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. 2019 May 1;11(5):407.
doi: 10.3390/v11050407.

Development of an E2 ELISA Methodology to Assess Chikungunya Seroprevalence in Patients from an Endemic Region of Mexico

Young Chan Kim  1   2 , César López-Camacho  3 , Nallely Garcia-Larragoiti  4   5 , Alan Cano-Mendez  6   7 , Karina Guadalupe Hernandez-Flores  8 , Carlos Alonso Domínguez-Alemán  9   10 , Maria Antonieta Mar  11 , Héctor Vivanco-Cid  12 , Martha Eva Viveros-Sandoval  13   14 , Arturo Reyes-Sandoval  15
Affiliations

Development of an E2 ELISA Methodology to Assess Chikungunya Seroprevalence in Patients from an Endemic Region of Mexico

Young Chan Kim et al. Viruses. .

Abstract

Chikungunya fever is a debilitating disease caused by Chikungunya virus (CHIKV) that can result in long-lasting arthralgias. The early diagnosis of CHIKV relies on PCR during the acute infection phase to allow differential diagnosis with other co-circulating arboviruses such as dengue and Zika. Alternatively, serology can support diagnosis and provide epidemiological information on current and past outbreaks. Many commercial serological ELISA assays are based on the inactivated whole CHIKV, but their sensitivity and specificity show great variability. We produced recombinant CHIKV E2 that is suitable for ELISA assays, which was used for the serodiagnosis of CHIKV infections occurring in an arbovirus endemic Mexican region within Michoacán state. A cross-sectional study was conducted in 2016-2017; sera was obtained from 15 healthy donors and 68 patients presenting undifferentiated febrile illness. Serum samples were screened by RT-PCR and by our in-house ELISA assay. Our results indicate that IgM and IgG anti-CHIKV E2 antibodies were detected with our ELISA assay with higher sensitivity than a commercially available CHIKV ELISA kit. Our simple and sensitive ELISA assay for the serodiagnosis of CHIKV infections can be applied to population-based seroprevalence surveys and has potential for monitoring vaccine immunogenicity in CHIKV vaccine clinical trials.

Keywords: CHIKV antibodies; Chikungunya virus; ELISA; Mexico; diagnosis; envelope protein 2; febrile patients.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The geographical location of study sites. (A–C). Map of Michoacan State in Mexico showing the locations of study sites (Lazaro Cardenas and Morelia) and illustration of the geographical differences between the two municipalities. (D). Graph showing the elevation, surface, precipitation, and temperature between the two municipalities.
Figure 2
Figure 2
Flow chart describing tests performed in samples using RT-PCR and two ELISA methodologies.
Figure 3
Figure 3
ELISA assays to assess the reactivity of human sera. Chikungunya virus (CHIKV)-infected patients (blue) confirmed by RT-PCR, sera obtained from healthy donors (HD) from a non-endemic region (red) and a CHIKV-positive control serum (green). (A) The graph of CHIKV E2 IgG OD405 against sera dilutions (left) and the corresponding endpoint log reciprocal titres (right). (B) The graph of CHIKV E2 IgM OD405 (left) and the corresponding endpoint log reciprocal titres (right). The black dotted line indicates the limit of detection (LoD), which corresponds to 1:300 serum dilution on a log scale.
Figure 4
Figure 4
Qualitative ELISA assays using the commercial kits to assess the reactivity of human serum samples. Mean standard unit (SU) for IgG (positive y-axis) and IgM (negative y-axis) are represented for all 68 serum samples. Positive samples are defined as >11 SU, and the negative samples are defined as <9. The SU of samples 9–11 are defined as in the grey zone (inconclusive results). RT-PCR positive samples are indicated by the (*) and color-filled bars.
Figure 5
Figure 5
In-house recombinant CHIKV envelope protein 2 (E2)-based ELISA. (A) IgG ELISA assay to assess the reactivity of all the human serum samples in this study. Positive CHIKV serology (blue), negative CHIKV serology (brown), and negative control serum samples from healthy volunteers (red). (B) Log reciprocal E2 endpoint titres for IgG (positive y-axis) and IgM (negative y-axis) are represented for all 68 serum samples. RT-PCR positive samples are indicated by the (*) and color-filled bars.
Figure 6
Figure 6
Correlation between the endpoint titres from in-house ELISA assay and mean standard unit (SU) from the commercial CHIKV kit. (A) Correlation between the in-house IgG endpoint titres and the commercial IgG mean SU. (B) Correlation between the IgM endpoint titres and the commercial IgM mean SU. The p values and R2 values reflect Pearson correlation tests. A positive cut-off is defined as >11 SU.
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