Detection and Quantification of Cryptosporidium in HCT-8 Cells and Human Fecal Specimens Using Real-Time Polymerase Chain Reaction

Parr, Jonathan B.; Sevilleja, Jesus Emmanuel; Samie, Amidou; Alcantara, Cirle; Stroup, Suzanne; Kohli, Anita; Fayer, R.; Lima, Aldo A. M.; Houpt, Eric R.; Guerrant, Richard L.

doi:10.4269/ajtmh.2007.76.938

Cited by 39 publications

(29 citation statements)

References 11 publications

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“…parvum-specific CP15 gene to determine shedding of organisms in stool or tissue burden (19). Additionally, stool DNA was probed for murine β-actin to measure the amount of host-shed cells in the feces.…”

Section: Methodsmentioning

confidence: 99%

Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model

et al. 2016

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Malnutrition and cryptosporidiosis form a vicious cycle and lead to acute and long-term growth impairment in children from developing countries. Insights into mechanisms underlying the vicious cycle will help to design rational therapies to mitigate this infection. We tested the effect of short-term protein malnutrition on Cryptosporidium parvum infection in a murine model by examining stool shedding, tissue burden, and histologic change and explored the mechanism underlying the interaction between malnutrition and cryptosporidiosis through immunostaining and immunoblotting. Protein malnutrition increased stool shedding and the number of intestine-associated C. parvum organisms, accompanied by significant suppression of C. parvum-induced caspase 3 activity and expression of PCNA and Ki67, but activation of the Akt survival pathway in intestinal epithelial cells. We find that even very brief periods of protein malnutrition may enhance (or intensify) cryptosporidiosis by suppressing C. parvum-induced cell turnover and caspase-dependent apoptosis of intestinal epithelial cells. This implicates a potential strategy to attenuate C. parvum's effects by modulating apoptosis and promoting regeneration in the intestinal epithelium.

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Section: Methodsmentioning

confidence: 99%

Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model

et al. 2016

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“…Real-time PCR assays have been developed independently for the detection and quantification of some enteropathogens, Helicobacter pylori (12), Clostridium difficile (12), Campylobacter (13), Cryptosporidium (14), Salmonella (11), enteropathogenic E. coli (15), and enterohemorrhagic E. coli (16). Although the PCR conditions were standardized, when we tested our conventional PCR in the real-time thermocycler, we saw that the melting temperatures (T m ) of the products of Campylobacter spp.…”

Section: Discussionmentioning

confidence: 99%

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

et al. 2013

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Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ؎ 0.05°C for invA, 85.56 ؎ 0.28°C for ipaH, and 89.21 ؎ 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10 4 CFU g ؊1 ; however, the limit of detection was 10 3 CFU g ؊1 . This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens. Infectious diarrhea is a global health problem that is still responsible for thousands of deaths worldwide, especially in children (1). It can be classified based on its clinical presentation as one of two syndromes-noninflammatory or inflammatory diarrhea (2). Among cases of inflammatory diarrhea, Shigella is the most common cause, followed by Campylobacter and Salmonella (3). These invasive organisms primarily target the lower bowel; they invade the intestinal mucosa to induce an acute inflammatory reaction and activate cytokines and inflammatory mediators (4).Because the time frame in which treatment choices must be made is short and the conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. We searched for DNA sequences that were highly conserved between the different species of each genera, and we selected the following genes as targets, invA (invasion A gene) for Salmonella spp., ipaH (invasion plasmid antigen H) for Shigella spp., and 16S rRNA for Campylobacter spp., to develop a fluorescence-based real-time PCR procedure to simultaneously identify these enteropathogens. In this method the post-PCR products are identified based on melting-point curve analysis. We have also standardized the technique to quantify these bacteria directly from stool samples. MATERIALS AND METHODSBacterial strains. A total of 147 enteropathogenic strains (Table 1) were analyzed, including clinical isolates representative of Salmonella spp., Shigella spp., and Campylobacter spp., as well as other enteropathogens. These clinical strains had previously been identified based on serology, biochemical assays, and real-time PCR for the diarrheagenic Escherichia coli (5). In add...

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“…Current diagnostic methods include microscopic examination of stool for Cryptosporidium species oocysts with acid-fast stains (modified Ziehl-Neelsen, Auramine-O, or Kinyoun’s), direct fluorescent-antibody (DFA) tests, antigen-based enzyme immunoassays (EIAs), PCR, and lateral-flow immunochromatographic “strip” tests (Johnston et al, 2003; White, 2010). The sensitivity of these tests range from 10 3 –10 5 oocysts/g of stool with PCR being the most sensitive and acid-fast microscopy the least, often requiring examination of multiple stool samples in order to report a negative test result (Weber et al, 1991; Huang and White, 2006; Parr et al, 2007; White, 2010; Calderaro et al, 2011; Centers for Disease Control, 2013). With the exception of lateral-flow tests which provide the advantage of a simple qualitative result (Garcia et al, 2003), existing diagnostic techniques remain confined to clinical laboratories due to the high instrumentation and reagent costs, and the need for a highly skilled technician or pathologist.…”

mentioning

confidence: 99%

Amplification-Free Detection ofCryptosporidium parvumNucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies

Weigum

Castellanos-González

White

et al. 2013

Journal of Parasitology

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This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a red-shift in the wavelength of light scattered by the gold nanoparticles. The limit-of-detection was between 4 × 105 and 4 × 106 copies of RNA per μL reaction mix, when a short synthetic target or full length in vitro transcribed target were employed. With total nucleic acids purified from C. parvum oocysts spiked into 100 mg stool, as few as 670 oocysts/μL reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

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Detection and Quantification of Cryptosporidium in HCT-8 Cells and Human Fecal Specimens Using Real-Time Polymerase Chain Reaction

Cited by 39 publications

References 11 publications

Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model

Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model

Multiplex Real-Time PCR for Detection of Campylobacter, Salmonella, and Shigella

Amplification-Free Detection ofCryptosporidium parvumNucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies

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