RESUMEN
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.
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Gammapapillomavirus/genética , Variación Genética , Gammapapillomavirus/fisiología , Humanos , Carga ViralRESUMEN
Molluscum contagiosum (MC) manifests as small, umbilicated papules caused by the molluscum contagiosum virus (MCV). The extent of clinical misdiagnosis of MC is unknown. Combined clinical, histopathological, and virological evaluation of 203 consecutive patients with clinical diagnosis of MC treated at a university hospital during a 5-year period showed the correct clinical diagnosis in 188 of 203 (92.6%) patients. All 15 clinically misdiagnosed MC lesions were histopathologically and virologically confirmed as either common or anogenital warts caused by different human papillomaviruses. The MCV1/MCV2 subtypes ratio was 1.54:1, and the distribution of MCV subtypes differed across patients' age and anatomical location of lesions.
RESUMEN
Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.
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Genoma Viral , Genómica , Molusco Contagioso/virología , Virus del Molusco Contagioso/genética , Quimiotaxis/inmunología , Biología Computacional/métodos , Evolución Molecular , Variación Genética , Genómica/métodos , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunidad , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Anotación de Secuencia Molecular , Molusco Contagioso/inmunología , Virus del Molusco Contagioso/inmunología , Mosaicismo , Filogenia , Recombinación Genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Carga ViralRESUMEN
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 (pâ¯=â¯0.0047, pâ¯=â¯0.0172, pâ¯=â¯0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (pâ¯=â¯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.
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Gammapapillomavirus/genética , Gammapapillomavirus/aislamiento & purificación , Queratosis Actínica/virología , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Gammapapillomavirus/clasificación , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Adulto JovenRESUMEN
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 60â% (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.
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Betapapillomavirus/aislamiento & purificación , Cartilla de ADN/química , ADN Viral/genética , Gammapapillomavirus/aislamiento & purificación , Genotipo , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Adulto , Secuencia de Bases , Betapapillomavirus/clasificación , Betapapillomavirus/genética , Cartilla de ADN/metabolismo , Cejas/virología , Gammapapillomavirus/clasificación , Gammapapillomavirus/genética , Folículo Piloso/virología , Humanos , Masculino , Tipificación Molecular/métodos , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Filogenia , Prevalencia , Sensibilidad y Especificidad , Eslovenia/epidemiologíaRESUMEN
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; 89.5â%) 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.
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Variación Genética , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/veterinaria , Phodopus , Canal Anal/virología , Animales , Enfermedades Asintomáticas , Genoma Viral , Boca/virología , Neoplasias/veterinaria , Neoplasias/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Prevalencia , Infecciones del Sistema Genital/veterinaria , Infecciones del Sistema Genital/virología , Análisis de Secuencia de ADNRESUMEN
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 47â% 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.
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Betapapillomavirus/clasificación , Betapapillomavirus/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Piel/efectos de la radiación , Piel/virología , Adulto , Análisis por Conglomerados , ADN Viral/química , ADN Viral/genética , Femenino , Genotipo , Voluntarios Sanos , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Filogenia , Prevalencia , Estaciones del Año , Análisis de Secuencia de ADN , Luz Solar , Rayos UltravioletaRESUMEN
HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.
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Genes Virales , Mupapillomavirus/genética , Mupapillomavirus/fisiología , Filogenia , Tropismo Viral , Humanos , Mupapillomavirus/clasificación , Sistemas de Lectura Abierta , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
INTRODUCTION: The present study describes the development and evaluation of the first multiplex type-specific quantitative real-time PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction. RESULTS: The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR. CONCLUSION: The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.
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ADN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Niño , Preescolar , Femenino , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
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.
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Variación Genética , Genoma Viral , Papillomavirus Humano 11/genética , Infecciones por Papillomavirus/virología , Evolución Molecular , Genómica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Papillomavirus Humano 11/clasificación , Papillomavirus Humano 11/aislamiento & purificación , Humanos , Funciones de Verosimilitud , Sistemas de Lectura Abierta , Filogenia , Polimorfismo de Nucleótido Simple , Alineación de SecuenciaRESUMEN
Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens.
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Alphapapillomavirus/aislamiento & purificación , ADN Viral/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , ARN Viral/aislamiento & purificación , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Alphapapillomavirus/clasificación , Alphapapillomavirus/genética , ADN Viral/genética , Femenino , Fijadores/química , Formaldehído/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pruebas de ADN del Papillomavirus Humano , Humanos , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/virología , Adhesión en Parafina/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Fijación del Tejido/métodos , Neoplasias del Cuello Uterino/complicaciones , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/complicaciones , Displasia del Cuello del Útero/virologíaRESUMEN
Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.
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Pruebas de ADN del Papillomavirus Humano/estadística & datos numéricos , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Juego de Reactivos para Diagnóstico/provisión & distribución , Displasia del Cuello del Útero/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Detección Precoz del Cáncer/métodos , Femenino , Pruebas de ADN del Papillomavirus Humano/clasificación , Humanos , Papillomaviridae/clasificación , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/virología , Sistemas de Atención de Punto , Juego de Reactivos para Diagnóstico/estadística & datos numéricos , Neoplasias del Cuello Uterino/complicaciones , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/complicaciones , Displasia del Cuello del Útero/virologíaRESUMEN
The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.
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Canal Anal/virología , Gammapapillomavirus/aislamiento & purificación , Genoma Viral , Genómica/métodos , Nasofaringe/virología , Infecciones por Papillomavirus/virología , Adolescente , Adulto , Anciano , Niño , Preescolar , ADN Viral/genética , Femenino , Gammapapillomavirus/genética , Gammapapillomavirus/patogenicidad , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Virales/genética , Adulto JovenRESUMEN
INTRODUCTION: Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source for diagnostic purposes when fresh clinical material is unavailable, and also for molecular and epidemiological studies. The recovery of nucleic acids from FFPE tissues is particularly challenging, and several in-house methods have been developed for this purpose over the last three decades. Recently, several commercial kits specifically developed for DNA and/or RNA extraction from FFPE tissues have been introduced to the market, but their inventory is not available in peer-reviewed literature. METHODS: This article provides the first comprehensive inventory of commercial FFPE DNA/RNA extraction kits currently available on the market and describes their basic characteristics and features. RESULTS: A total of 69 commercial kits from 43 companies were identified. Thirty-five kits were developed specifically for DNA extraction, 22 for RNA extraction, and 12 for both DNA and RNA extraction. Only two commercial kits allow full automation of the entire nucleic acid extraction procedure. The tissue deparaffinization step is omitted in many protocols by melting paraffin directly in a tissue lysis buffer. Purification of the released nucleic acids is mainly based on silica or resin adsorption technology. A formalin reverse cross-linking step to increase the quality of extracted DNA and RNA is an intrinsic part of over half of the kits identified. CONCLUSIONS: It is hope that this comprehensive list of available commercial kits for extracting nucleic acids from FFPE will encourage researchers to strongly consider using them in diagnostic and research settings instead of old-fashioned, crude, and probably less effective in-house methods.
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Ácidos Nucleicos/aislamiento & purificación , Adhesión en Parafina , Juego de Reactivos para Diagnóstico , Fijación del Tejido , Automatización , ADN/aislamiento & purificación , Formaldehído , ARN/aislamiento & purificaciónRESUMEN
Human papillomaviruses (HPVs) are small, non-enveloped viruses with a circular double-stranded DNA genome, etiologically associated with various benign and malignant neoplasms of the skin and mucosa. As of May 30, 2015, 201 different HPV types had been completely sequenced and officially recognized and divided into five PV-genera: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. The Mupapillomavirus genus currently consists of only two HPV types: HPV1 and HPV63, identified in 1980 and 1993, respectively, both associated with sporadic cases of cutaneous warts. In this preliminary study, we announce the complete genome sequence of a novel HPV type, now officially recognized as HPV204. Based on preliminary data, the genome of HPV204 comprises a total of 7,227 bp and contains five early open reading frames (E1, E2, E4, E6, and E7) and two late ORFs (L1 and L2). No E5 ORF could be identified. Preliminary HPV204 clusters to the Mu-PV genus, species Mu-3.
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Genoma Viral/genética , Papillomaviridae/clasificación , Papillomaviridae/genética , Infecciones por Papillomavirus/clasificación , Infecciones por Papillomavirus/virología , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Datos de Secuencia MolecularRESUMEN
Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient's facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients' facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens, suggesting its dual tissue tropism.
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Gammapapillomavirus , Genes Virales , Trasplante de Riñón , Verrugas , Secuencia de Bases , Gammapapillomavirus/genética , Gammapapillomavirus/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Verrugas/genética , Verrugas/virologíaRESUMEN
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.
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Carcinoma de Células Escamosas/virología , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/epidemiología , Carcinoma de Células Escamosas/epidemiología , ADN Viral/análisis , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Papillomaviridae/genética , ARN Mensajero/metabolismo , Sistema de Registros , Distribución por Sexo , Eslovenia/epidemiologíaRESUMEN
OBJECTIVE: To determine the prevalence of a broad spectrum of human papillomavirus (HPV) types in conjunctival papillomas and a possible difference in clinical and histopathological presentation of HPV-positive and HPV-negative papillomas. METHODS: Formalin-fixed, paraffin-embedded papilloma tissue specimens obtained from 25 patients were analysed using six different PCR-based methods targeting 87 HPV types from four different papillomavirus (PV) genera: α-PV, ß-PV, γ-PV and µ-PV, and in situ hybridisation for HPV-6/HPV-11. Slides were reviewed for pedunculated or sessile growth, the presence of goblet cells, keratinising or non-keratinising epithelium, elastosis, atypia and koilocytes. RESULTS: α-PV types HPV-6 and HPV-11 were detected in 19/25 (76%) conjunctival papilloma tissue specimens, 9 (47%) of which were also HPV-6/HPV-11 positive with in situ hybridisation. Six different ß-PV types-HPV-9, HPV-12, HPV-20, HPV-21, HPV-22, HPV-24-were additionally detected in four cases, all of which were also HPV-6/HPV-11 positive. No γ-PVs or µ-PVs were found in any of the tested tissues samples. Extralimbal location (p=0.021), presence of goblet cells (p=0.005), non-keratinising squamous epithelium (p=0.005), and absence of elastosis (p=0.005) were associated with the presence of HPV-6/HPV-11. CONCLUSIONS: We demonstrated that certain clinical and histological features are more frequently associated with HPV infection and that HPV genera other than α-PV are most probably not significant factors in conjunctival papilloma occurrence.
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Neoplasias de la Conjuntiva/patología , Infecciones Virales del Ojo/patología , Papiloma/patología , Infecciones por Papillomavirus/patología , Adulto , Anciano , Neoplasias de la Conjuntiva/virología , ADN Viral/análisis , Infecciones Virales del Ojo/virología , Femenino , Genotipo , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , Papiloma/virología , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga ViralRESUMEN
INTRODUCTION: HPV-52 is one of the most frequent human papillomavirus (HPV) genotypes causing significant cervical pathology. The most widely used HPV genotyping assay, the Roche Linear Array HPV Genotyping Test (Linear Array), is unable to identify HPV-52 status in samples containing HPV-33, HPV-35, and/or HPV-58. METHODS: Linear Array HPV-52 analytical specificity was established by testing 100 specimens reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but not with the HPV-33, HPV-35, and/or HPV-58 individual probes, using an HPV-52-specific real-time PCR assay with a detection level of 3.9 DNA copies/reaction. In addition, we established the prevalence of HPV-52 in 49 samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but containing either HPV-33, HPV-35, and/or HPV-58 using the same approach. RESULTS: The HPV-52-specific assay detected HPV-52 in all 100 samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but not with the HPV-33, HPV-35, and/or HPV-58 probes. The presence of up to six other HPV genotypes did not influence the HPV-52 analytical performance of Linear Array. Only 6/49 (12.2%) samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but containing either HPV-33, HPV-35 or/and HPV-58, were HPV-52 positive. CONCLUSIONS: Linear Array is a highly reliable test for detecting HPV-52 in the absence of genotypes HPV-33, HPV-35, and HPV-58. Additional testing using an HPV-52-specific test is necessary when HPV-33, HPV-35, and/or HPV-58 are present in the sample.