Identification Of Nontuberculous Mycobacteria Research Articles

Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China.

Diseases caused by M. tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) have similar clinical symptoms but require different treatments. Rapid and accurate identification of MTB and NTM is essential for proper patient management and treatment. To develop and assess a multiplex real-time fluorescence PCR (Multiplex PCR) method for rapid identification of MTB, M. avium complex (MAC), M. chelonae-M. abscessus group (MCAG), and M. kansasii in clinical samples. The specificity and limit of detection (LOD) were tested using standard strains and clinical isolates. The accuracy of the Multiplex PCR was validated with DNA from 228 known clinical samples confirmed by Targeted next-generation sequencing (tNGS). Additionally, 901 consecutive clinical samples were assessed to evaluate the Multiplex PCR's diagnostic performance in detecting mycobacteria in pulmonary and extrapulmonary samples. LOD of the four mycobacteria ranged from 11.7 to 360.0CFU/mL in water, 43.8-922.0CFU/mL in sputum, and 53.2-859.3CFU/mL in sputum mixed infection, with 98.7% sample detection accuracy and 100% strains identification accuracy. Based on the composite reference standard, the sensitivity of the Multiplex PCR for detecting tuberculosis in pulmonary and extrapulmonary samples was comparable to Xpert (p>0.05) and higher than Culture, especially in extrapulmonary samples (P<0.0001). For NTM detection in pulmonary samples, the sensitivity was slightly lower than Culture (P>0.05) but higher than CapitalBio RT-PCR (P<0.05). Overall, the Multiplex PCR showed significantly higher sensitivity for mycobacterial diseases compared to the other three methods (P<0.01), with a specificity of 96%. The Multiplex PCR demonstrated excellent diagnostic performance in both pulmonary and extrapulmonary samples, offering a low-cost, rapid identifying tool for major pathogenic mycobacteria in eastern China.

Identification of Nontuberculous Mycobacterium and Mycobacterium tuberculosis Complex in Sputum Patients with Suspected Tuberculosis.

Pulmonary tuberculosis (TB) is predominantly caused by Mycobacterium tuberculosis complex (MTBC) and can also involve nontuberculous mycobacteria (NTM). These pathogens pose significant global health challenges, particularly in developing countries. Differentiating between MTBC and NTM in clinical specimens is often difficult using conventional acid-fast staining methods, leading to an underestimation of NTM prevalence in TB-endemic regions. This study aims to identify mycobacterial species in sputum samples from patients suspected of having TB, utilizing polymerase chain reaction (PCR) assays and gene sequencing techniques. We collected 111 sputum samples from patients at Dr. Wahidin Sudirohusodo Central General Hospital, Hasanuddin University Hospital, and Makassar Community Lung Health Center. The samples were analyzed at the Clinical Microbiology Laboratory of Hasanuddin University using standard microscopy and molecular detection techniques. Descriptive statistics were employed to summarize patient demographics, infection characteristics, and outcomes. We collected sputum from suspected TB patients with an average age of 50.86 years. We found 16.2% (n = 18) acid-fast bacteria in 111 patients with suspected pulmonary TB, and molecularly, we identified 17.1% (n = 19) Mycobacterium species by multiplex PCR. Three sputum samples tested positive for NTM. Phylogenetic analysis, based on 16S rRNA gene sequencing, revealed similarities between the samples and known mycobacterial species. The study underscores the challenges in differentiating between MTBC and NTM, highlighting the necessity for molecular diagnostic approaches. Notably, we found NTM in sputum samples from patients previously treated for TB. These findings can serve as a reference for improving diagnostic accuracy and preventing misdiagnosis of mycobacterial infections.

Isolation and identification of non-tuberculous mycobacteria from aquarium fish in Ilam, Iran

Non-tuberculous mycobacteria (NTM) are among the most important pathogens in wild, captive, marine, and freshwater fish species. So, it is important to consider fish as the primary source of infection for aquarium fish and humans. The present study analyzed the occurrence of NTM in aquarium fish in Ilam, west of Iran. In total, 50 samples of infected fish were collected from different aquariums. Following initial sample processing, sediment of each sample was inoculated into Lowenstein-Jensen and Herrold egg media. The positive colonies were investigated with, growth rate, pigmentation, colony morphology, niacin accumulation, nitrate reduction, catalase activity, urease activity, and arylsulfatase activity. Also, molecular identification was carried out by sequencing of heat shock protein 65 kD gene (hsp65) sequence analysis. According to our results, NTM were isolated from 13 samples (26%), comprising 6 (46.2%) rapid growing, and 7 (53.8%) slow growing mycobacteria. In addition, Mycobacterium marinum was the most common NTM isolated in ornamental fish, which is potentially dangerous for both fish and humans. In conclusion, the current study indicates that ornamental fish play a significant role as a source of NTM.

Retrospective and prospective evaluation of the FluoroType®-Mycobacteria VER 1.0 assay for the identification of mycobacteria from cultures in a French center.

Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER1.0kit. The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER1.0kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER1.0kit. The good performance of the FluoroType®-Mycobacteria-VER1.0kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.

Rapid and accurate identification and differentiation of Mycobacterium tuberculosis and non-tuberculous mycobacteria using PCR kits available in a high-burden setting.

Infections caused by mycobacteria, including Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), are a major public health issue worldwide. An accurate diagnosis of mycobacterial species is a challenge for surveillance and treatment, particularly in high-burden settings usually associated with low- and middle-income countries. In this study, we analyzed the clinical performance of two commercial PCR kits designed for the identification and differentiation of MTBC and NTM, available in a high-burden setting such as Ecuador. A total of 109 mycobacteria isolates were included in the study, 59 of which were previously characterized as M. tuberculosis and the other 59 as NTM. Both kits displayed great clinical performance for the identification of M. tuberculosis, with 100% sensitivity. On the other hand, for NTM, one of the kits displayed a good clinical performance with a sensitivity of 94.9% (CI 95%: 89-100%), while the second kit had a reduced sensitivity of 77.1% (CI 95%: 65-89%). In conclusion, one of the kits is a fast and reliable tool for the identification and discrimination of MTBC and NTM from clinical isolates.

Profile of non-tuberculous mycobacteria amongst tuberculosis presumptive people in Cameroon

BackgroundCameroon is a tuberculosis (TB) burden country with a 12% positivity among TB presumptive cases. Of the presumptive cases with a negative TB test, some are infected with Non-tuberculous Mycobacteria (NTM). However, the diagnosis of NTM infections remains difficult due to the lack of tools in many laboratories, particularly in resource limited laboratories and remote setting. The present study was undertaken to determine NTM profile and associated comorbidities among TB presumptive people.MethodsA retrospective study was conducted from December 2018 to December 2019 in the Tuberculosis-National Reference Laboratory (TB-NRL) for Bacteriological analysis of samples and Jamot Hospital of Yaounde (JHY) for clinical evaluation of confirmed NTM patients.We included in this study data of 5267 TB presumptive people previously diagnosed using three consecutive samples and having culture and SD Bioline results with or without Microscopy and reverse hybridization-based Line Probe Assay(LPA) results. The data on co-morbidities or history of people infected with NTM were then collected from the three participants with available clinical data.ResultsWe collected data of 5267 presumptive TB people. Among them, 3436 (65.24%), have a positive culture with 3200 (60.75%) isolates belong to Mycobacterium tuberculosis Complex (MBTC) and 236 (4.48%) to NTM. Our results showed that, 123 (52.11%) NTM were isolated from people with negative microscopy and 113 (47.88%) from people with positive microscopy. Among the 236 NTM, 108 (45.8%) isolates were identified using LPA. M. fortuitum was the most represented species (32.41%) followed by M. intracellulare (19.44%). Sputum had the highest proportion of NTM (56%), followed by bronchial aspirations (31%). The extra-pulmonary samples presented lower proportions of isolates compared to pulmonary samples. Some patients affected with NTM presented comorbidities as HIV infection, Pulmonary tuberculosis, Type 2 diabetes, Chronic bronchitis and Alveolar pneumonia.ConclusionsOur study showed the presence of NTM strains among presumptive TB people with a predominance of M. fortuitum and M. intracellulare. It is important to implement a surveillance system of NTM in TB burden country and also to develop a point-of-care test for NTM identification in limited-resource settings.

Employing Multicolor Melting Curve Analysis to Rapidly Identify Non-Tuberculous Mycobacteria in Patients with Bronchiectasis: A Study from a Pulmonary Hospital in the Fuzhou District of China, 2018-2022.

Non-tuberculous mycobacteria (NTM) infection is common in bronchiectasis, with rising incidence globally. However, investigation into NTM in bronchiectasis patients in China remains relatively limited. This work aimed to identify and understand the features of NTM in bronchiectasis patient in Fuzhou district of China. The pulmonary samples were collected from 281 bronchiectasis patients with suspected NTM infection in Fuzhou, 2018-2022. MPB64 antigen detection was employed for the preliminary evaluation of NTM. Further NTM identification was realized using gene chip and gene sequencing. Among 281 patients, 172 (61.21%) patients were NTM-positive (58.72%) according to MPB64 antigen detection, with females (58.72%) outnumbering males (41.28%) and the highest prevalence in the age group of 46-65 years. In total, 47 NTM single infections and 3 mixed infections (1 Mycobacterium tuberculosis complex-M. intracellulare, 1 M. avium-M. intracellulare, and 1 M. abscessus-M. intracellulare) were identified through multicolor melting curve analysis (MMCA), which was compared with gene sequencing results. Both methods suggested Mycobacterium (M.) intracellulare, M. abscessus, and M. avium as the primary NTM species affecting bronchiectasis patients. M. intracellulare and M. abscessus were more frequent in females than males with the highest prevalence in the age group of 46-65 years according to MMCA. This research provides novel insights into the epidemiological and clinical features of NTM in bronchiectasis patients in Southeastern China. Significantly, M. intracellulare, M. abscessus, and M. avium were identified as the major NTM species, contributing to a better understanding and management of bronchiectasis accompanied by NTM infection.

Incorporating direct molecular diagnostics in management algorithms for nontuberculous mycobacteria: Is it high time?

Nontuberculous mycobacteria (NTM) are a group of acid-fast mycobacteria other than Mycobacterium tuberculosis complex (MTBC) that cause pulmonary disease that is similar to the disease caused by MTBC. International guidelines for the diagnosis of pulmonary NTM disease are rigid and have remained unchanged for nearly 2 decades. In this opinion piece, we provide a new perspective on the traditional criteria by suggesting a diagnostic algorithm that incorporates direct molecular identification of NTM performed on raw sputum specimens (using Sanger or targeted deep sequencing approaches, among others) paired with traditional culture methods. Our approach ensures a more rapid diagnosis of pulmonary NTM disease, thus, facilitating timeous clinical diagnosis, and prompt treatment initiation, where indicated, and leverages recent advances in novel molecular techniques into routine NTM identification practice.

Review of methods for detection and characterization of non-tuberculous mycobacteria in aquatic organisms.

Mycobacteriosis is an emerging and often lethal disease of aquatic organisms caused by several non-tuberculous mycobacteria (NTM) species. Early diagnosis of mycobacteriosis in aquaculture and aquatic settings is critical; however, clinical diagnoses and laboratory detection are challenging, and the available literature is scarce. In an attempt to fill the gap, here we review the most relevant approaches to detect and characterize mycobacteria in clinical specimens of aquatic organisms. Emphasis is given to recent advances in molecular methods used to differentiate NTM species spanning from targeted gene sequencing to next-generation sequencing. Further, given that there are major gaps in our understanding of the prevalence of the different NTM species, partially because of their distinct requirements for in vitro growth, we also reviewed the most relevant NTM species reported to cause disease in aquatic organisms and their specific in vitro growth conditions. We also highlight that traditional bacterial culture continues to be relevant for NTM identification, particularly in non-automated laboratories. However, for NTM species discrimination, a high level of accuracy can be achieved with MALDI-TOF MS and molecular approaches, especially targeted gene sequencing applied from clinical specimens or from pure NTM isolates.

Isolation and identification of nontuberculous mycobacteria from raw milk and traditional cheese based on the 16S rRNA and hsp65 genes, Tehran, Iran.

As an important source of human food, milk can be a carrier of human pathogenic bacteria, including tuberculous and nontuberculous mycobacteria (NTM), in its raw and unpasteurized state. In this research, 175 raw milk samples and 175 traditional cheese samples were collected from traditional dairy stores in 22 regions of Tehran in a 9- month period from August 2019 to May 2020. Samples were prepared and transferred to a specialized laboratory, where they were inoculated in Lowenstein-Jensen (LJ) medium containing glycerol or sodium pyruvate, as well as Herrold's egg-yolk with and without Mycobactin J. to determine the sample's identity of samples. The recommended 16S rRNA (1436bp) and hsp65 (644bp) gene fragments from the positive isolates identified in Ziehl-Neelsen (Z-N) staining were amplified and sequenced using PCR and compared with the sequences of the gene fragments of reference strains available in the global GenBank database. No mycobacterial species were isolated from traditional cheese samples in microbial culture. In case of raw milk samples, a total of four bacteria were collected, all of which were found in the genetic differential testing to be NTM, including n = 1 Mycobacterium heraklionense, n = 2 Mycolicibacterium fortuitum, and n = 1 Mycobacterium thermoresistibile. The analysis of the results obtained by isolate sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities in the identification of the isolates than the hsp65 gene.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

Evaluation of Two Different Preparation Protocols for MALDI-TOF MS Nontuberculous Mycobacteria Identification from Liquid and Solid Media.

Nontuberculous mycobacteria (NTM) identification is essential for establishing the relevance of the isolate and for appropriate antimicrobial therapy. Traditionally, NTM identification is performed by using Line Probe Assays (LPA), a costly and time-consuming technique requiring trained personnel. MALDI-TOF MS is a promising tool for NTM identification, and its use is rapidly growing. We evaluated the newly introduced MBT Mycobacteria kit (MBT) and the MycoEx preparation protocol (Bruker Daltonics, Germany) for NTM MALDI-TOF MS identification using LPA results as a reference. Fifty NTM grown on 7H11 agar and MGIT broth were analyzed with both protocols using the Bruker Microflex® LT MALDI-TOF MS (Bruker Daltonics) instrument. MBT and MycoEx provided identification results in 97.0% and 95.0% of the cases, respectively. With both protocols, 100% of the provided results agreed with LPA with no registered mismatch. MBT achieved an elevated number of highly probable identifications (88.0% vs. 83.0%) and a higher reproducibility rate of correct results (86.6% vs. 75.8%) in comparison to MycoEx. This study provides results about MBT performance for liquid and solid media, underlining the strengths and weakness under different conditions. Our results suggest that MALDI-TOF MS could provide a great advantage for timely and cost-saving NTM identification with potential implications for patient outcome.

Nontuberculous mycobacteria otomastoiditis with facial palsy.

Nontuberculous mycobacteria otomastoiditis with facial palsy.

Screening and Drug Resistance Analysis of Non-Tuberculous Mycobacteria in Patients with Suspected Pulmonary Tuberculosis on the Hainan Island, China

China has seen a drastic increase in the incidence of non-tuberculous mycobacteria (NTM) infection, which is a notable public health issue. Due to a lack of reliable epidemiological surveillance information, there is a need to gather accurate epidemiological and surveillance data, which can help clinicians effectively treat NTM patients. Moreover, drug susceptibility testing for NTM is not frequently performed in China. This retrospective study, therefore, determined the prevalence and resistance characteristics of NTM to provide a reference to control the NTM epidemic. Sputum, alveolar lavage fluid, and other respiratory specimens were collected from 3025 patients with suspected pulmonary tuberculosis attending The Second Affiliated Hospital of Hainan Medical University from January 2014 to December 2021. Strain identification and species distribution of NTM were performed by DNA chip technology and gene sequencing, and the drug resistance of NTM isolates was evaluated by calculating the minimum inhibitory concentration through antimicrobial susceptibility testing for NTM. From 2014 to 2021, 373 strains of NTM were isolated and identified from respiratory specimens of 3025 suspected tuberculosis patients. Except in 2014, NTM-infected patients accounted for more than 10% of suspected tuberculosis patients in other years. The median age of patients with NTM infection was 62.0 years (53.0, 71.0), and the male-to-female ratio among these patients was 0.79:1. Among culture-positive strains, 12.3% (373/3040; 95% CI 11.1-13.4%) were identified as NTM comprising forty species of NTM. The forty species of NTM included 23 slow-growing mycobacteria (SGM) and 17 rapidly-growing mycobacteria (RGM). Among the NTM isolates, 58.7% (219/373; 95% CI 53.7-63.7%) were SGM and 41.3% (154/373; 95% CI 36.3-46.3%) were RGM. M.avium complex(MAC)(41.3%; 95% CI 36.3-46.3%) and M.abscessus complex (MABC)(33.2%; 95% CI 28.4-38.0%) were the most frequently detected species, followed by M.simiae Complex (11.8%; 95% CI 8.5-15.1%), M.fortuitum group (5.1%; 95% CI 2.9-7.3%), and others. Drug sensitivity test results showed that most of the NTM isolates were susceptible to amikacin and clarithromycin with a drug resistance rate of less than 10%. However, clarithromycin could induce drug resistance, followed by linezolid and moxifloxacin, and their drug resistance rate was less than 50%. During 2014-2021, the number of NTM isolates detected in the respiratory specimens of the study patients in The Second Affiliated Hospital of Hainan Medical University increased year by year. M. intracellulare is the most common pathogenic NTM species, and there is a high incidence of NTM infection on Hainan Island. Our findings might be of great importance for diagnosing and treating this patient population in Hainan.

Evaluation of the influence of the cultivation medium on the result of identification of microorganisms from the group of acid-resistant bacteria of the order actinomycetales by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Problem with mass spectrometers is the difficulty in identifying some genera of acid-fast bacteria (AFB). Due to the peculiarities of the colony architectonics (the formation of dry colonies with a complex structure) and the structure of the cell wall, the probability of obtaining a sufficient amount of ribosomal proteins is significantly reduced. This problem is partially solved using an extended method of direct application and extraction with formic acid, which can significantly improve the quality of the identification. The study analyzed strains of microorganisms obtained during the examination of patients with suspected tuberculosis. In total, 287 strains of nontuberculous mycobacteria (NTM) were obtained. In addition, 63 strains of the most common bacteria from the AFB group were analyzed. Matrix-assisted laser desorption/ionization (MALDI) was used. Three main methods of sample preparation of microorganisms according to the manufacturer's recommendations for use with the MALDI-time-of-flight (ToF) mass spectrometry method were used in the work: Direct coating method, extended direct coating method, and formic acid extraction method. When evaluating the influence of the cultivation medium on the result of NTM identification by MALDI-ToF mass spectrometry, statistically significant results of the influence of the medium on the result of NTM identification were revealed for all compared parameters. Optimization of sample preparation protocols and assessment of the impact on the identification of new methods of cultivating microorganisms can significantly improve the quality of the identification of both clinically significant microorganisms from the AFB group and saprophytic microflora, the clinical significance of which has not been proven at the moment.

Isolation and Antimicrobial Susceptibility of Nontuberculous Mycobacteria in a Tertiary Hospital in Korea, 2016 to 2020.

There is a global increase in isolation of nontuberculous mycobacteria (NTM). The aim of the study was to analyze longitudinal trends of NTM identification and pattern of antimicrobial susceptibility testing. NTM recovery rates, distribution of NTM species identification, and antimicrobial susceptibility pattern of NTM at Pusan National University Yangsan Hospital between January 2016 and December 2020 were retrospectively analyzed. A total of 52,456 specimens from 21,264 patients were submitted for mycobacterial culture, of which 2,521 from 1,410 patients were NTM positive over five years (January 2016 to December 2020). NTM isolation showed an increasing trend from 2016 to 2020 (p&lt;0.001, test for trend) mainly caused by Mycobacterium avium complex. The vast majority of M. avium complex were susceptible to key agents clarithromycin and amikacin. For Mycobacterium kansasii, resistance to rifampin and clarithromycin is rare. Amikacin was the most effective drug against Mycobacterium abscessus subspecies abscessus and Mycobacterium subspecies massiliense. Most of M. subspecies massiliense were susceptible to clarithromycin, while the majority of M. abscessus subspecies abscessus were resistant to clarithromycin (p&lt;0.001). There was an increasing trend of NTM isolation in our hospital. Resistance to key drugs was uncommon for most NTM species except for M. abscessus subspecies abscessus against clarithromycin.

Methods of isolation and identification of nontuberculous mycobacteria from environmental samples: A scoping review

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment. Some species of NTM are pathogenic and cause lung disease in susceptible persons. Epidemiologic studies of environmental NTM infection risk rely on both culture-dependent and culture-independent techniques for NTM isolation and identification. In this review, we summarized current methods used to isolate and identify NTM from the environment. We searched PubMed, Embase, Scopus, Web of Science: Core Collection, and Global Health (CAB Direct) for peer-reviewed studies from the last 12 years. We identified 1685 unique citations and 110 studies met our inclusion and exclusion criteria. Approximately half (55%) of the studies identified in this review used a combination of culture-independent and culture-dependent methods. The most common environmental substrate analyzed was water (n = 90). Identification of current, common methods for the isolation and identification of NTM from environmental samples may contribute to the development of standard methodological practices in the future. The choice of isolation method is based on the research question, environment, and species. A summary of common methods may contribute to the development of standard practices for isolation and identification of NTM from environmental samples, which may lead to more robust and comparable results.

Rapid and Accurate Identification of Nontuberculous Mycobacteria Directly from Positive Primary MGIT Cultures by MALDI-TOF MS.

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture.

Mitigation of nontuberculous mycobacteria in hospital water: challenges for infection prevention.

The purpose of this review is to summarize recent literature on nontuberculous mycobacteria in water of healthcare systems. Despite improvement in identification techniques and emergence of infection prevention and control programs, nontuberculous mycobacteria remain present in hospital water systems, causing outbreaks and pseudo-outbreaks in healthcare settings. Waterborne outbreaks and pseudo-outbreaks of nontuberculous mycobacteria continue to affect hospitals. Improvements in methods of identification and investigation, including MALDI-TOF and whole genome sequencing with evaluation of single nucleotide polymorphisms, have been used successfully in outbreak and pseudo-outbreak investigations. Recent studies have shown control of outbreaks in immunocompromised patients through the use of sterile water for consumption, as well as control of pseudo-outbreaks by using sterile water for procedures. Construction activities have been implicated in outbreaks and pseudo-outbreaks of nontuberculous mycobacteria. Water management programs are now required by the Joint Commission, which will likely improve water risk mitigation. Improvement in detection and identification of nontuberculous mycobacteria has led to increasing recognition of waterborne outbreaks and pseudo-outbreaks. Water management programs are of vital importance in infection prevention.

Identification of nontuberculous mycobacteria isolated from household showerheads of patients with nontuberculous mycobacteria

This study aimed to examine whether nontuberculous mycobacteria (NTM) inside household showerheads are identical to those in patients with NTM-pulmonary disease (PD) since household water is one of the potential NTM sources. Samples were obtained from 32 household showerheads of patients with NTM-PD recruited through the Pulmonary Outpatient Department at the Severance Hospital between October 2018 and October 2019. All isolates from patients with NTM-PD were diagnosed using a reverse-hybridization line probe assay based on the ropB gene. To determine the mycobacterial compositions, the washing fluids were collected and investigated using multiplex polymerase chain reaction assay and NTM culture; suspected microbial isolates in these fluids and culture were identified using sequencing analysis of 16S rRNA gene. NTM species causing the PD in the patients were Mycobacterium avium, M. intracellulare, M. abscessus, M. massiliense, and M. fortuitum complex. The mycobacteria isolated from the showerhead were M. lentiflavum, M. gordonae, M. triplex, M. phocaicum, M. mucogenicum, M. florentinum, M. gilvum, M. llatzerense, and M. peregrinum. However, the species identified in the showerheads did not match those of the patients. Despite NTM species in the showerheads, clinical implications in the main pathogenesis associated with the disease in the patients studied were not elucidated.