用于痰液样本下游分子处理以检测结核分枝杆菌的标本运输培养基的实验室评估。

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis.

作者信息

Omar Shaheed V, Peters Remco P H, Ismail Nazir A, Dreyer Andries W, Said Halima M, Gwala Thabisile, Ismail Nabila, Fourie P Bernard

机构信息

Faculty of Health Sciences, Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa; Centre for Tuberculosis, National TB Reference Laboratory, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa.

Faculty of Health Sciences, Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa; Anova Health Institute, Johannesburg, South Africa.

出版信息

J Microbiol Methods. 2015 Oct;117:57-63. doi: 10.1016/j.mimet.2015.07.010. Epub 2015 Jul 14.

DOI:10.1016/j.mimet.2015.07.010

PMID:26183764

Abstract

BACKGROUND

Modern molecular-based approaches for the detection of Mycobacterium tuberculosis in sputum samples promise quicker and more accurate detection of cases. However, processing sputum samples at central diagnostic facilities provides a diagnostic approach, but requires a safe and efficient system that is not affected by transport delays and ambient temperature to be feasible. We evaluated the technical properties of PrimeStore®-Molecular Transport Medium (PS-MTM) for its ability to inactivate mycobacteria, ensuring stability of DNA over time at ambient temperatures and to assess the compatibility of the transport medium with DNA extraction systems.

METHODS

Assessment of the transport medium for application of sputum samples processed for the detection of M. tuberculosis included the inactivation of M. tuberculosis in spiked sputum samples, compatibility of the medium with three commercial nucleic extraction systems and stability of DNA in the medium at ambient temperature over 28 days. We further performed a clinical laboratory evaluation on 256 sputum specimens sent for tuberculosis investigation.

RESULTS

Complete inactivation of M. tuberculosis occurred within 30 min of exposure at a ratio of 1:3 for sputum to PS-MTM. Sputum specimen in PS-MTM showed very good compatibility with automated bead-based extraction systems, producing high DNA output (estimated lower limits of detection: ~170 CFU/ml). Furthermore, PS-MTM samples remained stable over 28 days at ambient temperature displaying no significant change over time in Ct-values (<5% on a mean starting value of 22.47). Of the 256 clinical sputum specimens, 10.2% were culture positive and 11.0% were positive by real-time PCR of PS-MTM samples.

CONCLUSIONS

Collecting and transporting sputum from TB suspects in PS-MTM offer safe transport at ambient temperature, DNA stability for extended periods without cooling and specimens directly suitable for molecular testing. This novel approach may support introduction and further scale-up of molecular diagnostics for TB in resource-limited settings.

摘要

背景

基于现代分子技术的痰液样本中结核分枝杆菌检测方法有望实现更快速、准确的病例检测。然而，在中央诊断设施处理痰液样本是一种诊断方法，但需要一个安全、高效且不受运输延迟和环境温度影响的系统才可行。我们评估了PrimeStore®分子转运培养基（PS-MTM）的技术特性，包括其灭活分枝杆菌的能力、确保DNA在环境温度下随时间的稳定性以及评估转运培养基与DNA提取系统的兼容性。

方法

对用于检测结核分枝杆菌的痰液样本处理转运培养基的评估包括在加标痰液样本中灭活结核分枝杆菌、该培养基与三种商用核酸提取系统的兼容性以及DNA在该培养基中在环境温度下28天的稳定性。我们还对256份送检进行结核病调查的痰液标本进行了临床实验室评估。

结果

在痰液与PS-MTM比例为1:3的情况下，结核分枝杆菌在暴露30分钟内完全灭活。PS-MTM中的痰液标本与基于磁珠的自动化提取系统显示出非常好的兼容性，产生高DNA产量（估计检测下限：~170 CFU/ml）。此外，PS-MTM样本在环境温度下28天内保持稳定，Ct值随时间无显著变化（在平均起始值22.47的基础上<5%）。在256份临床痰液标本中，10.2%培养阳性，PS-MTM样本实时PCR检测11.0%阳性。