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

Background. Conventional methods for the identification of yeasts of the Candida genus require a lot of time, they need an important quantity of the sample and, in many cases, it is necessary to cultivate.
Goals. To evaluate a pair of primers for the identification of yeasts of the genus Candida and to demonstrate their potential application for the diagnosis of intraocular infections.
Materials and methods. Seventeen reference strains, 29 clinical isolates of vaginal discharge, 7 samples of aqueous humor and one of vitreous humor were used. We evaluated three methods for breaking the cell wall: freeze thawing, sonication, and the enzyme liticase. For the purification of the nucleic acid, the Wizard Genomics commercial kit was used in all three cases; in the PCR reaction, the Go Taq Green Master Mix commercial kit and the ITS3 and ITS4 primers at a concentration of 0.5 μM.
Results Of the three methods, the best results were the use of the enzyme liticase plus the commercial kit. With the ITS3 and ITS4 primers it was possible to identify the yeasts only at the genus level and there was no cross reaction with other microorganisms commonly found in different human tissue samples. The sensitivity was 100 fg. All isolates were identified by PCR from vaginal discharge and a sample of humor accus. For the other samples of aqueous humor the diagnosis was for other causative agents, among them, Toxoplasma sp.
Conclusion. We consider that this is an adequate methodology, which allows to identify yeasts of the genus