A New Dengue Rapid Test to Simultaneously Detect All Four Dengue Virus Serotypes

Abstract

Dengue virus (DENV) is an enveloped, positive- and single-stranded RNA virus belonging to the Flaviviridae family. The Flaviviridae family includes more than 70 human-disease causing pathogens, such as Zika virus, Yellow fever virus, West Nile virus, Japanese Encephalitis virus, Tick-Borne Encephalitis virus, besides DENV [1]. There are four antigenically distinct DENV serotypes, namely DENV-1, DENV-2, DENV-3, and DENV-4. Like many flaviviruses, DENV is an arbovirus that is primarily transmitted by the bite of infected Aedes aegypti mosquitoes [2]. DENV infection is responsible for one of the most common mosquito-borne infections, causing an estimated 100–400 million infections per year globally [2]. The incidence of DENV infection has risen dramatically over the past decade, with 2023 recording the highest number of cases, affecting over 80 countries [3]. DENV is now endemic in more than 100 countries across the Americas, Africa, Southeast Asia, and the Eastern Mediterranean and Western Pacific. The expanding geographic range of the arbovirus vector Aedes aegypti, driven by rising temperatures and high humidity, is putting more countries at risk [4].

DENV infection presents with a wide range of clinical manifestations, from asymptomatic or mild febrile illness to severe, life-threatening disease. The World Health Organization (WHO) classifies DENV infection into three categories: Dengue without warning signs, dengue with warning signs, and severe dengue (SD) [5]. Severe dengue can lead to increased vascular permeability and coagulopathy, resulting in complications such as hypovolemic shock, organ failure, and severe bleeding. With treatment, the mortality rate is approximately 2%–5%, but it can rise to as high as 20% without timely medical intervention [6].

Due to the nonspecific clinical symptoms presented during the early phase of DENV infection and the limited availability of diagnostics, DENV is often underdiagnosed in endemic regions around the world. During the early stage of infection (< 5 days), diagnosis of DENV infection relies on viral isolation, genome or antigen detection using reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR) [7]. However, these techniques require technical expertise and expensive laboratory reagents and equipment, which are often not available in resource-limited settings [2]. After this early stage, DENV RNA or antigens may no longer be detectable in the patient, necessitating serological antibody detection [7]. However, the high protein-sequence similarity among DENV serotypes and other flaviviruses can lead to antibody cross-reactivity, posing a significant challenge for accurate diagnosis [2].

In a recent study entitled “RT-RPA assisted CRISPR/Cas12a based one-pot rapid and visual detection of the Pan-Dengue virus” [8], the authors developed an innovative and simple assay system called one-pot RT-RPA CRISPR/Cas12a Integrated Detection of Pan-DENV serotypes (ORCID-PAD) for nucleic acid amplification and detection of DENV 1–4 using recombinase polymerase amplification (RPA) to amplify the targeted viral RNA and coupled it with the CRISPR/Cas12a and the integrated fluorescent-based detection (Figure 1). The ingenuity of the system has to do with the simplicity of the assay that is dependent on the Isothermal amplification (ISamp) technology that operates at a constant temperature, allowing for faster target gene amplification and improved diagnostic efficiency [9]. Unlike PCR, ISamp can use ssDNA, dsDNA, or RNA as a template, making it a more versatile diagnostic platform. The use of RPA improves the limit of detection, while CRISPR/Cas12a, an RNA-guided DNase enzyme, provides the high levels of sensitivity and specificity required to differentiate between the different DENV serotypes [9]. Additionally, the single-use tube design of the ORCID-PAD assay minimizes the risk of aerosol (or cross) contamination.

The ORCID-PAD assay also aligns more closely with the World Health Organization REASSURED (Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable) criteria for point-of-care diagnostics compared to the current diagnostic tools [10]. Since it does not rely on RT-PCR, it eliminates the need for expensive laboratory equipment and specialized technical expertise, while delivering significantly faster results. The authors showed that ORCID-PAD could achieve 93.7% sensitivity and 100% specificity, accurately identifying positive samples with a low false-negative rate. Overall, the assay demonstrated 98.7% accuracy for Pan-DENV detection, with no cross-reactivity with other closely related viral pathogens responsible for acute febrile illnesses [8]. Additionally, it requires only a minimal amount of DENV template RNA (with a detection limit of 10 ng for DENV-1 and DENV-4 genomes and 0.5 ng for DENV-3 and DENV-4 genomes) for accurate diagnosis, enabling detection during the acute phase of viral infection in the patient [8]. It is worth noting that the ORCID-PAD assay is designed as a single-reaction test to detect the presence of DENV 1–4 for point-of-care diagnostic, but it does not distinguish between the four serotypes, as the assay relies on fluorophore emission. However, if reagents are available, the reaction can be split into four separate tubes, potentially enabling precise serotyping of individual DENV serotype. The ORCID-PAD system offers significant advantages over existing DENV detection assays, including rapid turnaround time, ease of use, and minimal equipment requirements, making it highly suitable for point-of-care settings [8].

Currently, Dengvaxia is the only FDA-approved vaccine for dengue, but it is recommended only for individuals with a laboratory-confirmed previous dengue infection [11]. For severe dengue cases, supportive care remains the sole treatment option [6]. Because of this, accurate testing and early diagnosis are essential to reduce the rates of mortality, particularly in resource-limited areas. The ORCID-PAD assay described in a recent publication [8] offers a unique and accurate diagnostic solution that enables rapid, cost-effective, and highly specific detection of all four serotypes of DENV, making it a valuable tool for early diagnosis and disease management, especially in resource-limited settings.

The authors have nothing to report.

The authors declare no conflicts of interest.