The Comparison of Metagenomic Next-Generation Sequencing with Conventional Microbiological Tests for Identification of Pathogens and Antibiotic Resistance Genes in Infectious Diseases

Abstract

Background: Metagenomic next-generation sequencing (mNGS) has been widely studied, due to its ability of detecting all the microbial genetic information unbiasedly in a sample at one time and not relying on traditional culture. However, the application of mNGS in the diagnosis of clinical pathogens remains challenging.

Methods: From December 2019 to March 2021, 134 specimens including Broncho alveolar lavage fluid (BAFL), blood, sputum, cerebrospinal fluid (CSF), bile, pleural fluid, pus, were continuously collected in The First Hospital of Qinhuangdao, and their retrospective diagnoses were classified into infectious disease (128, 95.5%) and noninfectious disease (6, 4.5%). The pathogen-detection performance of mNGS was compared with conventional microbiological tests (CMT) and culture method. In addition, the antibiotic resistance genes (ARGs) and evolutionary relationship of common drug-resistant A. baumannii were also analyzed.

Results: Compared with CMT and culture methods, mNGS showed higher sensitivity in pathogen detection (74.2% vs 57.8%; P < 0.001 and 66.3% vs 31.7%; P < 0.001, respectively). Importantly, for cases that mNGS-positive only, 18 (35%) cases result in diagnosis modification, and 7 (23%) cases confirmed the clinical diagnosis. In 17 cases that A. baumannii were both detected in mNGS and culture, ade genes were the most frequently detected ARGs (from 13 cases), followed by sul2 and APH(3")-Ib (both from 12 cases). High consistency was observed among these ARGs and the related phenotype (100% for ade genes, 91.6% for sul2 and APH(3")-Ib). A. baumannii strains were classified into three groups, and most were well-clustered. It suggested those strains may be the epidemic strains.

Conclusion: In our study, mNGS had a higher sensitivity than CMT and culture method. And the result of ARGs frequency and cluster analysis of A. baumannii was of great significance to the anti-infective therapy.

Keywords: antibiotic resistance genes; conventional microbiological tests; infection; metagenomic next-generation sequencing; sensitivity.

Figure 1

Flow chart of cases inclusion and exclusion.

Figure 2

Schematic of metagenomic next-generation sequencing and analysis. (A) The wet lab pipeline including nucleic acid extraction, library construction and sequencing. (B) Dry lab pipeline including quality control, human host subtraction, alignment to reference database and report generation.

Figure 3

Distribution of the infectious diseases of clinical specimens. The pie chart demonstrated the cause of the retrospective diagnosis in ID (95.5%) and NID (4.5%) groups. Lower respiratory tract infection accounted for 70.9% of cases in ID groups, followed by bloodstream infection (7.5%), central nervous system infections (5.2%), intra-abdominal infection (4.5%) multifocal infections (3.9%), fever of unknown origin (3.1%), and skin and soft tissue infections (0.8%).

Figure 4

Diagnostic performance comparison of mNGS and CMT. (A) Positive and negative samples (y-axis) for pairwise mNGS and CMT is plotted against ID, NID group (x-axis). (B) Contingency tables showed the sensitivity and specificity of mNGS and CMT respectively. mNGS increased the sensitivity in comparison with that of culture (P < 0.001) while there were no differences in specificity between them (P > 0.05). (C) Pie chart demonstrated the positivity distribution of mNGS and CMT results from two groups. For the double-positive subset, a proportion of complete matching (22/65) and partial matching (at least 1 pathogen identified in the test was confirmed by the other) (24/65) was observed, with 19 conflicts between mNGS and CMT. (D) For microbes only identified in mNGS, the primary empirical diagnosis was confirmed (23%) or modified (35%) according to mNGS, whereas 13% of the samples were considered unreliable (diagnosis unsupported) and 29% were uncertain.

Figure 5

The positivity rate between mNGS and culture for different sample types. (A) Compared with the culture, mNGS increased the overall sensitivity by 34.6% (66.3% vs 31.7%). In cases of BALF and blood samples, mNGS detection had significantly higher sensitivity than the culture method (P = 0.034 for BALF, P < 0.01 for blood). (B) In all the 39 cases of BALF sample performed both mNGS and culture, high concordance for pathogen detection of BALF was shown in 16 double-positive cases of mMGS and culture. Only one case of mismatch was observed (2.6%), while 10 cases were total matched (25.6%).

Figure 6

The overlap of positivity in pathogen between mNGS and culture. A total of 40 different pathogens were detected in the infectious disease group with their corresponding frequencies plotted in histograms. The proportion of mNGS positive samples was significantly higher than that of CMT positive samples in terms of MTB, NTM, virus, and fungus (P < 0.05).

Figure 7

Frequency of ARGs detected from 17 cases that A. baumannii was both positive in mNGS and CMT. A total of 33 antiARGs were detected in 14 cases except 3 negative cases; of which adeL, sul2, and APH(3”)-Ib showed high detection frequency (13/14).

Figure 8

Evolutionary analysis of A. baumannii strains both positive in mNGS and CMT. Among the 17 cases, 15 were clustered in group B or group C, strain XH731 (7 cases of 17, 41.18%) and AB07 (5 cases of 17, 29.41%) were dominant strains.