Dziedzinska Radka, Okunkova Jana, Kralik Petr, Svobodova Jana, Mala Miriam, Slana Iva
Laboratory of Neurobiology and Pathological Physiology, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czechia.
Microbiology and Antimicrobial Resistance, Veterinary Research Institute, Brno, Czechia.
J Med Microbiol. 2022 Dec;71(12). doi: 10.1099/jmm.0.001611.
. 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 complex (MAC) and complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.. 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.. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.. 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 subsp. (MAM).. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between subsp. /subsp. (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.. 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.
囊性纤维化（CF）是一种具有多系统临床体征的严重疾病，极易且频繁地并发细菌感染。最近，非结核分枝杆菌作为CF的继发污染物的患病率有所增加，其中鸟分枝杆菌复合群（MAC）和脓肿分枝杆菌复合群（MABSC）最为常见。MABSC包括具有重要临床意义的亚种，主要是因为它们对抗生素具有抗性。缺乏用于常规临床实验室早期检测和区分MABSC成员及MAC复合群的灵敏方法。需要一种基于从痰液中直接分离DNA的方法，该方法使用临床实验室的标准设备，并能发现可能的样本抑制（假阴性结果）。这种方法的可用性将允许准确、快速地检测MABSC成员并及时进行有针对性的治疗。为开发一种用于快速、灵敏地鉴定和区分MABSC成员及MAC复合群的实时多重检测方法。开发并优化了直接从患者痰液中分离DNA然后进行四重实时定量PCR（qPCR）检测的方法。使用人工添加已知量脓肿分枝杆菌亚种（MAM）的人痰液样本确定qPCR的灵敏度和检测限（LOD）。该方法可以区分MAC和MABSC成员，同时区分脓肿分枝杆菌亚种/马赛分枝杆菌亚种（MAAb/MAB）和MAM。使用61株培养分离株以及来自CF和非CF患者的痰液样本对该系统进行了验证，结果显示MAAb/MAB占29.5%，MAM占14.7%，MAC占26.2%。痰液样本中MAM细胞的LOD确定为1490个，DNA分离效率为95.4%。测序验证qPCR结果显示同源性为100%。所开发的四重qPCR检测方法，在无需培养的情况下直接从患者痰液中提取DNA，显著改善并加速了CF患者的整个诊断过程，因此特别适合在常规实验室中使用。
