Applications and advances in molecular diagnostics: revolutionizing non-tuberculous mycobacteria species and subspecies identification.

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.

Application of a new designed high resolution melting analysis for mycobacterial species identification.

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.

Phylogenetic Profile of Nonulcerans and Nontuberculous Environmental Mycobacteria Isolated in Côte d'Ivoire.

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.

Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria.

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.

Comparison of the sputum microbiome between patients with stable nontuberculous mycobacterial pulmonary disease and patients requiring treatment.

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.

Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

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.

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

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.

Advances in diagnosis and treatment of non-tuberculous mycobacterial lung disease.

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.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

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.

Development of a nucleic acid chromatography assay for the detection of commonly isolated rapidly growing mycobacteria.

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.

Evaluation of a new assay for nontuberculous mycobacteria species identification in diagnostic material and cultures.

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%.

Direct Molecular Characterization of Acid-Fast Bacilli Smear of Nontuberculosis Mycobacterium Species Causing Pulmonary Tuberculosis in Guna Yala Region, Panama.

Mycobacterium tuberculosis (MTB) stands out as the main causative agent of pulmonary tuberculosis (TB). However, nontuberculous mycobacteria (NTM) species also have the potential to infect and cause TB in susceptible individuals. The objective of this study was to identify NTM species that cause public health problems in remote areas. The study was carried out using 105 sputum smears obtained from patients from the Guna Yala Region of Panama with clinical signs suggestive of TB. DNA was extracted from sputum smears. Nontuberculous mycobacteria and MTB were characterized using polymerase chain reaction restriction analysis (hsp65, rpob) and an evaluation of 24-mycobacterial interspersed repetitive units-variable number of tandem repeats loci. Twenty-six Mycobacterium species were characterized; 19 (18%) were identified as MTB, and 7 (6.7%) were identified as NTM (four M. avium complex, two M. haemophilum, one M. tusciae). These results suggest that at least one in five cases of pulmonary TB among this population is caused by an NTM. Thus, identifying the bacteria causing pulmonary disease is key even in remote regions of the world where standard diagnosis and culture are not available. Strengthening the laboratory capacity within the Guna Yala Region is needed to identify NTM infections promptly.

Combining comparative genomic analysis with machine learning reveals some promising diagnostic markers to identify five common pathogenic non-tuberculous mycobacteria.

Non-tuberculous mycobacteria (NTM) can cause various respiratory diseases and even death in severe cases, and its incidence has increased rapidly worldwide. To date, it's difficult to use routine diagnostic methods and strain identification to precisely diagnose various types of NTM infections. We combined systematic comparative genomics with machine learning to select new diagnostic markers for precisely identifying five common pathogenic NTMs (Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium chelonae, Mycobacterium abscessus). A panel including six genes and two SNPs (nikA, benM, codA, pfkA2, mpr, yjcH, rrl C2638T, rrl A1173G) was selected to simultaneously identify the five NTMs with high accuracy (> 90%). Notably, the panel only containing the six genes also showed a good classification effect (accuracy > 90%). Additionally, the two panels could precisely differentiate the five NTMs from M. tuberculosis (accuracy > 99%). We also revealed some new marker genes/SNPs/combinations to accurately discriminate any one of the five NTMs separately, which provided the possibility to diagnose one certain NTM infection precisely. Our research not only reveals novel promising diagnostic markers to promote the development of precision diagnosis in NTM infectious, but also provides an insight into precisely identifying various genetically close pathogens through comparative genomics and machine learning.

The lung microbiota in Korean patients with non-tuberculous mycobacterial pulmonary disease.

BACKGROUND: The microbiota of the lower respiratory tract in patients with non-tuberculous mycobacterial pulmonary disease (NTM-PD) has not been fully evaluated. We explored the role of the lung microbiota in NTM-PD by analyzing protected specimen brushing (PSB) and bronchial washing samples from patients with NTM-PD obtained using a flexible bronchoscope. RESULTS: Bronchial washing and PSB samples from the NTM-PD group tended to have fewer OTUs and lower Chao1 richness values compared with those from the control group. In both bronchial washing and PSB samples, beta diversity was significantly lower in the NTM-PD group than in the control group (P = 2.25E-6 and P = 4.13E-4, respectively). Principal component analysis showed that the PSBs and bronchial washings exhibited similar patterns within each group but differed between the two groups. The volcano plots indicated differences in several phyla and genera between the two groups. CONCLUSIONS: The lower respiratory tract of patients with NTM-PD has a unique microbiota distribution that is low in richness/diversity.

Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples.

Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to existing methods. In total, 223 environmental water samples, including potable and non-potable water, were cultured for NTM using three culture media. In addition to direct culture on RGM medium, each sample was cultured on Middlebrook 7H10 medium and Mitchison 7H11 medium after pre-treatment with 0.2M KCl-HCl. Additionally, 33 distinct species of NTM were inoculated onto RGM medium and 7H10 medium in parallel to directly compare their growth. The use of RGM medium alone without pre-treatment provided a sensitivity (91%) comparable to that offered by culture on both 7H10 and 7H11 with acid pretreatment (combined sensitivity; 86%) with significantly less overgrowth and interference from other organisms on RGM medium. The average concentration of NTM observed on RGM medium alone was comparable to or greater than the NTM concentration on either medium alone or combined. Thirty-three species were examined in parallel and all tested strains of 27 of these species successfully grew on RGM medium, including 19 of 21 from the CDC's healthcare-associated infections species list. RGM medium was successful at recovering environmental NTM without a pre-treatment, greatly reducing labor and materials required to process samples. Simplification of culture processing for environmental NTM will allow for a better assessment of their presence in building water systems and the potential for reduced exposure of susceptible populations.

Prevalence and speciation of non-tuberculous mycobacteria among pulmonary and extrapulmonary tuberculosis suspects in South India.

BACKGROUND AND OBJECTIVES: Non tuberculous mycobacteria (NTM) is an emerging opportunistic pathogen increasing globally and indistinguishable from tuberculosis (TB), which remains a challenge particularly in developing countries. This study aimed to identify the prevalence and diversity of NTM among both pulmonary TB (PTB) and extrpulmonary TB (EPTB) clinical isolates from south India. METHODOLOGY: A total of 7633 specimens from TB suspects (PTB, n = 4327 and EPTB, n = 3306) were collected during the study period (July 2018-March 2020) in a tertiary care hospital. The study specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine phenol (AP) staining followed by Lowenstein-Jensen (LJ) and mycobacteria growth indicator tube (MGIT) culture. The MPT64 immunochromatographic test (ICT) was performed among mycobacterial cultures and ICT negative isolates were subjected to Line Probe Assay (LPA). In addition, 53 (PTB, 48 and EPTB, 5) NTM MGIT positive cultures were collected from Intermediate Reference Laboratory (IRL), Puducherry and subjected to LPA for speciation. RESULTS: Of the 7633 TB suspects, 0.6% were diagnosed as NTM diseases and 5.5% with Mycobacterium tuberculosis (MTBC). NTM infection was observed among 0.7% (31/4327) of PTB and 0.4% (14/3306) of EPTB. MTBC was detected among 6.1% (264/4327) of PTB and 4.6% (153/3306) of EPTB. Among 98 NTM cultures, 80.6% of isolates were recovered from PTB and 19.4% from EPTB specimens. Among pulmonary specimens, Mycobacterium intracellulare (26.6%), Mycobacterium abscessus (17.7%) and Mycobacterium kansasii (12.7%) were the most frequently detected species, while Mycobacterium intracellulare (21.1%), Mycobacterium scrofulaceum (15.8%) and Mycobacterium fortuitum (10.5%) were common in extrapulmonary specimens. CONCLUSION: The frequency of NTM infection among TB suspects was low at a South Indian tertiary care hospital. The most predominant NTM species isolated from both pulmonary and extrapulmonary specimens was M. intracellulare.

Application of acid-fast staining combined with GeneXpert MTB/RIF in the diagnosis of non-tuberculous mycobacteria pulmonary disease.

OBJECTIVE: To evaluate the clinical diagnostic value of positive acid-fast staining combined with negative GeneXpert MTB/RIF in the diagnosis of non-tuberculous mycobacteria pulmonary disease (NTM-PD). METHODS: A total of 133 inpatients with confirmed NTM-PD were included consecutively between January 1, 2018 and December 31, 2019, at Tongji Hospital and Jinyintan Hospital, Tongji Medical College of Huazhong University of Science and Technology, in Wuhan, China. One hundred patients with confirmed pulmonary tuberculosis (PTB) were randomly included as the control group. RESULTS: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of positive acid-fast staining combined with a negative GeneXpert MTB/RIF result were 51.13% (95% confidence interval (CI) 42.52-59.73%), 97.00% (95% CI 93.60-100.40%), 95.78% (95% CI 90.98-100.57%), and 59.88% (95% CI 52.25-67.51%), respectively. When subjects were limited to patients with positive acid-fast staining, the sensitivity of a negative GeneXpert MTB/RIF result was 88.31% (95% CI 80.97-95.65%). When acid-fast staining was conducted ≥3 times, the sensitivity of this combination diagnosis method increased to 61.67% (95% CI 49.00-74.33%). CONCLUSIONS: Positive acid-fast staining combined with a negative GeneXpert MTB/RIF result could be an effective and time-saving method for the diagnosis of NTM-PD.

Mycobacterial infections in wild boars (Sus scrofa) from Southern Switzerland: Diagnostic improvements, epidemiological situation and zoonotic potential.

The occurrence of mycobacterial infections in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. In addition to the well-known pathogenic members of the Mycobacterium tuberculosis - complex (MTBC), over 180 non-tuberculous mycobacteria (NTM) species have been described. Although the large majority of the NTM is assumed to be non-pathogenic to most individuals, an increasing trend in NTM infections has been observed over the last decades. The reasons of such augmentation are probably more than one: improved laboratory diagnostics, an increasing number of immunocompromised patients and individuals with lung damage are some of the possible aspects. Mandibular lymph nodes of 176 hunted wild boars from the pre-Alpine region of Canton Ticino, Switzerland, were collected. Following gross inspection, each lymph node was subjected to culture and to an IS6110 based real-time PCR specific for MTBC members. Histology was performed of a selection of lymph nodes (n = 14) presenting gross visible lesions. Moreover, accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) species identification was compared with sequence analysis of a combination of housekeeping genes. Mycobacteria of the MTBC were detected in 2.8% of the wild boars (n = 5; CI95% 1.2-6.5) and were all confirmed to be Mycobacterium microti by molecular methods. In addition, based on the examined lymph nodes, NTM were detected in 57.4% (n = 101; CI95% 50.0-64.5) of the wild boars originating from the study area. The 111 isolates belonged to 24 known species and three potentially undescribed Mycobacterium species. M. avium subsp. hominissuis thereby predominated (22.5%) and was found in lymph nodes with and without macroscopic changes. Overall, the present findings show that, with the exception of undescribed Mycobacterium species where identification was not possible (3.6%; 4/111), MALDI-TOF MS had a high concordance rate (90.1%; 100/111 isolates) to the sequence-based reference method.

Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin.

Nontuberculous mycobacteria (NTM) compose a group of mycobacteria that do not belong to the Mycobacterium tuberculosis complex group. They are frequently isolated from environmental samples such as water, soil, and, to a lesser extent, food samples. Isolates of NTM represent a major health threat to humans worldwide, especially those who have asthma or are immunocompromised. Human disease is acquired from environmental exposures and through consumption of NTM-contaminated food. The most common clinical manifestation of NTM disease in human is lung disease, but lymphatic, skin and soft tissue, and disseminated disease are also important. The main objective of the current study was to profile the farm-level contamination of cow milk with NTM by examining milk filters and bulk tank milk samples. Five different NTM species were isolated in one dairy herd in Wisconsin, with confirmed 16S rRNA genotypes including Mycobacterium fortuitum, Mycobacterium avium ssp. hominissuis, Mycobacterium abscessus, Mycobacterium simiae, and Mycobacterium avium ssp. paratuberculosis (Mycobacterium paratuberculosis). In tank milk samples, M. fortuitum was the predominant species in 48% of the samples, whereas M. chelonae/abscessus and M. fortuitum were the only 2 species obtained from 77 and 23% of the examined filters, respectively. Surprisingly, M. avium ssp. hominissuis, M. paratuberculosis, and M. simiae were isolated from 16.7, 10.4, and 4% of the examined milk samples, respectively, but not from milk filters. Interestingly, NTM isolates from human clinical cases in Wisconsin clustered very closely with those from milk samples. These findings suggest that the problem of NTM contamination is underestimated in dairy herds and could contribute to human infections with NTM. Overall, the study validates the use of bulk tank samples rather than milk filters to assess contamination of milk with NTM. Nontuberculous mycobacteria represent one type of pathogens that extensively contaminate raw milk at the farm level. The significance of our research is in evaluating the existence of NTM at the farm level and identifying a simple approach to examine the potential milk contamination with NTM members using tank milk or milk filters from dairy operations. In addition, we attempted to examine the potential link between NTM isolates found in the farm to those circulating in humans in Wisconsin.