Identification Of Mycobacteria Research Articles

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.

Illumina and Nanopore sequencing in culture-negative samples from suspected lower respiratory tract infection patients.

To evaluate the diagnostic value of metagenomic sequencing technology based on Illumina and Nanopore sequencing platforms for patients with suspected lower respiratory tract infection whose pathogen could not be identified by conventional microbiological tests. Patients admitted to the Respiratory and Critical Care Medicine in Shanghai Ruijin Hospital were retrospectively studied from August 2021 to March 2022. Alveolar lavage or sputum was retained in patients with clinically suspected lower respiratory tract infection who were negative in conventional tests. Bronchoalveolar lavage fluid (BALF) samples were obtained using bronchoscopy. Sputum samples were collected, while BALF samples were not available due to bronchoscopy contraindications. Samples collected from enrolled patients were simultaneously sent for metagenomic sequencing on both platforms. Thirty-eight patients with suspected LRTI were enrolled in this study, consisting of 36 parts of alveolar lavage and 2 parts of sputum. According to the infection diagnosis, 31 patients were confirmed to be infected with pathogens, while 7 patients were diagnosed with non-infectious disease. With regard to the diagnosis of infectious diseases, the sensitivity and specificity of Illumina and Nanopore to diagnose infection in patients were 80.6% vs. 93.5% and 42.9 vs. 28.6%, respectively. In patients diagnosed with bacterial, Mycobacterium, and fungal infections, the positive rates of Illumina and Nanopore sequencer were 71.4% vs. 78.6%, 36.4% vs. 90.9%, and 50% vs. 62.5%, respectively. In terms of pathogen diagnosis, the sensitivity and specificity of pathogens detected by Illumina and Nanopore were 55.6% vs. 77.8% and 42.9% vs. 28.6%, respectively. Among the patients treated with antibiotics in the last 2 weeks, 61.1% (11/18) and 77.8% (14/18) cases of pathogens were accurately detected by Illumina and Nanopore, respectively, among which 8 cases were detected jointly. The consistency between Illumina and diagnosis was 63.9% (23/36), while the consistency between Nanopore and diagnosis was 83.3% (30/36). Between Illumina and Nanopore sequencing methods, the consistency ratio was 55% (22/42) based on pathogen diagnosis. Both platforms play a certain value in infection diagnosis and pathogen diagnosis of CMT-negative suspected LRTI patients, providing a theoretical basis for clinical accurate diagnosis and symptomatic treatment. The Nanopore platform demonstrated potential advantages in the identification of Mycobacterium and could further provide another powerful approach for patients with suspected Mycobacterium infection.

Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Evaluation of the Use of Distilled Water as a Sodium Hydroxide Wash in Antimicrobial Cultures

Background: Respiratory specimens subjected to mycobacterial detection were initially pretreated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) to remove the mucus and normal flora.Next, they were washed and neutralized with a phosphate-buffered solution (PBS). The effectiveness of distilled water (DW) compared to PBS as a washing neutralizer during the identification of mycobacteria was evaluated in this study. Methods: We analyzed the results of the mycobacterial test conducted at a general hospital in Gwangju from October 2016 to September 2018. PBS and DW were used as a respiratory sample-washing agent for one year each. Results:The positive culture rate for the culture of mycobacteria was 12.7% (1,843/14,532) and 14.7% (2,095/14,291), when PBS and DW were used, respectively. The recovery rate of the mycobacteria growth indicator tubes (MGIT) and the separation rates of the Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM) showed no significant change. However, in the 2% Ogawa medium, as the NTM culture increased from 47.4% (399/841) to 56.1% (630/1,122),the recovery rate increased from 45.6% (841/1,843) to 53.6% (1,122/2,095). The MGIT contamination rate decreased from 6.5% to 4.1%. Conclusion: DW as a washing agent for NALC-NaOH increased the recovery rate of Ogawa medium and reduced the contamination rate of MGIT. Therefore, the use of DW instead of PBS as a washing neutralizer during the identification of mycobacteria might be useful.

Species identification and drug susceptibility testing of non-tuberculous mycobacteria by Line Probe Assay in Lambaréné, Gabon—a cross-sectional study

BackgroundNon-tuberculous mycobacteria (NTM) are a group of bacteria that cause rare lung infections and are increasingly recognized as causative agents of opportunistic and device-associated infections in humans. In Gabon, there is a lack of data on NTM species identification and drug susceptibility. The aim of this study was to identify the frequency of NTM species and their genotypic susceptibility pattern to commonly used antibiotics for NTM infections in Gabon.MethodsA cross-sectional study was conducted at the CERMEL TB laboratory from January 2020 to December 2022, NTM subspecies identification and drug susceptibility testing to macrolides and aminoglycosides were performed using the genotype NTM-DR kit.ResultsThe study found that out of 524 culture-positive specimens, 146 (28%) were NTM, with the predominant group being Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABC). All MAC isolates were fully susceptible to macrolides and aminoglycosides, while five MABC isolates carried mutations indicative of reduced susceptibility to macrolide and aminoglycoside drugs.ConclusionsThese findings suggest that clinicians may use macrolides and aminoglycosides to manage NTM infections caused by MAC, but further investigation is required to determine MABC drug susceptibility.

Molecular identification and infection pathology of Mycobacterium spp. in captive wild animals in Pakistan.

Animal tuberculosis is an infectious, chronic, granulomatous, and debilitating disease affecting animals as well as humans. However, in recent decades, there have been many endemic geographic localities where animal tuberculosis has been identified in wildlife reservoirs, limiting the eradication program in cattle. This study aimed to identify animal tuberculosis in captive zoo animals in Pakistan. In total, 185 morbid zoo animals were brought for postmortem examination at a veterinary postmortem facility. During the macroscopic examination, these animals were thoroughly examined for the presence of suggestive gross lesions of animal tuberculosis (granulomas/tubercles), and the pattern and distribution of these lesions in different organs. The Ziehl-Neelsen (ZN) staining was performed on smears prepared from granulomatous lesions of lung tissue followed by molecular identification of M. bovis and M. tuberculosis DNA using polymerase chain reaction (PCR). The postmortem examination revealed that 8.1% (15/185) of animals had gross tuberculosis lesions on the lungs and lymph nodes. The ZN staining of tissue smears showed 5.40% positivity while M. bovis and M. tuberculosis DNA was identified in 3.78 % and 1.1% of investigated animals, respectively. The study showed that animal tuberculosis is prevalent among wildlife in Pakistan and it may pose serious public health concerns to the people visiting these zoos and wildlife parks.

Evaluation of the mycobacteria MBT kit for identification of nontuberculous mycobacteria by MALDI-TOF Biotyper (Bruker)

Identification of mycobacteria is crucial for clinical management of patients. The new MBT Mycobacteria kit from the Easy MycoEx protocol (Bruker) is used for the identification of mycobacteria by MALDI Biotyper. The Easy MycoEx protocol was compared to the MycoEx protocol (1) for identification of various mycobacterial isolates collected from samples in 2021, (2) for prospective identification on primary culture during 2 periods. For 44 isolates in MGIT broth, identification rates were high and similar for both protocols (98% vs 95% at cut-off 1.6 and 91% vs 82% at cut-off 1.8). For 42 mycobacteria on Coletsos agar, identification rates were 88% versus 90% at cut-off 1.6 and 76% for both protocols at cut-off 1.8. For slow-growing mycobacteria in MGIT, reproducibility of deposit results was superior with Easy MycoEx. No difference of score was observed between 2 protocols performed on primary culture. Clinical laboratories can easily implement the Easy MycoEx protocol.

Identification of mycobacteria isolated from the biomaterial of animals and environmental objects in Western Siberia

Identification of mycobacteria isolated from the biomaterial of animals and environmental objects in Western Siberia

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.

Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization

BackgroundBovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle. MethodsWe developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears. ResultsFour out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAVTAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection. ConclusionPNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

Improving the identification and diagnostic efficiency of Metagenomic Next-Generation Sequencing for mycobacterial granuloma on postoperative formalin-fixed paraffin-embedded specimens

ObjectiveMetagenomic Next-Generation Sequencing (mNGS) has been validated to have an important role in the diagnosis of mycobacterium infection. The study aimed to further explore the mycobacteria identification ability of mNGS on formalin-fixed paraffin-embedded（FFPE）tissues from postoperative specimens. MethodsPatients who underwent surgical biopsy or resection for clarifying the diagnosis and whose initial postoperative pathology indicated granulomatous lesions were included. Fresh tissues were sent for mycobacterium culture and Xpert MTB/RIF (Xpert) to establish the diagnosis. FFPE specimens were sent for mNGS and molecular pathology，the diagnostic values were compared between the two methods. ResultsA total of 65 cases with definite diagnoses were finally included in the study. 31 cases were confirmed as mycobacterium granuloma using the fresh specimen etiology as diagnostic criteria. The overall sensitivity and specificity of mNGS on FFPE specimens in the diagnosis of mycobacterium granuloma were 100% and 88.24%, respectively. In 19 cases diagnosed as tuberculous granulomas, the sensitivity (100% vs47.37%) and negative predictive value (NPV, 100%vs 82.14%) of mNGS were both significantly higher than that of molecular pathology on the FFPE section（both p 0.00）while the positive predictive value (PPV) and specificity were not significantly different. In 12 cases diagnosed as Non-tuberculous mycobacterium (NTM）granuloma, the sensitivity of mNGS was also significantly higher than that of molecular pathology on FFPE section (100% vs 66.67%, p 0.00) while the specificity, PPV and NPV were all not significantly different. ConclusionsThe mNGS could be used for one-time detection of pathogens on FFPE sections with high sensitivity. It could be recommended as a supplementary method for the identification of pathogenic bacteria in the diagnosis of postoperative granuloma lesions.

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol.

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.

Molecular Identification of Non-tuberculous Mycobacteria in Suspected Tuberculosis Cases in Central India.

Introduction Mycobacterium tuberculosis complex (MTBC), the primary cause of tuberculosis (TB), must be accurately identified to implement effective patient management and control strategies. Non-tuberculous mycobacteria (NTM) in suspected TB cases can result in erroneous diagnoses and needless treatment. Objective The study aimed to identify NTM in patients suspected of TB at a tertiary care hospital in central India using molecular methods. Methods This prospective study enrolled 400 suspected pulmonary and extra-pulmonary TB patients. Patients between the age of two to 90 years, of either gender, new and previously treated cases, Culture positive, patients with immune-compromised status, patients not responding to ATT, HIV positive and negative, and willing to give consent were included in the study. Liquid culture via the Mycobacterial growth indicator tube (MGIT) system was used to culture mycobacteria from clinical samples. The SD Bioline Ag MPT64 Test (Standard Diagnostics, South Korea) and in-house multiplex-PCR (mPCR) were used to differentiate between Mycobacterium tuberculosis complex and NTM species for the molecular identification of NTM GenoType® Mycobacterium Common Mycobacteria (CM) assay kit (HAIN Life Science, Nehren, Germany) was used following the manufacturer's protocol. Results Only 59/400 (14.7%) of the samples produced a positive result in MGIT culture, indicating the presence of mycobacteria, and 85.25% of the remaining 341 samples were negative for mycobacterial growth. Further investigation of these 59 cultures with mPCR and SD Bioline Ag MPT64 test showed that 12 (20.33%) cultures were determined to be NTM, while the remaining 47 (79.67%) were identified as MTBC. Genotype characterization with GenoType® mycobacterium CM assay kit revealed that five of the 12 NTM isolates (41.67%) showed patterns that were consistent with Mycobacterium (M.) fortuitum, three (25%) showed patterns that were consistent with M. abscessus, and four (33.33%) showed patterns that were consistent with M. tuberculosis. Conclusion These resultsemphasizethe value of molecular methods for precisely identifying mycobacterial species, particularly in suspected TB cases. The high prevalence of NTM in positive cultures emphasizes the significance of differentiating between MTBC and NTM to prevent misdiagnosis and ensure proper care. Understanding the epidemiology and clinical significance of these organisms in central India is made possible by the identification of particular NTM species.

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI‐TOF mass spectrometry

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.

MGIT Enriched Shotgun Metagenomics for Routine Identification of Nontuberculous Mycobacteria: a Route to Personalized Health Care.

Currently, nontuberculous mycobacteria (NTM) are identified using small genomic regions, and species-level identification is often not possible. We introduce a next-generation sequencing (NGS) workflow that identifies mycobacteria to (sub)species level on the basis of the whole genome extracted from enriched shotgun metagenomic data. This technique is used to study the association between genotypes and clinical manifestations to pave the way to more personalized health care. Two sets of clinical isolates (explorative set [n = 212] and validation set [n = 235]) were included. All data were analyzed using a custom pipeline called MyCodentifier. Sequences were matched against a custom hsp65 database (NGS-hsp65) and whole-genome database (NGS-WG) created based on the phylogeny presented by Tortoli et al. (E. Tortoli, T. Fedrizzi, C. J. Meehan, A. Trovato, et al., Infect Genet Evol 56:19-25, 2017, https://doi.org/10.1016/j.meegid.2017.10.013). Lastly, phylogenetic analysis was performed and correlated with clinical manifestation. In the explorative set, we observed 98.6% agreement between the line probe assay and the NGS-hsp65 database. In the validation set, 99.1% agreement between the NGS-WG and NGS-hsp65 databases was seen on the complex level. We identified a cluster of Mycobacterium marinum isolates not represented by the Tortoli et al. phylogeny. Phylogenetic analysis of M. avium complex isolates confirmed misclassification of M. timonense and M. bouchedurhonense and identified subclusters within M. avium although no correlation with clinical manifestation was observed. We performed routine NGS to identify NTM from MGIT enriched shotgun metagenomic data. Phylogenetic analyses identified subtypes of M. avium, but in our set of isolates no correlation with clinical manifestation was found. However, this NGS workflow paves a way for more personalized health care in the future.

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.

ЭТИОЛОГИЧЕСКАЯ ВЕРИФИКАЦИЯ МИКОБАКТЕРИОЗА ЛЕГКИХ ПРИ ИССЛЕДОВАНИИ ОПЕРАЦИОННОГО МАТЕРИАЛА У ПАЦИЕНТОВ С ДИАГНОЗОМ А16.0 – РЕАЛЬНАЯ ПРАКТИКА

Objective: to evaluate effectiveness of etiological verification of pulmonary mycobacteriosis (PM) in patients with diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) as per ICD-10 and species diversity of nontuberculous mycobacteria (NTM) in resection samples from the studied cohort. Materials and methods.We analyzed test results from 28 patients with initially diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) as per ICD-10 and verified PM according to resection sample studies. The microbiological study (at least two sputum samples, one bronchobiopsy sample, and one resection sample) included luminescent microscopy (LUM), extraction of M. tuberculosis DNA or NTM DNA by polymerase chain reaction (PCR), culture growth on liquid media in the ВАСТЕС MGIT 960 system, identification of mycobacteria by molecular genetic methods: real-time PCR for M. tuberculosis DNA detection (Syntol, Russia) and DNA-strip technology (GenoType Mycobacterium CM/AS Hain Lifescience). Results. The analysis of referral diagnoses established that in more than half of patients with diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) changes in the lungs had been referred to tuberculoma (64.3%) or infiltrative TB (21.4%); 28.6% of patients had received TB treatment as per chemotherapy regimen no. 3, and 17.8% of patients – broad-spectrum antibiotics. TB treatment duration varied from 4 to 28 months. Atypical lung resections were performed in most cases (75%). NTM DNA detection in resection samples was the most informative method of etiological verification of PMB (100% of cases). NTM culture growth was observed in 17.9% of cases. Only slowly-growing NTM were represented in the studied cohort as etiologically significant. Conclusion. The diagnosis verification is based on data obtained by microbiological and/or histological, cytological studies. Studies of resection samples from TB suspects significantly improve diagnostic effectiveness.

Performance Evaluation of MolecuTech REBA Myco-ID Using HybREAD480 for Identification of Nontuberculous Mycobacteria.

Rapid and accurate identification of nontuberculous mycobacteria (NTM) species is essential for the diagnosis and treatment of NTM disease. MolecuTech REBA Myco-ID (YD Diagnostics, Yongin, Korea) is a line probe assay for identification of NTM species and can be performed using HybREAD480, an instrument for automating the post-PCR steps. In this study, we assessed the performance of MolecuTech REBA Myco-ID using HybREAD480. Seventy-four reference strains, including 65 Mycobacterium strains and nine non-Mycobacterium strains within the order Mycobacteriales, were used to determine the analytical specificity of MolecuTech REBA Myco-ID. The clinical performance of this assay was evaluated with 192 clinical Mycobacterium strains, and the assay results were compared to those of multigene sequencing-based typing. The accuracy of MolecuTech REBA Myco-ID for the 74 reference strains and 192 clinical strains was 77.0% (57/74; 95% confidence interval [CI], 65.8 - 86.0%) and 94.3% (181/192; 95% CI, 90.0 - 97.1%), respectively. Although some rarely isolated NTM species are misidentified, the most commonly isolated NTM species, including M. avium complex, M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. fortuitum com-plex, were all correctly identified. Of note, all M. lentiflavum strains tested (reference strain, n = 1; clinical strain, n = 10) were misidentified as M. gordonae. MolecuTech REBA Myco-ID using HybREAD480 was accurate for identifying commonly isolated NTM species and for discriminating between M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. However, the main limitations of this assay, including misidentification of some rarely isolated NTM species and cross-reactivity between M. lentiflavum and M. gordonae, should be considered.

Rapid and Comprehensive Identification of Nontuberculous Mycobacteria.

Next-generation sequencing is a powerful tool to accurately identify pathogens. The MinION sequencer is best suited for the rapid identification of bacterial species due to its real-time sequence output. In this chapter, we introduce a method to identify nontuberculous mycobacteria (NTM) in one sequencing analysis from culture isolates using the MinION sequencer. NTM disease is now recognized as a growing global health concern due to its increasing incidence and prevalence. There are over 200 NTM species, of which the major pathogens are further classified into many subspecies showing different antibiotic susceptibilities. Therefore, identifying the pathogens at the subspecies level of NTM is necessary to select an appropriate treatment regimen. The protocol described here includes DNA extraction by lysis using silica beads, library preparation, sequencing by the MinION sequencer, and analysis of multilocus sequence typing using the software "mlstverse" and enables rapid and comprehensive identification of 175 species of NTM at the subspecies level with high sensitivity and accuracy.