Epidemic 2014 enterovirus D68 cross-reacts with human rhinovirus on a respiratory molecular diagnostic platform

Abstract

Enterovirus D68 (EV-D68) is an emerging virus known to cause sporadic disease and occasional epidemics of severe lower respiratory tract infection. However, the true prevalence of infection with EV-D68 is unknown, due in part to the lack of a rapid and specific nucleic acid amplification test as well as the infrequency with which respiratory samples are analyzed by enterovirus surveillance programs. During the 2014 EV-D68 epidemic in the United States, we noted an increased frequency of "low-positive" results for human rhinovirus (HRV) detected in respiratory tract samples using the GenMark Diagnostics eSensor respiratory viral panel, a multiplex PCR assay able to detect 14 known respiratory viruses but not enteroviruses. We simultaneously noted markedly increased admissions to our Pediatric Intensive Care Unit for severe lower respiratory tract infections in patients both with and without a history of reactive airway disease. Accordingly, we hypothesized that these "low-positive" RVP results were due to EV-D68 rather than rhinovirus infection. Sequencing of the picornavirus 5' untranslated region (5'-UTR) of 49 samples positive for HRV by the GenMark RVP revealed that 33 (67.3%) were in fact EV-D68. Notably, the mean intensity of the HRV RVP result was significantly lower in the sequence-identified EV-D68 samples (20.3 nA) compared to HRV (129.7 nA). Using a cut-off of 40 nA for the differentiation of EV-D68 from HRV resulted in 94% sensitivity and 88% specificity. The robust diagnostic characteristics of our data suggest that the cross-reactivity of EV-D68 and HRV on the GenMark Diagnostics eSensor RVP platform may be an important factor to consider in making accurate molecular diagnosis of EV-D68 at institutions utilizing this system or other molecular respiratory platforms that may also cross-react.

Fig 1. PCR amplification of a portion of the 5’-UTR from GenMark eSensor RVP samples.

cDNA synthesized from RNA extracted from de-identified and masked RVP samples was used for traditional PCR amplification of the 5’-UTR. Primers were described by Oberste, et. al., for the sequencing of EV-D68 [2]. Agarose-ethidium bromide electrophoresis demonstrated that 49 of 62 samples analyzed yielded a 396 base pair amplicon compatible with EV-D68. Results shown corresponded to samples 2–4, 6–13, and 15–17, and are representative of the range of band intensities observed for all positive samples. NTC: no template control.

Fig 2. Detection of enterovirus in sample 13 using the GeneXpert enterovirus assay.

As independent confirmation of the sequencing results for sample 13, we analyzed extracted nucleic acid on the GeneXpert platform. Sample 13, which had a RVP signal for HRV of 21.3 nA, gave a positive signal (blue line) with a C_T of 24.2 and an excellent amplification curve consistent with positive results obtained from CSF samples, whereas the internal control (CIC) amplified less efficiently, as expected, because of competition (gray line).

Fig 3. GenMark eSensor RVP signal strength for HRV in samples stratified by sequence results.

Un-masking of the RVP results for all 62 samples revealed that the 49 positive PCR amplicons identified were derived from those samples that were also positive for HRV on the GenMark panel. Sequencing of these amplicons revealed that 33 were EV-D68 and 16 were HRV. The mean signal strength in nA of the RVP results for those samples identified as EV-D68 was significantly lower than for the HRV samples (20.3 nA versus 129.7 nA, respectively, p < 0.00001).