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

BackgroundDengue 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 produce similar clinical manifestations, especially in the early stages of infection which poses a significant challenge for timely diagnosis and improper disease management. In India, diagnosis of Dengue and Chikungunya relies on ELISA-based tests, which often lead to false negatives and under estimation of the disease burden. MethodsA multiplex, quantitative, real-time PCR assay, DENCHIK was developed for simultaneous detection of DENV serotypes and CHIKV.A total of 903 sera samples were screened from suspected febrile patients across 161 public health centers in 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. FindingsUsing DENCHIK assay,36% infections were DENV, 17% CHIKV and 8% were DENV CHIKV co-infections. 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% 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 to September 2022, coinciding with the monsoon season. There was no significant difference observed in the prevalence of DENV and CHIKV infections across genders and ages. 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. ConclusionDENCHIK assay successfully enabled, simultaneous amplification of all four DENV serotypes and Chikungunya, from clinical samples. 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, using 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. Author SummaryDengue and Chikungunya are most common arboviral illnesses affecting more than half of the worlds population. Both the viral diseases have overlapping symptoms, which poses a challenge for accurate differential diagnostics in low-resource setting. Infection with one or more different serotypes of DENV results in a phenomenon, known as antibody-dependent enhancement (ADE), wherein antibodies against one serotype, instead of protecting against DENV infection caused by other serotypes, aids in the viral uptake by the host immune cells, resulting in severe dengue. Rapid antigen tests targeting NS1, and IgG/IgM are the most common methods used to detect DENV and CHIKV infections. However, there are several limitations of serological assays: a) ELISA cannot differentiate DENV serotypes, b) depending on the stage of infection, ELISA-based tests often provide false-positives or false-negatives. This warrants a need for a reliable molecular method which can differentiate between DENV serotypes and across Dengue and Chikungunya with reasonable sensitivity and specificity. Bengaluru has highest dengue burden in Southern India. There is high infestation of Aedes aegypti and Aedes albopictus in diverse breeding habitat and year-round circulation of four serotypes. Currently, Dengue and Chikungunya testing relies on ELISA (NS1, IgM and IgG) often leading to under estimation of disease burden. To address this gap, a cost-effective multiplex qRT-PCR assay, DENCHIK was developed for simultaneous detection of four DENV serotypes and CHIKV. The sensitivity and specificity of DENCHIK assay was tested across months and days from onset of febrile symptoms and compared with ELISA and two commercially available kits. We suggest implementation of molecular methods and using DENCHIK assay in urban health centres would help reduce underestimation of cases, actual estimates of disease burden across seasons and help in better clinical management of Dengue and Chikungunya.