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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.

SUBMITTER: Li F

PROVIDER: S-EPMC8267249 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSevere pneumonia due to lower respiratory tract infections (LRTIs) is a significant cause of morbidity and mortality in children. Noninfectious respiratory syndromes resembling LRTIs can complicate the diagnosis and may also make targeted therapy difficult because of the difficulty of identifying LRTI pathogens. In the present study, a highly sensitive metagenomic next-generation sequencing (mNGS) approach was used to characterize the microbiome of bronchoalveolar lavage fluid (BALF) in children with severe lower pneumonia and identify pathogenic microorganisms that may cause severe pneumonia. The purpose of this study was to use mNGS to explore the potential microbiomes of children with severe pneumonia in a PICU.MethodsWe enrolled patients meeting diagnostic criteria for severe pneumonia admitted at PICU of the Children's Hospital of Fudan University, China, from February 2018 to February 2020. In total, 126 BALF samples were collected, and mNGS was performed at the DNA and/or RNA level. The pathogenic microorganisms in BALF were identified and correlated with serological inflammatory indicators, lymphocyte subtypes, and clinical symptoms.ResultsmNGS of BALF identified potentially pathogenic bacteria in children with severe pneumonia in the PICU. An increased BALF bacterial diversity index was positively correlated with serum inflammatory indicators and lymphocyte subtypes. Children with severe pneumonia in the PICU had the potential for coinfection with viruses including Epstein-Barr virus, Cytomegalovirus, and Human betaherpesvirus 6B, the abundance of which was positively correlated with immunodeficiency and pneumonia severity, suggesting that the virus may be reactivated in children in the PICU. There was also the potential for coinfection with fungal pathogens including Pneumocystis jirovecii and Aspergillus fumigatus in children with severe pneumonia in the PICU, and an increase in potentially pathogenic eukaryotic diversity in BALF was positively associated with the occurrence of death and sepsis.ConclusionsmNGS can be used for clinical microbiological testing of BALF samples from children in the PICU. Bacterial combined with viral or fungal infections may be present in the BALF of patients with severe pneumonia in the PICU. Viral or fungal infections are associated with greater disease severity and death.

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

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