Comparison of metagenomic next-generation sequencing using cell-free DNA and whole-cell DNA for the diagnoses of pulmonary infections

Abstract

Although the fast-growing metagenomic next-generation sequencing (mNGS) has been used in diagnosing infectious diseases, low detection rate of mNGS in detecting pathogens with low loads limits its extensive application. In this study, 130 patients with suspected pulmonary infections were enrolled, from whom bronchoalveolar lavage fluid (BALF) samples were collected. The conventional tests and mNGS of cell-free DNA (cfDNA) and whole-cell DNA (wcDNA) using BALF were simultaneously performed. mNGS of cfDNA showed higher detection rate (91.5%) and total coincidence rate (73.8%) than mNGS of wcDNA (83.1% and 63.9%) and conventional methods (26.9% and 30.8%). A total of 70 microbes were detected by mNGS of cfDNA, and most of them (60) were also identified by mNGS of wcDNA. The 31.8% (21/66) of fungi, 38.6% (27/70) of viruses, and 26.7% (8/30) of intracellular microbes can be only detected by mNGS of cfDNA, much higher than those [19.7% (13/66), 14.3% (10/70), and 6.7% (2/30)] only detected by mNGS of wcDNA. After in-depth analysis on these microbes with low loads set by reads per million (RPM), we found that more RPM and fungi/viruses/intracellular microbes were detected by mNGS of cfDNA than by mNGS of wcDNA. Besides, the abilities of mNGS using both cfDNA and wcDNA to detect microbes with high loads were similar. We highlighted the advantage of mNGS using cfDNA in detecting fungi, viruses, and intracellular microbes with low loads, and suggested that mNGS of cfDNA could be considered as the first choice for diagnosing pulmonary infections.

Keywords: BALF; MNGs; cell-free DNA; pulmonary infection; whole-cell DNA.

Figure 1

Potential pathogen profiles detected by mNGS of cfDNA and wcDNA. (A), Microbial composition revealed by mNGS of cfDNA. (B), Microbial composition revealed by mNGS of wcDNA. (C), Comparison of mNGS using cfDNA and wcDNA in detecting microorganisms. The number in the box is the total number of patients from which some microorganism was detected.

Figure 2

Performance of mNGS using cfDNA/wcDNA and conventional methods. (A, B) show the coincidence rates, sensitivity, specificity, PPV, and NPV of conventional methods, mNGS of cfDNA, and mNGS of wcDNA. PPV and NPV represent positive predictive value and negative predictive value, respectively. (C), Comparison of mNGS using cfDNA and wcDNA at bacterial, fungal, and viral levels. *P < 0.05; ns: no significance.

Figure 3

Comparison of the RPM detected by mNGS of both cfDNA and wcDNA. (A), All microbes detected by mNGS. (B), Bacteria detected by mNGS. (C), Fungi detected by mNGS. (D), Viruses detected by mNGS. The number of microbes and base-2 logarithm of reads per million (RPM) detected by mNGS of cfDNA divided by that detected by mNGS of wcDNA for the same microbes were used to draw scatter plot, and the dots of >0 represented that the numbers of RPM detected by mNGS of cfDNA was higher than that detected by mNGS of wcDNA for the microbes. The microbes only detected by mNGS of cfDNA or wcDNA were summarized in Table 3 .

Figure 4

The detection of mNGS using cfDNA and wcDNA at different thresholds (RPM of 200, 100, 50, and 25). (A) Detection summary of fungi, viruses, and intracellular microbes by mNGS of both cfDNA and wcDNA at high and low loads. The number in the bubble above the horizontal axis is the ratio of the microbes with more RPM by mNGS of cfDNA than mNGS of wcDNA to the whole microbes at this threshold, while the number in the bubble below the horizontal axis is the ratio of the microbes with more RPM by mNGS of wcDNA than mNGS of cfDNA. Besides, vertical axis coordinate is the ratio of the numbers in the bubble. (B) Detection summary of fungi, viruses, and intracellular microbes only detected by mNGS of cfDNA or wcDNA at high and low loads. The column is the ratio of the microbes only detected by mNGS of cfDNA or wcDNA to the whole microbes at this threshold. The number above the column is the ratio of the microbes only detected by mNGS of cfDNA divided by that only detected by mNGS of wcDNA.