Molecular study of the presence and transcriptional activity of HPV in semen.

PURPOSE: Human Papillomavirus (HPV) in semen represents a controversial topic. Recent evidence suggests a correlation with poor semen quality, but its detection is still unstandardized in this biological fluid. Thus, the aims of this study were to verify the ability of nested PCR to reveal HPV-DNA in semen; to evaluate association of seminal HPV with sperm parameters and risk factors for infection; to investigate the rate of HPV-DNA positivity in patients with and without risk factors; to assess HPV transcriptional activity. METHODS: We enrolled sexually active men and collected clinical and anamnestic data during andrological and sexually transmitted infections (STIs) evaluation. For each patient, we performed semen analysis and nested PCR to detect HPV-DNA in semen. In positive semen samples, we proceeded with genotyping and RNA quantification to detect HPV transcriptional activity. RESULTS: We enrolled 185 men (36.0 ± 8.3 years), of which 85 with (Group A) and 100 without HPV risk factors (Group B). Nested PCR was able to reveal HPV-DNA in semen, discovering a prevalence of 8.6% (11.8% in Group A and 6% in Group B, respectively). We observed no correlation between sperm quality and seminal HPV. Genital warts and previous anogenital infection were significantly associated with the risk of HPV positivity in semen. Moreover, no viral transcriptional activity was detected in positive semen samples. CONCLUSIONS: Our study suggests that searching for seminal HPV could be important in patients both with and without risk factors, especially in assisted reproduction where the risk of injecting sperm carrying HPV-DNA is possible.

Genetic diversity and its impact on disease severity in respiratory syncytial virus subtype-A and -B bronchiolitis before and after pandemic restrictions in Rome.

OBJECTIVES: To scrutinize whether the high circulation of respiratory syncytial virus (RSV) observed in 2021-2022 and 2022-2023 was due to viral diversity, we characterized RSV-A and -B strains causing bronchiolitis in Rome, before and after the COVID-19 pandemic. METHODS: RSV-positive samples, prospectively collected from infants hospitalized for bronchiolitis from 2017-2018 to 2022-2023, were sequenced in the G gene; phylogenetic results and amino acid substitutions were analyzed. Subtype-specific data were compared among seasons. RESULTS: Predominance of RSV-A and -B alternated in the pre-pandemic seasons; RSV-A dominated in 2021-2022 whereas RSV-B was predominant in 2022-2023. RSV-A sequences were ON1 genotype but quite distant from the ancestor; two divergent clades included sequences from pre- and post-pandemic seasons. Nearly all RSV-B were BA10 genotype; a divergent clade included only strains from 2021-2022 to 2022-2023. RSV-A cases had lower need of O2 therapy and of intensive care during 2021-2022 with respect to all other seasons. RSV-B infected infants were more frequently admitted to intensive care units and needed O2 in 2022-2023. CONCLUSIONS: The intense RSV peak in 2021-2022, driven by RSV-A phylogenetically related to pre-pandemic strains is attributable to the immune debt created by pandemic restrictions. The RSV-B genetic divergence observed in post-pandemic strains may have increased the RSV-B specific immune debt, being a possible contributor to bronchiolitis severity in 2022-2023.

Infants hospitalized for Bordetella pertussis infection commonly have respiratory viral coinfections.

BACKGROUND: Whether viral coinfections cause more severe disease than Bordetella pertussis (B. pertussis) alone remains unclear. We compared clinical disease severity and sought clinical and demographic differences between infants with B. pertussis infection alone and those with respiratory viral coinfections. We also analyzed how respiratory infections were distributed during the 2 years study. METHODS: We enrolled 53 infants with pertussis younger than 180 days (median age 58 days, range 17­109 days, 64. 1% boys), hospitalized in the Pediatric Departments at "Sapienza" University Rome and Bambino Gesù Children's Hospital from August 2012 to November 2014. We tested in naso-pharyngeal washings B. pertussis and 14 respiratory viruses with real-time reverse-transcriptase-polymerase chain reaction. Clinical data were obtained from hospital records and demographic characteristics collected using a structured questionnaire. RESULTS: 28/53 infants had B. pertussis alone and 25 viral coinfection: 10 human rhinovirus (9 alone and 1 in coinfection with parainfluenza virus), 3 human coronavirus, 2 respiratory syncytial virus. No differences were observed in clinical disease severity between infants with B. pertussis infection alone and those with coinfections. Infants with B. pertussis alone were younger than infants with coinfections, and less often breastfeed at admission. CONCLUSIONS: In this descriptive study, no associations between clinical severity and pertussis with or without co-infections were found. TRIAL REGISTRATION: Policlinico Umberto I: protocol 213/14, 3085/13.02.2014, retrospectively registered. Bambino Gesù Children's Hospital: protocol n. RF-2010-2317709.

Frequent detection of high human papillomavirus DNA loads in oral potentially malignant disorders.

Human papillomavirus (HPV) is estimated to be the cause of 40--80% of the squamous cell carcinoma of the oropharynx but only of a small fraction of the oral cavity cancers. The prevalence of oral HPV infection has significantly increased in the last decade, raising concerns about the role of HPV in progression of oral potentially malignant disorders (OPMD) toward squamous cell carcinomas. We sought to study HPV infection in patients with oral lesions, and in control individuals, using non-invasive and site-specific oral brushing and sensitive molecular methods. HPV DNA positivity and viral loads were evaluated in relation to patient data and clinical diagnosis. We enrolled 116 individuals attending Dental Clinics: 62 patients with benign oral lesions (e.g. fibromas, papillomatosis, ulcers) or OPMD (e.g. lichen, leukoplakia) and 54 controls. Oral cells were collected with Cytobrush and HPV-DNA was detected with quantitative real-time PCR for the more common high-risk (HR) and low-risk (LR) genotypes. HPV detection rate, percentage of HR HPVs and HPV-DNA loads (namely HPV16 and in particular, HPV18) were significantly higher in patients than in controls. Lichen planus cases had the highest HPV-positive rate (75.0%), hairy leukoplakia the lowest (33.3%). This study detected unexpectedly high rates of HPV infection in cells of the oral mucosa. The elevated HR HPV loads found in OPMD suggest the effectiveness of quantitative PCR in testing oral lesions. Prospective studies are needed to establish whether elevated viral loads represent a clinically useful marker of the risk of malignant progression.

Immunodeficiency-associated viral oncogenesis.

Several viruses with different replication mechanisms contribute to oncogenesis by both direct and indirect mechanisms in immunosuppressed subjects after solid organ transplantation, after allogeneic stem cell transplantation, or with human immunodeficiency virus (HIV) infection. Epstein-Barr virus (EBV), human papillomavirus (HPV), Kaposi sarcoma herpesvirus (KSHV), human T-cell lymphotropic virus type 1 (HTLV-1) and Merkel cell polyoma virus (MCV) are the main viruses associated with the development of cancer in immunosuppressed patients. Besides being a main cause of immunodeficiency, HIV1 has a direct pro-oncogenic effect. In this review, we provide an update on the association between the condition of acquired immunodeficiency and cancer risk, specifically addressing the contributions to oncogenesis of HPV, MCV, KSHV, HTLV-1, and EBV.

Rapid spread of the novel respiratory syncytial virus A ON1 genotype, central Italy, 2011 to 2013.

Respiratory infections positive for human respiratory syncytial virus (RSV) subtype A were characterised in children admitted to hospitals in Rome and Ancona (Italy) over the last three epidemic seasons. Different strains of the novel RSV-A genotype ON1, first identified in Ontario (Canada) in December 2010, were detected for the first time in Italy in the following 2011/12 epidemic season. They bear an insertion of 24 amino acids in the G glycoprotein as well as amino acid changes likely to change antigenicity. By early 2013, ON1 strains had spread so efficiently that they had nearly replaced other RSV-A strains. Notably, the RSV peak in the 2012/13 epidemic season occurred earlier and, compared with the previous two seasons, influenza-like illnesses diagnoses were more frequent in younger children; bronchiolitis cases had a less severe clinical course. Nonetheless, the ON1-associated intensive care unit admission rate was similar, if not greater, than that attributable to other RSV-A strains. Improving RSV surveillance would allow timely understanding of the epidemiological and clinicopathological features of the novel RSV-A genotype.

Virological and clinical characterization of respiratory infections in children attending an emergency department during the first autumn-winter circulation of pandemic A (H1N1) 2009 influenza virus.

To characterize respiratory virus infections during the first autumn-winter season of pandemic A (H1N1) 2009 influenza virus (A/H1N1/2009) circulation, a prospective study in children attending a paediatric emergency department at the Sapienza University hospital, Rome, was conducted from November 2009 to March 2010. By means of both nasal washings and pharyngeal swabs, enrolled children were checked for 14 respiratory viruses. The majority of acute respiratory infections resulted from viral pathogens (135/231, 58%). Overall, the most common was respiratory syncytial virus (RSV), in 64% of positive samples; A/H1N1/2009 was the only influenza virus found in 16% and rhinovirus (RV) in 15%. Virus-positive children did not differ significantly from virus-negative children in signs and symptoms at presentation; of the virus groups, RSV-infected children were younger and more frequently admitted to intensive-care units than those infected with A/H1N1/2009 and RV. Of the hospitalized children, stratified by age, both infants and children aged >1 year with RSV were most severely affected, whereas A/H1N1/2009 infections were the mildest overall, although with related pulmonary involvement in older children. Children with RV infections, detected in two flares partially overlapping with the A/H1N1/2009 and RSV peaks, presented with bronchiolitis, wheezing and pneumonia. Leukocytosis occurred more frequently in RV-infected and A/H1N1/2009-infected children, and numbers of blood eosinophils were significantly elevated in RV-infected infants. Given the fact that clinical and epidemiological criteria are not sufficient to identify viral respiratory infections, a timely virological diagnosis could allow different infections to be managed separately.

Rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up.

The association between bronchiolitis and recurrent wheezing remains controversial. In this prospective study, we assessed risk factors for recurrent wheezing during a 12-month follow-up in 313 infants aged <12 months hospitalised for their first episode of bronchiolitis. Demographic, clinical and laboratory data were obtained with a questionnaire and from medical files. A total of 14 respiratory viruses were concurrently assayed in nasal washings. Parents were interviewed 12 months after hospitalisation to check whether their infants experienced recurrent wheezing. The rate of recurrent wheezing was higher in infants with bronchiolitis than in controls (52.7 versus 10.3%; p<0.001). Multivariate analysis identified rhinovirus (RV) infection (OR 3.3, 95% CI 1.0-11.1) followed by a positive family history for asthma (OR 2.5, 95% CI 1.2-4.9) as major independent risk factors for recurrent wheezing. In conclusion, the virus most likely to be associated with recurrent wheezing at 12 months after initial bronchiolitis is RV, a viral agent that could predict infants prone to the development of recurrent wheezing.

Detection of respiratory viruses in the 2009 winter season in Rome: 2009 influenza A (H1N1) complications in children and concomitant type 1 diabetes onset.

We investigated clinical characteristics and complications, particularly type 1 diabetes onset, in children hospitalized for 2009 pandemic influenza A (H1N1) virus and compared number of consultations, rate of hospitalization and virus identification in children hospitalized for acute respiratory symptoms (ARS) during the winter season 2009-2010 and 2004-2005. Patients were tested for 2009 H1N1 virus and 14 respiratory viruses on pharyngeal brush/nasal aspirates, using a RT-PCR or nested PCR assays. Consultations and hospitalizations were extracted from operative system GIPSE. The total number of consultations increased by 12%, consultation rate for ARS by 13% and number of hospitalizations by 56% from 2004-2005 to 2009-2010. In 2004-2005, Influenza A virus was identified in only 7 percent of hospitalized children, while in 2009-2010 the 2009 H1N1 virus was identified in 21%. Three children attending the hospital for ARS and 2009 H1N1 infection had ketoacidosis as the onset manifestation of type 1 diabetes. By comparing the number of new diabetes diagnoses among the two winter seasons, we found a higher number of new diagnoses in October 2009-January 2010 than in the same period in 2004-2005 (19 vs 10). Six children (13%), all presenting with pre-existing diseases, were admitted to the pediatric intensive care unit. No children died. The outbreak of this novel virus has increased pediatric consultation rates and hospitalizations compared with previous winters without causing deaths. The children at highest risk for severe infection are those with comorbidities. The 2009 H1N1 virus seems in some way involved in the pathogenesis of type 1 diabetes.

Interferon-induced gene expression in cervical mucosa during human papillomavirus infection.

The aim of this study is to monitor type I interferon (IFN) activation in the cervical mucosa of Human Papillomavirus (HPV)-infected and uninfected women attending a routine gynaecologic clinic. The expression of three IFN-induced genes (MxA coding for human Mixovirus resistance protein A, ISG15 Interferon Stimulated Gene coding for a 15 kDa ubiquitin-like protein and UBP43 coding for the ISG15 isopeptidase) was determined as the mRNA copy number in cervical cells, normalized to the mRNA ones of the beta-glucuronidase gene. Type-specific HPV-DNA load was concurrently determined in the HPV-positive samples. Out of 127 samples tested, 54 were sufficient for both DNA and RNA extraction. The type-specific HPV-DNA copy numbers in the 34 HPV-positive samples varied widely. No significant association was found between copy numbers of MxA, ISG15, UBP43 and HPV status or viral load. However, despite a marked inter-individual variability, ISG15 expression was significantly higher when low-risk HPV infections were compared with HPV-negative samples, while high-risk HPV infections had very low ISG15 levels. The lack of ISG15 activation in high-risk HPV-infected cervical cells could be due to the lack of p53-mediated induction or to HPV-directed specific inhibition of type I IFN pathways. This study approach might be of value in clarifying the role of type I IFN activation in determining the clearance or persistence of HPV infections.

Virological diagnosis of respiratory virus infection in patients attending an emergency department during the influenza season.

To investigate the burden of influenza-like illness (ILI), patients attending an emergency department during the influenza season were tested for several common respiratory viruses, using PCR-based methods. Influenza A viruses were detected in 25 of 103 recruited patients (24%), rhinoviruses in 15%, and respiratory syncytial virus in only one. The data suggest that triage criteria based on ILI case definitions would not contain the spread of the influenza virus during pandemic alerts and could lead to unnecessary isolation of patients with other infections. Application of broader triage criteria followed by timely molecular diagnosis could be effective in preventing new respiratory agent transmission.

Respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants.

OBJECTIVE: To investigate the prevalence of 14 viruses in infants with bronchiolitis and to study demographic and clinical differences in those with respiratory syncytial virus (RSV), human bocavirus (hBoV) and rhinovirus (RV) infection. METHODS: 182 infants aged <12 months hospitalised for bronchiolitis were enrolled. Infants underwent nasal washing for the detection of RSV, influenza virus A and B, human coronavirus OC43, 229E, NL-63, HUK1, adenovirus, RV, parainfluenza 1-3, human metapneumovirus and hBoV. Demographic, clinical and laboratory data were obtained from parents and from patient medical files. Main outcome measurements were age, breastfeeding history, family smoking habits, family history for asthma and atopy, blood eosinophil count, chest radiological findings, clinical severity score and number of days of hospitalisation. RESULTS: A virus was detected in 57.2% of the 182 infants. The most frequently detected viruses were RSV (41.2%), hBoV (12.2%) and RV (8.8%). Infants with dual infections (RSV and hBoV) had a higher clinical severity score and more days of hospitalisation than infants with RSV, RV and hBoV bronchiolitis (mean+/-SD: 4.7+2.4 vs 4.3+/-2.4 vs 3.0+/-2.0 vs 2.9+/-1.7, p<0.05; and 6.0+/-3.2 vs 5.3+/-2.4 vs 4.0+/-1.6 vs 3.9+/-1.1 days; p<0.05). Infants with RV infection had higher blood eosinophil counts than infants with bronchiolitis from RSV and hBoV (307+/-436 vs 138+/-168 vs 89+/-19 n/mm(3); p<0.05). CONCLUSIONS: Although the major pathogen responsible for bronchiolitis remains RSV, the infection can also be caused by RV and hBoV. Demographic characteristics and clinical severity of the disease may depend on the number of viruses or on the specific virus detected.

Presence and environmental circulation of enteric viruses in three different wastewater treatment plants.

AIMS: The aim of the work was to evaluate the circulation of the viruses and to determine a correlation between faecal indicators and viruses. METHODS AND RESULTS: Raw wastewater and effluent samples were collected from three wastewater treatment plants, during three sampling periods, and analysed, using cultural and molecular methods, to determine bacteria and virus presence. The results show a removal of bacterial indicators, but a limited reduction of the phages. The viral analysis displays the circulation of cultivable enteroviruses and differences in the seasonal-geographical distribution. Hepatitis A virus was found with only two genotypes: IA-IB. Rotavirus was present in 11.11%, 24.14%, 2.78% of the samples in the 1st, 2nd and 3rd sampling periods; Astrovirus in 33.33%, 6.9%, 25%; Adenovirus in 7.41%, 3.45%, 2.78%; Norovirus in 7.41%, 10.34%, 5.56% respectively. Adenovirus was never identified in plants B and C as Rotavirus in plant C. CONCLUSIONS: The presence of faecal indicators was not predictive of the enteric virus presence, whereas a different circulation of Enteroviruses was found in the wastewater treatment plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows the importance and the usefulness of molecular methods to evaluate the virus circulation and the genetic variability of Enteroviruses.

Cutaneous human papillomaviruses as recurrence factor in actinic keratoses.

Actinic keratoses (AK) are common, premalignant lesions cause mainly by UV DNA damage. Progression into squamous cell carcinoma may be influenced by other several factors such as chronic chemical exposure or viral infection. A carcinogenic role of Human Papillomaviruses (HPV) in early steps of skin tumour development was recently hypothesized; moreover the presence of HPV DNA seems to be higher in cancer precursor lesions. The aim of this work is to identify the presence of HPV DNA in biopsies from Actinic Keratoses (AK) and from normal skin samples collected from dermatological healthy subjects in Italy, in order to evaluate the severity and the clinical evolution of the HPV positive lesions. The DNA test revealed 37% HPV positivity in AK patients versus 0% in the control group; many different genotypes and variants were identified by direct sequencing of PCR product. The HPV positive AK were usually clinically indistinguishable from the HPV negative. All AK lesions were removed by laser treatment, but AK lesions recurred in all HPV positive patients after a period of 45-60 days whereas the same disappeared in the HPV negative ones. These data permit to hypothesize that the presence of HPV DNA could be an aggravating factor for AK lesion severity and recurrence.

Markers of human papillomavirus infection and their correlation with cervical dysplasia in human immunodeficiency virus-positive women.

Human papillomavirus (HPV) genotypes and HPV DNA load were analysed in cervical smears from 76 human immunodeficiency virus (HIV)-positive and 54 HIV-negative women. The prevalence of genotypes was similar for all women, with the exception of HPV62, which was over-represented in HIV-positive samples. HIV-positive women showed a higher prevalence of multiple genotypes that correlated neither with CD4(+) T-cell counts nor with cervical dysplasia. No significant differences were observed in terms of total or single-type HPV DNA load. The HPV DNA load in both HIV-positive and HIV-negative women was significantly higher in squamous intra-epithelial lesions than in negative Pap smears.

Detection of human papillomavirus DNA, p53 and ki67 expression in penile carcinomas.

Our study is aimed at evaluating the presence of p53 and Ki67 expression by immunohistochemistry in a series of 11 paraffin-embedded penile carcinomas. We also investigated the presence of Human Papillomavirus (HPV) DNA in these tumours and performed an accurate typing by DNA sequencing on positive samples. Immunohistochemistry (IHC) was performed with the anti-p53 and Ki67 mouse monoclonal antibodies. DNA extracted from small sections of each specimen was submitted to amplification with HPV specific general primers; PCR products of the proper length were purified and sequenced. IHC demonstrated nuclear accumulation of mutated p53 and Ki 67 expression in 10/11 tumour samples (90.9%). The prevalence of HPV DNA was 72.7%; the most prevalent type was HPV16. Sequencing analysis revealed the presence of HPV53 (12.5%), HPV18 (25%) and HPV16 (62.5%). Out of the p53 or Ki67 positive carcinomas the percentage of HPV positives was 80% and 70% respectively. Our results indicate that penile carcinoma is frequently associated to high risk HPV and with diffuse p53 and Ki67 expression.

Interstitial cystitis and infectious agents.

Interstitial cystitis (IC) is a syndrome consisting of severe refractory bladder symptoms of unknown etiology. The disease tends to affect Caucasian women with a mean age of 40 years, with 25% of patients under the age of 30. Few population based epidemiological studies of IC have been performed. We analyzed a case of interstitial cystitis in a 42-year-old non-smoker woman. In two biopsy samples the presence of viral DNA of human polyomavirus BK (BKV), human herpes virus type 1 and type 2 (HHV- 1 and HHV-2), adenovirus, human papillomavirus (HPV) and bacterial DNA (Chlamydia trachomatis and Mycoplasma genitalium) were evaluated by means of polymerase chain reaction (PCR). Both samples resulted positive only for BKV and HPV DNA. HPV genotyping revealed the presence of HPV-66 that is associated with a high risk of cancer development. Thus the finding of a viral co-infection could support the hypothesis of the multi-factorial origin of this pathology.

Identification of human papilloma viruses in male dysplastic genital lesions.

The association of Human Papillomaviruses (HPV) DNA with female genital lesions has been widely documented whereas little has been reported about male genital pathologies. The aim of this work was to investigate the presence of HPV DNA and the genotype involved in male dysplastic genital lesions. All samples were analysed by polymerase chain reaction (PCR) to amplify HPV E1 and L1 genes. The PCR products were subjected to restriction fragment length polymorphism (RFLP) to determine the HPV genotype. We analysed 209 male genital biopsies from different lesions: mostly from acuminate condylomata and from Buschke-Lowenstein tumours, Bowen papulosis, leukoplakia of the glans, scrotal lymphangioma, penile horn and penile/perianal verrucous carcinoma. Our results revealed the constant presence of viral DNA in genital condylomata, mainly associated with low risk HPV; the presence of the same genotypes was also detected in some of the examined rare pathologies.

Delayed post-traumatic aneurysms of aorta, carotid and innominate arteries.

We report here, to our knowledge, the first successful case of combined treatment (surgical and by interventional neuroradiology) in a patient with delayed post-traumatic aneurysms of the aorta, carotid and innominate arteries.

Aortico-left ventricular tunnel: two new cases with a long-term follow-up.

Aortico-left ventricular tunnel is a rare congenital communication between the ascending aorta and the left ventricle. Its hemodynamic effect is severe aortic incompetence. Surgery is the only treatment and should be performed before aortic incompetence or ventricular dilation develops. Two neonates with aortico-left ventricular tunnel were operated on at our institution, with closure of the aortic end of the tunnel with a Gore-Tex patch. The 2 patients were discharged in good conditions, and at 112 and 42-month follow-up respectively they are in good health, without medication and with a normal echocardiographic pattern. Aortico-left ventricular tunnel should be treated surgically as soon as possible in order to prevent any damage to the aortic valve and the left ventricle. The operative risk is not low, but results are very encouraging.