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Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
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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. Vol. 64, 1053-1062 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-111507DOI: 10.1099/jmm.0.000129ISI: 000363356800016PubMedID: 26296348OAI: oai:DiVA.org:umu-111507DiVA: diva2:875357
Available from: 2015-12-01 Created: 2015-11-13 Last updated: 2017-03-14Bibliographically approved
In thesis
1. Genetic subtypes in unicellular intestinal parasites with special focus on Blastocystis
Open this publication in new window or tab >>Genetic subtypes in unicellular intestinal parasites with special focus on Blastocystis
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
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:nbn:se:umu:diva-132441 (URN)978-91-7601-682-4 (ISBN)
Public defence
2017-04-07, Hörsal D, byggnad 1D, Norrlands universitetssjukhus, Umeå, 09:00 (English)
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Available from: 2017-03-17 Created: 2017-03-14 Last updated: 2017-03-15Bibliographically approved

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