Time to prepare for dengue testing?

May 1, 2011

Mosquitoes are more than just a nuisance in the United States. Some transmit viruses that sicken people, agricultural animals, and wildlife. Perhaps no one knows this better than Lillian Stark, PhD, MPH, MS, virology administrator at the Florida Bureau of Laboratories (FBL) and adjunct faculty member at the University of South Florida College of Public Health. Below, she explains the problem and what the FBL is doing to help control it.

Nancy Maddox:

You are a virologist who studies mosquito-borne viruses, also known as arboviruses. What kind of data do you collect, and how is it used?

Lillian Stark, PhD, MPH, MS: Florida has a warm, even subtropical, climate, and mosquitoes may be present all year. Only certain types of mosquitoes, however, carry arboviruses, and transmission does not occur all the time. Our job is to perform surveillance testing to determine when transmission is occurring, so we know when the disease risk is increased for humans and horses. We send weekly reports to partners in county health departments and mosquito-control agencies so they can take action to reduce the level of risk when necessary.

Maddox: What arboviruses are present in Florida?

Stark: West Nile virus (WNV), St. Louis encephalitis (SLE), eastern equine encephalitis (EEE), and now all four types of dengue viruses (DEN1-4).

Maddox: Is it possible or even likely that these viruses will spread to other parts of the United States?

Stark: WNV and SLE occur throughout the U.S. already. EEE occurs primarily east of the Mississippi River, with the closely related western equine encephalitis in the West.

Maddox: How serious are these mosquito-borne diseases, especially dengue?

Stark: Any of the mosquito-borne diseases are serious for those who contract them. From a public-health standpoint, arboviruses are a concern.

Lillian Stark, PhD, MPH, MS

Dengue is only recently spreading into the U.S., chiefly a result of travelers bringing the disease into the country with them and then meeting up with the right type of local mosquito, Aedes aegypti or Aedes albopictus.

A primary case of dengue may cause only mild flu-like symptoms. But there is a reason dengue is also known as “breakbone fever.” Severe cases can include excruciating body and joint aches, high fever, severe headache with pain behind the eyes, nausea, vomiting, and a rash. Unfortunately, infection with one of the four types of dengue does not confer immunity against the other three. On the contrary, each subsequent infection with a different strain is more likely to prompt severe illness.

Maddox: Tell us about the dengue outbreak that began in Key West in 2009 and is still ongoing.

Stark: In September 2009, the first locally acquired case of dengue in the state of Florida in decades was detected in Key West, prompting a massive investigation by the Florida Department of Health, the Centers for Disease Control and Prevention, and Monroe County Mosquito Control. Retrospective surveillance testing detected 22 additional cases. Molecular assays picked up DEN1 virus in two human serum samples and in two groups of mosquitoes collected from the center of the transmission area. Local transmission of DEN1 so far has been restricted to Key West. In 2010, however, a single case of locally-acquired DEN3 was detected in another south Florida county and a case of locally-acquired DEN2 was found in a third. Neither of these strains has spread … yet.

Maddox: What kind of testing does the state perform in the laboratory to track arboviruses?

Stark: In 2010, the Florida Bureau of Laboratories tested 265 clinical serum specimens for dengue, with 103 positives, compared to 53 tests and 18 positives in 2009. This may not seem like a lot, but each specimen is tested for IgM and IgG antibodies against DEN1-4, WNV, and SLE. Antibodies to all of these viruses may cross react to varying degrees in the assays. Moreover, when an individual is suffering a second dengue infection, after previously being infected with a different strain, the IgM response may be quite low. Since infections with all of these viruses are possible and symptoms may be similar, we need to do the comparative testing to be sure.

If serum is collected less than five days after onset of fever, antibodies may not yet have developed, but the virus may be detected in the patient’s blood. We use a nested reverse transcriptase polymerase chain reaction [RT-PCR] assay to detect the virus and are in the process of validating a test we developed for real-time RT-PCR, which is faster, easier, and less prone to problems than the nested procedure. We also perform genetic sequencing of detected viruses so we can confirm epidemiologic linkages and new importations.

Molecular assays are sensitive, specific, and fast. They work best for a specimen drawn during acute illness — less than five days post-onset. This requirement limits their usefulness when dengue is not quickly recognized as a possible diagnosis. The new NS1-protein test may be able to detect the virus a few days longer, and antibody tests are useful for specimens collected after acute illness, when the virus is undetectable.

Maddox: Is arbovirus testing widely performed in hospital and commercial laboratories?

Stark: No. There are not many test requests in the U.S. Keep in mind, when the prevalence of disease is low, the predictive power of a single positive test is low. Only a few test kits are FDA approved. This new commercial NS1-protein assay for dengue is recently available, but its diagnostic potential has not been fully determined. The PCR assay and gene sequencing we perform are laboratory-developed tests.

Maddox: Can you speculate on likely reasons for the recent uptick in arboviruses in the U.S.?

Stark: Transmission of all these viruses occurs through a complex ecological cycle; the virus has to replicate in both its vertebrate hosts and in the mosquito carrier. In addition, the effectiveness of transmission can be impacted by weather patterns. Some years, there is only a little transmission; other years, more. Studies to determine how these viruses interact with the ecosystem will help us figure out the triggers for effective transmission to people. We are even now working with university researchers on such studies.

FDA approves first U.S. test to diagnose dengue fever

A test to help diagnose people with symptoms of the dengue fever has been approved by the U.S. Food and Drug Administration (FDA). The DENV Detect IgM Capture ELISA test detects antibodies to dengue virus in blood samples from patients with signs and symptoms of dengue fever or dengue hemorrhagic fever. The test, based on technology developed by the U.S. Centers for Disease Control and Prevention, is manufactured by Inbios of Seattle. This is the first FDA-approved test for direct detection of dengue virus. The FDA cautions that the assay detects an IgM antibody response that is not detectable until three to five days after the onset of fever.

Nancy Maddox, public-health communications specialist, is a consultant located in Alexandria, VA.