Bangladesh Journal of Pathology 2008, 23(2):2-3

Editorial

Latest updates for Laboratory diagnosis of Dengue

Dr. Rosy Sultana & Prof. Md. Tahminur Rahman

Introduction
Currently, dengue fever is the most important re-emerging mosquito-borne viral disease, with the major proportion of the target population residing in the developing countries of the world including Bangladesh.
It is one of the leading public health concerns in over a hundred tropical and subtropical countries in South East Asia, the Caribbean, Central and South America. The World Health Organization (WHO) receives reports of about 5,00,000 dengue fever cases each year, but estimates that as many as 50 million people are infected annually1.
Dengue virus is an enveloped; single stranded positive sense RNA virus of the genus Flavivirus. About 40% of the global population is estimated to be at risk of dengue infections. There are four serologically related but antigenically and genetically distinctive dengue viruses (Den-1,2,3 and 4)2.
The infection is accompanied with biphasic fever, headache, eye pain, myalgia, arthralgia, prostration, rash, lymphadenopathy, and leukopenia3 and often agonizing limb pains that have earned the disease its sobriquet 'break-bone fever'. Persons infected with dengue virus are generally mild or asymptomatic, but may also present with undifferentiated fever, classic dengue fever (DF), and even dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)4,5. DHF and DSS are the two main severe clinical manifestations of dengue; continue to be major causes of human morbidity and mortality in tropical areas6. Several large epidemics of dengue/ DHF have been reported in Taiwan7. Effective surveillance and efficient control depend on rapid and accurate laboratory diagnosis. Additionally, qualitative and quantitative information of dengue viral RNA, proteins, and antibodies will be very helpful in clarifying the epidemiology patterns of dengue virus infection, dengue fever and dengue hemorrhagic fever/ dengue shock syndrome2.
Laboratory diagnosis
Laboratory tests essential for confirmatory diagnosis of dengue infection include: a) isolation of the virus,
b) demonstration of a rising titre of specific serum dengue antibodies, and
c) demonstration of a specific viral antigen or RNA in the tissue or serum8.
d) Hematological test
a) Isolation of the Dengue virus
Isolation of the virus is the most definitive approach, but the techniques presently available require a relatively high level of technical skill and equipment. Isolation of most strains of dengue virus from clinical specimens can be accomplished in a majority of cases provided the sample is taken in the first few days of illness and processed without delay. Specimens that may be suitable for virus isolation include acute phase serum, plasma or washed buffy coat from the patient, autopsy tissues from fatal cases, especially from liver, spleen, lymph nodes and thymus, and mosquitoes collected in nature. Tissues and pooled mosquitoes are triturated or sonicated prior to inoculation. The choice of methods for isolation and identification of dengue virus will depend on local availability of mosquitoes, cell culture, and laboratory capability. Inoculation of serum or plasma into mosquitoes is the most sensitive method of virus isolation, but mosquito cell culture is the most cost-effective method for routine virologic surveillance. In order to identify the different dengue virus serotypes, mosquito head squashes and slides of infected cell cultures are examined by indirect immunofluorescence using serotype-specific monoclonal antibodies.

b) Serological tests for the diagnosis of DF/DHF
Serological tests are simpler and more rapid, but cross-reactions between antibodies to dengue and other flaviviruses may give false positive results. Six basic serological tests are used for the diagnosis of dengue infection ; few of them are routinely used:
i) Haemagglutination-inhibition (HI)- ideal for seroepidemiologic study, most frequently used, sensitive, easy to perform, requires only minimal equipment and very reliable if properly done. Themajor disadvantage of the HI test is lack of specificity, which makes the test unreliable for identifying the infecting virus serotype.
ii) Complement fixation test (CFT)- not widely used for routine dengue diagnostic serology. The complement fixation test is useful for patient with current infections, but is of limited value for seroepidemiologic studies where detection of persistent antibodies is important. It is more difficult to perform and require highly trained personnel.
iii) Neutralization test (NT)- is the most specific and sensitive serologic test for dengue viruses. Since dengue viruses produce cytopathic effect in susceptible cell cultures, this cytopathic effect is neutralized by the presence of specific antibodies. In general, neutralizing antibodies rise at about the same time or at a slightly slower rate than HI antibodies, but more quickly than CF, and persist for at least 50 years or longer. The NT can be used to identify the infecting virus in primary dengue infections, provided the serum samples are properly timed. In secondary and tertiary infections, it is not possible to reliably determine the infecting virus serotype by NT. Because of long persistence of neutralizing antibodies, the test may also be used for seroepidemiologic studies. The major disadvantages are the expense, time required to perform the test, and technical difficulty. It is therefore not routinely used in most laboratories.
iv) IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA)- It has become widely used in past few years. It is a simple, rapid test that requires very little sophisticated equipment. MAC-ELISA is based on detecting the dengue-specific IgM antibodies in the test serum by capturing them using anti-human IgM that was previously bound to the solid phase. If the patient serum contain IgM antibody, it will bind to the solid phase. It can be detected by subsequent addition of an enzyme labelled anti-dengue antibody, which may be human or monoclonal antibody. An enzyme substrate is added to give a colour reaction. The anti-dengue IgM antibody develops a little faster than IgG, and is usually detectable by day five of the illness. IgM antibody titers in primary infections are significantly higher than in secondary infections. MAC- ELISA is slightly less sensitive than the HI test for diagnosing dengue infection. MAC-ELISA has become an invaluable tool for surveillance of DF/DHF/DSS. In areas where dengue is not endemic, it can be used in clinical surveillance for viral illness or for random, population-based serosurveys, with the certainty that any positives detected are recent infections.v) Indirect IgG ELISA - an indirect IgG ELISA has been developed that compares well to the HI test. This test can also be used to differentiate primary and secondary dengue infections. The test is simple and easy to perform, and is thus useful for high volume testing. The IgG ELISA is very non-specific and exhibits the same broad cross- reactivity among flaviviruses as the HI test; it cannot be used to identify the infecting dengue serotype. However, it has a slightly higher sensitivity than the HI test.
vi) ICT (Immunochromatographic Test): This method utilises a test device for rapid detection of dengue IgM and IgG antibodies in patients sample. It is a qualitative membrane based immunoassay for the detection dengue antibodies in whole blood., serum or plasma. This test consists of two components. In the IgG antibodies to dengue, a coloured line will appear in the test line region 1. In the IgM component, anti ligand is coated in the test line region 2. During testing the specimen reacts with ligand antihuman IgM and the dengue antigen coated particles line in test region 2. Therefore if the specimen contains dengue IgM or IgG antibodies, coloured lines will appear in test line regions assigned for the specific antibodies. No coloured line in either of the test line regions indicate negative result. To sere as a procedural control, a coloured line always appear at central region or it change its colour, indicating that the proper volume of specimen has been added and membrane wicking has occurred. For the primary and secondary infection, the overall sensitivity is 95.3% and the overall specificity is >90% and the overalll accuracy is 99.3%.
c) Demonstration of a specific viral antigen or RNA in the tissue or serum- In addition, accurate identification of the infecting dengue virus serotype is not possible with most serological methods. New technologies avalilable for the laboratory diagnosis of dengue infection include immunohisto-chemistry on autopsy tissues and polymerase chain reaction (PCR) to detect viral RNA in the tissue or serum.
d) Hematological test Platelet count.
i. Platelet count: This is a very important but simple test particularly to assess the bleeding manifestations in a dengue fever patient.
ii) PCV (Hematocrit); This is also an important and simple test in dengue fever where hemoconcentration is a feature9.
iii) Total count of WBC. Usually normal in early stage, but there may be leucopenia in later stages along with thrombocytopneia.

Conclusion

The main and latest laboratory tests for dengue fever are described. The nature and type of tests should be determined by the physician depending on the need, type of tests, management of the patient depending on economic status and clinical condition of the patient.

References

1. WHO : Dengue and Dengue Hemorrhagic Fever. Fact Sheet. 200;117
2. Chuan-Liang Kao, Chwan-Chuen King, Day - Yu Chao, Hui-Lin Wu, Gwong-Jen J. Chang. Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health. J Microbiol Immunol Infect 2005; 38: 5-16.
3. Nimmannitya S. Clinical spectrum and management of dengue haemorrhagic fever. Southeast Asian J Trop Med Public Health 1987; 18: 392-3976.
4. Kalaynarooj S, Vaughn DW, Nimmannitya S, green S, Suntayakorn S, Kunentrasai N, et al. Early clinical and laboratory indicators of acute dengue illness J Infect Dis 1997; 176: 313-321.
5. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998; 11:480-496.
6. Sinniah M, Igarashi A. Dengue hemorrahagic fever. Rew Med Virol 1995; 5: 193-203.
7. King CC, Wu Ye, Chao DY, Lin TH, Chow L, Wang HT, et al. Major epidemics of dengue in Taiwan in 1981-2000: related to intensive virus activities in Asia. Dengue Bulletin 2000; 24: 1-10.
8. Dengue/DHF Laboratory diagnosis-regional Guidelines on Dengue/DHF Prevention and control (Regional publication 29/1999)
9. Davidson' Principle & Practice of Medicine. Editor. Boon NA, Colledge NR, Walker BR. 20th edition, 2002, Publisher. Churchill Livingstone, UK.