Department of Microbiology and Infectious Disease, Royal Children's Hospital,1 ... The diagnosis of Mycobacterium ulcerans infection is hampered by the slow ...
JOURNAL OF CLINICAL MICROBIOLOGY, July 1997, p. 1696–1700 0095-1137/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 35, No. 7
Development of a PCR Assay for Rapid Diagnosis of Mycobacterium ulcerans Infection B. C. ROSS,1* L. MARINO,1 F. OPPEDISANO,1 R. EDWARDS,2 R. M. ROBINS-BROWNE,1,3 1 AND P. D. R. JOHNSON † Department of Microbiology and Infectious Disease, Royal Children’s Hospital,1 and Department of Microbiology, University of Melbourne,3 Parkville, and Mycobacterium Reference Laboratory, Victorian Infectious Diseases Reference Laboratory, Melbourne,2 Australia Received 22 October 1996/Returned for modification 13 December 1996/Accepted 27 March 1997
The diagnosis of Mycobacterium ulcerans infection is hampered by the slow growth of the bacterium in culture, resulting in a delay of several months before a specific diagnosis can be obtained. In addition, M. ulcerans cannot be isolated from water even when there is convincing epidemiological evidence implicating this as the source of infection. The aim of the present study was to develop a PCR assay to circumvent the problems of delayed diagnosis and insensitivity of standard bacterial culture for M. ulcerans. For the PCR, we isolated an M. ulcerans-specific DNA fragment, 1,109 bp long, which is repeated at least 50 times throughout the genome. Use of this sequence as a target for PCR allowed us to detect as few as 2 molecules of genomic DNA in vitro. The PCR was used to detect M. ulcerans DNA in fresh tissue and paraffin-embedded sections from all seven patients with culture-confirmed cases of infection. of homology between different mycobacterial species (3, 7) and may not be an ideal target for specific amplification. The utility of 16S rRNA sequencing has been demonstrated in a case of suspected M. ulcerans infection in which a single nucleotide difference was detected between M. ulcerans and M. marinum (12). However, a recent report indicated variability among the 16S rRNA sequences of a number of different M. ulcerans strains (23). These data suggested that the existing DNA sequences lack the appropriate characteristics for the development of a simple, sensitive, and specific PCR-based assay. The aim of this study was to identify a specific DNA sequence in M. ulcerans which could be used as a template for the PCR amplification of DNA in clinical and environmental samples.
Mycobacterium ulcerans is a slowly growing mycobacterium which causes chronic progressive skin ulcers in humans (8). The first clinical description of infection with this bacterium was published in 1948 after it was identified in an ulcerative lesion of a patient from Bairnsdale, Australia (17), although the disease had previously been known in Africa (16). M. ulcerans-associated disease occurs mostly in tropical regions of the world including tropical Africa, Central and South America, and Southeast Asia (9). In recent years the incidence of this infection has been reported to be increasing in Africa (18, 20) and Australia (14), where an outbreak of infection occurred in a small country town (5, 13). A number of epidemiological studies of M. ulcerans-associated disease suggest that the source of infection is slowly flowing or stagnant water (9, 18, 19). However, despite numerous attempts, M. ulcerans has never been isolated from the environment (9, 19, 21, 22). A provisional diagnosis of M. ulcerans-associated disease can be achieved by detecting acid-fast bacilli (AFB) in smears or biopsy material from clinically suspicious ulcers, but the specific cause can only be identified by culturing the bacteria, a process which may take several months (26). Therefore, there is a need for a rapid and sensitive diagnostic assay for M. ulcerans which could be used to facilitate the diagnosis of infection and which may also allow for the identification of the environmental source of infection. Because PCR has been used to facilitate the detection and identification of a number of different Mycobacterium species in clinical and environmental samples, we sought to develop a PCR for the detection of M. ulcerans. Among the possible targets for a PCR-based assay for M. ulcerans are published DNA sequences for a 65-kDa heat shock protein antigen and the 16S and 23S rRNAs. However, the genus-specific 65-kDa antigen demonstrates a high degree
MATERIALS AND METHODS Cultivation of mycobacteria and purification of DNA. The origins of the mycobacterial strains used in this study are listed in Table 1. Mycobacterial reference strains other than M. ulcerans were grown in 50-ml cultures of Middlebrook 7H9 medium at 37°C with constant shaking until the mid-logarithmic phase, determined by visual estimation, before adding D-cycloserine to a final concentration of 1 mg/ml and incubating for a further 72 h. M. ulcerans strains were grown in 50 ml of Middlebrook 7H9 medium which was heavily inoculated with confluent growth on egg yolk agar incubated at 31°C. After incubation for 1 week at 31°C, D-cycloserine was added to a final concentration of 1 mg/ml before incubation for an additional 3 to 7 days at 31°C. The DNA was then extracted and purified as described previously (24). Southern blot hybridization. The Southern blot hybridization procedure was the same for restriction enzyme-digested chromosomal DNA or PCR product, except for the electrophoresis conditions. Approximately 1 mg of chromosomal DNA was digested with 10 U of each restriction enzyme for 5 h at 37°C. Electrophoresis was performed through a 0.7% agarose gel (Bio-Rad, Richmond, Calif.) in TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA) for 16 h at 60 V (24-cm gel). PCR products were separated by electrophoresis through 2.0% agarose gel (Nu-Sieve GTG; FMC BioProducts) in TAE (40 mM Tris acetate, 2 mM EDTA) at 100 V for 3 h (10-cm gel). The DNA fragments were transferred to nylon membranes (Boehringer GmbH) by using a vacuum transfer apparatus (Hybaid, London, United Kingdom) with a vacuum of 40-cm H2O in alkaline transfer buffer (0.5 M NaOH, 1.5 M NaCl). DNA was fixed to the nylon filter by baking at 120°C for 30 min, and the filter was then prehybridized in hybridization solution containing 53 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% (wt/vol) N-lauroylsarcosine, 0.02% (wt/vol) sodium dodecyl sulfate (SDS), and 1% skim milk powder for 2 h at 68°C. Heat-denatured DNA probe or RNA probe in hybridization solution was then added, and the filter was incubated overnight at 68°C. After hybridization the filter was washed twice for 5 min each time at room temperature with 23 SSC–0.1% SDS. For whole chromosomal probing, the
* Corresponding author. Present address: Microbiology Research, Research & Development, CSL Ltd., 45 Poplar Rd., Parkville 3052, Australia. Phone: 61 3 9389 1063. Fax: 61 3 9388 2063. E-mail: bross @csl.com.au. † Present address: Department of Infectious Disease, Monash Medical Center, Melbourne, Australia. 1696
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VOL. 35, 1997 TABLE 1. Origin of mycobacterial strains used in this study Mycobacterium species
M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M. M.
Strain or source
ulcerans..........................................ATCC 19423 marinum ........................................NCTC 2275 kansasii ..........................................NCTC 10268 tuberculosis ....................................H37Rv avium .............................................ATCC 25291 gastri...............................................Patient isolate, Queensland, Australia simiae.............................................ATCC 25295 malmoense.....................................NCTC 11298 haemophilum ................................CAP strain E8 szulgai ............................................NCTC 10831 gordonae ........................................NCTC 10267 flavescens .......................................Patient isolate, Queensland, Australia nonchromogenicum ......................NCTC 10424 terrae ..............................................NCTC 10856 chelonae.........................................ATCC 10269 fortuitum ........................................Patient isolate, Queensland, Australia phlei ...............................................ATCC 11758
filters were washed twice with 0.13 SSC–0.1% SDS for 15 min at 68°C. For probing with clone pA2, the filters were washed twice with 0.13 SSC–0.1% SDS for 15 min at 78°C. The presence of digoxigenin-labelled DNA probe was detected with alkaline phosphatase-conjugated antibody as described in the Genius nonradioactive nucleic acid detection kit (Boehringer GmbH). Molecular cloning. To clone restriction fragments from AluI-digested chromosomal M. ulcerans DNA, 10 mg of DNA was digested with AluI and was electrophoresed on a 1% preparative agarose gel. The required section of the preparative gel was identified by comparison with the migration of molecular size standards and was then excised. DNA was purified from the agarose with a Sephaglas BandPrep Kit (Pharmacia Biotech). For cloning into bacteriophage M13, EcoRI linkers were ligated to the AluI-digested DNA fragments. Ligated fragments were then digested with EcoRI, the excess linkers were removed by using Sephacryl S-400 spin columns (Promega, Madison, Wis.), and the DNA was ligated into EcoRI-digested M13mp18 replicative-form DNA (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). Ligated DNA was then transformed into Escherichia coli NM522 by electroporation in a 50-ml volume with a 0.2 mm cuvette at 2.5 kV, 25 mFD, and 200 W in a GenePulser apparatus (Bio-Rad). The transformation mixture was then added to an agar overlay, plated for bacteriophage plaques, and screened by DNA hybridization (1). DNA sequencing. Cloned DNA fragments in bacteriophage M13 were subcloned into the plasmid vector pT7T3 (Pharmacia Biotech) and then sequenced by using primers to the T7 and T3 promoters. Sequencing was performed by the direct incorporation protocol of the fmol DNA Sequencing System (Promega). Purification of DNA from skin biopsy specimens for PCR. DNA was extracted from clinical samples by an alkaline lysis procedure modified from a previously described method (2). Skin biopsy specimens were diced in 1 ml of phosphatebuffered saline (PBS) and then ground in a handheld Teflon homogenizer. The homogenate was pelleted by centrifugation at 2,000 3 g for 5 min, washed twice with PBS, and resuspended in 200 ml of alkaline lysis buffer (50 mM NaOH, 0.2% SDS). The lysate was heated at 95°C for 15 min and neutralized with 30 ml of 1 M Tris-HCl. The DNA was then purified by extraction with phenol-chloroform and precipitation with ethanol. With each tissue extraction, a negative control consisting of sterile PBS was treated as a mock specimen and was passed through all extraction steps including homogenization in the same apparatus. PCR amplification. For PCR, 2-ml samples of purified DNA were amplified in a buffer supplied by the manufacturer of Taq polymerase (Promega) in 20-ml reaction mixtures containing 1 U of Taq polymerase, 1 mM primers, 1.5 mM MgCl2, and 200 mM (each) deoxynucleoside triphosphates. The reactions were performed in an automated thermal cycler (MJ Research). After an initial denaturation at 94°C for 2 min, DNA was amplified by 35 cycles of 1-min steps at 94, 66, and 72°C. Nucleotide sequence accession number. The nucleotide sequence of the AluI fragment from clone pA2 has been allocated GenBank accession no. U38540.
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of this band suggested that the M. ulcerans genome contains a repetitive DNA element which, if specific, would be a potentially sensitive target for PCR amplification. To isolate this fragment, DNA of approximately 1.1 kb was purified from the agarose gel, cloned into bacteriophage M13, and screened with the genomic M. ulcerans probe. Those clones containing repetitive DNA sequences were expected to yield a stronger hybridization signal than those with nonrepetitive DNA due to the larger number of labelled repetitive fragments in the chromosomal probe. Accordingly, a fragment from a phage which displayed a strong hybridization signal was selected and subcloned into the EcoRI site of plasmid pT7T3. The resultant plasmid was designated pA2. When the insert of pA2 was used to probe Southern blots of AluI-digested M. ulcerans DNA, it hybridized to a 1.1-kb fragment of the same apparent molecular size as the band identified with the whole chromosomal probe (Fig. 2). The cloned fragment also hybridized to two lower-molecular-mass fragments of approximately 0.6 and 0.5 kb. This was subsequently shown to represent copies of the 1.1-kb fragment containing an internal AluI restriction site (data not shown). Hybridization of the pA2 probe with SacIdigested M. ulcerans DNA resulted in a complex banding pattern, confirming that the probe sequence is a repeated DNA fragment (Fig. 2, lane 3). The copy number of this repeat was difficult to estimate after SacI digestion (Fig. 2, lane 3) due to the large number of bands, but we estimate it to be present in at least 50 copies per genome. Characterization of the M. ulcerans-specific fragment. Southern blot hybridization of genomic DNA from 17 different Mycobacterium species with the insert from pA2 as a probe suggested that the fragment was specific to M. ulcerans (Fig. 3). Nucleotide sequencing of the 1.1-kb fragment was performed with primers for the T7 and T3 promoters in the plasmid vector and internal primers (Fig. 4). The 1,109-bp sequence demonstrated no significant homology to known DNA sequences. PCR amplification. The primers MU1 and MU2 used for DNA sequence analysis were also used for PCR amplification (Fig. 4). To assess the sensitivity of the PCR with MU1 and MU2, M. ulcerans DNA was serially diluted and then subjected to PCR amplification. This investigation showed that the sen-
RESULTS Cloning of M. ulcerans-specific DNA. To develop a specific PCR for M. ulcerans we intended to screen a genomic library of fragments after digestion with AluI and HaeIII. To determine the average fragment size after digestion, Southern blot hybridization was performed with a genomic M. ulcerans probe (Fig. 1). This revealed an intensely reacting discrete band of approximately 1.1 kb in the AluI-digested DNA. The presence
FIG. 1. Characterization of genomic M. ulcerans DNA by Southern blot hybridization after digestion with AluI (lane 1) or HaeIII (lane 2). The filter was probed with labelled genomic M. ulcerans DNA. The locations of HaeIII-restricted fX174 DNA standards (in base pairs) are indicated.
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FIG. 2. Genomic M. ulcerans DNA digested with AluI (lanes 1 and 2) or SacI (lane 3) and probed with whole chromosomal probe (lane 1) or clone pA2 (lanes 2 and 3). The sizes of DNA molecular weight standards (in kilobase pairs) are indicated (lane M).
sitivity of the PCR was approximately 2 molecules of genomic DNA when the product was detected by ethidium bromide staining of agarose gels (data not shown). The specificity of the assay was assessed by performing PCR with 10 ng of DNA from the mycobacterial species listed in Fig. 3 as the target. All species other than M. ulcerans were negative by this assay (data not shown). To enhance the specificity and sensitivity of the PCR for use
FIG. 4. Nucleotide sequence of AluI fragment from clone pA2 indicating the locations of the primers MU1 and MU2 (underlined) and the probe used to detect PCR product (boldface).
with clinical specimens, a probe was prepared from a subcloned fragment, representing nucleotides 325 to 524 of the AluI fragment (Fig. 4). Skin biopsy specimens from three patients with culture-confirmed cases of clinical M. ulcerans infection were investigated by the PCR. A 568-bp DNA band was amplified from all three samples (Fig. 5A), and this DNA was confirmed to be M. ulcerans DNA by Southern blot hybridization (Fig. 5B). To assess the utility of the PCR with paraffin-embedded sections, samples from a series of patients with culture-confirmed M. ulcerans infections and control tissues were examined by PCR (Fig. 6). All control tissues were negative by both ethidium bromide staining (Fig. 6A) and Southern blot hybridization, whereas the biopsy specimens from patients with culture-confirmed cases of infection were positive (Fig. 6B). DISCUSSION
FIG. 3. Southern blot analysis of SacI-digested genomic DNA from various mycobacterial species were hybridized with a cloned repetitive element from M. ulcerans. The origin of these strains are listed in Table 1. Lanes: 1, M. ulcerans; 2, M. marinum; 3, M. kansasii; 4, M. tuberculosis; 5, M. avium; 6, M. gastri; 7, M. simiae; 8, M. malmoense; 9, M. haemophilum; 10, M. szulgai; 11, M. gordonae; 12, M. flavescens; 13, M. nonchromogenicum; 14, M. terrae; 15, M. chelonae; 16, M. fortuitum; and 17, M. phlei. The locations of molecular weight standards (in kilobase pairs) are indicated.
The laboratory diagnosis of infections with M. ulcerans is hampered by the slow growth rate of the bacterium, which may delay its identification for several months. Although a preliminary diagnosis can be made by detecting AFB in smears or biopsy material from ulcerative lesions, a number of other mycobacterial species can also cause skin lesions (19). Accordingly, the detection of AFB does not establish M. ulcerans as the cause of the illness. Furthermore, although M. ulceransinduced lesions may display characteristic histopathological features, including a lack of inflammatory infiltrate, the presence of arterial occlusions and necrosis of subcutaneous fat, in some cases culture is negative and is therefore not able to confirm the clinical and histological findings (10).
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FIG. 5. PCR amplification of DNA extracted from skin biopsy material from patients with culture-confirmed cases of M. ulcerans infection. Following electrophoresis through a 1% agarose gel, PCR product was visualized by staining with ethidium bromide (A) and Southern blot hybridization (B) with an internal fragment of the amplicon as the probe (Fig. 4). Lanes contain M. ulcerans DNA positive controls (lanes 1 to 3), mock specimen negative controls (lanes 4, 6, and 8), and extractions from skin biopsy material (lanes 5, 7, and 9). The expected size of the PCR amplicon is 568 bp. The locations of HaeIII-restricted fX174 DNA standards (in base pairs) are indicated (lanes M).
Difficulties in the cultivation of M. ulcerans have also prevented the identification of the environmental source of infection. Epidemiological data indicate that most cases occur in close proximity to water sources such as rivers, swamps, and lakes, with the incidence falling as the distance from the water source increases (9, 18, 19). Hayman (9) has suggested that M. ulcerans may cause infection after contamination of the skin with aerosols generated by decomposition of organic matter in contaminated water, but numerous attempts to cultivate M. ulcerans from environmental samples have failed (9, 19, 21, 22). In many instances, this failure may be due to other environmental mycobacteria overgrowing the cultures before M. ulcerans reaches sufficient numbers for detection. It is also possible that M. ulcerans occupies a specialized niche in contaminated water such as in water-filtering aquatic organisms (22). PCR amplification of M. ulcerans DNA would circumvent many of the problems concerning the specific diagnosis and detection of this organism. Previously, there has been a single case report of M. ulcerans infection in which PCR amplification and sequencing of 16S rRNA was used to obtain a diagnosis (12). However, this case was not culture confirmed and relied upon a single nucleotide difference between M. ulcerans and M. marinum for species identification. Such a method is not amenable to a routine laboratory and could yield misleading results if a strain of M. ulcerans developed a point mutation at the critical nucleotide position. In this study, we identified a species-specific 1,109-bp DNA fragment which appears to be repeated at least 50 times in the M. ulcerans genome and is an ideal target for the PCR. Although it shares no significant homology with nucleotide sequences in genetic databases, it is likely that a repeated fragment of this size could represent part of an insertion sequence element. In mycobacteria, such elements are characterized by multiple copies per genome and species specificity (6), which makes them suitable targets for
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PCR amplification. We are attempting to sequence outward from the AluI sites to determine if it possesses the characteristic terminal direct and inverted repeats of an insertion sequence (6). A number of insertion sequences have been found in other mycobacterial species (4, 11, 15, 25) and used as templates for PCR amplification (25, 27). A major advantage of using these elements is high sensitivity due to the presence of multiple targets per genome. The PCR for M. ulcerans appeared to detect as few as 2 molecules of M. ulcerans DNA (representing approximately 100 copies of the element) when the amplified product was detected by using agarose gel electrophoresis and staining with ethidium bromide. After hybridization of the product with a labelled DNA probe, the sensitivity of detection would be likely to improve to a sensitivity of 1 molecule, enabling the detection of a single bacterium in a sample. For clinical diagnosis, the PCR provides a sensitive and specific assay that reduces the time required for a specific diagnosis from months to a few days. As a result, decisions relating to patient management, such as surgical resection, can be made in the light of a specific diagnosis. The PCR may also be beneficial in elucidating the spectrum of clinical disease in which a number of mild, self-limiting skin conditions that were previously undiagnosed may be attributed to M. ulcerans. Another application of this assay is the potential to detect M. ulcerans in environmental samples, thus permitting identification of the source of infection for the first time. We are using this assay with concentrated water samples from a previously described outbreak (13, 18) to determine if the PCR can be adapted to detect M. ulcerans in the environment.
FIG. 6. PCR amplification of DNA extracted from paraffin sections of skin biopsy material from culture-confirmed cases of M. ulcerans infection and controls. Following electrophoresis through a 1% agarose gel, PCR product was visualized by staining with ethidium bromide (A) and Southern blot hybridization (B) with an internal fragment of the amplicon as probe (Fig. 4). Lanes contain M. ulcerans DNA-positive control (lane 1), negative control (lane 2), material from patients with culture-confirmed cases of infection (lanes 3 to 6), and negative control tissues (lanes 7 to 11). The negative control tissues were from patients with basal cell carcinoma (lanes 7 and 10), squamous cell carcinoma (lane 8), venous ulcer (lane 9), and cutaneous M. haemophilum infection (lane 11). The expected size of the PCR amplicon is 568 bp. The locations of DNA standards (in base pairs) are indicated (lanes M).
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ACKNOWLEDGMENTS This work was generously supported by funding from the Royal Children’s Hospital Research Foundation and the Victorian Government Department of Human Services.
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REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1995. Current protocols in molecular biology. John Wiley & Sons, Inc. New York, N.Y. 2. Beige, J., J. Lokies, T. Schaberg, U. Finckh, M. Fischer, H. Mauch, H. Lode, B. Kohler, and A. Rolfs. 1995. Clinical evaluation of a Mycobacterium tuberculosis PCR assay. J. Clin. Microbiol. 33:90–95. 3. Buchanan, T. M., H. Nomaguchi, D. C. Anderson, R. A. Young, T. P. Gillis, W. J. Britton, J. Ivanyi, A. H. J. Kolkh, O. Closs, B. R. Bloom, and V. Mehara. 1987. Characterization of antibody-reactive epitopes on the 65kilodalton protein of Mycobacterium leprae. Infect. Immun. 55:1000–1003. 4. Collins, D. M., and D. M. Stephens. 1991. Identification of an insertion sequence, IS1081, in Mycobacterium bovis. FEMS Microbiol. Lett. 67:11–15. 5. Flood, P., A. Street, P. O’Brien, and J. Hayman. 1994. Mycobacterium ulcerans infection on Phillip Island, Victoria. Med. J. Aust. 160:160. 6. Galas, D. J., and M. Chandler. 1989. Bacterial insertion sequences, p. 109– 162. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 7. Hance, A. J., B. Grandchamp, V. Levy-Frebault, B. Lacossier, J. Rausier, D. Bocart, and B. Gicquel. 1989. Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol. Microbiol. 3:843–849. 8. Hayman, J. 1985. Clinical features of Mycobacterium ulcerans infection. Aust. J. Dermatol. 26:67–73. 9. Hayman, J. 1991. Postulated epidemiology of Mycobacterium ulcerans infection. Int. J. Epidemiol. 20:1093–1098. 10. Hayman, J., and A. McQueen. 1985. The pathology of Mycobacterium ulcerans infection. Pathology 17:594–600. 11. Hernandez-Perez, M., N. G. Fomukong, T. Hellyer, I. N. Brown, and J. W. Dale. 1994. Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium. Mol. Microbiol. 12:717–724. 12. Hofer, M., B. Hirschel, P. Kirschner, M. Beghetti, A. Kaelin, C. A. Siegrist, S. Suter, A. Teske, and E. C. Bottger. 1993. Brief report: disseminated osteomyelitis from Mycobacterium ulcerans after a snakebite. N. Engl. J. Med. 328:1007–1009. 13. Johnson, P. D., M. G. Veitch, P. E. Flood, and J. A. Hayman. 1995. Myco-
16. 17. 18.
19. 20. 21. 22. 23.
24. 25.
26. 27.
bacterium ulcerans infection on Phillip Island, Victoria. Med. J. Aust. 162: 221–222. Johnson, P. D., M. G. Veitch, D. E. Leslie, P. E. Flood, and J. A. Hayman. 1996. The emergence of Mycobacterium ulcerans infection near Melbourne. Med. J. Aust. 164:76–78. Kunze, Z. M., S. Wall, R. Appelberg, M. T. Silva, F. Portaels, and J. J. McFadden. 1991. IS901, a new member of a widespread class of atypical insertion sequences. Mol. Microbiol. 5:2265–2272. Lunn, H. F., D. H. Connor, and N. E. Wilks. 1965. Buruli (mycobacterial) ulceration in Uganda. East Afr. Med. J. 12:275–288. MacCallum, P., J. Tolhurst, G. Buckle, and H. Sissons. 1948. A new mycobacterial infection in man. J. Pathol. Bacteriol. 60:93–102. Marston, B. J., M. O. Diallo, C. R. Horsburgh, I. Diomande, M. Z. Saki, J. M. Kanga, G. Patrice, H. B. Lipman, S. M. Ostroff, and R. C. Good. 1995. Emergence of Buruli ulcer disease in the Daloa region of Cote d’Ivoire. Am. J. Trop. Med. Hyg. 52:219–224. Meyers, W. M. 1994. Mycobacterial infections of the skin. In G. Seifert (ed.), Tropical Dermatology. Springer-Verlag, Heidelberg, Germany. Muedler, K. 1988. Buruli ulcer in Benin. Trop. Doctor 18:53. Portaels, F. 1978. Etude d’Actinomycetales isole ´es de l’homme et de son environment en Afrique Centrale. Ph.D. thesis. Faculte ´ des Sciences, Universite´ Libre de Bruxelles, Brussels, Belgium. Portaels, F. 1989. Epidemiology of ulcers due to Mycobacterium ulcerans. Ann. Soc. Belg. Med. Trop. 69:91–103. Portaels, F., P.-A. Fonteyne, H. De Beenhouwer, P. De Rijk, A. Gue´de´non, J. Hayman, and W. M. Meyers. 1996. Variability in the 39 end of 16S rRNA sequence of Mycobacterium ulcerans is related to geographic origins of isolates. J. Clin. Microbiol. 34:962–965. Ross, B. C., K. Jackson, M. Yang, A. Sievers, and B. Dwyer. 1992. Identification of a genetically distinct subspecies of Mycobacterium kansasii. J. Clin. Microbiol. 30:2930–2933. Thierry, D., A. Brisson-Noel, V. Vincent-Levy-Frebault, S. Nguyen, J. L. Guesdon, and B. Gicquel. 1990. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J. Clin. Microbiol. 28:2668–2673. Tsang, A. Y., and E. R. Farber. 1973. The primary isolation of Mycobacterium ulcerans. Am. J. Clin. Pathol. 59:688–692. Vary, P. H., P. R. Andersen, E. Green, J. Hermon-Taylor, and J. J. McFadden. 1990. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. J. Clin. Microbiol. 28:933–937.
