Sensitive in vitro system to assess morphological and biochemical effects of praziquantel and albendazole on Taenia solium cysts

Abstract

Neurocysticercosis resulting from Taenia solium infections is a major cause of adult-acquired seizures worldwide. Disease is caused by larval cysts, and treatment consists of the anthelmintic drugs albendazole or praziquantel. There are no standard methods to assess drug activity to T. solium cysts in vitro. Morphological, functional, and biochemical changes that might reflect damaging (inhibiting, cytotoxic) drug effects were analyzed after exposure of cysts to albendazole sulfoxide (ABZ-SO), the major active metabolite of the drug in vivo, praziquantel (PZQ), or combinations of both. PZQ exposure led to a decrease in cyst size and inhibition of evagination, whereas ABZ-SO exposure resulted in minimal changes. Alkaline phosphatase (AP) is normally secreted by cysts, and both drugs inhibited AP secretion at concentrations of 5 and 50 ng/ml for PZQ and ABZ-SO, respectively. Some combinations of both drugs resulted in additive and/or synergistic activities. Parasite-specific antigen, detected in the cerebrospinal fluid and blood of infected patients, is also normally secreted by T. solium cysts. Antigen secretion was similarly inhibited by ABZ-SO and PZQ and a combination of both drugs, suggesting that inhibition of secretion is a common downstream consequence of the activities of both drugs. These studies establish quantitative methods to measure in vitro anthelmintic activity and suggest combination therapy with ABZ-SO and PZQ may have clinical benefit.

FIG. 1.

In vitro bile-stimulated evagination of cysts. Cysts of T. solium were cultured in triplicate wells for 6 days (see Materials and Methods), and on each of the indicated days (x axis), the percentage of cysts that evaginated after exposure to bile for 18 h was determined. The graph depicts the total percentage of cysts that evaginated on the indicated day of culture, a sum of spontaneously evaginated and bile-induced evagination, in control medium (RPMI medium alone), RPMI with DMSO (0.00005%), or ethanol (0.00025%), corresponding to the concentration of solvents used for dissolution of ABZ-SO (DMSO) or PZQ (ethanol). Each data point represents the mean of triplicate wells, and the bars indicate the standard errors of the mean (SEM). The asterisk indicates significantly different values compared to day 1.

FIG. 2.

Changes in the sizes of parasites during treatment with anthelmintics. Ten cysts were cultured in triplicate wells (see Materials and Methods) with ABZ-SO at a final concentration of 25 to 250 ng/ml (A), PZQ at a final concentration of 25 to 250 ng/ml (B), a fixed concentration of PZQ (25 ng/ml) with the indicated concentrations (25 to 250 ng/ml) of ABZ-SO (C), and a fixed concentration of ABZ-SO (25 ng/ml) with the indicated concentrations (25 to 250 ng/ml) of PZQ (D). The symbols represent the mean, and the error bars indicate the SEM. Asterisks signify conditions that are significantly different from the untreated.

FIG. 3.

Effect of ABZ-SO and PZQ on cyst evagination. Ten cysts of T. solium were cultured in triplicate wells for 4 days in medium alone, ABZ-SO (50, 75, or 100 ng/ml), or PZQ (1, 5, or 10 ng/ml), and the total percentages of cysts that evaginated spontaneously (A) and total evaginated cysts (spontaneous plus after bile) after stimulation with bile (B) for 18 h were determined. Each data point represents the mean of triplicate wells, and the bars indicate the SEM.

FIG. 4.

Inhibition of alkaline phosphatase (AP) release in supernatants of cysts by ABZ-SO and PZQ. AP levels were measured colorimetrically (see Materials and Methods). (A) Release of AP in medium containing the solvents used to solubilize the drugs, ABZ-SO (DMSO) and PZQ (ethanol). (B to D) Release of AP (shown as the corrected OD at 450 nm [OD₄₅₀]; see Materials and Methods) in the supernatants of cysts treated with ABZ-SO alone (B), both ABZ-SO and PZQ (C), or PZQ alone (D). The data shown are means and SEM of triplicate wells from one of three representative experiments.

FIG. 5.

Inhibition of parasite antigen secretion by anthelmintic treatment. The levels of parasite antigens in the supernatant of T. solium cysts in culture were assayed by ELISA using MAb B158C11A10 (see Materials and Methods). The graph depicts the mean antigen levels in supernatants from days 1 and 3 of culture, expressed as a ratio of the OD₄₉₂ values for each test condition to the corresponding control. The differences between days 1 and 3 are statistically significant for ABZ-SO and ABZ-SO plus PZQ (P < 0.05 [Kruskal-Wallis test]).