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Updated: May 1, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis
Jesse J Waggoner1, Janaki Abeynayake2, Ilana Balassiano3
1Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, USA.
Abstract:
Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.
Insights
A new multiplex nucleic acid amplification test accurately detects dengue virus (DENV), Leptospira, and Plasmodium species in travelers. This UFI assay improves diagnosis of undifferentiated febrile illness (UFI) in returning travelers.
Area of Science:
- Tropical Medicine
- Molecular Diagnostics
- Infectious Diseases
Background:
- Undifferentiated febrile illness (UFI) in travelers returning from developing nations is frequently caused by dengue virus (DENV), Leptospira, and malaria.
- These infections pose significant risks and can lead to life-threatening complications.
Purpose of the Study:
- To develop and evaluate an internally controlled multiplex nucleic acid amplification test (UFI assay) for simultaneous detection of DENV, Leptospira, and Plasmodium species.
- To improve diagnostic capabilities for common febrile illnesses in travelers.
Main Methods:
- Development of a multiplex nucleic acid amplification test (UFI assay) targeting DENV, Leptospira, and Plasmodium species (including Plasmodium falciparum).
- Analytical evaluation for dynamic range and limit of detection.
- Clinical evaluation using 210 previously tested samples and a panel of 66 negative clinical samples.
Main Results:
- The UFI assay demonstrated high sensitivity: 98% for DENV, 100% for Leptospira, and 100% for malaria (all five human Plasmodium species).
- 100% specificity was observed with negative clinical samples, and no cross-reactivity with related pathogens.
- The assay detected Plasmodium in plasma samples, even at low parasitemia levels.
Conclusions:
- The syndrome-based UFI assay offers a significant advancement over individual diagnostic tests for these pathogens.
- The assay's high sensitivity and specificity provide a reliable tool for diagnosing febrile illnesses in travelers.
- This multiplex approach enhances the timely and accurate diagnosis of critical infectious diseases.
