New micromethod to study the effect of antimicrobial agents on Toxoplasma gondii: comparison of sulfadoxine and sulfadiazine individually and in combination with pyrimethamine and study of clindamycin, metronidazole, and cyclosporin A

Abstract

An in vitro method by which reagents, cells, and Toxoplasma gondii trophozoites are conserved (microniethod) was developed to quantitate the effect of antimicrobial agents on T. gondii. Sulfadoxine alone had no effect on T. gondii in vitro when evaluated with a macromethod, the new knicromethod, or visual inspection of Giemsa-stained preparations. Sulfadoxine combined with pyrimethamine inhibited T. gbndit more than did pyrimethamine alohe, but the combination of sulfadoxine plus pyrimethamine was slightly less active than was the combination of sulfadiazine plus pyrimethamine. Neither clindamycin nor metronidazole, alone or in combination with sulfadiazine or pyrimethamine and sulfadiazine, had any effect on intracellular T. gondii. Brief exposure (10 min before and during challenge) to elindamycin had no effect on extraceUular T. gondii when clindamycin was studied alone or with sulfadiazine or pyrimethamine plus sulfadiazine. Cyclosporin A inhibited T. gondii replication at concentrations of ca. -2 ,ug/ml.In vitro assays have been used to evaluate the effects of atitimicrobial agents on Toxoplasma gondii (2, 8, 9), but none of these methods have been designed to minimize the amounts of cells, T. gondii trophozoites, and reagents used. We developed such a system and then utilized this system to study the effects of a number of antimicrobial agents. Pyrimethamine and sulfadiazine were studied to determine whether the tnicromethod would demonstrate the inhibitory effects on 1. gondii that have been described clinically and for other in vitro and in vivo assay methods. Clindamycin was studied because of its efficacy in the treatment of toxoplasmosis in mouse and rabbit models (1, 7, 10), and metronidazole was studied because it is effective against another protozoan, Entamoeba histolytica. Sulfadoxine was studied because it has a half-life that is substantially longer than those of sulfadiazine and the triple sulfonamides and it is available in combination with pyrimethamine, which has a half-life of days. Cyclosporin A was studied because of its antimalarial effect in vitro and in vivo (11) (GIBCO, Grand Island, N.Y.) that contained 10% heatinactivated (60 tnin, 56°C) fetal calf serum (GIBCO), penicillin (100 U/ml), and streptomycin (100 ,ug/ml) (M199-FCS).(ii) Macromethod. A 0.5-ml amount of the cell suspension was placed into each well of 24-well plates (2 cm2; Linbro Scientific Inc., Hamden, Conn.) and four-chamber Lab-Tek slides (2 cm2; Miles, Naperville, Ill.) which were then * Corresponding author. incubated at 37°C for 1 to 4 h to permit macrophages to adhere. Nonadherent cells were then removed, and cultures were washed with Hanks balanced salt solution (GIBCO).(iii) Micromethod. A 0.1-ml amount of the cell suspension was placed into each well of 96-well plates (0.28 cm2; Linbro Scientific Inc.). Cultures were incubated for 1 to 4 h at 37°C before challenge. Nonadherent cells were not removed from the cultures because eliminating this step simplified performance of the assay and did not alter the resu...

Delivery of antimicrobials into parasites

“…gondii Parasites. Tachyzoites of the RH strain were maintained and used in experiments as described (12,13). GFP-, plastidlabeled RH strain parasites were kindly provided by B. Streipen and D. Roos (University of Pennsylvania, Philadelphia).…”

In vitro effects of artemisinin ether, cycloguanil hydrochloride (alone and in combination with sulfadiazine), quinine sulfate, mefloquine, primaquine phosphate, trifluoperazine hydrochloride, and verapamil on Toxoplasma gondii

“…Calcium and calmodulin antagonists have been shown to inhibit Plasmodium falciparum in vitro (21). We have shown previously that the calmodulin inhibitor cyclosporin A inhibits T. gondii in vitro (12). Verapamil was studied as the prototype calcium channel blocker, and trifluoperazine hydrochloride appears to be one of the more potent calmodulin inhibitors active against malaria (21).…”

“…Since toxic effects from this therapy include bone marrow suppression and allergy to sulfonamides (especially in AIDS patients), there is currently a great need for alternative, effective antimicrobial agents. Therefore, we evaluated in vitro several antimalarial agents, as well as the calcium channel blocker verapamil and the phenothiazine trifluoperazine hydrochloride by using our previously described assay (12).…”

Effect of clindamycin on pneumonia from reactivation of Toxoplasma gondii infection in mice

“…In contrast, clindamycin has not prevented the replication of T. gondii in tissue culture systems (14,20).…”

“…For example, a trial comparing the combination of clindamycin and pyrimethamine with the combination of sulfadiazine and pyrimethamine suggested that the efficacies of the two combinations were not significantly different (10). Clindamycin therapy has produced conflicting results in animal models of primary T. gondii infections (3,12,13,15,21,25) and in cell cultures challenged with T. gondii tachyzoites (14,20). Clindamycin has not been used to treat reactivated chronic infections in experimental animals.…”