Diagnóstico simultáneo de enfermedades infectocontagiosas de importancia para la industria porcina : desarrollo de un ensayo Multiplex RT-PCR en tiempo real para la detección del virus del Síndrome reproductivo y respiratorio porcino y el virus de Influenza A.
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Fecha
2018
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
El Complejo respiratorio porcino (CRP) se caracteriza por ser una enfermedad respiratoria de
origen multifactorial, la cual afecta principalmente la ganancia de peso diaria de los animales
generando pérdidas significativas en la producción de las empresas. Se ha reportado que el virus
del Síndrome Reproductivo y Respiratorio Porcino (PRRSV) y el virus de Influenza A (IAV),
son los principales virus que generan enfermedades respiratorias e inciden en el desarrollo del
CRP en planteles industriales en Estados Unidos. Actualmente en Chile, IAV circula de forma
endémica en los planteles porcinos, y a pesar de que PRRSV se encuentra bajo un programa de
erradicación, aún hay planteles donde persiste la infección. Debido a la relevancia y pérdidas que
generan éstos virus en los planteles industriales, se han diseñado kit comerciales que permiten
realizar la detección de IAV o PRRSV, no obstante, actualmente no existe una herramienta
metodológica que permita diagnosticar simultáneamente estos patógenos en las empresas
productoras de cerdos. El objetivo principal de este trabajo fue desarrollar un ensayo Multiplex
de RT-PCR en tiempo real para la detección simultánea de PRRSV e IAV en cerdos. El ensayo
Multiplex demostró tener una alta especificidad y sensibilidad analítica, detectando 10-100
Unidades formadora de placa/ml (UFP/ml) de IAV y 1 UFP/ml de PRRSV. Además, se realizó la
validación clínica del ensayo utilizando 803 muestras de campo provenientes de diferentes
planteles industriales de Chile. Del total de muestras, se utilizaron 680 para la construcción de
una tabla de contingencia de 2x2 en donde se confrontaron los resultados de detección de IAV
obtenidos a través de la prueba de referencia versus los resultados del ensayo Multiplex RTqPCR. De modo que se determinaron los valores de sensibilidad diagnóstica 71,79% (IC 63,21 a
80,38%), especificidad diagnósticas 99,47% (IC 98,79 a 100%), valor predictivo positivo
96,55% (IC 92,14 a 100%) y valor predictivo negativo 94,44% (IC 92,51 a 96,36%). Además, se
calculó el índice de Youden (IY) que dio un valor de 0,71 (IC 0,63 a 0,79), todos estos
parámetros determinan un rendimiento robusto del ensayo Multiplex en la detección de IAV. Lo
cual se confirmó desde un ámbito clínico cuando se analizaron las razones de verosimilitud, que
dieron un RV+ de 134,74 (IC 43,34 a 418,88) y RV- de 0,28 (IC 0,21 a 0,38), determinado la
fuerte capacidad predictiva de la prueba diagnóstica. El ensayo Multiplex logró detectar
simultáneamente ambos patógenos en muestras clínicas y se validó con una alta capacidad
diagnóstica para IAV. Este estudio confirma el valor clínico de tener un ensayo que proporciona
un diagnóstico diferencial y oportuno de dos patógenos relevantes para la industria porcina en
Chile.
The Porcine respiratory disease complex (PRDC) is a multifactorial disease characterized by respiratory syndrome and poor growth in fattening swine, which has a negative impact on the swine industry. It has been reported that infections or coinfections with the virus of the Porcine Reproductive and Respiratory Syndrome (PRRSV) and Influenza A virus (IAV) play an important role in the development of PRDC in industrial facilities in the United States. Currently in Chile, IAV circulates endemically in swine farms, and although PRRSV is under an eradication program, there are still farms where infection persists. Despite the relevance of PRRS and IAV in the disease, there are no commercial kits that allow the simultaneous detection of both pathogens. Here, we developed a Multiplex real-time RT-PCR assay by designing primers and probes for the simultaneous detection of PRRSV and IAV in swine samples. This Multiplex assay demonstrated a high analytical specificity and sensitivity in the diagnosis of these viruses, detecting 10 - 100 plaque forming units/ml (PFU/ml) of IAV and 1 PFU/ml of PRRSV. To validate the assay with clinical samples, the assay was performed using 803 swine field samples obtained from different farms of Chile. Of the clinical samples 680 were used to form a 2x2 table to compare the results with a gold standard test used to detect IAV and estimated its diagnostic accuracy. The diagnostic sensitivity was 71.79% (CI 63.21 to 80.38%); whereas the diagnostic specificity was 99.47% (CI 98.79 to 100%), which validated the discriminative property of the test. The positive and negative predicted values was 96.55% (CI 92.14 to 100%) and 94.44% (CI 92.51 to 98.17%), respectively. In addition, the Youden’s index was 0.71 (CI 0.63 to 0.76), all these parameters indicate a robust performance of the assay. This was confirmed when we analyzed the likelihood ratios, which resulted in an LR+ of 134.74 (CI 43.34 to 418.88) and an LR- of 0.28 (CI 0.21 to 0.38), demonstrating the test’s strong predictive capacity. The Multiplex assay developed detects simultaneously both pathogens in clinical samples, and was validated to have a high capacity to make an accurate diagnosis. This study confirms the clinical value of having an assay that provides a differential and timely diagnosis of two relevant pathogens for the porcine industry in Chile.
The Porcine respiratory disease complex (PRDC) is a multifactorial disease characterized by respiratory syndrome and poor growth in fattening swine, which has a negative impact on the swine industry. It has been reported that infections or coinfections with the virus of the Porcine Reproductive and Respiratory Syndrome (PRRSV) and Influenza A virus (IAV) play an important role in the development of PRDC in industrial facilities in the United States. Currently in Chile, IAV circulates endemically in swine farms, and although PRRSV is under an eradication program, there are still farms where infection persists. Despite the relevance of PRRS and IAV in the disease, there are no commercial kits that allow the simultaneous detection of both pathogens. Here, we developed a Multiplex real-time RT-PCR assay by designing primers and probes for the simultaneous detection of PRRSV and IAV in swine samples. This Multiplex assay demonstrated a high analytical specificity and sensitivity in the diagnosis of these viruses, detecting 10 - 100 plaque forming units/ml (PFU/ml) of IAV and 1 PFU/ml of PRRSV. To validate the assay with clinical samples, the assay was performed using 803 swine field samples obtained from different farms of Chile. Of the clinical samples 680 were used to form a 2x2 table to compare the results with a gold standard test used to detect IAV and estimated its diagnostic accuracy. The diagnostic sensitivity was 71.79% (CI 63.21 to 80.38%); whereas the diagnostic specificity was 99.47% (CI 98.79 to 100%), which validated the discriminative property of the test. The positive and negative predicted values was 96.55% (CI 92.14 to 100%) and 94.44% (CI 92.51 to 98.17%), respectively. In addition, the Youden’s index was 0.71 (CI 0.63 to 0.76), all these parameters indicate a robust performance of the assay. This was confirmed when we analyzed the likelihood ratios, which resulted in an LR+ of 134.74 (CI 43.34 to 418.88) and an LR- of 0.28 (CI 0.21 to 0.38), demonstrating the test’s strong predictive capacity. The Multiplex assay developed detects simultaneously both pathogens in clinical samples, and was validated to have a high capacity to make an accurate diagnosis. This study confirms the clinical value of having an assay that provides a differential and timely diagnosis of two relevant pathogens for the porcine industry in Chile.
Notas
Proyecto de título (Ingeniero en Biotecnología)
Palabras clave
Enfermedades Transmisibles, Enfermedades de los Cerdos, Diagnóstico, Síndrome Reproductivo y Respiratorio Porcino, Virus de la Influenza Porcina