Incidence of anogenital warts in Liuzhou, south China: a comparison of data from a prospective study and from the national surveillance system.

To determine the incidence of anogenital warts (AGWs) in the Chinese general population, we compared the data from a prospective study and from the National Notifiable Disease Report System (NNDRS). A cohort study including 2378 women and 2309 men aged 18-55 years old enrolled from Liuzhou, China, was conducted with three scheduled visits at 6-month intervals from May 2014 to March 2016. And, a questionnaire survey was performed to collect the diagnosis history of AGWs at the enrollment visit. The data on reported AGW cases of Liuzhou in the NNDRS from 2006 to 2015 were also analyzed. Overall, the incidence rates of AGWs in the prospective study, in the self-reported diagnosis during past 12 months and in the NNDRS were 1.26 per 1000 person-years (95% confidence interval (CI): 0.16-2.37), 2.35 (95% CI: 1.17-4.20) and 0.183 (95% CI: 0.178-0.187), respectively. Human papillomavirus 6 or 11 were found in all the AGW biopsy samples (10/10). The onset time of AGWs in women was earlier, and the cumulative risk increased more quickly at a young age along with each subsequent younger birth cohort (P<0.0001), whereas slight differences were observed in the different male birth cohorts (P=0.0785). The sexual behavior of individuals and their sexual partners had a strong relationship with self-reported AGWs. Our study indicates that the incidence of AGWs in China is as high as that in developed countries, and the data based on the national surveillance system seriously underestimate the real disease burden of AGWs.

HPV-6 Molecular Variants Association With the Development of Genital Warts in Men: The HIM Study.

Background: Human papillomavirus type 6 (HPV-6) and HPV-11 are the etiological agents of approximately 90% of genital warts (GWs). The impact of HPV-6 genetic heterogeneity on persistence and progression to GWs remains undetermined. Methods: HPV Infection in Men (HIM) Study participants who had HPV-6 genital swabs and/or GWs preceded by a viable normal genital swab were analyzed. Variants characterization was performed by polymerase chain reaction sequencing and samples classified within lineages (A, B) and sublineages (B1, B2, B3, B4, B5). Country- and age-specific analyses were conducted for individual variants; odds ratios and 95% confidence intervals for the risk of GWs according to HPV-6 variants were calculated. Results: B3 variants were most prevalent. HPV-6 variants distribution differed between countries and case status. HPV-6 B1 variants prevalence was increased in GWs and genital swabs of cases compared to controls. There was difference in B1 and B3 variants detection in GW and the preceding genital swab. We observed significant association of HPV-6 B1 variants detection with GW development. Conclusions: HPV-6 B1 variants are more prevalent in genital swabs that precede GW development, and confer an increased risk for GW. Further research is warranted to understand the possible involvement of B1 variants in the progression to clinically relevant lesions.

FRET-based detection and genotyping of HPV-6 and HPV-11 causing recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a potentially life-threatening disease caused by human papillomavirus (HPV), usually HPV types 6 and 11. The conventional method used for detection and typing the RRP isolates in our laboratory is the polymerase chain reaction (PCR) and DNA sequencing method. A real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe technology was developed for the detection and rapid genotyping of HPV-6 and-11 isolates from biopsy material. The primers and probes were designed using multiple alignments of HPV-6 and HPV-11 partial E6 and E7 sequences that included prototypic and non-prototypic variants. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of HPV-6 and HPV-11. HPV-6 and HPV-11 amplicons were used to determine detection limits and inter- and intra-assay variability. The detection limit of the assay was 12.8 DNA copies for HPV-6 and 22.5 DNA copies for HPV-11. A total of 60 isolates were genotyped using the FRET real-time PCR assay and a 100% concordance was obtained when results were compared with genotyping based on conventional DNA sequencing. The real-time PCR assay based on FRET technology was able to detect and rapidly genotype HPV from tissue biopsy obtained from patients with RRP. The assay reduces the time required for genotyping from three working days to less than a day.

Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil.

There are few studies about the distribution of natural molecular variants of low-risk HPVs. Our aim was to evaluate the E6 early gene variability among HPV-6 and HPV-11 isolates detected in recurrent respiratory papillomatosis (RRP) samples obtained in a cohort of Brazilian patients. We also performed a phylogenetic analysis in order to compare nucleotide sequences identified in our study with previously reported isolates from different anatomic sites (laryngeal papillomas, genital warts, cervical cancer and anal swabs) obtained from other parts of the world to determine the phylogenetic relationships of variants detected in Brazil. The complete coding region of the E6 gene of 25 samples was cloned and sequenced: 18 isolates of HPV-6 (72%) and 7 isolates of HPV-11 (28%). A total of four different HPV-6 genomic variants and two HPV-11 genomic variants was identified. It was not possible to correlate specific variants with disease severity. Phylogenetic trees for both HPV types were constructed enclosing both E6 sequences detected in our study and formerly published sequences. In both phylogenetic trees, the sequences from Brazil did not group together. We could not establish a geographical association between HPV-6 or HPV-11 variants, unlike HPV-16 and HPV-18.

Identical human papillomavirus (HPV) genomic variants persist in recurrent respiratory papillomatosis for up to 22 years.

Seventy initial and 125 follow-up tissue specimens of laryngeal papillomas, obtained from 70 patients who had had recurrent respiratory papillomatosis for from 1-22 years, were investigated for the presence of human papillomavirus (HPV) DNA and HPV E5a, LCR and/or full-length genomic variants. HPV-6 was found in 130/195, HPV-11 in 63/195, and HPV-6/HPV-11 in 2/195 samples. Within 67/70 (95.7%) patients, all follow-up HPV isolates genetically matched completely initial HPV isolate over the highly variable parts of the genome or over the entire genome. Frequent recurrence of laryngeal papillomas is a consequence of long-term persistence of the identical initial HPV genomic variant.

Simultaneous detection and differentiation of human papillomavirus genotypes 6, 11, 16 and 18 by AllGlo quadruplex quantitative PCR.

BACKGROUND: Human papillomaviruses (HPV) are classified into high-risk HPV and low-risk HPV. The most common high-risk HPV types in cervical cancer are HPV 16 and 18, and the most common low-risk types causing genital warts are HPV 6 and HPV 11. In this study, applying novel AllGlo fluorescent probes, we established a quadruplex quantitative PCR method to simultaneously detect and differentiate HPV 6, 11, 16 and 18 in a single tube. METHODS: The specificity, the sensitivity, the detection limit, the reproducibility and the standard curve of this method were examined. Finally, clinical samples that had been tested previously by TaqMan PCR and HPV GenoArray (GA) test were used to verify the accuracy and sensitivity of the method. RESULTS: The assay has a sensitivity of 10(1) to 10(2) copies/test and a linear detection range from 10(1) to 10(8) copies/test. The mean amplification efficiencies for HPV 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r(2)) of each standard curve was above 0.99 for plasmid templates ranging from 10(3) to 10(7) copies/test. There was 100% agreement between the AllGlo quadruplex quantitative PCR, HPV GA test and TaqMan uniplex qPCR methods. CONCLUSIONS: AllGlo quadruplex quantitative PCR in a single tube has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. The convenient single tube format makes this assay a powerful tool for the studies of mixed infections by multiple pathogens, viral typing and viral load quantification.

Classification and nomenclature system for human Alphapapillomavirus variants: general features, nucleotide landmarks and assignment of HPV6 and HPV11 isolates to variant lineages.

BACKGROUND: Papillomaviruses constitute a family of viruses that can be classified into genera, species and types based on their viral genome heterogeneity. Currently circulating infectious human Alphapapillomaviruses (alpha-PVs) constitute a set of viral genomes that have evolved from archaic times and display features of host co-speciation. Viral variants are more recently evolved genomes that require a standardized classification and nomenclature. OBJECTIVES: To describe a system for the classification and nomenclature of HPV viral variants and provide landmarks for the numbering of nucleotide positions. METHODS: The complete 8 kb genomes of the alpha-9 species group and HPV6 and 11 types, collected from isolates throughout the world were obtained from published reports and GenBank. Complete genomes for each HPV type were aligned using the E1 start codon and sequence divergence was calculated by global and pairwise alignments using the MUSCLE program. Phylogenetic trees were constructed from the aligned sequences using a maximum likelihood method (RAxML). RESULTS: Pairwise comparisons of nucleotide differences between complete genomes of each type from alpha-9 HPV isolates (HPV16, 31, 33, 35, 52, 58 and 67) revealed a trimodal distribution. Maximum heterogeneity for variants within a type varied from 0.6%-2.3%. Nucleotide differences of approximately 1.0%-10.0% and 0.5%-1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Analysis of 43 HPV6 complete genomes indicated the presence of 2 variant lineages, whereas 32 HPV11 isolates were highly similar and clustered into 2 sublineages. A table was constructed of the human alpha-PV landmark nucleotide sequences for future reference and alignments. CONCLUSIONS: A proposed nomenclature system for viral variants and coordination of nucleotide positions will facilitate the comparison of variants across geographic regions and amongst different populations. In addition, this system will facilitate study of pathogenic, tissue tropism and functional differences amongst variant lineages of and polymorphisms within HPV variants.

Pre-vaccination genomic diversity of human papillomavirus genotype 6 (HPV 6): a comparative analysis of 21 full-length genome sequences.

Comparative analysis of 21 full-length genome sequences of human papillomavirus genotype 6 (HPV 6): 18 determined in this study and three sequences available in nucleotide sequence databases, revealed more than 98% nucleotide similarity to the HPV 6 prototype isolate. The minimum and maximum genomic distance between the full-length genomic variants and the prototype sequence was three nucleotide substitutions, and 122 nucleotide substitutions and three insertions, respectively. Detailed sequence analysis of early viral genes E7, E1, E2 and E4, late viral gene L2, and three non-classic non-coding genomic regions (NNCR) revealed the existence of at least four E7, twelve E1, eleven E2, six E4, eleven L2, two NNCR1, two NNCR2, and three NNCR3 genomic variants. In addition, several novel, potentially important amino acid mutations were identified. A phylogenetic tree calculated from viral genome sequences was dichotomic, separating all isolates into HPV 6b (prototypic) and HPV 6a/6vc (non-prototypic) genetic lineages. This study, which contributed the largest number of full-length HPV 6 genome sequences to date, confirmed that HPV 6 diversifies virtually equally across the entire genome by nucleotide (amino acid) exchanges in coding regions and additional nucleotide insertions/deletions in non-coding regions. However, this diversification trend was more evident in non-coding regions LCR and NNCR3 and early viral genes E4, E5a and E5b.

Molecular variants of human papilloma viruses type 16 and 6 in women with different cytological results detected by RFLP analysis.

HPV infections are common and the presence of the same high-risk type in cervical specimens can be due to reinfection or persistence. Persistent infection is the most important predictor for development of cervical carcinoma. The aim of this study was to validate PCR-RFLP with two sets of primers: MY09/MY11 that amplify a fragment of L1 and P1/P2 that amplify a fragment of E1 ORF. PCR product of MY09/MY11 was digested with a set of 6 restriction enzymes (RE) and PCR product of P1/P2 with a set of 12 RE. Cervical samples from 110 women patients of the University Gynecologic Clinic CHC Zagreb were analyzed. There were 98 (89.1%) PCR positive samples detected with P1/P2 primers, and 94 (85.5%) PCR positive samples detected with MY09/MY11 primers. Seven HPV types were detected with P1/P2-RFLP technique and 17 with MY09/MY11-RFLP PCR positive samples amplified with both primer pairs agreed with each other in 82 samples; 16 samples were only positive with P1/P2 and 12 samples were only positive by MY09/MY11. HPV 16 was detected in 39 samples with MY09/11-RFLP, out of these two variants (two different patterns) were found with P1/P2 using Dde I, Hae III and Eco I. HPV 6 was detected in 9 samples with MY09/11-RFLP, out of these two variants were found with P1/P2 using HinfI. Combining these two PCR-RFLP methods subtypes of HPV 16 and HPV 6 were detected.

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6).

Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6) was established by sequencing 3798 bp of 77 clinically important HPV 6 isolates obtained from 45 and 32 patients with genital warts and laryngeal papillomas, respectively. By analyzing pooled L1, LCR, E6, E2, and E5 nucleotide data of an individual isolate, a total of 36 different genomic variants were identified, of which six (12 isolates), one (one isolate) and 29 (64 isolates) corresponded to HPV 6b, HPV 6a, and HPV 6vc genetic lineages, respectively. Several novel, potentially important mutations were identified. Non-prototypic HPV 6vc genomic variants were found in the majority of genital warts and laryngeal papillomas included in the study. The presence of serious HPV 6 genome sequence errors was confirmed and novel sequence errors were identified in sequence repositories.

Human papilloma virus type and recurrence rate after surgical clearance of anal condylomata acuminata.

BACKGROUND: Anal condylomata acuminata (ACA) are caused by human papilloma virus (HPV) infection which is transmitted by close physical and sexual contact. The result of surgical treatment of ACA has an overall success rate of 71% to 93%, with a recurrence rate between 4% and 29%. The aim of this study was to assess a possible association between HPV type and ACA recurrence after surgical treatment. METHODS: We performed a retrospective analysis of 140 consecutive patients who underwent surgery for ACA from January 1990 to December 2005 at our tertiary University Hospital. We confirmed ACA by histopathological analysis and determined the HPV typing using the polymerase chain reaction. Patients gave consent for HPV testing and completed a questionnaire. We looked at the association of ACA, HPV typing, and HIV disease. We used chi, the Monte Carlo simulation, and Wilcoxon tests for statistical analysis. RESULTS: Among the 140 patients (123 M/17 F), HPV 6 and 11 were the most frequently encountered viruses (51% and 28%, respectively). Recurrence occurred in 35 (25%) patients. HPV 11 was present in 19 (41%) of these recurrences, which is statistically significant, when compared with other HPVs. There was no significant difference between recurrence rates in the 33 (24%) HIV-positive and the HIV-negative patients. CONCLUSIONS: HPV 11 is associated with higher recurrence rate of ACA. This makes routine clinical HPV typing questionable. Follow-up is required to identify recurrence and to treat it early, especially if HPV 11 has been identified.

Human papillomavirus type distribution and correlation with cyto-histological patterns in women from the South of Italy.

Human papillomavirus (HPV) type-specific distribution was evaluated in genital samples collected from 654 women from the South of Italy undergoing voluntary screening and correlated with cyto-histological abnormalities. HPV DNA was detected in 45.9% of the samples, 41.7% of which had multiple infection and 89.0% had high-risk HPV infection. The prevalence of HPV infection and the rate of multiple infections decreased with age, suggesting natural selection of HPV types with better fitness. In line with other Italian studies, the most common HPV types were HPV-6 and HPV-16, followed by HPV-51, HPV-31, HPV-53, and HPV-66, in women with both normal and abnormal cytology. Cervical intraepithelial lesions grade 2 or 3 were associated with high-risk HPV-16, HPV-18, HPV-31, and HPV-51 infection. These data indicate that prophylactic HPV vaccination is expected to reduce the burden of HPV-related cervical lesions in this population, but also suggest the potential utility of new vaccines with larger type coverage.

Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR.

A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.

[Study on the genotyping of human papillomavirus using a new DNA liquid chip in women of high-risk group of Shandong province].

OBJECTIVE: To evaluate the diagnostic applicability of human papillomavirus (HPV) liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong province. METHODS: To detect HPV genotypes on a 96-well plate with the liquid chip which can simultaneously detect and identify 26 common HPV genotypes in a total of 2925 cervical scrapes obtained from gynecological outpatients as well as to analyze the relationship between HPV types and different cervical diseases by studying the distribution of HPV genotypes and pathologic diagnosis. RESULTS: Among 639 cases who performed pathologic/cytological and histological diagnoses, 184 cases are in group of normal cytology, 266 cases in group of, 77 cases in group of cervical intra-epithelial neoplasia (CIN) I, 7 cases in group of CIN I - II, 46 cases in group of CIN I - II, 46 cases in group of CIN I - II and 13 cases in group of cervical cancer. The overall incidence of HPV in our samples is 36.0% (1054/2925) and 23 types of all 26 types on liquid chip are found. The most common genotypes found are HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18 (8.87%) and HPV-11 (6.94%). Among all the positive types, 87.32% are high-risk HPV and 13.68% are low-risk HPV genotypes. Both single and multiple types are easily identified, showing 66.22% ( n = 698) single type and 33.78% ( n = 356) multiple types. Of all the 1054 HPV-positive cases, 261 (24.8%) is occupied by women 21 to 25 years of age and progressively lower by older age groups, reaching 4.9% by women between 51 to 67 years old. The incidence of HPV in our samples is 23.37%, 33.08%, 54.54%, 57.14%, 82.61%, 91.30% and 100% for normal cytology, inflammation,CIN I ,CIN I - II, CIN II ,CIN III, and carcinomas specimens, respectively. Infections with more that one virus are common, accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I - II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II, III are 88.57%, 76.63%, 68.89% and 92.16% respectively. Conclusion The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. The HPV-positive rate increased along with the increase of grading on cervical lesions. There are more younger women among all the HPV-positive ones. Multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.

Penile cancer associated with so-called low-risk human papilloma virus. Report of five cases from rural Venezuela.

Penile cancer is uncommon in western countries. It has been related to human papilloma virus (HPV) types 16 and 18. We describe five cases of carcinoma of the penis in men from a rural Venezuelan town that were related to HPV types 6, 11, 53, which represent a rather low risk for cancer.

Molecular and serological evidence of the epidemiological association of HPV 13 with focal epithelial hyperplasia: a case-control study.

BACKGROUND: Focal epithelial hyperplasia is a benign proliferative condition that is more frequently found in children of certain ethnic groups. Human papillomavirus 13 and 32 DNA has been consistently detected in these lesions. OBJECTIVE: To demonstrate the epidemiological association of HPV 13 with FEH in the Emberá-Chamí community of Antioquia, Colombia. METHODS: A population-based, case-control study was conducted. One hundred and thirty-eight children were screened and 17 clinical and histologically-confirmed cases were sex and age-matched with 27 controls. Biopsies from FEH lesions and mouth washes from controls were obtained for DNA analysis. HPV 13 DNA was identified using a previously described type-specific PCR test. HPV 13 VLPs were produced by cloning of L1 from the HPV 13 cloned genome and seroreactivity against HPV 13 VLPs of sera from cases and controls were evaluated by ELISA. RESULTS: Among the whole population the prevalence of FEH was 13%. One-hundred-percent of the cases and 29.6% of the controls were HPV 13 positive. There was a significant difference in HPV DNA status between cases and controls (one-tailed Fisher exact test: P<0.0001). Antibodies against HPV 13 VLPs were found in 58.8% of cases and in 33.3% of controls, this difference was not statistically significant (P=0.089 Fisher exact test). However, the median of the ODs of the ELISA positive sera of the cases was 0.596 (interquartile range: 0.5075-0.8245) versus 0.452 (interquartile range: 0.337-0.479) in the controls and this was significantly different (P=0.0041 Man-Whitney test). CONCLUSIONS: We demonstrated a risk for association of FEH with infection with HPV 13. The higher level of antibodies against HPV 13 VLPs in cases may suggest the requirement of higher viral load or viral persistence for disease development.