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Journal of Clinical Microbiology, August 2005, p. 4286-4287, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4286-4287.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Infections Due to the Newly Described Species Mycobacterium parascrofulaceum

Regional Reference Center for Mycobacteria, Microbiology and Virology Laboratory,¹ Cytogenetics and Genetics Unit,⁴ Microbiological and Virological Serum-Immunology Laboratory, Careggi Hospital, Florence,⁶ Infectious Diseases Unit, Niguarda Hospital, Milan,² Social Hygiene Dispensary, Turin,³ Microbiology Laboratory, Bambino Gesù Hospital, Rome,⁷ Clinical Laboratory, Sant'Anna Hospital, Como, Italy,⁸ Microbiology Laboratory, Vall d'Hebron Hospital, Autonoma University, Barcelona, Spain⁵

Received 29 March 2005/ Returned for modification 29 April 2005/ Accepted 18 May 2005

	ABSTRACT

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We report on four cases of infection by the recently described species Mycobacterium parascrofulaceum. In two cases the mycobacterium was isolated from AIDS patients, while in the others it was responsible for pulmonary disease in elderly men. Our findings suggest that M. parascrofulaceum is an opportunistic pathogen, like many other nontuberculous mycobacterial species.

	CASE REPORTS

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Case 1. A 35-year-old male with AIDS presented, in 1997, along with a very low CD4 lymphocyte count (7/ml) cerebral neurotoxoplasmosis, cytomegalovirus disease, and systemic cryptococcosis. His major symptoms included chorea, fever, and diarrhea. From his sputa, which were smear negative, a nontuberculous mycobacterium, not identified at that time (FI-97251), was isolated twice. The patient was treated with ethambutol and rifabutin until death, which came 1 month later.

Case 2. An unidentified mycobacterium (FI-00121) was grown in 2000 from a blood culture of a 40-year-old AIDS patient. The man was severely immunocompromised (39 CD4 lymphocytes/ml) and presented esophageal candidosis, cytomegalovirus infection, and fever. He was not treated with antimycobacterial drugs and died 6 months later.

Case 3. A 67-year-old Spanish male with epidermoid carcinoma of the left lung and mediastinum presented with chronic obstructive pulmonary disease and emphysema since 1997. Between August 2003 and April 2004, a nontuberculous mycobacterium (FI-03077), at first identified as Mycobacterium scrofulaceum, was isolated seven times from the sputum, which was microscopically negative for acid-fast bacilli. No information is available about treatment and follow-up.

Case 4. A 63-year-old patient, previously subjected to lobectomy because of bronchiectasis, presented in 2004 a cavitation of the left lung. A clinical sample obtained by means of bronchial aspiration was strongly positive for acid-fast bacilli and yielded in culture a strain (FI-04199) which was initially identified as M. scrofulaceum. Treatment with isoniazid, ethambutol, and rifampin was undertaken but did not produce any radiological improvement, the inability of the patient to produce sputum did not allow a microbiological follow-up. The man died 4 months later because of worsening respiratory insufficiency.

An a posteriori reexamination of a cluster of mycobacteria showed that the four strains reported above belong to the newly described species M. parascrofulaceum.

The description of M. parascrofulaceum was based on a thorough analysis of multiple genetic regions of unidentified mycobacteria (7) previously named MCRO 33 (3). It was realized that several strains of unnamed mycobacteria reported in the literature or listed in public-domain nucleic acid databases in fact belong to the newly established species (7), to which also must be assigned several reference strains previously assigned to the species M. scrofulaceum and M. simiae. (7).

We were stimulated to investigate the presence of M. parascrofulaceum by the paper describing the new species (7), in which the authors refer to two strains of ours reported in an article concerning unidentified mycobacteria (5). In addition to the above strains, others were detected by sequencing the first 500 bp of the 16S rRNA gene (8) and the gene encoding the 65-kDa heat shock protein (hsp65) (7) in a group of 45 clinical mycobacteria collected in our laboratory since 1987. This group had been selected by screening the collection database on the basis of a number of phenotypic traits: slow growth at 25 and 37°C but not at 45°C, scotochromogenicity, negative Tween 80 hydrolysis, positive urease, and a high-performance liquid chromatography profile (HPLC) characterized by three peak clusters as reported by Turenne and coworkers for M. parascrofulaceum (7). Forty-five strains fit this pattern; 6 of them were unidentified, while 39 had been assigned to the species M. scrofulaceum, mostly on the basis of the identification obtained with the commercial inverse hybridization test INNO-LiPA Mycobacteria (LiPA; Innogenetics, Ghent, Belgium).

Four strains turned out to belong to the new species M. parascrofulaceum; two of these had not been identified before, while two had been previously considered to belong to the species M. scrofulaceum. With LiPA, all such strains hybridized, in addition to the genus specific probe, also with the ones recognizing the group M. avium-M. intracellulare-M. scrofulaceum and the species M. scrofulaceum. With another commercial inverse hybridization assay, GenoType Mycobacterium CM (Hain, Nehren, Germany), the line probes specific for gram-positive bacteria characterized by high guanidine-cytosine content were positive, as was the one recognizing the whole genus Mycobacterium and lines 9 and 10, which pattern is considered distinctive of M. scrofulaceum.

Differentiation of M. parascrofulaceum from M. scrofulaceum is a major problem. The biochemical and cultural tests are not discriminative, and the same is true for HPLC of mycolic acids. Both commercial DNA probes intended for the identification of M. scrofulaceum assign M. parascrofulaceum to the latter species; this is surprising, as they are aimed at different genetic targets, the 16S-23S internal transcribed spacer in LiPA (6) and the 23S rRNA gene in GenoType (2). Genetic sequencing seems therefore the only approach providing reliable identification, whatever the region used (the 16S rRNA gene, the 23S rRNA gene, the internal transcribed spacer, or hsp65) (7). In the first 500 bp of the 16S rRNA gene, the most popular sequencing target for identification purposes, the distinction between M. parascrofulaceum and M. scrofulaceum is clear-cut (Fig. 1), with 4 mismatches in a short tract between nucleotides 80 and 90 and as many as 18 in hypervariable region B (positions 444 to 480). In our opinion, genetic sequencing of the strains identified biochemically, by HPLC, or by commercial DNA probes as M. scrofulaceum is advisable. In fact, the probability that such strains actually belong to the species M. parascrofulaceum is about 10%.

View larger version (5K): [in this window] [in a new window]   FIG. 1. Alignment of traits of the 16S rRNA gene suitable for differentiation of M. parascrofulaceum from M. scrofulaceum. Base pair position is indicated according to the Escherichia coli sequence.

From the present cases, little can be said about the response to antimycobacterial treatment. Therefore, the value of the in vitro susceptibility pattern, showing sensitivity to rifampin, clarithromycin, and amikacin (7) in all the strains investigated so far, remains uncertain.

	FOOTNOTES

 
* Corresponding author. Mailing address: Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Piastra dei Servizi, Ospedale di Careggi, viale Morgagni 85, 50134 Firenze, Italy. Phone: 39-055-4279199. Fax: 39-055-4279830. E-mail: e.tortoli{at}libero.it.

	REFERENCES

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3. Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Böttger. 1996. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J. Clin. Microbiol. 34:296-303.[Abstract]
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6. Tortoli, E., A. Mariottini, and G. Mazzarelli. 2003. Evaluation of INNO-LiPA MYCOBACTERIA v2: improved reverse hybridization multiple DNA probe assay for mycobacterial identification. J. Clin. Microbiol. 41:4418-4420.[Abstract/Free Full Text]
7. Turenne, C. Y., V. J. Cook, T. V. Burdz, R. J. Pauls, L. Thibert, J. N. Wolfe, and A. Kabani. 2004. Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae. Int. J. Syst. Evol. Microbiol. 54:1543-1551.[Abstract/Free Full Text]
8. Turenne, C. Y., L. Tschetter, J. Wolfe, and A. Kabani. 2001. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J. Clin. Microbiol. 39:3637-3648.[Abstract/Free Full Text]
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