An upward trendiin dna p16ink4a methylation patern and high risk hpv infectionn according to the severity of the cervical lesion / Padrão de metilação do gene P16INK4A e de infecções por HPV de alto risco acompanham a evolução de lesões cervicais

SUMMARY High-risk human papillomavirus (hr-HPV) infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP). HPV frequency was 62.4% (88/141). The most common HPV were HPV16 (37%), HPV18 (16.3%) and HPV33/45(15.2%). An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%), low grade lesions (22.9%), high grade (57.1%) and carcinoma (93.1%) (p < 0.0001). A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001), hr-HPV (p = 0.01), HSIL (p < 0.0007) and malignant lesions (p < 0.0001). Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer. .

RESUMO É reconhecido que infecções por papilomavírus humanos de alto risco (HPV) são causa necessária, mas não suficiente para o desenvolvimento do câncer cervical. Recentemente, estudos de silenciamento gênico apontaram que a hipermetilação do gene p16INK4A é importante co-fator para a carcinogênese cervical, eliminando a função supressora de tumor da proteína p16 em lesões malignas. Entretanto poucos estudos avaliaram a relação da metilação com a progressão da doença. Nosso objetivo foi investigar o padrão de metilação do gene P16INK4A em diferentes graus de lesão cervical e sua associação com a infecção por diferentes tipos de HPV. Nosso estudo de corte transversal avaliou 141 amostras cervicais de pacientes atendidas no Hospital Moncorvo Filho, Rio de Janeiro. A detecção e tipagem do HPV foi realizada pela técnica de reação em cadeia da polimerase (PCR), e a metilação do gene P16INK4A pela PCR-metilação específica em formato nested (MSP). A frequência de HPV foi de 62,4% (88/141). O tipo mais prevalente foi o HPV16 (37%), seguido pelo HPV18 (16,3%) e HPV33/45 (15,2%). Curva ascendente foi observada quanto ao padrão de metilação do gene P16INK4A e o grau da lesão: a metilação foi identificada em somente 10,7% das amostras de epitélio normal, em 22,9% das lesões de baixo grau, em 57,1% das lesões de alto grau e em 93,1% dos carcinomas (p < 0,0001). Foram feitas análises univariada e multivariada a fim de correlacionar metilação, idade, exposição ao tabaco, infecção e genótipo de HPV. Foi encontrada correlação da metilação com a infecção pelo HPV (p < 0,0001), genótipos de alto risco (p = 0,01), ...

Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70 percent of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7 percent. Of the men who tested positive, 27 presented with HPV 16 (29.7 percent), five with HPV 18 (5.5 percent), 21 with HPV 45 (23.1 percent) and nine with HPV 6 (9.9 percent). Seven mixed infections were detected (9.2 percent), while 11 cases remained untyped (13.4 percent). Regarding EBV positivity, 46.7 percent of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6 percent). More than 23 percent of the men were co-infected with both HPV and EBV, while 35 percent presented exclusively with HPV DNA and 20 percent presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.

Detection of human herpesvirus 7 infection in young children presenting with exanthema subitum

In this study, we assessed the prevalence of human herpesvirus-7 (HHV-7) in 141 serum samples from children less than four years of age with exanthematic disease. All samples were negative for measles, rubella, dengue fever and parvovirus B19 infection. Testing for the presence of human herpesvirus-6 (HHV-6)-specific high avidity IgG antibodies by indirect immunofluorescence assay (IFA) revealed two main groups: one composed of 57 patients with recent primary HHV-6 infection and another group of 68 patients showing signs of past HHV-6 infection. Another 16 samples had indeterminate primary HHV-6 infection, by both IgG IFA and IgM IFA. Serum samples were subjected to a nested polymerase chain reaction to detect the presence of HHV-7 DNA. Among patients with a recent primary HHV-6 infection, HHV-7 DNA was present in 1.7 percent of individuals; however, 5.8 percent of individuals tested positive for HHV-7 DNA in the group with past primary HHV-6 infection. Among the 16 samples with indeterminate diagnosis, 25 percent (4/16) had HHV-7 DNA (p < 0.002). We hypothesise that HHV-7 might be the agent that causes exanthema. However, a relationship between clinical manifestations and the detection of virus DNA does not always exist. Therefore, a careful interpretation is necessary to diagnose a primary infection or a virus-associated disease. In conclusion, we detected HHV-7 DNA in young children from the state of Rio de Janeiro, Brazil.

Detection of human herpesvirus 6 and 7 DNA in saliva from healthy adults from Rio de Janeiro, Brazil

In this study, we aimed to evaluate virus shedding in the saliva of healthy adults from the metropolitan region of the city of Rio de Janeiro, Brazil, in order to verify the prevalence of both human herpesviruses 6 and 7 (HHV-6, HHV-7). The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Saliva specimens were subjected to a multiplex polymerase chain reaction (PCR) to detect the presence of HHV-6A, HHV-6B and HHV-7. The total Roseolovirus DNA prevalence was 22.4 percent. The PCR detected a HHV-6 prevalence of 9.8 percent, with HHV-6A detected in 7.1 percent of the samples and HHV-6B in 2.7 percent. HHV-7 DNA was revealed in 12.6 percent of the studied cases. Multiple infections caused by HHV-6A and 7 were found in 2.1 percent of the samples. No statistical differences were observed regarding age, but for HHV-7 infection, an upward trend was observed in female patients. Compared to studies from other countries, low prevalence rates of herpesvirus DNA were detected in saliva from the healthy individuals in our sample. PCR methodology thus proved to be a useful tool for Roseolovirus detection and it is important to consider possible geographic and populations differences that could explain the comparatively low prevalence rates described here.

Comparação de dois pares de oligonucleotídeos utilizados na reação em cadeia da polimerase para detecção de Papilomavírus Humanos em esfregaços cervicais / Comparison of two pairs of primers used in polymerase chain reaction for the detection of Hunan Papillomaviruses in cervical smears

Introdução: o câncer de colo de útero é a neoplasia mais frequente em mulheres de países em desenvolvimento. Grande parte desses casos é causada por infecção persistente com diferentes tipos de papilomavírus humano (HPV) classificados em alto e baixo risco de acordo com o potencial oncogênico. Atualmente, acredita-se que a melhor rotina de identificação e acompanhamento das lesões por HPV de forma a prevenir o câncer cervical é a combinação da técnica de Papanicolaou com a reação em cadeia por polimerase (PCR) na qual ocorre a amplificação de regiões conservadas do genoma viral, com uso de primers degenerados e em seguida identificação do tipo viral. O primer mais utilizado em todo o mundo é o MY09/11, com boa sensibilidade e especificidade. Recentemente, foi descrito na literatura um novo conjunto de primers consensuais denominado PGMY reformulando o primer MY e adicionando um novo primer HMBO visando diminuir perdas do MY (falso negativo). Objetivo: comparar os dois pares de primers, MY e PGMY, a fim de apontar aquele mais adequado para o rastreamento de infecções causadas por HPV no colo uterino pela técnica de reação em cadeia da polimerase. Métodos: avaliamos 116 amostras de esfregaços cervicais. Após a extração do DNA, a técnica de PCR foi realizada de acordo com os protocolos descritos na literatura. Resultados: através desse trabalho, observamos que o par de primers PGMY apresenta maior sensibilidadee especificidade na detecção do DNA do HPV quando comparado com o par de primers MY, além de melhores valores preditivos negativo e positivo. Conclusão: o novo par de primers PGMY, deve ser usado para substituir o par MY a fim de melhorar a detecção do DNA viral.

Introduction: cervical cancer is the most frequent neoplasia among the women of countries in development. Great part of those cases is caused by persistent infection with different types of Human Papillomavirus (HPV) classified as high and low risk, according to the risk of cervical cancer development. Nowadays, it is believed that the best identification routine and follow up of the lesions in order to prevent the malignant transformation is the combination of the technique of Papanicolaou with the polymerase chain reaction (PCR), in which the amplification of conserved areas of the viral genome occurs, with use of degenerate primers, followed by type identification. The degenerate primers MY 09/11 are used worldwide, presenting good sensibility and specificity. Recently, a new group of consensual primers denominated PGMY was described in the literature reformulating the primer MY and adding a new primer HMBO seeking to reduce losses of MY (false negative). Objective: compare two pairs of primers, MY and PGMY, to discorer the most appropriate for the diagnosis of infections caused by HPV in the uterine cervix for the technique of Polymerase chain reaction. Methods: a hundred and sixteen samples from cervical smears were evaluated. After DNA extraction, the PCR was done according to the protocols described in the literature. Results: we observed that the primers pair PGMY presents better sensibility and specificity in the detection of DNA of HPV when compared to the primers pair MY, it also presents better negative and positive predictive values. Conclusion: the new primers pair PGMY should be used to substitute the pair MY to improve the detection of the viral DNA.

Avaliação do uso combinado do teste de triagem de papanicolaou e da reação em cadeia da polimerase para identificação de pacientes em risco de câncer cervical / Evaluation of the combined use of papanicolaou screen test and polymerase chain reaction for the identification of patients at risk of cervical cancer

Introduction: in the last few years, the interest on Human Papillomavirus (HPV) has emerged due to the evidence of their carcinogenic potential,especially on the female genital tract. In the last fi fty years, the screening diagnosis has been carried out by Papanicolaou test (Pap). Nevertheless,literature describes a high rate of false-negative and false-positive samples. Objective: since an early detection is crucial to diminish the risk of cervical cancer, we aimed to analyze the use of HPV DNA detection by PCR as complementary test to routine screening. Methods: nearly 450 female smears were obtained from the DNA Bank of the Virological Diagnosis Laboratory from UFF. HPV prevalence was evaluated by using MY09/11 consensus primers and was compared to Pap test. Results: our results showed that 67.5% of the studied samples were normal tissues, among them 85.6% were negative by PCR but 14.4% were HPV infected. The remaining 32.8% were altered by Pap test. Among them, HPV DNA detection by PCR revealed a prevalence of 56.7% in ASCUS, 87.5% in LSIL and 66,6% in carcinoma. Kappa index showed a good agreement between tests (0.80). The Positive Predictive Value was considered low (58%) pointing out that important cases may be misdiagnosed as false-negatives. On the other hand, the Negative Predictive Value was considered high (90,5%) indicating that PCR should not be used as a screening test, but as a complementary one, revealing true negatives when associated to a normal result in Pap test. HPV positive samples detected by MY PCR were typed and the prevalence obtained for the different types was: 48% HPV 16, 17% HPV 33, 13% HPV 18, 18% HPV 6 and 19% were undetermined because of non tested primers or technical problems. Conclusion: analyzing the results we concluded the combination of both tests is the best diagnostic procedure, allowing a more effi cient evaluation of cancer risk and thus helping in prevention programs.

Introdução: nos últimos anos, o interesse pelo estudo dos papilomavírus humanos (HPV) aumentou, uma vez demonstrada a evidência de seu potencial oncogênico, especialmente no trato genital feminino. A partir de 1950, o rastreamento das lesões genitais foi feito pelo teste de Papanicolaou (colpocitologia oncótica). Entretanto, a literatura o atribui altas taxas de falso-positivos e negativos. Objetivo: uma vez que a detecção precoce é crucial para prevenção do câncer cervical, analisamos o uso do PCR para detecção do DNA do HPV como teste complementar à atual rotina diagnóstica. Métodos: cerca de 450 esfregaços cervicais foram obtidos do banco de amostras do Laboratório de Diagnóstico Virológico da UFF. A prevalência do HPV foi avaliadapelo uso de primers consensuais MY09/11. Resultados: nossos resultados mostraram que 67,5% das amostras eram normais e dentre eles, 85,6% eram negativas pelo PCR, mas 14,4% estavam infectadas. As 32,5% amostras restantes tinham preventivo alterado e com alta prevalência de infecções por HPV, oscilando de 56,7% em ASCUS a 87,5% em LSIL. O índice Kappa apresentou boa concordância entre os testes (0,80). O valor preditivo positivo foi baixo (58%) indicando que casos relevantes podem ser subdiagnosticados como falso-negativos. Por outro lado, o valor preditivo positivo foi elevado (90,5%) revelando que o PCR, embora não deva ser usado como método de triagem, tem importante papel complementar ao preventivo, apontando os verdadeiros negativos, quando associado à colpocitologia oncótica normal. Amostras positivas para HPV pelo PCR-MY foram tipadas e a prevalência obtida revelou 48% HPV 16, 17% HPV 33, 13% HPV 18, 18% HPV 6 e 19% de HPV indeterminados. Conclusão: nossos resultados indicam que o uso combinado de ambos os testes parece ser a conduta diagnóstica mais adequada, permitindo avaliação efi ciente do risco de câncer e assim colaborar em programas de prevenção.