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Open Access Highly Accessed Research article

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

Cleison Ledesma Taira1, Thelma Suely Okay2, Artur Figueiredo Delgado3, Maria Esther Jurfest Rivero Ceccon3, Margarete Teresa Gottardo de Almeida4 and Gilda Maria Barbaro Del Negro1*

Author Affiliations

1 Laboratory of Medical Mycology (LIM-53), Clinical Dermartology Division, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) and Instituto de Medicina Tropical da Universidade de São Paulo (IMT-USP), Av. Dr. Enéas Carvalho de Aguiar, 500, Andar térreo, Predio 2, CEP, 05403-900 São Paulo, SP, Brazil

2 Laboratory of Seroepidemiology and Immunobiology, IMT-USP, São Paulo, SP, Brazil

3 Pediatric and Neonatal Intensive Care Units, Instituto da Criança, HCFMUSP, São Paulo, SP, Brazil

4 Microbiology Laboratory, Department of Dermatologic, Infectious and Parasitic Diseases, Faculdade de Medicina de São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil

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BMC Infectious Diseases 2014, 14:406  doi:10.1186/1471-2334-14-406

Published: 21 July 2014

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.

Keywords:
Candidaemia; Candida spp; Multiplex PCR; ICU; Paediatric; Diagnosis