Technology Corner
Gazing at the Crystal Rods of Malaria Molly Webster1 and Vikram Sheel Kumar2*
Hemozoin, the black– brown crystalline byproduct from a parasite’s digestion of hemoglobin, aided the detection of malaria over a century ago when Ronald Ross won a Nobel Prize for his discovery of the transmission of malaria. Hemozoin was useful back then because little was understood of the parasite and hemozoin could be seen without a stain. For years the gold standard for diagnosing malaria has been simple microscopy that, while cheap, remains labor-intensive and requires significant training and experience. Rapid diagnostic tests have emerged for fast and easy use, but as Dr. David Sullivan of the John Hopkins Malaria Research Institute remarked in an interview to us, “There are disadvantages to tools on the market, or we wouldn’t be looking for new ones; if they were perfect, we’d stick just to them.” “The outdated idea that any febrile patient from a malaria endemic area should be treated with antimalarials has been replaced with the recommendation to test each patient with a rapid diagnostic test (when available),” explains Dr. Jeffrey D. Dvorin of the Division of Infectious Diseases at Boston Children’s Hospital. “Furthermore, a recent investigation of the etiology of fevers in a malaria endemic region of Tanzania demonstrated that malaria was not the cause of illness in the majority of patients. Although vaccine development and new antimalarial discovery remain important, accurate diagnostic tests that are field deployable (and cheap) are critical for malaria eradication,” he says. Available technologies may be limited in their ability to detect parasite levels below 200 parasites/L of blood. In the quest for a more sensitive test and better tools to exploit its physical properties, hemozoin is seeing a resurgence in its diagnostic applications. One diagnostic approach, using magneto-optics, can operate on batteries and be manufactured at a low cost, giving it the potential for use in resource-poor areas where the problem of malaria is most profound.
Science writer and producer, Brooklyn, NY; 2 Boston Children’s Hospital, Boston, MA. * Address correspondence to this author at: 390 Commonwealth Ave., Apt. 605, Boston, MA 02215. E-mail [email protected]. Received June 27, 2014; accepted July 3, 2014. © 2014 American Association for Clinical Chemistry
What Is the Innovation? Hemozoin is paramagnetic, so when an external magnetic field is applied, its crystals align. This property has been used to concentrate erythrocytes containing malarial parasites and to improve detection (1 ). Because of its crystal structure and a phenomenon known as optical dichroism, hemozoin also absorbs light John Lewandowski along its longer axis. In a cycling magnetic field, crystals that are suspended in blood will alternate between a random distribution that permits incident light to transmit freely, and an alignment perpendicular to the magnetic field lines that blocks the transmitted light in proportion to the amount of crystals present. Because the rod shape of the crystals is being detected, this approach is specific and less susceptible to confounding from other diseases that may produce hemozoin. This simple principle is the basis for the magnetooptical devices being developed for malaria. In 2008, Dr. Dave Newman from the University of Exeter in the United Kingdom developed the first such device (2, 3). More recently, John Lewandowski and his advisor Dr. Brian Grimberg at Case Western Reserve University developed a magneto-optical diagnosDr. David Sullivan tic called the rapid assessment of malaria (RAM)3 device (4 ) that, according to Lewandowski, has “different optical and magnetic configurations, yielding potentially very large cost and sensitivity differences and advantages.”
1
3
Nonstandard abbreviations: RAM, rapid assessment of malaria; RBC, red blood cell.
Clinical Chemistry 60:10 (2014) 1353
Technology Corner
Fig. 1. A prototype of the RAM device.
“Our innovation is not the biomarker itself. It is its reading and manipulation in an inexpensive and effective way,” says Lewandowski, who is now a graduate student in mechanical engineering at the Massachusetts Institute of Technology. The major components of the RAM include a standard laser diode, polarizer, and beamsplitter, with signal processing on a circuit board (Fig. 1). The design of the device with effectively “off-the-shelf” components should allow it to leverage reducing costs for these components, which are widely used in consumer electronics such as DVD players. The device, which Lewandowski believes can be manufactured for close to $50, has a consumable plastic sample holder that would cost a penny or two at scale. “We have the razor-blade model where the consumable amortizes the cost of the device,” he says. Where Can This Technology Fit in the Clinic? “The benefits of an accurate, sensitive, and cheap malaria diagnostic test are that antimalarial therapies can 1354 Clinical Chemistry 60:10 (2014)
be smartly deployed and that other significant causes of fever are not missed in patients,” says Dvorin. Early ring forms of malaria have small amounts of hemozoin, raising doubts that hemozoin-based detection would be able to pick up the earliest of infections. Sullivan is hopeful. He is also a self-identified member of the hemozoin choir and has been involved in the development of a noninvasive diagnostic that uses nearinfrared picosecond laser pulses to generate a hemozoin vapor “nanobubble” that can be detected through its acoustic signals (5 ). He and his colleagues were able to reliably distinguish ring-stage red blood cells (RBCs) from uninfected RBCs “despite the very small size of hemozoin, 50 –100 nm.” “If this technology can be made cheap and field deployable,” says Dvorin, “I think that it might really change the field of malaria diagnostics.” That technology depends on microlasers that at this point are costlier than the RAM components. Regarding the ability of the RAM to work at the early spectrum of the disease, Sullivan says, “It is possible to capture hemozoin in the ring stage, and optimize the de-
Technology Corner vice.” “Theoretically, there is no minimum crystal size that we could detect given further development and better signal processing,” says Lewandowski. “Practically and shown in the laboratory, the single crystal that we have been able to show so far is on the order of 100 nm.” In a patient study, the RAM was able to diagnose malaria at levels down to 39 parasites/L of blood within 1 min, and showed a sensitivity of 97% and specificity of 81% compared to PCR. “A lot of people are looking at fish in a bucket, or fish in a barrel—patients with very high parasitemia,” stresses Sullivan. Performance at a level of 39 parasites/L means that there needs to be 39 000 parasitized cells in a millimeter of blood for detection. “The need is low density, 1 to 10 parasites/L,” says Sullivan. PCR is able to perform at that level. “We can get down to that level and be a consistent indicator,” says Lewandowski. “The current version is not quite there, but could get there. I am cautiously optimistic,” says Sullivan.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
References 1. Nalbandian RM, Sammons DW, Manley M, Xie L, Sterling CR, et al. A molecular-based magnet test for malaria. Am J Clin Pathol 1995;103:57– 64. 2. Newman DM, Heptinstall J, Matelon RJ, Savage L, Wears ML, Beddow J, et al. A magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data. Biophys J 2008;95:994 –1000. 3. Mens PF, Matelon RJ, Nour BYM, Newman DM, Schallig H. Laboratory evaluation on the sensitivity and specificity of a novel and rapid detection method for malaria diagnosis based on magneto-optical technology (MOT). Malar J 2010;9:207. 4. Lewandowski JR, Condit WC, Deissler RJ, Bihary RF, Lewandowski MR, Jones JE, et al. Rapid detection of malaria infections with low-cost magneto-optical device. Poster presented at: 62nd Annual Meeting of the American Society of Tropical Medicine and Hygiene; 2013 Nov 13–17; Washington, DC. Deerfield (IL): American Society of Tropical Medicine and Hygiene; 2013. 5. Lukianova-Hleb EY, Campbell KM, Constantinou PE, Braam J, Olson JS, Ware RE, et al. Hemozoin-generated vapor nanobubbles for transdermal reagentand needle-free detection of malaria. Proc Natl Acad Sci U S A 2014; 111:900 –5.
DOI: 10.1373/clinchem.2013.218248
Clinical Chemistry 60:10 (2014) 1355
Gazing at the Crystal Rods of Malaria Molly Webster1 and Vikram Sheel Kumar2*
Hemozoin, the black– brown crystalline byproduct from a parasite’s digestion of hemoglobin, aided the detection of malaria over a century ago when Ronald Ross won a Nobel Prize for his discovery of the transmission of malaria. Hemozoin was useful back then because little was understood of the parasite and hemozoin could be seen without a stain. For years the gold standard for diagnosing malaria has been simple microscopy that, while cheap, remains labor-intensive and requires significant training and experience. Rapid diagnostic tests have emerged for fast and easy use, but as Dr. David Sullivan of the John Hopkins Malaria Research Institute remarked in an interview to us, “There are disadvantages to tools on the market, or we wouldn’t be looking for new ones; if they were perfect, we’d stick just to them.” “The outdated idea that any febrile patient from a malaria endemic area should be treated with antimalarials has been replaced with the recommendation to test each patient with a rapid diagnostic test (when available),” explains Dr. Jeffrey D. Dvorin of the Division of Infectious Diseases at Boston Children’s Hospital. “Furthermore, a recent investigation of the etiology of fevers in a malaria endemic region of Tanzania demonstrated that malaria was not the cause of illness in the majority of patients. Although vaccine development and new antimalarial discovery remain important, accurate diagnostic tests that are field deployable (and cheap) are critical for malaria eradication,” he says. Available technologies may be limited in their ability to detect parasite levels below 200 parasites/L of blood. In the quest for a more sensitive test and better tools to exploit its physical properties, hemozoin is seeing a resurgence in its diagnostic applications. One diagnostic approach, using magneto-optics, can operate on batteries and be manufactured at a low cost, giving it the potential for use in resource-poor areas where the problem of malaria is most profound.
Science writer and producer, Brooklyn, NY; 2 Boston Children’s Hospital, Boston, MA. * Address correspondence to this author at: 390 Commonwealth Ave., Apt. 605, Boston, MA 02215. E-mail [email protected]. Received June 27, 2014; accepted July 3, 2014. © 2014 American Association for Clinical Chemistry
What Is the Innovation? Hemozoin is paramagnetic, so when an external magnetic field is applied, its crystals align. This property has been used to concentrate erythrocytes containing malarial parasites and to improve detection (1 ). Because of its crystal structure and a phenomenon known as optical dichroism, hemozoin also absorbs light John Lewandowski along its longer axis. In a cycling magnetic field, crystals that are suspended in blood will alternate between a random distribution that permits incident light to transmit freely, and an alignment perpendicular to the magnetic field lines that blocks the transmitted light in proportion to the amount of crystals present. Because the rod shape of the crystals is being detected, this approach is specific and less susceptible to confounding from other diseases that may produce hemozoin. This simple principle is the basis for the magnetooptical devices being developed for malaria. In 2008, Dr. Dave Newman from the University of Exeter in the United Kingdom developed the first such device (2, 3). More recently, John Lewandowski and his advisor Dr. Brian Grimberg at Case Western Reserve University developed a magneto-optical diagnosDr. David Sullivan tic called the rapid assessment of malaria (RAM)3 device (4 ) that, according to Lewandowski, has “different optical and magnetic configurations, yielding potentially very large cost and sensitivity differences and advantages.”
1
3
Nonstandard abbreviations: RAM, rapid assessment of malaria; RBC, red blood cell.
Clinical Chemistry 60:10 (2014) 1353
Technology Corner
Fig. 1. A prototype of the RAM device.
“Our innovation is not the biomarker itself. It is its reading and manipulation in an inexpensive and effective way,” says Lewandowski, who is now a graduate student in mechanical engineering at the Massachusetts Institute of Technology. The major components of the RAM include a standard laser diode, polarizer, and beamsplitter, with signal processing on a circuit board (Fig. 1). The design of the device with effectively “off-the-shelf” components should allow it to leverage reducing costs for these components, which are widely used in consumer electronics such as DVD players. The device, which Lewandowski believes can be manufactured for close to $50, has a consumable plastic sample holder that would cost a penny or two at scale. “We have the razor-blade model where the consumable amortizes the cost of the device,” he says. Where Can This Technology Fit in the Clinic? “The benefits of an accurate, sensitive, and cheap malaria diagnostic test are that antimalarial therapies can 1354 Clinical Chemistry 60:10 (2014)
be smartly deployed and that other significant causes of fever are not missed in patients,” says Dvorin. Early ring forms of malaria have small amounts of hemozoin, raising doubts that hemozoin-based detection would be able to pick up the earliest of infections. Sullivan is hopeful. He is also a self-identified member of the hemozoin choir and has been involved in the development of a noninvasive diagnostic that uses nearinfrared picosecond laser pulses to generate a hemozoin vapor “nanobubble” that can be detected through its acoustic signals (5 ). He and his colleagues were able to reliably distinguish ring-stage red blood cells (RBCs) from uninfected RBCs “despite the very small size of hemozoin, 50 –100 nm.” “If this technology can be made cheap and field deployable,” says Dvorin, “I think that it might really change the field of malaria diagnostics.” That technology depends on microlasers that at this point are costlier than the RAM components. Regarding the ability of the RAM to work at the early spectrum of the disease, Sullivan says, “It is possible to capture hemozoin in the ring stage, and optimize the de-
Technology Corner vice.” “Theoretically, there is no minimum crystal size that we could detect given further development and better signal processing,” says Lewandowski. “Practically and shown in the laboratory, the single crystal that we have been able to show so far is on the order of 100 nm.” In a patient study, the RAM was able to diagnose malaria at levels down to 39 parasites/L of blood within 1 min, and showed a sensitivity of 97% and specificity of 81% compared to PCR. “A lot of people are looking at fish in a bucket, or fish in a barrel—patients with very high parasitemia,” stresses Sullivan. Performance at a level of 39 parasites/L means that there needs to be 39 000 parasitized cells in a millimeter of blood for detection. “The need is low density, 1 to 10 parasites/L,” says Sullivan. PCR is able to perform at that level. “We can get down to that level and be a consistent indicator,” says Lewandowski. “The current version is not quite there, but could get there. I am cautiously optimistic,” says Sullivan.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
References 1. Nalbandian RM, Sammons DW, Manley M, Xie L, Sterling CR, et al. A molecular-based magnet test for malaria. Am J Clin Pathol 1995;103:57– 64. 2. Newman DM, Heptinstall J, Matelon RJ, Savage L, Wears ML, Beddow J, et al. A magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data. Biophys J 2008;95:994 –1000. 3. Mens PF, Matelon RJ, Nour BYM, Newman DM, Schallig H. Laboratory evaluation on the sensitivity and specificity of a novel and rapid detection method for malaria diagnosis based on magneto-optical technology (MOT). Malar J 2010;9:207. 4. Lewandowski JR, Condit WC, Deissler RJ, Bihary RF, Lewandowski MR, Jones JE, et al. Rapid detection of malaria infections with low-cost magneto-optical device. Poster presented at: 62nd Annual Meeting of the American Society of Tropical Medicine and Hygiene; 2013 Nov 13–17; Washington, DC. Deerfield (IL): American Society of Tropical Medicine and Hygiene; 2013. 5. Lukianova-Hleb EY, Campbell KM, Constantinou PE, Braam J, Olson JS, Ware RE, et al. Hemozoin-generated vapor nanobubbles for transdermal reagentand needle-free detection of malaria. Proc Natl Acad Sci U S A 2014; 111:900 –5.
DOI: 10.1373/clinchem.2013.218248
Clinical Chemistry 60:10 (2014) 1355
