P019 A 5-year study on prevalence and molecular determinants of fluconazole -resistance in C. parapsilosis spp. complex
Authors: Sharma Vrinda , Paul Raees A. , Kaur Harsimran , Das Sourav , Choudhary Hansraj , Rudramurthy Shivaprakash M. , Chakrabarti Arunaloke , Ghosh Anup K. 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Objectives
Candida parapsilosis species complex is among the leading agents of invasive candidiasis, notably in neonates and transplant recipients. It exhibits intrinsically reduced susceptibility to echinocandins with increasing reports of acquired resistance to fluconazole from many centers. We evaluated antifungal susceptibility and molecular mechanisms of azole resistance in C. parapsilosis species complex, isolated in the last 5 years.

Methods
The isolates of C. parapsilosis species complex causing infections over a 5-year period (2017-2021) were included in the present study. Species identification of the isolates was performed using MALDI-TOF MS and sequencing of Internal Transcribed Spacer, ITS1. Antifungal susceptibility testing was performed by CLSI broth microdilution in accordance with standard operating procedure described in M27-A3 document. For amphotericin B, itraconazole, and posaconazole, the interpretation of the susceptibility data was done using epidemiological cut-off values provided in CLSI M59 document. The entire coding sequence of ERG11 gene in fluconazole-resistant isolates was PCR-amplified in four overlapping fragments. The sequencing of each fragment was performed by Sanger's bidirectional sequencing method using BigDye termination ready-reaction kit, 3.1. The sequences were assembled and proofread using Seqman software (Laser DNA, Applied Biosystems). The nucleotide sequences were translated into ERG11 protein using nucleotide translation tool, https://web.expasy.org/translate. Multiple sequence alignment with C. parapsilosis reference sequence was performed using BioEdit Sequence Alignment editor.

Results
A total of 580 C. parapsilosis complex clinical isolates were evaluated for antifungal susceptibility. C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis accounted for 457 (78.8%), 86 (14.8%), and 37 (6.3%), respectively. The isolation distribution revealed an increasing temporal trend over the years. A total of 40 (6.9%) isolates of the species complex exhibited reduced susceptibility to fluconazole, of which 23 were resistant (MIC ≥8 μg/ml) while 17 isolates exhibited susceptible dose-dependent phenotype (MIC, 4 μg/ml). Only two of the fluconazole-resistant isolates were cross-resistant to voriconazole. The resistant isolates were predominantly from adult patients [Median age, IQR; 47 (16-70) years.] The crude mortality rate in resistant cases was 56.5% (13/23). Interestingly, all the fluconazole-resistant isolates were C. parapsilosis sensu stricto, while C. orthopsilosis and C. metapsilosis isolates were susceptible. The fluconazole-resistance rate in the species complex and within the C. parapsilosis sensu stricto was 3.9% (23/599), 4.9% (23/466), respectively. For amphotericin B, the proportion of non-wildtype isolates was 17% (70/411), 25% (9/36), and 0% in C. parapsilosis sensu stricto, C. metapsilosis, and C. orthopsilosis, respectively. For itraconazole, the non-wildtype percentage was 1.7% (7/407), 1.35% (1/74), and 0% in C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis, respectively. For posaconazole, the non-wildtype percentage was 3.8% (16/413), 2.7% (2/74), and 0% in parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, respectively. Sequencing analysis of ERG11 gene revealed two homozygous mutations, Y132F mutation, and R398I in fluconazole-resistant isolates.

Conclusions
C. parapsilosis species complex infections are on the rise. The increasing azole resistance in C. parapsilosis with higher mortality is a great concern in clinical settings.