Viral Pathogen Detection by Metagenomics and Pan-Viral Group Polymerase Chain Reaction in Children With Pneumonia Lacking Identifiable Etiology

Abstract

Background: Community-acquired pneumonia (CAP) is a leading cause of pediatric hospitalization. Pathogen identification fails in approximately 20% of children but is critical for optimal treatment and prevention of hospital-acquired infections. We used two broad-spectrum detection strategies to identify pathogens in test-negative children with CAP and asymptomatic controls.

Methods: Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. Association of viruses with CAP was assessed by adjusted odds ratios (aOR) and 95% confidence intervals controlling for season and age group.

Results: RNA-seq/PVG PCR detected previously missed, putative pathogens in 34% of patients. Putative viral pathogens included human parainfluenza virus 4 (aOR 9.3, P = .12), human bocavirus (aOR 9.1, P < .01), Coxsackieviruses (aOR 5.1, P = .09), rhinovirus A (aOR 3.5, P = .34), and rhinovirus C (aOR 2.9, P = .57). RNA-seq was more sensitive for RNA viruses whereas PVG PCR detected more DNA viruses.

Conclusions: RNA-seq and PVG PCR identified additional viruses, some known to be pathogenic, in NP/OP specimens from one-third of children hospitalized with CAP without a previously identified etiology. Both broad-range methods could be useful tools in future epidemiologic and diagnostic studies.

Keywords: RNA sequencing (RNA-seq); metagenomics; pan-viral group polymerase chain reaction (PVG PCR); pneumonia..

Figure 1.

Detection of additional human viruses by RNA sequencing (RNA-seq) and pan-viral group polymerase chain reaction (PVG PCR) in children with community-acquired pneumonia (n = 63) and control subjects (n = 52) with positive pathogen-specific tests using the Etiology of Pneumonia in the Community (EPIC) study protocol. Human viruses detected by RNA-seq and PVG PCR that were not targeted in EPIC included human parechovirus, human bocavirus, Ebstein-Barr virus, human herpesvirus 6, and human herpesvirus 7. Abbreviatons: EBV, Epstein-Barr virus; HBoV, human bocavirus; HHV6, human herpesvirus 6; HHV7, human herpesvirus 7; HPeV, human parechovirus; PVG PCR, pan-viral group polymerase chain reaction; RNA-seq, RNA sequencing.

Figure 2.

Viruses detected by RNA sequencing and/or pan-viral group polymerase chain reaction in children with pneumonia with no identifiable etiology (n = 70; red) and asymptomatic control subjects (n = 90; blue). A total of 20 different human viruses were detected in nasopharyngeal/oropharyngeal samples. In addition, Chlamydia trachomatis was detected in 1 newborn child with pneumonia. Fifteen viruses were more frequently detected in patients than control subjects (odds ratios >1), with human bocavirus (P < .001) having significant associations with community-acquired pneumonia. Abbreviations: ADV, adenovirus; aOR, adjusted odds ratio (adjusted for season and age group); C. trachomatis, Chlamydia trachomatis; CI, confidence interval; CMV, cytomegalovirus; HBoV, human bocavirus; HHV6, human herpesvirus 6; HHV7, human herpesvirus 7; HPeV, human parechovirus; HPIV-4, human parainfluenza virus type 4; HRV-A, human rhinovirus A; HRV-C, human rhinovirus C; HSV, herpes simplex virus; OR, odds ratio.

Figure 3.

Putative pathogens by RNA sequencing and/or pan-viral group polymerase chain reaction in children with pneumonia with no identifiable etiology (n = 70; red) and asymptomatic control subjects (n = 90; blue). In 31% of detections, other putative pathogens were codetected (hashed bars), whereas no other putative pathogen was detected in the remaining samples (monodetection). Odds ratios (ORs) and 95% confidence intervals (CIs) are shown. Only monodetection of human bocavirus was significantly associated with community-acquired pneumonia. Abbreviations: HBoV, human bocavirus; HPeV, human parechovirus HPIV-4, human parainfluenza virus type 4; HRV-A, human rhinovirus A; HRV-C, human rhinovirus C.

Figure 4.

An abundant bacterial flora (>95% of sequencing reads) dominated by a single potential pathogen was detected by RNA sequencing in nasopharyngeal/oropharyngeal (NP/OP) samples of 2 children with community-acquired pneumonia and no identified pathogen by the Etiology of Pneumonia in the Community study protocol. A, In a 23-month-old patient, 94.6% of sequencing reads generated from the NP/OP sample was identified as Pseudomonas fluorescens, covering 35% of the genome of strain LBUM223 (NCBI accession number CP_011117) at a mean of 332X (data analyzed as described in [14]). B, In a 10-month-old patient, 89.7% of sequencing reads were derived from Serratia marcescens, covering 1.5% of the genome sequence of strain FGI94 (NCBI accession number NC_020064) at a mean of 537X.