Identification Of Mycobacteria Research Articles

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.

Comparison of laboratory methods for identifying members of the family Mycobacteriaceae.

The introduction of a method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF mass spectrometry) into the practice of laboratories significantly increased the identification of acid-resistant bacteria (ARB). Seventy-four nontuberculous mycobacteria (NTM) cultures identified by deoxyribonucleic acid (DNA) hybridization, polymerase chain reaction, Sanger sequencing, and MALDI-ToF mass spectrometry. Analysis of the identification results obtained by the methods of DNA hybridization and Sanger sequencing showed a complete match only for 67.6% of samples of the total number of cultures included in the study. The partial match of the identification results was 68.9%. When comparing the results of the identification of 74 samples obtained by MALDI-ToF mass spectrometry to the results obtained by sequencing, full match of identification of Mycobacterium chimaera/Mycobacterium intracelullare, Mycobacterium porcinum/Mycobacterium peregrinum and Mycobacterium tuberculosis complex was found for 90.5% of the samples; the partial match of the results - for 4.1%.. DNA hybridization as a method for identifying NTM showed acceptable sensitivity and specificity; however, for mass spectrometry, a significantly higher sensitivity with comparable specificity was determined. Mass spectrometry is an important element in the modern system of species identification of microorganisms. The optimization of sample preparation protocols and assessment of the impact on the identification of new techniques of cultivation of microorganisms can significantly improve the quality of identification of microorganisms from the ARB group. In this case, accurate species identification and the development of algorithms for its application will improve the diagnosis of diseases caused by ARB.

Identification Of Nontuberculous Mycobacterium Isolates in Suspected Pulmonary Tuberculosis Patients

According to World Health Organization, in the global tuberculosis ranking Pakistan is in 5th position. Mycobacterium tuberculosis bacterium is responsible for this dreadful disease, but there are other nontuberculous mycobacteria species that could also be the possible cause of this disease. Scanty data is available on the incidence and distribution of species responsible for this infection. Local studies on non-tuberculous mycobacterium species would be of great support in targeted therapy. Methods: This study was designed to investigate the incidence and distribution of non-tuberculous mycobacteria-associated infection in pulmonary suspected tuberculosis patients. Sputum samples were processed for microscopy and culturing on Lowenstein–Jensen regardless of age and gender suspected TB patients. Positive cultures were then processed for detection of non tuberculous mycobacteria species using commercially available Geno Type Mycobacterium CM hybridization strips. Results: A total of 1560 sputum samples were tested for Mycobacteria by culturing, 215 were positive, 71 contaminated and 1274 were negative. All 215 culture positive isolates were exposed to Geno Type Mycobacterium CM kit revealed, 55 cultures as Non-tuberculous mycobacteria and 160 as Mycobacterium complex. Adults between aged 40 to 60 years and male were predominantly (61.81%) infected than females (38.18%). Conclusions: Scanty data is available about the contributions of non tuberculous mycobacteria to tuberculosis-like disease, and noteworthy geographical distribution, clinical and molecular epidemiology-related knowledge gaps exist in the areas with a high burden of disease caused by mycobacterium tuberculosis complex. Isolation of nontuberculous mycobacteria from clinical specimens should promptly be evaluated for their clinical significance.

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.

Performance evaluation and clinical validation of optimized nucleotide MALDI-TOF-MS for mycobacterial identification.

To evaluate the performance and validate the diagnostic value of a nucleotide matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) with the analysis process optimized in identification of mycobacterium species. The optimized analysis process was used for mycobacterial identification in the nucleic MALDI-TOF-MS.108 samples were used for assessing the performance of nucleic MALDI-TOF-MS, including 25 reference standards, 37 clinical isolates, 37 BALF, and 9 plasmids. The BALF of 38 patients suspected of pulmonary mycobacterial infection was collected for validation. Clinical etiological diagnosis was used as the gold standard to evaluate the diagnostic value of nucleotide MALDI-TOF-MS. The sensitivity, specificity, and accuracy of the nucleotide MALDI-TOF-MS in mycobacterial identification were 96.91%, 100% and 97.22%, respectively, and the limit of detection for mycobacterium tuberculosis (MTB) was 50 bacteria/mL. Among 38patients suspected of pulmonary mycobacterial infection, 33 were diagnosed with pulmonary tuberculosis infection, and 5 with non-mycobacterial infection. In clinical validation, the positive rates of MALDI-TOF-MS, Xpert MTB/RIF, culture and AFS in BALF of patients diagnosed with tuberculosis infection were 72.7%, 63.6%, 54.5% and 27.3%, respectively. The sensitivity/specificity of MALDI-TOF-MS, Xpert, culture and AFS in diagnosing MTB were 72.7%/100%, 63.6%/100%, 54.5%/100%, 27.3%/100%, with the areas under the curve of 0.864, 0.818, 0.773, and 0.636, respectively. Optimized nucleotide MALDI-TOF-MS has satisfactory sensitivity, specificity and low LOD in the identification of mycobacteria, which may serve as a potential assay for mycobacterial identification.

Pulmonary Granulomas and Mycobacterial Infection: Concordance between the Results of Special Stains Performed on Lung Tissue Sections and Tissue Cultures.

The most common cause of infectious pulmonary granulomas worldwide is Mycobacterium tuberculosis. The diagnosis is based on clinical presentation, histopathologic findings, detection of acid-fast bacilli (AFB) in tissue or sputum using special stains, and/or isolation of mycobacteria in cultures or via PCR-based methods. Different studies have shown that high levels of discrepancy exist between these diagnostic approaches in lung tissue specimens. To assess the degree of concordance between the results of special stains and cultures on lung tissue specimens in the diagnosis of mycobacterial infections. Eighty-seven patients with a diagnosis of granulomas (necrotizing and non-necrotizing) on lung tissue specimens were identified. Cohen's kappa was used to measure the general concordance between the results of the histopathological examination (special stains) and bacteriological tissue cultures. With Kinyoun acid-fast stains, 8/48 (16.7%) cases were positive for AFB. With FITE stains, 10/57 (17.5%) cases were positive for AFB. There was strong agreement between Kinyoun acid-fast and FITE stains (Kappa = 0.806; p-value < 0.001). Tissue cultures were performed on 38/87 cases (43.7%), and 10/38 (26.3%) of the cultures were positive for mycobacteria. There was no concordance between Kinyoun acid-fast stains or FITE stains and tissue cultures results. Our observations represent an initial step in the process of reviewing the two methods used at our institution to diagnose mycobacterial infections on lung tissue specimens and highlight the need of incorporating more advanced diagnostic methods such as PCR to confirm mycobacterial infections and improve patient management. Importantly, species-level identification of mycobacteria is necessary to guide treatment.

Using Vitek MS v3.0 To Identify Nontuberculous Mycobacteria in Liquid Media in a Clinical Microbiology Laboratory.

Recently, the incidence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing worldwide, especially in immunocompromised patients and those with potential chronic lung disease. Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and reliable method for identifying mycobacteria in clinical laboratories. This study aimed to evaluate the performance of Vitek MS v3.0 by isolating NTM directly from automated liquid medium systems using patient samples. A total of 855 Mycobacterium growth indicator tube (MGIT)-positive liquid cultures were investigated. Among them, 658 (77.0%) liquid cultures were correctly identified to the species, group, or complex level, 192 (23.0%) resulted in no identification, and 5 (0.6%) were misidentified at the species level. DNA sequencing identified 855 NTM isolates from liquid cultures, comprising 316 isolates of rapidly growing mycobacteria (RGM) and 539 isolates of slow-growing mycobacteria (SGM). Using the Vitek MS system, the RGM integral identification rate (276/316 [87.34%]) was higher than the SGM rate (381/539 [70.69%]) (P < 0.01). It was also higher than the SGM rate for all MGIT report-positive periods. These results indicate that the Vitek MS v3.0 system can rapidly identify NTM species from liquid cultures. Further validation using molecular techniques is required. IMPORTANCE Rapid and accurate identification of nontuberculous mycobacteria (NTM) is essential for diagnosis, appropriate therapy, and infection control. Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and reliable method for identifying mycobacteria in clinical laboratories. This study reported a clinical validation of the Vitek MS V3.0 system for identification of NTM isolates from 855 MGIT-positive liquid cultures which contained relatively large NTM types. Vitek MS v3.0 showed a promising rate for identification NTM isolates in positive liquid cultures. Vitek MS v3.0 had a better performance with RGM than with SGM. Vitek MS v3.0 results included "unidentified" or "misidentified" NTM isolates, which would also serve as an important reference for future optimization of this system. Vitek MS v3.0 represented a valuable technique for NTM identification from positive liquid cultures.

530 Airway volatile organic compounds can achieve culture-independent identification of Nontuberculous mycobacteria in people with cystic fibrosis

530 Airway volatile organic compounds can achieve culture-independent identification of Nontuberculous mycobacteria in people with cystic fibrosis

A glimpse into the genotype and clinical importance of non tuberculous mycobacteria among pulmonary tuberculosis patients: The case of Ethiopia.

Laboratory identification of nontuberculous mycobacteria (NTM) species is not regularly performed while, they have a public health importance with a prevalence of more than 5% among pulmonary tuberculosis (PTB) patients in Ethiopia. Hence, this study aimed to identify the NTM species and their clinical significance among PTB patients. A retrospective study was conducted at the Ethiopian Public Health Institution’s (EPHI’s) national TB referral laboratory. Stored NTM isolates were genotyped using GenoType Mycobacterium CM/AS kit (Hain Life science, Germany). Data pertinent to the study was extracted from the EPHI’s database and patients’ medical records. Between January 2 & December 28 of 2017, a total of 3,834 samples were processed from 698 TB patients of whom 50% were female. Among 3,317 samples with mycobacterial culture results 7.3% were NTM and majority of them were identified from smear negative TB patients. M. simiae was the /predominant NTM among the genotyped isolates. All the studied NTM species were not clinically important however, considering the similarity of clinical and radiologic findings between NTM and MTBC infected patients, integrating NTM species identification in the routine TB laboratory diagnosis may augment clinicians’ decision particularly in DR-TB patients. Additional similar prospective study with a larger sample size is recommended. Moreover, urgent improvements on patients’ record keeping practice are required in the studied hospitals.

Identification and Characterisation of Nontuberculous Mycobacteria in African Buffaloes (Syncerus caffer), South Africa.

Diagnosis of bovine tuberculosis (bTB) may be confounded by immunological cross-reactivity to Mycobacterium bovis antigens when animals are sensitised by certain nontuberculous mycobacteria (NTMs). Therefore, this study aimed to investigate NTM species diversity in African buffalo (Syncerus caffer) respiratory secretions and tissue samples, using a combination of novel molecular tools. Oronasal swabs were collected opportunistically from 120 immobilised buffaloes in historically bTB-free herds. In addition, bronchoalveolar lavage fluid (BALF; n = 10) and tissue samples (n = 19) were obtained during post-mortem examination. Mycobacterial species were identified directly from oronasal swab samples using the Xpert MTB/RIF Ultra qPCR (14/120 positive) and GenoType CMdirect (104/120 positive). In addition, all samples underwent mycobacterial culture, and PCRs targeting hsp65 and rpoB were performed. Overall, 55 NTM species were identified in 36 mycobacterial culture-positive swab samples with presence of esat-6 or cfp-10 detected in 20 of 36 isolates. The predominant species were M. avium complex and M. komanii. Nontuberculous mycobacteria were also isolated from 6 of 10 culture-positive BALF and 4 of 19 culture-positive tissue samples. Our findings demonstrate that there is a high diversity of NTMs present in buffaloes, and further investigation should determine their role in confounding bTB diagnosis in this species.

Corticoids identification in a lipolytic product promoted by two influencers

Two influencers have consulted for abscesses requiring surgery on the faces and thighs which began, respectively, one month and ten days following injections by a Russian cosmetologist, of the so-called “Light Fit”, a lipolytic product. The “Light Fit” is made in South Korea and is available on the internet market. It claims that the composition includes different mineral salts and natural extracts of chestnut and walnut with exception of any other products. This product is frequently used in Russia for its indirect lipolytic action based on the promotion of “rapid breakdown of excessive fat”. Following the surgery of the two patients, the microbiological examination of the specimens allowed the identification of an atypical mycobacterium. We reported here the investigation of the chemical composition of this lipolytic product, which have led to side effects for two internet influencers promoting it, using both high-resolution targeted and untargeted approach. A saddled sample of the “Light Fit” product was furnished by the two influencers. Plasma and urine samples of the two patients were collected during the medical consultation, several weeks following the last injection of the lipolytic product. A sample preparation based on liquid-liquid extraction using acetonitrile and methyl tert-butyl ether was performed for each sample. Subsequently, extracts were analyzed by targeted and untargeted screening using liquid chromatography hyphenated to high-resolution tandem mass spectrometry with electrospray as ion source operating in polarity switching mode and a Q-Exactive Focus® Orbitrap mass spectrometer. The qualitative analysis of the lipolytic injection product allowed identifying glucocorticoids including triamcinolone acetonide and dexamethasone. The other detected chemicals were theophylline, lidocaine and tramadol using both targeted and untargeted approach. The “Light Fit” product was determined to be a normo-osmolar solution. Regarding the biological samples, triamcinolone acetonide was detected as trace amounts in the plasma of the two influencers. Furthermore, paracetamol was detected in urine samples as it was administered to the patients during medical consultation. The qualitative analysis of the lipolytic injection product invalidated the claim of a strictly natural composition. It is nevertheless known that theophylline and lidocaine are widely used as lipolytic agents. A conclusion of the natural or synthetic origin of theophylline, an alkaloid found in tealeaves, cannot be drawn. The synthetic glucocorticoid triamcinolone acetonide detected in the product is known to be a local anti-inflammatory steroid. The detection of trace amounts of triamcinolone acetonide in the plasma of the two patients may be related to the half-life of the compounds, which is higher than 20 days (Shen et al. Ophtalmology 2010;117:2375–2371; Schweitzer et al. Neth J Med 2000;56:12–16). Furthermore, the skin lesions observed at the injection site may be related to the infection by the atypical mycobacterium, highly favored by the local injection of triamcinolone acetonide, which, like other glucocorticoids, decreases immune response to pathogens. This study confirmed the usefulness of high-resolution screening to qualitatively characterize these types of products, a needed step prior to quantification. These two cases highlight the health risk of buying products on the internet with a falsified claim, even for people who promote these products like these two influencers.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

Beyond tuberculosis: Diversity and implications of non-tuberculous mycobacteria at the wildlife-livestock interface.

Non-tuberculous mycobacteria (NTM) circulate between the environment, animals and humans entailing a double concern: their ability to interfere with tuberculosis diagnosis and their potential to cause infections in their hosts. However, published records on NTM infections in animals are still scarce. The aims of the present study were to describe the diversity of NTM circulating among wild and domestic species from Spain and to analyze their implications as potential pathogenic microorganisms or as sources of interferences in the diagnosis of bovine tuberculosis. Overall, 293 NTM isolates of 277 animals were obtained from tissue samples collected between 2012 and 2019, and analyzed through a multigene approach for mycobacteria identification. Thirty-one species were identified, being Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah), but also M. bouchedurhonense, M. nonchromogenicum and M. lentiflavum, the most abundant ones. Maa and M. lentiflavum were isolated in several animals showing tuberculosis-like lesions. Maa, Mah and M. nonchromogenicum were recovered from many cattle that had reacted to the tuberculin skin test. Other NTM were also associated to these phenomena. These four mycobacterial species were geographically associated between wild boar and other hosts. The findings of the present study suggest that a high diversity of NTM circulates among wildlife and livestock. Wild boar and M. avium seem to play a relevant role in this epidemiological scenario.

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.

A New Artificial Intelligence-Based Method for Identifying Mycobacterium Tuberculosis in Ziehl-Neelsen Stain on Tissue.

Mycobacteria identification is crucial to diagnose tuberculosis. Since the bacillus is very small, finding it in Ziehl–Neelsen (ZN)-stained slides is a long task requiring significant pathologist’s effort. We developed an automated (AI-based) method of identification of mycobacteria. We prepared a training dataset of over 260,000 positive and over 700,000,000 negative patches annotated on scans of 510 whole slide images (WSI) of ZN-stained slides (110 positive and 400 negative). Several image augmentation techniques coupled with different custom computer vision architectures were used. WSIs automatic analysis was followed by a report indicating areas more likely to present mycobacteria. Our model performs AI-based diagnosis (the final decision of the diagnosis of WSI belongs to the pathologist). The results were validated internally on a dataset of 286,000 patches and tested in pathology laboratory settings on 60 ZN slides (23 positive and 37 negative). We compared the pathologists’ results obtained by separately evaluating slides and WSIs with the results given by a pathologist aided by automatic analysis of WSIs. Our architecture showed 0.977 area under the receiver operating characteristic curve. The clinical test presented 98.33% accuracy, 95.65% sensitivity, and 100% specificity for the AI-assisted method, outperforming any other AI-based proposed methods for AFB detection.

Evaluation of the MeltPro Myco Assay for the Identification of Non-Tuberculous Mycobacteria.

PurposeThe purpose of this study was to evaluate a new commercial kit for Mycobacterium species identification and compare its results with that of the commonly used GenoType Mycobacterium CM assay. In addition, we were committed to identifying the main frequent species of nontuberculous mycobacteria (NTM) in Fujian.MethodsA total of 261 clinical strains, collected at the Center for Disease Control and Prevention of Fujian Province, China, were preliminarily identified as NTM based on p-nitrobenzoic acid (PNB) growth test. The genomic DNA of all 261 strains was extracted and subjected to species identification using MeltPro Myco assay and GenoType Mycobacterium CM assay. The results of the latter were used as a control to calculate the positive agreement, negative agreement, agreement and the total agreement of the former. For samples with inconsistent detection results, sequencing was performed for verification.ResultsCompared to GenoType Mycobacterium CM assay, the total agreement of MeltPro Myco assay was 96.55% (252/261 strains). Both the positive and negative agreement of MeltPro Myco assay in identifying M. intracellulare, M. abscessus, M. kansasii, M. fortuitum, M. avium, M. gordonae, M. scrofulaceum, and mixed infections were higher than 99.00%, but the positive agreement of M. chelonae was relatively low at only 33.33%. In Fujian, the predominant strain of NTM was M. intracellulare (64.36%, 130/202 strains), followed by M. abscessus (19.31%, 39/202 strains), M. kansasii (4.46%, 9/202 strains), and M. fortuitum (3.96%, 8/202 strains).ConclusionThe reliability of MeltPro Myco assay for identifying mycobacterium species was strongly demonstrated in this study, which greatly supports its usage for the clinical identification of mycobacteria. The present study also showed that the distribution of mycobacteria in Fujian, China, was significantly different from that in other regions and provided important data for future epidemiological study of NTM.

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.

Breath biomarkers associated with nontuberculosis mycobacteria disease status in persons with cystic fibrosis: a pilot study

Pulmonary infections caused by mycobacteria cause significant mortality and morbidity in the human population. Diagnosing mycobacterial infections is challenging. An infection can lead to active disease or remain indolent with little clinical consequence. In patients with pulmonary nontuberculosis mycobacteria (PNTM) identification of infection and diagnosis of disease can take months to years. Our previous studies showed the potential diagnostic power of volatile molecules in the exhaled breath samples to detect active pulmonary M. tuberculosis infection. Herein, we demonstrate the ability to detect the disease status of PNTM in the breath of persons with cystic fibrosis (PwCF). We putatively identified 17 volatile molecules that could discriminate between active-NTM disease (n = 6), indolent patients (n = 3), and those patients who have never cultured an NTM (n = 2). The results suggest that further confirmation of the breath biomarkers as a non-invasive and culture-independent tool for diagnosis of NTM disease in a larger cohort of PwCF is warranted