Detection of Dientamoeba fragilis in animal faeces using species specific real time PCR assay

Chan, Douglas; Barratt, Joel; Roberts, Tamalee; Phillips, Owen R.; Šlapeta, Jan; Ryan, Una; Marriott, Deborah; Harkness, John; Ellis, John; Stark, Damien

doi:10.1016/j.vetpar.2016.07.025

Cited by 25 publications

(28 citation statements)

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“…Therefore, PCR is considered to be the reference method for D. fragilis detection [ 22 ]. However, available molecular assays are at risk of false positivity because of cross-reactions [ 6 ]. In our cohort, two samples were positive whereas microscopic examination of the stool sample was negative and these patients had no previous history of dientamoebiasis.…”

Section: Discussionmentioning

confidence: 99%

Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa

et al. 2018

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Although microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections, these techniques are time-consuming and require operators who are experienced and well trained. Molecular biology seems to offer performances at least equivalent in terms of sensitivity and specificity for certain parasites. This study aimed to compare three multiplex PCR assays on 93 prospectively collected positive stools (prospective cohort) and a panel of 12 more Cryptosporidium-positive samples (Cryptosporidium panel). On the prospective cohort, the sensitivity was 89%, 64% and 41% for Giardia sp. detection for BD MaxTM, G-DiaParaTM and RIDA®GENE, respectively and 75%, 100% and 100% for C. parvum/hominis detection. The sensitivity of the RIDA®GENE assay for all Cryptosporidium species was 100%, and for D. fragilis 71%. All the techniques obtained the same results for E. histolytica detection, with one positive sample. All species in the Cryptosporidium panel were identified by the RIDA®GENE PCR. The BD MaxTM and G-DiaParaTM assays detected only C. parvum/hominis with the exception of one positive sample for C. meleagridis. No assay showed satisfactory results for all parasites simultaneously, and the DNA extraction seems to be the critical step. More studies are needed to standardize this procedure.

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Section: Discussionmentioning

confidence: 99%

Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa

et al. 2018

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“…Eukaryotic OTUs in group 2 were predominantly limited to a number of fungi and Chordata, likely remnants from the patient's diet. The laboratory-developed real-time assay was shown to be cross-reactive with T. foetus (11) although no evidence of T. foetus sequences could be found, which is not surprising considering that zoonotic transmission to humans is opportunistic and uncommon (39). Ultimately, 18S diversity profiling ruled out the presence of any closely related trichomonad species that could account for cross-reactivity within the samples.…”

Section: Discussionmentioning

confidence: 92%

“…Considering that four samples from group 2 returned D. fragilis sequences, it was apparent that the Genetic Signatures EasyScreen assay initially missed these samples during routine screening. These four samples from group 2 were identified only using the laboratory-developed real-time assay on the SmartCycler II (Cepheid) platform, although C T values were relatively late, with values between 32 and 37, suggesting a very low parasite load in the original clinical sample, as C T values decrease with dilution of trophozoite concentration (11). The multiple positive results from group 2 highlight the importance of sensitivity cutoff values and fluorescence thresholds being implemented where possible, and where not possible, these thermocyclers and associated analysis platforms must be avoided for particular assays.…”

Section: Discussionmentioning

confidence: 99%

“…These assays offer a highly sensitive alternative to traditional diagnostic methods; however, this high degree of sensitivity brings with it its own difficulties. Cross-reactivity of real-time PCR primers often becomes an issue, as was previously demonstrated by Chan et al (11), who showed that the laboratory-developed real-time PCR assay was not useful for the study of animal specimens because the assay was shown to be nonspecific for D. fragilis, cross-reacting with Trichomonas foetus in animal stools (11).…”

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Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR Assays

2019

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Dientamoeba fragilis is a gastrointestinal trichomonad parasite whose pathogenicity is yet to be determined. The difficulty involved in microscopically diagnosing D. fragilis in feces led to the development of real-time PCR methodologies for the detection of D. fragilis in stool samples. Prevalence studies in Europe show much higher levels of infection where a laboratory-developed real-time assay is the predominant assay for the detection of Dientamoeba fragilis than in regions that use the EasyScreen assay for detection of gastrointestinal pathogens. The aim of this study was to compare a commercially available Dientamoeba fragilis assay (Genetic Signatures EasyScreen assay) to a widely used laboratory-developed real-time PCR method. Two hundred fifty fecal samples were screened using the laboratory-developed real-time assay on four real-time PCR platforms producing a number of discrepant results. Limit-of-detection studies were undertaken to attempt to resolve sensitivity for each platform tested. The presence or absence of Dientamoeba fragilis DNA in discrepant samples was shown using PCR amplicon next-generation sequencing. Eukaryotic 18S diversity profiling was conducted on discrepant samples to identify the presence or absence of additional protozoan species in samples that may be responsible for cross-reactivity seen in these samples. The results revealed the potential for multiple false-positive results when using the laboratory-developed real-time assay across multiple real-time platforms using manufacturer default settings. This report provides recommendations to resolve these issues where possible and suggestions for future prevalence studies, and it emphasizes the EasyScreen assay as the molecular method of choice as well as the need for standardization of detection assays across all nations screening for D. fragilis.

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“…Infection may also be able to affect non-human primates and pigs. D. fragilis has emerged as a neglected cosmopolitan intestinal protozoa [1]. A prevalence of 0-82% has been recorded for the infection worldwide [2].…”

Section: Introductionmentioning

confidence: 99%

Dientamoeba fragilis Infection in Patients with Digestive and Non-Digestive Symptoms: A Case-Control Study

et al. 2020

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In most developing countries, Dientamoeba fragilis infection is an obscure protozoan infection. We aimed to determine a frequency and clinical importance of D. fragilis infection in Taif, Saudi Arabia. A 1-year case control study included patients with gastrointestinal (cases, n=114) or non-gastrointestinal symptoms (controls, n=90). The fecal samples were examined with the classical parasitological methods for intestinal protozoa, and by real time PCR for D. fragilis. The infection by D. fragilis was detected in 5.8% by PCR and in 4.4% patients by microscopy. The infection was identified more in control group (n=9) than in cases (n=3); a sole infection in 11 patients and mixed with Giardia in 1 patient. The other enteric parasites detected were Blastocystis sp. (8.3%), Giardia sp. (5.3%), Cryptosporidium sp. (2.9%), Entamoeba histolytica (1.4%), Entamoeba coli (0.9%) and Hymenolepis nana (0.4%). Our results tend to reinforce the need to increase awareness of D. fragilis infection in Saudi Arabia.

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Detection of Dientamoeba fragilis in animal faeces using species specific real time PCR assay

Cited by 25 publications

References 40 publications

Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa

Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa

Comparison and Recommendations for Use of Dientamoeba fragilis Real-Time PCR Assays

Dientamoeba fragilis Infection in Patients with Digestive and Non-Digestive Symptoms: A Case-Control Study

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