A correction has been published: N Engl J Med 1996;335(23):1771.
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Case Report
The patient, born in November 1985, was the fourth child of a farmer from West Africa. In 1989 he was bitten on his right leg by a snake. Gangrene developed, and two months later he lost his right foot. From March 1990 to March 1991, he was hospitalized in Cotonou, Benin, where the amputation was extended to the midtibial level of the right leg. Later, other bone lesions of his arms and legs appeared. Multiple chronic abscesses in the subcutaneous tissue drained to the skin. Therapy with norfloxacin, co-trimoxazole (trimethoprim-sulfamethoxazole), chloramphenicol, ofloxacin, ketoconazole, and rifampin produced little improvement.
In April 1991 the boy was transferred to our hospital. On admission, he had multiple skin lesions on the sternum and limbs. The lesions were ulcerative and connected to fistulas, with copious discharge. The patient was febrile (temperature, 38.6 °C) and weighed 14 kg (less than the 5th percentile for his age).
Radiographs showed multiple destructive lesions with areas of radiolucency surrounded by bone sclerosis, sequestra, and periosteal areas of regeneration. The lesions were most common on the metaphyses of the right tibia and left femur, but were found on almost all the bones of the limbs (Figure 1). Laboratory studies revealed evidence of inflammation: an elevated erythrocyte sedimentation rate, elevated levels of C-reactive protein, and polyclonal hypergammaglobulinemia. Tests for antibodies against human immunodeficiency virus types 1 and 2 were negative. The function of the neutrophils was normal. The numbers of B lymphocytes (6 percent) and T lymphocytes (27 percent CD4+ cells [926 per cubic millimeter] and 30 percent CD8+ cells [1029 per cubic millimeter]) were within normal limits. A tuberculin test with 2 units of purified protein derivative (tuberculin RT 23) was positive, with an area of erythema and induration measuring 11 mm in diameter, and the lymphocyte response to purified protein derivative was markedly increased (stimulation index, 101). Phagocytic-cell function was normal, as judged by the degree of myeloperoxidase staining, the response to a nitroblue tetrazolium test, and a study of activation markers on monocytes.
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Antituberculous therapy was started with isoniazid at a dose of 15 mg per kilogram of body weight per day, rifampin at a dose of 15 mg per kilogram per day, ethambutol at a dose of 15 mg per kilogram per day, and pyrazinamide at a dose of 30 mg per kilogram per day. The specimens, cultured on Middlebrook 13A medium (Bactec, Becton Dickinson Diagnostic, Towson, Md.) and Lowenstein medium at 30 °C and 37 °C,2 remained negative after an incubation of up to eight months.
DNA was extracted from three samples obtained during drainage of different bone lesions on three occasions. Bacterial ribosomal DNA (rDNA) genes were amplified,3,4 and the nucleic acid sequence was determined. The positive controls consisted of DNA amplified from cultures of M. tuberculosis, M. avium complex, and M. genavense4. The negative controls consisted of the medium, buffers, and enzymes used for DNA amplification; tissue extracts without evidence of infection with mycobacteria; and extracts of medium used to culture mycobacteria. No amplification signal was observed in any of these samples. All controls were run in parallel and interspersed with the patient's samples. The sequence determined was identical in all samples investigated and was subsequently identified as M. ulcerans5 on the basis of the nucleotide present at position 1248 (Figure 2).
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Discussion
The child we describe had a multifocal osteomyelitis with extensive and progressive lesions (Figure 1). Cultures of bone remained sterile (even after antibiotics were discontinued), ruling out an infection with common bacteria, such as staphylococci or enterobacteriaceae. Chronic multifocal osteomyelitis, which is a self-limited, benign disease, was incompatible with this severe course. Negative serologic tests for syphilis excluded the possibility of treponematosis.
The diagnosis of mycobacterial osteomyelitis was made three weeks after admission because of the presence of acid-fast bacilli on smears from intraoperative bone samples. The differential diagnosis of mycobacteria known to cause skin and bone lesions includes M. fortuitum, M. chelonei, and M. marinum, all of which grow well and quickly; M. avium complex, which is rare in the immunocompetent host; M. terrae complex associated with indolent chronic disease and occasionally with hematogenous infection; M. kansasii; and M. simiae1. The cultures remained negative, and the mycobacterium was identified as M. ulcerans by amplifying the genes coding for 16S ribosomal RNA (rRNA).
M. ulcerans, the etiologic agent of an indolent, necrotizing skin disease that is endemic in some tropical areas, requires 6 to 12 weeks for initial isolation and may even fail to grow on primary medium. In contrast, M. marinum, which causes cutaneous granuloma that is usually self-limiting, readily grows at 30 °C to 32 °C in vitro2. The presence of identical phenolic mycosides6 has suggested a close link between M. marinum and M. ulcerans. This suggestion is supported by the finding that M. ulcerans and M. marinum possess a nearly identical 16S rRNA gene sequence (99.9 percent homology)5,7. The determination of the 16S rDNA sequence of five isolates of both M. marinum and M. ulcerans (kindly provided by F. Portaels, Antwerp, Belgium; H.R. Chang, Geneva, Switzerland; and K.H. Schroder, Borstel, Germany) revealed an identical sequence even in regions that are characteristically hypervariable in mycobacteria5. Within the complete molecule, only a single nucleotide, at position 1248 of the 16S rRNA (corresponding to the numbering sequence for Escherichia coli), differs in these two species (Figure 2). M. ulcerans is characterized by a guanine, whereas M. marinum is characterized by an adenine. Material from all three biopsy specimens examined had a guanine at this position, thus identifying the acid-fast bacteria as M. ulcerans. The failure of the organism to grow in vitro and the clinical presentation also strongly argued for M. ulcerans, rather than M. marinum.
The mode of transmission of M. ulcerans is not known, but some researchers believe that infection occurs by pricks or bites, or is waterborne8. In our patient, the disease seems to have followed a snakebite, which we suspect led to the inoculation of the mycobacteria into the subcutaneous tissue of the right foot. The reasons for the subsequent spread remain mysterious, because disseminated infections with nontuberculous mycobacteria occur almost exclusively in immunodeficient hosts9,10,11. The results of tests of the patient's immune function were normal, including a strong in vitro reactivity of lymphocytes to purified protein derivative, which is known to cross-react with atypical mycobacteria.
Infections with M. ulcerans are difficult to treat. Some report that antibiotics are ineffective12. In contrast, others are optimistic about the efficacy of combined medical and surgical therapy8. Many drugs are active against M. ulcerans in vitro but have limited value in vivo8. Co-trimoxazole and rifampin are often given. Clarithromycin, a macrolide with increased efficacy against mycobacteria such as M. avium complex,13 may perhaps be a promising new therapy.
More than 30 years ago, van der Hoeven et al. described a child with fatal multifocal osteomyelitis, in whom acid-fast bacilli were seen at autopsy and cultured, but not identified11. In our case, although cultures were negative, amplification of rRNA genes with nucleic acids extracted from biopsy specimens established the diagnosis. Analysis of 16S rRNA is especially suited to microorganisms that are difficult to grow in culture and has been used to clarify the cause of bacillary angiomatosis,14 Whipple's disease,15 and disseminated infection with M. genavense4.
Supported in part by a grant from the Niedersachsischer Verein zur Bekampfung der Tuberkulose e.V.
We are indebted to M. Kiekenbeck for expert technical assistance, to Dr. J.-D. Piguet for attempts to culture the mycobacterium, and to Dr. J. Garcia for help in describing the radiographic findings.
Source Information
From the Departement de Pediatrie (M.H., C.-A.S., S.S.), Division de maladies infectieuses (B.H.), and Unite d'orthopedie pediatrique (M.B., A.K.), Hopital Cantonal Universitaire, Geneva, Switzerland, and the Institut fur Medizinische Mikrobiologie, Medizinische Hochschule, Hannover, Germany (P.K., A.T., E.C.B.).
Address reprint requests to Dr. Hirschel at the Division of Infectious Diseases, Hopital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland.
References
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Related Letters:
Mycobacterium ulcerans and Osteomyelitis
Kristjansson M., Arbeit R. D., Weed H. G., Hirschel B., Bottger E.
Extract |
Full Text
N Engl J Med 1993;
329:582-583, Aug 19, 1993.
Correspondence
Mycobacterium ulcerans Osteomyelitis: A Closer Look at the X-Ray Films
Hu L. B., Hofer M., Hirschel B.
Extract |
Full Text
N Engl J Med 1996;
335:1771-1772, Dec 5, 1996.
Correspondence
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