria Angela BIASOLO, Massimo RUGGE, Carmelo MILITELLO, Giorgio PAL a and Luisa BARZO

Immunogenicity, safety and tolerability of a bivalent human papillomavirus vaccine in adolescents with juvenile idiopathic arthritis

Aims: To evaluate the immunogenicity, safety and tolerability of the bivalent HPV vaccine in female patients with juvenile idiopathic arthritis (JIA). Methods: Twenty-one patients with JIA aged 12-25 years and 21 healthy controls were enrolled and received three doses of the bivalent HPV vaccine. Results: All of the subjects were seronegative at baseline and seroconverted after the scheduled doses. The JIA patients showed significantly lower HPV16 neutralising antibody titres than controls 1 month after the administration of the third dose (p < 0.05), whereas no significant difference was observed in HPV18 neutralising antibody titres. Local and systemic reactions were similarly frequent in the patients and controls, and there were no significant changes in 27-joint juvenile arthritis disease activity score or laboratory tests. Conclusion: The bivalent HPV vaccine is safe in patients with stable JIA and regardless of the use of medications the vaccine assures an adequate degree of protection for a certain time

Neutralizing and cross-neutralizing antibody titres induced by bivalent and quadrivalent human papillomavirus vaccines in the target population of organized vaccination programmes.

Aim of this investigator-initiated study was to evaluate and compare the titres of neutralizing and cross-neutralizing antibodies (NAbs) induced by the bivalent (Cervarix®) and quadrivalent (Gardasil®) HPV vaccines in a cohort of girls aged 11-13 years from organized vaccination programmes. To this aim, HPV16 and HPV18 NAbs were measured by pseudovirion-based neutralization assays in serum collected at 1-6 months after the third vaccine dose in 107 girls vaccinated with Cervarix® and 126 vaccinated with Gardasil®, while HPV31 and HPV45 cross-NAbs were tested in the first 50 consecutive girls of both vaccine groups. The results of this study demonstrated that all vaccinated girls developed HPV16 and HPV18 NAbs, with the exception of two Gardasil® vaccinees with undetectable HPV18 NAbs. Geometric mean titres (GMTs) of both HPV16 and HPV18 NAbs were significantly higher in Cervarix® than in Gardasil® vaccinees [HPV16 NAb GMT 22,136 (95% CI, 18,811-26,073) vs 5092 (4230-6151), respectively; P<0.0001; HPV18 NAb GMT 11,962 (9536-14,363) vs 1804 (1574-2110), respectively; P<0.0001]. Cross-NAbs to HPV31 and HPV45 were detected more frequently Cervarix® (HPV31 NAb positivity rates 92.7% and 36%, respectively; P<0.05) than in Gardasil® vaccinees (HPV45 NAb positivity rates 56% and 6%, respectively; P<0.0001). The titres of cross-NAbs against HPV31 and HPV45 were also significantly higher in Cervarix® than in Gardasil® vaccinees [HPV31 NAb GMT 157.2 (95% CI, 92-269) vs 13.0 (6.5-25.8), respectively; P<0.0001; HPV45 NAb GMT 4.7 (2.1-10.2) vs 1.3 (0.3-3.1), respectively; P<0.01].In conclusion, in adolescent girls vaccinated within organized vaccination programmes, HPV vaccines drive the generation not only of NAbs to HPV vaccine types, but also of cross-NAbs. The bivalent vaccine induced significantly higher HPV16 and HPV18 NAb titres and more frequently and at higher titre HPV31 and HPV45 cross-NAbs than the quadrivalent vaccine

Immunogenicity, safety and tolerability of a bivalent human papillomavirus vaccine in adolescents with juvenile idiopathic arthritis.

Aims: To evaluate the immunogenicity, safety and tolerability of the bivalent HPV vaccine in female patients with juvenile idiopathic arthritis (JIA). Methods: Twenty-one patients with JIA aged 12-25 years and 21 healthy controls were enrolled and received three doses of the bivalent HPV vaccine. Results: All of the subjects were seronegative at baseline and seroconverted after the scheduled doses. The JIA patients showed significantly lower HPV16 neutralising antibody titres than controls 1 month after the administration of the third dose (p < 0.05), whereas no significant difference was observed in HPV18 neutralising antibody titres. Local and systemic reactions were similarly frequent in the patients and controls, and there were no significant changes in 27-joint juvenile arthritis disease activity score or laboratory tests. Conclusion: The bivalent HPV vaccine is safe in patients with stable JIA and regardless of the use of medications the vaccine assures an adequate degree of protection for a certain time