Geographic Linkage and Variation in Cryptosporidium hominis

Geographic Linkage and Variation in Cryptosporidium hominis


The Study
A total of 115 C. hominis isolates were collected and confi rmed during a case-control study of human cryptosporidiosis in Wales and northwest England (2). To identify subtypes, we analyzed the DNA sequences of an ≈850-bp region of the GP60 gene encompassing the polyserine tract (variable numbers and forms of a repeating sequence of 3 nucleotides coding for the amino acid serine) and the hypervariable downstream region (5). We used a nested PCR protocol with primary PCR primers AL3531 and AL3535 and secondary primers AL3532 and AL3534. PCR products were sequenced in both directions.
The microsatellite triplet codons were categorized according to the number of trinucleotide repeats (TCA, TCG, or TCT) coding for the amino acid serine (6), and the nomenclature was expanded for subtype family Ia to include the number of repeats (e.g., R1, R2) of the sequence AA(A/ G)ACGGTGGTAAGG after the microsatellite region (7). Sequence data for representative isolates were deposited in GenBank (accession nos. EU161648-EU161655, EF214734, and EF214735). We then investigated subtypes for relationships with reported exposures by using singlevariable analysis performed in SPSS 12.0 version (SPSS Inc., Chicago, IL, USA).
Of 115 C. hominis isolates, 14 were not typeable at the GP60 locus (12 did not amplify and 2 gave equivocal reactions); typeability was 87.8%. Nine subtypes were identifi ed but 92 (91.1%) typeable isolates were IbA10G2. Each of the other identifi ed types contained only 1 isolate member except for IgA24, which contained 2. This resulted in a low discriminatory power of 0.171.
More persons with subtypes other than IbA10G2 had a history of recent foreign travel (5/9, 55.6%) than did those with IbA10G2 (

Conclusions
Although GP60 sequence typing had very low discriminatory power for UK C. hominis isolates, our fi ndings are in agreement with previous fi ndings based on multiple loci that C. hominis appears to be highly conserved in the United Kingdom (3,4). DNA sequencing of a substantial proportion of the GP60 gene, including the microsatellite region, provides higher resolution data than investigating microsatellite length polymorphisms, which may mask differences in sequence (8) (9)(10)(11)(12) and occur worldwide (12). The conclusion of Cohen et al. (11), that Ib is the predominant C. hominis allele associated with waterborne outbreaks, is explained if this is the most common allele causing human cryptosporidiosis in Europe, as it is in the United Kingdom, and is therefore predominant in human sewage.
In nonindustrialized countries, a greater variety of C. hominis subtypes have been reported (7,8,13,14). Of the 3 isolates found in case-patients returning from Pakistan, IaA12R3 had been isolated from a patient from Nepal (Gen-Bank accession no. AY167595); IaA22R2 and IaA30R3 had not been reported previously. Subtype IaA25R3 was found in a case-patient returning from Kenya and was homologous to a C. hominis reference strain (TU502) of Ugandan origin (GenBank accession no. XM_663000). Notably, of the 4 case-patients with non-IbA10G2 subtypes who did not report foreign travel, 1 had the IgA24 subtype, which matched an isolate from Northern Ireland (GenBank accession no. EF214734), and may well circulate in the United Kingdom; IaA23R4 was homologous to isolates from the United States (GenBank accession no. AF164504) and Canada (GenBank accession no. DQ192510); and IfA12G1 had been identifi ed in Australia (12).
C. hominis is highly conserved in indigenous UK casepatients, and subtypes other than IbA10G2 are linked to re-cent foreign travel outside Europe. It is not possible to predict whether this apparent stability will remain or whether it will be infl uenced by international travel.