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