Working toward improved monitoring of Cryptosporidium and Giardia (oo)cysts in water samples: testing alternatives to elution and immunomagnetic separation from USEPA Method 1623.1

Abstract

Objective This study was designed to find a method to enhance the recovery of Cryptosporidium spp. and Giardia spp. parasites from water samples for research purposes compared to the results that can be achieved with USEPA Method 1623.1. Four different approaches were used to test water samples that were artificially spiked with parasites. The approaches were: (i) Method 1623.1 itself, (ii) elution of Method 1623.1 combined with microfiltration, (iii) an elution technique based on grinding the filter membrane in a blender before the eluent was concentrated by immunomagnetic separation, and (iv) the blender elution followed by microfiltration. Fluorescence microscopy was used to determine which approach led to the highest parasite recovery rates. Results Method 1623.1 gave the best results for Giardia, while all four approaches were statistically equivalent for Cryptosporidium. We evaluated the costs and laboratory time requirements for each protocol to give readers a complete comparison of the methods tested. Elution of Method 1623.1 combined with microfiltration resulted in lower costs and less laboratory work time without compromising the recovery of the parasites.

Methods used for concentrating oocysts of Cryptosporidium spp., spores Encephalitozoon spp. and Enterocytozoon spp. and their occurrence in Slovak water samples

“…Light microscopic examination of stained clinical smears, especially stool samples, is an inexpensive method for the detection of microsporidia spores, although it does not allow their identification to the species level. The most commonly used staining technique is the Chromotrope 2R method or its modifications (Feng et Li, 2017).…”

“…Transmission electron microscopy (TEM) has long been the gold standard for the identification of microsporidia based on polar filament observation in organisms and is still important for observing and describing the ultrastructural features of developing and mature organisms, but it is too expensive, timeconsuming, and unsuitable for routine diagnosis (Weber, Deplazes, Schwartz, 2000;Feng et Li, 2017). Weber's chromotropic staining was also used by Galván et al for the examination of microsporidia.…”

“…Over the past 20 years, PCR-based molecular methods for amplification of gene targets have been developed and increasingly applied to improve sensitivity and species specificity in the diagnosis of both cryptosporidiosis and microsporidiosis (Feng et Li, 2017;Dashti et al, 2022). Compared to traditional methods based on microscopy, these molecular methods can offer potential advantages such as increased sensitivity, higher specificity, faster time to result and easier interpretation.…”

Toward waterborne protozoa detection using sensing technologies

“…Magnetic iron oxide NPs are employed for biomedical applications such as magnetic separation (Wu et al, 2015). For example, immunomagnetic separation (IMS) is now employed as a standard method for the detection and separation of Cryptosporidium spp., and G. lamblia oocysts/cysts from 10 to 150 L water samples (USEPA, 2012), although several studies have been conducted to increase the recovery rate of the method using either additional concentration methods or alternative elution (Hu et al, 2004;Fradette and Charette, 2022).…”