Reply to “Ranking Filter Methods for Concentrating Pathogens in Lake Water”

Abstract

A ccurately comparing filtration methods is indeed difficult. Our method (1) and the method described by Borchardt et al. for determining recoveries are both acceptable approaches; however, each is designed to achieve a different research goal. Our study was designed to compare recoveries of multiple microorganisms in surface-water samples. Because, in practice, water-matrix effects come into play throughout filtration, concentration, and detection processes, we felt it important to incorporate those effects into the recovery results.In our study, the concentrations of microorganisms were measured prior to seeding the test sample. The concentrations of the seed organisms were determined in the absence of any matrix effect from the water sample. This method of determining the denominator of the recovery calculation does not invalidate the results and has been used by other researchers in the field (2, 3, 4), including Borchardt (4). In fact, we were not able to find references, other than those associated with Borchardt et al. (5,6), in which the concentration of the seed was determined in a seeded negative final concentrate created by filtering unseeded water with the same source and volume as the recovery test.Although for some experiments the recovery trials were done over several days, we took great pains to collect new samples for each trial day and ensure that water quality did not change over the course of the trial (no rain event, high waves, etc.). We also do not believe that mixing seeded and naturally occurring microorganisms makes the filter comparisons uninterpretable, as Borchardt et al. suggest. All the filters in our trials were treated the same in that they had all had the same matrix effects and the same microorganisms (seeded and naturally occurring). Although there were more trials for enterococci and Escherichia coli for the glass wool and automatic ultrafiltration (UF) (because these filters were used for unseeded controls) and fewer trials for protozoan pathogens for the NanoCeram, we do not believe that the numbers of trials need to be exactly the same to validate our results.We assume that the matrix effect and inhibition comments by Borchardt et al. are in regard to virus analyses since they refer to quantitative PCR (qPCR) determinations of the seed and qPCR inhibition values (which were only measured for viruses). Inhibition of qPCR was measured for each sample in our study. Instead of using hepatitis G virus (HGV) armored RNA as an inhibition control and assuming that inhibition of HGV is similar to that of other viruses, as was done by Lambertini et al. (5), we chose to seed a subsample of the final unseeded concentrate with the actual DNA and RNA viral targets. Multiple dilutions of these control samples were analyzed which permitted us to assess qPCR inhibition and choose an appropriate dilution of test sample to analyze.We do not disagree that the variability rank score (RCV) is an ad hoc measure and that filtration methods with better recoveries are more resistant to shifts in th...