Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Natural history of human papillomavirus infection of sun-exposed healthy skin of immunocompetent individuals over three climatic seasons and identification of HPV209, a novel betapapillomavirus.

We present the first longitudinal study reporting the natural history of human papillomavirus (HPV) infection in sun-exposed skin of healthy individuals living in a geographical area in which solar UV radiation is influenced by the ozone content of the atmosphere. During three climatic seasons, skin swab samples were obtained from 78 healthy individuals and the prevalence of cutaneous HPVs was assessed with broad-spectrum FAP and CUT primers and determined at 54, 45 and 47 % in spring, summer and winter, respectively. Frequencies of mixed HPV infections were significantly higher in spring with respect to summer and winter (P=0.02). Seventy-one different HPV types/putative types were identified. While 62 volunteers were HPV-infected in at least one season, 23 had persistent infections. ß-PVs (ß-1) were the most prevalent and persistent. Age was associated with both the infection status (P=0.01) and the type of HPV infection (no infection, indeterminate/transient, persistent P=0.02). The molecular/phylogenetic analysis of the newly identified ß-PV, officially designated as HPV209, showed that the virus has a typical genomic organization of cutaneous HPVs with five early (E6, E7, E1, E2 and E4) and two late genes (L2 and L1), which clusters to the species ß-2. This provides useful data on cutaneous HPV infections in high UV-exposed regions.

Molecular characterization, prevalence and clinical relevance of Phodopus sungorus papillomavirus type 1 (PsuPV1) naturally infecting Siberian hamsters (Phodopus sungorus).

Phodopus sungorus papillomavirus type 1 (PsuPV1), naturally infecting Siberian hamsters (Phodopus sungorus) and clustering in the genus Pipapillomavirus (Pi-PV), is only the second PV type isolated from the subfamily of hamsters. In silico analysis of three independent complete viral genomes obtained from cervical adenocarcinoma, oral squamous cell carcinoma and normal oral mucosa revealed that PsuPV1 encodes characteristic viral proteins (E1, E2, E4, E6, E7, L1 and L2) with conserved functional domains and a highly conserved non-coding region. The overall high prevalence (102/114; 89.5 %) of PsuPV1 infection in normal oral and anogenital mucosa suggests that asymptomatic infection with PsuPV1 is very frequent in healthy Siberian hamsters from an early age onward, and that the virus is often transmitted between both anatomical sites. Using type-specific real-time PCR and chromogenic in situ hybridization, the presence of PsuPV1 was additionally detected in several investigated tumours (cervical adenocarcinoma, cervical adenomyoma, vaginal carcinoma in situ, ovarian granulosa cell tumour, mammary ductal carcinoma, oral fibrosarcoma, hibernoma and squamous cell papilloma) and normal tissues of adult animals. In the tissue sample of the oral squamous cell carcinoma individual, punctuated PsuPV1-specific in situ hybridization spots were detected within the nuclei of infected animal cells, suggesting viral integration into the host genome and a potential etiological association of PsuPV1 with sporadic cases of this neoplasm.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

UNLABELLED: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.

Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences.

UNLABELLED: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Incidence trends in head and neck squamous cell carcinoma in Slovenia, 1983-2009: role of human papillomavirus infection.

An increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) was observed in several population-based registries and has been attributed to human papillomavirus (HPV) infection. In the present study, we aimed to assess the contribution of HPV infection to the burden of mucosal head and neck squamous cell carcinoma (HNSCC) in Slovenia. For this purpose, data from the nationwide Cancer Registry of Slovenia for cases diagnosed between 1983 and 2009 were analyzed to determine time trends of age-adjusted incidence rates and survival in terms of annual percentage change (APC) for HNSCC in potentially HPV-related and HPV-unrelated sites. In addition, determination of p16 protein, HPV DNA and E6/E7 mRNA was performed in a cohort of OPSCC patients identified from the prospective database for the years 2007-2008. In total, 2,862 cases of HNSCC in potentially HPV-related sites and 7,006 cases in potentially HPV-unrelated sites were identified with decreased incidence observed over the time period in both groups (-0.58; 95 % CI -1.28 to -0.13 and -0.90; 95 % CI -1.23 to -0.57). Regardless of the group, incidence trends for both genders showed a significant decrease in men and increase in women. In a cohort of 99 OPSCC patients diagnosed between 2007 and 2008, 20 (20.2 %) patients had HPV positive tumors and exhibited a superior outcome compared to HPV-negative patients. In conclusion, results of the epidemiologic and histopathologic study confirmed that HPV infection had no major impact on the incidence trends in the Slovenian patients with HNSCC and, specifically, OPSCC during the studied period.

Verrucous carcinoma of the head and neck - not a human papillomavirus-related tumour?

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α-PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin-fixed, paraffin-embedded samples of 30 VC and 30 location-matched normal tissue samples were analysed, by using six different polymerase chain reaction-based methods targeting DNA of at least 87 HPV types from α-PV, ß-PV, γ-PV and µ-PV genera, and immunohistochemistry against p16 protein. α-PV, γ-PV and µ-PV were not detected. ß-PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV-19, -24 and -36 were identified in VC, and HPV-5, -9, -12, -23, -24, -38, -47, -49 and -96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α-PV, γ-PV and µ-PV are not associated with aetiopathogenesis of VC of the head and neck. ß-PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation.

Genomic diversity of low-risk human papillomavirus genotypes HPV 40, HPV 42, HPV 43, and HPV 44.

In order to investigate the genomic diversity of low-risk human papillomavirus (HPV) genotypes, a total of 108 isolates of HPV 40, HPV 42, HPV 43, or HPV 44, obtained from anal swabs or tissue specimens of patients with anogenital warts, and cervical swabs of women with cervical intraepithelial neoplasia of different grades, were analyzed. The characterization of genomic variants was established by sequencing one third of the viral genome and analysis of three different genomic regions: L1, LCR, and E6. Maximum variant divergence accounted for 0.4-1.1% of the investigated genomic segments. Several novel, potentially important nucleotide substitutions, deletions, and insertions are described. Altogether, among 14 HPV 40 isolates, a total of nine different genomic variants were identified, composed of eight L1, five LCR, and four E6 genomic variants. Among 49 HPV 42 isolates, a total of 30 genomic variants were identified, composed of 20 L1, 18 LCR, and four E6 genomic variants. Among 10 HPV 43 isolates, distributed into two major genomic variant lineages with clearly defined nucleotide signatures, three genomic variants were identified, composed of three L1, two LCR, and two E6 genomic variants. Among 35 HPV 44 isolates, a total of eight HPV 44 and 11 subtype HPV 44 genomic variants were identified, composed of 13 L1, 14 LCR, and 6 E6 genomic variants. A similar level of genomic diversity of HPV 44 and its subtype was identified in our geographic region as has been reported previously on isolates collected worldwide.

Prevalence of the Helicobacter pylori babA2 gene and correlation with the degree of gastritis in infected Slovenian children.

The aims of our study were to determine the prevalence of the babA2 gene within Helicobacter pylori strains circulating in the Slovenian pediatric population, to further clarify its significance in causing inflammation of gastric mucosa in children and to verify whether cagA, vacA, iceA and babA genes work independently or synergistically in causing gastritis. A total of 163 H. pylori isolates obtained from the same number of children were tested for the presence of cagA, vacA and iceA genes using previously established methods, while the babA2 gene was determined using novel polymerase chain reaction assay targeting a 139-bp fragment of the central region of babA2. The babA2 gene was detected in 47.9% of H. pylori samples. The presence of the babA2 gene was strongly associated with cagA, vacA s1 and vacA m1 genotype. The babA2 status correlated positively with bacterial density score, activity of inflammation and chronic inflammation of gastric mucosa. No significant correlation was found between the babA2 status and the presence of atrophy or intestinal metaplasia. In addition, the activity of gastric inflammation and density score were significantly associated with the coexpression of the cagA, vacA s1, vacA m1 and babA2 genes. The study, which included the largest number of pediatric H. pylori samples to date, confirmed that babA2 gene plays an important role in the pathogenesis of H. pylori gastritis in children. Furthermore, our results suggest that babA2, cagA and vacA s1 and m1 gene products may work synergistically in worsening the inflammation of gastric mucosa.

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.

Characterization of human papillomavirus type 120: a novel betapapillomavirus with tropism for multiple anatomical niches.

Recent studies indicate that human papillomaviruses (HPVs) from the genera Betapapillomavirus and Gammapapillomavirus are abundant in the human oral cavity. We report the cloning and characterization of a 7304 bp HPV120 genome from the oral cavity that is related most closely to HPV23 (L1 ORF, 83.7 % similarity), clustering it in the genus Betapapillomavirus (ß-PV). HPV120 contains five early and two late genes, but no E5 ORF. HPV120 was detected from heterogeneous human biological niches, including the oral cavity, eyebrow hairs, anal canal and penile, vulvar and perianal warts. Characterization of the clinical spectrum of HPV120 infections indicates a broader spectrum of epithelial tropism than appreciated previously for HPV types from the genus ß-PV.

Tumor-specific and gender-specific pre-vaccination distribution of human papillomavirus types 6 and 11 in anogenital warts and laryngeal papillomas: a study on 574 tissue specimens.

Anogenital warts and laryngeal papillomas are two most important benign tumors etiologically linked with HPV. In the study, which included both the largest number of laryngeal papilloma tissue specimens (152 specimens from 152 patients) to date and the largest number of prospectively collected and histologically confirmed tissue specimens of anogenital warts obtained from both genders (422 specimens from 315 patients), HPV DNA was detected in 413/422 (97.9%) of anogenital warts and 139/152 (91.4%) of laryngeal papillomas. HPV-6 and/or HPV-11 were detected in 291/315 (92.4%) patients with anogenital warts and in 138/152 (90.8%) patients with laryngeal papillomas, indicating that the great majority of both tumors could be prevented with prophylactic quadrivalent vaccine. The HPV-6 gender-specific distribution in both anogenital warts and laryngeal papillomas was not statistically significant. In contrast, HPV-11 was found almost three times more often in males than in females with anogenital warts (16.5% vs. 6.3%; P = 0.008), with a gender neutral HPV-11 type distribution in laryngeal papillomas. The overall HPV DNA prevalence in anogenital warts was significantly different from that in laryngeal papillomas (97.1% vs. 91.4%; P = 0.01). In the first comparison of the HPV-6/HPV-11 type-specific distribution between patients suffering from anogenital warts and laryngeal papillomas with the same geographic and ethnic background, a significant imbalance in tumor-specific distribution of HPV-6 and HPV-11 was identified: HPV-6 was statistically more often present in anogenital warts than in laryngeal papillomas (79.0% vs. 59.2%; P = 0.000013), whereas HPV-11 was statistically more frequent in laryngeal papillomas than in anogenital warts (28.9% vs. 12.4%; P = 0.00003).

Prevaccination genomic diversity of human papillomavirus genotype 11: a study on 63 clinical isolates and 10 full-length genome sequences.

Prevaccination genomic diversity of human papillomavirus genotype 11 (HPV 11) was established by sequencing 40% of the genome of 63 clinical isolates obtained from an ethnogeographically closed Caucasian cohort, and full-length genome sequencing of the ten most divergent isolates. In the study, which included the largest number of isolates to date, by analyzing pooled L1, LCR, E6, E5a, and E5b sequences (3,217 bp) of an individual isolate, a total of 23 genomic variants were identified, of which three (5 isolates) and twenty (58 isolates) corresponded to prototypic and non-prototypic variant groups, respectively. Several novel, potentially important mutations are described. Full-length genome sequences of ten isolates revealed more than 99% similarity to the HPV 11 prototype isolate. The minimum genomic distance between the full-length sequences of genomic variants and the prototype was 3 point mutations and 2 inserts and the maximum distance 31 point mutations, one insertion and one deletion. Within the ethnogeographically closed cohort investigated in this study, HPV 11 was shown to be less polymorphic in comparison to the majority of HPV genotypes studied to date.

The genetic diversity of human papillomavirus types from the species Gammapapillomavirus 15: HPV135, HPV146, and HPV179.

OBJECTIVES: To determine the prevalence, viral load, tissue tropism, and genetic variability of novel human papillomavirus (HPV) type 179, which is etiologically associated with sporadic cases of common warts in immunocompromised patients, and phylogenetically related HPV types 135 and 146. METHODS: The representative collection of 850 HPV-associated clinical samples (oral/nasopharyngeal/anal, archival specimens of oral/oropharyngeal/conjunctival/cervical/skin cancer, benign lesions of the larynx/conjunctiva/skin, and eyebrows), obtained from immunocompetent individuals, was tested for the presence of HPV179, HPV135, and HPV146 using type-specific real-time PCRs. To assess the genetic diversity of the HPVs investigated in the non-coding long control region (LCR), several highly sensitive nested PCR protocols were developed for each HPV type. The genetic diversity of HPV179 was additionally determined in 12 HPV179 isolates from different anatomical sites of an only immunocompromised patient included in the study. RESULTS: HPV179, HPV135, and HPV146 were detected in 1.4, 2.0, and 1.5% of the samples tested, respectively, with no preference for cutaneous or mucosal epithelial cells. One (with five single nucleotide polymorphisms; SNPs), four (with one to six SNPs), and four (with one to eight SNPs) genetic variants of HPV179, HPV135, and HPV146, respectively, were identified among eligible samples. HPV179 isolates from the immunocompromised patient exhibited the identical LCR nucleotide sequence, suggesting that HPV179 can cause generalized HPV infections. CONCLUSIONS: HPV179, HPV135, and HPV146 have a mucocutaneous tissue tropism and are associated with sporadic infections in immunocompromised and immunocompetent individuals. Because the majority of mutations were found outside the major functional domains of the respective LCRs, we assume that HPV179, HPV135, and HPV146 genetic variants pathogenically do not differ from their prototypes. In addition, no association was found between specific HPV179, HPV135, and HPV146 genetic variants and anatomical sites of infection and/or specific neoplasms.

Genomic variants of human papillomavirus genotypes 16, 18, and 33 in women with cervical cancer in Slovenia.

AIM: Our aim was to analyze genomic variants of the three most common human papillomavirus (HPV) genotypes found in Slovenian women with cervical cancer (CC). MATERIAL & METHODS: A total of 40 isolates of HPV 16, 20 isolates of HPV 18 and 11 isolates of HPV 33 were included in the study. The genomic diversity of HPV 16, HPV 18 and HPV 33 isolates was investigated within the long control region (LCR), and E6 and E7 genomic regions using direct polymerase chain reaction-sequencing. RESULTS: A total of 26 genomic variants of HPV 16, consisting of 22 LCR, 10 E6 and 5 E7 variants were identified. Thirty-eight (95%) HPV 16 isolates belonged to the European branch, one (2.5%) to the African 2 branch and one (2.5%) to the Asian-American branch. A total of 18 genomic variants of HPV 18 consisting of 18 LCR, two E6 and four E7 variants were identified: 19 (95%) HPV 18 isolates belonged to the European branch and one (5%) to the African branch. A total of seven genomic variants of HPV 33 consisting of seven LCR, two E6 and three E7 variants were identified: five (45.5%) HPV 33 isolates belonged to prototypic and six (54.5%) to non-prototypic HPV 33 genomic variants. CONCLUSIONS: The majority of HPV 16 and HPV 18 isolates from Slovenian patients with CC analyzed in this study belonged to European branches. Prototypic and non-prototypic HPV 33 genomic variants were equally distributed among Slovenian patients with CC. Several novel mutations were identified in all three genotypes examined.

Human papillomavirus genotype specificity of hybrid capture 2 low-risk probe cocktail.

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.

Genomic diversity of human papillomavirus (HPV) genotype 38.

Human papillomavirus (HPV) genotype HPV 38 is a HPV genotype associated with skin cancer and is classified taxonomically in the beta-PV genus-species 2. At least six genomic variants of HPV 38, including prototype isolate and its subtype FA125, have been characterized so far. In order to investigate further the genomic diversity of HPV 38, a total of 39 HPV 38 positive samples obtained from hairs plucked from pubic, scrotal, perianal or eyebrow regions from 31 immunocompetent healthy male individuals were analyzed. The characterization of genomic variants was based on analysis of L1, E6, and E7 genomic regions. Sequence analysis revealed the presence of a single genomic variant in 35 samples and the presence of at least two different HPV 38 genomic sequences in four samples. A total of nine, nine, and five L1, E6, and E7 genomic variants were identified among 35 isolates, respectively. After combining nucleotide variations in all three genomic regions for a particular isolate, 13 different variants were identified, of which 6 and 7 corresponded to HPV 38 and FA125, respectively. In addition to 5 genomic variants identified previously (prototype isolate, subtype FA125, putative subtype AF091444, isolates U21875 and AF091443), 12 novel genomic variants were characterized. A sixth genomic variant described previously (L38917) was found to be identical with prototype HPV 38 isolate. Taking into account the results of this and previous studies, at least seventeen HPV 38 genomic variants exist today, 12 of which are described for the first time in this study.

Prevalence and clinical relevance of cagA, vacA, and iceA genotypes of Helicobacter pylori isolated from Slovenian children.

BACKGROUND: Although infection with Helicobacter pylori in children mostly induces asymptomatic chronic gastritis, the clinical outcome of H pylori infection is generally unpredictable. To identify the risk subgroup of infected children who can progress toward serious gastrointestinal disease, we assessed the prevalence of H pylori virulence genes cagA, vacA, and iceA in children from southeastern Europe and correlated their presence with the severity of histological changes in the stomach. MATERIALS AND METHODS: A total of 165 children (age range 4-18 years, mean 13 years) with H pylori infection were studied for a 6-year period. Virulence genes were determined by polymerase chain reaction from biopsy samples. RESULTS: The cagA gene was present in 61.2% of patients. The predominant vacA genotype was s1m1 (42%), followed by s1m2 (28%), and s2m2 (24%). IceA genotypes iceA1 and iceA2 were detected in 62% and 31% of the samples, respectively. Multiple genotypes were found in 11.5% of isolates. The H pylori density score, the degree of chronic and acute inflammation, correlated with a cagA-positive status (P < 0.01, P < 0.01, P = 0.01, respectively). Higher bacterial infiltration (P < 0.01) and degree of chronic inflammation (P = 0.03) were detected in vacA s1-positive samples. CONCLUSION: CagA, vacA s1m1, and iceA1 genotypes are the predominant genotypes of H pylori isolated from the southeastern European pediatric population. CagA and vacA s1 are important virulence determinants of H pylori in children, but were not found associated with an increased incidence of precancerous gastric lesions.

The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2 HPV DNA test.

BACKGROUND: The Abbott RealTime High Risk HPV test (RealTime) is a novel assay designed to detect 14 high-risk human papillomavirus genotypes (hr-HPV) and concurrently distinguish HPV-16 and HPV-18 from other hr-HPV within a single test. OBJECTIVE: To evaluate analytical specificity and clinical sensitivity for cervical carcinoma and cervical intraepithelial neoplasia grade 3 (CIN3) of the RealTime test in comparison with the Digene Hybrid Capture II Test (hc2). MATERIALS AND METHODS: Analytical specificity of the RealTime assay was evaluated on 37 samples with previously determined hc2 false-positive results due to cross-reactivity of the hc2 high-risk probe cocktail with untargeted low-risk HPV genotypes. All 37 samples were negative for 14 hr-HPV using the RealTime test. Clinical sensitivity of RealTime was evaluated in comparison to hc2 on 95 and 267 archived routine cervical specimens collected from women with histologically confirmed cervical carcinoma and CIN3 lesions, respectively. Archived specimens were selected for the present study after linkage with the Slovenian national registry of CIN3 and cervical cancer to obtain histology data. RESULTS: Concordant results between RealTime and hc2 were obtained in 90/95 cervical cancer samples (94.7% agreement) and in 250/267 CIN3 samples (93.6% agreement). Clinical sensitivity of RealTime and hc2 for cervical cancer in the total study cohort was 88.4% (95% confidence interval (CI): 80.3-93.6%) and 87.3% (95% CI: 79.0-92.8%), respectively, and analytical sensitivity for samples containing at least one targeted hr-HPV was 98.8% (95% CI: 93.0-100.0%) and 95.3% (95% CI: 88.2-98.5%), respectively. Clinical sensitivity of RealTime and hc2 for CIN3 lesions of the total study cohort was 91.8% (95% CI: 87.8-94.5%) and 89.1% (95% CI: 84.8-92.3%), respectively, and analytical sensitivity for samples containing at least one targeted hr-HPV was 96.4% (95% CI: 93.3-98.2%) and 92.5% (95% CI: 88.5-95.2%), respectively. CONCLUSION: The RealTime test showed excellent analytical specificity and no cross-reactivity with low risk HPV genotypes that tested positive with hc2. Clinical sensitivity of the RealTime assay using archived routine cervical specimens was comparable to hc2. The RealTime test is an important new method applicable to cervical carcinoma screening and management of cervical precancerous lesions.