J Med Microbiol 56 (2007), 598-602; DOI: 10.1099/jmm.0.46855-0
© 2007 Society for General Microbiology
ISSN 1473-5644
A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory
K. J. Williams1,
C. L. Ling1,
C. Jenkins1,
S. H. Gillespie2 and
T. D. McHugh2
1 Department of Microbiology, Royal Free Hospital, London NW3 2QG, UK
2 Centre for Medical Microbiology, Hampstead Campus, University College London, London NW3 2PF, UK
Correspondence
T. D. McHugh
t.mchugh{at}medsch.ucl.ac.uk
Received 26 July 2006
Accepted 10 January 2007
The aim of this study was to improve the identification of Mycobacterium species in the context of a UK teaching hospital. Real-time PCR assays were established to enable the rapid differentiation between Mycobacterium tuberculosis (MTB) complex and Mycobacterium species other than tuberculosis (MOTT), followed by 16S rRNA gene sequencing for the speciation of MOTT. Real-time PCR assays gave comparable results to those from the reference laboratory. The implementation of these PCR assays using an improved bead extraction method has enhanced the mycobacterial diagnostic service at the Royal Free Hospital by providing a rapid means of differentiating between MTB complex and MOTT, and would be simple to implement in similar laboratories. Sequence analysis successfully identified a range of Mycobacterium spp. representative of those encountered in the clinical setting of the authors, including Mycobacterium avium complex, Mycobacterium fortuitum group, Mycobacterium chelonae–Mycobacterium abscessus group, Mycobacterium xenopi and Mycobacterium gordonae. It provides a useful tool for the identification of MOTT when clinically indicated.
Abbreviations: MOTT, Mycobacterium species other than tuberculosis; MTB, Mycobacterium tuberculosis.
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INTRODUCTION
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The rapid detection and identification of clinically important Mycobacterium spp. is essential for patient management and infection control. Mycobacterium tuberculosis (MTB) remains one of the leading causes of morbidity and mortality worldwide, but Mycobacterium species other than tuberculosis (MOTT) are increasingly important pathogens. Speciation is particularly important when choosing antibiotic regimens for immunocompromised patients, in whom the presence of any acid-fast bacilli may be considered significant (Katoch, 2004).
In recent years, the development of molecular techniques has had a major impact on the diagnosis of mycobacterial infections (Davies et al., 1999; Conaty et al., 2005). Methods for the detection and identification of mycobacteria include nucleic acid probes (Arnold et al., 1989), conventional PCR amplification, PCR hybridization with species-specific probes (Fiss et al., 1992; Zolg & Philippi-Schulz, 1994; De Beenhouwer et al., 1995; Portaels et al., 1997; Hong et al., 2004), PCR RFLP analysis (Vaneechoutte et al., 1993; Kasai et al., 2000; Lee et al., 2000; Roth et al., 2000; Kim et al., 2005) and nucleic acid sequence analysis. Genes that have been targeted for sequence analysis include hsp65 (Ringuet et al., 1999), 32 kDa protein gene (Soini et al., 1994), gyrB (Kasai et al., 2000), recA (Blackwood et al., 2000), rpoB (Kim et al., 1999; Adekambi et al., 2003) and the 16S rRNA gene (Han et al., 2002; Hall et al., 2003; Pauls et al., 2003). Duplex, multiplex and real-time PCR assays for the identification of mycobacteria have also been described (Broccolo et al., 2003; Shrestha et al., 2003; Tanaka et al., 2003; Kim et al., 2004; Kurabachew et al., 2004).
Several commercial systems using various technologies for the detection and identification of Mycobacterium spp. are in routine use, including the INNO-LiPA Mycobacteria assay (Innogenetics; Tortoli et al., 2001), GenoType Mycobacterium (Hain Diagnostika; Padilla et al., 2004), the Cobas Amplicor PCR system (Roche; Bogard et al., 2001), the LCx Mycobacterium tuberculosis ligase chain reaction assay (Abbott Laboratories; Lindbrathen et al., 1997), the BD ProbeTec strand displacement amplification (Becton Dickinson; McHugh et al., 2004) and the amplified Mycobacterium tuberculosis direct test (Gen-Probe Inc; O'Sullivan et al., 2002).
The Royal Free Hospital is a UK teaching hospital and tertiary referral centre for transplantation and HIV, receiving 146 mycobacterium isolates in 2005. Due to our large immunocompromised population, a significant proportion (35 %) of these were MOTT and it is essential that these are rapidly identified to enable appropriate patient management. All new isolates are currently sent to a reference laboratory (Mycobacterial Reference Unit, Dulwich, UK, now at Barts & Royal London, Queen Mary School of Medicine and Dentistry, London, UK) for identification. We proposed to improve the mycobacterial diagnostic service at the Royal Free Hospital by the validation and implementation of real-time PCR assays for the differentiation between MTB complex and MOTT, and 16S rRNA gene sequence analysis for the speciation of MOTT isolates.
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METHODS
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Bacterial strains.
A total of 194 isolates from 194 patients were analysed. Group 1 (n=166) was retrospectively tested, and included all available isolates from different patients between September 2004 and May 2006, and group 2 (n=28) included all isolates prospectively collected between April 2006 and June 2006. The real-time PCR assays were performed for all 194 isolates. Sequencing was performed for 32 isolates from group 1.
DNA extraction
In our routine diagnostic practice, sodium hydroxide decontaminated samples were inoculated into an automated culture system (BacT Alert; bioMérieux) that reads positive when growth is detected. Selective 7H9 broths and two LJ slopes (one with pyruvate and one without) were then inoculated and DNA was extracted.
Crude extraction method.
This was performed for all group 1 isolates (n=166). Volumes of 500 µl 7H9 broth culture or a 10 µl loop full of cell growth in 500 µl Tris-EDTA (TE) (10 mM Tris, 0.1 mM EDTA, pH 8.0) buffer were centrifuged at 7500 g for 3 min, pellets were resuspended in 500 µl TE buffer and incubated at 80 °C for 1 h.
Chloroform extraction method.
Isolates producing no results using the real-time PCR assays were retested following chloroform extraction (n=21). Briefly, heat killed bacteria were incubated with lysozyme (Sigma) for 1 h at 37 °C, followed by digestion with 50 µg proteinase K (Sigma) in 10 % SDS for 10 min at 65 °C. A further incubation with 1 % (w/v) cetyltrimethylammonium bromide in 
0.5 M NaCl for 10 min at 65 °C was followed by partition using chloroform/isoamyl alcohol (24 : 1, v/v) (Gillespie et al., 2000).
Bead extraction method.
This was performed for all isolates in group 2 (n=28). Volumes of 500 µl 7H9 broth culture or a 10 µl loop full of cell growth in 500 µl TE buffer were centrifuged at 14 243 g for 3 min, pellets were resuspended in 200 µl TE buffer and transferred into microfuge tubes containing 0.1 g 80 mesh glass beads and 0.1 g 425–600 micron glass beads (Sigma). These were vortexed for 5 min, incubated at 80 °C for 20 min, centrifuged at 14 243 g for 5 min and 100 µl supernatant was retained.
MTB rpoB real-time PCR assay.
Primer set Tbc1 and TbcR5 (Kim et al., 2004) was used to amplify a 235 bp region of the MTB complex rpoB gene in a reaction volume of 25 µl containing 12.25 µl ABsolute QPCR SYBR green (ABgene), 2.5 µl extracted DNA, 0.5 mM each primer and PCR quality water.
MOTT rpoB real-time PCR assay.
Primer set M5 and RM3 (Kim et al., 2004) was used to amplify a 136 bp region of the MOTT rpoB gene in a final volume of 25 µl containing 12.25 µl ABsolute QPCR SYBR green, 2.5 µl extracted DNA, 1 mM each primer and PCR quality water. PCR cycles for both assays were performed using a Rotor-Gene (Corbett Research) and consisted of 15 min at 95 °C, followed by 30 cycles of 95 °C for 15 s and 72 °C for 30 s, with a final extension at 72 °C for 10 min. Post-amplification melt curves were generated and analysed using the Rotor-Gene software to determine the melting temperature of the PCR products, which is based on their size and composition. A positive MTB complex identification was defined as amplification in the quantification channel with the normal fluorescence threshold set at 0.5 and a melt curve peak at 90–91 °C. A positive MOTT identification was defined as amplification in the quantification channel with the normal fluorescence threshold set at 0.5 and a melt curve peak at 88–90 °C.
Mycobacterium 16S rRNA gene PCR.
The primer set described by Han et al. (2002) was used to amplify a 640–665 bp region of the 16S rRNA gene. PCR reactions were 100 µl in volume, consisting of 1x Taq buffer, 2 mM MgCl2, 0.1 mM dNTPs, 2 units Taq polymerase (Invitrogen), 1 mM each primer, 20 µl extracted DNA and PCR quality water. PCR cycles used were 95 °C for 5 min, followed by 40 cycles of 94 °C for 20 s, 55 °C for 20 s and 75 °C for 40 s.
DNA sequence analysis.
Amplicons purified with a QIAquick purification kit (Qiagen) were quantified by comparison with a Bioline Hyperladder I (Bioline) and sequenced using the Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems). Briefly, 2 ng cleaned-up DNA was added to 3 µl buffer, 1 µl cycle sequencing ready reaction mix, 0.16 mM forward or reverse primer and PCR quality water. The reaction parameters were 96 °C for 1 min, followed by 25 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min (Techne). For each isolate, two forward and two reverse cycle sequencing reactions were performed. Cycle sequencing products were precipitated, rehydrated in loading buffer and analysed using an ABI 377 automated sequencer according to manufacturer’s instructions (Applied Biosystems). Chromatograms were visualized using Chromas software (http://www.technelysium.com.au/chromas.html). Reverse compliments were generated using the Sequence Manipulation Site (http://www.ualberta.ca/~stothard/javascript/index.html) and forward sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw). GenBank BLAST searches (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) were performed for species identification.
Identification of mycobacterial species at the Mycobacterium Reference Unit.
At the beginning of the study phenotypic methods based on biochemical tests, growth characteristics and drug susceptibility profiles were used (Collins et al., 1997). During the course of the study these were replaced by a commercially available identification assay that uses PCR followed by reverse hybridization of the amplified products to a probe array (GenoType Mycobacterium; Hain Diagnostika; Padilla et al., 2004).
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RESULTS AND DISCUSSION
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Using real-time PCR assays 172 (89 %) isolates gave concordant results with the reference laboratory identifications and 22 (11 %) isolates failed to amplify for both real-time assays (Table 1
). The real-time PCR assays were able to detect a wide range of different Mycobacterium spp. including 4 Mycobacterium abscessus, 24 Mycobacterium avium complex, 1 Mycobacterium celatum, 4 Mycobacterium chelonae, 20 Mycobacterium fortuitum, 7 Mycobacterium gordonae, 5 Mycobacterium kansasii, 1 Mycobacterium lentiflavum, 2 Mycobacterium mucogenicum, 4 Mycobacterium peregrinum, 1 Mycobacterium simiae, 1 Mycobacterium szulgai, 6 Mycobacterium xenopi and 2 Mycobacteria spp. as identified by the reference laboratory. Of the 22 isolates failing to give results, 21 had been extracted using the crude and chloroform DNA extraction methods. A further seven isolates had failed to give results following the crude extraction method but these were resolved by re-extracting DNA using the chloroform extraction method (Table 2). The majority of isolates (19 of the 21) failing to give an identification following chloroform extraction were from 2005, it is therefore likely that the age of the isolates affected the results. Chloroform extraction methods are time consuming and labour intensive, and therefore not suitable for rapid identification assays. To address this, in 2006 a bead extraction method was adopted and used to test samples prospectively. Using this method all samples except one (96 %) were identified as MTB or MOTT (Table 2). The real-time PCR assays have proved to be a rapid and reliable method of differentiating between MTB complex and MOTT. They demonstrated a specificity value of 100 %, and a sensitivity value of 83 % using the crude extraction method, which was improved to 96 % using the bead extraction method although this was with a smaller sample number (Table 2). Using the bead extraction method and the real-time PCR assays, an identification of MTB complex or MOTT can be achieved in 2.5 h. We have found the combination of bead extraction and real-time PCR to be a rapid method that is user friendly and reagent costs (approx. £3 per patient) are relatively inexpensive compared to commercially available tests (approx. £15 per patient depending on assay selected). It can therefore be easily implemented in any diagnostic laboratory with a real-time amplification platform to differentiate Mycobacterium spp. from culture.
During this study 16S rRNA sequence analysis was successfully performed for 30 MOTT isolates giving results comparable to a reference laboratory (Table 3). Two isolates failed to give a 16S rRNA PCR product. Sequence analysis of 16S rRNA did not differentiate between M. kansasii and Mycobacterium gastri; M. chelonae–M. abscessus group and ‘Mycobacterium fuerth'; M. mucogenicum and ‘Mycobacterium ratisbonense’; or M. peregrinum, Mycobacterium septicum and M. fortuitum. The inability to differentiate between M. kansasii and M. gastri using 16S rRNA gene sequence analysis has been noted previously, but these species can be distinguished by culture characteristics (Han et al., 2002). Although treatment is the same for both species, M. kansasii isolates are usually regarded as clinically significant, but there have been few reported cases of clinically significant M. gastri infections (Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society, 2000; Velayati et al., 2005). ‘M. fuerth’ and ‘M. ratisbonense’ are newly described species previously shown to be identical to M. chelonae and M. mucogenicum, respectively, using 16S rRNA gene sequence analysis (Turenne et al., 2001), and there is no evidence that either cause infection in humans. There are reports of M. peregrinum and M. septicum causing infection in immunocompromised patients but there are no guidelines for treatment (Schinsky et al., 2000; Sakai et al., 2005). Both species have been proposed as members of the M. fortuitum group (Brown-Elliott & Wallace, 2002). In this study the identification was taken as the closest match generated by GenBank BLAST search analysis. Similarities ranged from 96 to 100 % homology over fragment sizes of 448 to 635 bp. Currently there are no standards for the interpretation and analysis of sequence data in a diagnostic setting; therefore, all results should be interpreted on an individual basis in conjunction with the clinical information.
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Table 3. Comparison of 16S rRNA gene sequence analysis for group 1 isolates (n=32) with reference laboratory identifications
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As demonstrated in a recent study conducted by Yam et al. (2006) 16S rRNA sequencing is an effective tool for the identification of Mycobacterium spp. It has been proposed that sequence analysis of several genes such as hsp65, rpoB and sod, in addition to the 16S rRNA gene, increases the robustness and power of discrimination to provide a more accurate identification (Devulder et al., 2005). However, the increased commitment in time and labour must be weighed against the clinical value of increased discrimination in the context of our setting. Sequence analysis of the 16S rRNA gene has proven to be clinically useful in our hospital; however, the cost (£18 per patient) and time commitment (18 h total time, including 4 h hands on time) of sequencing is only justified when the clinical circumstance requires a rapid identification for patient management. For example, in November 2004 Mycobacteria spp. were isolated from the blood cultures of three haematology patients on the same ward over a period of 10 days. There were concerns that this may due to transmission within the ward. Sequencing of the isolates identified two as M. chelonae and one as M. fortuitum, and following analysis of the M. chelonae sequences the isolates were considered to be unrelated. This indicated that the three positive blood cultures were independent episodes with no cross transmission.
From our analysis of all specimens submitted to the Royal Free Hospital TB service over a 21 month period we have adopted a model for the rapid discrimination between MTB and MOTT, appropriate to a clinical practice with low levels of MTB in the community but a substantial immunosuppressed population at risk of MOTT infection. Implementation of a real-time PCR assay has provided us with a rapid means of identifying MTB in-house, and 16S rRNA gene sequence analysis provides early diagnostic input to the management of patients who are often on complex treatment regimens.
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ACKNOWLEDGEMENTS
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The authors would like to acknowledge the contribution of M. Yates of the HPA Mycobacterium Reference Unit, Barts & Royal London, Queen Mary School of Medicine and Dentistry, London, UK, and the Clinical TB service at the Royal Free Hospital.
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REFERENCES
|
|---|
Adekambi, T., Colson, P. & Drancourt, M. (2003). rpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 41, 5699–5708.[Abstract/Free Full Text]
Arnold, L. J., Hammond, P. W., Wiese, W. A. & Nelson, N. C. (1989). Assay formats involving acridinium-ester-labeled DNA probes. Clin Chem 35, 1588–1594.[Abstract/Free Full Text]
Blackwood, K. S., He, C., Gunton, J., Turenne, C. Y., Wolfe, J. & Kabani, A. M. (2000). Evaluation of recA sequences for identification of Mycobacterium species. J Clin Microbiol 38, 2846–2852.[Abstract/Free Full Text]
Bogard, M., Vincelette, J., Antinozzi, R., Alonso, R., Fenner, T., Schirm, J., Aubert, D., Gaudreau, C., Sala, E. & other authors (2001). Multicenter study of a commercial, automated polymerase chain reaction system for the rapid detection of Mycobacterium tuberculosis in respiratory specimens in routine clinical practice. Eur J Clin Microbiol Infect Dis 20, 724–731.[Medline]
Broccolo, F., Scarpellini, P., Locatelli, G., Zingale, A., Brambilla, A. M., Cichero, P., Sechi, L. A., Lazzarin, A., Lusso, P. & Malnati, M. S. (2003). Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays. J Clin Microbiol 41, 4565–4572.[Abstract/Free Full Text]
Brown-Elliott, B. A. & Wallace, R. J. (2002). Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 15, 716–746.[Abstract/Free Full Text]
Collins, E. H., Grange, T. M. & Yates, M. D. (1997). Tuberculosis Bacteriology: Organisation and Practice, 2nd edn, London: Butterworth Heinemann.
Conaty, S. J., Claxton, A. P., Enoch, D. A., Hayward, A. C., Lipman, M. C. I. & Gillespie, S. H. (2005). The interpretation of nucleic acid amplification tests for tuberculosis: do rapid tests change treatment decisions?. J Infect 50, 187–192.[CrossRef][Medline]
Davies, A. P., Newport, L. E., Billington, O. J. & Gillespie, S. H. (1999). Length of time to laboratory diagnosis of Mycobacterium tuberculosis infection: comparison of in-house methods with reference laboratory results. J Infect 39, 205–208.[CrossRef][Medline]
De Beenhouwer, H., Liang, Z., de Rizk, P., van Eekeren, C. & Portaels, F. (1995). Detection and identification of mycobacteria by DNA amplification and oligonucleotide specific capture plate hybridization. J Clin Microbiol 33, 2994–2998.[Abstract]
Devulder, G., Perouse de Montclos, M. & Flandrois, J. P. (2005). A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. Int J Syst Evol Microbiol 55, 293–302.[Abstract/Free Full Text]
Fiss, E. H., Chehab, F. F. & Brooks, G. F. (1992). DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory. J Clin Microbiol 30, 1220–1224.[Abstract/Free Full Text]
Gillespie, S. H., Dickens, A. & McHugh, T. D. (2000). False molecular clusters due to non-random association of IS6110 with Mycobacterium tuberculosis. J Clin Microbiol 38, 2081–2086.[Abstract/Free Full Text]
Hall, L., Doerr, K. A., Wohlfiel, S. L. & Roberts, G. D. (2003). Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 41, 1447–1453.[Abstract/Free Full Text]
Han, X. Y., Pham, A. S., Tarrand, J. J., Sood, P. K. & Luthra, R. (2002). Rapid and accurate identification of mycobacteria by sequencing hypervariable regions of the 16S ribosomal RNA gene. Am J Clin Pathol 118, 796–801.[Abstract/Free Full Text]
Hong, S. K., Kim, B. J., Yun, Y. J., Lee, K. H., Kim, E. C., Park, E. M., Park, Y. G., Bai, G. H. & Kook, Y. H. (2004). Identification of Mycobacterium tuberculosis by PCR-linked reverse hybridization using specific rpoB oligonucleotide probes. J Microbiol Methods 59, 71–79.[CrossRef][Medline]
Kasai, H., Ezaki, T. & Harayama, S. (2000). Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences. J Clin Microbiol 38, 301–308.[Abstract/Free Full Text]
Katoch, V. M. (2004). Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 120, 290–304.[Medline]
Kim, B. J., Lee, S. H., Lyu, M. A., Kim, S. J., Bai, G. H., Kim, S. J., Chae, G. T., Kim, E. C., Cha, C. Y. & Kook, Y. H. (1999). Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 37, 1714–1720.[Abstract/Free Full Text]
Kim, B. J., Hong, S. K., Lee, K. H., Yun, Y. J., Kim, E. C., Park, Y. G., Bai, G. H. & Kook, Y. H. (2004). Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J Clin Microbiol 42, 1308–1312.[Abstract/Free Full Text]
Kim, H., Kim, S.-H., Shim, T.-S., Kim, M.-N., Bai, G.-H., Park, Y.-G., Lee, S.-H., Cha, C.-Y., Kook, Y.-H. & Kim, B.-J. (2005). PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp. J Microbiol Methods 62, 199–209.[CrossRef][Medline]
Kurabachew, M., Enger, O., Sandaa, R., Skuce, R. & Bjorvatn, B. (2004). A multiplex chain reaction assay for genus-, group- and species-specific detection of mycobacteria. Diagn Microbiol Infect Dis 49, 99–104.[CrossRef][Medline]
Lee, H., Park, H.-J., Cho, S.-N., Bai, G.-H. & Kim, S.-J. (2000). Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol 38, 2966–2971.[Abstract/Free Full Text]
Lindbrathen, A., Gaustad, P., Hovig, B. & Tonjum, T. (1997). Direct detection of Mycobacterium tuberculosis from patients in Norway by ligase chain reaction. J Clin Microbiol 35, 3248–3253.[Abstract]
McHugh, T. D., Pope, C. F., Ling, C. L., Patel, S., Billington, O. J., Gosling, R. D., Lipman, M. C. & Gillespie, S. H. (2004). Prospective evaluation of BD ProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples. J Med Microbiol 53, 1215–1219.[Abstract/Free Full Text]
O'Sullivan, C. E., Miller, D. R., Schneider, P. S. & Roberts, G. D. (2002). Evaluation of Gen-Probe amplified Mycobacterium tuberculosis direct test by using respiratory and non-respiratory specimens in a tertiary care center laboratory. J Clin Microbiol 40, 1723–1727.[Abstract/Free Full Text]
Padilla, E., Gonzalez, V., Manterola, J. M., Perez, A., Quesada, M. D., Gordillo, S., Vilaplana, C., Pallares, M. A., Molinos, S. & other authors (2004). Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and GenoType mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains. J Clin Microbiol 42, 3083–3088.[Abstract/Free Full Text]
Pauls, R. J., Turenne, C. Y., Wolfe, J. N. & Kabani, A. (2003). A high proportion of novel mycobacteria species identified by 16S rDNA analysis among slowly growing AccuProbe-negative strains in a clinical setting. Am J Clin Pathol 120, 560–566.[CrossRef][Medline]
Portaels, F., Aguiar, J., Fissetle, K., Fonteyne, P. A., de Beenhouwer, H., de Rijk, P., Guedenon, A., Lemans, R., Steunou, C. & other authors (1997). Direct detection and identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide specific capture plate hybridization. J Clin Microbiol 35, 1097–1100.[Abstract]
Ringuet, H., Akoua-Koffi, C., Honore, S., Varnerot, A., Vincent, V., Berche, P., Gaillard, J. L. & Pierre-Audigier, C. (1999). hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol 37, 852–857.[Abstract/Free Full Text]
Roth, A., Reischl, U., Streubel, A., Naumann, L., Koppenstedt, R. M., Habicht, M., Fischer, M. & Mauch, H. (2000). Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S–23S rRNA gene spacer and restriction endonucleases. J Clin Microbiol 38, 1094–1104.[Abstract/Free Full Text]
Sakai, T., Kobayashi, C. & Shinohara, M. (2005). Mycobacterium peregrinum infection in a patient with AIDS. Intern Med 44, 266–269.[CrossRef][Medline]
Schinsky, M. F., McNeil, M. N., Whitney, A. M., Steigerwalt, A. G., Lasker, B. A., Floyd, M. M., Hogg, G. G., Brenner, D. J. & Brown, J. M. (2000). Mycobacterium septicum sp. nov., a new and rapidly growing species associated with catheter-related bacteraemia. Int J Syst Evol Microbiol 50, 575–581.[Abstract]
Shrestha, N. K., Tuohy, M. J., Hall, G. S., Reischl, U., Gordon, S. M. & Procop, G. W. (2003). Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real time PCR. J Clin Microbiol 41, 5121–5126.[Abstract/Free Full Text]
Soini, H., Bottger, E. C. & Viljanen, M. K. (1994). Identification of mycobacteria by PCR-based sequence determination of the 32-kioldalton protein gene. J Clin Microbiol 32, 2944–2947.[Abstract/Free Full Text]
Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society (2000). Management of opportunistic mycobacterial infections: Joint Tuberculosis Committee guidelines 1999. Thorax 55, 210–218.[Free Full Text]
Tanaka, I. I., Anno, I. S., Andrade Leite, S. R., Cooksey, R. C. & Leite, C. Q. F. (2003). Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria. Microbiol Immunol 47, 307–312.[Medline]
Tortoli, E., Nanetti, A., Piersimoni, C., Chichero Farina, C., Mucignat, G., Scarparo, C., Bartolini, L., Valentini, R., Nista, D. & other authors (2001). Performance assessment of new multiplex probe assay for identification of mycobacteria. J Clin Microbiol 39, 1079–1084.[Abstract/Free Full Text]
Turenne, C. Y., Tschetter, L., Wolfe, J. & Kabani, A. (2001). Necessity of quality controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 39, 3637–3648.[Abstract/Free Full Text]
Vaneechoutte, M., Beenhouwer, H. D., Claeys, G., Verschraegen, G., De Rouck, A., Paepe, N., Elaichouni, A. & Portaels, F. (1993). Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol 31, 2061–2065.[Abstract/Free Full Text]
Velayati, A. A., Boloorsaze, M. R., Farnia, P., Mohammadi, F., Karam, M. B., Soheyla-Zahirifard, M. D. & Masjedi, M. R. (2005). Mycobacterium gastri causing disseminated infection in children of same family. Pediatr Pulmonol 39, 284–287.[CrossRef][Medline]
Yam, W.-C., Yuen, K.-Y., Kam, S.-Y., Yiu, L.-S., Chan, K.-S., Leung, C.-C., Tam, C.-M., Ho, P.-O., Yew, W.-W. & other authors (2006). Diagnostic application of genotypic identification of mycobacteria. J Med Microbiol 55, 529–536.[Abstract/Free Full Text]
Zolg, J. W. & Philippi-Schulz, S. (1994). The superoxide dismutase gene, a target for detection and identification of Mycobacteria by PCR. J Clin Microbiol 32, 2801–2812.[Abstract/Free Full Text]
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INTRODUCTION
METHODS
