Molecular Epidemiology Of Amoebiasis In Malaysia Biology Essay

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Currently, information on species-specific Entamoeba is unavailable in Malaysia and is restricted worldwide due to re-description of pathogenic, E. histolytica and non-pathogenic, E. dispar and E. moshkovskii. Given the importance of accurate identification of Entamoeba species, this cross-sectional study was conducted aimed at providing the first documented data on true prevalence of these three species by single-round PCR assay. Furthermore, this study also aimed to determine the association of potential risk factors associated with E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communities in Malaysia. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica/E. dispar/E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%). The present findings reported low prevalence rate of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistic regression analysis indicated that the dynamic of transmission of the three Entamoeba species was different. Of six statistically significant variables observed in the univariate analysis, three retained as significant risk factors for E. histolytica infection in the logistic regression model. There were not washing hands after playing with soil or gardening (OR = 4.7; 95% CI = 1.38, 16.14; P = 0.013), indiscriminate defecation in the river or bush (OR = 5.7; 95% CI = 1.46, 21.95; P = 0.012) and close contact with domestic animals (OR = 5.4; 95% CI = 1.36, 2.51; P = 0.017). However, subjects who had family members infected with E. histolytica/E. dispar/E. moshkovskii (OR = 3.8; 95 CI = 2.11, 6.86; P < 0.001) and those being a consumer of raw vegetables (OR = 1.8; 95% CI = 1.01, 3.23; P = 0.047) were more likely to be infected with E. dispar. On the other hand, no associated factor was identified with E. moshkovskii infection. Nevertheless, diarrhoea (P = 0.002) and other gastroenteritis symptoms (P < 0.001) were only associated with E. histolytica infection. The present study provides new insight into the distribution and risk factors of E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communites in Malaysia. Identifying the different risk factors of E. histolytica and E. dispar infections will help in the planning of specific strategies in the control and prevention of each infection in the communities. Although the prevalence of E. histolytica infection report in this present study is low, a significant association of diarrhoea and other gastroenteritis symptoms with this infection warrant the need for further study to determine the cause and effect relationship. Moreover, it also emphasizes the need of molecular method to determine the species-specific prevalence of Entamoeba species.

Keywords: Amoebiasis; Entamoeba histolytica; Entamoeba dispar; Risk factors; Orang Asli; Malaysia

1. Introduction

Amoebiasis caused by the protozoan parasite Entamoeba histolytica was first recognized as a deadly disease by Hippocrates who described a patient with fever and dysentery (460 to 377 B.C.). With the application of a number of new molecular biology-based techniques, tremendous advances have been made in our knowledge of the diagnosis, natural history and epidemiology of amoebiasis. Amoebiasis remains as important health problem in tropical countries where sanitation infrastructure and health are often inadequate (Ximenez et al., 2009). Clinical features of amoebiasis range from asymptomatic colonization to amoebic colitis (dysentery or diarrhoea) and invasive extraintestinal amoebiasis, which is manifested most commonly in the form of liver abscess (Fotedar et al., 2007). Current WHO estimates of 40-50 million cases of amoebic colitis and amoebic liver abscess (ALA) and up to 100,000 deaths annually, place amoebiasis second only to malaria in mortality (WHO, 1997).

Global statistics on the prevalence of E. histolytica infection indicates that 90% of individuals remain asymptomatic while another 10% develop clinically overt disease (Jackson et al., 1985; Haque et al., 1999). Although all the deaths could be due to invasive E. histolytica infection, the value for the prevalence of E. histolytica is an overestimate since it dates from before the separation of the pathogenic E. histolytica from the non-pathogenic Entamoeba dispar (Diamond and Clark, 1993). Recently however, Entamoeba moshkovskii, a morphologically identical species has been detected in individuals inhabiting endemic areas of amoebiasis (Ali et al., 2003; Parija and Khairnar, 2005; Khairnar et al., 2007; Fotedar et al., 2008; Anuar et al., 2012) and could be contributing to the prevalence figures. Thus, the reclassification of E. histolytica into the three morphologically identical yet genetically different species has further added to the complexity of the epidemiology of amoebiasis since they cannot be differentiated by microscopy that is the most commonly used diagnostic method particularly in tropical countries where resources are limited. To date, many highly sensitive and specific techniques such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed for the accurate identification and detection of E. histolytica in various clinical samples (Ackers, 2002).

The prevalence of E. histolytica/E. dispar/E. moshkovskii in Malaysia has been reported by many researchers, from way back in the sixties and the prevalence ranged from 1% to 61% (Tengku and Norhayati, 2011). As almost all surveys previously done relied on stool analysis by microscopy, these studies did not differentiate E. histolytica from E. dispar and E. moshkovskii. To the best of our knowledge, only one community study that has been carried out in Malaysia to differentiate the two species (Noor Azian et al., 2006). In their study, the prevalence of E. histolytica and E. dispar were 13.2% and 5.6%, respectively. The prevalence of E. histolytica, E. dispar and E. moshkovskii in the Orang Asli communities therefore remains unknown.

Given the great importance both for diagnosis and epidemiological studies of differentiating species that belong to the Entamoeba species and the fact that the prevalence of species-specific is unknown, this study aimed to assess the true prevalence of E. histolytica, E. dispar and E. moshkovskii by single-round PCR assay. Furthermore, the present study also aimed to determine the association of potential risk factors caused by pathogenic and non-pathogenic species among Orang Asli in Malaysia.

2. Materials and methods

2.1. Study area and sampling

The cross-sectional study was conducted between June to December 2011 among 500 subjects living in 6 villages from three different states (Jelebu, Gerik and Temerloh) in suburban and remote areas of West Malaysia (Fig. 1). Sample selection was achieved using a two-stage sampling method: (i) random selection of village and (ii) random selection of 10 to 15 household per village. All village entry has been approved by the Ministry of Rural and Regional Development Malaysia. With an expected prevalence of E. histolytica/E. dispar/ E. moshkovskii in the study area between 10% and 20% (Nor Aza et al., 2003; Hartini and Mohamed Kamel, 2009), the 95% confidence interval and an absolute precision of 0.05 (Lwanga and Lemeshow, 1991), the appropriate sample size for the study was calculated to be approximately 138 to 246 subjects for each state.

Within each village, subjects over 2 years of age and provided consent to participate were included in this study. Exclusion criteria included children below 2 years old and refusal to participate. Although 795 questionnaires and stool containers were distributed, only 500 (62.9%) subjects were present during the survey, respond to the questionnaire and provide stool samples. Hence, 253 (31.8%) failed to submit stool samples and/or were absent during parasitological survey, 13 (1.6%) subjects had no PVA-fixed stool sample and 29 (3.7%) subjects were absent during the household-based interviews, were excluded from the study (Fig. 2).

2.2 Structured questionnaire

The rationale and procedures of the study were explained and an informed consent sheet was signed by the head of the household or a designated literate substitute. A trained research team interviewed each subject using a previously tested, structured questionnaire that sought information regarding the following groups of variables: (i) demographic data (i.e., age, gender and education level); (ii) socioeconomic background (i.e., occupation, household income and educational status); (iii) behavioural risks (i.e., personal hygiene such as hand washing and food consumption); (iv) environmental sanitation and living condition characteristics (i.e., types of water supply, latrine system, sewage disposal system and presence of domestic animals); (v) diarrhoea and symptoms of gastroenteritis (i.e., vomiting, nausea, abdominal pain, watery stools and blood or mucus stools). This questionnaire was first designed in English and then translated and pretested in Malay language, which is the national language for Malaysia and well understood by the subjects. For children, the questionnaire was completed by interviewing their parents or guardians who signed the informed consent. Individuals who participated in this study were honoured with a small token of appreciation.

2.3. Stool sample collection

Following the administration of the questionnaire, a wide mouth screw-capped container pre-labelled with the subject's name and code was distributed to all subjects for the collection of stool sample the next day. Their ability to recognize their names was counter-checked. Each subject was instructed to scoop a thumb size stool sample using a provided scoop into the container. Then, the container was placed into zip-locked plastic bag. Parents and guardians were instructed to monitor their children during the sample collection to ensure that they placed their stool samples into the right container. All study subjects were asked to provide sufficiently large stool sample (at least 10 grams) so that both microscopic techniques and molecular method could be performed. This study had to rely on a single stool collection because of the limitation of resources and the cultural belief of the Orang Asli against giving of their stool samples.

2.4. Microscopic examination

Stool samples were processed in the designated area of work in the study village within a minimum of four hours after collection by experienced laboratory technicians. Approximately, 5 grams of each stool sample was kept into a 15 ml centrifuge tube containing 3 ml Polyvinyl Alcohol (PVA). PVA-fixed samples were forwarded to the parasitological department of the Faculty of Medicine, Universiti Kebangsaan Malaysia. The samples were subjected to modify trichrome staining. Briefly, the smear cover slip was stained as follows: (i) iodine alcohol (15 minutes); (ii) 70% alcohol (10 minutes); (iii) trichrome stain (10 minutes); (iv) acid alcohol (3 seconds); (v) 95% alcohol (5 minutes); (vi) absolute alcohol (5 minutes); (vii) Wintergreen oil (5 minutes) (Salleh et al., 2012). The cover slip was mounted using DPX and examined under light microscope at magnifications of 1000x. Additionally, another half of the samples were kept unpreserved and stored at 4°C upon arrival at the laboratory for further analysis by formalin-ether sedimentation and DNA extraction. Briefly, 2 grams of stool sample was mixed with 7 ml of formalin and 3 ml of ether, centrifuged, stained with Lugol's iodine, and finally examined under light microscope at magnifications of 400x (Fleck and Moody, 1993). Samples were considered microscopically positive if cysts and/or trophozoites were detected in at least one of the two techniques, and negative if negative in all two techniques. All stool samples were further characterized using molecular procedures.

2.5. Genomic DNA extraction

Genomic DNA was extracted directly from all stool samples using QIAamp Stool DNA extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Briefly, approximately 0.2 grams of stool was added into the microcentrifuge tube, incubated at 70°C for 5 minutes with the presence of cell lysis and disruption agent provided by the manufacturer. This was then subjected to homogenization and lysis procedure for complete cell lysis by mechanical shaking (vortexing) using Silent Crusher S (Heidolph, Germany). The final DNA elution was made in 70 µl of elution buffer and stored at -20°C until required for PCR amplification.

2.6. DNA amplification by PCR

The single-round PCR assay was performed according to a protocol described by Hamzah et al. (2006) based on the reported E. histolytica, E. dispar, and E. moshkovskii small-subunit rRNA gene sequences. The forward primer sequence (EntaF) was derived from the central region of the small-subunit rRNA gene that was conserved in all three Entamoeba species, whereas the reverse primers EhR, EdR, and EmR specific for E. histolytica, E. dispar, and E. moshkovskii. The primer sequences used were as follows: for EntaF, 5'- ATG CAC GAG AGC GAA AGC AT -3'; for EhR, 5'- GAT CTA GAA ACA ATG CTT CTC T -3'; for EdR, 5'- CAC CAC TTA CTA TCC CTA CC -3'; and for EmR, 5'- TGA CCG GAG CCA GAG ACA T -3'. The forward primer in combination with the appropriate reverse primer generates a 166-bp PCR product with E. histolytica DNA, a 752-bp PCR product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA.

The PCR amplification reaction was performed in a final volume of 50 µl in 0.2 ml PCR tubes by using Eppendorf Pro-S thermal cycler (Hamburg, Germany). The reaction mixture contained 200 µM of each deoxynucleoside triphosphate, 0.1 µM of each forward and reverse primer, 6 mM MgCl2, 0.5 U of Taq polymerase (Nano Helix, South Korea), 1X Taq buffer (Nano Helix, South Korea) and 10 µl of extracted DNA samples. Amplification of each species-specific DNA fragment started with an initial denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 1 minute, 58°C for 1 minute and 72°C for 1 minute, with a final extension at 72°C for 7 minutes. Amplified products were analyzed by electrophoresis in 1.5% agarose gels and stained with GelRed (0.1 µl/mL: Biotium). DNA isolated from axenically grown E. histolytica HM-1:IMSS, E. dispar SAW 760, and E. moshkovskii Laredo were used as positive controls in this study. All of these controls DNA were a gift from Dr. Graham Clark (London School of Hygiene and Tropical Medicine).

2.7. Sequencing of PCR product

The positive amplicons were then purified using the SolGent™ kit (South Korea) according to the manufacturer's instruction. All purified amplicons were sequenced in both directions using the same primer sets as in the respective PCR assay with an ABI 3730XL sequencer (Bioncer Corporation, South Korea). Sequence chromatograms were viewed using Sequence Scanner version 1.0 program (Applied Biosystem, USA). Forward and reverse sequences were edited, manually aligned and the consensus sequence was created for each sample using the BioEdit Sequence Alignment Search Tool (BLAST) (Hall, 1999) to the National Centre for Biotechnology Information (NCBI) reference sequences. The following reference sequences were used in the analysis: (i) E. histolytica (GenBank accession number X56991); (ii) E. dispar (GenBank accession number Z49256); (iii) E. moshkovskii (GenBank accession number AF149906).

2.8. Statistical analysis

Data was entered in a Microsoft Access and was cross-checked by technical staff in order to ensure that data were entered correctly. Statistical analysis was performed using the SPSS software (Statistical Package for the Social Sciences) program for Window version 20 (SPSS, Chicago, IL, USA). Prevalence of E. histolytica, E. dispar and E. moshkovskii were determined on the basis of microscopic examination and molecular method. Only those subjects who had formalin-ether sedimentation, trichrome staining and PCR assay results together with complete questionnaire data were included in the final analyses.

For descriptive analysis, rate (percentage) was used to describe the characteristics of the studied population, including the prevalence of E. histolytica. E. dispar and E. moshkovskii. A Chi-squares test (χ2) was used to test the associations between the variables. In the univariate analysis, the dependent variable was prevalence of E. histolytica, E. dispar and E. moshkovskii, while the independent variables were demographic and socioeconomic factors, behaviour risks, environmental sanitation, living condition characteristics, diarrhoea and gastrointestinal symptoms. All univariate models were used to assess potential associations between E. histolytica, E. dispar and E. moshkovskii infections and the potential associated factor characteristics. The level of statistical significance was set at P < 0.05 and for each statistically significant factor, an odds ratio (OR) and 95% confidence interval (CI) were computed for both univariate and multivariate logistic regression analysis. All factors that were significant in univariate model were included in a logistic multivariate analysis to determine which factors could be dropped from the multivariable model.

2.9. Ethical considerations and treatment

The study protocol (Reference Number: UKM 1.5.3.5/244/FF-165-2011) was reviewed and approved by the Ethics Committee of Universiti Kebangsaan Malaysia Medical Centre (UKMMC) and permission for field work was obtained from the Ministry of Rural and Regional Development Malaysia before starting the study. Village meeting were held and village authorities and villagers were handed detailed explanations about the aims, procedures, potential risks and benefit from the study. During the meeting, they were also informed that their identity and personal particular would be kept strictly confidential and they could withdraw from the study at any point of time without citing reasons for doing so. If they agreed to participate, their consent was obtained in written form (signature or thumbprint for those who were illiterate) or parents were approached for consent on behalf of their children. At the end of the study, each subject confirmed with E. histolytica by polymerase chain reaction was treated with metronidazole according to the Ministry of Health Malaysia for free of charge.

3. Results

3.1. Characteristics of the study population

Single stool samples were randomly collected from a total of 500 subjects. Of the 500 stool, 150 (30%) samples were from the Proto-Malay tribe, 139 (27.8%) from the Negrito tribe and 211 (42.2%) from the Senoi tribe. With regards to the age groups, 221 (44.2%) were aged less than 15 years while 279 (55.8%) aged ≥15 years old with a median age of 18 years [interquartile range (IQR) 9-34.5]. There were 219 (43.8%) males and 281 (56.2%) females participated in this study.

More than half (75%) of the parents have low level of education i.e., less than 6 years of formal education. Meanwhile, majority of the parents did odd jobs such as selling forest products without any stable income. Some were daily wage earners working in rubber or palm oil plantations, unskilled labourers in factories or construction sites. Therefore, 52% of the households belonged to earned less than RM500 per month (<US$159.90), the poverty income threshold in Malaysia (Department of Statistics Malaysia., 1997. Profile of Orang Asli in Peninsular Malaysia, Kuala Lumpur) which is inadequate to maintain a good living standard. Although 71.4% if the houses have provision of basic infrastructure such as treated water supply and 61.6% have pour flush toilet, at least 28.6% are still using untreated water originating from a nearby river for their domestic needs and 38.4% still indiscriminate defecation in the river or bush. 56.4% of the households kept dogs, cats and poultry as their domestic animals. Most of these domestic animals are left to roam freely. The villagers have very close contact with the dogs and cats. Occasionally, these animals also slept, defecated indoors and accompanied the villagers into the forest to harvest forest products. Interestingly, some villagers had monkeys and birds as pets.

3.2. Prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii

Table 1 showed 3.2% (16/500) and 13.4% (67/500) of the individuals were infected with E. histolytica and E. dispar respectively. E. moshkovskii was detected in 1% (5/500) of individuals. Meanwhile, 3.6% (18/500) of the subjects had mixed infections. The prevalence of E. histolytica and E. moshkovskii infections was not significantly associated with age groups and gender. Similarly, the prevalence of E. dispar infection was not significantly difference with gender. However, the prevalence of E. dispar infection was significantly higher in individuals age group of less than 15 years (P = 0.027). With regards to tribe, it was observed that the Negrito presented a greater risk of E. histolytica infection than the Proto-Malay (Negrito versus Proto-Malay: χ2 = 15.877; P < 0.001) and the Senoi tribes (Negrito versus Senoi: χ2 = 15.991; P < 0.001). On the other hand, there were no significant differences in the prevalence of E. dispar and E. moshkovskii infections among these three tribes.

3.3. Associated factors for Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii infections

The association of E. histolytica, E. dispar and E. moshkovskii infections and sociodemographic characteristics is shown in Table 2. The results showed that drinking untreated water (OR = 4.78; 95% CI = 1.30, 17.50; P < 0.001), bathing and washing in the river (OR = 3.90; 95% CI = 1.41, 10.83; P < 0.001), not washing hands after playing with soil or gardening (OR = 2.47; 95% CI = 1.06, 5.79; P = 0.003), close contact with domestic animals (OR = 2.37; 95% CI = 1.05, 6.60; P = 0.042), indiscriminate defecation in the river or bush (OR = 3.36; 95% CI = 1.21, 9.34; P < 0.001) and low household income (OR = 7.90; 95% CI = 1.18, 52.82; P = 0.001) were significantly associated with E. histolytica infection. Meanwhile, ten factors were found to be associated with E. dispar infection which include children less than 15 years (OR = 1.33; 95% CI = 1.00, 1.78; P = 0.027), drinking untreated water (OR = 1.30; 95% CI = 1.03, 1.70; P = 0.033), bathing and washing in the river (OR = 1.23; 95% CI = 1.05, 1.51; P = 0.023), not washing hands after playing with soil or gardening (OR = 1.52; 95% CI = 1.14, 2.03; P < 0.001), outdoor sewage disposal (OR = 1.34; 95% CI = 1.03, 1.81; P = 0.039), consuming raw vegetables (OR = 1.47; 95% CI = 1.06, 2.02; P = 0.008), low level of mother's education i.e., less than 6 years of formal education (OR = 2.46; 95% CI = 1.04, 5.80; P = 0.023), non working mother's (OR = 1.46; 95% CI = 1.02, 2.15; P = 0.029), low household income (OR = 1.70; 95% CI = 1.17, 2.49; P = 0.001) and the presence of other family members infected with E. histolytica/E. dispar/ E. moshkovskii (OR = 1.72; 95% CI = 1.33, 2.24; P < 0.001). However, there was no associated factor was identified with E. moshkovskii infection.

3.4. Aetiological factors associated with Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii infections

Logistic regression analysis confirmed that individuals who have close contact with domestic animals i.e., dogs and cats were 5.4 times (95% CI = 1.36, 2.51; P = 0.017) more likely to be infected with E. histolytica as compared to those who do not keep domestic animals as their pet. Not washing hands after playing with soil or gardening had a 4.7 times higher (95% CI = 1.38, 16.14; P = 0.013) risk of getting E. histolytica infection. Subjects who indiscriminate defecation in the river or bush were 5.7 times (95% CI = 1.46, 21.95; P = 0.012) more likely to be infected with E. histolytica as compared to those who have proper pour flush toilet (Table 3). In addition, individuals who had family members infected with E. histolytica/ E. dispar/E. moshkovskii and those being a consumer of raw vegetables were 3.8 (95% CI = 2.11, 6.86; P < 0.001) and 1.8 times (95% CI = 1.01, 3.23; P = 0.047) more likely to be infected with E. dispar.

3.5. Clinical significance of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii infections

Table 4 shows the association of clinical symptoms with E. histolytica and non-pathogenic, E. dispar and E. moshkovskii in Orang Asli. Diarrhoea was significantly associated with the E. histolytica infection (χ2 = 9.266; P = 0.002). Similarly, other gastroenteritis symptoms such as abdominal pain, loss of weight, loss of appetite, vomiting and nausea (χ2 = 14.800; P < 0.001) also showed positive significant association. On the other hand, E. dispar and E. moshkovskii did not show any association either with diarrhoea or other gastroenteritis symptoms.

3.6. Molecular characterization of Entamoeba species

Out of 500 stool samples, 106 (21.2%) samples were successfully amplified by single-round PCR assay and 93 (18.6%) of stool samples found to be positive microscopically. Although most Entamoeba-positive subjects were infected with single infections of E. histolytica, E. dispar and E. moshkovskii, only 2% (10/500) had mixed infections with E. dispar and E. histolytica, 1.2% (6/500) of E. dispar and E. moshkovskii and 0.4% (2/500) had triple infections with E. histolytica, E. dispar and E. moshkovskii (Fig. 3). Representative PCR products were sequenced in both directions from all of the 16 samples of E. dispar, 15 samples of E. histolytica and 5 samples of E. moshkovskii. The sequences of all the 16 E. dispar amplicons showed 100% homologous to previously published sequences of E. dispar (GenBank accession number Z49256), whereas 15 E. histolytica sequences showed high similarity (98% to 99%) to the E. histolytica sequences in GenBank (accession number X56991). All five single isolation of E. moshkovskii amplicons showed 99% to 100% similarity to the E. moshkovskii sequences in GenBank (accession number AF149906).

4. Discussion

Most of epidemiological studies for E. histolytica infection were performed before of the re-description of three species; E. histolytica, E. dispar and E. moshkovskii. Hence, there is a clear need to perform new epidemiological studies to distinguish these three species of Entamoeba and to determine the true prevalence rate of E. histolytica. To the best of our knowledge, this is the first report on the distribution of E. histolytica, E. dispar and E. moshkovskii in Malaysia since the re-description of the three species was established. The present study showed the prevalence rate of 3.2%, 13.4% and 1% for E. histolytica, E. dispar and E. moshkovskii, respectively among Orang Asli communities. High prevalence of E. dispar infection in these Orang Asli communities may be explained by the occurrence of human as a source infection as other family members who were infected with E. histolytica/E. dispar/ E. moshkovskii was identified as a risk factor of E. dispar infection. It can also be postulated that the infection occurred within the family most likely through direct transmission person-to-person and the infected family members acted as a carrier of E. dispar infection. The higher prevalence rate of E. dispar as compared to other two species observed in this present study was in agreement with most of studies carried out worldwide (Verweij, et al., 2003; Kebede et al. 2004; Calderaro et al., 2005; Lebbad and Svard, 2005; Ramos et al., 2005; Samie et al. 2005; Fotedar et al., 2007). Nonetheless, the only reported findings on E. histolytica and E. dispar infections in Malaysia (Noor Azian et al., 2006) has documented that the actual prevalence of E. histolytica (13.2%) was higher than E. dispar (5.6%). In their study, the prevalence of these two species was determined by nested-PCR and restriction enzyme digestion. Similarly, in the Philippines a study in mental institution, reported 74 individuals (65.5%) were positive for E. histolytica and only 6 individuals (5.3%) were positive for E. dispar (Rivera et al., 2006). In the Gaza strip, Palestine, E. histolytica was identified by PCR in 64 (69.6%) of the samples and that of E. dispar in 21 (22.8%) (Al-Hindi et al., 2005). Based on molecular identification, E. histolytica was more prevalent than E. dispar in Thai/Myanmar border region of Thailand (Intarapuk et al., 2009).

Results of the present study indicated non-significant difference of E. histolytica prevalence between genders. Similar findings were observed between E. dispar and E. moshkovskii infections. Rivera et al. (1998) reported that there was non-significant difference in the gender distribution of E. histolytica in 14 communities in the northern Philippines. Similar observations have been reported in most of community-based studies (Okafor and Azubike, 1992; Magambo et al., 1998; Hamze et al., 2004; Sharma et al., 2004; Zahida et al., 2010). However, most of hospital-based studies reported gender preference of E. histolytica infection. Ozyurt et al. (2007) reported 67% prevalence of E. histolytica in males and 33% in females among patients attending training hospital. Ohnishi and Murata (1997) studied prevalence of E. histolytica in the area of Tokyo. Out of 28 study cases, 26 were males and none of the females were infected. On the other hand, 64% (16/25) prevalence of E. histolytica for females and 36% (9/25) for males have been reported among attendees health care centre in Turkey (Ozgumus and Efe, 2007). Hospital-based study in Pakistan, observed significant high prevalence rate of E. histolytica infection in females (31.5%) as compared to males (19.6%) (Ejaz et al., 2011).

The present study observed high infection rate of E. histolytica and E. dispar in individual's age group less than 15 years, whereas E. moshkovskii was observed more in individuals 15 years old and more. However, the association was not statistically significant. This finding did not corroborate the results reported by Shetty et al. (1990), Waqar et al. (2003), Sayyari et al. (2005) and Zahida et al. (2010), who found significant association of E. histolytica infection with age and infection was mostly seen in young children. Nevertheless, this present study showed that children less than 15 years were at higher risk of being infected with E. dispar. In this present study, other family members infected with E. histolytica/E.dispar/E. moshkovskii was identified as a risk factor of E. dispar infection. Thus, this may explain high infection rate of this infection in children as they may be infected from close contact with other family members who were infected. The result of this finding was in agreement with a study conducted by Rivera et al. (1998) which showed that E. dispar prevalence rate was particularly high in 5-14 years old age group, with an observable decrease in positivity with age.

This present study also observed very encouraging trends in the dynamic transmission of E. histolytica and E. dispar infections. Individuals from the Negrito tribe have a significant greater risk of being infected by E. histolytica and E. dispar infections as compared with subjects from the Proto-Malay and Senoi tribes. This was attributed to the poor housing condition, provision of basic amenities and poor sanitary practices in that community. However, this finding was contradicted with the reported prevalence of Giardia intestinalis infection carried out in the same communities which observed a high prevalence rate of giardiasis in the Proto-Malay tribe that have better housing condition and basic amenities (Anuar et al., 2012). It indicates that sanitary practices and poor provision of basic amenities play important role in the transmission of E. histolytica in the Negrito tribe.

It is interesting to note that this present study identify different risk factors of E. histolytica and E. dispar infections. E. histolytica infection was significantly associated with a variety of risk factors that are related to sanitary practices of the individual, such as not washing hands after playing with soil or gardening, indiscriminate defecation in the river or bush and close contact with domestic animals. On the other hand, E. dispar infection was strongly associated with the presence of other family members infected with E. histolytica/ E. dispar/E. moshkovskii besides being a consumer of raw vegetables. E. histolytica infection is a faecal-oral disease, thus improper hygienic practice plays a major role in E. histolytica transmission (Rivera et al., 1998). In this study, individuals who do not washed their hands properly using soap and water after exposed with soil or gardening was at 4.7 higher risk of being infected with E. histolytica. Similar findings were observed by Seppo et al. (2005) and Naelah et al. (2011), showed that individuals who do not practice proper hand washing before eating are at two fold higher risk of getting E. histolytica infection. Study conducted in Vietnam also showed that transmission of E. histolytica was more than three-fold risk increase if hands are not properly washed (Pham Duc et al., 2011).

Another aspect analyzed in this study was indiscriminate defecation in the river or bush. Observations made in the present study noted that a quarter of the residents did not have proper sanitary facilities at home. Children and even adults were allowed to defecate indiscriminately around their houses. Hence, it was not surprising that indiscriminate defecation outdoors constituted a significantly higher risk of being infected with E. histolytica as compared to those who have adequate proper latrine. Our findings showed individuals who had no access to adequate sanitary infrastructure demonstrated 1.2 greater odds of infection, compared to having access to a flush latrine.

The role of companion animals as reservoirs for zoonotic diseases has been known as a significant health problem worldwide (Schantz, 1994). The probable zoonotic risk of E. histolytica is of great concern to public health as the amoebiasis is not a zoonotic disease. This present study once again highlighted the significant association of close contact between humans and animals especially dogs and cats. Comparable results were also observed in other endemic areas in Yemen (Naelah et al., 2011) and Vietnam (Pham Duc et al., 2011). The cysts of E. histolytica could be deposited on the surface (fur) of the animals during close contact with infected humans or from environment and then later transmitted to a next person. In order to support this hypothesis, the presence of E. histolytica cysts in fur must be documented. Unfortunately, we could not conduct this verification during our field work.

Although it is still unclear whether E. histolytica infection is zoonotic or not, this parasite has also been isolated from macaques. In the Philippines, E. histolytica was detected among captive macaques in a primate facility. In the same study, they found that 23 E. histolytica isolates using polymerase chain reaction were identical to human E. histolytica (Rivera et al., 2010). Similarly, in Ethiopia, a study on baboon and Cercopithecus (old world monkey) found that the prevalence of E. histolytica was 24.4% (Legesse and Erko, 2004), highlighting a possibility of zoonotic transmission.

Epidemiologic studies have shown that being a consumer of raw vegetables has 1.8 fold higher risks for acquiring E. dispar and this finding is consistent with a previous study from Iran (Shahnazi and Jafari-Sabet, 2010). Another study in Brazil revealed that participants who ate raw vegetables had a risk of 1.6 acquiring the infection when compared with those who do not eat them (Benetton et al., 2005). By contrast, recent findings from Kenya, Tajikistan, Yemen and Vietnam found no association between E. dispar infection and the consumption of raw vegetables (Nyarango et al., 2008; Matthys et al, 2011; Naelah et al., 2011; Pham Duc et al., 2011). Based on our observation, tapioca shoots, wild fern shoots and locally planted leaves are the main raw vegetables as salad by the Orang Asli communities. They seldom washed these vegetables before eating. Therefore, the current result highlights the potential of unwashed raw vegetables in transmission of E. dispar infection to the communities.

The present study also highlighted that those family members infected with E. histolytica/E. dispar/E. moshkovskii were 3.8 times more likely to get infected with E. dispar. This finding indicates that the possibility of infected family members as the source of infection and direct transmission occur within household. It has also been demonstrated in Mexico whereby 40% of contacts with E. dispar carriers were also infected (Ruiz-Palacios et al., 1992). Under such circumstances, children may be at constant risk of infection as their parents or guardians might be the source of infection and this can be observed in this present study where the prevalence is high in children less than 15 years. Some authors also claim that person-to-person transmission is the most important determinant of infection (Ostan et al., 2007). Although we found no associated risk factor with E. moshkovskii infection, the prevalence of E. moshkovskii was higher among those aged ≥15 years. This present study found a single isolation of E. moshkovskii which was shown to be positive by both microscopic techniques and single-round PCR. This individual and two other individuals infected with mixed infections of E. moshkovskii and E. dispar presented with gastroenteritis symptoms such as nausea, loss of appetite and loss of weight (Anuar et al., 2012). However, more studies are necessary to define the exact role of E. moshkovskii in gastroenteritis disorders and the possible virulence of this organism. Therefore, E. moshkovskii should not be dismissed as a non-pathogenic parasite and the detection of this species should be considered in the diagnosis of patients presenting with diarrhoea and other gastroenteritis symptoms, especially in cases where other pathogens, such as bacteria and viruses are not detected.

Clinical amoebiasis may mimic functional bowel disease when it has a sub-acute onset with symptoms of mild diarrhoea and abdominal pain. Almost 90% of E. histolytica infections were reported as being asymptomatic (WHO, 1997). In our study, we found significant association between E. histolytica infection and gastroenteritis symptoms including diarrhoea. Similarly, a most recent study conducted among 161 patients with chronic diarrhoea and also from 157 healthy controls in Pakistan (Yakoob et al., 2011), E. histolytica was significantly associated with diarrhoea. Likewise, study among 283 patients from the Netherlands (Visser et al., 2006) also demonstrated that three quarters of E. histolytica carriers reported abdominal complaints or diarrhoea. Similar observations have been reported in study among public hospitals and primary schools in Thailand (Haghighi et al., 2003) and South Africa (Samie et al., 2006). However, there is a disparity between this present finding with a previous study, which found a non-significant association between gastroenteritis symptoms and E. histolytica infection. A study carried out by Rivera et al. (1998) revealed that all the E. histolytica-positive subjects were asymptomatic. This is consistent with several reports from endemic areas that showed most E. histolytica infections are asymptomatic (Jackson et al., 1985; Ravdin et al., 1990).

In conclusion, this study provides new insight into the distribution and risk factors of E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communities in Malaysia. High prevalence rate of E. histolytica and E. dispar infections is observed among the Negrito tribe. The present study findings confirm transmission of E. histolytica and E. dispar infections are related to contamination of hands and vegetables with human faeces. Infected family members served as the source of E. dispar infection and person-to-person contact is postulated as the main mode of transmission. Thus, provision of basic safe water supply and sewage disposal together with health education pertinent to good personal and food hygiene practice are the main strategies in controlling and preventing of these infections in the communities.

Acknowledgements

The authors sincerely thank Dr. Graham Clark (London School of Hygiene and Tropical Medicine) for providing us with lyophilized DNA of standard cultures of E. histolytica HM-1:IMSS, E. dispar SAW 760, and E. moshkovskii Laredo.The authors gratefully acknowledge the Ministry of Rural and Regional Development Malaysia for granting us permission to conduct this research. We also thank all the participants from Parit Gong village, Pasu village, Pian village, Bagan Balak village, Sungai Banun village, Desa Damai village, Sungai Raba village, and Pengkalan Permai village for their commitment and contribution in providing their stool samples. The work presented in this paper was funded by the UKMMC Fundamental Research Grant (FF-165-2011) and Special Research University Grant (UKM-GUP-2011-316) from the Universiti Kebangsaan Malaysia. During this project, TSA also received fellowship support from the Ministry of Higher Education, Malaysia and Graduate Research Assistantship scheme from the Universiti Kebangsaan Malaysia.

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