Detection and Identification of Candida auris and Candida kefyr from Different Clinical Samples by Conventional and Molecular Methods with Determination of Their Antifungal Responses Using Sanger Sequencing

Abstract

Background: Candidiasis is an opportunistic fungal disease causing local or systemic invasive infections, especially in hospitalized and immunocompromised patients. Some of Candida species exhibit greater resistance to antifungals and their misidentification by available laboratory diagnostic methods lead to difficulty in control and treatment.

Aim of the study: To search for pathogenic C. auris and C. kefyr isolates from specimens taken up from inpatients and outpatients from different hospitals or private laboratory in Kirkuk city, to test their susceptibilities to various antifungals and the genetic background for their resistance pattern to the tested antifungals.

Materials and methods: The study was carried out in the period of  March/ 2022 to August/ 2023, comprising the collection of 1047 samples from four major hospitals and a private laboratory in Kirkuk city. Samples were cultured on Sabouraud dextrose agar plates, then all isolated Candida species were identified morphologically by direct and indirect methods, and the detection confirmed by molecular methods.  Antifungal susceptibility testing was performed using disc diffusion method, Etest and Vitek 2 system.

Molecular techniques that included conventional polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing were accomplished by targeting internal transcribed spacer region 1 (ITS1)-ITS2, domain 1 (D1) -D2 regions of 28S ribosomal DNA (rDNA), and ITS2- 28S rDNA for C. auris recognition from pink-colored Candida species. Futherly, ITS1-ITS4 region was amplificated for analysis of all isolates that had been identified as C. kefyr previously by phenotypic techniques. Ergosterol2 (ERG2), ERG3, ERG6 and ERG11 genes were subjected for studying the resistance profile of C. Kefyr isolates genotypically.  

Results: Among the total samples, 215 were positive for Candida species.      The pink-colored isolated species by Vitek 2 system were: C. rugosa 6/15 (40%), C. lipolytica 5/15 (33.3%), and C. kefyr 4/15 (26.7%). Finally after application of molecular analysis, C. auris has not been detected, and four isolates of C. kefyr were found, which represent 1.9% of the total 215 Candida isolates; two isolates from urine samples, and the others from wound swabs.

The disc diffusion method displayed amphotericin resistance of all C. kefyr isolates. Regarding azoles, all isolates were sensitive excluding one isolate that was found to be voriconazole and 5-flucytosine resistant.

The molecular analysis demonstrated that ERG3, ERG6 and ERG11 genes were expressed in all isolates. The ERG2 was not found to be expressed in isolate number 3, contrary to the FUR1 gene expression which was noted only in this isolate. On the other hand, isolate number 4 harbored one and two mutations in ERG2 and ERG6 genes separately. The ERG3 gene was highly mutated and included two, three and five mutations in isolates number 1, 2 and 4 respectively; while one and two mutations were found in isolates number 2 and 1 independently in the ERG11.

Conclusion: C. auris was not present. The incidence of Candidiasis caused by C. kefyr is low. There is extreme resistance of all four C. kefyr isolates to amphotericin B, which occurred by the contribution of mutations of at least more than one gene of the following: ERG2, ERG3, ERG6 and ERG11. Voriconazole and 5-flucytosine were inactive against one isolate; the expression of the FUR1 gene was responsible for resistance against the last antifungal.