Blood is the most important sample for dengue diagnosis. As dengue virus is sensitive to heat, pH, lipid solvents and proteolytic enzymes, sample for virus isolation and viral RNA detection should be transported in ice to the lab. The samples can be stored at 4°C for not more than 24 hours at peripheral hospital and should be transported in ice to the lab within that period. For prolonged storage, samples are stored at below –70°C.

Figure 1 depicts the timeline of dengue NS1 antigen, viremia, IgM and IgG antibody with symptoms.

Fig1. Kinetics of dengue virus infection (schematic)

Methods of Lab Diagnosis

Virus Isolation

 Following three methods are used for dengue virus isolation.

1. Mice inoculation: 1 to 3 days old newborn swiss albino mice is used for this purpose. Patient’s serum is inoculated through intracerebral route. In positive cases, mice develops hunch back, less intake of food, fine tremor, and paralysis by 8–10 days post inoculation (Fig. 2). Mice needs to be observed for development of symptoms for 2–3 weeks before declaring negative. The test is slow, time consuming, requires animal facility, and poorly sensitive. Other arboviruses that cause dengue-like illness can also be isolated in the mice host system. Because of restriction in use of lab animals and poor sensitivity, the technique is no more recommended.

Fig2. Dengue virus isolation in mice

Identification of virus can be done from the harvested mice brain.

• Dengue antigen can be detected in mice brain by direct or indirect fluorescent staining using specific polyclonal or monoclonal antibody.

• RT-PCR for dengue virus RNA detection.

2. Mosquito cell culture: This is presently the most common method and considered as the gold standard for dengue virus diagnosis. Various mosquito cell lines like C6/36 clone of Aedes albopictus, AP-61 from Aedes pseudocutellaris and TRA-284 from Toxorhynchities amboinensis are used for this. Rounding and clustering of infected cells may be observed as cytopathic effect. Cytopathic effects may be apparent in second or third passage.

Identification of virus:

• Infected cells are harvested for identification by direct or indirect fluorescent staining using specific polyclonal or monoclonal antibody.

• RT-PCR from cell culture supernatant.

3. Mosquito inoculation: Intrathoracic route of inoculation in mosquito is considered as the most sensitive method for dengue virus isolation. Four mosquito species have been used for this test; Aedes aegypti, Aedes albopictus, Toxorhynchities amboinensis and Toxorhynchities splendens. Virus grows rapidly to high titer in this method as compared to other methods of isolation.

Identification of virus: Antigen detection or RT-PCR from mosquito head-squash preparation.

Serological Tests

Hemagglutination inhibition (HI), complement fixation, neutralization, enzyme-linked immunosorbent assay (ELISA), and immunochromatographic tests are used for detection of dengue antibody.

Hemagglutination Inhibition (HI)

Principle: This is based on the principle of agglutination of dengue virus with chick red blood cells. The presence of dengue specific antibody in the patient’s serum inhibits the agglutination. The antigen employed in HI test is prepared either from dengue virus infected suckling mouse brain with sucrose and acetone extraction or infected mosquito cell culture concentrate.

Primary infection: HI antibody begins to appear from five days of illness onwards and the titer goes up to 640 in convalescent phase serum and persists for 2–3 months.

Secondary infection: The titer rises rapidly and becomes 5120 or more.

Interpretation: HI titer more than 1280 in acute phase serum is often considered as the presumptive diagnosis of acute dengue infection.

Rise in fourfold titer between acute and convalescent sample is confirmative.

Use: As HI antibody persists for a long time, the test is often used for serosurveillance. The test is sensitive but lacks specificity.

Complement Fixation Tests (CFT)

The test is based on the principle of consumption of complement during antigen antibody reactions.

CF antibodies appear later than HI antibody and do not persist for long periods.

Therefore, positive CF antibody titer indicates acute or recent infection and not used for sero-surveillance.

The CF antibody is more specific and often monotypic as compared to broad specificity of HI antibody and secondary dengue infection.

However, CFT requires vigorous standardization and difficult to perform.

Neutralization

The principle is based on the production of cytopathic effect or plaques in susceptible cell lines. When serum containing dengue antibody is mixed with the virus, it causes inhibition in the number of plaques produced by the virus.

Neutralizing antibody appears later than HI antibody but earlier than CF antibody and persists for a long time. This test is most sensitive and specific for dengue virus.

These above mentioned serological tests are not used these dates for patients diagnosis because of difficulty in standardization, tedious to perform and requirement of both acute and convalescent serum sample to demonstrate fourfold rise of titer.

Newer Serological Tests

Detection of IgM antibody: IgM antibody detection by capture ELISA is known as MAC ELISA. The sensitivity and specificity of the test has been reported to be >90% and >95% respectively.

In primary infection: IgM antibody appears from 5 days of illness and persists for about 3 months.

In secondary infection, IgM appears slightly earlier as compared to primary infection.

Detection of specific IgM antibody in single sample indicates on-going or recent infection.

Seroconversion of IgM antibody in the convalescent sample depicts acute infection (negative in acute sample and positive in convalescent sample).

Because of requirement of single serum sample, ease of the procedure, wide availability, capability of testing a large number of samples and low cost, MAC ELISA is the recommended method for IgM antibody detection.

Detection of dengue IgG antibody: This can be done by various format of ELISA like IgG capture ELISA (GAC ELISA), indirect ELISA, etc.

Demonstration of seroconversion or four fold rise in titer in acute and convalescent sample indicates acute infection.

Presence of IgG indicates exposure to the virus and thus used for serosurveillance.

IgG in acute sample indicates previous infection and helps to differentiate between primary and secondary infections.

Kinetics of dengue IgM and IgG antibody:

 • Primary dengue infection is characterized by a low and delayed antibody response.

• IgM antibody is the first antibody to appear.

• Anti-dengue IgG appears after IgM at the end of first week of illness.

• During secondary dengue infection, both IgG and IgM antibodies are detected in the first week of illness in most of the cases.

Differentiation between primary and secondary infection: As per the WHO recommendation, the ratio between IgM and IgG (IgM/IgG) antibodies against dengue virus E/M protein is considered to differentiate between primary and secondary infections as below:

• Primary infection: Ratio of IgM/IgG OD is >1.2 (serum dilution 1/100) or >1.4 (serum dilution 1/20).

• Secondary infection: Ratio of IgM/IgG OD is < 1.2 (serum dilution at 1/100) or < 1.4 (serum dilution at 1/20).

Detection of dengue NS1 antigen: Dengue NS1 is a highly conserved glycoprotein. During infection, NS1 is associated with intracellular organelles or transported to the cell surface through secretory pathway. The antigen has been found to be circulated in patients’ blood during acute phase of illness and presently considered as an acute marker of dengue virus infection.

The ELISA and rapid immunochromatographic tests are commercially available for detection of NS1 antigen in blood sample.

Dengue NS1 can be detected in the blood of the patient from day 1 to day 9 or more and used as early diagnostic marker.

Sensitivity: 75%; Specificity: 100%.

 In secondary infection, it may disappear early due to early appearance of dengue IgM.

Rapid immunochromatographic test: These test formats are available for detection of dengue IgM, dengue IgG antibody as well as for dengue NS1 antigen. The combined antigen antibody format is available by several manufacturers.

Rapidity and ease of use are its main advantages for which these tests are widely used.

Molecular Tests

Various formats of molecular tests are available for the detection of dengue virus RNA from clinical sample, like reverse transcriptase PCR (RT-PCR) (Fig. 3), real time reverse transcriptase PCR, nested RT PCR, single tube RT-PCR, and loop mediated isothermal amplification (LAMP). All these formats are used both for detection as well as for dengue virus type determination. Pre membrane (prM) gene is most commonly targeted for detection of dengue virus RNA. Test is applicable only during the first 3–5 days of illness, due to short period of viremia. The sensitivity and specificity of molecular tests is near 100%.

Fig3. Agarose gel analysis of the dengue type specific RT-PCR

Real-time RT-PCR for dengue 1–4 has been developed by CDC. The test has been found to be highly reproducible with high sensitivity and specificity and has been approved by FDA.

The advantages and disadvantages of molecular test are mentioned in Table 1.

Table1. Advantages and disadvantages of molecular tests for dengue diagnosis

LAMP assay: LAMP assay is a relatively new method of nucleic acid amplification based on the principle of strand displacement and stem loop structure that amplifies the target under isothermal condition. The assay generates large amount of target DNA production along with magnesium pyrophosphate as by product causing turbidity.

The method has also been developed as multiplex assay for detection along with chikungunya infection. Table 2 describes the advantages of LAMP assay.

Table2. Advantages of LAMP assay

Figure 4 depicts the practical approach to dengue virus infection diagnosis in patient and Table 3 summarizes the methods used for dengue diagnosis.

Fig4. Practical approach to lab diagnosis of dengue

Table3. Summary of methods of lab diagnosis

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