Corinne S. L. Ong

Novel Cryptosporidium Genotypes in Sporadic Cryptosporidiosis Cases: First Report of Human Infections with a Cervine Genotype

In this study, we genotyped parasites from the fecal specimens of sporadic cryptosporidiosis cases in British Columbia from 1995 to 1999. Genotyping was conducted by polymerase chain amplification of the internal transcribed spacer region, a hypervariable region in the 18S rRNA gene and the Cryptosporidium oocyst wall protein gene. Subsequent analysis was by restriction fragment length polymorphism and DNA sequencing. We identified two new Cryptosporidium genotypes in humans. One of these genotypes has been found recently in deer in New York state. The other genotype has not been identified in humans or animals. These results have important implications for drinking water quality strategies, especially for communities that obtain drinking water supplies from surface sources located in forested regions with deer populations.

Outbreak of cyclosporiasis in British Columbia associated with imported Thai basil

Sporadic outbreaks of cyclosporiasis, a common cause of protracted diarrhoea in underdeveloped countries, are often undetected and undiagnosed in industrial countries. In May 2001, an outbreak of Cyclospora cayetanensis gastroenteritis was identified in British Columbia, Canada, with 17 reported cases. We conducted a case-control study involving 12 out of the 17 reported and confirmed case patients. Eleven (92%) of the patients had consumed Thai basil, an essential ingredient in Vietnamese cuisine, compared to 3 out of 16 (19%) of the control patients (P = 0.003). Trace-back investigations implicated Thai basil imported via the United States as the vehicle for this outbreak. This is the first documented sporadic outbreak of cyclosporiasis linked to Thai basil in Canada, and the first outbreak of cyclosporiasis identified in an ethnic immigrant population. This outbreak provides the opportunity to increase our understanding of this emerging pathogen and improve on our prevention and control for future outbreaks.

Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

ABSTRACT Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence ofCryptosporidium infection in the population.

CORRELATION BETWEEN MARKERS OF STRAIN VARIATION IN CRYPTOSPORIDIUM PARVUM : EVIDENCE OF CLONALITY

Abstract Cryptosporidium parvum has been shown, by molecular studies, to be heterogeneous in a manner linked to the host of isolation. However, no assessment of the degree of correlation between different typing systems or of the impact of geographical variation on the observed heterogeneity has been made. In this report we describe a new marker, based on point mutations in the sequence of the dihydrofolate reductase (DHFR) gene, that employed a polymerase chain reaction/endonuclease restriction protocol. The system was first tested on three well-studied isolates (a human isolate exhibiting ‘human’ markers, a human isolate exhibiting ‘animal’ markers and an animal isolate exhibiting ‘animal’ markers — no animal isolates exhibiting ‘human’ markers have been detected to date). The human isolate carrying human markers showed a unique DHFR sequence while both the animal isolate and the second human isolate (showing animal markers in other typing systems) showed a different sequence. The new system was used successfully to type 14 C. parvum isolates from three continents. The complete agreement of this new typing system with others described earlier and the apparent linkage of alleles at unrelated loci suggested that C. parvum might have a clonal population structure.

Molecular Forensic Profiling of Cryptosporidium Species and Genotypes in Raw Water

ABSTRACT The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.

Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities

Cryptosporidium and Giardia were characterized in a watershed in southern Ontario, Canada, over a 2½ year period. River samples were collected every two weeks, primarily near a municipal drinking water treatment plant intake. Cryptosporidium and Giardia were frequently detected with an overall occurrence rate of 88 and 97%, respectively. Giardia concentrations were higher than Cryptosporidium, with median values of 80 cysts 100 L(-1) and 12 oocysts 100 L(-1), respectively. Although pathogens rarely show a significant relationship with fecal or water quality indicators, this study determined that Cryptosporidium, but not Giardia, was significantly correlated with Escherichia coli, turbidity and river flow. There was no correlation between the two types of protozoa, and only Giardia showed a seasonal trend with higher concentrations at cold water temperatures. Cryptosporidium genotyping of all samples found that farm animals and wildlife were an important contributor of oocysts in the watershed, and that Cryptosporidium strains/genotypes of medium to high risk for human infection (C. hominis, C. parvum and C. ubiquitum) were detected in 16% of samples. This study was able to identify Cryptosporidium host sources and human health risk, and to identify differences between Cryptosporidium and Giardia occurrence in the watershed.

Challenges of Investigating Community Outbreaks of Cyclosporiasis, British Columbia, Canada

Investigations of community outbreaks of cyclosporiasis are challenged by case-patients’ poor recall of exposure resulting from lags in detection and the stealthy nature of food vehicles. We combined multiple techniques, including early consultation with food regulators, traceback of suspected items, and grocery store loyalty card records, to identify a single vehicle for a cyclosporiasis outbreak in British Columbia, Canada, in 2007.

Cyclospora spp. in herbs and water samples collected from markets and farms in Hanoi, Vietnam

Objective  To determine the prevalence of Cyclospora spp. oocysts in herb and water samples as well as in fecal specimens of clinical cases of diarrhoea in Hanoi, Vietnam.

Perspectives on Emerging Zoonotic Disease Research and Capacity Building in Canada

Zoonoses are fundamental determinants of community health. Preventing, identifying and managing these infections must be a central public health focus. Most current zoonoses research focuses on the interface of the pathogen and the clinically ill person, emphasizing microbial detection, mechanisms of pathogenicity and clinical intervention strategies, rather than examining the causes of emergence, persistence and spread of new zoonoses. There are gaps in the understanding of the animal determinants of emergence and the capacity to train highly qualified individuals; these are major obstacles to preventing new disease threats. The ability to predict the emergence of zoonoses and their resulting public health and societal impacts are hindered when insufficient effort is devoted to understanding zoonotic disease epidemiology, and when zoonoses are not examined in a manner that yields fundamental insight into their origin and spread. Emerging infectious disease research should rest on four pillars: enhanced communications across disciplinary and agency boundaries; the assessment and development of surveillance and disease detection tools; the examination of linkages between animal health determinants of human health outcomes; and finally, cross-disciplinary training and research. A national strategy to predict, prevent and manage emerging diseases must have a prominent and explicit role for veterinary and biological researchers. An integrated health approach would provide decision makers with a firmer foundation from which to build evidence-based disease prevention and control plans that involve complex human/animal/environmental systems, and would serve as the foundation to train and support the new cadre of individuals ultimately needed to maintain and apply research capacity in this area.

Rare Cryptosporidium hominis Subtype Associated with Aquatic Center Use

To the Editor: Cryptosporidiosis is the most frequently reported gastrointestinal illness in outbreaks associated with treated (disinfected) recreational water venues in the United States (1). In 2003, an increased number of cryptosporidiosis cases occurred in the Tri-Cities area of the Lower Mainland region (near Vancouver), in British Columbia, Canada. Although all cases were associated with the use of a community aquatic center, their onset dates were spread over a 3-month period, and the link between cases was unclear. The aim of this study was to determine if the cases in this disease cluster were related. Although suitable molecular markers had yet to be defined at the time of the outbreak, recent reports on the use of the gp60 gene for subtyping in molecular epidemiologic studies (2,3) have enabled us to reanalyze the isolates and report these results. Fifteen laboratory-confirmed cases were identified from October 15 to December 5, 2003. This number was in excess of the anticipated incidence rate for this community, which averaged 5 reported cryptosporidiosis cases per year. During the period of investigation, an incident of fecal contamination at the aquatic center on October 10, 2003, was documented and remediation involved increasing the free chlorine concentration. Because the regional health authority was concerned about the increased number of cases, the facility closed voluntarily on December 5 for further remediation. However, recorded free chlorine concentrations did not exceed 2.0 ppm at any time during the investigative period (October 5–December 31). The health authority released a public advisory encouraging those who used the facility to submit fecal specimens for laboratory testing. The health authority also sent letters to family physicians in the area, informing them of the disease cluster and requesting that unpreserved stool specimens be collected, in addition to the formalin-fixed specimens, for routine diagnostic testing. Nine fecal specimens were collected from clinically symptomatic case-patients with histories of exposure to the implicated aquatic center. Five specimens were selected by using the criteria that they were from patients with laboratory-confirmed cryptosporidiosis cases from 5 separate households. The specimens were then coded for anonymity before subsequent molecular analysis. Genomic DNA was extracted from purified Cryptosporidium oocysts by freeze-thawing, and the species was determined by PCR amplification and sequencing of the 18S rRNA gene as described previously (4). The gp60 gene was also amplified by PCR by using primers described by Ong and Isaac-Renton (5). DNA sequences of amplicons were determined by cycle sequencing and assembled as described previously (4,5). The gp60 allele and subtype were identified by multiple sequence alignment with GenBank reference sequences and phylogenetic analysis that used ClustalX version 1.8 (www.clustal.org) as well as manual quantification of microsatellite repeats. The 18S rRNA and gp60 genes were amplified successfully from 4 specimens. On the basis of the 18S rRNA gene sequence, all case-patients were infected with Cryptosporidium hominis, a species associated primarily with human-to-human transmission. The gp60 sequences from all 4 case-patients were identical and were subtype IdA19, a rarely reported subtype of C. hominis. Globally, most reports of the gp60 Id allele, such as 9 reported cases from Australia, have identified the IdA15G1 subtype (3). Another subtype, IdA18, was isolated from 5 case-patients in a 1997 foodborne outbreak in Spokane, Washington (6). To date, the IdA19 subtype has been identified in only 1 sporadic case, in northern Ontario (7), and a subset of cases (seven 1dA19 and 2 mixed IdA19 and IbA10G2) in the 2001 waterborne outbreak in North Battleford, Saskatchewan (5,8). The IdA19 subtype is identical in sequence to the IdA18 subtype except for 1 extra TCA repeat in the microsatellite region. Neither subtype has been reported anywhere in the world except in Canada and the Pacific Northwest. Because cases from all previous C. hominis outbreaks of cryptosporidiosis in British Columbia have been caused by the IbA10G2 subtype, the most prevalent subtype in sporadic and outbreak cases around the world (2,5,9,10), our results indicate the presence of a new subpopulation of C. hominis parasites that could cause future disease outbreaks. The identification of the same subtype in all 4 case-patients with cryptosporidiosis associated with the use of a community aquatic center was consistent with their exposure history and confirmed that all cases were linked epidemiologically. However, the association between the single northern Ontario sporadic case and the larger number of Saskatchewan and British Columbia outbreak cases is uncertain. The association with the IdA18 subtype in the Washington foodborne outbreak is also unknown. Further research is needed to determine the distribution and prevalence of gp60 subtypes in Canada as well as other parts of the world before we can more clearly understand the transmission of the IdA19 subtype.