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RESEARCH NOTE
The identification of mycobacteria from identifications are restricted to a few species [4– solid media and directly from VersaTREK 6]. High-performance liquid chromatography Myco bottles using the Sherlock (HPLC) is an alternative identification method, Mycobacteria Identification HPLC system but is generally performed from solid media and requires manual inspection of mycolic acid chro- V. J. LaBombardi1,2, R. Katariwala1 and 1 matographic profiles, as well as extensive exper- G. Pipia tise. The purpose of the present study was to investigate the use of a commercially available 1Department of Laboratories, St Vincent’s Hos- HPLC system with pattern recognition software pital-Manhattan, New York and 2Department of for the identification of mycobacteria obtained Pathology, New York Medical College, Valhalla, from solid media and directly from positive broth NY, USA culture bottles. Clinical specimens received for mycobacterial culture were processed by standard laboratory ABSTRACT protocols and inoculated on to Middlebrook 7H11 The Sherlock Mycobacteria Identification HPLC agar plates (BBL, Sparks, MD, USA) and into system correctly identified to the species level 61 VersaTREK Myco bottles containing growth sup- (67.8%) of 90 isolates growing on solid media, plement and PVNA (polymyxin B, vancomycin, and 73 (45.3%) of 161 isolates directly from nalidixic acid, amphotoricin B) antibiotic mixture positive VersaTREK Myco bottles. When these (TREK Diagnostic Systems, Cleveland, OH, USA). data were re-analysed with a revised database, These bottles were incubated in the ESP Culture correct identifications increased to 91.1% and System II (TREK Diagnostic Systems) and the 83.2%, respectively. All Mycobacterium tuberculosis VersaTREK system when available. Bottles were isolates were identified correctly, regardless of the monitored continuously for growth and an inoculum source or database used. The use of the aliquot was removed for identification when a revised database with isolates obtained directly positive culture was detected. All clinical isolates from positive VersaTREK Myco bottles allows the were identified initially using nucleic acid probes identification of most isolates within clinically as described previously [5]. If the isolates could relevant time-frames. not be speciated using probes, they were identi- Keywords Diagnosis, HPLC, identification, myco- fied by biochemical testing [7]. New clinical bacteria, Mycobacterium tuberculosis, VersaTREK isolates were tested by HPLC in real-time as the cultures became positive. In total, 90 isolates were Original Submission: 20 May 2005; Revised Submis- tested from solid media (45 from concurrent sion: 24 August 2005; Accepted: 23 September 2005 positive cultures and 45 from subcultured frozen Clin Microbiol Infect 2006; 12: 478–481 stock cultures), and 161 isolates were tested 10.1111/j.1469-0691.2006.01373.x directly from positive VersaTREK Myco bottles (120 from concurrent positive cultures and 41 The use of continuously monitored liquid-based seeded from subcultured stock cultures). culture systems for mycobacteria has resulted in For the HPLC assay, a sterile applicator stick was decreased times to detection for positive cultures. used to pick up a barely visible amount of growth In addition, various species of clinically relevant, from solid media for processing, or the pellet from non-tuberculous mycobacteria are now isolated 3 mL of broth, centrifuged at 3000 g for 30 min, routinely with increasing frequency [1–3]. Several was used from a positive VersaTREK Myco bottle. investigators have used nucleic acid probes to Saponification, extraction and derivatisation identify mycobacteria directly from positive cul- were performed according to the manufacturer’s ture bottles or tubes, but these probe-based instructions (MIDI Inc., Newark, DE, USA). An Agilent 1100 Series HPLC system (Agilent Technologies, Wilmington, DE, USA) with a C18 Corresponding author and reprint requests: V. J. LaBombardi, reverse phase column was used to analyse the St Vincent’s Hospital-Manhattan, 153 W 11th Street, New York, mycolic acid content of the samples. A calibration NY 10011, USA standard (in duplicate) and the specimen samples E-mail: [email protected]