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The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. KEY POINTS: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy.

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BACKGROUND: Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. METHODS: Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. RESULTS: Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. CONCLUSIONS: PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.

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Failure in recognizing non-tuberculous mycobacteria (NTM) leads to misdiagnosis of multidrug-resistant Mycobacterium tuberculosis Complex (MTBC). There is an unmet need for diagnostic tools that can differentiate between NTMs and MTBC, and that are affordable for Low- and Middle-income Countries (LMIC). Earlier we developed a strip-based CrfA assay technology to detect the Carbapenem Resistance Factor A (CrfA) enzyme present only in MTBC. However, the strip-based CrfA assay had low turnaround time and lacked high-throughput capabilities. In this current research, we have developed a 96well-formatted CrfA assay for high-throughput detection of MTBC and differentiation with NTMs. This 96well-formatted CrfA assay displays a low turnaround time of 6-8 h with 100 % specificity and 93.75 % sensitivity on clinical samples. Based on these attributes, this 96well-formatted assay represents a valuable complementary tool to mitigate the misdiagnosis of chronic pulmonary tuberculosis with non-tuberculous mycobacteria in poorer nations.

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Nontuberculous mycobacteria (NTM) are emerging as notable causative agents of opportunistic infections. To examine clinical significance, species distribution, and temporal trends of NTM in Denmark, we performed a nationwide register-based study of all unique persons with NTM isolated in the country during 1991-2022. We categorized patients as having definite disease, possible disease, or isolation by using a previously validated method. The incidence of pulmonary NTM increased throughout the study period, in contrast to earlier findings. Mycobacterium malmoense, M. kansasii, M. szulgai, and M. avium complex were the most clinically significant species based on microbiologic findings; M. avium dominated in incidence. This study shows the need for surveillance for an emerging infection that is not notifiable in most countries, provides evidence to support clinical decision-making, and highlights the importance of not considering NTM as a single entity.

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Non-tuberculous mycobacteria (NTM) infections pose a significant public health challenge worldwide, affecting individuals across a wide spectrum of immune statuses. Recent epidemiological studies indicate rising incidence rates in both immunocompromised and immunocompetent populations, underscoring the need for enhanced diagnostic and therapeutic approaches. NTM infections often present with symptoms similar to those of tuberculosis, yet with less specificity, increasing the risk of misdiagnosis and potentially adverse outcomes for patients. Consequently, rapid and accurate identification of the pathogen is crucial for precise diagnosis and treatment. Traditional detection methods, notably microbiological culture, are hampered by lengthy incubation periods and a limited capacity to differentiate closely related NTM subtypes, thereby delaying diagnosis and the initiation of targeted therapies. Emerging diagnostic technologies offer new possibilities for the swift detection and accurate identification of NTM infections, playing a critical role in early diagnosis and providing more accurate and comprehensive information. This review delineates the current molecular methodologies for NTM species and subspecies identification. We critically assess the limitations and challenges inherent in these technologies for diagnosing NTM and explore potential future directions for their advancement. It aims to provide valuable insights into advancing the application of molecular diagnostic techniques in NTM infection identification.

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The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

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BACKGROUND: Environmental mycobacteria are involved in several infections ranging from lung to skin infections. In Côte d'Ivoire, apart from Mycobacterium ulcerans and Mycobacterium tuberculosis, little information exists on other species. The culture of these species, a real challenge, especially in developing countries like Cote d'Ivoire, limits their identification. However, there are reports in literature of infections caused by these mycobacteria, and few species have never been described in human or animal infections. These are difficult cases to treat because of their resistance to most antituberculosis antibiotics. The aim of our work was to study the diversity of potentially pathogenic mycobacterial species in wastewater drainage channels in different townships and in two hospital effluents in the city of Abidjan. METHODS: Wastewater samples were cultured, followed by conventional polymerase chain reaction (PCR) targeting mycobacterial 16S ribonucleic acid (16S RNA) using PA/MSHA primers. 16 S RNA identified were sequenced by Sanger techniques. Sequences obtained were analyzed, and a phylogenic tree was built. RESULTS: Fast-growing mycobacteria, including Mycobacterium fortuitum, Mycobacterium phocaicum, Mycobacterium sp., and others presence, were confirmed both by culture and molecular techniques. M. fortuitum strain was the same in effluents of the Treichville University Hospital and in the wastewater of the township of Koumassi. New species never isolated in Côte d'Ivoire, such as M. phocaicum, have been identified in wastewater of the township of Yopougon. CONCLUSION: This study showed that the sewer network in the city of Abidjan is colonized by both potentially pathogenic mycobacteria and saprophytic environmental mycobacteria.

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Nontuberculous mycobacteria (NTM) is a large group of mycobacteria other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. Epidemiological investigations have found that the incidence of NTM infections is increasing in China, and it is naturally resistant to many antibiotics. Therefore, studies of NTM species in clinical isolates are useful for understanding the epidemiology of NTM infections. The present study aimed to investigate the incidence of NTM infections and types of NTM species. Of the 420 samples collected, 285 were positive for M. tuberculosis, 62 samples were negative, and the remaining 73 samples contained NTM, including 35 (8.3%) only NTM and 38 (9%) mixed (M. tuberculosis and NTM). The most prevalent NTM species were Mycobacterium intracellulare (30.1%), followed by Mycobacterium abscessus (15%) and M. triviale (12%). M. gordonae infection was detected in 9.5% of total NTM-positive cases. Moreover, this study reports the presence of Mycobacterium nonchromogenicum infection and a high prevalence of M. triviale for the first time in Henan. M. intracellulare is the most prevalent, accompanied by some emerging NTM species, including M. nonchromogenicum and a high prevalence of M. triviale in Henan Province. Monitoring NTM transmission and epidemiology could enhance mycobacteriosis management in future.

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Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

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BACKGROUND: We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics. METHODS: We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups. RESULTS: In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013). CONCLUSIONS: Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.

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PURPOSE: 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 VER 1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. METHODS: 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 VER 1.0 kit. RESULTS: The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER 1.0 kit 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 VER 1.0 database 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 VER 1.0 database 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-VER 1.0 kit. CONCLUSION: The good performance of the FluoroType®-Mycobacteria-VER 1.0 kit 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.

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Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

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The prevalence of Non-tuberculous Mycobacterial Pulmonary Disease (NTM-PD) is increasing worldwide. The advancement in molecular diagnostic technology has greatly promoted the rapid diagnosis of NTM-PD clinically, and the pathogenic strains can be identified to the species level through molecular typing, which provides a reliable basis for treatment. In addition to the well-known PCR and mNGS methods, there are numerous alternative methods to identify NTM to the species level. The treatment of NTM-PD remains a challenging problem. Although clinical guidelines outline several treatment options for common NTM species infections, in most cases, the therapeutic outcomes of these drugs for NTM-PD often fall short of expectations. At present, the focus of research is to find more effective and more tolerable NTM-PD therapeutic drugs and regimens. In this paper, the latest diagnostic techniques, therapeutic drugs and methods, and prevention of NTM-PD are reviewed.

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Objetivo. Conocer la incidencia y epidemiología de mico bacterias no tuberculosas (MNT) en nuestra área y la preva lencia de comorbilidades en pacientes con infección por MNT. Como objetivos secundarios, estudiamos la distribución por es pecies de MNT, las formas de enfermedad objetivadas y el tipo de muestra empleada para su diagnóstico. Material y métodos. Estudio retrospectivo en el que se incluyeron todos los aislamientos de micobacterias realizados por el Laboratorio de Microbiología del Hospital Clínico Uni versitario Lozano Blesa de Zaragoza durante el periodo com prendido entre el 1 de enero de 2011 y el 31 de diciembre de 2018. Resultados. Se aislaron un total de 533 micobacterias, de las cuales 295 (55,35%) eran micobacterias tuberculosas (MTB) y 238 (44,65%) MNT. Del total de aislamientos de MNT, el 15,54% fueron considerados clínicamente significativos. Se identificaron 21 especies y las más frecuentes fueron: M. gor donae (26,89%), M. fortuitum (19,75%) y M. avium (16,39%). El 32,72% de los aislamientos de MNT se realizaron en mayores de 70 años. Conclusiones. Podemos confirmar que el número de ais lamientos de MNT en nuestra área está siendo mayor que en periodos previos. La infección por MNT es más frecuente en varones y mayores de 70 años. La epidemiología, especialmen te los factores de riesgo, de la enfermedad por MNT está cam biando (AU)

Objectives. The main objective of our investigation was to know the incidence and epidemiology of non-tuberculous mycobacteria (NTM) in our area and the prevalence of comor bidities in patients with MNT infection. As secondary objec tives, we studied the distribution by species of MNT, the forms of disease and the type of sample used for its diagnosis. Material and methods. A retrospective study was carried out in which all the isolates of mycobacteria carried out by the microbiology laboratory of the Hospital Clínico Universitario Lozano Blesa of Zaragoza during the period between January 1, 2011 and December 31, 2018 were included. Results. A total of 533 mycobacteria were isolated, of which 295 (55.35%) were tuberculosis (MTB) and 238 (44.65%) were MNT. Of the whole MNT isolates, only 15.54% were con sidered clinically significant. Twenty-one species were identi fied being the most frequent: M. gordonae (26.89%), M. for tuitum (19.75%) and M. avium (16.39%). 32.72% of the MNT isolates were found in people over 70 years of age. Conclusions. We can confirm that the reported number of MNT isolates in our area is higher than in previous periods. MNT infection is more common in men and those older than 70 years. The epidemiology, especially the risk factors, of MNT disease is changing (AU)

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Introducción El objetivo de nuestro estudio fue evaluar la frecuencia de aislamiento de la infección respiratoria por micobacterias no tuberculosas (MNT) y analizar las características clínico-epidemiológicas de los pacientes infectados por MNT. Métodos Estudio observacional retrospectivo de 83 muestras respiratorias con aislamiento de MNT de 62 pacientes entre los años 2015 y 2021 en el Hospital General Universitario Doctor Balmis. Resultados Se cumplían criterios de infección respiratoria por MNT en 15 pacientes (24,2%). Las MNT más frecuentemente aisladas en los pacientes que cumplieron criterios de infección fueron las pertenecientes al complejo Mycobacterium avium complex (M. avium complex). De los 15 pacientes infectados, 11 (73,3%) presentaban comorbilidad respiratoria y la comorbilidad respiratoria más frecuente en los pacientes infectados fueron las bronquiectasias (5 pacientes; 45,5%). De los pacientes infectados se pautó tratamiento antibiótico dirigido en el 83,3% de los casos. Conclusión Uno de cada 7 pacientes con aislamiento por MNT cumplen criterios de infección. Se corrobora el papel principal de las especies de M. avium complex y la relevancia del daño estructural pulmonar en el desarrollo de enfermedad pulmonar por MNT (AU)

Introduction The objective of our study was to evaluate the frequency of isolation of respiratory infection by non-tuberculous mycobacteria (NTM) and to analyze the clinical-epidemiological characteristics of patients infected with NTM. Methods Retrospective observational study of 83 respiratory samples with NTM isolation from 62 patients between 2015 and 2021 at the Doctor Balmis General University Hospital. Results MNT respiratory infection criteria were met in 15 patients (24.2%). The most frequently isolated NTM's in patients who met infection criteria were those belonging to the Mycobacterium avium complex. Of the 15 infected patients, 11 (73.3%) had respiratory comorbidity and the most frequent respiratory comorbidity in infected patients was bronchiectasis (5 patients; 45.5%). Of the infected patients, targeted antibiotic treatment was prescribed in 83.3% of the cases. Conclusion One in 7 patients with NTM isolation meets infection criteria. The main role of the species of Mycobacterium avium complex is corroborated, and the relevance of lung structural damage in the development of lung disease due to NTM (AU)

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La identificación proteómica de micobacterias no tuberculosas (MNTs) mediante MALDI-TOF presenta una mayor complejidad debido a la especial composición de su pared celular, que complica la extracción de proteínas. Un total de 106 aislamientos pertenecientes a diferentes especies de MNTs procedentes de muestras clínicas del Complejo Asistencial Universitario de León recogidas durante los años 2019 y 2020 se han identificado por un método proteómico abreviado (MALDI-TOF Biotyper Bruker®) desarrollado en nuestro laboratorio. La identificación se ha comparado con la realizada en paralelo en el Centro de Referencia de Majadahonda. Se analizaron un total de 22 especies diferentes de MNTs obteniendo una concordancia del 91,5%. Las nueve discrepancias detectadas se dieron entre especies pertenecientes al mismo grupo taxonómico. En el 67,92% de las identificaciones el score fue superior a 1,8. En el tiempo de procesamiento se obtuvo un ahorro aproximado de 24 minutos con respecto al recomendado por el fabricante.(AU)

Proteomic techniques relaying upon mass spectrometry (MALDI_TOF) applied to nontuberculous mycobacteria (NTM) identification, constitute a difficult goal. Cell wall structure features complicates the protein extraction procedure. A total of 106 isolates belonging to a variety of MNTs species isolated from clinical samples taken at the Complejo Asistencial Universitario de León for a two years period (2019-20) were identified following a simplified method (MALDI-TOF Biotyper Bruker®) developped in our laboratory. The resultant identification was compared to a parallel one ruled on the Centro de Referencia de Majadahonda. A total of 22 different MNTs species were tested, obtaining an agreement of 91,5%. Only 9 minor discrepancies between species belonging to the same taxonomic group of MNTs were detected. The score obtained in the 67.92% of the cases was higher than 1.8. A time-saving of 24 minutes compared to the manufacturer‘s proceeding was achieved.(AU)

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The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

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Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM).Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal.Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM.Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense, Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium peregrinum. The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay.Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1.Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.

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The purpose of the present study was to evaluate a real-time PCR system for 12 nontuberculous mycobacteria (NTM) species identification developed by Central Tuberculosis Research Institute (CTRI; Moscow, Russia) in cooperation with Syntol LLC (Moscow, Russia). NTM cultures (210 strains, 19 species), Mycobacterium tuberculosis complex (MTBC) cultures (21 strains, 2 species), non-mycobacterial microorganisms (18 strains, 13 species) were used for the first stage of the assay evaluation. Clinical samples (sputum, N = 973) positive for smear microscopy and MTBC/NTM DNA by a PCR-based screening assay collected from 819 patients were used for specificity and sensitivity evaluation. Sensitivity for determining the NTM species directly from diagnostic material was 99.71%, with the specificity of 100%. The sensitivity and specificity for NTM species identification in cultures was 99.67% and 100%, respectively. Both sensitivity and specificity for determining MTBC in cultures was 100%.

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