Methods for parasitic protozoans detection in the environmental samples

Abstract

Summary :The environmental route of transmission of many parasitic protozoa and their potential for producing large numbers of transmissive stages constitute persistent threats to public and veterinary health. Conventional and new immunological and molecular methods enable to assess the occurrence, prevalence, levels and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne cysts and oocysts. These steps have been optimized to such an extent that low levels of naturally occurring (oo)cysts of protozoan can be efficiently recovered from water. Ten years have passed since the United States Environmental Protection Agency (USEPA) introduced the 1622 and 1623 methods and used them to concentrate and detect the oocysts of Cryptosporidium and cysts of Giardia in water samples. Nevertheless, the methods still need studies and improvements. Pre-PCR processing procedures have been developed and they are still improved to remove or reduce the effects of PCR inhibitors. The progress in molecular methods allows to more precise distinction of species or simultaneous detection of several parasites, however, they are still not routinely used and need standardization. Standardized methods are required to maximize public health surveillance. Résumé : MÉTHODES DE DÉTECTIONS DES PROTOZOAIRES PARASITES DANS L'ENVIRONNEMENT Les voies de transmissions de nombreux protozoaires parasites dans l'environnement et leur aptitude à y produire un nombre élevé de formes transmissibles constituent des menaces persistantes tant en santé humaine qu'animale. Des méthodes conventionnelles et de nouvelles méthodes moléculaires et immunologiques permettent d'évaluer la fréquence, la prévalence, les niveaux et les origines des protozoaires transmis par l'eau. La concentration, la purification et la détection sont les trois étapes clés de toutes les méthodes agréées pour la surveillance de routine des kystes et des oocystes transmis par l'eau. Ces étapes ont été optimisées à un niveau d'efficacité tel que les faibles concentrations naturelles d'(oo)cystes présents dans l'eau sont efficacement détectées. Dix ans se sont écoulés depuis l'introduction par l'United States

Food safety issues in fresh produce: Bacterial pathogens, viruses and pesticide residues indicated as major concerns by stakeholders in the fresh produce chain

“…However, although NoV genomic sequences have been regularly detected in fresh produce in several countries, the actual risk from NoV positive produce is still unknown (Baert et al, 2011b). In the case of parasites, standardized methods for detection in fresh produce are currently not available (Skotarczak, 2009) and they were also not notified to the RASFF-systems in the categories fruits, vegetables and herbs (table 3). These may be one of the elements why although several parasite outbreaks (e.g.…”

Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

“…The LAMP assay has been used for detecting Cryptosporidium on the basis of amplification of the gp60 and hsp70 genes of C. parvum; however, further fine tuning of this method is required, particularly for species identification and differentiation, which requires an adequate range of species-specific DNA sequences from GenBank. The major advantage of this method is its insensitivity to commonly occurring PCR-inhibiting factors in environmental samples (Notomi et al, 2000;Karanis et al, 2007;Bakheit et al, 2008;Skotarczak, 2009). Evaluation of this method with varying matrices may be the first step towards its application to water quality monitoring programs.…”

“…In NASBA, the isothermal reaction takes place at a temperature of 41 u C and does not require denaturation. Consequently, it avoids amplification of other genomic DNA, which often occurs when testing environmental samples (Skotarczak, 2009;Notomi et al, 2000;Mens et al, 2006). NASBA has prospective applications as a Cryptosporidium detection technique (viable and nonviable oocysts) from environmental samples.…”

“…Although PCR is an efficient tool for tracking the source of community-acquired diseases and for epidemiological and transmission studies, its specificity and sensitivity are commonly affected by the sample matrix, which may contain inhibitory factors that particularly affect the DNA extraction stage. PCR amplification of DNA extracted from Cryptosporidium oocysts has also been found to be inhibited by environmental factors such as formaldehyde, potassium dichromate and humic acids, which are generally carried over through the extraction and purification stages (Bakheit et al, 2008;Skotarczak, 2009). …”

“…The second primer, which is supplemented with a T7 promoter sequence, then hybridizes to the cDNA and transcription begins with the third enzyme and RNA polymerase. Each transcript is a template for creating another cDNA with a T7 promoter, which, in turn, is a template for RNA transcripts (Skotarczak, 2009).…”