Dataset Information

Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica.

ABSTRACT: We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with Entamoeba histolytica, as well as other aspects of the organism's epidemiology.

SUBMITTER: Ali IK

PROVIDER: S-EPMC1317169 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica.

Ali Ibne Karim M IK Zaki Mehreen M Clark C Graham CG

Journal of clinical microbiology 20051201 12

We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and ...[more]

PMID: 16333065

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Project description:This study aimed to determine the frequency of Entamoeba histolytica and Entamoeba dispar infection in school children in the community of Tlaltizapan, in order to understand the dynamics of infection within the school and family spheres of this population. Amoebiasis is an unsolved public health problem and an endemic disease in Mexico. The incidence rate varies depending on the state; the most affected states show the highest numbers of new cases of amoebiasis per year. Previously, we reported the molecular frequency of infection with E. histolytica and/or E. dispar in other rural communities of the state of Morelos.Children from 3 schools were studied to estimate the frequency of intestinal parasites through microscopic examination of fresh stool samples. The number of studied individuals were 309 school children. The molecular characterization of E. histolytica or E. dispar was carried out by Polymerase Chain Reaction (PCR) using species-specific primers to amplify short tandem repeats (STR) in non-coding sequences associated with the tRNA gene; the amplified fragments were sequenced and analyzed.Eight different genotypes were obtained from E. dispar isolates with the molecular marker NKD3-D5. None of the cases in which the species E. histolytica was detected developed symptoms attributable to an invasive process of disease. Moreover, the parasitized condition appeared to have no significant impact on the development or nutritional status of affected children. Genotype 1, which corresponds to the reference strain E. dispar SAW760, considered a non-pathogenic amoeba, was the most prevalent.The comparison of the genotypes of Entamoeba species did not show a correlation between children and their relatives. In this community, the species Entamoeba dispar genotype 1 was the most widespread. Based on the indicators of growth, development and nutrition status, the studied community seems to be reasonably adapted to constant exposure to intestinal parasites, since there were no evidences of a serious impact of the parasitized condition on the children's health.

Project description:BACKGROUND: The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains. RESULTS: We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies. CONCLUSIONS: Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

Dataset Information

Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica.

Publications

Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica.

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