Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen
            <i>Cryptosporidium parvum</i>
            In Vitro

Cai, Xiwen; Woods, Keith M.; Upton, Steve J.; Zhu, Guangxi

doi:10.1128/aac.49.11.4437-4442.2005

Cited by 73 publications

(83 citation statements)

References 31 publications

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“…Though microscopy can provide qualitative information about infection, a more precise means of quantification of live parasites is necessary to validate the model system. We quantified cDNA (derived from RNA) rather than DNA to obtain a more accurate representation of viable parasites, as C. parvum 18S rRNA degrades rapidly, within 3 h after parasite death (27,28). The results for the controls, consisting of either no reverse transcriptase or no template, were negative.…”

Section: Resultsmentioning

confidence: 99%

“…Five nanograms of RNA, quantified by use of a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA), was used to synthesize cDNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative reverse transcription-PCR (qRT-PCR) was then performed using C. parvum 18S rRNA gene-specific primers (27) and QuantiTect SYBR green master mix (Qiagen). Quantitative PCRs (qPCRs) were performed in 96-well plates using an Mx3000P qPCR system (Agilent Technologies, Santa Clara, CA).…”

Section: Preparation Of C Parvum Oocysts For Infectionmentioning

confidence: 99%

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Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

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Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of hostparasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites. KEYWORDS 3D model, Cryptosporidium, in vitro culture, intestinal epithelial cells C ryptosporidium spp. are apicomplexan parasites of global importance that cause diarrheal disease in humans and animals worldwide (1, 2). Though immunocompetent individuals experience self-limiting or asymptomatic infection, immunocompromised hosts, such as untreated patients with HIV infection or AIDS (3) and malnourished children (1, 2) in resource-limited settings, may experience severe diarrhea, wasting, and death. In a recent case-control study, Cryptosporidium was one of four pathogens responsible for moderate to severe diarrhea in children under the age of 5 years and was associated with an increased risk of death in children from 1 to 2 years of age in seven countries in sub-Saharan Africa and South Asia (4). The only FDA-approved drug for the treatment of cryptosporidiosis is nitazoxanide, a drug with broad antiparasitic properties (5). However, this drug is not effective in immunocompromised patients (6) and has not been widely tested in malnourished children in resource-limited countries. Though significant improvements in water purification methods have occurred (7) since the outbreak of waterborne cryptosporidiosis in Milwaukee, WI, in 1993 (8), industrialized countries are seeing increased numbers of cases, largely due to recreational water outbreaks (9).Unlike many other apicomplexan parasites which often require more than one host to complete their life cycles (10), Cryptosporidium completes its entire life cycle in a

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Section: Resultsmentioning

confidence: 99%

Section: Preparation Of C Parvum Oocysts For Infectionmentioning

confidence: 99%

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

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“…NITAZOXANIDE Nitazoxanide (NTZ), first-in-class thiazolide is a 5-nitrothiazolyl salicylamide derivative with broad activity against protozoa and helminths. NTZ and its two metabolites, tizoxanide (TZ) and tizoxanide-glucuronide (TZglu) were shown to inhibit the growth of C. parvum at concentrations lower than 10 mg/L (Theodos, 1998;Gargala, 2000;Cai, 2005). In vivo, it was subsequently found to be effective against C. parvum in suckling mice, nude mice, gerbils, rats and piglets (Theodos, 1998;Blagburn, 1998;Li, 2003;Baishanbo, 2005).…”

Section: Roxithromycinmentioning

confidence: 99%

Drug treatment and novel drug target againstCryptosporidium

Gargala

2008

Parasite

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Summary :Cryptosporidiosis emergence triggered the screening of many compounds for potential anti-cryptosporidial activity in which the majority were ineffective. The outbreak of cryptosporidiosis which occurred in Milwaukee in 1993 was not only the first significant emergence of Cryptosporidium spp. as a major human pathogen but also a huge waterborne outbreak thickening thousands of people from a major city in North America. Since then, outbreaks of cryptosporidiosis are regularly occurring throughout the world. New drugs against this parasite became consequently urgently needed. Among the most commonly used treatments against cryptosporidiosis are paromomycin, and azithromycin, which are partially effective. Nitazoxanide (NTZ)'s effectiveness was demonstrated in vitro, and in vivo using several animal models and finally in clinical trials. It significantly shortened the duration of diarrhea and decreased mortality in adults and in malnourished children. NTZ is not effective without an appropriate immune response. In AIDS patients, combination therapy restoring immunity along with antimicrobial treatment of Cryptosporidium infection is necessary. Recent investigations focused on the potential of molecular-based immunotherapy against this parasite. Others tested the effects of probiotic bacteria, but were unable to demonstrate eradication of C. parvum. New synthetic isoflavone derivatives demonstrated excellent activity against C. parvum in vitro and in a gerbil model of infection. Newly synthesized nitroor non nitro-thiazolide compounds, derived from NTZ, have been recently shown to be at least as effective as NTZ against C. parvum in vitro development and are promising new therapeutic agents.

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“…For infection experiments, the parasite burden was quantified by RT-PCR from infected PECs obtained at different time points (see below). For RT-PCR amplification, we used 100 ng of RNA as a template, and we used the method and the specific primers previously reported to detect 18S rRNA (19). To analyze the number of parasites, we used the ABI Prism software using as a reference the threshold cycle (C T ) values obtained from a standard curve prepared with known numbers of C. parvum oocysts ranging from 10 2 to 10 6 organisms.…”

Section: Methodsmentioning

confidence: 99%

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

et al. 2013

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The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of hostparasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens.

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Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

Cited by 73 publications

References 31 publications

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Drug treatment and novel drug target againstCryptosporidium

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

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