environmental samples. Thus, currently, molecular characterization is very prominent in the published literature.
3.4.2 Collection, Concentration, and Purification Techniques
The original methods for concentrating the parasites included grab samples followed by centrifugation and flotation methods. The Ascaris methods for assessing sludge have followed this approach and use a flotation method in a density gradient of mag- nesium sulfate (USEPA, 2003). However, it was clear that low-level detection required larger-volume processing. Thus, filtration methods began to be used. The new Gelman parasite filter was designed specifically for Cryptosporidium and Giardia collection and recovery, as part of EPA method 1623 (USEPA, 2001). More recently, ultrafil- tration has been used and has improved recovery. The use of hollow-fiber ultrafilters pretreated with sodium polyphosphate (NaPP, 0.01%) and Tween 80 (0.01%) with cross-flow tangential processing decreased clogging, allowing larger volumes of water with higher turbidity to be processed (Hill et al., 2007; Polaczyk et al., 2008).
Further concentration and purification has been undertaken with immunomagnetic separation techniques (USEPA, 2001), which then produces a concentrate that is used for microscopy or molecular testing. This, in particular, has decreased interferences when PCR methods are used (Johnson et al., 1995).
For Naegleria, up to 1 L of water is generally filtered through membranes (0.65-μm DVPP Milipore filters), after which the filter is cut up and eluted with 10 mL of PBS via vortexing and sonication, and finally, concentrated by centrifugation (Behets et al., 2007).
As is the case with any molecular techniques, PCR inhibitors are significant prob- lems with parasites. Unlike the bacteria and viruses, however, it is much more difficult to open up and extract DNA from the oo(cysts) and ova. Several procedures have been used after IMS or flotation, including (1) freeze–thaw and or bead-beating and (2) extraction with Ultraclean soil DNA (Behets et al., 2007) or a FastDNA SPIN soil kit (Hill et al., 2007; Xiao and Ryan, 2008). In other studies, both human and water samples have been evaluated after using the QIAamp DNA stool kits (Bertrand and Schwartzbrod, 2007).
3.4.3 Molecular Techniques for Parasite Detection
Molecular tools have been developed over the last 20 years to detect and differen- tiate the parasites at the species, genotype, and subtype levels. This has led to an improvement in understanding the diversity of the parasites, their epidemiology, and their pathogenicity. A real-time quantitative PCR (qPCR) method has been reported on and used to determine the levels of total and viable Ascaris eggs in laboratory solu- tions (Pecson et al., 2006). This procedure targeted the internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. Currently, microscopy remains the method of choice for sludges.
The PCR methods for Entamoeba histolytica and E. dispar were developed in the 1990s targeting the ribosomal RNA genes (Clark and Diamond, 1991), which resulted from an awareness that one type of infection in humans was seen as benign (Lebbad and Sv¨ard, 2005). In fact, two separate species were suggested as early as 1925, but it was not until 1993 that E. dispar , which causes asymptomatic colonization, was finally
NOVEL METHODS FOR DETECTION OF PARASITES IN WATER 73
described (Clark and Diamond, 1991). These methods have assisted in the clinical laboratory, determining those patients that require treatment (Lebbad and Sv¨ard, 2005) but have yet to be used in water. A multiplex-PCR method has also been utilized for clinical samples (Santos et al., 2007), targeting tandem sequences repeated in the estrachromosomal circular DNA found in these two species, detecting as few as five trophozoites (the replicating form of the parasite, which is also excreted in stools). The PCR methods could have great application as biomonitoring tools in sewage, examining risks at the community level associated with E. histolytica.
Cryptosporidium is the most highly studied parasite of all enteric pathogens in the last 20 years. A recent book and several reviews have described the developments in molecular characterization of the genetic variation, in diagnosis, and in detection in the environment, particularly for the water environment (Smith et al., 2006; Fayer and Xiao, 2008; Jex et al., 2008; Xiao and Fayer, 2008). In many studies and publications, both Cryptosporidium and Giardia are addressed, with identification of species and the genotypes using the 18S subunit of the ribosomal DNA. Unlike Giardia, though, Cryptosporidium reproduces by both sexual and asexual means, and early on it was recognized that C. parvum, one of the key species, was zoonotic and could be transmit- ted readily between mammals and humans. PCR tools targeted the 18S rDNA, COWP (cell outer wall protein), and in some cases the Hsp70 (heat shock protein), and using RFLP or sequencing, these have been used to describe the human-specific species, C. hominis, and other species associated with other animals [C. andersonii (in cattle) C. muris (in mice), and C. baileyi (in birds)] during outbreaks and in other investigations of water (Smith et al., 2006). New approaches to examine Cryptosporidium (and Gia- rdia) by quantitative PCR have not gained much traction until recently. In 2003, Guy et al. published a qPCR procedure focused on the COWP gene (and theβ-giardin gene for Giardia). This could be multiplexed and provided numbers that were consistent with oo(cyst) counts under the microscope from sewage samples. These researchers also suggested the need for Chelex 100 and poly(vinylpyrrolidone) (PVP3600 2%) as pretreatment to decrease the impact of inhibitors on the PCR reaction. Finally, Hill et al. 2007 developed a qPCR method that has been used successfully with ultrafiltration to quantify the oocysts from tap water.
Giardia methods are very tied to the methods that have been used for Cryptosporid- ium; this is due partially to the USEPA methods. Early studies had shown that the epidemiology of the two protozoans was similar and that the methods could readily be multiplexed with fluorescently labeled antibodies with microscopy to examine waters for both protozoans. These parasites are also found in similar sources, animals and humans, albeit not at the same prevalence. Molecular studies have focused on devel- opment of PCR for both theβ-giardin and gdh genes, which have been used in water studies and in outbreak investigations (Robertson et al., 2006). Giardia duodenalis is, however, described by seven assemblages, A through G. Bertrand and Schwartzbrod (2007) have developed specific real-time PCR assays to examine the G. duodenalis assemblages A, B, and E, which they applied in studies of wastewater. The targets were areas in the tpi gene and primers amplified at 148 and 81 bp for A and B, respectively. While assemblages A and B were considered to cause most of the infec- tions in humans, these have been found in a number of other animals. In wastewater, G. duodenalis assemblage B was found most often at higher concentration and then, secondarily, assemblage A, but E (associated with animals) was not detected.