Josephine Ng-Hublin

Specific and quantitative detection and identification of Cryptosporidium hominis and C. parvum in clinical and environmental samples.

Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by Cryptosporidium parvum and Cryptosporidium hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1-10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no cross-reactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4%, respectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4-9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18S locus. This qPCR assay should be a valuable tool for the detection and differentiation of C. hominis and C. parvum in both clinical and environmental samples.

Longitudinal prevalence, oocyst shedding and molecular characterisation of Cryptosporidium species in sheep across four states in Australia.

The prevalence of Cryptosporidium in sheep in the eastern states of Australia has not been well described, therefore a study of the prevalence, oocyst concentration, species and subtypes of Cryptosporidium were assessed from lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected from approximately 1182 lambs across the four states and screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) at the actin locus. Positives were typed at the 18S locus and at a second locus using C. parvum and C. hominis specific qPCR primers. The overall prevalence was 16.9% (95% CI: 15.6-18.1%) and of the 576 positives, 500 were successfully genotyped. In general, the prevalence of Cryptosporidium was higher in WA than the eastern states. Cryptosporidium prevalence peaked at 43.9% and 37.1% at Pingelly (WA2) and Arthur River (WA1), respectively during weaning and at Pingelly (WA2) during pre-slaughter (36.4%). The range of oocyst shedding at weaning overall across all states was 63-7.9×10(6) and the median was 3.2 × 10(4) oocysts g(-1). The following species were identified; C. xiaoi (69%-345/500), C. ubiquitum (17.6%-88/500), C. parvum (9.8%-49/500), C. scrofarum (0.8%-4/500), mixed C. parvum and C. xiaoi (2.4%-12/500), C. andersoni (0.2%-1/500) and sheep genotype 1 (0.2%-1/500). Subtyping of C. parvum and C. ubiquitum isolates identified IIa and IId subtype families within C. parvum (with IId as the dominant subtype) and XIIa within C. ubiquitum. This is the first published description of C. parvum subtypes detected in lambs in Australia.

Identification of novel and zoonotic Cryptosporidium species in fish from Papua New Guinea

There is still limited information on the distribution of Cryptosporidium species and genotypes in fish. The present study investigated the prevalence of Cryptosporidium species in cultured freshwater (n=132), wild freshwater (n=206) and wild marine (n=276) fish in Papua New Guinea (PNG) by PCR screening at the 18S rRNA locus. A total of seven fish (2 cultured freshwater, 1 wild freshwater and 4 wild marine fish) were identified as positive for Cryptosporidium. Specifically, Cryptosporidium was found in four different host species (Nile tilapia, Oreochromis niloticus; silver barb, Puntius gonionotus; mackerel scad, Decapterus maracellus and oblong silver biddy, Gerres oblongus), giving an overall prevalence of 1.14% (95% CI: 0.3-2%, n=7/614). Of the seven positive isolates, five were identified as C. parvum and two were a novel piscine genotype, which we have named piscine genotype 8. Piscine genotype 8 was identified in two marine oblong silver biddies and exhibited 4.3% genetic distance from piscine genotype 3 at the 18S locus. Further subtyping of C. parvum isolates at the 60 kDa glycoprotein (gp60) locus identified 3 C. parvum subtypes (IIaA14G2R1, IIaA15G2R1 and IIaA19G4R1) all of which are zoonotic and a C. hominis subtype (IdA15G1). The zoonotic Cryptosporidium were identified in fish samples from all three groups; cultured and wild freshwater and wild marine fish. Detection of Cryptosporidium among aquaculture fingerlings warrants further research to gain a better understanding of the epidemiology of Cryptosporidium infection in cultured fish. The identification of zoonotic Cryptosporidium genotypes in fish from PNG has important public health implications and should be investigated further.

Molecular characterization of Cryptosporidium spp. from wild rats and mice from rural communities in the Philippines

In order to examine the prevalence of Cryptosporidium in wild rodents in the Philippines and understand the role wild rodents play in the transmission of this parasite to humans and livestock, 194 fecal samples from wild rats and mice from Luzon and Mindoro islands were examined. Molecular screening at the 18S and actin gene loci identified an overall prevalence of 25.8% (95%CI: 19.8, 32.5). Sequence and phylogenetic analysis of both loci identified C. parvum, C. muris, C. scrofarum, rat genotypes I-IV and a C. suis-like genotype in the rat-derived isolates and is the first report of C. suis-like and C. scrofarum in rats. Mixed infections were identified in 24% of the Cryptosporidium positive isolates. Rat genotypes II, III and IV showed high intragenotypic variation at the 18S gene locus compared to the actin locus.

Human Cryptosporidiosis Diagnosed in Western Australia: a Mixed Infection with Cryptosporidium meleagridis, the Cryptosporidium Mink Genotype, and an Unknown Cryptosporidium Species

ABSTRACT This report describes a case of cryptosporidiosis from an immunocompetent patient from Perth, Western Australia, suffering from diarrhea and a spectrum of other symptoms. Molecular identification revealed that this patient was infected with three Cryptosporidium species—Cryptosporidium meleagridis, the Cryptosporidium mink genotype, and an unknown Cryptosporidium species.

Investigation of a swimming pool-associated cryptosporidiosis outbreak in the Kimberley region of Western Australia

Cryptosporidiosis is a gastroenteric disease caused by the protozoan parasite Cryptosporidium, which manifests primarily as watery diarrhoea. Transmitted via the faecal-oral route, infection with the parasite can occur through ingestion of water, food or other fomites contaminated with its infective oocyst stage. In the months of November and December 2012, there were 18 notified cases of cryptosporidiosis from Broome, Western Australia. The 5-year average for the Kimberley region for this period is <1 case. Interviews conducted by Broome local government staff on the notified cases revealed that 11/18 cases had been swimming at the Broome public swimming pool. Molecular analyses of extracted DNA performed on 8/18 microscopy-positive faecal samples from interviewed cases and three water samples from different locations at the hypervariable glycoprotein 60 (gp60) gene, identified the C. hominis IbA10G2 subtype in all human samples and one water sample.

Differences in the occurrence and epidemiology of cryptosporidiosis in Aboriginal and non-Aboriginal people in Western Australia (2002 − 2012)

Cryptosporidiosis is a diarrhoeal illness caused by the protozoan parasite Cryptosporidium. In Australia, very little is known about the epidemiology of cryptosporidiosis in Aboriginal peoples. The present study analysed long-term cryptosporidiosis patterns across Western Australia (WA) (2001-2012), combined with genotyping and subtyping data at the 18S and glycoprotein 60 (gp60) loci respectively. Comparison of cryptosporidiosis notifications between Aboriginal and non-Aboriginal people in WA, revealed that notification rates among Aboriginal people were up to 50 times higher compared to non-Aboriginal people, highlighting the burden of the disease in this population. More than 90% of notifications were in Aboriginal children aged 00-04years, who had a notification rate 20.5 times higher than non-Aboriginal children in the same age group. Cryptosporidium hominis was the predominant species infecting both Aboriginal and non-Aboriginal people. However, Aboriginal people were mainly infected with the C. hominis IdA15G1 subtype, whereas non-Aboriginal people were predominantly infected with the IbA10G2 subtype. To control cryptosporidiosis in Aboriginal populations in Australia, effective health interventions/promotions need to be a priority for public health research and action.