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Honing the Sensitivity of Malaria Diagnosis

By: Carol A. Rouzer, VICB Communications
Published: May 6, 2014

A simple, inexpensive sample processing device markedly improves the performance of rapid diagnostic tests used to diagnose malaria.

A major challenge facing the world-wide effort to eliminate malaria is the need for rapid, sensitive, and inexpensive diagnostic tests that can be used at the point of patient care. Although malaria is a common cause of illness with fever in areas such as sub-Saharan Africa, it is not the major cause. Yet, patients are frequently diagnosed and treated for malaria on the basis of symptoms alone. Consequently, many are treated unnecessarily, wasting resources, while at the same time not receiving appropriate care for the real cause of their fever. Alternatively, others who truly have malaria may be misdiagnosed and not receive the therapy they need. The gold standard for malaria diagnosis, microscopic examination of a stained blood smear, is not practical in many areas where the disease is prevalent. A potential answer to this problem is the rapid diagnostic test (RDT), a relatively inexpensive device that can be used by minimally trained caregivers at the patient’s bedside. However, experience with RDTs has led to the conclusion that they can be unreliable and often lack adequate sensitivity to detect asymptomatic carriers of the malaria parasite who must be found and treated if the disease is to be eliminated. Now, Vanderbilt Institute of Chemical Biology member David Wright, his collaborator Rick Haselton (Department of Biomedical Engineering), and their laboratories present a simple and economical way to improve the performance of malaria RDTs in the field [K.M. Davis, L.E. Gibson, et al. (2014) Analyst, published online April 17, doi:10.1039/c4an00338a].

Figure 1. Example of how a typical RDT detects malaria. The RDT uses an antibody that detects a protein from the malaria parasite. The antibody is labeled with a colored tag and bound to one end of a nitrocellulose strip. The blood sample is added to this end of the strip, which also contains an agent that lyses the red blood cells, releasing parasite proteins. The protein binds to the tagged antibody. Then a buffer is added to elute the antibody-protein complex along the strip. A second unlabeled antibody that also recognizes the malaria protein is present about half-way up the strip. It traps the tagged antibody-protein complex, which is visible as a colored line due to the tag on the first antibody. A second tagged antibody serves as a control and is trapped at a second line further along the strip. Image reproduced by permission from Macmillan Publishers Ltd, from D. Bell, et al. (2006) Nat. Rev. Microbiol., 4, 682, copyright 2006.

Most RDTs are lateral immunochromatographic devices that detect the presence of a malaria protein using an antibody labeled with a colored tag (Figure 1). There are currently 200 different types of RDTs sold under 60 different brand names, but only about 10% of these can detect the levels of parasitemia (≤200 parasites/μL blood) that are found in asymptomatic carriers. The protein most commonly detected by commercial RDTs is the histidine-rich protein 2 from the malaria parasite Plasmodium falciparum (pfHRP2). In previous work (K. M. Davis, et al. (2012) Anal. Chem., 84, 6136), the Wright and Haselton groups developed a highly effective and inexpensive device to capture and concentrate pfHRP2 from blood samples. This device (Figure 2) consists of a piece of Tygon tubing about 24 cm in length and 1.6 mm in diameter containing, in succession, three 100 μL aliquots of wash buffer and one 10 μL aliquot of elution buffer separated by 25 μL “valves” of mineral oil. An Eppendorf tube attached to one end of the device serves as a chamber in which 200 μL of blood is mixed with magnetic nickel nitrilotriacetic acid agarose particles. pfHRP2 in the blood sample binds to the magnetic particles through a strong interaction between nickel nitriloactetic acid and histidine residues in the protein. Then, a donut magnet is used to trap and move the magnetic particles along the Tygon tubing, sequentially exposing them to each of the wash buffers and finally the elution buffer, which frees the protein from the particles. The now purified and concentrated sample of pfHRP2 is ready for detection by an RDT device.


Figure 2. Diagram of the pfHRP2 extraction device. A blood sample is combined with the nickel nitriloacetic acid (Ni(II)NTA) magnetic particles in the incubation chamber. A donut magnet surrounding the device moves the particles along the tubing through each wash solution to remove nonspecific contaminants. Movement into the elution chamber frees the protein for analysis in an RDT device. K. M. Davis, et al. (2012) Anal. Chem., 84, 6136. Copyright 2012, American Chemical Society.

The prior work had shown that the pfHRP2 extraction device substantially improved the performance of a commercially available RDT. To confirm this finding, the investigators selected six different RDTs that exhibited a range of performance characteristics from excellent to poor in World Health Organization (WHO) tests. They prepared pooled blood samples containing malaria parasites at six different levels of parasitemia from 0 to 200 parasites/μL, and examined the ability of each RDT to detect the parasites in samples of unextracted blood and samples of concentrated pfHRP2 obtained following processing through their extraction device. The results (Figure 3) showed that prior extraction substantially improved the ability of all devices to detect low levels of the parasite. For example, only one device was capable of detecting 12.5 and 25 parasites/μL in unextracted blood, while 5 out of 6 of the devices exhibited this level of sensitivity for the extracted samples. In fact, prior extraction increased the signal generated by samples containing 200 parasites/μL by 4- to 13-fold and improved the limit of detection of the devices by a similar amount.

Figure 3. The number of RDT tests that detected pfHRP is plotted against the concentration of parasites per microliter of blood in the sample. Data are provided for unextracted blood (green) and blood processed through the extraction device. Note that a much higher number of RDTs gave positive tests when the blood had been previously extracted.

To determine if variations between blood samples would affect the performance of the extraction device, the investigators acquired blood from five different donors and spiked the samples with P. falciparum at levels of 10 and 100 parasites/μL. They processed the samples through the extraction device and then compared the ability of three selected RDTs to detect the presence of the parasite in the processed samples versus unextracted blood. For this experiment, the researchers chose one RDT that was highly rated by the WHO, one that was rated in the middle range, and one that was poorly rated. They discovered that none of the RDTs could detect 10 parasites/μL in unprocessed blood, while the most highly rated RDT could detect 100 parasites/μL in all five blood samples, and the moderately rated and poorly rated RDTs detected the parasites in 3 and 0 of the 100 parasites/μL samples, respectively. In contrast, all three RDTs detected 100 parasites/μL in every extracted blood sample, and the most highly rated and moderately rated RDTs detected 10 parasites/μL in 5 and 4 of the extracted samples, respectively. The most poorly rated RDT did not detect 10 parasites/μL in any of the extracted samples. Importantly, there was no obvious effect of blood sample source on the ability of the extraction device to improve RDT performance.

Together the data confirm that processing blood samples with the pfHRP2 extraction device substantially improves the performance of even the least sensitive and least reliable RDTs. Use of the extraction device promises to increase the reliability of RDT-based diagnosis, especially with regard to reducing false negative results. These findings are particularly important with regard to the detection of asymptomatic carriers of P. falciparum, who must be identified and treated in order to eliminate the parasite from the human population. Recently, members of the Wright and Haselton groups tested the effectiveness of a modified version of their extraction device in the mountains of Haiti (Figure 4). The results of these studies bring the benefits of their process one step closer to use in the clinic.

Figure 4.  Wright/Haselton lab members Nick Adams, Keersten Davis, and Phillip Budge with village children in Gobert, Haiti. Image kindly provided by David Wright.






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