(306e) Malaria Smartphone Diagnostic Using Isothermal Amplification

Title: Malaria Smartphone Point-of-Care
Diagnostic Using Isothermal Amplification

Ashlee J. Colbert¹, Katherine N. Clayton^1,2,Tamara
L. Kinzer-Ursem¹, Jacqueline C. Linnes¹

¹Weldon
School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
47907 ²School of Mechanical Engineering, Purdue University, West
Lafayette, IN 47907

Malaria contributes to over 400,000 deaths a year.
Despite advances in technologies, developing countries are still endemic to
malaria. Advanced laboratory facilities in malaria endemic countries/regions
may be inaccessible, causing an increase in malaria related deaths.
Furthermore, many low resource areas cannot afford the necessary infrastructure
to maintain the equipment for malaria diagnoses. Indeed, there is a need for a
simple, robust point of care diagnostic for physicians to accurately diagnose
malaria. The current technologies for malaria detection include white light
microscopy, polymerase chain reaction (PCR), and rapid diagnostic tests (RDTs).
Microscopy is considered the gold standard method for detecting malaria, however
the technique is time consuming, requires stained blood smears, and advanced
microscopists. Microscopy is cheap and can determine the species of malaria
parasites. However, developing countries do not always have access to expert
microscopists and will falsely identify levels of parasites in the blood. On
the other hand, rapid diagnostic tests use antibodies for detection of malaria
antigens instead of DNA in blood and are easy to use. Yet, RDTs are inconsistent
and have low sensitivity when parasite levels are near 100 parasites/μl. Finally,
PCR methods have increased sensitivity, but are costly, time consuming, require
advanced laboratory equipment, and advanced scientific training for technicians.

We have designed a loop mediated isothermal
amplification (LAMP) technique for specific and robust detection of the malaria
strain, Plasmodium falciparum. LAMP uses six primers to displace and aid
in the exponential amplification of malaria DNA. The amplified blood sample is
placed into a small microfluidic chip containing 400 nm streptavidin coated fluorescent
beads. One of the six primers is biotinylated to bind the amplified products to
the beads. This chip is placed into our smartphone device where a thirty second
video is taken. The change in diffusivity of the particles in the presence of
LAMP amplification products is monitored by visually tracking the Brownian
motion of the beads via particle diffusometry (PD). PD diffusion coefficients decrease
when Brownian motion is hindered, indicating the presence of amplified target
DNA.

We have found that LAMP-PD with a smartphone device
can detect as low as 100 parasites/µl (corresponding to 0.0025% parasitemia) in
blood. The diagnostic device has also been tested against other infectious
disease DNA and was found to be specific to P. falciparum, one of
the deadliest strains of the malaria parasites.  The PD-LAMP smartphone
platform can detect malaria in an hour, making the technique promising for
sensitive and robust point-of-care malaria detection.

Figure 1.  PD-LAMP
is specific to malaria when tested against Chikungunya Virus and Dengue Virus (Type
3) at the same concentrations (top). A significant difference is shown between
positive samples and controls. There is no significant difference between
controls when using PD-LAMP. 2% agarose gel confirms LAMP amplicons are present
only for targeted malaria DNA (bottom).