giardiasis causes significant worldwide morbidity with an estimated 184 million symptomatic cases annually ( pires et  al . , 2015 ) and an associated 171,100 disability - adjusted life years ( dalys ) ( kirk et  al . , 2015
while giardiasis is more prevalent in the developing world it is also a burden in developed countries , with hospital based treatments in the united states of america costing $ 34.4 million ( usd ) annually ( collier et  al . , 2012 )
giardiasis is commonly associated with clinical symptoms including nausea , vomiting and acute diarrhoea ( nash et  al . , 1987 , farthing , 1996 ) .
however it can manifest as a chronic disease and cause malabsorption , weight loss and failure to thrive in children ( al - mekhlafi et  al .
, 2005 , al - mekhlafi et  al . , 2013 , bartelt et  al . ,
there is also mounting evidence that giardia infection may be linked to irritable bowel syndrome , food allergies and obesity ( di prisco et  al . , 1998 , hanevik et  al . , 2009 ,
as there is no currently available vaccine for humans , the control of giardiasis is dependent on chemotherapy .
current chemotherapeutic options are limited to a small number of compounds which are associated with treatment failures and clinical resistance ( reviewed in ansell et  al . , 2015 ) .
the 5-nitroimidazole class of compounds , typically metronidazole , are the most commonly used treatment agents ( watkins and eckmann , 2014 ) .
however , these compounds have reported clinical failure rates of up to 40% ( oren et  al . , 1991 , farthing , 1996 ; reviewed in watkins and eckmann , 2014 , nabarro et  al . , 2015 ) and can also cause significant side - effects including neurological disorders and sudden death ( escobedo and cimerman , 2007 ) .
however , the efficacy of these drugs varies widely ( e.g. hall and nahar , 1993 , escobedo et  al . ,
in addition , the benzimidazole drugs appear particularly susceptible to the development of drug resistance , with data suggesting that parasite resistance can be easily selected in  vitro ( gardner and hill , 2001 ) . new anti - giardia agents with improved efficacy and toxicity
a number of low to high throughput in  vitro assays have been developed to identify new compounds active against giardia .
however , most rely on metabolic indicators or manual cell counting . activity assays that rely on manual cell counting via microscopy have the advantage of permitting the assessment of growth at multiple time - points and provide useful morphological information , but are time consuming and may be subjective . while the more automated assays that make use of growth indicators including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ( mtt ) , 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ( xtt ) , resazurin ( alamarblue ) h - thymidine , atp content or the assessment of glucuronidase activity in transgenic parasites ( mller et  al .
, 2009 ) are more rapid , they inherently increase assay cost , provide limited activity / morphology information and permit only single time - point of assessment .
activity assays reliant on transgene expression are also limited to assessing activity against genetically manipulated parasites .
efforts to improve current growth assay methods have included combining microscopy with automated image analysis software to decrease time limitations associated with manual enumeration methods ( bonilla - santiago et  al . , 2008 , faghiri et  al . , 2011 , gut et  al . , 2011 ) .
( 2011 ) parasites are stained with 4,6-diamidino-2-phenylindole ( dapi ) to automatically distinguish and enumerate living trophozoites without bias .
while this significantly reduces assay evaluation time , parasites must still be fixed and stained which necessitates extra handling and eliminates the possibility of multiple time - point evaluations . in this study
, we developed an automated live - cell digital phase - contrast microscopy assay to assess the activity of compounds against giardia trophozoites in  vitro .
the perkin - elmer operetta , with its associated harmony and phenologic software , was used to exploit the power of automated digital phase - contrast microscopy and image analysis as a mechanism to identify and enumerate parasites based on their morphology without the need for a cell marker .
a particular advantage of this approach is the ability to assess parasite growth at multiple time - points .
this assay was used to assess the anti - giardia activity of compounds from the  malaria box  .
the  malaria box  , a set of compounds with known activity against mammalian cells ( kaiser et  al . , 2015 ) multiple parasite species including p.  falciparum ( spangenberg et  al . ,
2013 ) , toxoplasma gondii , entamoeba histolytica ( boyom et  al . , 2014 ) , cryptosporidium parvum ( bessoff et  al . , 2014 ) , leishmania major ( khraiwesh et  al . , 2016 ) and trypanosoma spp .
( kaiser et  al . , 2015 ) has never previously been assessed for anti - giardia activity .
g.  duodenalis ( strain bris/91/hepu/1279 ; metronidazole sensitive ; assemblage b ( upcroft et  al . , 1995 , nolan et  al . , 2011 ) ) was grown axenically ( 3% o2 5% co2 , in n2 at 37 
c ) in kiesters - modified tyi - s-33 media in 8  ml borosilicate vials ( pyrex glass , no . 9825 ; vwr ) as previously described ( keister , 1983 , meloni and thompson , 1987 ) .
media was prepared on a weekly basis and stored at 4  c . when required for use ,
aliquots were supplemented with 10% foetal bovine serum , 100 units / ml penicillin and 100  g / ml streptomycin .
albendazole , metronidazole and furazolidone were obtained from sigma - aldrich , usa and prepared in 100% dmso to stock concentrations of 1050  mm .
malaria box compounds were obtained from the medicines for malaria venture ( mmv ; www.mmv.org ) as 10  mm stocks prepared in 100% dmso .
giardia parasites were grown in stock 8  ml borosilicate tubes ( section 2.1 ) to    80% confluence .
after detachment , parasites were collected , counted using a haemocytometer and seeded in 96-well micro titre plates ( corning costar 3596 ; total volume 200  l ; 2    10 to 5    10  cells / well ) .
outside wells of plates contained phosphate - buffered saline to reduce evaporation ( pbs ; 200  l ) .
plates were incubated at 37  c in sealed , activated anaerocult c mini bags as per manufacturer instructions as previously described ( upcroft and upcroft , 2001 ) .
growth of parasites seeded in triplicate wells on two separate occasions was assessed at 24 and 48  h by digital phase - contrast microscopy , enumerated using harmony and phenologic software ( section 2.6 ) and by the manual counting of bright - field images .
data from all experiments were combined ( mean parasite count/1.7  mm    sd ) and manual versus automated counts were compared using a student 's t - test ( graphpad prism 7 ) .
after detachment , parasites were collected , counted using a haemocytometer and seeded in 96-well micro titre plates ( corning costar 3596 ; total volume 200  l ; 6    10 to 5    10  cells / well ) . however , as a reliable source of anaerocult c mini bags ( merck , millipore ) could not be obtained , microaerophilic conditions were established by incubating plates at 37  c in air - tight chambers filled with 3% o2 5% co2 in n2 as previously described ( gut et  al . , 2011 ) .
growth of parasites was assessed at 24 and 48  h by digital phase - contrast microscopy and enumerated using harmony and phenologic software ( section 2.6 ) .
data are presented as mean trophozoite count    sd of 4 separate experiments , each carried out in triplicate wells .
the average doubling time between 24 and 48  h for each seeding concentration was calculated using the equation , t  = ( 24 )    log ( 2)/log ( c / c ) where t  =  doubling time , c was the average 24  h count and c was the average 48  h count .
as assay plates were outside of anaerobic conditions during imaging ( section 2.6 ; 20  min ) and then returned to culture post - imaging for further incubation and assessment , the impact of imaging on parasite growth was assessed . in these assays two identical 96-well micro titre plates were prepared .
one plate was imaged at 24 and 48  h and the other only at 48  h. in brief trophozoites were seeded into 96-well plates ( 3    10 to 3.75    10 parasites / well in 200  l ) and incubated in 3% o2 5% co2 , in n2 at 37  c until imaging ( section 2.6 ) .
the 24 and 48  h imaged plate was returned to culture conditions after imaging at 24  h and re - imaged again at 48  h whereas the 48  h only plate remained in microaerophilic conditions until imaging at 48  h. each cell seeding concentration was plated in six technical replicates on a single plate and each assay was repeated on three separate occasions .
data are presented as mean parasite count/1.7  mm    sd and cell counts were compared using a student 's t - test ( graphpad prism 7 ) .
each compound was serially diluted in triplicate wells ( 100  l ; 8 point dilution series for albendazole and furazolidone and 15 point dilution series for metronidazole ) , and all wells except media only controls , were seeded with 1.5    10 
 giardia trophozoites ( 100  l ; 200  l final volume ) .
plates were incubated in 3% o2 5% co2 , in n2 at 37  c until imaging and growth analysis ( section 2.6 ) at 24 and 48  h. each assay included no drug with vehicle ( 0.2% dmso ) and no vehicle controls and in each case three independent assays were carried out .
the concentration of dmso in drug dilutions was kept constant at 0.2% and as previously shown ( johns et  al . , 1995 )
mean percentage growth inhibition compared to vehicle ( 0.2% dmso ) and background controls was determined for each assay .
ic50 values were calculated using log - linear interpolation ( huber and koella , 1993 ) .
all malaria box compounds were screened for activity against g.  duodenalis bris/91/hepu/1279  at a final concentration of 10  m in singlicate , in two independent experiments .
each plate included , background media , vehicle ( 0.2% dmso ) , no vehicle and albendazole ( 10  m ) controls .
assays were performed under the same conditions as those used to assess the activity of control anti - giardia compounds ( section 2.4 ; 1.5    10 parasites / well in final volume 200  l ; imaged at 24 and 48  h ) .
compounds demonstrating greater than 50% inhibition at this concentration were assessed for activity at 5  m in duplicate ( n  =  2 ) .
z - factors were calculated for each plate of each screening assay as previously described ( zhang et  al . , 1999 ) .
compounds showing 50% inhibition at 5  m were further investigated to determine ic50 values as described for control compounds ( section 2.4 ; each titration was performed in duplicate on three occasions ; 8 point dilution series ; compound concentration range 10,00078  nm ) .
plates were removed from incubation and each well was imaged using brightfield and phase - contrast microscopy ( total area imaged 1.7  mm < 20  min / plate ) before being returned to incubation if required .
brightfield images were taken 1  m from the base of each well , with exposure set to 100  ms .
digital phase - contrast images were taken with 40  ms exposure between  5  m and 5  m , with a speckle scale of 10  m .
images were automatically analysed and parasites enumerated with harmony and phenologic software manually trained to identify and count trophozoites .
manual training was performed within harmony and phenologic using the  select population  building block and the  linear classifier  method .
after training , an algorithm to identify trophozoites based on properties including their size and morphology was generated and used to assess all subsequent images .
growth assessment experiments showed that enumeration of giardia parasites by digital phase - contrast microscopy and automated image analysis is as least as effective as examining parasite growth by manual cell counting ( fig .  1 ) .
1 c and d ) and there was no statistically significant difference in parasite numbers when assessed manually or automatically using the harmony and phenologic software ( fig . 
data describing trophozoite growth in assay culture conditions suggested that 1.5    10  cells / well was the highest initial cell density able to maintain adequate bris/91/hepu/1279 growth for 48  h , the duration of planned screening assays ( fig . 
when seeded at higher or lower cell concentrations mean parasite doubling times increased ( fig . 
imaging was also found to have no significant impact on parasite growth as growth of parasites on plates imaged at 24 and 48  h was not significantly different to those grown on plates imaged only at 48  h ( fig .  2b ; p  =  0.68 ) .
the in  vitro anti - giardia activity of albendazole , metronidazole and furazolidone was assessed using digital phase - contrast microscopy and automated enumeration ( table  1 ) .
none of the ic50 values determined using the assay described here fell further than a single standard deviation from the published range at either 24  h or 48  h ( table  1 ) .
preliminary screens of the malaria box compound set identified 122 compounds with > 50% inhibition at 24 or 48  h when assessed at 10  m ( fig .  3a and b ; table  s1 ) .
the z factor of all assays plates in the 10  m screen was > 0.5 ( average    sd ; 0.74  +  0.11 ) . further analysis of the 122 compounds identified 22 with > 50% growth inhibition at 5  m ( fig . 
the z factor of all assays plates in 5  m assays was also > 0.5 ( average    sd ; 0.73  +  0.10 ) . further dose response analysis of the 22 compound with > 50% inhibition at 5  m identified three compounds ( mmv007384 , mmv019690 and mmv006203 ) with sub-m ic50 values ( table  2 , table  s1 & fig . 
we have developed an in  vitro medium throughput assay that permits giardia drug susceptibility testing in real - time without any need to stain parasites .
this assay is unique in that it harnesses the power of digital phase - contrast microscopy and dedicated analysis software to identify and count parasites thereby permitting speedy , multi - time point analysis of live parasite numbers .
a comparison of automatically generated parasite counts with manual counts demonstrates that the system can quickly and reliably assess trophozoite numbers ( fig .  1 ) .
as this assay permits the activity of compounds to be assessed at multiple time - points without any impact on parasite growth ( fig . 
additional information regarding the time course of compound activity and morphological effects , which can be derived from acquired images , may aid in compound triage and mechanism of action studies .
further reductions in cost and additional data acquisition may also be possible given that the assay is likely to be amenable to miniaturization and longer assessment periods ( up to 72  h ( upcroft and upcroft , 2001 , kulakova et  al .
while a potential limitation of the current assay may be in its assessment of parasite number rather than a metabolic parameter linked to viability , compounds with static activity can be of use therapeutically ( pankey and sabath , 2004 ) .
in addition , metabolic assays can be associated with the same liability in the case of dormancy or when the compounds assessed interfere with the metabolic process used to quantitate inhibition ( collier and pritsos , 2003 , ulukaya et  al . , 2004 ) . indeed , as a result of continued growth and the enhanced metabolic activity of controls over time , both assay types are likely to identify compounds with static activity as inhibitors .
more specialized methods designed to assess mode of action are therefore more adequately placed to examine the nature of compound activity , post - identification .
an additional limitation of the current assay that should be considered is its assessment of parasite number based on adherence .
while this is an inherent limitation of other assays including those that require the removal of culture media and well - washing prior to activity assessment , this would mean that the assay is likely to identify compounds that effect attachment in addition to compounds that effect replication .
although the consequences of this anti - attachment activity in the in  vivo setting may be limited , more specialized assays would be required to discriminate between compounds that effect attachment versus those that inhibit replication .
nevertheless , the ability of the current image - based assay to effectively examine the activity of compounds against giardia parasites was demonstrated by assessing the activity of control anti - giardia compounds albendazole , metronidazole and furazolidone , with ic50 values generated by the automated imaging and enumeration system being within the range of previously published studies ( table  1 ) .
the suitability of the assay as a mechanism to identify compounds with activity against giardia parasites was also demonstrated by assessing the malaria box compound set for potential anti - giardia activity .
the mean z factor for all plates in these assays ( 0.74 in 10  m and 0.73 in 5  m assays ) suggest that the assay is of excellent quality ( zhang et  al . , 1999 ) and a promising new tool for giardia parasite drug discovery . of interest a previously described image - based giardia assay which is dependent on parasite
staining and hence an end - point assay , reported a z factor of 0.54 ( gut et  al . , 2011 ) .
the identification of anti - giardia compounds within the malaria box set that have structural similarities to known anti - giardia compounds provides additional evidence that the current assay is suitable for compound activity assessment .
mmv007384 , the most potent of the anti - giardia hits identified ( fig . 
s1 , table  2 ; 24  h ic50 0.8  m and 48  h ic50 0.6  m ) is a benzimidazole .
in addition mmv667492 ( table  s1 ; 24  h ic50 3.7  m and 48  h ic50 2.6  m ) is a napthoquinone similar to menadione that has been shown to have promising efficacy against g.  duodenalis trophozoites and cysts in  vitro ( paget et  al . , 2004 ) .
two malaria box compounds , in addition to mmv007384 were identified to have sub m ic50 values against giardia parasites in the current study .
these compounds were mmv019690 ( table  2 ; 24  h ic50 2.8  m and 48  h ic50 0.9  m ) and mmv006203 ( table  2 ; 24  h ic50 3.1  m and 48  h ic50 0.7  m ) . while the selectivity index for mmv019690 ( 4.8 ; table  2 ) , generated using ic50 data against mrc-5 fibroblasts ( kaiser et  al . ,
2015 ) suggest this compound may be associated with toxicity , the selectivity index of mmv006203 , ( 25.7 ; table  2 ) , was more favourable , falling within recently published lead criteria range ( katsuno et  al . , 2015 ) .
importantly , the identification of cell debris in images acquired during the assessment of mmv006203 and mmv019690 ( fig . 
the current study has validated digital - phase contrast microscopy and automated parasite enumeration as a method to investigate giardia drug susceptibility and identified new chemical scaffolds with anti - giardia activity that may warrant further investigation .
unlike previously published image - based assessment giardia assays , the method described in this study negates the need for cell staining and permits multiple - time point activity assessment which can improve screening costs and only add value to current drug discovery efforts .
 giardia duodenalis is an intestinal parasite that causes giardiasis , a widespread human gastrointestinal disease . 
 treatment of giardiasis relies on a small arsenal of compounds that can suffer from limitations including side - effects , variable treatment efficacy and parasite drug resistance . 
 thus new anti - giardia drug leads are required . the search for new compounds with anti - giardia activity currently depends on assays that can be labour - intensive , expensive and restricted to measuring activity at a single time - point . here 
 we describe a new in  vitro assay to assess anti - giardia activity . 
 this image - based assay utilizes the perkin - elmer operetta and permits automated assessment of parasite growth at multiple time points without cell - staining . using this new approach 
 , we assessed the  malaria box  compound set for anti - giardia activity . 
 three compounds with sub-m activity ( ic50 0.60.9  m ) were identified as potential starting points for giardiasis drug discovery . 
Introduction
Materials and methods
Results
Discussion