BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.

• Klíčová slova
• Giardia intestinalis, Aneuploidy, Chromosomes, Copy number variation, Digital PCR, Flavohemoglobin, Flavohemoprotein, Metronidazole,
• MeSH
• antiprotozoální látky * farmakologie   MeSH
• Giardia lamblia * genetika   účinky léků   MeSH
• giardiáza * parazitologie   farmakoterapie   MeSH
• léková rezistence * genetika   MeSH
• lidé MeSH
• metronidazol * farmakologie   MeSH
• protozoální proteiny genetika   MeSH
• variabilita počtu kopií segmentů DNA * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiprotozoální látky * MeSH
• metronidazol * MeSH
• protozoální proteiny MeSH

The level of genetic variability of Giardia intestinalis clinical isolates is an intensively studied and discussed issue within the scientific community. Our collection of G. intestinalis human isolates includes six in vitro-cultured isolates from assemblage B, with extensive genetic variability. Such variability prevents the precise genotype characterisation by the multi-locus genotyping (MLG) method commonly used for assemblage A. It was speculated that the intra-assemblage variations represent a reciprocal genetic exchange or true mixed infection. Thus, we analysed gene sequences of the molecular clones of the assemblage B isolates, each representing a single DNA molecule (haplotype) to determine whether the polymorphisms are present within individual haplotypes. Our results, which are based on the analysis of three standard genetic markers (bg, gdh, tpi), point to haplotype diversity and show numerous single nucleotide polymorphisms (SNPs) mostly in codon wobble positions. We do not support the recombinatory origin of the detected haplotypes. The point mutations tolerated by mismatch repair are the possible cause for the detected sequence divergence. The precise sub-genotyping of assemblage B will require finding more conservative genes, as the existing ones are hypervariable in most isolates and prevent their molecular and epidemiological characterisation.

• Klíčová slova
• Assemblage B, Genetic variability, Giardia intestinalis, Haplotypes, Molecular cloning,
• MeSH
• feces parazitologie   MeSH
• fylogeneze MeSH
• genotyp MeSH
• Giardia lamblia klasifikace   genetika   izolace a purifikace   MeSH
• giardiáza parazitologie   MeSH
• haplotypy MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• protozoální DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální DNA MeSH