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© 1999 The Journal of Bone and Joint Surgery, Inc. Mycobacterium bovis Osteomyelitis as a Complication of Bacille Calmette-Guérin (BCG) Vaccination: Rapid Diagnosis with Use of DNA Sequencing Analysis. A Case Report*  , TAINAN, TAIWANInvestigation performed at National Cheng Kung University Medical College, Tainan
Vaccination with bacille Calmette-Guérin (BCG) has been used for the prevention of tuberculosis in many areas of the world. It is estimated that more than three billion people worldwide have been vaccinated with live BCG since 19459. In almost all children, inoculation with BCG is harmless. However, it must be understood that BCG, like other vaccines, such as that for poliomyelitis and even that for measles, mumps, and rubella, occasionally produces adverse reactions5,11,21-23. Despite these rare complications, BCG has proved to be effective for preventing miliary tuberculosis and tubercular meningitis in many developing countries. Disseminated complications were observed in association with approximately one per million vaccinations, and severe local reactions occurred in approximately 2 percent of individuals who were vaccinated11,22,23. BCG osteomyelitis has been reported often in Europe but relatively rarely elsewhere1-3,6,7,13,25; however, it may not be as rare as it seems. It is possible that the diagnosis may be missed unless the treating physician has a high index of suspicion and unless a sensitive technique of identification is available. This complication deserves greater attention because it has been reported in association with intravesical administration of BCG for the treatment of carcinoma of the bladder8,24. The reason why the importance of BCG osteomyelitis has often been neglected may be related to the difficulty of confirming the diagnosis. Traditionally, the definitive diagnosis is made on the basis of a positive identification of the BCG strain of Mycobacterium bovis on tissue culture, which is by no means simple22. However, the task has been made easier by advances in molecular biology and the application of molecular biological techniques to clinical situations. Polymerase chain reaction has been used to rapidly identify organisms of the Mycobacterium tuberculosis complex10,14,27. We report the case of a patient who had BCG osteomyelitis that was confirmed rapidly with use of a recently developed diagnostic protocol of molecular analysis in which polymerase-chain-reaction-mediated direct DNA-sequencing analysis for pncA was used29. PncA is a recently identified gene with many polymorphic nucleotides encoding pyrazinamidase or nicotinamidase in the tubercle bacillus. A single nucleotide difference distinguishing Mycobacterium bovis from Mycobacterium tuberculosis can be used to differentiate BCG osteomyelitis from tuberculous osteomyelitis.
An eight-month-old girl was referred to our clinic because of a progressive inability to move the left upper limb. Approximately one month before the child was seen by us, the parents had noted that she was reluctant to move the arm through a complete range of motion and that she would cry when the arm was abducted. The parents sought medical attention when the child stopped moving the arm. No history of trauma was elicited, although the referring doctor suspected an occult fracture. The patient was the product of a full-term pregnancy and had normal growth and development. All vaccinations had been performed on a regular schedule; these had included a BCG vaccination in the proximal part of the left upper extremity at the age of one week, which had not resulted in any obvious immediate untoward reaction. There was no history of fever or infection. Physical examination revealed no obvious swelling, erythema, tenderness, or any other skin lesion in the area of the left shoulder except for a vaccination mark. No active motion of the shoulder was observed. The passive range of motion was limited by painful withdrawal at 20 degrees of flexion, 15 degrees of abduction, 20 degrees of external rotation, and 30 degrees of internal rotation. The general activity of the child was otherwise normal, and she did not appear to be acutely or chronically ill. Routine laboratory studies revealed a white blood-cell count of 22,000 per cubic millimeter (22.0 x 109 per liter) (normal, 3400 to 9100 per cubic millimeter [3.4 to 9.1 x 109 per liter]), with 24 percent segmented neutrophils (normal, 43 to 64 percent), 0 percent bands (normal, 0 to 5 percent), 1 percent eosinophils (normal, 0 to 6 percent), 0 percent basophils (normal, 0 to 1 percent), 4 percent monocytes (normal, 3 to 9 percent), and 70 percent lymphocytes (normal, 27 to 47 percent). The erythrocyte sedimentation rate was thirteen millimeters per hour (normal, zero to fifteen millimeters per hour), the level of C-reactive protein was less than 1.40 milligrams per deciliter (14.00 milligrams per liter) (normal, less than sixty milligrams per deciliter [less than 600.00 milligrams per liter]), the level of calcium was 11.1 milligrams per deciliter (2.77 millimoles per liter) (normal, 8.1 to 10.1 milligrams per deciliter [2.02 to 2.52 millimoles per liter]), the level of phosphorus was 6.5 milligrams per deciliter (2.10 millimoles per liter) (normal, 2.5 to 5.0 milligrams per deciliter [0.81 to 1.61 millimoles per liter]), the level of IgA was nineteen milligrams per deciliter (0.19 gram per liter) (normal, ninety to 450 milligrams per deciliter [0.90 to 4.50 grams per liter]), the level of IgG was 821 milligrams per deciliter (8.21 grams per liter) (normal, 800 to 1500 milligrams per deciliter [8.00 to 15.00 grams per liter]), and the level of IgM was 86.2 milligrams per deciliter (0.86 gram per liter) (normal, sixty to 200 milligrams per deciliter [0.60 to 2.00 grams per liter]). The test for the human immunodeficiency virus was negative. Other laboratory studies were normal, including those for polymorphous neutrophil function and lymphocyte proliferation. A radiograph revealed a pathological fracture, mild soft-tissue swelling, an osteolytic lesion in the proximal aspect of the humerus, periosteal reaction, and juxta-articular osteoporosis (Fig. 1-A). Three-phase bone-scanning performed with technetium-99m methylene diphosphonate showed increased blood flow and pooling around the left shoulder. A T2-weighted magnetic resonance image (gradient echo, fast low-angle shot) revealed a hyperintense lesion in the proximal aspect of the humerus, with expansion of the cortex (Fig. 1-B). The differential diagnosis included osteomyelitis, a cystic bone lesion, histiocytosis, and a metastatic lesion. An open biopsy, performed through an anterolateral approach, revealed grayish, sandy, jellylike material. A window measuring approximately one by one centimeter was made in the cortex, and a curet was used to excise a specimen of tissue from the lesion. Histological examination showed an epithelioid-cell type of granulomatous inflammation with infiltration by lymphocytes, histiocytes, and plasma cells (Figs. 2-A and 2-B). There was also caseating necrosis with necrotic bone, suggesting tuberculosis. No tubercle bacilli were identified with either acid-fast or rhodamin stain.
We then performed polymerase chain reaction together with direct DNA sequencing to identify polymorphic nucleotides in pncA, a recently identified gene encoding pyrazinamidase or nicotinamidase in the tubercle bacillus. A single nucleotide difference distinguishing Mycobacterium bovis from Mycobacterium tuberculosis confirmed that the diagnosis was BCG osteomyelitis rather than tuberculous osteomyelitis (Fig. 3). As the immediate postoperative diagnosis had been tuberculosis, the patient had been started on a regimen consisting of pyrazinamide (ten milligrams per kilogram of body weight per day), rifampin (ten milligrams per kilogram of body weight per day), isoniazid (fifteen milligrams per kilogram of body weight per day), and vitamin B6 (fifty milligrams per day). Once the definite diagnosis of BCG osteomyelitis had been established with use of polymerase chain reaction, the treatment regimen was changed rapidly to isoniazid (fifteen milligrams per kilogram of body weight per day), rifampin (ten milligrams per kilogram of body weight per day), ethambutol (fifteen milligrams per kilogram of body weight per day), and streptomycin (twenty milligrams per kilogram of body weight per day intravenously for one month). Pyrazinamide was eliminated because it is generally considered ineffective for the treatment of BCG osteomyelitis.
Only after two months did both the pathogen grown on culture and its biochemical characteristics reveal that an acid-fast rod identical to the strain used for the BCG vaccine was responsible for the problem. The radiograph that had been made four months after treatment showed a so-called head-within-a-head appearance, formation of callus, and periosteal reaction (Fig. 4). The medications were given for a full year.
Ten months after treatment, a radiograph of the chest and both arms showed remodeling of the proximal aspect of the left humerus (Fig. 5). The function of the left arm and shoulder was normal in terms of range of motion and activity.
BCG is used worldwide as a vaccine against tuberculosis. The first dose usually is given soon after birth, and the other dose is administered at the age of approximately ten years. The vaccine is not entirely harmless. Many Scandinavian and other European authors have reported osteomyelitis and infectious arthritis following vaccination with BCG in children who have normal immunity as well as in those who have immune defects1-3,6,7,13,17,25. Those reports should have aroused attention elsewhere, as these types of infections also have been reported as complications of intravesical administration of BCG for the treatment of carcinoma of the bladder8,24. The instillation of BCG into the bladder was reported to decrease the rate of cancer of the bladder and to slow the progression of superficial cancer of the bladder after operative intervention. In a recent report of thiry-five patients managed with BCG, twenty-five (71 percent) were free of tumor and only seven (20 percent) had a recurrent lesion or did not respond20. However, the reported complications have made the use of such a live vaccine a concern to urologists. The frequency of BCG osteomyelitis is considered to range from one in 80,000 to one in 100,000 vaccinations1,2. It has been suggested that actual but unverified cases of BCG osteomyelitis are four times more frequent than laboratory-confirmed cases13. Berges et al. found that the first symptoms of BCG osteomyelitis generally appeared approximately twelve months (range, three to twenty-six months) after vaccination with BCG3. Bergdahl et al. reported that the symptoms might appear over a wide time-period (range, five months to five years)2. The clinical signs of BCG osetomyelitis generally include limited motion, as in the case of our patient. Otherwise, the affected child appears constitutionally and generally well but may have a low-grade fever1. BCG osteomyelitis most commonly involves the bones of the extremities and, less commonly, the vertebrae, ribs, sternum, and clavicle, in descending order of frequency1,12. Of the lesions that occur in the long bones, 80 percent3,15 (exact numbers not given) are in the epiphysis or the metaphysis. It is important to make a precise diagnosis rapidly because different mycobacteria (Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis BCG) necessitate different regimens of treatment. The history and laboratory data are essential for establishing the diagnosis expeditiously. Several pitfalls may be encountered during the process of making the diagnosis. First, because BCG osteomyelitis is not a common condition, it may not be included in the initial differential diagnosis. Second, there is a long latent period (approximately twelve months) after the vaccination with BCG before the appearance of symptoms. Without considering this diagnosis, the physician may not elicit either a pertinent positive history such as vaccination with BCG or pertinent negative information such as the absence of contact with individuals who have active tuberculosis. Third, the symptoms of BCG osteomyelitis evolve insidiously. The clinical course may be deceptively benign. Indices of inflammation, such as the erythrocyte sedimentation rate and the C-reactive protein level, are only moderately elevated. Radiographic changes are helpful in making the diagnosis but are not pathognomonic1,3,12. When present, the radiographic findings include a well demarcated, eccentric, osteolytic lesion in the metaphysis, with cortical infarctions and soft-tissue swelling. These findings are also associated with pyogenic, tuberculous, syphilitic, and fungal osteomyelitis. Eosinophilic granuloma also may share at least some of these radiographic features. Pathological studies are helpful for establishing the diagnosis of mycobacterial infection; however, differentiating among various types of mycobacterial infections is difficult if not impossible26,28. In the past, a positive culture for acid-fast bacilli was the only dependable method of confirming the diagnosis. However, a negative culture is not proof of the absence of the condition. Moreover, cultures are time-consuming, resulting in a delay in the initiation of the specific treatment. Berk et al., in 1996, reported false-negative findings in more than one-half of patients4. The polymerase-chain-reaction method that was used in the current study can overcome most of these difficulties10,14,27,29. A combination of polymerase chain reaction and direct DNA sequencing for pncA allowed us to differentiate BCG osteomyelitis from other mycobacterial infections and thus enabled us to make a specific microbiological diagnosis. We found that Mycobacterium bovis BCG caused the osteomyelitis in our patient, and we strongly believe that this method is useful for the rapid diagnosis of Mycobacterium bovis BCG osteomyelitis. The advent of this effective method is timely because an increasing number of cases of this condition are being reported in the literature5,7,11,18,19,24-26,28. The pathogenesis of this disorder remains unknown. Possible risk factors include the strain used for the BCG vaccine and the method of vaccination. However, no single cause has been established3,18. The most effective chemotherapy regimen for the treatment of Mycobacterium bovis BCG osteomyelitis has not yet been determined. However, pyrazinamide should not be used, as all strains of Mycobacterium bovis are resistant to it; therefore, this standard antituberculous agent has no role in the treatment of this condition16. Kroger et al. suggested a treatment protocol that was similar to the one described in the present report; the regimen recommended by those authors included administration of streptomycin combined with ethionamide and isoniazid for one month, isoniazid combined with ethionamide or rifampin for four more months, and isoniazid alone for twelve months19. In summary, the present report illustrates a rarely encountered form of infective osteomyelitis. In the case described here, the infective process was determined to have been the sequela of a BCG vaccination that had been performed eight months previously. Treatment was started early, before the results of culture were available, because the diagnosis was made rapidly with use of polymerase chain reaction, which is now available in many centers as a clinical test. Direct inoculation was considered to be the cause of the disorder in our patient because the site of the lesion corresponded to the site of the vaccination. We believe that BCG osteomyelitis, although rare, should be kept in mind as a possible complication of vaccination. Confirmation of the pathogen with the help of molecular analysis will become both practical and crucial for the adequate treatment of this problem.
*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in support of this study. The funding source was the National Science Council of Taiwan, Grant 88-2314-B-006-050.
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