Elsevier

Infection, Genetics and Evolution

Volume 36, December 2015, Pages 389-395
Infection, Genetics and Evolution

Genetic analysis of Giardia and Cryptosporidium from people in Northern Australia using PCR-based tools

https://doi.org/10.1016/j.meegid.2015.08.034Get rights and content

Abstract

To date, there has been limited genetic study of the gastrointestinal pathogens Giardia and Cryptosporidium in northern parts of Australia. Here, PCR-based methods were used for the genetic characterization of Giardia and Cryptosporidium from 695 people with histories of gastrointestinal disorders from the tropical North of Australia. Genomic DNAs from fecal samples were subjected to PCR-based analyses of regions from the triose phosphate isomerase (tpi), small subunit (SSU) of the nuclear ribosomal RNA and/or the glycoprotein (gp60) genes. Giardia and Cryptosporidium were detected in 13 and four of the 695 samples, respectively. Giardia duodenalis assemblages A and B were found in 4 (31%) and 9 (69%) of the 13 samples in persons of < 9 years of age. Cryptosporidium hominis (subgenotype IdA18), Cryptosporidium mink genotype (subgenotype IIA16R1) and C. felis were also identified in single patients of 11–21 years of age. Future studies might focus on a comparative study of these and other protists in rural communities in Northern Australia.

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

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    Note: Nucleotide sequences reported in this paper are available from the GenBank database under accession codes: KT123172 to KT123189.

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