Sviluppo di un metodo accurato per l'identificazione e genotipizzazione del papillomavirus umano basato sul sequenziamento 454 di nuova generazione

Accurate HPV typing is essential for evaluation an monitoring HPV vaccines, as second line testing in cervical cancer screening and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons. We set up a first protocol by using modified degenerated primer MY09/11 to analyze 164 cervical samples. This protocol represent a proof of principle that NGS can be used for HPV typing, but had an intrinsic weakness conceivably due to mismatches between the sequence of the MY09/11 primers. For this reason we generate a new protocol by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12,000-fold coverage, the NGS method allowed to correctly identify all high-risk HPV types, either in single and multiple infection, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative, and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types and variants

Health Surveillance and Response to SARS-CoV-2 Mass Testing in Health Workers of a Large Italian Hospital in Verona, Veneto

Italy presented the first largest COVID-19 outbreak outside of China. Veneto currently ranks fourth among the Italian regions for COVID-19 confirmed cases (~19,000). This study presents health surveillance data for SARS-CoV-2 in 6100 health workers (HW) employed in a large public hospital. Workers underwent oropharyngeal and nasopharyngeal swabs, with a total of 5942 participants (97.5% of the population). A total of 11,890 specimens were tested for SARS-CoV-2 infection using PCR, identifying the viral genes E, RdRP, and N. Positive tests were returned for 238 workers (cumulative incidence of 4.0%, similar in both COVID and nonCOVID units). SARS-CoV-2 risk was not affected by gender, age, or job type, whereas work setting and occupation were both predictors of infection. The risk was higher in medical wards (OR 2.7, 95% CI 1.9\u20133.9) and health services (OR 4.3, 95% CI 2.4\u20137.6), and lower in surgical wards and administration areas. To our knowledge, this study represents the largest available HW case list swab-tested for SARS-CoV-2, covering almost the total workforce. Mass screening enabled the isolation of HW, improved risk assessment, allowed for close contacts of and infected HW to return to work, provided evidence of SARS-CoV-2 diffusion, and presented solid ground to prevent nosocomial SARS-CoV-2 infections. The ongoing concurrent sero-epidemiological study aims to enable the improvement of health surveillance to maintain the safety of HWs and the communities they serve

Synthesis and characterization of CdS nanoparticles embedded in a polymethylmethacrylate matrix

CdS nanopowder capped with sodium bis(2-ethylhexyl)sulfosuccinate was synthesized by using water-in-oil microemulsions. The CdS nanoparticles of about 5 nm obtained were embedded in polymethylmethacrylate matrix by a photocuring process. The transparent yellow solid compound was characterized by optical absorption and emission spectroscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The properties of this compound were compared with those of the nanopowder dispersed in heptane and in methylmethacrylate. The results obtained indicate that the nanoparticles are homogeneously dispersed in the matrix and do not change in size during the embedding process. Even if the surface slightly changes its luminescence properties, as a consequence of the different new chemical environment, the final product seems to be suitable for practical applications

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics

Sviluppo di un metodo accurato per l'identificazione e genotipizzazione del papillomavirus umano basato sul sequenziamento 454 di nuova generazione

Accurate HPV typing is essential for evaluation an monitoring HPV vaccines, as second line testing in cervical cancer screening and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons. We set up a first protocol by using modified degenerated primer MY09/11 to analyze 164 cervical samples. This protocol represent a proof of principle that NGS can be used for HPV typing, but had an intrinsic weakness conceivably due to mismatches between the sequence of the MY09/11 primers. For this reason we generate a new protocol by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12,000-fold coverage, the NGS method allowed to correctly identify all high-risk HPV types, either in single and multiple infection, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative, and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types and variants.Un' accurata tipizzazione di HPV è essenziale per la valutazione e il monitoraggio della vaccinazione, come test di secondo livello nello screening del carcinoma cervicale e per le indagini epidemiologiche. In questo studio, è stato messo a punto e testato su campioni clinici un nuovo metodo di tipizzazione di HPV basato sul sequenziamento 454 next-generation (NGS) dell' amplicone L1 di HPV. A tale scopo è stato sviluppato un primo protocollo che utilizzava dei primer ottenuti modificando i primer degenerati MY09/11; tale protocollo è stato testato su 164 campioni citologici cervicali fornendo una prima dimostrazione che il NGS può essere utilizzato per la tipizzazione di HPV. Il protocollo presentava però alcuni limiti legati soprattutto al disegno dei primer. E' stato quindi studiato un secondo protocollo. Tale protocollo utilizzava un set di primer PGMY modificati in modo da ottenere sensibilità migliorata per alcuni tipi di HPV che non erano riconosciuti dai primer PGMY standard. Usando una median coverage di 12,000-fold, il metodo NGS è arrivato a identificare correttamente tutti i tipi di HPV ad alto rischio sia in infezioni singole che multiple, con una sensibilità di 50 genomi equivalenti, come dimostrato testando il pannello di campioni del WHO LabNet EQA. L' analisi di miscele di linee cellulari positive per HPV16 e per HPV18 ha dimostrato che il metodo NGS può quantificare in modo riproducibile la proporzione con cui ogni tipo virale è presente nelle infezioni multiple con un ampio range. Nell'analisi di campioni clinici il metodo NGS ha identificato correttamente un ampio numero di tipi di HPV nelle infezioni multiple ed è stato anche utile nell'analisi di campioni cervicali con risultati discordanti al test hybrid capture 2 e line probe assays. Il metodo ha permesso inoltre di riconoscere la presenza di varianti intratipo (sostituzioni nucleotidiche) nello stesso campione clinico. In conclusione, è stato messo a punto un nuovo metodo di tipizzazione dell' HPV basato sul pirosequenziamento 454. Questo metodo è sensibile, specifico, quantitativo, e preciso sia nelle infezioni singole che nelle infezioni multiple. Permette inoltre di identificare un ampio range di tipi di HPV e potenzialmente consente di scoprire nuovi tipi di HPV e varianti

Timing and pharmacological support in the surgical treatment of multiple perianal fistulas in Crohn's Disease

We present a case report on the management of a young woman affected by Crohn's Disease ever since childhood, complicated by complex, multiple perianal fistulas. In literature, there is increasing evidence to support the treatment of perianal fistulas using a combined association of medical and surgical strategies. In the case of our patient, the choice of surgery in association with pharmacological treatment was supported by the consideration of the fact that intervening during a quiescent phase of the disease, from the symptomatic, clinical-biohumoral and endoscopic standpoint, would have reduced the risk of complications and thus promoted healing. (www.actabiomedica.it)

Hernioplasty in Elderly High-Risk Adults: Efficacy of Fibrin Glue

Background: Risk factors as cirrhosis, diabetes, coagulopathies, advanced age, have often been considered a contraindication to perform an inguinal hernioplasty. The aim of this study was to establish whether fibrin glue application to fix the mesh during a Lichtenstein inguinal hernioplasty is useful to prevent complications in high risk patients. Methods: From March 2004 to September 2009, 87 high risk patients underwent hernioplasty using fibrin glue to fix mesh prosthesis. Patients were evaluated for immediate and late postoperative pain, presence of haematomas and seromas, infiltration of ascitic fluid, and wound complications. Results: Among our patients (mean age 73.6\ub112.4 years) 38 (44%) had diabetes, 33 (38%) had coagulopathies and 16 (18%) had liver cirrhosis (2 C Child\u2019s grade). During a follow-up period ranging from 12 to 24 months, no complications nor significant post-operative pain were observed. Conclusions: This preliminary observational study indicates that fibrin glue application can be considered a very useful way to obtain good results by open hernioplasty in high risk patients

Comparison of INNO-LiPA Genotyping Extra and Hybrid Capture 2 assays for detection of carcinogenic human papillomavirus genotypes

BACKGROUND: Accurate HPV detection and genotyping tests are useful for management of women with HPV infection and for monitoring HPV vaccine efficacy. OBJECTIVES: To evaluate the performance of the INNO-LiPA HPV Genotyping Extra assay (SPF10-LiPA) for the detection of carcinogenic HPV types in women referred for opportunistic cervical cancer screening by comparison with the Hybrid Capture 2 (HC2) assay. STUDY DESIGN: Cross-sectional analysis from baseline data of HC2 and SPF10-LiPA testing in cervical specimens collected from 1580 consecutive women and correlation with cervical cytology and histology data, when available. RESULTS: The two assays showed a good agreement for detection of carcinogenic HPV types and reported the same prevalence of carcinogenic HPV infections in different age groups. Stratification of study subjects by cervical cytology interpretation and histology results demonstrated that the two tests gave very similar results in the different cytology interpretation groups and in CIN2 and CIN3 samples, while in <CIN2 samples the SPF10-LiPA assay provided a significantly lower number of carcinogenic HPV-positive results than the HC2 test. A comparative analysis of the two assay for individual HPV types showed that HC2 identified as positive between 73% and 100% of specimens with carcinogenic HPV types detected by SPF10-LiPA and, in particular, approximately 90% and 80% of HPV16- and HPV18-positive samples, respectively. CONCLUSIONS: A good agreement was observed between HC2 and SPF10-LiPA for carcinogenic HPV type detection, that supports further evaluation of the clinical performance of the new version of SPF10-LiPA in cervical cancer screening protocols

WU and KI Polyomaviruses Remain Orphans in Adults Reply

Comment on the presence of the newly discovered polyomaviruses in human tissues