Nontuberculous Mycobacteria Strains Research Articles

Isolation of Staphylococcus aureus, Uropathogenic Escherichia coli, and Nontuberculous Mycobacteria Strains from Pasteurized Cheeses and Unpasteurized Cream Sold at Traditional Open Markets in Mexico City

Fresh cheeses and cream are important garnishes of traditional Mexican food, often purchased at street or itinerant open markets or tianguis. However, there is scarce information regarding the microbiological quality of cheeses and cream sold in tianguis. For 2 years, three dairy stalls from three tianguis in Mexico City were visited once each season, trading practices were registered, and 96 dairy products were purchased. In total 72 fresh pasteurized cheeses that were hand-cut to order (24 Panela, 24 Canasto, and 24 Doble Crema) and 24 unpasteurized Crema de Rancho samples were collected. All dairy products remained without refrigeration for 8 h. Based on the National Guidelines limits, 87.5% of cheeses and 8% of Crema de Rancho samples were of low microbiological quality, and 1 sample of each type of cheese and 3 samples of Crema de Rancho exceeded the guidelines limits for Staphylococcus aureus. All dairy products were negative for Salmonella, Listeria monocytogenes, and all diarrheagenic Escherichia coli pathotypes, including Shiga toxin-producing E. coli. Among the 96 dairy samples, the prevalence of uropathogenic E. coli (UPEC) and of mycobacteria strains were determined because food items contaminated with these strains have been associated with urinary tract infections and mycobacteriosis, respectively. UPEC strains were isolated from 43% of cut-to-order cheeses and 29% of Crema de Rancho samples. Nontuberculous mycobacteria (NTM) strains were identified in 12.5% of Doble Crema cheese samples and 21% of Crema de Rancho samples. From the eight NTM-positive samples, 10 strains were identified (3 strains of Mycolicibacterium fortuitum, 2 of Mycobacteroides abscessus, 2 of Mycobacteroides chelonae, 2 of Mycolicibacterium porcinum, and 1 of Mycolicibacterium rhodesiae). All produced biofilms, and 70% had sliding motility (both virulence traits). Trading practices of cut-to-order pasteurized cheeses and unpasteurized Crema de Rancho in tianguis increase the risk of microbiological contamination of these products, including with human pathogens, and their consumption may cause human illness.

16S-ITS-23S rRNA operon segment sequencing provides necessary and sufficient conditions for bacterial species-specific identification

Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2nd generation sequencing)-based technology does not allow analysis beyond the 16S rRNA gene. The taxonomic verification level of samples usually remains at the genus or even family level. Currently, there have been proposed the latest versions of long-read technologies (Oxford Nanopore MinION, PacBio) for amplicon sequencing of near-complete ribosomal operon, including genes 16S, 23S, 5S, and internal transcribed spacer (ITS). At the moment, this approach has not been sufficiently studied, in addition, it involves PCR amplification of a very extended DNA region (more than 4000 bp-long). Materials and methods. The collection of non-tuberculous mycobacteria strains and their primary identification was carried out in the years 20192021. The strains were obtained by inoculation of positive cultures from the Bactec MGIT 960 bacteriological analyzer lacking MPT64 antigen in the MGIT TB Identification Test (Becton Dickinson, USA) on Lowenstein-Jensen medium. Preliminary species strain identification was carried out with the Speed-oligo Mycobacteria kit (Vircell, Spain) according to the manufacturers protocol. In this work, both known and newly developed universal bacterial primers flanking the near-complete 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene are used. In the present study, both known and newly developed universal bacterial primers are used to flank the near-complete 16S rRNA gene, ITS, and start of the 23S rRNA gene. Results and discussion. Sanger sequencing of the amplicons (about 2000 bp) obtained shows the taxonomic level sufficient to determine species up to 8 strains of non-tuberculous mycobacteria isolated from humans that caused clinically and bacteriologically confirmed diseases. The method proposed for PCR amplification of a bacterial operon a fragment containing most of the 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene is considered by us as an approbation of a methodological approach to study microbial communities in material with a high degree of degradation (necrotic foci, etc.). The results obtained indicate a significantly higher resolution of the approach used than the classical 16S rRNA gene sequencing.

Clinical Characteristics of Nontuberculous Mycobacterium Infection Cases in Sichuan, China in 2016-2021: A Retrospective Study

To study the distribution of nontuberculous mycobacterium (NTM) strains, clinical characteristics and drug sensitivity data of NTM infections so as to provide support for the prevention and treatment of diseases caused by NTM infection in Sichuan. The clinical data of NTM infection cases treated at the Public Health Clinical Center of Chengdu between July 2016 and July 2021 were collected and the characteristics of the infections were retrospectively reviewed. There were differences in sex, age and underlying diseases among the NTM infection cases in Sichuan. Specifically, young and middle-aged men aged between 20 and 40 were susceptible to AIDS, older men aged over 60 were susceptible to lung diseases, and middle-aged and older women over 40 were susceptible to bronchiectasis. Respiratory tract was the main route of NTM infection. The dominant strain in Sichuan was M. chelonae/ abscessus. The drug resistance rate of M. avium and M. chelonae/ abscessus were relatively higher. For NTM infection patients with different demographic characteristics and underlying diseases, the NTM infection sites, strains, and drug resistance are also different. Definite etiological diagnosis is essential to the treatment of NTM infection. We should highlight the importance of adopting individualized treatment for different NTM infections.

Risk Factors and Mental Health Status in Patients With Non-Tuberculous Mycobacterial Lung Disease: A Single Center Retrospective Study.

According to the existing data, the incidence and prevalence of non-tuberculous mycobacteria (NTM) are increasing worldwide. The risk factors and mental health status of patients with NTM lung disease are important and deserve our attention. A total of 180 patients with NTM lung disease hospitalized from January 2018 to December 2021 were selected as the NTM group, and 90 patients with non-severe community-acquired pneumonia (CAP) who were hospitalized during the same period were selected as the control group. The Symptom Checklist-90 (SCL-90) was used to assess the mental health status of the patients. The data were analyzed using descriptive statistics, logistic regression, and receiver operating characteristic (ROC) curves. There were no significant differences in age, sex, marital status, or smoking history between the two groups (p > 0.05), but there were significant differences in educational level, underlying diseases, occupation, living environment, and body mass index (BMI) (p < 0.01). According to the classification of basic diseases, bronchiectasis was found in 82 (45.6%) patients, followed by hypertension in 66 (36.7%) patients, and chronic obstructive pulmonary disease (COPD) in 39 (21.7%) patients. The NTM strains were identified M. intercelleulare caused 41 cases (22.8%), followed by Mycobacterium avium and Mycobacterium gordonae, each with 35 cases (19.4%), and Mycobacterium abscessus with 32 cases (17.8%). The SCL-90 found that 160 (88.9%) of 180 patients with NTM lung disease had developed mental health problems, among which the four highest-scoring factors were anxiety (ANX: 29.4%), depression (DEPR: 18.8%), sleep and diet (SD: 16.9%), and somatization (SOM: 11.3%). Through multivariate logistic regression analysis, it was found that educational level, underlying diseases, living environment, and BMI were independent risk factors for the occurrence of NTM lung disease (p < 0.01). The Hosmer–Lemeshow test was used to check the model's fitness. The ROC curve showed that the area under the curve (AUC) was 0.896, the sensitivity was 83.3%, and the specificity was 85.6%. Patients with NTM lung disease have many risk factors and prominent mental health problems that may require interventions during the process of clinical diagnosis and treatment.

Trends and Species Diversity of Non-tuberculous Mycobacteria Isolated From Respiratory Samples in Northern China, 2014-2021.

BackgroundPulmonary non-tuberculous mycobacteria (NTM) infection has become a public health concern in China and around the world. The objective of this study was to describe the longitudinal changes in the frequency and diversity of NTM in northern China.MethodsWe retrospectively analyzed data on mycobacterium species in Beijing Chest Hospital from January 2014 to December 2021. The isolates were identified to species level by targeted DNA sequencing.ResultsAfter excluding duplicates, 1,755 NTM strains were analyzed, which were from 27 provinces in China over 8 years. Among all mycobacteria, the proportion of NTM increased each year, from 4.24% in 2014 to 12.68% in 2021. Overall, 39 different NTM species were identified, including 23 slow growing mycobacteria (SGM) and 16 rapid growing mycobacteria (RGM). The most common species were M. intracellulare (51.62%), M. abscessus (22.22%), M. kansasii (8.32%), M. avium (7.75%) and M. fortuitum (2.05%). The number of NTM species identified also increased each year from 9 in 2014 to 26 in 2021. Most species showed stable isolation rates over the years; however, the proportion of M. avium increased from 3.85 to 10.42% during the study period. Besides, 81 non-mycobacteria strains, including Gordonia (21 isolates), Nocardia (19 isolates) and Tsukamurella (17 isolates), etc., were also discovered.ConclusionThe proportion of NTM and species diversity increased considerably in northern China from 2014 to 2021. M. intracellulare was the most common NTM isolated among respiratory specimens, followed by M. abscessus and M. kansasii. Rare NTM species and non-mycobacteria pathogens also need attention.

Effect of alkaloid berberine on the susceptibility of nontuberculous mycobacteria to antibiotics.

Nontuberculous mycobacteria (NTM) have recently emerged as important bacterial pathogens of both animals and humans. In this study, we aimed to evaluate the effect of a combination of ten antibiotics with an inhibitor of efflux pumps (EPI), i.e. berberine (BER), against 6 strains of NTM. Our results showed that the BER potentiated the anti-mycobacterial activities of the antibiotics. Overall, our findings show the importance of BER in increasing the efficacy of antibiotics in NTM.

In vitro activity of fidaxomicin against nontuberculosis mycobacteria.

Introduction. Nontuberculous mycobacteria (NTM) infections are increasing worldwide and are relatively resistant to many of the first- and second-line drugs to treat tuberculosis. Macrolide antibiotics, such as clarithromycin and azithromycin, are the key drugs for treating NTM infections. Fidaxomicin is a macrolide antibiotic that is widely used in treating Clostridium difficle (C.difficile) infections, and has high in vitro activity against Mycobacterium tuberculosis especially multidrug-resistant tuberculosis (MDR-TB) and has no cross-resistance with rifampicin.Hypothesis. Fidaxomicin may have in vitro activity against NTM strains.Aim. To find that whether the macrolide antibiotic fidaxomicin has in vitro activity against NTM strains.Methodology. Fidaxomicin used in this study was firstly tested on C. difficile reference strains and has shown to be effective and workable. And then 28 rapidly growing mycobacteria (RGM), 12 slowly growing mycobacteria (SGM) reference strains and 103 NTM clinical isolates were tested by the microplate-based AlamarBlue assay (MABA) method to determine the MICs. Fidaxomicin, rifampicin and clarithromycin were tested against M. abcessus complex subspecies 14 M. abscessus and 5 M. massiliense strains for inducible resistance determination.Results. In total, 21 out of 28 RGM and 9 of 12 SGM reference strains have the MICs of fidaxomicin at or below 1 µg ml-1. Fidaxomicin also showed low MIC values for some clinical isolates including M. abscessus complex, M. avium complex, M. fortuitum, M. kansasii and M. parascrofulaceum. Fidaxomicin also has no inducible macrolide resistance in M. abscessus complex in comparison with clarithromycin.Conclusion. Fidaxomicin has high in vitro activity against most of the NTM reference strains and some prevalent NTM clinical isolates. This promising finding warrants further investigation on the actions of fidaxomicn in vivo and as a potential antibiotic for NTM treatment.

Quinoxaline-based efflux pump inhibitors restore drug susceptibility in drug-resistant nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) comprise several ubiquitous, environmentally localized bacteria that may be responsible for serious human diseases. NTM-associated pulmonary infections largely affect individuals with underlying respiratory disease or chronic disease and immunosuppressed patients. Mycobacterium simiae and M. abscessus are two NTMs responsible for lung disease in immunocompetent and immunocompromised individuals. In this study, two NTM strains were isolated from two patients admitted to an Italian hospital and were identified as M. simiae and M. abscessus. The two NTMs were tested for drug susceptibility against different antibiotics. To restore drug susceptibility, a new series of 2-aryl-3-phenoxymethyl-quinoxaline derivatives (QXs) was designed, synthesized, and investigated as efflux pump inhibitors (EPIs) against two clinical isolates of the above-cited NTMs, evaluating how EPIs can influence the drug minimal inhibitory concentration values and, therefore, the activity. The different\ resistance levels tracked in the clinical strains were reduced by EPIs, and in several cases, the susceptibility was completely restored. QXs also resulted as potential chemical probes to be used in drug susceptibility tests to identify the resistance origin when detected.

Evaluation of antimicrobial susceptibilities of non-tuberculous mycobacteria against linezolid and tigecycline

Mycobacterial susceptibility testing is important for the management of nontuberculous mycobacteria (NTM) infections. The aim of the study is to determine the susceptibilities of tigecycline (TGC) and linezolid (LZD) against NTM. The study was carried out using stocks of NTM strains in the tuberculosis department of the microbiology laboratory. It was designed a retrospective study. LZD and TGC sensitivities of study isolates were analyzed by microdilution. Forty NTM isolates have been studied. LZD and TGC sensitivities varied according to the NTM type. It is concluded that each isolate should be individually evaluated due to variable susceptibilities to LZD and TGC.

Zn(II) complexes with a new isoniazid ligand: synthesis, structural characterization and antimycobacterial activity

The present work reports the synthesis of two new zinc(II) complexes based on the 2-acetylfuranisonicotinichydrazone ligand (HL). The structure of the hydrazone and [ZnCl2(HL)]·MeOH (1) and [Zn(L)2] (2) have been elucidated by single-crystal X-ray diffraction method, physicochemical, and spectroscopic techniques. The hydrazone is a bidentate ligand, forming two new mononuclear complexes with zinc(II). Non-covalent interactions in the structures of the compounds were evaluated with Hirshfeld surface analysis, which showed the presence of hydrogen bonds in HL and 1 and also a π···π stacking interaction in 1. The antimycobacterial potentials of the compounds were tested against tuberculous and non-tuberculous mycobacteria strains.

Distribution of nontuberculous mycobacteria in patients with and without HIV/AIDS in Chongqing.

In this study, the distribution of nontuberculous mycobacteria (NTM) strains in patients with and without HIV/AIDS in Chongqing, China was evaluated. A retrospective study was performed in January-December 2020 at Chongqing Public Health Medical Center. NTM strains were assessed by a multi locus phylogenetic analysis. The distribution of NTM strains in HIV/AIDS and non-HIV/AIDS groups was compared. CD4+ cell counts, imaging changes, and characteristics of mycobacterial species were determined. In total, 324 patients with NTM infection (50 patients with HIV/AIDS and 274 patients without HIV/AIDS) were included. The most common etiological agent was M.abscessus (29%), followed by M.paraintracellulare (12%) andM.colombiense (11%). Predominant NTM species were M.avium (26%), M.colombiense (24%), and M.kansasii (18%) in patients with HIV/AIDS and wereM.abscessus (32%), M.paraintracellulare (13%), M.fortuitum (10%), and M. intracellulare (10%) in patients without HIV/AIDS. For a CD4+ cell count of <200/μl, the predominant species were M.aviumin the HIV/AIDS group and M.abscessus in the non-HIV/AIDS group. With respect to radiologic characteristics, different NTM strains were associated with distinct imaging manifestations; for example, M.marseillense, M.kansasii, and M.parasenchytosis were more likely to induce cavities. Imaging cavities, bronchiectasis, and acinar-like changes were more common in the non-HIV/AIDS groups. The infection rates of HIV and NTM in Chongqing are high, while M.abscessus, M.paraintracellulare, and M.colombiense are the main pathogens causing NTM diseases in Chongqing, and NTM strains differed significantly between patients with and without HIV/AIDS. Monitoring these indicators can help develop prevention strategies.

Chemical susceptibility testing of non-tuberculous mycobacterium strains and other aquatic bacteria: Results of a study for the development of a more sensitive and simple method for the detection of NTM in environmental samples

The methods employed to detect non-tuberculous mycobacteria on environmental samples are essentially those classically used in clinical microbiology, which envisage a decontamination step to reduce the overgrowth of non-mycobacterial organisms before plating them on the culture medium. The aim of this study was to propose alternative culture techniques to improve non-tuberculous mycobacteria detection in environmental samples. We used artificially contaminated samples to compare the membrane filter washing procedure against direct plating of membrane filters on culture media in relation to M.chimaera and M.chelonae recovery efficiency. Moreover, we compared the efficacy of NTM Elite agar in inhibiting the growth of aquatic bacteria with that of cetylpyridinium chloride and N-acetyl-L-cysteine sodium hydroxide decontamination treatments. The washing procedure yielded a low release of both mycobacterium strains (6.6% for Mycobacterium chimaera and 7.5% for Mycobacterium chelonae) from the membrane filters; on the contrary, direct plating of membrane filters led to a 100% cell recovery. Water sample pretreatment with N-acetyl-L-cysteine sodium hydroxide (1%), despite achieving complete suppression of non-acid fast bacilli, caused a reduction in mycobacteria growth. Decontamination with cetylpyridinium chloride (0.005%) was found to be ineffective against Methylobacterium spp. and Burkholderia multivorans. NTM Elite agar was ineffective against B. multivorans, but it inhibited the growth of all other aquatic bacteria.Our results indicate that NTM Elite agar provides a valid alternative method of recovering non-tuberculous mycobacteria from environmental samples. It does not involve a decontamination step and provides greater recovery efficiency by skipping the washing step and directly plating the filters on the media.

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.

Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Isolates derived from clinical specimens were subcultured in the Lowenstein-Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.

Healthcare-associated links in transmission of nontuberculous mycobacteria among people with cystic fibrosis (HALT NTM) study: Rationale and study design.

BackgroundHealthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been reported and is of increasing concern. No standardized epidemiologic investigation tool has been published for healthcare-associated NTM outbreak investigations. This report describes the design of an ongoing observational study to standardize the approach to NTM outbreak investigation among pwCF.MethodsThis is a parallel multi-site study of pwCF within a single Center who have respiratory NTM isolates identified as being highly-similar. Participants have a history of positive airway cultures for NTM, receive care within a single Center, and have been identified as part of a possible outbreak based on genomic analysis of NTM isolates. Participants are enrolled in the study over a 3-year period. Primary endpoints are identification of a shared healthcare-associated encounter(s) among patients in a Center and identification of environmental isolates that are genetically highly-similar to respiratory isolates recovered from pwCF. Secondary endpoints include characterization of potential transmission modes and settings, as well as incidence and prevalence of healthcare-associated environmental NTM species/subspecies by geographical region.DiscussionWe hypothesize that genetically highly-similar strains of NTM among pwCF cared for at the same Center may arise from healthcare sources including patient-to-patient transmission and/or acquisition from environmental sources. This novel study design will establish a standardized, evidence-based epidemiologic investigation tool for healthcare-associated NTM outbreak investigation within CF Care Centers, will broaden the scope of independent outbreak investigations and demonstrate the frequency and nature of healthcare-associated NTM transmission in CF Care Centers nationwide. Furthermore, it will provide valuable insights into modeling risk factors associated with healthcare-associated NTM transmission and better inform future infection prevention and control guidelines. This study will systematically characterize clinically-relevant NTM isolates of CF healthcare environmental dust and water biofilms and set the stage to describe the most common environmental sources within the healthcare setting harboring clinically-relevant NTM isolates.Trial registrationClinicalTrials.gov NCT04024423. Date of registry July 18, 2019.

Nontuberculous Mycobacteria under Scrutiny in the Geneva Area (2015–2020)

Background: Nontuberculous mycobacteria (NTM) are increasingly identified in industrialized countries, and their role as pathogens is more frequently recognized. The relative prevalence of NTM strains shows an important geographical variability. Thus, establishing the local relative prevalence of NTM strains is relevant and useful for clinicians. Methods: Retrospective analysis (2015–2020) of a comprehensive database was conducted including all results of cultures for mycobacteria in a University Hospital (Geneva, Switzerland), covering a population of approximately 500,000 inhabitants. All NTM culture-positive patients were included in the analyses. Patients’ characteristics, NTM strains, and time to culture positivity were reported. Results: Among 38,065 samples analyzed during the study period, 411 were culture-positive for NTM, representing 236 strains, and 231 episodes of care which occurred in 222 patients. Patients in whom NTM were identified were predominantly female (55%), with a median age of 62 years, and a low BMI (median: 22.6 kg/m²). The Mycobacterium avium complex (MAC) was the most frequently identified group (37% of strains) followed by Mycobacterium gordonae (25%) and Mycobacterium xenopi (12%) among the slowly growing mycobacteria (SGM), while the Mycobacterium chelonae/abscessus group (11%) were the most frequently identified rapidly growing mycobacteria (RGM). Only 19% of all patients were treated, mostly for pulmonary infections: the MAC was the most frequently treated NTM (n = 19, 43% of cases in patients treated) followed by RGM (n = 15, 34%) and M. xenopi (n = 6, 14%). Among those treated, 23% were immunosuppressed, 12% had pulmonary comorbidities, and 5% systemic comorbidities. Cultures became positive after a median of 41 days (IQR: 23; 68) for SGM and 28 days (14; 35) for RGM. Conclusions: In Western Switzerland, M. avium and M. gordonae were the most prevalent NTM identified. Positive cultures for NTM led to a specific treatment in 19% of subjects. Patients with a positive culture for NTM were mostly female, with a median age of 62 years, a low BMI, and a low prevalence of immunosuppression or associated severe comorbidities.

The key factors contributing to the risk, diagnosis and treatment of non-tuberculous mycobacterial opportunistic infections

Abstract The incidence and prevalence of diseases caused by non-tuberculous mycobacteria (NTM) have been steadily increasing worldwide. NTM are environmental saprophytic organisms; however, a few strains are known to produce diseases in humans affecting pulmonary and extra-pulmonary sites. Although the environment is a major source of NTM infection, recent studies have shown that person-to-person dissemination could be an important transmission route for these microorganisms. Structural and functional lung defects and immunodeficiency are major risk factors for acquiring NTM infections. Diagnosis of NTM diseases is very complex owing to the necessity of distinguishing between a true pathogen and an environmental contaminant. Identification at the species level is critical due to differences in the antibiotic susceptibility patterns of various NTM strains. Such identification is mainly achieved by molecular methods; additionally, mass spectrometry (e.g., MALDI-TOF) is useful for NTM species determination. Natural resistance of NTM species to a wide spectrum of antibiotics makes prescribing treatment for NTM diseases very difficult. NTM therapy usually takes more than one year and requires multi-drug regimens, yet the outcome often remains poor. Therefore, alternatives to antibiotic therapy treatment methods is an area under active exploration. NTM infections are an active global health problem imposing the necessity for better diagnostic tools and more effective treatment methods.

Prevalence, Strains, and Drug Susceptibility of Nontuberculous Mycobacteria Isolates from Tuberculous Suspects

Background: The prevalence of nontuberculous mycobacteria (NTM) infection has been increasing globally. Many cases of NTM infection are misdiagnosed as Mycobacterium tuberculous (MTB) because of similar clinicoradiological features. Objectives: To determine the burden and characteristics of NTM infection, this study was done to evaluate clinical isolates collected from tuberculous (TB) suspects in a population from Northwest China. Methods: From January to December 2020, the clinical samples of 9,142 TB suspects were collected for the PCR-fluorescent probe and mycobacterial culture. The PCR-fluorescent probe-positive nucleic acid samples were further subjected to a DNA microarray for confirmation and species identification. Drug susceptibility testing (DST) was also carried out using the micropore plate method (MicroDSTTM) on isolates from NTM patients. Results: Of 9,412 TB suspects, 85 cases (0.9%) were clinically diagnosed with NTM infection according to the American Thoracic Society (ATS) guidelines. For the laboratory samples, a total of 169 NTM strains, identified by molecular biology methods, were classified into 10 species. The most common species were M. chelonae/ M. abscessus (64/169, 37.7%) and M. intracellulare (40/169, 23.7%). All strains showed the highest resistance to imipenem/cilastatin (85/85, 100%) and the highest susceptibility to linezolid (4/85, 4.7%). In comparison with the rapidly growing mycobacteria (RGM) group, the slowly growing mycobacteria (SGM) group showed a lower resistance and a shorter hospital inpatient stay (t = 6.66, P &lt; 0.001 and t = 2.40, P = 0.020, respectively). Conclusions: Mycobacterium chelonae/M. abscessus and M. intracellulare were the most frequently detected NTM pathogens in Northwest China. The differences in drug sensitivity and clinical characteristics were giant for different strains. Timely identification and accurate DST play important roles in NTM management.

The value of gene chip detection of bronchoalveolar lavage fluid in the diagnosis of nontuberculous mycobacterial lung disease.

Nontuberculous mycobacteria (NTM) are mycobacteria other than mycobacterium tuberculosis complex (MTBC) and mycobacterium leprae. NTM can cause infection in many human tissues and organs and is most commonly seen in the lungs. Clinically, the symptoms and signs of nontuberculous mycobacteria lung disease (NMLD) are very similar to those of tuberculosis (TB). Because most NTMs are resistant to conventional anti-TB drugs, the rapid diagnosis of NMLD is the key to treatment. This study aimed to use gene chip technology to examine bronchoalveolar lavage fluid (BALF) from NMLD patients to explore the value of this technique for the rapid diagnosis of NMLD in BALF. A retrospective analysis of 308 patients with NMLD treated at Fuzhou Pulmonary Hospital from January 2018 to June 2020 was performed. BALF was collected from the patients. Gene chip detection (Capital Bio Corporation, Chengdu, China) and BACTEC MGIT960 (Becton, Dickinson and Company, MD, USA) liquid culture were performed to compare the NTM positive detection rates between the two methods. The NTM strain isolated from liquid culture were identified by rDNA sequencing and the results of identification were compared with those of gene chip detection using BALF specimens. A total of 221 cases of NTM were detected in 308 BALF specimens by the gene chip method; the positive rate was 71.75% (221/308). A total of 218 cases of NTM were detected by the liquid culture method, and the positive rate was 70.78% (218/308). There was no significant difference in the positive rate of NTM detected in BALF specimens between the two methods (χ2=0.138 P=0.804>0.05); 187 cases were detected with both sequencing and gene chip detection, and the coincidence rate of strain identification with the two methods reached 96.79% (181/187). Sequencing of 218 strains of NTM was carried out; eight species were identified, and the top four species were M. intracellulare (131/218, 60.09%), M. avium (48/218, 22.02%), M. abscessus (27/218, 12.38%), and M. kansasii (5/218, 2.29%). Gene chip technology can rapidly detect NTM in BALF and accurately identify bacterial species. It has important clinical value in the early diagnosis and treatment of NMLD.

Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex.

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.

Characterization of Clinical Isolates of Mycobacterium simiae Using Drug Susceptibility Tests and Molecular Analyses.

Mycobacterium simiae is an emerging nontuberculous mycobacterium (NTM) and an opportunistic pathogen which is described mainly in Asia and presents in the environment that can cause pulmonary infection. The objective of this study is to characterize M. simiae clinical isolates using mycobacterial interspersed repetitive unit variable-number tandem repeats (MIRU-VNTR) typing for the differentiation of the strains. A total of 169 clinical isolates of NTM were recovered from patients suspected of having tuberculosis (TB)-like and related infections. After isolation and identification of mycobacterial strains by conventional biochemical and PCR-based tests, M. simiae strains were confirmed using restriction fragment length polymorphism (RFLP)-based identification assay. Furthermore, drug susceptibility and MIRU-VNTR typing was performed using on the clinical isolates of M. simiae. Out of 169 NTM strains, 92 (54.4%) isolates were identified as M. simiae. Antibiotic susceptibility experiments indicated that all 92 M. simiae isolates were resistant to first line antimycobacterial agents. Moreover, 8 (8.6%) M. simiae isolates were resistant to ciprofloxacin; and 6 (6.5%) were resistant to both amikacin and kanamycin, while the remaining were susceptible to second line antimycobacterial agents. MIRU-VNTR analysis showed that the M. simiae isolates were classified in four distinct M. simiae clusters and two single types. The minimum spanning analysis revealed that the isolates were grouped in three complexes. The data suggested that MIRI-VNTR typing is useful for typing of M. simiae isolates, however, MIRU-16 locus was absolutely absent in M. simiae.