Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

Abstract

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.

Introduction

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 cells^3,4. 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 prognosis^6,7. 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.

Protocol

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

1. 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).
2. Material preparation (see Table of Materials).
   1. 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).

2. M-ROSE workflow

1. Preparation of cytological slides
   NOTE: There are many ways to prepare slides, and the following are several commonly used preparation methods.
   1. Roll slides
      NOTE: Ensure minimal loss of tissue sample during this process.
      1. Extract tissue particles by using a disposable syringe needle with a capacity of 2.5-5 mL (Figure 2A).
      2. 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.
   2. 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.
      1. 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.
   3. Spray slides
      1. Position the puncture needle at one-third of the stained end of a sterile cytology slide with robust cell adhesion.
      2. 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.
2. Cytological slide staining
   NOTE: The World Health Organization (WHO) recommends rapid staining of ROSE cytological slide using Diff'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.
   1. 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).
   2. Next, immerse the slide in Diff B solution for 20-40 s (Figure 2E).
   3. 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.

Representative Results

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)-β-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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 3: Chest CT images. (A) At admission. (B) On the day of the transfer to the ICU. (C) After the treatment of sulfamethoxazole Please click here to view a larger version of this figure.

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 Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

Interventional pulmonology is an valuable branch of modern respiratory disease; in particular, it has been widely used in the diagnosis of lung diseases^8,9. 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 diseases^10,11,12. 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 "real-time accompany technique" 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 advancements^14,15,16,17. 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 fungi^3,18. 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'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'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'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.

Materials

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