Laboratory diagnosis of dengue infection

TU 2063

Laboratory diagnosis of dengue infection

The clinical diagnosis of dengue, both the uncomplicated dengue fever form and DHF(dengue hemorrhagic fever)/DSS( dengue shock syndrome), is often unreliable. Dengue fever may resemble clinically a variety of acute febrile illnesses, although the severe muscle and bone pain is suggestive of dengue.

Similarly, DHF may resemble other causes of haemorrhagic fever, although thrombocytopaenia with haemoconcentration and signs of a moderate consumptivecoagulopathy is suggestive of dengue.

The most widely used serological test is the HI test, detecting antibodies as early as 4 days post-onset. A specific diagnosis of dengue can be made early in primary infections, but cross-reactions with other flaviviruses occur in late primary or secondary infections. The IgM antibody capture ELISA (MAC ELISA) is being used during outbreaks of dengue, and there are now rapid assays available for the detection of dengue IgG and IgM, although crossreaction may occur with the IgG assay. The IgM antibodies may persist for over 3 months, so the test is also useful for retrospective studies, but this persistence may cause diagnostic problems in areas where dengue is endemic. A combined IgG and IgM assay, detecting high levels of IgG indicating secondary infection, is useful in dengue endemic areas. Immunofluorescent assays have been used successfully to detect dengue IgG and IgM antibodies. The CF test is more specific than the HI test, but the antibodies detected by this assay appear later and disappear earlier. The NT and PRNT tests are the most specific and sensitive but are difficult to perform and thus tend to be used only for specific purposes.

Virus isolation, the only definitive way of being able to type isolates, is difficult and if mice are used, a number of blind passages are usually required. Intracerebral inoculation of adult or larval Toxorhynchites spp. is a sensitive and rapid method for the isolation of dengue virus, giving results in 2–3 days. The use of a rapid centrifugation method may increase sensitivity and reduce time scales. Intrathoracic inoculation of mosquitoes is easier and just as sensitive, but head squashes cannot be made or tested for specific dengue antigen for at least 7 days post-inoculation.

The most commonly used system of virus isolation is the inoculation of mosquito cell lines, viz. A. albopictus (C6-36), A. pseudoscutellaris (AP-61) and Toxorhynchites

ambionensis (TRA-248), which are almost as sensitive as mosquito inoculation, which allows specific results to be obtained within 2–3 days using fluorescent- labelled monoclonal antibodies. A combination of MAC ELISA and RT-PCR on peripheral blood

Leukocytes have been shown to give high levels of sensitivity and specificity.

Laboratory diagnosis of dengue infection

(JAWETZ)

Reverse transcriptase- polymerase chain reaction based methods are available for rapid identification and serotyping of dengue virus in acute- phase serum, roughly during the period of fever. Isolation of the virus is difficult. The current favored approach is inoculation of a mosquito cell line with patient serum, coupled with nucleic acid assay to identify a recovered virus.

Serological diagnosis is complicated by cross- reactivity of IgG antibodies to heterologus falvivirus antigens. A variety of methods are available; the most commonly used methods are E/M viral protein- specific capture IgM or IgG ELISA and the hemagglutination inhibition test. IgM antibodies develop within a few days of illness. Neutralizing and hemagglutination- inhibiting antibodies appear within a week after onset of dengue fever. Analysis of paired acute and convalescent sera to show a significant rise in antibody titer is the most reliable evidence of an active dengue infection.