Abstract
Nowadays, many commercial kits allow the polymerase chain reaction (PCR) detection of Cryptosporidium deoxyribonucleic acid (DNA) in stool samples, the efficiency of which relies on the extraction method used. Mechanical pretreatment of the stools using grinding beads has been reported to greatly improve this extraction step. However, optimization of this key step remains to be carried out. Indeed, many parameters could influence the pretreatment performances, among which the modulation of the speed and duration of the grinding step, in addition to the physicochemical features of the grinding beads, have never been evaluated to date. In this study, eleven commercial mechanical pretreatment matrixes (Lysis matrix tubes®, MP Biomedical, Irvine, CA, USA) composed of beads with different sizes, shapes, and molecular compositions, were evaluated for their performances in improving Cryptosporidium parvum oocyst DNA extraction before amplification by using our routinely used real-time PCR method. As expected, the eleven commercial mechanical pretreatment matrixes showed varying performances depending on the composition, size, and shape. All in all, the best performances were obtained when using the Lysing matrix, including ceramic beads with a median size (diameter of 1.4 mm).
Highlights
Cryptosporidium sp. is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts, including humans
We recently evaluated six extraction protocols associated with various mechanical pretreatments for C. parvum oocyst deoxyribonucleic acid (DNA) extraction in a multicenter comparative study [20]
The performances in C. parvum DNA amplification were variable depending on the mechanical lysis matrix tested
Summary
Cryptosporidium sp. is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts, including humans. Is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts, including humans. Transmission occurs through the fecal–oral route, by the ingestion of viable oocysts excreted in the environment by infected hosts. Because of their directly contaminating feature, ingestion of drinking water or food contaminated by Cryptosporidium oocysts can lead to epidemics affecting numerous people. Cryptosporidiosis is asymptomatic or expresses as a diarrhea most often self-resolving. Severe and prolonged infections are observed in immunocompromised patients and children under five [1,2]. The most frequent species isolated in humans are C. parvum and C. hominis, encompassing more than
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