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* From the Departments of Pulmonary and Infectious Diseases (Drs. Sasabayashi, Tsushima, and Hatayama), Endoscopy (Dr. Yamazaki), and Laboratory Medicine (Dr. Okabe), Shinshu University School of Medicine, Matsumoto, Japan.
Correspondence to: Yoshitaka Yamazaki, MD, PhD, Department of Endoscopy, Shinshu University School of Medicine, Asahi, Matsumoto, 390-8621, Japan.
Abstract
Background: Bronchoscopic microsampling (BMS) is a method in which a device consisting of a wire with a polyester probe at the tip is used to collect bronchial epithelial lining fluid with bronchoscopy. In this study, we bacteriologically investigated sample collection using BMS to incorporate BMS into diagnosis of respiratory infection.
Methods: Strains of Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, and Mycobacterium avium complex (MAC), were used for experiments. In the standard sampling procedure using BMS, the probe coming out of the sheath was immersed in approximately 6 x 106 cfu/mL bacterial suspension for 30 s and cut into a tube containing 1 mL of normal saline solution. The tube was stirred for 1 min using a vortex. The sampling rate was calculated by the following equation: (actual amount of bacteria collected by BMS [colony forming units per milliliter])/(bacterial amount in suspension for sampling [colony forming units per milliliter]) x 100 (percentage).
Results: The sampling rate of S pneumoniae, H influenzae, and MAC showed no significant difference among three bacteria, but the sampling rate of P aeruginosa was higher. The shortened time of sampling, stirring, and the reduced bacterial amount in the suspension (1/100) did not significantly affect the rates of standard procedure. In contrast, in comparison with a protected specimen brush (PSB), the recoveries of S pneumoniae, H influenzae, and MAC using PSB were significantly lower than those by BMS, but the recovery of P aeruginosa was not significantly different.
Conclusion: This in vitro study might suggested the usefulness of BMS as a new diagnostic technique capable of quantitative and stable sampling.
Key Words: BAL • bronchial epithelial lining fluid • bronchoscopic microsampling • protected specimen brush
Bacteriologic tests such as sputum culture and smear are important particularly for the diagnosis of bacterial pneumonia among respiratory infections. In contrast, for patients with specific pathologic conditions, such as severe pneumonia and ventilator-associated pneumonia (VAP), bronchoscopic detection of the pathogen is recommended.12345 BAL used for this test is prepared by infusing 50 to 150 mL of normal saline solution through a bronchoscope, and collecting the saline solution with bacteria. Bacteria are collected by lavage of the bronchus over the alveolar area, but lavage is frequently contaminated with oral indigenous bacteria. In the diagnosis of VAP, diagnostic criteria based on bacterial amounts are employed to exclude oral and skin indigenous bacteria. Bacteria present at concentrations of 
1 x 103 cfu/mL in a protected specimen brush (PSB) sample and/or 1 x 104 cfu/mL in BAL are considered to be pathogenic. The level in BAL required for diagnosis is one-order higher, suggesting that PSB is more useful.26
There have been some reports4567 on the usefulness of PSB for the diagnosis of VAP. Characteristics of PSB are as follows: (1) the sampling brush is covered by a sheath, and (2) the tip of the sheath is plugged, which reduces contamination by indigenous bacteria even though the brush is inserted through the channel for forceps of a bronchoscope. Bronchoscopic microsampling (BMS) is covered by a sheath but not plugged.
Since PSB is capable of sampling pus components from lesions by inserting the brush with bronchoscopic guidance, it may increase the amount of sampled bacteria in clinical cases.7 Wimberly et al7 described that the weight determinations of PBS before and after based on quantitative cultures indicated that the brush accumulates approximately 0.001 mL (1 µL) of sample. It is possible that the brush may accumulate different quantities of purulent secretions. However, even a 10-fold variation in specimen size would give a difference of only one log unit with quantitative bacterial counts.
BMS is a new procedure for bronchoscopic diagnosis, capable of collecting local bronchial epithelial lining fluid (ELF).8910 A polyethylene-absorbable probe 30 mm in length and 1.1 mm in diameter is attached to a wire tip, and the wire is stored in a plastic guide sheath (Fig 1 ). BMS is performed while observing the bronchial lumen using a bronchofiberscope; when the target site is reached, the probe is pushed out of the sheath tip and absorbs bronchial epithelial fluid present in the bronchial lumen. Ishizaka et al8910 analyzed various cytokines in patients with ARDS using this method, and found that BMS was as useful as BAL for effective and less-invasive sampling of bronchial ELF.
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Materials and Methods
Bacterial Strains and Culture
The bacterial strains used were S pneumoniae (IID553 [NYSDHDP-2]), H influenzae (ATCC9833), P aeruginosa (ATCC27107), and MAC 104 strain (provided by Dr. L.E. Bermudez, Oregon State University). Cryopreserved (– 78°C) bacteria were thawed, and S pneumoniae and P aeruginosa were cultured on a blood agar plate (Becton Dickinson; Franklin Lakes, NJ), H influenzae was cultured on a chocolate agar plate (Becton Dickinson), and MAC was cultured on Middlebrook 7H11 medium (Becton Dickinson) at 37°C in 5% CO2. S pneumoniae and P aeruginosa were subcultured overnight and used. MAC was subcultured for 10 days and used.
Preparation of Bacterial Suspension
S pneumoniae, H influenzae, P aeruginosa, and MAC were collected using a cotton swab and suspended in a plastic tube containing 3 mL of normal saline solution. The turbidity was adjusted using saline solution and an McF assay meter (bioMerieux; Marcy l’Etoile, France), and the concentration of bacterial suspensions for sampling was adjusted to approximately 6 x 106 cfu/mL. The suspension (2 mL) was added to a 3-mL plastic tube, and 100 µL was collected using a pipette and combined with 900 µL of saline solution for 1/10 dilution, and 100 µL of each dilution was inoculated and homogeneously spread using a Conradi stick on an agar plate. We particularly paid attention to the two points listed below: (1) the probe was completely immersed in bacterial suspension, and (2) all procedures were performed on a clean bench to exclude contamination.
Standard Procedure
A probe (polyethylene, 30 mm in length, 1.1 mm in diameter) attached to the tip of a microsampling device (BC-401C; Olympus; Tokyo, Japan) was pushed 5 cm out of the sheath. The probe was placed in the bacterial suspension, pulled out after 30 s, and stored back in the sheath. The probe was then pushed out of the sheath, and the sponge region was cut with ethanol-disinfected scissors. The probe was placed in 1 mL of saline solution in an Eppendorf tube. The tube was immediately stirred for 1 min using a vortex. Serial dilutions were prepared and inoculated on agar plates, and colonies were counted. The bacterial sampling rate was calculated using the following equation: BMS sampling rate = actually sampled bacterial amount (colony forming units per milliliter)/bacterial amount in suspension for sampling (colony forming units per milliliter) x 100 (percentage).
Revision of the Study Parameters
Sampling and Stirring Times:
Shortening of the treatment time in the standard BMS method was investigated. The duration of immersing a probe into bacterial suspension for sampling (approximately 6 x 106 cfu/mL) was shortened to 5 s (30 s in the standard method). Next, the stirring time of the probe after sampling bacteria using a vortex was shortened to 10 s (1 min in the standard method).
Sampling Amount of Bacteria: To investigate whether BMS is capable of stably collecting bacteria when the sampling amount is changed, the bacterial suspension for sampling was diluted to 1/100. This solution was also subjected to colony counting. The sampling time was 30 s, and stirring, preparation of serial dilutions, and colony counting were performed by the standard procedure.
Time of Standing (Storage): Since samples collected by BMS may not be processed immediately after sampling in clinical application, the stability of bacteria sampled by BMS within the storage time was investigated. After sampling by the standard procedure, the probe was kept in an Eppendorf tube at room temperature for 3 h, followed by stirring, preparation of serial dilutions, and colony counting by the standard procedure.
Surfactant: It is important to investigate any possible inconsistency in the bacterial amount due to bacterial adhesion to the sampling device, which reduces the apparent bacterial number. Physiologic saline solution containing 0.1% surfactant (Tween80; Sigma; St. Louis, MO) was prepared to inhibit bacterial adhesion to probes and tubes. Tween solution (1 mL) was added to the Eppendorf tube prepared by the standard procedure and stirred for 1 min using a vortex, followed by preparation of serial dilutions with Tween solution and colony counting.
Current Test Using a PSB
The PSB is a device with a brush covered with a sheath and distal occlusion composed of polyethylene glycerol. In the present study, we used a sheath brush (disposable cytology brush, BC-202D-2010; Olympus) immersed in a bacterial suspension for sampling (6 x 106 cfu/mL) for 30 s and stored in the sheath. The brush was then pushed out of the sheath, cut with ethanol-disinfected scissors, and placed in an Eppendorf tube containing 1 mL of saline solution. After stirring, the preparation was serially diluted, and colonies were counted by the standard procedure.
Statistical Analysis
All experiments were repeated three times, and the means ± SD were calculated. For between-group comparison, the Mann-Whitney U test was used; p < 0.05 was regarded as significant.
Results
Investigation of the Standard Procedure
The bacterial amount in the suspension for sampling was 5.90 ± 2.42 x 105 cfu/mL in S pneumoniae suspension, 6.87 ± 4.04 x 106 cfu/mL in H influenzae suspension, 7.83 ± 1.80 x 106 cfu/mL in P aeruginosa suspension, and 3.47 ± 1.97 x 106 cfu/mL in MAC suspension. The bacterial amounts collected on the BMS probe were 9.10 ± 4.19 x 104, 9.17 ± 4.91 x 104, 23.7 ± 8.62 x 104, and 4.27 ± 3.57 x 104 cfu/mL, respectively. The sampling rates were 1.41 ± 0.02%, 1.40 ± 0.17%, 3.29 ± 1.64%, and 1.22 ± 0.53%, respectively, showing no significant difference among the S pneumoniae, H influenzae, and MAC sampling rates, but the P aeruginosa sampling rate was significantly higher than the other three species (p < 0.05) [Table 1
]. Colonies formed on agar medium were carefully observed, but no contaminating environmental bacteria or fungi were noted.
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Changes in the Bacterial Amount in Suspension for Sampling: The investigation was performed using 1/100-diluted bacterial suspensions for sampling. The amounts of S pneumoniae, H influenzae, P aeruginosa, and MAC in the dilutions were 5.57 ± 3.41 x 104, 1.97 ± 0.42 x 104, 4.17 ± 1.31 x 104, and 1.67 ± 0.60 x 104 cfu/mL, respectively. The amounts of bacteria collected on the probe were 12.9 ± 8.15 x 102, 7.37 ± 3.07 x 102, 5.83 ± 0.80 x 102, and 1.23 ± 0.97 x 102 cfu/mL, respectively. The sampling rates were 2.40 ± 1.23%, 3.88 ± 1.80%, 1.86 ± 0.58%, and 0.66 ± 0.32%, respectively, showing no significant difference among the four species (Table 2 ).
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Surfactant (0.1% Tween Solution): The S pneumoniae, H influenzae, P aeruginosa, and MAC sampling rates were 1.94 ± 0.65%, 1.79 ± 0.35%, 4.64 ± 0.93%, and 1.97 ± 0.82%, respectively, showing that the P aeruginosa sampling rate was significantly higher among the four species (p < 0.05), but no significant differences from the those obtained by the standard procedure were noted.
Stirring Time Using a Vortex: The stirring time was shortened to 10 from 60 s in the standard procedure. The S pneumoniae, H influenzae, P aeruginosa, and MAC sampling rates were 1.47 ± 0.15%, 2.70 ± 1.60%, 3.37 ± 2.25%, and 1.37 ± 0.72%, respectively, showing no significant difference from those obtained by the standard procedure.
Comparison With PSB: When PBS was used for sampling, the S pneumoniae, H influenzae, P aeruginosa, and MAC sampling rates were 0.09 ± 0.02%, 0.42 ± 0.33%, 2.85 ± 0.48%, and 0.04 ± 0.02%, respectively, showing that the S pneumoniae, H influenzae, and MAC sampling rates obtained by the standard BMS procedure were significantly higher (p < 0.05), but the P aeruginosa sampling rate was not significantly different between sampling using BMS and PSB (Table 3 ).
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This in vitro bacteriologic study was performed to introduce BMS as a diagnostic aid for infectious diseases in vitro, and the procedure of extracting various cytokines from bronchial ELF reported by Ishizaka et al89 was used as the standard procedure: (1) sampling time was 30 s; (2) the probe was cut and placed in a tube containing saline solution; (3) the probe was stirred for 60 s using a vortex to dislodge bacteria adsorbed to the probe; and (4) 1/10 serial dilutions were prepared and subjected to colony counting. The ratio of the amount of bacteria sampled by the standard BMS procedure to the amount in suspension for sampling was calculated, and the value was almost constantly 1 to 3%, indicating that when 33 to 100 bacterial cells are present per 1-mL sample, one bacterium can be detected using BMS. Since 1 x 104 cfu/mL bacteria are present in BAL from patients with VAP collected using a bronchoscope, bacteria are theoretically detectable using BMS.118
Regarding the relationship between the amounts of bacteria and water collected by BMS, interestingly, the amount of bacteria was proportional to the collectable water amount. In BMS, bacteria are adsorbed together when water is adsorbed to the BMS probe. According to Ishizaka et al,89 one probe adsorbs 2 to 20 µL, which corresponds to 0.2 to 2% of 1 mL, and the sampling rate of approximately 1 to 3% in our experiments was consistent. BMS is capable of collecting 20 µL of bronchial ELF and all bacteria in the ELF, which may contain mucus and pus depending on the local lesion. Thus, detection using BMS is sufficiently quantitative. Regarding the sampling rate of each bacterial species, no significant difference was noted among S pneumonia, H influenzae, and MAC, but the P aeruginosa sampling rate was significantly higher than the other three species. Although the reason was not clarified, P aeruginosa may easily adhere to polyethylene tubes.
The amount of bacteria for sampling, sampling time, stirring time, and use of surfactant were investigated by modifying the standard procedure using the four bacterial species. Regarding the bacterial amount for sampling, when the suspension was diluted to 1/100, the sampling rate did not change, showing that the bacterial amount in suspension does not affect the sampling rate using BMS. When the sampling time was changed from 30 to 5 s, and the stirring time was changed from 60 to 10 s, the sampling rate was not affected, indicating that bacteria are easily dislodged from the probe. A surfactant was added during stirring to investigate its effect on bacterial recovery, but the sampling rate was not changed compared to stirring in saline solution alone. Thus, the shortened standard procedure may provide equivalent results and make the procedure more easily applicable in clinical practice.
Investigation of storage conditions (standing time) after sampling is also important. Transport of samples to the site of bacterial testing may often take time in clinical practice. When the probes after sampling were kept in tubes containing saline solution for 3 h, the bacterial amounts of S pneumoniae, H influenzae, P aeruginosa, and MAC were not changed compared to those processed by the standard procedure, clarifying that processing of samples within 3 h after sampling is acceptable for clinical practice. Although we did not investigate storage overnight or at 4°C, according to Forceville et al,19 when bacteria in bronchoscopic samples were quantified after storage at 4°C for 48 h, no changes were noted in the amounts of Staphylococcus, Enterobacteriaceae, and Pseudomonas, showing that storage is possible, but Haemophilus decreased after storage. Since Haemophilus easily perishes at low temperature, and storage conditions vary among species, the understanding of individual bacterial characteristics is necessary to evaluate the results.
The sampling rates using PSB and BMS were compared in vitro. The usefulness of PSB for diagnosis of respiratory infections has been reported.1 PSB is similar to BMS in the following ways: the brush is protected by a sheath and comes out of the sheath only during sampling, avoiding contamination with oral indigenous bacteria, and bacteria are directly collected from the lesion. In the experiment using PBS, the sampling rate was not constant among the bacterial species, and the P aeruginosa sampling rate was markedly high. On comparison with BMS, the recoveries of S pneumoniae, H influenzae, and MAC using PBS were significantly lower than those using BMS, but the recovery of P aeruginosa was similar. Since only bacteria adhered to the PBS brush are recovered when bacteria are directly sampled from bacterial suspension, the amount of the three bacterial species collected using PBS were naturally lower those collected using BMS, but P aeruginosa was highly adherent to the brush. The sampling rate may be higher when purulent secretion is collected from local lesions of infection.
This in vitro study clarified that BMS is capable of the quantitative sampling of bacteria. Further studies are necessary to clarify its applicability as a diagnostic device, as have been performed for PSB and BAL, which have been applied to clinical cases and reported to be useful.
Footnotes
Abbreviations: BMS = bronchoscopic microsampling; ELF = epithelial lining fluid; MAC = Mycobacterium avium complex; PSB = protected specimen brush; VAP = ventilator-associated pneumonia
Dr. Yamazaki was partially supported by a research grant-in-aid from the Japanese Foundation for Research and Promotion of Endoscopy (2006). The other authors have no conflicts of interest to disclose.
Received for publication April 18, 2006. Accepted for publication August 31, 2006.
References
This article has been cited by other articles:
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  E. Kipnis, B. Guery, and J. Wiener-Kronish Promises and Limitations of the Bronchoscopic Microsampling Probe Chest, October 1, 2007; 132(4): 1414 - 1414. [Full Text] [PDF]
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