Genotype Characteristics of Giardia duodenalis in Patients Using High Resolution Melting Analysis Technique in Khorramabad, Iran
-
Akram SEPAHVAND
,
Ahmad HOSSEINI-SAFA
,
Hossein Ali YOUSOFI
,
Mohammad Hassan TAJEDINI
,
Reza PAHLAVAN GHAREHBABAH
,
Nader PESTEHCHIAN
Abstract
Background: We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a cost-effective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared.
Methods: Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software.
Results: The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea.
Conclusion: The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.
1. Sayyari AA, Imanzadeh F, Bagheri Yazdi SA, et al. Prevalence of intestinal parasitic infections in the Islamic Republic of Iran. East Mediterr Health J. 2005;11:377-383.
2. Inpankaew T, Schar F, Odermatt P, et al. Low risk for transmission of zoonotic Giardia duodenalis from dogs to humans in rural cambodia. Parasit Vectors. 2014;7:412.
3. Zhang P, Liu Y, Alsarakibi M, et al. Application of hrm assays with evagreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res. 2012;111:2157-2163.
4. Olson ME, O'Handley RM, Ralston BJ, et al. Update on cryptosporidium and Giardia infections in cattle. Trends Parasitol. 2004;20:185-191.
5. Thompson RC, Palmer CS, O'Handley R. The public health and clinical significance of Giardia and Cryptosporidium in domestic animals. Vet J. 2008;177:18-25.
6. Bundy DA, Hall A, Medley GF, et al. Evaluating measures to control intestinal parasitic infections. World Health Stat Q. 1992;45:168-179.
7. Crompton DW, Savioli L. Intestinal parasitic infections and urbanization. Bull World Health Organ. 1993;71:1-7.
8. Hazrati Tappeh K, Manafi G, Asgharzadeh M, et al. Incidence of Giardia lamblia subspecies by pcr-rflp in stool specimens of hospitalized children at Urmia Mutahhari Hospital, west azerbaijan province, Iran. Iran J Parasitol. 2014;9:541-547.
9. Thompson RC, Monis PT. Variation in Giardia: Implications for taxonomy and epidemiology. Adv Parasitol. 2004;58:69-137.
10. Fallah E, Nahavandi K, Jamali R, et al. Genetic characterization of Giardia intestinalis strains from patients having sporadic giardiasis by using PCR assay. J Med Sci. 2008;8:310-315.
11. Fakhar M, Kialashaki E, Sharif M. An overview on the present situation of giardiasis in Iran and the world with emphasis on zoonotic aspects. J Mazandaran Univ Med Sci. 2014;24:235-251.
12. Pestehchian N, Rasekh H, Babaei Z, et al. Identification of genotypes of Giardia duodenalis human isolates in Isfahan, Iran, using polymerase chain reaction - restriction fragment length polymorphism. Adv Biomed Res. 2012;1:84.
13. Thompson RA, Monis PT. Taxonomy of Giardia species. Giardia. Springer; 2011:3-1
14. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24:110-140.
15. Lasek-Nesselquist E, Welch DM, Sogin ML. The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol. 2010;40:1063-1074.
16. Ryan U, Caccio SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43:943-956.
17. Rayani M, Zasmy Unyah N, Hatam G. Molecular identification of Giardia duodenalis isolates from Fars Province, Iran. Iran J Parasitol. 2014;9:70-78.
18. Monis PT, Mayrhofer G, Andrews RH, et al. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology. 1996;112 ( Pt 1):1-12.
19. Caccio SM, Beck R, Lalle M, et al. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages a and b. Int J Parasitol. 2008;38:1523-1531.
20. Eligio-Garcia L, Cortes-Campos A, Jimenez-Cardoso E. Classification of Giardia intestinalis isolates by multiple polymerase chain reaction (multiplex). Parasitol Res. 2008;103:797-800.
21. Babaei Z, Oormazdi H, Akhlaghi L, et al. Molecular characterization of the Iranian isolates of Giardia lamblia: Application of the glutamate dehydrogenase gene. Iran J Public Health. 2008;37:75-82.
22. Skhal D, Aboualchamat G, Al Nahhas S. Giardia duodenalis in damascus, syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method. Acta Trop. 2016;154:1-5.
23. Almeida A, Pozio E, Caccio SM. Genotyping of Giardia duodenalis cysts by new real-time pcr assays for detection of mixed infections in human samples. Appl Environ Microbiol. 2010;76:1895-1901.
24. Pelayo L, Fraga J, Nunez FA, et al. Genetic characterization by random amplified polymorphic DNA analysis (rapd) of 18 isolates of Giardia lamblia obtained from day care children. Exp Parasitol. 2003;104:162-166.
25. Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol. 2008;85:50-58.
26. Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics. 2007;8:597-608.
27. Herrmann MG, Durtschi JD, Bromley LK, et al. Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes. Clin Chem. 2006;52:494-503.
28. Price EP, Smith H, Huygens F, et al. High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni. Appl Environ Microbiol. 2007;73:3431-3436.
29. Hosseini-Safa A, Mohaghegh MA, Pestechian N, et al. First report of tasmanian sheep strain (g2) genotype isolated from Iranian goat using high resolution melting (hrm) analysis. Gastroenterol Hepatol Bed Bench. 2016;9:70-74.
30. Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using lcgreen. Clin Chem. 2003;49:853-860.
31. Bienz M, Siles-Lucas M, Muller N. Use of a novel DNA melting profile assay for the identification of pcr-amplified genomic sequences encoding for variant-specific surface proteins from the clonal gs/m-83-h7 line of Giardia lamblia. Parasitol Res. 2001;87:1011-1015.
32. Dryden MW, Payne PA, Smith V. Accurate diagnosis of Giardia spp and proper fecal examination procedures. Vet Ther. 2006;7:4-14.
33. Sepahvand A, Pestehchian N, Yousefi HA, et al. Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for pcr targeting the tpi gene. J Parasit Dis. 2017;41:263-267.
34. Pestechian N, Hosseini Safa A, Tajedini M, et al. Genetic diversity of Echinococcus granulosus in center of Iran. Korean J Parasitol. 2014;52:413-418.
35. Safa AH, Harandi MF, Tajaddini M, et al. Rapid identification of Echinococcus granulosus and E. canadensis using high-resolution melting (HRM) analysis by focusing on a single nucleotide polymorphism. Jpn J Infect Dis. 2016;69:300-305.
36. Tamura K, Stecher G, Peterson D, et al. Mega6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725-2729.
37. Tan L, Yu X, Abdullahi AY, et al. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia. Parasitol Res. 2015;114:4081-4086.
38. Meloni BP, Lymbery AJ, Thompson RC. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: Implications for reproductive biology, population structure, taxonomy, and epidemiology. J Parasitol. 1995;81:368-383.
39. Helmy MM, Abdel-Fattah HS, Rashed L. Real-time pcr/rflp assay to detect Giardia intestinalis genotypes in human isolates with diarrhea in Egypt. J Parasitol. 2009;95:1000-1004.
40. Manouchehri Naeini K, Hosseini SA, Gholipour A, et al. Genotyping of Giardia duodenalis isolates in individuals with and without chronic diarrhea using Polymerase Chain Reaction. Journal of Mazandaran University of Medical Sciences. 2012:22:39-46. (In Persian)
41. 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: 17:61-71. (In Persian)
42. Bahrami F, Zamini GH, Haghighi A, et al. Detection and molecular identification of human Giardia isolates in the West of Iran. Biomed Res India. 2017; 28(13):5687-5692.
43. Minetti C, Lamden K, Durband C, et al. Determination of Giardia duodenalis assemblages and multi-locus genotypes in patients with sporadic giardiasis from England. Parasit Vectors. 2015;8:444.
44. Read C, Walters J, Robertson ID, et al. Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol. 2002;32:229-231.
45. Pestechian N, Rasekh H, Rostami-Nejad M, et al. Molecular identification of Giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population? Gastroenterol Hepatol Bed Bench. 2014;7:168-172.
46. Sahagun J, Clavel A, Goñi P, et al. Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis. 2008; 27(1):81-3.
2. Inpankaew T, Schar F, Odermatt P, et al. Low risk for transmission of zoonotic Giardia duodenalis from dogs to humans in rural cambodia. Parasit Vectors. 2014;7:412.
3. Zhang P, Liu Y, Alsarakibi M, et al. Application of hrm assays with evagreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res. 2012;111:2157-2163.
4. Olson ME, O'Handley RM, Ralston BJ, et al. Update on cryptosporidium and Giardia infections in cattle. Trends Parasitol. 2004;20:185-191.
5. Thompson RC, Palmer CS, O'Handley R. The public health and clinical significance of Giardia and Cryptosporidium in domestic animals. Vet J. 2008;177:18-25.
6. Bundy DA, Hall A, Medley GF, et al. Evaluating measures to control intestinal parasitic infections. World Health Stat Q. 1992;45:168-179.
7. Crompton DW, Savioli L. Intestinal parasitic infections and urbanization. Bull World Health Organ. 1993;71:1-7.
8. Hazrati Tappeh K, Manafi G, Asgharzadeh M, et al. Incidence of Giardia lamblia subspecies by pcr-rflp in stool specimens of hospitalized children at Urmia Mutahhari Hospital, west azerbaijan province, Iran. Iran J Parasitol. 2014;9:541-547.
9. Thompson RC, Monis PT. Variation in Giardia: Implications for taxonomy and epidemiology. Adv Parasitol. 2004;58:69-137.
10. Fallah E, Nahavandi K, Jamali R, et al. Genetic characterization of Giardia intestinalis strains from patients having sporadic giardiasis by using PCR assay. J Med Sci. 2008;8:310-315.
11. Fakhar M, Kialashaki E, Sharif M. An overview on the present situation of giardiasis in Iran and the world with emphasis on zoonotic aspects. J Mazandaran Univ Med Sci. 2014;24:235-251.
12. Pestehchian N, Rasekh H, Babaei Z, et al. Identification of genotypes of Giardia duodenalis human isolates in Isfahan, Iran, using polymerase chain reaction - restriction fragment length polymorphism. Adv Biomed Res. 2012;1:84.
13. Thompson RA, Monis PT. Taxonomy of Giardia species. Giardia. Springer; 2011:3-1
14. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24:110-140.
15. Lasek-Nesselquist E, Welch DM, Sogin ML. The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems. Int J Parasitol. 2010;40:1063-1074.
16. Ryan U, Caccio SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43:943-956.
17. Rayani M, Zasmy Unyah N, Hatam G. Molecular identification of Giardia duodenalis isolates from Fars Province, Iran. Iran J Parasitol. 2014;9:70-78.
18. Monis PT, Mayrhofer G, Andrews RH, et al. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology. 1996;112 ( Pt 1):1-12.
19. Caccio SM, Beck R, Lalle M, et al. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages a and b. Int J Parasitol. 2008;38:1523-1531.
20. Eligio-Garcia L, Cortes-Campos A, Jimenez-Cardoso E. Classification of Giardia intestinalis isolates by multiple polymerase chain reaction (multiplex). Parasitol Res. 2008;103:797-800.
21. Babaei Z, Oormazdi H, Akhlaghi L, et al. Molecular characterization of the Iranian isolates of Giardia lamblia: Application of the glutamate dehydrogenase gene. Iran J Public Health. 2008;37:75-82.
22. Skhal D, Aboualchamat G, Al Nahhas S. Giardia duodenalis in damascus, syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method. Acta Trop. 2016;154:1-5.
23. Almeida A, Pozio E, Caccio SM. Genotyping of Giardia duodenalis cysts by new real-time pcr assays for detection of mixed infections in human samples. Appl Environ Microbiol. 2010;76:1895-1901.
24. Pelayo L, Fraga J, Nunez FA, et al. Genetic characterization by random amplified polymorphic DNA analysis (rapd) of 18 isolates of Giardia lamblia obtained from day care children. Exp Parasitol. 2003;104:162-166.
25. Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol. 2008;85:50-58.
26. Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics. 2007;8:597-608.
27. Herrmann MG, Durtschi JD, Bromley LK, et al. Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes. Clin Chem. 2006;52:494-503.
28. Price EP, Smith H, Huygens F, et al. High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni. Appl Environ Microbiol. 2007;73:3431-3436.
29. Hosseini-Safa A, Mohaghegh MA, Pestechian N, et al. First report of tasmanian sheep strain (g2) genotype isolated from Iranian goat using high resolution melting (hrm) analysis. Gastroenterol Hepatol Bed Bench. 2016;9:70-74.
30. Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using lcgreen. Clin Chem. 2003;49:853-860.
31. Bienz M, Siles-Lucas M, Muller N. Use of a novel DNA melting profile assay for the identification of pcr-amplified genomic sequences encoding for variant-specific surface proteins from the clonal gs/m-83-h7 line of Giardia lamblia. Parasitol Res. 2001;87:1011-1015.
32. Dryden MW, Payne PA, Smith V. Accurate diagnosis of Giardia spp and proper fecal examination procedures. Vet Ther. 2006;7:4-14.
33. Sepahvand A, Pestehchian N, Yousefi HA, et al. Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for pcr targeting the tpi gene. J Parasit Dis. 2017;41:263-267.
34. Pestechian N, Hosseini Safa A, Tajedini M, et al. Genetic diversity of Echinococcus granulosus in center of Iran. Korean J Parasitol. 2014;52:413-418.
35. Safa AH, Harandi MF, Tajaddini M, et al. Rapid identification of Echinococcus granulosus and E. canadensis using high-resolution melting (HRM) analysis by focusing on a single nucleotide polymorphism. Jpn J Infect Dis. 2016;69:300-305.
36. Tamura K, Stecher G, Peterson D, et al. Mega6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725-2729.
37. Tan L, Yu X, Abdullahi AY, et al. Development of a rapid HRM genotyping method for detection of dog-derived Giardia lamblia. Parasitol Res. 2015;114:4081-4086.
38. Meloni BP, Lymbery AJ, Thompson RC. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: Implications for reproductive biology, population structure, taxonomy, and epidemiology. J Parasitol. 1995;81:368-383.
39. Helmy MM, Abdel-Fattah HS, Rashed L. Real-time pcr/rflp assay to detect Giardia intestinalis genotypes in human isolates with diarrhea in Egypt. J Parasitol. 2009;95:1000-1004.
40. Manouchehri Naeini K, Hosseini SA, Gholipour A, et al. Genotyping of Giardia duodenalis isolates in individuals with and without chronic diarrhea using Polymerase Chain Reaction. Journal of Mazandaran University of Medical Sciences. 2012:22:39-46. (In Persian)
41. 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: 17:61-71. (In Persian)
42. Bahrami F, Zamini GH, Haghighi A, et al. Detection and molecular identification of human Giardia isolates in the West of Iran. Biomed Res India. 2017; 28(13):5687-5692.
43. Minetti C, Lamden K, Durband C, et al. Determination of Giardia duodenalis assemblages and multi-locus genotypes in patients with sporadic giardiasis from England. Parasit Vectors. 2015;8:444.
44. Read C, Walters J, Robertson ID, et al. Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol. 2002;32:229-231.
45. Pestechian N, Rasekh H, Rostami-Nejad M, et al. Molecular identification of Giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population? Gastroenterol Hepatol Bed Bench. 2014;7:168-172.
46. Sahagun J, Clavel A, Goñi P, et al. Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis. 2008; 27(1):81-3.
| Files | ||
| Issue | Vol 15 No 2 (2020) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v15i2.3302 | |
| Keywords | ||
| Giardia duodenalis Genotyping Triosephosphate isomer-ase gene High resolution melting; Iran | ||
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How to Cite
1.
SEPAHVAND A, HOSSEINI-SAFA A, YOUSOFI HA, TAJEDINI MH, PAHLAVAN GHAREHBABAH R, PESTEHCHIAN N. Genotype Characteristics of Giardia duodenalis in Patients Using High Resolution Melting Analysis Technique in Khorramabad, Iran. Iran J Parasitol. 2020;15(2):204-213.
