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Published: May 2012Print Record of Viewing
More than 2.5 billion people are at risk of contracting dengue fever. There are an estimated 50–100 million infections per year, and 500,000 hospitalizations due to severe disease. Dengue virus is a member of the flaviviridae family, transmitted to a human host by Aedes aegypti and Aedes albopictus. Initial infections may be mild, or even asymptomatic, but secondary infections lead to more severe disease. Dr. Hata provides an overview of dengue fever and describes viral transmission, epidemiology, and pathogenesis of the infection. She also explains laboratory testing for diagnostic markers of the illness as well as recent molecular-based testing.
Presenter: D. Jane Hata, PhD, D(ABMM)
- Director of the Clinical Microbiology Laboratory in the Department of Laboratory Medicine and Pathology at Mayo Clinic Florida
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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.
Our presenter for this program is Dr. Jane Hata, Director of the Clinical Microbiology Laboratory in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Jacksonville, Florida. Dr. Hata presents a review of dengue fever, including transmission, presentation, and diagnostic methods.
Thank you, Sharon.
In this presentation we will discuss: The virus which results in dengue fever, and history of the disease, epidemiology and pathogenesis, the symptoms of dengue, treatment modalities, and diagnostic methods and future considerations.
Dengue virus infection is the most common mosquito-borne infection worldwide – even surpassing malaria. This is a member of the flaviviridae. Other members of the flaviviridae family include: yellow fever, West Nile virus, St. Louis and Japanese encephalitis.
The virus in this electron micrograph consists of a 40 – 50 nm sphere, surrounded by a lipopolysaccharide envelope.
There are 4 serotypes (1 – 4), all of which have the same clinical manifestations. We have seen a shift in serotypes; in the 1970s, dengue 1 – 4 was limited to Asia, whereas types 1 – 2 were seen in South America and Africa. Today we generally see all 4 serotypes distributed worldwide throughout the “dengue belt” which has traditionally extended in a zone where winter temperatures average 50 degrees Fahrenheit. Generally speaking, this covers the area between 30 degrees north and south latitude.
Structure of Dengue Virus
Dengue is a positive-sense, single-stranded RNA virus which is approximately 11 kilobases in length.
The genome depicted here contains regions encoding the viral capsid (C), the membrane (M), and the envelope (E), as well as 7 nonstructural proteins; labeled NS in the figure.
The capsid, membrane, and envelope are all associated with viral structure and subsequent uptake of virus by host cells.
Envelope glycoproteins are associated with binding to host receptors, agglutination of erythrocytes, development of neutralizing antibody, and immune response.
Each subtype of dengue is 65% genetically related to one another.
This figure is taken from an excellent review from Guzman et al, which is cited in the references at the end of this presentation. I strongly recommend this paper if you are interested in additional information on dengue.
Disease due to what we believe was dengue, was first described in a Chinese medical encyclopedia between AD 265 – 420.This included a disease description and association with “poison water”. Association with flying insects was also noted.
Several epidemics occurred between 1635 – 1699 in the Caribbean, Asia, Africa, and North America.
During this period, sailing vessels carrying mosquitos were primary means of transporting this vector from endemic regions to new areas.
This is an engraving is from the National Library of Medicine of Dr. Benjamin Rush. Not only was he a signatory of the Declaration of Independence, but a professor of chemistry and medical theory at the University of Pennsylvania.
During the Philadelphia epidemic of 1779 – 1780, Dr. Rush first described the dramatic symptoms of dengue as "Breakbone Fever."
Jump forward to the late 1930s, where WWII facilitated spread of dengue through Asia/Pacific region. Approximately 80,000 military personnel serving in the Pacific theater were diagnosed with dengue between 1942 and 1945.
Epidemiology of Dengue
There are 2.5 billion people at risk of dengue due to living in an endemic area, in addition to the actual living conditions. There are an estimated 50 – 100 million infections per year, and 500,000 hospitalizations due to severe disease.
The case fatality rate from severe dengue approaches 5%, however the risk of fatal disease is <1% with appropriate therapy.
There is a seasonal component to dengue. Peak frequency depends on where you are in the world; generally speaking, warm and wet conditions favor development of mosquitoes. This will vary between countries, and it is essential to remember in certain areas, the risk of dengue exists year-round.
Finally, there are worldwide outbreaks every 20 – 40 years.
Vectors of Dengue
Let us now turn our attention to the very important discussion of the mosquito vectors for the dengue virus.
Aedes aegypti is the primary vector associated with transmission of dengue – you can see the lyre-shaped dorsal pattern on the thorax of the mosquito, and silver bands on the legs.
This is a day biter, and preferentially feeds on humans, which makes it more likely to transmit dengue. It likes to both rest and feed indoors.
This species survives in the 50 degree Fahrenheit band; basically Mexico to Southern Brazil, but it can also survive where human conditions allow, thus is not solely dependent on climate. Aedes aegypti has historically persisted in Memphis, Tennessee, which was also the site of a major epidemic of yellow fever, another flavivirus, in the late 1800s.
Note I mentioned vectors – plural; we have an additional mosquito species now associated with transmission of dengue.
Aedes albopictus is also known as the Asian Tiger mosquito – you can see why by the striking white bands on the legs. A single dorsal stripe on the thorax differentiates this species from Aedes aegypti.This species moves in when Aedes aegypti declines in population. It has markedly different habits in that it is an aggressive biter, both day and night, and prefers to live outside. Humans are not its sole source of food, in that it bites a variety of other wild and domestic vertebrates as well.
Aedes albopictus is a vector for eastern equine encephalitis, in addition to dengue.
This species arrived in United States at the port of Houston in the 1980s via a shipment of old tires.
This mosquito migrates further from its breeding sites than Aedes aegypti, and thus can extend its range.
Life Cycle of Dengue Virus
The life cycle of dengue virus is strictly dependent on only mosquitoes and humans. Once the mosquito feeds on a viremic human, there is a 10-day extrinsic incubation period in which the virus develops and passes from the mosquito intestinal tract to its salivary glands.
Human infection results from the bite of the infected mosquito.
The mosquito remains infectious for its entire 1-month life, and can transmit infection with as few as 100 viral particles.
Mosquito eggs actually can survive desiccation for several months, and repopulation will occur once they are rehydrated.
Pathogenesis of Dengue
In the human host, after a bite, the virus infects immature dendritic cells of immune system.
The infected dendritic cells then migrate to the lymph nodes with activation of the cellular and humoral immune response.
Viral replication occurs in macrophages of lymph nodes, liver, spleen, parenchymal cells, as well as peripheral blood monocytes.
The host will then become viremic within 3 to 6 days.
Classification of Dengue Disease
Disease due to dengue virus was originally classified as dengue fever and dengue hemorrhagic fever. This placed an excessive emphasis on actual hemorrhage, whereas the most important manifestation was actually that of vascular permeability. Consequently, the World Health Organization (WHO) revised the disease classification in 2009; replacing 1997 recommendations. Disease is divided into 2 syndromes, dengue fever (DF) and severe dengue, which may include dengue shock syndrome (DSS), and hemorrhage.
Initial infections may be mild, or even asymptomatic, but secondary infections lead to more severe disease particularly if an infection with DENV serotype 1 is followed by DENV serotypes 2, 3 or an infection with DENV serotype 3 is followed by DENV serotype 2.
Age and nutritional status also affects severity of disease.
Antibody-Dependent Enhancement (ADE)
It is well established that the primary risk factor of development of the shock and hemorrhagic syndrome is evidence of a previous infection with dengue fever. But why are secondary infections more severe? Antibodies formed in response to a dengue infection are not cross-protective against other subtypes of the virus. In fact they may result in more severe disease due to a phenomenon known as antibody-dependent enhancement or ADE.
This occurs when there is a formation of immune complexes between dengue virus and existing non-neutralizing antibodies. Non-neutralizing antibodies result from previous dengue infection, or low level of maternal antibodies in infant sera.
Mononuclear phagocytes are infected through their Fc receptors by immune complexes and severe disease then results by suppression of innate immune response secondary to ADE via release of inflammatory cytokines and chemokines, resulting in enhanced disease.
Symptoms of Dengue
There are 3 phases of disease due to dengue virus infection, the febrile phase, critical phase, and recovery phase.
The febrile phase is characterized by high fever, headache, generalized arthralgia and myalgia; thus the "breakbone" description. Progressive leukopenia is noted and petechiae and bleeding from mucous membranes may occur.
In the critical phase we see defervescence, progression of leukopenia, and thrombocytopenia.
Importantly, we see an increase of capillary permeability leading to a plasma leakage – this is primary indicator of the severity of disease.
Abdominal pain is often a sign of plasma leakage, if uncorrected by intravenous rehydration, plasma leakage can lead to metabolic acidosis and disseminated intravascular coagulation, resulting in a high risk of death.
During the recovery phase, we see a resorption of leaked plasma.
The haemodynamic status stabilizes, and the urinary output increases. There is an overall clinical improvement. It is very important to note that the prevention of fluid overload during the recovery phase is critical, as this is a preventable cause of death in severe dengue.
There are warning signs that indicate that the patient has progressed during the critical phase to what is classified as "severe dengue" disease which may be fatal. This is characterized by: hypovolemic shock, bleeding from GI tract, liver failure, neurologic manifestations such as encephalopathy and weakness, bradycardia and multiorgan failure.
Treatment of Dengue
The treatment of both dengue fever and severe dengue consists of supportive care only, no antiviral agents are available for treatment. Acetaminophen is recommended for pain and fever; corticosteroids, aspirin or other nonsteroidal anti-inflammatory agents should be avoided.
The patient should be carefully monitored for hydration status and for development of a plasma leak, as demonstrated by a rise in hematocrit.
There is no vaccine for dengue currently available, although mosquito control is key. The best prevention to avoid dengue is to avoid mosquito bites!
Diagnosis of Dengue
Although we will discuss the laboratory diagnosis of dengue, this may not always be utilized. In 75% of cases, the disease may be asymptomatic. In many cases, dengue may be diagnosed by a combination of clinical signs in addition to travel history.
In addition, many other differential diagnoses need to be ruled out. Dengue presentation may be similar to: influenza, malaria, typhoid fever, leptospirosis, and other acute febrile syndromes.
Diagnostic Markers for Dengue
As with many other infectious diseases, an understanding of the sequence in which viral markers are expressed may help guide us in selection of the best test to use for diagnosis. You can see that the period of actual viremia is fairly short – 5 to 6 days after onset of illness. IgM starts to appear as viremia declines, and peaks approximately 14 days after onset of disease. IgM may persist up to 3 months.
IgG appears at the end of the first week of illness and slowly increases. IgG may be detectable over the lifetime of the individual. In secondary infections, high levels of IgG are detectable even in the acute phase of illness, whereas IgM levels are significantly lower. The structural protein, NS1 is expressed during the first 10 days of illness. All of these markers have been proven useful for diagnostic methods.
Laboratory Confirmation of Dengue
Viral cell culture for the dengue virus is performed using the C6/36 cell line of Aedes albopictus for growth – however, this cell line is very unlikely to be available in clinical laboratories.
Viral cell culture is confirmatory, and identifies serotypes via immunofluorescence with monoclonal antibody.
An acute serum sample collected within the first 5 days of symptoms is required for best yield of virus. Growth of dengue virus does not differentiate between primary and secondary infections.
Viral culture is generally performed only at research laboratories or certain state department of health laboratories. Cell culture facilities are required, and testing will usually take at least a week before results are available.
There are several molecular formats that have proven useful for the confirmation of dengue virus in patient samples.
Nucleic acid amplification tests (NAAT) are based on a nested PCR, which initially detects a highly conserved region of the virus, followed by a serotype-specific secondary PCR reaction.
Reverse transcriptase PCR and nucleic-acid sequence based amplification (NASBA) formats are also used.
Real-time PCR has an advantage due to its rapid turnaround time. Like other molecular tests it is useful for blood collected during the first 5 days of symptoms.
Real-time PCR has been reported to have 80 – 90% sensitivity; >95% specificity.
It is recommended that negative results from a molecular test are followed up with a serologic method.
Unfortunately, at this time, there are no FDA-approved tests available yet using these molecular methodologies.
The IgM capture ELISA method, also known as MAC-ELISA, can be used for confirmation of infection, as well as serotyping of infection. In this method, an antihuman IgM bound to a microtiter plate captures IgM in the patient sample. A serotype-specific dengue antigen (protein E envelope glycoprotein) is added and detected with an enzyme-conjugated, dengue specific monoclonal antibody.
False-positive results due to cross-reactivity with other flaviviruses such as yellow fever, West Nile virus, and St. Louis encephalitis can occur.
Reported sensitivity of MAC-ELISA varies between 61.5 – 99%; specificity varies between 79.9 – 97.8%. IgM can persist up to 3 months.
An FDA-approved test using this format became available in April of 2011.
The IgG ELISA is also available as a confirmatory test, if multiple specimens, collected during the course of disease, are used.
This test uses the same antigens as does the MAC-ELISA, the protein E membrane glycoprotein.
The IgG ELISA can differentiate primary or secondary infections, however you must have both acute and convalescent serum specimens. A negative IgG followed by a positive IgG is indicative of a primary infection. A 4-fold rise between acute and convalescent specimens is indicative of a secondary dengue infection. This assay cannot be used to serotype dengue infections.
Commercial tests are available; but they are not FDA-approved at this time.
An additional method that has been used for determination of dengue infection is the detection of antigen and antibody to NS1.
Remember, this is a structural protein which is secreted by all flaviviruses, and is detectable up to 10 days after onset of illness. The NS1 antigen will disappear once seroconversion has occurred in the host.
Both antigen-capture ELISAs and lateral flow-antigen detection methods are used, as well as NS1-specific IgM and IgG assays.
Many commercial assays available, however they are not FDA approved. As a whole, NS1 tests have excellent specificity; although sensitivity may vary (60 – 90%). Some test formats can be used for serotyping.
As I alluded to in the previous slide, a number of immunochromatographic lateral flow tests (cassette tests) are available for the detection of dengue infections. These tests are based on detection of IgM,or IgG antibody, or detection of the NS1 antigen.
Recall that the NS1 structural protein antigen is expressed within the first 10 days of illness. This provides a good marker for early detection of infections. However, since NS1 is detected in response to infections due to other flaviviruses, cross-reactivity can be an issue. These tests are simple to perform. They take 15 minutes to result, and have assumed a very important role in outpatient screening or other field work. Studies in Southeast Asia have indicated use of both antibody and antigen rapid tests may increase sensitivity for acute dengue and allow earlier detection of infection.
However, these tests are not available for US use.
Laboratory Confirmation of Dengue
Finally, the plaque reduction and neutralization assay, the PRNT assay, is the most specific serologic tool for the detection of dengue.
This test will subtype dengue infections, and measures titer of neutralizing antibodies. It is labor intensive and requires maintenance of very specific cell lines, thus it is primarily limited to research laboratories and certain state health departments. The CDC website listed on the slide gives a up-to-date overview of available dengue tests, which you may find helpful.
Let’s now shift gears and discuss what we may expect from dengue in the future.
Why Are We Concerned About Dengue?
Why are we so concerned about the occurrence of dengue in the United States?
For one thing, range of Aedes species has expanded, moving out of the southern states and persisting. We have a greater frequency of travel to endemic areas, and we have the ability to travel long distances in a relatively short period of time. We have had local outbreaks of dengue in the United States: in Hawaii in 2001 – 2002, and Texas in 2005. Notably, outbreaks have occurred in relief workers returning from Haitian earthquake in January 2010. There is an ongoing epidemic in Puerto Rico, accounting for 21,000 cases in 2010.
Key West, Florida 2009 – 2010
In the last several years, there has been an interesting turn of events in that we have had several self-sustaining epidemics of dengue fever in the continental United States.
In the fall of 2009, a 34-year-old female from Rochester, NY diagnosed with dengue had just returned from 1 week in Key West, Florida on day of illness onset. Shortly thereafter, a 48-year-old male resident from Key West was also diagnosed with dengue, however, he had no history of travel. Key West is 396 miles from Jacksonville, Florida as the mosquito flies.
In the follow-up investigation, the Florida department of health, working with the CDC conducted a serosurvey of Key West, which resulted in identification of 26 additional cases of dengue. Mosquito pools tested positive for DENV serotype 1. A separate study in 2009 indicated seroprevalence of 3 – 5% among residents. This indicated that dengue had indeed become established in the population.
Why Re-emergence of Dengue in Florida?
Signs point to a re-emergence of dengue in Florida – many of the characteristics apply to other areas of the US as well. We have lots of travelers going to and from endemic areas. We have a prevalence of Aedes species; both Aedes aegypti, and Aedes albopictus. Both species can easily overwinter here in Florida. Many parts of the US have recently experienced one of the warmest winters in history. We have a largely nonimmune population. We certainly have the opportunity for mosquito exposure; very few visitors come to Florida and stay inside for the entire week. The take-home message is that dengue should now be included in differential diagnosis of acute febrile illness, especially among those who live or travel in the Southern United States or tropics.
So, to summarize today’s presentation:
Remember that dengue virus is a single-strand RNA flavivirus, with 4 serotypes. It is responsible for the most common mosquito-borne viral infection. Dengue is transmitted by Aedes mosquitoes; Aedes aegypti and Aedes albopictus, and the incubation period after a mosquito bite is approximately 3 – 6 days.
Two types of disease are caused by the virus:
Dengue, and severe dengue, which can occur if the host has had a previous dengue infection.
Shock, plasma leak and hemorrhage can be ominous clinical signs. There are 3 phases of disease; febrile, critical, and recovery, and supportive care is the only recommended treatment.
Dengue can be diagnosed using the methods you see listed on this slide. Availability of these tests may be limited. Check with your local department of health or contact CDC for the most up to date information on testing. All of these methods have strengths and weaknesses, and you need to be aware of the phase of disease the patient is in, in order to make the best use of these diagnostic tests.
Finally, we discussed US outbreaks of dengue. An overall increase of dengue prevalence, and locally sustained infections have been attributed to travel, prevalence of mosquito populations, and a largely nonimmune population. Importantly, dengue should be considered as part of differential diagnosis in an acute febrile illness in travelers to tropical regions, or even residents in certain parts of the United States. For additional information consult http://www.cdc.gov/Dengue/.
This is an excellent resource that provides up to date information, and an interactive map which provides locations of recent outbreaks.