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The nontuberculous mycobacteria are typically environmental organisms residing in soil and water. These microorganisms can cause a wide range of clinical diseases; pulmonary disease is most frequent, followed by lymphadenitis in children, skin and soft tissue disease, and rare extra pulmonary or disseminated infections. Mycobacterium avium complex is the second most common cause of pulmonary mycobacterioses after M. tuberculosis. This review covers the clinical and laboratory diagnosis of infection caused by the members of this complex and particularities for the treatment of different disease types and patient populations.

ABSTRACT:The presence of mycobacteria was studied in Bull Rock Cave ("Byci skala") and the water catchment area of Jedovnice Brook ("Jedovnicky potok") using direct microscopy after Ziehl-Neelsen (ZN) staining, culture examination and molecular techniques. Mycobacteria were detected in 47.1% of a total of 68 samples. The mycobacterial genes hsp65 and dnaA were detected and sequenced in 37 (74.0%) out of the 50 cave environmental samples and in 10 (55.6%) out of the 18 samples of water catchment sediments. Nine species of slowly growing mycobacteria (M. terrae, M. arupense, M. gordonae, M. lentiflavum, M. parascrofulaceum, M. parmense, M. saskatchewanense, M. simiae and M. xenopi) and two subsp. (M. avium subsp. avium and M. avium subsp. hominissuis) were detected. Fourteen species of rapidly growing mycobacteria (M. chelonae, M. chubuense, M. poriferae, M. flavescens, M. fortuitum, M. porcinum, M. rhodesiae, M. gilvum, M. goodii, M. peregrinum, M. mageritense, M. vanbaalenii, M. gadium and M. insubricum) were detected. The highest mycobacterial presence was documented by ZN staining and/or culture examinations in earthworm castings and bat guano (73.3% positivity out of the 15 samples) in the cave environment and in the water sediments collected under the outflow from the wastewater treatment plants (77.8% positivity out of nine samples). The highest total organic carbon (TOC) was detected in wooden material and earthworm castings with pH values between 5.0 and 7.7 in the cave environment and in water sediments collected under the outflow from the wastewater treatment plants with pH between 5.8 and 7.0. It could be concluded that the karst cave environment with its running surface water contaminated with different microorganisms or chemical substances creates favourable conditions not only for animals (especially earthworms) but also for mycobacteria. This fact is also demonstrated by the presence of these mycobacteria in the cave environment mainly in earthworm castings and bat guano.

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9%% in Cluster R and 12.6%% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum, and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

Environmental mycobacteria (EM) constitute a health risk, particularly for immunocompromised people. Workers in heavy industry and in collieries represent an at-risk group of people as their immunity is often weakened by long-term employment in dusty environments, frequent smoking and an increased occurrence of pulmonary diseases. This study was concerned with the presence of EM in non-drinking water used for the hygiene of employees in six large industrial companies and collieries. Over a period of ten years, 1096 samples of surface water treated for hygiene purposes (treated surface water) and treated surface water diluted with mining water were examined. EM were detected in 63.4 and 41.5% samples of treated surface water and treated surface water diluted with mining water, respectively. Mycobacterium gordonae, M. avium-intracellulare and M. kansasii were the most frequently detected species. Adoption of suitable precautions should be enforced to reduce the incidence of mycobacteria in shower water and to decrease the infectious pressure on employees belonging to an at-risk group of people.

Mycobacterium kansasii is an emerging non-tuberculous mycobacterial (NTM) pathogen capable of causing severe lung disease. Of the seven currently recognized M. kansasii genotypes (I-VII), genotypes I and II are most prevalent and have been associated with human disease, whereas the other five (III-VII) genotypes are predominantly of environmental origin and are believed to be non-pathogenic. Subtyping of M. kansasii serves as a valuable tool to guide clinicians in pursuing diagnosis and to initiate the proper timely treatment. Most of the previous rapid diagnostic tests for mycobacteria employing the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology focused on species-level identification. The purpose of this study was to establish MALDI-TOF MS reference spectra database for discrimination of M. kansasii at the genotype level. A panel of 32 strains, representatives of M. kansasii genotypes I-VI were selected, whole cell proteins extracted and measured with MALDI-TOF MS. A unique main spectra (MSP) library was created using MALDI Biotyper Compass Explorer software. The spectra reproducibility was assessed by computing composite correlation index and MSPs cross-matching. One hundred clinical M. kansasii isolates used for testing of the database resulted in 90% identification at genus-level, 7% identification at species-level and 2% identification was below the threshold of log score value 1.7, of which all were correct at genotype level. One strain could not be identified. On the other hand, 37% of strains were identified at species level, 40% at genus level and 23% was not identified with the manufacturer's database. The MALDI-TOF MS was proven a rapid and robust tool to detect and differentiate between M. kansasii genotypes. It is concluded that MALDI-TOF MS has a potential to be incorporated into the routine diagnostic workflow of M. kansasii and possibly other NTM species.

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental bacteria that can induce pulmonary and non-pulmonary diseases in susceptible persons. It is reported that the prevalence of NTM diseases is increasing in developed countries, but this differs by regions and countries. NTM species distribution and the rate of diseases caused by NTM vary widely in the historical territories of Moravia and Silesia (Czech Republic). This epidemiologic study of NTM diseases covers the period 2012–2018, reviews isolates obtained from patients with clinical disease and investigates correlations with related socio-economic and environmental factors. Individual NTM patients were included only once during the studied period and results were presented as incidence rate per year. The most frequently isolated NTM meeting the microbiological and clinical criteria in the study were the Mycobacterium avium-intracellulare complex, followed by Mycobacterium kansasii and Mycobacterium xenopi. A previously described endemic incidence of M. kansasii in the Karviná district and M. xenopi in the Ostrava district was also observed in this study. The incidence of NTM patients in the whole studied territory was 1.10/100,000 inhabitants (1.33/100,000 in men and 0.88/100,000 in women). The annual incidence of lymphadenitis in children (≤5 years of age) was 2.35/100,000 of the population of children during the 7 year period but increased in the year 2018 to 5.95/100,000. The rate of human tuberculosis in the studied area was 1.97/100,000 inhabitants. The incidence of NTM pulmonary diseases correlated with a lower socio-economic status (r = 0.63) and a higher concentration of benzo[a]pyrene pollution in the air (r = 0.64).

A total of 281 guano samples were collected from caves (N = 181) in 8 European countries (Bulgaria, Czech Republic, France, Hungary, Italy, Romania, Slovakia and Slovenia) and attics in the Czech R. (N = 100). The correlation of detection of mycobacteria between Ziehl-Neelsen (ZN) microscopy and culture examination and qPCR was strong. ZN microscopy was positive in guano from caves (58.6%) more than double than positivity in guano from attics (21.0%; P <0.01). From 89 mycobacterial isolates (73 isolates from cave guano and 16 isolates from attics’ guano) 68 (76.4%) isolates of 19 sp., ssp. and complex were identified as members of 3 Groups (M. fortuitum, M. chelonae, and M. mucogenicum), and 4 Complexes (M. avium, M. terrae, M. vaccae, and M. smegmatis). A total of 20 isolates (22,5%) belonged to risk group 1 (environmental saprophytes), 48 isolates (53.9%) belonged to risk group 2 (potential pathogens), and none of isolates belonged to risk group 3 (obligatory pathogens). When comparing bat guano collected from caves and attics, differences (P<0.01; Mann-Whitney test) were observed for the electrical conductivity, total carbon, total organic and total inorganic carbon. None difference (P> 0.05; Mann-Whitney test) was found for pH and oxidation-reduction potential parameters.