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. 2022 Sep 27:12:1021320.
doi: 10.3389/fcimb.2022.1021320. eCollection 2022.

Clinical value of metagenomic next-generation sequencing by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid in suspected community-acquired pneumonia patients

Jing Zhang  1 , Lin Gao  1 , Chi Zhu  2 , Jiajia Jin  1 , Chao Song  2 , Hang Dong  2 , Zhenzhong Li  2 , Zheng Wang  1 , Yubao Chen  1 , Zhenhua Yang  1 , Yan Tan  1 , Li Wang  1
Affiliations

Clinical value of metagenomic next-generation sequencing by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid in suspected community-acquired pneumonia patients

Jing Zhang et al. Front Cell Infect Microbiol. .

Abstract

At present, metagenomic next-generation sequencing (mNGS) based on Illumina platform has been widely reported for pathogen detection. There are few studies on the diagnosis of major pathogens and treatment regulation using mNGS based on Illumina versus Nanopore. We aim to evaluate the clinical value of metagenomic next-generation sequencing (mNGS) by Illumina and Nanopore for the detection of pathogens in bronchoalveolar lavage fluid (BALF) in suspected community-acquired pneumonia (CAP) patients. BALF samples collected from 66 suspected CAP patients within 48 hours of hospitalization were divided into two parts, one for conventional culture and the other for mNGS by two platforms (Illumina and Nanopore). The clinical value based on infection diagnosis, diagnostic performance for main pathogens and treatment guidance were assessed. More types of species were detected by Nanopore than Illumina, especially in viruses, fungus and mycobacterium. Illumina and Nanopore showed similar detectability in bacterium except for mycobacterium tuberculosis complex/nontuberculosis mycobacteria. Pathogenic infection was established or excluded in 53 of 66 patients. There was little difference in the coincidence rate between Illumina and Nanopore with the clinical diagnosis, but both were superior to the culture (57.81%, 59.38%, 25%, respectively). Compared with Illumina, the diagnostic area under the curve of Nanopore was higher in fungi, but lower in bacteria and Chlamydia psittaci. There was no statistically significant difference between Illumina and Nanopore in guiding drug treatment (56.1% vs. 50%, p=0.43), but both were superior to the culture (56.1% vs. 28.8%, p=0.01; 50% vs. 28.8%, p=0.01). Single inflammatory indicators could not be used to determine whether the patients with culture-negative BALF were established or excluded from infection. The species detected at 1 h and 4 h by Nanopore were consistent to some extent, and its turn-around time (TAT) was significantly shorter than Illumina (p<0.01). Illumina and Nanopore both have its own advantages in pathogenic diagnosis and play similar roles in infection diagnosis and guiding clinical treatment. Nanopore has a relatively short TAT, which may be promising in rapid etiological diagnosis of acute and critically ill patients.

Keywords: Illumina; bronchoalveolar lavage fluid; community-acquired pneumonia; diagnostic value; metagenomic next-generation sequencing; nanopore; pathogenic identification.

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Conflict of interest statement

Author CZ, CS, HD, ZL were employed by Jiangsu Simcere Diagnostics Co. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Genus distribution of bacterium, fungus, viruses and chlamydiae detected by Illumina, Nanopore and culture. EBV, Epstein-Barr virus; CMV, Human cytomegalovirus; MTB, Mycobacterium tuberculosis; NTM, Non-tuberculous mycobacteria.
Figure 2
Figure 2
The consistency of pathogens detected by Illumina, Nanopore and culture.
Figure 3
Figure 3
Distribution of infection diagnosis in suspected CAP patients. (A), Diagnostic distribution and etiological classification of infection established in patients with suspected CAP. (B), Specific pathogens detected in bronchial alveolar lavage fluid from patients with established infection.
Figure 4
Figure 4
The clinical diagnosis based on Illumina, Nanopore and culture. (A), A Venn diagram of diagnosis based on the three methods. None accepted, none results of the three methods were accepted by clinicians. None confirmed, none results of the three methods were consistent with clinical diagnosis. (B), Comparison of the coincidence rate between the three methods with the clinical etiological diagnosis.
Figure 5
Figure 5
Comparison of diagnostic performance of Illumina and Nanopore for the main pathogens in BALF of patients with suspected CAP. (A), Confirmation/exclusion of pathogenic infection in 53 patients using Illumina, Nanopore and culture. *: The results were consistent with the clinical etiological diagnosis. (B), Contingency tables for the Illumina and Nanopore in detecting different classes of pathogenic microorganisms. Clinical etiological diagnosis was used as a reference method. (C). ROC curves stratified by classes of microbes.
Figure 6
Figure 6
Evaluation of the clinical utility of the three methods. (A), Distribution of antibiotic therapy and pathogens for targeted drug use. Continuous empirical medication, including unnecessary drug use for a specific pathogen and empiric treatment for an undefined pathogen. Transfer, Infectious pathogens detected should be treated at designated hospitals. Other, Drug modification according to other primary diseases. (B), Comparison of different methods to guide the treatment.
Figure 7
Figure 7
Diagnostic value of inflammatory indicators in culture-negative BALF of suspected CAP patients. (A), Comparison of inflammatory markers in different types of pathogenic infection. (B, C), ROC curves for inflammatory indicators. NLR, Neutrophil to lymphocyte count ratio.
Figure 8
Figure 8
Comparison of genus distribution at an hour and 4 hours using Nanopore technology. “*” represents targeted sequencing detection results.
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