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

BACKGROUND: Human 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. METHODS: Here, 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. RESULTS: Overall 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. CONCLUSIONS: Our 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.

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.

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.

Analysis of the genetic diversity and phylogenetic relationships of putative human papillomavirus types.

More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.

Respuesta inflamatoria genital en la detección de alteraciones por virus del papiloma humano / The inflammatory genital reaction in the detection of alterations by human papilloma virus / Reagào inflamatoria genital na detecgào de alteragòes por Papilomavírus Humano

Disfunción vaginal (DV) (vaginosis/vaginitis) es el síndrome genérico de mayor prevalencia, alcanzando el 50% de todas las mujeres en edad fértil (sintomáticas y asintomáticas). El virus del Papiloma Humano (HPV) se detecta en 30 a 40% de mujeres en edad fértil (sintomáticas y asintomáticas) y se asocia a alteraciones pre-neoplásicas y a carcinoma invasor del cuello uterino. El diagnóstico sindrómico de DV y alteraciones inducidas por HPV es ineficiente y en la actualidad la morfología (macro y microscópica) es el gold standard, pero requiere ordenamiento. El Estudio del Contenido Vaginal es la prueba de laboratorio bacteriológico de mayor solicitud luego del urocultivo. BACOVA normatiza el diagnóstico de vaginosis/vaginitis y ERIGE aumenta el valor predictivo de células que alertan sobre alteraciones epiteliales. Desde 2007 al presente en los talleres BACOVA ERIGE (tinción de Giemsa) se evaluó la sensibilidad de la detección de células anormales exfoliadas. Un 99% de los participantes coincidió con la detección de koi-locitos. BACOVA/ERIGE no reemplaza al Papanicolaou de ninguna manera, pero puede y debe realizarse en laboratorios periféricos, con lo que además del diagnóstico de vaginosis/vaginitis con 100% de valor predictivo, aumentan la cobertura preventiva de estados proliferativos.

Vaginal dysfunction (DV) (vaginosis/vaginitis) is the generic syndrome of major prevalence, reaching 50% of all women in fertile age (symptomatic and asymptomatic). The Human Papillomavirus (HPV) is detected in 30-40% of women in fertile age (symptomatic and asymptomatic) and is associated to pre-neoplastic lesions and invading carcinoma of the uterine cervix. The diagnosis for the symptoms of DV and the alterations induced by HPV are inefficient and at present, the morphology (macroscopic and microscopic) is the standard gold, but it needs better classification. The Study of the Vaginal Content is the test of major request after urocultives in bacteriological laboratories. BACOVA establishes the procedure for the diagnosis of vaginosis/vaginitis and ERIGE increases the predictive value of cells that give the alarm on epithelial alterations. From 2007 to the present sensitivity in the detection of abnormal exfoliated cells from vagina and uterine cervix was evaluated during the BACOVA - ERIGE, (Giemsa's stain) workshops, 99% of the participants coincided with the detection of koilocytes. BACOVA/ERIGE does not replace the Papanicolaou by any means, but it can and must be performed in peripheral laboratories, where apart from the diagnosis of vaginosis/vaginitis with 100% of predictive value, it is possible to increase the detection of precocious proliferative changes of the squamous epithelium.

Disfungào vaginal (DV) (vaginose / vaginite) é a síndrome mais prevalente genérica, atingindo 50% de todas as mulheres em idade fértil (sintomáticas e assintomáticas). O Papilomavírus Humano (HPV) é detectado em 30-40% das mulheres em idade fértil (sintomáticas e assintomáticas) e está associado a alteragòes pré-neoplásicas e a carcinoma invasivo do colo do útero. O diagnóstico sindrómico de DV e alteragòes induzidas pelo HPV é ineficiente e atualmente a morfologia (macroscópica e microscópica) é o padrào ouro, mas precisa de ordenamento. O Estudo do Conteúdo Vaginal é o exame de laboratòrio bacteriológico mais solicitado, seguido da urocultura. BACOVA normatiza o diagnòstico de vaginose/ vaginite e ERIGE aumenta o valor preditivo de células que alertam a respeito de alteragòes epiteliais. Desde 2007 até hoje, nos workshops BACOVA/ERIGE (coloragào de Giemsa), foi avaliada a sensibili-dade da detecgào de células anormais esfoliadas. 99% dos participantes coincidiram com a detecgào de coilócitos. Bacova/Erige nào substitui o Papanicolaou de forma alguma, mas pode e deve ser feito em laboratórios periféricos, com o qual além do diagnóstico de vaginose / vaginite com 100% de valor preditivo, aumentam a cobertura preventiva de estados proliferativos.

Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.

Randomized trial of HPV4 vaccine assessing the response to HPV4 vaccine in two schedules among Peruvian female sex workers.

Two hundred female sex workers (FSWs) in Lima, Peru were randomized to receive HPV4 vaccine in the standard (0, 2, 6 months) or a modified schedule (0, 3, 6 months). One hundred and eighty four (92%) participants completed 3 doses of vaccine. Baseline seropositive rates were 58% for HPV6, 22.5% for HPV11, 41.5% for HPV16, and 13% for HPV18. The final geometric mean antibody titer (GMT) following vaccination was significantly greater for women who were seropositive at baseline compared to seronegative women: HPV6 (GMT ratio=2.3, p<0.01), HPV11 (GMT ratio=2.7, p<0.01), HPV16 (GMT ratio=1.3, p=0.04), and HPV18 (GMT ratio=2.4, p<0.01). Antibody titers in the modified schedule were not inferior to those in the standard schedule, suggesting the modified schedule may be paired with required STD visits. Although all women benefit from vaccination, administration at a younger age and before sexual debut is needed to achieve maximum protection from vaccine.

[RNA interference: biogenesis molecular mechanisms and its applications in cervical cancer]. / RNA de interferencia: biogénesis, mecanismos moleculares y sus aplicaciones en cáncer cervical.

RNAi (RNA interference) is a natural process by which eukaryotic cells silence gene expression through small interference RNAs (siRNA) which are complementary to messenger RNA (mRNA). In this process, the siRNA that are 21-25 nucleotides long and are known as microRNA (miRNA), either associate with the RNA-induced silencing complex (RISC), which targets and cleaves the complementary mRNAs by the endonucleolytic pathway, or repress the translation. It is also possible to silence exogenous gene expression during viral infections by using DNA templates to transcribe siRNA with properties that are identical to those of bioactive microRNA. Persistent human papillomavirus (HPV) infection is the main etiological agent during cervical cancer development and the HPV E6 and E7 oncogenes, which induce cellular transformation and immortalization, represent strategic targets to be silenced with siRNA. In several in vitro and in vivo studies, it has been demonstrated that the introduction of siRNA directed against the E6 and E7 oncogenes in human tumoral cervical cells transformed by HPV, leads to the efficient silencing of HPV E6 and E7 oncogene expression, which induces the accumulation of the products of the p53 and pRb tumor suppressor genes and activates the mechanism of programmed cell death by apoptosis; thus, the progression of the tumoral growth process may be prevented. The goal of this review is to analyze the microRNA biogenesis process in the silencing of gene expression and to discuss the different protocols for the use of siRNA as a potential gene therapy strategy for the treatment of cervical cancer.

Circumcision and sexual behavior: factors independently associated with human papillomavirus detection among men in the HIM study.

There is growing interest in understanding human papillomavirus (HPV) infection and related disease among men. To date there have been numerous studies reporting HPV DNA prevalence among men from several different countries, however, few have incorporated multivariable analyses to determine factors independently associated with male HPV detection. The purpose of this study was to assess the factors independently associated with HPV detection in men ages 18-70 years residing in Brazil (n = 343), Mexico (n = 312), and the United States (US) (n = 333). In samples combined from the coronal sulcus, glans penis, shaft, and scrotum, we evaluated factors associated with any, oncogenic, and nononcogenic HPV infections. In multivariable analyses, detection of any HPV infection was significantly associated with reported race of Asian/Pacific Islander, lifetime and recent number of sexual partners, and having sex in the past 3 months. Oncogenic HPV detection was independently associated with lifetime and recent number of sexual partners, and having sex in the past 3 months. NonOncogenic HPV infection was independently associated with lifetime number of sexual partners. Circumcision, assessed by clinical examination, was associated with reduced risk of HPV detection across all categories of HPV evaluated. HPV detection in men in the current study was strongly related to sexual behavior and circumcision status. Interventions such as circumcision may provide a low-cost method to reduce HPV infection.

High HPV genetic diversity in women infected with HIV-1 in Brazil.

The present study on genetic diversity of human papillomaviruses in women infected by HIV in Brazil describes the frequency, the genotypes, and five new variants of HPV. One hundred fifty cervical smears of HIV-positive women were subjected to cytological examination, and the DNA samples obtained were assayed by MY09/MY11 amplification, followed by RFLP typing. The overall HPV-DNA-positive rate was 42.7%. One hundred twenty-two samples (81.3%) had benign cellular alterations or normal cytological results, and HPV DNA frequency among them was 30.3%. Otherwise, 96.4% of samples with altered cytology were positive for HPV DNA. A high diversity of genotypes was observed. HPVs-16 and 81 were the most prevalent (14.1%) and were followed by HPVs 52, 35, 62, 33, 53, 56, 66, 70, 18, 58, 6b, 11, 31, 39, 40, 61, 71, 32, 54, 59, 67, 68, 85, and 102. Five new variants of the high-risk HPVs 18, 33, 53, 59, and 66 were detected. Possible associations between the detection of HPV genotypes and the cytological classification, HIV viral load, CD4 count, and antiretroviral treatment were also examined. We observed that a high proportion of HIV-infected women are infected with HPV and may carry oncogenic genotypes, even when cytological evaluation shows normal results.