薄层凝胶TLA+PNB法培养和鉴定结核杆菌的应用评价

Evaluation of a method for the rapid culture and identification of mycobacterial species-TLA+PNB

目的通过与L-J固体培养,16SrDNA核酸序列测序方法比较,评价一种快速结核分枝杆菌培养和鉴定方法(薄层凝胶TLA+PNB)的应用价值。方法实验样本来源于北京胸科医院疑似结核病例,包括痰样本和气管分泌物。所有的样本用NaOH/NALC方法前处理,然后离心用PBS中和后接种于L-J固体培养基和TLA+PNB固体培养基上。对培养阳性的标本,水煮法提取DNA后PCR扩增16S rDNA核酸序列并测序。最后分别计算2种培养基的敏感度,特异度,培养时间,污染率和TLA+PNB菌种鉴定的敏感度,特异度。结果实验共包含211份临床样本。对于抗酸染色阳性的标本,培养阳性率为77.4%,而对于抗酸染色阴性的标本,L-J和TLA培养阳性率均为8.2%。TLA和L-J敏感度均为84.2%,特异度均为100%。TLA+PNB培养基在48例培养阳性的标本中发现8例NTM,敏感度为88.9%,特异度为100%。污染率TLA和L-J分别为4.7%和1.2%。平均报告阳性结果时间TLA和L-J分别为10.6 d和22.9 d(P<0.001)。结论 TLA+PNB培养和鉴定方法是一种快速、可靠、廉价的结核分枝杆菌诊断方法。在基层实验室中,有很好的推广价值。

Objective evaluate the clinical value of thin-layer agar(TLA+PNB) on the diagnosis of TB and identification of mycobacterial species. Methods 211 samples including sputum and extra-pulmonary specimens from suspects with a clinical diagnosis of TB were decontaminated using NaOH/NALC,centrifuged,and then stained with Ziehl-Neelsen for the detection of acid-fast bacilli(AFB),cultured on L-J medium and TLA+PNB medium and identified by 16S rDNA sequencing.Sensitivity and specificity,growth detection time and contamination rate were calculated for both media. Results Of AFB-positive samples,77.4% were culture positive.Of AFB-negative samples,8.2% were both L-J and TLA culture positive.The sensitivity and specificity of both L-J and TLA culture were 84.2% and 100%,respectively.8 non-tuberculosis mycobacteria of 48 cuture-positive samples were detected by TLA+PNB,in which the sensitivity and specificity were 88.9% and 100%,respectively.Contamination rates were 4.7% for TLA and 1.2% for L-J.Median time of a positive culture was 10.6 days for TLA and 22.9 days for L-J(P<0.001). Conclusions The TLA+PNB method is an inexpensive,rapid and reliable alternative for detecting M.tuberculosis.