Traditionally, molecular typing of G. duodenalis isolates has been largely based on sequence analyses of three marker genes, namely those encoding for beta-giardin (BG), triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) [11, 12]. This typing scheme allows the discrimination of different assemblages and sub-assemblages of the parasite, but lacks the resolution required for epidemiological applications, such as tracing of outbreaks and zoonotic transmission.
In 2018, Ankarklev et al. published a novel MLST scheme for G. duodenalis assemblage A, one of the two assemblages infecting humans . The scheme, based on six highly polymorphic genes located on different chromosomes, demonstrated good resolution, with 42 different MLST types identified in 61 isolates. The authors demonstrated the applicability of the scheme to infer zoonotic transmission and to support outbreak investigation . More recently, Woschke et al. applied this typing scheme to human isolates from Germany and identified 15 novel MLST types . Importantly, the authors reported the presence of an identical isolate type in samples collected longitudinally from patients with chronic infection.
In the present study, we aimed at providing additional data from isolates of human and animal origin representing infections acquired in additional European countries or on other continents. Sequencing of the six markers (CID1, HCMP6372, RHP26, DIS3, HCMP22547, NEK15411) from 65 isolates identified only five novel sequence variants at five of the six markers; this slight increase suggests that a large fraction of the existing variability has been sampled. On the other hand, the number of different MLST increased from 57  to 78, showing additional combinations of sequence types.
Zoonotic transmission of G. duodenalis has been an open research question for many years [7, 17, 21]. A recent review reassessed the prevalence and distribution of genotypes in animals, based on the bg, tpi and gdh loci . The authors confirmed that host-adapted assemblages (C to G) are largely more prevalent than the potentially zoonotic assemblages A and B, and that sub-assemblage AI is the dominant type in both livestock and companion animals infected with G. duodenalis assemblage A . This latter finding is confirmed by our data, as all animal isolates belonged to sub-assemblage AI, with the exception of a cat isolate of sub-assemblage AIII. Only eight human isolates clustered with sub-assemblage AI, and only a single MLST type was shared between humans and animals. These results suggest low rates of zoonotic transmission, although well-designed epidemiologic studies, particularly in areas where close contact between humans and animals is common, are needed to draw robust conclusions.
Giardia is a major cause of waterborne  and foodborne  outbreaks of enteric disease in industrialized nations, and genotyping strains in outbreak situations is of epidemiological relevance. In this study, we show that 15 isolates from an outbreak in Italy all shared the same MLST, which was not found in any other isolate included in this study. It is noteworthy that isolates from a Swedish outbreak  also shared an identical MLST type, yet distinct from that characterizing the Italian outbreak. Besides outbreak investigation, the MLST may help to attribute the main source of human assemblage A infections in endemic countries, and elucidate the proportion of person-to-person transmission versus infection via food/water in specific local settings.
The population structure of G. duodenalis at the assemblage level is not clear, in part because the commonly used bg, tpi and gdh markers are too conserved. The new typing scheme for assemblage A  is based on six polymorphic single-copy genes identified by whole-genome comparison of just three assemblage A isolates, namely WB (AI), AS98 and AS175 (both AII). Therefore, broader genomic sequence variation within assemblage A isolates likely exists [13, 15]. In comparison to sub-assemblage AI, which is found in animals and humans, sub-assemblage AII is only found in humans and shows a larger genetic variability within the analysed dataset. However, it should be noted that this observation might be biased by the fact that the MLST type of all in vitro-cultured sub-assemblage AI strains was identical, whereas the AI isolates typed from faeces were more diverse. As these isolates had no obvious epidemiological connection, this indicates that the axenization of assemblage AI isolates may introduce a bias and select specific variants. Whether this is indeed the case and the identity of the underlying biological mechanisms need to be addressed in future studies using larger and more defined datasets.
Genotyping of G. duodenalis from faecal samples by classical MLST methods, as shown in the present study, poses several potential issues. First, due to the tetraploid nature of the genome , heterozygote organisms can occur, from which blurred typing results are generated due to ASH. It has been reported that the occurrence of ASH is lower in sub-assemblage A than in assemblage B [10, 13, 15], and indeed in most assemblage A isolates no ASH was detected [10, 13, 15]. This indicates that assemblage A isolates are mainly homozygotic organisms. However, it should be noted that a minor fraction of assemblage AII isolates have heterozygote nucleotides in some marker sequences [10, 13, 15]. Second, it is important to note that the majority of the samples included in the present study are clinical faecal samples from either infected humans or animals. An infection originates from a population of parasites that potentially may have different MLST types. It is also been suggested that recombination occurs in Giardia , which also may add to the occurrence of several MLST types in a single sample. Hence, whether observed variability within a sample is due to true heterozygotes, populations or to mixed infections in the faecal sample is not known and we excluded those isolates with unclear genotypes from the analysed dataset.