Evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in India, a dengue-endemic country
- Jaspreet Jain†1Email author,
- Tamaki Okabayashi†2, 3, 4,
- Navjot Kaur5,
- Emi Nakayama6,
- Tatsuo Shioda4, 6,
- Rajni Gaind5Email author,
- Takeshi Kurosu7Email author and
- Sujatha Sunil1Email author
© The Author(s). 2018
Received: 8 February 2018
Accepted: 7 May 2018
Published: 11 May 2018
Chikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses that share the same Aedes mosquito vector, and there is much overlap in endemic areas. In India, co-infection with both viruses is often reported. Clinical manifestations of Chikungunya fever is often confused with dengue fever because clinical symptoms of both infections are similar. It is, therefore, difficult to differentiate from those of other febrile illnesses, especially dengue fever. We previously developed a CHIKV antigen detection immunochromatography (IC) rapid diagnosis kit . The current study examined the efficacy of previously mentioned IC kit in India, a dengue-endemic country.
Sera from 104 CHIKV-positive (by qRT-PCR) and/or IgM-positive (ELISA) subjects collected in 2016, were examined. Fifteen samples from individuals with CHIKV-negative/DENV-positive and 4 samples from healthy individuals were also examined. Of the 104 CHIKV-positive sera, 20 were co-infected with DENV.
The sensitivity, specificity and overall agreement of the IC assay were 93.7, 95.5 and 94.3%, respectively, using qRT-PCR as a gold standard. Also, there was a strong, statistically significant positive correlation between the IC kit device score and the CHIKV RNA copy number. The IC kit detected CHIKV antigen even in DENV-co-infected patient sera and did not cross-react with DENV NS1-positive/CHIKV-negative samples.
The results suggest that the IC kit is useful for rapid diagnosis of CHIKV in endemic areas in which both CHIKV and DENV are circulating.
Chikungunya fever (CF) is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus and is transmitted to humans by infected mosquitoes. CF is an acute febrile disease with symptoms that include arthralgia, myalgia, headache, vomiting, backache and diffused maculopapular rashes, which are similar to those of dengue fever (DF) [2, 3]. Therefore, it is difficult to diagnose whether an individual is infected with CHIKV or with both CHIKV and dengue virus (DENV). Co-infection of humans and mosquitoes with CHIKV and DENV has been reported in India [4–6]. It is also of concern that several acute febrile diseases such as malaria, influenza, leptospirosis, rickettsiosis, rubella, mycoplasma infections and other febrile diseases are also prevalent in areas in which CF is found; this makes accurate and confident diagnosis of acute febrile diseases more difficult.
Although, several chikungunya rapid diagnostic kits are commercially available, their sensitivity does not always correlate with that of RT-PCR because all of them detect host-derived anti-CHIKV IgM antibodies. Detection of IgM antibodies is less sensitive than detecting antigen as the antibodies are produced later during the course of infection, thereby affecting prompt diagnosis and eventually disease management . Recently, we developed a rapid diagnostic immunochromatography (IC) test kit based on mouse-derived anti-CHIKV monoclonal antibodies that react with a CHIKV East Central South African genotype (ECSA) isolated from patient sera obtained during a CHIKV outbreak in Thailand in 2010 [1, 8]. However, we did not examine the reactivity of these IC kits with serum samples taken from other febrile patients, including those with DF.
The present study was aimed to examine the suitability of the IC kit as a tool for rapid diagnosis of CHIKV in an endemic area, India. For this purpose, we tested the kit during a recent CHIKV outbreak that occurred in New Delhi in 2016.
Virus, cell culture and titrations
CHIKV strain CP10 (ECSA genotype) was propagated in Vero cells maintained in Minimum Essential Medium (Life Technologies, Inc., USA) supplemented with 10% (v/v) heat-inactivated Foetal Bovine Serum (Life Technologies). The virus was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) , using a laboratory-generated strain (IND/DEL/2010–01) cloned in pGEM-T vector and serially diluted from 100 ng to 1 pg as reference to determine viral copy number of CHIKV viral RNA isolated from patients sera using the formula.
Number of VNA copies = (amount of VNA in nanograms × 6.022 × 1023) / (length of VNA amplicon (in basepairs) × 1 × 109 × 330).
The CHIKV strain CP10 was also titrated using standard plaque assay .
Patient recruitment and sample collection
Blood samples were collected from a cohort of suspected dengue and chikungunya patients, with history of fever with joint pains, present within 1 to 15 days of illness and referred to the Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India. In addition, samples from patients suffering from other febrile diseases were collected and used as negative controls for specificity. Also, samples from healthy volunteers were collected as negative controls. The study was jointly funded by the Department of Science and technology (DST), Government of India and Japan Agency for Medical Research and Development (AMED), and all patients and controls signed a consent form approved by the institutional ethical board (IEC/VMMC/SJH/Project/February-2016/574, ICGEB/IEC/2014/01, Version 3). Onset of fever and other clinical features were documented at the time of patient recruitment. Sera were separated from whole blood and stored in − 80 °C.
Diagnosis of samples and study design
Anti-chikungunya IgM antibodies were detected using Chikungunya-IgM capture ELISA kit (MAC-ELISA; NIV-Pune, India). Also, all samples were subjected to qRT-PCR analysis . Samples that were positive for IgM and/or positive in the qRT-PCR were grouped according to the presence of viral RNA and/or antibodies. The groups are explained in the additional figure file (Additional file 1). Group 1 included all CHIKV samples that tested positive by CHIKV qRT-PCR and/or positive for IgM by ELISA. These samples were then sub-grouped as follows: Group 2, samples positive for CHIKV RNA irrespective of the presence of CHIKV antibodies; Group 3, samples positive for antibodies irrespective of the presence of CHIKV RNA; and Group 4, samples positive for both CHIKV RNA and IgM, DEN IgM and NS1. Samples for other febrile diseases were collected retrospectively after being were diagnosed by following detection methods; Malaria: rapid card test and confirmation by microscopy, Salmonella: Vidal, IgM immunochromatography test and culture, HIV: 4th generation ELISA, Leptospirosis: rapid IC test and Influenza: PCR. These samples were used to detect cross-reactivity of the IC kits.
Testing the IC kit
Thirty microliters of serum or ten-fold serially diluted CHIKV culture supernatant was placed in a tube and mixed with 30 μl of IC kit extraction buffer (supplied by ARKRAY, Inc. Kyoto, Japan). The IC kit was inserted into the tube and developed chromatographically. After 15 min, two independent researchers examined the control and test lines visually and in a blinded manner. Then, an IC Reader C10066–10 (Hamamatsu Photonics K.K., Japan) was used to measure the actual intensity of the test lines.
The correlation between the test device score and the CHIKV RNA copy number measured by qRT-PCR was analysed and the significance of the correlations was estimated using the Pearson correction; P < 0.05 was considered to be significant. All data were analysed using statistical software R 3.3.3 (The R Foundation, https://www.r-project.org/).
Testing the IC kit using clinical samples
Sensitivity, specificity and over all agreement with real time RT-PCR of immunochromatography kit for chikungunya virus antigen using clinical serum samples in various clinical categories
Criteria of CHIKV positive
qRT-PCR or IgM
qRT-PCR and/or IgM
qRT-PCR and IgM
Comparison of IC kit performance with that of qRT-PCR and IgM ELISA using confirmed CF samples
Correlation between the test device scores and the results from the IC kit
Detection of CHIKV antigen, RNA and IgM antibodies with respect to the time of fever onset
Specificity test using samples from patients with dengue
Use of the immunochromatography kit to detect chikungunya virus antigen in sera co-infected with dengue virus
IC kit Positive
CHIKVq RT-PCR + ve
/CHIKV IgM + ve
CHIKVq RT-PCR + ve
/CHIKV IgM + ve
Since its first report in Delhi in 2009, a total of 272,384 confirmed CF patients have been reported . Apart from these CF outbreaks that occur from time to time, the additional burden of co-infections with DENV makes patient management a huge concern  The current approach of CF diagnosis is either by detecting the viral RNA using quantitative PCR or by detecting the antibodies against the virus using ELISA. Both the above-mentioned methods have their own caveats making early diagnosis of CF cumbersome and/or ineffective, especially during outbreaks involving large number of individuals. Through the present report we provide evidence of the antigen-based IC kit as a valuable tool for early diagnosis of CF in Indian patients that can replace the current approach of qRT-PCR detection. While the kit did not cross-react with serum samples from patients infected with other febrile diseases including DENV, we show that this kit is effective in detecting CHIKV in patients co-infected with DENV. Whereas the detection rate of the IC kit was similar to that of qRT-PCR, the IC kit provides a CF diagnosis in between than 15–30 min making it much faster than qRT-PCR (or ELISA) and making this kit comparable to other early diagnostic tools available for other febrile fevers such as dengue . Owing to its rapid detection time, this kit may also be used as a point-of-care kit especially in outbreak situations.
The detection rate of the IC kit fell at 6 days post-fever onset (Fig. 3). It is probably because the IC kit detects viral envelop protein which generally drops after 4–5 days post-infection. At the same time, the production of IgM increases as a host response that could probably hamper antigen detection. As most patients visit the clinic early while they are still in the pyrexic phase of the disease, we believe that the detection rates of the IC kit as recorded in this study are satisfactory to be used during the outbreaks in the chikungunya endemic countries.
The MAb used in the IC kit was produced by immunization of the ECSA strain isolated in Thailand . Mutation analysis of this strain revealed some substitutions located within the epitope region of E1 gene for anti-CHIKV antibody used in the IC kit. Results from the current study reveal that these changes do not impact the kit performance. Analysis of the E1 gene from the Indian isolates reveal that these variations were not present in these isolates thereby providing confidence to the utility of this kit in Indian patients . Our results suggest that the IC kit could be used for early CF diagnosis not only in Southeast Asian areas but also Indian Ocean areas as well, where CHIKV ECSA is endemic.
The IC kit tested herein detected ECSA genotypes of CHIKV present in India during the early phase of the disease suggesting that this kit could be used during outbreak situations in endemic regions. The IC kit did not cross-react with sera from patients infected with DENV alone and it detected CHIKV antigen in co-infected cases. Thus, the IC kit may be useful in areas in where the CHIKV ECSA genotype is endemic and have high probability to occur as co-infections with dengue.
We thank Juthamas Phadungsombat, at Mahidol-Osaka Center for Infectious Diseases, for her help. ARKRAY Inc. (Kyoto, Japan) provided the IC kits evaluated in this study. ARKRAY had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. The manuscript was proofread by BioEdit (Manchester, UK).
This work was supported by Strategic International Research Cooperative Program under the DST (India) and Japan Initiative for Global Research Network on Infectious Diseases from AMED.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.
SS, TK and RG designed the study. JJ and TO performed the study. JJ, TO, NK, TS, and TK analysed the data. TO and EN provided reagents for the study. TK, TO, JJ and SS were involved in manuscript preparation and review. RG and NK were involved in recruitment of patients, clinical and lab diagnosis, and follow up of patients. SS, TK and RG supervised the overall study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All patients and controls signed a consent form approved by the institutional ethical board (IEC/VMMC/SJH/Project/February-2016/574, ICGEB/IEC/2014/01, Version 3).
Consent for publication
All authors have given their consent for the publication of this work. We have obtained consent to publish from the participant (or legal parent or guardian for children) to report individual patient data.
The authors declare that they have no competing interests.
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