RESUMEN
BACKGROUND: Intestinal cells grown in microgravity produce a three-dimensional tissue assembly, or "organoid," similar to the human intestinal mucosa, making it an ideal model for enteric infections such as cryptosporidiosis. METHODS: HCT-8 cells were grown in a reduced-gravity, low-shear, rotating-wall vessel (RWV) and were infected with Cryptosporidium parvum oocysts. Routine and electron microscopy (EM), immunolabeling with fluorescein-labeled Vicia villosa lectin and phycoerythrin-labeled monoclonal antibody to a 15-kD surface-membrane protein, and quantitative polymerase chain reaction (qPCR) using probes for 18s rRNA of C. parvum and HCT-8 cells were performed. RESULTS: The RWV allowed development of columnar epithelium-like structures. Higher magnification revealed well-developed brush borders at the apical side of the tissue. Incubation with C. parvum resulted in patchy disruption of the epithelium and, at the surface of several epithelial cells, in localized infection with the organism. EM revealed irregular stunting of microvilli, foci of indistinct tight junctions, and areas of loose paracellular spaces. qPCR showed a 1.85-log (i.e., 70-fold) progression of infection from 6 h to 48 h of incubation. CONCLUSION: The HCT-8 organoid displayed morphologic changes indicative of successful and quantifiable infection with C. parvum. The HCT-8 organoid-culture system may have application in interventional in vitro studies of cryptosporidiosis.
Asunto(s)
Criptosporidiosis/patología , Cryptosporidium parvum/fisiología , Células Epiteliales/parasitología , Organoides/citología , Organoides/parasitología , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Criptosporidiosis/parasitología , Células Epiteliales/patología , HumanosRESUMEN
Specific polymerase chain reaction (PCR) protocols were used to determine the prevalence of toxigenic Clostridium difficile in Vhembe, South Africa. Of 322 stool samples collected, toxigenic C. difficile was found in 23 (7.1%) cases and was significantly associated with diarrhea 20 (11.4%) compared with 3 (2%) in non-diarrheal samples (chi(2) = 426, P = 0.001), intestinal inflammation in 18 (12.1%) compared with 5 (2.9%) in lactoferrin-negative samples (chi(2) = 10.194, P = 0.001), and occult blood in 19 (16%) compared with 4 (2%) in occult blood-negative samples (chi(2) = 22.157, P < 0.001). Toxigenic C. difficile was more common among individuals > 50 years of age (20%), followed by those between 30 and 39 years of age (19%) and was not associated with HIV infections (chi(2) = 0.289, P = 0.591). Co-infection with other pathogens was common. Multivariate analysis indicated that toxigenic C. difficile was associated with E. bieneusi (P = 0.028), C. parvum (P = 0.007), and Enteroaggregative Escherichia coli (EAEC) (P = 0.007) in diarrheal samples. This study confirms the usefulness of PCR methodologies in the detection of toxigenic C. difficile and suggests that C. difficile is responsible for a small, but underappreciated, proportion of diarrheal cases in the region, and further study is warranted in this area.