Development and Evaluation of a Real-Time PCR Assay for Detection and Quantification of Blastocystis Parasites in Human Stool Samples: Prospective Study of Patients with Hematological Malignancies

Abstract

Blastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts-a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders.

Prevalence and Subtype Identification of Blastocystis sp. in Healthy Individuals in the Tunis Area, Tunisia

“…However, the real prevalence of the parasite in this geographic area could greatly be underestimated because nonmolecular methods have a low diagnostic sensitivity compared with qPCR. 5 The present study confirms this observation, since 17 of 100 DLMnegative samples were subsequently shown to be positive for Blastocystis sp. by qPCR.…”

“…1 It has also been shown that prevalence data may depend on detection methods, especially direct-light microscopy (DLM) of fecal smears versus polymerase chain reaction (PCR) assays. 5 For many years, Blastocystis sp. was considered with no clinical relevance mainly because asymptomatic colonization is very common.…”

Frequency of Blastocystosis and Its Association with Clinical Symptoms in 2 Years of Surveillance at "Pedro Kourí" Institute

“…However, prevalence data are largely dependent on the methods used for detection, quantitative PCR being the most sensitive method, meaning that infections by Blastocystis sp. are likely underestimated [7,15].…”

Update on the Molecular Epidemiology and Diagnostic Tools for Blastocystis sp

“…The first study [21] was able to detect ST1, ST3 and ST4 but it is unknown whether other subtypes are able to be detected using this assay and therefore the specificity and sensitivity is unknown. The second study designed a genus-specific PCR which was able to detect all known subtypes so far identified in humans [22]. This test only had 95% specificity though, and the amplicon size of 339 bp is much longer than usually desired for RT-PCR sensitivity.…”

“…There have been two studies where ST1 was the predominant subtype with 51.4% of positive from this group belonging to this group in China, 41% in Brazil [17,33,34] and three others where ST4 was the predominant subtype with 63% in France, 84% in Nepal and 94.1% in Spain, with no ST3 identified in the Spain population [22,35,36]. From South American countries it is seen that there is a predominance of ST1, ST2 and ST3 with no other STs being isolated.…”