Fulminant Cryptosporidiosis after Near-Drowning: a Human Cryptosporidium parvum Strain Implicated in Invasive Gastrointestinal Adenocarcinoma and Cholangiocarcinoma in an Experimental Model Gabriela Certad,a,b Sadia Benamrouz,a,c Karine Guyot,a Anthony Mouray,d Thierry Chassat,d Nicolas Flament,d Laurence Delhaes,a,e Valerie Coiteux,f Baptiste Delaire,g Marleen Praet,h Claude Cuvelier,h Pierre Gosset,g Eduardo Dei-Cas,a,e and Colette Creusyg Laboratory Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d’Infection et d’Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8402, Université Lille Nord de France, Lille, Francea; Cátedra de Parasitología, Escuela de Medicina José María Vargas, Universidad Central de Venezuela (UCV), Caracas, Venezuelab; Environnement et Santé, FLST, Université Catholique de Lille, Université Lille Nord de France, Lille, Francec; Plateau d’Expérimentation Animale, Institut Pasteur de Lille, Lille, Franced; Parasitologie-Mycologie, Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, Lille, Francee; Service des Maladies du Sang, Hôpital Huriez, Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, Lille, Francef; Service d’Anatomie et de Cytologie Pathologiques, Groupe Hospitalier de l’Université Catholique de Lille, Lille, Franceg; and Academic Department of Pathology, Ghent University, Ghent, Belgiumh
In the present work, we report the characterization of a Cryptosporidium parvum strain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient’s stools was identified as C. parvum II2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulent C. parvum isolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derived C. parvum isolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model.
C
ryptosporidium parvum, an intracellular parasite ubiquitous in nature, is a significant health risk to humans and animals. It causes self-limited watery diarrhea in immunocompetent persons but has devastating effects in immunocompromised patients with AIDS, hematological malignancies, organ transplantation, or cancer or those undergoing chemotherapy (9). Contaminated water is the major source of Cryptosporidium infections for humans. More than 160 waterborne outbreaks have been reported globally, implicating contaminated drinking and recreational water (14, 15, 21). The ingestion of as few as 10 oocysts can cause infection in immunocompetent persons (13). These parameters, together with the well-known resistance of the parasite to chlorine disinfection at concentrations typically applied in drinking water plants, are a risk for water transmission of Cryptosporidium spp. (15, 21). Interestingly, C. parvum has been correlated with digestive carcinogenesis. An epidemiologic study in Poland reported a frequency of 18% of cryptosporidiosis in patients with colorectal cancer (17). However, in this report it was unclear whether C. parvum behaved as a carcinogenesis factor or simply as an opportunistic agent whose development was enhanced by host immunosuppression. More consistent with a potential tumorigenic role of this parasite, we recently showed that IOWA and TUM1 strains of C. parvum of animal origin induced digestive neoplasia in a rodent model (4–6). We report herein the first evidence of the ability of a human-derived C. parvum strain to induce gastrointestinal cancer in mice.
had undergone allogeneic stem cell transplantation (HSCT). Briefly, on day 19 posttransplantation, extensive skin lesions documented as graftversus-host disease (GVHD) grade II occurred, requiring intense immunosuppression with methylprednisolone and tacrolimus. The skin lesions evolved favorably. The patient left the hospital on day 49 posttransplantation. Five days later, he plunged into the Deule River (Lille, France) with his car to commit suicide. He was rescued after nearly drowning and admitted to the hospital. Two days after hospitalization, he developed abdominal pain and severe diarrhea (losing 1 to 2 liters/day). An endoscopy ruled out digestive GVHD. The presence of Cryptosporidium sp. was documented in biopsy specimens of the stomach, duodenum, colon, and rectum, as well as in the stools. Stool microbiological tests for bacteria and viruses were negative. Nitazoxanide was administered, reducing the number of evacuations per day. On day 85 posttransplantation, he developed abdominal pain and distension accompanied by fecal vomiting and interruption of intestinal transit. An abdominal computed tomography scan with contrast revealed pancolitis, without either fluid collection or free air in the peritoneal cavity. Intestinal GVHD was ruled out after colonoscopy. Intestinal transit began spontaneously 2 days later. Stool analysis revealed an uncontrollable cryptosporidial infection (about 500,000 oocysts/g of feces). Fecal cultures were negative for bacteria and viruses. At that time, white blood cell counts were as low as 1,610 cells/mm3. On day 91 posttransplantation, he developed acute respiratory failure with severe hypoxemia, which was treated with imipenem and amikacin. While microbio-
Received 8 August 2011 Accepted 5 January 2012 Published ahead of print 13 January 2012 Address correspondence to Gabriela Certad,
[email protected].
MATERIALS AND METHODS
G.C. and S.B. contributed equally to this article.
C. parvum isolate. The Cryptosporidium isolate was recovered from stool samples from a 51-year-old man with acute lymphoblastic leukemia type B who nearly drowned after being discharged from the hospital where he
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logical tests (bacterial, fungal, and viral cultures, as well as PCRs of blood and fluid samples) remained negative, diarrhea intensity increased again to more than 2 liters/day. He deteriorated rapidly and became confused despite intensive supportive therapies. The patient died on day 100 posttransplantation. Molecular identification of the Cryptosporidium isolate. Genomic DNA was isolated from oocysts present in the patient’s stools, and a fragment of the 18S rRNA gene was amplified by nested PCR (20) and sequenced for isolate identification. Subtyping of C. parvum was based on sequence analysis of the GP60 gene (1). Inoculation of SCID mice with the C. parvum isolate. In order to evaluate the virulence and tumorigenic potential of this C. parvum isolate of human origin, oocysts purified from the patient’s feces were inoculated in SCID mice treated or not with 4 mg/liter dexamethasone (Dex) (Merck, Lyon, France) via drinking water (4–6). Seven-week-old CB17 SCID mice were obtained from a colony bred and regularly controlled for assessment of microbial (including Helicobacter spp.) or parasitological infections at the Pasteur Institute of Lille (France). Animals were housed in groups in covered cages and maintained under aseptic conditions in an isolator with standard laboratory food and water ad libitum. Before inoculation, the samples were screened for the presence of culturable bacteria by plating onto selective or nonselective culture media (Trypticase soy, Trypticase soy and blood, Hektoen, Tergitol 7 with triphenyl tetrazolium chloride [TTC], Difco Pseudomonas isolation agar, and Sabouraud agar). Oocyst viability was assessed by testing excystation (5, 6), and the infectivity of parasites was verified by a preliminary oral inoculation of four SCID mice. These animals developed C. parvum infection and were followed throughout the experiment. Infective doses of 105 oocysts were prepared as described previously (4–6) and inoculated by oral-gastric gavage. Control mice were inoculated with only phosphate-buffered saline (PBS). In addition, the possibility that biotic (e.g., virus) or abiotic factors present in the inoculum could induce neoplastic lesions was ruled out by administering to six Dextreated SCID mice an inoculum from which oocysts were previously removed by filtration using a Nanosep MF tube with a 0.45-m-pore-size membrane. Fecal specimens were collected and processed as previously described (5). Periodically or when signs of imminent death appeared, mice were euthanatized by carbon dioxide inhalation. Experiments were conducted in the animal facility of the Institut Pasteur de Lille (research accreditation no. A59107). Animal protocols were approved by the French regional ethical committee (approval no. CEEA 112011). Histology and immunohistochemistry. Stomach, liver, duodenum, samples of proximal, medium, and distal parts of jejunum, the ileocecal region, and colon were removed, fixed in 10% buffered formalin, processed using standard histological techniques, and embedded in paraffin. Sections 5 m thick were stained with hematoxylin and eosin (H&E). Parasite load in digestive sections was scored as follows: 0, no parasites; ⫹1, small number of parasites, focally distributed; ⫹2, moderate number of parasites, widely distributed; and ⫹3, abundant parasites present, widely distributed throughout the section (4, 5). Lesions at different sites were scored as described previously (4, 5), with slight modifications as follows: 0, no lesion; 1, inflammation and/or regenerative changes; 2, low-grade intraepithelial neoplasia (LGIEN); 3, high-grade intraepithelial neoplasia (HGIEN); 4, suspicion of invasive adenocarcinoma or invasive adenocarcinoma (penetration of dysplastic glands through the muscularis mucosae with desmoplastic stromal response); and 5, adenocarcinoma with the invasion through the submucosa and deeper. (Note that in category 3, adenoma with HGIEN, carcinoma in situ [limited to the epithelium], and intramucosal adenocarcinoma [invasion of the lamina propria] were also included.) The Volgens-Gomori stain (Reticulin) (3) was employed for assessment of basement membrane integrity. A mouse monoclonal antibody to
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cytokeratin (without dilution) (AM071-5 M; Biogenex, Netherlands) was used to detect epithelial cells. An anti-alpha smooth muscle actin monoclonal antibody (dilution 1:100) (M0851; Dako, Denmark) was used to stain muscle fibers. Sections were examined using a Leica DMRB microscope equipped with a Leica digital camera connected to an Imaging Research MCID analysis system (MCID software, Cambridge, United Kingdom). Histological diagnoses were made independently by three pathologists. In order to analyze results, mice were divided into four groups considering postinfection (p.i.) delay at euthanasia or death, as follows: group 1, days 40 to 50 p.i.; group 2, days 51 to 60 p.i.; group 3, days 61 to 90 p.i.; group 4, ⬎90 days p.i. (Table 1).
RESULTS
Numerous oocysts and life cycle stages of Cryptosporidium sp. were observed in the patient’s stools and digestive biopsy specimens, respectively. Sequencing was performed, and C. parvum subtype IIaA15G2R1was identified. After experimental inoculation of Dex-treated SCID mice with oocysts from this patient, numerous parasites were detected in mouse feces from day 1 p.i. until the end of the experiment. The mice presented clinical manifestations, such as bloody diarrhea, ruffled coat, hunched posture, and lethargy, after day 40 p.i. One mouse had a rectal prolapse. A total of 10 Dex-treated mice out of 20 (50%) exhibiting these signs died before planned euthanasia (Table 1). After spontaneous or programmed death, organs were removed for histological examination. Microscopic examination of tissue sections showed intense cryptosporidial presence (scored by microscopic analysis [Table 1]) in the ileocecal region and between moderate and intense presence in the stomach, duodenum, colon, and biliary tree. The tumorigenic potential of this isolate was assessed: in 18 out of 18 (100%) Dex-treated or untreated mice submitted to histological analysis, neoplastic lesions were found in one organ or more (Table 1). Gastric neoplastic lesions located in the antropyloric area were observed in 13 out of 13 (100%) Dex-treated or untreated mice. Lesions evolved from low- and high-grade dysplasia to invasive carcinoma (Fig. 1A to D). Variable C. parvum colonization of the duodenum, intrahepatic bile ducts, and colon was found, and the presence of parasites in these organs coincided with the presence of neoplasia. Particularly, a well-differentiated cholangiocarcinoma associated with a high load of parasites was observed in two Dex-untreated SCID mice (Fig. 1E and F). In agreement with our previous studies (4–6), the most severe lesions were found in the ileocecal region. In this organ, lesions were characterized by an irregularly thickened mucosa containing areas of low- and high-grade dysplasia (dysplasia-associated lesion or mass [DALM]) observed mainly in group 1 (earliest euthanasia) (Fig. 2A), progressing to adenocarcinoma invading beyond the submucosa (latest euthanasia) (Fig. 2E). After day 60 p.i., all Dex-treated mice had invasive adenocarcinoma in the submucosa and deeper (Fig. 2D). In addition, one mouse from group 3 exhibited vascular tumor emboli in the submucosa (Fig. 2F). The invasive nature of lesions was strengthened by the observation of the penetration of dysplastic glands through the muscularis mucosae associated with a desmoplastic stromal response (Fig. 2D). In the group of Dex-untreated mice, ileocecal neoplastic lesions varied in severity, confirming previous observations (4–6). In one Dex-untreated mouse, a well-differentiated adenocarcinoma invading the subserosa was detected (Fig. 2E and Table 1). In addition to these neoplastic lesions, inflammation as well as
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TABLE 1 Development of C. parvum II2A15G2R1 in SCID mice shown by parasite distribution and associated lesions Day of death p.i.b: a
Mouse
Dex treatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Nod No No No
By euthanasia
Histopathological lesion score(s) (parasite score)c Before planned euthanasia
Stomach
Duodenum
Ileocecal region
Colon
Biliary tree
40 40 40
NDe ND ND 3 (⫹3) ND ND 2 (⫹2) ND ND ND 3 (⫹3) 3 (⫹3) ND ND 3 (⫹3) ND 2 (⫹2) 1, 5 (⫹3) 1, 3 (⫹3) 1, 5 (⫹3) 1, 5 (⫹3) 1, 3 (⫹3) 1, 5 (⫹3) 1, 3 (⫹2)
ND ND 1 (⫹3) 1 (0) ND ND 1 (⫹2) ND ND ND 1 (⫹2) 1 (0) 1 (0) 1 (0) 1 (0) ND 1 (0) 3 (⫹3) 3 (⫹3) 2 (⫹2) 1 (⫹3) 0 (0) 0 (⫹2) 0 (0)
ND ND 1, 2 (⫹3) 1, 4 (⫹3) ND 1, 4 (⫹3) 1, 4 (⫹3) 1, 4 (⫹3) ND ND 1, 4 (⫹3) 1, 5 (⫹3) 1, 4 (⫹3) 1, 4 (⫹3) 1, 4 (⫹3) ND 1, 4 (⫹3) 1, 5 (⫹3) 1, 5 (⫹3) 1, 5 (⫹3) 1, 3 (⫹3) 1, 5 (⫹3) 1, 3 (⫹3) 1, 3 (⫹3)
ND ND ND 0 (0) ND 2 (⫹2) 1 (⫹2) ND ND ND 3 (⫹3) 1, 3 (⫹3) 0 (0) 0 (0) 0 (0) ND 0 (0) 3 (⫹3) 1, 2 (⫹2) 2 (⫹2) 0 (0) 3 (⫹3) 3 (⫹3) 2 (⫹2)
ND ND ND 0 (0) ND ND 2 (⫹1) 2 (⫹1) ND ND 0 (⫹1) 0 (0) 0 (0) 0 (0) 2 (⫹1) ND 3 (⫹3) 2 (⫹2) 2 (⫹1) 2 (⫹1) 1, 2 (⫹1) 1, 4 (⫹3) 1, 4 (⫹3) 0 (0)
41 47 48 48 49 49 52 54 55 55 55 55 59 59 62 62 62 91 91 91 91
a
Dexamethasone (Dex; 4 mg/liter of drinking water) administration commenced 2 weeks prior to inoculation and was maintained throughout the experiment. For histopathological analysis, mice were divided into four groups considering p.i. delay at euthanasia or death, as follows: group 1, days 40 to 50; group 2, days 51 to 60; group 3, days 61 to 90; and group 4, ⬎90 days. c Lesions at different sites were scored as follows: 0, no lesion; 1, inflammation and/or regenerative changes; 2, low-grade intraepithelial neoplasia (LGIEN); 3, high-grade intraepithelial neoplasia (HGIEN); 4, suspicion of invasive adenocarcinoma or invasive adenocarcinoma (penetration of dysplastic glands through the muscularis mucosae with desmoplastic stromal response); and 5, carcinoma with the invasion of the submucosa and deeper. (Note that in category 3, adenoma with HGIEN, carcinoma in situ [limited to the epithelium], and intramucosal adenocarcinoma [invasion of the basal membrane of glands] were also included.) Numbers in parentheses include the results of the histological semiquantitative assessment of C. parvum organisms in the host tissues. The presence of parasites in the tissues was scored as follows: 0, no parasites; ⫹1, small number of parasites, focally distributed; ⫹2, moderate number of parasites, widely distributed; and ⫹3, abundant parasites present, widely distributed throughout the tissue. d Oocyst viability before inoculation was assessed by an excystation test followed by a preliminary inoculation of four Dex-untreated SCID mice. These animals were infected and were also followed until the end of the experiment. Control mice (uninfected Dex-treated SCID mice and Dex-treated SCID mice inoculated with an inoculum from which Cryptosporidium oocysts were removed by filtration) were euthanatized periodically as experimental mice. None of the mice in the control groups developed Cryptosporidium infection or histological gastrointestinal lesions. e ND, not done (organ not included in the histological examination). b
degenerative and regenerative changes were observed. A diffuse inflammatory cell infiltrate with clusters of polymorphs involving the lamina propria adjacent to the crypts, the epithelium of the crypts, the crypt lumen, and the submucosa was observed in the ileocecal region. Congestion and dilatation of capillary blood vessels was noted. Ulcerations of the mucosa were covered by a fibrinous exudate and inflammatory cells (similar aspects to those found in the patient’s biopsy specimens). No parasites in the feces, tissues, or lesions were detected in control mice at any time. DISCUSSION
We report herein the isolation and characterization of a virulent C. parvum strain. On the basis of clinical, parasitological, and epidemiological evidence, it can be assumed that this isolate induced a severe cryptosporidiosis in the HSCT recipient as a consequence of near-drowning. No diarrhea or other digestive symptom was noted before the submersion episode. Furthermore, the finding of C. parvum subtype IIaA15G2R1 as a cause of the infection is consistent with a possible ingestion of water contaminated
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with stools from C. parvum-infected animals, even though a mixed infection with other Cryptosporidium species cannot be completely discarded. In fact, the subtype we identified is very common in many areas of the world, including Europe, and human cryptosporidiosis cases due to this subtype are usually linked to zoonotic exposure (7, 19). For example, some studies reported that IIaA15G2R1 is predominant in calves in Portugal, Slovenia, and The Netherlands and is also the major C. parvum subtype in humans in these countries (19). Finally, Cryptosporidium was identified as a causal agent in 67% of recreational water outbreaks of gastroenteritis in the United States between 2005 and 2006 (21). To our knowledge, this is the first evidence of near-drowning as a cause of cryptosporidiosis. In near-drowning events, in addition to the complications directly caused by the submersion, there are indirect causes of morbidity and mortality, including infections. However, reports on the association between neardrowning and gastrointestinal infections are scarce, although a high risk for gut infection would be expected after swallowing water from a contaminated aquatic environment. The fatal outcome of this case, 6 weeks after the submersion
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FIG 1 Gastric and hepatic neoplastic lesions in SCID mice infected with C. parvum IIaA15G2R1 isolated from an HSCT recipient patient. (A) High-grade intraepithelial neoplasia in the antropyloric region of a Dex-treated SCID mouse 51 to 60 days postinfection (p.i.) showing important cellular atypias and presence of numerous parasites (arrows) inside the glands. Stain, hematoxylin and eosin. Bar, 50 m. (B) Intramucosal adenocarcinoma in the stomach of a Dex-treated SCID mouse 51 to 60 days p.i. showing architectural distortion, gland fusion with buds of the glandular epithelium into the lamina propia, and loss of the basement membrane. Stain, Volgens-Gomori. Bar, 50 m. (C) Well-differentiated adenocarcinoma in the gastric region of a Dex-treated SCID mouse 51 to 60 days p.i. showing major cellular atypias (arrow) and numerous mitoses. Stain, hematoxylin and eosin. Bar, 100 m. (D) Well-differentiated gastric adenocarcinoma invading (arrow) through the muscularis (m) in a Dex-treated SCID mouse 61 to 70 days p.i. Stain, hematoxylin and eosin. Bar, 100 m. (E) Well-differentiated bile duct adenocarcinoma of the hepatohilar region of the liver (l) in a Dex-untreated SCID mouse 90 days p.i. Proliferation of glands disposed in desmoplastic stroma was observed. Stain, hematoxylin and eosin. Bar, 400 m. (F) Well-differentiated bile duct adenocarcinoma of the hepatohilar region in a Dex-untreated SCID mouse 90 days p.i. showing cellular atypias, architectural distortion, and parasites (arrow) in the lumen of the glands. Stain, hematoxylin and eosin. Bar, 100 m.
event, may be due to the patient’s immunosuppression status, which could favor C. parvum infection. This diagnosis and its course are in agreement with (i) the fact that cryptosporidiosis remains an opportunistic infection causing significant lifethreatening diarrhea (2), (ii) the observation of passage of more than 2 liters of stool per day as a criterion of fulminant disease (9), and (iii) a rapid progression, comparable with the median of 5 weeks reported in patients with fulminant cryptosporidiosis (2). On the other hand, the extremely high parasite shedding in this patient allowed us to purify a large amount of infective oocysts from stools and to infect SCID mice to test the tumorigenic po-
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tential of a human-derived C. parvum isolate. We recently showed that C. parvum strains of animal origin induce digestive cancer in SCID mice (4–6). After experimental inoculation with the C. parvum clinical isolate, high mortality was observed (Table 1). Additionally, as confirmed by immunohistochemistry, several animals developed adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels. This neoplastic process associated with a C. parvum isolate of high virulence seems to be a good illustration of the well-known dysplasia-cancer pathway (11).
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FIG 2 Ileocecal lesions of SCID mice infected with C. parvum IIaA15G2R1 isolated from an HSCT recipient patient. (A) Intramucosal adenocarcinoma in a Dex-treated SCID mouse 40 to 50 days PI. The mucosa is irregularly thickened, with intraluminal budding in an adenomatous fashion around the entire circumference of the cecum. Stain, hematoxylin and eosin. Bar, 1,500 m. (B) Well-differentiated adenocarcinoma in a Dex-treated SCID mouse 51 to 60 days PI. Architectural distortion of glands and loss of gland differentiation with epithelial atypias consisting of loss of normal polarity, nuclear stratification, prominent nucleoli, and irregularly scattered chromatin were observed. The presence of parasites (arrows) inside the glands is shown. Stain, hematoxylin and eosin. Bar, 50 m. (C) Dex-treated SCID mouse 40 to 50 days PI. Volgens-Gomori stain shows architectural distortion, fusion of glands, loss of the basal membrane, and presence of the neoplastic cells in the lamina propria. Bar, 50 m. (D) Dex-treated SCID mouse 40 to 50 days PI. Interruption (arrows) of the muscularis mucosae (mm) and invasion into the submucosa (sm) by the neoplastic glands are shown (immunohistochemical stain for alpha smooth muscle actin). Bar, 400 m. (E) Dex-treated SCID mouse 90 days PI. A well-differentiated adenocarcinoma invading the subserosa (ss) through the muscularis (m) is shown (immunohistochemical stain for cytokeratin). Bar, 200 m. (F) Dex-treated SCID mouse 51 to 60 days PI. Vascular tumor emboli (arrow) were detected in the submucosa (sm). Stain, hematoxylin and eosin. Bar, 50 m.
In particular, dysplastic changes in the biliary tree after C. parvum infection have been reported in experimental rodents (4, 16). However, this is the first time that well-developed cholangiocarcinoma lesions were found in C. parvum-infected mice. Interestingly, a possible association between human cryptosporidiosis and liver cancer (bile duct carcinoma) was suggested in patients with X-linked hyper-IgM syndrome (8, 18). Development of cholangiocarcinoma, like most tumors, is probably a multistep process dependent on the interaction between host genetics and environmental factors, including infection. Thus, it is widely accepted that metazoan parasites such as Opisthorcis viverrini and Clonorchis sinensis could act as triggers of such carcinoma processes (10).
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In conclusion, C. parvum IIaA15G2R1 was isolated and identified as a cause of fulminant cryptosporidiosis in a near-drowning victim who had undergone HSCT before the submersion episode. Additionally, the results of this study showed for the first time the ability of an isolate of C. parvum of human origin to cause gastrointestinal and biliary adenocarcinomas in an experimental model, providing supplementary evidence of a direct role of this parasite in the induction of digestive cancer (4–6). Furthermore, these findings demonstrate that C. parvum-induced neoplasia is an invasive process that can evolve rapidly in immunosuppressed hosts. We finally highlight that this study is of special interest, con-
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sidering the exposure of large number of animals and persons to this protozoan, which is highly tumorigenic when inoculated in an experimental model. Since mouse models of cryptosporidiosis mimic the course of human cryptosporidiosis (12), a serious exploration of this type of process in humans is needed.
8.
9.
ACKNOWLEDGMENTS This work was supported by the Ministry of Research, France (EA3609/ 4547 Université de Lille 2), Federative Institute of Research 142 (Institut Pasteur de Lille), and the French National Research Agency (grants ANR09-ALIA-009 and ANR-10-ALIA-004). G.C. was supported by a scholarship from the Consejo de Desarrollo Científico y Humanístico of the Universidad Central de Venezuela. S.B. was supported by a scholarship from the Catholic Institute of Lille. Special thanks are extended to David Buob and Emmanuelle Leteurtre (Centre of Biology, Pathology and Cytogenetics, CHRU Lille, France), to Jean-Pierre De Cavel (Platform of Animal Experimentation, Institut Pasteur de Lille, France), and to Elizabeth Baumelou (ICL, Lille, France) for advice and discussion.
REFERENCES 1. Alves M, et al. 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J. Clin. Microbiol. 41:2744 –2747. 2. Blanshard C, Jackson AM, Shanson DC, Francis N, Gazzard BG. 1992. Cryptosporidiosis in HIV-seropositive patients. Q. J. Med. 85:813– 823. 3. Bulckaen H, et al. 2008. Low-dose aspirine prevents age-related endothelial dysfunction in a mouse model of physiological aging. Am. J. Physiol. Heart Circ. Physiol. 294:H1562–H1570. 4. Certad G, et al. 2010. Fulminant cryptosporidiosis associated with digestive adenocarcinoma in SCID mice infected with Cryptosporidium parvum TUM1 strain. Int. J. Parasitol. 40:1469 –1475. 5. Certad G, et al. 2010. Development of Cryptosporidium parvum induced gastro-intestinal neoplasia in SCID mice: severity of lesions is correlated with infection intensity. Am. J. Trop. Med. Hyg. 82:257–265. 6. Certad G, et al. 2007. Cryptosporidium parvum, a potential cause of colic adenocarcinoma. Infect. Agent Cancer 2:22. 7. Chalmers RM, Smith RP, Hadfield SJ, Elwin K, Giles M. 2011. Zoonotic
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10. 11.
12.
13.
14.
15.
16.
17. 18.
19. 20.
21.
linkage and variation in Cryptosporidium parvum from patients in the United Kingdom. Parasitol. Res. 108:1321–1325. Hayward AR, et al. 1997. Cholangiopathy and tumors of the pancreas, liver, and biliary tree in boys with X-linked immunodeficiency with hyperIgM. J. Immunol. 158:977–983. Hunter PR, Nichols G. 2002. Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin. Microbiol. Rev. 15:145–154. Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. 2005. Cholangiocarcinoma. Lancet 366:1303–1314. Luperchio SA, Schauer DB. 2001. Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia. Microbes Infect. 3:333–340. Mead JR, Arrowood MJ, Sidwell RW, Healey MC. 1991. Chronic Cryptosporidium parvum infections in congenitally immunodeficient SCID and nude mice. J. Infect. Dis. 163:1297–1304. Okhuysen PC, Chappell CL, Crabb JH, Sterling CR, DuPont HL. 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J. Infect. Dis. 180:1275–1281. Ramirez NE, Ward LA, Sreevatsan S. 2004. A review of the biology and epidemiology of cryptosporidiosis in humans and animals. Microbes Infect. 6:773–785. Rowan NJ. 27 September 2011, posting date. Defining established and emerging microbial risks in the aquatic environment: current knowledge, implications, and outlooks. Int. J. Microbiol. doi:10.1155/2011/462832. Stephens J, Cosyns M, Jones M, Hayward A. 1999. Liver and bile duct pathology following Cryptosporidium parvum infection of immunodeficient mice. Hepatology 30:27–35. Sulzyc-Bielicka V, et al. 2007. Cryptosporidiosis in patients with colorectal cancer. J. Parasitol. 93:722–724. Tomizawa D, et al. 2004. Allogeneic hematopoietic stem cell transplantation for seven children with X-linked hyper-IgM syndrome: a single center experience. Am. J. Hematol. 76:33–39. Xiao L. 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp. Parasitol. 124:80 – 89. Xiao L, et al. 1999. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl. Environ. Microbiol. 65:3386 – 3391. Yoder JS, Beach MJ. 2010. Cryptosporidium surveillance and risk factors in the United States. Exp. Parasitol. 124:31–39.
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