The online version of this article (doi:10.1186/1475-2875-11-267) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
DL and EG carried out the experiments. RS prepared all cell media and reagents for the experiments. EG conceived the idea of using THG by haemozoin to reduce parasitaemia in malaria. EG has previously worked on the related technique of photodynamic therapy (PDT). All authors have read and approved the final version of the paper.
Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite’s food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated.
Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU).
Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with ~4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (~2-log reduction). A ~1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 μM synthetic haemozoin and a ~2-log reduction in the presence of 10 μM haemozoin. Minimal (≤15%) cell kill was observed in the presence of 10 μM haemin.
Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient’s blood through the skin or through dialysis tubing with a NIR laser.
Additional file 1: Theory of third harmonic generation (THG). Light propagating through a vacuum will obey the principle of superposition, however this is not generally true for light propagating through condensed media. (DOCX 17 KB)12936_2012_2200_MOESM1_ESM.docx
Additional file 2: Black-box diagram for third harmonic generation. (top panel) A black-box diagram illustrating the principle of THG. ωo is the fundamental laser frequency. (bottom panel) Optical transitions between the different energy levels are indicated by gray arrows. The darker the shade of the arrow the larger the probability of that transition. The frequency of light emitted by THG is 3ωo. NLOC, non-linear optical crystal. (PDF 165 KB)12936_2012_2200_MOESM2_ESM.pdf
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Keller CC, Kremsner PG, Hittner JB, Misukonis MA, Weinberg JB, Perkins DJ: Elevated nitric oxide production in children with malarial anemia: haemozoin-induced nitric oxide synthase type 2 transcripts and nitric oxide in blood mononuclear cells. Infect Immun. 2004, 72: 4868-4873. PubMedCentralCrossRefPubMed
WHO: Guidelines for the treatment of malaria. 2010, World Health Organization, Geneva, 2
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