Background
Community-acquired pneumonia (CAP) is a common respiratory disease in children with significantly higher morbidity and mortality rates in developing countries than that in developed countries [
1]. According to the World Health Organization (WHO) report, the incidence of CAP in children under 5 years of age in developing countries is 0.28 times/child/year, accounting for 95% of all cases of CAP in children worldwide, and the mortality rate is 1.3–2.6% [
2]. In North America and Europe, the incidence rate among pre-schoolers is 36 per 1,000 children per year [
3], placing a significant economic burden on families and society. Children with CAP are prone to develop pleural effusion, lung abscess, pericarditis, meningitis, and other pulmonary and external complications [
4], which eventually lead to severe community-acquired pneumonia (SCAP), respiratory and circulatory failure, or even life-threatening complications. For the pathogen diagnosis of CAP, clinical work is carried out in various microorganisms, including viruses, bacteria, and atypical pathogen combination detection to avoid missed diagnosis and mixed infection, although pathogens cannot be identified or detected in 22–57% of children [
5‐
7] and 13–62% of adult with pneumonia [
8,
9]. Culture, the gold standard for bacterial and fungal infection diagnosis, has high accuracy but a long detection time and low detection rate and is not suitable for the identification of pathogens that are difficult to cultivate. Viruses and atypical pathogens can be detected by polymerase chain reaction (PCR) and antigen detection, but the positive rate varies greatly and suitable for detecting only the known, limited target pathogens. In addition, some unknown pathogens in nature cannot be detected. Therefore, there is an urgent need for rapid and accurate diagnostic methods to identify pathogens in children with SCAP [
10].
Metagenomic next-generation sequencing (mNGS) is a method for whole nucleic acid detection using patient samples directly [
11], which can simultaneously detect various microorganisms. It has the characteristics of being less affected by antibiotics, taking a wide range of samples, and does not need to set gene sequences in advance. Therefore, it greatly applicable in diagnosis of new and rare infectious diseases [
10,
12‐
14]. Currently, mNGS has been applied in the diagnosis of infectious diseases of the lungs, blood system, and central nervous system in adults [
15‐
17]. Sepsis, encephalitis, and bone and joint infections have also been reported in children [
18‐
20]. However, no study has been conducted on bronchoalveolar lavage fluid (BALF) in children with SCAP. In this study, 84 children with SCAP were enrolled for bronchoscopic lavage. BALF was used as the sample source to evaluate the value of mNGS in the diagnosis of SCAP in children by comparing it with conventional technology (CT).
Discussion
Our study comprehensively evaluated the application of mNGS in children with SCAP. In this study, pathogen detection rate of mNGS was higher than that of the CT (83.3% vs. 63.1%, P = 0.003), especially for S. pneumoniae, H. influenzae, and fungi. There was no significant difference between mNGS and CT in the detection of tuberculosis and virus. However, MP detection rate was lower for mNGS than that for CT (52.4% [11/21] vs. 95.2% [20/21], P = 0.004). In addition, mNGS can detect pathogens that cannot be detected using CT, which is of great significance for the adjustment of antimicrobial therapy.
BALF was used as the sample source in this study because it was collected from infected sites and was less contaminated by oral colonisation bacteria [
27,
28]. Regarding bacterial detection, our study found that mNGS had a higher detection rate for
S. pneumoniae than that of the CT (89.2% vs. 44.8%,
P = 0.001). In a study on children with CAP by Farnaes et al., 6.7% (1/15) of
S. pneumoniae were detected by culture and cell-free plasma next-generation sequencing (CFPNGS); pneumococcus was only detected using CFPNGS in the other eight cases [
29]. There was another study on invasive pneumococcal disease in 96 children; positive rate of culture method was 27.1% (
n = 26), while that of mNGS test was 62.5% (
n = 60) (blood, cerebrospinal fluid, and pleural effusion samples) [
30]. These findings suggest that when considering
S. pneumoniae infection in children with SCAP, mNGS should be actively performed to clarify the aetiology and guide anti-infection treatment. In this study, the detection of
S. aureus using mNGS was lower than that of traditional methods (41.7% vs. 83.3%). However, Ren et al.'s study collected noted that mNGS (blood, cerebrospinal fluid, and BALF) was more sensitive than that of the culture method (77.8% vs. 44.4%) in adult patients with sepsis [
24]. We utilised both culture and PCR, which may have enhanced the rate of
S. aureus identification by conventional methods. This may also be associated with the different immune systems of children and adults.
For Gram-negative bacteria, our study found that the mNGS detection rate was significantly higher than that of the CT, especially for
H. influenzae (91.7% vs. 33.3%,
P < 0.005). mNGS also had a greater detection rate for
P. aeruginosa,
K. pneumoniae, and
E. coli than that of the CT in our study. The study by Wu et al. on adult SCAP also revealed that the rate of
H. influenzae identification using mNGS was higher than that of the conventional detection techniques (100% vs. 33.3%) in BALF [
31]. In a study on adult sepsis, Ren et al. discovered that mNGS had a greater detection, n rate of
P. aeruginosa and
K. pneumoniae than that of the culture method (75.4% vs. 43.5%, 81.6% vs. 26.3%, respectively), using blood, cerebrospinal fluid, and BALF samples [
24]. According to the aforementioned study, mNGS should be actively performed whenever Gram-negative bacterial infection is considered. However, a previous study revealed that after paediatric haematopoietic stem cell transplantation, the detection rate of
P. aeruginosa and
K. pneumoniae using conventional methods is comparable to that of the mNGS using BALF [
32]. These differences may be due to the different specimen sources and immune functions.
Regarding virus detection, mNGS showed no difference compared with that of the CT in the detection of ADV, EBV, and influenza A viruses in this study. When children with SCAP caused by the above pathogens are clinically suspected, traditional methods should be considered first for pathogen detection. A previous study showed that mNGS has a low sensitivity for influenza A viruses [
33]. Real-time fluorescence quantitative PCR is more sensitive than mNGS for the detection of influenza virus infection in adult SCAP [
34]. In another study, mNGS (RNA-SEQ) did not detect any of the eight positive ADV samples for paediatric pneumonia but had high sensitivity to rhinovirus and RSV (100% and 96%, respectively) [
35]. The potential of mNGS to identify additional viruses and subtypes that cannot be detected by conventional methods has also been confirmed in previous studies [
33,
36]. In contrast, mNGS assisted in identifying cytomegalovirus (CMV) infection and not merely colonisation. In our study, mNGS and CT eventually detected three children with CMV infection, including one patient with congenital heart disease. Based on the findings of the mNGS, two cases were treated with antiviral medications, and the patients were cured. In a study, one child with leukaemia received antiviral treatment after CMV was detected using mNGS, and their symptoms improved [
37]. This demonstrates that CMV should be appraised not only based on colonisation but also on the child’s underlying illness and clinical characteristics.
The fungal detection rate of mNGS was significantly higher than that of CT alone. In a study on the detection of fungal pneumonia in adults, the culture method was only used in 1/21 samples, while mNGS was used in 19/21 samples [
38]. In a study of children after haematopoietic stem cell transplantation, mNGS (BALF) detected nine cases of fungal pneumonia (
Pneumocystis jiroi [
n = 6], Aspergillus [
n = 2], and mucor [
n = 1]), while none was detected using traditional methods [
32]. In this study, the positive rate of fungal pneumonia detected using mNGS was 81.8% (9/11), while only 18.2% (2/11) used the traditional pathogen detection method. Among them, 54.5% (6/11) had underlying diseases, including congenital heart disease, primary ciliary dyskinesia, chronic granuloma, pelvic mass, diabetes, and congenital cleft palate. Two kinds of
Aspergillus, Aspergillus aspergillus and
Aspergillus niger, were also detected using mNGS, but not by traditional methods. Huang et al. also reported that mNGS detected various
A. fumigatus, A. niger, A. flavus, and A. oryzae in immunosuppressed adult patients diagnosed with fungal pneumonia [
39]. Therefore, mNGS should be actively applied to children with severe pneumonia and underlying diseases or immunodeficiency. In our study, three cases of
C. albicans infection were confirmed, and the detection rates of mNGS and CT were similar, among which two cases had underlying diseases. Liu et al. also reported that
C. albicans was one of the fungi most frequently detected in BALF cultures [
40]. However, another study found that the mNGS detection rate of
C. albicans was lower than that of the traditional method (70.6% vs. 82.4%) in ventilators-associated pneumonia in adults [
25]. Therefore, traditional methods should be used for pathogen detection in children with
C. albicans infection. Interestingly, patient 26 tested negative on mNGS and CT but positive on the 1,3-D glucan test, which combined clinical symptoms and imaging to diagnose fungal infection. Therefore, culture, mNGS, galactomannan (GM) test, and 1, 3-D glucan test should be combined in the diagnosis of fungal pneumonia to avoid missing positive samples.
The detection sensitivity of mNGS for MTB was similar to that of CT in our study. Previous studies have also confirmed that the diagnostic ability of mNGS for MTB is similar to that of the traditional detection methods [
41,
42]. Therefore, when clinically considering the presence of MTB infection, traditional methods rather than mNGS should be considered first. MTB, a type of intracellular bacteria, releases less nucleic acid into the extracellular environment, and insufficient wall fracture can also affect the extraction of DNA; therefore, fewer sequences can be detected [
43,
44]. mNGS combined with MTB-Xpert can be used to improve diagnostic rates [
11,
43].
Surprisingly, we found that mNGS was inferior to CT in the detection of atypical pathogens (50.0% vs. 91.7%), especially for MP (52.4% vs. 95.2%). This has never been mentioned in previous studies. Only studies on children with severe unresponsive pneumonia indicated that mycoplasma detection rates of mNGS and PCR were equal (15.6% vs. 18.8%) [
37]. In this study, the detection rate of MP-PCR was 57.1% (12/21), which was similar to that by mNGS. The possible reasons are the combination of PCR and two immunological methods, which increased the sensitivity of the traditional method in this study. Therefore, when considering MP infections in children with SCAP, it is not necessary to conduct mNGS examinations.
The patient received empirical anti-infective medication in the absence of knowledge of its precise aetiology. After identifying the illness' pathogenic microorganisms, 26 people (31.0%) modified their course of treatment according to the mNGS results, and their symptoms improved. In a study of adults with severe respiratory diseases, mNGS led to a change in treatment in 34.4% (11/32) of patients [
13]. Combining mNGS with conventional detection technologies can improve the ability to target drug use. On the other hand, the culture process takes at least 3 days [
45], while mNGS can obtain test results within 24–48 h [
36]. Although PCR is quick, its reach is constrained and may not even detect the causal agent [
33]. These results suggest that mNGS is important for the detection and treatment of SCAP in children.
Our study has several limitations. First, the sample size of our study was modest; hence, the number of pathogens identified was limited. Second, because of the high sensitivity of mNGS, pathogens of colonisation and contamination will also be discovered; therefore, there is no universal standard for the report's issuance and interpretation. Finally, because this was a single-centre observational study, a case selection bias may have occurred. Further prospective, multicentre, randomised controlled studies are needed to confirm the sensitivity, specificity, and clinical value of mNGS.
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