To our knowledge, no study has investigated the prevalence and genetic diversity of Entamoeba species among children under 5 years in the Vhembe District, Limpopo, South Africa. In the present study, we have shown for the first time that the infection rate with Entamoeba species is 8% using molecular tools. Compared to PCR and sequencing, vegetative and/or cyst forms were found in 24.3% (130/534) by direct wet mount microscopy as Entamoeba cysts highlighting the trouble many laboratory technicians face in identifying and differentiating morphologically similar cysts and/or trophozoites of Entamoeba genus such as E. histolytica, and other uni-nucleated cysts including immature cysts of E. histolytica [14–16]. To investigate the genetic diversity of Entamoeba species in the study population, twenty samples were Sanger sequenced resulting in four Entamoeba species, E. coli (60%), E. polecki (33.3%), and E. muris and E. hartmanni (8.3%). Although the identified species might be less pathogenic in the case of a single infection, coinfections with other pathogens including bacterial, fungal and viral infection may augment the severity of the disease [17].
A large genetic distance exists between the un-, tetra-, and octanucleated cyst forming Entamoeba species as described by Silberman et al (1999) [18]. As presented in the phylogenetic tree, all Entamoeba polecki isolates clustered with E. polecki (AB845671) and E. coli (AB845674) reference sequence. On the other hand, they are widely separated from E. coli (AB444953), E. muris (FN396613) and the tetra-nucleated cyst forming E. histolytica and E. moshkovskii. Interestingly, possibly two variants of E. polecki are clearly distinguishable in the phylogenetic tree (Fig. 2). Isolate 46 is further away in the tree from the other three E. polecki samples suggesting that possibly two variants of E. polecki are identified in the present study. It has previously been proposed that variants of E. polecki exist since there is no host specificity and no known difference except for small amounts of sequence divergence [6, 19].
Sequencing revealed more E. coli positive samples clustered in two distant parts of the phylogenetic tree suggesting that they may be different species/types/stains of E. coli. Figure 2 shows that isolates 22, 24 and 27 clustered with E. coli (AB845674) reference sequence whereas isolates 28, 5 and 16 all clustered together and were widely separated from the other E. coli samples and reference sequence. Stensvold et al. (2011a) reported that E. coli samples from humans group into two clusters, which have been named subtypes 1 and 2 (ST1 and ST2) with ST1 widespread among humans [19]. Whether this variation is a result of the possible source of infection, human or animal origin, remains to be established. Sample 3, Entamoeba hartmanni, s tetra-nucleate cyst producing Entamoeba visibly branches out separately in the phylogenetic tree away from the other tetra-nucleated Entamoeba species (Fig. 2).
Studies done by Stensvold et al., (2011b) demonstrated human infections with E. polecki, in which a novel 18S rRNA gene sequence was identified in a species of Sulawesi macaque [20]. However, in many cases, the local prevalence of these species may vary significantly based on the different geographical regions. A study done in South Africa reported that E. polecki (90%) were more prevalent as compared to E. coli with (10%) [21]. Furthermore, another study was reported in India which reported about 49.5% of E. polecki and only 7.4% with E. coli and E. moshkovskii [21]. Entamoeba polecki is mostly isolated from domestic animals especially pigs [22]. Therefore, looking at the study population setting we can also suggest that the infection might be transmitted from pigs to water than humans. Only one sample (#23) returned 100% identity with E. muris (FN396613) and E. coli (AB444953) suggesting the sample could either be infected from an animal or a human source. Both E. muris and E. coli are producers of octa-nucleated cysts and both look identical morphologically under the microscope.