Gastroenteritis caused by the Cryptosporidium hedgehog genotype in an immunocompetent

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1 JCM Accepts, published online ahead of print on 16 October 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved Title Gastroenteritis caused by the Cryptosporidium hedgehog genotype in an immunocompetent man Running title Human Cryptosporidium hedgehog genotype infection Names of authors Martin Kváč 1,2,*, Kamila Saková 3, Dana Kv to ová 1, Marta Kicia 4, Maria Wesołowska 4, John McEvoy 5, Bohumil Sak 1 Author affiliations: 1 Biology Centre Academy of Sciences of the Czech Republic, České Bud jovice, Czech Republic; 2 University of South Bohemia, Faculty of Agriculture, České Bud jovice, Czech Republic; 3 Hospital Tábor, Tábor, Czech Republic; 4 Department of Biology and Medical Parasitology, Wrocław Medical University, Poland; 5 Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND, USA *Corresponding author: Mailing address: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, České Bud jovice, Czech Republic; Phone: Fax: kvac@paru.cas.cz Abstract The Cryptosporidium hedgehog genotype, which has been reported previously in hedgehogs and horses, was identified as the cause of the diarrheal disease cryptosporidiosis in an immunocompetent man in the Czech Republic. This is the first report of human illness caused by the Cryptosporidium hedgehog genotype. CASE REPORT 1

2 De-identified diarrheal and non-diarrheal human stool samples from immunocompetent (n=2,956) and immunodeficient patients (n=561; HIV/AIDS and oncology patients) were obtained from public hospitals in the Czech Republic and Poland. In 2012, a 26-year-old man from an urban area, who was HIV negative and had no history of immunodeficiency was admitted to hospital in the Czech Republic with gastroenteritis. The patient had abdominal pain and a non-bloody stool of soft consistency. The classification of the stool as soft was based on criteria described by Rossignol et al. (13). The individual also was examined for the presence of exogenous protozoan stages, helminth eggs, Shigella, Salmonella, Campylobacter, Clostridium, amoebas, rotaviruses, and noroviruses. No other bacterial, viral, or parasitic pathogens were detected (data not shown). The stool sample was examined for the presence of Cryptosporidium oocysts using aniline-carbol-methyl violet staining and light microscopy at 1,000 magnification (11), and the infection intensity was estimated using a method described previously (9). Cryptosporidium oocysts with typical staining characteristics were identified in fecal smears. The infection intensity of Cryptosporidium was estimated to be 30,000 oocysts per gram. To identify the Cryptosporidium sp., DNA was extracted and a nested PCR protocol was used to amplify a partial sequence of the small subunit (SSU) rrna (8), Cryptosporidium 60- kda glycoprotein (gp60) (1) and actin genes (15). PCR amplicons were sequenced directly in both directions using an ABI 3130 sequence analyzer (Applied Biosystems, Foster City, CA). The identity of obtained sequences was determined by a BLAST search ( The relationships of identified sequences to those from known species and genotypes were inferred from Neighbor-Joining (NJ) phylogenies constructed using the software Treecon. Actin, gp60, and SSU rrna sequences have been deposited in GenBank under the accession numbers KF055452, KF055453, and KF055454, respectively. 2

3 The SSU rrna sequence was identical to a sequence from a Cryptosporidium hedgehog genotype isolated from horses in Algeria (accession no. KC305650) (Figure 1). The actin sequence clustered with sequences previously obtained from European hedgehogs (accession no. GQ and GQ214079), sharing % similarity (Figure 1). An established nomenclature (14) was used to describe subtype families and subtypes of gp60 gene sequences. Sequences of gp60 clustered with the XIIIa subtype family originally reported from European hedgehogs (accession nos. GQ259140, GQ214085, and GQ214081) and from a horse (KC305644) (13, 14) (Figure 1). The gp60 subtype of the identified hedgehog genotype had ten repeats of the sequence ACATCA immediately following 20 serine-coding TCA repeats. This subtype has not been detected previously in hedgehogs, horses, or other hosts. The study was approved by the ethics committee of the Institute of Parasitology, Biology Centre Academy of Sciences of the Czech Republic, České Bud jovice, Czech Republic (protocol 1/2012). Zoonotic pathogens are responsible for about 75% of new diseases affecting humans (10). Cryptosporidium is a globally distributed protozoan parasite, infecting humans, domestic animals, and many wild vertebrates (7). It is a significant cause of disease in developing and developed countries. In most cases, including those involving humans, Cryptosporidium causes an enteritic infection leading to a gastrointestinal illnesses characterized by severe diarrhea (17). Although more than 90% of human infections are caused by two species, Cryptosporidium hominis and Cryptosporidium parvum, the increasing application of molecular diagnostic tools during the last decade has uncovered greater than 17 other Cryptosporidium species and genotypes capable of infecting humans (2, 5, 6, 12, 16, 18). Furthermore, infection by these atypical cryptosporidia can occur in immunocompetent hosts (3). In the present study, we report an unusual case of human cryptosporidiosis caused by the Cryptosporidium hedgehog genotype. 3

4 Gastroenteritis caused by the Cryptosporidium hedgehog genotype was detected in an immunocompetent 26-year-old man from the Czech Republic. The hedgehog genotype has previously been reported in hedgehogs and horses; however, it may not be pathogenic for these non-human hosts. Horses in Algeria showed no clinical signs of infection. Although a high prevalence of Cryptosporidium infection was reported in diarrheal hedgehogs in Germany, any association between diarrhea and the hedgehog genotype was unclear due to the frequent occurrence of coinfections with other parasite species (4). The patient in the present study did not report contact with hedgehogs or horses, and the source of infection is unknown. Nevertheless, occurrence of this genotype in horses, hedgehogs, and humans indicates broader host specificity than previously thought. Passive transport of oocysts through the digestive tract is unlikely considering the high infection intensity and manifestation of clinical disease. The infection intensity, disease symptoms, and stool consistency are similar to those described in a previous report of human cryptosporidiosis caused by C. parvum and C. tyzzeri (12). Moreover, identification of the infection in an immunocompetent patient underlines the zoonotic potential of this genotype, particularly among people who have contact with horses or hedgehogs. Although it is possible that the hedgehog genotype can be transmitted indirectly via food or water, there is currently no evidence to support these transmission routes. Generally, the importance of unusual genotypes in humans warrants further investigation. Acknowledgments This study was funded by grant of the Ministry of Education, Youth and Sports of the Czech Republic (LH11061) and by grant of the National Science Centre, Poland (DEC- 2012/05/D/NZ6/00615). References 4

5 Alves, M., L. Xiao, I. Sulaiman, A. A. Lal, O. Matos, and F. Antunes Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J. Clin. Microbiol. 41: Azami, M., D. D. Moghaddam, R. Salehi, and M. Salehi The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rrna gene. Mol. Biol. (Mosk). 41: Chalmers, R. M., K. Elwin, A. L. Thomas, and D. H. Joynson Infection with unusual types of Cryptosporidium is not restricted to immunocompromised patients. J. Infect. Dis. 185: Dyachenko, V., Y. Kuhnert, R. Schmaeschke, M. Etzold, N. Pantchev, and A. Daugschies Occurrence and molecular characterization of Cryptosporidium spp. genotypes in European hedgehogs (Erinaceus europaeus L.) in Germany. Parasitology 137: Elwin, K., S. J. Hadfield, G. Robinson, and R. M. Chalmers The epidemiology of sporadic human infections with unusual cryptosporidia detected during routine typing in England and Wales, Epidemiol. Infect. 140: Elwin, K., S. J. Hadfield, G. Robinson, N. D. Crouch, and R. M. Chalmers Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, Int. J. Parasitol. 42: Fayer, R Taxonomy and species delimitation in Cryptosporidium. Exp. Parasitol. 124: Jiang, J., K. A. Alderisio, and L. Xiao Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl. Environ. Microbiol. 71:

6 Kváč, M., Z. Ondráčková, D. Kv to ová, B. Sak, and J. Vítovec Infectivity and pathogenicity of Cryptosporidium andersoni to a novel host, southern multimammate mouse (Mastomys coucha). Vet. Parasitol. 143: Merianos, A Surveillance and response to disease emergence. Curr. Top. Microbiol. Immunol. 315: Miláček, P., and J. Vítovec Differential staining of cryptosporidia by anilinecarbol-methyl violet and tartrazine in smears from feces and scrapings of intestinal mucosa. Folia Parasitol. (Praha). 32: Rašková, V., D. Kv to ová, B. Sak, J. McEvoy, A. Edwinson, B. Stenger, and M. Kváč Human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum isolates presumably transmitted from wild mice. J. Clin. Microbiol. 51: Rossignol, J. F., A. Ayoub, and M. S. Ayers Treatment of diarrhea caused by Cryptosporidium parvum: a prospective randomized, double-blind, placebo-controlled study of Nitazoxanide. J. Infect. Dis. 184: Sulaiman, I. M., P. R. Hira, L. Zhou, F. M. Al-Ali, F. A. Al-Shelahi, H. M. Shweiki, J. Iqbal, N. Khalid, and L. Xiao Unique endemicity of cryptosporidiosis in children in Kuwait. J. Clin. Microbiol. 43: Sulaiman, I. M., A. A. Lal, and L. Xiao Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus. J. Parasitol. 88: Waldron, L. S., C. Cheung-Kwok-Sang, and M. L. Power Wildlife-associated Cryptosporidium fayeri in human, Australia. Emerg. Infect. Dis. 16: Wielinga, P. R., A. de Vries, T. H. van der Goot, T. Mank, M. H. Mars, L. M. Kortbeek, and J. W. van der Giessen Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands. Int. J. Parasitol. 38:

7 Xiao, L Molecular epidemiology of cryptosporidiosis: an update. Exp. Parasitol. 124: Figure 1. Phylogenetic relationships between Cryptosporidium hedgehog genotype (highlighted) and other Cryptosporidium spp. as inferred by a neighbor-joining analysis of (A) gp60 (743 base positions in the final dataset), (B) the SSU (750 base positions in the final dataset), and (C) actin (782 base positions in the final dataset) genes. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). Numbers at the nodes represent bootstrap values for the nodes gaining more than 50% support. Scale bar included in each tree 7

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