Molecular Identification and Subtype Analysis of Blastocystis

Abstract

Significance Statement: The clinical and public health significance of the single-celled intestinal parasite Blastocystis remains unclear. Multiple subtypes of Blastocystis are known, and molecular characterization of strains in samples from patients, healthy carriers, nonhuman hosts, and the environment is critical to obtaining an increased understanding of Blastocystis epidemiology, including whether subtypes (or strains within subtypes) are potentially linked to disease. Analysis and comparison of data, however, require that standardized methods be employed for generating such data. Lately, "barcoding" (relying on small subunit ribosomal DNA sequencing) coupled with online sequence analysis has gained popularity, appearing robust and simple to use. Here, we provide the protocol for barcoding and an introduction to using online databases for small subunit ribosomal RNA gene sequence analysis.ABSTRACT: Several methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to our knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites. Recently, a publicly available online facility was developed for quick and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes) based on sequence data obtained by barcoding PCR. Moreover, a modified barcoding approach is now available using nested PCR, which enables detection of mixed subtype infections.

Blastocystis sp. Subtype Diversity in Wild Carnivore Species from Spain

“…The high number of positives by PCR that could not be successfully identified as Blastocystis when using primers RD5 and BhRDr could be related to their ability to amplify other eukaryotes (mostly fungi), with no obvious differences in PCR product size, especially when screening DNA extracted directly from faeces (Stensvold 2013). Therefore, barcoding primers are better suited for molecular characterization of already known positive samples and not for screening (Stensvold and Clark 2016). The Sant ın et al (2011) primers perform better for screening faecal specimens for Blastocystis as they produce fewer false positives without compromising specificity and sensitivity.…”

Intestinal parasites in hemodialysis patients from developing countries: A systematic review and meta‐analysis

“…as another parasite with high prevalence rates in hemodialysis patients, which is in line with recent studies conducted in various population groups . Blastocystis is a molecularly divergent microorganism with 17 subtypes (STs) reported among human and animal hosts . Despite the availability of several articles on the distribution pattern of Blastocystis and the risk factors for the infection, there is no strong proof signifying the pathogenic nature of this protozoan in human cases .…”

Genetic diversity and pathogenicity of Blastocystis

“…In recent times, a publicly available online facility was developed for fast and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes), based on sequence data acquired by barcoding PCR [58]. Furthermore, a modified barcoding method is now available using nested PCR, which allows detecting mixed subtype infections [6,58].…”

“…In recent times, a publicly available online facility was developed for fast and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes), based on sequence data acquired by barcoding PCR [58]. Furthermore, a modified barcoding method is now available using nested PCR, which allows detecting mixed subtype infections [6,58]. 'DNA barcoding' is the term used to describe a method proposed for constructing a unique identifier for all living species [59]; Scicluna et al [21] investigated the barcoding region for Blastocystis.…”