Diagnosis and insight into the unique lung microbiota of pediatric pulmonary tuberculosis patients by bronchoalveolar lavage using metagenomic next-generation sequencing

Abstract

Background: Although previous studies have reported the dysregulation of respiratory tract microbiota in infectious diseases, insufficient data exist regarding respiratory microbiota imbalances in the lower respiratory tracts of children with pulmonary tuberculosis (PTB). In this study, we assessed the value of mNGS in the pathogen diagnosis and microbiome analysis of PTB patients using bronchoalveolar lavage fluid (BALF) samples.

Methods: A total of 64 participants, comprising 43 pediatric PTB and 21 pediatric pneumonia patients were recruited in the present study. BALF samples were collected from the above participants. Parallel comparisons between mNGS and conventional microbial test (CMT) pathogen detection were performed. Moreover, the diversity and structure of all 64 patients' lung BALF microbiomes were explored using the mNGS data.

Results: Comparing to the final clinical diagnosis, mNGS in BALF samples produced a sensitivity of 46.51%, which was lower than that of TB-PCR (55.00%) and Xpert (55.00%). The diagnostic efficacy of PTB can be highly enhanced by mNGS combined with TB-PCR (AUC=0.8140, P<0.0001). There were no significant differences in the diversity either between patients with TB and pneumonia. Positive mNGS pathogen results in pediatric PTB patients significantly affect the β-diversity of the pulmonary microbiota. In addition, significant taxonomic differences were found in BALF specimens from patients with PTB and pneumonia, both of which have unique bacterial compositions.

Conclusions: mNGS is valuable in the etiological diagnosis of PTB, and can reveal pulmonary microecological characteristics. For pediatric PTB patients, the mNGS should be implemented early and complementary to CMTs.

Keywords: bronchoalveolar lavage fluid; conventional microbial test; metagenomic next-generation sequencing; microecology; pediatric pulmonary tuberculosis.

Figure 1

Culture, TB-PCR, AFB, X-pert results and MTBC Reads of mNGS for all subjects. C, culture; P, TB-PCR; A, AFB; X, X-pert;/, NA, unavailable; +, positive; -. negative.

Figure 2

Comparison of α-diversity and β-diversity of the total microbial community in the lungs of pediatric patients with PTB and NTB. (A-C) α-diversity indexes of the total microbial community of the lungs between the two groups at genus level; (D-F) α-diversity indexes of the total microbial community of the lungs between the two groups at species level; (G-I) β-diversity indexes of the total microbial community of the lungs between the two groups at genus level; (J-L) β-diversity indexes of the total microbial community of the lungs between the two groups at species level.

Figure 3

The difference in the distribution of pathogens between PTB and NTB patients. (A, B) The relative abundance of microorganisms between the two groups at the genus and species level; (C, D) Anosim analysis between the two groups at the genus and species level; (E) ALDEx2 analysis was performed to compare microbiome data differences after central log-ratio transformation of data; (F, G) LEfSe analysis was performed to identify differentially abundant taxa, which are highlighted on the phylogenetic tree in cladogram format (F) and for which the linear discriminant analysis scores are shown (G).

Figure 4

Comparison of α-diversity and β-diversity of the total microbial community in the lungs of pediatric patients with Positive-PTB and NTB. (A-C) α-diversity indexes of the total microbial community of the lungs between the two groups at genus level; (D-F) α-diversity indexes of the total microbial community of the lungs between the two groups at species level; (G-I) β-diversity indexes of the total microbial community of the lungs between the two groups at genus level; (J-L) β-diversity indexes of the total microbial community of the lungs between the two groups at species level.

Figure 5

The difference in the distribution of pathogens between Positive-PTB and NTB patients. (A, B) Anosim analysis between the two groups at the genus and species level; (C, D) LEfSe analysis was performed to identify differentially abundant taxa, which are highlighted on the phylogenetic tree in cladogram format.