in excess of one billion people are infected with helminths in developing countries , where these diseases cause substantial morbidity and hundreds of thousands of deaths annually ( may , 2007 ; albonico et  al . , 2008 ; hotez , 2011 ; bardosh , 2014 ) . helminths also plague livestock in developing and developed countries , with the global anthelmintic market for livestock and companion animals valued at $ us 3.7 billion in 2002 ( evans and chapple , 2002 ) . whereas chemotherapy is available for infection with most parasitic helminths , widespread use of anthelmintics in agriculture has resulted in the emergence of drug - resistant parasites ( james et  al . , 2009 ; molento , 2009 ) . concern remains about the emergence of drug resistance in human helminth parasites , and as mass drug administration campaigns increase this worrisome prospect becomes more likely ( abdul - ghani et  al . this is due in part to the high cost and limited financial return from drug development , particularly for pathogens of medical importance . a decade ago , the major component of this cost limitation was the lack of objective high - throughput screening methods for assessing drug effectiveness ( kotze et  al . , 2006 ; abdul - ghani et  al . , 2009 ; keiser , 2009 ) . the gold standard for measuring drug effectiveness for helminth parasites was the costly , laborious and subjective in  vitro assessment of worm motility , as measured visually via microscopy and larval development assays ( kotze et  al . , 2004 ; abdulla et  al . , 2009 ; keiser , 2009 ) . indeed , there are multiple requests in the peer - reviewed literature for high - throughput screening methods to facilitate drug development and to detect emerging resistance ( morel , 2003 ; sommerfeld and oduola , 2007 ; keiser , 2009 ) . in response , the tropical diseases research network ( tdr ) of the who ( http://apps.who.int/tdr/ ) developed an international resistance screening network , but due to the limitations of available techniques , screening deployed to date has utilized only low - to medium - throughput approaches ( hopkins et  al . , 2007 ; abdulla et  al . , recently , a range of screening techniques have emerged , often adapted from methods to monitor cultured cells , which allow monitoring of several helminth parasite species and developmental stages by video ( paveley et  al . , 2012 ) , impedance ( smout et  al . , 2010 ) , enzymatic ( mansour and bickle , 2010 ) , colorimetric ( tritten et  al . , 2012 ) , fluorescence ( peak et  al . , 2010 ) and other mechanisms ( howe et  al . , 2015 ; lalli et  al . , 2015 ) . we have previously adapted the impedance - based real time cell assay  the xcelligence system  to monitor the viability of a range of human and livestock parasitic helminths ( smout et  al . , 2010 ) . the xcelligence unit conventionally measures cell growth relaying on gold electrodes embedded in the base of tissue culture microplates that monitor changes in conductivity due to contact of the cells with the electrodes ( vistejnova et  al . with many species of parasitic helminths , the wild type phenotype is motile in tissue culture , and the movement of the worm is detected by change in the conductivity on the electrodes . the xworm approach is simple , objective , high - throughput , relatively inexpensive , and applicable to many species of parasitic worms ( smout et  al . , 2010 ) . the technique has been favorably received in the field for its sensitivity , broad applicability and adaptability ( peak and hoffmann , 2011 ; silbereisen et  al . , 2011 ; , 2012 ; you et  al . , 2013 ; zeraik et  al . , 2014 ) . however , alternate viability monitoring methods , such as energy metabolism and membrane permeability , have been shown to be more suitable for specific life cycle stages , notably the intra - mammalian larval stages of the human schistosome blood flukes ( mansour and bickle , 2010 ; peak et  al . , 2010 ; howe et  al . xworm has been adapted for a range of applications that rely on measurement of a phenotypic change , including responses of genetically manipulated parasites ( you et  al . , 2013 ) . moreover , developmental stages that originally were not envisioned suitable for the assay , such as eggs of schistosomes that require fresh water to hatch , have been successfully employed ( zeraik et  al . , 2014 ) . these new applications were successful but required further optimization as a low concentration buffered salt solution of 0.1 phosphate buffered saline ( pbs ) was necessary for egg hatching . in addition the signal : noise ratio at the default 10  khz frequency was sub - optimal for sensitive detection . discussions with the developers of the xcelligence system ( acea ) , revealed alternate frequency settings of 25 and 50  khz available for monitoring cells  or in our case monitoring parasitic helminths . in order to improve the sensitivity of the xworm assay we employed strictly standardized mean difference prime ( ssmd ) statistical analysis to compare different monitoring frequencies , i.e. the default 10  khz and alternate 25  khz and 50  khz . in addition , low concentration salt motility assay facilitated accurate measurement of motility of cercariae , the infective stage of the schistosome for humans and other mammals . mice infected with schistosoma mansoni were obtained from the biomedical research institute ( bri ) , rockville , md and housed at the animal research facility of the george washington university medical school , which is accredited by the american association for accreditation of laboratory animal care ( aaalac no . 000347 ) and has an animal welfare assurance on file with the national institutes of health , office of laboratory animal welfare , olaw assurance number a3205 - 01 . all procedures employed were consistent with the guide for the care and use of laboratory animals . maintenance of the mice and recovery of schistosomes were approved by the institutional animal care and use committee of the george washington university . biomphalaria glabrata snails and swiss - webster mice infected with the nmri ( puerto rican ) strain of s.  mansoni were supplied by the biomedical research institute , rockville , maryland usa under nih - niaid contract hhsn272201000005i . four developmental stages collected and maintained as described ( mann et  al . , 2010 ) were investigated : adults , eggs / miracidia , cercariae and schistosomula . the motility assay was performed using the xcelligence dp platform ( acea biosciences , san diego , ca ) at 10  khz as described ( zeraik et  al . , 2014 ) , and with the addition of 25 and 50  khz frequencies . all motility index readings were monitored with sweeps at intervals of 15  s by the rtca software . prior to the addition of parasites to e - plates , baseline motility using media alone was determined overnight in e - plate wells containing 100  l of relevant media but without parasites ( see sections 2.42.7 below ) . treatments of forchlorfenuron in dmso ( fcf , sigma  aldrich ) or dmso ( untreated vehicle controls ) were included when specified for the different life cycle stages . developmental stages of s.  mansoni were heat killed , 80  c for 15  min , immediately before the assay . , 2010 ) , and egg hatching / miracidia motility was assessed as described ( zeraik et  al . , 2014 ) . briefly 5000 eggs were seeded into 180  l of 0.1 pbs , ph 7.2 ( 13.7  mm nacl , 0.27  mm kcl , 1  mm nah2po4/na2hpo4 , 0.2  mm kh2po4 ) per well and supplemented with 20  l of dmso vehicle control , 50  m or 200  m of fcf ( final concentration ) in duplicate . the eggs were induced to hatch under bright light at 23  c for 16  h. a replicate of the experiment was run in parallel in a 96-well microtitre plate for direct visual observation of the egg hatching using an axio observer a.1 inverted microscope fitted with an axiocam icc3 camera ( zeiss ) . cercariae shed from b. glabrata snails infected with s.  mansoni in water under bright light for 2  h  at room temperature were washed three times in 0.1 pbs and 2% antibiotic / antimycotic ( life technologies ) , transferred to 0.1 pbs and dispensed in duplicate to e - plate wells in two - fold decreasing serial dilution of cercariae from 9000 - 281 cercariae / well . the motility of the larvae was monitored overnight for 20  h  at 23  c . schistosomula were obtained by mechanical transformation of cercariae ( mann et  al . , 2010 ) and cultured in modified basch 's medium under 5% co2 at 37  c ( dalton et  al . , 1997 ; mann et  al . , 2010 ; zeraik et  al . , 2014 ) . serial two - fold dilutions of 5000 - 78 schistosomula in 100  l of modified basch 's medium were added to e - plate wells containing 100  l of the same medium . two wells containing 200  l of modified basch 's medium alone were included as empty well controls . the parasites were monitored every 15  s  at 37  c under 5% co2 for 16  h. the experiment was performed in duplicate . adult worms were recovered from the hepatic veins of infected mice by portal perfusion ( mann et  al . , 2010 ; zeraik et  al . , 2014 ) . thereafter , adult worms were washed and cultured in dulbecco 's modified eagle 's medium ( dmem - life technologies ) , supplemented with 10% fetal bovine serum and 2% antibiotic / antimycotic ( invitrogen , carlsbad , ca , catalogue no . individual adult schistosomes were dispensed into wells of e - plates and the pre - treatment motility of flukes was registered for a minimum of 6  h before addition of fcf to 1  mm or dmso vehicle control with 68 replicates per condition . the motility index for each e - plate well , i.e. individual flukes , 5000 eggs , schistosomula or cercariae , was calculated in excel ( microsoft ) as the standard deviation over 800 data points of the cell index ( ci ) difference from the rolling average over 30 data points as described ( smout et  al . , 2010 ) . the absolute ssmd value is the ratio of the difference of the sample mean to the standard deviation of the sample data and was generated as described by zhang ( 2011 ) . treatment and negative control samples ( untreated parasites ) for egg hatch and cercariae motility have mean values designated as xt and xn with standard deviations of st and sn respectively . for egg hatch and cercariae motility , the total motility index over 16 and 20  h respectively was used for graphing and ssmd calculations.absolutessmd=|xtxnst2+sn2| ssmd prime ( ssmd ) is based upon the positive / negative controls and replaces xt with the heat - killed parasite positive control . outliers encountered with the adult fluke assay require the use of robust ssmd value ( ssmd * ) , which uses a similar ssmd formula as shown below , where the mean and standard deviations are replaced with median ( x ) and median absolute deviation ( mad ) values ( s2 ) . adult worm motility used the average motility index over 5  h for generation of ssmd * and ssmd* scores.absoluterobustssmd=|xtxnst2+sn2| the heat - killed parasites used as a positive control were an  extremely strong control  and used the most stringent category , shown in table  1 , for determination of assay quality . treatment magnitude in table  2 delineates ssmd ranges for effect quality as recommended by zhang ( 2011 ) . the xcelligence motility assay was developed ( smout et  al . , 2010 ) for measuring motility of hookworms and adult blood flukes ( schistosomes ) ( fig .  1 ) , and adapted later to several helminth species and developmental stages ( silbereisen et  al . you et  al . , 2013 ; zeraik et  al . , 2014 ) . the schistosome egg hatch assay requires 0.1 pbs as an approximation for the salinity of fresh water ( zeraik et  al . , 2014 ) ; tests using buffer alone ( 0.1 pbs ) at 25  khz and 50  khz showed both a lower and more temporally stable background motility index than the default 10  khz xcelligence frequency output ( fig . assessment of the three frequencies with egg hatching showed an improvement for both 25  khz and 50  khz frequencies compared to 10  khz ( 68.9% and 70.4% total motility index respectively ) , although 50  khz resulted in higher variability ( fig . furthermore , inhibition of egg hatching with 200  m fcf , reported by zeraik et  al . ( 2014 ) measured at 10  khz , resulted in a similar 60% reduction of signal across all frequencies ( fig .  2c ) . although it is worth noting the readings obtained at the two higher frequencies , 25 and 50  khz , demonstrated marginally steeper slopes than that at 10  khz ( fig . this may represent the frequency influencing hatching rates and miracidial motility , and/or the higher sensitivity of the assay reflects improved detection resolution of normal hatching . these xworm experiments were designed to determine which frequency generates the highest quality control parameters  that is the largest difference between positive and negative controls relative to the variation within each group . comparing assay quality using classic null hypothesis statistical significance tests with p - values is problematic ( cohen , 1994 ; kirk , 1996 ) given that the magnitude of the difference between groups is not measured , and only confirms that the difference is not zero ( zhang , 2007 ; goktug et  al . , a range of statistical assessments has been developed for high - throughput assays , many compatible with xworm analysis ( birmingham et  al . z factor was initially used for quality control , but over the past decade the more meaningful strictly standardized mean difference ( ssmd ) parameter has become more commonplace ( zhang , 2007 ; goktug et  al . , 2012 ) . the ssmd value is the difference between groups relative to the variance within each group . hence a high ssmd value represents a low variance within each group relative to the difference between the groups . this allows the output to measure the magnitude of impact more effectively that other metrics based on null hypothesis ( zhang , 2007 ; birmingham et  al . , 2009 ; kozak , 2012 ) . when used to evaluate the assay quality , the ssmd prime ( ssmd ) value is generated to compare the positive and negative control groups , i.e. heat killed and untreated parasites , respectively . zhang and colleagues determined ssmd ranges to asses assay quality based upon the predicted effect size of the positive control ( zhang , 2011 ) . as heat - killed control parasites were expected to be completely immobile , the strictest assay criteria were selected , and a summary is shown in table  1 ( zhang , 2011 ) . an absolute ssmd of 57 is considered a  good assay  rating , and values > 7 indicate an  excellent assay  meaning that the difference in motility between groups is > 7-fold the square root of sum of the variance of the groups . although the 25 and 50  khz groups had similar total motility indices ( fig .  2c ) , the lower variation obtained with the 25  khz frequency resulted in an  excellent assay screening of schistosome eggs in the presence of different compounds to detect a significant reduction in hatching / motility relies on the comparison of untreated negative control and treatment groups , in this case the drug fcf . a positive hit for a significant motility reduction from a treatment is any ssmd > 1.645 , as mathematically this is the equivalent of the conventional t - test p  <  0.05 ( zhang et  al . , 2010 ; zhang , 2011 ) . we observed that the ssmd values for parasites treated with 200  m fcf were significant ( > 1.645 ) at every frequency ; the 10 and 50  khz ssmd scores of 35 are considered  strong hits  ( table  2 ) and the 25  khz score ( > 5 ) was considered an  extremely strong hit  ( fig .  2d ) . comparison of control flukes and those treated with 50  m fcf did not reach significance at any frequency ( fig . 2 ) , the adult motility index score showed greater variability within groups at all the tested frequencies ( fig . 3a ) , probably due to differences in size / activity levels among individual worms . as each worm generally displays similar motility index scores over time , this variability was reduced when post - treatment motility index scores were converted to percentages of pre - treatment values as previously described ( fig . 2010 ) . because the occasional parasite was overly active or subdued in the untreated groups we utilized median scores to counter outliers . the subsequent ssmd values generated with median values have been designated robust ssmd ( zhang , 2011 ) and distinguished with an asterisk ( ssmd * and ssmd* ) . while the 10  khz frequency assay reached the  good assay  ( ssmd* 35 ) category with a score of 4.0 , both the 25 and 50  khz frequency reached the  excellent assay  range ( ssmd* > 7 ) with 7.3 and 7.9 scores respectively ( fig . the complete ablation of schistosome movement when flukes were cultured in 1  mm fcf was observed at all three frequencies in fig . 3b . both the 25 and 50  khz frequencies scored  extremely strong hits  ( ssmd *  > 5 ) with ssmd * scores of 6.9 and 8.8 respectively , while the 10  khz frequency scored ssmd *  =  4.1 and is categorized a  very strong hit  ( ssmd *  =  35 ) . given the success of xworm for measuring egg hatch in low salt conditions , we also tested the assay with the highly motile cercariae of s.  mansoni , the fork - tailed , free - swimming larval stage that is infective to humans . normally , when maintained in fresh water this developmental stage has minimal contact with the gold electrodes at the base of the wells , since it actively swims in the water column , and hence does not generate a substantial signal . however , when cultured in 0.1 pbs rather than water , the cercariae still twitched rapidly ( as seen during swimming ) , but sunk to the base of the wells making contact with the sensors of the e - plate . optimal numbers of cercariae per well , 2819000/well , were assessed at several frequencies . as with hatching of schistosome eggs , the motility was initially high but slowly declined over time ( fig . the area under the curve was employed to generate a total motility index ( fig . 4b ) , further emphasizing the higher scores generated at 25 or 50  khz compared to 10  khz . as with the egg hatching , the downward motility index slope tended to increase as frequency 4c ) we further demonstrated that 25  khz with 4500 cercariae / well scored the highest ssmd value of 42.5  substantially greater than the indeed , all three frequencies tested with 22509000 cercariae / well all scored above the  excellent assay  threshold ( table  2 ) . whereas the highest ssmd score would be optimal for applications requiring high sensitivity levels , a high - throughput screen that seeks to test thousands of samples would ideally consume fewer parasites / well . our data showed that a  good assay  score in the assay ( ssmd 57 ) was achievable at 562 cercariae / well with 25 and 50  khz frequencies . schistosomula of s.  mansoni move in the bottom of the well by an expansion and contraction process rather than the period of rapid twitching exhibited by the cercariae in the water column since the tails of schistosomula were shed during the mechanical transformation . testing of 1505000 schistosomula at the three discrete frequencies was unsuccessful , with minimal signals detected ( not shown ) . ( ssmd  <  3 ) and only 625 schistosomula at 25  khz stimulated a score of 3.03 , the  poor assay  category ( ssmd  =  35 ) . this score did allow for detection of motility , but not at levels for reliable testing ( data not shown ) . we previously developed a motility assay for adult and larval parasitic helminths ( smout et  al . our assay has been adapted by a number of other laboratories for a range of anthelmintics screens and other explorations of drug  helminth interactions ( mansour and bickle , 2010 ; silbereisen et  al . , 2011 ; tritten et  al . , 2012 ; you et  al . , 2013 ; zeraik et  al . , 2014 ) . more recently the assay was adapted to monitor hatching of eggs of the human schistosome , s.  mansoni , measuring a combination of direct miracidial electrode contact , the erratic movement of eggs induced by miracidial hatching as well as agitation of eggs by the highly motile hatched miracidia in the microplate well ( zeraik et  al . , 2014 ) . a low salt buffer is essential for schistosome egg hatching , as it mimics the natural fresh water body where hatching occurs . however , xworm requires buffered salts for conductivity measurements by the xcelligence system . the alternate frequency option of 25  khz and 0.1 pbs was verified by ssmd comparisons as the optimal combination to measure egg hatching using xworm . when assessing the utility of xworm to monitor motility of cercariae , the 25  khz frequency was determined to be optimal with 4500 cercariae / well ; with  good assay  motility scores with as few as 562 cercariae / well . although studying cercariae out of the natural fresh water environment is artificial , the motility assay in 0.1 pbs with the xcelligence system represents a surrogate approach to monitor motility of this developmental stage . despite this , caution should be taken as the artificial conditions may confound interpretation of results . despite the improvements to the xworm sensitivity , the range of xcelligence systems that incorporate the three frequency options ( 10 , 25 and 50  khz ) required for this optimized xworm assay span from small lab scale ( dual - plate unit  3    16 wells and single plate unit  96 wells ) to larger systems ( multi - plate unit  6    96 well and high - throughput unit  384 wells ) . the xworm assay is a robust yet sensitive technique that can now be predicted to be widely applicable across a range of species and life cycle stages , but recent progress has greatly expanded the available options to researchers seeking reliable methods to monitor parasitic helminths ( peak and hoffmann , 2011 ) . initially , employing the model free - living helminth caenorhabditis elegans , methods that allowed microfluidics - based detection of motility from an individual worm were developed ( rohde et  al . subsequent approaches were adapted from techniques originally designed to count mammalian cells  either based on mitochondrial enzymes ( lai et  al . , 2014 ) or computer algorithms that automated detection in bright field images ( white et  al . , 2013 ) . these algorithmic and enzymatic techniques have been shown to be efficacious for monitoring the viability of schistosomula , the ideal developmental stage for high - throughput anthelmintic drug screening . a range of cell health indicators are now available for monitoring schistosomula viability , such as the colorimetric alamar blue ( mansour and bickle , 2010 ) and the dual fluorogenesis assay using propidium iodide and fluorescein diacetate ( peak et  al . , 2010 ; marxer et  al . , 2012 ; rinaldi et  al . , progress has been accomplished in developing assays to detect and quantify metabolism , notably luminescent quantitation of atp ( lalli et  al . , 2015 ) and lactate  the end product of glycolysis ( howe et  al . , 2015 ) . these provide a low - cost approach to screening schistosomula with a standard microplate reader , and while effective at detecting reduced metabolism or impaired cellular membranes , these assays have difficulty in detecting phenotypic or motility distortion ( marxer et  al . , 2012 ; paveley et  al . , this impediment was overcome by the development of algorithms that detected schistosomula movement or phenotypic change by using automated image acquisition systems ( paveley et  al . progress towards the screening of other schistosome life cycle stages is less advanced , and the detection of lactate ( howe et  al . , 2015 ) is the only non - observational method that is also applicable to the adult schistosome . in terms of other flatworm genera , there has been some progress , such as screening for secreted enzymes in tapeworms ( mahanty et  al . , so while xworm is not suitable for measuring the motility of the schistosomula , our assay complements the current range of schistosomula viability assays with wide applicability for the remaining major aspects of the schistosome life cycle ( eggs , cercariae , adults ) . the widespread use of high - throughput methods to screen for new therapeutics or emerging drug resistance will facilitate greater capacity to drug screens to parse small compound libraries for novel interventions . monitoring the efficacy of anthelmintic medicines and screening for new drugs was previously hindered by the lack of objective , high - throughput approaches . here we present a more sensitive xworm assay for s.  mansoni adults and egg hatch monitoring and expanded capabilities to monitor cercariae . we focused on s.  mansoni , but the xworm assay is envisaged to be applicable for the majority of helminth species and developmental stages where egg hatch assays and/or motility are deployed presently to evaluate parasite viability . additionally , this assay further improves high - throughput helminth viability screening and will be an asset in the fight against the wide range of biomedical and veterinary helminths . mjs helped conceive , design and analyze experiments , provided project oversight and wrote the manuscript . gr and mjs have received travel funds to present at an xcelligence workshop , and the 2014 the international congress of parasitology meeting through the competitive acea travel award program , respectively . acea played no role in performing the experiments , analyzing the data , or developing the manuscript other than providing raw data for fig .