Treffon Janina, Isserstedt-John Nicole, Klemm Richard, Gärtner Claudia, Mellmann Alexander
University Hospital Münster, Institute of Hygiene, Münster, Germany.
microfluidic ChipShop GmbH, Jena, Germany.
Microbiol Spectr. 2024 Nov 6;12(12):e0086224. doi: 10.1128/spectrum.00862-24.
Bacterial infection clusters cause high mortality rates and healthcare costs due to excessive therapy and hygiene measures. The aim of this study was to develop an automated real-time PCR-based point-of-care (POC) system with a customized microfluidic chip that facilitates fast detection of bacterial cluster isolates by targeting cluster-specific single-nucleotide polymorphisms (SNPs). For cluster detection of , , and , nine TaqMan real-time PCR assays targeting cluster-specific SNPs were designed. Additionally, for DNA input control, a universal PCR amplifying the 16S rDNA was constructed. All reactions were implemented into a microfluidic chip that was analyzed by a POC prototype enabling automated sample processing, fluid handling, and signal detection. Performance of the prototype was evaluated using 45 chips loaded with defined bacterial solutions, including swab eluates. For seven PCRs, implementation into the microfluidic chip was successful, leading to correct identification of all SNPs specific for and cluster isolates and delineation of all non-cluster strains within 70 min. The remaining three reactions failed in the chip, which resulted in misidentification of the cluster. Sensitivity, specificity, and accuracy of the prototype were 43%, 88%, and 55%, respectively. The detection limit was PCR dependent and ranged between 10 and 10 colony-forming units/mL. Once optimized, the microfluidic POC system for cluster detection could be applied as time-saving and easy-to-use method to complement whole-genome sequencing efforts during screening for bacterial clusters.
Especially in medical facilities, where morbid people are nursed in close distance to each other, pathogenic bacteria can accumulate and spread. To contain such infection clusters, usually time- and labor-intensive large-scale screening assays are conducted, where patients and patient-side surfaces are sampled, and PCR or whole-genome sequencing analyses are conducted to confirm or deny cluster affiliation of cultivated bacteria. Hence, fast solutions with easy application are required to complement the current state-of-the-art technology for cluster surveillance. Here, we developed a fully automated microfluidic point-of-care prototype that identified bacterial cluster isolates within 70 min from bacterial solutions, including swab eluates. The system requires only low hands-on time and can be applied apart from laboratory infrastructures near the patient, which considerably reduces the time from sampling to result. This ensures fast implementation of hygiene measures and quick containment of the infection cluster, which would enhance patients' safety and outcome.
细菌感染聚集会因过度治疗和卫生措施导致高死亡率和医疗成本。本研究的目的是开发一种基于自动化实时聚合酶链反应（PCR）的即时检测（POC）系统，该系统配备定制的微流控芯片，通过靶向聚集特异性单核苷酸多态性（SNP）来促进细菌聚集分离株的快速检测。为了对[具体细菌名称1]、[具体细菌名称2]和[具体细菌名称3]进行聚集检测，设计了9种针对聚集特异性SNP的TaqMan实时PCR检测方法。此外，为了进行DNA输入控制，构建了一种扩增16S核糖体DNA（rDNA）的通用PCR。所有反应都在一个微流控芯片中进行，该芯片由一个POC原型进行分析，能够实现自动样品处理、流体处理和信号检测。使用45个装载有确定细菌溶液（包括拭子洗脱液）的芯片对该原型的性能进行了评估。对于7种PCR反应，成功地在微流控芯片中实现，从而在70分钟内正确鉴定了所有针对[具体细菌名称1]和[具体细菌名称2]聚集分离株的特异性SNP，并区分了所有非聚集菌株。其余3种反应在芯片中失败，导致对[具体细菌名称3]聚集的错误鉴定。该原型的灵敏度、特异性和准确性分别为43%、88%和55%。检测限取决于PCR，范围在10⁴至10⁵菌落形成单位/毫升之间。一旦优化，用于聚集检测的微流控POC系统可作为一种节省时间且易于使用的方法，以补充细菌聚集筛查期间的全基因组测序工作。
特别是在医疗机构中，患病人员彼此近距离护理，病原菌可能会聚集和传播。为了控制此类感染聚集，通常会进行耗时且费力的大规模筛查检测，对患者及其周边表面进行采样，并进行PCR或全基因组测序分析，以确认或否定培养细菌的聚集归属。因此，需要快速且易于应用的解决方案来补充当前用于聚集监测的先进技术。在此，我们开发了一种全自动微流控即时检测原型，可在70分钟内从包括拭子洗脱液在内的细菌溶液中鉴定出细菌聚集分离株。该系统只需很少的人工操作时间，并且可以在靠近患者的非实验室基础设施旁应用，这大大缩短了从采样到得出结果的时间。这确保了卫生措施的快速实施以及感染聚集的快速控制，从而提高患者的安全性和治疗效果。
