Quantitative Detection of Cryptosporidium parvum after In Vitro Excystation by LightCycler PCR

Abstract

Cryptosporidium parvum is a protozoan parasite (protist) which can infect a wide variety of vertebrates including man. It can cause a severe but usually self-limiting diarrhoea. It is transmitted by the fecal—oral route. Human infections and epidemics have been linked to the consumption of contaminated drinking water, swimming pool water, recreational water, milk, cider, and berries [1]. The infection starts with the oral uptake of infective sporulated oocysts. Inside each oocyst are four sporozoites which actively penetrate the oocyst wall when certain physiologic triggers such as temperature, changes in pH, and the presence of bile salts and pancreatic enzymes are present. This biological process can be simulated in vitro and is described as “in vitro excystation”. In vitro excystation has been widely used as a surrogate marker for viability and infectivity of Cryptosporidium parvum oocysts [2–8]. Usually the quantification of in vitro excystation is achieved by a microscopic evaluation of the excystation rate. On the other hand, various PCR protocols, including a TaqMan protocol, have been described in combination with in vitro excystation; they lack, however, a quantification of the PCR results [9–17].