Estandarización de la prueba de amplificación isotérmica mediada por bucles (LAMP) para la detección molecular del virus del chikungunya
Cargando...
Fecha
2023
Título de la revista
ISSN de la revista
Título del volumen
Editor
Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
Las enfermedades transmitidas por vectores (ETVs) afectan a millones de personas a nivel mundial, y se estima que por eventos migratorios y cambio climático la situación epidemiológica podría empeorar. Aedes aegypti es un mosquito capaz de transmitir virus tales como, Dengue, Mayaro, Zika y Chikungunya, generando impactos significativos en salud pública. Estas enfermedades generan un espectro clínico similar, lo que complica su rápido diagnóstico por criterio clínico. Por su parte, estas infecciones pueden ocasionar brotes epidémicos que afectan a comunidades enteras, lo que conlleva a la sobrecarga de los sistemas de salud locales, agotando sus recursos y dificultando la respuesta a otras emergencias médicas. Este impacto se hace evidente al observar que, en el período comprendido entre el 1 de enero y el 4 de marzo del 2023, se reportaron 113,447 casos de chikungunya en América latina, con un total de 51 fallecidos, este número representa un aumento cuatro veces mayor en comparación con el mismo periodo de 2022 [1]. El diagnóstico molecular y definitivo del virus del chikungunya (VCHIK) usualmente se lleva a cabo por qRT-PCR, una metodología que a pesar de su sensibilidad y especificidad es impráctica en centros de salud primarios, y está centralizado al Instituto Nacional de Salud. En este trabajo nos propusimos estandarizar una metodología que fuera capaz de detectar la presencia del virus del VCHIK de manera rápida, sencilla, precisa y económica. Para ello, se optimizó la técnica de amplificación isotérmica mediada por bucles con transcripción reversa (RT- LAMP) para identificar al VCHIK mediante una reacción colorimétrica que vira de rosado a amarillo en presencia del genoma viral. En una etapa inicial de validación se evaluaron varias diluciones de ARN del VCHIK que fueron generadas in vitro, con el objetivo de determinar el límite de detección de la prueba. Adicionalmente, como una forma de validación se simuló una infección adicionando diferentes diluciones del ARN del VCHIK sintetizado in vitro en una muestra de sangre total extraída de un paciente. Luego de contar con la sangre infectada se realizó una extracción del mismo ARN de VCHIK combinado con esta; a partir de esto se determinó el límite de detección de la prueba RT-LAMP reconociendo que esta es efectiva hasta concentraciones de 0,5 atogramo (ag) en un tiempo de 30 minutos. El experimento demostró que es posible identificar el virus en un entorno clínico real. Como controles en cada una de las pruebas realizadas se incluyó ARN del virus del dengue para estimar la especificidad de la prueba, se observó que en ningún caso la prueba RT-LAMP resultó positiva para dengue. A pesar de ser un estudio preliminar y en curso, este trabajo muestra el gran potencial de la prueba RT-LAMP para
la identificación molecular del virus en individuos febriles con diagnóstico presuntivo de VCHIK, lo que convierte a esta prueba en una valiosa herramienta para la vigilancia virológica de este virus, especialmente en áreas con recursos tecnológicos limitados y una infraestructura de salud insuficiente.
Vector-borne diseases (VTE) affect millions of people worldwide, and it is estimated that due to migratory events and climate change the epidemiological situation could worsen. Aedes aegypti is a mosquito capable of transmitting viruses such as Dengue, Mayaro, Zika and chikungunya, generating significant impacts on public health. These diseases generatea similar clinical spectrum, which complicates their rapid diagnosis based on clinical criteria. For their part, these infections can cause epidemic outbreaks that affect entire communities, which leads to the overload of local health systems, depleting theirresources and making it difficult to respond to other medical emergencies. This impact isevident when observing that, in the period between January 1 and March 4, 2023, 113,447 cases of chikungunya were reported in Latin America, with a total of 51 deaths, this number represents a four-fold increase. higher compared to the same period in 2022 [1]. The molecular and definitive diagnosis of CHIKV is usually carried out by qRT-PCR, a methodology that despite its sensitivity and specificity is impractical in primary health centers, and is centralized to the National Institute of Health. In this work we set out to standardize a methodology that would be capable of detecting the presence of the CHIKV virus quickly, simply, accurately and economically. To this end, the loop-mediated isothermal amplification technique with reverse transcription (RT-LAMP) was optimized to identify CHIKV through a colorimetric reaction that turns from pink to yellow in the presence of the viral genome. In an initial validation stage, several dilutions of CHIKV RNA that were generated in vitro were evaluated, with the objective of determining the limit of detection of the test. Additionally, as a form of validation, an infection was simulated by adding different dilutions of CHIKV RNA synthesized in vitro to a whole blood sample taken from a patient. After having the infected blood, an extraction of the same CHIKV RNA combined with it was carried out; From this, the detection limit of the RT-LAMP test was determined, recognizing that it is effective up to concentrations of 0.5 atogramo (ag) in a time of 30 minutes. The experiment demonstrated that it is possible to identify the virus in a real clinical setting. Dengue virus RNA was included as controls in each of the tests performed to estimate the specificity of the test. It was observed that in no case was the RT-LAMP test positive for dengue. Despite being a preliminary and ongoing study, this work shows the great potential of the RT-LAMP test for the molecular identification of the virus in febrile individuals with a presumptive diagnosis of CHIKV, which makes this test a valuable tool for the virological surveillance of this virus, especially in areas with limited technological resources and insufficient health infrastructure.
Vector-borne diseases (VTE) affect millions of people worldwide, and it is estimated that due to migratory events and climate change the epidemiological situation could worsen. Aedes aegypti is a mosquito capable of transmitting viruses such as Dengue, Mayaro, Zika and chikungunya, generating significant impacts on public health. These diseases generatea similar clinical spectrum, which complicates their rapid diagnosis based on clinical criteria. For their part, these infections can cause epidemic outbreaks that affect entire communities, which leads to the overload of local health systems, depleting theirresources and making it difficult to respond to other medical emergencies. This impact isevident when observing that, in the period between January 1 and March 4, 2023, 113,447 cases of chikungunya were reported in Latin America, with a total of 51 deaths, this number represents a four-fold increase. higher compared to the same period in 2022 [1]. The molecular and definitive diagnosis of CHIKV is usually carried out by qRT-PCR, a methodology that despite its sensitivity and specificity is impractical in primary health centers, and is centralized to the National Institute of Health. In this work we set out to standardize a methodology that would be capable of detecting the presence of the CHIKV virus quickly, simply, accurately and economically. To this end, the loop-mediated isothermal amplification technique with reverse transcription (RT-LAMP) was optimized to identify CHIKV through a colorimetric reaction that turns from pink to yellow in the presence of the viral genome. In an initial validation stage, several dilutions of CHIKV RNA that were generated in vitro were evaluated, with the objective of determining the limit of detection of the test. Additionally, as a form of validation, an infection was simulated by adding different dilutions of CHIKV RNA synthesized in vitro to a whole blood sample taken from a patient. After having the infected blood, an extraction of the same CHIKV RNA combined with it was carried out; From this, the detection limit of the RT-LAMP test was determined, recognizing that it is effective up to concentrations of 0.5 atogramo (ag) in a time of 30 minutes. The experiment demonstrated that it is possible to identify the virus in a real clinical setting. Dengue virus RNA was included as controls in each of the tests performed to estimate the specificity of the test. It was observed that in no case was the RT-LAMP test positive for dengue. Despite being a preliminary and ongoing study, this work shows the great potential of the RT-LAMP test for the molecular identification of the virus in febrile individuals with a presumptive diagnosis of CHIKV, which makes this test a valuable tool for the virological surveillance of this virus, especially in areas with limited technological resources and insufficient health infrastructure.
Descripción
Palabras clave
Diagnóstico molecular, Arbovirus, Virus del Chikungunya (VCHIK), Prueba isotérmica, RT- LAMP, Molecular diagnosis, Arbovirus, Chikungunya Virus (CHIKV), Isothermal test, RT-LAMP
