Genetic analysis of Giardia and Cryptosporidium from people in Northern Australia using PCR-based tools☆
Introduction
Gastrointestinal pathogens of humans cause diseases of major socio–economic importance worldwide. For example, in the developed world, waterborne protists, such as Giardia and Cryptosporidium, can be responsible for many cases of diarrhea in some countries (Feng and Xiao, 2011, Jex et al., 2011, Fletcher et al., 2012, Koehler et al., 2014a). Diarrhea has been recognized by the World Health Organization (WHO) as the second leading cause of mortality in children, and a leading cause of malnutrition in those of less than five years of age (e.g., Kosek et al., 2003). Giardia and Cryptosporidium were included in the WHO's Neglected Diseases Initiative (cf. Savioli et al., 2006), in an effort to improve the diagnosis and control of these pathogens worldwide.
The accurate diagnosis of Giardia and Cryptosporidium infections and the diseases that they cause (i.e. giardiasis and cryptosporidiosis) is central to studying their epidemiology. Classical techniques routinely used for diagnosis rely mostly on the microscopic detection of cyst or oocyst stages in feces (e.g., Jex et al., 2008a, Chalmers, 2009, Garcia, 2009, Koehler et al., 2014a). These coproscopic methods are not able to unequivocally identify or distinguish different parasite species based on the morphology of cysts/oocysts, because of a lack of distinguishing morphological features (Cacciò and Pozio, 2006, Stensvold and Nielsen, 2012). Moreover, immunological assays, such as commercial coproantigen detection methods, are often not entirely specific (e.g., Johnston et al., 2003, Weitzel et al., 2006, Jex et al., 2008a, Ndao, 2009, ten Hove et al., 2009, van Lieshout and Verweij, 2010, Koehler et al., 2014a). The limited sensitivity and/or specificity of some of these phenetic methods represent a constraint for the routine diagnosis of infections.
The use of DNA techniques can circumvent this limitation. Some polymerase chain reaction (PCR)-coupled techniques have proven to be useful for the genetic identification and characterization of pathogens from tiny amounts (pg to fg) of parasite DNA in biological matrices, such as feces (cf. Gasser, 2006). While many methods are available, mutation scanning-coupled sequencing (Gasser et al., 2006) can provide a practical tool for both genetic analysis and diagnosis if suitably informative genetic markers are utilized. Markers in the triose phosphate isomerase (tpi) (Giardia), the 60 kDa glycoprotein (gp60) and the small subunit (SSU) of the nuclear ribosomal RNA genes (Cryptosporidium) are particularly reliable for the specific/genotypic identification or characterization of these protists in fecal DNA (reviewed by Jex et al., 2008a, Jex et al., 2011, Koehler et al., 2014a, Abeywardena et al., 2015). In the present study, we used PCR-based mutation scanning and/or sequencing of these markers to genetically characterize Giardia and Cryptosporidium in the feces from a cohort of 695 human outpatients, from parts of the tropical North of Australia, with histories of apparent gastrointestinal illnesses.
Section snippets
Study area, ethics and samples
The study regions were in Northern Australia within the Tropic of Capricorn (Fig. 1). The climate ranges from dry- to wet-tropical. These regions cover an area of 449,190.9 km2 (25.9% of Queensland) and have a population of 269,730 people (5.8% of Queensland), residing predominantly in Townsville (population: 229,210) and Mount Isa (population: 22,628), but also in many small rural and remote communities (Regional Development Australia — http://www.rdatanwq.org.au; Australian Bureau of
Results and discussion
Using three distinct PCRs, amplicons were produced from 695 genomic DNAs from individual fecal samples from humans with histories of gastrointestinal disorders. In total, 13 fecal DNA samples were test-positive for Giardia and three for Cryptosporidium (sample nos. 74 and 441 for pgp60; nos. 74, 334 and 441 for pSSU) (Table 1); none of the test-positive samples had both Giardia and Cryptosporidium. All of the ptpi amplicons were subjected to REF analysis (see Fig. 2); all REF profiles were
Acknowledgments
Funding from the Department of Health (Study Education & Research Committee, SERC), Pathology Queensland, Health Support Queensland, Queensland Government, Australia (project no. 5030) (G.R. et al.) as well as the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) of Australia, Melbourne Water Corporation (R.B.G. et al.) is gratefully acknowledged. Travel support from the University of Veterinary Medicine, Vienna, is gratefully acknowledged (J.E.). This
References (49)
- et al.
A perspective on Cryptosporidium and Giardia, with an emphasis on bovines and recent epidemiological findings
Adv. Parasitol.
(2015) Molecular tools — advances, opportunities and prospects
Vet. Parasitol.
(2006)- et al.
Genetic richness and diversity in Cryptosporidium hominis and C. parvum reveals major knowledge gaps and a need for the application of “next generation” technologies—research review
Biotechnol. Adv.
(2010) - et al.
Cryptosporidium—biotechnological advances in the detection, diagnosis and analysis of genetic variation
Biotechnol. Adv.
(2008) - et al.
Giardia/giardiasis — a perspective on diagnostic and analytical tools
Biotechnol. Adv.
(2014) - et al.
Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children
Trans. R. Soc. Trop. Med. Hyg.
(2008) - et al.
Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008
Exp. Parasitol.
(2010) - et al.
Molecular detection of Cryptosporidium cuniculus in rabbits in Australia
Infect. Genet. Evol.
(2010) - et al.
Molecular-based investigation of Cryptosporidium and Giardia from animals in water catchments in southeastern Australia
Water Res.
(2013) - et al.
High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans
Mol. Cell. Probes
(2009)
Correlation between genotype of Giardia duodenalis and diarrhoea
Int. J. Parasitol.
The role of companion animals in the emergence of parasitic zoonoses
Int. J. Parasitol.
Giardia and Cryptosporidium join the ‘neglected diseases initiative’
Trends Parasitol.
Cryptosporidiosis and Cryptosporidium species in animals and humans: a thirty colour rainbow?
Int. J. Parasitol.
Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples
Clin. Microbiol. Infect.
Molecular epidemiology of cryptosporidiosis: an update
Exp. Parasitol.
High prevalence Giardia duodenalis assemblage B and potentially zoonotic subtypes in sporadic human cases in Western Australia
Int. J. Parasitol.
Advances in the epidemiology, diagnosis and treatment of cryptosporidiosis
Expert Rev. Anti-Infect. Ther.
Advances in diagnosis: is microscopy still the benchmark?
jModelTest 2: more models, new heuristics and parallel computing
Nat. Methods
MUSCLE: multiple sequence alignment with high accuracy and high throughput
Nucleic Acids Res.
Zoonotic potential and molecular epidemiology of Giardia species and giardiasis
Clin. Microbiol. Rev.
Enteric protozoa in the developed world: a public health perspective
Clin. Microbiol. Rev.
Practical Guide to Diagnostic Parasitology
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Note: Nucleotide sequences reported in this paper are available from the GenBank database under accession codes: KT123172 to KT123189.