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Microorganisms
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Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace...
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Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Parasitology".

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 13856

Special Issue Editor

Prof. Dr. Florence Robert-Gangneux

E-Mail Website
Guest Editor
CHU Rennes, Inserm, EHESP IRSET (Institut de Recherche en Santé Environnement et Travail) - UMR_S 1085, University of Rennes, 35000 Rennes, France
Interests: molecular diagnosis of parasitic and fungal infections; immune response to Leishmania parasites; liver parasites; antiparasitic treatment; toxoplasmosis; pneumocystis pneumonia; genotyping

Special Issue Information

Dear Colleagues,

In the era of multiplex panels to screen pathogens in various clinical settings, an increasing number of molecular assays is being marketed to detect intestinal parasites in stools. Some of them offer a completed automated process, including DNA extraction, plate setup, and amplification, whereas others only consist in a multiplex qPCR amplification kit. Regardless, DNA extraction from parasites, particularly cysts and eggs, is a critical step, which needs evaluation before widespread use in clinical laboratories. These assays are very attractive because conventional stool examination for parasites needs immediate direct examination for vegetative forms and concentration methods, which are time-consuming and require microscopic expertise and trained staff. Clinical evaluations of these assays, whether prospective or retrospective, are still scarce, and their performances must be validated before routine use. Additionally, clinical microbiologists should be aware of the potential gaps in parasite species detection, to select the most appropriate procedures according to their local epidemiology, patient profile, and clinical setting.

In this Special issue, I encourage all research or clinical studies evaluating multiplex molecular methods compared to conventional microscopy by trained labs to provide accurate and reliable data on their efficiency and help nonspecialized microbiologists to choose the best-suited system for their own lab and activity.

Prof. Dr. Florence Robert-Gangneux
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • intestinal parasites
  • protozoa
  • helminthes
  • stool
  • molecular diagnosis
  • detection
  • microscopy

Published Papers (5 papers)

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Research

11 pages, 1071 KiB  
Article
Comparative Study of Eleven Mechanical Pretreatment Protocols for Cryptosporidium parvum DNA Extraction from Stool Samples
by Laure Claudel, Nicolas Valeix, Louise Basmaciyan, Bruno Pereira, Damien Costa, Anne Vincent, Stéphane Valot, Loic Favennec and Frederic Dalle
Microorganisms 2021, 9(2), 297; https://doi.org/10.3390/microorganisms9020297 - 2 Feb 2021
Cited by 10 | Viewed by 2730
Abstract
Nowadays, many commercial kits allow the polymerase chain reaction (PCR) detection of Cryptosporidium deoxyribonucleic acid (DNA) in stool samples, the efficiency of which relies on the extraction method used. Mechanical pretreatment of the stools using grinding beads has been reported to greatly improve [...] Read more.
Nowadays, many commercial kits allow the polymerase chain reaction (PCR) detection of Cryptosporidium deoxyribonucleic acid (DNA) in stool samples, the efficiency of which relies on the extraction method used. Mechanical pretreatment of the stools using grinding beads has been reported to greatly improve this extraction step. However, optimization of this key step remains to be carried out. Indeed, many parameters could influence the pretreatment performances, among which the modulation of the speed and duration of the grinding step, in addition to the physicochemical features of the grinding beads, have never been evaluated to date. In this study, eleven commercial mechanical pretreatment matrixes (Lysis matrix tubes®, MP Biomedical, Irvine, CA, USA) composed of beads with different sizes, shapes, and molecular compositions, were evaluated for their performances in improving Cryptosporidium parvum oocyst DNA extraction before amplification by using our routinely used real-time PCR method. As expected, the eleven commercial mechanical pretreatment matrixes showed varying performances depending on the composition, size, and shape. All in all, the best performances were obtained when using the Lysing matrix, including ceramic beads with a median size (diameter of 1.4 mm). Full article
(This article belongs to the Special Issue Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy)
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6 pages, 241 KiB  
Article
Multicenter Evaluation of an ELISA for the Detection of Cryptosporidium spp. Antigen in Clinical Human Stool Samples
by Romy Razakandrainibe, Célia Mérat, Nathalie Kapel, Marc Sautour, Karine Guyot, Gilles Gargala, Jean-Jacques Ballet, Patrice Le Pape, French Cryptosporidiosis Network, Frédéric Dalle and Loïc Favennec
Microorganisms 2021, 9(2), 209; https://doi.org/10.3390/microorganisms9020209 - 20 Jan 2021
Cited by 3 | Viewed by 1839
Abstract
Human cryptosporidiosis remains underdiagnosed, and rapid/accurate diagnosis is of clinical importance. Diagnosis of the Cryptosporidium oocyst in stool samples by conventional microscopy is labor-intensive, time-consuming, and requires skillful experience. Thus, we aimed to evaluate the usefulness of a coproantigen enzyme-linked immunosorbent assay (ELISA) [...] Read more.
Human cryptosporidiosis remains underdiagnosed, and rapid/accurate diagnosis is of clinical importance. Diagnosis of the Cryptosporidium oocyst in stool samples by conventional microscopy is labor-intensive, time-consuming, and requires skillful experience. Thus, we aimed to evaluate the usefulness of a coproantigen enzyme-linked immunosorbent assay (ELISA) test in detecting Cryptosporidium spp. from fecal specimens. For this aim, we evaluated the performances of a commercial ELISA (CoproELISA Cryptosporidium kit, Savyon Diagnostics, Israel) for the detection of Cryptosporidium spp. in random clinical stool samples through a multicenter study. The sensitivity and specificity for coproantigen ELISA were 98.86% and 94.32%, respectively. The coproantigen ELISA results indicate that the simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis, and may be useful for large-scale epidemiological studies of cryptosporidiosis. Full article
(This article belongs to the Special Issue Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy)
8 pages, 675 KiB  
Article
Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
by Céline Nourrisson, Julie Brunet, Pierre Flori, Maxime Moniot, Virginie Bonnin, Frédéric Delbac and Philippe Poirier
Microorganisms 2020, 8(11), 1768; https://doi.org/10.3390/microorganisms8111768 - 11 Nov 2020
Cited by 3 | Viewed by 1961
Abstract
Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and [...] Read more.
Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation. Full article
(This article belongs to the Special Issue Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy)
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16 pages, 535 KiB  
Article
Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples
by Nicolas Valeix, Damien Costa, Louise Basmaciyan, Stéphane Valot, Anne Vincent, Romy Razakandrainibe, Florence Robert-Gangneux, Céline Nourrisson, Bruno Pereira, Emilie Fréalle, Philippe Poirier, Loic Favennec and Frederic Dalle
Microorganisms 2020, 8(9), 1450; https://doi.org/10.3390/microorganisms8091450 - 22 Sep 2020
Cited by 18 | Viewed by 3392
Abstract
Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium [...] Read more.
Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods. Full article
(This article belongs to the Special Issue Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy)
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9 pages, 804 KiB  
Article
Evaluation of the AllplexTM Gastrointestinal Panel—Parasite Assay for Protozoa Detection in Stool Samples: A Retrospective and Prospective Study
by Brice Autier, Jean-Pierre Gangneux and Florence Robert-Gangneux
Microorganisms 2020, 8(4), 569; https://doi.org/10.3390/microorganisms8040569 - 15 Apr 2020
Cited by 18 | Viewed by 3117
Abstract
This study aims at evaluating the performances of the multiplex PCR AllplexTM Gastrointestinal Panel-Parasite Assay (GIPPA), which detects G. duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, B. hominis, and C. cayetanensis, by comparison to microscopy. A retrospective [...] Read more.
This study aims at evaluating the performances of the multiplex PCR AllplexTM Gastrointestinal Panel-Parasite Assay (GIPPA), which detects G. duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, B. hominis, and C. cayetanensis, by comparison to microscopy. A retrospective evaluation was conducted on a series of positive clinical samples (n = 99) stored at −80 °C or at +4 °C. A five-month prospective study was then conducted on all samples sent to our lab for parasite detection (n = 586). In the retrospective cohort, sensitivity was 81% for both G. duodenalis (26/32) and D. fragilis (21/26) and 100% for Cryptosporidium spp. (26/26, including 6 different species), B. hominis (26/26), and C. cayetanensis (4/4). During the prospective study, 95 samples were positive by microscopy and 207 by multiplex PCR assay. The molecular assay showed a significantly higher sensitivity of PCR, especially for G. duodenalis (100% vs. 60.7%, p < 0.01), D. fragilis (97.2% vs. 14.1%, p < 0.001), and B. hominis (99.4% vs. 44.2%, p < 0.001) but also for E. histolytica (100% vs. 50.0%). The sensitivity of the AllplexTM GIPPA on the first stool sample was equivalent to the sensitivity of microscopy on multiple stool samples but inferior to multiplex PCR on multiple stool samples. Taken together, the AllplexTM GIPPA is suitable for the routine detection of protozoa in fecal samples. Full article
(This article belongs to the Special Issue Diagnosis of Intestinal Parasites in Humans: Can Molecular Diagnosis Replace Microscopy)
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