Acta Parasitologica

, Volume 58, Issue 2, pp 132–138 | Cite as

PCR amplification and DNA sequence analysis of parasitic intestinal protozoa in specimens stained with Chlorazol Black E

  • Norihito Morimoto
  • Masataka Korenaga
  • Yoshie Nishida
  • Hiroaki Takeuchi
  • Yoshitaka Kumon
  • Tetsuro Sugiura
Article
  • 213 Downloads

Abstract

Chlorazol Black E (CBE) stain has been used for the detection and identification of intestinal parasitic protozoa. In recent years, genotyping of protozoa has been performed to examine pathogenicity and for epidemiologic analysis. In this study, protozoan DNA was amplified from preserved human fecal specimens stained with CBE that were positive for Giardia intestinalis (syn. G. lamblia and G. duodenalis), Chilomastix mesnili, Pentatrichomonas hominis, and Entamoeba histolytica. DNA was amplified from 11 of the 12 (91.6%) samples examined. DNA from CBE-stained smears of G. intestinalis, E. histolytica, and P. hominis was amplified, whereas any amplification product could not be obtained from one of three smears of C. mesnili. Storage term and protozoan number had no association with results of PCR amplification. In genotyping of G. intestinalis, four out of six (66.7%) samples were of genotype AI, while the remaining two (33.3%) samples were of genotype B. The amplified DNA sequences showed high similarity (>99%) with that of G. intestinalis in the GenBank database. These results suggest that DNA remains stable in CBE-stained smears for long term. The present study demonstrates that nuclear extracts from specimens stained with CBE can be amplified by PCR and suggests that specimens stored for extended periods could be applied to genetic and prospective epidemiologic analyses.

Keywords

Chlorazol Black E intestinal protozoa Giardia intestinalis genotyping Thermococcus kodakaraensis KOD1 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abe N., Kimata I., Tokoro M. 2005. Genotyping of Giardia isolates from humans in Japan using the small subunit ribosomal RNA and glutamate dehydrogenase gene sequences. Japanese Journal of Infectious Diseases, 58, 57–58.PubMedGoogle Scholar
  2. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search Tool. 1990. Journal of Molecular Biology, 215, 403–410. DOI: 10.1016/s0022-2836(05)80360-2.PubMedGoogle Scholar
  3. Amar C., Pedraza-Díaz S., McLauchlin J. 2001. Extraction and genotyping of Cryptosporidium parvum DNA from fecal smears on glass slides stained conventionally for direct microscope examination. Journal of Clinical Microbiology, 39, 401–403. DOI: 10.1128/JCM.39.1.401-403.2001.PubMedCrossRefGoogle Scholar
  4. Amar C.F., Dear P.H., Pedraza-Diaz S., Looker N., Linnane E., McLauchlin J. 2002. Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. Journal of Clinical Microbiology, 40, 446–452. DOI: 10.1128/JCM.40.2.446-452.2002.PubMedCrossRefGoogle Scholar
  5. Bhattacharya D., Haque R., Singh U. 2005. Coding and noncoding genomic regions of Entamoeba histolytica have significantly different rates of sequence polymorphisms: implications for epidemiological studies. Journal of Clinical Microbiology, 43, 4815–4819. DOI: 10.1128/JCM.43.9.4815-4819.2005.PubMedCrossRefGoogle Scholar
  6. Gleason N.N., Healy G.R. 1965. Modification and evaluation of Kohn’s one-step staining technic for intestinal protozoa in feces or tissue. American Journal of Clinical Pathology, 43, 494–496.Google Scholar
  7. Hashimoto H., Nishioka M., Fujiwara S., Takagi M., Imanaka T., Inoue T., Kai Y. 2001. Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. Journal of Molecular Biology, 306, 469–477. DOI: 10.1006/jmbi.2000.4403.PubMedCrossRefGoogle Scholar
  8. Hayashida M., Iwao-Koizumi K., Murata S., Kinoshita K. 2009. Singletube genotyping from a human hair root by direct PCR. Analytical Sciences, 25, 1487. DOI: 10.2116/analsci.25.1487.PubMedCrossRefGoogle Scholar
  9. Kim Y.A., Kim H.Y., Cho S.H., Cheun H.I., Yu J.R., Lee S.E. 2010. PCR detection and molecular characterization of Pentatrichomonas hominis from feces of dogs with diarrhea in the Republic of Korea. Korean Journal of Parasitology, 48, 9–13. DOI: 10.3347/kjp.2010.48.1.9.PubMedCrossRefGoogle Scholar
  10. Kohn J. 1960. A one stage permanent staining method for faecal protozoa. Dapim Refuiim 19: IV–V.Google Scholar
  11. Lalle M. 2010. Giardiasis in the post genomic era: treatment, drug resistance and novel therapeutic perspectives. Infectious Disorders Drug Targets, 10, 283–294. DOI: 10.2174/187152610791591610.PubMedCrossRefGoogle Scholar
  12. Matsubayashi M., Kimata I., Abe N. 2005. Identification of genotypes of Giardia intestinalis isolates from a human and calf in Japan. Journal of Veterinary Medical Science, 67, 337–340. DOI: 10.1292/jvms.67.337.PubMedCrossRefGoogle Scholar
  13. Molina N., Polverino D., Minvielle M., Basualdo J. 2007. PCR amplification of triosephosphate isomerase gene of Giardia lam blia in formalin-fixed feces. Revista Latinoamericana de Microbiología, 49, 6–11.PubMedGoogle Scholar
  14. Ramos F., Zurabian R., Morán P., Ramiro M., Gómez A., Clark C.G., Melendro E.I., García G., Ximénez C. 1999. The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene, 93, 335–336. DOI: 10.1016/S0035-9203(99)90045-7.PubMedCrossRefGoogle Scholar
  15. Tachibana H., Kobayashi S., Takekoshi M., Ihara S. 1991. Distinguishing pathogenic isolates of Entamoeba histolytica by polymerase chain reaction. Journal of Infectious Diseases, 164, 825–826. DOI: 10.1093/infdis/164.4.825.PubMedCrossRefGoogle Scholar
  16. Takagi M., Nishioka M., Kakihara H., Kitabayashi M., Inoue H., Kawakami B., Oka M., Imanaka T. 1997. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Applied and Environmental Microbiology, 63, 4504–4510.PubMedGoogle Scholar
  17. Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882. DOI: 10.1093/nar/25.24.4876.PubMedCrossRefGoogle Scholar
  18. Traub R.J., Monis P.T., Robertson I., Irwin P., Mencke N., Thompson R.C. 2004. Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology, 128, 253–262. DOI: 10.1017/S0031182003004505.PubMedCrossRefGoogle Scholar
  19. Troll H., Marti H., Weiss N. 1997. Simple Differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. Journal of Clinical Microbiology, 35, 1701–1705.PubMedGoogle Scholar
  20. Van der Giessen J.W., de Vries A., Roos M., Wielinga P., Kortbeek L.M., Mank T.G. 2006. Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates. International Journal for Parasitology, 36, 849–858. DOI: 10.1016/j.ijpara.2006.03.001.PubMedCrossRefGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2013

Authors and Affiliations

  • Norihito Morimoto
    • 1
  • Masataka Korenaga
    • 2
  • Yoshie Nishida
    • 1
    • 3
  • Hiroaki Takeuchi
    • 3
  • Yoshitaka Kumon
    • 3
  • Tetsuro Sugiura
    • 1
    • 3
  1. 1.Clinical Laboratory, Kochi Medical School HospitalKochi UniversityKohasu, Oko-cho, Nankoku, KochiJapan
  2. 2.ParasitologyKochi UniversityKohasu, Oko-cho, Nankoku, KochiJapan
  3. 3.Clinical Laboratory Medicine, Kochi Medical SchoolKochi UniversityKohasu, Oko-cho, Nankoku, KochiJapan

Personalised recommendations