This book provides a comprehensive review of the current state of analytical techniques for the detection of Cryptosporidium, as well as looking at likely future developments.

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Cryptosporidium: The Analytical Challenge

By M. Smith, K.C. Thompson

The Royal Society of Chemistry

Cryptosporidium: The Analytical Challenge H. V. Smith and A. Ronald, 1,
Molecular Epidemiology and Systematics of Cryptosporidium parvum Una M. Morgan, Lihua Xiao, Ronald Fayer, Altaf A. Lal and R. C. Andrew Thompson, 44,
Molecular and Phenotypic Analysis of Cryptosporidium parvum Oocysts of Human and Animal Origin G. Widmer, X. Feng, D. Akiyoshi, S. M. Rich, B. Stein and S. Tzipori, 51,
Complying with the New Cryptosporidium Regulations P. Jiggins, 56,
Cryptosporidiosis in Healthy Adult Volunteers C. L. Chappell and P. C. Okhuysen, 62,
Trial of a Method for Continuous Monitoring of the Concentration of Cryptosporidium Oocysts in Treated Drinking Water for Regulatory Purposes D. P. Casemore, B. Hoyle, P. Tynan and Mark S. Smith with Members of PHLS project team, 73,
A Dielectrophoresis System for Rapid Analysis of Cryptosporidium parvum A. P. Brown and W. B. Betts, 84,
A Review of Methods for Assessing the Infectivity of Cryptosporidium parvum Using In-vitro Cell Culture P. A. Rochelle and R. De Leon, 88,
Applications of MALDI-TOF Mass Spectrometry in the Analysis of Cryptosporidium K. Hall, M. A. Claydon, D. J. Evason, M. S. Smith and J. Watkins, 96,
Some Observations on Factors which Affect Recovery Efficiency in Cryptosporidium Analysis A. P. Walker, 101,
Development of a Novel Method for the Capture, Recovery and Analysis of Cryptosporidium Oocysts from High Volume Water Samples A. C. Parton, A. Parton, B. Brewin, K. Bergrnann, E. Hewson and D. Sartory, 111,
The Experience of the Leap Proficiency Scheme With Respect to Cryptosporidium Testing K. Clive Thompson, Barry May, Diane Corscadden and John Watkins, 120,
An Evaluation of the Current Methods for the Detection and Enumeration of Cryptosporidium in Water Carol Francis, Diane Corscadden, John Watkins and Mark Wyer, 133,
Automated Detection and Viability Assessment D. A. Veal, M. R. Dorsch and B. C. Ferrari, 143,
Can We Believe Our Results? Frank W. Schaefer, III, 155,
Subject Index, 162,

CHAPTER 1

CRYPTOSPORIDIUM: THE ANALYTICAL CHALLENGE

H. V. Smith and A. Ronald

Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK. corresponding author. Tel: +44 (0)141 201 3028, Fax: +44 (0)141 558 5508

1 INTRODUCTION

In the last twenty years, the protozoan parasite, Cryptosporidium has been widely recognised as a cause of waterborne disease. Its transmissive stage, the oocyst, is a frequent inhabitant of raw water sources used for the abstraction of potable water and its importance is heightened because, coupled to its low infectious dose, conventional water treatment processes, including chemical disinfection, cannot guarantee to remove or destroy oocysts completely. Furthermore, because of their chlorine insensitivity, the coliform standard cannot be relied upon as an indicator of either the presence or viability of waterborne Cryptosporidium oocysts. For these reasons, robust, sensitive and specific methods are required for the recovery and identification of oocysts in water concentrates.

2 THE PARASITE

Cryptosporidium has a complex life cycle, involving both asexual and sexual reproductive cycles, which it completes within an individual host (monoxenous). Transmission from host to host is via an environmentally robust oocyst which is excreted in the faeces of the infected host. C. parvum is the major species responsible for clinical disease in man and domestic mammals (Current and Garcia, 1991), although infections with species other than C. parvum have been described in both immunocompetent and immunocompromised human hosts (Morgan et al., 2000; Pedraza-Diaz et al., 2000, 2001).

By the nature of its characters, the genus Cryptosporidium (Kingdom, Protozoa; Phylum, Apicomplexa (Sporozoa); Class, Coccidea; Order, Eimeriida) belongs to the Family Cryptosporidiidae. These include: "development just under the surface membrane of the host cell or within its striated border and not in the cell proper. Oocysts and meronts with a knob-like attachment organelle at some point on their surface. Oocysts without sporocysts, with four naked sporozoites. Monoxenous. Microgametes without flagella" (Levine, 1973). Currently, the genus Cryptosporidium contains 10 valid named species. C. parvum, C. muris, C. felis, C. andersoni and C. wrairi infect mammals, C. baileyi and C. meleagridis infect birds, C. serpentis and C. saurophilum infect reptiles and C. nasorum infect fish.

C. parvum infects the epithelia of the intestinal tract (enterocytes) of various mammals. Exposure to the environments of the gastrointestinal tract trigger the poorly understood process of excystation, whereby sporozoites are actively released through the suture of the oocyst. Known triggers include temperature (37�C), acidity (~ pH 2), slight alkalinity (~ pH 7.6) exposure to bile salts and trypsin (Fayer and Leek, 1984; Reducker and Speer, 1985; Hill et al., 1991; Robertson et al., 1993a). During the course of symptomatic infection, up to 1010 oocysts are shed into the environment, which are capable of prolonged survival in moist microclimates. Figure 1 describes the life cycle of C. parvum.

Waterborne transmission is well documented (Smith and Rose, 1990, 1998; Smith and Nichols, 2001), and can affect numerous individuals. Smith and Rose (1998) stated that more than an estimated 427,100 individuals had been affected in 19 documented waterborne outbreaks. The numerous contributors to waterborne oocysts, and the small size, environmental robustness and chlorine insensitivity of oocysts are factors that enhance their presence and likelihood of surviving in water treatment processes. The second report of the UK Joint Group of Experts on Cryptosporidium in water supplies stated that "... absence of Cryptosporidium from drinking water can never be guaranteed ..., criteria for 'best practice' for operating water treatment works could be identified and should be adopted" (Anon., 1995a). Well operated treatment works can achieve better than 2 log.10 of oocyst removal and for disinfectants which could be used within current UK guidelines or legislation, ozone and UV have potential (Hall and Pressdee, 1995; Anon., 1995; Croll and Hall, 1997).

3 GETTING TOUGH ON CRYPTOSPORIDIUM OOCYSTS – A MULTINATIONAL APPROACH

Many requirements, rules and regulations are in place to attempt to address the control of Cryptosporidium which threatens the safety of drinking water. The current European Union 'drinking water' directive (Anon., 1980) requires that 'water intended for human consumption should not contain pathogenic organisms' and 'nor should such water contain: parasites, algas, other organisms such as animalcules'. The proposed revision to this directive, to be implemented in 2003 (Anon., 1995b), in recognising the impracticality of the current zero standard, will make it a general requirement 'that water intended for human consumption does not contain pathogenic micro-organisms and parasites in numbers which constitute a potential danger to health'. No numerical standard for Cryptosporidium is proposed.

The Unites States Environmental Protection Agency (USEPA) has issued...

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