Molecular Characterization of E. Histolytica Strains Based on the Serine Rich Entamoeba Histolytica Protein (Srehp) And Chitinase Genes.

BACKGROUND: Even though E. histolytica is recognized as an effective pathogen, what determines the outcome of this infection is still not well understood. The present study was carried out to determine the genetic characteristics of E. histolytica isolates from two different regions in South Africa. METHOD: Diarrheal and non-diarrheal stool samples were collected from patients of all ages from Giyani and Pretoria. Different PCR protocols were used to identify E. histolytica and amplify the serine rich E. histolytica protein (SREHP) and chitinase genes. The proles obtained were compared among the different samples. RESULTS: Out of 111 stool samples collected, 51 were positive by either PCR or microscopy and 14 samples were positive by both methods. The serine- rich E. histolytica protein was amplied in 26 samples. Out of the 26 samples (19) different SREHP proles were obtained. SREHP #2 was obtained in 5 different samples, 4 from Pretoria and 1 from Giyani (2 diarrheal and 3 non-diarrheal). The chitinase gene was amplied from 51 samples and 22 different chitinase proles were obtained. Of all the proles, prole #4 was found in 6 different isolates, 5 from Giyani and 1 from Pretoria (3 symptomatic and 3 asymptomatic). However, prole # 18 was only found in formed stools from Giyani. CONCLUSIONS. The results obtained in this study have further conrmed the genetic heterogeneity of E. histolytica for the SREHP and chitinase genes which might have a signicant inuence in the outcome of amebic infection, depending on the genetic prole of the infecting strain.


Background
Entamoeba histolytica is an anaerobic parasite known as the most common pathogenic parasite in the genus entamoeba, and is the major cause of morbidity and mortality in both humans and animals [1]. It is estimated that 50 million people get invasive amebiasis every year due to E. histolytica [2,3]. The organism may colonize the host's bowel system for years without inducing clinical symptoms of the disease, in most cases, only 10% of the infected individuals develop symptomatic diseases and the remaining 90% do not show symptoms [4][5][6]. In symptomatic patients, the trophozoites may spread to extra-intestinal organs such as lungs, brain and liver, which results in liver abscess [7]. It remains puzzling why only a subset of infected individuals develops invasive diseases. Therefore, the factors that govern the transition from colonization to invasion remain to be answered.
Some studies suggests that this may be due to the differences in the pathogenic capability of the infecting strains [8], or due to the differences in the host immune response against the infection [9]. One of the possible explanations for this is that genetic subgroups exist within E. histolytica that give rise to infection with different outcomes [6]. However, the role of genetic diversity within E. histolytica to human disease is not clear. Another study has shown that the structural speci city of the genome might play an important role in the genetic variability of E. histolytica and possibly its strain related virulence [10]. Understanding the nature of amoebic virulence is important, several studies have shown that the genetic packs (4 0 C) and transported to the Parasitology laboratory at the University of Venda for further analysis.
Upon arrival at the laboratory, all the samples were observed under a light microscope for the presence of E. histolytica cysts or trophozoites, the samples were then aliquoted and stored at -80 0 C for further analysis.
Data collection.
At the time of sample collection, demographic as well as some clinical data were collected using a questionnaire. Information collected included origin, age, and sex. Information on the type of sample was also collected (loose, bloody, watery, or mucous).
Detection of E. histolytica cysts and trophozoites in fecal specimens by microscop y All fecal samples were screened for Entamoeba cysts by microscopy. A portion of each unpreserved stool specimen was placed on a glass slide and iodine was added to the stool smears and covered with a coverslip and examined under a light microscopy at 40x magni cation. The identi cation of E. histolytica was based on morphologic characteristics of the cysts and trophozoites. Fecal samples containing amebic cells were stored at -20ºC until DNA extraction was performed.
Extraction of total genomic DNA from stool samples.
Total genomic DNA of E. histolytica was puri ed from the stool specimens. The fecal samples were subjected to QIAaMP DNA stool mini kit (QIAGEN, Hilden, Germany). The extraction procedure was performed following the manufacturer's instructions. The DNA was kept frozen at -20ºc until needed for ampli cation analysis [14].
Identi cation of Entamoeba species by PCR.
The detection of E. histolytica was performed using genus-speci c primers (E1 and E2) and E. histolytica (EH1 and EH2) primers (15) ( Table 1). In the initial PCR, the ampli cation was carried out in a total of 25µl reaction volume containing 12.5 µl of dreamtaq, 0.25µl of BSA, 0.6µl of each primer and 6.05µl deionized water. An amount of 5µl stool DNA was mixed with 20µl of master mix to a nal volume of 25 µl. The cycling conditions were as follows: initial denaturation at 96ºC for 2 minutes, followed by 35 cycles of PCR performed with denaturation at 92ºC for 60 seconds, annealing at 56ºC for 60 seconds and extension at 72ºC for 90 minutes and nal extension 72ºC for 7 minutes. In the second round, the PCR product was used as a template with the same cycling conditions, except the annealing temperature which was changed to 60ºC (15). The PCR products were separated by electrophoresis in 1.5% agarose gel at 100 V for 45 min in Tris-acetate buffer and visualized by UV-transilluminator.
Genotyping of the Serine rich E. histolytica protein gene by PCR.
All the samples positive by PCR were used to amplify the serine rich protein of E. histolytica. The ampli cation was performed in 25 µl reaction mixture containing 12.5µl of dreamtaq, 0.6µl of each primer (SREHP1 and SREHP2) ( Table 1), 0.25µl of BSA, 6.05 µl of nuclease free water and 5µl of genomic DNA. The cycling conditions were as follows: initial denaturation at 94 0 C for 5 minutes followed by 35 thermal cycles of 94 0 C for 1 minute, 48 0 C for 1 minute, and 72 0 C for 1 minute, followed by a nal extension at 72 0 C for 5 minutes.
Genotyping of E. histolytica based on the chitinase gene.
The samples that were positive by PCR were selected for the ampli cation of the chitinase gene. PCR was performed in 25 µl reaction mixture containing 12.5µl of dreamtaq, 0.6µl of each primer (CEH1 and CEH2) ( Table 1), 0.25µl of BSA, 6.05 µl of nuclease free water and 5µl of genomic DNA. The cycling conditions were as follows: initial denaturation at 94 0 C for 5 minutes followed by 35 thermal cycles of 94 0 C for 1 minute, 48 0 C for 1 minute, and 72 0 C for 1 minute, followed by a nal extension at 72 0 C for 5 minutes. Assuming that, the data followed a normal distribution, comparison of proportions and statistical signi cance was tested using the Chi-square test. The genotype associations between the positive and negative groups were analyzed by using chi-square (χ 2 ) analysis. A p-value of < 0.05 was considered statistically signi cant.

Results
Demographic data of the study population.
A total of one hundred and eleven (111) stool samples were tested in this study, of which 84 (75.7%) were from Giyani and 27 (24.3%) were from Pretoria. Of all the samples collected 50 (45.0%) were males and 61 (55.0%) were females. Most of the patients were aged between 0 to 25 (19.8%), followed by 6 (5.4%) patients aged 26 to 45, only 2 patients were aged 46 to 65 (1.8%) and the age of about 81 (73.0%) was unknown. A total 28 (25.2%) were formed, 69 (62.2%) loose and 14 (12.6%) were watery stools. (Table 2). positive by either method (microscopy or PCR) and 14 (12.6%) samples were positive by both methods (microscopy and PCR). Figure 1 shows the samples positive for Entamoeba ampli ed by EH1 and EH2 primers with the product size of 450bp.
Genetic diversity of Entamoeba histolytica based on the SREHP pro les.
Out of the 111 stool samples tested, the SREHP1 and SREHP2 primers speci cally ampli ed the serine- Genetic diversity of E. histolytica in stool samples from Giyani and Pretoria.
Out of the 65 samples positive for E. histolytica by either PCR or microscopy, the SREHP successfully ampli ed 26 samples. The SREHP PCR reaction gave different banding patterns (Tables 3 and 4). Table 4 Clearly shows the distribution of the SREHP genetic pro les in the study population. The band sizes varied between 210bp and 1600bp, the bands-300, 500, 400 and 600 were seen in most of the samples. This ampli cation revealed 19 SREHP pro les out of the 26 samples positive for the SREHP. Twenty-two (22) isolates were from Giyani and 4 from Pretoria. Pro le #2 was observed in 3 different isolates, interestingly, these isolates were from the same area (Giyani), these patients may possibly have been infected with the same strain, however, according to the observations of this study, this pro le is not related to diarrhea, since it was found in patients with formed stools. Another pro le (#18) was found mostly in isolates from Pretoria and in one isolate from Giyani, two of the isolates were symptomatic and the other 4 were asymptomatic. Therefore, this pro le, is more distributed in Pretoria than Giyani and it was related to diarrhea since it was found in samples with diarrhea.    Out of the stool samples tested, the CEH1 and CEH2 (Chitinase) primers speci cally ampli ed the chitinase and band sizes varying between 100 and 1,200bp ( Fig. 3A and B) were obtained with other additional amplicons of 200, 300, 400, 600 and 1000bp.
Out of the 65 samples positive for E. histolytica by both PCR and microscopy, the chitinase successfully ampli ed 59 samples out of the 65 samples tested. The chitinase PCR reaction gave different banding patterns as represented in Table 5 and 6 below. Table 7 Clearly shows the distribution of the chitinase genetic pro les in the study population. This ampli cation revealed 22 SREHP pro les out of the 59 samples positive for the chitinase. Forty-seven (47) isolates were from Giyani and 12 from Pretoria. Pro le #4 was observed in 6 different isolates, 5 of these isolates were from the same area (Giyani), and 1 from Pretoria. The pro le is distributed in both the areas, but more diverse in Giyani.

Discussion
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 ndings have been reported [14].
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 con rm 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 speci cally 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 ampli ed 19 isolates out of 65 E. histolytica positive samples. Some of the isolates successfully ampli ed for the 700bp SREHP gene, while the other isolates ampli ed 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 [18] and in the Thai/Myanmar border region [14]. However, our study showed a high number SREHP pro les compared to these studies. These pro les were also different from those obtained previously [13,19,20]. The obtained pro les were compared with the stool type, and it was found that three asymptomatic patients from Giyani were infected with the same pro le (#2), suggesting that this pro le 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 pro le (#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 pro le was more prevalent in Pretoria than in Giyani, with high distribution in males than females. These nding corroborated previous ndings 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 [21]. Similar results were obtained in United Arab, where the same pro le was shown in four isolates [18].
Clustering of pro les 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 pro le, which is pro le 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 pro le number were low compared to the pro le 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 pro le. It is also possible to use the pro les 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 rst time the diversity of this parasite based on the chitinase gene in these two areas.
The chitinase gene successfully ampli ed 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 [14]. The high number of the chitinase pro les observed in this study is an indicative that E. histolytica is prevalent in the study population. High diversity of the chitinase pro les were obtained mostly in Giyani than Pretoria.
Interestingly, the results of this study showed that ve asymptomatic patients (formed stools) from Giyani were infected with the same pro le (#18), of these ve patients 3 were males and 2 were females.
This pro le was not associated with diarrhea since it was obtained only in asymptomatic patients, further con rming that the presentation of amebic infection depends on the pro le of the infecting organisms.
Another interesting nding of this study is the occurrence of the same pro le 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 nger printing individual isolates [22]. The ndings of the same pro les affecting individuals in the same area was also reported in United Arab Emirates [18]. Clustering of pro les was observed in both study sites, indicating that some patients were infected with more than one pro le. Concerning the geographical distribution, the most occurring pro les, 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 signi cance and if appropriate care is not taken, it could result in epidemic situation. The nding 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 con rmed the genetic heterogeneity of E. histolytica for the SREHP and chitinase genes which might have a signi cant in uence in the outcome of amebic infection, depending on the genetic pro le of the infecting strain. Therefore, more studies are needed to understand in depth the role of these pro les in the outcome of amebic infection.