Desarrollo y validación de una reacción en cadena de la polimerasa múltiple para la identificación de los serogrupos B, C2, D y E de Salmonella enterica / Development and validation of a multiplex polymerase chain reaction for molecular identification of Salmonella enterica serogroups B, C2, D and E

Introducción. El esquema Kaufmann-White para la serotipificación de Salmonella, reconoce 46 antígenos O y 119 antígenos H, los cuales han permitido la caracterización de 2.541 serovares. La serotipificación es una herramienta epidemiológica útil en la identificación de serovares circulantes y estudio de brotes, sin embargo, presenta limitaciones técnicas, de interpretación de resultados y alto costo. Objetivo. Desarrollar una prueba de reacción en cadena de la polimerasa múltiple (PCR-M) como alternativa para identificar los serogrupos B, C2, D y E de Salmonella enterica. Materiales y métodos. Se desarrolló una PCR-M para detectar los genes rfbJ de los serogrupos B y C2 y wzx de los serogrupos D y E. Para estandarizar la PCR-M se probaron cepas de referencia de Salmonella pertenecientes a los serogrupos de estudio. Se incluyó el gen invA específico del género Salmonella como control interno de amplificación. La técnica fue validada con un estudio ciego que incluyó 400 aislamientos de Salmonella previamente serotipificados. Resultados. La PCR-M permitió identificar los serogrupos de Salmonella con resultados reproducibles (índice kappa=0,95). La sensibilidad de la prueba estuvo entre 98% y 100% y la especificidad entre 96% y 100%. Conclusiones. El polimorfismo de los genes rfbJ y wzx permitió desarrollar un método de tipificación molecular sensible, específico y reproducible, que podría servir de apoyo a la serotipificación para identificar serogrupos de Salmonella.

Introduction. The scheme Kauffman-White (KW) for serotyping of Salmonella recognizes 46 O antigens, and 119 H antigens, thereby permitting the characterization of 2,541 serotypes. The serotyping is a useful epidemiological tool in identifying circulating serotypes and to characterize outbreaks. However, the method presents technical limitations, difficulty in interpretation of results and high costs. Objective. A multiplex polymerase chain reaction test (M-PCR) was developed as an alternative method for the identification of serogroups B, C2, D, and E of Salmonella enterica. Materials and methods. The M-PCR detected Salmonella genes rfbJ of serogroups B and C2 and wzx of serogroups D and E. To standardize the M-PCR, reference strains of Salmonella serogroups were compared. Amplification of invA gender-specific gene of Salmonella was included as internal control of amplification. To validate the test, a blind study was conducted to identify by M-PCR 400 isolates that had been previously characterized by serology. Results. The M-PCR detected Salmonella serogroups with reproducible results (Kappa index=0.95). The sensitivity of the test was between 98% to 100% and specificity between 96% to 100%. Conclusions. The polymorphisms in the Salmonella genes rfbJ and wzx permitted the development of a method for molecular typing of Salmonella serogroups that was sensitive, specific and reproducible.

[Development and validation of a multiplex polymerase chain reaction for molecular identification of Salmonella enterica serogroups B, C2, D and E]. / Desarrollo y validación de una reacción en cadena de la polimerasa multiple para la identificación de los serogrupos B, C2, D y E de Salmonella enterica.

INTRODUCTION: The scheme Kauffman-White (KW) for serotyping of Salmonella recognizes 46 O antigens, and 119 H antigens, thereby permitting the characterization of 2541 serotypes. The serotyping is a useful epidemiological tool in identifying circulating serotypes and to characterize outbreaks. However, the method presents technical limitations, difficulty in interpretation of results and high costs. OBJECTIVE: A multiplex polymerase chain reaction test (M-PCR) was developed as an alternative method for the identification of serogroups B, C2, D, and E of Salmonella enterica. MATERIALS AND METHODS: The M-PCR detected Salmonella genes rfbJ of serogroups B and C2 and wzx of serogroups D and E. To standardize the M-PCR, reference strains of Salmonella serogroups were compared. Amplification of invA gender-specific gene of Salmonella was included as internal control of amplification. To validate the test, a blind study was conducted to identify by M-PCR 400 isolates that had been previously characterized by serology. RESULTS: The M-PCR detected Salmonella serogroups with reproducible results (Kappa index = 0.95). The sensitivity of the test was between 98% to 100% and specificity between 96% to 100%. CONCLUSIONS: The polymorphisms in the Salmonella genes rfbJ and wzx permitted the development of a method for molecular typing of Salmonella serogroups that was sensitive, specific and reproducible.

[Development and evaluation of a PCR method for diagnosis of Salmonella enteric fever, based on DNA sequences of the hilA gene]. / Desarrollo y evaluación de una prueba de reacción en cadena de la polimerasa (PCR), utilizando la secuencia del gen hilA para diagnóstico de fiebre entérica por Salmonella spp.

Typically, diagnosis of enteric fever due to Salmonella spp. is by bacterial isolation from blood culture; however, the blood culture method is slow, not always available, and not informative in patients with antibiotic treatment. Salmonella spp. uses the hilA gene (component of the pathogenicity island I) to invade epithelial cells and produce infection. Using the hilA gene sequence a PCR test was designed to detect Salmonella in blood samples. The sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR method were obtained by testing the blood samples from 34 patients with suspected of enteric fever. Presence of S. typhi was confirmed by blood culture. Blood samples were also tested from 35 patients with infections due to other non-Salmonella pathogens, again corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4; Enterobacter cloacae, 4). Control samples were obtained from 150 healthy volunteers. The S, SP, PPV and NPV for the PCR method were all 100%. The lowest number of colony forming units/ml detected by PCR in blood samples was 10.