The use of high resolution molecular tools to study Cryptosporidium parvum and Cryptosporidium hominis intra-species variation is becoming common practice, but there is currently no consensus in the methods used. The most commonly applied tool is partial gp60 gene sequence analysis. However, multi-locus schemes are acknowledged to improve resolution over analysis of a single locus, which neglects potential re-assortment of genes during the sexual phase of the Cryptosporidium life-cycle. Multi-locus markers have been investigated in isolates from a variety of sampling frames, in varying combinations and using different assays and methods of analysis. To identify the most informative markers as candidates for the development of a standardised multi-locus fragment size-based typing (MLFT) scheme to integrate with epidemiological analyses, we examined the published literature. A total of 31 MLFT studies were found, employing 55 markers of which 45 were applied to both C. parvum and C. hominis. Of the studies, 11 had sufficient raw data, from three or more markers, and a sampling frame containing at least 50 samples, for meaningful in-depth analysis using assessment criteria based on the sampling frame, study size, number of markers investigated in each study, marker characteristics (>2 nucleotide repeats) and the combinations of markers generating all possible multi-locus genotypes. Markers investigated differed between C. hominis and C. parvum. When each scheme was analysed for the fewest markers required to identify 95% of all MLFTs, some redundancy was identified in all schemes; an average redundancy of 40% for C. hominis and 27% for C. parvum. Ranking markers, based on the most productive combinations, identified two different sets of potentially most informative candidate markers, one for each species. These will be subjected to technical evaluation including typability (percentage of samples generating a complete multi-locus type) and discriminatory power by direct fragment size analysis and analysed for correlation with epidemiological data in suitable sampling frames. The establishment of a group of users and agreed subtyping scheme for improved epidemiological and public health investigations of C. parvum and C. hominis will facilitate further developments and consideration of technological advances in a harmonised manner.
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