Mycobacterium triplex pulmonary disease with acquired macrolide resistance in immunocompetent patients
Authors: S. Matsuda, S. Suzuki, K. Morimoto, A. Aono, K. Nishio, T. Asakura, Y. Sasaki, H. Namkoong, T. Nishimura, H. Ogata, N. Hasegawa, A. Kurashima, M. Ishii, K. Tatsumi, S. Mitarai, H. Goto
Journal: Clinical Microbiology and Infection (2018)

Mycobacterium triplex, a slowly growing, non-pigmented mycobacterium, i.e. a Runyon group III organism, was named after the triple-peak cluster pattern of mycolic acid obtained by HPLC in 1996 [[1]Floyd M.M. Guthertz L.S. Silcox V.A. Duffey P.S. Jang Y. Desmond E.P. et al.Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov.J Clin Microbiol. 1996; 34: 2963-2967Crossref PubMed Scopus (69) Google Scholar]. Mycobacterium triplex resembles Mycobacterium simiae or Mycobacterium avium-intracellulare complex (MAC) in biochemical tests. The American Thoracic Society and the Infectious Disease Society of America state that M. triplex was previously identified as MAC by using conventional biochemical identification methods and that the organism might have minimal pathogenicity [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. Documentation of the clinical characteristics of uncommon non-tuberculous mycobacterial infection is crucial for better management of patients. Pulmonary M. triplex disease is rare, though several cases have been reported in the last two decades. We report two cases of pulmonary M. triplex infection in immunocompetent women with acquired clarithromycin resistance after cessation of ethambutol, resulting in disease progression. In 2005, Patient 1, a woman in her fifties, presented to Fukujuji Hospital complaining of 1 week of chest discomfort. She did not have any history of smoking or immunosuppressive drug use but had had pulmonary TB at the age of 21 years. Chest X-ray showed infiltrations in the left middle and lower lung fields. Chest CT scans revealed nodular opacities and bronchiectatic changes in the lingular segment and the left lower lobe (see Supplementary material, Fig. S1). Although her three consecutive sputum smears were negative for acid-fast bacilli, the cultures became positive thereafter. The isolates were identified as M. triplex by 16S rRNA sequencing. Because her symptoms resolved spontaneously before non-tuberculous mycobacteria identification, she was followed up once every 3 months without treatment. One year later, she presented with productive cough and haemoptysis, and her bronchial washing specimen culture again became positive for M. triplex. Her chest X-ray findings also worsened, so combination chemotherapy with clarithromycin (600 mg), rifampicin (300 mg) and ethambutol (750 mg) was initiated. After 1 year of treatment, she complained of visual impairment, although her respiratory symptoms were relieved, and the chest X-ray findings improved slightly. Considering the optical neuropathy, the physician replaced ethambutol with sitafloxacin. The MIC of clarithromycin against the isolate at that time was 1 mg/L. After changing the regimen, her chest X-ray findings gradually deteriorated. Follow-up CT scans showed multiple cavitary lesions in the lingular segment and left lower lobe of the lung while she was treated with the same regimen for 5 years. Sputum cultures were consecutively positive for M. triplex, and no other non-tuberculous mycobacterium was isolated. Five years after the cessation of ethambutol, the MIC increased to >32 mg/L, and the case was considered to have acquired clarithromycin resistance (Table 1).Table 1rrl sequence analysis of stock strains of the two Mycobacterium triplex cases as well as MICsStrain16S rRNArrl (23S rRNA)Antibiotics MIC (mg/L)CLRMFXAMKPatient 1, (2006)M. triplex (100%)wild-type1216Patient 1, (2017)M. triplex (100%)A 2058 C>32>816Patient 2, (2014)M. triplex (100%)A 2058 T>32432M. triplex (DSM44626)10.516Abbreviations: CLR, clarithromycin; MFX, moxifloxacin; AMK, amikacin.Note: We analysed the stock strains, which were collected before initiating treatment (in 2006) and after the development of macrolide resistance (in 2017).The strain at the time of diagnosis in patient 2 was not collected. DSM44626 is the type strain. Open table in a new tab Abbreviations: CLR, clarithromycin; MFX, moxifloxacin; AMK, amikacin. Note: We analysed the stock strains, which were collected before initiating treatment (in 2006) and after the development of macrolide resistance (in 2017). The strain at the time of diagnosis in patient 2 was not collected. DSM44626 is the type strain. In 2004, Patient 2, a woman in her fifties, was diagnosed with pulmonary MAC disease and treated with a standard three-drug combination regimen. She discontinued the treatment after 18 months due to optical neuropathy. Six years later, her sputum culture became positive again, and the isolates were identified as M. triplex; the MIC of clarithromycin was 8 mg/L. Treatment with clarithromycin, rifampicin and levofloxacin without ethambutol was initiated 1 year after diagnosis. Her symptoms persisted for 34 months as CT findings showed progression even after treatment initiation, and the MIC of clarithromycin against M. triplex increased to >32 mg/L (Table 1). To confirm the observed acquired resistance to clarithromycin, we performed sequence analysis of rrl using three isolates [[3]Meier A. Kirschner P. Springer B. Steingrube V.A. Brown B.A. Wallace Jr., R.J. et al.Identification of mutations in 23S rRNA gene of clarithromycin-resistant Mycobacterium intracellulare.Antimicrob Agents Chemother. 1994; 38: 381-384Crossref PubMed Scopus (166) Google Scholar]. The isolate from patient 1 at diagnosis had no mutation in rrl, whereas the isolate in 2017 harboured a mutation (A2058C). The isolate from patient 2 in 2014 also had a mutation in rrl (A 2058 T) (Table 1). The present cases show that M. triplex can cause chronic pulmonary disease in immunocompetent hosts, with a refractory clinical course possibly due to acquired clarithromycin resistance. Clarithromycin resistance was acquired due to the regimen without ethambutol, which is one of the major causes of macrolide resistance in MAC [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. Nine cases of M. triplex infections have been reported to date; five extra-pulmonary, two paediatric cervical lymphadenitis, one polyserositis in liver transplantation, and two disseminated infections in severe immunosuppression due to human immunodeficiency virus infection. Among four cases of pulmonary diseases [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar], three patients were immunocompetent. The other patient was infected with human immunodeficiency virus but had retained CD4 cells (514 cells/μL) on antiretroviral therapy. The described cases suggest that M. triplex can cause a pulmonary infection, even in an immunocompetent host. Additionally, all the individuals reported with pulmonary M. triplex disease have particular characteristics similar to those of pulmonary MAC disease; such as female gender, 50 years of age or older, and nodular bronchiectasis (see Supplementary material, Table S1) [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar]. No standard treatment regimen for M. triplex infection has been established. Two of four previously reported pulmonary infections were susceptible to clarithromycin, and treatment with clarithromycin and ethambutol resulted in good clinical outcomes [4Piersimoni C. Zitti P. Mazzarelli G. Mariottini A. Nista D. Zallocco D. Mycobacterium triplex pulmonary disease in immunocompetent host.Emerg Infect Dis. 2004; 10: 1859-1862Crossref PubMed Scopus (14) Google Scholar, 5Campos C.E. Dias C.F. Unis G. de Souza Caldas P.C. Redner P. de Carvalho L.D. et al.First detection of Mycobacterium triplex in Latin America.Int J Mycobacteriol. 2016; 5: 89-91Crossref PubMed Scopus (6) Google Scholar]. In this report, patient 1 had been successfully treated until ethambutol cessation. However, acquisition of clarithromycin resistance, probably due to ethambutol cessation, led to the poor outcome. The present cases suggest that pulmonary M. triplex disease may respond poorly to the combination of clarithromycin, rifampicin and fluoroquinolone, similar to pulmonary MAC disease. The treatment of macrolide-resistant MAC disease with surgical therapy along with aminoglycosides recommended by American Thoracic Society and the Infectious Disease Society of America could also be suitable for macrolide-resistant M. triplex disease [[2]Griffith D.E. Aksamit T. Brown-Elliott B.A. Catanzaro A. Daley C. Gordin F. et al.An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.Am J Respir Crit Care Med. 2007; 175: 367-416Crossref PubMed Scopus (4303) Google Scholar]. In conclusion, treatment of M. triplex infection should be conducted using a multidrug regimen based on clarithromycin and ethambutol at the recommended doses. Moreover, the physician must understand that the modified regimen without ethambutol for any reason may lead to the development of CLR resistance. All authors report no conflicts. This work was supported by the Japan Agency for Medical Research and Development, AMED (project number 17fk0108116h0401).