Low-Cost Tetraplex PCR for the Global Spreading Multi-Drug Resistant Fungus, Candida auris and Its Phylogenetic Relatives

Abstract

Candida auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii are closely related and highly multidrug resistant yeast pathogens. The high cost and low accuracy of current diagnostics may underestimate their prevalence, especially in medical resource-limited regions. In this study, we used 172 C. auris stains and its relatives and 192 other fungal strains to establish and validate a novel multiplex end-point PCR. A prospective and a retrospective clinical screenings using this assay were further performed in China and Iran respectively. We identified the first isolate of C. pseudohaemulonii in China and the first isolate of C. haemulonii in Iran from 821 clinical isolates in total, without any false positive. Animal models of C. auris and C. haemulonii were established for validation. The overall positive rates of the assay for mice blood and tissue were 28.6 and 92.9%, respectively. Compared with previously developed assays, our assay is more available and affordable to the developing countries, and may contribute to a better understanding of the epidemiology of C. auris and its relatives in these regions.

Keywords: Candida auris; animal model; clinical validation; end-point PCR; molecular diagnosis; multi-drug resistance.

FIGURE 1

Primer sequences and positions used in the tetraplex PCR. Uni-F: Universal forward primer for Candida auris, C. duobushaemulonii, C. haemulonii, and C. pseudohaemulonii; Du-R: C. duobushaemulonii specific reverse primer; Au-R: C. auris specific reverse primer; Ps-R: C. pseudohaemulonii specific reverse primer; Ha-R: C. haemulonii specific reverse primer.

FIGURE 2

Agarose gel electrophoresis (2% agarose) of PCR amplicons. Lanes 1–4 are C. auris (CBS 10913 type, 331 bp), C. duobushaemulonii (CBS 7798 type, 115 bp), C. haemulonii (CBS 5149 type, 696 bp) and C. pseudohaemulonii (CBS 10004 type, 576 bp), respectively. Lane M, HyperLadder^TM 100 bp (Bioline, London, United Kingdom).

FIGURE 3

Lanes B1–B7 are PCR results of blood sample from seven mice infected with C. auris; Lanes B8-B14 are PCR results of blood sample from seven mice infected with C. haemulonii; Lanes B15-B21 are PCR results of blood sample from seven mice infected with C. albicans; Lane BC1–4 are PCR results of four cultured strains from blood samples of all the 21 mice; Lanes T1–T7 are PCR results of kidney tissue sample from seven mice infected with C. auris; Lanes T8–T14 are PCR results of kidney tissue sample from seven mice infected with C. haemulonii; Lanes T15–T21 are PCR results of kidney tissue sample from seven mice infected with C. albicans; Lane TC1–7 are PCR results of seven cultured strains from kidney samples of mice infected with C. auris; Lane TC8–13 are PCR results of six cultured strains from kidney samples of mice infected with C. haemulonii; Lane TC14–19 are PCR results of six cultured strains from kidney samples of mice infected with C. albicans; Lane M, GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher, Waltham, MA, United States).