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.

Department of Infectious Diseases Istituto Superiore Di Sanita Rome Italy

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Prague Czech Republic

Zobrazit více v PubMed

Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ et al. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science. 2007;317:1921–6. 10.1126/science.1143837. 10.1126/science.1143837 PubMed DOI

Xu FF, Jex A, Svard SG. A chromosome-scale reference genome for Giardia intestinalis WB. Sci Data. 2020. 10.1038/s41597-020-0377-y. 10.1038/s41597-020-0377-y PubMed DOI PMC

Leger MM, Kolisko M, Kamikawa R, Stairs CW, Kume K, Cepicka I, et al. Organelles that illuminate the origins of trichomonas hydrogenosomes and Giardia mitosomes. Nat Ecol Evol. 2017;1:0092. 10.1038/s41559-017-0092. 10.1038/s41559-017-0092 PubMed DOI PMC

Tumova P, Dluhosova J, Weisz F, Nohynkova E. Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis. Int J Parasitol. 2019;49:463–70. 10.1016/j.ijpara.2019.01.003. 10.1016/j.ijpara.2019.01.003 PubMed DOI

Tumova P, Uzlikova M, Jurczyk T, Nohynkova E. Constitutive aneuploidy and genomic instability in the single-celled eukaryote Giardia intestinalis. Microbiologyopen. 2016;5:560–74. 10.1002/mbo3.351. 10.1002/mbo3.351 PubMed DOI PMC

Bernander R, Palm JE, Svard SG. Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol. 2001;3:55–62. 10.1046/j.1462-5822.2001.00094.x. 10.1046/j.1462-5822.2001.00094.x PubMed DOI

Jirakova K, Kulda J, Nohynkova E. How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection. Protist. 2012;163:465–79. 10.1016/j.protis.2011.11.008. 10.1016/j.protis.2011.11.008 PubMed DOI

Lagunas-Rangel FA, Yee J, Bermudez-Cruz RM. An update on cell division of Giardia duodenalis trophozoites. Microbiol Res. 2021;250:126807. 10.1016/j.micres.2021.126807. 10.1016/j.micres.2021.126807 PubMed DOI

Ankarklev J, Franzen O, Peirasmaki D, Jerlstrom-Hultqvist J, Lebbad M, Andersson J, et al. Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates. BMC Genomics. 2015;16:697. 10.1186/s12864-015-1893-6. 10.1186/s12864-015-1893-6 PubMed DOI PMC

Ey PL, Mansouri M, Kulda J, Nohynkova E, Monis PT, Andrews RH, et al. Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes. J Eukaryot Microbiol. 1997;44:626–35. 10.1111/j.1550-7408.1997.tb05970.x. 10.1111/j.1550-7408.1997.tb05970.x PubMed DOI

Jerlstrom-Hultqvist J, Franzen O, Ankarklev J, Xu FF, Nohynkova E, Andersson JO, et al. Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate. Bmc Genomics. 2010. 10.1186/1471-2164-11-543. 10.1186/1471-2164-11-543 PubMed DOI PMC

Zajaczkowski P, Lee R, Fletcher-Lartey SM, Alexander K, Mahimbo A, Stark D, et al. The controversies surrounding Giardia intestinalis assemblages A and B. Curr Res Parasitol Vector Borne Dis. 2021;1:100055. 10.1016/j.crpvbd.2021.100055. 10.1016/j.crpvbd.2021.100055 PubMed DOI PMC

Caccio SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J Parasitol. 2008;38:1523–31. 10.1016/j.ijpara.2008.04.008. 10.1016/j.ijpara.2008.04.008 PubMed DOI

Klotz C, Sannella AR, Weisz F, Chaudhry U, Sroka J, Tumova P, et al. Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power. Parasite Vector. 2022. 10.1186/s13071-022-05615-x.10.1186/s13071-022-05615-x PubMed DOI PMC

Monis PT, Caccio SM, Thompson RC. Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol. 2009;25:93–100. 10.1016/j.pt.2008.11.006. 10.1016/j.pt.2008.11.006 PubMed DOI

Seabolt MH, Roellig DM, Konstantinidis KT. Genomic comparisons confirm Giardia duodenalis sub-assemblage AII as a unique species. Front Cell Infect Mi. 2022. 10.3389/fcimb.2022.1010244.10.3389/fcimb.2022.1010244 PubMed DOI PMC

Wielinga C, Williams A, Monis P, Thompson RCA. Proposed taxonomic revision of Giardia duodenalis. Infect Genet Evol. 2023;111:105430. 10.1016/j.meegid.2023.105430. 10.1016/j.meegid.2023.105430 PubMed DOI

Xu F, Jimenez-Gonzalez A, Einarsson E, Astvaldsson A, Peirasmaki D, Eckmann L, et al The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites. Microb Genom. 2020. 10.1099/mgen.0.000402. 10.1099/mgen.0.000402 PubMed DOI PMC

Kamikawa R, Inagaki Y, Hashimoto T. Secondary loss of a cis-spliced intron during the divergence of Giardia intestinalis assemblages. BMC Res Notes. 2014;7:413. 10.1186/1756-0500-7-413. 10.1186/1756-0500-7-413 PubMed DOI PMC

Xue M, Chen B, Ye Q, Shao J, Lyu Z, Wen J. Sense-antisense gene overlap is probably a cause for retaining the few introns in Giardia genome and the implications. Biol Direct. 2018;13:23. 10.1186/s13062-018-0226-5. 10.1186/s13062-018-0226-5 PubMed DOI PMC

Adam RD, Nigam A, Seshadri V, Martens CA, Farneth GA, Morrison HG, et al. The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution. Bmc Genomics. 2010. 10.1186/1471-2164-11-424. 10.1186/1471-2164-11-424 PubMed DOI PMC

Rafferty S, Luu B, March RE, Yee J. Giardia lamblia encodes a functional flavohemoglobin. Biochem Biophys Res Commun. 2010;399:347–51. 10.1016/j.bbrc.2010.07.073. 10.1016/j.bbrc.2010.07.073 PubMed DOI

Rafferty SP, Dayer G. Heme proteins of Giardia intestinalis. Exp Parasitol. 2015;159:13–23. 10.1016/j.exppara.2015.08.001. 10.1016/j.exppara.2015.08.001 PubMed DOI

Poole RK. Flavohaemoglobin: the pre-eminent nitric oxide-detoxifying machine of microorganisms. F1000Res. 2020. 10.12688/f1000research.20563.1. 10.12688/f1000research.20563.1 PubMed DOI PMC

Mastronicola D, Testa F, Forte E, Bordi E, Pucillo LP, Sarti P, et al. Biochem Biophys Res Commun. 2010;399:654–8. 10.1016/j.bbrc.2010.07.137. 10.1016/j.bbrc.2010.07.137 PubMed DOI

Muller J, Braga S, Heller M, Muller N. Resistance formation to nitro drugs in Giardia lamblia: no common markers identified by comparative proteomics. Int J Parasitol Drugs Drug Resist. 2019;9:112–9. 10.1016/j.ijpddr.2019.03.002. 10.1016/j.ijpddr.2019.03.002 PubMed DOI PMC

Muller J, Hemphill A, Muller N. Physiological aspects of nitro drug resistance in Giardia lamblia. Int J Parasitol Drugs Drug Resist. 2018;8:271–7. 10.1016/j.ijpddr.2018.04.008. 10.1016/j.ijpddr.2018.04.008 PubMed DOI PMC

Lalle M, Hanevik K. Treatment-refractory giardiasis: challenges and solutions. Infect Drug Resist. 2018;11:1921–33. 10.2147/IDR.S141468. 10.2147/IDR.S141468 PubMed DOI PMC

Bourque DL, Neumayr A, Libman M, Chen LH. Treatment strategies for nitroimidazole-refractory giardiasis: a systematic review. J Travel Med. 2022. 10.1093/jtm/taab120. 10.1093/jtm/taab120 PubMed DOI

Carter ER, Nabarro LE, Hedley L, Chiodini PL. Nitroimidazole-refractory giardiasis: a growing problem requiring rational solutions. Clin Microbiol Infect. 2018;24:37–42. 10.1016/j.cmi.2017.05.028. 10.1016/j.cmi.2017.05.028 PubMed DOI

Morch K, Hanevik K. Giardiasis treatment: an update with a focus on refractory disease. Curr Opin Infect Dis. 2020;33:355–64. 10.1097/QCO.0000000000000668. 10.1097/QCO.0000000000000668 PubMed DOI

Krakovka S, Ribacke U, Miyamoto Y, Eckmann L, Svard S. Characterization of metronidazole-resistant Giardia intestinalis lines by comparative transcriptomics and proteomics. Front Microbiol. 2022;13:834008. 10.3389/fmicb.2022.834008. 10.3389/fmicb.2022.834008 PubMed DOI PMC

Saghaug CS, Klotz C, Kallio JP, Aebischer T, Langeland N, Hanevik K. Genetic diversity of the flavohemoprotein gene of Giardia lamblia: evidence for high allelic heterozygosity and copy number variation. Infect Drug Resist. 2020;13:4531–45. 10.2147/IDR.S274543. 10.2147/IDR.S274543 PubMed DOI PMC

Kubota R, Ishino T, Iwanaga S, Shinzawa N. Evaluation of the effect of gene duplication by genome editing on drug resistance in plasmodium falciparum. Front Cell Infect Microbiol. 2022;12:915656. 10.3389/fcimb.2022.915656. 10.3389/fcimb.2022.915656 PubMed DOI PMC

Lecova L, Weisz F, Tumova P, Tolarova V, Nohynkova E. The first multilocus genotype analysis of Giardia intestinalis in humans in the Czech Republic. Parasitology. 2018;145:1577–87. 10.1017/S0031182018000409. 10.1017/S0031182018000409 PubMed DOI

Medicine NLo: Primer-BLAST. 2024. https://www.ncbi.nlm.nih.gov/tools/primer-blast/. Accessed 08 Feb 2024.

Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem. 2013;59:892–902. 10.1373/clinchem.2013.206375. 10.1373/clinchem.2013.206375 PubMed DOI

Quan PL, Sauzade M, Brouzes E. dPCR: a technology review. Sensors-Basel. 2018. 10.3390/s18041271. 10.3390/s18041271 PubMed DOI PMC

Weisz F, Lalle M, Nohynkova E, Sannella AR, Dluhosova J, Caccio SM. Testing the impact of whole genome amplification on genome comparison using the polyploid flagellated Giardia duodenalis as a model. Exp Parasitol. 2019. 10.1016/j.exppara.2019.107776. 10.1016/j.exppara.2019.107776 PubMed DOI

Klotz C, Schmid MW, Winter K, Ignatius R, Weisz F, Saghaug CS, et al. Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes. Microb Genom. 2023. 10.1099/mgen.0.000963. 10.1099/mgen.0.000963 PubMed DOI PMC

Ghosh S, Kumar V, Verma A, Sharma T, Pradhan D, Selvapandiyan A, et al. Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani. Parasitol Res. 2022;121:3121–32. 10.1007/s00436-022-07645-x. 10.1007/s00436-022-07645-x PubMed DOI

Trost B, Walker S, Wang Z, Thiruvahindrapuram B, MacDonald JR, Sung WWL, et al. Am J Hum Genet. 2018;102:142–55. 10.1016/j.ajhg.2017.12.007. 10.1016/j.ajhg.2017.12.007 PubMed DOI PMC

Yoon ST, Xuan ZY, Makarov V, Ye K, Sebat J. Sensitive and accurate detection of copy number variants using read depth of coverage. Genome Res. 2009;19:1586–92. 10.1101/gr.092981.109. 10.1101/gr.092981.109 PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. 10.1093/bioinformatics/btu170. 10.1093/bioinformatics/btu170 PubMed DOI PMC

Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–60. 10.1093/bioinformatics/btp324. 10.1093/bioinformatics/btp324 PubMed DOI PMC

Broad Institute. 2019. https://broadinstitute.github.io/picard/. Accessed 08 Feb 2024.

Garcia-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, Gotz S, Tarazona S, et al. Qualimap: evaluating next-generation sequencing alignment data. Bioinformatics. 2012;28:2678–9. 10.1093/bioinformatics/bts503. 10.1093/bioinformatics/bts503 PubMed DOI

Bussotti G, Gouzelou E, Cortes Boite M, Kherachi I, Harrat Z, Eddaikra N, et al. Leishmania genome dynamics during environmental adaptation reveal strain-specific differences in gene copy number variation, karyotype instability, and telomeric amplification. Mbio. 2018. 10.1128/mBio.01399-18. 10.1128/mBio.01399-18 PubMed DOI PMC

Team D: DATAtab: Online Statistics Calculator. 2024. https://datatab.net. Accessed 08 Feb 2024.

Mazaika E, Homsy J. Digital droplet PCR: CNV analysis and other applications. Curr Protoc Hum Genet. 2014. 10.1002/0471142905.hg0724s82. 10.1002/0471142905.hg0724s82 PubMed DOI PMC

Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84:1003–11. 10.1021/ac202578x. 10.1021/ac202578x PubMed DOI PMC

Talevich E, Shain AH, Botton T, Bastian BC. CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing. Plos Comput Biol. 2016;12:e1004873. 10.1371/journal.pcbi.1004873. 10.1371/journal.pcbi.1004873 PubMed DOI PMC

Jager R. Special issue on whole genome amplification. Int J Mol Sci. 2023. 10.3390/ijms24119626. 10.3390/ijms24119626 PubMed DOI PMC

Deleye L, Tilleman L, Vander Plaetsen AS, Cornelis S, Deforce D, Van Nieuwerburgh F. Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Sci Rep. 2017;7:3422. 10.1038/s41598-017-03711-y. 10.1038/s41598-017-03711-y PubMed DOI PMC

Negreira GH, de Groote R, Van Giel D, Monsieurs P, Maes I, de Muylder G, et al. The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress. EMBO Rep. 2023. 10.15252/embr.202357413. 10.15252/embr.202357413 PubMed DOI PMC

Negreira GH, Monsieurs P, Imamura H, Maes I, Kuk N, Yagoubat A, et al. High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani. Nucleic Acids Res. 2022;50:293–305. 10.1093/nar/gkab1203. 10.1093/nar/gkab1203 PubMed DOI PMC

Tibayrenc M. Subspecific nomenclature of giardia duodenalis in the light of a compared population genomics of pathogens. Pathogens. 2023;12:249. 10.3390/pathogens12020249. 10.3390/pathogens12020249 PubMed DOI PMC

Association WM: Declaration of Helsinki. 2024. https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/i/. Accessed 08 Feb 2024.