Genetic diversity of Entamoeba species, and the impact of the Madi Drop on the occurrence of Entamoeba among children in Vhembe, South Africa

Abstract

Background: Amebiasis is a common parasitic disease that contributes to the burden of diarrhea in areas with poor sanitation and inadequate water supply worldwide. Water quality, therefore, plays an essential role in the transmission of amebiasis. The Madi Drop is a recent point of use water treatment tool that has not been tested for its efficiency against parasitic infections. Therefore, the present study sought to investigate the genetic diversity of Entamoeba species, and the impact of the Madi Drop utilization on the occurrence of Entamoeba among children in Vhembe, South Africa. Materials and methods: The present study is part of the Madi Trial project entitled: The effectiveness of low-cost use water treatment technology to prevent stunting in children in Limpopo province. The trial included 4 groups of children recruited in different households to whom a filter with either the Madi Drop or not was given as well as a control group. Stool samples were collected every 3 months from (month 0 to 24 months follow up). For the present study, a total of 534 stool samples (months 18 and 24) from 313 participants both males and females, aged 3 years and below were used. All the stool samples were examined microscopically for the presence of Entamoeba cysts and trophozoites. DNA was extracted from all the samples by QIAamp Fast DNA stool mini kit and was subjected to conventional PCR for the presence of Entamoeba genus. Positive amplicons were sequenced by the Sanger method and 15 were selected for Next-generation sequencing in the MiSeq platform based on the Entamoeba 18S rRNA gene, using a MiSeq v3 (600 cycles) kit. Data analysis was performed at Inqaba in-house developed data analysis. The phylogenetic trees were constructed to determine the species relatedness. Results: Of the 313 children recruited in the study, 163 were females and 150 were males, the age ranged between 1 to 3 years. Of 534 samples, 130/534 (24.3%) were microscopically positive for Entamoeba cysts. The month 18 sample had the highest presence of Entamoeba cysts 78/299 (26%) compared to month 24; 52/235 (22%). Entamoeba infections were high before the interventions of Madi Drop technology as compared to after intervention. Forty-three (8%) of the total samples were positive for Entamoeba genus by PCR. Twenty positive amplicons were sequenced by Sanger sequencing technologies. Of these, 11/20 (55%) were E. polecki 8(40%) followed by E. coli 2 (10%) and E. muris 1(5%). Next-generation sequencing showed a wide variety of organisms. Of note was the fact that about 3 different species of Entamoeba were found in most of the samples. The NGS results revealed that 14/15 (93%) of amplicon were positive for Entamoeba species, dominated by E. coli 9/15 (60%), followed by E. polecki 3/15 (20%) and E. moshkovskii 2/15 (13%). The phylogenetic tree showed the close relationship between isolated species and the ones in the GenBank. Conclusion: The present study showed that the use of Madi Drop helped reduce the number of Entamoeba infections among children in a rural community north of South Africa. Even though the identified species are regarded or known to be non-pathogenic, their presence indicates intense fecal-oral transmission. Therefore, water could play a very important role in the transmission of Entamoeba and possibly other parasites in the region. Furthermore, in some cases, E. polecki and E. moshkoviskii have been isolated from patients that encountered an episode of bloody diarrhea associated with many Entamoeba polecki and E. moshkovskii cysts in the stool. The results suggested that the usage of Madi Drop and filters may decrease the level of parasitic infections and improved drinking water quality in these communities.