Accurate laboratory confirmation of dengue infection currently relies on demonstration of virus presence by the methods described below. All of these methods have limitations and their performance depends on the stage of disease progression when sample collection was performed (Guzmán ; Teles ; WHO ).
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Viral isolation is laborious and requires between seven to 14 days to obtain results. Thus, it is an unsuitable assay for early diagnosis of dengue infection. Cell culture is the most widely used method for viral isolation. Mosquito cell lines of Ae. albopictus C6/36 and Ae. pseudoscutellaris AP61 are the cell lines of choice for routine diagnosis ( Race ; Tesh ; Kuno ; Nawa ). Virus identification is mostly performed using dengue specific monoclonal antibodies in immunofluorescence (IF) ( Henchal ; Gubler ). The World Health Organization (WHO) recommends that virus isolation samples are collected from suspected dengue cases within the first five days from onset of fever ( WHO ).
In summary, both viral isolation by culture and viral genome identification by NAATs require highly equipped laboratories, well'trained staff, and their use should be restricted to samples collected between five days from fever onset ( WHO ). The high logistical requirements mean these methods are only suitable for implementation in national laboratories in most of the dengue endemic countries. Moreover, the NAATs often operate on sophisticated and expensive devices and contamination can often undermine their performance in routine clinical settings.
This technique allows for a faster turnaround time (24 to 48 hours), but is an expensive and often unaffordable option for routine diagnostic purposes in endemic countries ( Guzmán ). For routine diagnosis of dengue virus infection, the most commonly used nucleic acid amplification tests (NAATs) are based on a single reverse transcriptase polymerase chain reaction (RT'PCR) assay ( Morita ; Lanciotti ), a nested RT'PCR assay ( Lanciotti ), or a one'step multiplex RT'PCR assay ( Harris ). The sensitivity of RT'PCR assays compared to virus isolation in mosquito cell culture varies between 25% and 79% ( Raengsakulrach ). The reported sensitivity is above 90% for several recent NAAT methodologies compared to virus isolation using the C6/36 cell line ( Callahan ; Houng ; Wu ; Hapugoda ). However, an external quality control assessment of the efficiency and accuracy of dengue NAAT methods applied by expert laboratories, reported low sensitivity of these assays even in laboratories where reliable real'time procedures were used ( Domingo ). Time of sample collection is crucial for viral genome identification by NAAT and the WHO recommends samples are collected within the first five days from onset of fever ( WHO ).
Currently these assays are the most widely used laboratory method for diagnosis of dengue infection. The presence of IgM and IgG antibodies in correlation with dengue progression and type of infection (primary versus secondary) has been extensively characterized (Gubler ; Innis ; Chanama ; Wahala ). Although less expensive and simpler to perform than viral isolation and RT'PCR, these assays require testing of paired sera (for example, one sample collected during the acute phase and the second collected during the convalescent phase, with the second sample collected ' 7 days from onset of fever) to provide a definitive diagnosis (WHO ). As results are retrospective, they are unsuitable for making clinical decisions.
IgM antibody'capture enzyme'linked immunosorbent assay (MAC'ELISA) (Innis ), E/M'specific capture IgM and IgG ELISA (Burke ; Bundo ), E/M antigen coated IgM and IgG ELISA and the haemagglutination inhibition test (HI) (Clark ) are the most commonly used serological techniques for the routine diagnosis of dengue virus infections (Shu ; Guzman a). Traditionally, the HI test was used to detect and differentiate primary and secondary dengue virus infections due to its simplicity and sensitivity. However, this test has recently become less popular and has gradually been replaced by the MAC'ELISA and E/M'specific capture IgM and IgG ELISA (Shu ). Over 50 commercial kits are available with variable sensitivity and specificity (Innis ; Blacksell ; Dussart ; Blacksell ; Kumarasamy a; Kumarasamy b; Bessoff ; Dussart ; Lapphra ; Hunsperger ; McBride ; Ramirez ; Shu ; Zainah ; Guzman c; Lima ; Osorio ; Pok ; Tricou ; Wang ; Blacksell ; Chaterij ; Ding ; Fry ; Najioullah ; Blacksell b).
Although simpler and less expensive than viral isolation and NAATs, MAC'ELISA (detecting IgM antibodies) has some important limitations. This test does not allow for early diagnosis of dengue infection as during primary infections IgM antibodies are detectable only in 50% of the patients between day 3 and 5 from onset of fever, increasing to 80% by day 5 and 99% by day 10 (PAHO ). Therefore, WHO recommend that this test is run on samples collected after five days from onset of fever (WHO ). In patients with secondary dengue virus infections the kinetics of IgM production are more variable and IgM levels are significantly lower than during primary infection (Gubler ; Chanama ). False'negative test results for dengue'specific IgM antibodies can occur during secondary infection (Chanama ). Also, some tests show non'specific reactivity in sera from patients with malaria and leptospirosis (Hunsperger ).
The IgG ELISA can be used to confirm a dengue infection if paired sera are collected and to distinguish between primary and secondary infection (Vaughn ; Guzmán ). In primary dengue infection, IgG is generally detectable at low titres at the end of the first week of illness, increasing slowly thereafter, with serum IgG still detectable after several months (and possibly for life). During a secondary infection, high levels of IgG antibodies are detectable during the acute phase. A fourfold or greater increase in IgG antibodies in acute and convalescent paired sera can be used to document recent infections (Shu ). However, IgG ELISA generally lacks specificity within the flavivirus serocomplex groups (Makino ). Also, IgG antibodies may persist for life which further complicates the serodiagnosis.
A dengue virus E/M protein'specific IgM/IgG ratio can be used to distinguish between primary and secondary dengue virus infections. IgM capture and IgG capture ELISAs are the most common assays for this purpose (Prince ). According to this method, a dengue infection is defined as primary if the IgM/IgG optical density (OD) ratio is higher than 1.2 (using patient sera at 1/100 dilution) or 1.4 (using patient sera at 1/20 dilution). The infection is secondary if the ratio is less than 1.2 (using patient sera at 1/100 dilution) or 1.4 (using patient sera at 1/20 dilution). (Kuno ; Shu ; Shu ). However, ratios may vary between laboratories, indicating the need for better standardization of test performance (Falconar ).
Based on WHO recommendations (WHO ), dengue virus diagnosis can be considered confirmed when at least one of the following diagnostic methods gives a positive result:
Viral isolation by culture;
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Identification of viral RNA by RT'PCR;
IgM seroconversion (from negative to positive) in paired sera ;
IgG seroconversion (from negative to positive) in paired sera or at least a fourfold increase of IgG titer in paired sera
However, a thorough diagnostic work'up for dengue infection should include both virus isolation procedures (by virus identification or genome detection) and detection of immune responses (WHO ).
Dengue fever is a viral infection transmitted to humans by mosquitoes that live in tropical and subtropical climates and carry the virus. Blood testing detects the dengue virus or antibodies produced in response to dengue infection.
According to the Centers for Disease Control and Prevention (CDC), dengue infections have been reported in more than 100 countries from parts of Africa, the Americas, the Caribbean, the Eastern Mediterranean, Southeast Asia, and the Western Pacific. It is a fast emerging infectious disease, according to the World Health Organization (WHO), with an increasing number of cases and countries affected throughout the world. The actual number is not known because about 75% of cases are asymptomatic, but a recent estimate put the number of annual dengue infections as high as 390 million. Approximately 50 to 100 million symptomatic cases occur annually worldwide.
In the U.S., the majority of dengue cases occur in travelers returning from areas where dengue is endemic. Most dengue cases in U.S. citizens occur in people who live in Puerto Rico, the U.S. Virgin Islands, Samoa and Guam. Outbreaks where a large number of cases occur in a defined area are rare in the U.S. In recent years, there have been small outbreaks in Texas and Hawaii and a few cases diagnosed in southern Florida.
Many individuals will develop no symptoms at all, or have only a mild illness when exposed to one of the four serotypes (1-4) of the dengue virus. For those who do develop symptoms, prognosis is still very good for full recovery within a few weeks. The most common initial symptoms are a sudden high fever (104°F or 40°C) and flu-like symptoms that appear roughly 4 to 7 days after being bitten by an infected mosquito (this is called the incubation period and can range from 3 to 14 days). Additional signs and symptoms may include severe headache, especially behind the eyes, muscle and joint pain, skin rash, nausea, vomiting, and swollen glands.
Some people who develop a fever will recover on their own with no lasting ill effects while others may progress to severe dengue fever (sometimes called Dengue Hemorrhagic Fever). If the disease progresses to this form, a new wave of symptoms will appear 3 to 7 days after initial symptoms and as the fever recedes. These may include nose bleeds, vomiting blood, passing blood in the stool, difficulty breathing and cold clammy skin, especially in the extremities. During the second phase, the virus may attack blood vessels (the vascular system), causing capillaries to leak fluid into the space around the lungs (pleural effusion) or into the abdominal cavity (ascites).
The loss of blood and fluid during the second phase, if untreated, can worsen and can be fatal. In order to avoid that complication (sometimes called Dengue Shock Syndrome), a healthcare practitioner may hospitalize a patient with severe dengue fever so that falling blood pressure and dehydration caused by the loss of blood and fluids can be managed while the disease runs its course ' generally a period of one to two weeks. During the following week of recovery, a person may develop a second rash that lasts a week or more.
Dengue fever is usually diagnosed via some combination of blood tests because the body's immune response to the virus is dynamic and complex. Laboratory tests may include:
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