Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM).Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus/subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients' sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.
- MeSH
- atypické mykobakteriální infekce * diagnóza farmakoterapie MeSH
- cystická fibróza * komplikace mikrobiologie MeSH
- DNA terapeutické užití MeSH
- intracelulární infekce bakterií Mycobacterium avium * epidemiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Mycobacterium abscessus * genetika MeSH
- Mycobacterium avium komplex genetika MeSH
- netuberkulózní mykobakterie MeSH
- sputum mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mycobacterial infections in pigs are caused particularly by the Mycobacterium avium complex (MAC) and these infections lead to great economic losses mainly within the countries with high pork meat production. The importance of the MAC infections in humans is rising because of its higher prevalence and also higher mortality rates particularly in advanced countries. In addition, treatment of the MAC infections in humans tends to be complicated because of its increasing resistance to antimicrobial agents. Several studies across Europe have documented the MAC occurrence in the slaughtered pigs - not only in their lymph nodes and tonsils, which are the most frequent, but also in the diaphragmas, other organs and not least in meat. This is why we need both more specific and more sensitive methods for the MAC infection detection. Different PCR assays were established as well as advanced intravital testing by the gamma interferon release test. On the other hand, tuberculin skin test is still one of the cheapest methods of mycobacterial infections detection.
- MeSH
- DNA bakterií genetika MeSH
- intracelulární infekce bakterií Mycobacterium avium diagnóza epidemiologie mikrobiologie veterinární MeSH
- lidé MeSH
- Mycobacterium avium komplex klasifikace genetika imunologie izolace a purifikace MeSH
- nemoci prasat diagnóza epidemiologie mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- prasata MeSH
- test pomocí interferonu gama metody MeSH
- tuberkulinový test MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Geografické názvy
- Evropa epidemiologie MeSH
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.
- MeSH
- intracelulární infekce bakterií Mycobacterium avium diagnóza etiologie terapie MeSH
- lidé MeSH
- mikrobiologie životního prostředí MeSH
- Mycobacterium avium komplex klasifikace účinky léků genetika MeSH
- rizikové faktory MeSH
- zdroje nemoci mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- MeSH
- DNA bakterií genetika chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- Mycobacterium avium komplex genetika izolace a purifikace růst a vývoj MeSH
- mykobakteriózy veterinární MeSH
- nemoci skotu mikrobiologie MeSH
- polymerázová řetězová reakce veterinární MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- skot MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Geografické názvy
- Česká republika MeSH
A cooperative study was conducted by the International Working Group on Mycobacterial Taxonomy to correlate the agglutination serovar designations of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum strains with the species ascriptions of these organisms according to molecular criteria and cultural properties and to assess the reproducibility of serovar determinations for a set of 63 reference strains of these species. Among the molecular criteria, the level of agreement between results obtained with nucleic acid probes and T-catalase serology results was 94% for strains of M. avium and M. intracellulare. Nucleic acid probes were not available for M. scrofulaceum, but none of the 10 strains ascribed to this species on the basis of catalase serology data reacted with a nucleic acid probe for M. avium or M. intracellulare. Ascription to a species on the basis of mycolic acid high-performance liquid chromatography patterns was in agreement with catalase serology results in 86% of the cases examined. Most strains belonging to serovars 1 through 6 and 8 through 11 were identified by molecular criteria as M. avium, most strains belonging to serovars 7, 12 through 20, 23, and 25 were identified as M. intracellulare, and most strains belonging to serovars 41 through 43 were identified as M. scrofulaceum, in agreement with common current practice. Evidence for assigning serovar 27 to M. scrofulaceum was obtained. However, two strains of a given serovar may, on occasion, be placed in different species. The dominant species assignments for strains belonging to serovars 21, 24, 26, and 28 remain unresolved.(ABSTRACT TRUNCATED AT 250 WORDS)
- MeSH
- aglutinační testy MeSH
- bakteriální proteiny analýza MeSH
- buněčné dělení MeSH
- katalasa analýza MeSH
- Mycobacterium avium komplex * genetika imunologie klasifikace MeSH
- Mycobacterium avium * genetika imunologie klasifikace MeSH
- Mycobacterium scrofulaceum * genetika imunologie klasifikace MeSH
- protilátky bakteriální imunologie MeSH
- RNA ribozomální * genetika MeSH
- Publikační typ
- multicentrická studie MeSH
- srovnávací studie MeSH
