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      Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa Translated title: Comparaison de trois kits commerciaux de PCR multiplex pour la mise en évidence de protozoaires intestinaux

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          Abstract

          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 Max TM, G-DiaPara TM 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 Max TM and G-DiaPara TM 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.

          Translated abstract

          Bien que l’examen microscopique des selles reste la méthode de référence pour le diagnostic des protozooses intestinales, ces techniques sont chronophages et demandent une grande expérience et des opérateurs entrainés. La biologie moléculaire semble offrir des performances au moins équivalentes en termes de sensibilité comme de spécificité pour certains parasites. Cette étude visait à comparer trois techniques de PCR multiplex sur une cohorte de 93 selles positives collectées prospectivement et un panel de 12 échantillons positifs à Cryptosporidium. Respectivement pour BD Max TM, G-DiaPara TM et RIDA ®GENE la sensibilité était de 89 %, 64 % et 41 % pour la détection de Giardia sp. et 75 %, 100 % et 100 % pour la détection de C. parvum/hominis. La sensibilité de la technique RIDA ®GENE pour l’ensemble des espèces de Cryptosporidium était de 100 % et de 71 % pour D. fragilis. Toutes les techniques ont obtenu les mêmes résultats pour la détection d’ E. histolytica (1 échantillon positif). Toutes les espèces de Cryptosporidium ont été détectées par la PCR RIDA ®GENE. Les techniques BD Max TM et G-DiaPara TM ont détecté seulement C. parvum/hominis en dehors d’un échantillon positif à C. meleagridis. Aucun essai n’a montré de résultats satisfaisants pour l’ensemble des parasites simultanément et l’extraction d’ADN semble être l’étape critique. Plus d’études sont nécessaires afin de standardiser cette procédure.

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          Most cited references 27

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          Global, regional, and national age–sex specific all-cause and cause-specific mortality for 240 causes of death, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013

          The Lancet, 385(9963), 117-171
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            Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

            Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.
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              Dientamoeba fragilis, the Neglected Trichomonad of the Human Bowel.

              Dientamoeba fragilis is a protozoan parasite of the human bowel, commonly reported throughout the world in association with gastrointestinal symptoms. Despite its initial discovery over 100 years ago, arguably, we know less about this peculiar organism than any other pathogenic or potentially pathogenic protozoan that infects humans. The details of its life cycle and mode of transmission are not completely known, and its potential as a human pathogen is debated within the scientific community. Recently, several major advances have been made with respect to this organism's life cycle and molecular biology. While many questions remain unanswered, these and other recent advances have given rise to some intriguing new leads, which will pave the way for future research. This review encompasses a large body of knowledge generated on various aspects of D. fragilis over the last century, together with an update on the most recent developments. This includes an update on the latest diagnostic techniques and treatments, the clinical aspects of dientamoebiasis, the development of an animal model, the description of a D. fragilis cyst stage, and the sequencing of the first D. fragilis transcriptome.
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                Author and article information

                Journal
                Parasite
                Parasite
                parasite
                Parasite
                EDP Sciences
                1252-607X
                1776-1042
                2018
                18 September 2018
                : 25
                : ( publisher-idID: parasite/2018/01 )
                Affiliations
                [1 ] Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Rennes 2 rue Henri Le Guilloux 35033 Rennes France
                [2 ] Laboratoire de Parasitologie-Mycologie, CNR Laboratoire expert Cryptosporidiose, Centre Hospitalier Universitaire de Rouen Rouen France
                Author notes
                Article
                parasite180081 10.1051/parasite/2018049
                10.1051/parasite/2018049
                6144649
                30230444
                © B. Autier et al., published by EDP Sciences, 2018

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 2, Tables: 3, Equations: 0, References: 25, Pages: 8
                Categories
                Research Article

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