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A case of pulmonary Mycobacterium heckeshornense infection in a healthy Japanese man

A 72-year-old man, healthy, smoker, with long-standing cough, was referred to our hospital and his chest X-ray (CXR) revealed a cavity lesion in the right upper lobe. Direct sputum smears, but not culture in solid medium, were positive for acid-fast bacilli (AFB) without tuberculosis DNA. The preliminary diagnosis was of a non-tuberculosis infection that progressed slowly, and the CXR showed the condition to worsen daily. Four years later, a commercialized mycobacteria growth indicator tube system was used to culture the colonies of AFB successfully in liquid medium, and the species Mycobacterium heckeshornense was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The patient responded well to triple therapy with rifampicin, ethambutol, and clarithromycin, the sputum cultures remained negative and the roentgenogram showed minor improvement over the following 6 months.


Introduction
A species of Mycobacterium (M.) heckeshornense was first described as a new isolated non-tuberculosis mycobacteria (NTM) which infects human. It was reported in a patient with pulmonary infiltrations in 2000 [1]. Except for pulmonary infectious diseases with M. heckeshornense [2][3][4][5][6][7][8][9], diseases such as spinal osteomyelitis and diskitis [10], lumbar spondylodiskitis [11], and lymphadenitis [12] were reported as systemic diseases. M. heckeshornense might be categorized as Runyon group II, as a slow-growing scotochromogenic mycobacteria [4,13], and be misdiagnosed as M. xenopi. The pathogenesis of infection by these two bacteria is similar, but M. xenopi can be distinguished by 16S ribosomal RNA (rRNA) and 16S-23S spacer gene sequences [2,6]. Today, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique was developed to easily identify bacterial species, including the species of M. heckeshornense [8,14]. We herein report a rare case of a healthy Japanese man with pulmonary M. heckeshornense infections by using modified liquid culture system and the MALDI-TOF MS techniques. Furthermore, triple anti-mycobacterial therapy with rifampicin (RFP), ethambutol (EB), and clarithromycin (CAM) was successful for the treatment of the progressive pulmonary disease.

Case report
A 72-year-old healthy man with chronic cough and CXR abnormalities was referred to our clinic. He was a current smoker and his smoking index was 92 pack-years. His CXR ( conditions showed no growth. His CXR abnormalities were diagnosed as non-tuberculosis mycobacteria infection with unknown causes. His home doctor followed up the patient, as the chronic cough was relieved with temporary cough medicines or spontaneously. Four years later, he revisited our clinic with cough recurrence and a weight loss of 5 kg/year with 16.4 kg/m 2 of body mass index. His vital signs were almost normal and his body temperature was 36.7 � C. His white blood cells count was 5000 cells/μL and serum C-reactive protein level was 2.26 mg/L. His chest CT revealed worsened cavity lesions with surrounding infiltrative shadows at the right upper lobe (Fig. 1C).
Direct sputum smears were positive for AFB, although MTB and MAC-DNA could not be detected by PCR techniques and standard AFB cultures; AFB was ultimately successfully cultured in the mycobacterium growth indicator tubes (MGIT) system (BD Biosciences, Sparks, MD, USA). A specie of M. heckeshornense was finally identified with the MALDI-TOF MS techniques, although no match of 18 mycobacterial species (not involving M. heckeshornense) were identified among the cultured AFB strains by the commercial kits of the DNA-DNA hybridization techniques (DDH Mycobacteria, Kyokuto, Japan). The progressive pulmonary M. heckeshornense infections were defined in the patient, because the species was isolated repeatedly from sputum samples on different days after fiberoptic bronchoscopy.
We summarized the characteristics of 11 patients (number of male, 6; median age at diagnosis, 52.6 years [range: 30-72 years]) with pulmonary M. heckeshornense infections based on our case and previous case reports [1][2][3][4][5][6][7][8][9] (Tables 1 and 2). Five patients were in the immunocompetent state, and the remaining 6 patients had some underlying diseases. Only two patients had no symptoms, although most patients had similar respiratory and general symptoms with other NTM infections. The number of patients diagnosed with lung M. heckeshornense infections were 7, by the 16S rRNA sequence analysis, 2 by the DNA strip assay and 2 by the MALDI-TOF MS techniques, respectively. The CXR and CT abnormalities of lung fields were limited to the right upper lobe in six patients and 6 had cavity lesions. The regimens for M. heckeshornense infections varied, although multidrug therapy was selected for all the patients including anti-tuberculous drugs, CAM, aminoglycosides, and some new quinolones. The limited lung lesions were successfully resected in 2 patients after anti-mycobacterial chemotherapies. Except for two patients, the number (population) of patients who had improved, not changed and worsened were 7 (64%), 2 (18%) and 1 (9%), respectively, after the treatments.
The indirect drug susceptibility of M. heckeshornense strain was tested according to the absolute concentration method using a microtiter technique developed by a commercial laboratory (BML. Tokyo, Japan). The minimum inhibitory concentrations (MIC) of isoniazid, RFP, EB, and streptomycin used were 0.2 g/mL, 40 g/mL, 2.5 g/ml, and 10 g/ml, respectively. According to the previous reports, M. heckeshornense was resistant to INH, but susceptible to RFP, EB, amikacin, CAM, and ciprofloxacin (1). Our case showed similar susceptibility to previous reports. Triple anti-mycobacterial therapy with RFP, EB and CAM at 450, 750, and 600 mg/day, respectively (his body weight was under 50 kg) was started against the progressive pulmonary M. heckeshornense infections based on the results of MIC and the regimens in the previous reports [1][2][3][4][5][6][7][8][9]. His symptoms and abnormal shadows on chest CT improved 6 months after the treatments without adverse events (Fig. 1D). Mycobacterial cultures remained negative.

Discussion
We experienced a rare case of pulmonary M. heckeshornense infection in a healthy Japanese man. Triple anti-mycobacterial therapy with RFP, EB, and CAM was successful for the treatment of progressive pulmonary M. heckeshornense infection. M. heckeshornense shows a very slow growth rate over 37-45 � C, and the differences in the growth temperature depends on the individual isolates of M. heckeshornence [1,3]. While we were initially unable to culture the sputum AFB on solid medium, the AFB were ultimately successfully grown using the MGIT system. According to the previous reports, isolates from 4 to 2 cases grew either in MGIT [2,5,7,8] or solid medium [3,9], respectively, while isolates from 2 additional cases grew in both conditions [3,4]. Curiously, paralleling our observation, other study reported an isolated whose culture was only achieved with MGIT, and not solid medium [5]. This is possibly because the MGIT method increased the culture-positive rate, as others reported: the culture-positive rate in smear-negative specimens was greatly increased [15]. It is possible that patients infected with M. heckeshornense have not been diagnosed correctly, because the MALDI-TOF MS techniques and 16s rRNA method are performed only in few laboratories. However, the use of MALDI-TOF MS techniques is becoming widespread in Japan and was helpful for the isolation of M. heckeshornense species [8].
The three most common NTM pathogens of lung diseases are M. gordonae (31.8%), M. abscessusnon (22.4%), and M. fortuitum (11.8%) Fig. 1. Findings of chest X-ray and computed tomography Note: A) Chest X-ray revealed infiltrations with pleural wall thickness in right upper lobe at initial visit. B) Chest CT revealed a cavity lesion with infiltration in right upper lobe at initial visit. C) Chest CT revealed worsened cavity lesions with infiltration in right upper lobe 4 years after initial visit. D) Chest CT showed that the abnormal shadows improved 6 months after treatments. Abbreviation: CT, computed tomography. except for MAC and M. kansasii in Japan [16]. To our knowledge, the pulmonary M. heckeshornense infections are rare in Japan as well as worldwide ( Table 1). The pathogenesis of M. heckeshornense is still unclear, although the species seems to infect both immunocompromised and immunocompetent patients [1][2][3][4][5][6][7][8][9][10][11][12].
With these clinical, radiologic, and microbiologic findings, the patient met the American Thoracic Society/Infectious Diseases Society of America diagnostic criteria of nontuberculous mycobacterial lung disease [13]. The DNA-DNA hybridization techniques did not cover M. heckeshornense in commercial kits in Japan. M. heckeshornense species could be isolated by the MALDI-TOF MS techniques in our patient. The MALDI-TOF MS techniques may be a useful method for identifying rare species of NTM, such as M. heckeshornense [8,14].
In conclusion, we experienced a rare case of pulmonary M. heckeshornense infection and summarized the previous reported cases. The widespread use of MALDI-TOF MS techniques and triple therapy with RFP, EB, and CAM may contribute to early diagnosis and good controls of M. heckeshornense infections.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest
The authors have no conflicts of interest to declare.