The clinical value of metagenomic next-generation sequencing in the diagnosis of pulmonary tuberculosis and the exploration of lung microbiota characteristics

Abstract

The lung microbiota plays a critical role in many important physiological processes and is linked with various pulmonary infectious diseases. The present study aimed to characterize the lung microbiota in patients with pulmonary tuberculosis (PTB), and to explore the association between the abundance of Mycobacterium tuberculosis complex (MTBC) and the lung microbiota. This retrospective study included 190 patients with MTBC infection. The enrolled patients were classified into three groups based on the abundance results of bronchoalveolar lavage fluid (BALF) mNGS: low [reads per ten million (RPTM) = 1 ~ 1000], medium (RPTM = 1001 ~ 10000) and high (RPTM > 10000). In the high-abundance group, there were more bilateral lobar involvement and symptoms of cavitation. In addition to mNGS, the highest positivity rates were T-spot (92.36%), GeneXpert (71.58%), culture (68.95%) and AFB smear (17.84%). The positive rates of culture, AFB smear and GeneXpert increased with the increase of MTBC abundance, and the positive rates were highest in the high-abundance MTBC group. Both the alpha and beta diversity showed significant difference between the three groups, with the high-abundance MTBC groups showed lowest alpha diversity. The increased abundance of MTBC positively associated with the longer time of hospital stay. To sum up, the lung microbiota of patients with PTB were significantly distinct between groups with different abundant levels of MTBC. Combined with imaging features, a high abundance of MTBC suggests the patient is more severely ill and has a poorer prognosis.

Keywords: Mycobacterium tuberculosis complex; Abundance; Lung microbiota; Pulmonary tuberculosis.

Fig. 1

Flow chart of patient enrollment. Abbreviations: mNGS, metagenomic next-generation sequencing; RPTM, reads per ten million; AFB, acid-fast staining; MTBC, Mycobacterium tuberculosis complex.

Fig. 2

Comparison of mNGS, BALF culture, AFB staining, T-spot and GeneXpert for the identification of MTBC in the patients. (A) The positive rate of the five methods for MTBC detection in low, medium and high-abundance MTBC groups. (B) Heatmap showed the performance of the five methods in the diagnosis of MTBC. Each column represents a patient. The red bars indicate positive MTBC results, the green bar indicate negative MTBC results, and the white bars indicate not performing the test.

Fig. 3

Comparison of microbial alterations in patients with different abundance of MTBC. (A) Alpha diversity (Shannon and Simpson index) analysis between the low, medium and high-abundance MTBC groups. (B) Beta diversity analysis of the low, medium and high-abundance MTBC groups, with plots based on the Bray-Curtis distance. (C) Bar plot showed the top 10 species with highest abundance between the different groups. (D) LEfSe used to identify essential differences in microorganism abundance (at species level) between the 3 groups.

Fig. 4

Spearman’s correlation analysis between MTBC abundance and clinical characteristics of the patients. Abbreviations: WBC, white blood cell count; ANC, absolute neutrophil count; LY, lymphocyte; GR%, granulocyte percentage; LY%, lymphocyte percentage; PLT, platelet; HBG, hemoglobin; TBIL, total bilirubin; LOHS, length of hospital stays.