Evaluación de los cebadores ITS3 e ITS4 para la detección de infecciones por Candida spp. en muestras de flujo vaginal y humor acuoso: Evaluation of ITS3 and ITS4 primers for detection of Candida spp. Candida spp. infections from vaginal simples and aqueous humor

Abstract

Background. Conventional methods for Candida identification are time consuming, require high amounts of the sample and in several cases the culture is mandatory. Aims. To evaluate two primers for the identification of Candida and to demonstrate their application for intraocular infections. Materials and methods. 17 Candida reference strains, 29 vaginal samples, 7 samples of aqueous humor and one of vitreous humor were studied. Three methods to lyse the Candida wall were analyzed: freezing-thawing, sonication and the use of lyticase. For nucleic acid purification the Wizard Genomics ?it was used; in the PCR the commercial kit Go Taq Green Master Mix and the primers ITS3 and ITS4 were used. Results. The best method for lysis was the enzymatic lysis with lyticase. The primers ITS3 and ITS4 identified all the Candida strains and there were no cross-reaction with other microorganisms. The sensitivity of the PCR was 100 fg. All the samples of vaginal flux and Candida strains were identified and only one sample of aqueous humor was positive for the genus Candida. In the other samples of aqueous humor toxoplasmosis was the definitive diagnosis. Conclusion. PCR amplification for Candida is a sensitive and specific technique for the diagnosis