IV Lab’s engineers and scientists are developing new malaria diagnostic techniques to support elimination and eventual eradication of malaria.

Malaria is a preventable and treatable parasitic disease, but diagnosing it can be a challenge in the developing countries where it’s most prevalent. Although diagnosis for case management is increasingly being provided by antigen-based rapid diagnostic tests (RDTs), the diagnostic needs for elimination and eventual eradication are much more demanding. A combination of performance, speed, cost per test, and ease-of-use, not currently available, will likely be required.  As part of IV’s Global Good program, IV Lab’s engineers and scientists seek to address this by developing new malaria diagnostic techniques.

One area of focus is in improving and simplifying the long-time gold standard in malaria diagnosis: optical microscopy. Giemsa smear microscopy was developed over a century ago and is still widely used to diagnose malaria. While it can be very effective, it is time consuming and prone to error in both the preparation and imaging of slides. We have developed a staining cartridge based on an alternate stain, Acridine Orange, which significantly simplifies sample preparation. While we continue to develop and test this cartridge, we are also exploring its utilization in a fully automated microscopy system.

Another area of interest is to develop a way to diagnose malaria through the detection of the malaria parasite’s waste product: hemozoin. We have explored various methods to detect hemozoin in infected red blood cells, including dark field cross polarization (DFxP) microscopy and nonlinear optical detection. One of the inherent challenges of working with hemozoin, however, is that the amount of hemozoin found in infected red blood cells varies during the different stages of the parasite’s 48-hour lifecycle. There is a latency between the time a parasite first invades a red blood cell and when the parasite starts to produce detectable quantities of hemozoin in its food vacuole. This presents a limit to using hemozoin to diagnose malaria, as often parasite ages are synchronized within an infected person. Because of this limitation, diagnosis based solely on detecting hemozoin in red blood cells is unlikely to achieve the sensitivity required to be a useful technique.

The images in the scroll bar above include thick and thin smear slides of plasmodium parasites used in the research and development of our Autoscope, an automated optical diagnostic microscope. For more information or to request access to additional images, please email M-Images@intven.com.

Publications:

Horning, Matthew P., Delahunt, Charles B., Sing S. Ryan, Garing Spencer H., and Nichols Kevin P. “A Paper Microfluidic Cartridge for Automated Staining of Malaria Parasites with an Optically Transparent Microscopy Window.” Lab on a Chip, 2014, 14 (12): p. 2040 - 2046

Delahunt ,Charles B., Horning, Matthew P., Wilson, Benjamin K., Proctor, Joshua L., and Hegg, Michael C. “Limitations of Haemozoin-based Diagnosis of Plasmodium falciparum Using Dark-field Microscopy.” Malaria Journal, 2014, 13:147 doi:10.1186/1475-2875-13-147

Wilson Benjamin K., Behrend Matthew R., Horning, Matthew P., and Hegg, Michael C. “Detection of Malarial Byproduct Hemozoin Utilizing its Unique Scattering Properties.” Optics Express, 2011, Vol. 19, Issue 13: p. 12190-12196

Publications

Published malaria diagnostics research.

Automated Staining
Hemozoin Diagnostic Limitations
Hemozoin Detection
A) A 3D view of cartridge showing blood being dispensed from the left, acridine orange dye filled paper where staining occurs, and the unobstructed, transparent coverslip region where imaging is conducted via microscopy. B) A top-down view of the cartridg

A Paper Microfluidic Cartridge for Automated Staining of Malaria Parasites with an Optically Transparent Microscopy Window

ABSTRACT EXCERPT: A paper microfluidic cartridge for the automated staining of malaria parasites (Plasmodium) with acridine orange prior to microscopy is presented. The cartridge enables simultaneous, sub-minute generation of both thin and thick smears of acridine orange stained parasites. Parasites are stained in a cellulose matrix, after which the parasites are ejected via capillary forces into an optically transparent chamber. The unique slanted design of the chamber ensures that a high percentage of the stained blood will be of the required thickness for a thin smear, without resorting to spacers or other methods that can increase production cost or require tight quality controls. smear region, containing multiple cell layers, for enhanced limit of detection.

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Typical DF haemozoin and non-haemozoin objects. Typical objects of interest. A: Haemozoin objects, 100× BF thumbnails (top row) and the corresponding 50× DF thumbnails (second row) showing the haemozoin within the parasites. (1–4) are rings,

Limitations of Haemozoin-based Diagnosis of Plasmodium falciparum Using Dark-field Microscopy

ABSTRACT EXCERPT: The algorithm distinguishes hemozoin from non-hemozoin objects in dark-field images with an object-level sensitivity of 95% and specificity of 97%. Ring stages older than about 6 hours begin to show detectable hemozoin, and rings between 10–16 hours reliably contain detectable hemozoin. However, dark-field microscopy coupled with the image-processing algorithm detect no hemozoin in rings younger than six hours.

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(a) Illustration of the selective capture of hemozoin scattering. (b) schematic of bench-top dark-field microscope used for variable aperture testing. VDFS = variable dark-field stop, PBS = polarizing beam splitter, BFP = objective back focal plane.

Detection of Malarial Byproduct Hemozoin Utilizing its Unique Scattering Properties

ABSTRACT: The scattering characteristics of the malaria byproduct hemozoin, including its scattering distribution and depolarization, are modeled using Discrete Dipole Approximation (DDA) and compared to those of healthy red blood cells. Scattering (or dark-field) spectroscopy and imaging are used to identify hemozoin in fresh rodent blood samples. A new detection method is proposed and demonstrated using dark-field in conjunction with cross-polarization imaging and spectroscopy. SNRs greater than 50:1 are achieved for hemozoin in fresh blood without the addition of stains or reagents. The potential of such a detection system is discussed.

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