Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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 ...

Dyskusje

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

Materiały

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