Molecular Diagnosis and Genotyping of Entamoeba spp Among Infected Egyptians

Abstract

An accurate diagnosis of intestinal amoebiasis is of a great clinical importance further more to detect and distinguish E. histolytica and E. dispar in stool samples for determining the true prevalence of pathogenic E. histolytica in the community. The present study used two molecular techniques for the discrimination of the pathogenic amoeba species, the first a nested multiplex PCR by species-specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii in the stool samples, the second is q-PCR using specific probes for both E. histolytica and E. dispar. In our study the nested multiplex PCR was negative in 157 out of 194 stool specimens that were positive for E. histolytica/E. dispar/E. moshkovskii complex trophozoites/cysts by microscopy. The negative PCR amplification result may be caused by the presence of potential PCR inhibitory materials in examined stool samples. On the other hand a second reason for the limited PCR positivity may be due to the presence of other Entamoeba species such as E. coli, E. hartmanni or other similarly looking Entamoeba species . The results of q-PCR detection level were less than that of the conventional PCR amplification with a total number of negative samples (164 out of 194 stool specimens). This result was expected based on the fact that the nested PCR amplification used in nested-multiplex PCR method has possibility to overcome the PCR inhibitory action in stool samples as well as the ability to detect lower parasite counts in some of the samples. It is well documented that clinical specimens such as stool often contain PCR inhibitors even after purification steps, despite the use of a specific DNA extraction kit from stool (Qiagen- QIAampDNA Stool Kit). In order to test the presence of inhibitory contaminants in the extracted DNA in our study, seeding experiment was performed for 20 PCR negative samples that were selected randomly where an aliquot of each sample was seeded with Entamoeba histolytica positive control then the same protocol of nested multiplex PCR was done for the original negative sample besides the seeded one; unexpectedly the result obtained was that the seeded samples were positive in all the examined samples (20/194; 12%) which diminishes the potential effect of inhibitors in those samples. The result of seeding experiment led us to believe that the exact prevalence of Entamoeba histolytica in the examined samples may be much lower than reported by the microscopic examination so we recommend to include the nested PCR amplification technique to confirm the diagnosis of E. histolytica infection. So, we conclude that in the absence of tests such as the nested multiplex PCR or q-PCR for specific detection of E. histolytica, most of the suspected infections would have been over estimated infection and treated unnecessarily with anti-amoebic drugs. We therefore, recommend the use of the nested PCR amplification to confirm the diagnosis of E. histolytica infection.