Chikungunya Fever

Laboratory Diagnosis of Chikungunya Fevers

The clinical manifestations of chikungunya fever resemble those of dengue fever. Laboratory diagnosis is critical to establish the cause of diagnosis and initiate specific public health response.

Types of Laboratory tests available and specimens required

Three main laboratory tests are used for diagnosing Chikungunya fevers: virus isolation, serological tests and molecular technique of Polymerase Chain Reaction (PCR). 

Virus isolation

Virus isolation is the most definitive tests. Between 2-5 ml of whole blood is collected during the first week of illness in commercial heparinzed tube and transported on ice to the laboratory. The CHIK virus produces cytopathic effects in a variety of cell lines including BHK-21, HeLa and Vero cells. The cytopathic effects must be confirmed by CHIK specific antiserum and the results can take between 1-2 weeks. Virus isolation must only be carried in BSL-3 laboratories to reduce the risk of viral transmission.  

RT-PCR

Recently, a reverse transcriptase, RT- PCR technique for diagnosing CHIK virus has been developed using nested primer pairs amplifying specific components of three structural gene regions, Capsid (C ), Envelope E-2 and part of Envelope E1.  PCR results can be available from within 1-2 days. Specimens for PCR is same as the virus isolation i.e. heparized whole bloodl

Serological diagnosis

For serological diagnosis between 10-15 ml of whole blood sera are required; an acute phase serum must be collected immediately after clinical onset and a convalescent phase serum10-14 after the disease onset. The blood specimen is transported at 4 degrees and not frozen to the laboratory immediately. If testing cannot be done immediately, the blood specimen is separated into sera that should be stored and shipped frozen.

Serologic diagnosis can be made by demonstration of fourfold increase in antibody in acute and convalescent sera or demonstrating IgM antibodies specific for CHIK virus. A commonly used test is the Immunoglobulin M Antibody (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA). Results of MAC-ELISA can be available within 2-3 days. Cross-reaction with other flavirus antibodies such as o’nyong-nyong and Semliki Forest occur in the MAC-ELISA; however, the latter viruses are relatively rare in South East Asia but if further confirmation is required it can be done by   neutralization tests and Hemagglutination Inhibition Assay (HIA).

Interpretation of results

Sero-diagnosis rests on demonstrating a fourfold increase in CHIK IgG titer between the acute and convalescent phase sera. However, getting paired sera is usually not practical.   Alternatively, the demonstration of IgM antibodies specific for Chikungunya virus in acute-phase sera is used in instances where paired sera cannot be collected.

A positive virus culture supplemented with neutralization is taken as definitive proof for the presence of Chikungunya virus.

PCR results for E1 and C genome either singly or together constitute a positive result for Chikungunya virus.

Existing Laboratory Network for Diagnosing Chikungunya

The virology laboratory network in South East Asia, from India, Indonesia, Myanmar, Sri Lanka and Thailand can perform many of the laboratory tests for CHIK virus. See box 1. The regional office of the South East Asia Region is prepared to advise and help with testing in the region, for further information contact Dr Caussy at Caussyd@searo.who.int  of Dr Bhatia at Bhatiaraj@searo.who.int.

Box 1: Existing Laboratory Network for Diagnosing Chikungunya