Application of nested multiplex polymerase chain reaction respiratory and pneumonia panels in children with severe community‐acquired pneumonia

Abstract

Community‐acquired pneumonia (CAP) is a serious clinical concern. A lack of accurate diagnosis could hinder pathogen‐directed therapeutic strategies. To solve this problem, we evaluated clinical application of nested multiplex polymerase chain reaction (PCR) in children with severe CAP. We prospectively enrolled 60 children with severe CAP requiring intensive care between December 2019 and November 2021 at a tertiary medical center. Nested multiplex PCR respiratory panel (RP) and pneumonia panel (PP) were performed on upper and lower respiratory tract specimens. We integrated standard‐of‐care tests and quantitative PCR for validation. The combination of RP, PP, and standard‐of‐care tests could detect at least one pathogen in 98% of cases and the mixed viral‐bacterial detection rate was 65%. The positive percent agreement (PPA), and negative percent agreement (NPA) for RP were 94% and 99%; the PPA and NPA for PP were 89% and 98%. The distribution of pathogens was similar in the upper and lower respiratory tracts, and the DNA or RNA copies of pathogens in the lower respiratory tract were equal to or higher than those in the upper respiratory tract. PP detected bacterial pathogens in 40 (67%) cases, and clinicians tended to increase bacterial diagnosis and escalate antimicrobial therapy for them. RP and PP had satisfactory performance to help pediatricians make pathogenic diagnoses and establish therapy earlier. The pathogens in the upper respiratory tract had predictive diagnostic values for lower respiratory tract infections in children with severe CAP.

Impact of Nested Multiplex Polymerase Chain Reaction Assay in the management of pediatric patients with acute respiratory tract infections: a single center experience

“…Positive BioFire results with negative bacterial culture may be related to multiple factors: low count of the isolate on culture plates compared to the cut-off which is therefore not reported, unavailability of culture media that support growth of fastidious bacteria, PCR may detect non-viable organisms, in addition to the antibiotic misuse which may inhibit growth on culture media, also the fact that the count released by BioFire is in the form of copies/ml which is greater when compared to the culture count in the form of CFU/ml [16]. This was discordant with Ginocchio et al who stated that the positive BioFire results (70.6%) were mainly bacterial (20.5%) rather than viral targets (2.9%) and Yen et al who stated that the three most common bacteria detected were S. aureus, Streptococcus pneumoniae, and Moraxella catarrhalis [11,13]. Also, Debbagh et al found that bacterial etiology was the most common (69.3%), represented mainly by M. catarrhalis (11.4%), followed by viral etiology (30.7%), with HR/EV as the most prevalent [18].…”

“…FilmArray testing (BioFire Diagnostics, LLC, Salt Lake City, UT, USA) Sample collection: ETA or BAL was collected from children who were intubated or underwent bronchoscopic examination. For other children, sputum samples were collected from children unless they were not able to expectorate, and a nasopharyngeal swab (NPS) was collected [11]. The NPS was immediately placed in viral transport media (VTM) (Disposable virus sampling swab kits, Bioteke corporation, Wuxi, Co., Ltd., China), sputum, ETA and BAL samples were collected in sterile containers.…”

“…The nasopharynx (NP) microbiome at the time of upper respiratory viral infections during infancy are a significant determinant of risk for the spread of infection to the lower airways and for the resultant expression of inflammatory symptoms marked by fever [19]. Yen and his team study showed that the concordance rate of pathogens detected in the upper and lower respiratory tract specimens was 96% and therefore, recommended that LRT specimens are more preferred to upper respiratory tract (URT) specimens, however, since obtaining highquality LRT specimens from children is difficult [11]. Therefore, URT specimens associated with molecular testing techniques could be a potential solution.…”

“…Ginocchio et al found that PPA for the single BPPP bacterial pathogens ranged from 50.0 -100% for BAL specimens and 81.0 -100% for sputum specimens. NPA for the single bacterial pathogens ranged from 90.2 -99.4% for BAL and 83.9 -99.2% for sputum, Westhuyzen et al reported 80% correlation between culture and BPPP results with ≥10 7 copies/ml and Yen et al demonstrated 89% PPA and 98% NPA for bacterial targets [11,13,28]. This difference in percent agreement (PA) can be attributed to the difference in the lower limit of reporting for bacteria by standard culture compared to BioFire, where, the PA increase as the count detected by BPPP increase to 10 6 -≥10 7 .…”