Skip to main content

Advertisement

SpringerLink
Log in

Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Dientamoeba fragilis is a parasite that has been recognized as a causative agent of gastrointestinal symptoms. The search for genetic variation in D. fragilis based on the small-subunit (SSU) rRNA gene using restriction fragment length polymorphism was found not useful for molecular epidemiology. In this study, genetic variability of different clinical isolates of D. fragilis was explored by high-resolution melting curve (HRM) following polymerase chain reaction (PCR) in a one-step closed-tube method. Thirty fecal samples from irritable bowel syndrome (IBS) patients having D. fragilis trophozoites and negative for other organisms were involved in this study. According to the type of diarrhea, eight patients had acute, 14 patients had chronic intermittent, and eight patients had diarrhea alternating with constipation. HRM proved that four profiles (subtypes) were present as detecting by scanning mutation. One of these profiles (profile 1) was predominant (50%). Profile 2 was present on 20%. Profiles 3 and 4 were present on 16.7% and 13.4%, respectively. No mixed profiles were detected among the samples. The melting curves characterized by \( T_{\text{m}} 1 = 77.17\; \pm \;0.29~^\circ {\text{C}} \) in profile 1, \( T_{\text{m}} 1 = 77.37\; \pm \;1.45~^\circ {\text{C}} \) in profile 2, \( T_{\text{m}} 1 = 74.24\; \pm \;0.08~^\circ {\text{C}} \) and \( T_{\text{m}} 2 = 79.64\; \pm \;0.06~^\circ {\text{C}} \) in profile 3, and \( T_{\text{m}} 1 = 75.51\; \pm \;0.09~^\circ {\text{C}} \) and \( T_{\text{m}} 2 = 79.42\; \pm \;0.09~^\circ {\text{C}} \) in profile 4. The relation between these profiles and types of diarrhea proved that the majority of patients having profile 1 (73.4%) and profile 4 (75%) had chronic intermittent diarrhea. All of the patients having profile 2 had acute diarrhea while all of the patients having profile 3 had diarrhea alternating with constipation. Although profile 1 was detected among all types of diarrhea, it was corresponding to 11/14 of patients with chronic intermittent diarrhea. All the differences were clinically and statistically significant. In conclusion, HRM following PCR was proved as a wide variation on D. fragilis genotypes that could be related to the characters of diarrhea among IBS patients. As the differences in HRM reflect different sequences of SSU RNA gene, thus, another study for identifying the sequences of these isolates (profiles) will be done and published later.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Ayadi A, Bahri I (1999) Dientamoeba fragilis: pathogenic flagellate? Ball Soc Pathol Exot 92(5):299–301

    CAS  Google Scholar 

  • Bart A, van der Hijeden H, Grevs S, Speijer D, Landman W, Van Gool T (2008) Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker. J Clinic Microbiol 46(10):3270–3275

    Article  CAS  Google Scholar 

  • Bienz M, Siles-Lucas M, Muller N (2001) Use of novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the cloned GS/M-83-H7 line of Giardia lamblia. Parasitol Res 87:1011–1015

    PubMed  CAS  Google Scholar 

  • Borody T, Warren E, Wettstein A, Robertson G, Recabarren P, Fontella A, Herdnman K, Surace R (2002) Eradication of Dientamoeba fragilis can resolve IBS-like symptoms. J Gastroenterol Hepatol 17(Suppl):A103

    Google Scholar 

  • Chan F, Guan M, Mackenzie A (1993) Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens. J Clin Microbiol 31(7):1710–1714

    PubMed  CAS  Google Scholar 

  • Chan F, Stewart N, Guan M, Robb I, Fuite L, Chan I, Diaz-Mitoma F, King J, MacDonald N, Mackenzie A (1996) Prevalence of Dientamoeba fragilis antibodies in children and recognition of a 39 kDa immunodominant protein antigen of the organism. Eur J Clin Microbiol Infect Dis 15:950–954

    Article  PubMed  CAS  Google Scholar 

  • Crotti D, D'Annibale M (2007) Intestinal infections caused by Dientamoeba fragilis and Giardia duodenalis in our experience. Recenti Prog Med 98(6):361–366

    PubMed  Google Scholar 

  • Crotti D, Sensi M, Crotti S, Grelloni V, Manuali E (2007) Dientamoeba fragilis in swine population: a preliminary investigation. Vet Parasitol 145(3–4):349–351

    Article  PubMed  CAS  Google Scholar 

  • Dwyer D (1974) Analysis of the antigenic relationships among Trichomonas, Histomonas, Dientamoeba and Entamoeba. 3. Immunoelectrophoresis technics. J Protozool 21:139–145

    PubMed  CAS  Google Scholar 

  • Erali M, Voelkerding K, Wittwer C (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58

    Article  PubMed  CAS  Google Scholar 

  • Essa A, El-Wakil S (2005) Dientamoeba fragilis and irritable bowel syndrome in Egypt: a mere coincidence or true association. Egypt J Med Sci 26(1):251–261

    Google Scholar 

  • Garcia LS (2001) Intestinal protozoa: coccidia and microsporidia. In: Garcia L (ed) Diagnostic medical parasitology, 4th edn. American Society Microbiology, Washington, D.C

    Google Scholar 

  • Gerbod D, Edgcomb V, Noel C, Zenner L, Wintjens R, Delgado-Viscogliosi P, Holder M, Sogin M, Viscogliosi E (2001) Phylogenetic position of the Trichomonad parasite of Turkys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence. J Eukaryot Microbiol 48:498–504

    Article  PubMed  CAS  Google Scholar 

  • Girginkardesler N, Coskun S, Cuneyt Balcioglu I, Ertan P, Ok Z (2003) Dientamoeba fragilis, a neglected cause of diarrhea, successfully treated with secnidazole. Clin Microbiol Infect 9:110–113

    Article  PubMed  CAS  Google Scholar 

  • Graham R, Liew M, Meadows C, Lyon E, Wittwer C (2005) Distinguishing different DNA heterozygotes by high-resolution melting. Clin Chem 51:1295–1298

    Article  PubMed  CAS  Google Scholar 

  • Grendon J, DiGiacomo R, Frost J (1995) Descriptive features of Dientamoeba fragilis infections. J Trop Med Hyg 98:309–315

    PubMed  CAS  Google Scholar 

  • Gundry C, Bernard P, Herrmann M, Reed G, Wittwer C (1999) Rapid F508del and F508C assay using fluorescent hybridization probes. Genet Test 3:365–370

    PubMed  CAS  Google Scholar 

  • Herrmann M, Durtschi J, Bromley L, Wittwer C, Voelkerding K (2006) Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem 52(3):494–503

    Article  PubMed  CAS  Google Scholar 

  • Jeffery N, Gasser R, Steer P, Noormohammadi A (2007) Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting curve analysis of the v1hA gene single-copy region. Microbiol 153:2679–2688

    Article  CAS  Google Scholar 

  • Johnson J, Clark G (2000) Cryptic genetic diversity in Dientamoeba fragilis. J Clin Microbiol 38:4653–4654

    PubMed  CAS  Google Scholar 

  • Johnson E, Windsor J, Clark G (2004) Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clin Microbiol Rev 17(3):553–570

    Article  PubMed  Google Scholar 

  • Krypuy M, Newnham G, Thomas D, Conron M, Dobrovic A (2006) High resolution melting analysis for rapid detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non small cell lung cancer. BMC Cancer 6:295

    Google Scholar 

  • Libman M, Gyorkos T, Kokosin E, MacLean J (2008) Detection of pathogenic protozoa in the diagnostic laboratory: result reproducibility, specimens pooling, and competency assessment. J Clin Microbiol 46(7):2200–2205

    Article  PubMed  CAS  Google Scholar 

  • Mangold K, Manson R, Koay E, Stephens K, Regner M, Thomson R, Peterson L, Kaul K (2005) Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43(5):2435–2440

    Article  PubMed  CAS  Google Scholar 

  • Millet V, Spencer M, Chapin M, Stewart M, Yatabe J, Brewer T, Garcia L (1983) Dientamoeba fragilis, a protozoan parasite in adult members of a semi-communal group. Dig Dis Sci 28:335–339

    Article  PubMed  CAS  Google Scholar 

  • Murray P, Baron E (2007) Manual of clinical microbiology, 9th edn. Washington, DC

    Google Scholar 

  • Nicolas L, Milon G, Prina E (2002) Rapid differentiation of old world Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51:295–299

    Article  PubMed  CAS  Google Scholar 

  • Norberg A, Nord E, Evengard B (2003) Dientamoeba fragilis—a protozoal infection which may cause severe bowel distress. Clin Microbiol Infect 9:65–68

    Article  PubMed  CAS  Google Scholar 

  • Ockert G (1990) Symptomatology, pathology, epidemiology and diagnosis of Dientamoeba fragilis. In: Honigberg BM (ed) Trichomonads parasitic in humans. Springer, New York

    Google Scholar 

  • Pangasa A, Jex A, Campbell B, Bott J, Whip M, Hogg G, Stevens A, Gasser B (2008) High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. Mol Cell Probes 23:10–15

    Article  PubMed  Google Scholar 

  • Peek R, Reedeker R, van Gool T (2004) Direct amplification and genotyping of Dientamoeba fragilis from human stool specimens. J Clin Microbiol 42(2):631–635

    Article  PubMed  CAS  Google Scholar 

  • Press W, Teukolsky S, Vetterling W, Flannery B (eds) (1992) Numerical recipes in C, 2nd edn. Cambridge University Press, New York, pp 650–655

  • Price E, Smith H, Huygen F, Giffard P (2007) High-resolution DNA melt curve analysis of the clustered regularly interspaced short palindromic-repeat locus of Campylobacter jejuni. Appl Environ Microbiol 73:3431–3436

    Article  PubMed  CAS  Google Scholar 

  • Reed G, Wittwer C (2004) Sensitivity and specificity of single-nucleotide polymorphism scanning by high resolution melting analysis. Clin Chem 50:1748–1754

    Article  PubMed  CAS  Google Scholar 

  • Reed G, Kent J, Wittwer C (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597–608

    Article  PubMed  CAS  Google Scholar 

  • Ririe K, Rasmusen R, Wittwer C (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Bioch 245:154–160

    Article  CAS  Google Scholar 

  • Robinson B, Saint C, Monis P (2006) Rapid, sensitive and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. App Environ Microbiol 72:5857–5863

    Article  CAS  Google Scholar 

  • Schuster H, Jackson B (2008) Prevalence of Dientamoeba fragilis amongst patients consulting complimentary medicine practitioners in the British Isles. J Clin Pathol 62(2):182–184

    Article  PubMed  Google Scholar 

  • Silberman J, Clark C, Sogin M (1996) Dientamoeba fragilis shares a recent common evolutionary history with the trichomonads. Mol Biochem Parasitol 76:311–314

    Article  PubMed  CAS  Google Scholar 

  • Stark D, Beebe N, Marriott D, Ellis J, Harkness J (2005a) Detection of Dientamoeba fragilis in fresh stool specimens using PCR. Int J Parasitol 35:57–62

    Article  PubMed  CAS  Google Scholar 

  • Stark D, Beebe N, Marriott D, Ellis J, Harkness J (2005b) Prospective study of the prevalence, genotyping and clinical relevance of Dientamoeba fragilis infections in an Australian population. J Clin Microbiol 43:2718–2723

    Article  PubMed  CAS  Google Scholar 

  • Stark D, van Hal S, Marriot D, Ellis J, Harkness J (2007) Irritable bowel syndrome: a review on the role of intestinal protozoa and the importance of their detection and diagnosis. Int J Parasitol 37(1):11–20

    Article  PubMed  CAS  Google Scholar 

  • Stensfold C, Arendrup Mølbak K, Nielsen H (2007) The prevalence of Dientamoeba fragilis in patients with suspected enteroparasitic disease in a metropolitan area in Denmark. J Clin Microbiol 13(8):839–842

    Google Scholar 

  • van Gool T, Dankert J (1996) Emerging protozoal infections in The Netherlands: Cyclospora, Dientameoba and Microdpora. Ned Tijdschr Geneesked 140(3):155–160

    Google Scholar 

  • Vandenberg O, Peek R, Soouayah H, Dedsite A, Buset M, Scheen R, Retore P, Zissis G, van Gool T (2006) Clinical and microbiological features of dientamoebiasis in patients suspected of suffering from a parasitic gastrointestinal illness: a comparison of Dientamoeba fragilis and Giardia lamblia infections. Int J Infect Dis 10:225–261

    Article  Google Scholar 

  • Windsor J, Johnson E (1999) More laboratories should test for Dientamoeba fragilis. B M J 318:735

    CAS  Google Scholar 

  • Windsor J, Macfarlane L (2005) Irritable bowel syndrome-the need to exclude Dientamoeba fragilis. Am J Trop Med Hyg 72(5):501

    PubMed  CAS  Google Scholar 

  • Windsor J, Rafay M, Shenoy A, Johnson E (1998) Incidence of Dientamoeba fragilis in faecal samples submitted for routine microbiological analysis. Br J Biomed Sci 55:172–175

    PubMed  CAS  Google Scholar 

  • Windsor J, Macfarlane L, Hughes-Thapa G, Jones S, Whiteside T (2003) Detection of Dientamoeba fragilis by culture. Br J Biomed Sci 60:79–83

    PubMed  CAS  Google Scholar 

  • Windsor J, Clark C, Macfarlane L (2004) Molecular typing of Dientamoeba fragilis. Br J Biomed Sci 61:153–155

    PubMed  CAS  Google Scholar 

  • Windsor J, Macfarlane L, Clark C (2006) Internal transcribed spacer dimorphism and diversity in Dientamoeba fragilis. J Eukaryot Microbiol 53(3):188–192

    Article  PubMed  CAS  Google Scholar 

  • Wittwer C, Reed G, Gundry C, Vandersteen J, Pryor J (2003) High resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860

    Article  PubMed  CAS  Google Scholar 

  • Zhou L, Wang L, Palais R, Wittwer C (2005) High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51:1770–1777

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Parasitology Department, Faculty of Medicine, Suez Canal University, P.O. Box 41152, Ismailia, Egypt

    Eman M. Hussein

  2. Medical Microbiology and Parasitology Department, College of Medicine, King Faisal University, P.O. Box 400, Al-Ahsa, 31982, Saudi Arabia

    Eman M. Hussein & Hamdan I. Al-Mohammed

  3. Bio-physics Department, Faculty of Science, Al-Azhar University, Cairo, Egypt

    Abdalla M. Hussein

Authors
  1. Eman M. Hussein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hamdan I. Al-Mohammed
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abdalla M. Hussein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eman M. Hussein.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hussein, E.M., Al-Mohammed, H.I. & Hussein, A.M. Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis. Parasitol Res 105, 1053–1060 (2009). https://doi.org/10.1007/s00436-009-1515-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-009-1515-9

Keywords

Search

Navigation