The present study reported an overall prevalence of Entamoeba species as determined by microscopy as 17.6% (75/426). In Malaysia, previous studies have also reported high prevalence of Entamoeba infection with prevalence rates ranging from 9.4% to 21.0% among rural communities [23–26, 28]. In contrast, the most recent study among rural communities in Malaysia demonstrated 10.2% of the participants were infected with Entamoeba species . The high prevalence of Entamoeba infection may be due to the transmission and pathogenesis as well as other risk factors which favor the persistence of this infection. Given its faecal-oral route, habits related to eating, defecation, personal hygiene, cleanliness and level of education may have an impact on the prevalence rates. The findings of this study confirmed a trend of high risk of infection with Entamoeba species among the rural population as shown by other local studies [21–23, 25], where prevailing poverty, poor socioeconomic condition, low standards of sanitation and hygiene and lack of education attainment may contribute to high prevalence of Entamoeba infection.
Additionally, the use of untreated river water as a source for household needs could also lead to the increase in the transmission of Entamoeba infection. A study in Thailand conducted to determine the occurrence of Entamoeba species in water samples using molecular techniques has indicated that water is a possible source for transmission of Entamoeba to humans . From our personal observation, the majority of the households have no toilet facilities. They often defecate indiscriminately in the bushes and nearby river at the back of their houses. These water sources may be highly polluted especially in rainy seasons, by rain runoff contaminated with cysts of parasites from human faeces. Another possible source of infection could also be from the consumption of water contaminated with Entamoeba cysts from faeces of infected wild or domestic animals as they may come to the river bank to drink and at the same time defecate in or near the river. This situation is further aggravated as the drinking of unboiled water is a common practice among this community. Therefore, these communities should be given health education and made aware of the danger of drinking unboiled or improperly boiled untreated water.
Our molecular technique showed that E. histolytica (75.0%) was found to be the most common species detected in this study, followed by E. dispar (30.8%) and E. moshkovskii (5.8%). Similarly, the only available species-specific study of Entamoeba species conducted in Malaysia found that E. histolytica (13.2%) was more prevalent compared to E. dispar (5.6%) . Interestingly, the high prevalence of E. histolytica in the present study was in contrast to the worldwide distribution of Entamoeba species, which indicated that E. dispar is perhaps 10 times more common than E. histolytica[1, 2, 30], however, the local prevalence may vary significantly, thus necessitating the assessment of prevalence in different geographical regions. Similar observation also reported that 70.8% of patients were infected with E. dispar, compared to 4.5% of E. histolytica and 61.8% of E. moshkovskii in Australia . A study in Brazil also showed that the prevalence of E. dispar (90%) was more frequent compared to E. histolytica (10%) among infected individuals . A study in India also showed parallel findings, where 49.5% patients were infected with E. dispar and only 7.4% with E. histolytica. Likewise, a study in the Netherlands also found 91.2% microscopic positive samples were identified as E. dispar while 6.7% were E. histolytica by PCR . In Canada, 97.1% of the examined samples contained E. dispar compared to 2.9% of E. histolytica by both PCR and ELISA assay .
To the best of our knowledge, the detection E. moshkovskii (5.8%) in this study was the first to be reported in Malaysia. Cases of humans infected with E. moshkovskii have been reported sporadically from different parts of the world including Thailand , India [9, 18, 20], Bangladesh [8, 32] and Australia . A study in Bangladesh highlighted that infection with E. moshkovskii was common in children aged 2 to 5 years  while a study in India found that E. moshkovskii infection was associated with dysentery . In our study, it was noted that all individuals infected with E. moshkovskii were children and were asymptomatic. Although amoebic liver abscess (65%) has been documented in patients admitted to an urban hospital in Malaysia , information from rural communities is not available as this infection can only be confirmed in a hospital setup. Therefore, future investigation which includes the clinical impact of E. moshkovskii and other Entamoeba species is imperative for a better understanding of a true pathogenic potential of E. moshkovskii.
Although every negative PCR sample was retested by PCR, each was again found to be negative. This result can potentially be explained by the presence of faecal inhibitor substances which were not completely eliminated prior to PCR reaction. Further study to optimize the reduction of these inhibitors is necessary during the extraction process in order to increase PCR sensitivity. It is also possible that the samples which were detected by microscopy but not PCR may belong to other Entamoeba species such as E. coli, E. hartmanni and E. polecki, or the contained a low number of parasites, which fell below the PCR detection limit. Therefore, a more sensitive method such as Real-Time PCR and employing primers for all Entamoeba species should be considered in future study. The failure to amplify samples could also be due to the fact that samples may contain only trophozoites that could have degenerated with time. Several studies have confirmed that the presence of Entamoeba cysts in the faecal samples, in contrast to trophozoites, somewhat increase the chances of the PCR assay [10, 34].