Potential Microorganisms from Bronchial Lavage Fluid in Bronchiectasis Patients: Bacteria, Nontuberculous Mycobacteria, and Fungi

2.1. Study Design and Eligible Patients

This study was an observational, multicenter study conducted at the endoscopy units of Cho Ray’s Hospital and University Medical Center Ho Chi Minh City, from January 2023 to February 2024. Adult patients were enrolled according to the inclusion criteria as follows: (1) the patient was diagnosed with bronchiectasis based on the criteria of the British Thoracic Society guidelines 2019 [11], (2) the patient’s disease was stable, which was defined as the absence of exacerbation [12] and no antibiotic treatments during the four weeks prior to their bronchoscopy, (3) the patients were given bronchoscopy for collecting BLF, and agreed to take part in this study. Exclusion criteria included retraction bronchiectasis, active pulmonary tuberculosis, or failure to undertake bronchoscopy. The study received approval from the Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (528/HĐĐĐ-ĐHYD). All participants provided informed consent to take part in this study.

2.2. Data Collection

Following enrollment in the study, clinical data were collected for each patient, including demographic characteristics, comorbidities, clinical symptoms, radiological findings (morphology and location of bronchiectasis), forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC), and complete blood count (CBC). The neutrophil-to-lymphocyte ratio (NLR) was calculated by dividing neutrophil count by lymphocyte count. The Bronchiectasis Severity Index (BSI) including features such as age, body mass index, FEV1% predicted, hospital admission before study, exacerbations before the study, modified medical research council dyspnea scale, Pseudomonas colonization, colonization with other organisms, and ≥ 3 involved lobes or cystic bronchiectasis was calculated by two pulmonologists. The modified Reiff score [13] assesses the radiological severity of bronchiectasis through the number of lobes involved and the degree of bronchial dilation (scoring 0-3 for each lobe, the patient’s lung having 6 lobes with the lingula segment considered as a lobe, and the maximum score 18). The etiologies of bronchiectasis were determined according to the European Respiratory Society guideline 2017 [1] and depended on the physician’s judgment.

2.3. BLF Collection

Flexible bronchoscopy was undertaken according to the conventional protocols of the University Medical Center Ho Chi Minh City and Cho Ray’s Hospital. Pre-procedure preparation included fasting for at least six hours and normal results for CBC and coagulation status. Pre-medication for bronchoscopy included subcutaneous atropine 0.5 mg and nebulizer with a combination of lidocaine 2% 2mL and salbutamol 5 mg/ 2.5 mL.

A bronchoscope (Olympus EVIS EXERA III CLV-190, Olympus Corporation, Tokyo, Japan) was inserted into the tracheobronchial tree via the nasal or oral route under local anesthesia with lidocaine 2%. The patients were monitored for both blood pressure and peripheral capillary oxygen saturation (SpO₂) throughout the procedure. BLF was collected from the target lesion in the third bronchial segment, as previously identified on the images of chest computed tomography (CT). A 60-80 mL volume of normal saline 0.9% was instilled into the targeted bronchial branch in aliquots, and fluid recovery was facilitated using a suction kit. The collected BLF was sent to the laboratory to evaluate acid-fast bacillus (AFB) smear, bacterial and fungal culture, and detection for mycobacterium tuberculosis (MTB) and NTM.

2.4. Bacterial and Fungal Culture

After collection, BLF samples underwent standard microbiological analysis, starting with a Gram stain to determine initial bacterial morphology. For bacterial culture, the samples were inoculated on sheep blood agar (SBA), chocolate agar (CA), and MacConkey agar (MC). SBA and CA plates were incubated at 35-37°C in 5% CO2 for 16-24 hours, while MC plates were incubated under similar conditions but in a standard aerobic incubator. Following initial morphology identification, automatic identification and antibiotic susceptibility testing were performed using the Vitek 2 Compact (bioMérieux, Marcy-l'Étoile, France). For fungal culture, the samples were inoculated on Sabouraud Chloramphenicol agar and incubated at 35°C for 7 days. Following initial morphology identification, yeasts were identified automatically using the Vitek 2 Compact, and moulds were stained with Lactophenol Cotton Blue and identified under the support of a microscope.

Multidrug-resistant (MDR) bacterium was defined as a pathogen acquired non-susceptibility to at least one agent in three or more antimicrobial categories [14], including Haemophilus influenzae and Achromobacter denitrificans. Pathogens with intrinsic multi-drug resistance (Chryseobacterium indologenes, Stenotrophomonas maltophilia) were classified as MDR isolates.

2.5. Mycobacterial Culture and Identification

After BLF collection, an AFB smear was performed. For culture, 0.5 mL BLF was inoculated into a BACTEC MGIT 960 tube (BD, Sparks, USA) and 0.2 mL onto two solid Lowenstein-Jensen (LJ) slants. MGIT tubes were incubated at 37 °C for up to 42 days and LJ slants were examined weekly for 8 weeks. Any positive MGIT growth or LJ colonies were confirmed as AFB by Ziehl–Neelsen staining and PCR for MTB; cultures without growth by the end of incubation were reported negative.

2.6. Molecular Detection and Identification of NTM and MTB

2.7. DNA Extraction and PCR Assay Protocol

DNA was then extracted from BLF samples using ^NKDNARNAprep-MAGBEAD kit (Nam Khoa Company, Ho Chi Minh City, Vietnam), employing the KingFisher system (Thermo Fisher Scientific, Waltham, MA, USA). This kit includes dedicated binding, washing, and elution buffers and follows a standardized five-step automated protocol that ensures consistent quality across extractions. The method has been validated for its efficiency and sensitivity across a range of clinical samples and pathogens, demonstrating non-inferior performance compared to commercial kits such as MagnaPure (Roche), QIAGEN, BOOM, and Trizol-LS.

5 µL of DNA extraction samples were added to 15 µL of each reaction mixture. To detect potential false negatives, we employed internal control, including the 16S rRNA gene for microbial presence. The PCR assays were performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The thermal cycling protocol began with DNA denaturation at 96°C for 10 minutes, followed by 35 cycles of denaturation at 98°C and incubating at 60°C for 30 seconds each, ending with a hold at 10°C.

To ensure contamination control, in addition to performing bronchoscopy strictly under institutional protocol, DNA extraction and PCR setup were carried out in physically separated pre- and post-PCR rooms. Work surfaces and equipment were disinfected and UV-irradiated before and after each session. Staff used aerosol-resistant filter tips, strict aseptic technique, and protective gloves. Every run included negative controls; any suspected assay was discarded and repeated. After amplification, samples were assessed for sensitivity, and positives were confirmed when all fluorescent channels reached a cycle threshold below 35.

2.8. Statistical Analysis

Based on Fujita et al. [15], who reported an NTM detection rate of approximately 47.8% in bronchiectasis patients, we calculated that enrolling 96 participants

would allow estimation of this prevalence with a ±10% margin of error at the 95% confidence level. We ultimately enrolled 99 patients, thereby satisfying this requirement. The normality of the data was evaluated using histograms and the Kolmogorov-Smirnov test. Means (± SD, standard deviation) were used to present normally distributed data, whereas medians [IQR, interquartile range] were used for data that were not normally distributed. Proportions or percentages describe categorical data. For normally distributed variables, the t-test was employed, and the Mann-Whitney U test was applied to variables that were not normally distributed. A two-sided p-value of less than 0.05 was considered statistically significant. SPSS statistical software (version 22.0, IBM Corp, Armonk, NY, 2017) was used to process the data.

3.1. Demographic and Clinical Characteristics of Study Subjects

Between January 2023 and February 2024, 112 patients were eligible for our study. Of these, 2 refused to participate and 11 were excluded after Mycobacterium tuberculosis was detected in the BLF specimens (either by AFB smear, PCR, or culture for tuberculosis), leaving a total of 99 cases included in the final analysis (Fig. 1). The mean age was 63 ± 12 years, with 60.6% of patients being over 60 years old. Females were slightly predominant, comprising 55.6% of the cases. Detailed demographic and clinical data are presented in Table 1. All cases had multiplex PCR for NTM and MTB detection in BLF, but bacterial and fungal cultures were performed on 86.9% (86/99) and 69.7% (69/99) of cases, respectively, depending on the physician’s adjustment. NLR showed an association with the severity of bronchiectasis defined by BSI (r = 0.373, p = 0.001).

3.2. Bacterial Profiles from BLF of Bronchiectasis Patients

Positive bacterial cultures were identified in 36 (41.9%) cases, with the frequency distribution of organisms detailed in Fig. (2). The most frequent isolates were Klebsiella pneumoniae (28.2%) and P. aeruginosa (25.6%). Polymicrobial isolations were observed in three cases: K. pneumoniae + H. influenzae, Staphylococcus aureus + H. influenzae, and Stenotrophomonas maltophilia + Chryseobacterium indologenes. Patients with dry cough had higher bacterial yields (p = 0.013). Additionally, older patients and those with higher BSI also showed higher positive culture (Table 2).

MDR bacteria were determined in 35.9% (14/39) of the isolated organisms. Supplement Table S2 describes antimicrobial resistance profiles of bacteria isolated from BLF. Patients with MDR isolate had higher BSI scores but no statistical significance (6.0 [3.5 – 11.0] vs. 4.0 [3.0 – 6.0], p = 0.123) and significantly higher episodes of hospitalization in the previous year (76.9% vs. 16.7%, p < 0.001) than those with susceptible isolates.

3.3. Non-tuberculous Mycobacteria in Patients with Bronchiectasis

NTM was found in 52.5% of patients, with 90.4% being slowly growing species. Patients with frequent expectoration were more likely to have NTM detected than those in the dry cough group (p = 0.029). There were six cases with two NTM species to be simultaneously detected (two cases with M. avium-intracellulare complex + M. szulgai, one case with M. avium-intracellulare complex + M. xenopi, two cases with M. szulgai + M. tilburgii, one case with M. abscessus complex + M. chelonae). Distribution of NTM species was presented in Fig. (3), in which M. xenopi and Mycobacterium avium-intracellulare complex (MAC) were the most common. Patients with lower BSI score had a significantly higher frequency of NTM detection (Table 3).

3.4. Fungal Characteristics in BLF from Bronchiectasis Patients

Among the 69 BLF samples for fungal culture were performed, fungal species were detected in 17 cases (14 cases with Candida spp, one case with both Candida spp and Penicillium spp, and two cases with Aspergillus spp). Fungi were detected more frequently in cases with greater bronchial dilation on chest CT according to the modified Reiff score (3.0 [2.0 – 5.0] vs. 2.0 [1.3 – 4.0], p = 0.044).

4.1. Bacterial species

In our study, P. aeruginosa and H. influenzae remained the frequently detected organisms in patients with stable bronchiectasis, consistent with findings from previous studies [18, 19]. These organisms were associated with more severe disease and a greater incidence of exacerbation [2, 20]. The antimicrobial resistance of these bacteria, a great concern, can develop in the follow-up study of Wagner et al [21] due to antibiotic therapy and chronic bacterial infection. However, they were still susceptible in our observational study, and one of the reasons could be that the majority of mild bronchiectasis cases were accounted for in our cohort.

K. pneumoniae emerged as the most common bacterium detected in BLF of our bronchiectasis patients, which was similar to the Indian bronchiectasis cohort [22]. Understanding its role in bronchiectasis patients still needs to be improved. Therefore, in-depth studies about the effects of K. pneumoniae in bronchiectasis patients is required. Moreover, K. pneumoniae resistant to carbapenems and fluoroquinolones was common in our study, along with another unusual bacterium (Acinetobacter baumannii). This finding should be interpreted in the context of nosocomial bacteria in a country with a high prevalence of antimicrobial resistance, such as Vietnam. Notably, 35.9% of isolated bacteria in BLF had antimicrobial resistance. These isolations may originate in hospital settings, as evidenced by the fact that these patients experience significantly more frequent exacerbations requiring hospitalization, which aligns with the previous study [23]. It is uncertain whether bronchiectasis patients with MDR bacteria have worsening long-term clinical outcomes. The high isolated rate of MDR strains indicates a need to revise infection control policies, especially by enhancing hygiene and disinfection protocols, selecting the appropriate strategy for antibiotic therapy (based on the antimicrobial susceptibility test, the preferred option of inhaled/nebulized antibiotics with a lower resistance rate) [23], and even propose the strict criteria of hospitalization for bronchiectasis patients in Vietnam.

4.2. NTM Detection

The globally estimated prevalence of NTM infection in bronchiectasis patients is about 10% [24], and this prevalence in the Asian population is 9.5% (4.6% - 18.7%) [25]. However, this rate varies due to multiple factors, including differences in microbiological detection methods, the practice preferences of each medical center regarding mycobacterial culture indications (routine testing vs. testing based on clinical suspicion), the types of respiratory samples used for analysis, and geographical influences [10, 26-28]. The NTM detection rate in bronchiectasis patients was higher in the United States at 63% (1158/1826) [10] and on the rising trend at European and Asian countries in recent years. The study by Suska et al in 2022 conducted in Italy had a prevalence of 26.1% (the previous rate 12.2%) [29]. Using multiplex PCR technique and BLF, our study demonstrated that NTM is commonly found in Vietnamese patients with bronchiectasis (52.5%), likewise a study in Japan employing BLF samples [15] and another study in China using newer microbiology detection techniques with MPB64 antigen [30] have reported higher detection rates (47.8% and 61.2%, respectively).

MAC and M. xenopi were the most common NTM species in our study. The distribution of NTM species changes according to geographic features, and MAC was documented as the most frequently detected mycobacteria in bronchiectasis patients, especially in the United States [10, 31-33]. On the other hand, M. xenopi was reported more commonly in Western European countries. However, NTM studies in Northern Vietnam and Cambodia revealed no M. xenopi detected [34, 35]. This finding could imply that the distribution of NTM species varies between regions in Vietnam.

The clinical implications of NTM detection in bronchiectasis patients remain a matter of active investigation, with mounting evidence suggesting that NTM may play a pathogenic role beyond mere colonization. Fujita et al. conducted a prospective cohort study comparing frailty in patients with NTM lung disease and bronchiectasis, demonstrating that those with NTM infection exhibited significantly higher frailty indices and worse functional outcomes, implicating NTM in disease progression and poorer clinical prognosis [15]. In our study, NTM was more frequently detected in milder cases of bronchiectasis, as measured by the BSI. This finding contrasts with the study from Italy, in which patients had the more severe disease and immunodeficiency as a common etiology of bronchiectasis [29]. This difference could result from discrepancies in the study population, the detection method of NTM, and the type of specimen employed. Our study used BLF via bronchoscopy, which can lead to more cases of milder bronchiectasis (dry cough and local bronchiectasis) being enrolled. The presence of NTM in mild patients suggests that NTM can exist from the earlier stages of the disease, potentially triggering airway inflammation and damage that later leads to more severe bronchiectasis. Detecting NTM in mild disease may serve as an “early warning” prompting closer monitoring of lung function, frailty, and quality of life. Routine BLF screening for NTM, regardless of disease stage, could enable earlier diagnosis and timely, species-specific multidrug therapy. Adding longitudinal follow-up with frailty assessments, serial imaging, and biomarkers can help distinguish true NTM disease from benign colonization and gauge the impact of treatment on exacerbations and radiological progression.

4.3. Fungal Profiles

Our findings also revealed that Candida spp and Aspergillus spp were the most frequently isolated fungal species. This aligns with results from other studies [31, 36] but did not show the association between previous pulmonary tuberculosis and Aspergillus spp detection, which was mentioned in the study of Yang et al [37]. The study of Cheng et al revealed that the Reiff score can be useful in predicting disease severity and prognosis of bronchiectasis patients [38]. Moreover, our study described the positive fungal culture existing in more severe bronchiectasis on radiography, as defined by higher modified Reiff scores. However, it remains unclear whether these fungi merely colonize the airways or contribute to bronchiectasis progression or trigger exacerbations. Further studies are needed to explore their role in bronchiectasis.

Our study has several limitations. First, the limited sample size prevented a detailed analysis. Second, bacterial and fungal cultures of BLF were conducted at the discretion of the physician rather than uniformly across the entire study sample. However, this approach reflects real-world practice in limited-resource settings like Vietnam, where tuberculosis diagnostics often take precedence with less concern for bacteria and fungi in patients with stable bronchiectasis. In addition, we did not analyze which subjects with NTM isolation have NTM pulmonary disease. We applied homogenized and strict protocols to collect BLF and to detect NTM in BLF, but the minimal contamination issue remains unavoidable. Finally, the microbiological results may reflect the local microbiological etiology in bronchiectasis, specific to Southern Vietnam, which may not represent the significant geographical diversity.