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Research Article
4 April 2011

Isothermal Microcalorimetry To Study Drugs against Schistosoma mansoni

ABSTRACT

Alternative antischistosomal drugs are required since praziquantel is virtually the only drug available for treatment and morbidity control of schistosomiasis. Manual microscopic reading is the current “gold standard” to assess the in vitro antischistosomal properties of test drugs; however, it is labor-intensive, subjective, and difficult to standardize. Hence, there is a need to develop novel tools for antischistosomal drug discovery. The in vitro effects of praziquantel, oxamniquine, artesunate, and mefloquine on metabolic activity and parasite motility of Schistosoma mansoni (newly transformed schistosomula [NTS] and 49-day-old adult worms) were studied using isothermal microcalorimetry (IMC). Results were compared to morphological readouts of viability. Results obtained for the four drugs tested with phenotypic evaluation by microscopy and IMC showed a good correlation, but IMC also identified drug effects that were not visible by microscopic evaluation, and IMC precisely determined the onset of action of the test drugs. Similar sensitivities on NTS and adult schistosomes were observed for praziquantel and mefloquine, while slight differences in the drug susceptibilities of the two developmental stages were noted with oxamniquine and artesunate. IMC is a useful tool for antischistosomal drug discovery that should be further validated. In addition, our data support the use of NTS in in vitro antischistosomal drug assays.

INTRODUCTION

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is an important health problem affecting 779 million people mainly in sub-Saharan Africa (36). Praziquantel is virtually the only drug available and is increasingly deployed in mass drug administration programs, advocated by the World Health Organization (11). The annual estimated need for praziquantel in Africa exceeds 400 million tablets (http://www.who.int/wormcontrol/newsletter/PPC7_Eng_min.pdf). Two alternative drugs, oxamniquine and metrifonate, exist; however, they are rarely used today as they have a rather narrow activity profile and multiple doses have to be administered (12, 37). The extensive use and reliance on one single agent have raised concerns about the emergence of praziquantel resistance (3, 8), and indeed, schistosome strains with increased drug tolerance have already been isolated from patients as well as selected in the laboratory (9, 26). Therefore, there is growing consensus that novel antischistosomal drugs should be discovered and developed (8, 15, 33).
Antischistosomal drug discovery at many academic institutions (e.g., the Special Programme for Research and Training in Tropical Diseases; screening centers in London, United Kingdom, and Cairo, Egypt; the University of California, San Francisco, Sandler Center in San Francisco, CA; and the Swiss Tropical and Public Health Institute [Swiss TPH] in Basel, Switzerland) is based on in vitro whole-organism drug screening assays followed by in vivo tests in infected mice (15, 31). Stages used for in vitro assays are 1- to 7-day-old schistosomula (newly transformed schistosomula [NTS]) and juvenile and adult schistosomes.
The current “gold standard” is to assess worm viability microscopically and to evaluate drug effects with regard to death, changes in motility, viability, and morphological differences (15). However, microscopic techniques are often very labor-intensive and hence do not allow medium or high throughput, have to be carried out by well-trained personnel, and have a subjective nature. In addition, they are difficult to standardize and do not provide any information about the possible drug target and/or the mechanism of action of the drugs (1). Several studies have therefore been launched in the recent past. These have explored novel tools to facilitate the readout of in vitro drug screening on schistosomes, such as fluorescence-labeled albumin (14), fluorophores (propidium iodide and fluorescein diacetate) (29), and colorimetric assays with alamarBlue (25).
In the present work, we studied the effects of the 4 schistosomicidal compounds, praziquantel, oxamniquine, artesunate, and mefloquine, on the metabolic activity and parasite motility of Schistosoma mansoni (NTS and 49-day-old adult worms) using isothermal microcalorimetry (IMC). IMC measures the heat flow of biological processes (endo- or exothermic reactions) over time and has already been applied in various disciplines, including studies on food deterioration, drug shelf life (34), microbial processes, and the effects of different antimicrobial agents (6, 39). We have already demonstrated the applicability of this method evaluating the antischistosomal properties of mefloquine isomers (23). The whole-organism microscopic assay served as a reference.

MATERIALS AND METHODS

Parasites.

Cercariae of S. mansoni were harvested from infected intermediate-host snails (Biomphalaria glabrata) maintained at laboratories at the Swiss TPH after exposure to light for 3 h. Female NMRI mice (n = 20; age, 3 weeks; weight, ∼35 g) were purchased from Harlan Laboratories (Horst, Netherlands).

Drugs.

Praziquantel was purchased from Sigma, oxamniquine was obtained from Q. Bickle (London School of Hygiene and Tropical Medicine, London, United Kingdom) and M. J. Doenhoff (University of Nottingham, Nottingham, United Kingdom), artesunate was the product of Dafra Pharma (Turnhout, Belgium), and mefloquine hydrochloride was provided by Mepha Pharma AG (Aesch, Switzerland).
Drugs were dissolved in dimethyl sulfoxide (DMSO; Fluka, Buchs, Switzerland) to obtain stock solutions of 10 mg/ml. Stocks were kept at 4°C for a maximum of 6 months.

Preparation of NTS.

Cercariae of S. mansoni were mechanically transformed into NTS (30) as described previously (15, 24). NTS were kept in medium 199 (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated fetal calf serum (iFCS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 5% CO2 in ambient air for a minimum of 3 to 12 h until use. This incubation period ensured that conversion from cercariae to NTS was completed.

Preparation of adult S. mansoni.

NMRI mice were infected subcutaneously with approximately 200 S. mansoni cercariae. After 7 to 8 weeks, mice were killed with CO2 and dissected, and all schistosomes were removed from the hepatic portal system and the mesenteric veins. Worms were washed 3 times with phosphate-buffered saline (PBS; pH 7.4), placed in RPMI 1640 culture medium, and kept at 37°C in an atmosphere of 5% CO2 until use. All culture media used were supplemented with 5% iFCS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Drug sensitivity assay with microscopic evaluation.

Stock solutions of praziquantel, oxamniquine, artesunate, and mefloquine were diluted (Costar) in culture medium (medium 199 for NTS and RPMI 1640 medium for adult worms supplemented with 5% iFCS, 100 U/ml penicillin, and 100 μg/ml streptomycin) in 24-well plates, yielding final concentrations of 100, 10, and 1 μg/ml. One hundred microliters of an NTS suspension containing 100 to 1,000 NTS or 2 adult male and 2 adult female worms were added to each well. Worms incubated in medium containing 1% DMSO served as controls. Parasite fitness was assessed before and 3, 17, 24, 48, and 72 h after drug incubation at 37°C in 5% CO2 using a dissecting microscope (magnification, 8- to 40-fold; Carl Zeiss AG, Germany). Drug effects were evaluated using a viability scale, as described previously (15, 24), which classifies parasite fitness and motility with scores ranging from 4 (hyperactive, increased motility) and 3 (normal activity, no morphological changes) to 0 (all worms dead). All experiments were carried out in duplicate and were repeated at least three times.

Microcalorimeter.

A 48-channel isothermal microcalorimeter (model TAM 48; TA Instruments, New Castle, DE) was used to measure the heat production of schistosomes over 96 h. The instrument was thermostated at 37°C at least 2 days before the experiment to achieve maximum stability. A simple drug injection system (Fig. 1) designed and manufactured by the Laboratory of Biomechanics and Biocalorimetry (University of Basel, Basel, Switzerland) was employed. It offers the advantage of minimal thermal disturbance of the sample and minimal loss of data compared to those that occur by removing the ampoule from the microcalorimeter, injecting the drug, and reintroducing the ampoule into the microcalorimeter.
Fig. 1.
Fig. 1. Injection system for IMC. 1, microcalorimeter tube; 2, rod with insulating rings; 3, prefilled 0.5-ml syringe (medium/drugs) (Becton Dickinson, NJ); 4, ampoule with schistosomes; 5, thermoelectric module.

Sample preparation.

Glass ampoules were filled with 800 μl medium 199 and 100 μl NTS suspension containing 100 to 1,000 NTS for initial validation experiments and 400 to 1,000 NTS for the main studies. For adult worm assay, ampoules were filled with 900 μl RPMI 1640 medium and 3 to 9 schistosomes for preliminary validation studies and 3 or 4 parasites for the main experiments. All materials used were autoclaved at 121°C for 20 min, and media and drug solutions were sterile filtered (pore size, 0.2 μm). Ampoules were sealed and placed into the measuring channels. After a stable signal (equilibration) was obtained, medium or drug dilutions (reaching final concentrations of 100, 10, and 1 μg/ml) were injected into the ampoules. Heat flows were recorded at a frequency of 1 data point per 10 min for a minimum of 96 h. All samples were assayed in triplicate.

Heat-flow analysis.

To investigate drug effects on the metabolic activity of schistosomes, heat-flow curves of NTS and adult S. mansoni were examined before and after drug addition. The levels of heat production of treated and untreated parasites (adjusted to their corresponding starting values) were compared at 24 and 72 h after drug injection.

Motor activity analysis.

We analyzed the amplitudes of the random oscillations occurring in heat-flow curves of adult S. mansoni to study drug effects on the motor activity of adult worms. Increased amplitudes compared to the machine average signal noise reflect worm motor activities (20, 23). The endpoint of worm motility (reflecting death of worms) was determined by the intersection of the sample amplitude curve (exponential decay) with the background signal noise of the microcalorimeter (0.4 to 0.55 μW). Since 3 or 4 schistosomes were present in each ampoule, the heat flow obtained and the motor activity observed in the calorimetric data reflect the overall motor activity of these worms, which is presented as delayed response due to the calorimeter time constant (21).

Statistics.

Heat-flow values and parasite motility (heat-flow random oscillations) of treated and untreated worms were calculated as means (± standard deviations) using Microsoft Excel software. The statistical significance of the means was assessed using the parametric paired t test at a 5% level of significance (StatsDirect statistical software, version 2.7.2.; StatsDirect Ltd., Cheshire, United Kingdom). Analyses of noise amplitudes were performed by using R software (32) and Microsoft Excel.

RESULTS

Drug sensitivity assay with NTS and microscopic evaluation.

The time-effect curves of praziquantel, oxamniquine, artesunate, and mefloquine against NTS evaluated microscopically for up to 72 h are shown in Fig. 2A to D. Morphological changes induced by these drugs are depicted in Fig. 3. Untreated control NTS were viable for at least 72 h. In the presence of praziquantel (Fig. 2A and 3A), NTS showed decreased levels of viability, granularity, and tegumental alterations, such as extensive blebbing, at all concentrations tested (100, 10, and 1 μg/ml), but none of the concentrations tested resulted in the complete absence of motility. Treatment of NTS with 100 and 10 μg/ml oxamniquine (Fig. 2B and 3B) increased worm motility for 24 h; at later examination points, motility was similar to that of the controls (100 μg/ml) or slightly reduced (10 μg/ml). Moderate granularity and swollen bodies were observed. Incubation with 1 μg/ml oxamniquine resulted in slightly decreased viability of NTS. None of the oxamniquine concentrations were lethal over the examination period of 72 h. NTS exposed to 100 μg/ml of artesunate (Fig. 2C and 3C) died within 17 h. Incubation with 10 μg/ml artesunate steadily reduced parasite viability and caused granularity and swollen bodies, resulting in minimal viability after 72 h. At 1 μg/ml, artesunate showed no effect. In the presence of 100 μg/ml mefloquine (Fig. 2D and 3D), all NTS died immediately. Incubation with a 10-fold lower concentration of mefloquine caused granularity and decreased viability and death of NTS at 24 h postincubation. No effect was observed at 1 μg/ml of mefloquine (23).
Fig. 2.
Fig. 2. Viability of S. mansoni NTS following in vitro treatment with praziquantel (A), oxamniquine (B), artesunate (C), and mefloquine (D) (100, 10, and 1 μg/ml) over time. Mean values of viability (viability score) were derived from a minimum of 6 experiments.
Fig. 3.
Fig. 3. Light microscopic observations of S. mansoni NTS after in vitro incubation with 10 μg/ml praziquantel, oxamniquine, and artesunate for 72 h and 10 μg/ml mefloquine for 24 h.

Adult S. mansoni.

Temporal effects of praziquantel, oxamniquine, artesunate, and mefloquine on adult S. mansoni are depicted in Fig. 4A to D. Control schistosomes remained viable over the entire observation period of 72 h. Schistosomes treated with praziquantel (Fig. 4A) showed immediate contractions at any of the concentrations tested (100, 10, and 1 μg/ml) and the absence of motility, and worms had a dark, shrunken appearance. Only basic movements such as occasional twitching of the female sucker were observed. Worms exposed to 100 μg/ml of oxamniquine (Fig. 4B) showed increased motor activity for 48 h, while concentrations of 10 and 1 μg/ml oxamniquine did not influence parasite viability. The highest artesunate concentration (100 μg/ml) tested caused death of adult worms after 72 h; however, worms treated with 10 and 1 μg/ml artesunate were vital without any morphological changes for up to 72 h following incubation (Fig. 4C). Exposure to 100 μg/ml of mefloquine (Fig. 4D) resulted in immediate death of all schistosomes. Mefloquine at 10 μg/ml highly affected worm viability within the first 24 h, and all worms were dead after 72 h of incubation. Incubation with the lowest mefloquine concentration (1 μg/ml) resulted in decreased activity after 72 h (23).
Fig. 4.
Fig. 4. Viability of adult S. mansoni following in vitro treatment with praziquantel (A), oxamniquine (B), artesunate (C), and mefloquine (D) (100, 10, and 1 μg/ml) over time. Mean values of viability (viability score) were derived from a minimum of 6 experiments.

Comparison of drug susceptibilities of NTS and adult schistosomes by microscopic evaluation.

Since antischistosomal drug discovery often relies on the schistosomular stage, we compared the in vitro responses (viability) of NTS and adult schistosomes to praziquantel, oxamniquine, artesunate, and mefloquine at 24 h and 72 h after drug incubation (Fig. 5). This comparison shows that drug sensitivities of NTS and adult worms were similar following praziquantel incubation, though praziquantel showed a slightly faster effect on adults. Similarly, comparable in vitro responses of NTS and adults were obtained following exposure to mefloquine. On the other hand, NTS were more affected by oxamniquine (hypermotility after 24 h, viability decrease after 72 h) and artesunate than adult worms.
Fig. 5.
Fig. 5. In vitro response (viability) of 2 developmental stages of S. mansoni (NTS and adults) to praziquantel, oxamniquine, artesunate, and mefloquine 24 h and 72 h after drug incubation. Asterisk, significant difference between control and drug of interest (P < 0.05).

IMC. (i) Preliminary studies.

To determine whether IMC is able to monitor the heat flow of schistosomes over time and therefore can be used for antischistosomal drug screening, initial experiments with different numbers of NTS and adult S. mansoni worms were carried out (in duplicate). Ampoules containing culture medium only or dead worms showed low signals of −1.4 μW to 0.4 μW. No difference in the thermogenic profiles of culture medium and medium containing dead worms was observed. One hundred to 200 NTS did not yield any detectable signal (comparable to that for medium alone). Increasing the number of NTS to 400 to 1,000 worms per vial resulted in heat-flow values of 0.4 to 4.3 μW (mean, 2.4 μW), respectively. NTS did not show any motility-related random oscillations in their thermogenic curves. Consistently higher heat-flow values of 5.4 to 9.6 μW were obtained following the examination of 3 to 9 adult S. mansoni worms. A 50% decrease in the heat flow of adult S. mansoni worms was observed after about 46 h, possibly due to the lack of oxygen (23). Adult worms showed random oscillations in the heat-flow signals for 88 h. After injection of medium to NTS or adult worms, heat flow as well as motility-related random oscillations remained stable. A low interindividual variability was observed. Based on these results, we decided to run experiments for 72 h after drug injection and to use 400 to 1,000 NTS or 3 or 4 adult worms per sample, as these numbers produced readily detectable signals and were in the same range as those in our microscope assays.

(ii) IMC of NTS.

Heat-flow curves of culture medium (background control), dead NTS (positive controls), untreated NTS (negative controls), and NTS exposed to praziquantel, oxamniquine, artesunate, and mefloquine (100, 10, and 1 μg/ml) are shown in Fig. 6.
Fig. 6.
Fig. 6. Heat-flow curves of S. mansoni NTS exposed to 3 different concentrations of praziquantel, oxamniquine, artesunate, and mefloquine in vitro. Black lines, negative control (untreated NTS); dark gray dotted lines, background control (culture medium); light gray dotted lines, positive control (dead NTS); dark gray lines, 100 μg/ml; medium gray lines, 10 μg/ml; light gray lines, 1 μg/ml. Data shown are mean values from 3 experiments. Arrows indicate the time of medium or drug injection.
Exposure to praziquantel at any of the 3 concentrations tested (100, 10, and 1 μg/ml) resulted in an immediate increase of heat flow (metabolic activation). At 72 h after injection of praziquantel, the metabolic activity of NTS was still detectable, with worms exposed to 100 μg/ml characterized by a decreased heat flow and worms treated with 10 and 1 μg/ml showing increased heat flow in comparison to that for control worms. When 100 μg/ml of oxamniquine was added to NTS, metabolic activation was observed for approximately 24 h; at later examination points, no effect on the heat flow was observed. Exposure to 10 and 1 μg/ml oxamniquine caused an alternating increase and decrease of heat production of approximately 0.7 μW over the entire course of the experiment. The heat flow of NTS increased significantly for about 5 h after injection of 100 μg/ml artesunate. From this time point onwards, the heat flow decreased rapidly, and at approximately 24 h after drug addition, only 10% metabolic heat production was detectable. Heat-flow curves of NTS treated with 10 and 1 μg/ml artesunate showed slightly higher signals than control curves. Following the addition of 100 μg/ml mefloquine to NTS, the heat production of NTS decreased immediately and worms died within 1 h. Metabolic activity of NTS was rapidly reduced by 50% at 2 h postexposure to 10 μg/ml of mefloquine. At 8 h following incubation with 10 μg/ml of mefloquine, NTS did not reveal any heat production (23). When 1 μg/ml of mefloquine was added to NTS, a slight decrease of heat production was observed, in particular, at 48 to 72 h after drug injection.

(iii) IMC of adult S. mansoni.

Heat-flow curves of medium (background control), dead schistosomes (positive controls), adult control worms (negative controls), and S. mansoni worms treated with praziquantel, oxamniquine, artesunate, and mefloquine (100, 10, and 1 μg/ml) are presented in Fig. 7.
Fig. 7.
Fig. 7. Heat-flow curves of adult S. mansoni worms treated with 3 different concentrations of praziquantel, oxamniquine, artesunate, and mefloquine in vitro. Black lines, negative control (untreated schistosomes); dark gray dotted lines, background control (culture medium); light gray dotted lines, positive control (dead schistosomes); dark gray lines, 100 μg/ml; medium gray lines, 10 μg/ml; light gray lines, 1 μg/ml. Data shown are mean values from 3 experiments. Arrows indicate the time of medium or drug injection.
No differences in the heat profiles of adult S. mansoni worms were seen following exposure to 100, 10, and 1 μg/ml of praziquantel. Heat-flow random oscillations, which reflect parasite motility, stopped immediately (P < 0.001) after praziquantel addition, and during the first 8 to 10 h after injection, the heat flow was reduced by approximately 30%. Afterwards the heat flow continued to decrease slowly. When 100 μg/ml oxamniquine was injected into the ampoules, metabolic activation was observed for approximately 4 h, followed by a steady decrease of heat production. Decreased heat flow was also observed for schistosomes exposed to 10 and 1 μg/ml oxamniquine. Oscillation analysis revealed a significant difference (P < 0.01) between 1 μg/ml oxamniquine-treated and control worms. Worms treated with 100 μg/ml artesunate were characterized by an absence of detectable random oscillations after 21 h (P < 0.01). Heat-flow curves of schistosomes treated with 10 and 1 μg/ml artesunate showed the same heat and oscillation profiles as the controls. Following injection of 100 μg/ml mefloquine, heat-flow curves decreased rapidly, reaching baseline values within 1 h. Random oscillations were not detectable (P < 0.001). Mefloquine at 10 μg/ml provoked a 50% heat-flow reduction during the first 24 h postexposure, whereas oscillations disappeared immediately after drug addition (P < 0.001). No effect on the heat flow of adult S. mansoni was observed with 1 μg/ml of mefloquine (23), while parasite motility was affected significantly (P < 0.01).

Comparison of drug susceptibilities of NTS and adult schistosomes by IMC.

Figure 8 illustrates the heat production of NTS and adult worms at 24 h and 72 h following incubation with the four drugs tested. Heat production of control worms was set to 100%. This comparison shows that praziquantel influenced the metabolic activity of NTS and adult schistosomes at all concentrations tested. However, differences in drug response were observed: while heat production of adult parasites decreased significantly (P < 0.05) by 43.1% (100 μg/ml), 34.5% (10 μg/ml), and 35.6% (1 μg/ml) during the first 24 h, the heat flow of NTS decreased only in the presence of 100 μg/ml praziquantel (P < 0.01). Lower praziquantel concentrations had no significant effect on the metabolic heat of NTS. Both developmental stages of S. mansoni also showed variations in the drug response toward oxamniquine: at 24 h postincubation, activated heat production was observed for NTS in the presence of 100 μg/ml and significant decreases were observed in the presence of 10 μg/ml (P < 0.05) and 1 μg/ml (P < 0.01). At 72 h, the metabolic activities were comparable to that of the controls. On the other hand, reduced heat flow was observed for adult worms in parallel to decreasing oxamniquine concentrations used. Differences between NTS and adult worms treated with artesunate were also observed and were particularly visible with the 2 lowest concentrations tested and at the 72-h examination point: while NTS showed only slight increases in heat production, adult S. mansoni worms revealed a strong increase in heat flow, which was significant for 10 μg/ml (P < 0.05). Finally, mefloquine showed a similar antischistosomal effect on NTS and adults using IMC, with NTS being slightly more susceptible to the drug.
Fig. 8.
Fig. 8. In vitro response (heat flow) of two developmental stages of S. mansoni (NTS and adults) to praziquantel, oxamniquine, artesunate, and mefloquine at 24 h and 72 h after drug addition. Asterisk, significant difference between control and drug of interest (P < 0.05).

DISCUSSION

Microscopy is the current gold standard to evaluate drug effects on schistosomes. However, the method is rather labor-intensive and less accurate due to the fact that it is prone to subjective results and interpretations (15). In the present study, we evaluated whether IMC, a highly sensitive, accurate, and simple tool to detect heat produced by microorganisms (6), could be used to study the in vitro activity of antischistosomal compounds. We were motivated to examine well-described antischistosomal drugs with IMC based on promising preliminary findings obtained with this technique analyzing the antischistosomal properties of 6 mefloquine isomers and racemates (23). In addition, in the framework of the present work, we thoroughly compared drug effects on the schistosomular and adult stages, as the former developmental stage is increasingly being used in antischistosomal drug screening assays (1, 15).
Comparing the performance of the two diagnostic techniques, IMC showed two key advantages over microscopy. First, the onset of action of all drugs tested could precisely be determined with IMC. For two of the drugs (mefloquine, artesunate), the onset of action on adult schistosomes was detected earlier by IMC than by microscopic evaluation. Heat-flow random oscillation analysis revealed an immediate parasite immobility following exposure of adult S. mansoni worms to 10 μg/ml mefloquine and after 21 h following exposure to 100 μg/ml artesunate. By microscopy, decreased viabilities were observed only after 24 h for mefloquine and after 72 h for artesunate. Second, drug effects, which were not visible by microscopic evaluation, were recorded by calorimetric measurements. For example, while an absence of motility of adult schistosomes was recorded by IMC 7 h after oxamniquine treatment (1 μg/ml), no effect was observed with microscopy. The faster and more accurate detection of drug activity by IMC is not surprising, as IMC provides a continuous real-time electronic signal proportional to the amount of heat produced (38), while worms are monitored by microscopy at selected time points only (in our study, 1 or 2 times per day). Third, IMC is characterized by a high sensitivity (200 nW) (5) and is therefore able to capture small changes in the thermal output of schistosomes, which corresponds to metabolism and motility, while morphological damages and death are the key criteria assessed by microscopy (31). Hence, while microscopy reliably detects severely damaging and lethal effects of drugs, IMC also readily detects more subtle drug effects, as demonstrated for oxamniquine and for low concentrations of mefloquine. Finally, another advantage of IMC, though not done in the present study, is that IC50s can be exactly determined by analyzing parasite motility (amplitudes in thermogenic curves), which would facilitate the comparison of antischistosomal activities of different standard and experimental drugs.
Although IMC seems to have many advantages in the evaluation of antischistosomal drugs, potential drawbacks have to be noted. First, all materials used need to be sterile, since contamination (e.g., bacterial growth) would be recorded by the calorimeter (6). Second, heat-flow curves obtained by IMC reflect the overall activity of the worms. It is not possible to distinguish between worm contractions and increased movement, because both physiological processes result in increased metabolic activity with heat production. Third, the present cost of a microcalorimeter is much higher than that of a regular microscope. Therefore, unless substantially less expensive instruments become available, it is unlikely that IMC will be widely used, and its use will be restricted to well-equipped laboratories in high-income countries. Fourth, in order to achieve analyzable signals, a large number of NTS (at least 400) were required per sample. Finally, the IMC used in this study allows the independent evaluation of just 48 measuring channels, and several hours of data must be accumulated. Hence, IMC does not allow high-throughput evaluation of drugs, i.e., thousands of samples per week.
An interesting difference observed between the two methods was that microscopy revealed more drug effects on NTS than calorimetric assessment. This finding might be explained by the nonspecificity of IMC, as described above, on the one hand (the net signal corresponds to the sum of all processes occurring in the ampoule), and the procedure of cercaria transformation into NTS, with a certain amount of cercariae and tails remaining, on the other hand. The impurities of cercariae and tails do not affect microscopy, as they can be disregarded during observation. However, cercariae and tails also produce heat-flow signals, which might disturb the heat-flow signal of the parasites of interest. Furthermore, the drug susceptibilities of cercarial stages can be different from those of schistosomula (13, 28), resulting in false-positive or -negative signals. Another explanation might be that the drugs analyzed impact motility and morphology (which are not detectable on NTS by IMC) rather than the metabolic activity of NTS.
Today, schistosomula are often used in in vitro studies, as large numbers of this developmental stage can be obtained in a cost-effective manner and the use of live animals is not required (1, 15). The transformation from cercariae to schistosomula by the vortexing (30) used in our labs is convenient and easy to perform. Similar advantages have been described for the syringe transformation (1). It is interesting to note that schistosomula show different viabilities depending on the transformation method (whether cercariae are mechanically transformed or actively penetrate skin) applied (data not shown). Schistosomula derived by vortex transformations lose some of their fitness and become granular and less active within 24 h posttransformation, but afterwards the viability remains relatively steady (Fig. 2). It is believed that the application of mechanical stress as the only key transformation factor might not be sufficient to induce different biochemical modifications, such as an emptying of acetabular glands or shedding of the glycocalyx (7, 35), which might be crucial for the further survival of the parasite. In our study, regardless of whether IMC or microscopy was used, the overall sensitivities to 2 of the 4 drugs tested (praziquantel and mefloquine) were similar on schistosomula produced by vortex transformation and adult S. mansoni worms. Differences in the drug susceptibilities were noted with oxamniquine and artesunate. NTS seemed to be slightly more susceptible to these drugs than adults. The slightly lower susceptibility of adult S. mansoni worms than NTS to artesunate might be due to the lack of hemin, a primary activator of the peroxide group, in the incubation medium (41). Overall, the findings on the individual parasite stages are consistent with previous in vitro studies with these drugs (2, 10, 24, 27, 40).
In conclusion, in line with previous studies (1), our data support the use of NTS in antischistosomal drug screening assays and indicate that IMC is a useful tool for antischistosomal drug discovery. IMC may be particularly helpful in the characterization of new hits. The method should be further validated using additional antischistosomal drugs, which ideally are characterized by distinct mechanisms of action (e.g., nilutamide or related hydantoin derivatives) at different concentrations (18). In order to overcome the limitation of the low throughput of the widely used 48-channel IMC, either chip calorimeters (4, 22) or prototypes of 96-well-plate calorimeters should be further developed and adapted. These calorimeters are presently used for different biophysical applications. IMC might also be useful to distinguish drug effects on resistant and sensitive schistosome strains, in line with a recently developed bioassay based on the praziquantel susceptibility of miracidiae (19). Finally, it is likely that IMC is widely applicable to a range of helminths and their developmental stages. Hence, drug effects on other helminths, for example, hookworms, should be monitored with IMC, as the number of drugs currently available to treat these infections is too small and drug screening approaches are limited (16).

ACKNOWLEDGMENTS

T.M. and J.K. are thankful to the 3R Research Foundation and Swiss National Science Foundation for financial support (project no. 110/08 and PPOOA-114941), and O.B. and his laboratory are grateful for the support of the Hardy & Otto Frey-Zund Foundation, Basel, Switzerland.
We all appreciate the pioneering work of A. U. Daniels in using microcalorimetry to evaluate the responses of living systems in culture and for excellent comments on the manuscript. We thank A. Trampuz for the support of calorimetric measurements and D. Wirz, who designed the injection system and helped with the original wavelet analysis.
We declare that we have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 49Number 4April 2011
Pages: 1217 - 1225
PubMed: 21270220

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Received: 24 November 2010
Returned for modification: 22 December 2010
Accepted: 18 January 2011
Published online: 4 April 2011

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Theresia Manneck
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland
University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
Olivier Braissant
Laboratory of Biomechanics and Biocalorimetry, c/o Biozentrum/Pharmazentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland
Yolanda Haggenmüller
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland
University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
Jennifer Keiser [email protected]
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland
University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland

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