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Journal of Clinical Microbiology, December 2002, p. 4705-4712, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4705-4712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Algorithm Identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis

Tomotada Iwamoto,^1* Toshiaki Sonobe,¹ and Kozaburo Hayashi²

Department of Bacteriology,¹ Department of Parasitic Agents, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan²

Received 10 June 2002/ Returned for modification 5 August 2002/ Accepted 17 September 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
An algorithm to directly identify multiple mycobacterial species in a sample by using fluorescence capillary electrophoresis (FCE)-based single-strand conformation polymorphism (SSCP) analysis was developed. Part of the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region in 37 reference strains and 73 clinical isolates representing 19 mycobacterial species and Mycobacterium tuberculosis complex was PCR amplified with a fluorescence-labeled mycobacterium-specific primer, 6-carboxyfluorescein-labeled primer Sp1f, and 5-hexachlorofluorescein-tagged Sp2r. FCE-SSCP analysis was applied to both undigested PCR products and the corresponding HaeIII-digested restriction fragments (RF) from each strain. The 23 resultant SSCP patterns distinguished all 19 species and M. tuberculosis complex. The technique is applicable for the detection of multiple mycobacterial species in a sample. It was demonstrated by analyzing two model mycobacterial communities consisting of five species with both rapidly and slowly growing species (model A) and four species commonly encountered in clinical practice (model B). The sensitivity study with spiked sputum samples with different amounts of M. tuberculosis H37Rv, M. avium, and M. intracellulare cells indicated that up to 25% of the amount of each mycobacterium could be detected relative to the two other species. Fifty-one sputum specimens analyzed by FCE-RF-SSCP were compared with the Amplicor assay (Roche Diagnostics GmbH). Species identified by both assays were always the same. Moreover, FCE-RF-SSCP could identify M. abscessus and M. kansasii, which are not targeted by Amplicor. FCE-RF-SSCP of sputum obtained from a patient with mixed M. avium and M. intracellulare infection gave SSCP patterns corresponding to these two species.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent increases in infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria are a serious hazard to public health (2, 6, 11-13, 20). More than 30 mycobacterial species are recognized as pathogenic or potentially pathogenic. Mixed infections with two or more species (1, 18, 29, 31) and nosocomial infections (3, 8, 19, 25, 27) are always troublesome. Therefore, timely identification of causative mycobacteria is imperative for appropriate therapy and epidemiological analyses.

Genotyping the sequence polymorphisms in the genus Mycobacterium can identify species quickly and unequivocally. PCR-restriction fragment polymorphism analysis (PRA) of the hsp65 gene (4, 21, 22, 28), 16S ribosomal DNA (rDNA) (5, 14, 15, 30), 16S-23S rDNA internal transcribed spacer (ITS) (23), dnaJ (26), rpoB (17), and gyrB (16) has been intensively studied. However, these methods are not applicable for a polymycobacterial sample because too many restriction fragments from multiple species of mycobacteria in it blur the analysis.

We developed a fluorescence capillary electrophoresis (FCE)-based restriction fragment-single-strand conformation polymorphism (FCE-RF-SSCP) technique which can simultaneously identify multiple mycobacterial species in a sample. We first established an algorithm for species identification by applying this technique to 110 defined mycobacterial strains. The technique was further validated on "spiked" specimens, clinical sputum specimens, and sputum obtained from a patient with mixed M. avium and M. intracellulare infection.

(This work was presented in part at the 102nd Annual Meeting of the American Society for Microbiology, Salt Lake City, Utah, 20 May 2002.)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mycobacteria and DNA preparation. Genomic DNAs used for the establishment of an algorithm were isolated from 37 reference strains and 73 clinical isolates representing 19 mycobacterial species and M. tuberculosis complex with Isoplant (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions (Table 1). Clinical isolates were identified by conventional biochemical tests, Accu-Probe (Gen-Probe, Inc., San Diego, Calif.), DDH Mycobacteria ‘Kyokuto’ (Kyokuto Pharmaceutical Ind. Co., Ltd., Tokyo, Japan), or partial 16S rDNA sequencing.

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TABLE 1. Strains used to establish an algorithm for species identification

Clinical sputum specimens. Fifty-one sputum specimens from patients with suspected M. tuberculosis or nontuberculous mycobacterial infections were obtained from Kobe City General Hospital. After decontamination with N-acetyl-L-cystein-NaOH, DNA was extracted from these specimens with a respiratory specimen preparation kit (Roche Diagnostics GmbH). The sputum specimens were also subjected to acid-fast staining and cultured. One frozen sputum sample obtained from a patient with mixed M. avium and M. intracellulare infection was retrospectively analyzed by FCE-RF-SSCP.

DNA amplification and restriction enzyme digestion. Genus Mycobacterium-specific primers Sp1f and Sp2r, designed by Roth et al. (23), were used to amplify part of the 16S-23S rRNA gene spacer. A reaction mixture (50 µl) containing 20 pmol of 6-carboxyfluorescein (FAM)-labeled Sp1f and 5-hexachlorofluorescein (HEX)-tagged Sp2r primers, 1.5 mM MgCl₂, 3.5 µl of dimethyl sulfoxide, and 1 to 20 µl of DNA solution was amplified with the Expand high-fidelity PCR system (Roche Diagnostics GmbH) following the thermal profile method of Roth et al. (23). Fivefold-diluted PCR product (9 µl) was digested at 37°C for 3 h in a buffer containing 5 U of HaeIII enzyme (Takara Shuzo Co., Ltd., Shiga, Japan).

Artificially constructed mycobacterial community. Two model mycobacterial communities were constructed. Model A represents a mixture of rapid- and slow-growing species consisting of M. abscessus ATCC 19977, M. avium ATCC 25291, M. chelonae ATCC 19237, M. intracellulare ATCC 35772, and M. xenopi ATCC 19250. Model B is a mixture of M. avium ATCC 25291, M. intracellulare ATCC 13950, M. tuberculosis H37Rv, and M. kansasii ATCC 12478, which is responsible for more than 95% of the cases of mycobacteriosis in Japan (24). Five nanograms of purified genomic DNAs from each species was suspended together in the PCR mixture and used for FCE-RF-SSCP analysis after the PCR.

Spiked specimen preparation. M. avium ATCC 25291, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were individually cultured in Middlebrook 7H9 broth until the turbidity equaled a McFarland no. 1 standard and was then adjusted to the same cell density with an appropriate dilution. Each of these liquid cultures (10 µl) was mixed with 100 µl of acid-fast bacillus-negative sputum sample that was diluted with an equivalent amount of phosphate-buffered saline. The amount of M. tuberculosis H37Rv was serially diluted twofold, while the other strains were kept constant to produce five levels: no dilution, twofold, fourfold, eightfold, and 16-fold dilutions. One hundred microliters of each mixture was used for DNA extraction after NALC-NaOH treatment.

SSCP electrophoresis. The PCR products were analyzed for SSCP on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.) by Gillman's method (7) with modifications: 0.5 µl of 0.3 N NaOH was added to the SSCP analysis master mix, the SSCP analysis mixture was then denatured by heating at 95°C for 5 min and left at room temperature, and the retention times of the fluorescently labeled fragments were determined against that of the 6-carboxy-X-rhodamine-labeled internal standard (Fig. 1). In addition, the conditions for electrophoresis were set as follows: 5-s injection time, 15.0-kV injection voltage, 13-kV electrophoresis voltage, 210-s syringe pump time, 30°C constant temperature of the capillary at a room temperature of 23 to 24°C, and a 23-min collection time. The PCR products and corresponding HaeIII restriction fragments were run in two separate sample tubes after appropriate dilution with sterilized water (in most cases, diluted 1:10).

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FIG. 1. FCE-RF-SSCP analysis of the internal standard, Mycobacterium avium ATCC 25291 undigested ITS fragment and HaeIII-digested fragment. 6-carboxy-X-rhodamine-labeled internal standard, FAM-labeled fragments, and HEX-labeled fragments are shown as red peaks, blue peaks, and green peaks, respectively. Arbitrary electrophoretic mobility values for internal standard were assigned as follows: peak 1, 3859; peak 2, 3960; peak 3, 4096; peak 4, 4125; peak 5, 4157; peak 6, 4219; peak 7, 4404; peak 8, 4426; peak 9, 4462; peak 10, 4493; peak 11, 4817; peak 12, 4880; peak 13, 4919; peak 14, 5150; peak 15, 5188; peak 16, 5454; peak 17, 5485; peak 18, 5538; peak 19, 5563; peak 20, 5675; peak 21, 5702; peak 22, 5716; and peak 23, 5739.

Top Abstract Introduction Materials and Methods Results Discussion References

 
FCE-RF-SSCP analysis of 19 mycobacterial species and M. tuberculosis complex showed 23 SSCP patterns that could be used as the algorithm to identify the species and subtypes of these mycobacteria (Table 2). M. tuberculosis H37Rv and M. bovis BCG Tokyo could not be discriminated because the 16S-23S rDNA ITS sequences are identical among M. tuberculosis complex isolates (9, 16). M. terrae and M. nonchronogenicum had multiple peaks (Table 2). The method is highly reproducible, with reasonable intraspecies stability of SSCP (Table 2). Based on the standard deviation of SSCP peak placement, we applied a tolerance level of ±5 data points in the algorithm for species identification.

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TABLE 2. Algorithm for the identification of Mycobacterium species by FCE-RF-SSCP

The established polymorphisms among SSCP patterns for the 19 species and M. tuberculosis complex suggest that species can be distinguished even when multiple mycobacterial species are found in a single sample. To test this hypothesis, two model mycobacterial communities representing a mixture of rapid- and slow-growing species (model A) and the most ubiquitous mycobacterial species found in clinical practice (model B) were constructed. The FCE-RF-SSCP analysis of the model communities yielded characteristics SSCP patterns (Fig. 2) from which all of the mixed strains were correctly identified (Table 3).

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FIG. 2. FCE-RF-SSCP analysis of 16S-23S rDNA ITS fragments amplified from artificially constructed mycobacterial communities. Electrophoretic mobility values are indicated at each peak on the electropherograms for FAM-labeled fragments (in blue) and HEX-labeled fragments (in green).

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TABLE 3. Identification of mycobacterial species from the SSCP patterns shown in Fig. 2a

The species belonging to each peak in Fig. 2 were identified as follows (Table 3). In the initial step, we listed all of the possible species under the peaks with the algorithm, then the species that appeared on all four peaks, undigested 6-FAM, undigested HEX, HaeIII 6-FAM, and HaeIII HEX, were judged as the existing species in a sample. The blue peak (3690.0 data points) and green peak (3886.1 data points) in the HaeIII fragments of model B are considered overlapping peaks originating from M. avium and M. intracellulare I. Besides these two model communities, our study of various combinations of three to five species demonstrated that this technique would work clinically (data not shown).

When sputum samples spiked with M. avium, M. intracellulare, and different amounts of M. tuberculosis H37Rv cells were analyzed, the peaks of single-stranded DNA from M. tuberculosis H37Rv were discernible in fourfold- but not in eightfold-diluted samples (Fig. 3). No unexpected peaks or interference from the spiked samples were observed. These results indicate that the detection limit for each mycobacterium is up to 25% of the amount relative to the other two species.

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FIG. 3. FCE-RF-SSCP analysis of PCR amplification products for sputum samples spiked with M. avium ATCC 25291, M. intracellulare ATCC 13950, and various amounts of M. tuberculosis H37Rv. The amount of M. tuberculosis H37Rv was serially twofold diluted, while that of the other strains was kept constant: no dilution (A), -fold (B), 4-fold (C), 8-fold (D), or 16-fold (E) dilution. Electrophoretic mobility values at each peak and their corresponding strains are indicated on the electropherograms: av, M. avium ATCC 25291; int, M. intracellulare ATCC 13950; TB, M. tuberculosis H37Rv.

Fifty-one sputum specimens from patients with suspected M. tuberculosis or nontuberculous mycobacterial infections were tested by FCE-RF-SSCP and the Amplicor assay (Table 4). These specimens were also subjected to acid-fast staining and cultured. Species identified by FCE-RF-SSCP, Amplicor, and the culture method were always the same. In addition to the three species targeted by Amplicor, FCE-RF-SSCP could identify M. abscessus and M. kansasii. Two specimens were negative by smear and culture tests but showed positive results only in the Amplicor (specimens 32 and 33); this may be due to the higher sensitivity of Amplicor than FCE-RF-SSCP or may represent false-positive results of the Amplicor test. The 51 sputum specimens were not amplified nonspecifically. The results also support the specificity of the primer set used in this study and warrant the application of our technique to the direct identification of mycobacterial species in a sputum specimen.

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TABLE 4. Comparison of FCE-RF-SSCP with Amplicor assay for direct identification of mycobacteria in sputum specimens

FCE-RF-SSCP of sputum obtained from a patient with mixed M. avium and M. intracellulare infection gave SSCP patterns corresponding to these two species (4421.3 data points of blue and 4480.0 of green for M. avium, and 4442.0 blue and 4515.9 green for M. intracellulare I). The blue peak (3687.0) and green peak (3886.2) in HaeIII are the overlapping peaks originating from these species (Fig. 4). This result demonstrates the effectiveness of FCE-RF-SSCP analysis in the direct identification of mixed infections from patient specimens.

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FIG. 4. FCE-RF-SSCP analysis of sputum from a patient with mixed mycobacterial M. avium and M. intracellulare infection. Electrophoretic mobility values at each peak are indicated on the electropherograms.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We developed a simple algorithm using FCE-RF-SSCP with genus Mycobacterium-specific primers (23) to simultaneously distinguish multiple mycobacterial species in a polymicrobial sample. This is the first report to expand the PRA method to this application. Compared with ordinary PRA methods, this technique is unique because it analyzes only terminal restriction fragments and undigested fragments. In this method, the troublesome gel-to-gel inconsistencies seen in agarose-based methods are avoided, as FCE gives high resolution and reproducible analyses by with an internal standard in each sample. Recently, Gillman et al. (7) used FCE for the identification of mycobacterial species by using four pairs of fluorescent primers of the 16S rRNA gene and subsequently analyzed the SSCPs. Without using a restriction enzyme, they obtained species-specific SSCP patterns for 30 of the common mycobacteria. However, since the PCR primers used in their method are not genus Mycobacterium specific, prior cultivation is required for the analysis.

Kim et al. (17) used PRA of rpoB DNA for direct identification of mycobacterial species in sputum samples and bronchoalveolar lavage specimens. Although this method showed improved identification of clinical specimens, application is limited to a single mycobacterial infection because too many fragments from a mixed infection blur the analysis. In our method, peaks from four types of fragments were obtained individually and used to identify each species. Since the number of SSCP peaks originating from each primer for a single species is theoretically one, multiple mycobacterial species in a sample can be identified with the algorithm. Multiple peaks obtained from M. terrae and M. nonchronogenicum are due to different stable conformations of the same fragment (7, 10) or the existence of multicopies of ITS in these species.

The newly developed technique, FCE-RF-SSCP, was validated on artificially constructed mycobacterial communities, clinical sputum specimens, and a sputum specimen from a patient with mixed M. avium and M. intracellulare infection. All results supported the ability of the technique to directly identify multiple mycobacterial species present in one specimen. Further investigations would accumulate more SSCP patterns from more Mycobacterium species and perform a multicenter study to ensure applicability in a laboratory setting.

 
We thank M. Okazaki and B. Umeda of Kobe City General Hospital for providing sputum samples and the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, for providing M. tuberculosis H37Rv and M. bovis BCG Tokyo.

 
* Corresponding author. Mailing address: Department of Bacteriology, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan. Phone: 81 78 302 6251. Fax: 81 78 302 0894. E-mail: kx2t-iwmt{at}asahi-net.or.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Branger, B., A. Gouby, A. Oules, J. P. Balcucci, G. Mourad, J. Fourcade, C. Mion, F. Duntz, and M. Ramuz. 1985. Mycobacterium haemophilum and Mycobacterium xenopi associated infection in a renal transplant patient. Clin. Nephrol. 23:46-49.[Medline]
2
2. Covert, T. C., M. R. Rodgers, A. L. Reyes, and G. N. Stelma, Jr. 1999. Occurrence of nontuberculous mycobacteria in environmental samples. Appl. Environ. Microbiol. 65:2492-2496.[Abstract/Free Full Text]
3
3. Dawson, D. J., J. F. Armstrong, and Z. M. Blacklock. 1982. Mycobacterial cross-contamination of bronchoscopy specimens. Am. Rev. Respir. Dis. 126:1095-1097.[Medline]
4
4. Devallois, A., K. S. Goh, and N. Rastogi. 1997. Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 mycobacterial species. J. Clin. Microbiol. 35:2969-2973.[Abstract]
5
5. Domenech, P., M. C. Menendez, and M. J. Garcia. 1994. Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species. FEMS Microbiol. Lett. 116:19-24.[CrossRef][Medline]
6
6. Falkinham, J. O., III. 1996. Epidemiology of infection by nontuberculous mycobacteria. Clin. Microbiol. Rev. 9:177-215.[Medline]
7
7. Gillman, L. M., J. Gunton, C. Y. Turenne, J. Wolfe, and A. M. Kabani. 2001. Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J. Clin. Microbiol. 39:3085-3091.[Abstract/Free Full Text]
8
8. Gubler, J. G., H. M. Salfinger, and A. von Graevenitz. 1992. Pseudoepidemic of nontuberculous mycobacteria due to a contaminated bronchoscope cleaning machine. Chest 101:1245-1249.[Abstract/Free Full Text]
9
9. Harmsen, D., J. Rothgänger, C. Singer, J. Albert, and M. Frosch. 1999. Intuitive hypertext-based molecular identification of micro-organisms. Lancet 353:291.[Medline]
10
10. Hayashi, K. 1991. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl. 1:34-38.[Medline]
11
11. Horsburgh, C. R., Jr. 1991. Mycobacterium avium complex infection in the acquired immunodeficiency syndrome. N. Engl. J. Med. 324:1332-1338.[Medline]
12
12. Horsburgh, C. R., Jr. 1992. Epidemiology of mycobacterial diseases in AIDS. Res. Microbiol. 143:372-377.[Medline]
13
13. Horsburgh, C. R., Jr., and R. Selik. 1989. The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). Am. Rev. Respir. Dis. 139:4-7.[Medline]
14
14. Hughes, M. S., R. A. Skuce, L.-A. Beck, and S. D. Neill. 1993. Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences. J. Clin. Microbiol. 31:3216-3222.[Abstract/Free Full Text]
15
15. Kanaujia, G. V., V. M. Katoch, C. T. Shivannavar, V. D. Sharma, and M. A. Patil. 1991. Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes. FEMS Microbiol. Lett. 61:205-208.[Medline]
16
16. Kasai, H., T. Ezaki, and S. Harayama. 2000. Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences. J. Clin. Microbiol. 38:301-308.[Abstract/Free Full Text]
17
17. Kim, B. J., K. H. Lee, B. N. Park, S. J. Kim, G. H. Bai, S. J. Kim, and Y. H. Kook. 2001. Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB). J. Clin. Microbiol. 39:2102-2109.[Abstract/Free Full Text]
18
18. Lévy-Frébault, V., B. Pangon, A. Buré, C. Katlama, C. Marche, and H. L. David. 1987. Mycobacterium simiae and Mycobacterium avium-M. intracellulare mixed infection in acquired immune deficiency syndrome. J. Clin. Microbiol. 25:154-157.[Abstract/Free Full Text]
19
19. Mbithi, J. N., V. S. Springthorpe, S. A. Sattar, and M. Pacquette. 1993. Bactericidal, virucidal, and mycobactericidal activities of reused alkaline glutaraldehyde in an endoscopy unit. J. Clin. Microbiol. 31:2988-2995.[Abstract/Free Full Text]
20
20. Peters, M., D. Schurmann, A. C. Mayr, R. Hetzer, H. D. Pohle, and B. Ruf. 1989. Immunosuppression and mycobacteria other than Mycobacterium tuberculosis: results from patients with and without HIV infection. Epidemiol. Infect. 103:293-300.[Medline]
21
21. Plikaytis, B. B., B. D. Plikaytis, M. A. Yakurus, W. R. Butler, C. L. Woodley, V. A. Silcox, and T. M. Shinnick. 1992. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis. J. Clin. Microbiol. 30:1815-1822.[Abstract/Free Full Text]
22
22. Ringuet, H., C. Akoua-Koffi, S. Honore, A. Varnerot, V. Vincent, P. Berche, J. L. Gaillard, and C. Pierre-Audigier. 1999. hsp65 sequencing for identification of rapidly growing mycobacteria. J. Clin. Microbiol. 37:852-857.[Abstract/Free Full Text]
23
23. Roth, A., U. Reischl, A. Streubel, L. Naumann, R. M. Kroppenstedt, M. Habicht, M. Fischer, and H. Mauch. 2000. Novel diagnostic algorithm for identification of mycobacteria with genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases. J. Clin. Microbiol. 38:1094-1104.[Abstract/Free Full Text]
24
24. Sakatani, M. 1999. Nontuberculous mycobacteriosis; the present status of epidemiology and clinical studies. Kekkaku 74:377-384.[Medline]
25
25. Spach, D. H., F. E. Silverstein, and W. E. Stamm. 1993. Transmission of infection by gastrointestinal endoscopy and bronchoscopy. Ann. Intern. Med. 118:117-128.[Abstract/Free Full Text]
26
26. Takewaki, S., K. Okuzumi, I. Manabe, M. Tanimura, K. Miyamura, K. Nakahara, Y. Yazaki, A. Ohkubo, and R. Nagai. 1994. Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species. Int. J. Syst. Bacteriol. 44:159-166.[Abstract/Free Full Text]
27
27. Takigawa, K., J. Fujita, K. Negayama, S. Terada, Y. Yahaji, K. Kawanishi, and J. Takahara. 1995. Eradication of contaminating Mycobacterium chelonae from bronchofibrescopes and an automated bronchoscope disinfection machine. Respir. Med. 89:423-427.[CrossRef][Medline]
28
28. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Böttger, and T. Bodmer. 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175-178.[Abstract/Free Full Text]
29
29. Torres, R. A., J. Nord, R. Feldman, V. LaBombardi, and M. Barr. 1991. Disseminated mixed Mycobacterium simiae-Mycobacterium avium complex infection in acquired immunodeficiency syndrome. J. Infect. Dis. 164:432-433.[Medline]
30
30. Vaneechoutte, M., H. De Beenhouwer, G. Claeys, G. Verschraegen, A. De Rouck, N. Paepe, A. Elaichouni, and F. Portaels. 1993. Identification of Mycobacterium species by with amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 31:2061-2065.[Abstract/Free Full Text]
31
31. Weiszfeiler, J. G., and V. Karasseva. 1981. Mixed mycobacterial infections. Rev. Infect. Dis. 3:1081-1083.[Medline]

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Journal of Clinical Microbiology, December 2002, p. 4705-4712, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4705-4712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Iwamoto, T. Articles by Hayashi, K. Search for Related Content PubMed PubMed Citation Articles by Iwamoto, T. Articles by Hayashi, K.