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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]; Associate Editor(s)-in-Chief: Alejandro Lemor, M.D. [2], Vendhan Ramanujam M.B.B.S [3]

Overview

Laboratory diagnosis can be accomplished by testing serum or plasma to detect virus, viral nucleic acid, or virus-specific immunoglobulin (Ig) M and neutralizing antibodies. Viral culture may detect virus in the first 3 days of illness; however, Chikungunya virus should be handled under biosafety level (BSL) 3 conditions. During the first 8 days of illness, Chikungunya viral RNA can often be identified in serum. Chikungunya virus antibodies normally develop toward the end of the first week of illness. Therefore, to definitively rule out the diagnosis, convalescent-phase samples should be obtained from patients whose acute-phase samples test negative.

Chikungunya virus testing is performed at CDC, a few state health departments, and one commercial laboratory. Test results are normally available 4 to 14 days after specimen receipt. Reporting times for test results may be longer during summer months when arbovirus activity increases. Receipt of a hard copy of the results will take at least 2 weeks after testing is completed. Initial serological testing will be performed using IgM-capture ELISA and IgG ELISA. If the initial results are positive, further confirmatory testing will be performed and it may delay the reporting of final results.

Other Diagnostic Studies Adapted from Preparedness and Response for Chikungunya Virus: Introduction in the Americas. PAHO © 2011.

Samples collected during the first week after onset of symptoms should be tested by both serological (IgM and IgG ELISA) and virological (RT-PCR and isolation) methods. Specimens are usually blood or serum, but in neurological cases with meningoencephalitic features, cerebrospinal fluid (CSF) may also be obtained. Limited information is available for the detection of virus by isolation or RT-PCR from tissues or organs. In suspected fatal cases, virus detection can be attempted on available specimens. Selection of the appropriate laboratory test is based upon the source of the specimen (human or field-collected mosquitoes) and the time of sample collection relative to symptom onset for humans.

Three main types of laboratory tests are used for diagnosing Chikungunya virus (CHIKV): virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), and serology.

Virus Isolation

Virus isolation can be performed on field collected mosquitoes or acute serum specimens (≤8 days). Serum obtained from whole blood collected during the first week of illness and transported cold (between 2°–8°C or dry ice) as soon as possible (within 48 hours) to the laboratory can be inoculated into a susceptible cell line or suckling mouse. CHIKV will produce typical cytopathic effects (CPE) within three days after inoculation in a variety of cell lines, including Vero, BHK- 21, and HeLa cells. Virus isolation can be performed in T-25 flasks or shell vials. Recent data suggest that isolation in shell vials is both more sensitive and produces CPE earlier than conventional isolation in flasks. CHIKV isolation must be confirmed either by immunofluorescence assay (IFA), using CHIKV-specific antiserum, or by RT-PCR of the culture supernatant or mouse brain suspension. Virus isolation must only be carried out in biosafety level 3 (BSL-3) laboratories to reduce the risk of viral transmission.

RT-PCR

Several RT-PCR assays for the detection of CHIKV RNA have been published. Real time, closed system assays should be utilized, due to their increased sensitivity and lower risk of contamination. The Arboviral Diagnostic Laboratory within the Division of Vector-Borne Diseases (DVBD) routinely utilizes the published assay, which demonstrates a sensitivity of less than 1 pfu or 50 genome copies. Serum from whole blood is used for PCR testing as well as virus isolation.

Serological Tests

For serological diagnosis, serum obtained from whole blood is utilized in enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization testing (PRNT). The serum (or blood) specimen should be transported at 2°–8°C and should not be frozen. Serologic diagnosis can be made by demonstration of IgM antibodies specific for CHIKV or by a four-fold rise in PRNT titer in acute and convalescent specimens. IgM antibodies specific for CHIKV are demonstrated by using the IgM antibody capture ELISA (MAC-ELISA), followed by the PRNT. As of 2010, there were no World Health Organization (WHO) validated commercial IgM ELISAs available. PRNT is required to confirm the MAC-ELISA results, since cross-reactivity in the MAC-ELISA between some members of the Semliki Forest virus (SFV) serogroup has been observed. PRNT testing, whether used to confirm the MAC-ELISA or to demonstrate a four-fold rise in acute/convalescent specimens, should always include other viruses within the SFV serogroup (e.g., Mayaro virus) to validate specificity of reactivity. In situations where the PRNT assay is not available, other serological tests (e.g. hemagglutination inhibition assay) can be used to identify a recent alphavirus infection; however, PRNT is required to confirm a recent CHIKV infection.

An acute phase serum should be collected immediately after the onset of illness and the convalescent phase serum 10–14 days later. CHIKV-specific IgM and neutralizing antibodies normally develop towards the end of the first week of illness. Therefore, to definitively rule out the diagnosis, convalescent samples should be obtained on patients whose acute samples test negative.

Collection, Storage, and Transportation of Samples

Collection of Samples for Serology, Isolation, and Molecular Diagnosis:

• Sample:

• Serum

• Time of collection:

• Acute — within the first eight days of illness
• Convalescent — 10–14 days after acute specimen collection

• To collect serum:

• Aseptically collect 4–5 ml of venous blood in a tube or a vial.
• Allow blood to clot at room temperature, centrifuge at 2,000 rpm to separate serum. Collect the serum in a clean dry vial.
• All clinical samples should be accompanied by their clinical and epidemiological information.

Other Types of Specimens for Laboratory Investigation:

• Specimens:

• CSF in meningoencephalitis cases.
• Synovial fluid in arthritis with effusion.
• Autopsy material – serum or available tissues. (Note: Mosquitoes collected in the field will also be handled using the same techniques described here.)

• Transportation of Samples:

• Transport specimens to the laboratory at 2°−8°C (icebox) as soon as possible.
• Do not freeze whole blood, as hemolysis may interfere with serology test results.
• If a delay greater than 24 hours is expected before specimens can be submitted to the laboratory, the serum should be separated and stored at refrigerated temperature.
• Serum samples for virus isolation and molecular diagnosis should be stored frozen (at –20°C for short-term storage or at –70°C for long-term storage).

Laboratory Surveillance

Prior to identification of CHIKV in a country, laboratory surveillance should be conducted on three sets of samples, as follows:

• Dengue-negative specimens where the patient exhibits severe joint pain
• Samples with clinically compatible illness from new geographic areas without active dengue circulation
• Clusters of febrile illness with severe joint pain

The following table outlines the ideal tests to be performed in various epidemiological settings.

Epidemiological scenario	Testing to be performed	Samples to test
No signs of transmission	IgM ELISA, IgG ELISA	All samples from patients exhibiting clinically compatible illness
Suspect CHIKV illness	IgM ELISA, IgG ELISA, real-time RT-PCR, virus isolation, PRNT	All samples from patients exhibiting clinically compatible illness
Continued transmission	IgM ELISA, IgG ELISA, real-time RT-PCR; limited virus isolation	Subset samples from classical CHIK cases, as determined by lab constraints and epidemiological status; Samples from all atypical or severe cases should be tested
Periodic outbreaks or active surveillance in areas near CHIKV transmission	IgM ELISA, IgG ELISA, real-time RT-PCR; limited virus isolation	Subset of samples from classical CHIK cases, as determined by lab constraints and epidemiological status; samples from all atypical or severe cases should be tested

During the initial introduction of CHIKV into a new region, comprehensive testing should be completed to confirm that CHIKV is the etiological agent. After CHIKV has been identified, limited testing (not testing all specimens or performing fewer assay types) can be considered depending upon the capacity of the lab and the epidemiological situation.

Interpretation and Reporting of Results

Courtesy: Center for Disease Control and Prevention

Typical results of samples tested at various time points Days post illness onset Virus testing Antibody testing Day 1–3 RT-PCR = Positive Isolation = Positive IgM = Negative PRNT = Negative Day 4–8 RT-PCR = Positive Isolation = Negative IgM = Positive PRNT = Negative >Day 8 RT-PCR = Negative Isolation = Negative IgM = Positive PRNT = Positive

The figure shows typical viremia and antibody response in humans following Chikungunya virus infection and the table describes the typical results of testing samples at various time points.

The following laboratory test results would confirm a recent CHIKV infection:

• Isolation of CHIKV, including confirmatory identification (either IFA, RT-PCR, or sequencing).
• Detection of CHIKV RNA by real time RT-PCR.
• Identification of a positive IgM result in a patient with acute symptoms of CHIKV, followed by the demonstration of CHIKV-specific antibody determined by PRNT with viruses in the SFV serogroup.
• Demonstration of seroconversion or a four-fold rise in PRNT, HI, or ELISA titers (again using other SFV serogroup viruses) between acute and convalescent specimens.

Autochthonous cases should be reported to WHO, in collaboration with an epidemiologist, according to International Health Regulations (IHR).

External Links

• CDC Chikungunya virus