Evaluating molecular diagnostics in candidemia: Challenges, limitations, and future directions for RT-PCR and...

Candidemia is a serious infection in critically ill patients, and early diagnosis remains challenging due to limitations of blood culture. Molecular methods such as real-time PCR (RT-PCR) and next-generation sequencing (NGS) offer rapid detection, but their performance in direct blood samples is not well established.This prospective diagnostic study included three groups: (i) 30 patients with culture-confirmed candidemia, (ii) 20 patients with suspected candidiasis who were β-d-glucan (BDG) positive but culture-negative, and (iii) 10 healthy controls. All samples were tested using TaqMan RT-PCR targeting Candida species. NGS was performed on RT-PCR-positive samples to assess pathogen detection, species identification, and antifungal resistance. Diagnostic performance was evaluated using blood culture as the reference standard. RT-PCR detected Candida DNA in 26/30 samples (sensitivity 86.7%; 95% CI: 69.3-96.2%), whereas NGS detected Candida DNA in 18/30 samples (sensitivity 60.0%; 95% CI: 40.6-77.3%) (p = 0.008). Species-level concordance between NGS and culture-based identification was observed in 10/30 blood samples (33.3%). In contrast, cultured isolates showed higher concordance, with 24/30 (80%) agreement between NGS and MALDI-TOF. ERG11 mutations associated with elevated fluconazole MICs were identified in some isolates, but not in direct blood samples. In BDG-positive, culture-negative cases, RT-PCR was positive in 3/20 (15%), while NGS detected no cases. All controls were negative. RT-PCR demonstrated higher sensitivity than NGS for Candida detection in blood and may serve as a rapid diagnostic tool. NGS showed limited sensitivity in blood but improved performance in isolates. Molecular methods should complement, not replace, blood culture, and BDG results require cautious interpretation.