Reacción cadena polimerasa en tiempo real para detección del virus papiloma humano (VPH) cáncer de cérvix / Real-time polymerase chain reaction for detection of human papillomavirus (HPV) cervical cancer

INTRODUCCIÓN: El cáncer de cérvix ha ocupa el primer lugar en las neoplasias con mayor número de casos nuevos registrados en el INEN durante los últimos 10 años. - El virus del papiloma humano (VPH) es fundamental para el desarrollo de neoplasias de cuello uterino y puede detectarse en el 99,7% de los cánceres de cuello uterino. - PCR y captura de híbridos (CH2) son dos técnicas aprobadas nacional y mundialmente para el diagnóstico de VPH. - El diagnóstico por PCR tiene la ventaja de diferenciar oncogenes de alto grado, especialmente 16 y 18 para VPH, en comparación con CH2. METODOLOGÍA: Se revisó literatura existente respecto a la técnica de diagnóstico por PCR para VPH. Se encontró una revisión sistemática y tres estudios clínicos primario. DISCUSIÓN: Ambas pruebas tienen una concordancia mayor del 80%, sin embargo, la prueba PCR diferencia los subtipos oncogénicos y no oncogénicos. - La evidencia es discordante, respecto a la sensibilidad y especificad de las pruebas de PCR y CH2, pues estas varían según la prevalencia de la enfermedad y la población evaluada. Sin embargo, muestra mayor especificidad para PCR. - En el Perú se realizan detección de VPH con PCR-TR. Las agencias reguladoras como la FDA han aprobado el uso de esta tecnología en diferentes plataformas de diagnóstico, así como el MINSA aprobado el uso de diagnóstico de VPH con técnicas moleculares. - Para la implementación de esta nueva tecnología se debe contar con los reactivos, profesional médico capacitado para la lectura de los resultados y contar con el personal médico que atienda a quienes den positivo a la prueba de VPH. - Se sugiere el uso de PCR-TR para el diagnóstico de VPH en el INEN. CONCLUSIONES: El virus del papiloma humano (VPH) es fundamental para el desarrollo de neoplasias de cuello uterino y puede detectarse en el 99,7% de los cánceres de cuello uterino. PCR y captura de híbridos (CHII) son dos técnicas aprobadas nacional y mundialmente para el diagnóstico de VPH. El diagnóstico por PCR tiene la ventaja de diferenciar oncogenes de alto grado, especialmente 16 y 18 para VPH, en comparación con CHII. Se revisó literatura existente respecto a la técnica de diagnóstico por PCR para VPH. Se encontró una revisión sistemática y algunos estudios clínicos primario. Ambas pruebas tienen una concordancia mayor del 80%, sin embargo, la prueba PCR diferencia los subtipos oncogénicos y no oncogénicos. La evidencia es discordante, respecto a la sensibilidad y especificad de las pruebas de PCR y CHII. Sin embargo, muestra mayor especificidad para PCR.

Viral non-coding RNAs: Stealth strategies in the tug-of-war between humans and herpesviruses.

Oncogenic DNA viruses establish lifelong infections in humans, and they cause cancers, often in immunocompromised patients, despite anti-viral immune surveillance targeted against viral antigens. High-throughput sequencing techniques allowed the field to identify novel viral non-coding RNAs (ncRNAs). ncRNAs are ideal factors for DNA viruses to exploit; they are non-immunogenic to T cells, thus viral ncRNAs can manipulate host cells without evoking adaptive immune responses. Viral ncRNAs may still trigger the host innate immune response, but many viruses encode decoys/inhibitors to counter-act and evade recognition. In addition, ncRNAs can be secreted to the extracellular space and influence adjacent cells to create a pro-viral microenvironment. In this review, we present recent progress in understanding interactions between oncoviruses and ncRNAs including small and long ncRNAs, microRNAs, and recently identified viral circular RNAs. In addition, potential clinical applications for ncRNA will be discussed. Extracellular ncRNAs are suggested to be diagnostic and prognostic biomarkers and, with the realization of the importance of viral ncRNAs in tumorigenesis, approaches to target critical viral ncRNAs are emerging. Further understanding of viral utilization of ncRNAs will advance anti-viral therapeutics beyond conventional medication and vaccination.

Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

This protocol describes the production of human papillomavirus (HPV)-derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government. Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells Alternate Protocol 2: Production of higher-purity quasivirus using the "Ripcord" method Support Protocol 1: Production of HPV minicircles Support Protocol 2: Production of recircularized HPV genomes Support Protocol 3: Screening of fractions for viral proteins Support Protocol 4: Screening of fractions for viral DNA Support Protocol 5: Measuring viral titer Support Protocol 6: Quantitation of quasivirions Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus Basic Protocol 3: HPV quasivirus transcription assay Basic Protocol 4: HPV quasivirus replication assay.

The association between human papillomavirus and oropharyngeal squamous cell Carcinoma: Reviewed according to the Bradford Hill criteria for causality.

There is emerging evidence of the association between human papillomavirus and a subset of head and neck cancers. However, the role of human papillomavirus as a causal factor is still debated. This review addresses the association between human papillomavirus and oropharyngeal squamous cell carcinoma using the Bradford Hill criteria. The strength of the association is supported by, detection of human papillomavirus infection and antibodies prior to oropharyngeal squamous cell carcinoma. This is furthermore reinforced by the absence of human papillomavirus DNA in healthy tonsils. The association is geographically consistent throughout the economically developed world. The presence and integration of high-risk human papillomavirus genome in tonsillar tumours, and expression of viral oncogenes, are specific and plausible. Analogous to human papillomavirus in cervical cancer, the rising incidence in human papillomavirus positive oropharyngeal squamous cell carcinoma is associated with sexual behaviour. These associations have been repeatedly observed and are in accordance with our current knowledge. The time relation between cause and effect remains the main challenge, due to the lack of well-defined premalignant lesions. However, a causal relationship between human papillomavirus infection and oropharyngeal squamous cell carcinoma seems evident.

The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

Type-specific persistence and associated risk factors of human papillomavirus infections in women living in central Italy.

OBJECTIVE: We examined persistence and clearance of human papillomavirus (HPV) infections and risk factors associated with persistence in 79 women based on the results of two sequential tests performed over 12-24 months. STUDY DESIGN: Between February 2008 and August 2009, 398 women aged 18-63 years were examined for presence of cervical HPV infection by cervical scrape specimen and PCR. Detection was performed using Linear Array (LA) HPV Genotyping Test. All women were interviewed, and a short questionnaire was administered to collect information on socio-demographic characteristics, sexual and reproductive history, smoking habits, oral contraceptive use, history of sexually transmitted diseases, and Chlamydia trachomatis or Mycoplasma spp. infections. Pearson's χ² test was used to verify the association between all independent variables with the response variable. RESULTS: Initially, high risk-HPV (HR-HPV) and low risk-HPV (LR-HPV) infection was detected in 69.6% and 30.4% of the women, respectively, whilst multiple infections occurred in 53.2%. HPV 16 was the most common (20.2%) high-risk type, followed by 52, 31 and 53. At follow-up, HR-HPV infection was detected in 50.6% of the women; among these, 67.5% had persistent infection, while 12.5% acquired other high-risk types, and 20.0% of those positive for LR-HPV at entry had a new HR-HPV infection. Multiple infections were detected in 38.0% of the women. HPV 16 and 31 were the most frequent types, followed by HPV 73. Type-specific HR-HPV persistence was found in 49.1% of women. HPV 31, 39 and 73 were the most frequently persistent types, whilst HPV 16 was the least persistent. No significant age difference between women with persistence or clearance was found. The highest HR-HPV persistence occurred in the 22-27 years old group, whereas clearance increased in women aged 28-33 years. No significant association between persistent HR-HPV infection and oral contraceptive use, smoking habits and history of sexually transmitted disease was detected both at entry and follow-up study. The association between C. trachomatis or Mycoplasma spp. and HPV persistence could not be investigated because of the low detection rate of these microorganisms. CONCLUSIONS: The persistence of HR-HPV infection level was similar to that reported elsewhere, and HPV 31, 39 and 73 showed the highest likelihood of persistence, partially in agreement with other studies. The clinical relevance of the low persistence of HPV 16 and other HR-HPV is unknown. Persistent HR-HPV infection in women aged 22-27 years was in agreement with other authors. To the best of our knowledge, this is the first report on persistence of HR-HPV infections in Italy in a general population, although we examined a small sample in a short follow-up time.

Transitional probability-based model for HPV clearance in HIV-1-positive adolescent females.

BACKGROUND: HIV-1-positive patients clear the human papillomavirus (HPV) infection less frequently than HIV-1-negative. Datasets for estimating HPV clearance probability often have irregular measurements of HPV status and risk factors. A new transitional probability-based model for estimation of probability of HPV clearance was developed to fully incorporate information on HIV-1-related clinical data, such as CD4 counts, HIV-1 viral load (VL), highly active antiretroviral therapy (HAART), and risk factors (measured quarterly), and HPV infection status (measured at 6-month intervals). METHODOLOGY AND FINDINGS: Data from 266 HIV-1-positive and 134 at-risk HIV-1-negative adolescent females from the Reaching for Excellence in Adolescent Care and Health (REACH) cohort were used in this study. First, the associations were evaluated using the Cox proportional hazard model, and the variables that demonstrated significant effects on HPV clearance were included in transitional probability models. The new model established the efficacy of CD4 cell counts as a main clearance predictor for all type-specific HPV phylogenetic groups. The 3-month probability of HPV clearance in HIV-1-infected patients significantly increased with increasing CD4 counts for HPV16/16-like (p<0.001), HPV18/18-like (p<0.001), HPV56/56-like (p = 0.05), and low-risk HPV (p<0.001) phylogenetic groups, with the lowest probability found for HPV16/16-like infections (21.60±1.81% at CD4 level 200 cells/mm(3), p<0.05; and 28.03±1.47% at CD4 level 500 cells/mm(3)). HIV-1 VL was a significant predictor for clearance of low-risk HPV infections (p<0.05). HAART (with protease inhibitor) was significant predictor of probability of HPV16 clearance (p<0.05). HPV16/16-like and HPV18/18-like groups showed heterogeneity (p<0.05) in terms of how CD4 counts, HIV VL, and HAART affected probability of clearance of each HPV infection. CONCLUSIONS: This new model predicts the 3-month probability of HPV infection clearance based on CD4 cell counts and other HIV-1-related clinical measurements.

Persistence of human papillomavirus infection: keys to malignant progression.

Human papillomaviruses (HPVs) are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Although prophylactic vaccines have been developed that target high-risk HPV types, there is a continuing need to understand better the virus-host interactions that underlie persistent benign infection and progression to cancer. In this review we summarize the molecular events that facilitate the differentiation-dependent HPV life cycle, how the life cycle is organized to facilitate virus persistence, and how the activities of HPV regulatory proteins result in malignancy.

Anogenital hairs are an important reservoir of alpha-papillomaviruses in patients with genital warts.

Human papillomaviruses (HPVs) were detected in 69 (43.7%) of 158 and in 7 (4.5%) of 155 anogenital hairs obtained from 53 patients with genital warts (GWs) and from 53 age-matched healthy control subjects, respectively. At least 1 hair sample was positive for 69.8% of patients and for 13.2% of control subjects. For patients, HPV was detected in 64.2%, 39.6%, and 26.9% of hairs plucked from the pubic, scrotal, and perianal regions, respectively. For 91.9% of patients, the same HPV genotype was identified in GWs and hairs from at least 1 sampling site. Having GWs was found to be strongly associated with the presence in anogenital hairs of the HPV genotype causing the GWs (range of odds ratios, 13.0-20.0).

Report on the 2007 International Workshop on Human Papillomaviruses and Consensus Recommendations for Cervical Cancer Prevention.

National and international experts in cervical cancer prevention met at the International Workshop on Human Papillomaviruses and Consensus Recommendations for Cervical Cancer Prevention to review the current evidence and assess the potential for improvement in cervical cancer prevention and to develop plans for implementation of cervical cancer prevention programmes in Croatia. Key recommendations were developed and adopted during the course of the meeting. The process of bringing national experts together with internationally recognized experts in an open forum for the development of consensus recommendations could serve as a model for other countries seeking to implement or improve cervical cancer prevention programmes.

Screening HIV-infected women for cervical cancer in Thailand: findings from a demonstration project.

OBJECTIVES: Although cervical cancer is an AIDS-defining illness, few HIV-infected women are routinely screened for cervical cancer in Thailand. We screened HIV-infected women for cervical cancer as a component of HIV care and assessed high-risk human papillomavirus (HPV) and cervical cancer prevalence. METHODS: From July 2003 through February 2004, HIV-infected women attending either an infectious disease clinic or a sexually transmitted infection (STI) clinic in Bangkok were tested for high-risk HPV types by Hybrid Capture 2 and screened for cervical cancer by Pap test; those with abnormal cervical cytology were referred for diagnosis and treatment. RESULTS: Two hundred ten HIV-infected women at an infectious disease clinic (n = 150) and an STI clinic (n = 60) received cervical cancer screening. The high-risk HPV prevalence was 38.6% and the prevalence of abnormal cervical cytology was 20.4%. Abnormal cervical cytology and high-risk HPV detection were associated (P < 0.001). We received pathology reports for 23 (53.5%) of 43 women, including all those with a Pap test showing high-grade squamous intraepithelial lesions; the cervical cancer prevalence was 1.9% (4 of 210; 95% confidence interval, 0.5-4.8%). CONCLUSION: The estimated prevalence of high-risk HPV and cervical cancer among HIV-infected women in Thailand was high. This emphasizes the need to integrate cervical cancer screening into HIV care.

Pre- and post-conization high-risk HPV testing predicts residual/recurrent disease in patients treated for CIN 2-3.

OBJECTIVE: To evaluate whether high-risk human papillomavirus (HR-HPV) detection and viral load prior to treatment and status of cone margins can predict residual/recurrent disease as well as the ability of current diagnostic tools to identify residual/recurrent disease during follow-up of high-grade cervical intraepithelial neoplasia (CIN) treated by conization using loop electrosurgical procedure (LEEP). METHODS: Two hundred and three women (mean age 38.6 +/- 9.7; range 22-83) with CIN2-3 treated by LEEP conization and confirmed in the surgical specimen, attending follow-up visits were included. Age, HR-HPV detection and viral load determined by HybridCapture 2, and cone margins were evaluated as possible predictors of residual/recurrent disease. Value of single and repeated cytology as well as HR-HPV detection and viral load during follow-up were analyzed as screening tools of recurrence. RESULTS: Residual/recurrent disease was demonstrated by colposcopy guided biopsy in 36 patients (17.7%). High HR-HPV load (>1000 RLU) prior to LEEP and positive cone margins were significantly associated with higher risk of recurrence (31.8% vs. 9.4%, P = 0.005; and 36.4% vs. 11.9%, P < 0.001 respectively). HR-HPV detection at 6-12 m after LEEP showed higher sensitivity than a single or repeated cytology (97.2% vs. 83.3% and 94.4% respectively) although it showed less specificity (81.4% vs. 92.2% and 82.6%). The combination of HR-HPV detection and the first cytology during follow-up detected all patients with residual/recurrent disease (sensitivity 100%, negative predictive value 100%) with an acceptable specificity (76.6%). CONCLUSION: The inclusion of HR-HPV testing with cytology in follow-up of patients treated for CIN2-3 would allow for fewer post-treatment visits and avoid unnecessary cytologies. High HR-HPV load prior to LEEP or positive margins should be considered as risk factors for developing residual/recurrent disease.