Karine Guyot

MOLECULAR CHARACTERIZATION OF CRYPTOSPORIDIUM ISOLATES OBTAINED FROM HUMANS IN FRANCE

ABSTRACT Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification ofCryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whomCryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes ofCryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range ofCryptosporidium species and genotypes.

Pneumocystis carinii f. sp. hominis Is Not Infectious for SCID mice

ABSTRACT The infectious power of Pneumocystis carinii f. sp. hominis was explored by inoculating SCID mice intranasally with either P. carinii f. sp. hominis or P. carinii f. sp. muris isolates. Only mice inoculated with mouse parasites developed Pneumocystis pneumonia, as assessed by microscopy and PCR. These results suggest that humans do not contract pneumocystosis from animals.

Molecular characterization of Cryptosporidium isolates from humans and animals in Iran.

ABSTRACT Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.

Cryptosporidium parvum, a potential cause of colic adenocarcinoma

BackgroundCryptosporidiosis represents a major public health problem. This infection has been reported worldwide as a frequent cause of diarrhoea. Particularly, it remains a clinically significant opportunistic infection among immunocompromised patients, causing potentially life-threatening diarrhoea in HIV-infected persons. However, the understanding about different aspects of this infection such as invasion, transmission and pathogenesis is problematic. Additionally, it has been difficult to find suitable animal models for propagation of this parasite. Efforts are needed to develop reproducible animal models allowing both the routine passage of different species and approaching unclear aspects of Cryptosporidium infection, especially in the pathophysiology field.ResultsWe developed a model using adult severe combined immunodeficiency (SCID) mice inoculated with Cryptosporidium parvum or Cryptosporidium muris while treated or not with Dexamethasone (Dex) in order to investigate divergences in prepatent period, oocyst shedding or clinical and histopathological manifestations. C. muris-infected mice showed high levels of oocysts excretion, whatever the chemical immunosuppression status. Pre-patent periods were 11 days and 9.7 days in average in Dex treated and untreated mice, respectively. Parasite infection was restricted to the stomach, and had a clear preferential colonization for fundic area in both groups. Among C. parvum-infected mice, Dex-treated SCID mice became chronic shedders with a prepatent period of 6.2 days in average. C. parvum-inoculated mice treated with Dex developed glandular cystic polyps with areas of intraepithelial neoplasia, and also with the presence of intramucosal adenocarcinoma.ConclusionFor the first time C. parvum is associated with the formation of polyps and adenocarcinoma lesions in the gut of Dex-treated SCID mice. Additionally, we have developed a model to compare chronic muris and parvum cryptosporidiosis using SCID mice treated with corticoids. This reproducible model has facilitated the evaluation of clinical signs, oocyst shedding, location of the infection, pathogenicity, and histopathological changes in the gastrointestinal tract, indicating divergent effects of Dex according to Cryptosporidium species causing infection.

Cryptosporidium Population Genetics: Evidence of Clonality in Isolates from France and Haiti

TRAMY NGOUANESAVANH, KARINE GUYOT, GABRIELA CERTAD, YVES LE FICHOUX, CHRISTOPHE CHARTIER, ROSE-IRENE VERDIER, JEAN-CHARLES CAILLIEZ, DANIEL CAMUS, EDUARDO DEI-CAS and ANNE-LAURE BAÑULS Ecologie du Parasitisme (EA 3609), IFR 142, Institut Pasteur de Lille, 1 rue du Professeur Calmette-BP245, 59019 Lille Cedex, France, and Cátedra de Parasitologı́a, Escuela de Medicina ‘‘José Marı́a Vargas’’, Universidad Central de Venezuela, Caracas, Venezuela, and Parasitologie-Mycologie, Centre Hospitalier Universitaire de Nice, France, and AFSSA Laboratoire d’études et de recherches caprines, Niort, France, and Centre GHESKIO, Port au Prince, Haı̈ti, and Laboratoire Environnement et Santé, Université Catholique de Lille, France, and Parasitologie-Mycologie, Centre Hospitalier Régional et Universitaire de Lille, France, and Génétique et Evolution des Maladies Infectieuses, IRD, Montpellier, France

Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

ABSTRACT 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.

Fulminant cryptosporidiosis associated with digestive adenocarcinoma in SCID mice infected with Cryptosporidium parvum TUM1 strain.

We recently demonstrated that Cryptosporidium parvum IOWA strain induces in situ ileo-caecal adenocarcinoma in an animal model. Herein, the ability of another C. parvum strain to induce digestive neoplasia in dexamethasone-treated SCID mice was explored. SCID mice infected with C. parvum TUM1 strain developed a fulminant cryptosporidiosis associated with intramucosal adenocarcinoma, which is considered an early histological sign of invasive cancer. Both evidence of a role of C. parvum in adenocarcinoma induction and the extended prevalence of cryptosporidiosis worldwide, suggest that the risk of C. parvum-induced gastro-intestinal cancer in humans should be assessed.

Cryptosporidium parvum Infection in SCID Mice Infected with Only One Oocyst: qPCR Assessment of Parasite Replication in Tissues and Development of Digestive Cancer

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 105 oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

Molecular Epidemiology of Blastocystis sp. in Various Animal Groups from Two French Zoos and Evaluation of Potential Zoonotic Risk.

Blastocystis sp. is a common intestinal parasite infecting humans and a wide range of animals worldwide. It exhibits an extensive genetic diversity and 17 subtypes (STs) have thus far been identified in mammalian and avian hosts. Since several STs are common to humans and animals, it was proposed that a proportion of human infections may result from zoonotic transmission. However, the contribution of each animal source to human infection remains to be clarified. Therefore, the aim of this study was to expand our knowledge of the epidemiology and host specificity of this parasite by performing the largest epidemiological survey ever conducted in animal groups in terms of numbers of species screened. A total of 307 stool samples from 161 mammalian and non-mammalian species in two French zoos were screened by real-time PCR for the presence of Blastocystis sp. Overall, 32.2% of the animal samples and 37.9% of the species tested were shown to be infected with the parasite. A total of 111 animal Blastocystis sp. isolates were subtyped, and 11 of the 17 mammalian and avian STs as well as additional STs previously identified in reptiles and insects were found with a varying prevalence according to animal groups. These data were combined with those obtained from previous surveys to evaluate the potential risk of zoonotic transmission of Blastocystis sp. through the comparison of ST distribution between human and animal hosts. This suggests that non-human primates, artiodactyls and birds may serve as reservoirs for human infection, especially in animal handlers. In contrast, other mammals such as carnivores, and non-mammalian groups including reptiles and insects, do not seem to represent significant sources of Blastocystis sp. infection in humans. In further studies, more intensive sampling and screening of potential new animal hosts will reinforce these statements and expand our understanding of the circulation of Blastocystis sp. in animal and human populations.

Cryptosporidium infection in a veal calf cohort in France: molecular characterization of species in a longitudinal study

Feces from 142 animals were collected on 15 farms in the region of Brittany, France. Each sample was directly collected from the rectum of the animal and identified with the ear tag number. Animals were sampled three times, at 5, 15 and 22 weeks of age. After DNA extraction from stool samples, nested PCR was performed to amplify partial 18S-rDNA and 60 kDa glycoprotein genes of Cryptosporidium. The parasite was detected on all farms. One hundred out of 142 calves (70.4%) were found to be parasitized by Cryptosporidium. Amplified fragments were sequenced for Cryptosporidium species identification and revealed the presence of C. parvum (43.8%), C. ryanae (28.5%), and C. bovis (27%). One animal was infected with Cryptosporidium ubiquitum. The prevalence of these species was related to the age of the animal. C. parvum caused 86.7% of Cryptosporidium infections in 5-week-old calves but only 1.7% in 15-week-old animals. The analysis of the results showed that animals could be infected successively by C. parvum, C. ryanae, and C. bovis for the study period. C. parvum gp60 genotyping identifies 6 IIa subtypes of which 74.5% were represented by IIaA15G2R1. This work confirms previous studies in other countries showing that zoonotic C. parvum is the dominant species seen in young calves.