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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

328 Views.  Chongqing Hospital of Traditional Chinese Medicine. 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.

Video: Author Spotlight: Advancing Pathogen Detection and Disease Assessment in Real-Time Using 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 ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing.
Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing.&#160;Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing.
In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus

Ultrasound is a safe bedside imaging tool that obviates the use of ionizing radiation diagnostic procedures. Due to its convenience, the lung ultrasound has received increasing attention from neonatal physicians. Nevertheless, clear reference standards and guideline limits are needed for accurate application of this diagnostic modality. This document aims to summarize expert opinions and to provide precise guidance to help facilitate the use of the lung ultrasound in the diagnosis of neonatal lung diseases.

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

Ultrasound is a safe bedside imaging tool that obviates the use of ionizing radiation diagnostic procedures. Due to its convenience, the lung ultrasound ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

High Content Screening in Neurodegenerative Diseases

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

An Improved Method for Rapid Intubation of the Trachea in Mice

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice