Broad-spectrum Primers Research Articles

Development of a touchdown droplet digital PCR assay for the detection and quantitation of human papillomavirus 16 and 18 from self-collected anal samples

ABSTRACT Broad-spectrum droplet digital polymerase chain reaction (ddPCR) assays can be used to quantitate multiple human papillomavirus (HPV) types. However, the presence of nucleotide mismatches at the primer binding sites may result in different optimal annealing temperatures and, consequently, under detection of some HPV types. Touchdown PCR uses decreasing annealing temperature over the first PCR cycles allowing for specific and efficient amplification of the target sequence. Here, as a proof-of-concept, we assessed if touchdown ddPCR (TD-ddPCR) using broad-spectrum oligonucleotides and genotype-specific probes targeting HPV L1 can be used to quantitate HPV. We tested our hypothesis using HPV positive cell lines and anal samples which potentially contain PCR inhibitors. Using serial dilutions of HPV16 and HPV18 DNA, the TD-ddPCR assays were performed with linearity across the dilution series (Pearson’s correlation r = 0.9999) and detected one copy of both HPV types, with no cross-reactivity to 27 other low-risk and high-risk HPV types. The PCRs were highly reproducible, with inter- and intra-assay coefficient of variations ranging 2.0–6.3% for HPV16 and 4.9–11.6% for HPV18. Testing self-collected anal samples positive for HPV16 ( n = 29) or HPV18 ( n = 33) and those negative for both HPV types ( n = 22) with the ddPCR assay and Seegene Anyplex II HPV28 (Seegene, TD-ddPCR) showed high agreements between the two assays. In conclusion, HPV broad-spectrum primers containing mismatches at their target binding sites and a touchdown thermal cycling approach resulted in sensitive and specific amplification of HPV genome using ddPCR. The assay has potential clinical diagnostic applications for detection, quantitation, and monitoring of anal HPV infections. IMPORTANCE The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

Distribution of human papillomavirus genotypes by severity of cervical lesions in HPV screened positive women from the ESTAMPA study in Latin America.

The proportion of HPV16 and 18-associated cervical cancer (CC) appears rather constant worldwide (≥70%), but the relative importance of the other HR-HPV differs slightly by geographical region. Here, we studied the HPV genotype distribution of HPV positive Latin American (LA) women by histological grade, in a sub-cohort from the ESTAMPA study; we also explored the association of age-specific HPV genotypes in severe lesions. Cervical samples from 1,252 participants (854 ≤CIN1, 121 CIN2, 194 CIN3 and 83 CC) were genotyped by two PCRs-Reverse Blotting Hybridization strategies: i) Broad-Spectrum General Primers 5+/6+ and ii) PGMY9/11 PCRs. HPV16 was the most frequently found genotype in all histological grades, and increased with the severity of lesions from 14.5% in ≤ CIN1, 19.8% in CIN2, 51.5% in CIN3 to 65.1% in CC (p < 0.001). For the remaining HR-HPVs their frequency in CC did not increase when compared to less severe categories. The nonavalent vaccine HR-types ranked at the top in CC, the dominant ones being HPV16 and HPV45. HR-HPV single infection occurs, respectively, in 57.1% and 57.0% of ≤CIN1 and CIN2, increasing to 72.2% and 91.6% in CIN3 and CC (p<0.001). No association between age and HPV type was observed in CC, although the risk of HPV16 infection in CIN3 cases increased with age. Results confirm the relevance of HPV16 in the whole clinical spectrum, with a strong rise of its proportion in CIN3 and cancer. This information will be relevant in evaluating the impact of HPV vaccination, as a baseline against which to compare genotype changes in HPV type-specific distribution as vaccinated women participate in screening in LA region. Likewise, these data may help select the best HPV testing system for HPV-based efficient, affordable, and sustainable screening programmes.

Multiple HPV infections among men who have sex with men engaged in anal cancer screening in Abuja, Nigeria

BackgroundAnal precancers and cancers can be detected during screening with high-resolution anoscopy (HRA). The sensitivity of HRA depends on the burden and duration of human papillomavirus (HPV) among those screened as well as anoscopist proficiency, which is highly correlated with prior screening experience. Our objective was to compare the identification and type of HPV and the likelihood of HRA-detected precancer for men who have sex with men (MSM) undergoing their first HRA-screening in Nigeria. MethodsMSM were recruited from an HIV test-and-treat cohort, TRUST/RV368, into a new anal cancer screening program. Anal swabs obtained during screening underwent Ion Torrent next-generation sequencing using barcoded HPV PCR broad-spectrum primers 5+/6+ to detect up to 161 HPVs. All high-risk (HR) HPVs and the most abundant low-risk (LR)-HPVs were evaluated as type-specific infections with some categorized as belonging to a multiple infection. HRA screening results included benign, low-grade squamous intraepithelial lesions (LSIL), or HSIL as detected by cytology or histology. Multivariable logistic regression was used to assess the association of HPV and other cofactors with any SIL. ResultsAmong 342 MSM, 60% were HIV-infected, 89% were under 35 years of age, and 51% had 8 or more years since anal coital debut. Of those with SIL, 89% had LSIL and only 11% had HSIL. Prevalence of any HPV and high-risk (HR)-HPV was 92% and 74%, respectively. The most prevalent genotypes in rank order were HPV6 (31%), HPV16 (23%), HPV42 (20%), HPV11 (18%), HPV45 (18%), and HPV51 (17%). For multiple HR-HPVs, 31% had a single HR-HPV, 32% had 2-3, and 10% had 4 or more. Low-risk HPVs, type 6 and/or 11, were common (42%) and were significantly associated with SIL (adjusted odds ratio [aOR]:1.8, 95% confidence interval [CI]: 1.1–3.1) together with perianal warts (aOR:6.7, 95% CI: 3.3–13.5). In contrast, HR-HPV and multiple HR-HPVs were not significantly associated with SIL (all p > 0.05). ConclusionsDetection of HSIL was low. Although HR-HPV was abundant, HSIL development also depends on the duration of HR-HPV infections and the anoscopist's level of experience. As our cohort ages and the anoscopist becomes more skilled, detection of HSIL will likely improve.

Assessing Gammapapillomavirus infections of mucosal epithelia with two broad-spectrum PCR protocols

BackgroundHuman papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites.MethodsHere, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR.ResultsOverall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma- (37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected.ConclusionsOur findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs’ tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.

Distribution of high risk Human Papilloma Virus genotypes among cervical cancer patients in a tertiary level hospital in Bangladesh

Background: Cervical cancer is one of the most common types of cancer affecting women worldwide. Persistent HPV infection plays a major role in cervical cancer. The risk of cervical cancer has increased in parallel with the incidence of certain genotypes of human papilloma virus (HPV).&#x0D; Methods: The study was aimed to detect the distribution of HPV genotypes among cervical cancer patients from a specialized hospital in Dhaka, Bangladesh. HPV DNA testing was done by polymerase chain reaction (PCR) using SPF-10 broad-spectrum primers followed by genotyping by reverse hybridization using the INNO-LIPA genotyping system at the Department of Virology, Bangabandhu Sheikh Mujib Medical University, Dhaka.&#x0D; Results: HPV 16 was more prevalent (72.0%) in cervical cancer patients from Bangladesh followed by type 18 (6.0%) and 45 (2.0%). Genotype 16 and 18 alone and as co-infection were detected in 88.0% cases. Multiple HPV infection was found in 20% patients. Along with high risk (HR) HPV 16, 18 and 45 HR HPV 56, 39, 31 and 58 were also prevalent in multiple infections.&#x0D; Conclusion: Thus the study concluded that HR HPV 16 and HPV 18 were more prevalent genotypes among cervical cancer patients in a specialised hospital in Bangladesh. Along with HR HPV 16 and HPV 18, HR HPV 45, 56, 39, 31 and 58 were also prevalent&#x0D; Bangladesh Med Res Counc Bull 2019; 45: 86-92

High prevalence of Gammapapillomaviruses (Gamma-PVs) in pre-malignant cutaneous lesions of immunocompetent individuals using a new broad-spectrum primer system, and identification of HPV210, a novel Gamma-PV type

Genus Gammapapillomavirus (Gamma-PV) is the most diverse and largest clade within the Papillomaviridae family. A novel set of degenerate primers targeting the E1 gene was designed and further used in combination with the well-known CUT PCR assay to assess HPV prevalence and genus distribution in a variety of cutaneous samples from 448 immunocompetent individuals. General HPV, Gamma-PV and mixed infections prevalence were significantly higher in actinic keratosis with respect to benign and malignant neoplasms, respectively (p = 0.0047, p = 0.0172, p = 0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (p = 0.002). The full-length genome sequence of a novel putative Gamma-PV type was amplified by ‘hanging droplet’ long-range PCR and cloned. The novel virus, designated HPV210, clustered within species Gamma-12. This study provides an additional tool enabling detection of HPV infections in skin and adds new insights about possible early roles of Gamma-PVs in the development of cutaneous malignant lesions.

A Three-Locus, PCR-based Method for Forensic Identification of Plant Material.

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant-derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA-based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad-spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL-F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species-targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.

Cervical HPV type-specific pre-vaccination prevalence and age distribution in Croatia.

The main etiological factor of precancerous lesion and invasive cervical cancer are oncogenic human papillomaviruses types (HPVs). The objective of this study was to establish the distribution of the most common HPVs in different cervical lesions and cancer prior to the implementation of organized population-based cervical screening and HPV vaccination in Croatia. In this study, 4,432 cervical specimens, collected through a 16-year period, were tested for the presence of HPV-DNA by polymerase chain reaction (PCR) with three sets of broad-spectrum primers and type-specific primers for most common low-risk (LR) types (HPV-6, 11) and the most common high-risk (HR) types (HPV-16, 18, 31, 33, 45, 52, 58). Additional 35 archival formalin-fixed, paraffin embedded tissue of cervical cancer specimens were analyzed using LiPA25 assay. The highest age-specific HPV-prevalence was in the group 18–24 years, which decreased continuously with age (P<0.0001) regardless of the cytological diagnosis. The prevalence of HR-HPV types significantly increased (P<0.0001) with the severity of cervical lesions. HPV-16 was the most common type found with a prevalence (with or without another HPV-type) of 6.9% in normal cytology, 15.5% in atypical squamous cells of undetermined significance, 14.4% in low-grade squamous intraepithelial lesions, 33.3% in high-grade squamous intraepithelial lesions, and 60.9% in cervical cancer specimens (P<0.0001). This study provides comprehensive and extensive data on the distribution of the most common HPV types among Croatian women, which will enable to predict and to monitor the impact of HPV-vaccination and to design effective screening strategies in Croatia.

Genotyping of high-risk anal human papillomavirus (HPV): ion torrent-next generation sequencing vs. linear array

BackgroundOur next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections.MethodsWe conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections.ResultsIn total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5–96.2; sensitivity: 93.1%, 95% CI: 83.3–98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1–87.7; sensitivity: 61.1, 95% CI: 43.5–76.9) (all p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4–95.4). The NPV and specificity were similar for high and low risk HPVs (all p > 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114).ConclusionsThe NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.

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. β-PVs (β-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 β-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 β-2. This provides useful data on cutaneous HPV infections in high UV-exposed regions.

Genetic and Manual Survey Methods Yield Different and Complementary Views of an Ecosystem

Given the rapid rise of environmental DNA (eDNA) surveys in ecology and environmental science, it is important to be able to compare the results of these surveys to traditional methods of measuring biodiversity. Here we compare samples from a traditional method (a manual tow-net) to companion eDNA samples sequenced at three different genetic loci. We find only partial taxonomic overlap among the resulting datasets, with each reflecting a portion of the larger suite of taxa present in the sampled nearshore marine environment. In the larger context of eDNA sequencing surveys, our results suggest that primer amplification bias drives much of the taxonomic bias in eDNA detection, and that the baseline probability of detecting any given taxon with a broad-spectrum primer set is likely to be low. Whether catching fish with different nets or using different PCR primer sets, multiple data types can provide complementary views of a common ecosystem. However, it remains difficult to cross-validate eDNA sequencing techniques in the field, either for presence/absence or for abundance, particularly for primer sets that target very wide taxonomic ranges. Finally, our results highlight the breadth of diversity in a single habitat, and although eDNA does capture a richer sample of the community than traditional methods of sampling, a large number of eDNA primer sets focusing on different subsets of the biota would be necessary to survey any ecological community in a reasonably comprehensive way.

High prevalence of genital HPV infection among long-term monogamous partners of women with cervical dysplasia or genital warts-Another reason for HPV vaccination of boys.

We conducted a cross-sectional study on the occurrence of a specific type of genital human papillomavirus (HPV) among long-term monogamous male partners of women with cervical dysplasia and genital warts. The purpose of the study was to improve knowledge with regards to the management of these couples. The presence of genital HPV-DNA was detected by PCR with broad spectrum primers followed by hybridization. 82 males met the study criteria, 41 in each group. Genital HPV-DNA prevalence was 67.5% in the genital warts group and 72.2% in the cervical dysplasia group. The prevalence of high risk HPVs was higher in the cervical dysplasia group, while low risk HPVs were more prevalent in the genital warts group (p < .05). The prevalence of HPV in males was independent of the duration of the relationship (73.5% for 6-24 months and 66.7% for longer relationships). In conclusion, our results suggest that the prevalence of the genital HPV infection in both groups of male partners is comparable and very high, but the spectrum of HPV types varies significantly. The presence of the genital HPV infection in male sexual partners seems to be independent of the duration of the relationship. Applying the HPV vaccination to boys may prevent this phenomenon.

Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

Characterization of an intracellular bacterium infecting the digestive gland of the South African abalone Haliotis midae

A Rickettsiales-like prokaryote was observed in digestive gland epithelium of farmed South African abalone Haliotis midae sampled for routine surveillance. Histological examination showed basophilic inclusions typical of rickettsial infections in shellfish (Mouton, 2000). This study aimed to identify the Rickettsiales-like prokaryote (RLP) colonising H. midae. Total genomic DNA was extracted from infected digestive gland sampled from cultured H. midae. PCR performed with “Candidatus Xenohaliotis californiensis” specific primers RA 5-1 and RA 3-6 did not yield positive results, indicating that the H. midae RLP is not the withering syndrome agent. The 16S rRNA gene sequence of the intracellular bacterium was determined following nested PCR using universal eubacterial primers fD1/Rp2 and the broad spectrum primers EHR16SD/EHR16SR which are specific for members of the Anaplasmataceae family within the Rickettsiales order. Phylogenetic and evolutionary distance analysis showed that the H. midae RLP is a unique member of the α-proteobacteria. The identity and location of the histologically observed intracellular bacterium was confirmed by in situ hybridization performed on sequential sections of infected H. midae digestive gland using a combination of three digoxigenin-labelled probes specific to various regions of the 16S rRNA gene of the RLP.Statement of relevanceThe manuscript shows that farmed abalone, showing no disease symptoms, can remain healthy despite the presence of intracellular bacteria colonising their digestive glands. The study shows that Rickettsiales-like prokaryotes detected in farmed abalone are not necessarily “Candidatus Xenohaliotis californiensis”, the causative agent of withering syndrome.

P235 Prevalence and risk factors associated with oral HPV among sti clinic attendees

Background Oral human papillomavirus (HPV) infection increases the risk of a sub-set of head and neck cancers. The epidemiology of oral HPV infection is not well understood. Aim To describe the prevalence and risk factors for oral HPV infection amongst STI clinic attendees. Methods Participants were recruited from a STI clinic, completed a risk factor questionnaire and provided oral samples for HPV DNA testing by a highly sensitive PCR using the SPF-10 broad spectrum primers. Overall positivity (prevalence) for any HPV was calculated. Chi-square test was used to determine the association between risk factors and oral HPV-positivity. Results Ninety-eight participants (50 men and 48 women) with a median age of 29 (range 20–52 years) were recruited. Overall, 67.4% (66 of 98) participants were positive. All participants reported a history of oral sex. Participants from a non-White ethnic group were more likely to be oral HPV-positive than Whites (63.1% vs. 92.9%, p = 0.03) and those who engaged in open mouth/deep kissing in the last 24 h were also more likely to be oral HPV-positive than those who did not (86.2% vs. 59.7%, p = 0.01). No statistically significant associations were found with recent history of oral sex, smoking, alcohol and cannabis use, or lifetime number of sexual partners. Conclusion Oral HPV infection is common among STI clinic attendees. It is unclear whether these are transient oral HPV infections or true persistent infections with oncogenic potential. Our limited data suggest that recent open mouth/deep kissing behaviour is associated with transmission of oral HPV.

FIRST REPORT OF PEPINO MOSAIC VIRUS ON TOMATO IN APULIA, ITALY

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrum potexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.

Human papillomavirus genotype distribution in invasive cervical cancer in Bosnia and Herzegovina

PurposeCountries of the former Yugoslavia bear some of the highest cervical cancer burden in Europe. In Bosnia and Herzegovina (B&H), data on human papillomavirus (HPV) genotype distribution among cervical cancer cases is scarce. This baseline information is critical in order to evaluate the impact of prophylactic HPV vaccines. This study aims to provide specific information for B&H. MethodsThe final analysis comprised 283 cases of invasive cervical cancer identified at the Polyclinic for Laboratory Diagnostic, University Clinical Center Tuzla in B&H between 1984 and 2004. HPV was detected through amplification of HPV DNA using SPF-10 broad spectrum primers followed by deoxyribonucleic acid enzyme inmunoassay and genotyping by reverse line probe assay (LiPA25, version 1). ResultsMost cases (92.2%) were histologically classified as squamous cell carcinoma (SCC). A total of 268 cases (94.7%) were positive for HPV. Infections were mainly present as single (95.5%) and HPV16 and 18 accounted for 77.8% of the positive cases. The next most common HPV types were HPV45 (4.4%), HPV33 (3.1%), HPV51 (2.3%) and HPV31 (2.2%). The mean age of cases infected with the seven most common types worldwide (HPV16/18/45/31/33/52/58) was 51.1 (SD=11.6), six years younger than the one for cases infected with other types (56.3, SD=12.9). ConclusionsAvailable HPV vaccines could potentially prevent 77.8% of Bosnian cervical cancer cases (i.e. those associated with HPV16/18). If the reported magnitude of the cross-protection of licensed vaccines for non-vaccine HPV types is long lasting, an additional 6 to 10% of cases could be prevented.

Evaluation of p16INK4a Overexpression in a Large Series of Cervical Carcinomas

The aims of this study were to assess the overexpression of p16 in formalin-fixed paraffin-embedded cervical carcinoma tissue blocks and to determine its concordance with the human papillomavirus (HPV) status using the SPF10-LiPA25 polymerase chain reaction System and its correlation with the histologic type of invasive cervical cancer (ICC) and individual HPV genotypes. A total of 205 retrospectively collected ICC cases were analyzed by p16 immunohistochemistry. HPV detection was performed by polymerase chain reaction using SPF10 broad-spectrum primers, followed by deoxyribonucleic acid enzyme immunoassay and genotyping by reverse hybridization line probe assay (LiPA25). Of 205, 188 analyzed (91.7%) ICC cases showed p16 overexpression, whereas 181 (83.3%) cases were HPV positive using HPV LiPA testing. One hundred and seventy four (84.9%) cases were both p16 and HPV LiPA positive, indicating a positive concordance of 89.9% between both techniques (κ index agreement of 0.43; P<0.001), and no statistically significant difference (McNemar test, P>0.05). Squamous cell carcinomas were strongly positive compared with the adenocarcinomas (93.6% vs. 75% of the cases, respectively). When performed on formalin-fixed paraffin-embedded cervical tissue specimens, the higher positivity rate of p16 immunohistochemistry as compared with HPV DNA testing may allow identifying HPV-related ICC cases in which HPV testing was negative.

Human Papillomavirus Type-Specific Prevalence in the Cervical Cancer Screening Population of Czech Women

BackgroundInfection with high-risk human papillomavirus (HPV)types has been recognized as a causal factor for the development of cervical cancer and a number of other malignancies. Today, vaccines against HPV, highly effective in the prevention of persistent infection and precancerous lesions, are available for the routine clinical practice.ObjectivesThe data on the prevalence and type-specific HPV distribution in the population of each country are crucial for the surveillance of HPV type-specific prevalence at the onset of vaccination against HPV.MethodsWomen attending a preventive gynecological examination who had no history of abnormal cytological finding and/or surgery for cervical lesions were enrolled. All samples were tested for the presence of HPV by High-Risk Hybrid Capture 2 (HR HC2) and by a modified PCR-reverse line blot assay with broad spectrum primers (BS-RLB).ResultsCervical smears of 1393 women were analyzed. In 6.5% of women, atypical cytological findings were detected. Altogether, 28.3% (394/1393) of women were positive for any HPV type by BS-RLB, 18.2% (254/1393) by HR HC2, and 22.3% (310/1393) by BS-RLB for HR HPV types. In women with atypical findings the prevalence for HR and any HPV types were significantly higher than in women with normal cytological findings. Overall, 36 different HPV types were detected, with HPV 16 being the most prevalent (4.8%). HPV positivity decreased with age; the highest prevalence was 31.5% in the age group 21-25 years.ConclusionsOur study subjects represent the real screening population. HPV prevalence in this population in the Czech Republic is higher than in other countries of Eastern Europe. Also the spectrum of the most prevalent HPV types differs from those reported by others but HPV 16 is, concordantly, the most prevalent type. Country-specific HPV type-specific prevalences provide baseline information which will enable to measure the impact of HPV vaccination in the future.

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BackgroundHuman papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. MethodsHistologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16INK4a by immunohistochemistry (CINtec histology kit, ROCHE).An IVC was considered HPV driven if both HPV-DNA and p16INK4a overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). ResultsOf 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16INK4a positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16INK4a positive (AP=69.5%, CI=63.6–74.8) versus KSCC (AP=11.5%, CI=9.7–13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). ConclusionCombined data from HPV-DNA and p16INK4a testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.