Charna Albert
September 2023—For the tickborne and mosquito-borne arboviruses, which are frequently more transient in blood and tissue than other pathogens, both molecular and serologic testing have a place in the diagnostic toolkit. But what test to order, and at what point during disease presentation, is often misunderstood, and it can lead to result misinterpretation, says Elitza Theel, PhD, D(ABMM), director of the infectious disease serology laboratory and co-director of the vector-borne diseases service line at Mayo Clinic.
“The common thread is you’ll have a positive serology and the PCR will be negative, if it’s even ordered. And then clinicians will ask, ‘Is this serology positive due to past infection? Wouldn’t we expect the PCR to also be positive if this is an acute infection?’ The teaching point is that for most of the arboviral diseases, whether it’s Powassan, West Nile virus, or even Zika, if you’re testing in blood in particular, that viremic period is fairly short and transient.” And the level of viremia is low to begin with, further hampering the sensitivity of PCR testing. After the viremic period, PCR testing likely will be negative despite the recent infection, she says.
“We wouldn’t necessarily expect to see serology and PCR positivity at the same time. That’s why we have two different markers,” she says, “because they should be used at different times in the disease course.”
In a session at this year’s Association for Diagnostics and Laboratory Medicine meeting (formerly AACC) and in a recent interview, Dr. Theel, who is also professor of laboratory medicine and pathology, Mayo Clinic College of Medicine and Science, brought clarity to the confusion around infectious disease serology in common clinical situations.
She shared the case of a 56-year-old woman from Minnesota who presented to the emergency department in July with a 10-day history of fever, increasing confusion, gait instability, and aphasia. “And she had a maculopapular rash on her torso,” she says. A lumbar puncture showed elevated protein (75 mg/dL) and a lymphocytic pleocytosis, suggesting a viral infection. “She had a gamut—and I do mean a gamut—of infectious disease testing on serum and spinal fluid, all of which came back negative, including cell-free metagenomic NGS testing on her plasma and spinal fluid.” An arboviral serology panel was ordered three days post-admission; the patient had equivocal IgM results for West Nile virus in CSF and serum. The lab was asked: Could this be consistent with a West Nile virus infection, and if it is West Nile, why is the PCR negative in both specimens?
At the time the patient presented, there were reports in the news about Powassan virus, an emerging tickborne virus found in the upper Midwest. “That caught our attention because Powassan is in the same family as West Nile virus, so we wondered if this could be causing cross-reactivity,” she says. At that time they were developing their own Powassan PCR as well as working with a company to develop a Powassan IgM EIA, and decided to test the patient’s samples by both. The Powassan virus IgM EIA came back positive in CSF and serum. The PCR was again negative. “We ended up sending the sample to the CDC for plaque reduction neutralization testing to confirm our results,” and the titers were elevated at 1:128 in CSF and 1:1024 in serum. “So we had a case of Powassan virus.”
Powassan virus is an Ixodes tick-transmitted flavivirus maintained in small rodents. It can be transmitted to humans within 15 minutes of tick attachment, Dr. Theel says, “which is super rapid compared to transmission of other tickborne diseases like Lyme, Anaplasma, and Babesia,” which take 24 to 48 hours.
About five percent of Ixodes ticks in the upper Midwest and Northeast have been found to carry Powassan, and the number of cases reported to the CDC has increased over the past decade. “But a lot of them are underdiagnosed due to limited clinician awareness and a general lack of diagnostics,” she says. “So we’ll be seeing more of this virus in the coming years.”
About a third of Powassan patients develop systemic symptoms, and a third of those go on to develop central nervous system involvement. “It’s a bad virus—there’s about a 10 percent fatality rate,” she says. Though no targeted treatment is available, a diagnosis makes it possible to discontinue unnecessary empiric antibiotics and to provide diagnostic and prognostic information to physicians and patients. “And it helps us continue to understand the epidemiology of this emerging virus.”
Test selection for the neuroinvasive arboviruses should be guided by when patients present after symptom onset. The incubation period ranges from two to 10 days. “We then see a short viremic period—it peaks anywhere from two to four days after symptom onset, and then that viremia drops off precipitously by the end of the first week. So your molecular testing, if you have it, is most useful in those first four to five days after symptom onset.” IgM develops at the end of the first week of symptoms, and IgG develops two to three days later. “In patients with more than that week or five to seven days of symptoms, serologic testing is going to be your best approach.”
Few patients with symptoms present immediately, she notes. “Nobody presents on day one of a fever—they typically wait it out, they don’t feel better, and then they present,” at which point they may no longer be viremic. Even in the viremic period, the level of viremia is low. “It’s not like other viruses, like HIV, where you get into the millions of copies, potentially,” she says. “With these arboviral infections, they don’t get that high because humans are not the natural, amplifying host.” If a naïve mosquito feeds on a human host infected with West Nile virus, for example, the mosquito will not be infected due to the low viral load. “We’re considered ‘dead-end’ hosts because the virus dies with the human. So they just don’t replicate as well.” In addition, limited molecular tests are available, “which is why we continue to rely on serology, but it has to be used at the right time.”
Six cases of Powassan had been documented in Minnesota by late July, Dr. Theel says. “I don’t actually like the word ‘emerging,’ because the virus is and has been out there. We are only now more aware of and able to detect it. We need to be aware of these viruses that aren’t in our daily differential diagnoses.”
Dr. Theel shared the case of a 52-year-old man who presented to his primary care physician with a two-week history of persistent severe fatigue and headaches, as well as on-and-off fever and shortness of breath.
He was otherwise healthy and had been camping in upper New York State four weeks prior to symptom onset. His routine labs showed anemia and thrombocytopenia; his neurology, cardiology, and rheumatology labs were normal. His physician ordered a panel of tickborne disease serology and molecular testing. “He was positive for all of the serologies, but negative entirely by PCR,” she says. The question for the laboratory was how to interpret it.
The patient’s serology workup was as follows: Babesia microti Ab: 1:64; Ehrlichia chaffeensis Ab: 1:64; Anaplasma phagocytophilum Ab: 1:1024; and Lyme disease: EIA(+), IgM blot (+, two bands), IgG blot (+, seven bands).
“When it comes to tickborne disease serology, anything with a titer of 1:64 or greater is considered positive,” Dr. Theel says, “but anything with a titer of 1:512 or higher is typically considered to indicate more recent and acute infection. With that background, for the Babesia and Ehrlichia we would say this is likely indicative of a past or more remote infection.” The patient’s elevated titer for Ehrlichia could be due to cross-reactivity to antibodies of Anaplasma, “because those are closely related organisms, so that might be bumping up the Ehrlichia a bit. But we were more concerned with a potential coinfection with Anaplasma and Lyme disease.” A negative PCR result for these doesn’t exclude infection.
With the tickborne diseases—other than Lyme disease, for which serologic testing is the preferred approach—timing is everything “when it comes to what test you’re going to use at what point after symptom onset in these patients,” she says. After a tick bite and infection, the incubation period is two to approximately seven days. Bacteremia (or in the case of Babesia, parasitemia) increases and peaks about four to seven days post-infection, and in otherwise healthy individuals, DNA levels decrease rapidly and may be negative two weeks after infection. “So molecular testing for tickborne diseases is most sensitive in that first seven to 10 days after symptom onset, with sensitivities ranging from 70 to 90 percent. They’re higher the sooner after onset you perform testing.”
IgM and IgG develop concurrently following the first week of symptoms. “We don’t recommend IgM testing for a number of reasons: one, cross-reactivity, but two, IgM is considered a marker of acute disease, and it’s not acute if it’s a week later,” she says. “And we have PCR testing,” which fills that role.
Mayo Clinic’s diagnostic algorithm for the tickborne diseases begins with establishing the appropriate clinical suspicions, Dr. Theel says. “What time of year is it? What are the symptoms? What are the exposure and pathogen risks?” If the patient’s risk factor is for Ixodes-transmitted pathogens, “we recommend considering testing for coinfections, because Ixodes can transmit multiple pathogens at the same time. And we also strongly encourage empiric treatment of patients while you’re waiting to get those Anaplasma or Ehrlichia results back.” For patients presenting with fewer than seven to 10 days of symptoms, the recommended approach is molecular testing for Anaplasma, Ehrlichia, and Babesia, and Lyme disease serology. “In patients with more than 10 days of symptoms we recommend starting with serology,” and adding on molecular testing in immunosuppressed patients. “We would expect the bacteremia or parasitemia to remain longer in those particular patients,” she explains. “In other patients, it wouldn’t be beyond the realm of possibility for the PCR to be negative that far out of symptom onset.”
The patient in this case was diagnosed with Lyme and Anaplasma coinfection and successfully treated, she says. “These coinfections are not uncommon—roughly 10 to 15 percent of patients with Lyme disease also have an Anaplasma infection. So for laboratories offering tickborne disease testing, it’s important to make sure you have panels put together for both your serology and your molecular tests, if you offer them, so we’re not missing these coinfections.”
In another of Dr. Theel’s cases, one related to herpes simplex virus 1/2 serologic testing, a 32-year-old woman presented to an STI clinic for screening.
There were no findings on the physical exam and the patient had no concerns. “Her clinician did the gamut of STI testing, all of which came back negative,” she says, “except he also ordered HSV serologies.” The IgG was negative and the IgM positive.
In this case, Dr. Theel says, “in the absence of any suspicious lesions which would be tested by PCR, the approach I suggested was a repeat serology, because if this were an acute HSV infection, that was asymptomatic, we would expect the IgG to seroconvert.” So the physician ordered a second HSV 1/2 antibody panel from an alternative laboratory, which came back negative. “Based on that, we would interpret that the IgM most likely was a false-positive,” she says. “The clinician attempted to tell the patient this was nothing to be concerned about—a false-positive on the lab side—but unfortunately she was convinced she had a newly acquired HSV infection, which she blamed a new boyfriend for and ended the relationship. So these results can have real-life consequences.”
In the U.S., 48 percent of those between 14 and 50 are seropositive for HSV-1, and 12 percent are positive for HSV-2, according to the 2015–2016 National Health and Nutrition Examination survey
(McQuillan G, et al. NCHS Data Brief. 2018;304:1–8). “Clinically we’re seeing an increasing number of HSV-1 anogenital infections, whereas many of those who have an HSV-2 infection are unaware, due to mild disease or unrecognized infection. But it’s important to identify these people to halt the transmission cycle,” Dr. Theel says.
HSV antibodies develop seven to 10 days post-infection, with IgM and IgG seroconverting to positive roughly concurrently. IgM decreases to undetectable in four to five months, while IgG in many patients persists indefinitely. HSV 1/2 IgM serologic assays are not type-specific, and cross-reactivity with parvovirus B19, varicella-zoster virus, rheumatoid factor, and SARS-CoV-2 can cause a false-positive (Vandervore L, et al. Diagn Microbiol Infect Dis. 2022;103[1]:115653). HSV-2 IgG assays are type-specific, based on HSV glycoprotein G (gG1 and gG2).
HSV-2 IgG testing is useful for a subset of patients, according to the Centers for Disease Control and Prevention 2021 STI treatment guidelines. They are patients with recurrent genital or atypical symptoms and a negative RT-PCR, women of childbearing age with lesions suspicious for HSV and a negative RT-PCR, those with a clinical diagnosis of genital herpes without laboratory confirmation, and those with a recently diagnosed partner, as well as patients who present for STI testing and have multiple partners or are living with HIV or at high risk. HSV-1 IgG testing, on the other hand, is less informative, Dr. Theel says. “There’s no distinction between site of infection,” and the seroprevalence rate is high.
The U.S. Preventive Services Task Force this year released its guidance reaffirming its 2016 recommendation against routine HSV-2 serologic screening in asymptomatic individuals (U.S. Preventive Services Task Force. JAMA. 2023;329[6]:502–507). “And the reason for that is you’re not going to treat these individuals,” she says. “There’s no clinical action you’re going to take on those test results.”
Moreover, IgM-based serologic assays often are used inappropriately to diagnose acute infection, with HSV IgM testing one of the most misused (Theel ES. Clin Chem. 2022; 68[1]:36–39). “HSV IgM testing is just flat-out not useful,” she says, noting that the American Academy of Pediatrics, the Infectious Diseases Society of America and American Society for Microbiology, and CDC all have released guidance stating that the testing should no longer be performed. “So we decided to look back on our utilization of HSV IgM testing in the laboratory, based on the case I presented and all of these guidance documents.”
Dr. Theel and her colleagues performed a retrospective review of all HSV tests ordered at Mayo Clinic Laboratories between May and July 2018, determining utilization and positivity rates for the following assays: HSV 1/2 PCR, HSV IgM only, HSV IgM and HSV 1/2 IgG serologic panel, and concurrent (within seven days) HSV IgM and HSV 1/2 PCR orders (Jung S, et al. J Appl Lab Med. 2020;5[1]:241–243). “At the time we were offering all sorts of HSV serology combinations, as well as HSV PCR,” she says. “During that three-month period, we performed almost 23,000 HSV tests, of which, to my shock and concern, 20 percent were for HSV IgM alone.”
During that period, the lab performed 9,999 HSV 1/2 RT-PCR tests, with a positivity rate of 15.7 percent and 14.2 percent for HSV-1 and HSV-2, respectively. Among the 4,584 HSV IgM-only orders, positivity was 2.3 percent. “If we’re thinking IgM is a marker of acute disease, that doesn’t jibe well with the 15 percent PCR positivity,” she says. Of the 8,100 IgM and IgG serologic panels performed, 0.6 percent were positive by IgM alone. And of 170 patients tested for HSV IgM and by HSV 1/2 RT-PCR within seven days, 49.7 percent were positive by RT-PCR only, 4.7 percent were positive by both RT-PCR and IgM, and only 1.2 percent were positive by IgM alone.
“Taking all this into consideration, we concluded that HSV IgM testing is significantly overutilized in our laboratory with questionable value added to patient care. Based on this and all the guidance out there, we decided there was only one solution to our HSV IgM problem and that was to discontinue the test, which we did in 2019, with a lot of education around our reasoning and alternative approaches for testing.”
Dr. Theel recommends that physicians and laboratories assess if HSV IgM testing is being sent out and why. “We have got to stop doing HSV IgM. There is no purpose for that test.”
“It was one of the most overutilized tests in my laboratory,” she says.
Charna Albert is CAP TODAY associate contributing editor.