Cryptosporidium — Biotechnological advances in the detection, diagnosis and analysis of genetic variation

Abstract

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal–oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as “genotypes” or “subgenotypes”) of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.

Introduction

The genus Cryptosporidium (Apicomplexa) includes important parasitic protists which are directly transmitted by the faecal–oral route and cause gastroenteritis in mammals and birds, respiratory disease in birds or gastritis in reptiles and possibly fish (O'Donoghue, 1995, Xiao et al., 2004a). In agricultural animals, Cryptosporidium parvum, C. andersoni and C. meleagridis have been reported to cause cryptosporidiosis outbreaks, leading to substantial morbidity and economic losses (de Graaf et al., 1999). In humans, cryptosporidiosis is mainly caused by C. hominis or C. parvum (see Morgan et al., 1999a, Xiao et al., 2004, Cacciò, 2005, Cacciò and Pozio, 2006), although species, such as C. meleagridis, C. felis or C. canis have been reported rarely (Guyot et al., 2001, Xiao et al., 2001, Cacciò et al., 2002, Chalmers et al., 2002, Gatei et al., 2002, Cama et al., 2003, Cacciò, 2005, Cama et al., 2006). The transmission of human cryptosporidiosis has been inferred to occur primarily through direct human-to-human (anthroponotic) transmission for C. hominis and C. parvum, and, less by animal-to-human (zoonotic) transmission for C. parvum and the other “rare” species (C. meleagridis, C. felis or C. canis) (Morgan et al., 1999a, Ramirez et al., 2004, Cacciò, 2005). Clinical cryptosporidiosis in humans is mainly associated with diarrhoea, dehydration, malabsorption, weight loss and/or wasting (Farthing, 2000, Kosek et al., 2001, Chen et al., 2002, Tzipori and Ward, 2002). Subclinical infections can occur in both immunocompetent (Checkley et al., 1997, Hellard et al., 2000, Al-Braiken et al., 2003) and immunodeficient (Houpt et al., 2005) humans, although such infections have been more commonly reported in animals (Lorenzo-Lorenzo et al., 1993, Skerrett and Holland, 2001). In many cases, clinical cryptosporidiosis resolves as immunity develops and eliminates or suppresses the infection (Riggs, 2002), although chronic infections can develop in high-risk host groups, particularly neonates and immuno-compromised individuals (with congenital or acquired immuno-deficiencies or those undergoing immuno-suppressive chemotherapy), which, due in part to the limited availability of highly effective chemotherapeutic treatment (Greif et al., 2001, Mead, 2002, Armson et al., 2003, Zardi et al., 2005, Cacciò and Pozio, 2006), may result in death.

Human cryptosporidiosis can occur sporadically (Hunter et al., 2004, Roy et al., 2004), but it is also commonly associated with outbreaks linked to child day-care centres, contaminated food (Graczyk and Fried, 2007, Greig et al., 2007, Smith et al., 2007), contaminated swimming pools and/or drinking water supplies (MacKenzie et al., 1994, MacKenzie et al., 1995a, Fayer et al., 2000, Glaberman et al., 2002, Cohen et al., 2006, Karanis et al., 2007). Waterborne cryptosporidiosis is particularly important because Cryptosporidium oocysts are resistant to disinfectants (such as chlorine) commonly used for water treatment (Peeters et al., 1989, Korich et al., 1990, Carpenter et al., 1999). Waterborne outbreaks have been reported in developed countries, including the USA and Canada, the most notable being the outbreak in Milwaukee in 1993, which resulted in more than 400,000 suspected and 5,000 confirmed cases of clinical cryptosporidiosis (MacKenzie et al., 1994, MacKenzie et al., 1995b, Cicirello et al., 1997, Corso et al., 2003). Given the relative ubiquity and resilience of oocysts in water and the environment (King and Monis, 2007), the limited availability and cost of chemotherapeutic compounds or regimens for treatment or vaccination in humans and other animals (Greif et al., 2001, Mead, 2002, Armson et al., 2003, Zardi et al., 2005, Cacciò and Pozio, 2006) and the socioeconomic impact of cryptosporidiosis (particularly in human populations in which HIV/AIDS is prevalent), cryptosporidiosis has been classified by the World Health Organization (WHO) as a ‘reference pathogen’ indicating water quality globally (Medema et al., 2006).

The specific diagnosis of cryptosporidiosis, including the precise identification and characterisation of Cryptosporidium species, is central to the control of this disease in humans and a wide range of other animals and to the understanding of the intricacies of its epidemiology. There have been significant limitations in specific diagnosis using traditional, microscopic, biochemical and serological techniques (Cacciò and Pozio, 2006), such that there has been a substantial need for improved molecular techniques. Using molecular tools, 15 recognized species and numerous additional genotypic variants have been reported from a range of vertebrate host groups (Alvarez-Pellitero and Sitja-Bobadilla, 2002, Morgan-Ryan et al., 2002, Fayer, 2004, Power et al., 2004, Ryan and O'Hara, 2004, Xiao et al., 2004a). These species and genotypes cannot be distinguished based on morphology or on host occurrence (Fall et al., 2003, Fayer, 2004, Xiao et al., 2004a). In the present article, we provide an account of selected microscopic, immunological and flow cytometric methods employed for the detection of Cryptosporidium, and describe advances in the development and evaluation of nucleic acid-based approaches for the specific diagnosis of cryptosporidiosis and the analysis of genetic variation within and among species of Cryptosporidium. We also discuss the advantages and disadvantages of selected techniques and emphasize the benefits of utilizing molecular tools toward achieving a better fundamental understanding of the systematics, epidemiology and population genetics of members within this genus, underpinning the prevention and control of cryptosporidiosis in human and non-human hosts. Readers interested in topics extending beyond the scope of the present article are referred to selected review articles of Cryptosporidium/cryptosporidiosis (Fayer, 2004, Xiao et al., 2004a, Xiao et al., 2004, Hunter and Thompson, 2005, Monis et al., 2005, Smith et al., 2005, Cacciò and Pozio, 2006, Boulter-Bitzer et al., 2007, Karanis et al., 2007, King and Monis, 2007).