Abstract
Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T m) was 73°C and 70°C, respectively. For E. hartmanni, the T m was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.
Highlights
The genus Entamoeba contains many species, six of which are found in the human intestinal tract: Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli, Entamoeba hartmanni, and Entamoeba polecki
Historical epidemiological data concerning E. histolytica infections can lead to overestimates because they were obtained before the formal separation of this parasite into two morphologically identical species: the potentially pathogenic E. histolytica and the nonpathogenic E. dispar [4]
The positive controls used in the SYBR Green multiplex real-time Polymerase Chain Reaction (PCR) were genomic DNA from the standard strain of E. histolytica HM1-IMSS and E. dispar P2
Summary
The genus Entamoeba contains many species, six of which are found in the human intestinal tract: Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli, Entamoeba hartmanni, and Entamoeba polecki. Of these species, only E. histolytica is associated with pathological injuries; the others are considered to be nonpathogenic species [1]. E. histolytica is responsible for approximately 50 million cases of amoebiasis worldwide each year, resulting in up to 100,000 deaths [2, 3] Because of these characteristics, E. histolytica is globally considered to be a leading parasitic cause of human mortality [1]. It is estimated that 90% of diagnosed infections are caused by E. dispar [5]
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