A 78-year-old HIV-negative Japanese man was admitted to our hospital for left upper meibomian gland resection of a sebaceous gland carcinoma in December 2012. He was referred to our respiratory center because of abnormal findings on chest X-ray and computed tomography (CT) scans. He had no subjective respiratory complaints. He had no past history of smoking or alcohol abuse, but had a past medical history of pulmonary tuberculosis at the age of 15 years. In 2008, some NTM were cultured from his sputum at another hospital, but the strain was not identified.
On the first visit to our center, physical examination revealed piping sounds in the right lung field. Laboratory tests showed a slightly elevated inflammatory response. A chest X-ray image showed a reticular shadow in the right upper lung field and pleural calcification of the left lung along with volume reduction (Fig. 1). A chest high-resolution computed tomography (HRCT) scan revealed moderately thickened bronchial walls in the upper right bronchus, centrilobular micronodules in the upper right lobe, infiltration in the right middle lobe, and pleural calcification of the left lung along with volume reduction (Fig. 1). Sputum smears and cultures for acid-fast bacilli (AFB) were negative, but we suspected a chronic respiratory tract infection and started the administration of a low dose of clarithromycin (CLR, 200 mg per day).
In September 2013, 9 months after the initiation of macrolide administration, his chest CT scan revealed deterioration of centrilobular micronodules and infiltration in the right lobe. He had a positive culture for AFB on an expectorated sputum sample. This isolate demonstrated slow-growing, non-pigmented, creamy and smooth colonies, consistent with the characteristics of Runyon III organisms. However, the strain could not be identified by both polymerase chain reaction (PCR) method to detect M. tuberculosis and DNA-DNA hybridization method (DDH Mycobacteria, Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) which is commonly used to identify 17 clinically isolated species of NTM in Japan. We also performed antimicrobial susceptibility testing of the isolates. We used BrothMIC NTM  for the broth microdilution method (Kyokuto Pharmaceutical Industrial Co., Ltd.) to determine the minimum inhibitory concentration (MIC). MICs of antibiotics for this strain were 0.06 mug/ml (CLR), 0.12 mug/ml (rifampicin), 0.03 mug/ml [rifabutin (RFB)], 0.25 mug/ml (streptomycin), <=0.5 mug/ml (amikacin), 1 mug/ml [levofloxacin (LVX)], 4 mug/ml [ethambutol (EMB)], and 8 mug/ml [ethionamide (ETO)]. In March 2014, an expectorated sputum smear for the AFB was positive, and the patient received a higher dose of CLR (600 mg per day) and rifampicin (RIF, 300 mg per day). Ethambutol (EMB) was not administered because of his glaucoma. Eventually, the pulmonary shadow partially reduced, but expectorated sputum smear and culture for the AFB remained positive.
We amplified and sequenced the 16S rRNA gene to identify the species of this NTM isolate. Next, we searched for homology between this sequence and various mycobacteria with the Basic Local Alignment Search Tool (BLAST ). Our results identified 2 species of NTM (M. triplex and M. florentinum) as superior candidates; however, they had the exact similarity score (Fig. 2). Therefore, we sequenced the hsp65 gene of the NTM isolate (Fig. 3), which resulted in a similarity score of 686 for M. triplex and 652 for M. florentinum. We finally identified this bacterium as M. triplex.
The HRCT findings of this patient were consistent with those of pulmonary NTM disease, and NTM strains were detected in 3 serial expectorated sputum samples during the clinical course. There were no other microorganisms that could cause respiratory infections detected. DNA sequencing analysis identified at least one of these NTM strains as M. triplex. In general clinical practice, it is extremely rare that NTM which cannot be identified with DDH are detected. Therefore, it was reasonable to consider this patient had respiratory infection caused by M. triplex. In December 2014, levofloxacin (LVX, 250 mg per day) was started. Eventually, the pulmonary shadow reduced further, and the sputum AFB culture became negative. Fig. 4 shows the clinical course of the present case including expectorated sputum smear and culture, identification results of bacterial species, chest CT images, and treatment details.
Chest image of the case patient. (B, C) Chest high-resolution CT (HRCT) showed centrilobular micronodules in the right upper lobe and pleural calcification of the left lung, along with volume reduction.