The diagnostic value of metagenomic next⁃generation sequencing in infectious diseases

Abstract

Background: Although traditional diagnostic techniques of infection are mature and price favorable at present, most of them are time-consuming and with a low positivity. Metagenomic next⁃generation sequencing (mNGS) was studied widely because of identification and typing of all pathogens not rely on culture and retrieving all DNA without bias. Based on this background, we aim to detect the difference between mNGS and traditional culture method, and to explore the relationship between mNGS results and the severity, prognosis of infectious patients.

Methods: 109 adult patients were enrolled in our study in Shanghai Tenth People's Hospital from October 2018 to December 2019. The diagnostic results, negative predictive values, positive predictive values, false positive rate, false negative rate, pathogen and sample types were analyzed by using both traditional culture and mNGS methods. Then, the samples and clinical information of 93 patients in the infected group (ID) were collected. According to whether mNGS detected pathogens, the patients in ID group were divided into the positive group of 67 cases and the negative group of 26 cases. Peripheral blood leukocytes, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil counts were measured, and the concentrations of IL-2, IL-4, IL-6, TNF-α, IL-17A, IL-10 and INF-γ in the serum were determined by ELISA. The correlation between the positive detection of pathogens by mNGS and the severity of illness, hospitalization days, and mortality were analyzed.

Results: 109 samples were assigned into infected group (ID, 92/109, 84.4%), non-infected group (NID, 16/109, 14.7%), and unknown group (1/109, 0.9%). Blood was the most abundant type of samples with 37 cases, followed by bronchoalveolar lavage fluid in 36 cases, tissue, sputum, pleural effusion, cerebrospinal fluid (CSF), pus, bone marrow and nasal swab. In the ID group, the majority of patients were diagnosed with lower respiratory system infections (73/109, 67%), followed by bloodstream infections, pleural effusion and central nervous system infections. The sensitivity of mNGS was significantly higher than that of culture method (67.4% vs 23.6%; P < 0.001), especially in sample types of bronchoalveolar lavage fluid (P = 0.002), blood (P < 0.001) and sputum (P = 0.037), while the specificity of mNGS was not significantly different from culture method (68.8% vs 81.3%; P = 0.41). The number of hospitals stays and 28-day-motality in the positive mNGS group were significantly higher than those in the negative group, and the difference was statistically significant (P < 0.05). Age was significant in multivariate logistic analyses of positive results of mNGS.

Conclusions: The study found that mNGS had a higher sensitivity than the traditional method, especially in blood, bronchoalveolar lavage fluid and sputum samples. And positive mNGS group had a higher hospital stay, 28-day-mortality, which means the positive of pathogen nucleic acid sequences detection may be a potential high-risk factor for poor prognosis of adult patients and has significant clinical value. MNGS should be used more in early pathogen diagnosis in the future.

Keywords: Diagnostic; Infection; Next-generation sequencing; Sensitivity; Survival.

Fig. 1

Flow diagram of cases inclusion and exclusion

Fig. 2

Flow diagram of Metagenomic Next-generation Sequencing and Analysis

Fig. 3

Patients composition and samples types. a. In samples of this study, 33.9% were from blood which was the most, 33.0% from BALF, 11.0% from tissue and the others were from sputum (8.3%), pleural fluid (6.4%), CSF (3.7%), pus (1.8%), bone marrow (0.9%) and nasal swab (0.9%). b. Patients were subdivided into ID (92/109, 84.4%), NID (16/109, 14.7%) and unknown (1/109, 0.9%) groups according to their diagnosis by conventional technique. c. Infection sites of patients in ID group. Most were respiratory system infections (73/109, 67.0%) and followed by bloodstream infections (10/109, 9.17%), pleural effusion (6/109, 5.50%), central nervous system infections (6/109, 5.50%), cardiovascular system infection (2/109,1.83%), eye, ear, nose, throat, or mouth infection (2/109,1.83%), skin and soft tissue infection (1/109, 0.92%), multifocal infection (1/109, 0.92%), urinary system infection (1/109, 0.92%). Abbreviations: CSF, cerebrospinal fluid; BALF, bronchoalveolar lavage fluid

Fig. 4

Diagnostic Performance Comparison of mNGS and Culture. a. Positive and negative cases in all, ID, NID and unknown group of mNGS and the culture, respectively. There were statistical differences between mNGS and culture of all (P < 0.01) and of ID group (P < 0.01), but no differences in NID and unknown group for the limited amounts(P > 0.05). b. Contingency tables showed the sensitivity and specificity of mNGS were 67.4 and 68.8%, while those of culture were 23.6 and 81.3%. mNGS increased the sensitivity in comparison with that of culture (P < 0.001) while there were no differences in specificity between them (P = 0.41). c. Pie chart demonstrated the positivity distribution of mNGS and culture for all samples from 3 groups. 53.21% were positive by mNGS, 4.59% by culture, 19.27% by both and 22.94% were both negative. Abbreviations: NPV, negative predictive values; PPV, positive predictive values

Fig. 5

The overlap of positivity between mNGS and culture in pathogen and sample types. a. 19 pathogens detected in ID group with their corresponding frequencies were showed in histograms. Klebsiella, bacteria without MTB/NTM, EBV, CMV, NTM, Anaerobes, Saccharomyces cerevisiae, Proteus, Pneumocystis carinii, Abiotrophia, Nocardia, Staphylococcus aureus, Enterococcu and Escherichia coli demonstrated a trend of higher positivity rate in mNGS than that in culture with no statistical differences (P > 0.05). Acinetobacter baumannii and MTB were found equally in two groups. b. The overall sensitivity of mNGS in the different sample types were significantly different (P = 0.03) while sample types did not affect the sensitivity of pathogens in culture. Interestingly, especially in the types of BALF, blood and sputum samples, mNGS had significantly higher sensitivity than the culture (P = 0.002 for BALF, P < 0.001 for blood, P = 0.037 for sputum). Abbreviations: BALF, bronchoalveolar lavage fluid; CSF, cerebrospinal fluid; mNGS, metagenomic next-generation sequencing; HSV, herpes simplex virus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; MTB, Mycobacterium tuberculosis; NTM, nontuberculous mycobacteria; ns, no significant difference

Fig. 6

The survival curves of positive and negative group of mNGS in ID. The survival curves suggested that the overall survival rate declined faster in the positive group, however, there was no statistically differences between the two groups