Rapid, Sample-to-Answer Host Gene Expression Test to Diagnose Viral Infection

Abstract

Objective: Distinguishing bacterial, viral, or other etiologies of acute illness is diagnostically challenging with significant implications for appropriate antimicrobial use. Host gene expression offers a promising approach, although no clinically useful test has been developed yet to accomplish this. Here, Qvella's FAST HR (Richmond Hill, Ontario, Canada) process was developed to quantify previously identified host gene expression signatures in whole blood in <45 minutes.

Method: Whole blood was collected from 128 human subjects (mean age 47, range 18-88) with clinically adjudicated, microbiologically confirmed viral infection, bacterial infection, noninfectious illness, or healthy controls. Stabilized mRNA was released from cleaned and stabilized RNA-surfactant complexes using e-lysis, an electrical process providing a quantitative real-time reverse transcription polymerase chain reaction-ready sample. Threshold cycle values (CT) for 10 host response targets were normalized to hypoxanthine phosphoribosyltransferase 1 expression, a control mRNA. The transcripts in the signature were specifically chosen to discriminate viral from nonviral infection (bacterial, noninfectious illness, or healthy). Classification accuracy was determined using cross-validated sparse logistic regression.

Results: Reproducibility of mRNA quantification was within 1 cycle as compared to the difference seen between subjects with viral versus nonviral infection (up to 5.0 normalized CT difference). Classification of 128 subjects into viral or nonviral etiologies demonstrated 90.6% overall accuracy compared to 82.0% for procalcitonin (P = .06). FAST HR achieved rapid and accurate measurement of the host response to viral infection in less than 45 minutes.

Conclusions: These results demonstrate the ability to translate host gene expression signatures to clinical platforms for use in patients with suspected infection.

Clinical trials registration: NCT00258869.

Keywords: gene expression profiling; infection; molecular diagnostic techniques; real-time polymerase chain reaction; virus diseases.

Published by Oxford University Press on behalf of Infectious Diseases Society of America 2019.

Figures

Figure 1.

The FAST HR process separates stabilized mRNA from debris in the PAXgene Blood RNA tubes and releases them in a nuclease-free and inhibitor-free liquid medium suitable for performing quantitative reverse transcription polymerase chain reaction (PCR) assays.

Figure 2.

Target Selection by Variance. A, sorted variance of all targets in the assay; dashed lines represent 95% empirical quantiles of the variance of the normalizing target (HPRT1). B, distribution of signature size obtained by leave-one-out cross-validation (LOOCV); the most frequent signature size was 10 and signatures smaller than 9 targets were never selected. C, frequency of targets selected by LOOCV; 10 targets were selected in more than 90% of the cases and 9 targets were selected fewer than 50% of the cases.

Figure 3.

Classification Performance. Whole blood from 128 subjects was collected in PAXgene Blood RNA tubes and used to measure host response gene expression in response to viral infection. A logistic regression equation was used to generate a probability of viral infection based on the level of expression of mRNAs in the signature. This signature classifies subjects as viral or nonviral. Therefore, all cases of nonviral infection (bacterial, healthy, and noninfectious illness (NI)) are expected to appear similar. Each symbol represents 1 subject. The box denotes the interquartile range. Plus signs (+) represent statistical outliers. The dashed line at p (Viral) = 0.27 represents the prevalence of viral ARI in the study cohort.