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Frequent occurrence of mixed Enterocytozoon bieneusi infections in humans
Giovanni Widmer1, Julia Dilo, James K Tumwine
1Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA.
Abstract:
Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.
Insights
Enterocytozoon bieneusi infections in Uganda show high genetic diversity within individuals. This finding complicates multilocus genotyping, suggesting infections with mixed populations of this human pathogen are common.
Area of Science:
- Microbiology
- Genetics
- Epidemiology
Background:
- Enterocytozoon bieneusi is a microsporidian human pathogen with a wide host range.
- Genome sequencing identified simple sequence repeats (SSRs) in E. bieneusi.
- SSRs offer potential for developing genotyping methods to track pathogen epidemiology.
Purpose of the Study:
- To develop and apply a multilocus genotyping method for Enterocytozoon bieneusi.
- To investigate the genetic diversity and population structure of E. bieneusi in Ugandan children with diarrhea.
Main Methods:
- Sequencing of simple sequence repeats (SSRs) from E. bieneusi isolates.
- Development of a multilocus genotyping method using SSRs and the internal transcribed spacer (ITS).
- Genotyping of E. bieneusi DNA from 197 fecal samples using PCR amplification, cloning, and sequencing of multiple amplicons.
Main Results:
- Extensive sequence polymorphism was identified among and within E. bieneusi isolates from Uganda.
- Most identified microsatellite sequences were unique to the Ugandan population.
- High intra-isolate polymorphism, potentially indicating heterogeneous E. bieneusi infections, precluded definitive multilocus genotyping.
Conclusions:
- Infections with genetically diverse Enterocytozoon bieneusi populations appear common in the studied Ugandan region.
- The high degree of intra-isolate polymorphism challenges the adequacy of sequencing uncloned PCR products for multilocus genotyping in high-prevalence areas.
- Further refinement of molecular methods is needed for accurate epidemiological studies of E. bieneusi in diverse settings.
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