Trypanosoma cruzi infectivity assessment in “in vitro” culture systems by automated cell counting

Accepted Manuscript Title: Trypanosoma cruzi infectivity assessment in “in vitro” culture systems by automated cell counting Author: Ana Liempi Christian Castillo Mauricio Cerda Daniel Droguett Juan Duaso Katherine Barahona Ariane Hern´andez Cintia D´ıaz-Luj´an Ricardo Fretes Steffen H¨artel Ulrike Kemmerling PII: DOI: Reference:

S0001-706X(14)00392-1 http://dx.doi.org/doi:10.1016/j.actatropica.2014.12.006 ACTROP 3515

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

14-7-2014 4-11-2014 9-12-2014

Please cite this article as: Liempi, Ana, Castillo, Christian, Cerda, Mauricio, Droguett, Daniel, Duaso, Juan, Barahona, Katherine, Hern´andez, Ariane, D´iaz-Luj´an, Cintia, Fretes, Ricardo, H¨artel, Steffen, Kemmerling, Ulrike, Trypanosoma cruzi infectivity assessment in “in vitro” culture systems by automated cell counting.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2014.12.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Trypanosoma cruzi infectivity assessment in “in vitro” and “ex vivo” culture systems by automated cell counting and Real time PCR Ana Liempia*, Christian Castilloa*, Mauricio Cerdaa, Daniel Droguetta,c, Juan Duasoa, Katherine Barahonaa, Ariane Hernándeza, Cintia Díaz-Lujánb, Ricardo Fretesb, Steffen Härtela and Ulrike Kemmerlinga,# a

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Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Región Metropolitana, Santiago de Chile, Chile. Zip code: 8380453 b Department of Histology and Embryology, Faculty of Medicine, Universidad Nacional de Córdoba-INICSA (CONICET), 5000 Córdoba, Argentina and IICSHUM and Cathedra of Histology, Embryology and Genetic, Health Department, Universidad Nacional La Rioja. 3300 La Rioja, Argentina. Zip code: X5003 c Departamento de Estomatología, Facultad de Ciencias de la Salud, Universidad de Talca, Av Lircay s/n, VII Región, Talca; Chile. Zip code: 3460000

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* Both authors contributed equally to this work

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# To whom correspondence should be addressed: Tel.: 56-2-9786261, Fax: 56-2-9786264, E-mail: [email protected]; [email protected]

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Highlights

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► Automated cell counting to determine Trypanosoma cruzi infection in cells ► Use of a commercial available software (Matlab®)

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Abstract Chagas disease is an endemic, neglected tropical disease in Latin America that is caused by the protozoan parasite Trypanosoma cruzi. In vitro and ex vivo models constitute the first

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experimental approaches to study the physiopathology of the disease and to assay potential new trypanocidal agents.

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Here, we report and describe clearly the use of commercial software (MATLAB®) to quantify T. cruzi amastigotes and infected mammalian cells (BeWo) and compared this

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analysis with the manual one. There was no statistically significant difference between the manual and the automatic quantification of the parasite; the two methods showed a correlation analysis r2 value of 0.9159. The most significant advantage of the automatic quantification was the efficiency of the analysis We also describe the use of the real-time PCR (qPCR) double quantification (∆∆Ct) method for human placental chorionic villi

explants (HPCVE). The drawback of this automated cell counting method was that some parasites were assigned to the wrong BeWo cell, however this data did not exceed 5% when adequate experimental conditions were chosen. We conclude that both parasite this quantification methods are constitute an excellent tools for evaluating the parasite load in cells or tissues and therefore constitutes an easy and reliable ways to study parasite infectivity.

Keywords

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Trypanosoma cruzi, infectivity, quantification

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Introduction

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Chagas disease is caused by the hemoflagellated protozoan Trypanosoma cruzi (Chagas, 1909) of the Kinetoplastidae order and Trypanosomatidae family and is a major public

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health concern in Latin America. Among vector-borne diseases, it is second to malaria in

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prevalence and mortality (Coura and Vinas, 2010).

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T. cruzi has an indirect heteroxenic life cycle. Hematophagous insects (Triatomids) serve as

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intermediary hosts, and mammals, including humans, serve as definitive hosts (De Souza,

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2002). The primary triatomid vectors of T. cruzi in South America are Triatoma infestans, Rhodnius prolixus, and Panstrongylus megistus (De Souza, 2002; Teixeira et al., 2006).

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The biological cycle of the parasite includes the three following morphologic forms, which

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are characterized by the relative positions of the flagellum, kinetoplast, and nucleus: trypomastigotes, epimastigotes, and amastigotes. Trypomastigotes are 20 µm in length and

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have subterminal kinetoplasts and fusiform nuclei. This form is infectious to humans and is found in mammalian blood and in the posterior intestine of triatomids. Trypomastigotes do not multiply. In mammals, this is the cellular form that disseminates the infection through the blood. Epimastigotes are 20 µm in length, have kinetoplasts that are anterior to the

nucleus, and display fusiform nuclei. They replicate in the intestine of the triatomid and are the predominant form in axenic cultures. Amastigotes are approximately 2 µm in diameter and are round with no emergent flagellum. They multiply within mammalian host cells until the host cells are ruptured by the parasites (after 8-9 cell divisions). Before their release from the host cells, amastigotes differentiate into trypomastigotes that invade the blood stream; they may then re-enter any other nucleated cell (De Souza, 2002).

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Amastigotes can be grown in cell culture within muscle cells, fibroblasts, and macrophages,

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and infectious forms of T. cruzi (trypomastigotes) can be harvested from these cells

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(Burleigh and Andrews, 1995; Yoshida, 2006).

Disease transmission primarily occurs via the bite of a T. cruzi-infected insect. Upon

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feeding on mammalian blood, the insect deposits feces containing infectious metacyclic

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trypomastigotes onto the skin. The parasites then enter the mammalian host through the

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skin by a mechanism that is facilitated by self-inflicted scratching and pore-forming lytic protein proteolytic enzymes, such as tryalisin which can permeabilize mammalian cells, in

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the insect’s saliva (Amino et al., 2002). Trypomastigotes are phagocytosed by antigen-

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presenting cells, and they then differentiate into amastigotes, the obligate cytoplasmic form.

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After a certain number of replications, amastigotes differentiate into trypomastigotes, escape into the circulation, and migrate towards target tissues, such as the myocardium,

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skeletal muscle, smooth visceral muscle, glial cells of the central nervous system, and the

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placenta (Kemmerling et al., 2010). Other important means of transmission are blood transfusions, organ transplants (10% of cases) (Coura and Borges-Pereira, 2012), oral infection through the ingestion of contaminated foods (Coura and Vinas, 2010; de Souza et al., 2010; Toso et al., 2011; Yoshida, 2006; 2008), and transplacental transmission in

chagasic mothers (Coura and Borges-Pereira, 2012; Duaso et al., 2012; Fretes and Kemmerling, 2012; Kemmerling et al., 2010). Although extensive public health measures to control vectors and prevent blood-borne transmission have decreased the incidence and prevalence of this disease, the currently available therapies do not possess high efficacies (Molina-Berrios et al., 2013). In vitro and ex vivo models are the primary experimental approaches that are used to assess

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potential new trypanocidal agents (Triquell et al., 2009). The quantification of infected

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mammalian cells and of intracellular parasites (amastigotes) in in vitro assays is a fundamental method used to determine the effect of anti-parasitic drugs (Faundez et al.,

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2005). However, the manual or semi-automatic quantification of infected cells and

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amastigotes requires trained and experienced observers and can take many hours. Today,

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the development of diverse algorithms and bioimaging software allows for the automated

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analysis of large data sets (Carpenter et al., 2012).

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Ex vivo infection models are also important tools to evaluate parasite infectivity. The main

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advantages of tissue explants are that the cells are maintained in a more physiological

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condition and that tissue damage can be evaluated (Fretes and Kemmerling, 2012). However, in the tissue model, parasites are not easily detected, and exact quantification is

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required (Duaso et al., 2010; Fretes and Kemmerling, 2012). The real-time PCR (qPCR)

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assay is highly sensitive and allows for the quantification of subtle changes in gene expression (Pfaffl, 2001) or parasite load in animal tissues (Caldas et al., 2012; Cummings

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and Tarleton, 2003). Consequently, changes in tissue explant T. cruzi DNA levels as a result of diverse therapeutic interventions can be quantified by a relative quantification (RQ) analysis with the double comparative (∆∆Ct) method (Castillo et al., 2012; Pfaffl, 2001).

Here we report the use of commercial software (MATLAB®) to quantify T. cruzi amastigotes in infected mammalian cells (BeWo) and the use of the qPCR ∆∆Ct method to assess the parasite burden in human placental chorionic villi explants (HPCVE). 2. Materials and Methods 2.1 Cell culture. Green Monkey (Cercopithecus aethiops) renal fibroblast like cells (VERO cells (ATCC® CCL-81)) were grown in RPMI medium supplemented with 5%

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fetal bovine serum (FBS) and antibiotics (penicillin-streptomycin) (Villalta and

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Kierszenbaum, 1982). BeWo cells (ATCC CCL-98) were grown in DMEM-F12K medium supplemented with 10% FBS, L-glutamine and antibiotics (penicillin-streptomycin)

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(Drewlo et al., 2008). The cells were grown at 37°C in a humidified atmosphere with 5%

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CO2. The culture medium was replaced every 24 hours.

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2.2. Production of the infective cellular form (trypomastigotes) of T. cruzi. Upon

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reaching confluence, VERO cells were incubated with a culture of Ypsilon strain

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epimastigotes (non-infective cellular form of the parasite) in the late stationary phase,

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which contains approximately 5% trypomastigotes (Contreras et al., 1985). The

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trypomastigotes were then allowed to invade fibroblasts VERO cells and replicate intracellularly as amastigotes. After 72 hours, the amastigotes transformed back into

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trypomastigotes and lysed the host cells. The parasites were recovered by low-speed

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centrifugation (500 x g) to separate the trypomastigotes in the supernatant from the amastigotes in the sediment (Villalta and Kierszenbaum, 1982).

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2.3 Infection of BeWo cells with T. cruzi trypomastigotes. BeWo cells were detached by trypsinization, sedimented, and resuspended in media containing 10% FBS. Then, 2 x 105 cells were seeded into 6-well plates. The cells were allowed to adhere to the bottom of the wells for 3 hours and were then challenged with the parasites at a BeWo cell:parasite ratio

of 1:1. The cells were analyzed 48 hours post-infection, and the parasites were identified based on nuclear size and morphology. 2.4 Microscopy analysis of BeWo cells and amastigote identification: BeWo cells were fixed in cold 90% methanol. The preparations were washed with PBS and incubated with 1 µg/ml 4´6´-diamidino-2-phenylindole (DAPI) (Molecular Probes). Then, the sections were mounted in Vectashield (ScyTek, ACA) and observed on an epifluorescence microscope

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(Motic BA310; Hong Kong, China). Amastigote were recognized by their morphology,

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including nuclear size and the presence of a kinetoplast (Liempi et al., 2014).

2.4 HPCVE cultures and infection with T. cruzi trypomastigotes. Healthy human full-

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term placentas were obtained from uncomplicated pregnancies following vaginal or

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caesarean deliveries. Informed consent for the experimental use of the placenta was given

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by each patient as stipulated by the Code of Ethics of the Faculty of Medicine of the

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University of Chile. The organs were collected in a cold, sterile saline-buffered solution

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(PBS) and processed no more than 30 minutes after delivery. The maternal and fetal

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surfaces were discarded, and villous tissue was obtained from the central part of the

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cotyledons. The isolated chorionic villi were washed with PBS to remove the blood, dissected into approximately 0.5-cm3 fragments, and co-cultured with 1 x 106 T. cruzi

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trypomastigotes for 48 hours in 1 ml of RPMI culture medium supplemented with

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inactivated FBS and antibiotics. T. cruzi infection was confirmed by detecting the parasite DNA using a real-time polymerase chain reaction (Castillo et al., 2013b).

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2.5 DNA amplification by real-time PCR (qPCR). Genomic DNA was extracted from placental tissue with a Wizard Genomic DNA Purification Kit (Promega®, USA) according to the manufacturer’s instructions. The resulting DNA was quantified with a µDrop Plate DNA quantification system in a Varioskan Flash Multimode Reader (Thermo Scientific,

USA). For amplification of human and parasite DNA, two specific primer pairs were used. A 100 bp human GAPDH sequence was amplified using the primers hGDH-F (5’TGATGCGTGTACAAGCGTTTT-3’)

and

hGDH-R

(5’-

ACATGGTATTCACCACCCCACTAT-3’), which were designed using Primer Express software (version 3.0; Applied Biosystems®). For T. cruzi DNA detection, a 182 bp sequence

of

satellite

DNA

was

amplified

using

the

primers

TCZ-F

(5’-

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GCTCTTGCCCACAMGGGTGC-3’) and TCZ-R (5’-CAAGCAGCGGATAGTTCAGG-

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3’) (Castillo 2012; Cummings and Tarleton 2003). Each reaction mix contained 200 nM of each primer (forward and reverse), 1 ng of DNA, 12.5 µL of SensiMix® SYBR Green

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Master Mix (Bioline®, USA) and H20 for a total volume of 25 µL. The amplification was

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performed in an ABI Prism 7300 sequence detector (Applied Biosystems®, USA). The

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cycling program was as follows: an initial incubation at 20°C for 2 min, a denaturation step

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at 95°C for 10 min and 40 amplification cycles of 95°C (15 s), 60°C (15 s) and 72°C (30 s).

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The final step was a dissociation stage that ranged from 60 to 95°C (105 s). The relative

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quantification analysis of the results was expressed as an RQ value determined using the

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comparative control (∆∆Ct) method (Pfaffl 2001; Castillo et al., 2012). 3. Results and Discussion

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3.1 Quantification of in vitro infection by T. cruzi using MATLAB® software

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T. cruzi can invade and infect any nucleated cell (De Souza, 2002; Diaz-Lujan et al., 2012). Here, we challenged the choriocarcinoma-derived cell line BeWo (ATCC CCL-98) with T.

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cruzi Ypsilon strain trypomastigotes at a parasite:cell ratio of 1:1 for 2, 4, 6, 12 and 24 hours. After 48 hours, the cell nuclei were stained with DAPI (4, 6-diamidino-2phenylindole, Molecular Probes; 1 µg DAPI/ml) (Duaso et al., 2010). The parasite forms were recognized by their morphology, including nuclear size and the presence of a

kinetoplast (Fig 1). The cells were observed on an epifluorescence microscope, and the images were captured as “tiff” files (Fig 2A). Importantly, all of the files were of the same quality (pixels) for later analysis. The color images were converted into gray scale (Fig 2A), equalized (Fig 2B) (contrast and brightness adjustment), and subjected to binarization (Fig 2C) (black and white scale). Afterwards, the small objects within the image (in this case, the parasites) were digitally

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eliminated (Fig 2D), and a “watershed” algorithm was applied (Fig 2E) to count the

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mammalian cell nuclei (Fig 2F) (Gudla et al., 2008; Nandy et al., 2011).

After counting the mammalian cell nuclei, the parasites were quantified. In the equalized

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and binarized images, the mammalian nuclei were digitally eliminated (Fig 2G), and the

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parasites were identified by the presence of smaller nuclei beneath a bright kinetoplast. The

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2I)(Gudla et al., 2008; Nandy et al., 2011).

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software then calculated the local variance (Fig 2H) and counted the parasites (Fig

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There was no statistically significant difference between the manual and the automatic

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quantification of the parasite (Fig 3A). The two methods showed a correlation analysis r2

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value of 0.9159 (Fig 3B). The most significant advantage of the automatic quantification was the efficiency of the analysis; it took approximately fifteen minutes to quantify the

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parasites and BeWo cell nuclei of 450 files. For the manual count, each evaluator required

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at least seven working days. Additionally, the automatic quantification eliminated the variability between the different individuals who performed the evaluations.

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To assign the parasites to their respective mammalian cells, we defined an infected cell as containing at least three parasites localized perinuclearly. The software used the Voronoi diagram (Wakamatsu et al., 2011; Yu et al., 2010) to assign amastigotes to each nucleus and hypothetical cell (Fig 4). The drawback of this method was that some parasites were

assigned to the wrong BeWo cell; between 2.55 and 4.6 percent of amastigotes were assigned to the wrong cell. The lowest percentage of wrong assigned parasite corresponds to conditions in which a low concentration of BeWo cells were seeded and challenged with the corresponding parasite:cell ratio (Fig 4B). Conditions in which the BeWo cells were confluent and challenged with a high parasite concentration, the percentage of wrong assigned parasite were above 10 percent (Fig 4A). Therefore, this analysis needs adequate

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experimental conditions, in which the concentration of mammalian cells and parasites must

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be carefully choosen. Only, experiments done with mammalian cells in semi-confluence can be considered reliable (Fig 4B). The use of immunofluorescence methods to determine

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cell limits should correct the false assignation of the parasites. However, this methodology

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would increase the duration and cost of the analysis. Currently, we are working on other

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experimental approaches, such as the combination of images with fluorescent nuclei and

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the respective phase contrast images, to improve the analysis.

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3.2 Quantification of parasite DNA in tissue by qPCR

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The ex vivo infection of HPCVE with parasites is a well-established and relatively simple way to study mechanisms of tissue invasion. However The explants can be kept in culture

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for several days to maintain the constituent cells and tissues in a more physiologically

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relevant condition than their isolated counterparts in monolayer cell culture models (Fretes and Kemmerling, 2012).

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We have established optimal conditions for the ex vivo infection of HPCVE with T. cruzi trypomastigotes (Duaso et al., 2010). The co-incubation of 103 or 106 trypomastigotes yields a reproducible infection of HPCVE (Castillo et al., 2013a; Duaso et al., 2010; Fretes and Kemmerling, 2012). However, parasite visualization and quantification are difficult in

this model. The qPCR assay constitutes a highly reliable tool to determine the exact amount of parasite DNA within tissues. Conventional PCR (or end-point PCR) gives only qualitative information regarding the infection. Upon completion of the amplification cycles in conventional PCR, the amount of amplified DNA is influenced by diverse variables, such as the sample input and technical variations that occurred during the reaction, which can lead to inconsistent results (Schefe et al., 2006). Other important

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variables that can affect the result of DNA amplification by conventional PCR include the

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volume of the processed samples, the DNA extraction procedure, and the region of T. cruzi DNA that is being amplified (Piron et al., 2007). In contrast, qPCR is a more sensitive and

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specific technique that allows for the continuous monitoring of the reaction and for high

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throughput results because the amplification and detection are performed simultaneously.

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Another advantage of this method is the reduced risk of sample contamination due to the

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reduced manipulation and the lower frequency of false negatives from DNA extraction

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failures (Caldas et al., 2012; Piron et al., 2007).

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qPCR can be performed by the SYBR Green method or the TaqMan method. In both cases,

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the fluorescent signal can be measured in the logarithmic phase of the polymerase chain reaction. The SYBR Green fluorescent dye specifically binds to the minor groove of

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double-stranded DNA (Pfaffl, 2001). The TaqMan method uses a fluorescent probe that is

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labeled at the 5’ end and contains a quencher molecule at the 3’ end that hybridizes to an additional conserved region within the target amplicon sequence (Smith and Osborn, 2009).

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However, it is important to consider the primer efficiency for the target and housekeeping genes since it is fundamental because it influences the accuracy of the infectivity measurement (Pfaffl, 2001).

The threshold cycle (Ct) value is defined as the number of cycles within the exponential phase in which the PCR kinetic curve reaches a previously defined threshold of fluorescence (Fig 5 A-B) (Schefe et al., 2006). For absolute quantification, the Ct value is compared to a calibration curve, whereas during relative quantification, the Ct values of the target gene and an endogenous control gene (or “housekeeping gene”) are compared (Pfaffl, 2001). In the case of two or more experimental conditions, the relative amount of

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parasite DNA in each condition is compared, constituting the double comparative (∆∆Ct)

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method (Fig 5C).

The quantification of parasite DNA by qPCR in HPCVE has been used to measure the

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effect of matrix metalloprotease inhibitors (Castillo et al., 2012), the role of the

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complement system in T. cruzi infectivity in placental tissue (Castillo 2013), and the effects

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of the classic anti-chagasic drugs benznidazole and nifurtimox (Rojo et al., 2014). In those

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studies, SYBR Green dye and the ∆∆Ct method were used. With the ∆∆Ct method, the

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changes in parasite DNA load were measured in a straightforward and reliable manner.

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Additionally, qPCR has been used to determine the effect of pharmacological treatments in

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experimental murine chronic chagasic heart disease. Parasite DNA loads in heart tissue have been determined by the SYBR Green method (Molina-Berrios et al., 2013) and by the

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TaqMan probe method (Fauro et al., 2013; Molina-Berríos et al., 2013).

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We conclude that both parasite this quantification methods are constitute an excellent tools for evaluating the parasite load in cells or tissues and therefore constitutes an easy and

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reliable ways to study parasite infectivity.

Acknowledgments. This study was supported by grants 1120230 (to UK) and 1120579 (to SH) from FONDECYT, Chile and by grants CONICYT-PBCT Anillo ACT 112 and ICM,

P09-015-F, from the Chile “Programa de Cooperación Científica Internacional” CONICYT/MINCYT 2011-595-CH/11/08 (to REF and UK).

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Figure Legends Figure 1 Infection of BeWo cells with T. cruzi amastigotes. BeWo cells were challenged with T. cruzi Ypsilon strain trypomastigotes at a parasite:cell ratio of 1:1 for 24 hours and were processed for DAPI staining after 48 hours. The arrows show BeWo cell nuclei, and the arrowheads show intracellular amastigotes. Scale bar: 10 µm. Figure 2 Quantification of BeWo cell nuclei. Tiff files from the different experimental conditions were converted into gray scale (A), the contrast and brightness were adjusted (equalization) (B), and images were subjected to binarization (C). Then, the small objects in the image (the parasites) (D) were digitally eliminated, and a “watershed” algorithm (E) was applied to count the mammalian cell nuclei, which were randomly colored (F). Subsequently, the BeWo cell nuclei were digitally eliminated (G), and the parasites were identified by the presence of smaller nuclei beneath a bright kinetoplast. The software calculated the local variance (H) to count the parasites (I). Figure 3 Quantification of infection of BeWo cells with T. cruzi. BeWo cells were challenged with T. cruzi Ypsilon strain trypomastigotes at a parasite:cell ratio of 1:1 for 2, 4, 6, 12 and 24 hours. After 48 hours, the cells were processed for DAPI nuclear staining. In A, the percentages of infected BeWo cells after different incubation times as determined automatically with MATLAB software (white bars) or manually (black bars) are shown. There was no statistically significant difference between the two quantification methods for

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any condition. The data were analyzed by Student´s t test. In B, the correlation analysis of both methods is shown. Figure 4 Assignment of the parasites to their respective BeWo cell. Images of the tiff files, which show the quantification of BeWo cell nuclei (See Fig 2I), were subjected to Voronoi diagram analysis. A: Image corresponding to a highly confluent culture. B: Image corresponding to a semi-confluent culture. Note that a significant number of parasites were assigned to the wrong BeWo cell (arrows) in A, but not in B. Figure 5 Quantification of parasite DNA in the tissue by the qPCR ∆∆Ct method. HPCVE were incubated with 2 x 104 trypomastigotes (left column) and an inhibitor (fluid-phase HuCRT (5 µg/ml) (right column) (Castillo et al., 2013a). Genomic DNA was extracted from HPCVE with the Wizard Genomic DNA Purification Kit (Promega®, USA) according to the manufacturer’s instructions. The DNA (1 ng) from each sample was subjected to amplification. A and B show representative plots of the amplification curves (each line is a different sample) for a T. cruzi gene (TCZ) and a housekeeping gene (hGAPDH), respectively. “Ct A,” “Ct B” and “Ct C” represent the threshold cycle numbers for the reference sample gene (infected control sample), the treated sample gene (inhibitor-treated sample) and housekeeping gene, respectively. Rn corresponds to the fluorescence emission intensity of the reporter dye (SensiMix® SYBR Green Master Mix, Bioline®, USA) divided by the fluorescence emission intensity of the passive reference dye (ROX). In C, the graph shows the relative quantification value (RQ) of the infected control sample and the sample treated with the inhibitor.