Clinical utility of SARS-CoV-2 subgenomic RT-PCR in a pediatric quaternary care setting

Abstract

Background: During active transcription, SARS-CoV-2 generates subgenomic regions of viral RNA. While standard SARS-CoV-2 RT-PCR amplifies region(s) of genomic RNA, it cannot distinguish active infection from remnant viral genomic material. However, screening for subgenomic RNA (sgRNA) by RT-PCR may aid in the determination of actively transcribing virus.

Objectives: To evaluate the clinical utility of SARS-CoV-2 sgRNA RT-PCR testing in a pediatric population.

Study design: Retrospective analysis was performed on inpatients from February-September 2022 positive for SARS-CoV-2 by RT-PCR with a concomitant order for sgRNA RT-PCR. Chart abstractions were conducted to determine clinical outcomes, management, and infection prevention and control (IPC) practices.

Results: Of 95 SARS-CoV-2 positive samples from 75 unique patients, 27 (28.4%) were positive by sgRNA RT-PCR. A negative sgRNA RT-PCR test allowed for de-isolation in 68 (71.6%) patient episodes. Regardless of age or sex, a positive sgRNA RT-PCR result significantly correlated with disease severity (P = 0.007), generalized COVID-19 symptoms (P = 0.012), hospitalization for COVID-19 (P = 0.019), and immune status (P = 0.024). Moreover, sgRNA RT-PCR results prompted changes in management in 28 patients (37.3%); specifically, therapeutic escalation in 13/27 (48.1%) positives and de-escalation in 15/68 (22.1%) negatives.

Conclusions: Taken together, these findings underscore the clinical utility of sgRNA RT-PCR testing in a pediatric population as we report significant associations between sgRNA RT-PCR results and clinical parameters related to COVID-19. These findings align with the proposed use of sgRNA RT-PCR testing to guide patient management and IPC practices in the hospital setting.

Keywords: Clinical utility; PCR; SARS-CoV-2; Subgenomic.

Fig. 1

(A) Subgenomic RNA RT-PCR assay design and workflow. From left to right: SARS-CoV-2 virion, SARS-CoV-2 genomic and subgenomic RNA, RT-PCR strategy with red arrows represent forward and reverse primers targeting a region within the E gene subgenomic RNA, real time RT-PCR amplification plot representative of the sgRNA RT-PCR assay. SARS-CoV-2 virus, genome, and subgenomic RNAs are not to scale. (B) Relative expression of subgenomic SARS-CoV-2 transcripts, descending from left to right .

Fig. 2

World Health Organization (WHO) severity score stratified by sgRNA PCR result. (A) Stacked bar chart representing WHO clinical severity for sgRNA PCR results of Not Detected (black) and Detected (red), N = 95 samples. Definitions of each severity score are described in Study Design sub-Section 2.1. (B) Violin plots demonstrating overall WHO severity scores by sgRNA PCR result. Solid horizontal lines represent the median whereas dotted lines represent quartiles, N = 95 samples. Statistical analysis was performed using unpaired, two-tailed t-test with Welch's correction. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Cycle threshold (C_T) value analysis. (A) Correlation between genomic SARS-CoV-2 RT-PCR C_T values and sgRNA RT-PCR C_T values for all specimens with sgRNA “detected”. One gene target represented per specimen. Line represents simple linear regression (B) Correlation between genomic SARS-CoV-2 RT-PCR C_T values and sgRNA RT-PCR C_T values for all specimens with sgRNA “detected”. For assays with multiple gene targets, all detected targets are shown. Lines represent simple linear regressions. (C) Distribution of genomic SARS-CoV-2 RT-PCR C_T values stratified by sgRNA RT-PCR qualitative result. C_T values shown are assay and platform dependent as represented in panel (B).