Objective:

Rapid identification of blood isolates is a key to the management of bacteraemia. The Gram stain is crucial, but a definite diagnosis must await subculture and supplementary tests. Identification within 3.5 h has recently become feasible by use of peptide nucleic acid (PNA) probes for fluorescence-in situ-hybridization (FISH). We have evaluated this technique for identification of S. aureus (Sau), E. coli (Eco), P. aeruginosa (Pae), and C. albicans (Cal).

Methods:

The evaluation was conducted May–November 2003. BacT/Alert (bioMérieux) was used for blood cultures (BCs) and a set with two aerobic and one anaerobic bottle was recommended for adults. PNA probes were provided by AdvanDx (Woburn, MA, USA), and PNA-FISH assays were carried out with flame-fixed slides. The Gram stain report determined which probes were used (the Sau probe for Gram-positive cocci in clusters, Eco and Pae probes for Gram-negative rods, and the Cal probe for yeasts). The observer was blinded to conventional identification. Positive and negative predictive values (PPN and PNV, respectively) were calculated for monomicrobial cultures and numbers of patients with homologous and heterologous isolates are given in [ ].

Results:

Among app. 9000 BC sets 1241 produced growth. Based on Gram stain results 789 BCs from 534 patients entered the evaluation. Diagnoses provided (or rejected) by PNA-FISH were in agreement with conventional diagnoses for the following groups of bacteraemic patients: S. aureus (60) vs. coagulase-negative staphylococci (127), E. coli (197) vs. other Gram-negative rods (145), and C. albicans (9) vs. non-C. albicans (10). Thus, PPV was 100% and NPV 100% for the Sau, Eco, and Cal probes. PNA-FISH with the Pae probe was positive in 18 of 19 patients with P. aeruginosa; weak to moderate staining was seen in four patients with other aerobic bacteria, and 316 patients with a broad range of Gram-negative rods were negative. The PPV for the Pae probe was 82% and NPV 99.7%.

Conclusion:

PNA-FISH is speedy and the diagnosis of P. aeruginosa is imperative in order to provide coverage for this pathogen. PNA-FISH was found to be highly reliable in the routine setting for diagnosis of four important blood-borne pathogens. For all four pathogens the NPV was > 99% and the slightly lower PPV for P. aeruginosa should not limit the clinical utility of PNA-FISH.