J 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.

Základní údaje

Originální název

Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR Subscribed

Autoři

DZIEDZINSKA, Radka (203 Česká republika, garant), Jana OKUNKOVA (203 Česká republika), Petr KRALIK (203 Česká republika), Jana SVOBODOVA (203 Česká republika), Miriam MALÁ (203 Česká republika, domácí) a Iva SLANA

Vydání

Journal Of Medical Microbiology, LONDON, MICROBIOLOGY SOC, 2022, 0022-2615

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10606 Microbiology

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

Impakt faktor

Impact factor: 3.000

Kód RIV

RIV/00216224:14110/22:00127503

Organizační jednotka

Lékařská fakulta

DOI

UT WoS

001129579900003

Klíčová slova anglicky

cystic fibrosis; mycobacteria; quadruplex quantitative PCR; sputum

Štítky

Příznaky

Mezinárodní význam, Recenzováno
Změněno: 19. 2. 2024 09:07, Mgr. Tereza Miškechová

Anotace

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.

Návaznosti

MUNI/A/1404/2021, interní kód MU
Název: Imunita u dětí po Covid-19 (Akronym: ImunCovid)
Investor: Masarykova univerzita, Imunita u dětí po Covid-19