Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay

Abstract

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP).Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR).Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1–3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as “Golden Standard” for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant (p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77–95.18%) and 100.0% (95% CI, 62.88–100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35–100%) and 64.2% (95% CI, 35.62–86.02%), respectively. While Multi-PCR results were used as “Golden Standard,” the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR (p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23–92.03%) and 100.0% (95% CI, 89.08–100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays.Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.

Mycoplasma pneumoniae and Adenovirus Coinfection Cause Pediatric Severe Community-Acquired Pneumonia

“…Meanwhile, the detection rates of MP, ADV, and FLUA by multi-PCR were as high as 96.7% (88/91), 79.1% (72/91), and 8.7% (8/91), respectively. Such high detection rates of MP and AVD from ALF samples has also been reported in previous studies based on next-generation high-throughput sequencing technologies ( 17 , 21 ). Hence, both the results of CT and multi-PCR had proved MP, ADV, and FLUA were the most common pathogens detected from the patients who received fiberscope intervention, consistent with previous findings ( 17 , 20 – 23 ).…”

“…Such high detection rates of MP and AVD from ALF samples has also been reported in previous studies based on next-generation high-throughput sequencing technologies ( 17 , 21 ). Hence, both the results of CT and multi-PCR had proved MP, ADV, and FLUA were the most common pathogens detected from the patients who received fiberscope intervention, consistent with previous findings ( 17 , 20 – 23 ). Additionally, we found that the total positive rate of MP-IgM in these cases were 63.7% (51/80), and supported the PCR results to a certain extent (Data set S2).…”

“…We previously demonstrated that different respiratory pathogens can be detected simultaneously in respiratory specimens of pneumonia in children ( 16 – 18 ). In this study, we used a multi-PCR compared with CT to detect pathogens from 91 ALF samples collected by bronchoscopy.…”

“…Consolidation and lung abscesses caused by mixed pulmonary pathogens can present as poor efficacy of antimicrobial therapy in the acute stage, and relate with complications including pleural effusion, necrotizing pneumonia, and even higher mortality ( 11 – 15 ). Our previous studies reported that MP and adenovirus (ADV) were important pathogens of pediatric pneumonia in Shanghai ( 16 – 18 ). However, the identification of pathogens in severe community-acquired pneumonia (SCAP) pediatric patients was unclear, especially in cases that required bronchoscopy intervention.…”

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010–2020: What Happened Before and After the COVID-19 Outbreak?

“…In this retrospective study, the total detection rate of eight respiratory viruses by DFA was 20.3%, especially the detection rate of 17.9% in severe CAP cases, which was far lower than the results of our previous studies using molecular biological methods [ 13 – 15 ]. We believe this was related to the low sensitivity of DFA to nucleic acid detection methods.…”