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Newly defined conditions for the in vitro cultivation and cryopreservation of Dientamoeba fragilis: new techniques set to fast track molecular studies on this organism

Published online by Cambridge University Press:  08 July 2010

J. L. N. BARRATT ,
G. R. BANIK ,
J. HARKNESS ,
D. MARRIOTT ,
J. T. ELLIS  and
D. STARK
J. L. N. BARRATT*
Affiliation:
Division of Microbiology, SydPath, St Vincent's Hospital, Darlinghurst, Australia University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
G. R. BANIK
Affiliation:
Division of Microbiology, SydPath, St Vincent's Hospital, Darlinghurst, Australia University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
J. HARKNESS
Affiliation:
Division of Microbiology, SydPath, St Vincent's Hospital, Darlinghurst, Australia University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
D. MARRIOTT
Affiliation:
Division of Microbiology, SydPath, St Vincent's Hospital, Darlinghurst, Australia University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
J. T. ELLIS
Affiliation:
University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
D. STARK
Affiliation:
Division of Microbiology, SydPath, St Vincent's Hospital, Darlinghurst, Australia University of Technology Sydney, Department of Medical and Molecular Biosciences, Broadway, Australia
*
*Corresponding author: Department of Microbiology, St Vincent's Hospital, Darlinghurst 2010, NSW, Australia. Tel: +61 2 8382 9196. Fax: +612 8382 2989. E-mail: [email protected]
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Summary

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42°C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis.

Keywords

Dientamoeba fragilisin vitroculturecultivationcryopreservationculture mediaoptimization
Type
Research Article
Information
Parasitology , Volume 137 , Issue 13 , November 2010 , pp. 1867 - 1878
Copyright
Copyright © Cambridge University Press 2010

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References

REFERENCES

Balamuth, W. (1946). Improved egg yolk medium for cultivation of Entamoeba histolytica and other intestinal protozoa. American Journal of Clinical Pathology 16, 380–384.CrossRefGoogle Scholar
Bart, A., van der Heijden, H. M., Greve, S., Speijer, S. D., Landman, W. J. and van Gool, T. (2008). Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker. Journal of Clinical Microbiology 46, 3270–3275.Google Scholar
Borchardt, K. A., Zhang, M. Z., Shing, H. and Flink, K. (1997). A comparison of the sensitivity of the InPouch TV, Diamond's and Trichosel media for detection of Trichomonas vaginalis. Genitourinary Medicine 73, 297–298.Google Scholar
Brug, S. L. (1938). Observations on Dientamoeba fragilis. Annals of Tropical Medicine and Parasitology 30, 441–452.CrossRefGoogle Scholar
Camp, R. R., Mattern, C. F. and Honigberg, B. M. (1974). Study of Dientamoeba fragilis Jepps & Dobell. I. Electronmicroscopic observations of the binucleate stages. II. Taxonomic position and revision of the genus. The Journal of Protozoology 21, 69–82.Google Scholar
Chan, F. T., Guan, M. X., Mackenzie, A. M. and Diaz-Mitoma, F. (1994). Susceptibility testing of Dientamoeba fragilis ATCC 30948 with iodoquinol, paromomycin, tetracycline, and metronidazole. Antimicrobial Agents and Chemotherapy 38, 1157–1160.Google Scholar
Clark, C. G. and Diamond, L. S. (2002). Methods for cultivation of luminal parasitic protists of clinical importance. Clinical Microbiology Reviews 15, 329–341.CrossRefGoogle ScholarPubMed
Cleveland, L. R. and Collier, J. (1930). Various improvements in cultivation of Entamoeba histolytica. American Journal of Hygiene 12, 606–613.Google Scholar
Dickinson, E. C., Cohen, M. A. and Schlenker, M. K. (2002). Dientamoeba fragilis: a significant pathogen. The American Journal of Emergency Medicine 20, 62–63.CrossRefGoogle ScholarPubMed
Dobell, C. (1940). Researches on intestinal protozoa in monkeys and man. X. The life history of Dientamoeba fragilis: observations, experiments and speculations. Parasitology 32, 417–461.CrossRefGoogle Scholar
Dwyer, D. M. and Honigberg, B. M. (1971). Freezing and maintenance of Dientamoeba fragilis in liquid nitrogen. The Journal of Parasitology 57, 190–191.CrossRefGoogle Scholar
Girginkardesler, N., Coskun, S., Cuneyt Balcioglu, I., Ertan, P. and Ok, U. Z. (2003). Dientamoeba fragilis, a neglected cause of diarrhea, successfully treated with secnidazole. Clinical Microbiology and Infection 9, 110–113.Google Scholar
Jacobs, L. (1953). The cultivation of Dientamoeba fragilis. Annals of the New York Academy of Sciences 56, 1057–1061.CrossRefGoogle Scholar
Jepps, M. W. and Dobell, C. (1918). Dientamoeba fragilis n.g., n. sp.: a new intestinal amoeba from man. Parasitology 10, 352–367.CrossRefGoogle Scholar
Johnson, E. H., Windsor, J. J. and Clark, C. G. (2004). Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clinical Microbiology Reviews 17, 553–570.CrossRefGoogle ScholarPubMed
Johnson, J. A. and Clark, C. G. (2000). Cryptic genetic diversity in Dientamoeba fragilis. Journal of Clinical Microbiology 38, 4653–4654.Google Scholar
Lagace-Wiens, P. R., VanCaeseele, P. G. and Koschik, C. (2006). Dientamoeba fragilis: an emerging role in intestinal disease. Canadian Medical Association Journal 175, 468–469.Google Scholar
Lamy, L. (1960). [Dientamoeba fragilis: detection, culture, incidence, interest and pathogenic properties.]. Bulletin de la Société de pathologie exotique et de ses filiales 53, 505–509.Google Scholar
Lesser, E. (1961). In vitro cultivation of Histomonas meleagridis free of demonstratable bacteria. Protozoology 8, 228–230.Google Scholar
Phillips, R. E., Boreham, P. F. and Shepherd, R. W. (1984). Cryopreservation of viable Giardia intestinalis trophozoites. Transactions of the Royal Society of Tropical Medicine and Hygiene 78, 604–606.Google Scholar
Robinson, G. L. (1968). Laboratory cultivation of some human parasitic amoebae. Journal of General Microbiology 53, 69–79.CrossRefGoogle ScholarPubMed
Robinson, G. L. and Ng, P. H. (1968). The size of Dientamoeba fragilis in culture. Transactions of the Royal Society of Tropical Medicine and Hygiene 62, 156–158.Google Scholar
Sawangjaroen, N., Luke, R. and Prociv, P. (1993). Diagnosis by faecal culture of Dientamoeba fragilis infections in Australian patients with diarrhoea. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 163–165.CrossRefGoogle ScholarPubMed
Stark, D., Beebe, N., Marriott, D., Ellis, J. and Harkness, J. (2005 a). Detection of Dientamoeba fragilis in fresh stool specimens using PCR. International Journal for Parasitology 35, 57–62.Google Scholar
Stark, D., Beebe, N., Marriott, D., Ellis, J. and Harkness, J. (2005 b). Prospective study of the prevalence, genotyping, and clinical relevance of Dientamoeba fragilis infections in an Australian population. Journal of Clinical Microbiology 43, 2718–2723.Google Scholar
Stark, D., Beebe, N., Marriott, D., Ellis, J. and Harkness, J. (2007). Dientamoeba fragilis as a cause of travelers' diarrhea: report of seven cases. Journal of Travel Medicine 14, 72–73.Google Scholar
Stark, D., Barratt, J., Ellis, J., Harkness, J. and Marriott, D. (2009). Repeated Dientamoeba fragilis infections: A case report of two families from Sydney, Australia. Infectious Disease Reports 1, 7–9.Google Scholar
Stark, D., Barratt, J., Roberts, T., Marriott, D., Harkness, J. and Ellis, J. (2010). Clinical and biological aspects of D. fragilis infections. A review of the clinical presentation of dientamoebiasis. American Journal of Tropical Medicine and Hygiene 82, 614–619.Google Scholar
Stark, D. J., Beebe, N., Marriott, D., Ellis, J. T. and Harkness, J. (2006). Dientamoebiasis: clinical importance and recent advances. Trends in Parasitology 22, 92–96.CrossRefGoogle ScholarPubMed
van Der Schee, C., van Belkum, A., Zwijgers, L., van Der Brugge, E., O'Neill, L., Luijendijk, E. A., van Rijsoort-Vos, T., van Der Meijden, W. I., Verbrugh, H. and Sluiters, H. J. (1999). Improved diagnosis of Trichomonas vaginalis infection by PCR using vaginal swabs and urine specimens compared to diagnosis by wet mount microscopy, culture, and fluorescent staining. Journal of Clinical Microbiology 37, 4127–4130.Google Scholar
Windsor, J. J., Clark, C. G. and Macfarlane, L. (2004). Molecular typing of Dientamoeba fragilis. British Journal of Biomedical Science 61, 153–155.Google Scholar
Windsor, J. J., Macfarlane, L., Hughes-Thapa, G., Jones, S. K. and Whiteside, T. M. (2003). Detection of Dientamoeba fragilis by culture. British Journal of Biomedical Science 60, 79–83.CrossRefGoogle ScholarPubMed
Zanoni, B., Garzaroli, C., Anselmi, S. and Rondinini, G. (1993). Modeling the growth of Enterococcus faecium in bologna sausage. Applied Environmental Microbiology 59, 3411–3417.Google Scholar