New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM).

Rapidly growing mycobacteria (RGM) cause an increasing international concern, mainly due to their natural resistance to many antibiotics. The aim of this study was to conduct species identification and determine the antimicrobial susceptibility profiles of RGM isolated in Poland. Antimicrobial susceptibility was tested using broth microdilution and the RAPMYCOI panel. A total of 60 strains were analysed, including the following species: M. fortuitum complex (30), M. abscessus subsp. abscessus (16), M. abscessus subsp. massiliense (7), M. chelonae (5), and M. mucogenicum (2). For 12 M. abscessus subsp. abscessus strains, the presence of the erm 41T28 genotype associated with inducible macrolide resistance and a functional erm gene was confirmed. A MUT2 mutation in the rrl gene (constitutive resistance) was identified for two strains from the subtype M. abscessus subsp. massiliense. Among the 15 tested antibiotics, amikacin and linezolid had the strongest antimycobacterial activity. Most of the tested strains were resistant to doxycycline and trimethoprim/sulfamethoxazole. Tigecycline MICs were low for all tested strains. Findings from our study highlight the importance of correct identification of clinical isolates and antimicrobial susceptibility testing.

Nontuberculous Mycobacterial Lung Disease in the Patients with Cystic Fibrosis-A Challenging Diagnostic Problem.

BACKGROUND: Cystic fibrosis (CF) is an autosomal, recessive genetic disorder, caused by a mutation in the cystic fibrosis transmembrane conductance receptor regulator (CFTR) gene. Dysregulated mucous production, and decreased bronchial mucociliary clearance, results in increased susceptibility to bacterial and fungal infections. Recently, nontuberculous mycobacteria (NTM) infections were identified as an emerging clinical problem in CF patients. AIM: The aim of the present study was to assess the frequency of NTM isolations in CF patients hospitalized in the pulmonary department, serving as a hospital CF center, and to describe challenges concerning the recognition of NTMLD (nontuberculous mycobacterial lung disease) in those patients. METHODS: Consecutive CF patients, who were hospitalized due to pulmonary exacerbations (PEX), in a single CF center, between 2010 and 2020, were retrospectively assessed for the presence of NTM in respiratory specimens. Clinical and radiological data were retrospectively reviewed. RESULTS: Positive respiratory specimen cultures for NTM were obtained in 11 out of 151 patients (7%), mean age-35.7 years, mean BMI-20.2 kg/m2, mean FEV1-58.6% pred. Cultures and phenotyping revealed the presence of Mycobacterium avium (M. avium)-in six patients, Mycobacterium chimaera (M. chimaera) in two, Mycobacterium kansasii (M. kansasii)-in one, Mycobacterium abscessus (M. abscessus)-in one, Mycobacterium lentifavum (M. lentiflavum)-in one. Simultaneously, respiratory cultures were positive for fungi in 91% of patients: Candida albicans (C. albicans)-in 82%, Aspergillus fumigatus (A. fumigatus)-in 45%. Clinical signs of NTMLD were non-specific, chest CT indicated NTMLD in five patients only. CONCLUSION: Due to non-specific clinical presentation, frequent sputum cultures for NTM and analysis of serial chest CT examinations are crucial for NTMLD recognition in CF patients. Further studies concerning the predictive role of fungal pathogens for NTMLD development in CF patients are needed.

Use of a FluoroType® System for the Rapid Detection of Patients with Multidrug-Resistant Tuberculosis-State of the Art Case Presentations.

According to the World Health Organization (WHO), there were 465,000 cases of tuberculosis caused by strains resistant to at least two first-line anti-tuberculosis drugs: rifampicin and isoniazid (MDR-TB). In light of the growing problem of drug resistance in Mycobacterium tuberculosis across laboratories worldwide, the rapid identification of drug-resistant strains of the Mycobacterium tuberculosis complex poses the greatest challenge. Progress in molecular biology and the development of nucleic acid amplification assays have paved the way for improvements to methods for the direct detection of Mycobacterium tuberculosis in specimens from patients. This paper presents two cases that illustrate the implementation of molecular tools in the recognition of drug-resistant tuberculosis.

From NTM (Nontuberculous mycobacterium) to Gordonia bronchialis-A Diagnostic Challenge in the COPD Patient.

In patients with chronic obstructive pulmonary disease, respiratory infections are of various aetiology, predominantly viral and bacterial. However, due to structural and immunological changes within the respiratory system, such patients are also prone to mycobacterial and other relatively rare infections. We present the 70-year old male patient with chronic obstructive pulmonary disease (COPD) and coexisting bronchial asthma, diagnosed due to cough with purulent sputum expectoration lasting over three months. The first microbiological investigation of the sputum sample revealed the growth of mycobacteria. The identification test based on protein MPT64 production indicated an organism belonging to NTM (nontuberculous mycobacterium). However, further species identification by genetic testing verified the obtained culture as not belonging to the Mycobacterium genus. Based on observed morphology, the new characterisation identified an aerobic actinomycete, possibly a Nocardia spp. The isolated strain was recultured on standard microbiological media. The growth of colonies was observed on Columbia blood agar plates and solid Löewenstein-Jensen medium. The Gram and Zhiel-Nielsen stains revealed the presence of Gram-positive acid-fast bacilli. The extraction protocol and identification were performed in two repetitions; the result was G. bronchialis, with a confidence value of 99% and 95%, respectively. The gene sequencing method was applied to confirm the species affiliation of this isolate. The resulting sequence was checked against the 16S ribosomal RNA sequences database (Bacteria and Archaea). The ten best results indicated the genus Gordonia (99.04-100%) and 100% similarity of the 16S sequenced region was demonstrated for Gordonia bronchialis. The case described indicates that the correct interpretation of microbiological test results requires the use of advanced microbiology diagnosis techniques, including molecular identification of gene sequences. From a clinical point of view, Gordonia bronchialis infection or colonization may present a mild course, with no febrile episodes and no significant patient status deterioration and thus, it may remain undiagnosed more often than expected.

Non-Tuberculous Mycobacteria in Respiratory Specimens of Patients with Obstructive Lung Diseases-Colonization or Disease?

Non-tuberculous mycobacteria (NTM) are increasingly a cause of human respiratory tract colonization and mycobacterial lung disease (NTM-LD), especially in patients with chronic lung diseases. The aim of the present study was to find the factors predictive of NTM-LD in patients with obstructive lung diseases and NTM respiratory isolates. A total of 839 isolates of NTM, obtained from 161 patients between 2010 and 2020 in a single pulmonary unit, have been retrospectively reviewed. Of these isolates, 73 concerned 36 patients with obstructive lung diseases (COPD-26, asthma-3, COPD/asthma overlap syndrome-7). NTM-LD was recognized according to the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) criteria in 17 patients, colonization in 19. Lower BMI, elevated body temperature on admission, infiltrative/cavitary lesions on chest CT, and NTM species other than Mycobacterium gordonae were the significant predictors of NTM-LD recognition. Based on the above-mentioned predictive factors, an original scoring system was implemented. The diagnostic utility of the scoring system was higher than that of single parameters. We conclude that NTM-LD prediction in patients with obstructive lung diseases and positive respiratory isolates is difficult. A scoring system based on clinical, radiological and microbiological characteristics was capable of facilitating the differential diagnosis, but it needs further validation in a larger study group.

Interferon Gamma Release Assays in Patients with Respiratory Isolates of Non-Tuberculous Mycobacteria - a Preliminary Study.

Interferon gamma releasing assays (IGRAs) are extensively used in the diagnosis of latent tuberculosis infections. Comparing to tuberculin skin test (TST) they lack false positive results in the populations vaccinated with BCG, and in most non-tuberculous mycobacteria (NTM) infections. Nevertheless, Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium szulgai may induce positive IGRAs due to RD1 homology with Mycobacterium tuberculosis. The aim of the study was to investigate the possible influence of NTM respiratory isolates on the results of IGRAs. 39 patients (23 females and 16 males) of median age 61 years, with negative medical history concerning tuberculosis, entered the study. Identification of NTM was performed using the niacin test and molecular method GenoType CM test (Hain Lifescience). QFT-Plus was performed in 17 patients, T-SPOT-Tb - in 23 patients. Chest X-rays and a high-resolution computed tomography of the chest have been reviewed by the experienced radiologist blinded to the results of IGRAs, in search of past tuberculosis signs. Positive IGRAs results were obtained in three out of 39 patients (8%): 22% of patients with M. kansasii isolates and 18% of patients with radiological signs on HRCT that might be suggestive of past tuberculosis. Positive IGRAs correlated with radiological signs suggestive of past tuberculosis (r = 0.32, p = 0.04), and on the borderline with isolation of M. kansasii (r = 0.29, p = 0.06). These findings may suggest that a positive IGRAs result, in our material, could depend mostly on asymptomatic past Tb infection. The cross-reactivity of M. kansasii isolates with IGRAs was less probable; nevertheless, it requires further investigations.Interferon gamma releasing assays (IGRAs) are extensively used in the diagnosis of latent tuberculosis infections. Comparing to tuberculin skin test (TST) they lack false positive results in the populations vaccinated with BCG, and in most non-tuberculous mycobacteria (NTM) infections. Nevertheless, Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium szulgai may induce positive IGRAs due to RD1 homology with Mycobacterium tuberculosis. The aim of the study was to investigate the possible influence of NTM respiratory isolates on the results of IGRAs. 39 patients (23 females and 16 males) of median age 61 years, with negative medical history concerning tuberculosis, entered the study. Identification of NTM was performed using the niacin test and molecular method GenoType CM test (Hain Lifescience). QFT-Plus was performed in 17 patients, T-SPOT-Tb ­ in 23 patients. Chest X-rays and a high-resolution computed tomography of the chest have been reviewed by the experienced radiologist blinded to the results of IGRAs, in search of past tuberculosis signs. Positive IGRAs results were obtained in three out of 39 patients (8%): 22% of patients with M. kansasii isolates and 18% of patients with radiological signs on HRCT that might be suggestive of past tuberculosis. Positive IGRAs correlated with radiological signs suggestive of past tuberculosis (r = 0.32, p = 0.04), and on the borderline with isolation of M. kansasii (r = 0.29, p = 0.06). These findings may suggest that a positive IGRAs result, in our material, could depend mostly on asymptomatic past Tb infection. The cross-reactivity of M. kansasii isolates with IGRAs was less probable; nevertheless, it requires further investigations.

Factors predisposing to non-tuberculous mycobacterial lung disease in the patients with respiratory isolates of non-tuberculous mycobacteria.

INTRODUCTION: An increasing incidence rate of respiratory isolates of non-tuberculous mycobacteria (NTM) has been noted recently in most European countries as well as in the US. Despite many publications, there is no consensus concerning the importance of different factors in promoting NTM lung disease (NTMLD). The aim of the present retrospective study was to analyse patients with positive NTM respiratory isolates in search of factors predisposing to NTMLD. MATERIAL AND METHODS: 73 patients, 23 males, 50 females, median age 62.2 years, in whom NTM have been cultured from respiratory specimen (sputum and/or bronchial washings), in the period 2010-2015, entered the study. RESULTS: NTMLD (according to ATS/IDSA) has been recognised in 36 patients, airways colonisation by NTM - in 37 patients. NTMLD was diagnosed more often in the patients infected with M. kansasii, M. abscessus and M. avium/M. intracellulare comparing to those infected with M.xenopi, M. gordonae and M. fortuitum (p < 0.0001). The proportion of females to males was significantly higher in the NTMLD group comparing to the colonisation group (p < 0.007). Previous tuberculosis or mycobacteriosis were noted significantly more frequently in the group of patients with NTMLD comparing to the colonisation group (28% vs 8%, p = 0.038). Univariate regression analysis revealed M. kansasii, female gender, and previous tuberculosis or mycobacteriosis as significant predictors of NTMLD. CONCLUSIONS: The risk factors of NTMLD recognition in the presented group of patients were the following: female gender, M. kansasii isolation, as well as past tuberculosis or mycobacteriosis.

Nontuberculous mycobacteria strains isolated from patients between 2013 and 2017 in Poland. Our data with respect to the global trends.

INTRODUCTION: During the last decades the prevalence of NTM infections has increased, especially in developed countries. The aim of the study was to provide an overview on all NTM isolated from clinical samples in Poland between 2013 and 2017. MATERIAL AND METHODS: The study comprised 2799 clinical specimens, mostly respiratory accessed in the reference laboratory of National Tuberculosis and Lung Diseases Research Institute in Warsaw and in the Wielkopolska Center of Pulmonology and Thoracic Surgery, Poland, 2013-2017. RESULTS: During the study period 35 species of NTM were isolated . The number of isolates increased almost 1.6-fold: from 420 in 2013 to 674 in 2017. M. kansasii, M. avium, M. xenopi, M. gordonae and M. intracellulare were the most common species. This NTM pattern was rather stable over the time. If the aggregated amount of all MAC species was taken into account they dominated over M. kansasii from 2015. M. avium and M. intracellulare were more often isolated from women, while M. kansasii, M. gordonae and M. xenopi predominated in men. Men and women were infected almost with the same frequency. In older patients 65+ women were in majority, quite opposite to those aged 25 to 64 years. CONCLUSION: In Poland, like in other countries increased the frequency of isolated NTM. M. kansasii and M. avium were the most frequently identified species from clinical samples. Men and women were infected with NTM with the same frequency.