DOI QR코드

DOI QR Code

PCR Diagnosis of Entamoeba histolytica Cysts in Stool Samples

  • Moon, Joung-Ho (Division of Malaria and Parasitic Diseases, Korea National Institute of Health) ;
  • Cho, Shin-Hyeong (Division of Malaria and Parasitic Diseases, Korea National Institute of Health) ;
  • Yu, Jae-Ran (Kon-Kuk University College of Medicine, Environmental and Tropical Medicine) ;
  • Lee, Won-Ja (Division of Malaria and Parasitic Diseases, Korea National Institute of Health) ;
  • Cheun, Hyeng-Il (Division of Malaria and Parasitic Diseases, Korea National Institute of Health)
  • Received : 2010.06.11
  • Accepted : 2011.08.17
  • Published : 2011.09.30

Abstract

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

Keywords

References

  1. Haque R, Petri WA Jr. Diagnosis of amebiasis in Bangladesh. Arch Med Res 2006; 37: 273-276.
  2. World Health Organization. Amoebiasis. WHO Weekly Epidemiol Rec 1997; 72: 97-100.
  3. Gonzalez-Ruiz A, Haque R, Aguirre A, Castanon G, Hall A, Guhl F, Ruiz-Palacios G, Miles MA, Warhurst DC. Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. J Clin Pathol 1994; 47: 236-239. https://doi.org/10.1136/jcp.47.3.236
  4. Tannich E, Burchard GD. Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. J Clin Microbiol 1991; 29: 250-255.
  5. Britten D, Wilson SM, McNerney R, Moody AH, Chiodini PL, Ackers JP. An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces. J Clin Microbiol 1997; 35: 1108-1111.
  6. Troll H, Marti H, Weiss N. Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. J Clin Microbiol 1997; 35: 1701-1705.
  7. Intarapuk A, Kalambaheti T, Thammapalerd N, Mahannop P, Kaewsatien P, Bhumiratana A, Nityasuddhi D. Identification of Entamoeba histolytica and Entamoeba dispar by PCR assay of fecal specimens obtained from Thai/Myanmar border region. Southeast Asian J Trop Med Public Health 2009; 40: 425-434.
  8. Bhattacharya S, Bhattacharya A, Diamond LS, Soldo AT. Circular DNA of Entamoeba histolytica encodes ribosomal RNA. J Protozool 1989; 36: 455-458.
  9. Mirelman D, Nuchamowitz Y, Stolarsky T. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and Entamoeba dispar. J Clin Microbiol 1997; 35: 2405-2407.
  10. Garcia LS, Brewer TC, Bruckner DA. A comparison of the formalin-ether concentration and trichrome-stained smear methods for the recovery and identification of intestinal protozoa. Am J Med Technol 1979; 45: 932-935.
  11. Garcia LS, Shimizu R. Comparison of clinical results for the use of ethyl acetate and diethyl ether in the formalin-ether sedimentation technique performed on polyvinyl alcohol-preserved specimens. J Clin Microbiol 1981; 13: 709-713.
  12. Petri WA Jr, Haque R, Lyerly D, Vines RR. Estimating the impact of amebiasis on health. Parasitol Today 2000; 16: 320-321. https://doi.org/10.1016/S0169-4758(00)01730-0
  13. Zindrou S, Orozco E, Linder E, Tellez A, Bjorkman A. Specific detection of Entamoeba histolytica DNA by hemolysin gene targeted PCR. Acta Trop 2001; 78: 117-125. https://doi.org/10.1016/S0001-706X(00)00175-3
  14. Khairnar K, Parija SC. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples. BMC Microbiol 2007; 7: 47. https://doi.org/10.1186/1471-2180-7-47
  15. Haque R, Ali IK, Akther S, Petri WA Jr. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. J Clin Microbiol 1998; 36: 449-452.
  16. Lantz PG, Matsson M, Wadström T, Rådström P. Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase systems for sample preparation prior to PCR. J Microbiol Methods 1997; 28: 159-167. https://doi.org/10.1016/S0167-7012(97)00979-2
  17. Korea National Institute of Health. Water and Food-borne Diarrheal Disease-Surveillance in Korea. 2008, p 73 (in Korean).

Cited by

  1. Utilization of ELISA Using Thioredoxin Peroxidase-1 and Tandem Repeat Proteins for Diagnosis of Schistosoma japonicum Infection among Water Buffaloes vol.6, pp.8, 2011, https://doi.org/10.1371/journal.pntd.0001800
  2. Frequency of amoebiasis and other intestinal parasitoses in a settlement in Ilhéus City, State of Bahia, Brazil vol.47, pp.1, 2011, https://doi.org/10.1590/0037-8682-0078-2012
  3. Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay vol.9, pp.None, 2016, https://doi.org/10.1186/s13071-016-1418-4
  4. Laboratory Diagnosis of Parasites from the Gastrointestinal Tract vol.31, pp.1, 2018, https://doi.org/10.1128/cmr.00025-17