Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623

Abstract

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r ‫؍‬ 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

Methods for parasitic protozoans detection in the environmental samples

“…Therefore, the authors emphasize that it seems reasonable to bring the MS procedure, not only when a new source of water is investigated, but also when the samples are collected in different streams of flowing water or at different turbidity than those samples collected earlier and investigated from the same place. Both methods allow to replace components with others more efficient or effective, provided that they comply with the criteria of quality control (Francy et al, 2004). And so, different filter systems are applied for the filtration required in these methods.…”

“…A logistic limitation is another restriction (degradation of oocysts during transfer), especially in the method, and finally the state of the science of Cryptosporidium testing in general (Weintraub, 2006). Because the 1623 method is commonly used to monitor sources of drinking water and is the required method in the programme of surface water treatment, the modifications for the improvement of recovery and reduction of analytical costs are needed (Francy et al, 2004;Weintraub, 2006 (Johnson et al, 1995;Loge et al, 2002). The environmental samples (water, soil, and food) are rich in PCR inhibitors, such as humic acids, potassium dichromate, formaldehyde which could be co-extracted with DNA during the DNA isolation and purification process and which therefore could interfere with the PCR amplification (Tebbe & Vahjen, 1993).…”

“…The analysis of MS is a determinant of the effectiveness of the applied analytical procedure (Francy et al, 2004). There are two analyzed samples, the number of viable added organisms (matrix spikes) is known in…”

“…Francy et al (2004) took into consideration the MS for every analyzed sample. They discovered, that in some cases, the recoveries between two samples collected from the same place were completely different, especially when turbidities of the water samples were different.…”

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

“…However, the recovery of oocysts from environmental water samples with the use of these both methods has been reported to be very variable (Simmons et al 2001;Kuhn and Oshima 2002;Wohlsen et al 2004;Krometis et al 2009;Karim et al 2010). With regard to this fact, matrix spikes are routinely analysed to determine the influence of environmental matrices on the effectiveness of the methods for detecting target organisms (Francy et al 2004).…”