Real Time PCR for Dientamoeba fragilis: a comparison between molecular and microscopical approach

Abstract

Real time PCR per Dientamoeba fragilis: confronto tra l'approccio molecolare e l'esame microscopico SUMMARY Introduction. Dientamoeba fragilis is an intestinal protozoan parasite that is attracting growing interest. Several authors have associated the presence of this faecal agent to numerous intestinal and systemic clinical symptoms, even though asymptomatic carriers have been reported.Actually, to perform the coproparasitology exam for the identification of D. fragilis it is necessary to recur to an expert microscopist. Recently, several real-time TaqMan assays for the detection of D. fragilis has been reported. In this work we validate an original real-time PCR (TaqMan), re-evaluating the traditional microscopic diagnosis. Moreover, the potential for quantitative information, intrinsic of this molecular technique, was preliminarly explored. Methods. Forty-six carriers of D. fragilis were selected from patients referred to the Service of Microbiology of Padova University Hospital. The parasite was preliminarily detected on several occasions in each patient using traditional microscopy. A control group included forty-two healthy volunteers. Real-time PCR was applied to a stool sample from each subject. On the same sample, gel-electrophoresis amplicon detection PCR and microscopic examination were also performed. Results. The sensitivity of real-time PCR was 100%, whereas microscopy showed 93% sensitivity (direct wet mount on fixed stools and trichrome stain), gel-electrophoresis PCR 76% and Giemsa stain 52%.The specificity of all methods was 100%. On the quantitative side, the frequency distribution of the protozoan load of the stools revealed a near to log-normal pattern is apparent for the data yielded by real-time PCR. Conclusions. The real-time PCR was clearly superior to either the gel electrophoresis PCR or the traditional microscopic examination. Just one unpreserved stool sample was adequate to find the parasite. The entire procedure could be performed within a few hours avoiding toxic dyes.

The effect of Blastocystis sp. and Dientamoeba fragilis on psychological symptom severity in a sample of clinically diverse males and females

“…or D. fragilis, these organisms may have been dead and therefore not exerting their typical influence. Despite this, PCR testing is thought to be best practice for detecting intestinal protozoa (De Canale et al, 2009;Garcia et al, 2018;Padukone et al, 2018). Roberts et al (2011) suggest that the results of studies that rely on microscopy alone should be interpreted with caution.…”

Talking of... Dientamoeba fragilis

“…L'infezione da parte di D. fragilis, così come di altri parassiti, potrebbe quindi avere un ruolo come possibile trigger di queste condizioni patologiche. Quanto da noi osservato conferma che questo protozoo appare in grado di causare manifestazioni cutanee (peraltro da chiarire e confermare) oltre che intestinali e che, nella nostra realtà epidemiologica, prevale in maniera assoluta rispetto a G. duodenalis, altro protozoo patogeno indubbiamente in grado di causare queste patologie (2,5,11). Riteniamo inoltre importante segnalare il frequente riscontro di dientamoebiasi all'interno dei nuclei familiari e di contatti stretti.…”

“…Recenti studi hanno documentato la potenziale patogenicità di questo organismo che causa prevalentemente sintomi gastro-intestinali, come diarrea, vomito e dolori addominali ma anche affaticabilità, prurito e rash cutanei orticarioidi (1, 2, 5, 11). Inoltre, questo parassita è uno dei pochi protozoi in grado di dare anche forme lievi di ipereosinofilia (2,5,11).…”

Amplicon-based next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection of single-celled parasites in human faecal samples

“…Several conventional laboratory methods are available for the detection and differentiation of intestinal eukaryotic protists ( Siala et al, 2015 ). Although considered the gold standard for many decades, microscopic examination is generally insensitive compared with DNA-based methods and require the assistance of highly experienced microscopists ( Blessmann et al, 2002 ; De Canale et al, 2009 ; Julio et al, 2015 ; Morio et al, 2018 ; Sarafraz et al, 2013 ; Stark et al, 2005 ; Verweij et al, 2007 ). Conventional PCRs have limitations such as PCR primer bias and inherent difficulty in detection of parasites in mixed-infection samples.…”