Plasmodium falciparum: a simple, rapid method for detecting parasite clones in microtiter plates.

The genomic mapping of Plasmodium falciparum and the analysis of mutant parasites produced by genetic manipulation requires a rapid, sensitive method to identify selected parasite clones. This report describes the utilization of a previously described parasite lactate dehydrogenase (pLDH) assay (Makler et al. 1993) for this purpose. The cloning procedure generally consists of diluting the selected parasite populations with uninfected erythrocytes in 96-well plates at less than one infected erythrocyte per well. After a 3-week period, a Giemsa-stained smear is prepared from each well and examined to determine which wells contain parasites. Parasitemias lower than 0.1% are detectable using this method. While this method is sensitive, it is very time consuming, severely limiting the number of plates that can be processed at any time. A single 96-well plate can take at least a day to analyze by this method. Recently, an improved method for parasite detection has been described (Kirkman et al. 1996). It utilizes the change in color of the phenol red indicator of RPMI 1640 culture medium from red to orange–yellow when the parasitemia is greater than 0.5%. Although faster than analyzing Giemsastained smears, this method has some disadvantages. First, the parasites are cultured under harsh conditions, as the yellow color of the medium is an indication of a low pH (due to lactic acid production). Under conditions of low pH, parasites would be in a glucose-deprived state. Second, the method requires a skilled technician to detect the subtle color changes. Third, the method can only detect parasitemias in excess of 0.5% (Kirkman et al. 1996). A parasite-specific lactate dehydrogenase assay was first described by Makler et al. (1993). The pLDH assay is specific for the malarial parasite enzyme and does not detect erythrocyte LDH. It is based on the principle that malaria LDH can utilize 3-acetylpyridine NAD (APAD) as an NAD analogue, whereas erythrocyte LDH cannot (Makler and Hinrichs 1993). The assay reagents, nitroblue tetrozolium (NBT) and phenylethyl sulfate (PES), were obtained from Sigma (St. Louis, MO). The Malstat reagent was obtained from Flow, Inc. (Portland, OR). To perform the pLDH assay, 20 ml of infected erythrocytes (1% hematocrit) in each well of a 96-well plate was mixed with 100 ml of Malstat reagent, 10 ml of 1 mg/ml NBT, and 10 ml of 0.1 mg/ml PES for 30 min at 20°C. The reaction was stopped by the addition of 100 ml of 5% acetic acid to each well. The absorbance at 650 nm (A650) was read on a plate reader. The A650 has been shown to be proportional to the parasitemia detected by Giemsa-stained blood smears (Makler et al. 1993). To test the utility of the pLDH assay for the identification of malarial clones, P. falciparum (pyrimethamine-sensitive strain 3D7) were transfected by an adaptation of the method of Wu et al. (1995), with a DNA construct containing the Toxoplasma gondii dihydrofolate reductase/thymidylate synthase gene flanked by regulatory regions from the malaria knob-associated histidine-rich protein (KAHRP) (Lanzer et al. 1992). The cells were grown in the presence of 15 ng/ml pyrimethamine for 5 weeks, after which drugresistant parasites were isolated. These parasites were diluted and transferred to 96-well microtiter plates that were maintained in a low-oxygen atmosphere (1% O2, 5% CO2, 94% N2) using a Modular Chamber Incubator (Vanguard, Neptune, NJ). The medium was changed after 1 week. After another week (14 days postseeding), the pLDH assay was performed on each sample in the microtiter plates. Some of the wells were weakly positive (A650 ∼ 0.1). The medium was changed and the cells were allowed to grow for an additional week, after which a pLDH assay was performed. The results of a typical plate are shown in Fig. 1. The purple color was detectable without the use of the plate reader in the wells that were positive the previous week. Giemsastained smears of positive and negative wells confirmed that the purple color arose from parasitized erythrocytes. After 3 weeks, the wells had a parasitemia of approximately 1%. 1 To whom correspondence should be addressed. Fax: (215) 923-2218. E-mail: tarasch1@jeflin.tju.edu. EXPERIMENTAL PARASITOLOGY 86, 158–160 (1997) ARTICLE NO. PR974156