Urine Becoming Preferred Zika Sample as Testing Industry Prepares for Summer Mosquito Season
Diagnosing Zika virus is challenging. While diagnosis previously relied on serology, it is difficult to differentiate Zika antibodies from other closely related viruses, including dengue. As a result, the World Health Organization (WHO) has encouraged test developers to move away from serology-based assays towards nucleic acid-based assays that are more specific and sensitive, if performed […]
Diagnosing Zika virus is challenging. While diagnosis previously relied on serology, it is difficult to differentiate Zika antibodies from other closely related viruses, including dengue. As a result, the World Health Organization (WHO) has encouraged test developers to move away from serology-based assays towards nucleic acid-based assays that are more specific and sensitive, if performed during the acute phase of infection. But, new evidence shows that the sensitivity of nucleic acid testing can be impacted by sample type.
Test developers around the world are working to quickly deploy tests for surveillance, clinical diagnosis and monitoring, and blood donation screening. Diagnostic Testing and Emerging Technologies (DTET) undertook a sampling of evolving testing recommendations, federal efforts to speed diagnostic development, and industry’s progress on bringing tests to market as the Southern sections of the United States brace for mosquito season and the possibility of local Zika transmission.
The U.S. Centers for Disease Control and Prevention (CDC) recommends testing for potentially exposed persons with signs or symptoms consistent with Zika virus infection and for asymptomatic pregnant women within 12 weeks of exposure.
From Jan. 3, 2016 to March 5, 2016, Zika virus testing was performed for 4,534 people in the United States who traveled to or moved from areas with active Zika virus transmission, with pregnant women accounting for nearly three-quarters of those tested (73.6 percent), according to a report from the CDC’s Epidemic Intelligence Service published April 22 in Morbidity and Mortality Weekly Report (MMWR). The authors say that while currently the likelihood of Zika infection among asymptomatic persons in the United States with a travel-related exposure history is low (less than one percent among pregnant women tested in the United States), providers should continue to offer testing to asymptomatic pregnant women with potential exposure due to the potential for adverse pregnancy and infant outcomes with perinatal Zika infection.
Preferred Sample Guidance
As more is understood about the biology of the Zika virus, modes of testing are changing. Significant development efforts are underway for improved molecular and serologic assays for detection of Zika infection. In addition to clinical diagnostics, assays are in development for blood, organ, and tissue donor screening, given the recent reports of several probable cases of transfusion transmission in Brazil.
“Nucleic acid testing is moving forward well with extremely high sensitivity and throughput, development efforts are very successful,” Michael Busch, M.D., Ph.D., co-director of the Blood Systems Research Institute in San Francisco, tells DTET. “On the serology size there are fundamental challenges, biological challenges, in populations with a history of dengue. In general there is good progress on the development of laboratory-based assays.”
In an interim guidance published May 13 in MMWR, the CDC says that real-time reverse transcription–polymerase chain reaction (rRT-PCR) is the preferred test for Zika virus infection because of its rapid turnaround and its high specificity. However, given emerging information, urine samples may be preferable to serum depending on the length of time from onset of symptoms.
Zika virus RNA is unlikely to be detected in serum after the first week of illness, but Zika virus RNA can be detected in urine for at least two weeks after onset of symptoms, the CDC says. Therefore, CDC recommends that Zika virus rRT-PCR be performed on urine collected less than 14 days after onset of symptoms in suspected patients and that rRT-PCR urine testing be performed in conjunction with serum testing if specimens are collected less than seven days after symptom onset.
This recommendation was based on findings of a Florida Department of Health study of multiple specimen types from persons with suspected Zika virus disease. Test results were classified by specimen type and number of days after symptom onset to determine the most sensitive and efficient testing algorithm for acute Zika infection. Results were published May 10 in MMWR.
Zika virus RT-PCR was performed at the state’s public health laboratory using a laboratory-developed test based on a previously published protocol using two RT-PCR targets. Serologic testing was performed on all serum specimens. Case definition criteria for Zika virus disease was based on serology and required Zika virus–specific IgM antibodies and no dengue virus–specific IgM antibodies in serum or cerebrospinal fluid samples. Urine and saliva RT-PCR tests were only used for surveillance purposes, while serum analysis was used for diagnostic purposes.
Urine specimens were collected from 70 patients with suspected Zika virus disease from zero to 20 days after symptom onset. The vast majority (93 percent) tested positive for Zika virus RNA using RT-PCR. For urine specimens collected from persons within five days of symptom onset, 95 percent (52 of 55) tested positive by RT-PCR compared to only 56 percent (31 of 55) of serum specimens collected on the same date. For specimens collected more than five days after symptom onset, 82 percent (9 of 11) of urine specimens tested positive by RT-PCR, compared to none of the serum specimens. Viral RNA was detectable in urine as early as the first day of symptoms and as late as 20 days after onset of symptoms.
Zika virus IgM antibodies were detectable in 40 percent (22 of 55) of the serum specimens, including two specimens collected one day after symptom onset. Among the 31 cases in which urine specimens tested positive by RT-PCR, but serum specimens tested negative, Zika virus IgM antibody was detected in 74 percent of the serum samples.
“Results of testing conducted at Florida Department of Health Bureau of Public Health Laboratories suggest that urine might be the preferred specimen type to identify acute Zika virus disease,” writes lead author Andrea M. Bingham, Ph.D.
Expanded Funding for Testing, Test Development
Federal agencies are accelerating efforts to combat the Zika epidemic on multiple fronts.
Increasing Sample Access - Industry cites a lack of access to positive Zika blood samples as a significant barrier to advancing test development. To speed the development of diagnostic tests for Zika virus infection, the U.S. Department of Health and Human Services’ Office of the Assistant Secretary for Preparedness and Response (ASPR) is supporting the collection of blood samples from people in the continental United States and Puerto Rico who have been infected with Zika virus. Under a six-month, $692,000 project funded by ASPR’s Biomedical Advanced Research and Development Authority, Clinical Research Management (Hinckley, Ohio), will collect blood samples from people with confirmed Zika virus infection, in coordination with state and local health departments and the CDC. These samples will be collected and made available to diagnostic companies for use in validating the performance of their tests.
Military Enhances Testing, Surveillance - The Armed Forces Health Surveillance Branch allocated $1.76 million in additional funding in mid-May to 10 Department of Defense (DoD) laboratories to enhance their Zika virus surveillance efforts worldwide. (This is in addition to the $51 million to support a range of emerging infectious disease surveillance programs already allocated this fiscal year to network partners.) The enhanced Zika virus surveillance will involve 10 projects in 18 countries and territories by four lab partners based in the United States and five located overseas. DoD labs will use the Zika money for three kinds of surveillance studies: retrospective examination for Zika virus exposure among DoD personnel through serum repository samples; expansion of clinic-based surveillance for Zika virus disease among DoD and civilian populations with febrile disease in California, Arizona and Texas; and expanded testing for Zika virus in mosquitoes in the Caribbean, East Africa, and Southeast Asia.
|FDA ’s First EUA for a RT-PCR Zika Assay|
|The U.S. Food and Drug Administration (FDA) granted Emergency Use Authorization (EUA) for multiple tests to detect patients who have been infected with Zika virus. The first RT-PCR assay granted EUA was the CDC’s Trioplex Real-time RT-PCR Assay for the qualitative detection and differentiation of RNA from Zika virus, dengue virus, and chikungunya virus. The test is intended for use with human sera or cerebrospinal fluid (collected alongside a patient-matched serum specimen), and for the qualitative detection of Zika virus RNA in urine and amniotic fluid (each collected alongside a patient-matched serum specimen). The FDA previously gave EUA to the CDC’s Zika Immunoglobulin M (IgM) Antibody Capture Enzyme-Linked Immunosorbent Assay.|
Sampling of Test Development Efforts
Below is a sampling of industry test development efforts.
Clinical Testing Devices
Tetracore (Rockville, Md.) deployed its Cirrus DX point of care molecular device (Tetracore T-COR 8 instrument and the Collectto- Test cartridge) for the detection of Zika, dengue, and chikungunya viruses in collaboration with the Naval Infectious Diseases Diagnostic Laboratory on the island of Grenada in late May. The T-COR 8 instrument can run up to eight samples at a time and has a built-in battery that can run the instrument up to four hours without power. The proprietary collection device enables samples to go directly from the finger stick into the C2T cartridge without additional processing. This is the first time the system is being used for clinical diagnostics. Previously, the company says, the Cirrus DX solution was used to detect bio-threat agents and for veterinary testing. The system is expected to be deployed on other islands during the summer, enabling sharing of information about the spread of Zika in the region, through the system’s connectivity capability that allows instrument access from anywhere in the world on a secure network.
altona Diagnostics (Germany) was granted EUA by the FDA in mid-May for its RealStar Zika Virus RT-PCR Kit, allowing the test to be used by CLIA High Complexity Laboratories. The molecular diagnostic tool can provide in vitro qualitative detection of RNA from the Zika virus in human serum or urine (collected alongside a patient-matched serum specimen) from individuals meeting CDC Zika virus clinical (symptoms) or epidemiologic (travel) criteria for testing. The kit can be used on multiple platforms.
Rheonix (Ithaca, N.Y.), in collaboration with New York University College of Dentistry (NYUCD), received funding to develop a rapid diagnostic for Zika virus infection. The $656,414 award supplements an existing Small Business Innovation Research grant to develop a fully automated screening and self-confirming assay to simultaneously detect and confirm the presence of Zika virus in a single, small sample of saliva or blood. The proposed approach is based upon previous success in which the Rheonix-NYUCD team developed a dual assay for the simultaneous detection of HIV antibodies and viral RNA in a single specimen. Richard Montagna, Ph.D., senior vice president for scientific and clinical affairs, tells DTET that the dual-assay, fully automated system can pick up early infections with viral RNA or later infections with antibodies, but that the company is still ensuring the assay can be adapted from HIV to Zika.
First Commercial Laboratory Clearance
Quest Diagnostics (Madison, N.J.) received FDA EUA for the Zika Virus RNA Qualitative Real-Time RT-PCR test at the end of April. The emergency clearance from the FDA is the first for a test developed by a commercial laboratory in the United States. The molecular test is intended for the qualitative detection of RNA from the Zika virus in human serum specimens. Quest Diagnostics revealed plans to make the new test broadly available to physicians for patient testing, including in Puerto Rico, early in May.
Investigational Approval for Blood Screening
Roche was granted approval to initiate collection and testing of blood samples for screening with the cobas Zika assay under an Investigational New Drug Application (IND) protocol. The cobas Zika test uses the cobas 6800/8800 Systems, to qualitatively detect Zika virus RNA in plasma specimens from human blood donors. Initially, the cobas Zika test will be deployed to screen blood donations collected locally in Puerto Rico, enabling the reinstatement of the blood services in Puerto Rico and reducing the reliance of blood importation from other areas in the United States. The second stage of deployment for the cobas Zika test will prepare for screening of blood donations collected by blood services in the southern United States, which could be impacted by the domestic spread in the virus during mosquito season.
Takeaway: As the Southern parts of the United States brace for summer mosquito season, the federal government and industry are trying to accelerate the readiness of clinical diagnostic and blood screening tests for Zika detection.
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