Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

Abstract

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. Dengue is a mosquito-borne viral infection in humans that is of global importance. An estimated 390 million infections occur each year, of which 96 million result in clinically apparent infections, and in some epidemics the mortality rate may reach 5% (1, 2). Dengue viruses (DENVs) are members of the family Flaviviridae and consist of four antigenically distinct serotypes which exhibit 65% to 70% sequence homology (3). Disease manifestations range from mild undifferentiated acute dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (4, 5). After an incubation period of 2 to 10 days, a primary infection presents with the acute onset of high fever (Ն40°C), accompanied by headache, retroorbital pain, generalized myalgias, arthralgias, and malaise. The early phase of DHF or DSS has similar clinical features; however, after defervescence, new abdominal pain, nausea, and vomiting, followed by thrombocytopenia and a rise in hematocrit, may progress to shock leading to multiorgan dysfunction, life-threatening hemorrhage, and death. DF symptoms may be clinically indistinguishable from acute febrile illness due to other infectious diseases such as influenza, malaria, measles, and chikungunya virus (CHIKV) infections. CHIKVs are alphaviruses belonging to the ...

Clinical and Serological Insights from the Asian Lineage Chikungunya Outbreak in Grenada, 2014: An Observational Study

“…In addition, a quantitative multiplex real-time polymerase chain reaction (PCR) for CHIKV and DENV was performed by the NIDDL using an in-house developed and Clinical Laboratory Improvement Amendments-validated assay with a cutoff for positivity < 37 cycle time. 21 Of all 493 cases in the study, 426 (86%) tested positive for CHIKV by either IgM or PCR, with 49 testing positive by both tests. By gender, 286 of 325 (88%) female cases and 140 of 165 (85%) male cases tested positive.…”

A novel multiplex PCR for virus detection by melting curve analysis

“…Compared with other virus detection techniques, this novel multiplex PCR has many advantages. First, the number of detectable targets is significantly increased in our 2D-label multiplex PCR when compared with those multiplex PCR assays using a label that involves either Tm or fluorophore alone (Wan et al, 2016;Simmons et al, 2016). In this study, 2 probes with discriminable Tm values were designed in each channel.…”

Chikungunya Virus

“…Reliance on PCR testing only is unlikely to accurately characterize incidence. [45][46][47] Commercially available kits are of variable quality. 48 PCR assays with favorable performance characteristics are documented in the literature and becoming commercially available for clinical use in other countries; however, they can only be obtained for research use in the United States.…”

“…Encouraging research studies suggest that this will be possible, first for PCR and perhaps later for the IgM assays. 46,51,52 TREATMENT There is presently no licensed targeted therapy for acute CHIKV infection. Treatment is primarily supportive care and includes the use of analgesic and anti-inflammatory medication, rehydration, and rest.…”