Mycobacterium shimoidei, a rare non‐tuberculous mycobacteria pathogen identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Non‐tuberculous mycobacteria (NTM) cause several infectious diseases in humans. This study reports on Mycobacterium shimoidei infection in an immunosuppressed 61‐year‐old male with a background of emphysema. His chief complaint was haemoptysis. Chest computed tomography showed a large, thin‐walled cavitary lesion in the upper right lobe. Although NTM were identified in two separate expectorated sputum samples, DNA–DNA hybridization (DDH) failed to identify the species. M. shimoidei was finally identified using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Following antimicrobial agent susceptibility tests, treatment with clarithromycin, levofloxacin, and ethambutol commenced. Six months post‐treatment, acid‐fast sputum culture was negative and repeat imaging demonstrated improvement of the radiographic abnormalities. This study aimed to assess the utility of MALDI‐TOF MS for successful identification of rare NTM species that are not identifiable by DDH. It is the first report of M. shimoidei from Okinawa, which is the only prefecture in Japan categorized as subtropical.


Introduction
Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and can cause various infectious diseases in humans. The prevalence of NTM-induced lung infections is increasing globally. Mycobacterium shimoidei is a slow-growing NTM that was first isolated in Japan in 1968, gaining species status in 1975 [1]. Since then, a number of cases of infection have been reported worldwide [1][2][3][4][5][6][7].

Case Report
A 61-year-old man from Okinawa, Japan, presented at his local hospital complaining of cough and bloody sputum during the preceding week. He was a current smoker with a history of chronic obstructive pulmonary disease (COPD), and rheumatoid arthritis that had been treated with prednisolone 7.5 mg per day and methotrexate 12 mg per day since 2005 by a primary care physician. He was a professional gardener. Chest X-ray demonstrated a thinwalled cavitary lesion in the upper right lobe. He was referred to our hospital for further evaluation. Chest computed tomography (CT) on the first visit showed a large, thin-walled cavitary lesion, pleural wall thickening, trabecular and linear shadows in the upper right lobe, and bronchiectasis in the upper and middle lobe ( Fig. 1A-C). Acidfast bacilli were found on Ziehl-Neelsen staining of two separate sputum smears, respiratory specimens were cultured with 2% Ogawa agar, and the resulting bacterial colonies were collected for species identification. Polymerase chain reaction for tuberculosis (TB) was negative. The resulting suspension liquid was tested using a DNA-DNA hybridization (DDH) method from a commercially available identification kit (Kyokuto Pharmaceutical Industrial Co. Ltd., Tokyo, Japan). Finally, an NTM was isolated from two separate expectorated sputum samples by DDH, which failed to identify the NTM species. The patient's haemoptysis symptoms improved spontaneously without treatment, and he was discharged from the hospital on his  own judgement. Although we encouraged him to attend regular follow-ups at the outpatient centre of our hospital, he declined due to personal reasons. Two years later, in December 2017, the patient was readmitted to our hospital with a recurrence of bloody sputum. The CT scan showed that the cavity in the upper right lobe had extended and the cavity wall had become thinner compared to the previous lesions. Microbiological work-up again isolated an NTM from two separate expectorated sputum samples, and DDH again failed to identify the bacterial species. The positive cultures were sent to a specialized microbiology laboratory for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and M. shimoidei was finally identified. The activities of antimicrobial agents were examined by a commercially available drug susceptibility kit for NTM (Kyokuto Pharmaceutical Industrial Co. Ltd., Tokyo, Japan), with the breakpoints derived using Mycobacterium kansasii as a reference. M. shimoidei proved susceptible to clarithromycin (CAM), ethambutol (EB), streptomycin, amikacin, and levofloxacin (LVFX); and resistant to rifampicin (RFP). After referring to published literature and the advice of an expert on acid-fast bacteria, the patient was started on a treatment regimen of CAM, EB, and LVFX in January 2018. His haemoptysis decreased gradually and the cavitary lesion improved (Fig. 1D, E). Six months after treatment, acid-fast culture of sputum was negative.

Discussion
The purpose of this study is to communicate the potential utility of MALDI-TOF MS for identification of M. shimoidei that is not identifiable by DDH. This is essential for choice of the appropriate antimicrobial treatment. Notably, this is also the first identification of M. shimoidei in Okinawa, the southernmost and only prefecture categorized as subtropical in Japan. Species isolated from patients with NTM lung disease are geographically diverse, although the Mycobacterium avium complex is the commonest species in most countries, including mainland Japan [8]. Recently, our group reported that Okinawa may be one of the few places where the Mycobacterium abscessus complex is the predominant pathogen causing NTM lung disease [9].
Previously published details of 25 cases and this case are summarized together in Table 1 [1][2][3][4][5][6][7], which is based on the format of the table in the study by Baird et al. [3] Cases with normal chest X-ray were excluded in Table 1. Previous reports of M. shimoidei infections have been predominantly in the lungs, and largely among male patients. The most commonly associated concurrent conditions were COPD, bronchiectasis, and past TB [1][2][3][4][5][6][7]. Common symptoms were cough or sputum production, weight loss, dyspnoea, fevers, or sweats. Radiology demonstrated cavitating disease in approximately 70% of patients (Table 1). The infection route for humans is unknown.
MALDI-TOF MS is a proteomics method that permits rapid and accurate identification of mycobacterium species from positive cultures [10]. Whereas only 18 acid-fast bacteria species have been identified using DDH, MALDI-TOF MS successfully identified 148 species from NTM isolates [10]. Furthermore, compared to other molecular techniques, MALDI-TOF MS is more cost effective, provides faster identification of mycobacterial isolates to the species level, and often facilitates earlier implementation of more appropriate therapies than is possible with previous bacterial identification methods [10,11].
Although more information about drug susceptibilities of M. shimoidei is required, it is known to be resistant to isoniazid and RFP, and susceptible to EB and rifabutin (RFB) [2,4,5]. Recently, Baird et al. suggested drug regimens combining RFB, EB, and CAM, with moxifloxacin/LVFX, sulfamethoxazole, pyrazinamide, and clofazimine also being potentially useful [3]. In the case presented here, susceptibility testing combined with expert opinion permitted to add LVFX to the treatment regimen for M. shimoidei. Increased recognition and understanding of this pathogen are necessary to expedite diagnosis and improve patient outcomes.

Disclosure Statement
Appropriate written informed consent was obtained for publication of this case report and accompanying images.