Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp.
ABSTRACT Blastocystis is a common intestinal parasite of unsettled clinical significance, which is not easily detected by standard parasitological methods. The genus comprises at least 13 subtypes (STs) (which likely represent separate species), 9 of which have been found in humans. Recent data indicate that at least one of the subtypes is associated with intestinal disease. A quantitative TaqMan 5' nuclease real-time PCR (TaqMan PCR) including an internal process control (IPC) was developed for the detection of Blastocystis and shown to be applicable to genomic DNAs extracted directly from feces. The assay enabled successful amplification of DNAs from all relevant subtypes within the genus (ST1 to ST9). For assay evaluation, 153 samples previously tested by xenic in vitro culture (XIVC) were screened by the TaqMan assay. A total of 49/51 samples positive by XIVC and 13/102 samples negative by XIVC were positive by the TaqMan assay; samples positive by the TaqMan assay and negative by XIVC were subsequently tested by conventional PCR, and amplicons could be identified to the subtype level by sequencing in 69% of the cases. Compared to the TaqMan assay, XIVC had a sensitivity of 79%. This is the first time that a genus-specific, probe-based, internal-process-controlled real-time PCR assay for the detection Blastocystis has been introduced.
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ABSTRACT: Blastocystis sp. is among the few enteric parasites with a prevalence that often exceeds 5% in the general population of industrialized countries and can reach 30–60% in developing countries. This parasite is frequently found in people who are immunocompromised (patients with human immunodeficiency virus/acquired immunodeficiency syndrome or cancer) and a higher risk of Blastocystis sp. infection has been found in people with close animal contact. Such prevalence in the human population and the zoonotic potential naturally raise questions about the impact of these parasites on public health and has increased interest in this area. Recent in vitro and in vivo studies have shed new light on the pathogenic power of this parasite, suggesting that Blastocystis sp. infection is associated with a variety of gastrointestinal disorders, may play a significant role in irritable bowel syndrome, and may be linked with cutaneous lesions (urticaria). Despite recent significant advances in the knowledge of the extensive genetic diversity of this species, the identification of extracellular proteases as virulence factors and the publication of one isolate genome, many aspects of the biology of Blastocystis sp. remain poorly investigated. In this review, we investigate several biological aspects of Blastocystis sp. (diversity and epidemiology, diagnosis tools and pathophysiology). These data pave the way for the following challenges concerning Blastocystis sp. research: deciphering key biological mechanisms and pathways of this parasite and clarification of its clinical impact in humans.09/2013; DOI:10.1177/2049936113504754
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ABSTRACT: Blastocystis is the most common non-fungal micro-eukaryote of the human intestinal tract and comprises numerous subtypes (STs), nine of which have been found in humans (ST1-ST9). While efforts continue to explore the relationship between human health status and subtypes, no consensus regarding subtyping methodology exists. It has been speculated that differences detected in subtype distribution in various cohorts may to some extent reflect different approaches. Blastocystis subtypes have been determined primarily in one of two ways: 1) sequencing of small subunit ribosomal RNA gene (SSU-rDNA) PCR products and 2) PCR with subtype-specific sequence-tagged site (STS) diagnostic primers.Here, STS primers were evaluated against a panel of samples (n=58) already subtyped by SSU-rDNA sequencing (barcode region), including subtypes for which STS primers are not available, and a small panel of DNAs from 4 other eukaryotes often present in faeces (n=18). While STS primers appeared highly specific, their sensitivity was only moderate, and our results indicate that some infections may go undetected when this method is used. False-negative STS results were not linked exclusively to certain subtypes or alleles and evidence of substantial genetic variation in STS loci was obtained. Since the majority of DNAs included in the study were extracted from faeces, it is possible that STS primers may generally work better with DNAs extracted from Blastocystis cultures. In conclusion, due to its higher applicability and sensitivity, and since sequence information is useful for other forms of research, SSU-rDNA barcoding is recommended as the method of choice for Blastocystis subtyping.Journal of clinical microbiology 10/2012; 51(1). DOI:10.1128/JCM.02541-12 · 4.23 Impact Factor
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ABSTRACT: Blastocystis is a common parasite of the human large intestine but has an uncertain role in disease. In this review, we appraise the published evidence addressing this and its weaknesses. Genetic diversity studies have led to the identification of numerous subtypes (STs) within the genus Blastocystis and, recently, methods for studying variation within STs have been developed, with implications for our understanding of host specificity. The geographic distribution of STs is summarised and the impact this may have on investigations into the role of the organism in disease is discussed. Finally, we describe the organelle and nuclear genome characteristics and look to future developments in the field.Advances in Parasitology 01/2013; 82:1-32. DOI:10.1016/B978-0-12-407706-5.00001-0 · 4.36 Impact Factor