Comparison of metagenomic next-generation sequencing (mNGS) technology with routine laboratory culture for bacterial and fungal detection in bronchoalveolar lavage fluid

Introduction

Pulmonary infections remain a serious public health issue worldwide, characterized by high incidence and mortality rates, particularly among the elderly and immunocompromised populations (1,2). Pulmonary infections can be caused by viruses, bacteria, parasites, and fungi, including community-acquired pneumonia, hospital-acquired pneumonia, bronchitis, lung abscesses, fungal infections, and tuberculosis (3). Timely diagnosis and effective treatment can lead to a favorable prognosis for patients.

Currently, traditional diagnostic methods for pulmonary infections have limitations in terms of sensitivity and turnaround time, requiring the development of more efficient pathogen detection technologies. The conventional diagnostic method widely used in clinical practice is culture method in the laboratory. However, this method has the limitation of a lengthy culture cycle and cannot detect pathogens that are not detected by conventional culture, such as viruses and intracellular bacteria. For slow-growing pathogens, such as filamentous fungi like Mucor, the positive detection rate is low, and false-negative results may occur (4,5). Chest radiography (CR) screening can be used to detect lung infections, but its detection effectiveness is limited. Low-dose computed tomography (LDCT) may be more suitable for screening chest diseases, especially for detecting vascular changes (6).

In recent years, next-generation sequencing (NGS) technology has evolved from scientific research to clinical applications, initially used for cancer treatment and genetic disease screening. Currently, it has gradually expanded into the field of infectious diseases for rapid pathogen identification (7). Among these, metagenomic next-generation sequencing (mNGS) does not require culture and can detect all nucleic acids in a sample without bias, enabling rapid and accurate identification of pathogenic bacteria. Timely and effective pathogen detection methods are crucial for improving diagnostic accuracy and enabling targeted antimicrobial therapy.

In this study, we conducted a retrospective analysis of 50 patients with pulmonary infections that underwent concurrent culture method and mNGS testing of bronchoalveolar lavage fluid (BALF) at our hospital’s respiratory department from September 20, 2024, to November 20, 2024. We compared the differences between culture method and mNGS in terms of pathogen detection. We present this article in accordance with the STARD reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1647/rc).

Methods

Patients and samples

In this study, we retrieved cases from the Department of Respiratory Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Patients diagnosed with pulmonary infection by clinicians based on symptoms, signs, and relevant auxiliary examinations between September 20 and November 20, 2024, were initially screened. Fifty patients who received both BALF culture and mNGS were enrolled. Within the entire study cohort, thirty-four patients (68.0%) were male and sixteen (32.0%) were female, and the average age of patients was 62.9 years. We retrospectively analyzed their test results. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (2018-S356). Due to the retrospective nature of the study, patient consent for inclusion was waived. We ensure that the use of anonymized data posed minimal risk to patient privacy.

Culture method

The collected BALF should be placed in a sterile container and immediately sent to the microbiology laboratory for culture. Quantitatively inoculate 10 µL of BALF using a pipette and inoculate separately on blood plate and chocolate plate. Incubate the blood plate and chocolate plate overnight in a 5–10% CO₂ incubator at 35 ℃. After culture, exclude normal oral bacteria from the samples, count the colonies of suspected pathogens, and identify them. Negative report: No bacterial growth or normal oral bacterial growth, no Haemophilus influenzae detected. Positive report: Name of bacteria detected, colony count CFU/mL.

mNGS analysis

The collected BALF is placed in a sterile container and immediately sent to the sequencing laboratory for testing. The nucleic acid in BALF was extracted, enriched, and purified using the nucleic acid purification kit from Huada Gene Co., Ltd. (Wuhan, China). The obtained nucleic acids were cyclized reaction using a universal sequencing kit from Mgi Tech Co., Ltd. (Wuhan, China) to prepare deoxyribonucleic acid nanoball (DNB), and finally sequenced and analyzed on a gene sequencer (MGISEQ-200). After the sequencing is completed, the program will generate a standard sequence file.

Data collection

Review the results of culture method and mNGS testing of the patients’ BALF through the hospital’s laboratory information management system.

Statistical analysis

We were only responsible for the specimens and did not consider that poor sampling by doctors may lead to false-negative results, and all results were included in the statistical analysis. Statistical analysis was performed using SPSS 26.0 statistical software. A 4×4 table was constructed to obtain all data, including the detection rate. The McNemar^,s χ² test for matched 4×4 tables were used to compare the results of culture method and mNGS testing.

Results

Data analysis of clinical samples

Review the results of 50 patients who underwent simultaneous mNGS and culture of BALF. Detailed information is shown in Table 1. As shown in Table 1, mNGS can detect multiple pathogens, including bacteria, fungi, and viruses, within the same patient, whereas culture method generally detects only a small number of bacteria or fungi, and viruses cannot be detected. Therefore, we summarized two methods for detecting the number of pathogens. Table 2 shows that mNGS detected 23 bacteria, 19 fungi, and 17 viruses, for a total of 59 pathogens. Culture method detected 3 bacteria, 2 fungi, and no viruses, for a total of 5 pathogens. Using a combination of mNGS and conventional culture, a total of 26 bacteria, 21 fungi, and 17 viruses were detected, for a total of 64 pathogens.

We analyzed culture-positive specimens separately and listed the relative abundance of pathogens detected by mNGS. As shown in Table 3, Klebsiella pneumoniae (Kpn), Pseudomonas aeruginosa (Pae), Corynebacterium striatum, Aspergillus flavus, and Candida albicans (Alb) detected by culture were all detected by mNGS. The number of sequences detected by mNGS were as follows: Kpn was detected in 945,760 and 17,465, Pae was detected in 127 and 2466, Corynebacterium striatum was detected in 2,547,721, Aspergillus flavus was detected in 46, and Alb was detected in 271.

In addition, mNGS has significant advantages in virus detection, as culture method is currently unable to detect viruses. Furthermore, for pathogens that cannot be cultured in vitro, such as Pneumocystis jirovecii, mNGS technology can be used for detection.

Comparison of mNGS and culture method

As shown in Table 4, the detection rate of mNGS for fungi or bacteria was 72.0% (36/50), while that of culture was 12.0% (6/50). The McNemar^,s χ² test showed that the results of mNGS and culture method were statistically significant (P<0.0001). Among 50 patient samples, only 6 cases (12%) tested positive using both mNGS and culture method, 30 cases (60%) tested positive using mNGS alone, none tested positive using culture method alone, and 14 cases (28%) tested negative using both methods. In this study, bacteria and fungi detected by culture method could all be detected by mNGS, while bacteria and fungi detected by mNGS could not necessarily be detected by culture method. This demonstrates that mNGS has significant advantages in the diagnosis of bacterial and fungal infections.

In addition, our review of cases revealed that clinicians adjusted treatment for 18 patients after the mNGS results were obtained. As shown in Table 5, eight cases added antifungal therapy, seven cases adjusted antibiotic treatment, two cases adjusted both antifungal and antibiotic treatment, and one case adjusted antifungal treatment. Furthermore, our review of sample 19’s case revealed that after empirical antimicrobial therapy, the patient’s pulmonary inflammation improved and blood inflammatory markers decreased significantly (the result was not shown). According to the patient’s symptoms, the clinician considered the small amount of Aspergillus detected by mNGS to be colonization and did not provide antifungal treatment. Therefore, the patient’s treatment must be determined and modified based on actual symptoms and relevant laboratory results.

This study indicates that traditional culture method cannot satisfy the requirements of clinical diagnosis. In this study, the detection rate and number of pathogens detected by mNGS were higher than culture method. In addition, mNGS has an absolute advantage in detecting pathogens that cannot be cultured in vitro or cannot be cultured by conventional methods.

Discussion

Compared with conventional culture methods, mNGS has obvious advantages in terms of the number and types of bacteria and fungi detected. In addition, for uncultivable pathogens, such as Pneumocystis jirovecii, or pathogens that cannot be cultured by conventional methods, such as viruses, the detection rate of mNGS is significantly higher than conventional culture. In terms of testing time, as bacteria and fungi need to be cultured overnight before identification, the culture results for BALF in our laboratory are available two days later, while mNGS provides a report the day after the sample is submitted. For critically ill patients, rapid test results enable doctors to make timely diagnoses and provide patients with more effective treatment. In this study, bacteria and fungi detected by conventional culture showed the highest relative abundance in the corresponding mNGS. This indicates that pathogens can only be detected through culture once they have multiplied to a specific level. In addition, errors in the amount of sample taken during inoculation and the quality of the alveolar lavage fluid sample may affect the results of conventional culture. However, mNGS technology can detect all microorganisms in the sample, including bacteria, fungi, and viruses, without bias, and has broad spectrum and high sensitivity. Viruses are considered an important cause of hospitalization for patients with unexplained pulmonary infections (4,8). Polymerase chain reaction (PCR) technology is limited by the large number of virus types and subtypes (9,10), while mNGS solves the problem of virus detection with its broad spectrum. Diagnosing pulmonary infections in immunocompromised patients is particularly difficult. A study has shown that in patients with pulmonary infections following allogeneic hematopoietic stem cell transplantation, the positive rate of mNGS of BALF is significantly higher than that of conventional microbiological testing (11). In the detection of pathogens causing unexplained pulmonary infections, mNGS has a significantly higher detection rate than conventional culture, providing strong support for precise antimicrobial therapy and thereby reducing the use of broad-spectrum antibiotics. Therefore, mNGS may reduce antibiotic resistance in patients with pulmonary infections by improving treatment efficacy, demonstrating significant clinical value (12). In patients with mixed infections, conventional culture may fail to detect pathogens due to growth competition between bacteria or fungi, whereas mNGS can comprehensively detect the presence of pathogens. There have been reports of a critically ill patient with a mixed infection involving bacteria, fungi, viruses, and atypical pathogens. Conventional culture primarily detects bacteria and fungi, while mNGS supplements the detection of viruses and atypical pathogens. By combining the results of both tests and administering targeted treatment, the patient’s condition improved and he was discharged from the hospital (4). For critically ill patients who may be infected with atypical pathogens or mixed viral infections, mNGS testing should be performed promptly to improve their prognosis. In addition, for patients with severe infections, clinicians typically use empirical treatment to quickly control the infection and prevent the condition from worsening when the pathogen is not clearly identified (13,14). Empirical treatment typically involves the selection of broad-spectrum antibiotics to cover possible pathogens and reduce the risk of treatment failure. However, antibiotic exposure can affect the detection rate of routine microbial culture. Studies indicate that as the duration of antibiotic exposure increases, the positivity rate in culture decreases significantly while the positivity rate in mNGS remains stable, suggesting that mNGS exhibits lower sensitivity to antibiotic exposure (15). Therefore, mNGS testing has higher sensitivity in identifying pathogens and is relatively less affected by antibiotic exposure due to empirical treatment.

Although mNGS technology can detect all microorganisms in a sample, it is difficult to determine which ones are true pathogenic agents and distinguish between colonization and infection (16). Clinicians need to make judgments based on the specific circumstances of each patient, which requires high levels of interpretation of mNGS test results. Furthermore, mNGS cannot distinguish whether the nucleic acids detected are from live bacteria, dead bacteria, or free nucleic acid fragments (17,18). During the testing process, the laboratory environment, reagents, or equipment may be polluted with nucleic acids, which may affect the mNGS test results. Nucleic acid contamination may also be one of the reasons for the significant differences between the two detection methods in this study. mNGS testing is relatively expensive, which may increase the financial burden on patients. Additionally, there are certain limitations in this study. Our study is a retrospective analysis with a small sample size and a relatively single type of infection among patients. Further in-depth research is needed to compare the detection rates of mNGS and culture method.

Conclusions

In summary, mNGS technology has a distinct advantage in the detection rate of bacteria and fungi, effectively supplementing the shortcomings of conventional culture method. Furthermore, mNGS can identify viruses that cannot be cultured using conventional methods. Combining mNGS with conventional culture method can significantly shorten the pathogen detection time, improve detection efficiency, and help clinicians diagnose patients’ conditions more accurately, thereby initiating targeted treatment earlier, improving patient prognosis, and increasing survival rates.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1647/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1647/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1647/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1647/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (2018-S356). Due to the retrospective nature of the study, patient consent for inclusion was waived. We ensure that the use of anonymized data posed minimal risk to patient privacy.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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