Abstract
BackgroundIn 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.MethodsFaecal 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).ResultsBased 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.ConclusionsThe 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.
Highlights
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
Prevalence of Entamoeba infection via microscopy A total of 426 samples were collected and 75 (17.6%; 95% confidence intervals (CIs) = 14.0-21.2%) samples were microscopically positive for Entamoeba cysts, either singly or in combination with other intestinal parasites (Table 1)
Infection was more prevalent in females (19.1%; 95% CI = 14.0-24.2%) compared to males (15.9%; 95% CI = 10.8-21.0%), it was not statistically significant
Summary
Study area and population The present study was carried out from 2009 to 2011 in five rural villages, namely Pos Iskandar (102.65°E longitude, 3.06°N latitude), Sungai Koyan (101.63°E longitude, 4.25°N latitude), Sungai Bumbun (101.42°E longitude, 2.85°N latitude), Bukit Serok (102.82°E longitude, 2.91°N latitude) and Sungai Layau (104.10°E longitude, 1.53°N latitude) (Figure 1). Their consent was taken either in written form (signed) or verbally followed by thumb prints (for those who were illiterate) or their parents/ guardians (on behalf of their children) after which prelabeled plastic containers for faecal collection were handed out to all participants. Their ability to recognize their names was checked. The secondary PCR had a similar cycling condition except that the annealing temperature (48°C instead of 56°C) and extension duration (1 min instead of 1 min 30 sec) were modified In both amplifications, samples were incubated in the MyCycler thermal cycler (BioRad, Hercules, USA). Associations between proportions were explored using Chi-Square X2 (test) and a P value of
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