A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients

Abstract

Background: Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients.

Methods: DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures.

Results: The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients.

Conclusions: Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

Figure 1

Agarose gel showing the nested multiplex PCR products obtained from patients after amplification with primers for assays 1 and 2. Lane L – 100 bp molecular weight marker (GE Healthcare); Lanes 1 and 8 – Negative controls; Lanes 2 and 9 – Patient 2 (C. albicans); Lanes 3 and 10 – Patient 8 (C. albicans); Lanes 4 and 11 – Patient 11 (C. tropicalis); Lanes 5 and 12 – Patient 13 (C. tropicalis and C. parapsilosis); Lanes 6 and 13 – Patient 7 (C. krusei); Lane 7 – Positive controls for assay 1: DNA from C. glabrata (423 bp), C. tropicalis (357 bp), C. albicans (272 bp) and C. lusitaniae (137 bp) reference strains; Lane 14 – Positive controls for assay 2: DNA from C. krusei (362 bp), C. parapsilosis (297 bp) and C. pelliculosa (160 bp) reference strains.