Análisis del impacto presupuestal en Colombia de la prueba de HPV con genotipificación comparada con la citología / Budget impact analysis of primary screening with the HPV test and genotyping against conventional cytology in Colombia

Introducción. La detección del virus del papiloma humano mediante la combinación de la prueba de HPV y otras técnicas como la citología, ha demostrado su eficacia en el diagnóstico y tratamiento oportuno de lesiones asociadas con el cáncer de cuello uterino. Objetivo. Estimar el impacto presupuestal de la estrategia de detección temprana del HPV mediante la prueba de genotipificación combinada con la citología en comparación con la citología convencional, en mujeres de 30 a 65 años participantes en el programa de tamizaje de cáncer de cuello uterino en una Entidad Administradora del Plan de Beneficios en salud (EAPB) en Colombia. Materiales y métodos. Utilizando un árbol de decisiones y un modelo de Markov, se estimaron las implicaciones clínicas y los costos directos anuales de dos ciclos de tamizaje, diagnóstico y tratamiento, en una cohorte de mujeres. Las prevalencias de los resultados clínicos y los costos se tomaron de la base de datos de una EAPB y la información de la progresión, persistencia y regresión de los estados de salud provinieron del estudio ATHENA. Resultados. El esquema de tamizaje con la prueba de HPV, la genotipificación y la citología resultó en un ahorro de costos comparado con la citología convencional. El costo promedio por ciclo de tamizaje con la prueba de HPV se estimó en COP $129'201.363 y con la citología en COP $186'309.952, es decir, un ahorro de COP $57'108.589 (30,7 %). Conclusión. La implementación de la estrategia de tamizaje evaluada sugiere que habría ahorros derivados de la detección temprana de los estados de salud asociados con el desarrollo de cáncer de cuello uterino.

Introduction: The detection of the human papillomavirus (HPV) through the combination of the HPV test and other techniques such as cytology has impacted the detection and timely treatment of lesions associated with cervical cancer. Objective: To estimate the budgetary impact of the strategy of early detection of HPV with DNA test genotyping with reflex cytology versus conventional cytology in women aged 30 to 65 years attending the cervical cancer screening program at a health benefit managing entity in Colombia. Materials and methods: Using a decision tree and a Markov model, the clinical implications and direct costs of screening, diagnosis, and treatment were estimated in a cohort of women. The analysis considered two screening cycles and their annual costs. The data on the prevalence of clinical results and the costs were taken from the health managing entity. The information on the progression, persistence, and regression of the health states were taken from the ATHENA study. Results: The screening scheme with the HPV test, genotyping, and reflex cytology compared to conventional cytology was cost-saving. The average cost per screening cycle with the HPV test was estimated at COP $ 129,201,363 and with cytology at COP $ 186,309,952, i.e., a saving of COP $ 57,108,589 (30.7%). Conclusion: The implementation of the screening strategy under evaluation suggests prospective savings derived from the early detection of health states associated with the development of cervical cancer.

Ocular surface findings in impression cytology after interferon a2b or mitomycin C in rabbits / Achados em citologia de impressão da superfície ocular após uso de interferon alfa-2b ou mitomicina C: estudo em coelho

ABSTRACT Objective: To describe ocular surface findings in impression cytology obtained from healthy rabbit conjunctiva treated with interferon alpha-2b eyedrop, and compare them to findings after use of mitomycin C 0.02%. Methods: An experimental study using a rabbit model was performed between September 2013 and October 2014 at the Faculdade de Medicina de Marília, Universidade Federal de São Paulo, Clínica de Olhos Moacir Cunha. Thirty New Zealand white rabbits were divided into 6 groups and received interferon alpha-2b or mitomycin C 0.02%. Impression cytology (IC) was performed prior to topical applications and at15, 30 and 60 days of use. The following variables were analyzed in impression cytology: goblet cells, cellularity, cell-to-cell adhesion, nucleus/cytoplasm ratio, nuclear chromatin, inflammatory cells keratinization, and cytomegaly. Results: The major findings in impression cytology after us of interferon alpha-2b included loss of goblet cells (50.8%), reduced cell-to-cell adhesion (26.2%), abnormal nucleus/cytoplasm ratio (20%) and reduced cellularity (15.4%). After use of mitomycin C 0.02%, the most common changes included loss of goblet cells (46.2%), abnormal nucleus/cytoplasm ratio (25.6%), less cell-to-cell adhesion (23.1%), and reduced cellularity (20.5%). There were no significant differences in any variable when comparing impression cytology after interferon alpha-2b and after mitomycin C 0.02%. Goblet cell loss was more pronounced at days 30 and 60, as compared to impression cytology at day 15 for both drugs. Conclusion: The loss of goblet cells, reduced cell-to-cell adhesion and cellularity, along with abnormal nucleus/cytoplasm ratio were the most common findings in impression cytology after use of interferon alpha-2b. These findings are similar to those described for use of mitomycin C 0.02%. ..

RESUMO Objetivo: Descrever os achados em citologia de impressão de conjuntiva sadia de coelho submetida ao uso de colírio de interferon alfa-2b e compará-los ao que foi encontrado após uso da mitomicina C 0,02%. Métodos: Estudo experimental realizado em modelo animal no período entre setembro de 2013 e outubro de 2014 nas dependências da Faculdade de Medicina de Marília, da Universidade Federal de São Paulo e da Clínica de Olhos Moacir Cunha. Trinta coelhos albinos da raça Nova Zelândia foram divididos em seis grupos e receberam interferon alfa-2b ou mitomicina C. A citologia de impressão foi realizada antes do início dos colírios e após 15, 30, 60 dias de seu uso. As seguintes variáveis foram analisadas na citologia de impressão: células caliciformes, celularidade, adesão intercelular, razão núcleo/citoplasma, cromatina, células inflamatórias, queratinização e citomegalia. Resultados: Os principais achados na citologia de impressão após o uso do interferon alfa-2b foram a redução de células caliciformes (50,8%), a diminuição da adesão intercelular (26,2%), a alteração da razão N/C (20%) e a redução da celularidade (15,4%). Após o uso da mitomicina C 0,02%, foram mais frequentes a redução das células caliciformes (46,2%), a alteração da razão N/C (25,6%), a adesão intercelular (23,1%) e a redução da celularidade (20,5%). Não houve diferença estatisticamente significante para nenhuma das variáveis estudas quando se compararam as citologias de impressão após interferon alfa-2b com as citologias de impressão após mitomicina C 0,02%. Independentemente da substância utilizada, as citologias colhidas 30 e 60 dias após início das drogas apresentaram maior redução de células caliciformes quando comparadas com as citologias de impressão colhidas após 15 dias. Conclusão: A redução das células caliciformes, a diminuição da adesão intercelular, a alteração da razão N/C e a diminuição da celularidade foram as alterações mais frequentes na citologia de impressão colhida após o uso de interferon alfa-2b. Os achados em citologias de impressão após o uso de interferon alfa-2b são semelhantes àqueles encontrados após o uso da mitomicina C 0,02%.

Infección por virus del papiloma humano en mujeres del Cantón Cañar, Ecuador / Human papillomavirus infection among women from Cañar Canton in Ecuador

Introducción: Existen pocos estudios sobre la circulación del virus del papiloma humano en mujeres ecuatorianas, particularmente residentes en el Cantón Cañar. Objetivo: Determinar la circulación del virus del papiloma humano, las alteraciones en la citología cérvico-vaginal de mujeres cañaríes y el comportamiento de algunas variables sociodemográficas y clínico-epidemiológicas. Métodos: Estudio analítico de corte transversal desde julio 2017-septiembre 2018. Se colectaron células cervicouterinas de 100 mujeres entre 15 y 55 años de edad para determinar la infección viral y alteraciones citológicas. Se investigó la asociación entre variables sociodemográficas y clínico-epidemiológicas con la infección viral. Resultados: El 51 por ciento (51/100) de las mujeres examinadas resultó positivo al virus, con predominio de los genotipos oncogénicos. El genotipo 31 fue el más frecuente (56,9 por ciento; 29/51), seguido por el genotipo 58 (43,1 por ciento; 22/51). Las mujeres mayores de 50 años, tenían una probabilidad menor de estar infectadas (3,9 por ciento; 2/51). La probabilidad de infección fue mayor en mujeres solteras, con antecedentes de infecciones de transmisión sexual, que padecían procesos cervicales inflamatorios, y en las fumadoras. La infección con genotipo 66 estuvo asociada al uso de anticonceptivos hormonales (53,3 por ciento; 8/15); p= 0,045, RP= 3,08 IC95 por ciento (1,00-9,46). Se obtuvo el 97 por ciento de citologías negativas para malignidad; no se diagnosticaron casos con lesiones de alto grado. Conclusiones: La elevada prevalencia de infección con genotipos oncogénicos en contraste con la baja frecuencia de citologías positivas, indica la necesidad de implementar programas eficientes para la detección precoz del cáncer cervicouterino en la población del Cañar y divulgar campañas de educación sexual y reproductiva(AU)

Introduction: Few studies are available about the circulation of human papillomavirus among Ecuadorian women, particularly those from Cañar Canton. Objectives: Determine the circulation of human papillomavirus, alterations in the cervical-vaginal cytology of women from Cañar Canton, and the behavior of some sociodemographic and clinical-epidemiological variables. Methods: An analytical cross-sectional study was conducted from July 2017 to September 2018. Cervical cells were collected from 100 women aged 15-55 years to determine viral infection and cytological alterations. An analysis was performed of the relationship of sociodemographic and clinical-epidemiological variables to viral infection. Results: Of the women examined, 51 percent; (51/100) tested positive for the virus, with a predominance of oncogenic genotypes. Genotype 31 was the most common (56.9 percent;; 29/51), followed by genotype 58 (43.1 percent; 22/51). Women aged over 50 years had a lesser probability of being infected (3.9 percent;; 2/51). Infection probability was greater among single women, with a history of sexually transmitted infections, who suffered from inflammatory cervical processes, and smokers. Infection by genotype 66 was associated to the use of hormonal contraceptives (53.3 percent;; 8/15); p= 0.045, PR= 3.08 CI95 percent; (1.00-9.46). Of the sample cytologies, 97 percent; were negative for malignancy; no case was diagnosed of high-grade lesions. Conclusions: The high prevalence of infection by oncogenic genotypes, as opposed to the low frequency of positive cytologies, points to the need to implement efficient programs aimed at early detection of cervical cancer in the population of Cañar Canton, as well as sexual and reproductive education campaigns(AU)

Factibilidad para implementar un sistema electrónico de registros de citología cervical / Feasibility to implement an electronic cervical cytology record system

Introducción: para garantizar la información y la atención de calidad, la Organización Mundial de la Salud, la Organización Panamericana de la Salud y las normas oficiales del cáncer cervicouterino y los sistemas de salud en México sugieren fortalecer los sistemas de información en salud con el uso de herramientas tecnológicas. Objetivo: el propósito de este estudio es conocer la factibilidad de un proyecto para la innovación de procesos, a través de un sistema electrónico para los registros de las citologías cervicales. Metodología: estudio observacional, transversal, prospectivo, realizado del 4 al 25 de junio de 2018 en la Unidad de Cuidados Integrales e Investigación en Salud de la Universidad Autónoma de San Luis Potosí, México. Se estudió al universo del personal sanitario que implementa el Programa de Detección Oportuna de Cáncer Cervico Uterino, con 7 participantes: personal directivo (2) y operativo (5). Resultados: las políticas y recursos institucionales permiten la implementación del proyecto, pues consideran tendría impacto positivo en la atención. Conclusiones: actualmente, la totalidad del personal cuenta con las competencias básicas para el manejo de sistemas electrónicos.

Introduction: To guarantee quality information and care, World Health Organization, Pan American Health Organization and the Official Standards of Cervical Cancer and Health Systems in Mexico suggest strengthening health information systems with the use of technological tools. Objective: The purpose of the study is to know the feasibility of a project for process innovation, through an electronic system for records of cervical cytologies. Methods: An observational, cross-sectional, prospective study conducted from June 4 to 25, 2018 in the Unit of Integral Care and Health Research of the Autonomous University of San Luis Potosí, Mexico. The universe of health personnel that implements the Timely Detection of Cervical Cancer Program was studied, 7 participants: managerial staff (2) and operative staff (5). Results: Institutional policies and resources allow for the implementation of the project, as they consider it would have a positive impact on care. Conclusions: Currently, all the staff has the basic skills for the management of electronic systems.

COVID-19 e o exame do líquor / COVID-19 and Cerebral Spinal Fluid analysis

O novo coronavírus (SARS-Coronavírus-2: SARS-Cov-2) foi inicialmente relatado em 2019, em Wuhan, China, onde o aparecimento de muitos casos inexplicáveis de pneumonia severa deixou em alerta todos os serviços de saúde do mundo. A Organização Mundial da Saúde (OMS) declarou recentemente uma pandemia, com mais de um milhão de casos relatados até o momento de COVID-19. A relação da fisiopatologia deste novo agente infeccioso associado ao comportamento dos achados laboratoriais do exame do líquor-LCR, através das descrições de casos e relatos, é importante para a análise, interpretação e apoio diagnóstico.

The new coronavirus (SARS-Coronavirus-2: SARS-Cov-2), was initially reported in 2019 in Wuhan, China, where the appearance of many unexplained cases of severe pneumonia has put all health services on alert of the world. The World Health Organization (WHO) recently declared a pandemic, with more than 1,000,000 cases reported to date as of COVID-19. The pathophysiological relationship of this new infectious agent associated with the behavior of laboratory findings of Cerebrospinal Fluid (CSF) examination, through case descriptions and reports, are important for analysis, interpretation and diagnostic support.

Estratégias Utilizadas para Melhorar a Qualidade dos Exames Citopatológicos / Strategies Used to Improve the Quality of Citopathological Examinations / Estrategias Utilizadas para Mejorar la Calidad de los Examenes Citopatológicos

Introdução: O exame citopatológico é utilizado para detecção precoce das lesões precursoras do câncer do colo uterino. Objetivo: Avaliar os indicadores de qualidade de acordo com o Manual de Gestão da Qualidade para Laboratório de Citopatologia. Método: Verificaram-se os laudos das fichas de requisição dos exames citopatológicos do laboratório clínico da Pontifícia Universidade de Goiás (LC-PUC-Goiás) entre janeiro de 2013 e dezembro de 2017. Resultados: Do total de 6.809 diagnósticos, observaram-se 99,4% (6.768/6.809) satisfatórios, sendo 91,3% (6.215/6.809) resultados negativos, 8,1% (553/6.809) diagnósticos com anormalidades citológicas e 0,6% (41/6.809) de exames insatisfatórios. O índice de positividade dos anos de 2013 a 2017 foram 10,5%, 7,9%, 8,6%, 6,8% e 5,3%, respectivamente. O percentual de exames compatíveis com lesão intraepitelial de alto grau (HSIL) entre os exames satisfatórios no ano de 2013 foi de 1,3%; 2014: 1,0%; 2015: 0,5%; 2016: 0,6%; e 2017: 0,7%, resultados dentro do estabelecido, ≥0,4%. A relação de células escamosas atípicas (ASC)/satisfatórios demostrou valores acima do estabelecido nos anos de 2013 com 6,8% e 2015 com 6,1%. Segundo o Manual de Gestão para Controle de Qualidade, espera-se que, no máximo, 4% a 5% de todos dos exames sejam classificados como ASC. Valores acima de 5% necessitam de uma atenção diferenciada. Conclusão: É de suma importância a educação continuada dos profissionais que participam de todas as etapas do processo, da fase pré-analítica à analítica, para que possíveis erros possam ser corrigidos e medidas preventivas tomadas para uma melhor qualidade na interpretação dos exames citopatológicos.

Introduction: The cytopathological examination is used for early detection of cervical cancer precursor lesions. Objective: Evaluate the quality indicators according to the Quality Management Manual for the Cytopathology Laboratory. Method: The results of the request forms of cytopathological examinations of the Clinical Laboratory of the Pontifical University of Goiás (LC-PUC-Goiás) were verified between January 2013 and December 2017. Results: Of 6,809 diagnoses in total, 99.4% (6,768/6,809) were satisfactory, 91.3% (6,215/6,809) were negative, 8.1% (553/6,809) presented cytological abnormalities and 0.6% (41/6,809) were unsatisfactory. The Positivity Index from 2013 to 2017 was 10.5%, 7.9%, 8.6%, 6.8% and 5.3%, respectively. The percentage of examinations with high-grade intraepithelial lesion (HSIL) among satisfactory examinations in 2013 was 1.3%, in 2014, 1.0%, in 2015, 0.5%, in 2016, 0.6 % and in 2017, 0.7%, results within the established ≥ 0.4%. The atypical squamous cells (ASC)/satisfactory ratio showed values above the standard figures in 2013 with 6.8% and 2015 with 6.1%. According to the Quality Control Management Manual, it is expected that, at the most, 4% to 5% of all exams be classified as ASC. Values above 5% demand differentiated attention. Conclusion: It is of the utmost importance the continued education of professionals who participate in all stages of the process, from the pre-analytical to the analytical phase, so that possible errors can be avoided and preventive measures taken for better quality interpretation of the cytopathological examinations.

Introducción: El examen citopatológico se utiliza para la detección temprana de lesiones precursoras de cáncer cervical. Objetivo: Evaluar los indicadores de calidad de acuerdo con el Manual de Gestión de Calidad para el laboratorio de citopatología. Método: Se verificaron los informes de los formularios de solicitud para los exámenes citopatológicos del Laboratorio Clínico de la Pontificia Universidad de Goiás (LC-PUC-Goiás), de enero de 2013 a diciembre de 2017. Resultados: Del total de 6.809 diagnósticos, se observarán 99,4% (6.768/6.809) diagnósticos satisfactorios, 91,3% (6.215/6.809) resultados negativos, 8,1% (553/6.809) diagnósticos con anomalías citológicas y 0,6% (41/6.809) exámenes insatisfactorios. El índice de positividad de 2013 a 2017 fue de 10,5%, 7,9%, 8,6%, 6,8% y 5,3% respectivamente. El porcentaje de exámenes compatibles con lesiones intraepiteliales de alto grado (HSIL) entre los exámenes satisfactorios en 2013 fue de 1,3%; 2014: 1,0%; 2015: 0,5%; 2016: 0,6%; y 2017: 0,7%, resultados dentro de lo establecido, ≥0,4%. La relación células escamosas atípicas (ASC)/satisfactorio fue más alta que la establecida en 2013 con 6,8% y 2015 con 6,1%. Según el Manual de Gestión de Control de Calidad, se espera que un máximo del 4% al 5% de todos los exámenes se clasifiquen como ASC. Los valores superiores al 5% requieren una atención diferente. Conclusión: Es de suma importancia la educación continua de los profesionales que participan en todas las etapas del proceso, desde la fase preanalítica hasta la analítica, para que se puedan corregir los posibles errores y se tomen medidas preventivas para una mejor calidad en la interpretación de los exámenes citopatológicos.

Pancreatic islet stereology: estimation of beta cells mass / Estereología de los islotes pancreáticos: estimación de la masa de células beta

Obesity and its comorbidities are becoming epidemic in the Western world. Beta cell mass estimation is an important indicator to track the progression of insulin resistance/type 2 diabetes, particularly in experimental studies, where it can be performed with stereological tools in an unbiased way. In this work, we present a simple protocol that can contribute to doing the practice of estimating the mass of beta cells more frequent and reproducible. As with any quantitative study, the necessary precautions regarding sampling and randomness must be respected.

La obesidad y sus comorbilidades se están convirtiendo en una epidemia en el mundo occidental. La estimación de la masa de células beta es un indicador importante para rastrear la progresión de la resistencia a la insulina/diabetes tipo 2, particularmente en estudios experimentales, donde se puede realizar con herramientas estereológicas de manera imparcial. En este trabajo presentamos un protocolo simple que puede contribuir a que la práctica de estimar la masa de células beta sea más frecuente y reproducible. Como en cualquier estudio cuantitativo, deben respetarse las precauciones necesarias con respecto al muestreo y la aleatoriedad.

Citología aspirativa con aguja fina transbroncoscópica guiada por ultrasonido endobronquial / Endobronchial ultrasound-guided transbronchoscopic fine needle aspiration cytology

Introducción: El enfrentamiento diagnóstico de las lesiones sospechosas de cáncer pulmonar ha cambiado en los últimos años. Objetivo: Describir el primer caso diagnosticado en Cuba de carcinoma de pulmón por aspiración transbronquial por aguja fina guiada por ultrasonografía endobronquial. Presentación del caso: Se presenta un caso de carcinoma indiferenciado de células pequeñas. El diagnóstico se obtiene mediante la realización de una citología aspirativa con aguja fina de ganglios mediastinales, transbroncoscópica y guiada por ultrasonido endobronquial. Conclusiones: El uso de EBUS-TBNA es una herramienta con un alto rendimiento diagnóstico, con escasas complicaciones descritas y debe considerarse como una posibilidad en el estudio de lesiones adyacentes a la vía aérea central(AU)

Introduction: The diagnostic confrontation of lesions suspected of lung cancer has changed in recent years. Objective: To describe the first case of lung carcinoma, diagnosed in Cuba by endobronchial ultrasound-guided transbronchoscopic fine needle aspiration cytology. Case report: A case of undifferentiated small cell carcinoma is reported. Diagnosis is obtained by performing a fine needle aspiration cytology of the mediastinal lymph nodes, transbronchoscopic and guided by endobronchial ultrasound. Conclusions: The use of EBUS-TBNA is a tool with high diagnostic yield, with few described complications and should be considered as a possibility in the study of lesions adjacent to the central airway(AU)

Brush Biopsy Sample Quality: Preliminary Investigation of a Metal Brush Prototype

Objective: To qualitatively investigate whether a prototype brush composed of metal bristles collects oral epithelial cells effectively for cytological evaluation of oral mucosal lesions. Material and Methods: Twenty patients with suspicious oral mucosal lesions were enrolled. Patients were asked to gargle with saline and to deposit the oral rinse into specimen cup. Then, oral mucosal cell samples were collected using a metal oral brush, via sweeping motion. Punch biopsy was performed for histological examination. All samples were evaluated with liquid based cytology (LBC) according to the cellularity, the depth of the epithelial layer, cellular integrity by an oral pathologist. Results: Oral rinse provided samples with 100% cellular integrity and cellularity, mostly from the intermediary layers. With metal brush, both inadequate cellularity and cellular integrity was observed in 25% of the cases. Cellular integrity was adequate in 65%, cellularity was adequate in 45% of the lesions. Samples were dominantly from the intermediary layers, but in one case, metal brush collected cells from the parabasal layer. Conclusion: The narrow spiral pitch and width of metal bristles may have resisted to release the cellular samples collected. With adjustment of the spiral pitch and diameter of metal brush bristles, its' efficacy could be enhanced.

Importancia del sistema de Bethesda en el diagnóstico citológico de lesiones precancerosas del cérvix / The importance of Bethesda system in the cytological diagnosis of the cervix pre-cancerous lesions

RESUMEN El cáncer cérvico uterino, causa alrededor de 250 000 muertes anuales en el mundo y alrededor de 400 en Cuba, a pesar del esfuerzo que realiza el MINSAP, a través del Programa de Pesquisaje. Con el mismo se puede obtener el diagnóstico de lesiones precursoras del cáncer de cuello uterino, este diagnóstico citológico se realiza en Cuba a través del método de Richard y Barron que demuestra que existe un progreso citológico aparente hasta llegar al cáncer, que comienza con neoplasia intraepitelial (NICI a NICIII y carcinoma in situ), hasta finalmente el cáncer invasor. Por otro lado existe el método de Bethesda que responde casi todas las interrogantes que la citología plantea para su enfrentamiento, evidentemente los mayores aportes y revisiones se enfocan al manejo de las citologías atípicas de significado incierto, ya que no sólo presentan un mayor número de posibles evaluaciones, sino que representan el mayor porcentaje de citologías alteradas y la inclusión del VPH en las lesiones de bajo grado. En Cuba todavía se clasifica por el método de Richard y no se utiliza el Bethesda. Por la alta incidencia de esta entidad el propósito de este trabajo es emitir consideraciones sobre la implementación del sistema de Bethesda en el diagnóstico citológico de lesiones precancerosas del cérvix.

ABSTRACT The cervical-uterine cancer causes almost 250 000 death a year around the world and around 400 in Cuba in spite of the efforts made by the Public Health Ministry through the Screening Program. With it, the diagnosis of lesions that are predecessors of the cervical cancer could be reached. This cytological diagnosis is carried out through the Richard and Barron method, showing that there is an apparent cytological progress leading to the cancer that begins with intraepithelial neoplasia (NICI and NICIII and carcinoma in-situ) and ends in the invasive cancer. From the other hand there is the Bethesda methods answering to all the questions cytology ask for confronting it. Obviously the biggest contributions and reviews are focused in the management of the atypical cytologies with uncertain significance since they not only have a higher number of possible evaluations, but also represent the highest percent of the altered cytologies and the inclusion of the HPV in low grade lesions. The classification in Cuba is still made by the Richard method and the Bethesda one is not used. Due to the high incidence of this entity, the aim of this article is exposing considerations on the implementation of the Bethesda system in the cytological diagnosis of cervix pre-cancerous lesions.

¿Permiten la citología, el genotipado y la anoscopia de alta resolución establecer un protocolo para la detección precoz del carcinoma epidermoide de canal anal en población de riesgo? / Do cytology, genotyping, and high resolution anoscopy allow to establish a protocol for early detection of squamous cell carcinoma of the anal canal in risk population?

Introducción: El cáncer anal ha experimentado un aumento de incidencia en los últimos años. Está mediado por el VPH y precedido de cambios precancerosos planteando la posibilidad de dirigir los esfuerzos preventivos hacia los grupos de alto riesgo. Sigue siendo controvertida la indicación de cribado y los métodos de detección ideales. Objetivo: Validar las pruebas de cribado implementadas en la actualidad comparadas con la biopsia como "gold standard". Material y Métodos: Estudio transversal con recogida de datos prospectiva, en una cohorte de hombres VIH+ que tienen sexo con hombres, pertenecientes al Hospital Gregorio Marañón e Infanta Leonor en un periodo de 2 años. Resultados: Se seleccionaron 179 pacientes con 286 visitas a la consulta de screening en las que se llevaron a cabo 3 pruebas de cribado en paralelo (citología anal, genotipado del VPH y anoscopia de alta resolución (AAR) con toma de biopsia dirigida sobre zona sospechosa o aleatoria). La sensibilidad y especificidad para la detección de displasia de alto grado y cáncer y su grado de concordancia con la biopsia fue la siguiente: citología 3,23%/94,43% (k: 0,03), genotipado de VPH de alto riesgo 90,32%/27,45% (k: 0,05), AAR 32,26%/87,45 (k: 0, 17) siendo el rendimiento diagnóstico de las tres pruebas muy bajo. Conclusión: La citología presenta un rendimiento diagnóstico muy bajo comparado con el genotipado que representa el mayor. A la luz de nuestros resultados, los protocolos clínicos tal y como vienen desarrollándose en la actualidad deberían de ser abandonados.

Introduction: The incidence of anal cancer has increased in recent years. It is mediated by HPV and preceded by precancerous changes, raising the possibility of directing preventive efforts towards high-risk groups. The indication of screening remains controversial and which methods would be the ideal ones. Objective: To validate the screening tests established actually, comparing it with the biopsy considered as the "gold standard". Materials and Methods: A cross-sectional study was performed, with prospective data collection in a cohort of VIH+ patients, who have male homosexual anal relations, belonging to Gregorio Marañón and Infanta Leonor Hospitals in a period of 2 years. Results: A total of 179 patients were selected with 286 visits to the screening Outpatient Clinic in which 3 parallel screening tests were performed (anal cytology, HPV genotyping and high resolution anoscopy (AAR) with a biopsy directed on a suspicious or random area). The sensitivity and specificity for the detection of high-grade dysplasia and cancer and their degree of agreement with the biopsy was as follows: cytology 3.23%/94.43% (k: 0.03), high HPV genotyping. risk 90.32%/27.45% (k: 0.05), AAR 32.26%/87.45 (k: 0, 17), the diagnostic accuracy of the three tests being very low. Conclusion: Cytology shows a very low diagnostic accuracy compared to the genotype that represents the highest one. In light of our results, clinical protocols as they are currently being developed should be abandoned.

Comparative study of metachromatic staining methods in assessing the exfoliative cell types during oestrous cycle in Sprague-Dawley laboratory rats / Estudio comparativo de los métodos de tinción metacromática en la evaluación de los tipos de células exfoliativas durante el ciclo estral en ratas Sprague-Dawley de laboratorio

This study was aimed at comparing the commonly used metachromatic stains viz., Papanicolaou stain, WrightGiemsa, Toluidine blue and Methylene blue in the assessment of cell types of the oestrous cycle in rats. Eight female Sprague-Dawley rats aged 8-9 weeks were used for this assessment. Cotton Swabs were gently inserted in the animals vagina to obtain cells from which they were then transferred to glass slides for staining and evaluation under microscopy. The different cell types were compared for their morphological features and clarity of cellular detail under all four stains. The application, advantages and limitations of all stains were then discussed. It was concluded that the selection of the most effective stain in the assessment of vaginal cytology depends on their application to clinical or research which was based on the cellular detail of interest, time, cost and availability of each staining procedure.

El presente estudio tuvo como objetivo comparar las tinciones metacromáticas comúnmente utilizadas, Wright's-Giemsa, azul de toluidina, azul de metileno y tinción de Papanicolaou, en la evaluación de los tipos de células del ciclo estral en ratas. El estudio se realizó en ocho ratas hembras SpragueDawley, con edades entre 8 y 9 semanas, y se usaron hisopos vaginales de algodón para preparar portaobjetos. Los diferentes tipos de células se compararon por sus características morfológicas y claridad en las cuatro tinciones. La aplicación, ventajas y limitaciones de todas las tinciones fueron discutidas. Se concluye que la selección de la tinción más efectiva en la evaluación de la citología vaginal depende de su uso, es decir, clínico o de investigación, el detalle celular de interés, tiempo, costo y disponibilidad.

Bacteriological, cytological and histopathological evaluation of the reproductive tract of slaughtered cows / Avaliação bacteriológica, citológica e histopatológica do trato reprodutivo de vacas abatidas

Reproductive diseases, mainly endometritis, are important hurdles in cattle raising, In the current study we evaluated gross, bacteriological, cytological, and histological findings from selected sites of the genital from 23 slaughtered cows and tested whether there is an association between these findings and the probability of reaching a reliable diagnosis. The results from the examinations of macroscopic aspects of uterine secretions, the cytological, bacteriological, and histopathological findings were then correlated. There was no significant correlation (P>0.05) of the statistical data from different parts of the genital tract. Trueperella pyogenes and Escherichia coli were isolated from the vagina in 3/23 cases. In only 2/23 samples Enterococcus faecalis and a gram-negative, oxidase-positive bacteria were isolated from the cervix uteri. Only Staphylococcus epidermidis, in 1/23 case, was isolated from the uterus. Histopathological findings in uterus from samples of Groups II (moderate lesions) and III (severe lesions) did not translated in grossly visible changes. Samples from reproductive tracts with secretion in the vagina and cervix uteri had no detectable changes in the other parameters analyzed from this portion. Uterus with positive bacterial culture had evidence of ascendant inflammation judging by the high granulocyte count in the three analyzed portions. This study reinforces that vaginitis and cervicitis in the cow diagnosed only by clinical examination does not reflect the real status of the uterine health. For this reason, treatment of uterine disorders should be conducted based on reliable tests to determine the appropriate therapy for each situation.(AU)

Doenças reprodutivas causam altas perdas econômicas nos rebanhos bovinos, sendo a endometrite uma das alterações mais relevantes. Os objetivos desta pesquisa foram avaliar a associação dos achados fisiopatológicos em exames macroscópicos, bacteriológicos, citológicos e histopatológicos nas porções do trato genital de 23 vacas abatidas, bem como avaliar a necessidade de associação destes exames para efetuar diagnóstico fidedigno. A avaliação macroscópica da secreção, os exames histopatológico, citológico e as bactérias identificadas foram correlacionados. Não houve associação (P>0,05) dos resultados nas diferentes porções do trato genital. Na vagina foram isoladas as bactérias Trueperella pyogenes e Escherichia coli. Na cérvix, em apenas 2/23 (8,6%) amostras isolou-se Enterococcus faecalis e gram negativo oxidase positiva. No útero houve isolamento apenas da bactéria Staphylococcus epidermidis. As amostras histopatológicas classificadas em grupo II e III não apresentaram alterações detectadas no exame macroscópico. As amostras com secreção não fisiológica na vagina e cérvix não apresentaram alterações nos outros exames na porção uterina. A amostra com cultura bacteriológica positiva no útero demonstrou uma infecção ascendente através da alta contagem de granulócitos nas três porções analisadas. O presente estudo reforça o conceito de que a vaginite e cervicite diagnosticadas pelo exame clinico na vaca não retratam o real status da saúde uterina e por esta razão, o tratamento do útero deve ser conduzido com critério, alicerçado nos exames complementares para definir a terapia adequada para cada situação.(AU)

Patologia morfológica e molecular aplicadas à inovação diagnóstica e vigilância da leishmaniose visceral / Morphological and molecular pathology applied to diagnostic innovation and surveillance of visceral leishmaniasis

O conceito de Saúde Única surgiu para ressaltar a união indissociável entre a saúde animal, humana e ambiental. Nesse contexto, a leishmaniose visceral americana (LVA) é considerada uma importante doença de saúde pública no Brasil, devido a sua crescente expansão geográfica e aumento na incidência de casos humanos. A LVA é uma doença parasitária, zoonótica, causada pela Leishmania (Leishmania) infantum (syn. chagasi) e transmitida por flebotomíneos do gênero Lutzomyia. Os cães são considerados os principais reservatórios do parasito nas áreas urbanas. O diagnóstico da LVA é baseado em aspectos epidemiológicos, clínicos e laboratoriais. A demonstração da presença do parasito através de exames diretos em tecidos biológicos do hospedeiro é o diagnóstico de escolha, principalmente, em municípios em que a transmissão de LVA ainda não tenha sido confirmada. Diversas metodologias podem ser aplicadas com essa finalidade. O objetivo desse trabalho é apresentar as técnicas citológicas, anatomo-patológicas e moleculares em amostras fixadas em formalina e incluídas em parafina para o diagnóstico da leishmaniose visceral em humanos e cães. Esses dados são complementares à apresentação realizada no I Simpósio Internacional de Leishmaniose Visceral, realizado nos dia 23 e 24 de Abril de 2018, e organizado pelo Instituto Adolfo Lutz em São Paulo-SP, Brasil.

The One Health concept emerged to highlight the inseparable link between animal, human and environmental health. In this context, American Visceral Leishmaniasis (AVL) is considered an important public health disease in Brazil, due to its increasing geographic expansion and in the incidence of human cases. AVL is a parasitic and zoonotic disease caused by Leishmania (Leishmania) infantum (syn. chagasi) and transmitted by sandflies of the genus Lutzomyia. Dogs are considered the main reservoirs of the parasite in urban areas. The diagnosis of AVL is based on epidemiological, clinical and laboratory aspects. The demonstration of the presence of the parasite through direct examinations in biological tissues of the host is the diagnosis of choice, mainly in municipalities where the transmission of AVL has not yet been confirmed. Several methodologies can be applied for this purpose. The objective of this work is to present the cytological, anatomopathological and molecular techniques in formalin fixed and paraffin embedded samples for the diagnosis of visceral leishmaniasis in humans and dogs. These data are complementary to the present study at the First International Symposium on Visceral Leishmaniasis, held on April 23 and 24, 2018, and organized by Adolfo Lutz Institute in São Paulo, Brazil.

Cumplimiento del flujo de decisiones clínicas de los algoritmos de derivación y confirmación diagnóstica del primer PAP atípico H1- Santiago Sur / Compliance with the flow of clinical decisions of the algorithms for referral and diagnostic confirmation of the first atypical PAP H1- Santiago Sur

RESUMEN Antecedentes: Un algoritmo puede definirse como un conjunto de operaciones y procedimientos que se deben seguir con el fin de resolver un problema. Objetivo: Conocer el cumplimiento del flujo de decisiones clínicas del protocolo preconizado en los algoritmos de derivación y de confirmación diagnóstica para la citología cervical atípica de significado indeterminado - H1, estipulados en el Programa Nacional de Búsqueda y Control del Cáncer Cervical de Chile. Métodos: Estudio epidemiológico de tipo descriptivo, observacional, cuantitativo y de seguimiento de una cohorte de mujeres portadoras de un primer informe de citología cervical atípica de significado indeterminado - H1, pertenecientes a los consultorios de Atención Primaria de Salud del área Metropolitana Sur de Santiago, Chile. El seguimiento fue a través de la aplicación de los "Algoritmo de derivación a especialista o U.P.C. del primer PAP atípico según la clasificación Bethesda 2001" y "Algoritmos para confirmación diagnóstica". Resultados: Se seleccionaron 446 mujeres con un primer informe H1. El cumplimiento de conductas de acuerdo al algoritmo de derivación fue de 42.4% y las conductas alternativas fue de 5.8%. La adherencia de conducta en los algoritmos para confirmación diagnóstica fue cumplida en un 89.6%. Conclusiones: El cumplimiento de las conductas ajustadas a algoritmo de derivación del primer Pap atípico fue realizado por la mitad de las mujeres y el cumplimiento del algoritmo de confirmación diagnóstica fue realizado por la gran mayoría.

SUMMARY Background: An algorithm can be defined as a set of operations and procedures that must be followed in order to solve a problem. Objective: Determine the compliance of the clinical decisions flow and procedures recommended by the ASCUS results derivation and diagnostic confirmation algorithms established in the National Program of Search and Control of Cervical Cancer of Chile. Methods: An epidemiological, descriptive, observational and quantitative study of a follow up investigation of a cohort of women with ASCUS cytological reports. These women belonging in the health care centers in the South Metropolitan area of Santiago de Chile. The algorithms "The Specialist Derivation Algorithm or UPC for the first atypical Pap according to the Bethesda 2001 classification" and "Diagnosis Confirmation Algorithm" were used for the follow-up process of these patients. Results: Women with a first ASCUS report were selected (n = 446). The compliance of the clinical decisions flow to derivation algorithm was 42.4% and alternative behaviors were 5.8%. In the diagnosis confirmation algorithm was 89.6% the compliance the recommended procedures. Conclusion: Half ASCUS carrier women followed the procedures recommended by the derivation algorithm. In the algorithms of diagnostic confirmation it is met mostly.

Lesiones epiteliales del cérvix en pacientes sin citología alterada en la Isla de la Juventud (2014-2015) / Cervix Epithelial Lesions in Patients with No Altered Cytology in the Isle of Youth (2014-2015)

Introducción: el estudio de las lesiones epiteliales del cérvix constituye una prioridad en la prevención de este cáncer que muestra un incremento evidente de la morbimortalidad, a pesar de programas de pesquiza poblacional. Objetivo: describir el comportamiento de las lesiones epiteliales según variables clínico - epidemiológicas de interés. Métodos: estudio descriptivo retrospectivo para caracterizar el comportamiento de lesiones epiteliales del cuello uterino en las 1 148 pacientes atendidas en la Consulta de Patología de Cuello en el Hospital General Docente de la Isla de la Juventud desde 2014 hasta 2015. Resultados: del total de pacientes, 71 por ciento tenían entre 25 y 51 años de edad, 44 por ciento procede de los Consejos Populares de Santa Fé y Pueblo Nuevo. El inicio de las relaciones sexuales supera los 16 años y el número promedio de partos y abortos no es mayor de dos. Del total, 42 % tenía una citología con infección por el virus del papiloma humano y 30 por ciento acudió por una lesión evidente del cérvix sin una citología alterada y 57 por ciento tenían un área lesional menor a 200 mm2. En los estudios colposcópico e histológico predominaron las lesiones de alto grado con más del 60 por ciento y las lesiones invasoras sólo representaron 4 por ciento. El número de pacientes jóvenes es significativamente alto. No se evidenció un comportamiento anormal de los factores de riesgo clásicos para esta enfermedad. Conclusiones: los resultados de este estudio pueden servir de base para trazar estrategias locales del Sistema de Salud encaminadas a la disminución de la morbimortalidad por cáncer en el territorio(AU)

Introduction: The study of cervix epithelial lesions is a priority in preventing this type of cancer because of the evident morbidity and mortality increase, despite population screening programs. Objective: To describe epithelial lesion behavior according to clinical - epidemiological variables of interest. Methods: A retrospective descriptive study was conducted to characterize the cervix epithelial lesion behavior in 1148 patients treated in the Consultation of Neck Pathology at the General Teaching Hospital of the Isle of Youth from 2014 to 2015. Results: 71 percent of the total number of patients aged between 25 and 51 years, 44 percent came from Santa Fé and Pueblo Nuevo territories. These subjects' first sexual relations were after they were 16 and the average number of deliveries and abortions is less than two. Forty-two percent had human papillomavirus infection confirmed by cytology. Thirty percent came to consultation for an obvious cervix lesion with no cytology altered. Fifty-seven percent had a lesion area less than 200 mm2. The colposcopy and histological studies, high-grade lesions predominated in more than 60 percent. Invasive lesions only represented 4 percent. The number of young patients is significantly high. There was no evidence of abnormal behavior of this disease's classic risk factors. Conclusions: The results of this study can serve as a basis to draw local strategies for the Health System aiming to reduce morbidity and mortality from cancer in the territory(AU)

Deep tissue culture and hemoculture in dogs with wounds and sepsis / Cultura de tecido profundo e hemocultura em cães com feridas e sepse

Contaminated and infected wounds occur very frequently in veterinary medicine and can cause systemic inflammatory response syndrome, sepsis, and death. This study aimed to test the feasibility of collecting wound material by deep-tissue or punch biopsy for microbial culture, determine the frequency of bacteria in the wound(s) and blood cultures and the susceptibility of these microbes to antimicrobials, and evaluate clinical parameters that could be related to prognosis. Thirty dogs with wounds and signs of SIRS/sepsis were included in this study. Bacteria were isolated from all wounds and 41 bacterial isolates could be identified based on culture of the materials collected by punch biopsy; 53.66% of the isolates were gram-negative, mainly involving Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus spp., and 46.34% were gram-positive bacteria such as Streptococcus spp., Enterococcus spp., and Staphylococcus spp. The survival rate was 66.67%. Based on blood culture analysis, we identified bacteremia in seven patients, predominantly of gram-negative bacteria, which negatively affected patient survival, as six dogs died. Hypoglycemia (≤60mg/dL) and severe hyperglycemia (≥180mg/dL) also negatively affected survival as 23.33% of the hypo/hyperglycemic dogs died. Factors such as blood lactate level at admission and hematocrit levels, and mean arterial pressure were not significantly correlated with death or survival of the dogs.(AU)

As feridas contaminadas e infectadas em cães ocorrem com grande frequência na medicina veterinária e podem causar síndrome da resposta inflamatória sistêmica, sepse e morte. Os objetivos do presente trabalho foram verificar a viabilidade da técnica de coleta de material da ferida por biópsia para realização de cultura microbiana, determinar a frequência das bactérias nas culturas das feridas e hemoculturas e a susceptibilidade destes agentes aos antimicrobianos, bem como avaliar parâmetros clínicos que pudessem ser relacionados ao prognóstico em 30 cães com feridas e sinais de SIRS/sepse. Foram isoladas bactérias de todas as feridas e a técnica de coleta de material para cultura por biópsia permitiu a obtenção de 41 agentes microbianos, sendo isoladas 53,66% bactérias Gram negativas e 46,34% Gram positivas, principalmente Pseudomonas aeruginosa, Klebsiella pneumoniae e Enterococcus spp. As bactérias gram positivas isoladas foram Streptococcus spp., Enterococcus spp. e Staphylococcus spp. A taxa de sobrevivência foi 66,67%. Na hemocultura constatou-se bacteremia em sete pacientes, com predominância de bactérias Gram negativas, o que influenciou negativamente na sobrevivência dos pacientes, pois seis cães vieram a óbito. A hipoglicemia (≤60mg/dL) ou hiperglicemia severa (≥180mg/dL), também influenciaram negativamente a sobrevivência, pois 23,33% dos pacientes hipo/hiperglicêmicos vieram a óbito. Já fatores como nível sérico de lactato na admissão do paciente, pressão arterial média (PAM) e hematócrito não apresentaram correlação estatística com o óbito ou sobrevivência destes pacientes.(AU)

Parâmetros citológicos e bioquímicos do líquido cefalorraquidiano coletado de bovinos sadios em dois momentos, com intervalo de 96 horas / Cerebrospinal fluid cytological and biochemical values of healthy cattle in two sampling times with an interval of 96 hours

A análise do líquido cefalorraquidiano (LCR) é uma importante ferramenta no diagnóstico das doenças neurológicas dos bovinos. A coleta do LCR em diferentes momentos facilita o monitoramento do quadro clínico e/ou a avaliação da eficácia de tratamentos estabelecidos. Todavia, os efeitos de coletas consecutivas sobre os parâmetros citológicos e bioquímicos do LCR de bovinos saudáveis ainda não foram analisados. Dessa forma, o objetivo do presente estudo foi avaliar potenciais diferenças citológicas e bioquímicas no LCR de bovinos sadios, obtidos em dois momentos, com intervalo de 96 horas. O LCR foi submetido à análise citológica e à determinação do teor de proteína total. Na segunda coleta, verificou-se aumento significativo da celularidade, porém ainda dentro do intervalo de referência para a espécie, diminuição de linfócitos e elevação de células mononucleares em relação à primeira coleta. No entanto, os números de hemácias (/µL), células nucleadas (/µL), neutrófilos (%), eosinófilos (%) e a concentração da proteína total (mg/dL) continuaram dentro dos valores de referência 96 horas após a realização da primeira punção. Conclui-se que os procedimentos de coleta de LCR consecutivos não provocaram inflamação local detectável 96 horas após a primeira punção, o que sugere que, quando necessário, coletas de LCR seriadas, por si só, não interfeririam nos resultados do exame.(AU)

The analysis of cerebrospinal fluid (CSF) is important for the diagnosis of neurological diseases in cattle. However, the effect of consecutive collections on the cytological and biochemical parameters of the CSF has not been evaluated yet. The objective of the present study was to evaluate potential differences on the cytological and biochemical analysis of CSF obtained from healthy cattle at two sampling times with 96 hours of interval in between. Total and differential cell counts, and total protein concentration were analyzed in the CSF. Increased cellularity and significant reduction of lymphocyte and elevated numbers of mononuclear cells were observed on the second collection. However, erythrocyte count (/µl), total leukocyte count (/µl), neutrophils (%), eosinophils (%) and total protein concentration (mg/dl) remained within the reference range 96 hours after the completion of the first puncture. In conclusion, consecutive CSF collections cause no detectable local inflammation 96 hours after the first puncture suggesting that repeated collections do not interfere in the CSF results.(AU)

Citocentrifugação e métodos convencionais na citologia uterina de éguas em estro e diestro / Cytocentrifugation and conventional methods in endometrial cytology of estrus and diestrus mares

A citologia uterina consiste em método prático e eficiente para o diagnóstico de afecções uterinas na égua, contribuindo para a conduta terapêutica e o prognóstico quanto à fertilidade das fêmeas. Assim, o objetivo deste trabalho foi comparar diferentes técnicas de coleta de material para citologia uterina em éguas, a fim de se verificarem possíveis diferenças qualitativas e quantitativas e a praticidade na execução das metodologias. Para tanto, 12 éguas multíparas foram submetidas ao exame citológico uterino, durante o estro e o diestro, por meio de escova ginecológica e lavado uterino de baixo volume. Com esse ensaio, observou-se que não houve diferença significativa (P>0,05) no resultado da citologia uterina entre as diferentes técnicas avaliadas. No entanto, quando comparadas as fases do ciclo estral, houve uma diferença significativa (P<0,05) quanto ao percentual médio de polimorfonucleares neutrófilos, sendo de 8,07±2,80 no estro e de 1,55±0,28 no diestro. Conclui-se, desse modo, que as técnicas estudadas apresentaram resultados igualmente eficazes para detecção de neutrófilos ao exame citológico do útero em ambas as fases avaliadas do ciclo estral, apesar de, na fase de estro, a detecção de maior percentual de neutrófilos possibilitar um diagnóstico e um prognóstico mais precoces de endometrite e, portanto, uma conduta terapêutica mais adequada.(AU)

The endometrial cytology consists in an efficient and practical method for the diagnosis of uterine disorders in mares, contributing for therapeutic conduct and prognostic evaluation regarding the females' fertility. Therefore, the aim of this study was to compare different techniques of material sampling for endometrial cytology in mares, in order to verify possible qualitative and quantitative differences in the practicality of implementation of this methodology. Twelve multiparous mares underwent an endometrial cytological exam, during estrus and diestrus, by means of a cytobrush and a low-volume uterine flush. With this test, no statistical difference was observed (P>0.05) regarding the result of endometrial cytology between the evaluated techniques. However, in comparison to the estrous cycle's phases, a significant difference was verified (P<0.05) on the average percentage of polymorphonuclear neutrophils: 8.07±2.80 in estrus and 1.55±0.28 in diestrus. Thus, we conclude that the studied techniques presented equally effective results for the detection of neutrophils in the cytological exam for both evaluated stages of the estrous cycle, whereas in estrus phase, the detection of a higher percentage of neutrophils allows a more precocious diagnostic and prognostic of endometritis and, therefore, a more adequate therapeutic approach.(AU)