SnackNTM: An Open-Source Software for Sanger Sequencing-based Identification of Nontuberculous Mycobacterial Species
2022; 42(2): 213-248
Ann Lab Med 2022; 42(3): 358-362
Published online May 1, 2022 https://doi.org/10.3343/alm.2022.42.3.358
Copyright © Korean Society for Laboratory Medicine.
1Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea; 2Department of Database Control, Nosquest Inc., Gyeonggi-do, Korea; 3Department of Laboratory Medicine, Gil Medical Center, Gachon University College of Medicine, Incheon, Korea
Correspondence to: Daewon Kim, M.D., Ph.D.
Department of Laboratory Medicine, Gil Medical Center, Gachon University College of Medicine, 21 Namdong-daero 774 beongil, Namdong-gu, Incheon 21565, Korea
Dongeun Yong, M.D., Ph.D.
Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Identifying Mycobacterium using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging. We evaluated the performance of MALDI-TOF MS in identifying nontuberculous mycobacteria (NTM) using the ASTA MicroIDSys system (ASTA Inc., Suwon, Korea) with the MycoDB v1.95s and upgraded MycoDB v2.0-beta databases. We tested 124 NTM isolates collected from Ogawa medium at Severance Hospital, Seoul, Korea, between January and April 2019. MicroIDSys scores were categorized into three groups: ≥140, reliable identification; 130–139, ambiguous identification; and <130, invalid identification. To validate the results, we used the reverse blot hybridization assay (Molecutech REBA MycoID, YD Diagnostics Corp., Korea). Initial analysis using MycoDB v1.95s resulted in 26.6% (33/124) reliable, 43.5% (54/124) ambiguous, and 29.8% (37/124) invalid identifications. Re-analysis using the upgraded MycoDB v2.0-beta database resulted in 94.4% (117/124) reliable, 4.0% (5/124) ambiguous, and 1.6% invalid (2/124) identifications. The percentage of reliable identifications that matched with the reference increased from 26.6% (33/124) with MycoDB v1.95s to 93.5% (116/124) with MycoDB v2.0-beta. The upgraded databases enable substantially improved NTM identification through deep learning in the inference algorithm and by considering more axes in the correlation analysis. MALDI-TOF MS using the upgraded database unambiguously identified most NTM species. Our study lays a foundation for applying MALDI-TOF MS for the simple and rapid identification of NTM isolated from solid media.
Keywords: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Nontuberculous mycobacteria, Database upgrade, Identification, Performance evaluation
More than one-third of nontuberculous mycobacteria (NTM) species are clinically relevant as they cause pneumonia, pediatric lymphadenitis, dermatitis, and systemic infections in patients with immunodeficiency [1, 2]. The treatment of NTM usually requires multidrug therapy, and the regimen is determined based on the NTM species identified [1, 3]. Rapid and accurate NTM identification is required to avoid drug toxicity or the development of drug resistance resulting from unnecessary treatment. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated for NTM identification in clinical laboratories . However, MALDI-TOF MS has limited applicability for
We evaluated the NTM identification performance of the ASTA MicroIDSys system (ASTA Inc., Suwon, Korea) using the most up-to-date database for
We acquired 124 NTM clinical strains from clinical samples (sputum, bronchoalveolar lavage, and pus) between January and April 2019 from the Department of Laboratory Medicine and the Research Institute of Bacterial Resistance of Severance Hospital. The clinical samples were decontaminated using acetyl-L-cysteine-sodium hydroxide, neutralized with phosphate buffer, centrifuged, and inoculated into mycobacteria growth indicator tube (MGIT) liquid culture (Becton Dickenson, Sparks, MD, USA) and 3% Ogawa solid culture systems (BANDIO Bio Science, Pocheon, Korea). The cultures were incubated at 37°C for 6–8 weeks. For positive cultures, PCR was performed to differentiate NTM from
The ASTA MicroIDSys MALDI-TOF MS system was used to acquire and analyze the spectra through laser exposure. The MycoDB v1.95s database was initially used to compare and analyze protein profiles, which were re-analyzed using the upgraded version MycoDB v2.0-beta. Both databases are based on the same dataset containing reference spectra from 71 mycobacterial species, including
Identification scores were categorized according to the manufacturer’s recommendation: a score of <130 was defined as an invalid identification, a score of 130–139 as ambiguous identification, and a score of ≥140 as reliable identification. To validate the results, we used the reverse blot hybridization assay (Molecutech REBA MycoID, YD Diagnostics Corp., Yongin, Korea), which is a modification of the reverse hybridization assay widely used as a reference method for identifying NTM species in previous studies [14, 15].
Of the 124 NTM isolates, 58 and 66 isolates were categorized as rapidly growing and slowly growing mycobacteria, respectively (Table 1). The results of the initial analysis with MycoDB v1.95s were as follows: 26.6% (33/124) isolates with a score of ≥140, 43.5% (54/124) isolates with a score of 130–139, and 29.8% (37/124) isolates with a score of <130. The percentage of MALDI-TOF MS results with a score ≥140 that matched with the reference reverse blot hybridization assay was 100.0% (33/33). Re-analysis with the upgraded MycoDB v2.0-beta database yielded 94.4% (117/124) isolates with a score of ≥140, 4.0% (5/124) isolates with a score of 130–139, and 1.6% (2/124) isolates with a score of <130 (Table 2). The isolates that scored <130 were two strains of
Previous studies reported that database upgrades can significantly increase the ability to identify NTM species. Rodriguez-Sanchez,
When applying MALDI-TOF MS in clinical microbiology, it is crucial to distinguish the unique signal peak for each microbial species from noise. This poses a significant obstacle, particularly in NTM identification due to artifact contamination from the culture medium during sample preparation and the limited amount of biomass that can be obtained. To overcome this obstacle, MycoDB v2.0-beta uses a strategy, in which additional weight is assigned to peaks that are likely to be true signals, while eliminating spectra in the database that include many peaks likely to represent noise from possible media contamination. Using MycoDB v2.0-beta, the 124 clinical NTM isolates were identified with high accuracy of 93.5%, as shown in Table 2. This is substantially better than most previously reported accuracies [6, 13, 16-18]. Notably, Yoo,
To our knowledge, only one study has used ASTA MicroIDSys MALDI-TOF MS for NTM identification . Along with this previous report, our study provides valuable information about the utility of ASTA MicroIDSys MALDI-TOF MS. One of the main strengths of our study is that the sample size was relatively large compared with that of the previous study. Second, the accuracy of identification was substantially higher than that reported in other studies using solid cultures, suggesting that improvement in the analytical algorithms can greatly enhance the identification ability of MALDI-TOF MS. This study also had some limitations. First, we used only isolates from solid cultures, although the ability of ASTA MicroIDSys to identify mycobacteria directly from positive MGIT liquid cultures was evaluated recently . Since mycobacterial growth would usually first be detected in liquid cultures, the clinical implications of this study are somewhat limited. Second, species that are rarely identified in clinical settings, including
In conclusion, our study demonstrated reliable NTM identification using ASTA MicroIDSys following MALDI-TOF MS, with substantially higher accuracy than previously reported. Our study lays a foundation for the future use of MALDI-TOF MS for the simple and rapid identification of NTM isolated from solid media.
Conception and design of study: Yong D; Data acquisition: Song JH; Data analysis, and interpretation: Song JH, Yoon S, In Y; Drafting of manuscript: Song JH; Final approval of manuscript: Kim D, Lee H, Yong D, Lee K.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI20C0951) and (HI21C0901); by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1324).