Discussion

Giardia duodenalis and Cryptosporidium spp. are intestinal protozoan parasites of humans and other mammals, and are common causes of diarrheal disease worldwide. Both parasites are genotyped to determine species, but also to perform epidemiological studies, source tracking, and outbreak investigations. For Giardia, either single-gene PCR typing or multi-locus sequence typing (MLST) are used for genotyping. The variability of the traditionally used markers (gdh, tpi and bg) are often high enough to discriminate subtypes of assemblage B isolates, but when applied on assemblage A isolates, low resolution is obtained. Recently, a set of six novel MLST markers were published, based on several whole genome sequences, and that can be used on Assemblage A isolates. These markers were shown to give a considerably higher resolution than the three commonly used loci, and were proven useful to discriminate between samples that were otherwise genotyped as identical. After species determination based on 18S sequencing sometimes, Cryptosporidium can be further subtyped using partial sequencing of the highly variable surface antigen gp60. However, this marker has limitations and a robust multi-locus typing scheme is desired. Recently a set of markers that can be used for fragment analysis was suggested for C. parvum. Similarly, a sequencing-based MLST scheme has been developed based on whole genomes sequences of many C. parvum isolates of both human and animal origin. An amplicon-based MLST scheme has been developed for the prevalent gp60 subtype, IbA10G2; this subtype that has previously proven difficult to type further as the variability on genome level is very low. In summary, genotyping of isolates contributes to the identification of parasite sources and routes of transmission. A well-designed multi-locus scheme should take into account recombination and needs to include unlinked markers, preferably located on different chromosomes. To maximise time and cost efficiency, it is useful to use a diversity index calculation to avoid using markers that provide redundant information.