Mycobacterium bohemicum cervical lymphadenitis

A lymph node excision was performed on a 2-year-old child with right submandibular swelling not responding to oral cefaclor therapy. Histology revealed granulomatous, partly necrotizing lymphadenitis and a large number of acid-fast bacilli subsequently identified by molecular techniques as Mycobacterium bohemicum. Prolonged cervical lymph node swelling is a frequent pediatric problem. Among infectious agents the members of the genus Mycobacterium are well-established causes of granulomatous lymphadenitis in immunocompetent children. The introduction of novel techniques to clinical mycobacteria laboratories made it clear that the complexity of this genus had been underestimated. This work illustrates the pathogenic potential of Mycobacterium bohemicum and emphasizes the importance of molecular methods for timely and accurate identification, particularly of the lesser known mycobacterial species. Case report. A previously healthy 2-year-old Indian girl who had been born and reared in Switzerland was referred to us by her private pediatrician because of a 1-month-history of right submandibular swelling and tenderness that had not responded to 10 days of oral cefaclor therapy (40 mg/kg divided into three daily doses). The patient was otherwise healthy. In particular there was no history of fever, cough, weight loss, excessive sweating or decreased appetite, and there were no such symptoms among family members. No recent animal contacts were reported. Physical examination revealed normal vital signs. Body weight and height were on the 10th and 25th percentiles, respectively. A 4- by 2-cm, firm, slightly tender, well-demarcated mass was visible and palpable in the right submandibular area. The overlying skin appeared normal. Small cervical and nuchal lymph nodes (<1 cm in diameter) were palpable bilaterally. Examination of ears, nose, oral cavity and throat were normal. The remainder of the physical examination was normal. Laboratory evaluation revealed a peripheral white blood cell count of 11.4 × 109/l with normal differential, and the erythrocyte sedimentation rate was 37 mm/h, C-reactive protein was <2 mg/l. Acute and convalescent (after 21 days) sera were negative for antibodies against Bartonella henselae, Toxoplasma gondii and human immunodeficiency virus types 1 and 2. Anti-cytomegalovirus IgG and anti-Epstein-Barr nuclear antigen-1 IgG documented past infections with cytomegalovirus and Epstein-Barr virus. A Mantoux test with 2 units of purified protein derivative RT23 (Statens Serum Institut, Copenhagen, Denmark) was negative. A chest radiograph was normal. Three gastric aspirates were negative for mycobacteria by microscopy, polymerase chain reaction and culture. Sonography of the right submandibular area demonstrated a conglomerate of multiple lymph nodes measuring up to 2 cm in diameter. There were no signs of liquefaction or calcification. Total lymph node excision was performed 6 weeks after the initial presentation. Histology revealed granulomatous, partly necrotizing lymphadenitis with multiple giant cells and perinodal fibrosis. Many acid-fast bacilli were seen in a biopsy specimen stained with auramine-rhodamine. Microbiology. The biopsy specimen was homogenized, decontaminated with sodium dodecyl sulfate-sodium hydroxide, neutralized, and the sediment was resuspended in phosphate-buffered saline. 1 Screening microscopy of 0.1 ml of this sediment stained with auramine-rhodamine confirmed the presence of numerous acid-fast bacilli. Two slants of Lowenstein-Jensen containing pyruvate and two BacT/ALERT Mycobacteria Process bottles with Mycobacteria Antibiotic Supplement (Organon Teknika Corp., Durham, NC) were inoculated with 0.2 and 0.5 ml of the resuspended sediment. Slants were incubated at 28°C and 35°C in ambient air for 8 weeks and inspected for growth twice weekly. The BacT/ALERT Mycobacteria Process bottles were incubated for 6 weeks at 28°C and 35°C with the BacT/ALERT Microbial Detection System (Organon Teknika Corp.) according to the manufacturer’s recommendations. Growth was detected after 14 days in the BacT/ALERT Mycobacteria Process bottle incubated at 35°C. After 32 days growth of a scotochromogenic mycobacterium was observed on the Lowenstein-Jensen slants. Biochemical testing 2 of the isolate revealed positive reactions for the activity of heat-stable catalase, urease and Tween 80 hydrolysis. Tests for niacin, nitrate, semiquantitative catalase activity, acid phosphatase and arylsulfatase tested after 3 and 14 days were negative. Partial sequencing of the 16S ribosomal DNA (rDNA) gene was performed using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems, Foster City, CA 3). The sequence was submitted to the Ribosomal Differentiation of Medical Microorganisms database 4 and showed a top scoring species identity of 100% with the type strain of M. bohemicum (44277 DSM). Cell wall mycolic acids were not analyzed. Antimicrobial susceptibility testing by broth microdilution method with Middlebrook 7H9 medium incubated at 35°C in ambient air showed the following MICs (milligrams/l): amikacin 4.0; clarithromycin <0.06; rifabutin <0.007; rifampin <0.015; ethambutol 4.0; and levofloxacin 0.25. Discussion. Prolonged cervical lymph node swelling caused by mycobacteria is a common pediatric problem. With the decreasing incidence of tuberculosis in industrial countries during the 20th century, infections caused by nontuberculous mycobacteria became more prevalent and now account for most such cases. Mycobacterium avium complex, Mycobacterium scrofulaceum, Mycobacterium kansasii and Mycobacterium fortuitum are well-known causes of lymphadenitis in children with the typical histologic findings of granulomatous lymphadenitis. The recent advent of more sophisticated techniques for identification and classification of mycobacteria in diagnostic laboratories revealed that standard identification methods underestimate the complexity of the genus Mycobacterium. Consequently the introduction of partial sequencing of the 16S rDNA gene into diagnostic routine led to the identification of novel nontuberculous mycobacterial species associated with cervical lymphadenitis in children, e.g. Mycobacterium triplex. 5 One of these newly defined species is M. bohemicum. In the first published case of M. bohemicum lymphadenitis, 6 the organism was initially classified as a mycobacterium closely related to M. scrofulaceum, from which it was indistinguishable by standard methods with the exception of semiquantitative catalase activity (M. scrofulaceum, +;M. bohemicum, −). High performance liquid chromatography of cell wall mycolic acids, however, disclosed some differences that led to the designation of a new species subsequently found to be identical with M. bohemicum. 7, 8 The first case of M. bohemicum lymphadenitis 6 involved a 2-year-old child with unilateral neck swelling (Table 1). Total lymph node excision was performed. An unusual mycobacterium was cultured and in retrospect 6, 7 identified as M. bohemicum. A second case was diagnosed in a 11-year-old patient with unilateral cervical lymphadenopathy. The organism was cultured from histologically uncharacterized lymph node material and was identified as M. bohemicum. 7 In our case lymph node histology demonstrating granulomatous and partly necrotizing inflammation at the site of isolation of M. bohemicum provided firm evidence for the pathogenic role of this organism.TABLE 1: Pediatric infections with Mycobacterium bohemicumStandard therapeutic management of cervical mycobacterial lymphadenitis in immunocompetent children consists of surgical lymph node excision. 9 Antimicrobial chemotherapy may be indicated if total excision is not feasible for anatomical reasons, if there is extension into adjacent skin structure or if a sinus tract has formed either spontaneously or after surgery. In the absence of clinical evidence, the choice of drugs and the duration of therapy are based on in vitro data and personal experience. Many experts recommend the combined use of clarithromycin and a rifamycin (rifampin or rifabutin). This combination is effective in vitro against M. avium complex which is the most common mycobacterial species causing nontuberculous mycobacterial lymphadenitis. 10 Drug susceptibility data presented here and in previous studies 6–8 indicate that the MICs to clarithromycin and the rifamycins are low and easily achievable in vivo. Hence if chemotherapy is considered in a case of M. bohemicum infection, the combination of clarithromycin and a rifamycin is a rational choice. We believe that M. bohemicum should be listed among the nontuberculous mycobacterial species capable of causing granulomatous lymphadenitis in immunocompetent children. Because of the weak biochemical activity and a close similarity to M. scrofulaceum, M. bohemicum can be misidentified unless partial sequencing of the 16 S rDNA gene is carried out. Acknowledgments. We thank Kim Mallard and Andrea Aebischer for their excellent technical assistance.