Human cryptosporidiosis diagnosed in Western Australia a mixed infection with

JCM Accepts, published online ahead of print on 1 May 2013 J. Clin. Microbiol. doi:10.1128/jcm.00424-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 Human cryptosporidiosis diagnosed in Western Australia a mixed infection with Cryptosporidium meleagridis, Cryptosporidium mink genotype and an unknown Cryptosporidium species. 4 5 Authors: Josephine S. Y. Ng-Hublin a*, Barry Combs b, Brian MacKenzie c, Una Ryan a 6 7 8 9 a School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch, WA, 6150, Australia b OzFoodNet Communicable Disease Control Directorate, Perth, Western Australia, Australia c PathWest Laboratory Medicine WA, Locked Bag 2009, Nedlands, WA 6909, Australia 10 11 12 13 14 15 *Corresponding author. Josephine Ng-Hublin Phone: +61 8 93602495 Fax: +61 8 9310 4144 E-mail: j.ng-hublin@outlook.com 16 17 18 19 20 21 Abstract This report describes a case of cryptosporidiosis from an immunocompetent patient from Perth, Western Australia suffering from diarrhoea and a spectrum of other symptoms. Molecular identification revealed that this patient was infected with three Cryptosporidium species - Cryptosporidium meleagridis, Cryptosporidium mink genotype and an unknown Cryptosporidium species.

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Case Report As part of a Cryptosporidium case control study currently being conducted by the Western Australia Department of Health, a 24 year old male resident tested positive for Cryptosporidium and was enrolled in the study. Screening for the presence of other gastroenteric pathogens carried out showed that the patient was negative for the presence of Salmonella spp., Campylobacter spp., Shigella spp., Vibrio spp., verotoxigenic E. coli, Yersinia spp., Plesiomonas spp. and rotavirus. The patient was interviewed using a 20 minute long questionnaire on his illness and known risk factors for Cryptosporidium. The case definition of diarrhoea was three or more loose stools in any 24 hour period and at the time of the interview, the patient had experienced intermittent diarrhoea for 12 days that was ongoing and had contained blood. The most number of stools the patient had in a 24 hour period was 6-10. Other symptoms included fever, chills, vomiting, stomach cramps, nausea, headaches and muscle body aches. The patient reported no history of heart diseases, high blood pressure, asthma, cancer, arthritis or any other chronic illness that may weaken the immune system and was not on any medications (i.e. antibiotics or antacids), suggesting that the patient was immunocompetent. The patient also reported being HIV negative, but it was unknown if a HIV test was actually performed. 39 40 41 42 43 44 45 46 The faecal sample (C011) was submitted to Murdoch University for molecular analysis. Total faecal DNA was extracted using a QIAamp stool DNA extraction kit (Qiagen, Germany). PCR amplification and bi-directional sequencing of the Cryptosporidium 18S rrna, actin and 60 kda-glycoprotein (gp60) gene loci were carried out as previously described (1, 2). Nucleotide sequence analysis was carried out using ChromasPro version v2.3 (http://www.technelysium.com.au) and aligned with reference sequences from GenBank using Clustal W (http://www.genome.jp/tools/clustalw). Phylogenetic analysis, based on evolutionary distances calculated using Kimura-2 parameters and grouped using neighbour

47 48 joining, was conducted using MEGA version 5.05 (3). Sequences generated from the present study have been submitted to GenBank under accession nos. JX472-JX475. 49 50 51 52 53 54 55 56 57 Sequence and phylogenetic analysis of the 18S rrna gene locus, showed % similarity to the Cryptosporidium mink genotype (GenBank accession no. EF428191) (data not shown). However, sequence and phylogenetic analysis at the actin locus identified a mixed infection; C. meleagridis and an unknown Cryptosporidium species, which formed a group with C. parvum, the Cryptosporidium hedgehog genotype, C. hominis, C. cuniculus and C. tyzzeri with genetic similarities of 97.7%, 98.1%, 97.7%, 97.7% and 98.5%, respectively (Figure 1). Sequence and phylogenetic analysis at the gp60 gene locus identified a single novel sequence that exhibited 92.7% similarity to the C. parvum IId subtype (Figure 2). 58 59 60 61 62 63 64 65 66 67 In the twelve days prior to onset of diarrhoea, the patient travelled to Cairns and Brisbane in Queensland and then overseas to Papua New Guinea (PNG). It is understood the patient travelled alone and no secondary cases were reported. In PNG, he trekked in a remote highland region where he drank the river water. During the PNG trip he also stayed on a rural property where he drank unboiled water, which was sourced from the local town s main water supply. At the time he was not aware of anyone else with diarrhoea living in the same residence but did share a toilet with 25 other people. He did not report any contact with domestic, farm or wild animals. In his exposure period he ate raw fruits, fruit juices and raw vegetables and at least some of these were home grown. He also swam in public and commercially operated swimming pools. 68 69 70 Cryptosporidiosis, a gastroenteric disease in humans and animals, is caused by infection with protozoan parasites of the genus Cryptosporidium. Transmission of this

71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 parasite is via the faecal-oral route, usually through ingestion of contaminated water or food. The disease mainly manifests itself as watery diarrhoea with varying severity, abdominal cramps, loss of appetite, nausea, vomiting and low-grade fever (4). Duration of cryptosporidiosis in immunocompetent persons may last from days to weeks and is eventually eliminated by a combined cellular and humoral immune response. For children <5 years old, the elderly or persons with compromised immune systems, the disease can be chronic and debilitating and can lead to severe dehydration and malnourishment (4). Most Cryptosporidium infections are caused by C. hominis and C. parvum, and to a lesser extent C. meleagridis, C. felis and C. canis with recent reports of C. cuniculus as an emerging human pathogen (4, 5). Other Cryptosporidium species and genotypes that have also been detected in humans include C. viatorum, C. fayeri, C. muris, C. andersoni, C. suis, C. bovis, C. ubiquitum, the chipmunk genotype I, the skunk genotype, and the horse genotype (4, 6). In Australia, sporadic cases of human cryptosporidiosis are mainly due to infection with C. hominis, C. parvum and to a lesser extent, C. meleagridis (1, 7). There has been one case of human cryptosporidiosis with C. fayeri, C. andersoni and C. bovis respectively in Australia (6, 8). It is likely that this patient was infected with Cryptosporidium during his travels to PNG through ingestion of contaminated water, however this cannot be confirmed. As three species of Cryptosporidium were identified in this patient, it is difficult to determine if the clinical symptoms were due solely to infection with C. meleagridis, which is a known human pathogen or if the Cryptosporidium mink genotype, the unknown Cryptosporidium species, or other unidentified pathogens were also contributing to the spectrum of symptoms reported. This is the first report of the Cryptosporidium mink genotype in a human as previously it had only been identified in mink (Mustela vison) (9). The unknown Cryptosporidium species identified at the actin gene locus in the present study, was genetically closest to C. tyzzeri (syn. Cryptosporidium mouse genotype I), which mainly

96 97 98 99 infects domestic mice and small rodents (10). Further studies are required to elucidate the identity of this unknown Cryptosporidium species and to determine if it is capable of causing disease in humans. As the gp60 sequence obtained did not match with either the mink genotype or C. meleagridis, it is likely that it corresponds to the unknown genotype identified at the actin locus, however this remains to be confirmed. 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115

116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 References 1. Ng J, MacKenzie B, Ryan U. 2010. Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008. Exp. Parasitol. 125:348-356. 2. Ng J, Pavlasek I, Ryan U. 2006. Identification of novel genotypes of Cryptosporidium from avian hosts. Appl. Environ. Microbiol. 72:7548-7553. 3. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evo. 28:2731-2739. 4. Xiao L, Ryan UM. 2008. Molecular Epidemiology, p. 119-170. In Fayer R, Xiao L (ed.), Cryptosporidium and Cryptosporidiosis, 2nd Edition ed. CRC Press, Boca Raton, FL. 5. Chalmers RM, Elwin K, Hadfield SJ, Robinson G. 2011. Sporadic human cryptosporidiosis caused by Cryptosporidium cuniculus, United Kingdom, 2007-2008. Emerg. Infect. Dis. 17:536-538. 6. Ng JSY, Eastwood K, Walker B, Durrheim DN, Massey PD, Porigneaux P, Kemp R, McKinnon B, Laurie K, Miller D, Bramley E, Ryan U. 2012. Evidence of Cryptosporidium transmission between cattle and humans in northern New South Wales. Exp. Parasitol. 130:437-441. 7. Ryan U, Power M. 2012. Cryptosporidium species in Australian wildlife and domestic animals. Parasitology 139:1673-1688. 8. Waldron LS, Dimeski B, Beggs PJ, Ferrari BC, Power ML. 2011. Molecular Epidemiology, Spatiotemporal Analysis, and Ecology of Sporadic Human Cryptosporidiosis in Australia. Appl. Environ. Microbiol. 77:7757-7765.

140 141 142 143 144 145 9. Wang R, Zhang L, Feng Y, Ning C, Jian F, Xiao L, Zhao J, Wang Y. 2008. Molecular characterization of a new genotype of Cryptosporidium from American minks (Mustela vison) in China. Vet. Parasitol. 154:162-166. 10. Ren X, Zhao J, Zhang L, Ning C, Jian F, Wang R, Lv C, Wang Q, Arrowood MJ, Xiao L. 2012. Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus). Exp. Parasitol. 130:274-281. 146 147 148 149 150 151 152 153 154 155 156 157 158 159

160 161 162 Figure 1: Phylogenetic relationships of C011 with other Cryptosporidium spp. at the actin gene locus inferred by neighbour-joining analysis based on genetic distances calculated using Kimura-2 parameters. Bootstrap values from 0 pseudoreplicates of >60% are shown. 163 164 165 166 167 Figure 2: Phylogenetic relationships of C011 and other Cryptosporidium spp. gp60 subtype families inferred by neighbour-joining (NJ) analysis based on genetic distances calculated using Kimura-2 parameters. C. fayeri gp60 sequence was used as an outgroup to generate the NJ tree. Bootstrap values from 0 pseudoreplicates of >50% are shown. 168 169

0.1 77 78 77 66 91 Plasmodium falciparum 96 66 99 87 Hedgehog genotype C. parvum C011 98 99 C. hominis C. cuniculus 94 C. tyzzeri Ferret genotype Mink genotype C. meleagridis C011 C. wrairi C. fayer i 87 60 78 C. baileyi Skunk genotype C. viatorum unknown Deer mouse genotype C. ubiquitum Mouse genotype II C. suis C. varanii Bear genotype Snake genotype C. felis Coyote genotype C. canis C. macropodum C. galli Fox genotype Goose genotype Pig genotype II Deer genotype C. xiaoi C. bovis Tortoise genotype Gecko genotype C. serpentis C. andersoni C. muris C. molnari C. ryanae

0.1 72 99 86 96 C. fayeri IVf Horse genotype VIb 79 Horse genotype VIa 71 C. parvum IIm 94 C. parvum IIi C. hominis Id 96 C. parvum IIl C. parvum IIg 59 C. parvum IIa 98 C. tyzzeri IXaA6 C. parvum IIf C. tyzzeri IXbA6 51 99 C. cuniculus Va 79 C. hominis Ih C. hominis Ig C. hominis Ia Mink genotype Xa 88 69 Ferret genotype VIIIa C. wrairi VIIa C. parvum IId 56 C011 79 C. parvum IIa C. cuniculus Vb 85 C. hominis IbA9G2 83 C. hominis IbA10G2 89 C. hominis Ie 97 C. hominis If 91 C. parvum IIh C. parvum IIc C. meleagridis IIIbA20G1 C. meleagridis IIIbA13G1 60 C. meleagridis IIIe C. meleagridis IIIa C. meleagridis IIId C. fayeri IVb C. fayeri IVa C. fayeri IVe C. fayeri IVd C. fayeri IVc