Development and Evaluation of a Genus-Specific, Probe-Based, Internal-Process-Controlled Real-Time PCR Assay for Sensitive and Specific Detection of Blastocystis spp.

Laboratory of Parasitology, Department of Microbiological Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
Journal of clinical microbiology (Impact Factor: 3.99). 03/2012; 50(6):1847-51. DOI: 10.1128/JCM.00007-12
Source: PubMed


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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    • "In this study, the primer set used might not have recognized other existing Blastocystis subtypes, thus, the sensitivity of the method was low. Recently, quantitative real-time PCR assays have been developed and reported to be highly sensitive compared to direct microscopy and culture methods [19] [20] . However, although PCR assays present many advantages, the cost of the assay serves as barrier in routine clinical detection of the parasite, especially in remote and underprivileged areas of developing countries. "
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    ABSTRACT: To compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection of Blastocystis sp. in human stool. Human stool samples were collected from a community in San Isidro, Rodriguez, Rizal, Philippines. These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystis sp. Of the 110 stool samples collected, 28 (25%) were detected positive for the presence of Blastocystis sp. by two or more tests. Culture method detected the highest number of Blastocystis-positive stool samples (n=36), followed by PCR of DNA extracted from culture (n=26), PCR of DNA extracted from stool (n=10), and direct fecal smear (n=9). Compared to culture, the sensitivity of the other detection methods were 66.7% for PCR from culture and 19.4% for both PCR from stool and direct fecal smear. Specificity of the methods was high, with PCR from culture and direct fecal smear having 97.3%, while PCR from stool at 95.9%. In this study, in vitro culture is the best method for detecting Blastocystis sp. in human stool samples.
    Preview · Article · Oct 2013 · Asian Pacific Journal of Tropical Medicine
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    • "A hypothesis to explain differences in the disease caused by Blastocystis sp. is its genetic diversity [Hussein et al. 2008; Tan, 2008; Tan et al. 2010; Scanlan, 2012], although no association was detected between symptoms and Blastocystis subtypes in several studies [Ozyurt et al. 2008; Dogruman- Al et al. 2009; Souppart et al. 2009; Jantermtor et al. 2013]. However, ST4 isolates are more common in symptomatic patients in Sweden, Denmark and Spain [Dominguez-Marquez et al. 2009; Stensvold et al. 2011; Forsell et al. 2012], arguing for an important role of this subtype that needs to be investigated further. "
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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.
    Full-text · Article · Sep 2013 · Therapeutic Advances in Infectious Disease
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    • "Techniques currently in use include microscopy, molecular detection, and xenic in vitro culture [3]. Recent studies suggest certain molecular methods to have highest sensitivities [26,32-34]. In this report we compare sequence-confirmed PCR to advanced microscopy, and describe the prevalence of Blastocystis infection and subtype determination in a large series of patients in The Netherlands. "
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    ABSTRACT: Blastocystis sp. are among the most commonly observed intestinal parasites in routine clinical parasitology. Blastocystis in humans consists of at least 9 genetic subtypes. Different subtypes of Blastocystis may be associated with differences in pathogenicity and symptomatology. Advanced microscopy on two samples and sequence-confirmed PCR on a third sample from the same individual were used for Blastocystis diagnosis and subtype analyses on routine clinical samples in a university hospital. With a combined gold standard of sequence-confirmed PCR and positive advanced microscopy, 107 out of 442 (24.2 %) patients were diagnosed with Blastocystis. infection, which is a high frequency of detection in comparison to previous reports from industrialized countries. The sensitivity of microscopy and sequence-confirmed PCR was 99.1 % (106/107) and 96.3 % (103/107), respectively.Among 103 typable samples, subtype 3 was most abundant (n = 43, 42%), followed by subtypes 1 and 2 (both n = 23, 22%), subtype 4 (n = 12, 12%), and single samples with subtypes 6 (1%) and subtype 7 (1%). The prevalence of Blastocystis infection was 38% in patients from the Department of Tropical Medicine and 18% in patients from other departments. A high prevalence of Blastocystis infection was found with both advanced microscopy and sequence-confirmed PCR in our patient population. Most cases were caused by subtypes ST1, ST2, ST3 and ST4. A significantly higher prevalence was found among patients with a history of recent travel to tropical countries.
    Full-text · Article · Aug 2013 · BMC Infectious Diseases
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