Scrambled eggs

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Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples. Nichola Calvani, Peter Windsor, ...

Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples Nichola Calvani, Peter Windsor, Russell Bush and Jan Šlapeta

The University of Sydney

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Fasciola hepatica and Fasciola gigantica  Fasciolosis is an important neglected tropical disease of humans and animals  91 million people considered at risk1  Production impacts expected to exceed US$2 billion/year1

 Ante-mortem diagnosis:  Traditional sedimentation and FEC or Kato-katz  Coprological ELISA for fluke antigen

The University of Sydney

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Fasciola hepatica and Fasciola gigantica

The University of Sydney

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Why scramble?  PhD: Diagnosis, prevalence and strategic control of Fasciolosis in Southeast Asian cattle  Requirements:  Ante-mortem diagnosis  Species differentiation  Sensitive quantification of change in faecal egg load No methods currently available meet these requirements The University of Sydney

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Why scramble? Despite several tools for the molecular identification of Fasciola spp...

No diagnostic workflow has been optimised for the sensitive and quantitative detection of Fasciola spp. eggs in faeces with low egg counts2,3

The University of Sydney

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Can we scramble?  Hard outer shell must be ruptured in  6 disruption approaches tested on order to extract DNA as proven for: clean F. hepatica eggs using FastPrep©-24 (MP Biomedicals,  Trichuris trichiura4 Australia):  Echinococcus multilocularis5

 Primers: 140bp amplicons of ITS-26

The University of Sydney

 4.0 m/s for 1, 2 and 3 x 40 seconds  6.0 m/s for 1, 2 and 3 x 40 seconds

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Can we scramble?  3 disruption approaches further tested on F. hepatica eggs in faecal samples (250EPG) using FastPrep©-24 (MP Biomedicals, Australia):  6.0 m/s for 1 x 40 seconds  6.0 m/s for 2 x 40 seconds  6.0 m/s for 3 x 40 seconds

 Primers: 140bp amplicons of ITS-26

The University of Sydney

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150 mg raw faeces

 Method 1:  Method 2:

6 g raw faeces

2 ml faecal sediment

150 ul vortexed sediment

 Method 3:

6 g raw faeces

2 ml faecal sediment

Entire faecal sediment

The University of Sydney

Disrupt

Can we scramble?

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Correlation with EPGs

The University of Sydney

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Analytical sensitivity Replicate

1.1

1.2

2.1

2.2

3.1

3.2

4.1

4.2

5.1

5.2

F. hepatica

35.16

34.23

34.35

37.67

37.83

-

32.18

32.68

35.29

34.99

F. gigantica 35.46

35.41

35.21

35.24

36.06

35.82

38.23

-

34.48

34.34

 Disrupted 5 replicates of a single egg at 6.0 m/s for 40 seconds  F. hepatica  F. gigantica

 Amplified in duplicate For each species 9/10 wells amplified demonstrating the limit of detection

The University of Sydney

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The new molecular diagnostic workflow Sediment Pellet Disrupt Isolate DNA Amplify!

The University of Sydney

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Back to Southeast Asia…  Need to be able to transport samples from areas with a lack of available infrastructure  Must meet requirements for importation:  70% alcohol, 10% formalin, 4% formaldehyde, ≥2% glutaraldehyde or plasinated

Preliminary investigation into different storage conditions:  Two replicates of 6 g raw faeces spiked with 2000 eggs stored at: 1. 4˚C 2. -20˚C 3. -20˚C + 70% EtOH 4. Room temperature + 70% EtOH

The University of Sydney

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Impact of storage conditions

2000 in 6g Treatment

~666 in 2g 1.

Mean EPG 340

~333 EPG

2.

3.

4.

412

356

337

No impact of storage condition on sedimentation and isolation outcomes

The University of Sydney

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Applications  Bead-beating approach can be adapted to align with the diagnostic procedures of individual labs  Samples can be stored in 70% EtOH and transported at room temperature prior to sedimentation and DNA extraction  Potential for species-specific probes to be employed for differentiation between F. hepatica and F. gigantica in areas of sympatry

The University of Sydney

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References 1. Keiser, J., Utzinger, J., 2005. Emerging foodborne trematodiasis. Emerg Infect Dis 11, 1507-1514. 2. Verweij, J.J., Stensvold, C.R., 2014. Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections. Clin Microbiol Rev 27, 371-418. 3. Ai, L., Chen, M.X., Alasaad, S., Elsheikha, H.M., Li, J., Li, H.L., Lin, R.Q., Zou, F.C., Zhu, X.Q., Chen, J.X., 2011. Genetic characterization, species differentiation and detection of Fasciola spp. by molecular approaches. Parasit Vectors 4, 101. 4. Kaisar, M.M.M., Brienen, E.A.T., Djuardi, Y., Sartono, E., Yazdanbakhsh, M., Verweij, J.J., Supali, T., L, V.A.N.L., 2017. Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites. Parasitology 144, 965-974. 5. Maksimov, P., Schares, G., Press, S., Frohlich, A., Basso, W., Herzig, M., Conraths, F.J., 2017. Comparison of different commercial DNA extraction kits and PCR protocols for the detection of Echinococcus multilocularis eggs in faecal samples from foxes. Veterinary parasitology 237, 83-93. 6. Alasaad, S., Soriguer, R.C., Abu-Madi, M., El Behairy, A., Jowers, M.J., Banos, P.D., Piriz, A., Fickel, J., Zhu, X.Q., 2011. A TaqMan real-time PCR-based assay for the identification of Fasciola spp. Veterinary parasitology 179, 266-271.

The University of Sydney

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Acknowledgements •

Jan Šlapeta



Russell Bush



Peter Windsor



David Emery



Graeme Brown



Peter Rolfe



Sarah George



Dominique Marendy



Katrina Gilchrist

The University of Sydney

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