SOLID PHASE CYTOMETRY AS A SINGLE ANALYTICAL PLATFORM FOR DETECTING PATHOGENS AND INDICATOR ORGANISMS IN DRINKING WATER

摘要

Current methods for detection of waterborne pathogens or indicator organisms are still open to improvement. Detection of pathogens such as Cryptosporidium and Giardia, although of major importance for protecting end-users' health, in the end still relies on completely manual results interpretation using epifluorescence and differential interference contrast (DIC) microscopy that is subject to operator fatigue and subjectivity. Moreover, the time required to analyze one slide is long, hence limiting the sample throughput. Similarly, speed of indicator organisms detection, the principal daily testing in each drinking water laboratory, is limited to 18 hours at best. Whilst this may not be a problem for routine monitoring, where more then 99% of all samples are negative, this is a major issue to the field of "emergency testing," e.g. in cases of water supply breakdown or after works, where rapid information about the potability of the water is indispensable. The recently introduced solid phase cytometry (SPC) technology brings within reach a solution to address the above flaws of existing methodologies with one analyzer. The ScanRDI (ChemScanRDI in Europe; Chemunex, Ivry-sur-Seine, France) has now proven its ability to form the basis of a single testing platform, now allowing the detection of Cryptosporidium, Giardia, Escherichia coli and total coliforms. The apparatus detects fluorescently labelled micro-organisms at the single cell level following excitation using a 488-nm argon-ion laser within 3 minutes. Results obtained are displayed both in table and graphical format, the latter allowing visual result confirmation using either epifluorescence or DIC microscopes linked directly to the ScanRDI and driven entirely by its software. Typically, less than 2 minutes are required to confirm the scan results as these data primarily represent detected microorganisms, as the vast majority of any interfering particulate material present in the concentrated, purified and labelled water sample has already been rejected by the system's discrimination software. Moreover, confirmation results are directly stored on the ScanRDI computer that is equipped with a CD-writer enabling storage and retrieval of previous results at any time. By applying this technology for Cryptosporidium and Giardia testing, speed (total detection time is typically less than 10 minutes compared to up to 40 or more minutes when using manual microscopy) and hence increased sample throughput, standardization and improved manpower use are guaranteed. The technique has been shown to produce accurate results that are comparable with manual microscopy, and is compatible with the concentration, purification and labelling methods described in EPA's Method 1623. For E. coli and total coliforms, the application of a novel test concept based on two-step enzymatic labelling of single cells allows their detection after 3.5 hours from initial sampling with the same efficiency (e.g. agreements of >90%) as growth based methods taking at least 18 hours. Aiming at a further expansion of the solid phase cytometry analytical platform, other micro-organisms of importance to increase consumers' health, e.g. enterococci and Legionella, are currently under investigation.