Detailed Information on Publication Record
2022
Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR Subscribed
DZIEDZINSKA, Radka, Jana OKUNKOVA, Petr KRALIK, Jana SVOBODOVA, Miriam MALÁ et. al.Basic information
Original name
Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR Subscribed
Authors
DZIEDZINSKA, Radka (203 Czech Republic, guarantor), Jana OKUNKOVA (203 Czech Republic), Petr KRALIK (203 Czech Republic), Jana SVOBODOVA (203 Czech Republic), Miriam MALÁ (203 Czech Republic, belonging to the institution) and Iva SLANA
Edition
Journal Of Medical Microbiology, LONDON, MICROBIOLOGY SOC, 2022, 0022-2615
Other information
Language
English
Type of outcome
Článek v odborném periodiku
Field of Study
10606 Microbiology
Country of publisher
United Kingdom of Great Britain and Northern Ireland
Confidentiality degree
není předmětem státního či obchodního tajemství
References:
Impact factor
Impact factor: 3.000
RIV identification code
RIV/00216224:14110/22:00127503
Organization unit
Faculty of Medicine
UT WoS
001129579900003
Keywords in English
cystic fibrosis; mycobacteria; quadruplex quantitative PCR; sputum
Tags
International impact, Reviewed
Změněno: 19/2/2024 09:07, Mgr. Tereza Miškechová
Abstract
V originále
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.
Links
| MUNI/A/1404/2021, interní kód MU |
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