PubMed2004
The accurate identification of a parasite at the species and/or genotype level has major implications for various aspects of human and veterinary parasitology, including the diagnosis, the taxonomy, the treatment and the control. The advent of molecular techniques, in particular those based on the in vitro amplification of nucleic acids, has dramatically improved our ability to detect infections caused by parasites. To illustrate the progress in molecular diagnostics, Cryptosporidium and Giardia are used here as examples of parasites for which both the diagnosis and the taxonomy have traditionally been problematic. These protozoan parasites, while very different for many aspects of their biology, shares a complex series of transmission routes, including anthroponotic and zoonotic transmission, as well as waterborne and foodborne transmission. The resistant stages produced by Cryptosporidium and Giardia (oocysts and cysts, respectively) are remarkably stable, and can survive for weeks to months in the environment. Further, the infective dose is low, and infectious dose studies and models suggest that even a single oocyst or cyst carries some probability of causing an infection. Finally, most faeces that contain (oo)cysts end up in the environment and can be spread to foods by irrigation or by direct contact, and can persist in the water, as routine treatments eliminate only a fraction of these stages. This situation explains the growing interest towards the development of methods that allows such stages to be detected with the highest sensitivity and specificity. A variety of Polymerase Chain Reaction (PCR) assays have been described for both Cryptosporidium and Giardia. The choice of a particular assay mainly depends on the amount of information carried by the genetic locus under analysis. Indeed, some assays can be used to identify the different species within a genus, while others allowed to distinguish between isolates of the same species (genotypes), and some can even be used for both purposes. Post-PCR analyses are usually based on the direct sequencing of the amplification products, or on the digestion with endonucleases followed by gel electrophoresis of the restriction fragments. In the last few years, the molecular characterization of a large number of isolates, collected from infected hosts and from the environment, has considerably changed our view of the epidemiology of cryptosporidiosis and giardiasis. Indeed, several species/genotypes have been established as human pathogens, and the nature of the parasites present in the water and in food have been investigated, allowing a better understanding of the complex circulation of the parasites in the environment, that may eventually led to implemented control measures. Finally, phylogenetic analysis of several nuclear genes is having a major impact in the revision of the taxonomy of Cryptosporidium and Giardia. The main limitation of PCR is that it doesn't provide information on the viability and infectivity of the pathogen. To obtain additional information on these important aspects, indirect methods, such as inclusion/exclusion assays using vital dyes or the Reverse-Transcriptase PCR (RT-PCR), can be used. Since RT-PCR relies on the integrity of mRNA, which usually has very short half-life (seconds), its use is thought to provide a more closely correlated indication of viability status compared to DNA-based methods. RT-PCR assays usually target the heat shock protein (hsp) 70 gene. The rationale behind this choice is that hsps are known to be synthesized with a high level of efficiency in stressed organisms; therefore, when (oo)cysts are exposed to a thermal shock, the induction of heat shock response provides both a level of amplification to increase detection sensitivity and an index of viability. Moreover, with the recent introduction of real-time PCR, that allows the continuous monitoring of amplicon formation throughout the reaction, quantitative aspect of the infection could be studied with exquisite sensitivity. This will, for example, allow (1) to detect carrier states, (2) to determine the number of oocysts/cysts present in a sample, (3) to study quantitative aspects of gene expression during the various phases of the infection.