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Genetic subtypes in unicellular intestinal parasites with special focus on Blastocystis
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of molecular tools for detection and typing of unicellular intestinal parasites has revealed genetic diversities in species that were previously considered as distinct entities. Of great importance is the genetic distinction found between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar, two morphologically indistinguishable species. Blastocystis sp. is a ubiquitous intestinal parasite with unsettled pathogenicity. Molecular studies of Blastocystis sp. have identified 17 genetic subtypes, named ST1-17. Genetically, these subtypes could be considered as different species, but it is largely unknown what phenotypic or pathogenic differences exist between them. This thesis explores molecular methods for detection and genetic subtyping of unicellular intestinal parasites, with special focus on Blastocystis.

We found that PCR-based methods were highly sensitive for detection of unicellular intestinal parasites, but could be partially or completely inhibited by substances present in faeces. A sample transport medium containing guanidinium thiocyanate was shown to limit the occurrence of PCR inhibition.

The prevalence of Blastocystis in Swedish university students was over 40%, which is markedly higher than what was previously estimated. Blastocystis ST3 and ST4 were the two most commonly found Blastocystis subtypes in Sweden, which is similar to results from other European countries.

Blastocystis sp. and Giardia intestinalis were both commonly detected in Zanzibar, Tanzania, each with a prevalence exceeding 50%. Blastocystis ST1, ST2, and ST3 were common, but ST4 was absent. While G. intestinalis was most common in the ages 2-5 years, the prevalence of Blastocystis increased with increasing age, at least up to young adulthood. We found no statistical association between diarrhoea and Blastocystis sp., specific Blastocystis subtype or G. intestinalis.

Metagenomic sequencing of faecal samples from Swedes revealed that Blastocystis was associated with high intestinal bacterial genus richness, possibly signifying gastrointestinal health. Blastocystis was also positively associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, and negatively associated with the genus Bacteroides.

Blastocystis ST4 was shown to have limited intra-subtype genetic diversity and limited geographic spread. ST4 was also found to be the major driver behind the positive association between Blastocystis and bacterial genus richness and the negative association with Bacteroides.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1889
Keyword [en]
Intestinal parasites, Blastocystis, Entamoeba, Giardia, molecular detection, PCR, subtype, intestinal microbiota, Sweden, Zanzibar
National Category
Microbiology in the medical area Infectious Medicine
Research subject
Clinical Bacteriology
Identifiers
URN: urn:nbn:se:umu:diva-132441ISBN: 978-91-7601-682-4 (print)OAI: oai:DiVA.org:umu-132441DiVA: diva2:1081616
Public defence
2017-04-07, Hörsal D, byggnad 1D, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-03-17 Created: 2017-03-14 Last updated: 2017-03-15Bibliographically approved
List of papers
1. Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples
Open this publication in new window or tab >>Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples
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2015 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 64, 1053-1062 p.Article in journal (Refereed) Published
Abstract [en]

Although PCR offers the potential for sensitive detection of parasites:there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

Place, publisher, year, edition, pages
Microbiology Society, 2015
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-111507 (URN)10.1099/jmm.0.000129 (DOI)000363356800016 ()26296348 (PubMedID)
Available from: 2015-12-01 Created: 2015-11-13 Last updated: 2017-03-14Bibliographically approved
2. Subtype analysis of Blastocystis isolates in Swedish patients
Open this publication in new window or tab >>Subtype analysis of Blastocystis isolates in Swedish patients
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2012 (English)In: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 31, no 7, 1689-1696 p.Article in journal (Refereed) Published
Abstract [en]

Blastocystis is a genetically diverse and widespread intestinal parasite of animals and humans with controversial pathogenic potential. At least nine subtypes of Blastocystis have been found in humans. The genetic diversity of Blastocystis was examined in stool samples from 68 patients from the Stockholm area, Sweden. Blastocystis was identified by light microscopy, and subtyped by sequencing the 5'-end of the small subunit ribosomal RNA gene. Five Blastocystis subtypes were identified in the 63 patients whose samples were successfully subtyped: ST1 (15.9%), ST2 (14.3%), ST3 (47.6%), ST4 (20.6%), and ST7 (1.6%). ST3 was more common in males compared to females (P = 0.049). Comparative molecular analysis of Blastocystis sequences revealed intra-subtype variations within the identified subtypes with the exception of ST4. Among ST4 sequences in this study, as well as in the majority of human GenBank sequences, a limited genetic diversity was found compared to what was found among the other common subtypes (ST1, ST2 and ST3). The relative prevalence of ST4 in this study was comparable to the overall distribution of ST4 in European cohorts (16.5%). This contrasts with the sparse reports of ST4 in studies from other continents, which may indicate that the distribution of this subtype is geographically heterogeneous.

Place, publisher, year, edition, pages
Heidelberg: Springer Berlin/Heidelberg, 2012
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-56679 (URN)10.1007/s10096-011-1416-6 (DOI)000304652800052 ()22350386 (PubMedID)
Available from: 2012-06-27 Created: 2012-06-25 Last updated: 2017-03-14Bibliographically approved
3. High occurrence of Blastocystis sp subtypes 1-3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania
Open this publication in new window or tab >>High occurrence of Blastocystis sp subtypes 1-3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania
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2016 (English)In: Parasites & Vectors, ISSN 1756-3305, E-ISSN 1756-3305, Vol. 9, 370Article in journal (Refereed) Published
Abstract [en]

Background: Blastocystis is a common intestinal parasite with worldwide distribution but the distribution of Blastocystis and its subtypes in East Africa is largely unknown. In this study, we investigate the distribution of Blastocystis subtypes in Zanzibar, Tanzania and report the prevalence of intestinal parasites using both molecular methods and microscopy.

Methods: Stool samples were collected from both diarrhoeic and non-diarrhoeic outpatients in Zanzibar. In addition to microscopy, real-time PCR for Blastocystis, Entamoeba histolytica and E. dispar, Giardia intestinalis, Cryptosporidium spp., and Dientamoeba fragilis was used. Blastocystis subtypes were determined by a conventional PCR followed by partial sequencing of the SSU-rRNA gene. Genetic assemblages of Giardia were determined by PCR with assemblage specific primers.

Results: Intestinal parasites were detected in 85 % of the 174 participants, with two or more parasites present in 56 %. Blastocystis sp. and Giardia intestinalis were the most common parasites, identified by PCR in 61 and 53 % of the stool samples respectively, but no correlation between carriage of Blastocystis and Giardia was found. The Blastocystis subtype distribution was ST1 34.0 %, ST2 26.4 %, ST3 25.5 %, ST7 0.9 %, and 13.2 % were positive only by qPCR (non-typable). The Giardia genetic assemblages identified were A 6.5 %, B 85 %, A + B 4.3 %, and non-typable 4.3 %. The detection rate with microscopy was substantially lower than with PCR, 20 % for Blastocystis and 13.8 % for Giardia. The prevalence of Blastocystis increased significantly with age while Giardia was most prevalent in children two to five years old. No correlation between diarrhoea and the identification of Giardia, Blastocystis, or their respective genetic subtypes could be shown and, as a possible indication of parasite load, the mean cycle threshold values in the qPCR for Giardia were equal in diarrhoeic and non-diarrhoeic patients.

Conclusions: Carriage of intestinal parasites was very common in the studied population in Zanzibar. The most commonly detected parasites, Blastocystis and Giardia, had different age distributions, possibly indicating differences in transmission routes, immunity, and/or other host factors for these two species. In the Blastocystis subtype analysis ST1-3 were common, but ST4, a subtype quite common in Europe, was completely absent, corroborating the geographical differences in subtype distributions previously reported.

Keyword
Zanzibar, Tanzania, Blastocystis, Subtype, Giardia, Assemblage, Real-time PCR, Genotyping
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-124236 (URN)10.1186/s13071-016-1637-8 (DOI)000378838200001 ()27356981 (PubMedID)
Available from: 2016-07-28 Created: 2016-07-28 Last updated: 2017-03-14Bibliographically approved
4. The relation between Blastocystis and the intestinal microbiota in Swedish travellers
Open this publication in new window or tab >>The relation between Blastocystis and the intestinal microbiota in Swedish travellers
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Blastocystis sp. is a unicellular eukaryote that is commonly found in the human intestine. Its ability to cause disease is debated and a subject for ongoing research. Many aspects of Blastocystis are not fully known, including its role in the gut microbiota. In this study, faecal samples from 35 Swedish university students were examined with shotgun metagenomics before and after travel to the Indian peninsula or Central Africa. We aimed at assessing the impact of travel on Blastocystis carriage and seek associations between Blastocystis and the bacterial microbiota. We found a prevalence of Blastocystis of 16/35 (46%) before travel and 15/35 (43%) after travel. The two most commonly found Blastocystis subtypes (STs) were ST3 and ST4, accounting for 65% (20/31) of the samples positive for Blastocystis. No mixed subtype carriage was detected. All ten individuals with a typable ST before and after travel maintained their initial ST. The community composition of the gut bacterial microbiota was not significantly different between Blastocystis-carriers and non-carriers. Interestingly, the presence of Blastocystis was associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, none of which regarded as normal residents of the human intestinal microbiota, but both could be linked to plants. This finding may imply that carriage of Blastocystis is linked to dietary vegetables. Blastocystis carriage was positively correlated to high bacterial genus richness, and negatively correlated to the Bacteroides enterotype. These correlations were both largely driven by ST4, a subtype most commonly described from Europe and Australia, and were not significant for the globally prevalent ST3. Whether the associations of Blastocystis with the bacterial microbiota as described in this study are resulting from the presence of Blastocystis, or are a prerequisite for colonization with Blastocystis, are interesting questions for further studies.

Keyword
Blastocystis; subtype; persistence; travel; microbiota; Sporolactobacillus; Candidatus Carsonella; transmission
National Category
Infectious Medicine Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-132437 (URN)
Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2017-03-15

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