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.Therapeutic advances in infectious disease. 09/2013;
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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.Asian Pacific Journal of Tropical Medicine 10/2013; 6(10):780-4. · 0.50 Impact Factor
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ABSTRACT: The aim of this study was to evaluate the EasyScreen™ Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia) for the detection and identification of 5 common enteric parasites: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis in human clinical samples. A total of 358 faecal samples were included in the study. When compared to real-time PCR and microscopy, the EasyScreen™ Enteric Parasite Detection Kit exhibited 92-100% sensitivity and 100% specificity and detected all commonly found genotypes and subtypes of clinically important human parasites. No cross reactivity was detected in stool samples containing various other bacterial, viral, and/or protozoan species. The EasyScreen™ PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the 5 most important diarrhoea-causing enteric parasites that infect humans. It should be noted, however, that the EasyScreen™ Kit does not substitute for microscopy or for additional PCRs as it does not detect the pathogenic Coccidia spp. Cystoisospora belli or Cyclospora cayetanensis and it does not differentiate between pathogenic and nonpathogenic Entamoeba spp. This study also highlights the lack of sensitivity demonstrated by microscopy; as such, molecular methods should be considered the diagnostic method of choice for enteric parasites.Diagnostic microbiology and infectious disease 10/2013; · 2.45 Impact Factor