Alternativas de procesos para la búsqueda de casos de tuberculosis bajo condiciones económicas limitadas. Cuba 2022-2025 / Process Alternatives for the Tuberculosis Case Finding under Limited Economic Conditions. Cuba 2022-2025

Ejecutar procesos efectivos de búsqueda de casos de tuberculosis es crucial para acele-rar el paso hacia su eliminación. El empeoramiento de las condiciones económicas mun-diales y nacionales no nos permite aplicar extensivamente las tecnologías rápidas mo-leculares idóneas de diagnóstico. Consideramos sensato entonces aplicar algoritmos alternativos que satisfagan las necesidades nacionales presentes hasta que las condi-ciones permitan la cobertura completa de las tecnologías moleculares recomendadas. Sugerimos introducir la radiografía digital para todos los algoritmos, utilizar mejor la microscopía de fluorescencia LED y la óptica convencional ya probadas. En conclusión, es preciso que este enfoque de trabajo, que procura optimizar la efectividad y eficiencia del programa, se introduzca en la práctica cotidiana hasta que lo idóneo sea permisible

Executing effective tuberculosis case-finding processes is crucial to accelerate the path towards elimination of the disease. The worsening of global and national economic conditions do not allow us to extensively apply rapid molecular diagnostic technolo-gies. We consider it sensible and necessary to apply alternative algorithms that meet the current national needs, until conditions allow full coverage of the recommended molecular technologies. We suggest introducing digital X-rays for all algorithms, bet-ter use of LED fluorescence microscopy and conventional optics already appropriate-ly tested. In conclusion, it is necessary that this approach that seeks to optimize the effectiveness and efficiency of the Cuban program be introduced into daily practice until the ideal is permissible

Identificación de Mycobacteria no pigmenteda de crecimiento rápido por el Método Molecular de Micobacterium de Genotipo CM / Identification of Fast-Growing Non-Pigmented Mycobacteria by the Genotype® Mycobacterium CM Molecular Method

Introducción: la micobacteria no terberculosa (NTM) forma un grupo heterogéneo de microorganismos que pueden causar infección en humanos. Las micobacterias no pigmentadas de rápido crecimiento (MNPCR) son de interés clínico debido al creciente número de pacientes infectados por ellos y a la dificultad del tratamiento. Dentro de este grupo, Mycobacterium fortuitum, Mycobacterium abscessus y Mycobacterium chelonae son reconocidos como patógenos potenciales; estas especies se han aislado de infecciones pulmonares y extrapulmonares. Objetivo: el objetivo de este trabajo es encontrar la frecuencia de aislamiento de especies micobacterianas de rápido crecimiento, específicamente el complejo Mycobacterium fortuitum, de muestras clínicas utilizando la técnica molecular de diagnóstico GenoType Mycobacterium CM. Material y Método: se analizaron 249 aislados de micobacterias no tuberculosas obtenidas de muestras pulmonares y extrapulmonares de pacientes sintomáticos en el período enero 2018-diciembre de 2022. La técnica molecular GenoType Mycobacterium CM se utilizó para identificar la especie. Resultados: Se obtuvieron 77 (3,9%) aislados de especies no pigmentadas de rápido crecimiento, estas se identificaron en orden decreciente: Mycobacterium fortuitum 65 (84,41%), Mycobacterium abcessus 9 (11,68%) y Mycobacterium chelonae 3 (4%). Conclusiones: los resultados reafirman que el complejo Mycobacterium fortuitum es responsable de la mayoría de las infecciones causadas por la micobacteria en rápido crecimiento en humanos. La técnica diagnóstica GenoType Mycobacterium CM es una herramienta útil para la rápida identificación de micobacterias; proporciona resultados precisos en menos tiempo, acortando significativamente el tiempo diagnóstico, permite la aplicación temprana de tratamiento específico, evitando así la propagación de la infección.

Introduction: non-tuberculous mycobacteria (NTM) form a heterogeneous group of mi-croorganisms that can cause infection in humans. Fast-growing non-pigmented my-cobacteria (MNPCR) are of clinical interest due to the increasing number of patients infected by them and the difficulty of treatment. Within this group, Mycobacterium fortuitum, Mycobacterium abscessus and Mycobacterium chelonae are recognized as potential pathogens; these species have been isolated from both pulmonary and ex-trapulmonary infections. Objective: the objective of this work is to find the frequency of isolation of fast-growing non-pigmented mycobacterial species, specifically the Myco-bacterium fortuitum complex, from clinical samples using the GenoType® Mycobacteri-um CM diagnostic molecular technique. Material and Method: 249 isolates of non-tu-berculous mycobacteria obtained from pulmonary and extrapulmonary samples from symptomatic patients in the period January 2018-December 2022 were analyzed. The G e n oTy p e® Mycobacterium CM molecular technique was used to identify the species. Results: 77 (30.9%) isolates of fast-growing non-pigmented species were obtained, these were identified in decreasing order: Mycobacterium fortuitum 65 (84.41%), Myco-bacterium abcessus 9 (11.68%) and Mycobacterium chelonae 3 (4%). Conclusions: the results reaffirm that the Mycobacterium fortuitum complex is responsible for most in-fections caused by fast-growing mycobacteria in humans. The GenoType® Mycobacte-riumCM diagnostic technique is a useful tool for the rapid identification of mycobacte-ria; it provides accurate results in less time, significantly shortening the diagnostic time, it allows the early application of specific treatment, thus avoiding the spread of infec-tion.

Implementación asistencial de estudios genómicos aplicados al diagnóstico molecular / Implementation of genomic studies applied to molecular diagnostics

El Hospital Garrahan ha sido pionero en el diagnóstico molecular de patologías pediátricas en Argentina. Los avances tecnológicos de las últimas décadas en el área de la biología molecular, sentaron las bases para la optimización y ampliación del diagnóstico molecular a partir de la secuenciación masiva en paralelo de múltiples genes. El presente trabajo describe el proceso de implementación de los estudios de secuenciación de nueva generación y el desarrollo de la Unidad de Genómica en un hospital público pediátrico de alta complejidad, así como su impacto en las capacidades diagnósticas de enfermedades poco frecuentes de origen genético. La creación del Grupo Interdisciplinario de Estudios Genómicos constituyó la vía institucional para la toma de decisiones que implican la implementación de nuevos estudios genómicos y el establecimiento de prioridades diagnósticas, extendiendo la disponibilidad del diagnóstico molecular a más disciplinas. La Unidad de Genómica trabaja en diseñar las estrategias que permitan la mayor optimización de los recursos con los que cuenta el hospital, teniendo en cuenta el equipamiento disponible, las prioridades establecidas y la frecuencia de las distintas patologías. Se demuestra el salto significativo operado en nuestras capacidades diagnósticas, tanto en la variedad de enfermedades como en el número de genes analizados, habiendo estudiado a la fecha alrededor de 2.000 pacientes, muchos de los cuales ven de este modo finalizada su odisea diagnóstica. Los estudios de NGS se han convertido en una herramienta de la práctica diaria para la atención de un número importante de pacientes de nuestro hospital. Continuaremos trabajando para ampliar su aplicación a la mayor cantidad de patologías, a través de los mecanismos institucionales ya existentes (AU)

The Garrahan Hospital has been a pioneer in the molecular diagnosis of pediatric diseases in Argentina. The technological advances of the last decades in the area of molecular biology have laid the foundations for the optimization and expansion of molecular diagnostics through massive parallel sequencing of multiple genes. This study describes the process of implementation of next-generation sequencing studies and the development of the Genomics Unit in a public pediatric tertiary hospital, and its impact on the capacity to diagnose rare diseases of genetic origin. The creation of the Interdisciplinary Group of Genomic Studies constituted the institutional pathway for decision-making involving the implementation of new genomic studies and the establishment of diagnostic priorities, extending the availability of molecular diagnostics to additional disciplines. The Genomics Unit is working to design strategies that allow for optimization of the resources available to the hospital, taking into account the equipment available, the priorities established, and the frequency of the different diseases. It demonstrates the significant leap in our diagnostic capabilities, both in the variety of diseases and in the number of genes analyzed. To date, around 2,000 patients have been studies, many of whom have thus completed their diagnostic odyssey. NGS studies have become a tool in daily practice for the care of a significant number of patients in our hospital. We will continue working to expand its application to as many diseases as possible, through the existing institutional mechanisms (AU)

Diagnóstico de precisão das lesões mamárias / Accurate diagnosis of breast lesions

PONTOS-CHAVE As lesões mamárias compreendem uma ampla variedade de diagnósticos que apresentam comportamentos diversos. As lesões mamárias podem ser classificadas como lesões benignas, de potencial de malignidade indeterminado (B3), carcinoma in situ e carcinoma invasor. Na era da medicina personalizada, individualizar e obter um diagnóstico preciso faz grande diferença no desfecho final da paciente, principalmente no caso do câncer de mama. Exames de imagem direcionados e de qualidade, métodos de biópsia adequadamente selecionados e análises de anatomopatologia convencional, imuno-histoquímica e até molecular são determinantes no diagnóstico e no manejo das pacientes.

Dogs naturally infected by Rangelia vitalii, Babesia canis vogeli, and Ehrlichia canis in São Paulo, Brazil / Cães naturalmente infectados por Rangelia vitalii, Babesia canis vogeli, e Ehrlichia canis em São Paulo, Brasil

Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)

As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)

Etiología de las infecciones de transmisión sexual (ITS) diagnosticadas por la técnica de PCR múltiple-hibridación en población colombiana de la ciudad de Medellín atendida en el Laboratorio Clínico VID / Etiology of sexually transmitted infections (STIs) diagnosed by multiplex PCR-hibridization assay technique in a Colombian population attending the VID Clinical Laboratory in Medellin

Introducción. Las infecciones de transmisión sexual (ITS) son y seguirán siendo un serio problema de salud pública en todo el mundo según los datos de la OMS, con el agravante que la mayoría de los casos son asintomáticos y, además, no existe otro reservorio distinto al humano. El diagnóstico se puede realizar con pruebas tradicionales y moleculares, estas últimas incluyen la reacción en cadena de la polimerasa (PCR), de las cuales existen varios tipos, entre ellas, la PCR múltiple que tiene la capacidad de detectar ITS polimicrobianas a partir de una sola muestra. El objetivo de este estudio fue establecer cuáles fueron las infecciones de transmisión sexual más frecuentes en diferentes grupos de pacientes, así como determinar la utilidad del uso de la técnica de PCR múltiple en el diagnóstico de las ITS. Metodología. Se trata de un estudio observacional de corte transversal realizado entre los años 2021 y 2022 con pacientes que acudieron al servicio de diagnóstico del Laboratorio Clínico VID por sospecha de ITS. Las muestras recolectadas fueron evaluadas utilizando una prueba comercial basada en la técnica de PCR múltiple e hibridación. Las muestras procesadas fueron: orina e hisopados de endocérvix, uretra, recto, faringe y úlceras. Resultados. Se estudiaron 1.027 pacientes, de estos, 228 (22,2 %) fueron positivos para diferentes agentes de trasmisión sexual, distribuidos así: 50 (21,9 %) mujeres, 129 (56,6 %) hombres heterosexuales y 49 (21,5 %) hombres que tenían sexo con hombres (HSH). La edad promedio de las mujeres fue 30 años, y la de ambos grupos de hombres fue 36 años. Los microorganismos más frecuentemente identificados en mujeres fueron: C. trachomatis (A-K) en 28,6 %, seguido de virus herpes simplex tipo 2 (VHS-2) en 26,8 % y N. gonorrhoeae en 17,9 %. En hombres heterosexuales fueron C. trachomatis (A-K) en 37,5 %, N. gonorrhoeae en 21,5 % y VHS-2 en 18,7 %. En HSH fueron C. trachomatis (L1-L3) en 32,7 %, seguido de N. gonorrhoeae en 27,6 %, y de C. trachomatis (A-K) y VHS-2, ambos en 13,8 %. En 11 hombres heterosexuales, 8 HSH y en 6 mujeres, se identificó infección polimicrobiana. Conclusiones. C. trachomatis (A-K) fue el microorganismo más prevalente causante de ITS, seguido de N. gonorrhoeae en ambos grupos de hombres, y de VHS-2 en las mujeres, muy similar a lo reportado a nivel mundial. La prueba de PCR múltiple permite la detección de infecciones polimicrobianas comúnmente asociadas a ITS y el diagnóstico es preciso y confiable, incluso en pacientes asintomáticos

Sexually transmitted infections (STIs) are and will continue to be a serious public health problem throughout the world according to WHO data, with the aggravating factor that most cases are asymptomatic and, furthermore, there is no other reservoir other than humans. The diagnosis can be made with traditional and molecular tests, the latter include the polymerase chain reaction (PCR), of which there are several types, among them, multiplex PCR that has the capacity to detect polymicrobial STIs from a single sample. The objective of this study was to establish which were the most frequent sexually transmitted infections in different groups of patients, as well as to determine the usefulness of the multiplex PCR technique in the diagnosis of STIs. Methodology. This is an observational, cross-sectional study carried out between 2021 and 2022 with patients who attended the VID Clinical Laboratory for suspected STIs. The collected samples were evaluated using a commercial test based on the multiplex PCR technique and hybridization. The samples processed were: urine and swabs from endocervix, urethra, rectum, pharynx, and ulcers. Results. The study included 1,027 patients, of these, 228 (22.2%) were positive for different sexually transmitted agents, distributed as follows: 50 (21.9%) women, 129 (56.6%) heterosexual men and 49 (21.5%) men who had sex with men (MSM). The average age of the women was 30 years, and that of both groups of men was 36 years. The microorganisms most frequently identified in women were: C. trachomatis (A-K) in 28.6%, followed by herpes simplex virus type 2 (HSV-2) in 26.8% and N. gonorrhoeae in 17.9%. In heterosexual men they were C. trachomatis (A-K) in 37.5%, N. gonorrhoeae in 21.5% and HSV-2 in 18.7%. In MSM they were C. trachomatis (L1-L3) in 32.7%, followed by N. gonorrhoeae in 27.6%, and C. trachomatis (A-K) and HSV-2, both in 13.8%. Polymicrobial infection was identified in 11 heterosexual men, 8 MSM, and 6 women. Conclusions. C. trachomatis (A-K) was the most prevalent STI-causing microorganism, followed by N. gonorrhoeae in both groups of men, and HSV-2 in women, very similar to that reported worldwide. The multiplex PCR test allows the detection of polymicrobial infections commonly associated with STIs and the diagnosis is accurate and reliable, even in asymptomatic patients

Reverse-Transcription Loop-Mediated Isothermal Amplification and alternative protocols for lower cost, large-scale COVID-19 testing: lessons from an emerging economy / Amplificación isotérmica mediada por bucle de transcripción inversa y protocolos alternativos para pruebas COVID-19 a gran escala y de bajo costo: lecciones de una economía emergente

Abstract Introduction: Most successful cases of COVID-19 pandemic mitigation and handling have relied on extensive reverse-transcription quantitative polymerase chain reaction (RT-qPCR). However, many emerging economies have struggled with current molecular testing demands due to economic, technical and technological constraints. Objective: To define a potential diagnostic protocol to increase testing capacity in current and post-pandemic conditions. Methods: We reviewed the literature, patents and commercial applications, for alternatives. Results: We found a good potential in saliva samples, viral inactivation and quick RNA extraction by heating; the use of an isothermal technology such as loop mediated isothermal amplification (LAMP) and naked eye test-result visualization by in-tube colorimetry or turbidity. Conclusions: Saliva samples with quick RNA extraction by heating and colorimetric LAMP are promising options for countries with economic and infrastructure limitations.

Resumen Introducción: La mayoría de los casos exitosos de mitigación y manejo de la pandemia de COVID-19 se han dado mediante pruebas basadas en la reacción en cadena de la polimerasa cuantitativa (RT-qPCR por sus siglas en inglés). Sin embargo, muchas economías emergentes han tenido problemas con las demandas actuales de pruebas moleculares debido a limitaciones económicas, técnicas y tecnológicas. Objetivo: Definir un protocolo de diagnóstico potencial para aumentar la capacidad de prueba en las condiciones actuales y posteriores a la pandemia. Métodos: Revisamos la literatura, las patentes y las aplicaciones comerciales, en busca de alternativas. Resultados: Encontramos un buen potencial en muestras de saliva, inactivación viral y extracción rápida de ARN por calentamiento; el uso de una tecnología isotérmica como la amplificación isotérmica mediada por horquillas (LAMP, por sus siglas en inglés) y la visualización del resultado de la prueba a simple vista mediante colorimetría o turbidez en el tubo. Conclusiones: Las muestras de saliva con extracción rápida de ARN por calentamiento y LAMP colorimétrico son opciones prometedoras para países con limitaciones económicas y de infraestructura.

Validación clínica de la prueba RT-LAMP para el diagnóstico rápido del SARS-CoV-2 / Clinical validation of the isothermal RT-LAMP test for rapid diagnosis of SARS-CoV-2

Introducción. Desde el primer reporte en la provincia de Wuhan (China) en el año 2019, el SARS-CoV-2 se ha diseminado por todo el mundo, provocando un enorme impacto en la salud pública. Para su diagnóstico, la Organización Mundial de la Salud ha incentivado el desarrollo de pruebas rápidas, de simple ejecución, sensibles y específicas, que complementan la RT-qPCR como prueba de referencia. La prueba RT-LAMP ha mostrado ser una excelente alternativa para la detección del SARS-CoV-2 en diferentes biofluidos. Objetivo. Validar la técnica RT-LAMP colorimétrica en muestras de hisopado nasofaríngeo previamente confirmadas por RT-qPCR, usando el protocolo Charité, Berlín, Alemania. Materiales y métodos. Un total de 153 muestras de hisopado nasofaríngeo de individuos con sospecha de COVID-19 se sometieron a RT-qPCR y RT-LAMP, usando un estuche comercial colorimétrico (NEB, Germany). La RT-LAMP se practicó con las muestras de ARN extraídas del hisopado nasofaríngeo y con muestras crudas sin previa extracción de ARN. El resultado fue evaluado por un simple cambio de color en la reacción. Resultados. La sensibilidad y especificidad de la técnica RT-LAMP para detectar el gen N del SARS-CoV-2 mediante un set de cebadores previamente reportados (set de Broughton), arrojó valores de 0,97 (0,85-1,00) y 0,81 (0,65-0,92), respectivamente, con un intervalo de confianza del 95%. Otro set de cebadores dirigidos contra otra región del mismo gen (set de Lalli) arrojó valores de sensibilidad y especificidad de 0,96 (0,78-1,00) y 0,77 (0,55-0,92), respectivamente. Sin previa extracción de ARN, se encontró que la sensibilidad fue del 0,95 (0,74-1,00) y la especificidad del 0,88 (0,64-0,99). Conclusiones. Estos resultados evidencian que la técnica RT-LAMP podría considerarse una prueba diagnóstica rápida, de fácil ejecución, libre de equipos sofisticados, sensible y específica, para el diagnóstico del SARS-CoV-2 en muestras de hisopados nasofaríngeos.

Introduction: Since the first report in Wuhan (China) in 2019, the SARS-CoV-2 virus has spread throughout the world, with a significant impact in public health. To contain its transmission, the WHO has encouraged the development of rapid, simple, sensitive and specific tests that complement qRT-PCR, as the gold standard. RT-LAMP has shown to be a good alternative to detect SARS-CoV-2 in different fluid samples. Objective: To validate the colorimetric RT-LAMP technique using two sets of primers targeting N gene of SARS-CoV-2 in 117 nasopharyngeal swab samples previously confirmed by RT-qPCR, using the Charité/Berlin protocol. Material and methods: A total of 153 nasopharyngeal swab samples from individuals with suspected COVID-19 were subjected to qRT-PCR and RT-LAMP using a commercial colorimetric kit (NEB, Germany). RT-LAMP was performed using both extracted RNA samples and raw samples without prior RNA extraction, and the result was assessed by a simple color change in the reaction. Results: Sensitivity and specificity for the previously reported RT-LAMP primers (Broughton set) targeting N gene of SARS-CoV-2 were 0.97 (0.85-1.00) and 0.81 (0.65-0.92) respectively, with CI95%. The Lalli primers targeting another region of the N gene used showed a sensitivity value of 0.96 (0.78-1.00) and a specificity of 0.77 (0.55-0.92). Without RNA extraction we found a sensitivity value of 0.95 (0.74, 1.00) and a specificity of 0.88 (0.64, 0.99). A sensitivity value of 0.95 (0.74-1.00) and a specificity 0.88 (0.64-0.99) were found without prior RNA extraction. Conclusion: Taking together, the results showed that RT-LAMP technique could be considered as a rapid diagnostic test, easy to perform, free of sophisticated equipment, sensitive and specific to diagnose SARS-CoV-2 in nasopharyngeal swabs with and without prior RNA extraction, allowing its implementation in places with scarce resources.

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

GeneXpert como método de diagnóstico de la tuberculosis en Santiago de Cuba / GeneXpert as diagnostic method of tuberculosis in Santiago de Cuba

Introducción: La tuberculosis es un problema de salud en el mundo, por lo que se necesitan métodos como el GeneXpert para realizar un diagnóstico rápido y seguro. Objetivo: Determinar la precisión del GeneXpert como método para el diagnóstico de la tuberculosis en Santiago de Cuba en relación con los estudios tradicionales. Métodos: Se efectuó un estudio descriptivo y transversal desde diciembre de 2018 hasta igual mes de 2019 de 31 pacientes a quienes se les realizaron los 3 métodos para el diagnóstico de la tuberculosis. Se utilizaron variables, tales como edad, sexo, muestras estudiadas, positividad de los métodos utilizados, así como concordancia entre el GeneXpert y el cultivo mediante el índice de Kappa. Resultados: En la serie predominaron los pacientes mayores de 50 años (48,4 %), el sexo masculino (66,7 %) y el esputo como muestra más representativa (80,6 %); asimismo, el cultivo resultó positivo en 32,3 % y el GeneXpert en 22,6 %. En tanto, se diagnosticó tuberculosis en 11 pacientes y el índice de Kappa fue de 0,58. Conclusiones: La determinación de GeneXpert en el diagnóstico de la tuberculosis es precisa, dada su sensibilidad y especificidad altas en relación con los estudios tradicionales de esputo y cultivo, lo cual se confirmó por la elevada concordancia del índice de Kappa.

Introduction: Tuberculosis is a health problem worldwide, reason why methods as the GeneXpert are needed to carry out a quick and safe diagnosis. Objective: To determine the accuracy of GeneXpert as a method for the diagnosis of tuberculosis in Santiago de Cuba in connection with the traditional studies. Methods: A descriptive and cross-sectional study was carried out from December, 2018 to the same month in 2019 of 31 patients to whom the 3 methods for the diagnosis of tuberculosis were carried out. Some variables were used, such as age, sex, studied samples, positivity of the used methods, as well as consistency between the GeneXpert and the culture by means of the Kappa index. Results: In the series there was a prevalence of the patients over 50 years (48.4 %), male sex (66.7 %) and sputum as more representative sample (80.6 %); also, the culture was positive in 32.3 % and GeneXpert in 22.6 %. As long as, tuberculosis was diagnosed in 11 patients and the Kappa index was 0.58. Conclusions: The determination of GeneXpert in the diagnosis of tuberculosis is necessary, given its high sensibility and specificity in connection with the traditional studies of sputum and culture, which was confirmed due to the high consistency of the Kappa index.

Plataforma laboratorial para monitorear el SARS-CoV-2 basada en la vigilancia de influenza y otros virus respiratorios en Perú / Laboratory platform for monitoring SARS-CoV-2 based on surveillance of influenza and other respiratory viruses in Peru

RESUMEN En el Perú, la pandemia de la COVID-19 ha evidenciado la utilidad de tener un sistema de vigilancia laboratorial estructurado y en funcionamiento desde hace 22 años, basado en la vigilancia de influenza; inicialmente en modalidad de unidades centinela, y después fortaleciéndose e innovándose, con recursos propios y con apoyo externo, para generar información de calidad. Se han implementado avances biotecnológicos para la confirmación diagnóstica e incrementado las capacidades de la red nacional de laboratorios, manteniendo la eficiencia, considerando las diversas y complejas realidades de los niveles regionales, y superando dificultades de comunicación y articulación entre instituciones. Resulta necesario consolidar este sistema, con trabajo colaborativo y coordinado entre sus componentes, impulsando su eficacia y oportunidad y promoviendo la vigilancia genómica de nuevos virus y variantes, como actualmente ocurre con el SARS-CoV-2.

ABSTRACT In Peru, the COVID-19 pandemic demonstrated the usefulness of having a structured laboratory surveillance system that has been operational for 22 years, based on influenza surveillance; initially in the form of sentinel units, and later strengthened and innovated, with its own resources and with external support, to provide quality information. Biotechnological advances have been implemented for diagnostic confirmation and the capacity of the national laboratory network has been expanded, maintaining efficiency, considering the diverse and complex realities of each region, and overcoming difficulties regarding communication and articulation between institutions. It is necessary to consolidate this system, with collaborative and coordinated work between its components, boosting its effectiveness and timeliness and promoting genomic surveillance of new viruses and variants, as is currently the case with SARS-CoV-2.

Reação em Cadeia da Polimerase (PCR) no diagnóstico complementar da toxoplasmose congênita: uma revisão bibliográfica / Polymerase Chain Reaction (PCR) in the complementary diagnosis of congenital toxoplasmosis: a bibliographic review

A toxoplasmose é uma doença de ampla distribuição geográfica, sendo um grave problema de saúde pública. A infecção primária durante a gravidez pode causar infecção congênita ao feto e gerar consequências graves. Neste contexto, a reação em cadeia da polimerase (PCR) é atualmente um dos métodos mais eficientes para investigação fetal em casos de suspeita de infecção. Portanto, o objetivo do presente estudo foi realizar uma revisão bibliográfica sobre a toxoplasmose congênita e seu diagnóstico molecular através da PCR. Para tanto, foram pesquisados artigos publicados no período de janeiro de 2010 a abril de 2021 em diferentes bases de dados usando os descritores "Reação em Cadeia da Polimerase", "Toxoplasma gondii" e "Toxoplasmose congênita". A partir da pesquisa, foi possível verificar que a combinação de métodos sorológicos com a realização da PCR, principalmente no líquido amniótico, constitui uma importante ferramenta para o diagnóstico antenatal e pós-natal da toxoplasmose congênita. Além disso, a PCR em tempo real parece não apresentar melhores resultados em comparação à PCR convencional. Não obstante, estudos voltados à padronização da técnica visando melhor sensibilidade são necessários, uma vez que o diagnóstico da toxoplasmose gestacional/congênita permite a implementação do tratamento a fim de minimizar as consequências da infecção ao neonato.

Toxoplasmosis is a disease of wide geographical distribution, being a serious public health concern. The primary infection during pregnancy may cause congenital infection of the infant and lead to serious consequences. In this context, the polymerase chain reaction (PCR) is currently one of the most efficient methods for fetal investigation in cases when infection is suspected. Therefore, the present study aimed to conduct a literature review on congenital toxoplasmosis and its molecular diagnosis by PCR. Thus, the research for papers published between January 2010 and April 2021 was conducted in different databases, using the terms "polymerase chain reaction", "Toxoplasma gondii" and "congenital toxoplasmosis". It was possible to verify that the combination of serological methods and PCR, mainly of the amniotic fluid, is a valuable tool the antenatal and post-natal diagnosis of congenital toxoplasmosis. Furthermore, real time PCR seems to have no better results in comparison to conventional PCR. Nevertheless, studies related to standardization of the technique aiming higher sensitivity are necessary since the diagnosis of gestational/congenital toxoplasmosis enables the treatment in order to minimize the consequences of the infection to the infant.

Implementación de un laboratorio de nivel de bioseguridad 2 para diagnóstico molecular de SARS-COV-2 en Puerto Iguazú, Misiones / Implementation of a biosafety level 2 laboratory for molecular diagnosis of SARS-COV-2 in Puerto Iguazú, Misiones

INTRODUCCIÓN: La pandemia de COVID-19 ha creado desafíos sin precedentes para los laboratorios de todo el mundo. Los gobiernos nacionales y subnacionales se vieron en la necesidad imperiosa de adaptar instituciones para la detección de SARS-CoV-2 en respuesta a la emergencia sanitaria declarada en 2020. Con el objetivo de aumentar la capacidad de diagnóstico en la provincia de Misiones, se implementó un laboratorio de diagnóstico de excelencia dentro del Instituto Misionero de Biodiversidad. Este artículo comparte los pasos realizados y los desafíos enfrentados, con el fin de que sirva de guía a otras instituciones. MÉTODOS: Las etapas para establecer el laboratorio fueron: adquisición de equipamientos/reactivos, adaptación/renovación del laboratorio, incorporación y capacitación del recurso humano, establecimiento de buenas prácticas y bioseguridad, selección de la metodología de extracción y detección, implementación de controles de calidad y habilitación. RESULTADOS: Desde su habilitación el Laboratorio de Análisis Integral procesó 1186 muestras biológicas de casos sospechosos de COVID-19 y se convirtió así en el segundo laboratorio de referencia provincial. DISCUSIÓN: La eficiente articulación entre el Instituto Misionero de Biodiversidad y el Ministerio de Salud de Misiones logró la rápida implementación de este laboratorio de alta complejidad en una región de importancia epidemiológica.

Investigation of the parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Amblyomma sculptum (Acari: Ixodidae) ticks in the municipality of Salto, São Paulo, Brazil

The presence of capybaras (Hydrochoerus hydrochaeris) in urban and periurban areas has caused increased numbers of cases of Brazilian spotted fever. With the aim of investigating the presence of the parasitoid Ixodiphagus hookeri in Amblyomma sculptum ticks in the municipality of Salto, state of São Paulo, samples were collected monthly from 14 sites. Thirty samples were placed in containers for observation of the emergence of microhymenopterans and 88 samples were subjected to molecular testing to identify the presence of I. hookeri DNA. Neither dissections nor observation of emergence indicated any presence of I. hookeri larvae in ticks. Samples subjected to polymerase chain reaction (PCR) amplification of the mCOX I region of I. hookeri did not reveal its presence, although fragments corresponding to mRNA 16S of Amblyomma sculptum ticks were amplified in all samples tested.

Estudo piloto de triagem usando a Análise da Curva de Dissociação de Alta Resolução Sensível à Metilação (MS -HRM) em swabs orais para detectar a Síndrome de Prader-Willi / Pilot screening study using Methylation Sensitive High Resolution Dissociation Curve Analysis (MS -HRM) on oral swabs to detect Prader-Willi Syndrome

A síndrome de Prader-Willi (SPW) é uma desordem genética complexa, caracterizada por deleções, dissomia uniparental materna ou defeito no centro de imprinting no alelo paterno do cromossomo 15. As perdas de funções de genes específicos da região 15q11 afetam múltiplos sistemas corporais. O diagnóstico da SPW é difícil de ser realizado com base apenas no exame clínico e envolve a realização de diversas técnicas de biologia molecular para a completa elucidação da etiologia genética, tornando todo o processo laborioso, demorado e custoso. A realização de um teste molecular que permita um diagnóstico rápido e preciso é de vital importância para um melhor prognóstico para paciente. A coleta bem-sucedida de amostras e a extração de DNA de swabs são alternativas não invasivas e confiáveis, tanto para os pacientes quanto para os profissionais que realizarão a coleta destas amostras. Neste trabalho foi possível demonstrar um método simples de coleta de amostras e extração de DNA, que possui baixo custo, é eficaz, fácil e rápido, que fornece uma quantidade e qualidade suficiente de DNA para a execução do MS-HRM, qPCR e sequenciamento. Uma comparação dos procedimentos de extração mostra que o método simples de extração de NaCl é o mais adequado para extração de DNA de amostra bucal coletada através de swab. Neste trabalho foi demonstrado um método simples de coleta de amostras através do swab e extração de DNA com baixo custo e boa qualidade do DNA.

Prader-Willi syndrome (PWS) is a complex disorder, uniparental by deletions, dissociated from no imprint defect in any of the chromosomes 15. As gene variants of the genetic region of the 15q11 region, the diagnosis of PWS is challenging to perform based on clinical examination alone. It involves the performance of several molecular biology techniques for the complete elucidation of genetics, determining the entire laborious, time-consuming, and costly process. The performance of a molecular test allows a quick diagnosis, which is vital for a better prognosis for the patient. Successful sample collection and DNA collection from swabs are non-invasive alternatives for patients and practitioners performing probable sample collection. In this work, it was possible to demonstrate a simple sample collection and DNA method, which has a low cost, is effective, easy, and fast, and provides a sufficient quantity and quality of DNA for the execution of MS-HRM, qPCR, and sequencing. A comparison of the extraction procedures shows that the simple NaCl extraction method is the most appropriate for extracting DNA from a buccal sample collected via swab. In this work, a simple method for swab sampling and extracting DNA was demonstrated, with low cost and good DNA quality.

Estudo exploratório da Síndrome Hipoplasia Cartilagem-Cabelo no Brasil: uma abordagem genômica, proteômica e de análise estrutural de RNA / Exploratory study of Hypoplastic Cartilage-Hair Syndrome in Brazil: a genomic, proteomic and RNA structural analysis approach

A Hipoplasia Cartilagem-Cabelo (do inglês, Cartilage Hair Hypoplasia; CHH) é uma doença autossômica recessiva descrita por McKusick et al., em 1964 em crianças da comunidade Amish. Clinicamente, os pacientes apresentam displasia óssea metafisária em diferentes graus de gravidade. Além disso, também pode ser observado hipotricose e imunodeficiência. Já no âmbito molecular, a condição se caracteriza por variantes patogênicas do gene RMRP. Apesar de ter sido delineada há quase 60 anos, ainda não existe uma correlação genótipo-fenótipo bem compreendida. Este trabalho é uma continuação do trabalho de mestrado realizado no IFF/Fiocruz, durante 2017 e 2018, no qual foi descrita uma coorte de 23 pacientes brasileiros com CHH em que foram encontradas diversas variantes patogênicas. Dentre estas, a variante g.196C>T destacou-se por sua elevada frequência no grupo estudado, diferentemente do observado em pacientes de outras nacionalidades, sugerindo um possível efeito fundador (EF) para a população brasileira. Esse trabalho teve por objetivo realizar diferentes ensaios moleculares para auxiliar na compreensão da CHH, além de investigar a hipótese de uma origem ancestral comum da variante g.196C>T. O estudo foi dividido em 4 eixos, sendo três relacionados às pesquisas de caráter exploratório dos mecanismos da doença e um dedicado à análise do EF. Dentro deste último, utilizando um painel de marcadores do tipo TAG SNPs cuidadosamente selecionados, foi observado que cromossomos de diferentes regiões brasileiras carregando o nucleotídeo T na posição196 do gene RMRP compartilharam o haplótipo T/C/G/A (16/17 haplótipos), apontando para uma origem comum desta substituição de base no gene RMRP. Adicionalmente, foram realizadas análises de proteômica comparativa, evidenciando que o perfil proteômico dos leucócitos de pacientes e controles expressam proteínas que traduzem vias moleculares distintas, além de apresentar diferenças de expressão de proteínas importantes relacionadas aos fenótipos clínicos da doença. Também foram realizados ensaios de RT-qPCR que mostraram que tanto os níveis do RNA RMRP, quanto dos dois pequenos RNAs derivados de RMRP, estavam significativamente reduzidos nos pacientes em relação ao grupo controle. Por fim, com o intuito de tentar prever o impacto das variantes na estrutura tridimensional do RNA, foi realizada uma análise in silico que mostrou que as alterações patogênicas identificadas nos pacientes ocorreram tanto em regiões conservadas entre espécies de mamíferos quanto em domínios essenciais para o complexo ribonucleoproteico. Duas alterações estão localizadas em regiões associadas à biogênese dos pequenos RNAs derivados de RMRP. Além disso, foi possível observar que certas variantes podem alterar o pareamento de bases e a topologia da estrutura das alças da molécula, o que poderia influenciar na montagem do complexo RNAse MRP. Em conjunto, os dados desta tese lançam luz sobre diversos pontos ainda não explorados para CHH, além de dar suporte para novos estudos que tenham por objetivo viabilizar uma medicina de precisão, contribuindo para minimizar os impactos da doença e para a promoção da qualidade de vida dos pacientes.

Cartilage Hair Hypoplasia (CHH) is an autosomal recessive disease described by McKusick et al. in 1964 in the Amish community. Clinically, patients present metaphyseal bone dysplasia in different degrees of severity. In addition, hypotrichosis and immunodeficiency may also be present. At the molecular level, the condition is characterized by pathogenic variants of the RMRP gene. Although CHH has been described more than 60 years ago, the genotype-phenotype correlation is still not well-understood. This work is a continuation of the dissertation carried out at IFF/Fiocruz, during 2017 and 2018, in which a cohort of 23 Brazilian patients with CHH was described and several pathogenic variants were found. Among these, the high frequency of g.196C>T variant in the studied group called our attention, unlike that observed in patients of other nationalities, suggesting a possible founder effect (EF) in the Brazilian population. This work aimed to perform different molecular assays to aid in the understanding of CHH, in addition to investigating the hypothesis of a common ancestral origin of the g.196C>T variant. The study was divided into 4 axes, three related to exploratory research on disease mechanisms and one dedicated to the analysis of EF. Within the latter, using a carefully selected panel of TAG SNPs markers, it was observed that chromosomes from different Brazilian regions carrying T at nucleotide 196 of the RMRP gene shared the T/C/G/A haplotype (16/17 haplotypes), indicating a common origin of this base substitution at position 196 of the RMRP gene. Additionally, comparative proteomics analysis was performed, showing that the proteomic profile of leukocytes from patients and controls express proteins that translate distinct molecular pathways, in addition to presenting differences in the expression of proteins related to important clinical phenotypes of the disease. RT-qPCR assays were also performed, which showed that both RMRP RNA levels and the two RMRP-derived small RNAs were significantly reduced in patients compared to the control group. Finally, in order to try to predict the impact of the variants on the three-dimensional structure of the RNA, an in silico analysis was performed which showed that the pathogenic alterations identified in the patients occurred both in regions conserved between mammalian species and in domains essential for the ribonucleoprotein complex. Two alterations are located in regions associated with the biogenesis of RMRP-derived small RNAs. In addition, it was possible to observe that certain variants can change the base pairing and the topology of the structure of the molecule's loops, which could influence the assembly of the RNAse MRP complex. Together, the data from this thesis shed light on several points not yet explored for CHH, in addition to providing support for new studies that aim to enable a precision medicine toward CHH patients, helping to minimize the impacts of the disease and to promote quality of life of patients.

Tuberculose pulmonar causada pelo uso de infliximab em paciente com Doença de Crohn / Pulmonary tuberculosis caused by the use of infliximab in a patient with Crohn's Disease

A doença de Crohn é uma patologia caracterizada pela inflamação transmural do trato gastrointestinal, compondo o espectro das doenças inflamatórias intestinais. Nos casos mais graves, dispõe de tratamento com uso de agentes biológicos e imunomoduladores que podem à reativação ou exacerbação de doenças infecciosas preexistentes. Este relato de caso trata de uma paciente do sexo feminino de 24 anos, diagnosticada com Doença de Crohn há 10 anos, evoluindo com necessidade de tratamento com infliximab e, após período de menos de 1 ano, apresentou odinofagia progressiva, dor abdominal e diarreia, além de perda ponderal, sudorese noturna e febre diária. Tomografia computadorizada de tórax evidenciou árvore em brotamento, sendo confirmado diagnóstico de tuberculose pulmonar pelo Teste Rápido Molecular no escarro e provável tuberculose laríngea e intestinal.

Crohn's disease is a pathology characterized by transmural inflammation of the gastrointestinal tract, comprising the spectrum of Inflammatory Bowel Diseases. In the most severe cases, treatment using biological agents and immunomodulators may be available, which can lead to the reactivation or exacerbation of preexisting infectious diseases. This case report is about a 24-year-old female patient, diagnosed with Crohn's disease 10 years ago, evolving in need of treatment with Infliximab and, after a period of less than 1 year, she presented progressive odynophagia, abdominal pain and diarrhea, in addition to weight loss, night sweats and daily fever. Chest computer tomography showed a tree in bud, and the diagnosis of pulmonary tuberculosis was confirmed by the Rapid Molecular Test in the sputum and probable laryngeal and intestinal tuberculosis.