Detecting Birefringence of Hemozoin to Diagnose MaIaria

Sheel M. Shah,
1
Andrew M. Davis,
1
Naman K. Shah,
2
Steven R. Meshnick
2,3
1
Department s of Physics,
2
Epidemiology,
3
Microbiology & mmunology, University of North Carolina, Chapel Hill, NC, USA
Malaria infects roughly half a billion people every year,
resulting in over a million deaths annually.
As a host of inexpensive drug therapies exist to treat malaria,
the key to survival is early diagnosis and treatment
7
.
Early diagnosis might be possible using the birefringence
property of hemozoin, a metabolic byproduct of the parasite.
We used a collimated polarized beam incident on an infected
sample to detect hemozoin with a polarizer completely out of
phase with the polarization of the beam before the CCD.
The increased sensitivity of the signal and ease of detection
lends itself to automated algorithms for diagnosis.
ABSTRACT
BACKGROUND
METHODS
CONCLUSIONS RESULTS
REFERENCES
ACKNOWLEDGEMENTS
Current Diagnosis Techniques Rapid Diagnostic Tests
(RDT) utilize antigen detection, but have variable sensitivity,
low shelf life, and high cost. Microscopy is limited by the
need for trained personnel to perform the test.
There is a need to develop an automated, high-throughput,
accurate, and inexpensive device that will diagnose malaria
rapidly and efficiently.
P.falciparum (3D7) parasites from in vitro culture were
reconstituted with packed RBC's. 30 L was dried on a slide
as a thick smear and methanol fixed for 30 seconds.
mages were taken using a Nikon TE2000 nverted
microscope with a 20x objective and a 10x eye piece.
mages were acquired using the DVC-1412 monochrome
CCD camera.
The collimated beam was polarized uniaxially and a
polarizer was inserted before the CCD in an orientation
completely out of phase with the polarization of the beam.
mage analysis was performed using software based on
mageJ (NH, Bethesda). Noise threshold levels were
calibrated using the images from the imaged P. falciparum
negative samples. The area used for particle counting was
based on the size of hemozoin
5
.
Annie Purfield and Jaina Patel for slide preparation and
maintaining parasite cultures. Dr. Micheal Chua and
Dr. Bob Bagnell for guidance in the microscopy
facilities. Dr. Robert Dennis of TESLa Laboratories for
providing engineer space and materials. Dr. Jungsang
Kim for general guidance with the project and feedback
of methods
1. Scott C, van Zyl D, Ho E, Meyersfeld D, Ruvio L, Mendelow B,
Coetzer T, 2003. Automated Detection of Malaria-Associated
ntraleucocytic Haemozoin by Cell-Dyn CD4000 Depolarization
Analysis. Clin Lab Haematol 25.2: 77--86.
2. de Langen A, van Dillen J, de Witte P, Muchetto S, Nagelkerke
N, Kager P, 2003. Detection of Malaria Pigment: Feasibility for
Malaria Diagnosing in an Area with Seasonal Malaria in
Northern Namibia. Trop Med Int Health 11.6: 809--816.
3. Noland G, Briones N, Sullivan D Jr, 2003. The Shape and Size
of Hemozoin Crystals Distinguishes Diverse Plasmodium
Species. Mol Biochem Parasitol 130.2: 91--99.
4. Lawrence C, 1999. Remembered: Malaria Haemozoin in
Leucocytes. Lancet 353.9167: 1852.
5. Ross N, Pritchard C, Rubin D, Dus A, 2006. Automated
mage Processing Method for the Diagnosis and Classification
of Malaria on Thin Blood Smears. Med Biol Eng Compu 44:
472--436.
Figure 1: Birefringent hemozoin superimposed on the same field as seen with ordinary
transmitted light.
Figure 2: nitial Device Prototype
Sensitivity of signal and ease of detection is greatly
increased with birefringence imaging
Based on the increased signal sensitivity of using
birefringent hemozoin
6
, images produced via hemozoin
birefringence could enable an automated, high-throughput,
low cost diagnosis device.
Diagnosis can be automated by translating a thick smear
sample using stepper motors and imaging random fields.
Figure 2 shows an image acquired over two channels. The
red channel carried the transmitted light image and the green
channel carried the polarized light image. The images were
superimposed using Adobe Photoshop
tm
.The hemozoin
crystals can be clearly seen as the green artifacts on the red
background. Machine detection of the presence of the
parasite in blood samples is greatly improved using
birefringence detection. We successfully imaged hemozoin
in samples with parasitemia as low as 0.1%.
Figure 2 shows the initial device prototype. The
detection of these crystals was easily automated using
few components:
Stepper motors and flexible drive shafts to provide
sample translation
An inexpensive microscope for imaging
Standard polarizing film.
FUTURE DIRECTIONS
Develop a full working prototype with images being
exported to image analysis software loaded onto blackfin
tm
(Analog Devices) Processor.
Determine correlation of the parasitemia level to hemozoin
density in the blood smear
Test the prototype with infected whole blood in the field.