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Transcript and References

Introduction

Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Dengue virus poses a significant public health threat worldwide. Rapid and accurate identification of the infection allows for directed monitoring of patients who may be at increased risk of developing hemorrhagic fever of dengue shock syndrome. In this month’s “Hot Topic,” my colleague, Dr. Elitza Theel, will provide information regarding the serologic and molecular testing Mayo Clinic Laboratories offers pertaining to the identification of infection with dengue virus. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients’ health care.

Thank you for the introduction and also to the viewers for joining us today. This Hot Topic will focus on the available diagnostic testing options for detection of dengue virus, with a brief review of the clinical manifestations of disease, treatment, and prevention.

Disclosures

Before we begin, I should mention that I have no financial or other conflicts to disclose.

Presentation Outline

During this presentation, I’ll provide some background on dengue virus and briefly review the clinical manifestations of infection. I’ll next focus on the available diagnostic methodologies, including testing by nucleic acid amplification tests, or NAATs, and evaluation of the serologic response to infection by detection of both antigen from and antibodies to dengue virus. Finally, I’ll conclude with a brief discussion of treatment and prevention of dengue virus infection.

 Overview

To begin, dengue virus is a member of the flavivirus family of viruses, which also include West Nile virus, St. Louis encephalitis virus, and yellow fever virus, among others. These are all enveloped, positive sense, single-stranded RNA viruses.

The dengue virus complex is primarily comprised of four different dengue virus serotypes, which are roughly 65% genetically identical to each other and all members are transmitted by Aedes species mosquitos, specifically Aedes aegypti and Aedes albopictus, both shown on the right. Importantly, humans are the primary reservoir for dengue virus, which limits the transmission cycle of this virus from mosquitos to humans and back to mosquitos. Following infection, the incubation period in humans can range from 3 to 12 days prior to development of symptoms.

Epidemiology

Dengue virus is most prevalent in the so-called “dengue belt,” which is roughly outlined by the red lines in the image shown here, laying between 30 degrees north and south latitude. These regions are typically in tropical and subtropical areas where winter temperatures do not fall below 50 degrees Fahrenheit. Within this belt, roughly 2.5 billion people are at risk of contracting the virus, among whom approximately 50 to 100 million are infected annually, and 500,000 patients develop severe disease. Over the last 50 years, according to the World Health Organization, there has been a 30-fold increase in the number of dengue infections, elevating the dissemination of this virus to a pandemic threat.

With regards to dengue virus infections in the United States, the vast majority of cases are acquired during travel to endemic regions. However, Aedes species mosquitos are endemic in the United States and prior outbreaks have been recorded in the southern region, including Texas and Florida, so the potential for additional outbreaks and further dissemination of dengue into the continental US is not insignificant.

Clinical Presentation

With regards to clinical presentation, following exposure to the virus, approximately 50% of individuals will remain entirely asymptomatic. The remaining 50% of patients can present with one of three disease manifestations. The first of these is an undifferentiated fever, which presents with nonspecific symptoms very similar to other causes of acute febrile illness. For many of these patients, unless specific dengue virus testing is performed, the diagnostic cause will remain unknown. These patients are typically younger children or those experiencing their first dengue virus infection and typically recover on their own without need of medical attention.

The second manifestation of disease is dengue fever, which can be with or without hemorrhage. These patients are typically older children or adults and present with prolonged high fever and severe headache, retro-orbital pain, and myalgia. While some mild epithelial hemorrhage may occur with dengue fever, these patients do not progress to significant plasma leakage, which is a hallmark of dengue hemorrhagic fever or dengue shock syndrome, which is the third potential clinical manifestation following infection with dengue virus. Individuals with dengue hemorrhagic fever progress through three different phases, including the febrile phase, which is indistinguishable from dengue fever, followed by the critical plasma leak and hemorrhage phase, which occurs around the time of fever defervescence. Some of the warning signs for progression to plasma leakage include increased capillary permeability, elevated hematocrit, pleural effusion, and hypovolemia. If these signs are noted and vascular permeability is not countered, patients can progress to shock, organ failure, and ultimately death. Finally, for patients who survive this stage, the third phase of dengue hemorrhagic fever is the convalescent phase, at which time fluids that have leaked from the intravascular space are reabsorbed.

Diagnostic Approaches

Diagnosis of dengue virus infections is multifactorial and dependent on the presence of appropriate clinical symptoms and a proper exposure history. There are also a number of laboratory methodologies available to confirm the diagnosis. These methods include culture, though this is available only through the CDC and as such, has limited clinical utility in the acute care setting. More commonly used testing methods include NAATs, which are limited to select reference laboratories, and serology for detection of both antigen from and antibodies to dengue virus.

Importantly, the decision of which test to choose among these is largely dependent on the duration of patient illness and understanding of the immune response to infection, which I will briefly review here.

At the time of mosquito transmission viremia rapidly spikes and is detectable by molecular methods for the first 4 to 6 days following symptom onset and during acute disease. During the viremic period and up to 10 days following symptom onset, the dengue virus NS1 antigen is released and also detectable. The IgM antibody response to dengue virus infection becomes detectable roughly 4 to 5 days following symptom onset and remains elevated for 2 to 3 months. Finally, IgG antibody levels become detectable 5 to 7 days following symptom onset and will remain elevated for years to decades.

NAATs for Dengue Virus Detection

So keeping these analyte profiles in mind, I’ll first focus on NAAT testing to detect dengue virus RNA. This methodology is most useful during acute disease, again typically within the first five days of symptom onset, during which time viral titers are still elevated. Notably, there are no FDA-cleared molecular assays for dengue virus; however, the CDC and a small number of larger reference laboratories have implemented their own laboratory-developed tests. In general, the sensitivity of molecular assays ranges from 80% to 90%, which is largely dependent on when the sample was collected with respect to symptom onset. These assays have a high specificity, however, of 95% or greater, and have the ability to differentiate between the various dengue virus serotypes.

Importantly, a negative NAAT result for dengue virus should not be used to rule out infection, and providers are strongly encouraged to follow up with serologic testing if risk of infection is high.

NS1 Antigen Detection

A second marker for detection of dengue virus infection is the NS1 antigen. NS1 is a conserved nonstructural glycoprotein that has approximately 80% amino acid similarity between the four dengue virus serotypes and is secreted from infected host cells.

Importantly, NS1 can be used as a marker of acute infection as it is detectable during the viremic period and prior to the development of a detectable IgM response to dengue virus. It also remains detectable for up to nine days following symptom onset. As you can see on the chart below, it nicely overlaps the window period during which time viremia is declining and antibodies have not yet developed sufficiently to be detectable and, as such, it is a suitable alternative to molecular assays for detection of acute disease.

Our own evaluation of a commercially available dengue virus NS1 enzyme immunoassay showed a sensitivity of 96% and specificity of 86% when compared to molecular detection of dengue virus RNA, further supporting the use of this marker to identify patients with recent dengue virus infection.

Finally, the NS1 marker is also detectable during reinfection with dengue virus, regardless of the serotype.

Antibody Detection

The final diagnostic method to discuss is standard serology for detection of IgM and IgG class antibodies to dengue virus.

Starting with IgM, again, it takes roughly three to five days following illness onset for this antibody class to become detectable, and as you can see from the graph on the right, they can remain detectable anywhere from 30 to even 90 days following infection. Additionally, in cases of reinfection, IgM levels are notoriously low and short lived, making diagnosis of subsequent dengue virus infections based on IgM antibodies alone challenging.

Many of the assays used currently at various reference laboratories and the CDC are laboratory developed and have variable sensitivity and specificity performance characteristics ranging from 60% to almost 100%. A recently FDA-cleared IgM assay has been introduced and is currently used at Mayo Clinic Laboratories with a sensitivity and specificity of approximately 88% and 100% respectively, compared to other commercially available laboratory assays.

Important to note, however, is that cross-reactivity with antibodies to other flaviviruses can occur, including with antibodies to West Nile virus, which is now endemic in the United States.

With regards to IgG antibodies, these usually become detectable roughly 10 days following symptom onset. The presence of IgG antibodies only is indicative of past exposure to dengue virus, and similar to the limitation of IgM antibodies, cross-reactivity of IgG class antibodies to other flaviviruses can occur.

Treatment and Prevention

Finally, as with all arboviruses, there is no targeted antiviral agent against dengue virus infection and treatment is entirely supportive. Clinical management includes proper fluid balance, is critical for patients with dengue hemorrhagic fever, and can decrease mortality to the virus by 50 to 100-fold.

Regarding infection prevention, it is important for patients who are viremic with dengue virus to avoid mosquitos during that period in order to minimize infection of native mosquitos to the virus. Also, personal protective measures against exposure to mosquitoes are critical to minimize infection. This includes use of mosquito repellants and wearing long-sleeved shirts and pants when venturing into areas with high mosquito burdens. Finally, while vaccines against dengue virus continue to be developed, in March of 2019, the FDA cleared the first dengue vaccine, Dengvaxia, for use in children aged 9 to 16 years who have had a prior, laboratory-confirmed dengue infection and who live in an endemic area.

Thank you for your attention.

References

1. Guzman MG, Halstead SB, Artsob H, et al: Dengue: a continuing global threat. Nature Rev Microbiol 2010;8:S7-S16
2. Anderson NW, Jespersen DJ, Rollins L, et al: Detection of the dengue virus NS1 antigen using an enzyme immunoassay. Diagn Microbiol Infect Dis 2014;79(2):194-197
3. www.cdc.gov
4. www.who.org