A simple and inexpensive haemozoin-based colorimetric method to evaluate anti-malarial drug activity

Chloroquine diphosphate, quinine sulphate, primaquine, clotrimazole, haemin chloride (haem), RPMI 1640 medium, hypoxanthine, and gentamycin were purchased from Sigma Aldrich Chemical Company (Tokyo, Japan). Albumax II (Gibco), and the other chemicals used in the present study were of a high grade. Plasmodium falciparum K1 (chloroquine resistant) and 3D7-9A (chloroquine susceptible) strains [23] were provided from Dr Osamu Kaneko and Dr Shusuke Nakazawa, respectively, from the Institute of Tropical Medicine, Nagasaki University, Japan.

Plasmodium falciparum K1 and 9A strains were maintained in vitro with continuous culture according to a previously described method with a slight modification [24]. The culture medium consisted of RPMI 1640 supplemented with 0.025 mg/ml gentamicin, 0.01 mM hypoxanthine, 23.8 mM NaHCO₃, 11 mM glucose and 0.5% albumax II, and adjusted to a pH of 7.3 to 7.4. The parasite was cultured and maintained in a tissue culture flask with complete culture medium containing 5% human erythrocytes. The parasite density was maintained at about 1.5% parasitaemia under an atmosphere of an AnaeroPack sachet (Mitsubishi Gas Chemical Company Inc, Tokyo, Japan) to create 20% CO₂ and remove O₂ (<0.1%) 37°C [25]. Every two days, infected erythrocytes were transferred into fresh medium containing 5% human erythrocyte. The level of parasitaemia was determined by light microscopy on a Giemsa-stained thin blood smear, and parasitized erythrocytes were diluted when parasitaemia was higher than 5% in erythrocytes contained at 5% in culture medium, in order to lower parasitaemia and allow continuous growth. Parasite culture was diluted with fresh uninfected erythrocytes and culture medium to achieve a starting parasitaemia of 2% and a haematocrit of 5%. This final parasite culture was immediately used for anti-malarial assay.

A culture of the P. falciparum K1 strain was serially diluted with uninfected erythrocytes in complete medium to yield a haematocrit of 5% and parasitaemia ranging from 0 to 10%. The serial culture containing 200 μl was prepared independently in triplicate in microtubes, followed by the addition of 800 μl of 2.5% sodium dodecyl sulphate in 0.1 M sodium bicarbonate pH 8.8, then the samples were mixed at room temperature for 15 min. After centrifugation at 13,000 rpm for 10 min, the supernatant was removed. The pellet was washed twice with 800 μl of 2.5% sodium dodecyl sulphate in 0.1 M sodium bicarbonate ₍pH 8.8), then 200 μl of 5% sodium dodecyl sulphate was added to 50 mM NaOH to convert the haemozoin into haem. After incubation at room temperature for 30 min, the sample (200 μl) was transferred to a 96-well microplate and scanned at 405/750 nm (A_{405 nm} minus A_{750 nm}) using an IMark microplate reader (Bio-Rad). After the background absorbance of haemozoin was purified of uninfected erythrocytes (5% haematocrit) then subtracted, the amount of haemozoin in the infected erythrocytes was presented as the absorbance at 405/750 nm and then plotted against parasitaemia.

The P. falciparum K1 and 9A strains were used to evaluate the anti-malarial activity of quinine, chloroquine, pyrimethamine, artemisinin and clotrimazole by using the haemozoin-based spectrophotometric method. Stocks of drugs were prepared in dimethyl sulphoxide or phosphate buffer saline (for chloroquine) and were then serially diluted with complete culture medium. To each well of a microplate, 10 μl of serially diluted drug solution was added into 200 μl of final asynchronous parasite culture. Dimethyl sulphoxide or phosphate buffer saline were also tested by adding a similar amount to control wells. The microplates were cultured 72 hr under the conditions described above. The haemozoin of infected erythrocytes was extracted, purified, and quantified, as described above. The 50% inhibitory concentration (IC₅₀) value was calculated by non-linear fitting of the absorbance at 405/750 nm against the logarithm of the drug concentration using the GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Sigmoidal doses with variable slope models were used with the following equation:

where y is the absorbance at 405/750 nm; x is the logarithm of the drug concentration, min is the absorbance at 405/750 nm measured at time zero (starting point of assay), and max is the maximal absorbance of a particular drug. The Hill slope is the steepness of the curve. The logarithm of the concentration at zero was defined at 2 log lower than the lowest concentration of a particular drug.

The relationship between the haemozoin amount in a parasitized erythrocyte and parasitaemia was revealed by measuring the absorbance of the haem content of the haemozoin obtained from the parasitized erythrocytes after degradation to monomer haem. As shown in Figure 1, the absorption of converted haem showed a direct and linear correlation with the level of parasitaemia. Low and unsynchronized parasitaemia (about 1%) could be detected using this haemozoin-based colorimetric method. The results indicate that this novel assay of parasites is applicable for monitoring parasite growth and for screening new anti-malarial compounds.

The IC₅₀ values of some anti-malarial drugs were determined with a dose-response experiment using a haemozoin-based colorimetric assay. The result showed that increasing the concentration of anti-malarial drugs resulted in a decreased absorbance at 405/750 nm (Figure 2). The data was best fitted by a typical sigmoidal dose-response model with a variable slope (four parameters) that agreed well with previous reports [26, 27].

Table 1 summarizes the results of the assay to determine IC₅₀ using the haemozoin-based colorimetric method. The P. falciparum K1 strain was primarily observed for quinine, chloroquine, clotrimazole, pyrimethamine and artemisinin, with IC₅₀ values of 0.258, 0.873, 0.805, 23.03, and 0.139 μM, respectively. For the P. falciparum 9A strain, pyrimethamine was not evaluated, and the IC₅₀ values were 0.398, 0.132, 1.67 and 0.483 μM in succession for quinine, chloroquine, clotrimazole and artemisinin, respectively. The IC₅₀ values for quinine, chloroquine, clotrimazole, and pyrimethamine were in a range that was similar to those observed in previous reports [28, 29]. On the other hand, the IC₅₀ values for artemisinine were higher than previous reports, probably due to asynchronous cultures, ring stage-specific target of artemisinine, accumulation of released hemozoin in the continuous cultures, or several rounds of continuous cultures and cloning of parasite strains in our laboratories. Therefore, further studies are required to compare the novel assay with recent developed methods to validate the accuracy in the screening new antimalarial compounds [30]. Another limitation of the novel method is that it is not easily adaptable for a high throughput screening of anti-malarial drug candidates, which is under-developed using 96-well filter plates [31].

In recent years, the number of laboratories, diagnosis centres and research institutes has risen in developing countries. However, most of them lack the modern equipment and expensive chemicals to apply new methods for screening anti-malarial candidates. In addition, some methods have potential risks of toxicity, so it is prudent to wear disposable gloves at all times when proceeding. Another obstacle is that many laboratories lack the facilities to treat toxic contamination before the toxin is discarded in the environment. The novel anti-malarial assay is safe, non-expensive and easy to apply in laboratories.