Name: microbiological rapid on-site evaluation for pulmonary infectious diseases
Uploaded: 2024-03-01T13:40:00.000+00:00
Duration: 3 min 22 s
Description: Read this Scientific Article Protocol about Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases at JoVE.com

感谢重庆市科研院所绩效激励指导项目（jxyn-2021-1-15 和 jxyn-2021-2-6）的财政支持。

临床试验已获得重庆市中医院审批委员会批准（编号 2022-KY-31）。典型病例涉及一名诊断为肺孢子菌肺炎的患者，并获得了患者的知情同意。
1. ROSE 的设备及材料要求
<ol><li>设备：有关本协议中使用的设备，请参阅 材料表 文件。 注意：必须使用专用的细胞学显微镜和图形成像系统（图 1A）。</li>
	<li>材料准备（见 材料表）。<ol><li>准备无菌细胞培养载玻片（具有很强的细胞粘附性）、吸水纸、无粉乳胶手套、一次性 2.5-5 mL 注射器针头，并将全套 Diff Quik （DQ） 染色溶液放入带有密封盖的玻璃染色罐中，以便于处理（图 1）。</li>
	</ol></li>
</ol>2. M-ROSE 工作流程
<ol><li>细胞学载玻片的制备 注意：制备玻片的方法有很多，以下是几种常用的制备方法。<ol><li>滚动幻灯片 注意：在此过程中确保组织样本的损失最小。<ol><li>使用容量为 2.5-5 mL 的一次性注射器针头提取组织颗粒（图 2A）。</li>
			<li>在无菌细胞学载玻片上，从染色端的内侧到外侧三分之一部分（表现出强烈细胞附着的末端）铺展一个直径约 1 厘米且厚度适中的圆形区域。</li>
		</ol></li>
		<li>画笔幻灯片 注：该方法适用于使用常规细胞刷、抗污染细胞刷或超细细胞刷获得的标本，以及痰液和粘性体液等半液体标本。<ol><li>伸出刷头，将其涂抹在无菌细胞学载玻片的远端三分之一处（载玻片具有很强的细胞粘附特性），以获得约 2 cm x 1 cm 的矩形区域，厚度适中。</li>
		</ol></li>
		<li>喷雾载玻片<ol><li>将穿刺针放置在具有强大细胞粘附的无菌细胞学载玻片染色端的三分之一处。</li>
			<li>对穿刺针的尖端施加气压，从内侧向外插入，形成直径约 1 cm 的中等厚度的圆形区域。</li>
		</ol></li>
	</ol></li>
	<li>细胞学玻片染色 注：世界卫生组织 （WHO） 建议使用 Diff 染料5 对 ROSE 细胞学玻片进行快速染色。Diff 染料可以重复使用，但不能多次重复。如有沉淀物，使用后应过滤。染色太深可以用甲醇或酒精适当脱色，最好不再染色。如果染色太深或太浅，应调整染色时间或工作液浓度;pH值对染色有一定影响，玻片应清洁无酸碱污染。Diff A 溶液和 Diff B 溶液均具有挥发性，使用后应密封保存。<ol><li>将载玻片浸入 Diff A 溶液中 10-30 秒（图 2B），然后在 PBS 染料浴中冲洗以去除多余的 Diff A 溶液并轻轻甩掉任何剩余的缓冲液（图 2C）。</li>
		<li>接下来，将载玻片浸入 Diff B 溶液中 20-40 秒（图 2E）。</li>
		<li>最后，在水染色槽中清洁载玻片（图 2F），用吸水纸吸干并擦拭载玻片的残留液体（图 2D），然后完成染色。 注：M-ROSE 能够直接观察病原微生物，并有助于评估呼吸道标本质量，能够区分感染和非感染以及感染和污染，这对评估感染严重程度和预后具有指导意义。讨论部分将涵盖这方面，因为它不涉及任何实验程序。</li>
	</ol></li>
</ol>

使用 M-ROSE 加强肺部感染治疗

<ol>
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S., Sorathia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palliative+care+and+interventional+pulmonology.">Palliative care and interventional pulmonology.</a> Clin Chest Med. 39 (1), 57-64 (2018).</li><li>Czarnecka, K., Yasufuku, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventional+pulmonology:+focus+on+pulmonary+diagnostics.">Interventional pulmonology: focus on pulmonary diagnostics.</a> Respirology. 18 (1), 47-60 (2013).</li><li>Schacht, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endobronchial+ultrasound-guided+transbronchial+needle+aspiration:+performance+of+biomedical+scientists+on+rapid+on-site+evaluation+and+preliminary+diagnosis.">Endobronchial ultrasound-guided transbronchial needle aspiration: performance of biomedical scientists on rapid on-site evaluation and preliminary diagnosis.</a> Cytopathology. 27 (5), 344-350 (2016).</li><li>Pearson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+on-site+evaluation+of+fine-needle+aspiration+by+non-cytopathologists:+A+systematic+review+and+meta-analysis+of+diagnostic+accuracy+studies+for+adequacy+assessment.">Rapid on-site evaluation of fine-needle aspiration by non-cytopathologists: A systematic review and meta-analysis of diagnostic accuracy studies for adequacy assessment.</a> Acta Cytol. 62 (4), 244-252 (2018).</li><li>Arimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiopsy+with+endobronchial+ultrasonography+using+a+guide+sheath+for+peripheral+pulmonary+lesions+and+DNA+analysis+by+next+generation+sequencing+and+rapid+on-site+evaluation.">Cryobiopsy with endobronchial ultrasonography using a guide sheath for peripheral pulmonary lesions and DNA analysis by next generation sequencing and rapid on-site evaluation.</a> Respir Investig. 57 (2), 150-156 (2019).</li><li>Gianella, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+rapid+on-site+cytologic+evaluation+during+endobronchial+ultrasound-guided+transbronchial+needle+aspiration+in+malignant+and+nonmalignant+disease.">Utility of rapid on-site cytologic evaluation during endobronchial ultrasound-guided transbronchial needle aspiration in malignant and nonmalignant disease.</a> Acta Cytol. 62 (5-6), 380-385 (2018).</li><li>Natella, V., Cozzolino, I., Sosa Fernandez, L. V., Vigliar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+nodes+fine+needle+cytology+in+the+diagnosis+of+infectious+diseases:+clinical+settings.">Lymph nodes fine needle cytology in the diagnosis of infectious diseases: clinical settings.</a> Infez Med. 20, 12-15 (2012).</li><li>Baughman, R. P., Spencer, R. E., Kleykamp, B. O., Rashkin, M. C., Douthit, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator+associated+pneumonia:+quality+of+nonbronchoscopic+bronchoalveolar+lavage+sample+affects+diagnostic+yield.">Ventilator associated pneumonia: quality of nonbronchoscopic bronchoalveolar lavage sample affects diagnostic yield.</a> Eur Respir J. 16 (6), 1152-1157 (2000).</li><li>Petrone, M. 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作者报告没有利益冲突。

介入肺病学是现代呼吸疾病的一个有价值的分支;特别是，它已广泛用于肺部疾病的诊断 8,9。近年来，由于肺部恶性肿瘤患病率的增加、下呼吸道发生的耐药病原体感染增加以及对莫名其妙和危重呼吸系统疾病的诊断要求要求，诊断介入肺病学一直在蓬勃发展 10,11,12.介入诊断效率取?...

快速现场评估技术 （ROSE） 是肺病手术领域采用的一种高效方法。它支持实时采样和诊断干预，便于即时细胞学分析1。这种创新方法包括将组织标本的一部分印记到载玻片上，同时保持其完整性。ROSE 的主要优势之一在于它能够通过专门的显微镜技术促进对临床信息的及时解释。这包括细胞形态分析、分类、定量、成分比例测定、排列评估、相关性分析、背景评估和异物鉴定。通过将所有这些数据与患者的临床信息相结合，ROSE 在评估采样充分性和指导实时介入程序和技术方面发挥着关键作用2。
作为 ROSE 技术的子公司，M-ROSE 技术主要侧重于获取目标病灶的微生物背景，而不是区分良性和恶性细胞 3,4。一方面，M-ROSE 能够对曲霉菌、隐球菌、肺孢子菌和念珠菌5 等病原体进行显微镜鉴定。另一方面，它在评估呼吸道标本质量、区分感染和非感染性疾病、区分感染和污染以及评估感染严重程度和预后方面具有重要的指导意义 6,7。例如，在呼吸道标本中，表现出相同形态的细菌与浸润的炎症细胞共存表明存在感染;相反，存在多种形态不同的细菌并伴有上皮细胞表明污染。全面分析临床信息和预测结果的能力使 M-ROSE 成为肺部疾病手术中的宝贵工具。
总之，ROSE 是一种重要的细胞学载体，可显着提高诊断肺部疾病的效率和准确性。其多方面功能通过实时采样和诊断干预技术确保及时干预和促进准确诊断，有助于改善患者预后。然而，重要的是要注意，这些进步依赖于获得合格的样品。在此，我们提出了一个标准化的 M-ROSE 方案，包括载玻片制备、染色技术和解释指南。该方案为临床医生建立准确的评估和治疗计划提供了宝贵的参考，同时有助于对目标标本的后续处理做出决策。

<table><tbody><tr><td>Cytological microscope</td><td>Olympus Corporation</td><td>CX43</td><td></td></tr><tr><td>Diff Quik (DQ) staining solutions</td><td>Besso Biotechnology Co. LTD</td><td>G1541</td><td></td></tr><tr><td>Disposable 2.5-5 mL syringe needles</td><td>Shandong Zhu Pharmaceutical Group</td><td>20183150304</td><td></td></tr><tr><td>Powder-free latex gloves</td><td>Henan Yadu Industrial Co., LTD</td><td>20182140728</td><td></td></tr><tr><td>Sterile cell culture slides</td><td>Jinan Preret industry and trade Co., LTD</td><td>7101</td><td></td></tr></tbody></table>

microbiological rapid on-site evaluation for pulmonary infectious diseases

对于表现不明原因的肺部感染患者，迅速开始经验性抗感染治疗至关重要。尽管影像学检查在临床实践中相对简单，但缺乏特异性通常需要额外的耗时检查，例如痰培养、支气管肺泡灌洗液培养或基因测序，以准确识别疾病的潜在病因。此外，经验性抗感染治疗的有限疗效可能导致抗生素滥用。在快速现场评估 （ROSE） 玻片上解释微生物背景的最新进展使临床医生能够通过支气管镜检查（例如，肺泡灌洗、粘膜刷洗、组织夹）及时获取样本，便于床边染色和解释，从而提供必要的微生物背景信息。因此，这为制定有针对性的抗感染治疗和个体化药物治疗计划奠定了基础。通过更好地了解哪些病原体实时引起感染，医生可以避免不必要的广谱抗生素导致抗生素耐药性。在呼吸内科或重症监护病房内建立快速、标准化的 M-ROSE 工作流程将极大地帮助医生为患者制定准确的治疗策略，具有重要的临床意义。

在一个典型病例中，一名 63 岁的男性因咳嗽、发热和胸痛到医院就诊。患者既往诊断为肾病综合征，以蛋白尿和水肿为特征，长期接受泼尼松和他克莫司治疗。初步实验室检查结果显示，白细胞计数为 10.49 x 109/L，中性粒细胞计数为 8.87 x 109/L，全血 C 反应蛋白和 （1-3）-β-D-葡聚糖浓度为 155.81 mg/L 和 249.7 pg/mL。胸部计算机断层扫描 （CT） 扫描显示磨玻璃样混浊，位于弥漫性、双?...

Read this Scientific Article Protocol about Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases at JoVE.com

作者聚焦：使用 M-ROSE 实时推进病原体检测和疾病评估

这里的方案展示了一种快速和标准化的微生物快速现场评估 （M-ROSE） 工作流程，包括三个步骤：载玻片制作、染色和解释。该协议将帮助医生快速做出临床决策。

肺部感染性疾病的微生物学快速现场评估

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging ...

我们的目的是引入一种实时快速评估方法 M-ROSE，以帮助医生为肺部感染患者制定准确的治疗策略。快速细胞染色技术和高清显微成像系统目前用于推进该领域的研究。关于 M-ROSE 的临床研究数量相对有限。最大的挑战是 M-ROSE 结果的解释，这需要人员和训练有素的人员，因为很难目视识别病原体并区分感染和污染。通过更好地了解实时引起感染的病原体，医生可以避免使用不必要的广谱抗生素，这些抗生素会导致抗生素耐药性并用于治疗肺部感染。M-ROSE 是实时且高效的。M-ROSE 能够对病原体进行显微镜鉴定，例如曲霉菌、隐球菌、许多和念珠菌。它在确定呼吸基本质量、区分感染与非感染性疾病、区分感染与污染以及评估感染、严重程度和预后方面具有重要的指导意义。

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Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

349 次观看.  Chongqing Hospital of Traditional Chinese Medicine. 我们的目的是引入一种实时快速评估方法 M-ROSE，以帮助医生为肺部感染患者制定准确的治疗策略。快速细胞染色技术和高清显微成像系统目前用于推进该领域的研究。关于 M-ROSE 的临床研究数量相对有限。最大的挑战是 M-ROSE 结果的解释，这需要人员和训练有素的人员，因为很难目视识别病原体并区分感染和污染。通过更好地了解实时引起感染的病原体，医生可以避免?...

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High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

科研

医学

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Xpert山地车/ RIF测试与诊断肺结核

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

免疫与感染

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

环境科学

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

激光内在人肺组织中的新型细菌特异探针的光学筛选

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

洁净室环境的微生物控制和监测策略

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

作者聚焦:洁净室环境和细胞疗法的微生物控制和监测策略  

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

洁净室环境和细胞疗法的微生物控制和监测策略

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...

生物工程

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

肺部感染性疾病的微生物学快速现场评估

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