Determinación de la carga viral de HPV-16 en muestras cervicales de mujeres con lesiones de alto y bajo grado

Tesis de Magíster en Bioquímica, Área de Especialización en Toxicología y Diagnóstico MolecularLa infección de transmisión sexual más común a nivel mundial, tanto en hombres como en mujeres es la producida por el Virus Papiloma Humano (HPV). Los diferentes HPV que afectan la zona anogenital se dividen en dos grupos: bajo y alto riesgo carcinogénico. La infección con HPV-AR ha sido detectada en más del 99,7% de los casos de cáncer cérvico-uterino. Este cáncer ocupa el sexto lugar dentro de las neoplasias malignas femeninas, produciendo alrededor de 600 muertes al año en Chile. En la actualidad no se dispone de biomarcadores adecuados para estimar el pronóstico de la infección viral en los pacientes. Por lo anterior, en esta investigación se evaluó la utilidad de la medición de la carga viral como predictor de progreso de la infección en mujeres infectadas con HPV-16. La hipótesis planteada fue que las pacientes infectadas con HPV-16 y con lesiones cervicales de alto grado presentan una mayor carga viral cuantificada a través de la amplificación de segmentos de los genes L1 y E7 en comparación con aquellas infectadas y con lesiones de bajo grado. El estudio se realizó con muestras cervicales de 60 mujeres controladas ginecológicamente en el Hospital San Juan de Dios. Todas las pacientes presentaban un examen citológico alterado: 40 estaban infectadas con HPV-16 y 20 fueron negativas a la presencia del virus. En todas las muestras se cuantificó la carga de DNA viral mediante PCR en tiempo real. El 28,3%, 18,3% y 53,3% de las pacientes presentaron lesiones NIE I, NIE II y NIE III, respectivamente. En las pacientes infectadas con HPV-16 se determinó que la mediana de la carga viral del ensayo utilizando L1 en LIBG es 1,4 veces mayor en comparación con la obtenida en las LIAG. La carga viral obtenida al amplificar un segmento del gen E7 se encontró que es 1,5 veces mayor en LIBG en comparación con LIAG. Además, los niveles de los amplicones de ambos genes en los diferentes grados de lesión mostraron una correlación lineal positiva. Estos resultados muestran que en LIBG existe una mayor carga viral en comparación con aquellas con lesiones LIAGThe most common worldwide sexually transmitted disease in both men and women is Human Papillomavirus (HPV). Different HPV infect the anogenital area and they are divided into two groups: low and high carcinogenic risk. High risk HPV have been found in 99,7% of cervical cancer cases. This cancer ranks sixth in female malignancies, causing about 600 deaths a year in Chile. Currently there are no good biomarkers to predict the viral infection in patients. Therefore, this study evaluated the usefulness of viral load as a predictor of disease progression in HPV-16-infected women. The hypothesis was that patients infected with HPV-16 and with high-grade cervical lesions have a higher viral load quantified through the amplification of segments of L1 and E7 genes compared to those infected with low-grade lesions. We studied vaginal samples of 60 women attending a gynecological control at San Juan de Dios Hospital. All patients had an altered cytology testing: 40 of them were HPV-16 infected and 20 non infected. Viral load of L1 and E7 genes were quantified in all samples by real time PCR. The overall detection of cervical intraepithelial neoplasia NIEI, NIEII and NIEIII was 28,3%, 18,3% and 53,3%, respectively. In LIBG patients infected with HPV-16 it was determined that the median viral load assay using L1 is 1,4 times higher compared with that obtained in LIAG. The viral load obtained by amplifying a segment of the E7 gene was found to be 1,5 times higher in patients with LIBG than LIAG. Furthermore, levels of L1 and E7 genes show a positive lineal correlation in all cervical lesions. These results indicate that lesions with low severity have higher viral load than high severit

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy control

Prevalence of human papillomavirus infection among women presenting for cervical cancer screening in Chile, 2014–2015

Cervical cancer is the fourth most common malignancy in women worldwide. In Chile, cervical cancer is the second leading cause of death among women of reproductive age, causing more than 600 deaths annually. This study was carried out to determine the burden and confirm the predominant human papillomavirus (HPV) genotypes among women presenting for cervical cancer screening in public health services in Chile. Women aged 18-64 years residing in the north and central areas covered by six primary care centers of Santiago, Chile, were invited to participate from March 2014 to August 2015. Cervical swabs were examined both HPV genotyping by PCR and Reverse Line Blot, and cervical cytology by Pap testing. A total of 1738 women were included in this study: 11.1 % were HPV positive, 9.7 % were high-risk types positive, 3.2 % were low-risk types positive, 1.4 % were Pap positive and 0.9 % were positive by both tests. The four most predominant genotypes were 16, 66, 51 and 59, with prevalence of 2.8, 1.4, 1.2 and 1.2 %, respectively. Multiple HPV infections were detected among 3.8 % participants. Age-specific prevalence of HPV showed a peak in HPV infection at younger ages (aecurrency sign30 years), declining to a plateau in middle age. Among women with normal cytology, the 9.4 % were HPV positive, while 58.3 % of women with abnormal cytology were HPV positive. These findings show new epidemiological data confirming HPV 16 and 66 as the most predominant genotypes in Chile. These data are important for design successful strategies for prevention of cervical cancer in Chile

Prevalence of Human Papillomavirus infection among Chilean women from 2012 to 2016

Here, we evaluated the prevalence of Human Papillomavirus (HPV) in two groups of Chilean women. The first group consisted of 3235 womenaged 18-64 years attended in six primary care centers of Santiago. The second group consisted of 456 women 18-85 aged who consulted the Gynaecology Department of the Reference Hospital of Santiago. Samples were collected from October 2012 to February 2016. Cervical swabs were analyzed both HPV genotyping by PCR and Reverse Line Blot, and cervical cytology by Pap testing. Results showed a prevalence of 12.0% HPV positive, 10.3% high-risk (HR) HPV types positive, 3.9% low-risk (LR) HPV types positive, and 1.0% Pap positive in group 1. The most frequent types were 16, 66, and 59, with a prevalence of 3.0%, 1.6%, and 1.5%, respectively. The prevalence were 71.9% HPV positive, 67.3%HR-HPVtypes positive, 13.6% LR-HPV types positive, and 62.5% Pap positive in group 2. The most frequent types were 16, 31, and 58, with prevalence of 33.6%, 10.5%, and 7.0%, respectively. Among infected women with HPV: 7.6% were infected with HPV16 or HPV18, 3.0% with HPV31, HPV33 or HPV45, and 6.7% with any other HR-HPV. These findings show great difference in HPV prevalence and types between primary care and reference center, and provide useful epidemiological information to assess the impact of HPV vaccination in the future

Human T-Lymphotropic Virus Type 1 and 2 Seroprevalence among first-time blood donors in Chile, 2011-2013

Infection with human T-lymphotropic virus type 1/2 (HTLV-1/2) is a major health problem. HTLV-1/2 infection is endemic in Chile but representative donor prevalence data are lacking. Data on all blood donors in a large network of Chilean blood centers were examined during 2011-2013. Screening of HTLV-1/2 antibodies were measured by enzyme immunoassay (EIA) at all blood banks. Blood samples with anticoagulants from initially reactive blood donors were analyzed by serological confirmation tests (immunofluorescence or recombinant immunoblot) at the HTLV National Reference Laboratory of the Public Health Institute of Chile. Additionally, detection of HTLV-1 and HTLV-2 provirus in peripheral blood mononuclear cells (PBMCs) was performed in all blood donors as confirmatory test. Prevalence rates were calculated. Among 694,016 donors, 706 were seropositive for HTLV-1 (prevalence, 1.02 cases per 1,000; 95% confidence interval [CI], 0.94-1.09), and 97 were seropositive for HTLV-2 (prevalence, 0.14 cases per 1,000; 95%CI, 0.11-0.17). Prevalence of HTLV-1 differed considerably by region, from 0.51 to 1.69 per 1,000. Prevalence of HTLV-2 was similar across the country (0.12-0.16). HTLV-1 prevalence was associated with female sex, older age, and residence in the north of Chile. HTVL-2 prevalence was associated with older age. The HTLV-1 prevalence among Chilean blood donors was relatively high and could be reduced by improving donor recruitment and selection in high prevalence areas. Blood center data may contribute to surveillance for HTLV-1 and HTLV-2 infections

Inicio del brote de Influenza A (H1N1) pandémica en Chile: caracterización genética de los primeros casos detectados

Background: Following the announcement of the Influenza A(H1N1) pandemic by the World Health Organization in April 2009, a surveillance program was carried out in Chile to detect the introduction of the virus in the country and to monitor its propagation and impact. Aim: To describe the onset of the outbreak and the genetic characterization of the pandemic H1N1 influenza virus in the first detected cases in Chile. Material and Methods: Analysis of18 clinical samples coming from suspicious patients, received in a National Reference Laboratory. RNA reverse transcription and real time influenza gene DNA amplification was carried out in a 7500 Fast and Step One Real Time PCR Systems of Applied Biosystems and MxPro-Mx3000P thermocycler from Stratagene. Super Script III Platinum One-Step Quantitative RT-PCR was used. Results: The virus was first detected in three persons returning from the Dominican Republic via Panamá and a child from the east zone of Santiago. Genetic characterization of the virus showed that the child was infected by a different variant of the pandemic virus than the three persons returning from the Caribbean. Conclusions: The onset of the Influenza outbreak in Chile apparently carne from two different epidemiological groups. The spread of the virus detected in the voyagers was limited immediately However the virus of the fourth case was found in different regions of Chile