Advertisement

Journal of Food Science and Technology

, Volume 56, Issue 11, pp 5000–5008 | Cite as

Efficiency of the Q3 lab-on-chip Real Time-PCR platform for detecting protozoan pathogens in bivalve mollusks

  • Annunziata GiangasperoEmail author
  • Marianna Marangi
  • Maria Stefania Latrofa
  • Giada Annoscia
  • Lorenza Putignani
  • Gioia Capelli
  • Lucia Bonassisa
  • Giovanni Normanno
  • Domenico Otranto
  • Marco Cereda
  • Francesco Ferrara
Original Article

Abstract

The zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum and Giardia duodenalis have been recorded worldwide in economically important edible shellfish, and are thus likely to represent a significant public health risk. Therefore, an innovative, user-friendly diagnostic tool is required in order to improve food safety control. The Q3 system is a miniaturized platform whose efficiency and applicability were investigated and compared with results obtained using standard Real-Time PCR. Tanks of saltwater containing acclimated Mytilus galloprovincialis, Ruditapes philippinarum and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). We then collected 30 specimens for each shellfish species from each group at 24 h and 72 h post-contamination. After DNA extraction, we tested all samples by Real-Time-PCR and Q3, and evaluated the sensitivity, specificity, predictive values, repeatability and concordance between the two systems. Concordance between Real-Time-PCR and Q3 was very good (p < 0.01), especially for Toxoplasma in M. galloprovincialis at both 24 h and 72 h after contamination, and in O. edulis at 72 h. The ability of Q3 to detect all the investigated pathogens was similar to that of Real-Time-PCR, and Q3 was efficient in detecting Toxoplasma in both M. galloprovincialis and O. edulis. This is the first study concerning the use of lab-on-chip technology in a food matrix, and in edible marine mollusks in particular.

Keywords

Protozoans Shellfish Real-Time PCR Lab-on-chip efficiency Food safety 

Notes

Acknowledgements

The study was funded by “New Strategies for Improvement of Food Safety: Prevention, Control, Correction” (S.I.Mi.S.A.)—PON02_00186_3417512—PON Ricerca e Competitività 2007–2013” (PO Puglia FESR 2007e2013 Asse I, Linea 1.2dPO Puglia FSE 2007e2013 Asse IV). The authors wish to thank Anna Lass of the Department of Tropical Parasitology, Medical University of Gdansk (Poland), and Anja Joachim of the Institute of Parasitology, University of Wien (Austria) for providing Toxoplasma oocyst strains, Tiziana Caradonna for her helpful work in the lab, Tommaso Marazia for his outstanding assistance in aquarium system management, and Alessandra Barlaam and Sarah Christopher for the English revision of the MS.

Authors’ contributions

AG, GN and DO conceived the study design; LP performed in vitro culture; FF and MC developed the Q3 system and trained for the Q3 analysis; MM, MSL and GA collected samples and performed the Real Time and Q3 analysis; GC performed the statistical analysis; AG, GN, MM, LP, MC and DO interpreted the data and wrote the paper. All authors contributed to editing the manuscript. All authors read and approved the final version of the manuscript.

Supplementary material

13197_2019_3972_MOESM1_ESM.ppt (3.2 mb)
Supplementary material 1 (PPT 3256 kb)
13197_2019_3972_MOESM2_ESM.doc (172 kb)
Supplementary material 2 (DOC 172 kb)

References

  1. Aksoy U, Marangi M, Papini R, Ozkoc S, Bayram Delibas S, Giangaspero A (2014) Detection of Toxoplasma gondii and Cyclospora cayetanensis in Mytilus galloprovincialis from Izmir Province coast (Turkey) by Real time PCR/High-Resolution Melting analysis (HRM). Food Microbiol 44:128–135CrossRefGoogle Scholar
  2. Baumgartner A, Marder HP, Munzinger J, Siegrist HH (2000) Frequency of Cryptosporidium spp. as cause of human gastrointestinal disease in Switzerland and possible sources of infection. Schweiz Med Wochenschr 130:1252–1258PubMedGoogle Scholar
  3. Biava M, Colavita F, Marzorati A, Russo D, Pirola D, Cocci A, Petrocelli A, Delli Guanti M, Cataldi G, Kamara TA, Kamara AS, Konneh K, Cannas A, Coen S, Quartu S, Meschi S, Valli MB, Mazzarelli A, Venditti C, Grassi G, Rozera G, Castilletti C, Mirazimi A, Capobianchi MR, Ippolito G, Miccio R, Di Caro A (2018) Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus. J Virol Methods 252:70–74CrossRefGoogle Scholar
  4. Coupe A, Howe L, Burrows E, Sine A, Pita A, Velathanthiri N, Vallée E, Hayman D, Shapiro K, Roe WD (2018) First report of Toxoplasma gondii sporulated oocysts and Giardia duodenalis in commercial green-lipped mussels Perna canaliculus in New Zealand. Parasitol Res 117:1453–1463CrossRefGoogle Scholar
  5. Dubey JP, Jones JL (2008) Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 38:1257–1278CrossRefGoogle Scholar
  6. El Sheikha AF, Montet D (2016) How to determine the geographical origin of seafood? Crit Rev Food Sci Nutr 56:306–317CrossRefGoogle Scholar
  7. FAO (2015) Fisheries and aquaculture statistics http://www.fao.org/3/a-i7989t.pdf/. Accessed 13 Dec 2018
  8. Giangaspero A, Cirillo R, Lacasella V, Lonigro A, Marangi M, Cavallo P, Berrilli F, Di Cave D, Brandonisio O (2009) Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy. Parasitol Int 58:12–17CrossRefGoogle Scholar
  9. Giangaspero A, Papini R, Marangi M, Koehler AV, Gasser RB (2014) Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets. Int J Food Microbiol 171:62–67CrossRefGoogle Scholar
  10. Graczyk TK, Fayer R, Lewis EJ, Trout JM, Farley CA (1999) Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay. Parasitol Res 85:518–521CrossRefGoogle Scholar
  11. Ghozzi K, Marangi M, Papini R, Lahmar I, Challouf R, Houas N, Ben Dhiab R, Normanno G, Babba H, Giangaspero A (2017) First report of Tunisian coastal water contamination by protozoan parasites using mollusk bivalves as biological indicators. Maar Pollut Bull 117:197–202CrossRefGoogle Scholar
  12. Hohweyer J, Dumètre A, Aubert D, Azas N, Villena I (2013) Tools and methods for detecting and characterizing Giardia, Cryptosporidium and Toxoplasma parasites in marine mollusks. J Food Prot 76:1649–1657CrossRefGoogle Scholar
  13. Kane AV, Ward HD, Keusch GT, Pereira ME (1991) In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency. J Parasitol 77:974–981CrossRefGoogle Scholar
  14. Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77:487–488CrossRefGoogle Scholar
  15. Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics 33(1):159–174CrossRefGoogle Scholar
  16. Marangi M, Giangaspero A, Lacasella V, Lonigro A, Gasser RB (2015) Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels. Mol Cell Probes 29:122–125CrossRefGoogle Scholar
  17. Marziliano N, Notarangelo MF, Cereda M, Caporale V, Coppini L, Demola MA, Guidorossi A, Crocamo A, Pigazzani F, Boffetti F, Del Giudice F, Orsini F, Pirola D, Cocci A, Manzalini C, Casu G, Bianchessi M, Ardissino D, Merlini PA (2015) Rapid and portable, lab-on-chip, point-of-care genotyping for evaluating clopidogrel metabolism. Clin Chim Acta 451:240–246CrossRefGoogle Scholar
  18. Miller WA, Gardner IA, Atwill ER, Leutenegger CM, Miller MA, Hedrick RP, Melli AC, Barnes NM, Conrad PA (2006) Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus). J Microbiol Methods 65:367–379CrossRefGoogle Scholar
  19. Mmbaga BT, Houpt ER (2017) Cryptosporidium and Giardia infections in children: a review. Pediatr Clin North Am 64:837–850CrossRefGoogle Scholar
  20. Nazeer JT, Khalifa KES, von Thien H, El-Sibaei MM, Abdel-Hamid MY, Tawfik RA, Tannich E (2013) Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt. Parasitol Res 112:595–601CrossRefGoogle Scholar
  21. Potasman I, Paz A, Odeh M (2002) Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective. Clin Infect Dis 35:921–928CrossRefGoogle Scholar
  22. Putignani L, Mancinelli L, Del Chierico F, Menichella D, Adlerstein D, Angelici MC, Marangi M, Berrilli F, Caffara M, di Regalbono DA, Giangaspero A (2012) Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG). Exp Parasitol 127:409–417CrossRefGoogle Scholar
  23. Robert-Gangneux F, Dardé ML (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. J Clin Microbiol 25:264–296CrossRefGoogle Scholar
  24. Robertson LJ (2007) The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: a review. Int J Food Microbiol 120:201–216CrossRefGoogle Scholar
  25. Ryan U, Zahedi A, Paparini A (2016) Cryptosporidium in humans and animals a one health approach to prophylaxis. Parasite Immunol 38:535–547CrossRefGoogle Scholar
  26. Sutthikornchai C, Popruk S, Chumpolbanchorn K, Sukhumavasi W, Sukthana Y (2016) Oyster is an effective transmission vehicle for Cryptosporidium infection in human. Asian Pac J Trop Dis 9:562–566CrossRefGoogle Scholar
  27. Thompson RC, Olson ME, Zhu G, Enomoto S, Abrahamsen MS, Hijjawi NS (2005) Cryptosporidium and cryptosporidiosis. Adv Parasitol 59:77–158CrossRefGoogle Scholar
  28. Yoon JY, Kim B (2012) Lab-on-a-chip pathogen sensors for food safety. Sensors (Basel) 12:10713–10741CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2019

Authors and Affiliations

  • Annunziata Giangaspero
    • 1
    Email author
  • Marianna Marangi
    • 1
  • Maria Stefania Latrofa
    • 2
  • Giada Annoscia
    • 2
  • Lorenza Putignani
    • 3
  • Gioia Capelli
    • 4
  • Lucia Bonassisa
    • 5
  • Giovanni Normanno
    • 1
  • Domenico Otranto
    • 2
  • Marco Cereda
    • 6
  • Francesco Ferrara
    • 6
  1. 1.Dipartimento di Scienze Agrarie, degli Alimenti e dell’AmbienteUniversità di FoggiaFoggiaItaly
  2. 2.Dipartimento di Medicina VeterinariaUniversità di BariBariItaly
  3. 3.Unità di Parassitologia e Unità di Ricerca di Microbioma umano, Bambino GesùOspedale Pediatrico e Istituto di RicercaRomeItaly
  4. 4.Istituto Zooprofilattico Sperimentale delle VenezieLegnaroItaly
  5. 5.BonassisaLabFoggiaItaly
  6. 6.STMicroelectronics Srl, Advanced System TechnologyAgrate BrianzaItaly

Personalised recommendations