A New Era in Parasitology

Jennifer O’ Leary

Dr Roy Sleator & Dr Brigid Lucey

Introduction

• Enteric parasitology encompasses the diagnosis of a wide range of species

• Cryptosporidiumspp & Giardia lamblia commonly encountered in Irish clinical microbiology labs

Cryptosporidium spp

• Obligate enteric protozoan parasite

• Faecal-oral transmission

• C. parvum & C. hominis –90%

• Cramps, fever, nausea, vomiting & diarrhoea.

• Ireland – highest infection rate in Europe

Giardia lamblia

• Flagellate protozoan parasite

• Broad spectrum of symptoms

• Chronic sequelae

• Contracted via faecal-oral route

• Travel associated infection

Current Diagnostic Techniques

• Brightfield & fluorescent staining for Cryptosporidium

• Wet-prep and special stains employed for other parasites

• Limits speciation

• Immuno-based methods also available

Molecular Diagnostics• Real time PCR

• Commercial kits currently limited to a small number of parasitic species

- Cryptosporidium spp- Giardia duodenalis

• Commercial kits and in-house assays (Hadfield et al., 2011; Manser et al., 2013)

• EntericBio® Gastro Panel 2 real time PCR kit, Clinical Microbiology Department, CUH

• Roche LightMix ® Modular assay

Microscopy vs Real Time PCR• GastroPanel II (1) Validation – 100% correlation

• 104 samples tested- 22 samples requested OCP: 17 tested, 5 lacked relevant

clinical data- 2 non-requested samples positive

• 6 positive samples detected by both PCR & microscopy- 5 cases of Giardia duodenalis- 1 case of Cryptosporidium parvum/hominis

• 4 positive samples detected by microscopy alone:- 3 Entamoeba coli cases detected - 1 co-infection of Entamoeba histolytica/dispar and Giardia duodenalis detected

• RIDA®GENE Parasitic stool panel II (2) & VIASURE (3) monoplex assays: 97.3 –100% correlation

Giardia duodenalis & Entamoeba spp

Microscopy vs Real Time PCRAdvantages Disadvantages

Microscopy Relatively low cost

Widely available

LOD: 1,000 oocysts /mL faeces

Time consuming

Skilled microscopist essential

Molecular

methods

LOD: 1 oocyst /mL of faeces

Exceptional sensitivity reported

Time efficient

Multiplex capabilities

Speciation

Expensive reagents and

instrumentation required

PCR inhibitors

Incorporation of Real Time PCR

Future Studies• Cryptosporidium research:- Speciating and genotyping

Cryptosporidium spp involved in clinically significant human infections

- Bioinformatics based approach to identify molecular bio-markers

- Relate findings to the clinical history and follow-up of patients

- Potential for diagnosis of a broader range of species – Continued collaboration with Serosep

• Giardia lamblia genotyping

Concluding Remarks

• Molecular diagnosis:

- Improved diagnostic capabilities

- Streamlined lab work flow

- Time efficiency

• May be equivalent when microscopy used by skilled microscopists

• Employed only when clinically indicated

• May impact on national notification figures

Acknowledgements

• I would like to express my gratitude to the staff of the Microbiology Department, Cork University Hospital, particularly Mr. Liam Blake and Dr. Dan Corcoran.