Viral Detection by Reverse Transcriptase Polymerase Chain Reaction in Upper Respiratory Tract and Metagenomic RNA Sequencing in Lower Respiratory Tract in Critically Ill Children With Suspected Lower Respiratory Tract Infection

Abstract

Objectives: Viral lower respiratory tract infection (vLRTI) contributes to substantial morbidity and mortality in children. Diagnosis is typically confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal specimens in hospitalized patients; however, it is unknown whether nasopharyngeal detection accurately reflects presence of virus in the lower respiratory tract (LRT). This study evaluates agreement between viral detection from nasopharyngeal specimens by RT-PCR compared with metagenomic next-generation RNA sequencing (RNA-Seq) from tracheal aspirates (TAs).

Design: This is an analysis of of a seven-center prospective cohort study.

Setting: Seven PICUs within academic children's hospitals in the United States.

Patients: Critically ill children (from 1 mo to 18 yr) who required mechanical ventilation via endotracheal tube for greater than or equal to 72 hours.

Interventions: We evaluated agreement in viral detection between paired upper and LRT samples. Results of clinical nasopharyngeal RT-PCR were compared with TA RNA-Seq. Positive and negative predictive agreement and Cohen's Kappa were used to assess agreement.

Measurements and main results: Of 295 subjects with paired testing available, 200 (68%) and 210 (71%) had positive viral testing by RT-PCR from nasopharyngeal and RNA-Seq from TA samples, respectively; 184 (62%) were positive by both nasopharyngeal RT-PCR and TA RNA-Seq for a virus, and 69 (23%) were negative by both methods. Nasopharyngeal RT-PCR detected the most abundant virus identified by RNA-Seq in 92.4% of subjects. Among the most frequent viruses detected, respiratory syncytial virus demonstrated the highest degree of concordance (κ = 0.89; 95% CI, 0.83-0.94), whereas rhinovirus/enterovirus demonstrated lower concordance (κ = 0.55; 95% CI, 0.44-0.66). Nasopharyngeal PCR was more likely to detect multiple viruses than TA RNA-Seq (54 [18.3%] vs 24 [8.1%], p ≤ 0.001).

Conclusions: Viral nucleic acid detection in the upper versus LRT reveals good overall agreement, but concordance depends on the virus. Further studies are indicated to determine the utility of LRT sampling or the use of RNA-Seq to determine LRTI etiology.

Figure 1.

Distribution of viruses detected by commercial multiplex reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal samples and RNA sequencing (RNA-Seq) of tracheal aspirate (TA) samples (n = 295). Within each virus type, samples with a single virus detected are represented in black, and samples with multiple viruses detected are designated in gray. Samples with multiple viruses detected are included in each individual virus count. RNA-Seq detected viruses not available on the RT-PCR panel in three samples (respiratory syncytial virus [RSV] + Influenza C, RSV + human herpesvirus 6, Parainfluenza 4 + cytomegalovirus). COV = coronavirus (non-SARS CoV-2), HMPV = human metapneumovirus, NP = negative percentage.

Figure 2.

Relative quantity of each virus presents in the 24 RNA sequencing samples that had multiple viruses detected. Each color represents a different virus and each color within a sample bar represents the fraction of total viral reads per million reads (rpm) in each. The total rpm per virus is detailed in Supplemental Table 5 (http://links.lww.com/PCC/C406). CMV = cytomegalovirus, HHV6 = human herpesvirus 6, HMPV = human metapneumovirus, RSV = respiratory syncytial virus.