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

Fei Li,Liyun Su,Qiguo Zhu,Yin Wang,Linfeng Cao,Jun Shen,Peng Shi,Roujian Lu,Roujian Lu,Yuhan Zhang,Wenjie Tan,Wenjie Tan,Libo Wang

doi:10.3389/fped.2021.659164

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.

Highlights

Severe pulmonary infection in children is still one of the main causes of infection-related deaths in children [1,2,3]
ALF samples were collected during fiberoptic bronchoscopy lavage treatment and divided into four parts: three were sent to clinical laboratories for routine tests, each one aliquot for gram staining and bacteriological culture, direct immunofluorescence antigen detection, conventional reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescent quantitative polymerase chain reaction; and one sample was stored in sterile containers frozen at −80◦C (Figure 1)
One case stayed in hospital only 1 day and referred to the outpatient department

Summary

Introduction

Severe pulmonary infection in children is still one of the main causes of infection-related deaths in children [1,2,3]. The Golden Standard for the etiological diagnosis of pulmonary infection is the conventional culture depended diagnositic tests (CDTs). Due to the low positive rate of culture, antigen-based and nucleic acid-based tests, such as DFA and PCR, are often used in clinical settings as “alternative Golden Standard” for etiological diagnosis. The development of cultureindependent diagnositic tests (CIDTs), such as serology, antigen detection, PCR, TAS, and metagenomic sequencing (mNGS), had helped the clinical practitioners upgrade understanding of pulmonary infection pathogens and pulmonary microbiome [6,7,8,9,10]. Multi-PCR might popular for some time [7,8,9, 11] In another point, children are the most vulnerable to respiratory infections. In this study we tried to use a TAS assay in pediatric clinical to the diagnosis of SCAP etiologies, and map the pathogens of SCAP in children by TAS compared with CT and Multi-PCR to facilitate pediatricians to improve the etiological diagnosis and treatment strategies

Methods

Results

Conclusion