title: Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.
abstract: Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.
authors.affiliation: Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France.|Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire d'Angers, France; Groupe d'Etude des Interactions Hôte-Pathogène, UPRES-EA 3142, UNAM Université, Université d'Angers, Angers, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Le Govic
authors.affiliation: Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France.
authors.forename: K
authors.initials: K
authors.lastname: Guyot
authors.affiliation: Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France.
authors.forename: G
authors.initials: G
authors.lastname: Certad
authors.affiliation: Unité de pneumologie-allergologie pédiatrique, pôle enfant, clinique de pédiatrie Jeanne de Flandre, CHRU de Lille, Université de Lille, Lille, France.
authors.forename: A
authors.initials: A
authors.lastname: Deschildre
authors.affiliation: Unité de Néphrologie Pédiatrique, CHRU de Lille, Lille, France.
authors.forename: R
authors.initials: R
authors.lastname: Novo
authors.affiliation: Aix-Marseille Université, Faculté de Médecine, UMR MD3, et APHM, Laboratoire de Parasitologie-Mycologie, Hôpital de la Timone, Marseille, France.
authors.forename: C
authors.initials: C
authors.lastname: Mary
authors.affiliation: Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France.|Inserm U995, Université de Lille, Lille, France.
authors.forename: B
authors.initials: B
authors.lastname: Sendid
authors.affiliation: Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France.
authors.forename: E
authors.initials: E
authors.lastname: Viscogliosi
authors.affiliation: Laboratoire de Parasitologie, EA 3800-IRIB, CHRU de Rouen, Rouen, France.
authors.forename: L
authors.initials: L
authors.lastname: Favennec
authors.affiliation: Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France.|Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France.
authors.forename: E
authors.initials: E
authors.lastname: Dei-Cas
authors.affiliation: Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France. emilie.frealle-2@univ-lille2.fr.|Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France. emilie.frealle-2@univ-lille2.fr.
authors.forename: E
authors.initials: E
authors.lastname: Fréalle
authors.affiliation: Laboratoire de Parasitologie-Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d'Ascq, France.
authors.forename: E
authors.initials: E
authors.lastname: Dutoit
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D003457:Cryptosporidiosis / Q000175:diagnosis*; D003458:Cryptosporidium / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005188:False Negative Reactions; D005260:Female; D006801:Humans; D008297:Male; D008853:Microscopy / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S