# Invasive Candidiasis: Real-Time PCR-Based Species Detection and Antifungal Susceptibility Profiling of Suspected Patients in the ICU Using the Disc Diffusion Method

## TL;DR

This study uses real-time PCR and disc diffusion to identify Candida species and their antifungal resistance in ICU patients, finding non-albicans species dominate and show higher resistance.

## Contribution

The study introduces a combined approach of real-time PCR and disc diffusion for rapid species identification and susceptibility profiling in ICU patients with suspected invasive candidiasis.

## Key findings

- Non-albicans Candida species, particularly C. parapsilosis, were most commonly detected in ICU patients.
- C. glabrata showed reduced fluconazole susceptibility, while C. krusei was resistant to azoles but sensitive to amphotericin B.
- Real-time PCR and disc diffusion provided rapid and accurate species identification and susceptibility profiling.

## Abstract

Background

Invasive candidiasis (IC) is a major cause of morbidity and mortality in critically ill and immunocompromised patients, with a rising predominance of non-albicans Candida (NAC) species and increasing antifungal resistance. Rapid species identification and timely antifungal susceptibility testing are crucial for appropriate management.

Objective

To determine the species distribution of Candida in suspected ICU cases using real-time PCR and to assess antifungal susceptibility patterns by the disc diffusion method.

Methods

This cross-sectional study included 60 ICU patients with clinical suspicion of IC from September 2022 to August 2023. Clinical suspicion of invasive candidiasis was defined based on the presence of at least one of the following criteria: persistent fever unresponsive to broad-spectrum antibiotics, sepsis, or other systemic symptoms, such as hypotension, and relevant risk factors, including mechanical ventilation, central venous catheters, or prolonged use of broad-spectrum antibiotics. Blood samples were processed through automated culture, conventional microscopy, and species-level identification using multiplex real-time PCR targeting the ITS rDNA region. Antifungal susceptibility testing was performed using the Clinical & Laboratory Standards Institute (CLSI)-recommended disc diffusion method for fluconazole, itraconazole, voriconazole, and amphotericin B.

Results

Of 60 samples, 38 (63.3%) were PCR-positive for fungus. Among those, 24 were Candida species, and the remaining 14 were fungi other than Candida spp. The most common species detected was Candida (C.) parapsilosis (37.5%), followed by C. albicans (33.33%), C. glabrata (25%), and C. krusei (4.1%). Antifungal susceptibility testing revealed 100% susceptibility among C. albicans isolates to all tested agents. C. glabrata showed reduced susceptibility to fluconazole (50%), whereas C. parapsilosis demonstrated variable susceptibility across all antifungals. C. krusei showed complete resistance to all azoles but remained sensitive to amphotericin B.

Conclusion

NAC species predominated among ICU patients with suspected invasive candidiasis and exhibited higher resistance rates, especially to azoles. Real-time PCR provided rapid and accurate species identification, while disc diffusion offered reliable antifungal susceptibility profiling. Integrating molecular diagnostics with routine susceptibility testing is essential for guiding timely, targeted therapy and improving clinical outcomes in critically ill patients.