Cell-Culture Based Techniques for the Determination of the Infective Status of Waterborne Pathogens - PATHOS
Institution
CRP Gabriel Lippmann
Partenaire(s) :
Cellon S.A. ,
Centre Hospitalier de Luxembourg ,
Laboratoire National de Santé ,
Syndicat des Eaux du Barrage d'Esch-sur-Sûre ,
University of Liège ,
Université Henri Poincaré, Nancy (F) ,
Ministère de l'Intérieur et à la Grande Région (L)
Du : 01/05/2009
Au : 30/04/2012
Budget : 540 000,00€
Contact(s) :
Cauchie Henry-Michel
Summary
The PATHOS project aims to develop original methods for the determination of the infective status of non-bacterial pathogens in waters. Among enteric parasites and viruses, Cryptosporodium parvum, noroviruses and adenoviruses were chosen as target pathogens because of their frequent occurrence in the surface waters used for drinking water production in Luxembourg. Moreover, these microorganisms of interest are today considered as emerging waterborne pathogens all over the world. The proposed scientific strategy is based on a combination of cell culture and highly sensitive molecular tools.
This approach is used to overcome most of the disadvantages associated with both conventional cell culture and direct PCR assays. The reduction of time needed for detection of infectious microorganisms is the major advantage of this methodological association. Research activities during 2009 focused on the parasite Cryptosporidium parvum. First, an integrated cell culture real-time PCR method to detect infectious Cryptosporidium was implemented in the laboratory. This technique was adapted from assays previously described in scientific publications with some optimisations compared to the initial protocol.
An internal positive control was added in the real-time PCR step. This control allows us to distinguish true target negatives from false negatives due to PCR inhibition. The analysis of artificially contaminated field samples has also given promising results. Secondly, work has been carried out on the detection of Cryptosporidium by immunofluorescence assay, recoveries being usually highly variable and matrix-dependent. Recovery rates were compared for two commercial detection kits. Similarly, surface water samples with different turbidity were spiked with Cryptosporidium and assessed for recovery rates. Another part of this project is dedicated to the study of the propagation of Cryptosporidium in drinking water bodies. The basin of the Esch-sur-Sûre reservoir was divided in successive compartments, which are known to behave differently from a hydraulic point of view.
The first field campaigns have been carried out in the Upper-Sûre watershed. Sub-basins of various land use have been sampled within the watershed in order to find out how land use may affect the contamination load with Cryptosporidium that enters into the Upper-Sûre drinking water reservoir. Some differences were observed between sub-basins. Moreover, the spatial distribution at fine scale of pathogens has also been addressed by the sampling of a transversal section of the water reservoir. Further sampling campaigns are planned in the near future and in order to refine these first results. Concerning viral pathogens, cell culture and molecular tools are currently developed for human adenoviruses.
Project Website:
Figure: Oocysts of the pathogenic parasite Cryptosporidium parvum isolated from surface waters (nuclei are dyed in blue using DAPI