**5.1 Compound storage and handling**

To test new compounds, including synthetic or natural products and crude extracts or fractions of a natural source, it is necessary to consider factors such as solubility and stability. The compounds are usually stored in hermetically sealed glass containers, covered with aluminium foil to protect the contents from light, and kept refrigerated at 8 ºC or at ambient temperature until used. The compounds are commonly dissolved in dimethyl sulfoxide (DMSO), which should not exceed a final concentration of 2% in the culture medium containing parasites. Control schistosomes are incubated in the presence of the highest concentration of solvent used.

For *in vitro* screening assays, there is not a set maximum concentration to evaluate the activity of compounds on schistosomes, as long as no toxicity occurs on mammalian cells. However, in our laboratory, *in vitro* screenings are performed at final maximum concentrations of up to 500 µg/ml for crude extracts or fractions and 1,000 µM for isolated compounds. The reference drug praziquantel is used as a positive control at final concentrations ranging from 5 to 10 µM (Figure 2).

#### **5.2 Assessment of parasite viability**

The effects of compounds on *S. mansoni* are commonly assessed by phenotypic changes. The parasites are kept for 5 days as described above, and monitored at different time points (e.g., 24, 48, 72, 96, and 120 h) to evaluate their general condition, using parameters such as motor activity, morphological changes, and mortality rate (Figure 2).

and in vaccine development and drug screening protocols (Abdulla et al., 2009; Gobert et al., 2007; Harrop & Wilson, 1993; Peak et al., 2010). There is evidence that mechanically transformed schistosomula are structurally similar to their lung schistosomula counterparts (Chai et al., 2006), and after 7 days in culture, the larvae have the morphological features of lung worms and are capable of maturation when introduced into the portal vein of mice (Harrop & Wilson, 1993). Furthermore, mechanically transformed schistosomula are able to develop steadily until adult worm pairing (Basch, 1981). Because of these reasons, drugscreening assays in our laboratory are based on mechanically transformed schistosomula of different ages *in vitro* (3-h-, 1-, 3-, 5- and 7-day-olds). It takes roughly 3 h for the cercariae to secrete the contents of their acetabular glands; the 1- to 7-day-olds correspond to the skin-

**5. Operating procedures for antischistosomal drug screening and the** 

In recent years, the search for new anthelmintics has intensified, but little significant progress has been made in developing new techniques. The *in vitro* drug screening approaches must take into account some specic concerns, particularly to be simple and inexpensive. Important methodologies can objectively and rapidly distinguish helminth viability or phenotype. *In vitro* screening could identify novel anthelmintics and could eventually translate into practical applications. Herein, a general overview is given of the most common methodologies used for screening antischistosomal compounds and their

To test new compounds, including synthetic or natural products and crude extracts or fractions of a natural source, it is necessary to consider factors such as solubility and stability. The compounds are usually stored in hermetically sealed glass containers, covered with aluminium foil to protect the contents from light, and kept refrigerated at 8 ºC or at ambient temperature until used. The compounds are commonly dissolved in dimethyl sulfoxide (DMSO), which should not exceed a final concentration of 2% in the culture medium containing parasites. Control schistosomes are incubated in the presence of the

For *in vitro* screening assays, there is not a set maximum concentration to evaluate the activity of compounds on schistosomes, as long as no toxicity occurs on mammalian cells. However, in our laboratory, *in vitro* screenings are performed at final maximum concentrations of up to 500 µg/ml for crude extracts or fractions and 1,000 µM for isolated compounds. The reference drug praziquantel is used as a positive control at final

The effects of compounds on *S. mansoni* are commonly assessed by phenotypic changes. The parasites are kept for 5 days as described above, and monitored at different time points (e.g., 24, 48, 72, 96, and 120 h) to evaluate their general condition, using parameters such as motor

and lung-stage schistosomula.

effects on the whole organism.

**5.1 Compound storage and handling** 

highest concentration of solvent used.

**5.2 Assessment of parasite viability** 

concentrations ranging from 5 to 10 µM (Figure 2).

activity, morphological changes, and mortality rate (Figure 2).

**techniques employed** 

Current methods utilised to assess schistosomal viability have recently been reviewed, and most of these methods involve microscopic techniques (Keiser, 2010; Ramirez et al., 2007). The phenotypic changes are scored by using a viability scale. For example, a scale of 0 – 4, where 4= normally active, 3= slowed activity, 2= minimal activity, 1= absence of motility apart from gut movements, and 0= total absence of mobility, is based on standard procedures for compound screening at the Special Programme for Research and Training in Tropical Diseases, World Health Organization, WHO-TDR (Ramirez et al., 2007). Alternatively, as described by Manneck et al. (2010, 2011), drug activity is defined as 3= totally vital, normally active, and no morphological changes; 2= slowed activity, primary morphological changes and visible granularity; 1= minimal activity, severe morphological changes and granularity; 0= all worms dead, severe morphological changes and granularity; the granularity is characterised only for schistosomula. The regular movement of both larval and adult schistosomes has proven to be a valuable trait in assessing schistosome viability *in vitro* because lack of movement is a good indicator of death. Worm death is usually dened as no movement observed for at least 2 min of examination (Manneck et al., 2010). In this context, the viability of worm during the culture period is also assessed by motor activity reduction, and it is defined as "slight " or "significant". This subjective criterion is commonly used by several research groups (Braguine et al., 2009; de Melo et al., 2011; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011, Parreira et al., 2010; Pereira et al., 2011; Xiao et al., 2007). Size measurements of parasites are also employed to study phenotypic changes.

In addition to the phenotypic approaches, another *in vitro* drug-screening assay method is based on microcalorimetry. Manneck et al. (2011) analysed the effects of drugs on the metabolic activity of schistosomula and adult *S. mansoni* by comparing their heat ow. In this study, a multi-channel isothermal microcalorimeter equipped with 48 measuring channels was used to monitor the heat production by schistosomes as a result of their metabolism over time. The results show that microcalorimetry can be a valuable tool to study antischistosomal drugs, and the microcalorimetric measurements conrmed, in part, the results of the phenotypic evaluation. However, the level of agreement between microscopy and microcalorimetry data requires further investigation (Manneck et al., 2011). In the following section, other methods are described that are used to determine the effect of drugs on schistosomula and adult *S. mansoni.* 

Phenotypic changes are determined as mentioned above. However, because of the lack of standardisation between laboratories, the replication of results obtained by microscopic means is not always possible. In an effort to avoid the subjective nature of quantifying schistosome viability from the microscopic examination of phenotype alone, further adaptations have been developed and are based on the differentiating potential of some colorimetric vital dyes. Diamidinophenylindole (DAPI) has been used as a differential stain of dead schistosomula during microscopy; in addition, the low DAPI concentration (1 µg/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence (Van Der Linden & Deelder, 1984). Trypan blue has also been shown to be a reliable dye for differentially staining dead schistosomula (Harrop & Wilson, 1993) and by means of a methylene blue dye exclusion test (Gold, 1997). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is also a vital dye that has been successfully used to assess the viability of worms. The use of this assay on helminths was pioneered by Comley et al. (1989), and several nematode

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 347

morphological alterations of the tegument of *S. mansoni* are also assessed by methods that involve microscopic analysis. Indeed, during the assay, the parasite is manipulated *in vitro*, and the effect of such manipulation is assessed by bright-field examination of the morphology of the parasite (Figure 2). The criteria used to assess morphological changes induced by a drug require visual scoring by skilled operators and is assessed subjectively. Morphological changes are usually defined qualitatively as "partial" or "extensive" (Braguine et al., 2009; de Melo et al., 2011; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011; Parreira et al., 2010; Pereira et al., 2011; Xiao et al., 2007). Therefore, the effect of anthelmintic drugs on the tegument of schistosome cannot be evaluated in a dose-dependent manner. In addition, a question remains: is the death of the parasite is

The inherent subjectivity of this qualitative analysis led Moraes et al. (2011) and de Moraes et al. (2011) to develop a quantitative method for evaluating the effect of drugs on the tegument of *S. mansoni* using confocal microscopy. In this quantitative analysis, areas of the tegument of male worms are assessed, and the numbers of tubercles are counted. Briefly, the parasites are fixed in Formalin-Acetic-Alcohol solution (FAA) and analysed under a confocal microscope at 488 nm (excitation) and 505 nm (emission), as described by Moraes et al. (2011) and de Moraes et al. (2011). During the microscopic analysis of the threedimensional images captured using LSM Image Browser software (Zeiss), areas of the tegument of parasite are assessed, and the numbers of tubercles on the dorsal surface of male helminths are counted in a 20,000 µm2 area, which is calculated using the same software image capture. Importantly, the area chosen is in the dorsal region of the male adult worm, close to the ventral sucker (acetabulum) region, because there is no significant variation in the number of tubercles in this region. Furthermore, schistosome and others trematodes are self-fluorescent (Moraes et al., 2009), and this fluorescence is increased when the parasites are in the FAA solution. This is advantageous because it allows images to be captured without using a fluorescent fluorophore. The FAA solution consists of a 2:9:30:59 mixture of acetic acid, formaldehyde, ethanol (95%) and distilled water. This methodology is

As previously mentioned, drug effects are currently assessed by observing morphological changes in parasites using light microscopy methods. However, these techniques do not allow the microscopic analysis of the tegument in detail. The quantitative analysis described by Moraes et al. (2011) and de Moraes (2011) must be performed with high-resolution microscopy, such as confocal or scanning electron microscopy. Therefore, the drug effects assessed by phenotypic changes, such as tegumental alterations, cannot be trusted. For example, Moraes et al. (2011) used confocal microscopy to evaluate the *in vitro* schistosomicidal activity of piplartine, an amide isolated from *Piper tuberculatum*, and demonstrated that the tegumental damage occurs after incubation with doses higher than the lethal concentrations, suggesting that worm death is caused by different mechanisms. Thus, tegumental damage may not always result in death, and a quantitative assessment technique is needed to understand the mechanisms of action of newly discovered antischistosomal drugs. In addition, the advent of screening methods that allow highthroughput, scalable, automated, and objective assays for helminth viability, combined with knowledge of the molecular biology of the schistosome to identify possible new drug

targets, will make drug development for schistosomiasis easier.

associated with damage to the coat?

summarised in Figure 3.

species have been assessed. Nare et al. (1991) used the MTT marker to evaluate the viability of adult schistosomes; currently, some *in vitro* bioassays used to study the effects of drugs have demonstrated the ability of MTT to assess the viability of adult worms (Braguine et al., 2009; de Melo et al., 2011; Magalhães et al., 2009, 2010; Parreira et al., 2010; Pereira et al., 2011).

Recently, Peak et al. (2010) validated a high-throughput system for detecting the viability of schistosomula using a microtiter plate-based method. In this study, the authors combined the use of propidium iodide with fluorescein diacetate to allow the easy assessment of the percent of viable schistosomula present in a sample. This helminth fluorescent bioassay was developed into a method of wide-scale application because it is sensitive, relevant to industrial high-throughput (384-well microtiter plate compatibility, 200 schistosomula/well) and academic (96-well microtiter plate compatibility, 1000 schistosomula/well) settings, translatable to drug screening assays, does not require a priori knowledge of schistosome biology or extensive training in parasite morphology, and is objective and quantitative.

The development of high-content screening systems is an important step for the assessment of parasite viability in a high-throughput format. A novel assay for anthelmintic drug screening by real-time monitoring of parasite motility was developed by Smout et al. (2010). This technological advance is based on the detection of changing electrical currents running through mini gold electrodes on the bottom of tissue culture plates. In this assay, the authors assessed the motility of *S. mansoni* using an xCELLigence system (Real Time Cell Assay, RTCA SP instrument), which monitors cellular events in real time without the incorporation of labels by measuring the electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture plates. This technology was applied to adult schistosome using one pair (one coupled male and female worm) in 200 µl per well of an E-plate, which is a 96-well plate for cell-based assays on the RTCA instruments. Because the real-time system measures changes in worm motility with the high level of precision necessary for high-throughput studies, it is widely applicable to a range of helminth species and developmental stages (Smout et al., 2010). This motility assay may provide a superior methodology to microscopy by removing the subjectivity from helminth phenotype characterisation and making available a technology that could allow the direct comparison of results from different laboratories. However, the initial cost of this RTCA system and E-plates may restrict its use, especially in an academic laboratory.

### **5.3 Assessment of changes in the tegument of parasites**

The tegument is the major interface between the schistosome and its external environment. In addition to providing protection, the tegument is an important site of the uptake of nutrients and other molecules. Moreover, the tegument is extremely important for infection success and survival in the host, and it has been a major target for the development of antischistosomal drugs (Skelly & Wilson, 2006; Van Hellemond et al., 2006). Therefore, most of the drugs currently used against schistosome act by damaging the worm tegument (Doenhoff et al., 2008; Fenwick et al., 2003; El Ridi et al., 2010; Keiser, 2010; Manneck et al., 2010, 2011; Mostafa et al., 2011; Xiao et al., 2000).

The schistosome tegument is often approached as a drug target in schistosomiasis and is associated with the subjective assessment of parasite viability described here. The

species have been assessed. Nare et al. (1991) used the MTT marker to evaluate the viability of adult schistosomes; currently, some *in vitro* bioassays used to study the effects of drugs have demonstrated the ability of MTT to assess the viability of adult worms (Braguine et al., 2009; de Melo et al., 2011; Magalhães et al., 2009, 2010; Parreira et al.,

Recently, Peak et al. (2010) validated a high-throughput system for detecting the viability of schistosomula using a microtiter plate-based method. In this study, the authors combined the use of propidium iodide with fluorescein diacetate to allow the easy assessment of the percent of viable schistosomula present in a sample. This helminth fluorescent bioassay was developed into a method of wide-scale application because it is sensitive, relevant to industrial high-throughput (384-well microtiter plate compatibility, 200 schistosomula/well) and academic (96-well microtiter plate compatibility, 1000 schistosomula/well) settings, translatable to drug screening assays, does not require a priori knowledge of schistosome biology or extensive training in parasite morphology,

The development of high-content screening systems is an important step for the assessment of parasite viability in a high-throughput format. A novel assay for anthelmintic drug screening by real-time monitoring of parasite motility was developed by Smout et al. (2010). This technological advance is based on the detection of changing electrical currents running through mini gold electrodes on the bottom of tissue culture plates. In this assay, the authors assessed the motility of *S. mansoni* using an xCELLigence system (Real Time Cell Assay, RTCA SP instrument), which monitors cellular events in real time without the incorporation of labels by measuring the electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture plates. This technology was applied to adult schistosome using one pair (one coupled male and female worm) in 200 µl per well of an E-plate, which is a 96-well plate for cell-based assays on the RTCA instruments. Because the real-time system measures changes in worm motility with the high level of precision necessary for high-throughput studies, it is widely applicable to a range of helminth species and developmental stages (Smout et al., 2010). This motility assay may provide a superior methodology to microscopy by removing the subjectivity from helminth phenotype characterisation and making available a technology that could allow the direct comparison of results from different laboratories. However, the initial cost of this RTCA system and E-plates may restrict its

The tegument is the major interface between the schistosome and its external environment. In addition to providing protection, the tegument is an important site of the uptake of nutrients and other molecules. Moreover, the tegument is extremely important for infection success and survival in the host, and it has been a major target for the development of antischistosomal drugs (Skelly & Wilson, 2006; Van Hellemond et al., 2006). Therefore, most of the drugs currently used against schistosome act by damaging the worm tegument (Doenhoff et al., 2008; Fenwick et al., 2003; El Ridi et al., 2010; Keiser, 2010; Manneck et al.,

The schistosome tegument is often approached as a drug target in schistosomiasis and is associated with the subjective assessment of parasite viability described here. The

2010; Pereira et al., 2011).

and is objective and quantitative.

use, especially in an academic laboratory.

2010, 2011; Mostafa et al., 2011; Xiao et al., 2000).

**5.3 Assessment of changes in the tegument of parasites** 

morphological alterations of the tegument of *S. mansoni* are also assessed by methods that involve microscopic analysis. Indeed, during the assay, the parasite is manipulated *in vitro*, and the effect of such manipulation is assessed by bright-field examination of the morphology of the parasite (Figure 2). The criteria used to assess morphological changes induced by a drug require visual scoring by skilled operators and is assessed subjectively. Morphological changes are usually defined qualitatively as "partial" or "extensive" (Braguine et al., 2009; de Melo et al., 2011; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011; Parreira et al., 2010; Pereira et al., 2011; Xiao et al., 2007). Therefore, the effect of anthelmintic drugs on the tegument of schistosome cannot be evaluated in a dose-dependent manner. In addition, a question remains: is the death of the parasite is associated with damage to the coat?

The inherent subjectivity of this qualitative analysis led Moraes et al. (2011) and de Moraes et al. (2011) to develop a quantitative method for evaluating the effect of drugs on the tegument of *S. mansoni* using confocal microscopy. In this quantitative analysis, areas of the tegument of male worms are assessed, and the numbers of tubercles are counted. Briefly, the parasites are fixed in Formalin-Acetic-Alcohol solution (FAA) and analysed under a confocal microscope at 488 nm (excitation) and 505 nm (emission), as described by Moraes et al. (2011) and de Moraes et al. (2011). During the microscopic analysis of the threedimensional images captured using LSM Image Browser software (Zeiss), areas of the tegument of parasite are assessed, and the numbers of tubercles on the dorsal surface of male helminths are counted in a 20,000 µm2 area, which is calculated using the same software image capture. Importantly, the area chosen is in the dorsal region of the male adult worm, close to the ventral sucker (acetabulum) region, because there is no significant variation in the number of tubercles in this region. Furthermore, schistosome and others trematodes are self-fluorescent (Moraes et al., 2009), and this fluorescence is increased when the parasites are in the FAA solution. This is advantageous because it allows images to be captured without using a fluorescent fluorophore. The FAA solution consists of a 2:9:30:59 mixture of acetic acid, formaldehyde, ethanol (95%) and distilled water. This methodology is summarised in Figure 3.

As previously mentioned, drug effects are currently assessed by observing morphological changes in parasites using light microscopy methods. However, these techniques do not allow the microscopic analysis of the tegument in detail. The quantitative analysis described by Moraes et al. (2011) and de Moraes (2011) must be performed with high-resolution microscopy, such as confocal or scanning electron microscopy. Therefore, the drug effects assessed by phenotypic changes, such as tegumental alterations, cannot be trusted. For example, Moraes et al. (2011) used confocal microscopy to evaluate the *in vitro* schistosomicidal activity of piplartine, an amide isolated from *Piper tuberculatum*, and demonstrated that the tegumental damage occurs after incubation with doses higher than the lethal concentrations, suggesting that worm death is caused by different mechanisms. Thus, tegumental damage may not always result in death, and a quantitative assessment technique is needed to understand the mechanisms of action of newly discovered antischistosomal drugs. In addition, the advent of screening methods that allow highthroughput, scalable, automated, and objective assays for helminth viability, combined with knowledge of the molecular biology of the schistosome to identify possible new drug targets, will make drug development for schistosomiasis easier.

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 349

The effects of natural or synthetic products on the reproductive fitness of *S. mansoni* have been previously reported in several studies (Braguine et al., 2009; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Mohamed et al., 2005; Moraes et al., 2011; Sanderson et al., 2002). To evaluate drug effects on schistosome during *in vitro* screening drug assays, cultures are continually monitored to assess the sexual fitness of worms treated with sublethal concentrations of drug. In this case, the following parameters are assessed: (1) changes in the pairing, an indicator of the mating process; (2) egg production, an indicator of egg

In the experiments, adult worm pairs (male and female coupled) are incubated in a 24-well culture plate, as previously described here, and parasites are monitored on daily basis for 5 days using an inverted microscope and a stereomicroscope (Figure 2). Therefore, it is important that, after collection by the perfusion technique, the parasites are carefully

Schistosome egg output *in vitro* is usually determined by counting the number of eggs. Egg development can be analysed quantitatively and scored as developed or undeveloped on the basis of the presence or absence of the miracidium (de Melo et al., 2011; Magalhães et al., 2009, 2010). This is a simple and recommended method because conventional light microscopy is able to distinguish morphologic differences in eggs. However, the characterisation of the viability of immature eggs is very difficult. Alternatively, the analysis of egg viability, distinguishing live immature eggs from dead immature ones, can be performed using a fluorescent label, as described by Sarvel et al. (2006). In this assay, the eggs obtained in culture are stained with the Hoescht 33258 probe and observed with fluorescent microscopy. The authors evaluated fluorescent labels and vital dyes, aiming at differentiating live and dead eggs, and showed the only the fluorescent Hoechst 33258 can

Finding a new compound capable of killing a parasite is not difficult. However, it is difficult to find a substance that can kill the parasite without affecting the host. Therefore, early *in vitro* studies of new compounds must include comparative cytotoxicity data from human or animal cells in tissue culture to establish that the compound has selective antischistosomal activity and may be a realistic prospect for future clinical use in humans. In our operating procedures for antischistosomal drug screening, mammalian cells are exposed to concentrations of at least two times higher than what is needed to elicit a schistosomicidal effect. Thus, the *in vitro* schistosomicidal activity of compounds cannot be associated with

General toxicity tests can be conducted in many cell types (e.g., fibroblasts and epithelial and hepatoma cells). Peripheral blood mononuclear cells and erythrocytes are widely used in *in vitro* studies to detect cytotoxicity or cell viability following exposure to antischistosomal compounds. Vero mammalian cells (African green monkey kidney broblasts) are also commonly used to examine whether natural or synthetic antiparasitic compounds are tolerated by mammalian cells (da Silva Filho et al., 2009; Moraes et al., 2011;

The crystal violet staining method and the neutral red and MTT assays are the most common methodologies used to detect cytotoxicity or cell viability following exposure to toxic substances. In our *in vitro* cytotoxicity assays with cultured cells, the crystal violet

**5.4 In vitro assessment of the reproductive fitness of adult worms** 

be considered a useful tool to differentiate between dead and live eggs.

output per worm; and (3) egg development.

**5.5 Cytotoxicity assays** 

cytotoxic effects.

Parreira et al., 2010).

washed to prevent the separation of the worm pairs.

Fig. 3. Dorsal region of a *Schistosoma mansoni* adult male worm, on which the effect of antischistosomal compounds on the tegument is evaluated quantitatively. The parasite was fixed in FAA solution, and fluorescent images were obtained using a confocal microscope. A: General view of the anterior helminth region showing, in red, the location where tubercles were counted. Bar = 500 µm. B: View of an area of 20,000 µm2, calculated with the Zeiss LSM Image Browser software, showing the tubercles. This image is a higher magnification of the dorsal region of the *S. mansoni* adult worm marked in red in panel A. X and Y: three-dimensional images obtained from laser scanning confocal microscopy. Os: oral sucker; Vs: ventral sucker; Tu: tubercles
