The aim of the present study was to gather information regarding the molecular epidemiology of Human papillomavirus (HPV) and related risk factors in a group of women with low- and high-grade cervical lesions and cancer from the coastal region of Ecuador. In addition, we studied the evolution of HPV variants from the most prevalent types and provided a temporal framework for their emergence, which may help to trace the source of dissemination within the region. We analyzed 166 samples, including 57 CIN1, 95 CIN2/3 and 14 cancer cases. HPV detection and typing was done by PCR-sequencing (MY09/MY11). HPV variants and estimation of the time to most recent common ancestor (tMRCA) was assessed through phylogeny and coalescence analysis. HPV DNA was found in 54.4% of CIN1, 74.7% of CIN2/3 and 78.6% of cancer samples. HPV16 (38.9%) and HPV58 (19.5%) were the most prevalent types. Risk factors for the development of cervical lesions/cancer were the following: three or more pregnancies (OR = 4.3), HPV infection (OR = 3.7 for high-risk types; OR = 3.5 for HPV16), among others. With regard to HPV evolution, HPV16 isolates belonged to lineages A (69%) and D (31%) whereas HPV58 isolates belonged only to lineage A. The period of emergence of HPV16 was in association with human populations (tMRCA = 91 052 years for HPV16A and 27000 years for HPV16D), whereas HPV58A preceded Homo sapiens evolution (322 257 years). This study provides novel data on HPV epidemiology and evolution in Ecuador, which will be fundamental in the vaccine era.

El objetivo del presente estudio fue aportar información sobre la epidemiología molecular del virus del papiloma humano (human papillomavirus [HPV]) y los factores de riesgo asociados al desarrollo de lesiones cervicales y cáncer en mujeres de la costa del Ecuador. Además, se estudiaron la evolución de las variantes de los HPV más prevalentes y el marco temporal de su emergencia, para ayudar a rastrear la fuente de dispersión en la región. Se analizaron 166 muestras, incluyendo 57 y 95 casos de neoplasia intraepitelial cervical tipo 1 (CIN1) y tipo 2/3 (CIN2/3), respectivamente, y 14 de casos de cáncer. La detección/tipificación de HPV se realizó por PCR-secuenciación (MY09/MY11). La caracterización de variantes y la datación del ancestro común más reciente (tMRCA) se realizaron mediante filogenia y coalescencia. Se encontró ADN de HPV en el 54,4% de las muestras de CIN1, el 74,7% de las muestras de CIN2/3 y el 78,6% de las muestras de cáncer. Los tipos HPV16 (38,9%) y HPV58 (19,5%) fueron los más frecuentes. Los factores de riesgo para el desarrollo de lesiones cervicales/cáncer fueron 3 o más embarazos (OR = 4,3) e infección por HPV (O = 3,7 para HPV de alto riesgo, OR = 3,5 para HPV16), entre otros. En cuanto a la evolución viral, los aislados del HPV16 pertenecían a los linajes A (69%) y D (31%), mientras que los aislados del HPV58 pertenecían únicamente al linaje A. El período de emergencia del HPV16 estuvo asociado a poblaciones humanas (tMRCA = 91.052 años para HPV16Ay 27.000 para HPV16D), mientras que el del HPV58A precedió a la evolución de Homo sapiens (322.257 años). Este estudio proporciona datos novedosos sobre la epidemiología y la evolución del HPV en Ecuador, los cuales serán fundamentales en la era de la vacuna.

The association between colorectal cancer and human papillomavirus (HPV) infection is still unproven. The aim of this study was to investigate the presence of high-risk HPV (HR-HPV) DNA in colorectal tissues from Cuban patients. A total of 63 colorectal formalin-fixed paraffin-embedded tissues were studied (24 adenocarcinoma, 18 adenoma, and 21 colorectal tissues classified as benign colitis). DNA from colorectal samples was analysed by quantitative real-time polymerase chain reaction to detect the most clinically relevant high HR-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58). Associations between histologic findings and other risk factors were also analysed. Overall, HPV DNA was detected in 23.8% (15/63) of the samples studied. Viral infections were detected in 41.7% of adenocarcinoma (10/24) and 27.7% of adenoma cases (5/18). HPV DNA was not found in any of the negative cases. An association between histological diagnosis of adenocarcinoma and HPV infection was observed (odd ratio = 4.85, 95% confidence interval = 1.40-16.80, p = 0.009). The only genotypes identified were HPV 16 and 33. Viral loads were higher in adenocarcinoma, and these cases were associated with HPV 16. This study provides molecular evidence of HR-HPV infection in colorectal adenocarcinoma tissues from Cuban patients.

The objective of the present study is to standardize a real-time based polymerase chain reaction system in order to detect and quantify 7 high risk human papillomavirus in different clinical samples from patients suspected of this type of infection. The validation of a 5´ exonuclease fluorescent probe real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, and 58) which account for over 87% of cervical carcinomas world-wide was carried out. Simultaneous PCR reactions are required to detect the designated HPV types. Specificity tests for each HPV type and other DNA viruses were performed. Standard external curve constructions were achieved, which allow determining the number of target DNA copies in the previously HPV tested samples. HPV 16 and 18 standard curves were obtained from purified genomic DNA of SiHa and HeLa cell lines, respectively. The pattern curves were constructed on the basis of each of the resulting standard DNA, which showed good linear correlation (r = -0, 99) and low error values. The lower detection limit was 10 copies for both HPV 16 and 18. No cross reactions between HPV types and other DNA viruses were observed. Real-Time Polymerase Chain Reaction system, standardized for 7 HPV types, proved to be a rapid, specific and highly sensitive system for better diagnosis and follow-up of patients with high grade intraepithelial lesions. In addition, this assay will allow the development of coming researches in relation with the prevalence and pathogenesis of human papillomavirus infections in different samples from Cuban patients.

El objetivo de este trabajo fue normalizar e implementar un sistema de reacción en cadena de la polimerasa en tiempo real, para determinar la carga viral de 7 genotipos de papilomavirus humano (PVH) de alto riesgo oncogénico. Se evaluó la especificidad del sistema y se construyeron las curvas estándar para PVH 16 y 18, que se emplearon para la cuantificación de ADN viral en diferentes muestras de pacientes identificados como positivos a PVH, mediante reacción en cadena de la polimerasa (RCP) cualitativa y secuenciación nucleotídica. Se obtuvieron dos curvas estándar para PVH 16 y 18, a partir del ADN genómico de las líneas celulares SiHa y HeLa, las que mostraron una buena correlación lineal ( r = -0,99) y valores bajos de error. El límite inferior de detección a partir del ADN de las líneas celulares fue de hasta 10 copias para ambos genotipos. No se obtuvo reacción cruzada entre los diferentes tipos de PVH ni con otros virus ADN. La reacción en cadena de la polimerasa en tiempo real (RCP-TR) normalizada probó ser un sistema simple, rápido, específico y altamente sensible. Además, permitirá desarrollar investigaciones sobre la prevalencia de infección por PVH en Cuba, con vistas a la aplicación de las vacunas que se encuentran disponibles en el mercado internacional, así como la evaluación de otros candidatos vacunales diseñados en el futuro.

INTRODUCTION: Herpesvirus infections are prevalent worldwide, but most run their course asymptomatically. Clinical presentations in symptomatic cases vary widely and include febrile and mononucleosis-like syndromes. In immunocompromised patients, herpetic infection can be lethal and routine laboratory tests are of little use. Use of novel techniques may provide important improvements in diagnosis and treatment of these patients. OBJECTIVE: Investigate association between different herpesviruses and the etiology of mononucleosis and febrile syndromes in Cuban immunocompetent and immunocompromised patients. METHODS: The study used multiplex nested polymerase chain reaction, enabling simultaneous detection of six herpesviruses-cytomegalovirus, herpes simplex (1 and 2), Epstein-Barr, varicella-zoster and human herpesvirus 6-to study 1157 samples (770 urine and 387 serum samples) from 1140 patients with mononucleosis-like syndrome or febrile syndrome, classified according to history of immunosuppressive disease. Samples were analyzed at the Laboratory for Sexually Transmitted Diseases (Virology) of the Pedro Kourí Tropical Medicine Institute from January 2006 through December 2009. SPSS statistical package was used and incidence rates calculated. RESULTS: Of samples studied, 20.1% were positive for some herpesvirus. Higher risk of developing active herpesvirus infections was detected in samples from immunocompromised patients with febrile syndrome compared to those of immunocompetent ones (OR 2.02, CI 1.20-3.42, p=0.007). Cytomegalovirus was the most frequently found herpesvirus in both mononucleosis-like syndrome (60.4%) and febrile syndrome (63.6%) and in both children (69.2%) and adults (55.2%), followed by Epstein-Barr virus. Cytomegalovirus was detected in 68.9% of positive urine samples and in just 47.2% of serum samples. CONCLUSIONS: This is the first Cuban study demonstrating the pathogenic role of herpesviruses, particularly cytomegalovirus, in patients with febrile or mononucleosis-like syndrome, in both immunocompetent and immunocompromised patients. Results highlight the importance of including molecular diagnosis of the herpesvirus family in investigating mononucleosis and febrile syndromes of unknown etiology and demonstrate that etiologic diagnosis would not have been feasible in many cases without the use of this diagnostic tool.

OBJECTIVE: To obtain recombinant clones expressing different dengue virus 2 proteins in an expression vector of eukaryote cells. METHODS: Cloning of prM genes, E envelope and 65 amino-acids (aa) of dengue virus serotype 2 NS1 proteins (Nueva Guinea strain) in an expression vector of pcDNA eukaryote cells (3.1). The prM/E/NS1 zone and the truncated prM/E zone at 100 aa genes were obtained by polymerase chain reaction (PCR). Possible recombinant clones were detected using PCR, enzyme restriction analysis and nucleotide sequencing. Transfection of the CHO cell line with each recombinant plasmid was performed. Indirect immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) determined the transient expression of cloned genes. RESULTS: Bands of 2 202 and 1 600 base pairs (bp) respectively were obtained. Twenty possible recombinant colonies were studied, 7 of them were pr-E-NSI-positive and 5 truncated prM/E-positive. Fluorescent cells emerged 48 hours after being transfected in addition to positive PCR, all of which indicated that the studied proteins were present in transfected cells. CONCLUSIONS: The used vector proved to be efficient for cloning and expression of the selected proteins; therefore, the obtained genetic constructions could be evaluated in animals as likely vaccinal candidates for a dengue virus DNA vaccine.

OBJETIVO: obtener clones recombinantes que expresen diferentes proteínas del virus dengue 2 en un vector de expresión en células eucariotas. MÉTODOS: se realizó el clonaje de los genes prM, envoltura (E) y 65 aminoácidos (aa) de la proteína NS1 del virus dengue serotipo 2 (cepa Nueva Guinea C) en un vector de expresión en células eucariotas pcDNA (3.1). La obtención de los genes correspondientes a la zona prM/E/NS1 y prM/E truncada en 100 aa se realizó mediante reacción en cadena de la polimerasa (RCP). La detección de los posibles clones recombinantes se llevó a cabo mediante las técnicas de RCP, análisis de restricción enzimática y secuenciación nucleotídica. Se realizó la transfección de la línea celular CHO con cada plásmido recombinante. Para determinar la expresión transciente de los genes clonados se empleó la técnica de inmunofluorescencia indirecta (IFI) y trascripción reversa-RCP (TR-RCP). RESULTADOS: se obtuvieron bandas de 2 202 y 1 600 pares de bases (pb), respectivamente. Se estudiaron 20 posibles colonias recombinantes, de las cuales, 7 resultaron positivas para prM-E-NS1 y 5 para prM/E truncada. Se obtuvieron células fluorescentes 48 h después de transfectadas, además una RCP positiva a ese mismo tiempo, lo que indicó la presencia de las proteínas en las células transfectadas. CONCLUSIONES: el vector empleado fue eficiente para el clonaje y la expresión de las proteínas seleccionadas, por lo que las construcciones genéticas obtenidas pudieran ser evaluadas en animales como posibles candidatos vacunales para la obtención de una vacuna de ADN contra el dengue.

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups.

The polymerase chain reaction technique was applied to detect sequences of human Papillomavirus (HPV) by controls of cellular lines of cervical cancer and of tissues obtained through biopsy with a HPV-positive clinical diagnosis. A set of consensus oligonucleotides, which are complementary to a highly conserved region within the open reading frame El of the viral genome of HPV affecting the cervical mucosa, was used. With these primers it was possible to amplify DNA sequences corresponding to HPV 6 and 11, considered in the low risk group, and to HPV 16, 18, 31 and 33, included in the high risk group. The study of the sensitivity of the amplification technique showed a level of detection of 3,5 viral particles per each cellular diploid genome.

Se aplicó la técnica de reacción en cadena de la polimerasa para la detección de secuencias de Papillomavirus humano (PVH) mediante controles de líneas celulares de cáncer cervical y tejidos obtenidos por biopsia con diagnóstico clínico positivo a PVH. Se utilizó un juego de oligonucleótidos consenso, que son complementarios a una región altamente conservada dentro del marco de lectura abierta E1 del genoma viral de los PVH que afectan la mucosa cervical. Con este juego de cebadores fue posible amplificar secuencias de ácido desoxirribonucleico (ADN) correspondientes a los PVH 6 y 11, considerados dentro del grupo de bajo riesgo y de los PVH 16, 18, 31 y 33 comprendidos en el grupo de alto riego. El estudio de la sensibilidad de la técnica de amplificación arrojó como resultado un nivel de detección de 3,5 partículas virales por cada genoma diploide celular.

The immune response of a group of patients with epidemical neuropathy and of controls was studied by the immunoblotting technique against proteins of the Coxsackie virus and the proteins of slow effect isolated in our laboratory. 13 sera of patients with epidemical neuropathy and 9 sera of controls were studied. Of the 13 sera studied, 8 (61.5 %) recognized protein VPI and 2 sera (15.3 %) protein VP0 of the strain 47.93. Of the 9 controls studied, 4 (44.4 %) recognized protein VPI and 3 (33.3 %) protein VP0 only. With the antigen prepared from the slow effect strain it was obtained a specific signal in 5 (38.5 %) sera of patients and in 2 sera (22.5 %) of controls. It should be stressed that in this last case the protein observed had a molecular weight of 41 300 D, and that its size was smaller than that of the preceeding protein detected against the strain 47.93 was of 45 000 D.

Se estudió la respuesta inmune de un grupo de pacientes con neuropatía epidémica y de individuos controles mediante la técnica de inmunoblotting frente a las proteínas del virus Coxsackie y a las proteínas de la cepa de efecto lento aislada en nuestro laboratorio. Se estudiaron 13 sueros de pacientes con neuropatía epidémica y 9 sueros controles. De los 13 sueros estudiados, 8 (61,5 %) reconocieron a la proteína VP1 y 2 sueros (15,3 %) a la proteína VP0 de la cepa 47/93. De los 9 controles estudiados, 4 (44,4 %) reconocieron la proteína VP1 y 3 sueros (33,3 %) la proteína VP0 solamente. Con el antígeno preparado a partir de la cepa de efecto lento, en 5 (38,5 %) sueros de pacientes y 2 sueros (22,5 %) de controles se obtuvo una señal específica. Es de destacar en este último caso que la proteína observada tenía un peso molecular de 41 300 D, era de menor talla que la proteína precursora detectada frente a la cepa 47/93, que fue de 45 000 D.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed