Multiplex Nested PCR Detects Bloodstream Candida Species

Nosocomial candidaemia is associated with high mortality rates in critically ill pediatric and neonatal patients and therefore the early detection and identification of the infectious fungal agent is crucial for successful medical intervention.

Clinical microbiologists at the University of Sao Paulo (Butanta, Brazil) enrolled 54 consecutive pediatric patients, among whom there were 24 neonates, admitted to the intensive care unit (ICU) of two pediatric referral hospitals in Sao Paulo State (Brazil). The patients presented with systemic inflammatory response syndrome (SIRS) and at least two predisposing conditions/risk factors for the development of candidaemia. A control group included 28 children that had no evidence of any type of bloodstream infection and had undergone minor surgical procedures during the same time.

Blood samples were taken and either cultured or had DNA extracted. The 54 DNA samples 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.

The extracted DNA samples were amplified and the reactions were carried out in a Veriti thermocycler (Applied Biosystems; Carlsbad, CA, USA). The nested PCR products were detected on 2.5% agarose gels and visualized under a UV transilluminator apparatus (UVITEC; Cambridge, UK).

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. Candida albicans was the most frequently isolated species in blood cultures (five out of eight positive results), while the multiplex nested PCR identified C. albicans, C. parapsilosis complex, and C. tropicalis at similar frequencies.

The authors concluded that multiplex PCR assay targeting the intergenic space region (ITS) of Candida ribosomal DNA was able to identify the main Candida species involved in bloodstream pediatric infections with a detection limit of less than 10 colony forming units (CFU)/mL. The assay had high specificity, and is as least as sensitive as blood cultures but with a shorter turnaround time. The use of the multiplex nested PCR method also allowed the detection and identification of one or more Candida species in the same reaction. The study was published on July 21, 2014, in the journal BMC Infectious Diseases.

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University of Sao Paulo
Applied Biosystems
UVITEC