Laboratory Diagnosis of Chikungunya Virus Infections and Commercial Sources for Diagnostic Assays

Abstract

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >5 days of illness. Commercially available CHIKV IgM-detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

Keywords: Chikungunya virus; IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA); arbovirus diagnostic testing; real-time RT-PCR.

Figure 1

Time course of chikungunya virus (CHIKV) viremia and immune response. Limit of detection (LOD) of real-time reverse transcription–polymerase chain reaction (RT-PCR) assay is approximately 100 plaque-forming units (PFU)/mL (approximately 1 RNA transcript/reaction); the LOD of the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (ELISA) positive-to-negative ratio (P/N) is >2. Abbreviations: Ab, antibody; IgG, immunoglobulin G.

Figure 2

The Centers for Disease Control and Prevention diagnostic testing algorithm for detection of chikungunya virus (CHIKV) infection. Serum is collected from patients meeting the clinical case definition of fever and arthralgia who have returned from a region where CHIKV is endemic or CHIKV infection is epidemic. All samples with positive and equivocal real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) results are repeated with a second set of primer/ probes for confirmation. Specimens with positive results of tests using both sets of primers and probes are considered to be confirmed CHIKV-positive specimens. Samples with positive or equivocal immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA) results are confirmed to be positive if plaque reduction neutralizing testing (PRNT) yields positive results. Abbreviations: Neg, negative result; Pos, positive result.