Lineage and phylogenetic analysis of HPV-16, -18 in saliva of HNSCC patients

Aim: Head and Neck Squamous Cell Carcinoma (HNSCC) is a global health problem whose incidence varies by geographic region and race according to risk factors. Human papillomavirus (HPV) infection is a significant risk factor for HNSCC. HPV-16 and HPV-18 are two forms of HPV that are carcinogenic. HNSCCs that are HPV positive have a better prognosis rather than HPV negative. The purpose of this research was to characterize HPV-16, -18 variations in the saliva of HNSCC patients by examining the genetic diversity of HPV-16, -18 utilizing the full E6, E7, and L1 genes. Methods:The case-control research included 15 patients with HNSCC and 15 healthy volunteers. Unstimulated entire saliva samples were obtained from the case and control groups by spitting method. Genomic DNA was isolated from all saliva samples. A PCR reaction was used to determine the presence of HPV in saliva. HPV-positive samples were genotyped and data were analyzed. We conducted a variant study on the HPV-16, -18 E6, and E7 genes. Results: Three patients with HNSCC were HPV-positive for two HPV genotypes out of 30 people diagnosed with HPV-DNA. HPV-16 and -18 were the most common genotypes. The HPV-16, -18 E6, and E7 genes were sequenced and compared to the HPV-16, -18 (E6, E7) prototype sequence. In all, HPV-16 lineages A1 and HPV-18 lineages A3 were discovered. Conclusion: Regarding the variation of HPV found in Iranian HNSCC patients, the need for further studies in HPV genotyping was seen. Sequencing HPV genes in HNSCC may help answer questions about HPV genotyping in the Iranian population. HPV genotype analysis aids in the development of vaccinations against HNSCC, halting disease progression and preventing HPV-associated HNSCC

Deteccion del virus del papiloma humano en biopsias de cancer de mama mediante PCR e inmunohistoquimica en el Hospital Santa Rosa durante el 2019 / Detection of human papilloma virus in breast cancer biopsies using PCR and immunohistochemistry at the Santa Rosa Hospital during 2019

El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real.

The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.

Prevalence and frequencies of human papilloma virus types in adenocarcinoma in situ of the uterine cervix / 中华病理学杂志

Objective: To examine the prevalence and frequencies of human papillomavirus (HPV) genotypes in cervical adenocarcinoma in situ (AIS). Methods: The cases of cervical AIS with concurrent tests of cytology and HPV typing from January 2007 to February 2020 in the Obstetrics and Gynecology Hospital of Fudan University were collected and analyzed. Results: A total of 478 cases of cervical AIS were obtained. The average age of the patients was 39.4 years (range, 19-81 years). The largest age group was 30-39 years (44.8%), followed by 40-49 years (34.7%). Among the 478 patients, 355 underwent high-risk HPV (hrHPV) testing and had a hrHPV-positive rate of 93.8%. Of the 355 patients, 277 also underwent HPV typing and were mostly positive for either or both HPV16 and HPV18 (93.1%), with 55.6% positive for HPV18 and 48.7% positive for HPV16. Among the 478 cases, 266 cases (55.6%) were diagnosed with both AIS and squamous intraepithelial lesion (SIL), while 212 cases (44.4%) were diagnosed with only AIS. Patients infected with HPV16 in the AIS and SIL group significantly outnumbered those in the AIS alone group (P<0.05). Moreover, the rate of positive cytology was 55.9% (167/299 cases), while that of negative cytology was 44.1% (132/299). Among the 109 patients with negative cytology results and co-tested hrHPV, there were 101 HPV-positive cases (92.7%), of which 88 cases were subject to HPV typing and showed an HPV16/18 positive rate of 94.3% (83/88 cases). Conclusions: The combination of HPV typing and cytological screening can maximize the detection rate of cervical AIS, and should continue to be utilized, ideally on a larger scale, in the future.

Performance of point-of-care testing for cervical cancer screening / 中华预防医学杂志

Objective: To evaluate the performance of point-of-care testing for cervical cancer and precancerous lesions screening. Methods: In September 2020, 197 and 273 women were selected by using simple random sampling method from "self-sampling" cohort and "physician-sampling" cohort established in Xiangyuan county, Shanxi Province, China, respectively. Cervical exfoliated cells were collected by women themselves or gynecologists. All samples were detected by POCT and women with positive result were directly referred for colposcopy. Subsequently, all the samples were detected by careHPV and PCR test. Colposcopy and punch biopsy were performed for women with POCT negative but careHPV or PCR test positive at another visit. Using histopathological diagnosis as the gold standard, we calculated sensitivity, specificity and drew the receiver operating characteristic (ROC) curves. The accuracy of POCT was analyzed and compared to that of careHPV and conventional PCR test in cervical cancer and precancerous lesions screening. Results: The median (Q1 , Q3) age of 470 women was 51 (45, 57) years old. Based on self-sampling, the sensitivity and specificity of POCT for CIN2+ were 100.00% (95%CI: 56.56%-100.00%) and 28.95% (95%CI: 22.97%-35.76%), respectively. Compared with POCT, POCT HPV16/18 test had similar sensitivity and higher specificity of 89.47% (95%CI: 84.30%-93.08%). Self-sampling POCT HPV16/18 test had an AUC of 0.947 (95%CI:0.910-0.985), which was higher than that of careHPV and PCR test. Physician-sampling POCT test had 100.00% sensitivity (95%CI: 64.57%-100.00%) and 55.85% specificity (95%CI: 49.83%-61.70%) for detecting CIN2+. POCT HPV16/18 test had lower sensitivity (71.43%, 95%CI: 35.90%-91.76%) and higher specificity (92.45%, 95%CI: 88.63%-95.06%). POCT HPV16/18 test generally showed similar AUC on both self-collected samples and clinician-collected samples (0.947 vs 0.819, P=0.217). Conclusion: POCT HPV16/18 test is an effective method with relatively high sensitivity and specificity for cervical cancer screening.

Sexually transmitted infections among women living with HIV in a Brazilian city

ABSTRACT Background: Clinical improvements following highly active antiretroviral therapy (HAART) may increase high-risk behaviors resulting in sexually transmitted infections (STI). Optimism related to the success of HAART in slowing disease progression, reducing viral load, and improving health status might be important factors for increasing sexual risk behaviors such as less use of condoms. Objective: To determine the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, hepatitis B and C, high-risk HPV, and cervical cytological abnormalities among women living with HIV (WLHIV) who attended a Reference Center for STI/AIDS in Brazil. Methods: A cross-sectional study was conducted among 151 WLHIV attending an STI Clinic in Vitória city, Brazil. A structured questionnaire, including demographic, behavioral, and clinical information, was used for data collection. Serological tests for HIV, syphilis, hepatitis C and B, CD4 counts, and viral load determination were performed. Cervical samples were collected for cytology and real-time PCR for HPV, Chlamydia, and Neisseria gonorrhoeae. Results: In this study, 59% of women had at least one diagnosed STI at the time of the first clinic visit; 31% had clinical forms of anogenital HPV, 10% syphilis, 8%Neisseria gonorrhoeae, 5.0% trichomoniasis, 3% Chlamydia trachomatis, 1% hepatitis B, and 1% hepatitis C; 6.7% of the women presented with cervical cytological abnormalities. Furthermore, 46.3% of women had HR-HPV, and 17.6% had HPV 16/18. Only 5% of the women had a CD4 count <200 cells/mm3, 61.6% had undetectable HIV viral load, and 81.3% were currently on HAART. Conclusion: A high prevalence of STI and HR-HPV infections were observed among HIV-infected women in this investigation. Prevention programs need to focus on counseling WLHIV and their regular partners with focused interventions such as couples counseling and education programs.

Assessment of HPV 16, HPV 18, p16 expression in advanced stage laryngeal cancer patients and prognostic significance / Avaliação da expressão do HPV 16, HPV 18, e p16 em pacientes com câncer de laringe em estágio avançado e significado prognóstico

Abstract Introduction: Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx. Objective: We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study. Methods: This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined. Results: Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (p= 0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years. Conclusion: The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques.

Resumo Introdução: O papilomavírus humano é um fator de risco etiológico para um subconjunto de carcinoma espinocelular de cabeça e pescoço. Tem sido demonstrado que o HPV é um poderoso biomarcador prognóstico para o câncer de orofaringe, mas seu papel na laringe ainda não foi explorado em profundidade. Os mecanismos de desenvolvimento dos carcinomas de laringe são bastante complexos e controlados por vários fatores. Tabagismo e álcool são os fatores de risco mais importantes. Estudos recentes indicam que a infecção pelo HPV também desempenha um papel importante nos carcinomas da laringe. Os carcinomas laríngeos relacionados ao HPV ocorrem especialmente na região supraglótica. Objetivo: Nosso objetivo foi determinar a frequência da positividade para o HPV / proteína 16 em pacientes com carcinoma da laringe e a associação da positividade para o HPV e /ou proteína 16 com variáveis como idade, sexo, tabagismo, localização do tumor, metástase linfonodal, recidiva e sobrevivência de carcinoma da laringe em estágio avançado em nosso estudo. Método: Este estudo retrospectivo incluiu 90 pacientes com carcinoma laríngeo avançado. O grupo controle incluiu 10 amostras de mucosa laríngea normal. A presença de HPV foi inves-tigada por anticorpo policlonal através de reação de polimerase em cadeia e a proteína 16 por método imunohistoquímico. Nos casos positivos para o HPV, a presença dos tipos 16 e 18 do foi avaliada por reação de polimerase em cadeia. As características demográficas dos pacientes foram observadas. A sobrevida dos pacientes e a associação com HPV / proteína 16 foram determinadas. Resultados: A positividade com anticorpo policlonal do HPV foi detectada em 11 (12,2%) dos 90 casos. Desses 11 casos, o HPV 16 foi positivo em 6, o HPV 18 em 4 e o HPV 16 e 18 foram positivos em 1. Em 18 (20%) dos casos, a proteína 16 foi positiva. Seis dos casos (6,6%) apresentaram positividade para HPV e proteína16. Nos casos positivos apenas para a proteína 16 ou quando HPV e a proteína 16 foram co-positivos, a ingestão de álcool foi menor e o tumor apresentou maior probabilidade de estar localizado na área supraglótica. Essas proporções foram estatisticamente significantes. A localização supraglótica do tumor foi maior em casos positivos para proteína 16. A correlação entre positividade para proteína 16 e localização da área supraglótica foi estatisticamente significante (p = 0,011). Dos casos positivos para proteína 16, 55,6% foram supraglóticos, 33,3% glóticos e 11,1% transglóticos. Embora a expectativa de vida acima de 5 anos tenha sido numericamente maior nos casos positivos para HPV e proteína 16, isso não foi estatisticamente significante. Não houve relação estatisticamente significante entre positividade do HPV e média de idade, diferenciação, tabagismo e uso de álcool, progressão tumoral, metástase linfonodal, localização, recidiva, causa de mortalidade e métodos de tratamento em nosso estudo. O período médio de seguimento de nossos pacientes foi de 6,7 anos. Conclusão: A estreita relação entre HPV e carcinoma espinocelular orofaríngeo não pôde ser demonstrada na laringe em muitos estudos, inclusive no nosso estudo. Nossos achados confirmam um papel limitado do HPV na carcinogênese da laringe. A proteína 16 não é um substituto confiável para o status do HPV nos cânceres de laringe e não é preditor da sobrevida do câncer de laringe. A localização supraglótica do tumor foi maior em casos positivos para proteína16. A correlação entre positividade para proteína 16 e localização na área supraglótica foi determinada como estatisticamente significante. Há necessidade de ensaios clínicos com amostras maiores, nos quais a proliferação neoplásica seja melhor demonstrada e a precisão dos resultados obtidos seja apoiada por diferentes técnicas.

Frequency of high-risk genotypes of human papilloma virus in oral lesions / Frecuencia de genotipos alto riesgo del virus papiloma humano en lesiones bucales

Some genotypes of the human papilloma virus (HPV) in the oral cavity cause genetic instability that may lead to cancer. Clinical and histological diagnoses are key tools; however, molecular techniques allow predicting, detecting and monitoring the disease. Objective: To identify the frequency of four high-risk HPV genotypes and their association with lesions in the oral cavity. Materials and Methods: Descriptive cross-sectional study with a sample of 48 patients diagnosed with hyperplastic lesions and others currently classified as potentially malignant disorders (PMDs) of the oral cavity, who underwent biopsies, histopathological analysis, and HPV16, 18, 31, and 45 detection and genotyping by polymerase chain reaction (PCR). Results: Epithelial hyperplasia was the most frequent lesion found in 45.8% (n=22) of patients. Nicotine palatinus and leukoplakia were found in 8.3% and 6.2%, respectively; oral cancer in 6.2%. The total frequency of HPV was 12.5% (6/48). Oral papilloma was found in 6.1% (3/48), and nicotine palatinus and oral cancer in 2.0% each (1/48). HPV16, HPV31, and HPV45 were detected, while HPV18 was not observed. HPV16 was the most frequent genotype found (4 out of 6 patients), while HPV31 and HPV45 were found in one patient each. Only one genotype per lesion was found. The presence of HPV was associated with lesions (χ2=11.810; p=0.0375). No significant association with age and gender was found. Conclusion: High-risk HPV continues to be present in oral lesions. The HPV16 viral genotype was the most frequent in the studied lesions.

Algunos genotipos del virus del papiloma (VPH) en boca, producen inestabilidad genética dando lugar al cáncer. El diagnóstico clínico e histológico son herramientas claves, sin embargo, técnicas moleculares permiten predecir, detectar y dar seguimiento a la enfermedad. Objetivo: Identificar la frecuencia de cuatro genotipos del VPH de alto riesgo y su asociación con lesiones en cavidad bucal. Material y Métodos: Estudio descriptivo de corte transversal con una muestra de 48 pacientes diagnosticados con lesiones hiperplásicas y otros clasificados actualmente como desordenes potencialmente malignos (DPM) de la cavidad bucal, a quienes se les realizó biopsias, análisis histopatológico y detección y genotipificación VPH16, 18, 31, y 45 mediante reacción en cadena a la polimerasa (PCR). Resultado: La hiperplasia epitelial fue la lesión más frecuente en 45,8% (n=22). La palatinitis nicotínica y la leucoplasia, se encontraron 8,3% y 6,2% respectivamente, cáncer oral, en 6,2%. La frecuencia total de VPH fue 12,5% (6/48). El papiloma oral estuvo en un 6,1% (3/48), palatinitis nicotínica y cáncer oral en 2,0% (1/48).Se detectó VPH16, VPH31 y VPH45, mientras que VPH18 estuvo ausente. ElVPH16 fue el de mayor frecuencia con 66,7% (4/6), el VPH31 y VPH45 se encontraron en 16,7% (1/6). No se evidenció más de un genotipo por lesión. La presencia de VPH estuvo asociado con las lesiones (χ2=11,810; p=0,0375). No se encontró asociación significativa con edad y género. Conclusión: El VPH de alto riesgo sigue estando presente en lesiones bucales. El genotipo viral VPH16 se encontró con mayor frecuencia en las lesiones estudiadas.

Correlation between DNA Methylation of ZNF772 Promoter Region and Cervical Cancer / 中国医学科学院学报

To investigate the DNA methylation in ZNF772 promoter region and its mRNA and protein expressions and analyze the clinical significance of DNA methylation of ZNF772 gene in cervical cancer. Cervical squamous cell carcinoma (SCC) tissues were harvested from three patients (SCC group),and normal cervical tissues from healthy individuals of the same age were used as the control group. Hyper-methylation and lower transcripts were screened by whole-genome bisulfite sequencing (WGBS) and RNA sequencing. Furthermore,in 40 cervical tissue samples in SCC group and 45 normal cervical tissues in the control group,DNA methylation status and mRNA expression of ZNF772 were measured by using real-time quantitative polymerase chain reaction (RT-qPCR) and bisulfite sequencing polymerase chain reaction (BSP). The protein expression was detected by immunohistochemistry. In the SCC group,the potential relationships of DNA methylation status in ZNF772 promoter and mRNA expression with the clinicopathological parameters of cervical cancer were analyzed. As shown by WGBS and RNA sequencing,the abnormal DNA methylated gene ZNF772 was associated with mRNA expression. RT-qPCR verified that the mRNA expression of ZNF772 was significantly lower in SCC group than in control group (=8.351,=0.016). Immunohistochemistry further confirmed that the positive expression of ZNF772 protein was down-regulated in SCC group (=3.802,=0.005). BSP showed that the DNA methylation rate of ZNF772 promoter region (-420,-422 locus) in SCC group was significantly higher than that in control group (=8.566,=0.038;=6.332,=0.043). Spearman correlation analysis showed that,in SCC group,DNA hypermethylation in ZNF772 promoter was negatively correlated with the mRNA expression (=-0.351,=0.045;=-0.349,=0.032) and was significantly correlated with HPV16/18 infection,tumor size,World Health Organization pathological grade,and International Federation of Gynecology and Obstetrics clinical stage (=0.018,=0.012,=0.009,and =0.035,respectively). The DNA hypermethylation in the promoter region of ZNF772 gene is involved in the occurrence and development of cervical cancer.

Molecular detection of oncogenic subtypes of human papillomavirus (HPV) in a group of women in the Amazon region of Brazil

The natural history of cervical cancer is strongly related to the presence of human papillomavirus (HPV) infection, with its relationship with cervical cancer being a matter of concern. It is estimated that 70% of all cervical cancers worldwide are caused by HPV 16 and 18. Accordingly, the present study aimed to contribute to the identification of HPV subtypes circulating in a group of women of Manaus-Brazil. Cervical samples were collected from 49 women, following the eligibility criteria of the study, and DNA was then extracted from the samples, which were analyzed for the presence of the virus in the genetic material through the polymerase chain reaction (PCR) using generic primers (GP05/06). Finally, identification of the viral subtypes was performed using specific primers for the detection of the main subtypes already examined (16 and 18). Positive HPV DNA was detected in 100% of the samples included in the study. Human papillomavirus 16 was the most prevalent subtype in the majority of lesions, accounting for 29 (59.2%) of the positive cases, and HPV 18 was detected in four (8.2%) women. In these 4 cases there was co-infection, with the presence of both HPV 18 and HPV 16. Therefore, 40.8% (20 cases) in which HPV DNA was detected presented infection with other subtypes of HPV not included in the study. This data has clinical implications related to cervical cancer prevention, as the current prophylactic HPV vaccines are only effective against high-risk HPV 16 and 18 subtypes.

Identificación molecular de los VPH oncogénicos mediante PCR en tiempo real con sondas Taqman / Molecular identification of oncogenic HPVS by real time PCR with Taqman probes

INTRODUCCIÓN: los Virus del Papiloma Humano (VPH) constituyen un diverso grupo de virus, siendo los genotipos 16 y 18 los más prevalentes y, por lo tanto, principales objetivos de varios estudios en infecciones del tracto anogenital. El presente estudio pretende determinar la infección por VPH de Alto Riesgo en muestras genitales, mediante la aplicación de tecnología molecular para la genotipificación de VPH16 y VPH18 por PCR en Tiempo Real. MATERIALES Y MÉTODOS: se trabajó con 151 muestras de hisopados genitales, las extracciones de ADN se realizaron mediante columnas de sílice y la identificación de los VPH de Alto Riesgo fue mediante la optimización de una PCR en Tiempo Real duplex para la genotipificación de VPH16 y 18, para lo cual se realizó el diseño de cebadores y sondas TaqMan por software especializado, y se determinó las concentraciones de reactivos y temperaturas de reacción ideales. RESULTADOS: en la identificación de los VPH de Alto Riesgo se obtuvo un total de 41 casos positivos, muestras que fueron seleccionadas para realizar la genotipificación de VPH16 y VPH18 mediante la técnica de PCR en Tiempo Real. La mayor presencia de estos virus oncogénicos se determinó en mujeres de 15 a 29 años (40% y 26,7% respectivamente). CONCLUSIÓN: mediante la optimización e implementación de nuevas técnicas moleculares se pretende mejorar el seguimiento epidemiológico sobre la presencia de los VPH de Alto Riesgo y así proporcionar una mejor guía sobre la diseminación y presencia de los distintos genotipos oncogénicos de VPH en nuestra población.

INTRODUCTION: Human Papillomavirus (HPV) constitute a diverse group of viruses, being genotypes 16 and 18 the most prevalent and main objectives of several studies on anogenital tract infections. The present study intends to determine the High Risk HPV infection in genital samples, through the application of molecular technology for the genotyping of HPV16 and HPV18 by Real Time PCR. MATERIALS AND METHODS: we worked with 151 samples of genital swabs whereDNA extractions were performed using silica columns and the identification of High Risk HPV was through the optimization of a duplex real-time PCR for genotyping of HPV16 and 18, for which primers and TaqMan probes were designed by specialized software, and ideal reagent concentrations and reaction temperatures were determined. RESULTS: in the identification of HPV High Risk, a total of 41 positive cases were obtained. These samples were selected to perform genotyping of HPV16 and HPV18 using the Real Time PCR technique. The greater presence of these oncogenic viruses was determined in women aged 15 to 29 years (40% and 26.7% respectively). CONCLUSION: through the optimization and implementation of new molecular techniques, it is intended to improve the epidemiological follow-up on the presence of High Risk HPV and thus provide a better guide on the dissemination and presence of the different oncogenic HPV genotypes in our population.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

Clonación y expresión en Escherichia coli de un gen L1 completo del virus del papiloma humano 18 aislado de una paciente cubana y variantes delecionadas / Cloning and expression in Escherichia coli of the full-length and deletion variants of a human papillomavirus 18 L1 gene isolated from a Cuban patients

Introduction: Human papillomavirus (HPV) vaccines are based on the L1 major capsid protein. Objectives: To clone the HPV-18 L1 gene from a Cuban female HPV-18-infected patient and to express the full-length and deletion variants of the cloned HPV-18 L1 gene in Escherichia coli. Methods: The full-length HPV-18 L1 gene was PCR-amplified from total DNA isolated from a Cuban patient, cloned and finally subcloned into the E. coli expression vector pET26b. Three deletion mutants were constructed, which encode truncated proteins lacking 30 amino acids at the C-terminus in combination with 5, 6 or none deleted residue at the N-terminus. Production of L1 proteins in E. coli BL21(DE3) and E. coli SHuffle T7 was assessed by SDS-PAGE and Western blotting. Results: The cloned HPV-18 L1 gene was 99.9 por ciento similar to the African variant EF202152 and probably shares a common origin with the B lineage of genotype 18. The three truncated variants of HPV-18 L1 were produced at higher levels than the full-length HPV-18 L1 protein, attaining higher levels in E. coli BL21(DE3) and higher solubility in E. coli SHuffle. The C-terminus-only truncated variant, L1∆C30, was produced at similar levels to the HPV-18 L1s truncated at both termini. E. coli SHuffle produced about three times more amounts of L1∆C30 when grown under autoinduction conditions with respect to conventional induction and thus, amounts were comparable to those obtained in E. coli BL21(DE3) under conventional induction. Conclusions: Truncation of thirty amino acid residues at the carboxy-terminus of the HPV-18 L1 made a major contribution to the production and solubility of this wild-type protein in E. coli. This is the first report about soluble production of HPV-18 L1 protein in an E. coli SHuffle strain. However, higher amounts of L1 are needed to scale-up its production for developing an HPV vaccine candidate(AU)

Introducción: Las vacunas contra el virus del papiloma humano (VPH) se fundamentan en la proteína principal de la cápsida L1. Objetivo: Clonar el gen L1 del VPH-18 a partir de una paciente cubana infectada con VPH-18 y expresar las variantes de longitud completa y delecionadas del gen L1 del VPH-18 en Escherichia coli. Métodos: El gen L1 del VPH-18 de longitud completa se amplificó por PCR a partir de ADN total aislado de un paciente cubana, se clonó y finalmente se subclonó en el vector de expresión de E. coli pET26b. Se construyeron tres mutantes de deleción, que codifican para proteínas truncadas que carecen de 30 aminoácidos por el extremo carboxilo, en combinación con 5, 6 o ningún residuo delecionado por el extremo amino. La producción de las proteínas L1 en E. coli BL21(DE3) y E. coli SHuffle T7 se evaluó mediante SDS-PAGE y Western blot. Resultados: El gen L1 del VPH-18 clonado fue 99.9 percent similar a la variante africana EF202152 y probablemente comparte un origen común con el linaje B del genotipo 18. Las tres variantes truncadas de la proteína L1 del VPH-18 se produjeron a mayores niveles que la proteína L1 del VPH-18 de longitud completa, alcanzando mayores niveles en E. coli BL21(DE3) y mayor solubilidad en E. coli SHuffle. La variante truncada solo por el extremo carboxilo, L1(C30, se produjo a niveles similares a las proteínas L1 del VPH-18 truncadas por ambos extremos. E. coli SHuffle produjo aproximadamente tres veces más cantidades de L1(C30 cuando creció en condiciones de autoinducción, con respecto a la inducción convencional y, por ende, las cantidades fueron comparables a las obtenidas por E. coli BL21(DE3) bajo inducción convencional. Conclusiones: La truncación de treinta residuos de aminoácidos por el extremo carboxilo de la proteína L1 del VPH-18 tuvo una importante contribución a la producción y solubilidad de la proteína L1 nativa en E. coli. Este es el primer informe sobre la producción soluble de la proteína L1 del VPH-18 en una cepa de E. coli SHuffle. Sin embargo, se necesitan mayores cantidades de la proteína L1 para escalar su producción para desarrollar un candidato vacunal contra el VPH(AU)

Detección y tipificación del Virus Papiloma Humano en el marco del tamizaje virológico para la detección de lesiones del cuello uterino en Asunción, Paraguay / Detection and typing of Human Papilloma Virus during a virological screening for detection of cervical lesions in Asunción, Paraguay

En Paraguay la incidencia de cáncer de cuello uterino (CCU) es superior a las observadas en otros países de la región. El agente etiológico asociado al CCU es el virus papiloma humano (VPH), esencialmente tipos de alto riesgo oncogénicos. El objetivo es describir aspectos epidemiológicos de la infección genital por el virus papiloma humano de alto riesgo (VPH-AR) en mujeres de 25 a 64 años que consultaron en servicios de Patología Cervical del MSPyBS, de mayo a diciembre de 2013. Se utilizó el Cobas 4800 HPV Test (Roche) que permite la detección individual de VPH-16 y VPH-18 y un pool de otros VPH-AR que incluye 12 genotipos de alto riesgo. Los otros VPH-AR fueron tipificados por hibridación reversa en línea (RLB). Entre las 495 mujeres incluidas, se detectaron 72 casos positivos (14,5%) de VPH-AR. Se identificaron 19 tipos virales; siendo el más frecuente VPH-16 (2,1%), seguido del VPH-31, 33, 58 y 66; el VPH-18 aparece en sexto lugar. Este trabajo aporta los primeros datos sobre la implementación de técnicas moleculares para detección y tipificación de VPH como parte del sistema de salud pública de Paraguay. El predominio de VPH-16, confirma su amplia circulación a nivel mundial y dado su mayor potencial oncogénico, representa una alerta a considerar, en especial en las mujeres mayores de 30 años portadoras de una infección persistente. Estos resultados apoyan la importancia de la implementación criteriosa y la utilización apropiada de las pruebas moleculares actualmente disponibles para la prevención y control del CCU(AU)

Expressão e caracterização da glicoproteína D do HSV-1 geneticamente fusionada às oncoproteínas E6 e E7 do HPV-16 e HPV-18 (gDE7E6) em células de mamífero / Expression and characterization of the genetically fused HSV-1 glycoprotein D to E6 and E7 oncoproteins HPV-16 e HPV-18 (gDE7E6) in mammalian cells

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais

Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes

The effect of a booster dose of HPV tetravalent vaccine after 51 months: implications for extended vaccination schedules / El efecto de un refuerzo de la vacuna tetravalente de VPH después de 51 meses: implicaciones para un esquema de vacunación extendido

Abstract: Objective: To asses the non-inferiority between two different vaccination schedules one month after the administration of the third dose. Materials and methods: We evaluated the anti-HPV 16/18 antibody titers induced by quadrivalent HPV vaccine administered using two different schedules in girls 9 to 10-year-old girls: a traditional (0-2-6) and an alternative (0-6-50). Blood samples were collected at month 7, 21 and 51. Results: The antibody geometric mean titer ratios one month after the application of the third dose -month 51 for the alternative and month 7 for the traditional- were 1.55 for HPV16 (95%CI, 1.15-2.08) and 1.53 for HPV18 (95%CI, 1.12-2.09). The seropositive rate was above 99% in both groups. Conclusions: The application of an alternative 3-dose schedule in 9 to 10-year-old girls induces a non-inferior immune response compared to the standard one month after the last dose. Further research is needed to understand the minimal number of doses and their timing to provide the best coverage for HPV infection.

Resumen: Objetivo: Evaluar la no inferioridad entre dos diferentes esquemas de vacunación un mes después de la administración de la tercera dosis. Material y métodos: Se evaluaron los títulos de anticuerpos anti-VPH 16/18 inducidos por la vacuna contra VPH tetravalente administrada en niñas de 9 a 10 años utilizando dos esquemas diferentes: tradicional (0-2-6) y alternativo (0-6-50). Se recolectaron muestras en los meses 7, 21 y 51. Resultados: La media geométrica de títulos de anticuerpos un mes después de la aplicación de la tercera dosis -mes 51 para la alternativa y mes 7 para el tradicional- fueron 1.55 para HPV16 (95% IC 1.15-2.08) y 1.53 para HPV18 (95% IC 1.12-2.09). La tasa de seropositividad fue superior a 99% en ambos grupos. Conclusiones: la aplicación de un esquema alternativo de tres dosis (0-6-50 meses) en niñas parece inducir una respuesta inmune no inferior al esquema tradicional un mes después de la última dosis. Se necesitan más estudios para determinar las dosis mínimas e intervalos óptimos para obtener la mejor cobertura para la infección por VPH.

Optimización de una técnica de PCR convencional para detección de virus de papiloma humano tipo 16 y 18 / Optimization of a conventional PCR technique for detection of human papillomavirus type 16 and 18

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU

Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)

Current Status of Human Papillomavirus Infection and Introduction of Vaccination to the National Immunization Program in Korea: an Overview

Human Papillomavirus (HPV) infection is the most common sexually transmitted infection and is associated with the development of cervical cancer. The purpose of this report is to provide the literature evidences on selecting the HPV vaccine for national immunization program (NIP) in Korea. To complete these tasks, we reviewed domestic and foreign literature on the current status of HPV infection, efficacy and effectiveness of HPV vaccine, safety of vaccine and cost effectiveness analysis of vaccination business. Given that the median age of first sexual intercourse is continuing to fall, this may have serious implications for HPV infection and cervical cancer incidence at the age of 20s. The World Health Organization recommends that the HPV vaccination should be included in the NIP being implemented in each country. Both the bivalent and quadrivalent vaccines have a 90% or greater preventive efficacy on cervical intraepithelial lesion 2–3 and cervical cancer by the HPV 16 or HPV 18. In the future, if HPV vaccination rate as part of NIP increases, it is expected that the incidence of HPV infection, genital warts, and cervical precancerous lesions will be decreased in the vaccination age group. Therefore, in order to increase the HPV vaccination rate at this point in Korea, social consensus and efforts such as the introduction and promotion of HPV vaccine to the NIP according to appropriate cost-effectiveness analysis are required.

Sequencing analysis of HPV-other type on an HPV DNA chip

OBJECTIVES: To identify the specific human papillomavirus (HPV) genotypes from HPV-other type on an HPV DNA chip test by sequencing. METHODS: Among 13,600 women undergoing a routine gynecology examination including Pap smear and/or HPV test by DNA chip test in the healthcare system at Gangnam Center from July 2012 to February 2013, we prospectively collected and performed sequencing for a total of 351 consecutive cervicovaginal samples consisting of 180 samples that tested positive for HPV-other type and 171 samples that tested positive for either high-risk HPV or low-risk HPV. RESULTS: Of a total of 351 samples, individual HPV genotypes were successfully sequenced in 215 cases: 119 HPV-other type, 82 HPV-high-risk, and 14 HPV-low-risk. Based on the sequencing for 119 HPV-other type samples, 91.6% were detected as HPV types that were not included on the DNA chip; however, 7.6% (9/119) were proven to be high-risk HPV types: HPV 18 (n=4), HPV 33 (n=3), HPV 35 (n=1), and HPV 59 (n=1). For correlation analysis of all high-risk and HPV 16/18, the correlation rate was 76.2% and 86.6% with kappa-value of 0.38 and 0.69, respectively. CONCLUSION: HPV-other type on DNA chip test may still have possibility of high-risk HPV, i.e., HPV 18 and thus the significance of HPV-other type in detecting cervical disease remains to be investigated.