Researchers at the University of Massachusetts Amherst’s New England Regional Center for Vector-Borne Diseases have devised a new, more accurate method for detecting the emerging Powassan virus in ticks, which can cause life-threatening neuroinvasive diseases such as encephalitis and meningitis. .
NEWVEC researchers found that this robust, real-time approach reduces the incidence of false-positive test results. The team describes the study in a special issue of the journal Viruses, titled “Tick-borne viruses: transmission and surveillance.”
“Powassan has been a growing concern in New England over the past several years and false positives can confound surveillance efforts,” says vector-borne disease expert Stephen Rich, professor of microbiology at UMass Amherst and principal investigator and executive director. by NEWVEC. “The development of sensitive detection methods for diagnosis and surveillance is essential.”
Powassan virus, named after the town in Ontario, Canada, where it was first identified in 1958 in a 5-year-old boy who died of encephalitis, is a flavivirus related to West Nile and other mosquito-borne viruses.
Although still rare, the incidence of Powassan virus is increasing dramatically in the US, predominantly in the Northeast and Great Lakes region. More than 10% of the record 290 cases reported in the United States in 2022 (compared to just one case per year between 2004 and 2006) resulted in death, and half of the survivors suffered long-term neurological damage. The virus is transmitted to humans mainly by Scapular Ixodesthe same blood-sucking deer ticks that transmit Lyme disease, babesiosis, and other tick-borne diseases.
The NEWVEC team, which brings together academic communities, public health professionals, and residents and visitors from across the Northeast in an effort to reduce tick- and mosquito-borne diseases, developed a real-time triplex PCR test for simultaneous detection and quantitative analysis of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis, or deer ticks. (The prototype of the Powassan virus is mainly found inIxodes cookei and Ixodes marxi ticks that feed almost exclusively on groundhogs in their burrows and rarely bite humans or human pets).
The NEWVEC team conducted a study of ticks in coastal and offshore Massachusetts, focusing on 13 sites in the highly endemic tick-borne disease regions of Cape Cod and Martha’s Vineyard. They tested ticks for the Powassan virus, comparing their new triplex PCR method with the standard commercially available Luminex xMap technology.
“The good news is that ours works just as well as the other one. In other words, everything that the other one could detect, we could detect,” explains Rich. “The good news is that we also overcome the problem of false negatives, which is what happens when a sample is not of sufficient quality for any test to detect the virus in it.”
The new triplex method achieves a reduction in false negatives through the use of “intelligent” quality control. Both tests seek to detect the presence of Powassan virus RNA. “But we also performed a pairwise search for tick RNA, which is present in all ticks, regardless of whether they have the virus or not,” Rich says. “And what that tells us is that if we can amplify the tick’s RNA, then we have some hope of being able to detect the virus’s RNA. If we don’t detect the tick’s DNA, then we have no hope of being able to detect the virus’s RNA. virus RNA of the virus.
“And before we developed that method, people were wondering, if they were curious, whether a negative result meant that the virus was not there or that the sample was not analyzable. So, we have ruled out that last possibility. And now we know with some certainty that when a tick tests negative, it is a true negative. It’s not that the sample is simply not good enough.”
In the areas surveyed, “we found pockets of high incidence of this virus,” says Rich.
Powassan virus was detected at four of six sites on Cape Code and two of seven sites on Martha’s Vineyard. Of 819 ticks collected, 33 (4.03%) tested positive for Powassan virus and 752 tested negative for Powassan, using the new triplex method. Thirty-four ticks (4.15%) failed the quality control RNA test. That showed that the standard Luminex method underestimated the overall prevalence of Powassan virus because those 34 ticks tested negative for Powassan. And only 30 ticks tested positive using the Luminex method, demonstrating that the triplex technique has a higher sensitivity for detecting virus RNA.
Infection rates reached as high as 10.43% at one site in Truro on Cape Cod, and were completely absent at seven other sites. All ticks that tested positive for Powassan virus also tested positive for lineage II deer tick virus.
Researchers say they hope this improved triplex PCR test will be useful in transmission studies and as a tool to monitor and prevent Powassan virus infections in Massachusetts and other areas where the virus has been reported.
“Powassan virus only poses a threat to people through a tick bite,” Rich says. “That’s why these highly accurate and sensitive tick tests are so valuable in assessing where and when the risk of exposure is greatest.”