The First Evaluation of Genetic Diversity of Human Giardia Duodenalis Isolates Using Multilocus Sequence Typing (MLST) in Khuzestan Province
DOI:
https://doi.org/10.22100/jkh.v19i3.3311Keywords:
Giardia duodenalis, Assemblage, MLST, KhuzestanAbstract
Introduction: Giardia duodenalis is one of the most important agents of water- and food-borne parasites. The genetic characteristics of this parasite play a crucial role in its pathogenesis. This study represents the first genotyping of Giardia duodenalis in humans using the MLST technique on housekeeping genes, performed in Khuzestan province.
Methods: The genomic DNA was extracted from cysts isolated from 129 human fecal specimens, and the diagnosis of G. duodenalis was verified by amplifying a 350 bp fragment of the SSU-rRNA gene. Nested PCR targeting the tpi and β-giardin genes, as well as semi-nested PCR of the gdh gene, were carried out to amplify the 530 bp, 511 bp, and 432 bp fragments of these genes, respectively, followed by sequencing of the amplified fragments. Phylogenetic analysis was then performed to determine the assemblages and sub-assemblages present.
Results: Among the 29 specimens that were successfully genotyped for one of the three genes, assemblages A and B were identified in 23 (79.31%) and 6 (20.69%) specimens, respectively. The present study found no significant correlations between assemblages and clinical symptoms, nor with the place of residence. Phylogenetic analysis indicated the presence of sub-assemblages AII and BIV.
Conclusion: The results showed that assemblage A and sub-assemblage AII are the dominant genotypes in Khuzestan province. The identification of sub-assemblages AII and BIV suggests an anthroponotic transmission route. However, further study is necessary to determine the transmission route of Giardia infection from animals to humans.
References
Rodney DA. Giardia duodenalis: Biology and Pathogenesis. Clinical Microbiology Reviews. 2021;34(4):e00024-19.
Thompson RC. The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Vet Parasitol 2004 Dec 9;126(1-2):15-35 doi: 101016/jvetpar200409008. 2004.
Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011 Jan;24(1):110-40 doi: 101128/CMR00033-10. 2011.
Costache C, Kalmár Z, Colosi HA, Baciu AM, Opriş RV, Györke A, et al. First multilocus sequence typing (MLST) of Giardia duodenalis isolates from humans in Romania. Parasit Vectors 2020 Jul 31;13(1):387 doi: 101186/s13071-020-04248-2. 2020.
Sprong H, Caccio SM, van der Giessen JWB, on behalf of the Zn, partners. Identification of Zoonotic Genotypes of Giardia duodenalis. PLOS Neglected Tropical Diseases. 2009;3(12):e558.
Asghari A, Mahdavi F, Karimi K, Mohammadi MR, Shamsi L, Asgari Q, et al. Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern. Parasite Epidemiol Control 2024 Apr 29;25:e00354 doi: 101016/jparepi2024e00354 eCollection 2024 May. 2024;25.
Abasian L, Talebi FdMfBUoMST, Bazyar A, Shirbazo S, Sayehmiri K, Ahmad N. Prevalence of Giardia lamblia in Iran: a systematic review and Meta analysis study. Pejouhesh dar Pezeshki (Research in Medicine). 2013;36(5):111-6. URL: http://pejouhesh.sbmu.ac.ir/article-1-1118-en.html.
Mowlavi G, Mirahmadi H, Rezaeian M, Kia E, Daryani N, Rokni M, et al. Prevalence of Intestinal Parasites in Tribal Parts of Khuzestan Province During 2005-2007. Govaresh. 2008;12:219-28.
Karanis P, Kourenti C, Smith H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 2007 Mar;5(1):1-38 doi: 102166/wh2006002. 2007.
Nygård K, Schimmer B, Søbstad Ã, Walde A, Tveit I, Langeland N, et al. A large community outbreak of waterborne giardiasis-delayed detection in a non-endemic urban area. BMC Public Health 2006 May 25;6:141 doi: 101186/1471-2458-6-141. 2006.
Cheun HI, Kim CH, Cho SH, Ma DW, Goo BL, Na MS, et al. The first outbreak of giardiasis with drinking water in Korea. Osong Public Health Res Perspect 2013 Apr;4(2):89-92 doi: 101016/jphrp201303003. 2013.
Fantinatti M, Cascais-Figueredo T, Austriaco-Teixeira P, Carvalho-Costa FA, Da-Cruz AM. Giardia lamblia-infected preschoolers present growth delays independent of the assemblage A, B or E. Mem Inst Oswaldo Cruz 2023 Aug 18;118:e230043 doi: 101590/0074-02760230043 eCollection 2023. 2023.
Hashemi-Hafshejani S, Meamar AR, Moradi M, Hemmati N, Solaymani-Mohammadi S, Razmjou E. Multilocus sequence typing of Giardia duodenalis genotypes circulating in humans in a major metropolitan area. Front Med (Lausanne) 2022 Oct 5;9:976956 doi: 103389/fmed2022976956 eCollection 2022. 2022.
Klotz C, Sannella AR, Weisz F, Chaudhry U, Sroka J, Tůmová P, et al. Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power. Parasites & Vectors. 2022;15(1):489.
Faria CP, Zanini GM, Dias GS, da Silva S, Sousa MDC. New multilocus genotypes of Giardia lamblia human isolates. Infect Genet Evol 2017 Oct;54:128-137 doi: 101016/jmeegid201706028 Epub 2017 Jun 29. 2017.
Plutzer J, Lassen B, Jokelainen P, Djurković-Djaković O, Kucsera I, Dorbek-Kolin E, et al. Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016. Euro Surveill 2018 Jan;23(4):16-00825 doi: 102807/1560-7917ES201823416-00825. 2018.
Hellard ME, Sinclair MI, Hogg GG, Fairley CK. Prevalence of enteric pathogens among community based asymptomatic individuals. J Gastroenterol Hepatol 2000 Mar;15(3):290-3 doi: 101046/j1440-1746200002089x. 2000;15(3).
Koehler AV, Jex AR, Haydon SR, Stevens MA, Gasser RB. Giardia/giardiasis — A perspective on diagnostic and analytical tools. Biotechnology Advances. 2014;32(2):280-9.
Wegayehu T, Karim MR, Li J, Adamu H, Erko B, Zhang L, et al. Multilocus genotyping of Giardia duodenalis isolates from children in Oromia Special Zone, central Ethiopia. BMC Microbiol 2016 May 21;16:89 doi: 101186/s12866-016-0706-7. 2016.
Rafiei A, Roointan ES, Samarbafzadeh AR, Shayesteh AA, Shamsizadeh A, Pourmahdi Borujeni M. Investigation of Possible Correlation between Giardia duodenalis Genotypes and Clinical Symptoms in Southwest of Iran. Iran J Parasitol 2013 Jul;8(3):389-95. 2013. PMID: 24454431.
Rayani M, Zasmy Unyah N, Hatam G. Molecular Identification of Giardia duodenalis Isolates from Fars Province, Iran. Iran J Parasitol 2014 Mar;9(1):70-8. 2014. PMID: 25642262.
Elham K, Mahdi S, Mahdi F, Ahmad D, Abdol Sattar P. Assessing Different Methods of DNA Extraction from Giardia Lamblia Cysts. Journal of Mazandaran University of Medical Sciences. 2013;22(98):67-74.
Asgarian F, Tavalla M, Teimoori A, Zebardast N, Cheraghian B. Evaluation of Three Protocols of DNA Extraction for Detection of Giardia duodenalis in Human Fecal Specimens. Jundishapur J Microbiol. 2018;11(4):e63096.
Rai AK, Chakravorty R, Paul J. Detection of Giardia, Entamoeba, and Cryptosporidium in unprocessed food items from northern India. World Journal of Microbiology and Biotechnology. 2008;24(12):2879-87.
Read CM, Monis PT, Thompson RC. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect Genet Evol 2004 Jun;4(2):125-30 doi: 101016/jmeegid200402001. 2004.
Cacciò SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol 2002 Jul;32(8):1023-30 doi: 101016/s0020-7519(02)00068-1. 2002.
Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Cacciò SM. Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardiaduodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol 2005 Feb;35(2):207-13 doi: 101016/jijpara200410022 Epub 2004 Dec 15. 2005.
Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 2003 Nov;9(11):1444-52 doi: 103201/eid0911030084. 2003.
Huey CS, Mahdy MAK, Al-Mekhlafi HM, Nasr NA, Lim YAL, Mahmud R, et al. Multilocus genotyping of Giardia duodenalis in Malaysia. Infection, Genetics and Evolution. 2013;17:269-76.
Minetti C, Lamden K, Durband C, Cheesbrough J, Fox A, Wastling JM. Determination of Giardia duodenalis assemblages and multi-locus genotypes in patients with sporadic giardiasis from England. Parasites & Vectors. 2015;8(1):444.
Uda-Shimoda CF, Colli CM, Pavanelli MF, Falavigna-Guilherme AL, Gomes ML. Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis. Diagnostic Microbiology and Infectious Disease. 2014;78(1):53-8.
Nantavisai K, Mungthin M, Tan-ariya P, Rangsin R, Naaglor T, Leelayoova S. Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens. J Clin Microbiol 2007 Feb;45(2):581-3 doi: 101128/JCM01823-06 Epub 2006 Nov 22. 2007.
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol 2012 Nov;113(5):1014-26 doi: 101111/j1365-2672201205384x Epub 2012 Jul 24. 2012.
Wilke H, Robertson LJ. Preservation of Giardia cysts in stool samples for subsequent PCR analysis. J Microbiol Methods 2009 Sep;78(3):292-6 doi: 101016/jmimet200906018 Epub 2009 Jul 2. 2009.
Broglia A, Weitzel T, Harms G, Cacció SM, Nöckler K. Molecular typing of Giardia duodenalis isolates from German travellers. Parasitol Res 2013 Oct;112(10):3449-56 doi: 101007/s00436-013-3524-y Epub 2013 Jul 28. 2013.
Thompson RCA, Ash A. Molecular epidemiology of Giardia and Cryptosporidium infections. Infect Genet Evol 2016 Jun;40:315-323 doi: 101016/jmeegid201509028 Epub 2015 Oct 11. 2016.
Babaei Z, Oormazdi H, Akhlaghi L, Rezaie S, Razmjou E, Arabshahi SKS-, et al. Molecular characterization of the Iranian isolates of Giardia lamblia: application of the glutamate dehydrogenase gene. Iranian Journal of Public Health. 2008;37(2).. 75-82. eISSN: 2251-6093.
Pestechian N, Rasekh H, Rostami-Nejad M, Yousofi HA, Hosseini-Safa A. Molecular identification of Giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population? Gastroenterol Hepatol Bed Bench 2014 Summer;7(3):168-72. 2014. PMID: 25120898.
Monis PT, Andrews RH, Mayrhofer G, Ey PL. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol 1999 Sep;16(9):1135-44 doi: 101093/oxfordjournalsmolbeva026204. 1999.
Pallant L, Barutzki D, Schaper R, Thompson RC. The epidemiology of infections with Giardia species and genotypes in well cared for dogs and cats in Germany. Parasit Vectors 2015 Jan 6;8:2 doi: 101186/s13071-014-0615-2. 2015.
Holberton DV, Marshall J. Analysis of consensus sequence patterns in Giardia cytoskeleton gene promoters. Nucleic Acids Res 1995 Aug 11;23(15):2945-53 doi: 101093/nar/23152945. 1995.
Baruch AC, Isaac-Renton J, Adam RD. The molecular epidemiology of Giardia lamblia: a sequence-based approach. J Infect Dis 1996 Jul;174(1):233-6 doi: 101093/infdis/1741233. 1996.
Jerez Puebla LE, Núñez FA, Santos LP, Rivero LR, Silva IM, Valdés LA, et al. Molecular analysis of Giardia duodenalis isolates from symptomatic and asymptomatic children from La Habana, Cuba. Parasite Epidemiol Control 2017 May 24;2(3):105-113 doi: 101016/jparepi201705003 eCollection 2017 Aug. 2017.
Lee MF, Auer H, Lindo JF, Walochnik J. Multilocus sequence analysis of Giardia spp. isolated from patients with diarrhea in Austria. Parasitol Res 2017 Feb;116(2):477-481 doi: 101007/s00436-016-5306-9 Epub 2016 Nov 25. 2017.
Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland - first report of Assemblage E. Parasitology 2017 Aug;144(9):1154-1161 doi: 101017/S0031182017000439 Epub 2017 May 9. 2017.
Cacciò SM, Sprong H. Giardia duodenalis: genetic recombination and its implications for taxonomy and molecular epidemiology. Exp Parasitol 2010 Jan;124(1):107-12 doi: 101016/jexppara200902007 Epub 2009 Feb 21. 2010.
Ankarklev J, Franzén O, Peirasmaki D, Jerlström-Hultqvist J, Lebbad M, Andersson J, et al. Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates. BMC Genomics. 2015;16(1):697.
Birky C. Giardia Sex? Yes, but how and how much? Trends in parasitology. 2009;26:70-4.
Sarkari B, Ashrafmansori A, Hatam GR, Motazedian MH, Asgari Q, Mohammadpour I. Genotyping of Giardia lamblia isolates from human in southern Iran. Trop Biomed 2012 Sep;29(3):366-71. 2012. PMID: 23018499.
Ramírez JD, Heredia RD, Hernández C, León CM, Moncada LI, Reyes P, et al. Molecular diagnosis and genotype analysis of Giardia duodenalis in asymptomatic children from a rural area in central Colombia. Infect Genet Evol 2015 Jun;32:208-13 doi: 101016/jmeegid201503015 Epub 2015 Mar 18. 2015.
Fallah E, Hatam-Nahavandi K, Rasul J, Poor B, Mohammad A. Molecular Identification of Giardia duodenalis Isolates from Human and Animal Reservoirs by PCR-RFLP. Journal of Biological Sciences. 2008;8.
Bahrami F, Zamini G, Haghighi A, Khademerfan MB. Detection and molecular identification of human giardia isolates in the west of Iran. Biomedical Research (India). 2017;28:5687-92.
Akbarian A, Sadraie J, Forozandeh M. Evaluattion of Giardia lamblia genetic differences in Khorramabad City and surrounding villages by use of PCR and sequencing. Scientific Journal of Kurdistan University of Medical Sciences. 2012;17(2):61-71. URL: http://sjku.muk.ac.ir/article-1-801-en.html.
Anuar TS, Nor Azreen S, Salleh FM, Moktar N. Molecular epidemiology of giardiasis among Orang Asli in Malaysia: application of the triosephosphate isomerase gene. BMC Infectious Diseases. 2014;14(1):78.
Abdel-Moein KA, Saeed H. The zoonotic potential of Giardia intestinalis assemblage E in rural settings. Parasitol Res 2016 Aug;115(8):3197-202 doi: 101007/s00436-016-5081-7 Epub 2016 Apr 25. 2016.
Molina N, Minvielle M, Grenóvero S, Salomón C, Basualdo J. High prevalences of infection with Giardia intestinalis genotype B among children in urban and rural areas of Argentina. Ann Trop Med Parasitol 2011 Jun;105(4):299-309 doi: 101179/136485911X12987676649665. 2011.
Yousefi HA, Asgarian F, Tavalla M, Ghafari S, Kohansal K. In vitro Culture and Multilocus Genotyping of Giardia duodenalis Trophozoites Obtained from Human Fecal Samples in Southwest of Iran. Curr Comput Aided Drug Des. 2024;20(2):145-54.
Puebla LJ, Núñez FA, Fernández YA, Fraga J, Rivero LR, Millán IA, et al. Correlation of Giardia duodenalis assemblages with clinical and epidemiological data in Cuban children. Infect Genet Evol 2014 Apr;23:7-12 doi: 101016/jmeegid201401007 Epub 2014 Jan 22. 2014.
Downloads
Published
Issue
Section
License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.