The main objective of the present study was to determine the molecular epidemiology of E. histolytica in Giyani and Pretoria and to elucidate the impact of parasite genetics on amebic infection. As a screening method, microscopy was used to examine all the samples for the presence of Entamoeba cyst [16, 17], and of the 111 samples screened, 65 samples were positive. The main aim of the screening was to check the presence of the entamoeba cyst in the samples. In fact, people in our study communities are often infected with different types of parasitic organisms not only entamoeba. Similar microscopy findings have been reported .
However, the limitation about microscopy is that it is not reliable and cannot be used to differentiate species. Therefore polymerase chain reaction is mostly used as the gold method to further confirm the microscopy results as well as to differentiate between species [16, 17]. Entamoeba species are undistinguishable by microscopy, for example, the morphology of Entamoeba histolytica and dispar is the same and microscopy cannot differentiate these two organisms, hence the present study used the PCR based method to specifically detect E. histolytica and to avoid possible false positive results. The PCR results of this study showed that E. histolytica is prevalent in the study population.
All the samples that were positive by both methods (Microscopy and PCR), were used to study the genetic diversity of Entamoeba histolytica based on the SREHP and chitinase genes. The SREHP gene successfully amplified 19 isolates out of 65 E. histolytica positive samples. Some of the isolates successfully amplified for the 700bp SREHP gene, while the other isolates amplified additional amplicons of approximately 300, 400,500, 600, 800 and 1200bp. The result obtained in this study are not very far from the ones obtained in United Arab  and in the Thai/Myanmar border region . However, our study showed a high number SREHP profiles compared to these studies. These profiles were also different from those obtained previously [13, 19, 20]. The obtained profiles were compared with the stool type, and it was found that three asymptomatic patients from Giyani were infected with the same profile (#2), suggesting that this profile might be associated with the asymptomatic carriage of the parasite and may not be the cause of diarrheal infections in the study population since it was only obtained in asymptomatic patients. Another profile (#5) was detected in both the locations (Giyani and Pretoria) from symptomatic and asymptomatic patients, one symptomatic and asymptomatic (Giyani), two asymptomatic and one symptomatic (Pretoria). The results analysis shows that this profile was more prevalent in Pretoria than in Giyani, with high distribution in males than females. These finding corroborated previous findings in a study carried out in Japan, which concluded that all isolates from different mental institutions were derived from a single source of E. histolytica . Similar results were obtained in United Arab, where the same profile was shown in four isolates .
Clustering of profiles from the same region (Giyani) was also obtained in the present study and no clustering was obtained from Pretoria isolates. The patients from Pretoria were mostly infected with one profile, which is profile no 2. The results obtained in the present study indicate that there is a possibility of the existence of the same strain infecting individuals from the same region. Due to the small sample size used in this study (65) the profile number were low compared to the profile numbers obtained by previous studies [13, 19]. The results of this study suggest that the SREHP might have a role in the outcome of amebic infection depending on the infecting profile. It is also possible to use the profiles for tracing the sources of contamination in a community.
The chitinase gene was also used to study the genetic diversity and molecular epidemiology of E. histolytica in the study population. In general, little is known about the extent of intestinal parasitic infections in Limpopo and Gauteng province, especially in Giyani and Pretoria, and no reports have been published on the genetic diversity of E. histolytica based on the chitinase gene in Limpopo and Gauteng. Therefore, we are reporting for the first time the diversity of this parasite based on the chitinase gene in these two areas.
The chitinase gene successfully amplified 22 isolates from 65 positive isolates included in the study. The target bands of 500 and 1200bp were obtained in a few isolates, with some additional amplicons of approximately 200, 300, 400, 500, 600, 800, 900 and 1000bp. The obtained bands in this study are similar with the bands obtained by other studies . The high number of the chitinase profiles observed in this study is an indicative that E. histolytica is prevalent in the study population. High diversity of the chitinase profiles were obtained mostly in Giyani than Pretoria.
Interestingly, the results of this study showed that five asymptomatic patients (formed stools) from Giyani were infected with the same profile (#18), of these five patients 3 were males and 2 were females. This profile was not associated with diarrhea since it was obtained only in asymptomatic patients, further confirming that the presentation of amebic infection depends on the profile of the infecting organisms. Another interesting finding of this study is the occurrence of the same profile in different isolates of different gender from different geographic areas. Six different isolates, one asymptomatic isolate from Pretoria, two symptomatic (watery stool) and three asymptomatic (formed) isolates from Giyani.
The result of this study reinforces that the polymorphic loci (chitinase) could serve as a tool to determine the diversity of E. histolytica and to finger printing individual isolates . The findings of the same profiles affecting individuals in the same area was also reported in United Arab Emirates . Clustering of profiles was observed in both study sites, indicating that some patients were infected with more than one profile. Concerning the geographical distribution, the most occurring profiles, 18 and 4, were more distributed in Giyani compared to Pretoria.
In conclusion, the prevalence of E. histolytica in this study is of public health significance and if appropriate care is not taken, it could result in epidemic situation. The finding that this parasite affects young and old people opens a new dimension to understand the source of infection in the African setting. It is therefore recommended that the populations living in the study areas be educated properly on hygiene more especially on personal hygiene. The results obtained in this study have further confirmed the genetic heterogeneity of E. histolytica for the SREHP and chitinase genes which might have a significant influence in the outcome of amebic infection, depending on the genetic profile of the infecting strain. Therefore, more studies are needed to understand in depth the role of these profiles in the outcome of amebic infection.