Desarrollo de un ensayo inmunoenzimatico (ELISA) para el diagnóstico de Paragonimus mexicanus
Fecha
2023
Autores
Zúniga Moya, María José
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Editor
Universidad Nacional, Costa Rica
Resumen
La paragonimiasis es una zoonosis parasitaria transmitida por alimentos y ocasionada por
trematodos del género Paragonimus. La enfermedad es considerada un serio problema de salud
pública en áreas endémicas. Costa Rica se considera región endémica para Paragonimus
mexicanus. Debido a que en Latinoamérica no se encuentra disponible una prueba diagnóstica
inmunológica el objetivo del presente estudio fue determinar la secuencia parcial de cisteínas
proteasas (CP) de P. mexicanus, a partir de ARN total, aislado de metacercarias y formas
adultas del parásito, para producir una CP recombinante, caracterizarla y estandarizar una
prueba inmunoenzimática (ELISA) para el diagnóstico de la paragonimiasis. En la primera
parte del trabajo se identificaron a partir de la secuenciación de 16 clones diez nuevas
secuencias de CPs, diferentes a todas las reportadas previamente en bases de datos públicas a
nivel mundial. Se subclonó, expresó y purificó una CP recombinante de 23 kDa de P.
mexicanus (PmCP), la cual presentó alto grado de similitud con la CP6 de Paragonimus
kellicotti. La PmCP6 recombinante fue utilizada como antígeno en Western blot, y se evaluaron
sueros de humanos positivos a Paragonimus westermani, P. mexicanus, Fasciola spp.,
Schistosoma spp., Clonorchis sp., Taenia solium, Toxocara canis, Toxocara spp., Spirometra
erinaceieuropaei, Leishmania spp., Entamoeba histolytica y sueros negativos a parasitosis. Los
sueros positivos a P. westermani y P. mexicanus reaccionaron con la proteína de 23 kDa,
formando una banda. La proteína no fue detectada por los sueros positivos a otras parasitosis,
tampoco los sueros negativos detectaron la proteína, concluyéndose que PmCP6 es específica
para ser utilizada en el diagnóstico serológico.
Para la estandarización del ELISA se utilizaron 39 sueros positivos a P. mexicanus y
Paragonimus westermani, 33 sueros positivos a otras parasitosis (fascioliasis,
esquistosomiasis, clonorchiasis, cisticercosis, toxocariasis, leishmaniosis, esparganosis y
amebiasis) y 140 sueros de pacientes sanos. El ELISA se estandarizó utilizando 0,125 μg/100μl
del antígeno PmCP6, bloqueando la placa con leche descremada al 10%, diluyendo los sueros
1:100 y utilizando el conjugado en una dilución de 1:40.000. La sensibilidad y especificidad
del ensayo fue 92,3% y 95,7%, respectivamente, se determinaron reacciones cruzadas con C.
sinensis (4/8), Spirometra (3/5) y E. histolytica (2/5). Esta es la primera vez que se reporta el
uso de una cisteína proteasa específica de P. mexicanus en una técnica inmunoenzimática. Se
recomienda utilizarla en el sistema salud para confirmar infecciones ectópicas de
paragonimiasis.
Paragonimiasis is a foodborne parasitic zoonosis caused by trematodes of the genus Paragonimus. The disease is considered a serious public health problem in endemic areas. Costa Rica is considered an endemic region for Paragonimus mexicanus. Since an immunological diagnostic test is not available in Latin America, the aim of this study was to determine the partial sequence of cysteine proteases (CP) of P. mexicanus from total RNA isolated from metacercariae and adult forms of the parasite, to produce a recombinant CP, characterize it and standardize an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of paragonimiasis. In the first part of the work, ten new CP sequences, different from all those previously reported in public databases worldwide, were identified from the sequencing of 16 clones. A 23 kDa recombinant CP from P. mexicanus (PmCP) was subcloned, expressed and purified, which showed a high degree of similarity to CP6 from Paragonimus kellicotti. Recombinant PmCP6 was used as antigen in Western blotting, and sera from humans positive for Paragonimus westermani, P. mexicanus, Fasciola spp, Schistosoma spp, Clonorchis sp, Taenia solium, Toxocara canis, Toxocara spp, Spirometra erinaceieuropaei, Leishmania spp, Entamoeba histolytica and parasitosis-negative sera were evaluated. The P. westermani and P. mexicanus positive sera reacted with the 23 kDa protein, forming a band. The protein was not detected by the sera positive to other parasitosis, neither the negative sera detected the protein, concluding that PmCP6 is specific to be used in the serological diagnosis. For ELISA standardization, 39 sera positive for P. mexicanus and Paragonimus westermani, 33 sera positive for other parasitoses (fascioliasis, schistosomiasis, clonorchiasis, cysticercosis, toxocariasis, leishmaniasis, sparganosis and amebiasis) and 140 sera from healthy patients were used. The ELISA was standardized using 0.125 μg/100μl of PmCP6 antigen, blocking the plate with 10% skim milk, diluting the sera 1:100 and using the conjugate at a dilution of 1:40,000. The sensitivity and specificity of the assay was 92.3% and 95.7%, respectively; cross-reactions with C. sinensis (4/8), Spirometra (3/5) and E. histolytica (2/5) were determined. This is the first time that the use of a P. mexicanus-specific cysteine protease is reported in an enzyme-linked immunosorbent assay. It is recommended for use in the health system to confirm ectopic infections of paragonimiasis.
Paragonimiasis is a foodborne parasitic zoonosis caused by trematodes of the genus Paragonimus. The disease is considered a serious public health problem in endemic areas. Costa Rica is considered an endemic region for Paragonimus mexicanus. Since an immunological diagnostic test is not available in Latin America, the aim of this study was to determine the partial sequence of cysteine proteases (CP) of P. mexicanus from total RNA isolated from metacercariae and adult forms of the parasite, to produce a recombinant CP, characterize it and standardize an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of paragonimiasis. In the first part of the work, ten new CP sequences, different from all those previously reported in public databases worldwide, were identified from the sequencing of 16 clones. A 23 kDa recombinant CP from P. mexicanus (PmCP) was subcloned, expressed and purified, which showed a high degree of similarity to CP6 from Paragonimus kellicotti. Recombinant PmCP6 was used as antigen in Western blotting, and sera from humans positive for Paragonimus westermani, P. mexicanus, Fasciola spp, Schistosoma spp, Clonorchis sp, Taenia solium, Toxocara canis, Toxocara spp, Spirometra erinaceieuropaei, Leishmania spp, Entamoeba histolytica and parasitosis-negative sera were evaluated. The P. westermani and P. mexicanus positive sera reacted with the 23 kDa protein, forming a band. The protein was not detected by the sera positive to other parasitosis, neither the negative sera detected the protein, concluding that PmCP6 is specific to be used in the serological diagnosis. For ELISA standardization, 39 sera positive for P. mexicanus and Paragonimus westermani, 33 sera positive for other parasitoses (fascioliasis, schistosomiasis, clonorchiasis, cysticercosis, toxocariasis, leishmaniasis, sparganosis and amebiasis) and 140 sera from healthy patients were used. The ELISA was standardized using 0.125 μg/100μl of PmCP6 antigen, blocking the plate with 10% skim milk, diluting the sera 1:100 and using the conjugate at a dilution of 1:40,000. The sensitivity and specificity of the assay was 92.3% and 95.7%, respectively; cross-reactions with C. sinensis (4/8), Spirometra (3/5) and E. histolytica (2/5) were determined. This is the first time that the use of a P. mexicanus-specific cysteine protease is reported in an enzyme-linked immunosorbent assay. It is recommended for use in the health system to confirm ectopic infections of paragonimiasis.
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Palabras clave
ENFERMEDADES INFECCIOSAS, INFECTIOUS DISEASES, SALUD PÚBLICA, PUBLIC HEALTH, ZOONOSIS, ZOONOSES, PARASITOLOGÍA VETERINARIA, VETERINARY PARASITOLOGY