College of American Pathologists
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As season starts, here’s the buzz on WNV

August 2003
Cover Story

Karen Titus

Nancy Cornish, MD, had barely finished wrapping up last season’s West Nile virus cases when it became time to start worrying about the disease anew.

The large volume of testing for West Nile virus buried laboratories last year, including Methodist and Children’s Hospitals, Omaha, where Dr. Cornish is director of microbiology. She and her colleagues initially sent their WNV specimens through the Nebraska Public Health Laboratory to the CDC’s Arbovirus Diseases Branch, in Fort Collins, Colo., for testing. “When they became overwhelmed, we had an agreement with the Colorado Department of Public Health to send our specimens there,” Dr. Cornish says. “They became overwhelmed as well.”

Thus, says Dr. Cornish, “We didn’t get our last results back until March.” Just in time to start contemplating the 2003 West Nile season.

There’s plenty to ponder, but little laboratories can do. Though the disease has been present in the United States since 1999, when it began its cross-country march from the East Coast, no treatment or vaccination exists. Mortality rates are relatively low but troublesome nonetheless. Older patients—50 or older—infected with the virus are more likely to die than younger ones. “But in Nebraska, we had the youngest death reported in the country last year—age 19,” says Dr. Cornish. “He was perfectly healthy otherwise. Nobody could figure out why he died of this.”

Likewise, testing options for clinical laboratories are still rather limited, as commercial kits for screening are only now making their way into the market; the confirmatory test is not routinely available. “There’s precious little diagnostic testing that can be done” in most hospital-based clinical labs, says John Roehrig, PhD, chief of the CDC’s Arbovirus Diseases Branch.

All of which leaves most laboratories simply trying to educate clinicians about the disease and fine-tuning their relationship with local public health laboratories as mosquito season revs up throughout much of the country.

In one sense, WNV is not so unusual. As is so often the case, “Physicians don’t really understand the testing issues,” says Dr. Cornish.

The CDC ( and state and local health laboratories screen with an IgM antibody capture ELISA, a microtitre plate-based assay that is quickly evolving.

“Last year it was very labor intensive,” reports Bill Becker, DO, MPH, medical director of the Ohio Department of Health Laboratory and clinical associate professor, Department of Pathology, Ohio State University. Plates had to be prepared a day in advance of the actual testing, “which added to our testing time significantly, as you can imagine.”

This year Focus Technologies Inc., of Cypress, Calif., has introduced a faster version of the test, which has initially been made available to public health laboratories. However, 510(k) documentation was submitted to the FDA in late June for the IgM ELISA, along with an IgG ELISA. The tests were on expedited status, according to Mary Kay Mosch, Focus’ vice president of marketing for laboratory services and diagnostics, and the company hoped the ELISAs would be commercially available as early as late July.

The most efficient diagnostic method is detecting IgM antibody to West Nile in serum or cerebrospinal fluid collected within eight days of clinical illness onset; the test is positive in 90 percent of infected people within that period. “The IgM antibody capture ELISA is basically designed to detect a serologic response at one of the earliest stages of clinical infection,” says Dr. Becker.

Patients who have had WNV in the past, or remotely, might be IgG positive only, he continues. “However, it is known that IgM positivity can remain for up to well over a year in patients after acute illness. So we’re aware that IgM persists for longer periods of time than we’re used to thinking about in other viral illnesses, making it a useful marker even well after a patient’s clinical illness resolves.”

Adds Wayne Hogrefe, PhD: “As far as the lab is concerned, an IgM case in the CSF is considered diagnostic of central nervous system involvement with West Nile. The sensitivity is extremely high—about 100 percent,” says Dr. Hogrefe, director, diagnostic product development, Focus Technologies.

In serum, an IgM positive is also considered a presumptive case of West Nile, he continues, and the sensitivity for IgM in serum also approaches 100 percent. “It’s very good. Something on the order of one out of the several thousand confirmed cases last year didn’t have IgM on the first sample looked at—it’s an unusually high number,” he says.

Though it can be used in both serum and CSF, the interpretation of the IgM test is much more straightforward in the latter case. “It’s much more indicative of acute infection,” says Dr. Hogrefe. The sample, of course, is determined by patient status. “If someone has symptoms of encephalitis or meningitis, they’re probably going to get a tap,” he says.

Most symptomatic cases of WNV infection, however, are very mild and do not progress to CNS involvement, as Dr. Becker points out. “These milder cases are being termed ‘West Nile Fever,’” he says, “and in many states these cases are not being reported at this time.” Indeed, says Dr. Hogrefe, the majority of WNV cases are asymptomatic or so mildly acute that they are essentially underdiagnosed. About 20 percent of those infected develop West Nile Fever, he estimates.

The need for an IgG ELISA is less clear-cut. The CDC, for one, does not include IgG testing in its algorithm. But at Focus Technologies, which, along with the CDC and public health laboratories, has performed nearly all the WNV testing done in the United States up until now, the test has a use, says Dr. Hogrefe.

“In our hands, 25 percent or so of the samples that have come in that are IgM positive have yet to show IgG,” he says, adding that an IgM-only positive makes people leery, wondering if it’s a false-positive. “So more than anything else, there’s an added comfort level of seeing the IgG there.” Moreover, Dr. Hogrefe says, when a second sample is obtained, IgG serum conversion from negative to positive is considered confirmatory of a West Nile virus infection.

At Integrated Regional Laboratories-Florida, serum or CSF IgG is part of the WNV testing protocol alongside serum or CSF IgM.

“We do it because there’s such a large pool of previously infected people,” says James Robb, MD, medical director, IRL-Florida and vice president, medical affairs, MDS Diagnostic Services, U.S. “And if you’ve got someone with Alzheimer’s or stroke, or metabolic dysfunction or a psychiatric problem, you’ll blame it on West Nile. So the IgG is used for the differential diagnosis.”

With both serum and CSF ELISA testing, there are caveats. Cross-reactions can occur with other, related flaviviruses in the WNV family, particularly St. Louis encephalitis virus. And patients who have been recently vaccinated or recently infected with other flaviviruses, such as yellow fever, dengue, or Japanese encephalitis, may also test positive.

The CDC’s Dr. Roehrig points to another issue. “It was shown, after the 1999 outbreak, that certain individuals will carry West Nile-specific IgM in their serum for periods of time, long enough that they could last from one transmission season in one year all the way through to the beginning of the transmission season next year.” When West Nile first appeared in the United States, it was easy to assume this marked everybody’s first exposure to it. “But as we get more and more people infected, including a lot of silent infections, we’re going to run into individuals who may have had West Nile one year—maybe it wasn’t even diagnosed, maybe it was a mild case—and then the next year come in with a headache or a fever due to something completely different. But it will look like West Nile based on the serological testing.” In those cases, he says, it’s important to look for increasing levels of antibody in acute and convalescent phase sera, as would happen in an active infection.

“That being said,” he adds, “if it’s a fatality, and there’s actually tissue available, it’s a pretty direct approach to identify West Nile in those specimens, so we can differentiate the etiologic agent fairly easily.”

The CDC and selected public health laboratories have been confirming WNV ELISA results using the plaque reduction neutralization test, or PRNT.

Though the demand for PRNTs was overwhelming last year, it will probably decline this year as the CDC refines its WNV surveillance methods. Last year PRNTs were run on all positive ELISAs. “This year, as West Nile is first detected in a particular area, the states will try to push all those cases through to confirmatory testing,” says Dr. Roehrig. Then, as West Nile activity is confirmed in that particular area and the number of cases there increases, the IgM capture ELISA test alone should suffice, he says.

Focus Technologies planned to begin offering PRNT testing, via its infectious disease reference lab service, by the end of July. “I think it will be something that’s used early in the outbreak of the season, but as time goes on, requests or the need to do confirmatory testing will diminish,” says Dr. Hogrefe. “Or, if you had a really difficult case and were trying to figure out exactly what’s going on with a particular patient, you might want to look at confirmatory testing. But it would be done more on a case-by-case basis.”

Unlike the WNV ELISAs, there appears to be little need for a commercially available PRNT assay. “It’s very tedious,” Dr. Hogrefe says. “It’s really a confirmatory tool for epidemiological studies more than anything else.”

Since WNV testing lies outside the purview of most clinical laboratories, at least for now, their responses will likely remain low-key this season.

“I think the best thing a local laboratory can do when they get a specimen that comes in from a case that’s suspicious of West Nile infection, would be to do the normal tests that they would do on an individual with meningoencephalitis to rule out herpes or enterovirus, if they have that testing in place,” says Dr. Roehrig. “And then contact their county or even state health department to determine ways to follow up with testing.” Followup testing is also critical with suspected WNV fatalities.

Dr. Robb’s laboratory is an MDS Diagnostic Services consolidated core lab for the 13 HCA hospitals in Southeast Florida. “Although we send all of our WNV specimens to the Florida Department of Health [FL-DOH], we are organized to rapidly notify our entire system about important infectious disease information,” including anthrax and malaria, Dr. Robb says. “In the case of the first WNV encephalitis cases here in South Florida last season, we received the first notification from the FL-DOH that the patients were positive for WNV, and we immediately alerted all of our infection control staff, pathologists, laboratory managers, administrators, and infectious disease physicians in our 13 hospitals that active human WNV encephalitis was present in our system. This occurred on a Thursday evening, and we wanted all of the above people to be aware of this event before the weekend.”

The speed at which WNV has moved throughout the country last year, with confirmed cases in 44 states, came as a surprise to most public health authorities, says Dr. Becker. “Going into 2002, I don’t think anybody anticipated the 4,000-plus cases we had.”

This year, he says, everyone should be better prepared, largely due to rapid advancements in testing. “It’s gone from almost totally handmade to a kit format, and eventually it’s going to be available for the clinical labs.”

At the same time, because the virus is so complex ecologically, it’s difficult to make predictions about its spread. Florida, for example, had its first WNV activity in 2000–2001, but last year’s activity was less than might be expected. The Midwest, on the other hand, was extremely hard hit last year, with the greatest number of cases reported in Illinois, Michigan, and Ohio.

Despite its rapid spread, WNV cases have been relatively rare, leaving pathologists to scratch their heads (if not their mosquito bites) a little over the amount of attention the virus has received.

“It’s not a real big player,” says Dr. Cornish. “It’s certainly the biggest arbovirus outbreak that we’ve ever seen in the Western hemisphere, but influenza is more important in terms of morbidity and mortality.” When she gave a talk about WNV to employees at her institution recently, more than 100 showed up—“which is very unusual,” she says. By the same token, only 50 percent of the employees at her hospitals took advantage of free influenza vaccinations that were offered last year. “There’s more anxiety about West Nile, when in fact fewer people were infected and many fewer people died. I’m not sure why that is, except maybe it’s a new disease and it’s been hyped in the press a lot.”

Dr. Robb notes that the risk appears to be extremely minimal in his area. “The only cases we had in my system last year were two men who were out extensively in the Everglades. But the risk exists all year-round, because our mosquitoes never rest. We have about eight different species of mosquitoes, two or three of which carry the virus, and huge mosquito flights come in from the Everglades every now and then. We also have a lot of horses and poultry in central Florida, creating a huge reservoir.”

“People shouldn’t be scared of it, but they should be aware of it,” Dr. Robb continues. “It’s the same with SARS. I try to keep things in perspective—but I had my N-95 mask with me when I went to Toronto during their outbreak.”

“It’s a scary disease,” Dr. Roehrig concedes. “You’re talking about encephalitis being transmitted by a route you have very little control over. No one likes to think about getting a brain infection from a crazy mosquito bite that you never had to worry about before.”

Karen Titus is CAP TODAY contributing editor and co-managing editor.