We appreciate the Chongqing Scientific Research institution&#39;s performance incentive and guidance project (jxyn-2021-1-15 and jxyn-2021-2-6) for financial support.

The clinical trial has been approved by the Approval Committee of Chongqing Hospital of Traditional Chinese Medicine (No. 2022-ky-31). The typical case involved a patient diagnosed with Pneumocystis pneumonia, and informed consent was obtained from the patient.
1. Equipment and material requirements for ROSE
<ol>
	<li>Equipment: Refer to the Table of Materials file for the equipment used in this protocol. 
	NOTE: Utilizing a dedicated cytological microscope and graphic imaging system is imperative (Figure 1A).</li>
	<li>Material preparation (see Table of Materials).
	<ol>
		<li>Prepare sterile cell culture slides (with strong cell adhesion), absorbent paper, powder-free latex gloves, disposable 2.5-5 mL syringe needles, and place the complete set of Diff Quik (DQ) staining solutions in glass staining jars with sealed lids for easy handling (Figure 1).</li>
	</ol>
	</li>
</ol>
2. M-ROSE workflow
<ol>
	<li>Preparation of cytological slides 
	NOTE: There are many ways to prepare slides, and the following are several commonly used preparation methods.
	<ol>
		<li>Roll slides 
		NOTE: Ensure minimal loss of tissue sample during this process.
		<ol>
			<li>Extract tissue particles by using a disposable syringe needle with a capacity of 2.5-5 mL (Figure 2A).</li>
			<li>Spread a circular area, approximately 1 cm in diameter and of moderate thickness, from the inner to the outer one-third section of the stained end (the end exhibiting strong cell attachment) on the sterile cytology slide.</li>
		</ol>
		</li>
		<li>Brush slides 
		NOTE: This method is applicable to specimens obtained using conventional cell brushes, anti-pollution cell brushes, or ultrafine cell brushes, as well as semi-liquid specimens such as sputum and viscous body fluids.
		<ol>
			<li>Extend the brush head and apply it on the distal third of the sterile cytological glass slide (the slide has strong cell adhesion properties) to obtain a rectangular area of about 2 cm x 1 cm with a moderate thickness.</li>
		</ol>
		</li>
		<li>Spray slides
		<ol>
			<li>Position the puncture needle at one-third of the stained end of a sterile cytology slide with robust cell adhesion.</li>
			<li>Apply air pressure to the tip of the piercing needle and insert it from the inner side outward, forming a circular area with medium thickness measuring approximately 1 cm in diameter.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cytological slide staining 
	NOTE: The World Health Organization (WHO) recommends rapid staining of ROSE cytological slide using Diff&#39;s dye5. The Diff dye can be reused but not repeated many times. If there is sediment, it should be filtered after use. Dyeing too deep can be properly decolorized by methanol or alcohol, preferably no longer dyed. If the dyeing is too deep or too shallow, the dyeing time or working liquid concentration should be adjusted; the pH value has a certain influence on dyeing, and the slide should be clean and free of acid and alkali pollution. Both Diff A solution and Diff B solution are volatile and should be sealed and stored after use.
	<ol>
		<li>Submerge the slide in Diff A solution for 10-30 s (Figure 2B), then rinse it in the PBS dye bath to remove excess Diff A solution and gently shake off any remaining buffer (Figure 2C).</li>
		<li>Next, immerse the slide in Diff B solution for 20-40 s (Figure 2E).</li>
		<li>Finally, clean the slide in the water dyeing tank (Figure 2F), blot and wipe the residual liquid of the slide with absorbent paper (Figure 2D), and finish dyeing. 
		NOTE: M-ROSE enables direct visualization of pathogenic microorganisms and facilitates the assessment of respiratory specimen quality, enabling discrimination between infection and non-infection as well as infection and contamination, which has guiding significance for the evaluation of infection severity and prognosis. The discussion section will cover this aspect as it does not involve any experimental procedures.</li>
	</ol>
	</li>
</ol>

Enhancing Pulmonary Infection Treatment Using M-ROSE

<ol>
	<li>Zhang, S., 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=Diagnostic+value+of+endoscopic+ultrasound-guided+fine+needle+aspiration+with+rapid+on-site+evaluation+performed+by+endoscopists+in+solid+pancreatic+lesions:+A+prospective,+randomized+controlled+trial.">Diagnostic value of endoscopic ultrasound-guided fine needle aspiration with rapid on-site evaluation performed by endoscopists in solid pancreatic lesions: A prospective, randomized controlled trial.</a> J Gastroenterol Hepatol. 37 (10), 1975-1982 (2022).</li><li>Bruno, 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=Efficacy+and+cost+effectiveness+of+rapid+on+site+examination+(ROSE)+in+management+of+patients+with+mediastinal+lymphadenopathies.">Efficacy and cost effectiveness of rapid on site examination (ROSE) in management of patients with mediastinal lymphadenopathies.</a> Eur Rev Med Pharmacol Sci. 17 (11), 1517-1522 (2013).</li><li>Li, T., 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=Microbiology+rapid+on-site+evaluation:+a+better+method+for+Mucoid+Pseudomonas+Aeruginosa+diagnosis+in+bronchiectasic+patients.">Microbiology rapid on-site evaluation: a better method for Mucoid Pseudomonas Aeruginosa diagnosis in bronchiectasic patients.</a> Eur Rev Med Pharmacol Sci. 26 (5), 1738-1742 (2022).</li><li>Tao, Y., 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=Application+of+microbiological+rapid+on-site+evaluation+in+respiratory+intensive+care+units:+a+retrospective+study.">Application of microbiological rapid on-site evaluation in respiratory intensive care units: a retrospective study.</a> Ann Transl Med. 10 (1), 7 (2022).</li><li>Yan, 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=The+value+of+microbiology+rapid+on-site+evaluation+of+sepsis+caused+by+pulmonary+infection.">The value of microbiology rapid on-site evaluation of sepsis caused by pulmonary infection.</a> Eur Rev Med Pharmacol Sci. 27 (12), 5862-5868 (2023).</li><li>Muri, R., Trippel, M., Borner, U., Weidner, S., Trepp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+rapid+on-site+evaluation+on+the+quality+and+diagnostic+value+of+thyroid+nodule+fine-needle+aspirations.">The impact of rapid on-site evaluation on the quality and diagnostic value of thyroid nodule fine-needle aspirations.</a> Thyroid. 32 (6), 667-674 (2022).</li><li>Wang, Z., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+rapid+on-site+evaluation+in+contemporary+pediatric+interventional+respiratory+diseases.">Application of rapid on-site evaluation in contemporary pediatric interventional respiratory diseases.</a> Chinese Journal of Practical Pediatrics. 12, 470-475 (2019).</li><li>Shah, P. L., Herth, F. J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+interventional+pulmonology.">Progress in interventional pulmonology.</a> Respiration. 95 (5), 287-288 (2018).</li><li>Hsia, D., Musani, A. I. <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.">Interventional pulmonology.</a> Med Clin North Am. 95 (6), 1095-1114 (2011).</li><li>Moore, A. J., Mercer, R. M., Musani, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+interventional+pulmonology.">Advances in interventional pulmonology.</a> Clin Chest Med. 39 (1), 271-280 (2018).</li><li>Majid, A., Fernandez-Bussy, S., Folch, E. <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+and+solitary+pulmonary+nodule.">Interventional pulmonology and solitary pulmonary nodule.</a> Arch Bronconeumol. 54 (10), 497-498 (2018).</li><li>Ali, M. 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. C., 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=Does+cytotechnician+training+influence+the+accuracy+of+EUS-guided+fine-needle+aspiration+of+pancreatic+masses.">Does cytotechnician training influence the accuracy of EUS-guided fine-needle aspiration of pancreatic masses.</a> Dig Liver Dis. 44 (4), 311-314 (2012).</li></ol>

The authors report no conflicts of interest.

Interventional pulmonology is an valuable branch of modern respiratory disease; in particular, it has been widely used in the diagnosis of lung diseases8,9. In recent years, diagnostic interventional pulmonology has been thriving due to the increased prevalence of pulmonary malignant tumors, more drug-resistant pathogen infections occuring in the lower respiratory tract, and demanding requests for diagnosis of baffling and critical respiratory diseases10,11,12. Interventional diagnostic efficiency depends on factors like size and the anatomic location of focal areas, complication rates and number of biopsy sites, and underlying diseases, promoting the clinical use of numerous advanced technologies and facilities13. ROSE has received remarkable attention and emerged quickly as a &#34;real-time accompany technique&#34; for diagnosis in interventional pulmonology.
ROSE is designed for rapid evaluation, preliminary diagnosis, and prioritization of specimen satisfaction during procedures such as puncture, biopsy, and brushing. It provides feedback to guide the subsequent technological advancements14,15,16,17. M-ROSE, a branch of ROSE, primarily focuses on evaluating the microbial background rather than assessing tissue cell malignancy5. The M-ROSE method is a rapid evaluation technique rather than a diagnostic tool thus its diagnostic efficacy should not be exaggerated. Its clinical application primarily encompasses the following aspects: visual identification of certain pathogenic microorganisms, differentiation between infection and contamination, and discrimination between infection and non-infection7.
Following rapid staining, the cell nucleus of the tissue exhibited a purplish-red hue, while the cytoplasm displayed a bluish-purple color. Bacteria and fungi appeared blue, resulting in a distinct contrast between tissue cells and pathogens7. M-ROSE could demonstrate a certain level of capability in identifying bacteria and fungi. Specifically, it exhibits a high positive rate for Aspergillus, cryptococcus, Pneumocystis jiroveci, and other fungi3,18. However, it does have limitations when it comes to recognizing pathogens with small volume and diameter or intracellular pathogenic agents. It is crucial to underscore that the diagnosis of any disease should be predicated upon a comprehensive evaluation encompassing the patient&#39;s fundamental condition, clinical manifestations, imaging examinations, and other factors rather than solely relying on the microscopic identification of pathogenic microorganisms.
M-ROSE also offers advantages in the identification of infection and contamination, such as assessing the quality of alveolar lavage fluid samples. In departments where conditions allow, bedside evaluation of lavage fluid can be conducted through centrifugation and staining. Qualified samples of alveolar lavage fluid demonstrate that the proportion of squamous cells among all cells (excluding red blood cells) under low power microscopy is less than 1%, columnar epithelial cells are less than 5%, and red blood cells are less than 10% (excluding factors related to trauma or bleeding)19. The microscopic examination of the cytology background will facilitate us in making timely assessments.
Furthermore, M-ROSE can aid in distinguishing between infected and non-infected lesions5. In infected lesions, there is often infiltration of neutrophils, macrophages, and lymphocytes by inflammatory cells, with occasional observation of neutrophil phagocytosis. Conversely, non-infected lesions typically lack inflammatory cell infiltration.
The protocol presented here demonstrates the operation process of M-ROSE; through the interpretation of the microbiological background, the preliminary diagnosis can be made or the differential diagnosis can be narrowed. As our findings suggest, the diagnosis of PCP requires confirmed evidence of Pneumocystis jiroveci, which should not be based on clinical manifestations and imaging, even in symptomatic high-risk patients. Microscopic identification of ascus and trophic forms is the most intuitive evidence. The diagnostic efficiency is largely determined by the staining method of the specimen. Historically, conventional stains such as Wright&#39;s-Giemsa, hexaamine silver, and toluidine blue are available for PCP but are time-consuming and have lower sensitivity. In this case, the immediate diagnosis of PCP was confirmed on the first day of the patient&#39;s transfer to ICU with the use of ROSE. Diff-quick has shown its advantage in identifying pneumocystis, characterized by a clear structure, sharp contrast, and easy identification of ascus (Figure 5A).
In the application of ethnic medicine, ROSE also holds significant value. For instance, in suppurative diseases, clinicians can identify a diverse range of inflammatory cells primarily dominated by neutrophils within the microbiological background5. Consequently, drugs with heat-clearing and detoxifying properties can be selected as potential treatments for these conditions, such as honeysuckle, dandelion, and patrinia, among others.
Since ROSE is an additional program that does not have to be selected in interventional pulmonology, carrying out ROSE is bound to increase labor and economic costs. It is debatable that ROSE should be used on a regular basis. In terms of the benefits of economics, time and labor costs are what we are concerned about. The main additional labor need is the evaluator. ROSE evaluation can be performed by a cytotechnologist, pathologist, or clinician. Cytotechnologists are the likeliest personnel for the ROSE procedure. However, in clinical practice, clinicians replace the role, but it may require a certain training basis15. A previous study showed that a training program could help to improve the accuracy of adequacy assessment performed by clinicians, and eliminate confounding variables20. In addition, M-ROSE, as a morphological diagnostic technique, has subjective factors in its results report. The clinicians should make a comprehensive assessment based on additional clinical information.
We recommend that hospitals with appropriate facilities and trained professionals consider implementing the ROSE procedure in their respiratory departments or ICUs, as it can facilitate the early evaluation of challenging respiratory critical illnesses and guide clinical treatment effectively.

The technique of rapid on-site evaluation (ROSE) is a highly efficient method employed in the field of pulmonary disease procedures. It enables real-time sampling and diagnostic intervention, facilitating immediate cytological analysis1. This innovative approach involves imprinting a portion of the tissue specimen onto a slide while preserving its integrity. One of the key advantages of ROSE lies in its capacity to facilitate prompt interpretation of clinical information through specialized microscopy techniques. This encompasses the analysis of cell morphology, classification, quantification, determination of constituent ratios, assessment of arrangement, correlation analysis, evaluation of background, and identification of foreign objects. By integrating all this data with the patient's clinical information, ROSE plays a pivotal role in evaluating sampling adequacy and guiding real-time interventional procedures and techniques2.
As a subsidiary of ROSE technology, M-ROSE technology primarily focuses on acquiring the microbiological background of target lesions rather than discerning between benign and malignant cells3,4. On one hand, M-ROSE enables the microscopic identification of pathogens such as Aspergillus, Cryptococcus, Pneumocystis, and Candida5. On the other hand, it holds significant guiding implications in assessing respiratory specimen quality, distinguishing infectious from non-infectious diseases, discriminating infection from contamination, as well as evaluating infection severity and prognosis6,7. For instance, within a respiratory specimen, the coexistence of bacteria exhibiting identical morphology alongside infiltrating inflammatory cells indicates an infection; conversely, the presence of multiple morphologically diverse bacteria accompanied by epithelial cells suggests contamination. The capacity to comprehensively analyze clinical information and predict outcomes renders M-ROSE an invaluable tool in pulmonary disease procedures.
In conclusion, ROSE serves as a crucial cytological carrier that significantly enhances the efficiency and precision of diagnosing lung diseases. Its multifaceted capabilities contribute to improved patient outcomes by ensuring timely intervention and facilitating accurate diagnosis through real-time sampling and diagnostic intervention techniques. However, it is important to note that these advancements rely on obtaining qualified samples. Herein, we present a standardized M-ROSE protocol encompassing slide preparation, staining techniques, and interpretation guidelines. This protocol serves as an invaluable reference for clinicians to establish accurate evaluation and treatment plans while facilitating decision-making regarding subsequent handling of target specimens.

<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

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

In a typical case, a 63-year-old man presented to the hospital with a cough, fever, and chest pain. The patient was previously diagnosed with nephrotic syndrome, characterized by proteinuria and edema, and received treatment with prednisone and tacrolimus for a long time. The initial laboratory findings demonstrated a white blood cell count of 10.49 x 109/L, neutrophil counts of 8.87 x 109/L, whole blood C-reactive protein and (1-3)-&#946;-D-glucan concentrations of 155.81 mg/L and 249.7 pg/mL. The chest computed tomography (CT) scan showed ground-glass opacification with a diffuse, bilateral and central location (Figure 3A). Initial anti-infective treatment with cefoperazone tazobactam was ineffective, and his symptoms were aggravated by cardiac tiredness, shortness of breath, and dyspnea after exercise; repeated CT imaging showed no significant improvement (Figure 3B). Then, the patient was transferred to the intensive care unit (ICU) for mechanical ventilation and underwent bronchoscopy. The focus sampling was accessed under the guidance of virtual navigation, and three forceps biopsy samples were collected (Figure 4). ROSE was available for immediate microscopic assessment of the tissue, and the diagnosis of Pneumocystis jiroveci was confirmed (Figure 5A). The patient immediately received antifungal treatment with sulfamethoxazole (1.92 g, q6h); subsequent staining (Figure 5B-E) confirmed this diagnosis. However, repeated CT imaging still showed no significant improvement (Figure 3C). The patient received carbofungin (the first day of 70 mg and followed by 50 mg daily), methylprednisolone (80 mg daily), and immunoglobulin (10 g daily) for fungal infection. The symptoms were relieved quickly, and the patient continued follow-up treatment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66059/66059fig01.jpg" /> 
Figure 1: Equipment and materials. (A) Cytological microscope and Graphic imaging system. (B) Diff Quik (DQ) staining solution A. (C) Diff Quik (DQ) staining solution B. (D) Powder-free latex gloves. (E) Sterile cell culture slides. (F) Disposable 2.5-5 mL syringe needles. (G) Absorbent paper. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66059/66059fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66059/66059fig02.jpg" /> 
Figure 2: M-ROSE workflow. (A) Printing the slide. (B) Submerge the slide in Diff A solution for 10-30 s. (C) Rinse the slide in the PBS dye bath to remove excess Diff A solution. (D) Blot and wipe the residual liquid off the slide with absorbent paper. (E) Immerse the slide in Diff B solution for 20-40 s. (F) Clean the slide in the water dyeing tank. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66059/66059fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66059/66059fig03.jpg" /> 
Figure 3: Chest CT images. (A) At admission. (B) On the day of the transfer to the ICU. (C) After the treatment of sulfamethoxazole <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66059/66059fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66059/66059fig04.jpg" /> 
Figure 4: Localization of the posterior basal segment of the lower lobe of the right lung by VBN. (A) Right principal bronchus. (B) Right middle bronchus. (C) Posterior basal segment of bronchus of the lower lobe of the right lung. (D) 3D Map of VBN. (E) Coronal map of chest CT. (F) Axial map of chest CT. (G) Sagittal map of chest CT <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66059/66059fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66059/66059fig05.jpg" /> 
Figure 5: Pictures documenting the appearance of pneumocystis in various staining methods. (A) ROSE 100 x 10; White arrow: cysts containing ascospores. (B) Hematoxylin and eosin (H-E), 60 x 10. (C) Alcian blue,60 x 10. (D) GIEMSA, 40 x 10. (E) Hexamine silver, 20 x 10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66059/66059fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Author Spotlight: Advancing Pathogen Detection and Disease Assessment in Real-Time Using M-ROSE

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

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

microbiological-rapid-on-site-evaluation-for-pulmonary-infectious

Nanyang Technological University

Research

JoVE Journal

Medicine

355 Views.  Chongqing Hospital of Traditional Chinese Medicine. Het is onze bedoeling om een real-time snelle beoordelingsmethode te introduceren, M-ROSE, om de artsen te helpen bij het formuleren van nauwkeurige behandelingsstrategieën voor patiënten met longinfecties. Snelle celkleuringstechnologie en een high-definition microscopisch beeldvormingssysteem worden momenteel gebruikt om het onderzoek in het veld te bevorderen. Het aantal klinische studies naar M-ROSE is relatief beperkt.De grootste uitdaging is de interpretatie van de M-ROSE-resul...

Video: Auteur in de schijnwerpers: Bevordering van de detectie van ziekteverwekkers en ziektebeoordeling in realtime met behulp van M-ROSE

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.

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

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.

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

Immunology and Infection

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.

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

Environment

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.

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.

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

Microbial Control and Monitoring Strategies for Cleanroom Environments

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).

Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

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.

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

Bioengineering

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.

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.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

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