Background

Invasive non-tuberculous mycobacteria (NTM) infections are emerging worldwide in patients undergoing open-chest cardiac bypass surgery exposed to contaminated heater-cooler units (HCUs). Although this outbreak has been investigated by culturing bacteria isolated from HCU aerosol and water samples, these conventional methods have low-analytic sensitivity, high rates of sample contamination and long turnaround time (TAT).

Aim

To develop a simple and effective method to detect NTM in HCUs by real-time PCR, with a short laboratory TAT and reliable culture results.

Methods

A total of 281 water samples collected from various HCUs at 7 Italian hospitals were simultaneously screened for NTM by a propidium monoazide (PMA)-PCR assay and by conventional culture testing. The results were analyzed with culture testing being the reference method.

Findings

i) The agreement between culture testing and PMA-PCR was 85.0% with a Ct cut-off value of < 38 vs.80.0% with a Ct of < 43, with a moderate Cohen's kappa coefficient; ii) the Ct cut-off value of < 42 was deemed more suitable for predicting positive specimens; iii) given the low concentration of target DNA in water samples, the minimum volume to be tested was 1 lt.

Conclusion

We highly recommend the use of PMA-PCR for fast detection of NTM from environmental samples in order to ascertain whether HCUs may represent a potential source of human exposure to NTM. This reliable and simple method reduces laboratory TAT compared to conventional methods (1-2 days vs. 8 weeks, respectively), thereby improving control strategies and effective management of HCUs.

中文翻译：

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通过单叠氮化丙锭实时定量PCR缩短加热器-冷却器单元中非结核分枝杆菌检测的周转时间

背景

在接受暴露于受污染的加热器冷却器（HCU）的开胸式心脏搭桥手术的患者中，世界范围内正在出现非侵入性非结核分枝杆菌（NTM）感染。尽管已经通过培养从HCU气溶胶和水样中分离出的细菌对这种暴发进行了调查，但这些常规方法的分析灵敏度低，样品污染率高且周转时间（TAT）长。

目的

开发一种简单有效的方法，通过实时PCR检测HCU中的NTM，具有较短的实验室TAT和可靠的培养结果。

方法

通过单叠氮化丙锭（PMA）-PCR分析和常规培养测试，同时对从7家意大利医院的各种HCU中收集的281个水样品进行了NTM筛查。以培养测试作为参考方法分析结果。

发现

i）培养测试和PMA-PCR之间的一致性为85.0％，Ct截止值<38，而Ct截止值为80.0％，Ct <43，Cohen卡伯系数适中；ii）Ct截止值小于42被认为更适合预测阳性样本；iii）鉴于水样品中目标DNA的浓度低，要测试的最小体积为1升。

结论

我们强烈建议使用PMA-PCR快速检测环境样品中的NTM，以便确定HCU是否可能代表人类暴露于NTM的潜在来源。与常规方法相比（1-2天这可靠且简单的方法减少了实验室TAT与分别为8周，），从而提高控制策略和HCUs的有效管理。