Introduction
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus belonging to the family
Togaviridae. This pathogen was first isolated in Tanzania, East Africa, in 1952 [
1,
2]. Since then, CHIKV has caused sporadic outbreaks throughout the African and Asian continents. Although only one serotype CHIKV exists, the virus is classified into three genotypes named after the geographical location where the respective genotype was first recognized: East/Central/South African (ECSA), West African (WA), and Asian [
3,
4]. CHIKV was considered a neglected tropical pathogen until a large outbreak was reported on Indian Ocean islands in 2005. The outbreak affected 244,000 individuals, one-third of the total population in this region [
5,
6] . Sequence analysis revealed a specific amino acid change that rendered the virus infective to
Aedes albopictus; this clade of the ECSA genotype therefore was designated as the Indian Ocean Lineage (ECSA-IOL) [
6,
7]. Subsequently, ECSA-IOL also was identified in India [
8] and South East Asia (including Thailand [
9], Cambodia [
10], and Malaysia [
11]), and was detected for the first time in European countries (including Italy [
12] and France [
13]). In contrast, non-IOL ECSA-genotype CHIKV was detected in Brazil in 2013 [
14,
15]. Asian-genotype CHIKV subsequently gave rise to a novel clade, designated as Asian/American [
16,
17]. This clade has become a public health problem in the Caribbean islands and the Central American mainland [
18,
19]. Worldwide, approximately one billion people are estimated to live in areas at risk of CHIKV outbreaks [
20].
More than 75% of CHIKV-infected individuals develop acute febrile symptoms such as high fever, headache, and muscle and joint pains [
21,
22] that are similar to those observed in patients infected by other mosquito-borne viruses such as dengue dengue and Zika viruses [
23]. However, the CHIKV illness often is associated with prolonged and incapacitating arthritis; for this reason, large CHIKV epidemics have considerable economic consequences, highlighting their significant public health impact. To distinguish CHIKV infection in the acute (viremic) phase, several nucleic acid detection methods are considered the gold standards [
24]. However, immunochromatographic (IC) rapid diagnostic tests (RDTs) are user-friendly and easy to store without the need to maintain a cold chain, while facilitating early diagnosis. Such RDTs are expected to increase the accessibility of laboratory diagnosis of CHIKV infection. Previously, Okabayashi et al. reported a prototype IC RDT with high sensitivity (68 out of 76, 89.4%) and specificity (34 out of 36, 94.4%) against clinical samples known to contain ECSA-genotype CHIKV in Thailand and Laos [
25]. That RDT also showed high sensitivity (74 out of 79, 93.7%) and specificity (42 out of 44, 95.5%) in India [
26]. However, another trial of that RDT with confirmed clinical samples of Asian-genotype CHIKV from the island of Aruba revealed low sensitivity (10 out of 30, 33.3%) [
27].
We previously showed that CK47, one of the monoclonal antibodies (MAbs) used in the RDT described by Okabayashi et al., has a limited ability to recognize the Asian-genotype E1 protein [
28]. Notably, the Asian-genotype CHIKV encodes an aspartic acid (D) at residue 284 of the predicted E1 protein (residue 350 of the 6 K-E1 polyprotein), a position that is encoded as a glutamic acid (E) in the predicted E1 proteins of certain ECSA-genotype CHIKVs [
28]. This polymorphism critically affected the utility of that diagnostic tool across the various CHIKV genotypes [
17]. To improve the sensitivity and specificity of the IC RDT, we obtained several anti-CHIKV MAbs targeting the E1 and capsid (CA) proteins of CHIKV [
29]. These MAbs were shown to exhibit broad reactivity towards all genotypes of CHIKV, and some of these MAbs lacked cross-reactivity to other alphaviruses, including eastern, western, and Venezuela equine encephalitis viruses. Here, we report the development and evaluation of an improved CHIKV antigen-detection diagnostic test that incorporates these new MAbs.
Discussion
In the present study, using newly obtained MAbs [
29], we developed new IC CHIKV-E1 antigen detection RDTs with improved sensitivity to Asian- and WA-genotype viruses compared to the previous version-A RDT [
28]. Although we tested only 5 samples of Asian-genotype CHIKV-infected cases (i.e., Aruban cases before anti-CHIKV IgM/IgG seroconversion), the 2nd generation CHIKV antigen detection RDT version B yielded positive results in 4 of these 5 samples (i.e., 80% sensitivity). Sample numbers are low in this Asian-CHIKV serum panel, but the results suggest increased sensitivity over the previous version of RDT [
27]. Furthermore, the 3rd generation RDT version O showed sensitivity of 92% (92 out of 100, 95%-CI 85.0–95.9) in sera from 100 ECSA-genotype CHIKV-infected patients from Bangladesh. The version-O RDT also showed higher sensitivity to cultured Asian- and WA genotype CHIKVs than the version-A RDT did. These results suggest that the version-O RDT will be better for worldwide application compared to the version-A and -B RDTs.
As described in Materials and Methods, the sampling dates after the onset of fever were median 7 days and the median Ct value and its IQR of CHIKV-detecting PCR were 35.29 (34.22–35.50) for the Aruba samples. The sampling days after the onset of fever did not differ significantly among 11 PCR-positive and RDT-positive (median 6 days), 15 PCR-positive and RDT-negative (median 7 days), and 9 PCR-negative and RDT-positive groups (median 7 days). Importantly, many of the samples from Aruba were collected after anti-CHIKV antibody seroconversion had occurred. We look forward to confirming our findings in a larger sample size of Asian-genotype CHIKV-infected patients, preferably before seroconversion. After seroconversion, detection of anti-CHIKV antibodies can be used for diagnosis. Therefore, it would be ideal to simultaneously detect both CHIKV antigen and anti-CHIKV antibodies for accurate diagnosis.
In terms of the false-positive rate in CHIKV PCR-negative samples, the specificity of the version-O RDT in samples in Bangladesh was 100%, while that of the version-B RDT was 83.3% (45 out of 54) in samples from Aruba. Specificity of the version-B RDT in the Aruba samples was 94.2% (33 out of 35) for anti-CHIKV IgM- and IgG-negative cases, while that in anti-CHIKV IgM- or IgG-positive cases was 63.2% (12 out of 19) (Table
2). As described above, the version-B RDT uses 3 clones of anti-CHIKV MAbs, while the version-O RDT uses 5 MAbs, including 3 MAbs used in the version-B RDT. Thus, it is unlikely that a specific component present only in the version-B RDT caused the false-positive reactions. Most of the Ct-values of CHIKV-detecting RT-PCR in Bangladesh samples were lower than 20, and the median Ct-value and its IQR was 17.02 (14.89–19.92) (see Additional file
7). Of course, it is not appropriate to directly compare Ct-values between Aruba and Dhaka patients given that these values were determined with different real-time PCR systems, but these Ct-values suggest that the CHIKV sera in Dhaka were collected earlier in the disease course. Results of ELISA detecting anti-CHIKV IgM also support this idea, since only one out of 100 CHIKV PCR-positive cases was anti-CHIKV IgM positive (Table
3, Additional file
7). In contrast, among 54 PCR-negative samples from Aruba, 19 were positive for either anti-CHIKV IgM or IgG (Table
2, Additional file
3). As the kinetics of chikungunya antigenemia are not well understood, it is possible that the IC RDT-positive but PCR-negative samples are not truly CHIKV negative. In other words, CHIKV structure proteins might have persisted longer than the viral genome in blood, since the structural proteins might be expressed from a sub-genomic or defective viral RNA without full-length genomic RNA replication. In fact, persistence of a defective alphavirus genome in infected cells has been reported previously [
37]. Indeed, three (3%) positive results were obtained when we tested 93 CHIKV PCR-negative and DENV PCR-negative but anti-CHIKV IgM-positive samples (Additional file
7).
The precise mechanisms associated with chronic CHIKV-associated joint disease are largely unknown, although the effects of abnormalities present prior to infection cannot be ruled out. In CHIKV-infected patients, high viral loads are common, and anti-CHIKV IgM or IgG has been observed to persist in patients for 18 months or longer [
38,
39]. These findings suggest that continuous immune stimulation, possibly by persistent or continuously expressed CHIKV antigens, could play a role in prolonged CHIKV-associated poly-arthralgia, although no tools are available to rapidly detect CHIKV antigens. It will be interesting to use the diagnostic RDT described in the present work for follow-up studies on antigen persistence in post-chikungunya chronic poly-arthralgia. Thus, it will be critical to further evaluate these false-positive cases using our revised CHIKV RDT.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.