Qualitative Detection of Enterovirus D68 from PrimeStore® Molecular Transport Medium: Implications for Home- and Self-Collection

The Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the utility and need of home- and self-collection methods for pathogen detection and surveillance. Having readily accessible tools for home- and self-collection is not only important clinically but can also be utilized for research studies of emerging pathogens with outbreak potential.

Enterovirus D68 (EV-D68) is a nonenveloped, single-stranded, positive-sense RNA virus that is part of the Picornaviridae family and causes infections most commonly in children [1]. Infections range from asymptomatic to respiratory or febrile illnesses, but in a subset of patients EV-D68 has also been associated with acute flaccid myelitis (AFM), a devastating polio-like illness that leads to acute onset of limb weakness [1,2]. Increases in AFM cases have coincided with biennial EV-D68 outbreaks in 2014, 2016, and 2018 and this association has been supported by the detection of EV-D68 in neurons of a patient with AFM [2], [3], [4], [5], [6], [7]. Unlike other enteroviruses, EV-D68 can be acid labile, less likely to be detected in stool, and more commonly detected in the upper respiratory tract [8]. However, EV-D68 shedding duration in the upper respiratory tract is unknown and would require serial testing to determine shedding time. With a lack of an expected EV-D68 outbreak in 2020, likely due to non-pharmaceutical measures of the COVID-19 pandemic, an outbreak was imminent [9,10]. Increase in acute respiratory illnesses and detection of EV-D68 was seen again late summer 2022 and likely will not be the last outbreak [11]. Therefore, increasing the available tools for pathogen detection will be vital for further understanding EV-D68.

In 2014 a real-time reverse transcriptase polymerase chain-reaction (rRT-PCR) test was developed by the Centers for Disease Control and Prevention (CDC) for detecting EV-D68, and an assay for qualitative EV-D68 detection was validated at Children's Hospital Colorado (CHCO) in 2015 for in-house clinical and surveillance testing [12,13]. Samples collected in clinical settings are placed in universal transport media (UTM, COPAN Diagnostics, Murrieta, CA), which has been shown to maintain genetic material stability for SARS-CoV-2, influenza A, adenovirus, respiratory syncytial virus (RSV), rhinovirus, and human metapneumovirus molecular detection up to 48 hours at room temperature (25°C) and seven days at refrigerate (4°C) [14]. However, this media has limitations for home- and self-collection since it requires adequate temperature stability, transit time, and biohazard precautions. To date, no temperature stable, inactivating transport medium has been validated for potential use in home- and self-collection samples and EV-D68 detection.

PrimeStore® molecular transport medium (MTM) is a novel transport medium that inactivates pathogens while preserving and stabilizing molecular material. It can also be stored at room temperature (RT), making it an appealing media to use for studies that require home- and self-collection sample shipment. This MTM has been utilized in prior studies showing similar detection compared to UTM of different respiratory viruses such as influenza, RSV, human metapneumovirus, and parainfluenza [15]. However, studies have not been performed on the use of MTM and EV-D68 detection. The objective of this study was to compare the performance characteristics of our validated, in-house EV-D68 rRT-PCR assay when samples are collected and stored in PrimeStore® MTM.

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