Huw V. Smith

Waterborne cryptosporidiosis: current status.

In the past ten years Cryptosporidium oocysts have been shown to be common contaminants of water, causing at least 19 waterborne outbreaks of cryptosporidiosis which have affected more than 427 000 individuals. Recommended methods for oocyst isolation and enumeration are time-consuming and inefficient and experts state that the absence of Cryptosporidium oocysts in drinking water can never be guaranteed. In the UK alone, a National Research Programme costing pound3 million has been undertaken. Here, Huw Smith and Joan Rose review the current status of knowledge and identify approaches taken by UK and USA Government regulatory bodies in order to reduce the likelihood of waterborne transmission.

How common is human toxocariasis? Towards standardizing our knowledge

Our understanding of the global impact and cost of human toxocariasis is poor because there is insufficient clinical awareness and no clear repository for the efficacy of clinical, laboratory and treatment interventions. Uniform clinical and laboratory investigative approaches maximize disease diagnosis. International collaboration is required to develop web-based, professional educational support, surveillance questionnaires and standardized serodiagnostic criteria. Determining clinical benefits and treatment outcomes using less crossreactive antigens will enhance clinical and treatment interventions. Increased liaison will identify realistic occurrence and prevalence data and cost benefits of intervention. Web-based centres of excellence and repositories of current knowledge, which augment current veterinary and public health educational sites, should be supported. Expected outcomes should be capable of addressing the clinical and financial burdens of this treatable disease.

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.

Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection

Blastocystis is a prevalent enteric protozoan that infects a variety of vertebrates. Infection with Blastocystis in humans has been associated with abdominal pain, diarrhea, constipation, fatigue, skin rash, and other symptoms. Researchers using different methods and examining different patient groups have reported asymptomatic infection, acute symptomatic infection, and chronic symptomatic infection. The variation in accounts has lead to disagreements concerning the role of Blastocystis in human disease, and the importance of treating it. A better understanding of the number of species of Blastocystis that can infect humans, along with realization of the limitations of the existing clinical laboratory diagnostic techniques may account for much of the disagreement. The possibility that disagreement was caused by the emergence of particular pathogenic variants of Blastocystis is discussed, along with the potential role of Blastocystis infection in irritable bowel syndrome (IBS). Findings are discussed concerning the role of protease-activated receptor-2 in enteric disease which may account for the presence of abdominal pain and diffuse symptoms in Blastocystis infection, even in the absence of fever and endoscopic findings. The availability of better diagnostic techniques and treatments for Blastocystis infection may be of value in understanding chronic gastrointestinal illness of unknown etiology.

Cryptosporidium: Detection in water and food

Water and food are major environmental transmission routes for Cryptosporidium, but our ability to identify the spectrum of oocyst contributions in current performance-based methods is limited. Determining risks in water and foodstuffs, and the importance of zoonotic transmission, requires the use of molecular methods, which add value to performance-based morphologic methods. Multi-locus approaches increase the accuracy of identification, as many signatures detected in water originate from species/genotypes that are not infectious to humans. Method optimisation is necessary for detecting small numbers of oocysts in environmental samples consistently, and further work is required to (i) optimise IMS recovery efficiency, (ii) quality assure performance-based methods, (iii) maximise DNA extraction and purification, (iv) adopt standardised and validated loci and primers, (v) determine the species and subspecies range in samples containing mixtures, and standardising storage and transport matrices for validating genetic loci, primer sets and DNA sequences.

Pursuing the clinical significance of Blastocystis – diagnostic limitations

The clinical significance of one of the most prevalent single-celled intestinal parasites worldwide, Blastocystis, remains unsettled. A plethora of clinical and epidemiological studies have been undertaken to generate data on its prevalence in different populations and investigate the role of the parasite as a cause of gastro- and extra-intestinal disease. In this article, we pinpoint limitations of studies that seek to determine the clinical significance of Blastocystis, based on shortcomings in our understanding of Blastocystis diagnosis and biology, and identify methodologies for further studies aimed at determining the molecular epidemiology and clinical impact of this parasite.

An outbreak of cryptosporidiosis associated with a swimming pool.

In August 1988 an increase was noted in the number of cases of cryptosporidiosis identified by the microbiology laboratory at Doncaster Royal Infirmary. By 31 October, 67 cases had been reported. Preliminary investigations implicated the use of one of two swimming pools at a local sports centre and oocysts were identified in the pool water. Inspection of the pool revealed significant plumbing defects which had allowed ingress of sewage from the main sewer into the circulating pool water. Epidemiological investigation confirmed an association between head immersion and illness. The pools were closed when oocysts were identified in the water and extensive cleaning and repair work was undertaken. The pool water was retested for cryptosporidial oocysts and found to be negative before the pool reopened.

Incidence of cryptosporidiosis species in paediatric patients in Malawi

We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR–RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.

Identification of Cryptosporidium spp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay

ABSTRACT We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

Natural Cryptosporidium hominis infections in Scottish cattle

G21 3UW M. Mallon, BSc, A. Macleod, BSc, PhD, A. Tait, BSc, PhD, FRSE, Wellcome Centre for Molecular Parasitology J. M. Wastling, BSc, PhD, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ W. J. Reilly, BSc, BVMS, DVSM, L. M. Browning, BSc, PhD, Scottish Centre for Infection and Environmental Health, Glasgow G3 7LN D. Gray, BVM&S, MSc, MRCVS, SAC Veterinary Services, Mill of Craibstone, Bucksburn, Aberdeenshire