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Journal of Clinical Microbiology, February 2001, p. 725-727, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.725-727.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mycobacterium interjectum as Causative Agent of Cervical Lymphadenitis

Departments of Microbiology,¹ Head and Neck Surgery,² and Pediatric Hematology/Oncology,⁴ Ghent University Hospital, Ghent, and Department of Microbiology, Institute of Tropical Medicine, Antwerp,³ Belgium

Received 20 June 2000/Returned for modification 4 September 2000/Accepted 21 October 2000

	ABSTRACT

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A mycobacterial strain isolated from a lymph node of a 3-year-old female with cervical lymphadenitis was identified as Mycobacterium interjectum by means of sequencing of the 16S rRNA gene. Analysis of this case and previously published cases demonstrates the importance of M. interjectum as a causative agent of cervical lymphadenitis in young children.

	TEXT

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Molecular techniques have made it possible to recognize previously overlooked mycobacterial species. A mycobacterial isolate from a lymph node of a child with lymphadenitis could not be identified with amplified ribosomal DNA restriction analysis (ARDRA), the molecular technique routinely used at our laboratory for identification of mycobacteria. Determination of the sequence of the 16S rRNA gene led to a final identification as Mycobacterium interjectum and prompted us to review previously described cases of infection due to this organism.

Case report. A 3-year-old female was admitted to Ghent University Hospital with cervical lymphadenitis lasting for a period of 8 weeks. In the left submandibular and left parotid areas, a firm nodular mass of 3 by 4 cm was palpable and the overlying skin was blue-red. There was discrete local pain but no systemic illness. A chest X-ray and routine hematological examination were normal. An intradermal skin test using purified protein derivative (PPD) (five tuberculin units) of both tuberculous and nontuberculous mycobacteria was applied, and an induration with a diameter of more than 10 mm was seen for M. avium complex. Because antibiotic treatment with azithromycin given for 3 weeks resulted in no response, complete surgical excision was performed.

During surgery, the affected skin was resected, including a left parotidectomy and resection of the submandibular gland with associated lymph nodes in the superior jugular area. Samples of the lymph nodes were preserved for laboratory investigation and culturing. Diffuse tuberculoid granulomatous lymphadenitis and caseous necrosis were seen throughout the specimen. M. interjectum was isolated from all resected lymph nodes. Drug susceptibility testing was performed by the proportion method with Lowenstein-Jensen (LJ) medium or Middlebrook 7H11 agar (7H11) and a single concentration of each drug as recommended before (1, 7). The isolate was found to be resistant to rifampin (40 µg/ml of LJ), isoniazid (0.2 µg/ml of LJ), ethambutol (2 µg/ml of LJ), para-aminosalicylic acid (0.5 µg/ml of LJ), streptomycin (4 µg/ml of LJ), kanamycin (6 µg/ml of 7H11), and capreomycin (10 µg/ml of 7H11) and susceptible to cycloserine (60 µg/ml of 7H11), ethionamide (10 µg/ml of 7H11), clarithromycin (1 µg/ml of LJ), rifabutin (40 µg/ml of LJ), and ofloxacin (4 µg/ml of 7H11).

After surgery, the patient recovered completely and no relapse was seen.

Preparation of the specimens for mycobacterial culturing started with N-acetyl-L-cysteine-NaOH-based decontamination, followed by auramine staining and inoculation of a liquid medium (MB BacT; Organon Teknika, Boxtel, The Netherlands) and a solid medium (Ogawa; Sanofi-Pasteur, Marnes-la-Coquette, France). The auramine staining (auramine obtained from Merck, Darmstadt, Germany) was negative, no growth was observed on the solid medium, but the liquid culture became positive after 30 days.

At our laboratory, identification of cultured mycobacteria is done by ARDRA (14), which consists of restriction digestion of the amplified 16S rRNA gene. For the isolate obtained here, the combination of CfoI restriction pattern 5, MboI pattern 4, and RsaI pattern 4 was observed; this profile did not correspond to any of the profiles of the mycobacterial species included in our reference panel (14; http://allserv.rug.ac.be /~mvaneech/ARDRA/Mycobacterium.html). Therefore, sequencing of the 16S rRNA gene was necessary to obtain final identification. Sequencing was carried out as described previously (6). The obtained sequence was compared with all known sequences of GenBank by use of Blast 2.0 (National Center for Biotechnology Information, Bethesda, Md. [http://www3.ncbi.nlm.nih.gov/BLAST/]) and showed 99.8% similarity with M. interjectum. Identification as M. interjectum was confirmed by cluster analysis performed by use of Genecompar (Applied Maths, Kortrijk, Belgium). The sequences of the following strains were used in the UPGMA (unweighted pair-group method using arithmetic averages) clustering: Ghent University Hospital clinical strain (GenBank accession no. AJ272088), four previously sequenced M. interjectum strains (GenBank accession no. AF014935, AF014936, AF014937, and X70961), an M. simiae strain (GenBank accession no. X52931), an M. heidelbergense strain (GenBank accession no. AJ000684) (in the latter two species, the 16S rRNA gene sequence clusters very closely with that of M. interjectum), and an M. tuberculosis strain (GenBank accession no. X52917) (Fig. 1).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   UPGMA clustering of the 16S rRNA gene sequence of the clinical isolate reported in this study with sequences obtained from GenBank. Lengths are shown in base pairs.

Further confirmation of the sequencing results was done by biochemical testing as described before (15). The strain was found to be a scotochromogenic, slowly growing mycobacterium, susceptible on LJ medium to NaCl (5%) and isoniazid (10 µg/ml) but resistant to carboxylic acid hydrazide (2 µg/ml), hydroxylamine (250 µg/ml), and para-nitrobenzoic acid (500 µg/ml).

The strain was found to be negative for semiquantitative catalase (i.e., less than 45-mm foam production) nitrate reduction, acid phosphatase, and niacin production but positive for Tween hydrolysis and urease. Thin-layer chromatography of the fatty acids revealed alpha-, methoxy-, and keto-mycolic acids. All of these biochemical characteristics fit with the identification as M. interjectum (10). The negative catalase reaction, the positive Tween hydrolysis, and the susceptibility to 10 µg of isoniazid per ml, as well as the mycolic acid pattern, differentiate this species from the phenotypically very similar species M. scrofulaceum (15).

M. interjectum, for which the species name refers to the intermediate phylogenetic position between rapidly and slowly growing mycobacteria, was first described in 1993 (9). Table 1 summarizes the clinical features of all published cases in which M. interjectum was isolated.

View this table: [in this window] [in a new window]   TABLE 1.   Summary of the clinical features of our case and previously reported cases of infection with M. interjectum

M. interjectum was described as the causative agent in five pediatric cases of cervical lymphadenitis. Four cases in adults have been described, but in only one case (Table 1, case 7) was M. interjectum considered clinically important. The four patients (three pediatric) treated with antibiotics alone and/or undergoing partial resection were not cured. Cure was obtained only after total resection of the infected region.

Nontuberculous mycobacterial lymphadenitis was traditionally associated with M. scrofulaceum (5). During the 1980s, the M. avium complex was predominant (16). More recently, however, a wide variety of mycobacterial species causing lymphadenitis in young children have been reported, including some previously unrecognized mycobacteria (3, 4, 9, 12, 13). At present, it is difficult to establish whether this observation reflects real changes in the prevalence of different mycobacterial species or is due to increased diagnostic capabilities and to refined mycobacterial taxonomy.

Full identification of the nontuberculous agents causing cervical lymphadenitis in young children is warranted to reveal the role of different mycobacterial species and may indicate an underestimation of the pathogenic role of species such as M. interjectum. Also, correct identification may be important to guide therapy, since present experience seems to indicate that total resection is the only cure for lymphadenitis caused by species such as M. interjectum. Identification of M. interjectum by phenotypic methods is slow and not always straightforward, since the species has been reported to have quite a few variable reactions (6). Also, with high-pressure liquid chromatography analysis of mycolic acids, differences among the patterns obtained for different M. interjectum strains have been reported (6, 10). Accurate identification of M. interjectum is possible by means of 16S rRNA gene sequencing and by means of ARDRA, which results in CfoI restriction pattern 5, MboI pattern 4, and RsaI pattern 4, a profile thus far observed only for M. interjectum.

	ACKNOWLEDGMENTS

We thank Leen Van Simaey for excellent technical assistance.

	FOOTNOTES

^* Corresponding author. Mailing address: Laboratory Bacteriology & Virology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. Phone: 32 9 240 36 92. Fax: 32 9 240 36 5. E-mail: Thierry.Debaere{at}rug.ac.be.

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