Adler, H.

Frei, R.

Straub, C.

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Eur J Clin Microbiol Infect Dis (2005) 24: 499–500DOI 10.1007/s10096-005-1362-2BRIEF REPORTH. Adler · C. Straub · R. FreiComparison of BacT/ALERT 3D, Lowenstein-Jensen mediumand Middlebrook 7H10/7H11 biplate for recovering mycobacteriafrom clinical specimensPublished online: 29 June 2005C© Springer-Verlag 2005Rapid and sensitive detection of mycobacteria is essen-tial for the successful treatment and control of mycobacte-rial infections. It is considered good laboratory practice tocombine a liquid medium with at least one solid mediumfor culturing mycobacteria from clinical specimens. Auto-mated systems with liquid media provide faster results andare more sensitive, while solid media help to detect mixedinfections and can detect mycobacteria that do not grow inthe broth medium. In order to evaluate the BacT/ALERT3D system (bioMe´rieux, Durham, NC, USA) and to findthe optimal combination of media, we compared the re-sults achieved with BacT/ALERT 3D, Lowenstein-Jensen(L-J) medium (Bio-Rad, Marne-la Coquette, France), andthe Middlebrook 7H10/7H11 biplate (Becton Dickinson,Sparks, MD, USA) on consecutive clinical specimens col-lected within a period of 26 months.A total of 6,250 clinical samples (4,051 respiratory spec-imens, 1,209 normally sterile body fluids, 292 biopsy spec-imens, 247 blood specimens, 344 urine specimens and 304other specimens) from 3,247 patients were tested for thepresence of mycobacteria. Specimens with a different panelof media (blood and urine) and specimens contaminatedwith bacteria other than mycobacteria were excluded fromthe evaluation.Tissue specimens were crushed and homogenized. Ho-mogenized tissue, respiratory specimens and other speci-mens from contaminated sites were decontaminated withsodium dodecyl sulfate-NaOH as described by Tacquetand Tisson [1]. Liquids from sterile sites were concen-trated by centrifugation (3000 g, 15 min). Aliquots of0.3–0.5 ml were inoculated onto a L-J slant, a Middle-brook 7H10/7H11 selective agar biplate with carbenicillin,polymyxin B, trimethoprim lactate, and amphotericin B,and into a BacT/ALERT MP bottle. For specimens fromH. Adler () · C. Straub · R. FreiMicrobiology Laboratory, University Hospital Basel,Petersgraben 4,4031 Basel, Switzerlande-mail: hadler@uhbs.chTel.: +41-61-2654248Fax: +41-61-2655355contaminated sites, 0.5 ml of MB/BacT Antibiotic Sup-plement (bioMe´rieux) was added to the BacT/ALERT MPbottle.The incubation time was 8 weeks. L-J slants were incu-bated at 36◦C in ambient air and 7H10/7H11 biplates wereincubated at 36◦C in 5% CO2. Both media were exam-ined visually once a week for the appearance of colonies.The BacT/ALERT MP bottles were handled according tothe manufacturer’s instructions. Detection of mycobacte-rial growth in the BacT/ALERT 3D system is based on thecolorimetric detection of CO2, and the cultures are contin-uously monitored by the automated system.Ziehl-Neelsen staining was used to confirm the presenceof acid-fast bacilli in all positive media. Mycobacteriumtuberculosis was identified using the Cobas AmplicorMTB system (Roche Diagnostics, Basel, Switzerland)combined with the following biochemical tests: productionof niacin and heat-stable catalase and reduction of nitrate.Nontuberculous mycobacteria (NTM) were identified us-ing nucleic acid probes (Gen-Probe, San Diego, CA, USA)or by 16S rDNA sequencing [2] with the MicroSeq 50016S rDNA Bacterial Sequencing Kit (Applied Biosystems,Foster City, CA, USA). Sequences were compared withthose available in RIDOM, a comprehensive 16S rRNAsequence database for mycobacteria [3].A total of 345 isolates of mycobacteria were evaluated.Seventeen specimens were contaminated with organismsother than mycobacteria and consequently excluded; twowere in BacT/ALERT MP bottles, one was on L-J mediumand 14 were on Middlebrook 7H10/7H11 biplates.Mycobacterium tuberculosis was identified in 285specimens collected from 100 patients. Sixty isolatesof NTM were recovered from 58 clinical specimens of33 patients. The NTM isolates comprised the followingspecies: M. avium/intracellulare (26), M. chelonae (1),M. flavescens (1), M. fortuitum (1), M. gordonae (7),M. kansasii (5), M. lentiflavum/simiae (1), M. malmoense(1), M. marinum (2), M. peregrinum (1), M. phlei (1),M. septicum/peregrinum (3), M. terrae complex (1),M. xenopi (7), unidentified Mycobacterium spp. (2). Mixedcultures with Mycobacterium avium and Mycobacterium500Table 1 Results achieved using different media to detect mycobacteriaMycobacteria BacT/ALERT L-J 7H10/7H11 BacT/ALERTplus L-JBacT/ALERTplus 7H10/7H11L-J plus7H10/7H11TotalM. tuberculosisSmear positive 158 (95%) 159 (96%) 153 (92%) 165 (99%) 163 (98%) 165 (99%) 166Smear negative 104 (87%) 97 (82%) 84 (71%) 116 (97%) 111 (93%) 107 (90%) 119Totala 262 (92%) 256 (90%) 237 (83%) 281 (99%) 274 (96%) 272 (95%) 285NTMb 48 (80%) 21 (35%) 38 (63%) 55 (91%) 55 (91%) 45 (75%) 60L-J, Lowenstein-Jensen; NTM, non-tuberculous mycobacteriaa Significant differences (p<0.05): BacT/ALERT vs 7H10/7H11, L-J vs 7H10/7H11, BacT/ALERT vs BacT/ALERT plus L-J, BacT/ALERTvs BacT/ALERT plus 7H10/7H11, L-J vs BacT/ALERT plus L-J, L-J vs BacT/ALERT plus 7H10/7H11, L-J vs L-J plus 7H10/7H11,7H10/7H11 vs BacT/ALERT plus L-J, 7H10/7H11 vs BacT/ALERT plus 7H10/7H11, 7H10/7H11 vs L-J plus 7H10/7H11, BacT/ALERTplus L-J vs L-J plus 7H10/7H11b Significant differences (p<0.05): BacT/ALERT vs L-J, BacT/ALERT vs 7H10/7H11, L-J vs 7H10/7H11, L-J vs BacT/ALERT plus L-J,L-J vs BacT/ALERT plus 7H10/7H11, L-J vs L-J plus 7H10/7H11, 7H10/7H11 vs BacT/ALERT plus L-J, 7H10/7H11 vs BacT/ALERTplus 7H10/7H11, BacT/ALERT plus L-J vs L-J plus 7H10/7H11, BacT/ALERT plus 7H10/7H11 vs L-J plus 7H10/7H11kansasii were detected twice. Isolation rates achieved witheach medium are given in Table 1.The BacT/ALERT 3D system is a fully automated sys-tem based on the detection of CO2 produced by the pro-liferating mycobacteria. The main differences betweenthe BacT/ALERT 3D model and its predecessor, theMB/BacT, are in the system’s electronic and data man-agement components. Only a few studies have evaluatedthe new BacT/ALERT 3D system, and they assessed theBacT/ALERT 3D system in comparison with either L-Jmedium alone [4–6] or L-J medium and the Bactec 460 TBSystem [7]. In those studies, the recovery rates reported forthe BacT/ALERT 3D system ranged from 85 to 100% forM. tuberculosis and 52 to 100% for NTM. However, thetwo studies that reported recovery rates of 100% for NTMincluded only five and eight isolates of NTM, respectively[4, 5]. In comparison, our study included a large numberof isolates, and we found recovery rates of 92% for M. tu-berculosis and 80% for NTM using the BacT/ALERT 3Dsystem.In summary, the BacT/ALERT 3D system proved to bea highly sensitive culture system when compared to solidmedia. There were no significant differences in the recov-ery rates achieved using BacT/ALERT 3D alone and thecombination of the two solid media, but the recovery ratesof BacT/ALERT 3D were enhanced for both M. tubercu-losis and NTM when the system was used in combinationwith a solid medium. The combination of BacT/ALERT3D plus a solid medium offers the additional advantageof facilitating detection in cases of contamination of onemedium or in cases of mixed mycobacterial cultures, whichare more readily detected on solid media.References1. Tacquet A, Tisson F (1961) Re´sultat de l’isolement desmycobacte´ries par le lauryl-sulfate de sodium. Ann Inst Pasteur100:676–6802. Rogall T, Flohr T, Bottger EC (1990) Differentiation of My-cobacterium species by direct sequencing of amplified DNA. JGen Microbiol 136:1915–19203. Harmsen D, Rothganger J, Frosch M, Albert J (2002) RIDOM:Ribosomal differentiation of medical micro-organisms database.Nucleic Acids Res 30:416–4174. Carricajo A, Fonsale N, Vautrin AC, Aubert G (2001) Evaluationof BacT/Alert 3D liquid culture system for recovery of mycobac-teria from clinical specimens using sodium dodecyl (lauryl)sulfate-NaOH decontamination. J Clin Microbiol 39:3799–38005. Alcaide F, Gali N, Dominguez J, Berlanga P, Blanco S, Orus P,Martin R (2003) Usefulness of a new mycobacteriophage-basedtechnique for rapid diagnosis of pulmonary tuberculosis. J ClinMicrobiol 41:2867–28716. Angeby KA, Werngren J, Toro JC, Hedstrom G, Petrini B,Hoffner SE (2003) Evaluation of the BacT/ALERT 3D systemfor recovery and drug susceptibility testing of Mycobacteriumtuberculosis. Clin Microbiol Infect 9:1148–11527. Piersimoni C, Scarparo C, Callegaro A, Tosi CP, Nista D,Bornigia S, Scagnelli M, Rigon A, Ruggiero G, Goglio A (2001)Comparison of MB/Bact alert 3D system with radiometricBACTEC system and Lowenstein-Jensen medium for recoveryand identification of mycobacteria from clinical specimens: amulticenter study. J Clin Microbiol 39:651–657

Comparison of BacT/ALERT 3D, Lowenstein-Jensen medium and Middlebrook 7H10/7H11 biplate for recovering mycobacteria from clinical specimens

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Comparison of BacT/ALERT 3D, Lowenstein-Jensen medium and Middlebrook 7H10/7H11 biplate for recovering mycobacteria from clinical specimens

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