Molecular Detection and Differentiation of Cryptosporidium Oocysts in Water: theChallenge and Promise

摘要

Because of the presence of host-adapted Cryptosporidium species and genotypes,molecular tools can help assess the source and hazardous potential of Cryptosporidium oocystsin water. The development and use of molecular tools in the analysis of environmental sampleshave gone through several phases. Earlier polymerase chain reaction (PCR) tools were designedfor the detection of Cryptosporidium oocysts in clinical samples. Subsequently, a genotypingcomponent was incorporated into many of these assays to differentiate Cryptosporidium oocystsof anthroponotic origins from zoonotic origins. These tools were mostly based on the sequencesof bovine C. parvum isolates, and were intended for the detection for C. parvum in clinicalsamples, thus they do not detect and differentiate many Cryptosporidium spp. And distant C. Parvum genotypes. More recently, new molecular tools that are Cryptosporidium genus-specificand have the ability to differentiate Cryptosporidium species and genotype have been introduced,which has resulted in the finding of five major Cryptosporidium parasites in humans: the C. Parvum human and bovine genotypes, C. meleagridis, C. felis, and C. canis. Current problems inmolecular detection of Cryptosporidium oocysts include (1) the availability of only a limitednumber of tools for species differentiation, most of which are based on the small subunit rRNAgene; (2) the nonspecificity of some species differentiation tools; (3) the misinterpretation of databecause of lack of information of recent findings; and (4) the existence of erroneous data in thedatabase and publications. Nevertheless, in conjunction with immunomagnetic separation (IMS),some PCR-based tools have been successfully used in the detection, differentiation, and trackingof Cryptosporidium oocysts in storm water, surface water, and wastewater. Results of thesestudies have shown that a significant proportion of Cryptosporidium oocysts in water do not havehigh human-infective potential, which would have been overestimated by the recommended ICRmethod or method 1622/1623. Despite the recent progress, much more needs to be done beforemolecular tools can be used in routine analysis of water samples. Specifically, (1) rigorousstandardization and testing have yet to be carried out in order to develop quality assurance andquality control procedures; (2) there is a need for the development of protocols that allows theextraction of PCR-quality nucleic acid without using the expensive and pathogen-specific IMS;(3) turnaround times have to be reduced to allow close to real-time detection; (4) quantitative andhigh resolution typing procedures need to be incorporated for analysis of samples in specialsituations (such as outbreaks or bioterrorism); and (5) there is a need to take advantage of newtechniques such as biosensors and microarrays. The use of molecular tools can potentiallygenerate data that are useful in the risk assessment of various types of water in differentenvironmental settings, and for watershed management and source water protection.