Integrated Point of Care Nucleic Acid Testing for Acute Dengue Virus Infection - Summary
Dengue virus (DENV) infection causes major health problems with increasing incidence in endemic regions.
Dengue fever, caused by any of the four DENV serotypes (DENV1-4), initially presents with non-specific
symptoms of febrile illnesses, thus clinical diagnosis is often inaccurate. Early accurate diagnosis is important to
monitor patients for severe complications due to internal hemorrhage and shock, which can be fatal without
appropriate clinical management. Current DENV diagnostic methods either require specialized laboratory
facilities, or have poor sensitivity in the acute phase. Antibody detection in the convalescent phase has poor
specificity due to cross-reactivity with other flaviviruses. Nucleic acid amplification testing (NAAT) provides high
sensitivity and specificity, but current technologies are incompatible with peripheral low resource healthcare
facilities that serve the majority of affected patients. The goal of this R21 project is to develop an initial prototype
for a portable, inexpensive, robust, easy to use, integrated nucleic acid testing device consisting of a disposable
cartridge and an inexpensive, compact, battery operated instrument that enables early, rapid, sensitive, and
specific diagnosis of acute DENV infection. We have already developed such an integrated prototype device for
diagnosis of pulmonary tuberculosis from sputum. For diagnosis of DENV infection from whole blood, we have
established proof of principle for processes required during sample preparation, amplification, and detection.
Through the proposed project, we will further develop and optimize these processes, and then incorporate them
into a cartridge and instrument prototype that leverages components of our existing NAAT device, but in a simpler
format. Aim 1 entails optimization of processes for plasma separation, sample preparation, reverse transcription
coupled to isothermal loop-mediated amplification (RT-LAMP), and visual lateral flow-based detection. In Aim 2
we will develop a prototype cartridge and instrument that integrates and automates these processes. Using the
prototype device, we will demonstrate detection of all four DENV serotypes spiked into whole blood. Successful
completion of this project entails demonstrating that the device can detect DENV1-4 in blood with limit of
detection comparable to the FDA-cleared DENV1-4 real time RT-PCR assay developed by the Centers for
Disease Control and prevention (CDC). In future efforts, once feasibility has been established, we will advance
the initial prototype to a final version of the device, conduct full analytical validation and a field evaluation study,
with the goal to obtain FDA clearance. Our long term goal is also to expand the multiplexing capabilities to enable
differential diagnosis of Zika, dengue, and chikungunya viruses.
