It started around this time last year, in August 2014. Young children with respiratory distress were arriving in unusual numbers to Kansas City’s Childrens Mercy Hospital. Soon the Comer Children’s Hospital at the University of Chicago had a similar report. By mid-October, 691 people, mostly young children, were associated with the outbreak, which was diagnosed as Entervirus D-68 (EV-D68) infection. Recent data counts at least 1153 confirmed cases that have resulted in 14 deaths, with the total number of infected individuals unknown (since most people with colds don’t go to the doctor, let alone have their cold-causing microbe sequenced).
Prior to this outbreak, EV-D68 was detected in only the most severe patients, with fewer than 10 cases diagnosed annually in the US. These were detected and diagnosed the same way as early cases in the 2014 epidemic: amplification and subsequent sequencing of EV-D68 genes. Such a small incidence created little demand for better diagnostic techniques (better = faster, more accurate, more specific, cheaper, or some combination of these qualities). The 2014 outbreak was the impetus to develop a better diagnostic assay developed at the CDC: one that detected viral genes directly through RT-PCR.
A second phenomenon during this time also increased demand for improved diagnostics: 118 strange cases of limb weakness, or acute flaccid myelitis, were reported, some concurrently with EV-D68 infection. While only two reports of cerebrospinal fluid testing positive for EV-D68 have been published, these flaccid paralysis cases bear similarity to other viral causes, including enteroviruses (among other viruses). Improved diagnostics could therefore not only help improve patient outcomes, but gain a better epidemiological sense of the virus and its sequelae.
Near the site of initial outbreak reports, doctors at Washington University of St. Louis continued refining the RT-PCR diagnostic technique. Now Wylie and colleagues have published their results in the August issue of the Journal of Clinical Microbiology.
Using the genome sequenced by this same group in January 2015, the researchers set out to make a specific test for EV-D68 nucleic acid. EV-D68 uses (+)ssRNA as its genomic material. They based the sequence on specificity to EV-D68 but found a region in the conventional “gold standard” of enterovirus sequencing, the VP1 gene. As clinicians in the center of the outbreak, the authors were able to test respiratory secretions of clinical samples (saliva and nasal drippings).
Most important was ensuring assay specificity to minimize misdiagnoses. The authors used patient samples confirmed to carry EV-D68 as positive controls, and several other related viruses (including other related enteroviruses) that could be present in patients with similar symptoms as negative controls.
The authors’ assay, called the WashU protocol, outperformed previous assays, including the assay released by the CDC in the midst of the outbreak. It accurately amplied EV-D68, but not the closely related EV-D70 sequence, from patient samples. The WashU assay was sensitive down to 4 copies of viral RNA per sample, making it more sensitive than all available assays except a Pan-Enterovirus assay (which doesn’t differentiate between different EV strains).
What does a better diagnostic test mean for clinical research? Clearly, it’s easier to pinpoint where an outbreak is when diagnosis is quick and easy. More hospitals will diagnose specific microbial infections if the test is inexpensive and fast, leading to better epidemiological data. This will allow more rigorous analysis of EV-D68 and its most severe symptoms.
Diagnostic tools may not seem inherently sexy. They are often reactionary: advances are often made after an outbreak has begun. But these are the tools that allow us to track epidemics, measure subclinical infections, and identify index cases. The Ebola outbreak has revolutionized diagnostics of contagious samples in limited facilities – and many of the articles are open access, to expedite spread of the important knowledge within. These tools are win-win for both patients and clinical researchers: patients get better care and researchers learn more about an outbreak.
Will these assays confirm a causative link between EV-D68 and acute flaccid myelitis? We don’t know for sure. As for the EV-D68 outbreak, the numbers have waned. Enteroviruses circulate year round, but can be especially present in late summer and early fall, and we have yet to see if this will be a new seasonal pattern. If it does reappear, at least doctors will have better tools in their arsenals to recognize it.
-- Julie Wolf