A Cryptosporidium hominis outbreak in north-west Wales associated with low oocyst counts in treated drinking water.

An outbreak in the autumn of 2005 resulted in 218 confirmed cases of Cryptosporidium hominis. The attack rate (relative risk 4.1, 95%CI 2.8-9.1) was significantly higher in the population supplied by Cwellyn Water Treatment Works (WTW). A case-control study demonstrated a statistically significant association (odds ratio 6.1, 95% CI 1.8-23.8) between drinking unboiled tap water and C. hominis infection. The association remained significant in a logistic regression analysis, with an adjusted odds ratio of 1.30 (95 CI 1.05-1.61) per glass of unboiled tap water consumed per day. This evidence together with environmental and associated microbiological investigations, and the absence of effective treatment to remove Cryptosporidium oocysts at the WTW, led to the conclusion that the outbreak was waterborne. Oocyst counts in final treated water at the WTW and at different points in the distribution system were consistently very low, maximum count in continuous monitoring 0.08 oocysts per 10 litres. Data from continuous monitoring and the epidemic curve is consistent with the hypothesis that low numbers of oocysts of C hominis were present in treated water continuously during the outbreak and these were of sufficient infectivity to cause illness. All surface water derived water supplies present a potential risk to human health and appropriate control measures should be in place to minimise these risks.

Managing a large outbreak of cryptosporidiosis: how to investigate and when to decide to lift a 'boil water' notice.

The largest outbreak of cryptosporidiosis reported in the United Kingdom, involving 575 confirmed cases (of which 474 met an agreed case definition), occurred in the county of Devon during August and September of 1995. The descriptive epidemiology supports the hypothesis that the outbreak was associated with the consumption of cold tap water in the area served by a particular water treatment works. Cryptosporidium oocysts were detected in treated water samples at the time of the outbreak. Although the epidemiological analysis provided strong circumstantial evidence of a waterborne outbreak, the data were not recorded in a manner that made them admissible in criminal proceedings taken by the Drinking Water Inspectorate against the water company involved. The need to carry out an analytical study in conjunction with the identification and characterisation of the pathogen in the drinking water and the practicalities of agreeing criteria for lifting a 'boil water' notice are discussed.

Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations.

The application of genotyping to clinical isolates of Cryptosporidium has increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in the C. parvum thrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated with C. parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.

Uncertainties in risk assessment for the determination of drinking water pollutant concentrations: Cryptosporidium case study.

There is a growing acknowledgement of the need to recognise uncertainties in the estimation of waterborne risks. In this paper, the strength of the science available to characterise Cryptosporidium risks in drinking water is investigated, in particular the determination of oocyst concentrations at various points in the chain of water supply. A recently developed scientific quality audit framework is used as a tool to guide this investigation. The framework systematically reveals the relatively greater strength of the science for the "upstream" elements (estimation of concentrations in environmental reservoirs) than for the "downstream" elements (concentrations in distribution). It is the downstream elements that have greater bearing on human health and public policy issues. It is noted that the adoption of new monitoring Regulations is likely to prompt action to strengthen knowledge of these downstream elements in the future.

PCR-ELISA: a new simplified tool for tracing the source of cryptosporidiosis in HIV-positive patients.

Cryptosporidium parvum is a major parasitic cause of death in end-stage AIDS patients that results from both zoonotic and person-to-person transmission. Recent studies have provided evidence that parasites causing zoonotic disease and those causing anthroponotic infection are genetically distinct. Isolates carrying "animal"-type genetic markers were presumed to be the result of zoonotic spread, either directly or through contaminated food and water. The need for a genotype-specific diagnostic tool that can provide clues as to the origin and possible modes of spread of C. parvum strains has been recognised. Here, we report the development of such a tool for C. parvum based on polymerase chain reaction-enzyme linked immunosorbent assay that enables the accurate typing of isolates from HIV-seropositive and HIV-negative patients presenting with diarrhoea from the United Kingdom and Canada. This study also showed that zoonotic transmission might be predominant in the HIV-positive patient group in the United Kingdom.

Molecular characterisation of Cryptosporidium parvum from two large suspected waterborne outbreaks. Outbreak Control Team South and West Devon 1995, Incident Management Team and Further Epidemiological and Microbiological Studies Subgroup North Thames 1997.

Polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the Cryptosporidium parvum outer wall protein (COWP) gene was applied to specimens collected from 95 patients with cryptosporidiosis associated with two suspected waterborne outbreaks, 46 sporadic human cases and 62 infected livestock from other areas, and 12 patients infected with other gastrointestinal parasites. Ninety-six per cent of C. parvum isolates from patients linked to the two suspected waterborne outbreaks were of genotype 1; all the isolates from livestock were of genotype 2. Isolates from 59% of the sporadic human infections were of genotype 1 and 35% were of genotype 2. Specimens from two patients yielded both genotypes. Specimens from patients infected with other parasites yielded no amplicons.

Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992-5.

Following the introduction of an improved surveillance system for infectious intestinal disease outbreaks in England and Wales, the Public Health Laboratory Service Communicable Disease Surveillance Centre received reports of 26 outbreaks between 1 January 1992 and 31 December 1995 in which there was evidence for waterborne transmission of infection. In these 26 outbreaks, 1756 laboratory confirmed cases were identified of whom 69 (4%) were admitted to hospital. In 19 outbreaks, illness was associated with the consumption of drinking water from public supplies (10 outbreaks) or private supplies (9 outbreaks). The largest outbreak consisted of 575 cases. In 4 of the remaining 7 outbreaks, illness was associated with exposure to swimming pool water. Cryptosporidium was identified as the probable causative organism in all 14 outbreaks associated with public water supplies and swimming pools. Campylobacter was responsible for most outbreaks associated with private water supplies. This review confirms a continuing risk of cryptosporidiosis from chlorinated water supplies in England and Wales, and reinforces governmental advice to water utilities that water treatment processes should be rigorously applied to ensure effective particle removal. High standards of surveillance are important for prompt recognition of outbreaks and institution of control measures. As microbiological evidence of water contamination may be absent or insufficient to implicate a particular water supply, a high standard of epidemiological investigation is recommended in all outbreaks of suspected waterborne disease.

The epidemiology of cryptosporidiosis: application of experimental sub-typing and antibody detection systems to the investigation of water-borne outbreaks.

A technique based on the analysis of banding patterns obtained by SDS-PAGE Western-blotting of an oocyst wall antigen obtained from faeces has been evaluated to subtype Cryptosporidium parvum Tyzzer, 1912. This technique appears to have sufficient stability to recognise multiple types of this parasite. A similar Western-blotting technique has also been used to assess antibody responses to cryptosporidial antigens in human sera. Two systems were developed: one against three antigens of apparent molecular weights 6, 14 and 17 kDa; the second against oocyst wall antigens of apparent molecular weights 57, 69, 75, 89, 128, 151 and 173 kDa. Antibodies to three antigens of apparent molecular weights 6, 14 and 17 kDa were most successful as diagnostic markers in that they were found in > 88% of convalescent phase sera from confirmed cryptosporidiosis patients and were uncommon (> or = 7%) in control subjects. Faecal samples from human and animal sporadic cases yielded a wide range of cryptosporidial antigen banding patterns. Samples from patients in a water-borne outbreak in South Devon (England) in 1995 also yielded a wide range of banding patterns including members within individual household family groups. These results are in contrast with those from samples collected from other defined geographical areas, including some from a second water-borne outbreak where much more homogeneous banding patterns were obtained. Sera collected for other purposes from apparently uninfected individuals 9 months after the South Devon 1995 outbreak were examined. Antibodies to the three antigens of molecular weights 6, 14 and 17 kDa were detected in 32-49% of individuals resident in the outbreak water supply area, and in 15-21% of those resident in an adjacent water supply area. The significance of these findings is discussed in relation to data obtained from epidemiological field studies.

Prevalence of cryptosporidiosis in AIDS patients with diarrhoea in Santa Maria Hospital, Lisbon.

We report the findings of a longitudinal observational study on HIV-infected patients grouped by presumed transmission group, who had diarrhoea. The purpose of this study was to assess the prevalence of and factors associated with Cryptosporidium infection on these patients. Modifiied formol-ether concentration followed by modified Ziehl-Neelsen and phenol-auramine/carbol-fuchsin staining techniques were used to identify Cryptosporidium from 465 patients. Cryptosporidiosis was reported in 36/465 (8% and 95% confidence interval 6, 10) patients. Of the positive patients 30 (83%) were men and 6 (17%) women. Prevalence of infection was higher among HIV-seropositive patients whose exposure category was through sexual contact (69%) than among patients in other HIV exposure categories (9%, Standard Z test, P < 0.001). Median CD4+ cell count/mm3 was 120 (range 3-600). Besides diarrhoea, the main clinical manifestations were fever and weight loss in 14 (39%) and 26 (72%) patients, respectively. Cryptosporidium infection was considered to be the first AIDS defining disease in 31% of the patients followed by tuberculosis in 19%, Pneumocystis carinii pneumonia in 14%, Salmonella sepsis in 6%, isosporiasis in 3%, toxoplasmic encephalitis in 3%, leishmaniasis in 3% and Kaposi's sarcoma in 3% of the patients. There was no significant difference (P = 0.82) in survival times for those given folate antagonists to treat other opportunistic infections. The decrease in prevalence of cryptosporidiosis observed from 1994 until May 1997 is not statistically significant (P = 0.11). Most cases of cryptosporidial infection in AIDS patients in Lisbon occurred in those whose HIV infection was assumed to have been acquired by the sexual route (hetero-, homo- and bisexual), with few cases occurring in drug-abusers.

Differentiation between human and animal isolates of Cryptosporidium parvum using molecular and biological markers.

Isolates of Cryptosporidium parvum obtained from infected humans, calves and lambs were typed using arbitrary primed polymerase chain reaction (AP-PCR) and isoenzyme electrophoresis. All animal isolates tested (n = 17) showed similar profiles in AP-PCR and isoenzyme typing. In AP-PCR assays, 9 out of 15 human isolates showed a distinct "human" profile while the remaining 6 isolates showed the "animal" profile. In isoenzyme typing, 5 human isolates which had shown "human" profiles in AP-PCR demonstrated a unique isoenzyme banding pattern, while 2 isolates which had shown "animal" profiles in AP-PCR gave the "animal" banding pattern. In a murine model of infection, all four animal isolates tested were highly infective but only one of four human isolates identified as "human" type in the AP-PCR and isoenzyme typing systems was infective. The good correlation between the data from the different typing systems supports the hypothesis that there are genetically distinct human and animal populations of C. parvum.

Outbreak of cryptosporidiosis associated with a swimming pool in Andover.

An outbreak of eight cases of cryptosporidiosis in Hampshire over a period of eight weeks in the summer of 1996 was linked to use of one swimming pool. Cryptosporidial oocysts were not isolated from samples of backwash, but the presence of enterobius ova indicated faecal contamination and a case control study including the first four primary cases suggested an association with immersion in the pool. Even in small outbreaks case control studies can provide useful supportive evidence as to the possible source of infection.

A simple SDS-PAGE immunoblotting technique using an enhanced chemiluminescence detection system to identify polyclonal antibody responses to complex cryptosporidial antigen preparations following a monoclonal antibody retest and image overlay technique.

SDS-PAGE immunoblotting is a standard immunochemical technique. We describe a modification of this technique to facilitate the identification of polyclonal antibody responses to complex antigen preparations. The technique utilises an enhanced chemiluminescence (ECL) detection technique where an immunoblot is initially tested with polyclonal antibodies. The polyclonal antibodies are 'stripped' from the nitrocellulose support and the immunoblot is retested with a monoclonal antibody. Polyclonal antibody responses to individual components also recognised by the monoclonal antibody are identified by superimposing the two photographic-film images. This technique has the advantage that antigens can be identified across an individual blot despite distortions which may have occurred. An example of the use of this technique is described in which antibody responses in human sera against antigens from the coccidian parasite Cryptosporidium are identified.

PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin.

Cryptosporidium wrairi was isolated from guinea pigs during a spontaneous outbreak of cryptosporidiosis. Despite the morphological and antigenic similarities to C. parvum, C. wrairi displayed a different host range and site of infection and may represent a separate species or sub-species. We used the polymerase chain reaction to clone two distinct 550 bp-long DNA fragments, Wc-I and Wc-II, of the gene encoding the Cryptosporidium oocyst wall protein (COWP) of C. wrairi, which showed 98% identity to the C. parvum homologue. Within Wc-I, polymorphic Rsal restriction sites were used to develop a polymerase chain reaction-restriction fragment length polymorphism method able to distinguish C. wrairi from C. parvum and to identify two groups of C. parvum isolates differentially associated with animal and human infections.

An outbreak of waterborne cryptosporidiosis associated with a public water supply in the UK.

In November and December 1992, an outbreak of waterborne cryptosporidiosis occurred in Bradford, a city in the North of England. In all 125 cases were involved, the majority of whom lived in part of the city which received its drinking water supply from a single water treatment works. A case control study demonstrated an association between illness and the consumption of tapwater from this source; those drinking large volumes being more likely to have been ill. Treated water from the defined source yielded cryptosporidial oocysts. Heavy rainfall was recorded in the catchment area of the reservoir supplying raw water to the treatment works, immediately prior to the probable time of infection, based on dates of onset of illness.

Waterborne cryptosporidiosis associated with a borehole supply.

From 1 April to 31 May 1993, 64 cases of cryptosporidiosis were diagnosed within one district health authority. Forty were classified as primary cases, 35 of whom were clustered in an area supplied by a discrete public water supply that supplied the majority of homes in a large town. Most of the water in this supply is abstracted from boreholes and some is filtered before distribution. Households that received mains water from this supply were 15 times more likely to be affected than households nearby that received water from other sources. A case control study demonstrated a dose response relationship between consumption of water obtained from the town supply and risk of illness. Very low concentrations of cryptosporidial oocysts were detected in the water supply on four occasions several weeks after the outbreak. Environmental investigation failed to reveal a likely mechanism for contamination of the water supply.

Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans.

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).