EMERGING TESTS

Testing Recommendations Expand as Zika Season Approaches

As warm weather sets in across the southern portion of the United States, public health officials are planning for the 2017 Zika season. This mosquito year experts have a better knowledge about the infection and its natural history, revised testing and laboratory guidance, and more available diagnostics. DTET conducted a comprehensive review of 2016 testing volumes, current clinical testing recommendations, remaining assay development challenges, and expectations for the upcoming season.

Zika Testing Volumes
There are no commercially available diagnostic tests cleared by U.S. Food and Drug Administration (FDA) for the detecting Zika virus. However, the FDA is working with Zika virus diagnostic developers to accelerate assay development programs. Over the past year there has been a significant increase in the number of Zika diagnostic products the FDA currently allows use of under emergency use authorization (EUA). Given the serious consequences of Zika, particularly during pregnancy, the FDA encourages laboratory developed test developers to submit a request for an EUA.

The first Zika diagnostics approved were developed by the U.S. Centers for Disease Control and Prevention (CDC)—the Immunoglobulin M (IgM) Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA; approved Feb. 26, 2016) and Trioplex real-time Reverse Transcription Polymerase Chain Reaction Assay (rtRT-PCR; approved March 17, 2016). While other tests from the private sector have received EUA, these initial two tests are still manufactured and distributed.

The CDC reported, as of January 2017 that it had received more than 165,000 samples for Zika testing across multiple laboratories, with the majority of samples tested using the MAC-ELISA assay.

Laboratory #Specimens Received # Specimens Tested by rtRT-PCR # Tested by Zika IgM MAC-ELISA
CDC-Atlanta 5,023 3,464 2,827
CDC-Fort Collins 18,262 3,926 15,571
CDC-San Juan 81,667 45,136 48,015
Laboratory Response Network 60,788 25,439 35,349

(As of 1/2017; Source: CDC Zika IMS Sustaining the Zika Response in 2017

AthenaResearch’s analysis of data from its athenahealth network (more than 5,000 ordered tests; October 2016, the end of the 2016 mosquito season) shows that Zika testing was limited. As might have been expected the majority of testing was conducted in women (87 percent) and most testing occurred in those aged 19 to 44 years (90 percent). Just over half of testing (52 percent) was initiated by obstetrician/gynecologists, while 23 percent of testing was ordered by primary care.

"Results of these [IgM] tests may not be able to determine whether women were infected before or after they became pregnant."

—CDC

Testing Recommendations
The U.S. Centers for Disease Control and Prevention (CDC) issued a Health Alert Notice on May 5 with updated guidance for testing women who live in or frequently travel to areas with a CDC Zika travel notice. The guidance includes an update on interpreting Zika virus IgM serological tests and a recommendation for nucleic acid test (NAT) testing at least once per trimester.

The CDC says its update reflects new data suggesting that Zika virus antibodies may stay in the body for months (beyond 12 weeks) in some infected individuals. Therefore, test results may not be able to determine how recently one was infected.

"Results of these [IgM] tests may not be able to determine whether women were infected before or after they became pregnant," the agency writes in the alert. "Although IgM persistence could affect IgM test interpretation for all infected people, it would have the greatest effect on clinical management of pregnant women."

To better determine infection timing, although possibly not conclusively, the CDC now recommends the following testing for pregnant women living in or frequently traveling to areas with Zika virus transmission or with a partner who tests positive for Zika virus infection:

  • Test pregnant women promptly, using NAT, if they develop symptoms at any point during pregnancy or if their sexual partner tests positive for Zika virus infection.
  • Consider Zika NAT testing at least once per trimester in asymptomatic women, in addition to IgM testing as previously recommended. The CDC warns, though, that a negative NAT test result does not rule out recent infection because viral ribonucleic acid (RNA) declines over time.
  • NAT testing of amniocentesis specimens may provide additional information to help determine whether positive IgM test results suggest a recent infection.
  • Consider IgM testing as part of pre-conception counseling to establish baseline IgM results before pregnancy.

Challenges to Diagnosis
Despite the revised testing guidance, diagnosis of Zika remains complicated for multiple reasons. First, clinically, most cases are mild infections with rather generic symptoms., which challenges identification of suspected cases.

From a laboratory perspective, cross-reactivity in assays between the Zika virus and other flaviviruses is common. Additionally, confirmation of Zika virus infection in patients presenting within one week of fever and consistent symptoms is based on detection of Zika virus RNA. However, experts say that during the acute phase of infection, Zika virus RNA levels in blood can be detected best between three and five days of symptom onset, but RNA concentrations are relatively low, with reported levels between 103 and 105 copies/mL.

Over the last year, researchers have looked at the effectiveness of other sample sources. Zika virus RNA was detectable in urine for up to 20 days after its clearance from blood, with RNA levels in urine as high as 106 copies/mL. Saliva may have some benefits for detecting active infections. But, experts say access to well-characterized clinical samples has been a major problem for manufacturers and has limited verification and validation studies of new assays.

Lingering concerns remain over viral persistence, assay specificity, turnaround time (CDC says theirs is seven to 10 days once samples are received), and the usefulness of plaque-reduction neutralization tests, which is the confirmatory test used due to its specificity in differentiating closely related viruses.

Ideally, experts say Zika assays would be multiplexed, allowing simultaneous detection of several pathogens (Zika, Chikungunya, and Dengue viruses) in a single sample. Additionally, a test that could simultaneously detect both Zika virus RNA and anti-Zika virus IgM could over the entire time period of acute Zika virus infection. Lastly, the ideal test would possess operational characteristics (in terms of storage, reagents, cost, etc.) could be used for public health in low- and middle- income countries.

The 2017 Zika Season
In 2016 Florida and Texas were the only continental U.S. states with local transmission, although California and New York experienced a large number of travel- related cases. May kicks off the 2017 transmission season. According to the Florida Department of Health, as of May 15, Florida had 44 Zika travel-related cases and four locally acquired Zika infections so far in 2017 (plus seven additional cases where transmission occurred in 2016, but testing happened in 2017). As of the week ending May 12, 2017, the Texas Department of State Health Services reported 12 Zika cases in the state, but with no breakdown of whether they were travel-related or locally transmitted cases. Additionally, Puerto Rico was especially hard hit in 2016 and has already seen 493 confirmed, symptomatic Zika virus cases (all locally transmitted), although there have been recent reports that the true number of cases in Puerto Rico may be under-reported.

To prepare, the CDC is maintaining surge planning for laboratory support. Additionally, the agency is planning to:

  • Refine the performance of diagnostic assays, including assessing the value of whole blood and urine in molecular diagnostics
  • Consider updates to the testing algorithm to allow increased flexibility and to simplify as appropriate
  • Assist as needed in moving testing to commercial laboratories
  • Conduct new research (e.g., developing a Zika virus multiplex bead assay, investigating more specific antibodies, developing rapid and specific IgM diagnostic test that uses mass spectrometry, and refining recombinant antigens in testing platforms to eliminate the need for inactivation of live virus)

Despite preparations, a report from the Public Broadcasting Service says that at an April meeting at the CDC, federal officials warned state health departments that Zika funding initially envisioned to last five years will likely run out this summer. The $1.1 billion that was approved in September 2016 was a one-time earmark to fight Zika and is now nearly depleted.

Takeaway: Experts hope that preparations for expanded laboratory capacity and an increasing number of EUA assays will help efforts to track and combat Zika virus this upcoming mosquito season. However, researchers remain engaged to improve upon current assays’ ability to determine timing of infection.

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