INTER-LABORATORY ROUND-ROBIN TESTING OF IMMUNO-MAGNETISABLE SEPARATION (IMS) FOR THE CONCENTRATION AND SEPARATION OF CRYPTOSPORIDIUM OOCYSTS FROM WATER SAMPLES FINAL REPORT OF CONTRACT NUMBER EPC 1/9/059
Report No DWI0764

SUMMARY

Immunomagnetisable separation (IMS) technology for the separation and concentration of target cells has been of increasing application within the bio-medical field, both for routine diagnostic and measurement use and also for application as a research tool, in recent years. Whilst the use of this technology for the concentration of Giardia lamblia cysts from water samples has been published (Bifulco and Schaefer, 1993) and the potential for the use of this technique for the separation and concentration of Cryptosporidium oocysts from water has been recognised, (Robertson and Smith, 1992; Smith et al, 1993, Parker and Smith, 1994), no full-scale testing of the actual practical application of this technique for the separation and concentration of parasites from water has been previously conducted.

In the work undertaken for this report, the use of this technique was tested in five laboratories which undertake routine analyses of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of a carefully designed IMS technique with those techniques in current use (the "Blue Book" Standing Committee of Analysts (SCA) method and flow cytometry). The parameters investigated included the use of a range of target seeds of oocysts (3.3, 13 and 33 oocysts), two different volumes of water (1 ml and 10 ml) and a range of different turbidities (clean water, 40-60 nephlometric turbidity units (NTU) and greater than 600 NTU). Furthermore, as well as allowing comparison between the recovery efficiencies of these three techniques, under the constraints of the various parameters summarised above, work was undertaken to identify whether or not the IMS technique affected the viability of oocysts and also to compare the morphology, fluorescence and uptake of 4'6 diamidino-2-phenylindole (DAPI) by the oocysts following this technique.

Whilst inter-laboratory variation occurred (with some laboratories consistently finding higher or lower numbers of oocysts with the different techniques), comparison of the performance of the analytical laboratories was not the subject of this study. The laboratories were anonymised by the use of code letters and in the results section of this report the results from the laboratories are combined to allow comparison between methods and other variables without being influenced by the relative recovery efficiencies of the laboratories at the different techniques.

In very low turbidity samples (clean water), the IMS technique appeared to be significantly better than both SCA and FCM methods at recovering oocysts both from 1 and 10 ml samples. Not only were higher recovery efficiencies reported, but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-positive samples than with the other two techniques. Furthermore, the simple acid desorption step for dissociating the oocysts from the beads was considered to be successful, with >90% of the oocysts dissociated from the beads.

However, when the water sample is turbid, the recovery efficiency of the IMS technique may be reduced. In one trial with turbid 1 ml samples, significantly less oocysts were recovered using the IMS technique than either of the other methods and in another trial with a 1 ml turbid sample the IMS technique recovered significantly less oocysts than the FCM technique. Assessment of all the results from 1 ml turbid samples indicates that whilst the recovery efficiency of the IMS technique may be reduced by suspended matter, when the turbidity is relatively low (between 40-60 NTU), all 3 techniques performed with similar efficiency. However, when the turbidity is high (>600 NTU), the efficiency of the IMS technique is significantly affected in some water types. These results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently least affected by interfering particulate matter. However, it should be noted that in trials with clean water or low turbidity water this technique was the one which consistently reported negative results in oocyst-positive seeded samples (for clean water, this difference was found to be statistically significant).

Attempts were made to address the problems experienced in the IMS technique in samples of high turbidity, by introducing blocking agents into the method protocol. Whilst some of the blocking agents showed promise, insufficient time was available for development of this improved methodology and subsequent testing by the participating laboratories.

Whilst the IMS technique was found not to have any detectable effect on the viability of oocysts, it did appear to result in significant differences in the morphology of the oocysts (if the oocysts were "old"), fluorescent antibody staining characteristics and uptake of DAPI into the sporozoite nuclei as compared to the SCA method. Following IMS of "old" oocysts, more broken, misshapen and 'pac man' shaped oocysts were noted, however this did not appear to hinder the operators' identification of the oocysts. Following the IMS technique the fluorescence antibody staining was reported to be improved, this could be because acidification of the oocysts increases the number of epitopes available for antibody binding. The use of DAPI to assist in identification of oocysts was considered to be more useful following the SCA method than following IMS; this might be due to the acidification during IMS, hydrochloric acid is known to affect nucleic acids. However, it should also be noted that these differences were also, in part, due to characteristics of the oocysts themselves and not necessarily due to the techniques per se.

Despite the potential difficulties with the IMS in turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the armoury of methods for the concentration of oocysts from water samples and was considered by the participants to be simple and user-friendly, all the participating laboratories indicated that they would be eager to use the IMS technique in routine analysis. Furthermore, with further research to address problems which may be encountered in specific water types, the potential for this technique may be realised to an even greater extent.

Copies of this report may be available as an Acrobat pdf download under the 'Pre 2000 Reports' heading on the DWI website.