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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
Thierry
De
Baere,1,*
Mieke
Moerman,2
Leen
Rigouts,3
Catharina
Dhooge,4
Hubert
Vermeersch,2
Gerda
Verschraegen,1 and
Mario
Vaneechoutte1
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 |
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 |
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).
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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.
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|
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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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.
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