LAUSR

Sir, Direct testing of blood cultures for molecular targets by polymerase chain reaction (PCR) assays is a valuable adjunct to the rapid identification methods currently applied for this purpose such as matrix assisted laser desorption ionisationtime of flight mass spectrometry (MALDI-TOF MS), the direct tube coagulase test and antigen testing. Genotypic features of isolates may have implications for diagnosis, antibiotic therapy, clinical management and timely public health intervention. However, testing blood cultures has been thwarted by the presence of PCR inhibitors notably sodium polyanethol sulfonate (SPS). The media used in the BacT/ALERT MDS (bioMérieux, France) blood system has been shown in this study to have properties inhibitory to PCR assays. This was demonstrated in an experiment where BacT/ALERT FA Plus and FN Plus culture bottles without added blood were each inoculated with four strains of Enterobacteriaceae containing known resistance genes (bla-SHV, -IMP, -CTX-M-1, OXA-48) and a strain of Pseudomonas aeruginosa bla-GES. The inoculum was prepared as a 1.0 mL suspension of individual strains in sterile saline (w McFarland 1) and bottles were incubated overnight at 37 C to promote microbial growth as confirmed by a gas-permeable sensor (at the base of the culture bottle). A 210 mL aliquot of culture broth from each of the five paired bottles was supplemented with 5 mL of a suspension Escherichia coli ACM 5185 (approximately 10 CFU/mL) for the internal control and extracted by a silica column extraction method (QIAamp DNA Blood Mini Kit; Qiagen, Germany) according to the manufacturer’s instructions. Subsequent PCR testing on the extractions failed to detect the respective antibiotic resistance genes or the added E. coli. A range of DNA extraction methods has been evaluated to circumvent the inhibitory action of SPS in blood culture media. As far as we are aware, the most efficient method evaluated to date is based on organic extraction using benzyl alcohol-guanididine hydrochloride. However, the complexity of the protocol and an extraction time of 1.8 h is impractical for use in a busy diagnostic laboratory. We investigated a strategy to harvest and purify bacteria from BacT/ALERT FA Plus and FN Plus culture bottles to enable direct testing by PCR assays. The objective was to develop a simple protocol for preparation of a bacterial suspension suitable for PCR testing that is reliable, rapid, inexpensive and amenable for use in a busy setting. Here, a centrifugation method involving several replacements of supernatant (washings) was devised to harvest and purify the bacteria, lyse red cells and eliminate chemical PCR inhibitors. The method was validated using one commercial PCR assay (LightCycler VRE Detection Kit; Roche Diagnostics, Switzerland) and 23 uniplex RT-PCR assays configured inhouse and based on TaqMan hydrolysis probes for product detection as described by McIver et al. The in-house assays