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Journal of Clinical Microbiology, July 2001, p. 2668-2671, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2668-2671.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Pulmonary Infection Caused by an Unusual, Slowly
Growing Nontuberculous Mycobacterium
Sini
Suomalainen,1,*
Pirkko
Koukila-Kähkölä,2
Eljas
Brander,3
Marja-Leena
Katila,4
Anneli
Piilonen,2
Lars
Paulin,1 and
Karin
Mattson2
Institute of Biotechnology, University of
Helsinki,1 Helsinki University Central
Hospital,2 and United
Laboratories,3 Helsinki, and Department
of Clinical Microbiology, Kuopio University Hospital,
Kuopio,4 Finland
Received 7 December 2000/Returned for modification 16 January
2001/Accepted 22 March 2001
 |
ABSTRACT |
Mycobacterium triplex, a recently described slowly
growing nontuberculous mycobacterium, was isolated from a Finnish
patient with pulmonary mycobacteriosis. The disease was successfully
treated with antimycobacterial drugs. The strain isolated, which was
similar to the type strain but differed slightly from the species
description, was regarded as a variant of M. triplex sensu
stricto. According to present knowledge this variant of the species has
never been isolated before.
| |
TEXT |
Recent advances in diagnostic
methodology for mycobacteria other than Mycobacterium
tuberculosis (MOTT), and new possibilities introduced for drug
therapy of the diseases they induce, have renewed interest in
mycobacterial research. The recognition that most patients with AIDS
are at high risk of complications and/or terminal illness caused by
MOTT has further stimulated research efforts. The principles of
diagnosis and therapy of diseases caused by MOTT have also recently
been updated by the American Thoracic Society (1). These
guidelines include recommendations for specific drug regimens which
recognize the major impact of the newer chemotherapeutic agents.
Improved diagnostic means allow enhanced detection and more-accurate
species identification of mycobacteria isolated from clinical
specimens, including the classification of species not recognized
earlier. In 1996, Floyd et al. (5) characterized a new
species of slow-growing MOTT, which they designated Mycobacterium triplex. The primary characterization of this novel species was based on conventional testing, but the conclusive genetic evidence relied on 16S rRNA analyses. Although the species is uncommon, it was
found to be geographically widespread in the United States (5). There is one very recent report of M. triplex in Europe (4).
We isolated M. triplex from a Finnish patient suffering from
pulmonary mycobacteriosis. M. triplex was the only
mycobacterium isolated from the specimens. As illustrated in this
paper, the strain found in this patient differed slightly from the
earlier description of the species. To our knowledge, this is the first report of a pulmonary disease associated with M. triplex.
Case report.
A 67-year-old, human immunodeficiency
virus-negative dentist presented in hospital with hemoptysis in 1995. On chest radiographs, thin fibrotic changes were detected in both lower
fields, but no cavitary changes were detected. High-resolution computed
tomography (HRCT), performed in December 1995, revealed multifocal
bronchiectases in both lungs and multiple small nodules in the middle
and lower lobes of the right lung and the lower lobe of the left lung,
indicating mycobacteriosis (1). Bronchoscopy showed
nonspecific inflammatory mucosal changes on the right side. Smears of
sputum and bronchoalveolar lavage specimens were negative for acid-fast
bacilli, but cultures of sputum samples taken on three successive days
grew a slow-growing mycobacterium. It was identified using conventional
tests, gas-liquid chromatographic (GLC) analysis of cellular fatty acid
and alcohol composition, and 16S rRNA gene sequencing as described
earlier in detail (6, 7, 11). The isolates met the
identification criteria for M. triplex (5).
Since this was the only organism growing in the three cultures, we
believe that the isolation of M. triplex represents
infection rather than colonization. Moreover, the patient has
bronchiectases throughout both lungs, which would predispose her to
this kind of lung infection. Treatment was initiated using a standard
antituberculous regimen for 2 months, to which the lesions responded
only partially. Treatment was continued with rifampin, ciprofloxacin,
ethambutol, and clarithromycin for a total of 10 months, with the
exception of a 3-week period in March and April 1996 due to a temporary
rise in liver enzymes. This regimen proved to be well tolerated. The
bacteriological and clinical responses were good. An HRCT scan in
October 1996, after 10 months of treatment, showed a good radiological
response (Fig. 1). The patient was
regarded as cured at follow-up, after taking antituberculosis
medication for 18 months.
The growth and biochemical characteristics of the patient's isolates
were most closely compatible with
M. triplex (Table
1)
(
5). By in vitro testing,
performed using the proportion method
in Löwenstein-Jensen
medium, the strain was susceptible to rifampin,
streptomycin, and
clarithromycin and moderately sensitive to ciprofloxacin,
but it had
decreased susceptibility to ethambutol, isoniazid,
and pyrazinamide.
The isolates failed to hybridize with the commercially
available
genetic probe for
Mycobacterium avium complex (Accuprobe;
Gen-Probe Inc., San Diego, Calif.). Two of the patient's isolates
were
analyzed by GLC for cellular fatty acid methyl esters and
alcohols,
prepared by acid methanolysis, and analyzed and identified
as described
earlier in detail (
10). These two isolates had
an
identical GLC profile, which closely resembled that of the
M. triplex type strain (ATCC 70071). However, repeated analyses
of
the fatty acid composition verified minor but constantly detectable
differences from the
M. triplex type strain, including a
higher
relative amount of tetracosanoate (24:0) and a low relative
amount
of tuberculostearic acid (10-Me-18:0) (Table
1). The fatty acid
profile of the
M. triplex type strain was found to be
indistinguishable
from that of
M. simiae (
3), a
slow-growing photochromogenic
species. Pigment production easily
separates these two species.
They both have GLC profile is grossly
similar to that of
M. tuberculosis.
As shown in Table
1, the
high relative content of tetradecanoate
(14:0) in both the type strain
of
M. triplex and the patient isolates
(7%) differentiated
them from
M. tuberculosis (<2%). Others have
verified the
same difference between
M. tuberculosis and
M. simiae (
3). Overall, it was difficult to identify the
species of the
isolated strains using conventional techniques.
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TABLE 1.
Biochemical tests and markers in the GLC profile of fatty
acids and mycolic acid cleavage products found useful in
differentiation of M. triplex and the isolate described
|
|
Sequencing of the 16S rRNA gene is widely used for species
identification of mycobacteria. The complete 16S rRNA gene was
amplified, and both strands of the amplified DNA were sequenced
as
described elsewhere (
6). A BLAST sequence similarity
search
(EBI, Hinxton, United Kingdom) of the 16S sequence obtained
showed
a 99.5% similarity to
M. triplex (Fig.
2A). The internal transcribed
spacer
(ITS) sequences have been shown to be useful phylogenetic
markers for
species differentiation and identification (
8,
9). The ITS
region of the patient isolate was amplified and
sequenced as described
previously (
10). Comparison of the sequences
showed 8 differences in the 16S rRNA gene and 14 differences in
the ITS region
between the patient isolate and the
M. triplex type strain
(Fig.
2A and B, respectively) (
5,
8).
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|
FIG. 2.
Comparison of ribosomal gene sequences. Dots indicate
nucleotide identity, and dashes indicate deletions. (A) Differences in
the 16S rRNA gene between the isolate (AJ276890) and M. triplex (U57362). Numbering is according to the reference
sequence. (B) Alignment of ITS region sequences of selected species
including the patient isolate. Reference sequence accession numbers are
as follows: M. triplex, Y14189; M. simiae,
AB026694; M. tuberculosis, AB026698.
|
|
The result of 16S rRNA sequencing, with 99.5% sequence similarity with
M. triplex, indicated that the isolate was very closely
related to
M. triplex. We found the sequencing of the ITS
region
necessary because of the eight differences in the 16S rRNA gene.
The result of the ITS sequencing confirmed the other results obtained
in this study, indicating that this organism was an
M. triplex-like
mycobacterium. According to currently available data,
the patient's
strain is regarded as a variant of
M. triplex. Further analyses
are needed to evaluate the relevance of
the differences detected
and to answer the open question of whether the
strain should be
regarded as a subspecies of
M. triplex or
as another as yet unclassified
but closely related
species.
Several recent reports describe infections caused by unusual
Mycobacterium species (
2,
12). The development
of molecular
biological methods has allowed more-accurate species
identification
and the study of species diversity. The clinical case
reported
here indicated that the
M. triplex variant
described had to be
regarded as a potential human pathogen. The results
also demonstrated
that the regimen often used to treat pulmonary
mycobacteriosis
caused by
M. avium is also sufficient for
the treatment of
M. triplex infections.
Nucleotide sequence accession number.
The 16S and ITS
sequences determined in this study have been deposited in the EMBL
nucleotide sequence databank under accession number AJ276890.
| |
ACKNOWLEDGMENTS |
S.S. was supported by a grant from the Oskar Öflund Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biotechnology, P.O. Box 56, FIN-00014 University of Helsinki, Finland. Phone: 358-9-191 58886. Fax: 358-9-191 58952. E-mail:
sini.suomalainen{at}helsinki.fi.
| |
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Journal of Clinical Microbiology, July 2001, p. 2668-2671, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2668-2671.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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