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The Truman Show for protozoan parasites: A review of in vitro cultivation platforms

['Smita Sutrave', 'German Federal Institute For Risk Assessment', 'Bfr', 'Department Of Biological Safety', 'Berlin', 'Martin Heinrich Richter']

Date: 2021-09

Abstract Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host–parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases.

Author summary In light of the far-reaching social and economic repercussions of communicable, zoonotic parasitic diseases on human health, it is imperative to continue to strive toward developing in vitro models for in-depth scrutiny and understanding of pathogenicity, as well as for innovations toward combating these infections. This review, to our knowledge, is the first to offer a qualitative summary of the existing models for culturing protozoan parasites with major relevance to human health in vitro. The present work aims to provide a reference guide on the current state of in vitro culture of these protozoan parasites and offers a foundation to facilitate exchange of expertise among researchers, clinicians, and healthcare workers. This comprehensive review would aid in enabling discussions on new intervention approaches to fill in the knowledge gaps in the field of parasitic diseases affecting the global population.

Citation: Sutrave S, Richter MH (2021) The Truman Show for protozoan parasites: A review of in vitro cultivation platforms. PLoS Negl Trop Dis 15(8): e0009668. https://doi.org/10.1371/journal.pntd.0009668 Editor: Claudia Ida Brodskyn, Centro de Pesquisa Gonçalo Moniz-FIOCRUZ/BA, BRAZIL Published: August 26, 2021 Copyright: © 2021 Sutrave, Richter. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist.

Introduction The availability and proper functioning of sanitation services are intricately linked to maintaining public health standards; however, this still remains to be achieved in all parts of the world [1–3]. Despite major strides made in improving early childhood health in recent years, the mortality rate for the age group under 5 years in part due to parasite-related diseases remains highly correlated with geographical location, i.e., low- and middle-income countries [4,5]. Tropical and subtropical climate zones with endemic insect vectors, lower standards of living, lack of access to basic amenities such as clean drinking water, and public sanitation facilities as well as areas with political instability show a higher burden of disease (BoD) associated with parasitic diseases [6]. However, parasitic disease outbreaks have also been reported in well-developed regions of the world, as shown by the outbreak of Cryptosporidium hominis after heavy flooding in Germany [7], a foodborne outbreak of Cryptosporidium parvum in Norway [8], and several outbreaks involving both species in the United Kingdom in recent years [9]. In the past decades, several governmental and nongovernmental organizations have been involved in implementing programs aimed toward prevention and treatment of life-threatening protozoan diseases [10–12]. Treatment options have been developed for some of these parasitic diseases such as malaria, the leading cause of parasitic disease-related deaths worldwide, but are not always readily available. The malarial parasite, Plasmodium spp., is a prime example of a protozoan receiving continued funding and scientific interest. However, other protozoan diseases such as Chagas disease and African trypanosomiasis have largely been neglected despite their severe disease profiles [13]. Additional protozoan parasites add to the range of severe and debilitating diseases. Young children and immunocompromised individuals are at risk for C. parvum infections, while Toxoplasma gondii infections during pregnancy pose serious risks to the unborn child [14,15]. Naegleria fowleri, an emerging acarpomyxean parasite known to thrive in warm water bodies, causes primary amebic encephalitis associated with rapid deterioration of patient health and a high fatality rate [16]. This disease is now being reported in regions with no prior cases, implicating climate change as an important driver for spread of the pathogen. Well-established in vitro models provide a valuable alternative to in vivo models by giving in-depth information on life cycle stages as well as circumventing the ethical issues of laboratory animal use. Another major advantage of in vitro models is the ready availability of parasites for further experiments without having to be passaged through an animal host. Factors challenging the culture of parasites in the laboratory include complexity of parasitic life cycles including several life cycle stages, asexual and sexual cycles needing specific, and varied hosts depending on the parasite in question. As with all in vitro models, one of the main concerns is in vitro–in vivo correlation. The more complex a parasitic life cycle, the more challenging this correlation becomes, proving to be one of the main hindrances in the progression of parasite in vitro culture. New technologies such as 3D tissue and organoid models, 3D tissue printing, hollow fiber technologies, and other novel technology approaches have provided well-founded hope to develop successful and correlating in vitro systems that are of particular interest for parasitic disease research. The current review is not a systematic listing of all publications on the vast topic of in vitro models for the maintenance of protozoan parasites relevant to human health. However, this review aims to provide a comprehensive overview of available in vitro models for protozoa listed as significant agents of morbidity and mortality in humans by WHO and CDC. To our knowledge, this is the first review summarizing in vitro models of protozoan parasites with a significant BoD. The protozoan parasites have been classified into 3 categories, members belonging to the Apicomplexan group, the trypanosomes, and other protozoans, respectively. Table 1 outlines the disease burden of protozoan parasites worldwide including number of cases, fatality rate, and treatments available for each protozoan parasite included in this review. Only parasitic diseases for which an in vitro system has been described in literature have been outlined in the table. Table 1 includes the disability-adjusted life year (DALY) metric that has been used by WHO Global Health Estimates (GHE) to measure the global burden of disease (GBD) on society due to various ailments starting in 2000 with the latest data published in 2016 [17]. For any given disease, one DALY is equivalent to one lost year of “healthy” life and is calculated by summing up the years of life lost (YLL) due to premature death and the years lived with disability (YLD) due to suboptimal health caused by that specific disease [18]. PPT PowerPoint slide

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TIFF original image Download: Table 1. List of protozoans and the associated diseases for which in vitro models are published. https://doi.org/10.1371/journal.pntd.0009668.t001

Literature search criteria Searches of PubMed using the search terms “in vitro model,” “in vitro culture,” and “in vitro cultivation” along with the scientific name for each parasite were used to identify the references cited in this review. Titles and abstracts were screened for recent advances and innovations in the field of in vitro culture for the 9 protozoan parasites included in this review.

Discussion This review highlights current research in the culturing of protozoan parasites associated with high morbidity and mortality. Recent advances in in vitro studies provide new insights into the life cycles of these parasites and contribute to a deeper understanding of pathogenicity potentially leading to more precise and effective treatments and prevention strategies. Current in vitro models provide alternatives to in vivo studies, helping to mimic host–parasite interaction without the need for large-scale animal studies. The protozoan parasites categorized here into apicomplexan, flagellates, and amoebas represent the agents responsible for some of the most debilitating, chronic, and, in some cases, fatal outcomes throughout the world. In recent years, in vitro models for protozoan candidates such as Cryptosporidium spp. and T. gondii have been well established. In addition to well-recognized agents with a high BoD in humans such as Plasmodium spp., this review includes 2 other apicomplexan parasites, Cryptosporidium spp. and T. gondii as well as G. duodenalis included in the flagellate group. All 3 trypanosomatids included here, T. cruzi, T. brucei, Leishmania spp., and Plasmodium spp. are transmitted by insect vectors and cause debilitating and sometimes lethal diseases especially in the subtropical or tropical regions of the world. The adverse effects of climate change also have consequences on the emergence of pathogens in regions far beyond endemic areas. N. fowleri, for example, thrives in warm waters, and, as global temperatures rise, it is predicted that the incidence of PAM would increase considerably [85]. According to WHO, Leishmania is another climate-sensitive parasite with changes in the environment directly affecting its epidemiology. Climate change has a direct effect on the increased incidence of insect vector-borne parasitic diseases such as those caused by Plasmodium spp. and Trypanosoma spp. [53] that are more prevalent in the developing world but are now being reported in industrialized nations likely due to migration and reemergence of these diseases. For instance, adverse weather events such as prolonged periods of heavy rainfall and milder winters have been known to aid the spread of malarial mosquitos [86]. Robust in vitro models would aid in the application of modern research tools such as transcriptomics to study protozoan pathogens. Due to the qualitative appraisal of the current literature in this review, particular care was taken in the selection of papers to provide a representative picture of the research published on in vitro models. Publications highlighting significant advancements and turning points in the study of each of the parasites were included. Even within the in vitro models available, there exists a broad variation in terms of complexity of the model system; resources needed; cell lines used as well as reproducibility. Advanced 3D in vitro models already exist for certain well-known parasites. In the case of Cryptosporidium spp., for instance, while some publications have reported using stem-cell derived air–liquid interface monolayers [36], others have demonstrated more complex models employing a 2D cell culture platform using COLO-680N cells [40] and a 3D organoid culture using hollow fiber technology [41] for the cultivation of parasites in the laboratory. On the other hand, there is a lack of funding and interest in the scientific community for other lesser known yet severe parasitic diseases to humans mainly affecting the developing regions of the world. The in vitro models summarized in this review are at various stages of development, with some being highly sophisticated 3D cultures, while others are simpler models with less progress made in the past decades. In summary, in vitro models offer the following advantages in the fight against complex parasitic infections: ready availability of various parasitic life cycle stages for further research; platforms for testing antiparasitic drugs and potential vaccines; provision of material for the exploration of pathophysiology and genetics; and alternatives to the ethical implications of using laboratory animals for long-term in vivo studies. Furthermore, organoid-based 3D models are amenable to high-throughput use, live cell imaging, and transcriptome analyses [46]. Global climate change and other anthropogenic stressors such as migration and wildlife trade contribute to the spread of zoonoses and disease vectors, thereby placing an urgency on the need for effective intervention strategies for the control of zoonotic parasitic diseases [53,87,88]. In vitro models are valuable tools toward in-depth studies of parasite biology simulating in vivo conditions in patients. The model systems detailed here may serve to provide ideas for in vitro culturing of other protozoan parasites not included in this review. For instance, due to their common ancestry, apicomplexan parasites show many similarities in genetics, molecular biology and pathogen-specific cellular processes [26]. Advanced 3D culture models elucidating the processes involved in parasite replication leading to host cell and tissue damage are essential for effective disease prevention strategies.

Future perspectives The long-standing use of cell monolayers for growth and maintenance of protozoan parasites in vitro is gradually being advanced by novel 3D technologies. Three-dimensional models such as organoids and spheroids serve as bridges between animal models and the microenvironment within the human host. Stem cell–derived intestinal organoids simulate natural host conditions and aid in the study of host–pathogen interaction. Continued advances in 3D cultures open up new avenues for investigating the immunological basis of chronicity, dormancy and asymptomatic disease progression. Standardization and harmonization of culture conditions for protozoa in in vitro settings would aid in drug development and vaccine trials for debilitating diseases affecting communities worldwide. Key learning points Protozoan parasites cause severe, debilitating, and fatal diseases worldwide.

Culturing protozoan parasites in vitro has been challenging due to their complex life cycles involving various stages. In vivo studies pose ethical considerations and are not always representative systems for human hosts.

In recent years, novel studies including 3D models have been published for culturing protozoa in vitro.

Climate change and socioeconomic factors such as migration emphasize the need to study the disease cycles in order to develop effective prevention and treatment strategies.

This review aims to give a comprehensive overview of current in vitro models for culturing protozoan parasites. Top five papers Wilke G, Funkhouser-Jones LJ, Wang Y, Ravindran S, Wang Q, Beatty WL, et al. A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability. Cell Host Microbe. 2019;26(1):123–34.e8. Derricott H, Luu L, Fong WY, Hartley CS, Johnston LJ, Armstrong SD, et al. Developing a 3D intestinal epithelium model for livestock species. Cell Tissue Res. 2019;375(2):409–24. Chua ACY, Ananthanarayanan A, Ong JJY, Wong JY, Yip A, Singh NH, et al. Hepatic spheroids used as an in vitro model to study malaria relapse. Biomaterials. 2019;216:119221. Rodriguez ME, Rizzi M, Caeiro L, Masip Y, Sanchez DO, Tekiel V. Transmigration of Trypanosoma cruzi Trypomastigotes through 3D Spheroids Mimicking Host Tissues. Methods Mol Biol. 2019;1955:165–77. Zaongo SD, Shaio MF, Ji DD. Effects of Culture Media On Naegleria fowleri Growth At Different Temperatures. J Parasitol. 2018;104(5):451–6.

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