Nontuberculous Mycobacterial Lung Disease Caused by Mycobacterium lentiflavum in a Patient with Bronchiectasis
A 54-year-old woman was referred to our hospital due to chronic cough and sputum, which developed three years earlier. She had been a healthy, non-smoker up to this point, with the exception of pulmonary tuberculosis 10 years prior. Laboratory results, including immunoglobulin levels, were normal with the exception of an elevated erythrocyte sedimentation rate to 82 mm/hr. She had no risk factors for human immunodeficiency virus infection.\nA chest radiography revealed bilateral multifocal tram-track signs. A computed tomography scan of the chest revealed bilateral bronchiectasis and bronchiolitis in the right middle lobe and the lingular division of the left upper lobe (). There was no evidence of cystic fibrosis or other common causes of bronchiectasis. NTM were isolated twice from three consecutively expectorated sputum specimens.\nInitial identification of the isolated NTM was unsuccessful using the polymerase chain reaction (PCR)-reverse blot hybridization assay (REBA) method based on the rpoB gene (REBA Myco-ID; M&D Inc., Wonju, Korea). No specific species could be assigned using this method due to a lack of reference sequences. Therefore, we used multilocus sequence analysis (MLSA) based on hsp65, rpoB, and 16S rRNA fragments to determine their taxonomic affiliations. Genomic DNA of the isolates was extracted using a commercially available kit (QIAamp DNA Mini kit; Qiagen, Hilden, Germany) and amplified using the appropriate pair of primers for each of the three genes. PCR conditions and primer sequences are described below. Partial amplification of the rpoB gene (723 bp) was performed using primers MycoF (5'-GGCAAGGTCACCCCGAAGGG-3') and MycoR (5'-AGCGGCTGCTGGGTGATCATC-3'). Partial amplification of the hsp65 gene (439 bp) was performed using primers Tb11 (5'-ACCAACGATGGTGTGTCCAT-3') and Tb12 (5'-CTTGTCGAACCGCATACCCT-3'). PCR conditions for these genes were as follows: 2 minutes at 95℃ followed by 35 cycles of 94℃ for 30 seconds, 60℃ for 30 seconds, and 72℃ for 2 minutes, with a final extension step at 72℃ for 5 minutes. A 564 bp 5'-region of the 16S rRNA gene was amplified using primers 16MycF (5'-CGTGCTTAACACATGCAAGTCG-3') and 16MycR (5'-GTGAGATTTCACGAACAACGC-3'). Conditions for 16S rRNA gene amplification were as follows: 2 minutes at 95℃ followed by 35 cycles of 94℃ for 30 seconds, 52℃ for 30 seconds, and 72℃ for 1 minute. The amplicons were cloned into the pGEM-TEasy vector (Promega, Madison, WI, USA), transformed into DH5α Escherichia coli and amplified (Invitrogen, Karlsruhe, Germany). Clones were sequenced using the ABI Prism d-Rhodamine dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were recorded with an ABI Prism 3100 DNA sequencer following the standard protocol of the supplier (Applied Biosystems). The sequences were compared by referring to publically available sequence in the National Center for Biotechnology Information GenBank database and the Ribosomal Differentiation of Medical Micro-organisms (RIDOM) database. Homogeneity values above 99% were considered significant. M. lentiflavum was subsequently identified as the most closely related species for which the sequences of the three genes were available in GenBank (accession nos. EU109300 for rpoB, AF547851 for hsp65, and GU142924 for 16S rRNA).\nFive months after initial sputum cultures, the patient continued to have a mild cough and sputum. Additional sputum culture 4 months after the initial sputum examinations was negative. She was reluctant to receive long-term antibiotic therapy and refused further follow-up. Therefore, the long-term outcome of this patient is not available.