Universal Multiplex Polymerase Chain Reaction-Restriction Fragment Length polymorphism (UMPCR-RFLP) for Rapid Detection and Species Identification of Fungal and Mycobacterial Pathogens

Abstract

Fungal and mycobacterial skin infections are common in immunocompromised patients and patients with febrile neutropenia, since the patients’ ability to control localized infection is diminished by the disease. The similarity of the lesions caused by these organisms conduces to difficulty of differential diagnosis. Although a histopathological examination and a microbial culture are standard methods for laboratory diagnosis of skin infection, the methods have drawbacks. Histopathological examination yields low positive results, while microbial culture is time-consuming and might result in no growth, causing delayed treatment. This study aimed to develop and evaluate an in-house rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the detection and identification of fungal and mycobacterial pathogens in resource-limited laboratories. A total of 26 typed species of human pathogenic fungi and 12 species of mycobacteria were used. Strain differentiation was analyzed by using multiplex PCR-RFLP. The internal transcript spacer region (ITS) of fungi and heat-shock protein 65 (hsp65) gene of mycobacteria were amplified using ITS1-ITS4 and Tb11-Tb12 universal primers, respectively. The RFLP patterns were examined at genus-specific and species-specific level. No cross-amplification was observed between fungal and mycobacterial tested strains, nor any specific binding between primers and human DNA. It was concluded that the multiplex PCR-RFLP method developed in this study can be used as a molecular diagnostic test for fungal and mycobacterial species identification. In the future, this technique may be useful for detecting fungal and mycobacterial infection directly from clinical specimens.