Investigation of Cryptosporidium clinical isolates and analysis with epidemiological data
- The purpose of this project was to investigate Cryptosporidium hominis and Cryptosporidium parvum
subtypes present in two different sample sets: (i) strains from cases
implicated in a drinking waterborne outbreak associated with water
sourced from Thirlmere and distributed via the Thirlmere aqueduct; (ii)
strains from a DWI-funded case-control study of sporadic
cryptosporidiosis undertakenin Wales and the North West of England to
identify risk factors for sporadiccryptosporidiosis. Analysis with the
epidemiological data was performed to identify trends in prevalence of
subtypes, clusters of cases, risk factors andsources of infection, and
measures for control.
- Multi-locus microsatellite fragment analysis was chosen on the
basis of in-house and international evaluative studies and consultation
with funders and the scientific community as the most suitable method
for subtyping the required number of samples to a high degree of
- Three microsatellite markers were analysed: microsatellite
locus (ML) 1, ML2 and the gp15 surface glycoprotein gene (synonymous
with gp60). Microsatellite regions were amplified by PCR and fragment
lengths determined using the CEQ™ 8000 Genetic Analysis System.
Fragment sizes for each locus were combined to produce a multi-locus
fragment type for each strain.
- C. parvum was found to be significantly more variable than C. hominis
in both the Thirlmere and case-control study sample sets. Therefore,
subtyping using multi-locus microsatellite fragment analysis is
therefore more suitable for studies of C. parvum variation than C. hominis variation.
- Multi-locus microsatellite fragment analysis of 190 strains from the case-control study group identified nine subtypes of C. hominis and 30 subtypes of C. parvum. 90% of C. hominis strains were of the same subtype. Three distinct clusters of the C. parvum strains were identified (SC1, SC2, SC3). Analysis with the epidemiological data revealed a significant association between C. parvum
SC1 and animal contact. Associations were also found between ML1, ML2
and gp15 microsatellite sizes and animal contact, ML1 size being the
most dramatic and having the potential to be a useful marker for
zoonotic transmission. The results support the hypothesis that there
may be specific clones of C. parvum that are adapted to a human-only life cycle. Phenographic analysis revealed significant associations between C. parvum SC1 cases and living in a rural area; this was also the case for the four most common C. parvum multi-locus fragment types, which were within SC1.
- Analysis of 99 Thirlmere outbreak strains identified one subtype of C. hominis and 17 subtypes of C. parvum.
Three clusters of C. parvum cases were identified (C1a, C1b, C1c),
corresponding to sub-clusters of SC1 from the case-control study group.
Analysis with the epidemiological data revealed significant
associations between species (C. hominis and C. parvum) and the frequency of consuming undercooked meat. The temporal incidence of C. hominis and C. parvum during the outbreak indicated that C. hominis
strains were not part of this general outbreak. There was a significant
relationship between living in a rural area and cases of C. parvum cluster 1a. Clustering of C. parvum
subtype P36 was observed around Preston and Chorley, whereas there were
no cases of P36 strains on the Fylde and only one in the Morecambe Bay
area, supporting the epidemiological information which suggested that
cases on the Fylde were not related to the Thirlmere supply and the
cases in the Morecambe Bay area were probably not waterborne.
- As frequency of consumption of undercooked meat was identified as a risk factor for C. hominis and C. parvum
infection, control measures should include recommendations to caterers
and vulnerable individuals to cook meat thoroughly and adhere to food
handling advice from the Food Standards Agency.
- As animal contact was identified as a risk factor for infection with certain subtypes of C. parvum,
control measures should include recommendation of comprehensive hygiene
and hand washing procedures for implementation during and after animal
handling to minimise risk of hand-to-mouth transmission.
Copies of this report may be available as an Acrobat pdf download under the 'Post 2000 Reports' heading on the DWI website.