INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites
[['Intracellular protozoan parasites are responsible for worldwide infectious diseases with a high impact in public health for developing countries .', 'Among them , Leishmania parasites are causative agents of leishmaniasis , a worldwide endemic disease with diverse clinical manifestations ranging from self-healing skin ulcers to fatal outcome , depending on parasite species and host immune status or genetics [1] .', 'In mammals , Leishmania parasites replicate as amastigotes within parasitophorous vacuoles ( PVs ) of macrophages .', 'Without a vaccine and taking into account the limited number of existing drugs , the screening of new anti-leishmanial compounds or characterization of new drug target candidates is therefore a research priority .', 'Drug resistance to available treatments has also been well documented in certain areas , and partially explained in natural or experimentally resistant laboratory strains [2] , [3] .', 'Parasites isolated from patients refractory to treatment confirm the presence of circulating resistant parasites [4] and imply a careful surveillance of parasite drug susceptibility .', 'All these research activities require the use of common calibrated and reliable procedures to monitor parasite proliferation inside host cell , maintaining physiological conditions as much as possible [5] .', 'The most popular method still consists of manually counting intracellular parasites after Giemsa staining [6] .', 'This direct counting approach is largely employed because Giemsa stain is cheap and only requires basic equipment ( i . e light microscope ) .', 'Microscopic counting is , however , a laborious task merely providing a global estimation of the parasite burden that is further highly prone to operator experience and subjectivity .', 'Numerous alternative indirect approaches have been proposed in Leishmania [7]–[11] .', 'Most are based on reporter genes assays that increase screening capacities while limiting human intervention .', 'Indirect reporter assays , however , average the biological response of thousands of cells without integrating critical factors such as the percentage of infection and the discrimination between intra- and extracellular parasites .', 'Homogenous expression of reporter genes over time and biological stages in Leishmania further require genomic integration [7] , [8] .', 'These transformation and selection steps can have a profound impact on the phenotypic traits of the original parasite population .', 'This is particularly relevant when testing susceptibility of parasite strains isolated from drug unresponding patients .', 'The development of image processing and analysis techniques ( together with the improvement of fluorescence microscopy instrumentation ) has recently emerged as a new powerful solution for drug screening and susceptibility tests against intracellular protozoan parasites .', 'High Content Screening ( HCS ) systems have been indeed successfully adapted to major intracellular protozoan parasites and allow direct analysis of both parasite and host cell responses to drug candidates [12]–[15] .', 'However , considering the cost of such equipment and the expertise required to manage HCS instrumentation and analysis , those approaches will remain restricted to few laboratories in the world .', 'Extracting and quantifying information from biological images is a well-defined and common task in image processing .', 'Some open source solutions such as ImageJ software ( http://rsb . info . nih . gov/ij ) are very helpful for biologists with more than 500 plugins available .', 'Nevertheless , mastering image processing concepts require specific competences and , to our knowledge , no versatile open-access solutions for biologists are available to automate intracellular parasite quantification .', 'We present INsPECT ( INtracellular ParasitE CounTer ) , the first open-source Java based software that uses fluorescent DNA staining and image processing framework to automate infection level measurement .', 'This can be done through a user-friendly interface , where image files obtained from any fluorescent microscope and magnification , can be processed individually or as a batch , automatically or in a custom mode , without any experience required on image analysis .', 'The software runs either with DNA fluorescent image files alone , or in combination with the corresponding phase contrast or DIC ( Differential Interference Contrast ) image set .', 'Providing this complementary information allows automatic cell boundaries detection and discrimination between intra- and extracellular parasites without any additional use of fluorescent cytoplasm/membrane marker .', 'Output files comprise annotated images and a report table with all information needed for most of experimental infection studies in vitro: total number of cells , total number of parasites , percentage of infected cells , mean number of parasite/cell , parasitic index .', 'Software robustness was validated for the calculation of Ec50 toward intramacrophagic L . infantum treated with pentavalent antimonials ( glucantime ) as a proof-of-concept study .', 'Alternative analyses performed on T . gondii infected fibroblasts further confirm that INsPECT software utilization may be enlarged to unrelated intracellular parasites .'], ['The image-processing pipeline for cell , parasite and cytoplasm detection is illustrated in figure 1 and detailed below .', 'Acquired fluorescent images first need to be saved in tiff format , 8bit type and inverted to obtain dark objects in white backgrounds .', 'These steps can be performed directly from the acquisition software , or lately by different image analyzing softwares .', 'A simple procedure with ImageJ software is described in INsPECT user manual ( File S1 ) .'], ['Screen shots of the INsPECT interface are shown in Figure 2 , all frames are designed in such a way that a non-experimented user in image processing can easily work with them .', 'A complete explanation of the software utilization can be found in the INsPECT user manual ( File S1 ) .', 'The software comes with a basic functional image viewer that helps to see particles in images easily ( Figure 2A ) .', 'Software can run either in an automatic or in a custom mode ( Figure 2B ) .', 'In the former case , parameters and options are set to some default values that are in agreement with the nature of most input images; however , end users can also adapt their intended parameters and options for cell , parasite and cytoplasm pipelines manually ( see details in user manual ) .', 'Users should then indicate input and output folders used for the rest of the analysis , and the nature of the images to analyze ( Figure 2C ) .', 'In the same window , users further specify which result files need to be saved .', 'A running log and progress bar is displayed during the analysis ( Figure 2D ) .', 'Basically , the software takes images from the input folder , applies proposed algorithmic pipeline to each image , extracts needed information and finally saves the results in both text and visual forms in the specified output folder .', 'If DAPI and phase contrast image pairs are available , the software extracts cell outlines to distinguish intra from extracellular parasites ( Figure 2E ) .', 'An example of the accuracy of the proposed cell edges detection algorithm is illustrated in figure S1 for both phase contrast and DIC microscopic images .', 'Alternatively , when DIC images are not available or of minor importance in final results , the software can deal with DAPI images only ( Figure 2E ) .', 'The processing of each image takes less than 30 seconds , without any need of user interaction .', 'To validate the software accuracy in detecting intracellular Leishmania parasites , we performed two parallel experiments with L . infantum infected macrophage THP-1 cell line treated by increasing concentration of glucantime ( 0-25-50-100 µg/ml ) , the mainstay treatment for leishmaniasis .', 'After infection , drug incubation and cell fixation , one series was stained with Giemsa for manual counting and the other with DAPI , both procedures taking approximately similar time ( 30 minutes ) .', 'In the manual counting , 300 cells ( 100 for each triplicate ) were counted under light microscope for each drug concentration to determine the parasitic burden ( parasitic index ) , calculated as the percentage of infected macrophages x the mean number of amastigotes per macrophage ( Table 1 ) .', 'In parallel , random DAPI images were acquired with fluorescent microscope ( 40× objective ) until approximately 300 cells were reached by condition ( corresponding to 31 images and 1402 cells in total ) , together with their respective phase contrast images for cytoplasm detection ( File S2 ) .', 'A batch analysis of all images was performed with INsPECT software ( automatic parameters ) and was completed in less than 7 minutes ( Table 1 ) .', 'Direct comparison of output results shows that the number of detected intracellular parasites , and the subsequent calculated parasitic index ( PI ) , is markedly higher with INsPECT software than with Giemsa method ( Figure 3A ) .', 'Verification of the INsPECT analyzed images in the output folder allows us to confirm that the detected particles accurately correspond to true parasites , excluding therefore an overestimation of the parasite load by the software .', 'Conversely , the human operator naturally tends to select fields and cells offering the best visual resolution with Giemsa , generally excluding highly infected cells in which parasites cannot be well discriminated .', 'The analysis of the “Cell Parasites Report” generated by INsPECT software confirms that highly infected cells are indeed frequently observed in untreated or low glucantime treatment conditions , and that this category of cells is mainly responsible for the differences observed between the two methods ( Figure S2 ) .', 'Once normalized to the non-treated infected control however , scaled PI values are very similar with calculated Ec50 of 73 . 97 µg/ml ( 95% confidence interval [CI] , 60 . 31 to 90 . 71 ) and 79 . 53 µg/ml ( 95% confidence interval [CI] , 54 . 7 to 115 . 6 ) for INsPECT and Giemsa methods , respectively ( Figure 3B ) .', 'To further validate software robustness against unrelated intracellular parasite , we performed analyses on HFF fibroblasts labeled with Hoechst fluorescent DNA stain and infected by fluorescent YFP-expressing T . gondii RH parasites ( File S3 ) .', 'All input images were obtained from HCS system designed for fully automated image acquisition and analysis ( see details in Methods section ) .', 'The analysis of representative images was performed with INsPECT software using only DNA fluorescent input images and choosing automatic mode .', 'The size and distribution of Toxoplasma parasites strongly differ from that of L . infantum amastigotes , with tachyzoites parasites being mostly grouped in small aggregates inside cell cytoplasm .', 'As shown in figure 4 , comparison of visual output annotated images to YFP labeled parasites confirms , however , a good correspondence between the two methods .'], ['Monitoring intracellular parasite proliferation is an essential and quotidian activity for many academic or clinical diagnostic laboratories .', 'The majority are still using manual microscopic counting , an unpopular and time-consuming task , that is further highly dependent on operator experience and objectivity thus leading to strong inter-laboratory variability .', 'These facts make intracellular parasites counting a natural assignment for automation .', 'In this study , we describe an innovative image analysis software allowing automatic detection of intracellular parasites and calculation of infection parameters such as the percentage of infected cells , the mean number of parasites per cell or the parasitic index .', 'In a concept comparative study , we show that INsPECT software performs the complete analyze of 1402 Leishmania infected cells in less than 7 minutes , while counting the equivalent number of cells under microscope with Giemsa staining can take hours of effective work .', 'This timesaving is furthermore directly proportional to the number of conditions/replicates to analyze .', 'Because images are stored annotated in the output folder , users have the possibility to verify whether recognition of parasites and cells is satisfying enough and can optimize detection parameters accordingly ( see user manual , File S1 ) .', 'The use of common and affordable fluorescent nucleic acid stains such as DAPI or Hoechst present the advantages to display a bright , homogenous and stable labeling of both intracellular parasite and host cell DNA .', 'Furthermore , they allow working under physiological conditions with any wild-type parasite species , including clinical isolates .', 'Leishmania , like all members of the kinetoplastid family , possess two large DNA containing organelles ( kinetoplast and nucleus ) .', 'Kinetoplast is an A-T rich DNA structure binding DAPI and Hoechst stains with more affinity [13] , [30] and that consequently appears highly brighter than parasite nucleic DNA .', 'Since kinetoplasts occupy very tiny portions of input images , and may be occasionally very similar to random generated noises , the task of extracting them with existing thresholding methods is complex because available methods often work efficiently for the objects with quite noticeable size and shape and investigable intensity functions .', 'For that , we propose a new method which thresholds parasites with maximum accuracy by defining further discrimination steps than just the particle size .', 'DNA structures of all kinetoplastids ( including T . cruzi and other Leishmania species ) appear very similar on images stained with DAPI [13] , [14] .', 'Accordingly , we expect INsPECT software to be as effective with any other intracellular kinetoplastid parasite as with L . infantum .', 'Besides , we confirm that input images of T . gondii intracellular parasites acquired with HCS instrumentation could also be efficiently processed , even in automatic mode , proving therefore that INsPECT software is versatile enough to work with a variety of unrelated parasites and host cells .', "Regarding host cell nuclei in DAPI images , illumination variance was handled well by firstly , fitting appropriate noise model which removes noise efficiently while preserving precious cell portions' edge data and secondly hiring adaptive threshold with logical and automatic assignment of local window size .", 'Overlapping cells were also detected and separated using state of the art quantification and segmentation approaches .', 'Cytoplasm boundaries of individual cells are visible structures in DIC or phase contrast images and we used these properties to create a new algorithm that automatically detects cell boundaries to discriminate intra- from extracellular parasites .', 'This function allows considering only intracellular parasites in final calculated infection levels .', 'This can be very useful in the case of Leishmania promastigotes infection since we experimented that numerous non-internalized parasites may stay attached to macrophages membrane following initial contact , even after extensive washes .', 'Because their DNA will be as well labeled with DAPI stain , they can therefore induce a bias if analyze is based only on DAPI images .', 'With Giemsa , these parasites are simply excluded from the counting by the human operator .', 'In the case of indirect reporter gene assays using microplate readers however , these parasites can be the source of a false positive signal .', 'In conclusion , we can state that INsPECT software accuracy and effectiveness is at least as reliable as the existing global estimation methods .', 'Considering its flexibility , simplicity and the state of the art perspectives introduced , it could compete with available commercial and academic packages to become a new helpful tool for the scientific community working with intracellular parasites .']]
['Introduction', 'Methods', 'Results', 'Discussion']
['Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries .', 'Leishmania sp .', ', Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals , including humans .', 'Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs , analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains .', 'Nevertheless , laborious manual microscopic counting of intracellular parasites is still the most commonly used approach .', 'Here , we present INsPECT ( Intracellular ParasitE CounTer ) , an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining .', 'It offers the possibility to choose between different types of analyses ( fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites ) , and software running modes ( automatic or custom ) .', 'A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI ( 4′ , 6-diamidino-2-phenylindole ) confirms a good correspondence between digital results and the “gold standard” microscopic counting method with Giemsa .', 'Interestingly , this software is versatile enough to accurately detect intracellular T . gondii parasites on images acquired with High Content Screening ( HCS ) systems .', 'In conclusion , INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites .']
['Research on intracellular parasites require using non-invasive technologies to follow up parasite proliferation inside their natural host cells by staying in the more physiological conditions as possible .', 'High Content Screening ( HCS ) technology has recently emerged as a powerful image-based approach to screen new anti-parasitic compounds or to test parasite susceptibility to existing drugs in vitro .', 'Nevertheless , such equipments will remain poorly accessible for most of academic and clinical diagnostic laboratories that mostly use more affordable , but laborious , microscopic counting procedures .', 'The current work proposes new image-based , open-source software which provides a fast and accurate solution for investigating intracellular parasite quantification .', "Through an easy-to-use interface , cells' and parasites' information are dug out from DNA fluorescent images , and host cells' boundaries are extracted from corresponding phase contrast image set .", 'Parasites are then reassigned to their related cells and intra/extracellular parasites are discriminated for each cell .', 'The software further automatically calculates all data required for most of experimental infection studies .', 'INsPECT software is proposed as a free substitute or complement to the available quantification methods for measuring Leishmania infection level in vitro .', 'It may be enlarged , however , to different intracellular trypanosomatids or unrelated parasites such as T . gondii .']
['medicine and health sciences', 'pathology and laboratory medicine', 'engineering and technology', 'signal processing', 'tropical diseases', 'microbiology', 'parasitic diseases', 'parasitology', 'neglected tropical diseases', 'veterinary science', 'software design', 'infectious diseases', 'computer and information sciences', 'veterinary diseases', 'zoonoses', 'protozoan infections', 'pathogenesis', 'toxoplasmosis', 'host-pathogen interactions', 'image processing', 'leishmaniasis', 'software engineering', 'biology and life sciences']