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Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis

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Abstract

Purpose

Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran.

Methods

Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2.

Results

Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene.

Conclusion

This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

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Data Availability

The datasets used and/or analyzed during the current study are available in the online version.

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Acknowledgements

The current study was a part of the PhD thesis of Ali Asghari, financed by the Vice-Chancellor for Research of Shiraz University of Medical Sciences (Grant No: 21882). Hereby, we would like to express our gratitude and appreciation for the comprehensive support of this center.

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Author notes

  1. Ali Asghari and Farzad Mahdavi equally participated in this study.

Authors and Affiliations

  1. Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

    Ali Asghari & Qasem Asgari

  2. Department of Medical Parasitology and Mycology, School of Medicine, Guilan University of Medical Sciences, Rasht, Gilan, Iran

    Farzad Mahdavi

  3. Department of Pharmaceutical Biotechnology, University of Pavia, Pavia, Italy

    Amirhosein Yousefi & Roya Badali

  4. Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

    Laya Shamsi

  5. Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Mohammad Reza Mohammadi

  6. Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran

    Hamid Irannejad

  7. Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran

    Behnam Mohammadi-Ghalehbin

  8. Department of Biology and Control of Disease Vectors, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran

    Saeed Shahabi

  9. Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Mohammad Hossein Motazedian

  10. Department of Genetics and Molecular Medicine, School of ParaMedicine, Ilam University of Medical Sciences, Ilam, Iran

    Saiyad Bastaminejad

Authors
  1. Ali Asghari
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  2. Farzad Mahdavi
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  3. Amirhosein Yousefi
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  4. Laya Shamsi
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  5. Roya Badali
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  6. Mohammad Reza Mohammadi
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  7. Hamid Irannejad
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  8. Behnam Mohammadi-Ghalehbin
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  9. Saeed Shahabi
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  10. Qasem Asgari
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  11. Mohammad Hossein Motazedian
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  12. Saiyad Bastaminejad
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Contributions

AA, MHM, and SB conceived and designed the study. AA, FM, AY, RB, LS, HI, BM, SS, MRM, and QA had a role in collecting samples and methodology. AA and FM performed the molecular analysis. AA wrote the manuscript. AA, SB, and FM critically revised the manuscript. All the authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Mohammad Hossein Motazedian or Saiyad Bastaminejad.

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The authors declare that they have no conflicts of interest.

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Asghari, A., Mahdavi, F., Yousefi, A. et al. Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis. Acta Parasit. 69, 1073–1077 (2024). https://doi.org/10.1007/s11686-024-00828-9

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  • DOI: https://doi.org/10.1007/s11686-024-00828-9

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