However, considering the fact that the parasite has two diploid nuclei and the level of ASH is surprisingly low: <0.01% in the sequenced assemblage A (WB) and E (P15) isolates and 0.5% in the assemblage B (GS) isolate, it must mean that the A-769662 in vitro parasites can actively reduce the level of ASH and that there must be some kind of communication between the two nuclei, as seen during the SAHA HDAC diplomixis process [30]. The striking differences in ASH levels between assemblage A and B isolates could imply that the different assemblages have different mechanisms

in exchanging genetic material. Another possibility is that assemblage B isolates can fuse in a process similar to the newly discovered sexual process in Candida albicans and other

pathogenic fungi [14]. In C. albicans, two diploid cells fuse and form CYC202 a tetraploid cell that undergoes parasexual reduction to diploid or often aneuploid cells [14]. Aneuploid Giardia trophozoites have been reported [33], which could be remnants of cell fusion and reduction events. Thus, it is possible that the relatively high ASH levels in assemblage B (0.5%) compared to assemblage A (<0.01%) could be due to higher frequencies of cell fusions (sex) in assemblage B isolates. Yet another possibility is that the very low levels of ASH in assemblage A isolates could be due to highly active meiotic components, efficient diplomixis or efficient DNA repair systems. Recent reports indicate that elevated levels of ASH in the pathogenic fungi, C. albicans, are linked to virulence and drug resistance [34, 35]. Levert and colleagues have brought light to polymorphisms within bacterial populations and how this may be linked to the generation of virulence phenotypes, such as growth, resistance to stress or resistance to antibiotics [13]. Patient Sweh207, who Ixazomib cost had a mixed assemblage A and B infection, was subject to treatment failure.

Interestingly, after treatment only the assemblage B parasites were present and sequencing indicated high levels of ASH both pre- and post- treatment in the assemblage B portion of the infection [8]. In the same study there were eight other reported cases of suspected treatment failure involving assemblage B infections, where sequencing of the parasites showed double peaks in several positions before and after treatment. Although this has to be further verified, the data brings forth a potential link between elevated levels of ASH and drug resistance in Giardia, as is the case in C. albicans. Conclusion We have developed a methodological pipeline that enables isolation and sequencing analyses of single G. intestinalis parasites. The presence of ASH was verified on the single cell level, both in cultured assemblage B trophozoites and in cysts from clinical samples.