Laboratory diagnosis in dengue infection
There are 2 main methods for dengue infection diagnosis.
1. Viral isolation (definitive approach, but high level of technical skills and equipment require)
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 liver, spleen, lymph nodes and thymus, and mosquitoes collected in nature.
For short periods of storage (up to 48 hours), specimens to be used for virus isolation can be kept at +4 to +8oC. For longer storage, the serum should be separated and frozen at -70oC, and maintained at such so that thawing does not occur. If isolation from leucocytes is to be attempted, heparinized blood samples should be delivered to the laboratory within a few hours. Whenever possible, original material (viraemic serum or infected mosquito pools) as well as laboratory-passaged materials should be preserved for future study.
Tissues and pooled mosquitoes are triturated or sonicated prior to inoculation. The different methods of inoculation and the methods of confirming the presence of dengue virus are show in table1
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. It is essential for health workers interested in making a diagnosis by means of virus isolation to make contact with the appropriate virology laboratory prior to the collection of specimens. The acquisition, storage and shipment of the samples can then be organized to have the best chance of successful isolation.
In order to identify the different dengue virus serotypes, mosquito head squashes and slides of infected cell cultures are examined by indirect immunoflourescence using serotype-specific monoclonal antibodies.
Table 1. Dengue virus isolation methods
| Recommended methods Confirmation of dengue virus infection
Inoculation of mosquitoes Presence of antigen in head squashes Inoculation of insect cells or a) Presence of antigen in cells demonstrated mammalian cultures by immunofluorescence b) Cytopathic effect and plaque formation in mammalian cells |
2. Serological diagnosis ( simpler&more rapid, but may have false positive result from cross reactivity between antibody to dengue and other flavivirus)
Five basic serologic tests are routinely used for the diagnosis of dengue infection. They are
1. Haemagglutination inhibition (HI) test
Of the above tests, HI has been the most frequently used for routine serologic diagnosis of dengue infections. It is sensitive, easy to perform, requires only minimal equipment, and is very reliable if properly done. Because HI antibodies persist for long periods (up to 50 years or longer), the test is ideal for seroepidemiologic studies. The HI test is based on the fact that the dengue viruses, under controlled conditions of pH and temperature, can agglutinate goose red blood cells, and this effect can be inhibited by specific antibodies. The antigens employed are prepared from infected suckling mice brains by extraction with sucrose and acetone to remove the lipids, or from infected mosquito cell cultures that have been concentrated or purified. Serum specimens must be treated to remove non-specific inhibitors and agglutinins.
The HI antibody usually begins to appear at detectable levels (titer of 10) by day five or six of illness, and antibody titers in convalescent-phase serum specimens are generally at or below 1:640 in primary infections, although there are exceptions. By contrast, there is an immediate anamnestic response in secondary and tertiary dengue infections, and antibody titers increase rapidly during the first few days of illness, often reaching 1:5,120 to 1:10,240 or more. Thus, a titer of 1:1,280 or greater in an acute-phase serum is considered a presumptive diagnosis of current dengue infection. High levels of HI antibody may persist for 2-3 months in some patients, but in most antibody titers will generally begin to wane by 30-40 days and fall below the 1:1,280 level.
The major disadvantage of the HI test is lack of specificity, which makes the test unreliable for identifying the infecting virus serotype. However, some primary infections may show a relatively monotypic HI response that generally correlates with the virus isolated.
2.Complement fixation (CF)test
The CF test is not widely used for routine dengue diagnostic serology. It is more difficult to perform and requires highly-trained personnel. The CF test is based on the principle that the complement is consumed during antigen-antibody reactions. Two reactions are involved, a test system and an indicator system. Antigens for the CF test are prepared in the same manner as those for the HI test.
CF antibodies generally appear later than HI antibodies, are more specific in primary infections, and usually persist for shorter periods, although low-level antibodies may persist in some persons. Because of the late appearance of CF antibodies, some patients may show a diagnostic rise by CF, but have only stable antibody titers by HI. The greater specificity of CF test in primary infections is demonstrated by the monotypic CF responses, whereas HI responses are broadly heterotypic. The CF test is not specific in secondary infections. The CF test is useful for patients with current infections, but is of limited value for seroepidemiologic studies where detection of persistent antibodies is important.
3.Neutralization test (NT)
The NT is the most specific and sensitive serologic test for dengue viruses. The most common protocol used in most dengue laboratories is the serum dilution plaque reduction neutralization test (PRNT). It is based on the fact that dengue viruses produce cytopathic effects (CPE) which can be observed as plaques in susceptible cell cultures. This CPE 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. Because NT is more sensitive, neutralizing antibodies may be detectable in the absence of detectable HI antibodies in some persons with past dengue infection.
The NT can be used to identify the infecting virus in primary dengue infections, provided the serum samples are properly timed. Relatively monotypic responses are observed in properly timed convalescent-phase serum. As noted above, the HI and CF tests may also give monotypic responses to dengue infection that generally agree with NT results. In those cases where the responses are monotypic, the interpretation is generally reliable. In secondary and tertiary infections, it is not possible to reliably determine the infecting virus serotype by NT. Because of the 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.
4.IgM-capture enzyme-linked immuno-sorbent assay (MAC-ELISA)
MAC-ELISA has become widely used in the 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 out of solution using anti-human IgM that was previously bound to the solid phase. If the IgM antibody from the patient’s serum is anti-dengue antibody, it will bind the dengue antigen that is added in the next step and 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. However, the rapidity with which IgM develops varies considerably among patients. Some patients have detectable IgM on days two to four after the onset of illness, while others may not develop IgM for seven to eight days after the onset. IgM antibody titers in primary infections are significantly higher than in secondary infections, although it is not uncommon to obtain IgM titers of 320 in the latter cases. In some primary infections, detectable IgM may persist for more than 90 days, but in most patients it wanes to an undetectable level by 60 days.
MAC-ELISA is slightly less sensitive than the HI test for diagnosing dengue infection. It has the advantage, however, of frequently requiring only a single, properly timed blood sample. Considering the difficulty in obtaining second blood samples and the long delay in obtaining conclusive results from the HI test, this low error rate would be acceptable in most surveillance systems. It must be emphasized, however, that because of the persistence of IgM antibody, MAC-ELISA positive results on single serum samples are only provisional and do not necessarily mean that the dengue infection is current. It is reasonably certain, however, that the person had a dengue infection sometime in the previous two to three months.
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(21). It is especially useful for hospitalized patients, who are generally admitted late in the illness after detectable IgM is already present in the blood.
5. 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. It is expected that as more data are accumulated on the IgG ELISA, it will replace the HI test.
(reference : http://www.searo.who.int/EN/Section10/Section332/Section554_2566.htm)
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