Association of viral load and physical status of HPV-16 with survival of patients with head and neck cancer.

INTRODUCTION: Head and neck cancers (NHCs) are of multifaceted origins, and tobacco and alcohol are the primary risk factors. Currently, other factors associated with the genesis of these tumours are being considered, among these viral infections, especially human papillomavirus (HPV) infection. OBJECTIVE: The objective was to evaluate HPV infection, HPV-16 E6 load and its physical status in patients with squamous cell carcinoma in the head and neck and evaluate its effects in the survival of these patients. METHODOLOGY: A total of 80 fresh biopsies of HNC were evaluated. The genetic material was extracted using the commercial kit QIAGEN. The detection and classification of HPV were carried out using INNO-LiPA, whereas the quantification and analysis of integration of the viral genome into the host cell were carried out using real-time PCR. RESULTS: The average age of the patients included was 60.34 ± 14.48 years, with a predominance of the male gender. The most frequent HPV infection was genotype 16 (52.8%), with an average of 10 copies of the HPV-16 E6/ß-globin gene. Furthermore, an integration of the viral genome in the host cell was observed in 86% of cases with a statistically significant relationship between the location of the tumour and the viral load (p < 0.05). CONCLUSIONS: HPV-16 is the most common infection, and its physical status in the host cell is the determining factor in establishing response to treatment. However, more studies are needed to demonstrate the role of HPV infection in carcinogenesis.

Evaluación de virus papiloma humano de alto y bajo riesgo oncogénico en la cavidad bucal de pacientes VIH positivos / Evaluation of human papillomavirus of high and low oncogenic risk in the oral cavity of HIV positive patients

Objetivo. Realizar la detección y tipificación de virus papiloma humano (VPH) en la cavidad bucal de un grupo de pacientes VIH positivos, atendidos en el Centro de Atención de Pacientes con Enfermedades Infectocontagiosas (CAPEI) de la Facultad de Odontología-Universidad Central de Venezuela. Métodos. Se evaluaron 31 hisopados bucales de pacientes VIH positivos para la infección por VPH, mediante el Sistema de Hibridación Reversa de INNO-LiPA que detecta 28 genotipos de VPH de alto y bajo riesgo oncogénico. Resultados. El 61,0% de las muestras evaluadas presentó infección por VPH. El genotipo 6 fue el más frecuente (73,68%), seguido de los genotipos 18, 11 y 16 con 63,16%, 37,0% y 32,0% respectivamente. El 74,0% de las muestras positivas para VPH presentaron infecciones múltiples, siendo más frecuente la coinfección mixta (35,70%) con los genotipos de VPH-6/18 de bajo y alto riesgo oncogénico, 21,40% con los genotipos 6/11/18 y 6/11, cada una. Seguido de 14,30% de las muestras que presentaron infección con VPH-6/11/16/18 y 7,10% con los genotipos 11/16 de bajo y alto riesgo oncogénico. Conclusiones. La alta frecuencia de infección con VPH de alto riesgo oncogénico y la presencia de múltiples genotipos observada en la cavidad bucal de individuos VIH positivos, que no presentaron lesiones compatibles con esta infección en el examen extra e intrabucal, indica que los métodos moleculares de diagnóstico son importantes en la detección de infecciones subclínicas y latentes, lo que puede permitir un mejor seguimiento y manejo más oportuno de estos pacientes con mayor riesgo de desarrollar neoplasias malignas en la cavidad bucal.

Objective. Perform human papilomavirus (HPV) detection and typing in the oral cavity in a group of HIV positive patients, treated at the Center for Care of Patients with Infectious Diseases (CAPEI) of the Faculty of Dentistry-Central University of Venezuela. Methods. Thirty-one oral swabs from HIV-positive patients were evaluated to detect HPV infection using the INNO-LiPA Reverse Hybridization System that detects 28 HPV genotypes of high and low oncogenic risk. Results. The 61.0% of the evaluated samples had an HPV infection. The low risk genotype 6 was the most frequent (73.68%), followed by genotypes 18, 11 and 16 with 63.16%, 37.0%, and 32.0%, respectively. Seventy-four percent of HPV positive samples had multiple infections, being the more frequent the mixed coinfection (35.70%), with HPV genotypes 6/18 of low and high oncogenic risk, 21.40% with genotypes 11/6/18 and 6/11, each one, followed by 14.30% of the samples presented infection with HPV-6/11/16/18 and 7.10% with the 11/16 genotypes of low and high oncogenic risk. Conclusions. The high frequency of infection with high oncogenic risk HPV types and the presence of multiple genotypes observed in the oral cavity of HIV positive patients, who did not present lesions compatible with this infection in the extra and intraoral examination, indicates that molecular diagnostic methods are important in the detection of subclinical and latent infections, which may allow better follow-up and more timely management of these patients with a higher risk of developing malignant lesions in the oral cavity

Lymphocyte subsets predictive value and possible involvement of human papilloma virus infection on breast cancer molecular subtypes.

AIM: To detect human papilloma virus (HPV) presence and to characterize cellular immune response in breast cancer patients. METHODS: A total of 74 women were included, of which 48 samples were from patients diagnosed with breast cancer and 26 patients with benign pathology of the breast. Molecular subtype classification was performed based on the immunohistochemical reports of the tumor piece. HPV genome detection and genotyping from fresh breast biopsies was performed using the INNO-LIPA HPV Genotyping Extra test (Innogenetics, Ghent, Belgium). CD3+, CD4+, CD8+ and natural killer (NK)+ cells levels from peripheral blood samples from patients with breast cancer and benign pathology were measured by flow cytometry. RESULTS: Luminal A was the most frequent breast cancer molecular subtype (33.33%). HPV was detected in 25% of the breast cancer patients, and genotype 18 was the most frequent in the studied population. The mean of CD3+, CD4+ and CD8+ subpopulations were decreased in patients with breast cancer, in relation to those with benign pathology, with a statistically significant difference in CD8+ values (P = 0.048). The mean of NK+ cells was increased in the benign pathology group. The average level of CD3+, CD4+, CD8+ and NK+ cells decreased as the disease progressed. HER2+ and Luminal B HER2+ tumors had the lowest counts of cell subsets. HPV breast cancer patients had elevated counts of cellular subsets. CONCLUSION: Determining level changes in cellular subsets in breast cancer patients is a useful tool to evaluate treatment response.

Cytokines and MMPs levels in gingival crevicular fluid from patients with chronic periodontitis before and after non-surgical periodontal therapy

The study of host response in periodontal disease may provide a mechanism to monitor disease progression. the purpose of the present research was to determine the levels of IL-1alfa, IL-1beta, TNF-alfa, IL-6, IL-6sR, IL-8, IL-10, MMP-3 and MMP-8 in gingival crevicular fluid (GCF), before and after non-surgical periodontal treatment (NSPT) in order to evaluate therapy response. methodology: eleven patients diagnosed with chronic periodontitis and eleven healthy subjects were selected for this study. clinical measurements, including probing depth (PD) and clinical attachment loss (CAL) were carried out in patients diagnosed with chronic periodontitis and periodontal healthy controls. the clinical indexes evaluated were: gingival index (GI) and plaque index (PI). samples of GCF were taken from one tooth per quadrant before and 45 days after NSPT. the levels of inflammatory mediators were measured by ELISA. results: the values of all clinical parameters decressed significsntly after treatment. the concentration levels of all cytokines and MMP-3 and MMP-8 in the GCF sample were higher in patients diagnosed with chronic periodontitis compared to the healthy group. all inflammatory mediators decreased after therapy, but did not reach control values; IL-6, Il-6sR, IL-10 and TNF-alfa, attained the highest reduction (70 percent -54 percent); the vales of MMP3, IL-1alfa, IL-1beta and IL-8 were reduced between 50 percent ­ 34 percent; and MMP-8 showed the lowest decrease (28 percent). conclusion: all clinical parameters and cytokines levels decreased after NSPT. the mediators TNF-alfa IL-6, IL-6sR, and IL-10 showed the largest variation between before and after NSPT and could thus be used to evaluate therapy response.

Comparación de la detección y tipificación del virus del papiloma humano en muestras citológicas y de orina en pacientes del sexo femenino / Comparison of the detection and typing of human papilloma virus in cytological samples vs. urine samples in female patients

El cáncer de cuello uterino, es el segundo más frecuente en mujeres a nivel mundial; la infección por Virus de Papiloma Humano (VPH) de alto riesgo oncogénico, es el principal factor etiológico de esta malignidad. La identificación viral se logra mediante métodos moleculares sensibles y específicos como la reacción en cadena de la polimerasa (PCR), que generalmente utiliza hisopados cervicales o biopsias como material biológico. Debido a las complicaciones e incomodidad que implica la toma de estas muestras, se realizan estudios en otras más accesibles como la orina. Por lo que se realizó la detección y tipificación de VPH en muestras de hisopados endocervicales y de orina, se compararon los resultados obtenidos y se evaluó la efectividad del uso de esta última. La extracción del material genético se realizó con el estuche comercial AXYGEN. Para la detección y la tipificación viral se empleó la técnica de PCR en tiempo final. La positividad para VPH fue 68,6% en los hisopados cervicales y 62,9% en las muestras de orina de, valores similares y comparables a estudios previos. Así mismo, la concordancia obtenida entre los resultados de las muestras empleadas respecto a los tipos virales identificados fue “moderada” (k=0,609), encontrándose además valores altos de sensibilidad y especificidad de 83,3% y de 81,8% respectivamente al usar muestras de orina. Estos resultados apuntan a la posibilidad de desarrollar un diagnóstico efectivo para VPH empleando muestras de orina, ya que reducirían la intervención de personal adiestrado para su toma, costos y la incomodidad para las pacientes.

Cervical cancer is the second most common cancer in women worldwide. Infection with high oncogenic risk Human Papillomavirus (HPV) is the main etiological factor of this malignancy. Viral identification is achieved by sensitive and specific molecular methods, such as polymerase chain reaction (PCR), which are generally used on cervical biopsies or swabs of biological material. Because of the complications and discomfort that taking these samples implies, studies are conducted with other samples obtained from less invasive methods, such as urine. Accordingly, detection and genotyping of HPV in endocervical swabs and urine were performed to compare results and to evaluate the effectiveness of using the latter samples. The genetic material was obtaining using the commercial kit Axygen. For viral detection and typing the conventional PCR technique was used. Positivity for HPV in cervical swabs was 68.6% and 62.9% in urine samples, similar values and comparable to previous studies. Likewise, the concordance obtained between the results of the samples used with respect to the identified viral types was “moderate” (k = 0.609), with high values of sensitivity and specificity of 83.3% and 81.8%, respectively, when urine samples were used. These results point to the possibility of developing an effective diagnosis for HPV using urine samples, because it would reduce the intervention of trained personnel, cost and discomfort for the patients.

Prevalence of HPV and EBV infection andtheir relationship with the p53 and PCNA expression in oral carcinoma patients

Introduction: Infection caused by potentially oncogenic viruses, such as HPV and EBV, favors the role of certain oncoproteins that can induce dysplasias and malignant lesions. Objective: To evaluate the prevalence of HPV and EBV and their relation with the expression of p53 and PCNA in patients with oral carcinoma. Methodology: Twenty-seven oral squamous cell carcinomas (OSCC) were evaluated; DNA extraction was conducted using the QIAamp DNA mini kit; viral detection was obtained using the INNO-LiPA kit for HPV, and nested PCR was used for EBV. The evaluation of molecular markers was performed through immunohistochemical staining. Results: The mean age of the patients was 60.55 +/- 13.94 years, and 52 percent of these were female. Of the patients, 59 percent were tobacco users and 63 percent were alcohol consumers. HPV was detected in 70 percent of the patients with the predominance of genotype 16 (60 percent). As for EBV infection, it was observed in 59 percent of cases. p53 and PCNA immunopositivity corresponded to 44 percent and 59 percent, respectively. The tongue was the anatomical location with highest positivity for both viruses as well as for the expression of molecular markers. The 48 percent of the cases presented infection by both viruses. Conclusion: HPV and EBV infection together with the expression of p53 and PCNA were more frequently observed in advanced stages of the disease, suggesting a more relevant role in the progression than in tumor genesis.

Detection of EBV, CMV and HSV-1 in subgingival samples of HIV positive and negative patients with chronic periodontitis / Detección de VEB, CMV y VHS-1 en muestras subgingivales de pacientes VIH positivos y negativos con periodontitis crónica

Abstract: objective: to detect the presence of infection by EBV (Epstein-Barr Virus), CMV (Cytomegalovirus) and HSV-1 (Herpes Simplex Virus type 1) in subgingival samples from HIV- positive patients under HAART (High Activity Antiretroviral Therapy), HIV- positive patients without HAART, HIV-negative patients with chronic periodontitis and healthy controls. Methodology: Crevicular fluid samples of 11 HIV+ patients on therapy were evaluated, 6 without antiretroviral therapy, 7 HIV- negative subjects with chronic periodontitis and 7 periodontally-healthy controls. PI (Plaque index), GI (Gingival Index), PD (probing depth) and CAL (Clinical Attachment Loss) were registered at six sites per each tooth in all teeth and subgingival plaque samples of a tooth were collected per quadrant. Nested PCR was used to detect EBV and endpoint PCR to detect infection by CMV and HSV-1. Results: Clinical parameters showed statistically significant differences between HIV-positive patients and subjects with chronic periodontitis compared with the control group (p<0.05). DNA of EBV was detected mainly in HIV-positive patients under HAART, 91 percent (10/11). DNA of CMV was detected mainly in patients without HAART, 67 percent (4/6), while HSV-1 was observed in 27 percent (3/11) of patients under HAART. In the control group no virus was detected. Coinfection was observed in 50 percent of HIV patients without HAART, 36 percent of HIV patients with HAART and 14 percent of HIV-negative with chronic periodontitis. Conclusion: Viral infection was prevalent in HIV patients under HAART and EBV was the primary viral infection detected in HIV-positive patients with chronic periodontitis.

Resumen: detectar la presencia de infección por VEB (Virus Epstein-Barr), CMV (Citomegalovirus) y VHS-1 (Virus Herpes simple tipo 1) en muestras subgingivales de pacientes VIH-positivos bajo HAART (Terapia Anti Retroviral de Alta Actividad), VIH-positivos sin HAART, pacientes VIH-negativos con periodontitis crónica y controles sanos. Metodología: Se evaluaron muestras de fluido crevicular de 11 pacientes VIH+ bajo terapia, 6 sin terapia antiretroviral, 7 sujetos VIH–negativo con periodontitis crónica y 7 controles periodontalmente sanos. Se registró el IP (Índice de placa), IG (Índice Gingival), PS (Profundidad del Sondaje) y NIC (Nivel de Inserción Clínica) en seis sitios por diente en todos los dientes y se recolectaron muestras de placa subgingival de un diente por cuadrante. Se empleó PCR anidada para detectar VEB y PCR punto final para identificar la infección con CMV y VHS-1. Resultados: Los parámetros clínicos mostraron diferencias estadísticamente significativas entre pacientes VIH-positivos y sujetos con periodontitis crónica comparados con el grupo control (p<0.05). El ADN de EBV fue detectado principalmente en pacientes VIH-positivos bajo HAART con 91 por ciento (10/11). El ADN de CMV se detectó principalmente en pacientes sin HAART, 67 por ciento (4/6), mientras que VHS-1 se observó en 27 por ciento (3/11) de los pacientes bajo HAART. En el grupo control no se detectó ningún virus. La coinfección fue observada en 50 por ciento de los pacientes VIH sin HAART, 36 por ciento de los VIH con HAART y 14 por ciento de los VIH negativos con periodontitis crónica. Conclusión: La infección viral fue predominante en los pacientes VIH bajo HAART y VEB fue la principal infección viral detectada en los pacientes VIH positivos y con periodontitis crónica.

Cytokine and metalloproteinases in gingival fluid from patients with chronic periodontitis / Niveles de citocinas y metaloproteinasas en fluido gingival de pacientes con periodontitis crónica

The purpose of the present research was to determine the levels of IL-1α, IL-1β, TNF-α, IL-6, IL-6sR, IL-8, IL-10, MMP-3 and MMP-8 in gingival crevicular fluid (GCF) of subjects with chronic periodontitis. Clinical measurements were carried out in 20 patients with chronic periodontitis and 11 periodontally healthy controls. The clinical indexes evaluated were: gingival index (GI), plaque index (PI), bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL); the measurements were taken at six sites per tooth in all teeth in each subject. GCF samples were taken from one tooth per quadrant, and the levels of mediators were measured using an ELISA test. Statistically significant differences were observed between patients and control group in relation to all clinical parameters evaluated (p<0.05). The gingival concentrations, in pg/mL, of IL-1α (patients: 239.06 ± 65.5 vs control: 97.79 ± 15.81), IL-1β (patients: 157.19 ± 36.4 vs control: 63.44 ± 19.04), TNF-α (patients: 10.87 ± 1.7 vs control: 1.15 ± 0.84), IL-6 (patients: 3.77 ±1.7 vs control: 0.43 ± 0.22), IL-6Sr (patients: 655.59 ± 185.8 vs control: 73.59 ± 23.18), IL-8 (patients: 496.3 ± 155.3 vs control: 206.13 ± 46.63), IL-10 (patients: 10.75 ± 3.6 vs control: 2.41 ± 0.57), MMP-3 (patients: 3531 ± 1558.2 vs control: 724.84 ± 289.51) and MMP-8 (patients: 8231.70± 1279.2 vs control: 1534.67± 814.90) were significantly greater in patients with periodontal disease than in the control group (p<0.001). The higher levels of the cytokines and metalloproteinases obtained in this study were significantly associated with the severity of the periodontal disease.

El propósito de la presente investigación fue determinar los niveles de IL-1α, IL-1β, TNF-α, IL-6, IL-6sR, IL-8, IL-10, MMP-3 and MMP-8 en fluido gingival crevicular (FGC) de sujetos con periodontitis crónica. Se evaluaron los parámetros clínicos en 20 pacientes con periodontitis crónica y 11 controles periodontalmente sanos. Los índices clínicos evaluados fueron: indice gingival (IG), indice de placa dental (IP), sangramiento al sondaje (SS), profundidad del saco (PS) y nivel de inserción (NI). Las muestras de FGC fueron tomadas de un diente por cada cuadrante y los niveles de los mediadores fueron medidos utilizando la prueba de ELISA. Se observaron diferencias estadísticamente significativas entre los pacientes y el grupo control en relación a todos los parámetros clínicos evaluados (p<0,05). Las concentraciones en fluido gingival en pg/mL de IL-1α(pacientes: 239,06 ± 65,5 vs control: 97,79 ± 15,81), IL-1β (pacientes: 157,19 ± 36,4 vs control: 63,44 ± 19,04), TNF-α (pacientes: 10,87 ± 1,7 vs control: 1,15 ± 0,84), IL-6 (pacientes: 3,77 ±1,7 vs control: 0,43 ± 0,22), IL-6Sr (pacientes: 655,59 ± 185,8 vs control: 73,59 ± 23,18), IL-8 (pacientes: 496,3 ± 155,3 vs control: 206,13 ± 46,63), IL-10 (pacientes: 10,75 ± 3.6 vs control: 2,41 ± 0,57), MMP-3 (pacientes: 3531 ± 1558,2 vs control: 724,84 ± 289,51) and MMP-8 (pacientes: 8231,70± 1279,2 vs control: 1534,67± 814,90), estuvieron significativamente mayores en pacientes con enfermedad periodontal que en el grupo control. (p<0,001). Los niveles elevados de citocinas y metaloproteinasas obtenidos en este estudio estuvieron significativamente asociados con la severidad de la enfermedad periodontal.

Cytokine and metalloproteinases in gingival fluid from patients with chronic periodontitis.

The purpose of the present research was to determine the levels of IL- 1a, IL- 1 P, TNF-a, IL-6, IL-6sR, IL-8, IL-10, MMP-3 and EMP-8 in gingival crevicular fluid (GCF) of subjects with chronic periodontitis. Clinical measurements were carried out in 20 patients with chronic periodontitis and 11 periodontally healthy controls. The clinical indexes evalua- ted were: gingival index (GI), plaque index (PI), bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL); the measurements were taken at six sites per tooth in all teeth in each subject. GCF samples were taken from one tooth per quadrant, and the levels of mediators were measured using an ELISA test. Statistically significant differences were observed between patients and control group in relation to all clinical parameters evaluated (p<0.05). The gingival concentrations, in pg/mL, of IL-la (patients: 239.06 ± 65.5 vs control: 97.79 ± 15.81), IL-10 (patients: 157.19 ± 36.4 vs control: 63.44 ± 19.04), TNF-a (patients: 10.87 ± 1.7 vs control: 1.15 ± 0.84), IL-6 (patients: 3.77 ± 1.7 vs control: 0.43 ± 0.22), IL-6Sr (patients: 655.59 ± 185.8 vs control: 73.59 ± 23.18), IL-8 (patients: 496.3 ± 155.3 vs control: 206.13 ± 46.63), IL-10 (patients: 10.75 ± 3.6 vs control: 2.41 ± 0.57), MMP-3 (patients: 3531 ± 1558.2 vs control: 724.84 ± 289.51) and MMiP-8 (patients: 8231.70 ± 1279.2 vs control: 1534.67± 814.90) were significantly greater in patients with periodontal disease than in the control group (p<0.00 1). The higher levels of the cytokines and metalloproteinases obtained in this study were significantly associated with the severity of the periodontal disease.

Papilomatosis laríngea juvenil y su relación con la infección genital por virus de papiloma humano durante el embarazo / Laryngeal papillomatosis youth and their relationship with infection Genital human papilloma virus during pregnancy

OBJETIVO: establecer la relación entre el diagnóstico de papilomatosis laríngea juvenil y la infección genital por virus de papiloma humano durante el embarazo. MÉTODOS: Se incluyeron 18 madres de hijos con diagnóstico de papilomatosis laríngea juvenil. Se revisaron las historias de los niños, se realizó anamnesis a las madres buscando antecedentes de infección por virus de papiloma humano durante el embarazo, se practicó evaluación ginecológica actual, citología, vulvoscopia, vaginoscopia, colposcopia y biopsia, de ser necesario. Se tomaron muestras para tipificación viral. RESULTADOS: Hubo 120 casos de papilomatosis laríngea juvenil entre 14 400 pacientes, para una frecuencia de 0,8 %. Entre los pacientes evaluados, predominó el sexo masculino (61,1 %). La edad al momento del diagnóstico fue de 5,7 ± 3,2 años. La enfermedad tenía una mediana de evolución de 2 años. La mediana del número de intervenciones quirúrgicas requeridas por paciente, fue 3. Las manifestaciones clínicas más frecuentes fueron disnea (83,3 %), y disfonía (61,1 %). El genotipo viral en las lesiones laríngeas fue 6 (50 %), 11 (11,1 %) y coinfección 6 y 11 (11,1 %). Se detectó virus de papiloma humano en 5 madres: 3 de alto riesgo y 2 no tipificables. La vía del parto vaginal fue la más frecuente con un 83,3 %. CONCLUSIONES: La papilomatosis laríngea es poco frecuente (0,8 %), genera un cuadro de severidad variable caracterizado por disnea y disfonía, es producido por papilomavirus humano 6 y 11 y se asocia al nacimiento vía vaginal. No hubo correlación entre los tipos virales de madres e hijos.

OBJECTIVE: to establish the relationship between the diagnosis of juvenile laryngeal papillomatosis and genital infection with human papilloma virus during pregnancy. METHODS: 18 mothers whose children’s diagnoses were juvenile laryngeal papillomatosis were included. The children’s history were checked, the mothers were given the anamnesis procedure looking for human papilloma virus infections during pregnancy, they were given actual gynecologic evaluations, citology, vulvoscopy, vaginoscopy, colposcopy and biopsy where needed. Samples were taken for their viral tipification. RESULTS: There were 120 cases of juvenile laryngeal papillomatosis in 14400 patients, an 0.8% frecuency. Amongst the evaluated patients, most were males (61.1 %). Age at diagnosis was 5.7 ± 3.2 años. The disease had an evolution median time of 2 years. The medical intervention median per patient was 3. The most frecuent clinical manifestations were disnea (83.3 %) and dysphonia (61.1%). The viral genotype on laryngeal lesions was 6 (50%), 11 (11.11 %) and coinfection 6 y 11 (11.1 %). Human papilloma virus was detected in 5 mothers: 3 high risk and 2 non classifiable. Vaginal birth was the most frequent with an 83.3 % rate. CONCLUSION: Juvenile laryngeal papillomatosis is not very frequent (0.8 %), generates a manifestation of variable severity characterized by dysnea and dysphonia, produced by human papillomavirus 6 and 11 and is associated to vaginal birth. There was no correlation between the viral types of mothers and children.

Persistent infection with high-risk human papilloma viruses: cohort study, Mérida, Venezuela.

Cervical lesions have been associated with infection by high-risk human papilloma virus (high-risk HPV). In 409 women aged >15 years high-risk HPV lesions were identified. In a cohort of this population persistent infection was compared with cytological, colposcopic, and histological lesions. Cervical scrapes were taken and DNA was isolated. HPV was detected by PCR in the E6/E7 region. Genotyping was performed by PCR nested multiple E6/E7. HPV was detected in a 37.40% (153/409), high-risk HPV in 86% (153/178), HPV18 46.64% (83/178), HPV16 34.28% (61/178). Among these 53.93% (96/178) were multiple infections, and HPV18/16 (30/96) was the most frequent 31.25%. The cytology showed changes in 15% of positive patients. A 49.67% in women positive for HPV infection showed abnormalities in the colposcopic study, a relationship that turned out to be statistically significant ( p < 0.0019 test χ(2)). Among all 85% of the women were younger than 45 years of age. Fifty-seven patients were evaluated 15 months after the base study, with initial prevalence of morbidity 49.12% (28/57) and at the end 10.53% (6/57), showing in 89.29% (25/28) negative for HR-HPV infection, 10.34% (3/28) showed persistence of infection, 17.54% (10/57) presented cytological alterations, with 80% of positivity for HPV, and a regression of 100% (10/10) of the previously identified lesions. With colposcopy, 50% (14/28) presented alterations related to HPV, of these 85.71% (12/14) showed regression of such an alteration. The cumulative incidence for HPV was 10.34% (3/29). The incidence rate was 4.23% (3/71), which is equal to 4.23 new cases of HPV infection per 100 people, per year of follow-up. In conclusion, the present work shows a high frequency of infection by high-risk HPV, with predominance of HPV18 and 16 and in general for multiple infections. Colposcopy was better predictor than the Pap smear for infection. The follow-up study revealed a low percentage of persistent infection, and a high frequency of negativity for viral infection, high regression of cytological and colposcopic lesions, a low cumulative and incidence rate similar to that reported by other Latin American countries and higher than the European countries.

Presence of human papillomavirus in breast cancer and its association with prognostic factors.

Breast cancer accounts for 16% of all female cancers worldwide, and in Venezuela, it is the leading cause of death among women. Recently, the presence of high-risk genotypes of human papillomavirus (HPV) has been demonstrated in breast cancer and has been associated with histopathological features of the tumours. In Venezuela, there is no study which determines the association between the presence of HPV in breast cancer and the histopathological features. The aim of this investigation is to evaluate the presence of HPV in the different types of breast cancer, according to their molecular classification, based on the expression of ER, PR, HER2 and Ki67. With this purpose in mind, we assessed the presence of the HPV genome in 24 breast cancer samples diagnosed with infiltrating ductal carcinoma, ductal carcinoma in situ (DCIS) and lobular carcinoma, by the INNO-LIPA genotyping extra kit and the evaluation of the markers ER, PR, HER2, and Ki67 by immunohistochemistry. The viral genome was found in 41.67% of the total number of samples, 51 being the most frequent genotype with 30.77%, followed by types 18 and 33, with 23.08%, respectively. Most tumours were found in the group of luminal A, with a low range of Ki67 expression. The presence of HPV in breast tumours could affect their growth pattern and metastatic power.

Posible transmisión vertical de virus de papiloma humano en niños cuyas madres presentan antecedentes de infección viral

Objetivo: Detección y tipificación de virus de papiloma humano en muestras de niños cuyas madres tienen historia asociada al virus. Métodos: Estudio transversal y descriptivo. Se procesaron 66 hisopados perianales de niños con un promedio de edad de 18 meses atendidos en el Servicio de Dermatología del Hospital Universitario de Caracas. La detección viral se realizó mediante PCR con iniciadores genéricos MY11 y MY09; la tipificación de las muestras positivas en la detección se llevó a cabo mediante PCR múltiple. Resultados: Se obtuvo un porcentaje de positividad de 54,5 % y se identificó VPH de bajo riesgo oncogénico en 47,2 %, ADN viral de alto riesgo en 13,88 % e infecciones mixtas con tipos de alto y bajo riesgo oncogénico en 5,55 % de las muestras positivas. El 33,33 % de estas muestras no pudo ser tipificado con la metodología utilizada. Conclusiones: Aunque no se tienen datos del tipo viral en las madres para hacer comparaciones con los tipos identificados en los niños, estos resultados ponen en evidencia la posible transmisión vertical del VPH, considerando los antecedentes maternos, la corta edad de los niños y que la condición de abuso sexual fue descartada. Es recomendable hacer el seguimiento de la población estudiada con la incorporación de la evaluación de muestras obtenidas de los padres.

Objective: Detection and typing of Human Papillomavirus in samples of children whose mothers have a history associated with the virus. Methods: Transversal and descriptive study. Perianal swabs of 66 children with an average age of 18 months treated in the Service of Dermatology, University Hospital of Caracas, were processed. Viral detection was performed by PCR with MY11 and MY09 generic primers; typing of positive samples was performed by multiplex PCR. Results: A positive percentage of 54.5 % was obtained and low oncogenic risk HPV identified in 47.2 %, high-risk viral DNA in 13.88 % and mixed infections with types of high and low oncogenic risk in 5.55 % of the positive samples. 33.33 % of these samples could not be typed with the used methodology. Conclusions: Although the viral type is unknown in mothers to make comparisons with the types identified in children, these results highlight the possible vertical transmission of HPV, considering maternal history, the young age of the children and that the condition of sexual abuse was ruled out. It is recommended to monitor the studied population including the evaluation of samples obtained from the parents.

Human papillomavirus detection in head and neck squamous cell carcinoma.

INTRODUCTION: Human Papillomavirus (HPV) has been associated with benign and malignant lesions in different epitheliums. The relationship between specific genotypes of high-risk HPV and some human cancers is well established. The aim of this work was to detect the HPV genotypes present in head and neck squamous cell carcinoma (HNSCC). METHODS: We evaluated 71 samples of patients with histopathological diagnosis of HNSCC. The DNA extraction was conducted with the QIAGEN commercial kit. HPV detection and genotyping were performed by reverse hybridisation (INNO-LiPA) following the commercial specifications. RESULTS: The mean age of the patients evaluated was 60.7 ± 13.11 years. The distribution of the lesions included 25 (35.20%) cases of squamous cell carcinoma (SCC) of the oral cavity, 23 (32.39%) of larynx, 16 (22.50%) of the oropharynx, 4 (5.63%) of paranasal sinus, and 2 (2. 80%) cases of SCC of the nostril. Of the patients, 78.9% were males, and of these 76% were tobacco users and 67.6% were alcohol consumers. The viral DNA was detected in 67.6% of the samples. The oral cavity and the larynx were the highest HPV-positivity sites with 35.40% and 29.10% respectively. The most frequent genotype was 16 as single infection (18.70%), or in combination with another HPV types. In the oral cavity and larynx the genotypes 16 or the combination 6 and 51 were present in 11.76% and 14.28%, respectively; and in the oropharynx the most frequent genotype was 16 in 22.50% of the cases, and in the paranasal sinus 50% presented infection with HPV-6. We observed that tumours with most advanced size and stage presented greater HPV positivity. CONCLUSIONS: This study shows a high percentage of HPV positivity in SCC is mainly associated with high-risk HPV. It is important to highlight that viral infection, especially HPV-16, could be a risk factor in HNSCC progression.

Expression of COX-2 and bcl-2 in oral lichen planus lesions and lichenoid reactions.

UNLABELLED: Oral lichen planus and lichenoid reactions are autoimmune type inflammatory conditions of the oral mucosa with similar clinical and histological characteristics. Recent data suggest that oral lichenoid reactions (OLR) present a greater percentage of malignant transformation than oral lichen planus (OLP). OBJECTIVE: To compare the expression of bcl-2 and COX-2 in OLP and OLR. METHODS: The study population consisted of 65 cases; 34 cases diagnosed as OLR and 31 as OLP. A retrospective study was done, and bcl-2 and COX-2 expression was semiquantitatively analysed. RESULTS: Fifty-three per cent (18/34) of the ORL samples tested positive for COX-2, whereas in the OLP group, 81% of the samples (25/31) immunostained positive for COX-2. The Fisher's exact test for the expression of COX-2 revealed that there are significant differences between the two groups, P = 0.035. With respect to the expression of the bcl-2 protein, 76% (26/34) of the samples were positive in OLR, while 97% (30/31) were positive in the group with OLP. The Fisher's exact test for the expression of bcl-2 revealed that there are significant statistical differences between the two groups, P = 0.028. CONCLUSIONS: The expression of bcl-2 and COX-2 was more commonly expressed in OLP when compared with OLR.

Detección del virus de papiloma humano en muestras de pacientes con ectropión cervical

Objetivo: Detección subclínica del virus de papiloma humano en muestras de pacientes diagnosticadas con ectropión cervical, para evaluar la prevalencia de la infección viral asociada a dicha condición. Métodos: El ADN fue extraído utilizando solventes orgánicos (fenol/cloroformo-alcohol isoamílico). La detección del virus de papiloma humano se realizó mediante PCR con iniciadores genéricos MY09/MY11 y para la tipificación de las muestras positivas se utilizó un kit comercial de PCR múltiple. Ambiente: Laboratorio de Genética Molecular-Instituto de Oncología y Hematología. Resultados: Se obtuvo una positividad de 26 % (13/50 muestras analizadas) para la presencia de ADN del virus de papiloma humano. De las muestras positivas, 38,45 % resultó virus de papiloma humano de alto riesgo oncogénico (tipo 16 o 18), mientras que otro 38,45 % correspondió a virus de papiloma humano de bajo riesgo (tipos 6, 11 o infección mixta 6/11) y 23,07 % no pudo ser tipificado con la metodología utilizada. Conclusión: Aunque este resultado no fue estadísticamente significativo, señala la necesidad de mayor seguimiento clínico de las pacientes positivas, especialmente aquellos casos correspondientes a virus de papiloma humano de alto riesgo oncogénico, ya que presentan mayor probabilidad de desarrollar cáncer cervical.

Objective: In this study was conducted subclinical human papillomavirus detection in samples from patients diagnosed with cervical ectropion to assess the prevalence of human papillomavirus infection associated with this condition. Methods: DNA was extracted with organic solvents (phenol /chloroform- isoamylic). Human papillomavirus detection was performed by PCR with generic primers MY09 and MY11 and the viral typing was performed using a commercial MPCR kit. Setting: Laboratorio de Genetica Molecular-Instituto de Oncologia y Hematologia; Results: The results showed that 26 % of the evaluated sample (13/50) was positive for the presence of human papillomavirus genome. Viral typing test identified high- oncogenic risk human papillomavirus (types 16 or 18) in 38.45.% of the positives cases. Likewise, 38.45 % was low oncogenic risk (types 6, 11 or mixed infection with 6/11 human papillomavirus) and 23.07 % could not be typified with the used methodology. Conclusions: Although this result is not statistically significant, the virus latent presence highlights the need for greater medical surveillance for positive patients, especially in cases where detected 16 and 18 high-risk oncogenic human papillomavirus types, because they may have increased risk of cervical cancer.

[MMP-3 and MMP-8 levels in patients with chronic periodontitis before and after nonsurgical periodontal therapy]. / Niveles de MMP-3 y MMP-8 en pacientes con periodontitis crónica antes y después del tratamiento periodontal no quirúrgico.

Immune-inflammatory processes are trigged in chronic periodontitis (CP), where matrix metalloproteinases (MMPs) are released and involved in the degradation of the extracellular matrix components that can be detected in the gingival crevicular fluid (GCF). The purpose of the study was to determine the levels of MMP-3 and MMP-8 in GCF, before and after nonsurgical periodontal treatment (NSPT), to evaluate disease activity and therapy response. Eleven patients with PC and eleven healthy controls were selected. Clinical measurements to evaluate gingival index (GI), plaque index (PI), probing depth (PD) and clinical attachment loss (CAL) were made in all the teeth of each individual and in six sites per tooth. GCF samples were taken from one tooth per quadrant, with a pocket depth > or =4 mm and a clinical attachment loss > or =5 mm, and the levels of MMP-3 and MMP-8 measured using an ELISA test. Statistically significant differences in clinical parameters were observed (p < 0.05) between patients with CP and control groups before the periodontal treatment, with significant decrease in all indexes after the NSPT. The initial concentrations of MMP-3 and MMP-8 were significantly higher than those obtained after the NSPT and in the control group, without observing a correlation between the clinical parameters and the levels of MMPs. Increased levels of MMP-3 and MMP-8 in the GCF of patients with PC declined significantly after NSPT, and the difference between the levels in healthy individuals and patients, suggests the important participation of these MMPs in tissue destruction in PC disease..

Niveles de MMP-3 y MMP-8 en pacientes con periodontitis crónica antes y después del tratamiento periodontal no quirúrgico / MMP-3 and MMP-8 levels in patients with chronic periodontitis before and after nonsurgical periodontal therapy

En la periodontitis crónica (PC) se desencadenan procesos inmunoinflamatorios, donde se liberan metaloproteinasas de la matriz (MMPs), enzimas involucradas en la degradación de la matriz extracelular, las cuales pueden ser detectadas en el fluido gingival crevicular (FGC). El propósito del estudio fue determinar los niveles de MMP-3 y MMP-8 en FGC, antes y después del tratamiento periodontal no quirúrgico (TPNQ) para evaluar actividad de la enfermedad y respuesta terapéutica. Once pacientes con periodontitis crónica y 11 controles sanos fueron seleccionados. Se evaluaron los parámetros clínicos: índice gingival, índice de placa, profundidad al sondaje y pérdida de inserción; en todos los dientes de cada individuo y en seis sitios por diente. Muestras de FGC fueron tomadas de un diente por cuadrante, con profundidad de saco ≥ 4mm y pérdida de inserción ≥ 5 mm, los niveles de MMP-3 y MMP-8 fueron determinados por ELISA. Diferencias estadísticamente significativas fueron observadas entre los parámetros clínicos del grupo control y los pacientes con PC antes del tratamiento, registrándose posterior al TPNQ disminución significativa de todos los índices. Las concentraciones iniciales de MMP-3 y 8 en el grupo con PC fueron significativamente mayores a las obtenidas luego del TPNQ y en el grupo control, sin observar correlación entre parámetros clínicos y niveles de MMPs. La disminución significativa de los valores de MMP-3 y 8 en FGC de los pacientes con PC, posterior al TPNQ, indican la participación importante de estas enzimas en la degradación del tejido, y la efectividad del tratamiento periodontal para su control.

Immune-inflammatory processes are trigged in chronic periodontitis (CP), where matrix metalloproteinases (MMPs) are released and involved in the degradation of the extracellular matrix components that can be detected in the gingival crevicular fluid (GCF). The purpose of the study was to determine the levels of MMP-3 and MMP-8 in GCF, before and after nonsurgical periodontal treatment (NSPT), to evaluate disease activity and therapy response. Eleven patients with PC and eleven healthy controls were selected. Clinical measurements to evaluate gingival index (GI), plaque index (PI), probing depth (PD) and clinical attachment loss (CAL) were made in all the teeth of each individual and in six sites per tooth. GCF samples were taken from one tooth per quadrant, with a pocket depth ≥ 4 mm and a clinical attachment loss ≥ 5 mm, and the levels of MMP-3 and MMP-8 measured using an ELISA test. Statistically significant differences in clinical parameters were observed (p < 0.05) between patients with CP and control groups before the periodontal treatment, with significant decrease in all indexes after the NSPT. The initial concentrations of MMP-3 and MMP-8 were significantly higher than those obtained after the NSPT and in the control group, without observing a correlation between the clinical parameters and the levels of MMPs. Increased levels of MMP-3 and MMP-8 in the GCF of patients with PC declined significantly after NSPT, and the difference between the levels in healthy individuals and patients, suggests the important participation of these MMPs in tissue destruction in PC disease.

Extracción de ADN de muestras incluidas en parafina sin el uso de xilol para la detección y tipificación de VPH / DNA extraction from paraffin embedded samples without using xylene for HPV detection and typing

En el presente trabajo se llevó a cabo la extracción de ADN de biopsias cervicales con informe citológico de lesión intraepitelial cervical, incluidas en bloques de parafina, utilizando el “QIAamp DNA mini kit” (QIAGEN, Alemania) y la detección del Virus de Papiloma Humano (VPH). En el procedimiento original se eliminó la incubación en xilol, reduciendo los lavados del material biológico; la calidad de los ADNs extraídos fue probada mediante PCR para la detección del gen de la β-globina y del VPH, obteniéndose 84,3% y 28,1% de positividad, respectivamente. Estos resultados fueron comparables a los de otros trabajos similares y evidencian la obtención de ADN celular amplificable por PCR a partir de muestras parafinadas, que pudo ser utilizado eficientemente en procedimientos moleculares, eliminando la toxicidad del xilol y disminuyendo la manipulación del material biológico al reducir los lavados.

In the present study, DNA from cervical biopsies with a cytological report of intraepithelial cervical lesion and embedded in paraffin blocks was extracted using the “QIAamp DNA mini kit” (QIAGEN, Germany), and the Human Papyloma Virus (HPV) was detected. In the original procedure, the xylene incubation was eliminated, therefore reducing the washes of the biological material; the quality of the DNA extracted was tested by PCR for detecting the β-globine gene and HPV, with 84.3% and 28.1% positivity, respectively. These results were comparable to those of other similar studies, and they demonstrate that it is possible to obtain PCR amplifiable cellular DNA from paraffin embedded samples which can be used efficiently for molecular procedures, eliminating xylene toxicity, and decreasing the manipulation of the biological material by reducing washes.

Cambios moleculares en los márgenes de resección en el carcinoma de células escamosas de cavidad oral / Molecular changes in the resections borders in squamous cell cancer of oral cavity

Evaluación molecular de márgenes de resección en pacientes con carcinoma de células escamosas de cavidad oral sometidos a cirugía. 16 pacientes con carcinoma escamoso de cavidad oral, en cualquiera de sus localizaciones, sin tratamientos previos, intervenidos quirúrgicamente en el 2011. La pieza operatoria fue procesada por anatomía patológica a través del método tradicional, realizándose cortes adicionales que incluían: tumor y 0,5 cm de margen no tumoral. Se realizó hematoxilina-eosina y complementó con inmunomarcaje para p53, PCNA, Ki-67, factor de crecimiento epidérmico y receptor de crecimiento endotelial vascular. De los 16 pacientes en estudio la mayoría eran del género masculino, la edad promedio fue cercana a los 60 años, la mayoría eran pacientes consumidores de tabaco y alcohol. La lengua fue la localización más frecuente y los tumores se encontraban en un estadio avanzado (estadio III y IV). Estudio molecular: todos los marcadores evaluados se encontraban positivos en los márgenes de resección en el 93,75% de los pacientes. Los marcadores de proliferación celular como el PCNA y Ki-67 así como el p-53 se encontraban positivos entre 1,5 cm a 2 cm del tumor con un marcaje intenso. Por el contrario, el factor de crecimiento epidérmico el receptor de crecimiento endotelial vascular se encontraban positivos hasta 1,5 cm pero con menor intensidad. En el cáncer oral podemos observar con frecuencia cambios moleculares en el tejido aparentemente sano que rodea el tumor hasta por lo menos 15 mm.

The molecular evaluation of resection margins in patients with squamous cell carcinoma of oral cavity who underwent surgery. Field of cancerization concept. We included 16 patients with oral cavity squamous cell carcinoma in any of their locations,without pre treatment, surgically treated in our hospital in the 2011 year. The surgical specimen was processed by the pathology department of our institution, through the traditional method, additional sectioned including the tumor and at least 0.5 cm margin non tumorigenic. Study was performed hematoxylin eosin and was supplemented with immunostaining for p53, PCNA, Ki-67, epidermal growth factor receptor and vascular endothelial growth factor receptor. The most important features of the 16 patients studied were: The majorities were male, the average age was around 60 years old; most of them were tobacco and alcohol consumers. The tongue was the most frequent location and most of the tumors were in an advanced stage (stage III y IV). In molecular evaluation all the markers were positive in the resection margins in 93.75% of all patients. The cell proliferation markers suchas PCNA and Ki-67 and the p-53 were positive 1.5 cm to2 cm tumor with intense staining. Conversely, epidermal receptor grow factors and vascular endothelial grow factor receptor were positive up to 1.5 cm but with less intensity. In oral cancer can often observe molecular changes in the apparently healthy tissue surrounding the tumor to at least 15 mm.