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Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk

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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.

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Acknowledgments

We wish to thank Michèle Wauquier and Filoména Naji (Parasitology–Mycology Laboratory of Lille University Hospital Center) for their technical assistance, and the members of the French ANOFEL Cryptosporidium National Network: Ahmed Abou Bacar CHU Strasbourg, Isabelle Accoceberry CHU Bordeaux, Patrice Agnamey CHU Amiens, Adela Angoulvant CHU Bicêtre Paris, Dominique Aubert CHU Amiens, Belkhadi Ghania CHU St Antoine, Paris, Antoine Berry CHU Toulouse, Denis Blanchet CHU Cayenne, Julie Bonhomme CHU Caen, Françoise Botterel CHU Créteil, Marie-Elisabeth Bougnoux CHU Necker, Paris, Pierre Buffet CHU Pitié, Paris, Frédéric Dalle CHU Dijon, Eric Dannaoui HEGP, Paris, Marie-Laure Dardé CHU Limoges, Ludovic De Gentile CHU Angers, Anne Debourgogne CHU Nancy, Monique Debruyne (Cerba, Paris), Brigitte Degeilh CHU Rennes, Magalie Demar CHU Cayenne, Nicole Desbois CHU Fort de France, Guillaume Desoubeaux CHU Tours, Pascal Delaunay CHU Nice, Pierre Flori CHU St Etienne, Gilles Gargala CHU Rouen, Agathe Goubard Biomnis Paris, Frédéric Grenouillet CHU Besançon, Samia Hamane CHU St Louis, Paris, Sandrine Houzé CHU Bichat, Paris, Jamet Deborah CHU Brest, Nathalie Kapel CHU Pitié, Paris, Franck Labbe CH Le Havre, Denis Leméteil Lab. St Valéry en Caux, Denis Magne CHU St Antoine Paris, Pierre Marty CHU Nice, Jean Menotti CHU St Louis, Paris, Laurence Millon CHU Besançon, Christelle Morelle CHU Montpellier, Florent Morio CHU Nantes, Jean-Benjamin Murat CHU Grenoble, Gilles Nevez CHU Brest, Muriel Nicolas CHU Guadeloupe, Philippe Poirier CHU Clermont-Ferrand, Méja Rabodonirina CHU Lyon, Marie-Hélène Rodier CHU Poitiers, Marc Sautour CHU Dijon, Marc Thellier CHU Pitié, Paris, Anne Totet CHU Amiens, Alexis Valentin CHU Toulouse, Isabelle Villena CHU Reims, Hélène Yera CHU Cochin, Paris.

This work was supported by grants from the Lille University Hospital Center, from the University of Lille, Pasteur Institute of Lille, “Centre National de la Recherche Scientifique” (CNRS), and “Institut National de la Santé et de la Recherche Médicale” (Inserm). It was presented at the First French North African Parasitology and Mycology meeting in Rabat, Morocco, in October 2013, and at the European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) in Barcelona, Spain, in May 2014.

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  1. Y. Le Govic

    Present address: 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 and Affiliations

  1. Laboratoire de Parasitologie–Mycologie, CHRU de Lille & Faculté de Médecine de Lille, Université de Lille, Villeneuve-d′Ascq, France

    Y. Le Govic, B. Sendid, E. Dei-Cas, E. Fréalle & E. Dutoit

  2. 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

    K. Guyot, G. Certad, E. Viscogliosi, E. Dei-Cas & E. Fréalle

  3. Unité de pneumologie–allergologie pédiatrique, pôle enfant, clinique de pédiatrie Jeanne de Flandre, CHRU de Lille, Université de Lille, Lille, France

    A. Deschildre

  4. Unité de Néphrologie Pédiatrique, CHRU de Lille, Lille, France

    R. Novo

  5. Aix-Marseille Université, Faculté de Médecine, UMR MD3, et APHM, Laboratoire de Parasitologie-Mycologie, Hôpital de la Timone, Marseille, France

    C. Mary

  6. Inserm U995, Université de Lille, Lille, France

    B. Sendid

  7. Laboratoire de Parasitologie, EA 3800-IRIB, CHRU de Rouen, Rouen, France

    L. Favennec

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  1. Y. Le Govic
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ANOFEL Cryptosporidium National Network

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Correspondence to E. Fréalle.

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Eduardo Dei-Cas is deceased.

E. Fréalle and E. Dutoit contributed equally to this work.

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Le Govic, Y., Guyot, K., Certad, G. et al. Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk. Eur J Clin Microbiol Infect Dis 35, 137–148 (2016). https://doi.org/10.1007/s10096-015-2519-2

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