LETTER
AccuProbe (Hologic, Marlborough, MA) is an FDA-approved commercial test used for the identification of Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium avium complex (MAC), Mycobacterium gordonae, and Mycobacterium kansasii from growth in solid or liquid mycobacterial culture media. Species identification occurs through transcription-mediated amplification of 16S rRNA followed by hybridization of a species-specific oligonucleotide DNA probe with target rRNA. The DNA:RNA complex is detected by chemiluminescence measured in relative light units (RLU). Manufacturers recommend an RLU value above 30,000 for reliable species identification (1).
A mycobacterial isolate from Veterans Affairs Hospital in Lake City, FL, was sent to the mycobacteriology reference laboratory at the West Haven Veterans Affairs Hospital (West Haven, CT) for further identification. Initial testing with the MAC probe was positive, with an RLU of 177,803. Subculture on solid media resulted in the growth of a scotochromogen with smooth yellow colonies. Small beaded acid-fast bacilli were seen on Kinyoun staining. Testing of the isolate using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics) suggested M. gordonae, but the score was below the cutoff of 1.7 necessary for reliable species identification.
Sequencing using Microseq 500 16S rDNA bacterial identification system (Thermo Fisher Scientific, Waltham, MA) demonstrated a 100% sequence alignment match with Mycobacterium paraense, Mycobacterium malmoense, and Mycobacterium celatum. Neither Mycobacterium malmoense nor Mycobacterium celatum is a scotochromogen, leaving Mycobacterium paraense as the most probable identification per CLSI guidelines (2). The next closest match was with Mycobacterium simiae, with 99% sequence alignment.
To confirm the false-positive MAC probe reaction, 5 separate colonies picked from the 1st culture were subcultured and retested. All 5 hybridized with the MAC probe, with values ranging from 333,799 to 498,144 RLU. MALDI-TOF MS once again assigned them an identity of M. gordonae, with scores ranging from 1.444 to 1.508, below the cutoff for a reliable identification.
Mycobacterium paraense, isolated from the sputum of a patient in Brazil, was described as a novel mycobacterial species in 2015. It belongs to the M. simiae complex and is most closely related to M. interjectum (3). Mycobacterium paraense was isolated from a patient in Zimbabwe in 2016 (4).
Sensitivity and specificity of Accuprobe vary by species and complex; the MAC probe has sensitivity of 95.25% and specificity of 96% (1). The MAC probe has been reported to cross-react with M. aroiense, M. chimaera, M. colombiense, M. nebraskense, M. palustre, M. paraffinicum, M. saskatchewanense, and M. vulneris (5).
We are reporting the first identification of Mycobacterium paraense from the United States. The isolation of a yellow scotochromogen with a high RLU value with MAC probe and low-score identification as M. gordonae on MALDI-TOF should raise suspicion of M. paraense.
REFERENCES
- 1.Jorgensen JH, Pfaller MA, Carroll KC, Funke G, Landry ML, Richter SS, Warnock DW (ed). 2015. Manual of clinical microbiology, 11th ed ASM Press, Washington, DC. doi: 10.1128/9781555817381. [DOI] [Google Scholar]
- 2.CLSI. 2014. Nucleic acid sequencing methods in diagnostic laboratory medicine; approved guideline-2nd ed. CLSI document MM09-A2, 2nd ed. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 3.Fusco da Costa AR, Fedrizzi T, Lopes ML, Pecorari M, Oliveira da Costa WL, Giacobazzi E, da Costa Bahia JR, De Sanctis V, Batista Lima KV, Bertorelli R, Grottola A, Fabio A, Mariottini A, Ferretti P, Di Leva F, Fregni Serpini G, Tagliazucchi S, Rumpianesi F, Jousson O, Segata N, Tortoli E. 2015. Characterization of 17 strains belonging to the Mycobacterium simiae complex and description of Mycobacterium paraense sp. nov. Int J Syst Evol Microbiol 65:656–662. doi: 10.1099/ijs.0.068395-0. [DOI] [PubMed] [Google Scholar]
- 4.Chin'ombe N, Muzividzi B, Munemo E, Nziramasanga P. 2016. Molecular identification of nontuberculous mycobacteria in humans in Zimbabwe using 16S ribosequencing. Open Microbiol J 10:113–123. doi: 10.2174/1874285801610010113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Tortoli E, Pecorari M, Fabio G, Messino M, Fabio A. 2010. Commercial DNA probes for mycobacteria incorrectly identify a number of less frequently encountered species. J Clin Microbiol 48:307–310. doi: 10.1128/JCM.01536-09. [DOI] [PMC free article] [PubMed] [Google Scholar]