Development of a cost-effective multiplex quantitative RT-PCR assay for early detection and surveillance of Dengue, Chikungunya, and co-infections from clinical samples in low-resource settings

Abstract

Abstract Background Dengue and Chikungunya are Aedes-borne diseases that are predominantly prevalent in tropical and subtropical regions, affecting public health globally. Dengue is caused by multiple antigenically different Dengue virus (DENV) serotypes (DENV-1 to DENV-4) in the Flaviviridae family and Chikungunya (CHIKV) in the Togaviridae family. Both viral diseases can produce similar clinical manifestations, especially in the early stages of infection which poses a significant challenge for timely diagnosis and improper disease management. Currently in India, diagnosis of Dengue and Chikungunya relies on ELISA-based tests, which often lead to false negatives and under estimation of the actual disease burden. The city of Bengaluru, in the state of Karnataka, in Southern India, witnesses spike in Dengue infection, and hence it is important to diagnose these arboviral infections early as well as accurately, to estimate the disease burden for precise epidemiological assessments. Methods A multiplex, quantitative, real-time PCR assay, DENCHIK was developed that can detect and differentiate between all four DENV serotypes and CHIKV infections simultaneously. Using DENCHIK assay, a total of 903 sera samples from suspected febrile patients were screened. The blood samples were collected across 161 public health centers, maternity homes and referral hospitals, in urban Bengaluru, between July 2022 - December 2022. The sensitivity and specificity of DENCHIK assay was compared with ELISA (NS1 antigen and Immunoglobulin M (IgM) antibodies) and two commercially available q RT-PCR assays for DENV and CHIKV. Findings Using DENCHIK assay, out of 903 samples, 36% infections were DENV, 17% CHIKV infections and 8% were coinfections with DENV-CHIKV. In contrast, ELISA detected 29.90% of DENV and 22.92% of CHIKV infections. We observed 9% prevalence of DENV infections using NS1 ELISA as compared to 24% as detected by IgM ELISA. DENV-1 was the predominant serotype followed by DENV-2, DENV-3 and DENV-4. There was an increase in the prevalence of DENV and CHIKV infections from June 2022 to September 2022, coinciding with the monsoon season. However, there was no significant difference observed in the prevalence of DENV and CHIKV infections across genders and age groups. The sensitivity and specificity of DENCHIK assay in DENV detection as compared to NS1 ELISA assay was observed to be 62.82% and 66.45%, respectively. In comparison to commercially available q RT-PCR assays for DENV detection, DENCHIK assay exhibited 99% and 98% sensitivity and specificity, respectively. Similarly, in case of CHIKV 26% sensitivity, 86% specificity and 98% sensitivity and specificity were observed, as compared to the IgM ELISA and commercial RT-PCR assays, respectively. Conclusion DENCHIK assay successfully enabled, simultaneous amplification of all four DENV serotypes and Chikungunya, in a single tube from clinical samples with high sensitivity and specificity. DENCHIK assay detected 7.6% of additional Dengue infections and 6.65% less of Chikungunya infections in clinical samples, as compared to detection by ELISA. As, compared to ELISA, DENCHIK demonstrates early and accurate detection of DENV and CHIKV with higher sensitivity and specificity as early as day one of symptom onset post infection. DENCHIK aids in estimating the exact prevalence of DENV and CHIKV infections, that are often misdiagnosed or overestimated, respectively, using routine serological assays such as ELISA. Molecular surveillance using targeted diagnostic assays such as DENCHIK could be used to determine the prevalence of multiple DENV serotypes, CHIKV and DENV-CHIKV Co-infections from clinical samples. The findings from the study shall be useful to inform and aid the public health authorities, to contain and curb the rapid spread of these diseases in the community.