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 Table of Contents  
Year : 2016  |  Volume : 5  |  Issue : 1  |  Page : 89-91

First detection of Mycobacterium triplex in Latin America

1 National Reference Laboratory for Tuberculosis, Centro de Referência Professor Hélio Fraga, Escola Nacional de Saúde Pública, Fiocruz, Rio de Janeiro, Brazil
2 Hospital Sanatório Partenon, Secretaria Estadual da Saude, Porto Alegre, Rio Grande do Sul, Brazil
3 Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, Rio Grande do Sul, Brazil
4 Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan, Italy

Date of Web Publication8-Feb-2017

Correspondence Address:
Jesus Pais Ramos
National Reference Laboratory for Tuberculosis, Estrada da Curicica 2000, Rio de Janeiro, RJ 22780192
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2015.09.004

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In this study we describe the first isolation of Mycobacterium triplex in Latin America. This species causes infections in humans, with very few reports from around the world. We isolated two sputum specimens of a patient with a 6-year history of human immunodeficiency and tuberculosis treatment failure. All tests used confirmed M. triplex and the patient responded well to drug therapy for 18 months.

Keywords: M. triplex, Nontuberculous mycobacteria, Pulmonary disease, Sequencing

How to cite this article:
Campos CE, Dias CF, Unis G, Caldas PC, Redner P, de Carvalho LD, Gomes AP, Ribeiro MO, Montes FC, Tortoli E, Ramos JP. First detection of Mycobacterium triplex in Latin America. Int J Mycobacteriol 2016;5:89-91

How to cite this URL:
Campos CE, Dias CF, Unis G, Caldas PC, Redner P, de Carvalho LD, Gomes AP, Ribeiro MO, Montes FC, Tortoli E, Ramos JP. First detection of Mycobacterium triplex in Latin America. Int J Mycobacteriol [serial online] 2016 [cited 2023 Mar 28];5:89-91. Available from: https://www.ijmyco.org/text.asp?2016/5/1/89/199749

  Introduction Top

Nontuberculous mycobacteria are ubiquitous and transmitted to humans through the environment from sources like water and soil, causing infections in different areas of the body, but mainly pulmonary, skin, soft tissue infections, and lymphadenitis [1].

Mycobacterium triplex is a slow growing, nonpigmented, nontuberculous mycobacterium, described in 1996 from clinical strains of different geographical locations in the United States [2]. This species is related to Mycobacterium simiae, Mycobacterium lentiflavum, and Mycobacterium sherrisii, but more closely to Mycobacterium genavense [3].

Only a few cases of M. triplex infections have been reported in the United States and Europe. To our knowledge, this is the first report of M. triplex infection in a country of Latin America. This study was approved by the ENSP–Fiocruz Committee for Ethical Research with human beings.

  Methods Top

The patient studied was clinically, laboratorial, and radiological assessed at diagnosis. Monthly cultures were performed to monitor the progress of treatment in Hospital Sanatório Partenon in Porto Alegre, Rio Grande do Sul state, Brazil.

Two sputum isolates were obtained according of American Thoracic Society microbiologic criteria for nontuberculous mycobacterial lung disease [4] and processed at the central laboratory of Rio Grande do Sul, Brazil, in the period of 2007–2008. After growth in Löwenstein–Jensen medium, the cultures were sent to the Centro de Referência Professor Hélio Fraga/ENSP/FIOCRUZ for identification, but the PRA-hsp65 methodology did not allow for identification at species level.

To clarify the identity and to characterize the isolates, phenotypic and sequencing assays were performed in the laboratory of Centro de Referência. The two isolates were designated as HF1226 and HF2396 and tested for pigment production and growth rate at 35–37°C. The following biochemical tests were performed: nitrate reduction, niacin production, heat-stable catalase (68°C), arylsulfatase activity (at 3 days), Tween 80 hydrolysis, and urease. The isolates were slow growing and nonpigmentated. The biochemical profile was positive for nitrate reduction, heat-stable catalase, and urease, and negative for niacin, 3 days arylsulfatase, and Tween 80 hydrolysis. Part of the RNA polymerase beta-subunit gene (rpoB), the 16S–23S internal transcribed spacer, and the 441 bp fragment of the 65 kDa heat shock protein gene (hsp65) were sequenced using previously published approaches [5],[6],[7].

  Results and discussion Top

The 51-year-old Caucasian woman studied had been treated for tuberculosis three times in the past. The patient presented with a productive cough and night sweats and her CD4 count was 514 cells/mL. She was being treated for human immunodeficiency virus with antiretroviral therapy (zidovudine, lamivudine, and tenofovir). The sputum smear microscopy showed acid-fast bacilli (+) in two samples. A chest X-ray revealed bilateral pulmonary lesions (nodular opacities and cavities). She was empirically prescribed amikacin, ofloxacin, ethambutol, and clarithromycin, since there was no identification of mycobacteria. No sensitivity test was conducted. During follow-up, her respiratory symptoms improved and periodic respiratory specimen cultures tested negative. She was considered cured after 18 months of treatment.

The 16S rDNA gene was sequenced using MicroSEQ Full Gene 16S rDNA Kit (Life Technologies, Carlsbad, CA, USA), as indicated by the manufacturer. All the sequences were compared on identity with the ones of the closest reference strains using a basic local alignment search tool (BLAST) search [8]. The sequences of reference type strains (retrieved from GenBank) and strain HF1226 (the older of the two isolates) were used to build a concatenated neighbor-joining tree [9] using the MEGA version 6 program [10]. Comparison with the reference strains present in GenBank through a BLAST search showed that the 16S rDNA (accession number KF856620), hsp65 (KF856621), and internal transcribed spacer (KF856623) sequences of the isolates have 100% identity with M. triplex type strain (ATCC 700071), but the rpoB (KF856622) gene presented two mismatches when compared with M. triplex type strain (99.72% identity). The next high similarity was that of Mycobacterium florentinum type strain DSM 44852 with nine mismatches and 95.92% identity. The phylogenetic tree revealed that strain HF1226 was clustered (with 100% bootstrap) to the type strain of M. triplex and distinct from other related species([Figure 1]).
Figure 1: Phylogenetic concatenated tree using 16S rDNA, hsp65, rpoB, and internal transcribed spacer sequences, constructed using neighbor-joining method and Kimura's two-parameter as the substitution model. The bootstrap values, calculated on 1000 replicates, indicated the significance of branches. Mycobacterium tuberculosis sequence was used as the outgroup. M. = Mycobacterium.

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All phenotypic results were the same as described for the M. triplex type strain [2]. The molecular and phylogenetic analysis of the two isolates from the Brazilian patient confirmed the identification as M. triplex. BLAST analysis also revealed that the polymorphisms identified in the rpoB gene of these two strains had never been described, as no identical sequence was found in the database.

M. triplex has been suggested as a cause of disease in severely immunocompromised patients, but can also affect immunocompetent individuals [11],[12]. Symptoms and radiological findings of the studied patient are similar to those in previous reports. There are few reported cases of M. triplex infections to date. In Europe, M. triplex has been reported as causing pulmonary infection in patients of countries such as Italy, Portugal, Ireland, and Finland [12],[13],[14],[15]. In the United States, a study described a case of M. triplex that was isolated from the pericardial and peritoneal fluid of a liver transplant patient [16]. Also, in the United States another study identified eight M. triplex strains from clinical M. simiae complex isolates [17]. There are extremely rare reports of patients infected by M. triplex outside Europe and the United States; one exception is a Nigerian human immunodeficiency virus positive man with central nervous system involvement [18].

The results presented in this study extend the range of geographical distribution of M. triplex. This finding increases the possibility of the diagnosis of new cases of M. triplex infection in Latin America, especially in Brazil.

Conflicts of interest

The authors have no conflict of interest.


This work was supported by the Health Ministry of Brazil. We thank Teca Galvão for revising the manuscript.

  References Top

M.M. Johnson, J.A. Odell, Nontuberculous mycobacterial pulmonary infections, J. Thorac. Dis. 6 (2014) 210–220.  Back to cited text no. 1
M.M. Floyd, L.S. Guthertz, V.A. Silcox, et al, Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov., J. Clin. Microbiol. 34 (1996) 2963–2967.  Back to cited text no. 2
J. van Ingen, E. Tortoli, R. Selvarangan, et al, Mycobacterium sherrisii sp. nov., a slow-growing non-chromogenic species, Int. J. Syst. Evol. Microbiol. 61 (2011) 1293–1298.  Back to cited text no. 3
D.E. Griffith, T. Aksamit, B.A. Brown-Elliott, et al, ATS Mycobacterial Diseases Subcommittee; American Thoracic Society; Infectious Disease Society of America. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases, Am. J. Respir. Crit. Care Med. 175 (2007) 367–416.  Back to cited text no. 4
T. Adeékambi, P. Colson, M. Drancourt, RpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria, J. Clin. Microbiol. 41 (2003) 5699–5708.  Back to cited text no. 5
A. Roth, M. Fischer, M.E. Hamid, et al, Differentiation of phylogenetically related slowly growing mycobacteria based on 16S–23S rRNA gene internal transcribed spacer sequences, J. Clin. Microbiol. 36 (1998) 139–147.  Back to cited text no. 6
A. Telenti, F. Marchesi, M. Balz, et al, Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis, J. Clin. Microbiol. 31 (1998) 175–178.  Back to cited text no. 7
S.F. Altschul, W. Gish, W. Miller, et al, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410.  Back to cited text no. 8
G. Devulder, M. Peérouse de Montclos, J.P. Flandrois, A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model, Int. J. Syst. Evol. Microbiol. 55(2005) 293–302.  Back to cited text no. 9
K. Tamura, G. Stecher, D. Peterson, et al, MEGA6: molecular evolutionary genetics analysis version 6.0, Mol. Biol. Evol. 30(2013) 2725–2729.  Back to cited text no. 10
G. Caruso, R. Angotti, F. Molinaro, et al, Cervical lymphadenitis by Mycobacterium triplex in an immunocompetent child: case report and review, Indian J. Microbiol. 53 (2013) 241–244.  Back to cited text no. 11
C. Piersimoni, P. Zitti, G. Mazzarelli, et al, Mycobacterium triplex pulmonary disease in immunocompetent host, Emerg. Infect. Dis. 10 (2004) 1859–1862.  Back to cited text no. 12
A. Amorim, R. Macedo, A. Lopes, et al, Non-tuberculous mycobacteria in HIV-negative patients with pulmonary disease in Lisbon, Portugal, Scand. J. Infect. Dis. 42 (2010) 626– 628.  Back to cited text no. 13
R. McMullan, J. Xu, M. Kelly, et al, Mycobacterium triplex pulmonary infection in an immunocompetent patient, J. Infect. 44 (2002) 263–264.  Back to cited text no. 14
S. Suomalainen, P. Koukila-Kähkölä , E. Brander, et al, Pulmonary infection caused by an unusual, slowly growing nontuberculous Mycobacterium, J. Clin. Microbiol. 39 (2001) 2668–2671.  Back to cited text no. 15
E. Hoff, M. Sholtis, G. Procop, et al, Mycobacterium triplex infection in a liver transplant patient, J. Clin. Microbiol. 39 (2001) 2033–2034.  Back to cited text no. 16
J. van Ingen, S.E. Totten, L.B. Heifets, et al, Drug susceptibility testing and pharmacokinetics question current treatment regimens in Mycobacterium simiae complex disease, Int. J. Antimicrob. Agents 39 (2012) 173–176.  Back to cited text no. 17
V. Zeller, A.L. Nardi, C. Truffot-Pernot, et al, Disseminated infection with a mycobacterium related to Mycobacterium triplex with central nervous system involvement associated with AIDS, J. Clin. Microbiol. 41 (2003) 2785–2787.  Back to cited text no. 18


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