Loop-mediated isothermal amplification: A rapid and simple method for detection of fluconazole resistant Candida albicans vaginitis

Abstract

Background:Candidiasis is one of the most important opportunistic fungal infections. Time-saving antimicrobial susceptibility testing (AST) and molecular methods were developed to detect antimicrobial resistance. Loop-mediated isothermal amplification (LAMP) is a molecular diagnostic method performed isothermally with Bst DNA polymerase and four or six primers. Objective: Vitek identification of Candida isolates and determination of their AST were performed. However, it is time consuming; therefore, rapid and sensitive LAMP-based assay was tested as a method for phenotypic detection of resistance of Candida albicans isolates to fluconazole. Methods: Identification of Candida spp. isolated from vaginal swabs of non-pregnant women with vaginitis and minimal inhibitory concentration (MIC) values were done by Vitek2 system. Sensitive C. albicans isolates were inoculated into sabouraud´s dextrose broth without fluconazole while resistant strains were inoculated into broth with fluconazole and incubated at 37˚C. At end of 2nd, 4th and 6th h, cultures were harvested by 10 min centrifugation at 2,000 rpm and DNA was extracted by boiling. For LAMP, specific primers of C. albicans alpha-INT1 gene were used. Results: Out of 250 samples, 117 (46.8 %) were positive for Candida infections. Candida albicans isolates (57 isolates) were grouped into fluconazole-sensitive (12 isolates) and fluconazole-resistant (45 isolates). Fluconazole-resistant C. albicans could grow in presence of drug and positive LAMP reaction was obtained after a time interval similar to sensitive isolates. Conclusion: LAMP reaction allowed phenotypic detection of behavior of resistant strains in presence of antifungal agent. LAMP based evaluation of AST is superior to conventional methods and molecular detection of resistance genes.

Keywords

• loop-mediated isothermal amplification
• azole resistance
• candida