High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity.

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

Human Papillomavirus and Male Infertility: What Do We Know?

In recent years, increasing attention has been paid to understanding the causes of infertility, which is being recognized as a growing health problem affecting large numbers of couples worldwide. Male infertility is a contributing factor in approximately 30-40% of cases, and one of its etiological causes is sexually transmitted infections (STIs). Among sexually transmitted pathogens, human papillomavirus (HPV) can contribute in various ways to the failure of spontaneous and assisted reproduction, acting in the different phases of conception, especially in the early ones. In particular, HPV infection can affect sperm DNA integrity, sperm motility, count, viability, and morphology and can induce the production of anti-sperm antibodies (ASAs). In this narrative review, we aimed to provide an overview of existing research on the potential adverse effects of HPV infection on male reproductive health. Furthermore, we analyzed how limiting the spread of the infection, particularly with gender-neutral vaccination, could be a possible therapeutic tool to counteract male and female fertility problems.

Orogenital Human Papillomavirus Infection and Vaccines: A Survey of High- and Low-Risk Genotypes Not Included in Vaccines.

Knowledge of human papillomavirus transmission from the genital tract to the oral mucosa remains unsatisfactory, with poor and often inconsistent literature results. The increase in HPV-associated oral malignancies prompts further analysis of the simultaneous detection of the virus in the two anatomical areas and on the identification of genotypes to be included in future vaccines. Therefore, in this retrospective study, we evaluated orogenital HPV concurrence, hrHPV, lrHPV and type-concordance in 337 samples, as well as the prevalence of the most common genotypes not included in HPV vaccines. Concurrence was found in 12.5% (31/248) of cases, hr-concordance in 61.3% (19/31) and lr-concordance in 12.9% (4/31). Finally, type-concordance was found in 32.3% (10/31) of concurrent infections. Regarding the identification of non-vaccine genotypes, the significantly prevalent genotypes in the anogenital area were HPV66 (12.6%, p < 0.0001), HPV53 (11.1%, p < 0.0001), HPV51 (8.7%, p < 0.0001), HPV42 (8.2%, p < 0.0001) and HPV68 (5.6%, p = 0.0034) in women and HPV66 (14.6%, p = 0.0058), HPV42 (12.2%, p = 0.0428), HPV51 (12.2%, p = 0.0428), HPV53 (12.2%, p = 0.0428), HPV70 (12.2%, p = 0.0428) and HPV73 (12.2%, p = 0.0428) in men. Considering the results of our study, we recommend including the high-risk genotypes HPV51, HPV68, HPV53 and HPV66 in future HPV vaccine formulations.

Helicobacter pylori and Epstein-Barr Virus Co-Infection in Gastric Disease: What Is the Correlation with p53 Mutation, Genes Methylation and Microsatellite Instability in a Cohort of Sicilian Population?

Genetic predisposition, environmental factors, and infectious agents interact in the development of gastric diseases. Helicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection has recently been shown to be correlated with these diseases. A cross-sectional study was performed on 100 hospitalized Italian patients with and without gastric diseases. The patients were stratified into four groups. Significant methylation status differences among CDH1, DAPK, COX2, hMLH1 and CDKN2A were observed for coinfected (Hp-EBV group) patients; particularly, a significant presence of COX2 (p = 0.0179) was observed. For microsatellite instability, minor stability was described in the Hp-HBV group (69.23%, p = 0.0456). Finally, for p53 mutation in the EBV group, exon 6 was, significantly, most frequent in comparison to others (p = 0.0124), and in the Hp-EBV group exon 8 was, significantly, most frequent in comparison to others (p < 0.0001). A significant positive relationship was found between patients with infection (Hp, EBV or both) and p53 mutation (rho = 0.383, p = 0.0001), methylation status (rho = 0.432, p < 0.0001) and microsatellite instability (rho = 0.285, p = 0.004). Finally, we observed among infection and methylation status, microsatellite instability, and p53 mutation a significant positive relationship only between infection and methylation status (OR = 3.78, p = 0.0075) and infection and p53 mutation (OR = 6.21, p = 0.0082). According to our analysis, gastric disease in the Sicilian population has different pathways depending on the presence of various factors, including infectious agents such as Hp and EBV and genetic factors of the subject.

Human Papillomavirus (HPV) Infection and Its Impact on Male Infertility.

Nowadays, the striking numbers of infertile couples that turn to assisted reproductive technologies (ART) drive the research toward a more comprehensive understanding of the underlying causes. Male factors contribute to the inability to conceive in half of the cases, and it has been suggested that sexually transmitted infections could have a role in the onset of spermatozoa impairments. Since the impact of HPV infection on sperm quality and sperm DNA integrity is debated, we wanted to analyze its impact on conventional seminal parameters and the sperm DNA fragmentation index (DFI). Therefore, 117 semen samples of patients undergoing IVF were evaluated for the following characteristics: HPV DNA detection and sperm DNA fragmentation, concentration, motility, and morphology. The results showed a higher rate of HPV-negative patients (59.8% vs. 40.2%) and no HPV-related effect on DFI, sperm concentration, total sperm number, and total motility. Only progressive motility and morphology were found as significantly influenced by HPV positivity. Moreover, we observed a statistically significant difference in DFI when comparing high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) genotypes. Our data suggest that the presence of any HPV type, whatever the exact localization of the virions, can impair some sperm parameters, while HR-HPVs specifically affect the integrity of spermatozoa DNA.

Attitudes of mothers of pre-adolescent girls on HPV vaccine in Italy. Do we need a turning point?

BACKGROUND: To investigate the knowledge, awareness and attitude of mothers of preadolescent girls regarding the HPV vaccination and cervical cancer, and to understand how to improve the efficacy of the Italian vaccination campaign through the gathered data. METHODS: A questionnaire-based survey was conducted in mothers of unvaccinated 9 to 12-year-old (yo) girls in Italy from November 2018 to July 2019, to evaluate their awareness and the attitude toward HPV, its vaccination and the information sources of the vaccination campaign. The selection of the distribution sites of the questionnaire was performed with randomization of 50 major places of aggregation located throughout the Italian territory. RESULTS: Three hundred mothers of unvaccinated girls were included in the study and divided into two groups (191 subjects <45yo, 109 subjects >45yo). Results showed that 79.6% of <45yo knew what HPV is, compared to 60.6% of >45yo (p-value <0.001); only 60.2% (<45yo) and 54.1% (>45yo) showed awareness about the HPV vaccine (p-value 0.03). The percentage of parents against vaccination in pre-adolescent was higher in the >45yo (29.4%); however, most of them appeared favorable to the information campaigns regarding the vaccine (p-value <0.001). CONCLUSIONS: Our study showed that mothers of unvaccinated pre-adolescent girls have suboptimal knowledge on the topic. Moreover, the implementation of communication strategies dedicated to the population segment appears as a central aspect. As HPV vaccination keeps being a public health concern, it is fundamental to understand which trigger should be managed by healthcare decision makers in order to boost the vaccination campaigns.

Predictive Role of the p16 Immunostaining Pattern in Atypical Cervical Biopsies with Less Common High Risk HPV Genotypes.

P16 immunostaining is considered a useful surrogate of transcriptionally active high-risk (hr) HPV infection. Only strong and widespread "block-like" immunoreactivity is considered specific, whereas weak/focal p16 positive immunostaining is considered not specific, and follow-up and HPV molecular detection is not indicated. The aim of the study was to evaluate the presence of HPV DNA and Ki67 immunostaining in 40 cervical atypical biopsies (CALs) with mild and focal histological features suggestive of HPV infection-20 cases with weak/focal p16 positive immunoreactivity and 20 cases negative for p16 expression. In 16/20 weak/focal p16 positive CALs (80%), the INNO-LiPA HPV genotyping detected hrHPV genotypes (HPV 31, 51, 56, 59, 26, 53, 66, 73, and 82). Co-infection of two or more hrHPV genotypes was often evidenced. HPV16 and 18 genotypes were never detected. Ki67 immunostaining was increased in 10/20 cases (50%). In 19/20 p16 negative CALs, hrHPV infection was absent and Ki67 was not increased. These results suggest that weak/focal p16 immunostaining represents the early stage of transcriptionally active infection, strongly related to the presence of less common hrHPV genotypes, probably with a slower transforming power, but with a potential risk of progression if the infection persists. HPV DNA genotyping and follow-up could be useful in these cases to verify if they are able to evolve into overt dysplastic changes and to improve knowledge of less common hrHPV genotypes.

Low Frequency of Human Papillomavirus in Strictly Site-Coded Oral Squamous Cell Carcinomas, Using the Latest NHI/SEER-ICD Systems: A Pilot Observational Study and Critical Review.

The aim of this study was to evaluate HPV status in oral squamous cell carcinoma (OSCC), as coded by the latest classifications and applying a combination of detection methods used in clinical practice. Forty-two patients with suspect OSCC were consecutively recruited. Patients underwent an incisional biopsy for histological OSCC diagnosis and HPV identification by PCR DNA and p16 IHC. All lesions were coded by the latest ICD-0-3.2 site/histology classifications, as proposed for OSCC by the National Cancer Institute Surveillance, Epidemiology and End Results Programs. Moreover, a comparative analysis review, critically evaluated by the same site-coded systems and HPV detection methods, was performed. In 40 confirmed cases of OSCC, the frequency of HPV infection was 10% (4/40). Among positive patients, two cases were PCR DNA/p16 IHC positive (high-risk HPV 51, high-risk HPV 67), two cases were PCR DNA positive/p16 IHC negative (high-risk HPV 31 + 68, high-risk HPV 66). Applying the latest site coding systems for OSCC, the frequency of HPV infection in this study and in similar, reviewed investigations was low (from 3.3% to 12.5%). These results suggested no significant HPV role in oral carcinogenesis, particularly where an updated site-coded classification of OSCCs (categorically excluding the base of the tongue) had been performed.

Socio-Demographic Characteristics and Sexual Behavioral Factors of Patients with Sexually Transmitted Infections Attending a Hospital in Southern Italy.

Sexually transmitted infections (STIs) are a serious global health problem. In Italy, data describing the vulnerability to STIs of specific sexual minorities and the influence of sociodemographic and behavioral determinants are limited, as most infections are not subject to mandatory notification. This retrospective study describes the sociodemographic profile and main sexual behaviors of patients attending a hospital in Palermo (Sicily, Italy) from January 2018 to March 2019 as predictors of STI risk. Patients were divided in subgroups: men-who-have-sex-with-men (MSM), men-who-have-sex-with-women (MSW), bisexual men and females. Data were obtained through an anonymous questionnaire. Patients were tested for chlamydia, syphilis, Mycoplasma genitalium infection, genital herpes and HPV infection. A total of 294 subjects with STIs (male/female ratio about 2:1) were screened. Of the total sample, 79.6% of patients were Italian. MSM accounted for 34.3%, MSW for 29.6%, bisexual men for 5.8% and females for 30.3%. A total of 44.5% of patients had a high education level, 42.5% reported irregular use of condoms, 20.7% reported having had 5-10 partners in the six months prior to the visit and 32.9% were HIV-positive. HPV infection and syphilis were the most prevalent STIs. Conclusions: The most common profile of patients attending our clinic was that of an adult, Italian man with a high level of education, poor use of condoms and a high number of partners. MSM had the highest sex-behavior-related risk for STIs. In addition, our results suggest that all STD teams need to implement counselling topics and recommendations to share with patients, as well as tips on how to approach sexual health education/counselling, thereby promoting patient-centered approaches and educational programs.

Potential impact of a nonavalent anti HPV vaccine in Italian men with and without clinical manifestations.

Human papilloma virus infection (HPV) is the most common sexually transmitted disease. Little is known about male infection. Nonavalent vaccine against types 6/11/16/18/31/33/45/52/58 was approved and neutral gender immunization programs have been proposed. This study evaluates the potential impact of nonavalent vaccine compared to quadrivalent in male living in Sicily (Italy). 58.7% of samples were HPV positive and forty-four types of HPV were identified. A significant higher estimated coverage of nonavalent vaccine than quadrivalent was observed (64.3% vs. 45.8%), with absolute and relative additional impact of 20.1% and 47.2%, respectively. Low impact of the vaccine were calculated as the empirical probability of HPV genotypes 6/11/16/18/31/33/45/52/58 alone or in combination; the high impact as empirical probability of HPV6/11/16/18/31/33/45/52/58 genotypes alone or in association with other genotypes. The potential impact of the nonavalent vaccine vs quadrivalent was significant for low and high impact (29.7% > 18:8%; 34:6% > 26.6%, respectively). Particularly, in men with lesions and risky sexual contact was significant only for low impact (35.5% > 29.7%; 31.4% > 19.7%, respectively). In partners with positive females was significant for low impact (26.3% > 15.1%) and high impact (33.7% > 23.2%). Nonavalent vaccine offers broader protection in men with HPV positive partners, who would have a potential role in the transmission of the infection.

'Secondary prevention' against female HPV infection: literature review of the role of carrageenan.

Introduction: Human papillomaviruses (HPVs) are common sexually transmitted pathogens, causally associated with cervical cancer and other anogenital cancers, as well as approximately 20% of head and neck cancers. The HPV vaccine is an exceptional primary prevention tool, but the question of adequate secondary-prevention strategies remains open. The aim of this review is to better clarify the role of carrageenan in HPV prevention-strategies. Areas covered: A comprehensive literature search was performed (PubMed/MEDLINE, Embase, Google Scholar, Cochrane Databases) to identify articles on the use of carrageenan against HPV infection. The studies were identified using combinations of the search terms 'carrageenan,' 'papillomavirus,' 'HPV,' including only English language papers. Expert opinion: Our review data confirmed the 'inhibitory role' of carrageenan against HPV- pseudoviruses infection on different cell types. The most accredited mechanism to explain this effect involves the direct binding of CG to the viral capsid creating a 'physical' obstacle. However, a different susceptibility to carrageenan for different HPV types has been highlighted, suggesting the possibility of other strategies to infect the host. There are also relevant emerging data regarding the possible role of carrageenan as an adjuvant for antigen-specific immune responses and antitumor effects.

Helicobacter pylori and Epstein-Barr Virus Infection in Gastric Diseases: Correlation with IL-10 and IL1RN Polymorphism.

INTRODUCTION: Helicobacter pylori and Epstein-Barr virus (EBV) infection have recently been shown to be associated with gastric diseases. Polymorphisms in genes encoding cytokines such as interleukin 10 (IL-10) and interleukin 1 Receptor (IL-1RN) influence cytokine secretion levels and appear to contribute to the risk of developing gastroduodenal diseases. To our knowledge, this is the first preliminary study to address the association of coinfection with H. pylori and EBV and their correlation with genetic predisposition in the development of gastric diseases. METHODS: Gastric biopsy samples of 96 patients with different gastric diseases were used. RESULTS: Our results showed that the rate of coinfection was higher in patients with gastric cancer than in patients with normal gastric mucosa, active chronic gastritis, and MALT lymphoma. As regards the characterization of H. pilory strains, the polymorphism s1m1i1 of vacA gene was more frequent in patients with MALT Lymphoma in comparison to others, while the polymorphism s2m2i2 was most frequent in patients with normal gastric mucosa. In addition, patients who tested positive for the cagA gene were more frequently those affected with gastric cancer than those with inactive chronic gastritis. Similarly, the patients with oipA gene ON were more frequently those with gastric cancer than those with inactive chronic gastritis. CONCLUSION: According to our analysis, there was no correlation between coinfection and polymorphisms in genes encoding IL-10 and IL-1RN. We conclude that various factors can be involved in the development of gastric diseases.

Potential impact of a nonavalent HPV vaccine on HPV related low-and high-grade cervical intraepithelial lesions: A referral hospital-based study in Sicily.

While bivalent and quadrivalent HPV vaccines have been used for about 10 years, a nonavalent vaccine against HPV types 6/11/16/18/31/33/45/52 and 58 has been recently approved by FDA and EMA and is now commercially available. The objective of our study was to evaluate the potential impact of the nonavalent vaccine on HPV infection and related low- and high-grade squamous intraepithelial lesions (LSIL, HSIL), compared to the impact of the quadrivalent vaccine, in a female population living in Sicily (Italy). Low estimates of HPV vaccine impact were calculated as prevalence of HPV 6/11/16/18/31/33/45/52 and 58 genotypes, alone or in association, but excluding presence of other HPV types; high estimates were calculated as prevalence of HPV 6/11/16/18/31/33/45/52 and 58 genotypes alone or in association, in the presence of other HPV types. The nonavalent HPV vaccine showed increased impact, compared to the quadrivalent vaccine. Estimates of potential impact varied from 30.9% (low estimate) to 53.3% (high estimate) for LSIL, and from 56.9% to 81,0% for HSIL. The proportion of additional cases potentially prevented by the nonavalent vaccine was 14.4%-23.8% for LSIL, and 19.0%-32.8% for HSIL. The benefit of the nonavalent vaccine compared to the quadrivalent vaccine was more than 80% for both low and high impact estimates for LSIL and more than 50% for both low and high impact estimates for HSIL. The present study confirms that the switch from a first generation HPV vaccines to a nonavalent vaccine would increase the prevention of cervical HSIL in up to 90% of cases.

Detection of oncogenic human papillomavirus genotypes on spermatozoa from male partners of infertile couples.

OBJECTIVE: To evaluate the prevalence of human papillomavirus (HPV) sperm infection and its correlation with sperm parameters in patients who attended a fertility clinic. DESIGN: Cross-sectional clinical study. SETTING: University-affiliated reproductive medicine clinic. PATIENT(S): A total of 308 male partners of couples undergoing in vitro fertilization techniques. INTERVENTION(S): Specimens of semen were collected from all patients. MAIN OUTCOME MEASURE(S): Sperm parameters were evaluated according to the World Health Organization manual. The presence of HPV DNA was researched by the combined use of two HPV assays and a highly sensitive nested polymerase chain reaction assay followed by HPV genotyping. To examine whether HPV was associated with the sperm, in situ hybridization (ISH) analysis was performed. RESULT(S): Results of HPV investigation were compared with sperm parameters and ISH analysis. Twenty-four out of 308 semen samples (7.8%) were HPV DNA positive, but HPV infection did not seem to affect semen quality. Moreover, ISH revealed a clear HPV localization at the equatorial region of sperm head in infected samples. CONCLUSION(S): Oncogenic HPV genotypes were detected on spermatozoa from asymptomatic subjects, but a role of the infection in male infertility was not demonstrated.

Geographical distribution and oncogenic risk association of human papillomavirus type 58 E6 and E7 sequence variations.

Human papillomavirus (HPV) 58 accounts for a notable proportion of cervical cancers in East Asia and parts of Latin America, but it is uncommon elsewhere. The reason for such ethnogeographical predilection is unknown. In our study, nucleotide sequences of E6 and E7 genes of 401 HPV58 isolates collected from 15 countries/cities across four continents were examined. Phylogenetic relationship, geographical distribution and risk association of nucleotide sequence variations were analyzed. We found that the E6 genes of HPV58 variants were more conserved than E7. Thus, E6 is a more appropriate target for type-specific detection, whereas E7 is more appropriate for strain differentiation. The frequency of sequence variation varied geographically. Africa had significantly more isolates with E6-367A (D86E) but significantly less isolates with E6-203G, -245G, -367C (prototype-like) than other regions (p ≤ 0.003). E7-632T, -760A (T20I, G63S) was more frequently found in Asia, and E7-793G (T74A) was more frequent in Africa (p < 0.001). Variants with T20I and G63S substitutions at E7 conferred a significantly higher risk for cervical intraepithelial neoplasia grade III and invasive cervical cancer compared to other HPV58 variants (odds ratio = 4.44, p = 0.007). In conclusion, T20I and/or G63S substitution(s) at E7 of HPV58 is/are associated with a higher risk for cervical neoplasia. These substitutions are more commonly found in Asia and the Americas, which may account for the higher disease attribution of HPV58 in these areas.

HPV genotype prevalence in cytologically abnormal cervical samples from women living in south Italy.

Human papillomavirus (HPV) infection is the commonest sexually transmitted infection, and high-risk HPV types are associated with cervical carcinogenesis. This study investigated: the HPV type-specific prevalence in 970 women with an abnormal cytological diagnosis; and the association of HPV infection and cervical disease in a subset of 626 women with a histological diagnosis. HPV-DNA was researched by nested PCR/sequencing and the INNOLiPA HPV Genotyping assay. The data were analysed by the chi-square test (p

HPV group- and type-specific concordance in HPV infected sexual couples.

The concordance of human papillomavirus (HPV) groups and types was evaluated in 45 sexual couples with both partners HPV infected, by analyzing cervical samples from women and three genital sites (penile brushing, urethral brushing, and semen) from men. When grouping HPV types, no significant HPV group sharing was found between partners, either considering samples from any male site (concordance: 55.5%; P = 0.11) or from each site (concordance by penile brushing, 37.8%; urethral brushing, 24.5%; semen, 22.3%; P > 0.05). Examining individual HPV types, using samples from any male site, concordance was found in 29 (64.4%; P = 0.036) couples; significant concordance was evident for 16 HPV genotypes, the most frequent being HPV-6, -66, -31, -51, and -53. Using samples from specific male sites, concordance was found by penile brushing in 24 (53.3%) couples, urethral brushing in 16 (35.5%), and semen in 7 (15.5%; P = 0.014). Among the 16 HPV types shared by examining samples from any male site, 9 (56.3%) were in common by penile brushing, 11 (68.7%) by urethral brushing, and 6 (37.5%; P = 0.48) by semen. Combined penile brushing and urethral brushing sampling identified all the 29 couples concordant by the three male sites; combined penile brushing and semen detected 26 (89.6%) concordant couples. The most adequate approach to the assessment of HPV concordance in sexual couples could be based on the analysis of individual HPV types and the sampling of men by penile brushing combined with urethral brushing. The high and significant degree of HPV type-specific concordance confirms HPV transmission between sexual partners.

Penile, urethral, and seminal sampling for diagnosis of human papillomavirus infection in men.

Methods that used specimens from three genital sites (penile brushing [PB], urethral brushing [UB], and the retrieval of semen [SE]) from 50 men were examined for human papillomavirus (HPV) DNA detection. The rates of detection by PB, UB, SE, PB and UB, and PB and SE were 88.9%, 50.0%, 33.3%, 100%, and 97.2%, respectively. The use of PB and UB appears to be the most accurate method; as an alternative to UB, the use of SE with PB could be used to improve the rate of HPV DNA detection in men.

Brushing of oral mucosa for diagnosis of HPV infection in patients with potentially malignant and malignant oral lesions.

INTRODUCTION: Adequate brushing of oral mucosa is important for accurate human papillomavirus (HPV) detection in potentially malignant (oral leukoplakia [OL], oral lichen planus [OLP]) and malignant (oral squamous cell carcinoma [OSCC]) lesions. Since various factors may limit the adequacy of oral brushing and, consequently, the accuracy of HPV detection, modified sampling procedures should be evaluated for their effect on HPV frequency and/or types detected. AIM: To compare the HPV frequency in samples obtained by brushing the lesion site with the frequency in samples obtained by brushing an apparently normal adjacent site. The correlation between HPV frequency and keratinization of the site affected by the lesion, as well as sociodemographic variables (age, sex, smoking and drinking habits), was also examined. METHODS: HPV DNA was detected in brushing samples from 50 patients with OL, 49 with OLP, and 17 with OSCC. Polymerase chain reaction (PCR) amplification was performed by MY09/MY11 and GP05+/GP06+ primers; the HPV type was identified by DNA sequencing and a reverse hybridization (line probe) assay. Data were analyzed by the Z test, the Fisher's exact test, the chi-square test, odds ratio (OR), and a logistic regression model. RESULTS: HPV DNA was detected in 22% of samples from lesion sites and in 16% of samples from adjacent sites (p = 0.22) in patients with OL, in 24.5% and 22.4% of samples from lesion and adjacent sites, respectively, in patients with OLP (p = 0.40), and in 35.3% and 41.2% of samples from lesion and adjacent sites, respectively, in patients with OSCC (p = 0.36). Lesions adjacent to HPV-positive normal sites had an increased rate of HPV detection (OR = 30; 95% CI 9.57, 94.1). HPV-18 was the most frequent genotype, followed by HPV-6, -16, -33, and -53. HPV prevalence was reduced in lesions at keratinized sites (14.5%) compared with non-keratinized sites (34.4%; p = 0.007; OR = 0.32; 95% CI 0.13, 0.81). DISCUSSION: In patients with OL, OLP, or OSCC, a high prevalence of HPV infection was shown in apparently normal sites adjacent to lesion sites infected by HPV. The lower HPV frequency in lesions at keratinized sites suggests that HPV detection by lesion brushing is affected by keratinization. The keratinized epithelium may be less susceptible to HPV infection or, alternatively, the highly proliferative activity in non-keratinized sites may predispose to HPV infection. CONCLUSION: Results from this study indicate that taking samples from normal sites adjacent to oral lesions may be of value in HPV detection, particularly when the lesions are located at keratinized sites. This sampling procedure may allow more accurate diagnosis of HPV infection compared with sampling only the lesion site, and may also represent a reliable method to investigate the biological characteristics of HPV infection and related oral carcinogenesis.

Use of fuzzy neural networks in modeling relationships of HPV infection with apoptotic and proliferation markers in potentially malignant oral lesions.

To evaluate in oral leukoplakia the relationship between HPV infection and markers of apoptosis (bcl-2, survivin) and proliferation (PCNA), also conditionally to age, gender, smoking and drinking habits of patients, by means of Fuzzy neural networks (FNN) system 21 cases of oral leukopakia, clinically and histologically diagnosed, were examined for HPV DNA presence, bcl-2, survivin and PCNA expression. HPV DNA was investigated in exfoliated oral mucosa cells by nested PCR (nPCR: MY09-MY11/GP5-GP6), and the HPV genotype determined by direct DNA sequencing. All markers were investigated by means of standardised immunohistochemistry procedure. Data were analysed by chi-square test, crude OR and the 95% CI; in blindness, FNN was applied. HPV DNA was found in 8/21 OL (38.1%); survivin, PCNA, and tobacco smoking were associated in univariate analysis (p = 0.04) with HPV DNA status. HPV-18 was the most frequently detected genotype (6/8), followed by HPV-16 (2/8). FNN revealed that survivin and PCNA, both being expressed in all of OL HPV+ve, were associated with HPV infection. In conclusion, the FNN allowed to hypothesise a model of specific variables associated to HPV infection in OL. The relevance of survivin and PCNA suggest that they may be involved in HPV-mediated deregulation of epithelial maturation and, conversely, that HPV may have a role in the expression level of these two markers. FNN system seems to be an effective tool in the analysis of correlates of OL and HPV infection.