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(PCR) in Rural Communities in Malaysia Romano Ngui, Lorainne Angal, Siti Aminah Fakhrurrazi, Yvonne Lim Ai Lian, Lau Yee Ling, Jamaiah Ibrahim and Rohela Mahmud*

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(PCR) in Rural Communities in Malaysia Romano Ngui, Lorainne Angal, Siti Aminah Fakhrurrazi, Yvonne Lim Ai Lian, Lau Yee Ling, Jamaiah Ibrahim and Rohela Mahmud* Ngui et al. Parasites & Vectors 2012, 5:187 http://www.parasitesandvectors.com/content/5/1/187 RESEARCH Open Access Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia Romano Ngui, Lorainne Angal, Siti Aminah Fakhrurrazi, Yvonne Lim Ai Lian, Lau Yee Ling, Jamaiah Ibrahim and Rohela Mahmud* Abstract Background: In this study, a total of 426 human faecal samples were examined for the presence of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii infection via a combination of microscopic examination and nested polymerase chain reaction (PCR) targeting 16S ribosomal RNA of Entamoeba species. Methods: Faecal sample were collected from 426 participants in five rural villages in Peninsular Malaysia. The faecal samples were processed by direct wet smear and formalin ethyl acetate concentration technique followed by iodine staining and examined via microscopy for the presence of Entamoeba species and other intestinal parasites. Microscopically positive samples for Entamoeba species cysts were further characterized using a Nested Polymerase Chain Reaction (Nested-PCR) targeting 16S-like ribosomal RNA gene. The data entry and analysis was carried out using the SPSS software (Statistical Package for the Social Sciences) program for Windows version 17 (SPSS, Chicago, IL, USA). Results: Based on single faecal examination, overall prevalence of Entamoeba infection was 17.6% (75/426). Females (19.1%) were more commonly infected compared to males (15.9%). Comparison by age groups showed that adults (23.9%) had higher infection rates than children (15.3%). The PCR results showed that 52 out of 75 microscopy positive samples successfully generated species-specific amplicons. The infection with E. histolytica (75.0%; 39/52) was the most common, followed by E. dispar (30.8%; 18/52) and E. moshkovskii (5.8%; 3/52). Of these, 33 (63.5%) were shown to contain only E. histolytica, 10 (19.2%) contained E. dispar and 3 (5.8%) contained only E. moshkovskii. Mixed infection with E. histolytica and E. dispar was found in 6 (11.5%) samples. Conclusions: The present study essentially emphasized the benefit of molecular techniques in discriminating the pathogenic Entamoeba species from the non-pathogenic for accurate diagnosis and better management of amoebiasis. The presence of E. moshkovskii is of great public health concern as it was the first time it has been reported in Malaysia. Background commensal parasites and do not cause human disease. The genus Entamoeba comprises six species, namely Ent- Amoebiasis which is caused by Entamoeba histolytica is a amoeba histolytica, E. dispar, E. moshkovskii, E. coli, E. global health problem as it is responsible for more than hartmanni and E. polecki that live in the human intestinal 100,000 deaths per year and is the second leading cause of lumen. Infections with Entamoeba species can result in ei- global death due to protozoa after malaria [1-3]. Major ther a harmless colonization of the intestine or invasion of symptoms of amoebiasis are abdominal pain, diarrhea, the colonic wall and damage of other host tissues such as nausea, vomiting and flatulence. This infection is more liver, lung and brain. Most of the Entamoeba species are preponderant in children compared to adults [4] and is commonly found in tropical and subtropical areas. It has been reported that 10% of the world’spopulation * Correspondence: [email protected] Department of Parasitology, Faculty of Medicine, University of Malaya, 50603, are infected with Entamoeba species, in which pathogenic Kuala Lumpur, Malaysia © 2012 Ngui et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ngui et al. Parasites & Vectors 2012, 5:187 Page 2 of 7 http://www.parasitesandvectors.com/content/5/1/187 E. histolytica constitute 10% of these infections and the In order to avoid unnecessary treatment of individuals remaining 90% are infected by non-pathogenic E. dispar with non-pathogenic Entamoeba species, it is important [5]. However, a recent study highlighted the existence of to discriminate these species from the pathogenic E. his- another species of Entamoeba known as E. moshkovskii tolytica [1]. Additionally, there is also a need for simpler which can also cause infection among humans [6]. E. his- and better identification of these infections, not only for tolytica, E. dispar and E. moshkovskii are morphologically diagnostic purposes and care management, where E. dis- identical but are different biochemically and genetically par and E. moshkovskii infected individuals could be [6-8]. Although a previous study showed E. moshkovskii to treated unnecessarily with antiamoebic chemotherapy, be a non-pathogenic parasite, intestinal symptoms includ- but also for a better understanding of the epidemiology ing diarrhea and other gastrointestinal disorders in indivi- of these parasites in the human population. Within this duals infected with this species have been reported [8-10]. context, we conducted this study to determine the This, however, has been rebutted by Al-Harthi and Jam- prevalence of Entamoeba species and the true E. histoly- joon (2007) [11] who claimed that E. moshkovskii has tica, E. dispar and E. moshkovskii infections in human never been associated with any disease. This may indicate faecal samples using molecular techniques. that perhaps humans are a true host for this putatively free-living amoeba and are not just transiently infected. Methods Therefore, the true prevalence of E. dispar and E. mosh- Study area and population kovskii infections need to be investigated in order to deter- The present study was carried out from 2009 to 2011 in mine their significant pathogenic potential in humans. five rural villages, namely Pos Iskandar (102.65°E longi- Traditionally, Entamoeba infections are diagnosed tude, 3.06°N latitude), Sungai Koyan (101.63°E longitude, through microscopic examination of fresh or fixed faecal 4.25°N latitude), Sungai Bumbun (101.42°E longitude, samples. However, very often E. histolytica cysts and tro- 2.85°N latitude), Bukit Serok (102.82°E longitude, 2.91°N phozoites cannot be morphologically differentiated from latitude) and Sungai Layau (104.10°E longitude, 1.53°N E. dispar and E. moshkovskii through microscopic exam- latitude) (Figure 1). In brief, the villages are inhabited by ination. Only when ingested red blood cells are present members of the indigenous ethnic groups. Each village in trophozoites of E. histolytica, the ability to distinguish has a small population and the number of residents in them from those of E. dispar and E. moshkovskii each village is estimated to be 80 to 100 inhabitants. They becomes easier. Recently, sensitive and specific sero- live in deprived circumstances where overcrowding, poor logical and molecular techniques that are able to distin- environmental sanitation, low levels of education and poor guish E. histolytica from E. dispar have been developed provision of safe water are widespread. Although provi- [10,12-20]. These include the detection of E. histolytica sions of basic facilities are provided by local authorities, antigen using an enzyme-linked immunosorbent assay most of them could not afford to pay their monthly utility (ELISA) [16,17,19], the detection of E. histolytica by bills leading to the termination of water supplies. There- monoclonal antibodies of E. histolytica that specifically fore, rivers located adjacent to the village remain their recognize E. histolytica antigen [12,15] and the use of main source of water for domestic needs such as drinking, the polymerase chain reaction (PCR) to amplify amoebic cooking, bathing and washing clothes. The condition of DNA [10,13,14]. More current approaches include the the surrounding environment of the village is also gener- discrimination of E. histolytica, E. dispar and E. mosh- ally poor with limited provision of latrine facilities there- kovskii by a simultaneous detection using multiplex fore encouraging defecation in and around bushes or nested PCR [18,20]. nearby rivers. Children usually defecate indiscriminately In Malaysia, intestinal parasitic infections (IPIs) in- around their houses without parental supervision. Cats, cluding Entamoeba infections are more prevalent in dogs and poultry are the most common domestic animals. rural areas especially among aboriginal communities Most of these domestic animals are left to roam freely compared to urban areas [21-23]. A recent study con- within the village and were observed to defecate in the ducted by Ngui et al. (2011) among communities living surrounding property of their owners. The villagers have in rural areas of Malaysia, showed that 10.2% of the par- very close contact with these animals, even sharing food ticipants were infected with Entamoeba. Another study from the same plate with them. showed that the infection rate of Entamoeba in rural communities in Malaysia was 21.0% [24]. Other local Consent, sample collection and laboratory procedures studies which include aboriginal groups reported preva- The study protocol was approved by the Ethics Commit- lence ranging from 9.4% to 18.5% [25,26]. However, tee of the University of Malaya Medical Centre, Malaysia these prevalence rates were based on
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