• 1.

    Gonzalez-Ruiz A, Haque R, Aguirre A, Castanon G, Hall A, Guhl F, Ruiz-Palacios G, Miles MA, Warhurst DC, 1994. Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. J Clin Pathol 47: 236239.

    • Search Google Scholar
    • Export Citation
  • 2.

    Haque R, Ali IK, Clark CG, Petri WA Jr, 1998. A case report of Entamoeba moshkovskii infection in a Bangladeshi child. Parasitol Int 47: 201202.

  • 3.

    Ali IK, Hossain MB, Roy S, Ayeh-Kumi PF, Petri WA Jr, Haque R, Clark CG, 2003. Entamoeba moshkovskii infections in children, Bangladesh. Emerg Infect Dis 9: 580584.

    • Search Google Scholar
    • Export Citation
  • 4.

    Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J, 2007. PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia. J Clin Microbiol 45: 10351037.

    • Search Google Scholar
    • Export Citation
  • 5.

    Tachibana H, Kobayashi S, Takekoshi M, Ihara S, 1991. Distinguishing pathogenic isolates of Entamoeba histolytica by polymerase chain reaction. J Infect Dis 164: 825826.

    • Search Google Scholar
    • Export Citation
  • 6.

    Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. J Clin Microbiol 35: 24052407.

    • Search Google Scholar
    • Export Citation
  • 7.

    Haque R, Ali IK, Akther S, Petri WA Jr, 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. J Clin Microbiol 36: 449452.

    • Search Google Scholar
    • Export Citation
  • 8.

    Evangelopoulos A, Spanakos G, Patsoula E, Vakalis N, Legakis N, 2000. A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces. Ann Trop Med Parasitol 94: 233240.

    • Search Google Scholar
    • Export Citation
  • 9.

    Nunez YO, Fernandez MA, Torres-Nunez D, Silva JA, Montano I, Maestre JL, Fonte L, 2001. Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples. Am J Trop Med Hyg 64: 293297.

    • Search Google Scholar
    • Export Citation
  • 10.

    World Health Organization, 1997. Amebiasis. WHO Wkly Epidemiol Rec 72: 97100.

  • 11.

    Clark CG, Diamond LS, 1991. The Laredo strain and other Entamoeba histolytica-like amoebae are Entamoeba moshkovskii. Mol Biochem Parasitol 46: 1118.

    • Search Google Scholar
    • Export Citation
  • 12.

    Hamzah Z, Petmitr S, Mungthin M, Leelayoova S, Chavalitshewinkoon-Petmitr P, 2006. Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay. J Clin Microbiol 44: 31963200.

    • Search Google Scholar
    • Export Citation
  • 13.

    Khairnar K, Parija SC, 2007. 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 7: 47.

    • Search Google Scholar
    • Export Citation
  • 14.

    Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP, 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22: 130138.

    • Search Google Scholar
    • Export Citation
  • 15.

    Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ, 1997. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22: 176181.

    • Search Google Scholar
    • Export Citation
  • 16.

    Blessmann J, Buss H, Nu PA, Dinh BT, Ngo QT, Van AL, Alla MD, Jackson TF, Ravdin JI, Tannich E, 2002. Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. J Clin Microbiol 40: 44134417.

    • Search Google Scholar
    • Export Citation
  • 17.

    Qvarnstrom Y, James C, Xayavong M, Holloway BP, Visvesvara GS, Sriram R, Da Silva AJ, 2005. Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis. J Clin Microbiol 43: 54915497.

    • Search Google Scholar
    • Export Citation
  • 18.

    Verweij JJ, Polderman AM, Clark GC, 2001. Genetic variation among human isolates of uninucleated cyst-producing Entamoeba species. J Clin Microbiol 39: 16441646.

    • Search Google Scholar
    • Export Citation
  • 19.

    Roy S, Kabir M, Mondal D, Petri WA Jr, Haque R, 2005. Real-time PCR assay for the diagnosis of Entamoeba histolytica infection. J Clin Microbiol 43: 21682172.

    • Search Google Scholar
    • Export Citation
  • 20.

    Singh A, Houpt E, Petri WA, 2009. Rapid diagnosis of intestinal parasitic protozoa, with a focus on Entamoeba histolytica. Interdiscip Perspect Infect Dis 547090. Epub 2009 Jun.

    • Search Google Scholar
    • Export Citation
  • 21.

    Verweij JJ, Vermeer J, Brienen EA, Blotkamp C, Laeijendecker D, van Lieshout L, Polderman AM, 2003. Entamoeba histolytica infections in captive primates. Parasitol Res 90: 100103.

    • Search Google Scholar
    • Export Citation
  • 22.

    Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EA, van Rooyen MA, van Lieshout L, Polderman AM, 2004. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42: 12201223.

    • Search Google Scholar
    • Export Citation
  • 23.

    Leiva B, Lebbad M, Winiecka-Krusnell J, Altamirano I, Tellez A, Linder E, 2006. Overdiagnosis of Entamoeba histolytica and Entamoeba dispar in Nicaragua: a microscopic, triage parasite panel and PCR study. Arch Med Res 37: 529534.

    • Search Google Scholar
    • Export Citation
  • 24.

    Pillai DR, Keystone JS, Sheppard DC, MacLean JD, MacPherson DW, Kain KC, 1999. Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity. Clin Infect Dis 29: 13151318.

    • Search Google Scholar
    • Export Citation
  • 25.

    Petri WA Jr, Haque R, Lyerly D, Vines RR, 2000. Estimating the impact of amebiasis on health. Parasitol Today 16: 320321.

 
 
 

 

 

 

 

 

 

Development of Multiplex Real-Time Polymerase Chain Reaction for Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in Clinical Specimens

View More View Less
  • Department of Protozoology, Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand

Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.

Author Notes

*Address correspondence to Porntip Chavalitshewinkoon-Petmitr, Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Bangkok 10400 Thailand. E-mail: tmppm@mahidol.ac.th

Financial support: This research grant was supported by Mahidol University, Bangkok, Thailand.

Authors' address: Zulhainan Hamzah, Ipoh Public Health Laboratory, Ministry of Health Malaysia, Perak, Malaysia, E-mail: zulhainan@hotmail.com. Songsak Petmitr, Department of Tropical Nutrition and Food Science, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mail: tmspm@mahidol.ac.th. Mathirut Mungthin and Saovanee Leelayoova, Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand, E-mails: mathirut@hotmail.com and s_leelayoova@scientist.com. Porntip Chavalitshewinkoon-Petmitr, Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mail: tmppm@mahidol.ac.th.

Save