1887

Journal of Medical Microbiology logo

Volume 72, Issue 6

A comparison of qPCR and microscopy for the detection and enumeration of oocysts from drinking water Open Access

Abstract

presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures.

The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR.

We aimed to develop and evaluate a qPCR for the detection and enumeration of in drinking water, and to compare the assay with the standard method used in the UK.

We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 oocysts in 10 l of artificially contaminated drinking water.

The results demonstrated that detection of by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy.

Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Cryptosporidium , drinking water , enumeration and qPCR
Funding
This study was supported by the:
  • Drinking Water Inspectorate (Award DWI 70/2/332)
    • Principle Award Recipient: RachelM Chalmers
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001715
2023-06-19
2024-05-02
Loading full text...

Full text loading...

/deliver/fulltext/jmm/72/6/jmm001715.html?itemId=/content/journal/jmm/10.1099/jmm.0.001715&mimeType=html&fmt=ahah

References

  1. Betancourt WQ, Rose JB. Drinking water treatment processes for removal of Cryptosporidium and Giardia. Vet Parasitol 2004; 126:219–234 [View Article] [PubMed]
     [Google Scholar]
  2. Ma JY, Li MY, Qi ZZ, Fu M, Sun TF et al. Waterborne protozoan outbreaks: an update on the global, regional, and national prevalence from 2017 to 2020 and sources of contamination. Sci Total Environ 2022; 806:150562 [View Article] [PubMed]
     [Google Scholar]
  3. World Health Organization Risk Assessment of Cryptosporidium in Drinking-water. World Health Organization 2009, Geneva. n.d https://apps.who.int/iris/handle/10665/70117 accessed 30 November 2022
  4. World Health Organization Guidelines for drinking-water quality. Fourth edition incorporating the first and second addenda World Health Organization 2022, Geneva. n.d https://www.who.int/publications/i/item/9789240045064 accessed 30 November 2022
  5. International Organization for Standardization ISO 15553:2006 Water quality Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water; 2006 https://www.iso.org/standard/39804.html accessed 30 November 2022
  6. United States Environmental Protection Agency Method 1623.1: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. Office of Water; 2012 https://nepis.epa.gov/Exe/ZyPDF.cgi/P100J7G4.PDF?Dockey=P100J7G4.PDF
  7. Standing Committee of Analysts The Microbiology of Drinking Water 2010) - Part 14 - Methods for the isolation, identification and enumeration of Cryptosporidium oocysts and Giardia. Standing Committee of Analysts; 2010 https://secureservercdn.net/160.153.137.128/fkp.e54.myftpupload.com/wp-content/uploads/Blue_Book_Library/234.pdf
  8. Efstratiou A, Ongerth J, Karanis P. Evolution of monitoring for Giardia and Cryptosporidium in water. Water Res 2017; 123:96–112 [View Article] [PubMed]
     [Google Scholar]
  9. Fradette MS, Culley AI, Charette SJ. Detection of Cryptosporidium spp. and Giardia spp. in environmental water samples: a journey into the past and new perspectives. Microorganisms 2022; 10:1175 [View Article] [PubMed]
     [Google Scholar]
  10. Dien Bard J, McElvania E. Panels and syndromic testing in clinical microbiology. Clin Lab Med 2020; 40:393–420 [View Article] [PubMed]
     [Google Scholar]
  11. Nehra M, Kumar V, Kumar R, Dilbaghi N, Kumar S. Current scenario of pathogen detection techniques in agro-food sector. Biosensors 2022; 12:489 [View Article] [PubMed]
     [Google Scholar]
  12. Standing Committee of Analysts The determination of Legionella bacteria in waters and other environmental samples (2019) – Part 3 – Method for their detection and quantification by polymerase chain reaction (qPCR) and protocol for method validation. Standing Committee of Analysts; 2019 https://secureservercdn.net/160.153.137.128/fkp.e54.myftpupload.com/wp-content/uploads/Blue_Book_Library/266.pdf
  13. United States Environmental Protection Agency Method 1609.1: Enterococci in Water by TaqMan ® Quantitative Polymerase Chain Reaction (qPCR) with Internal Amplification Control (IAC) Assay Office of Water; 2015 https://nepis.epa.gov/Exe/ZyPDF.cgi/P100MZ8D.PDF?Dockey=P100MZ8D.PDF
  14. Fout GS, Cashdollar JL, Griffin SM, Brinkman NE, Varughese EA et al. EPA method 1615. Measurement of enterovirus and norovirus occurrence in water by culture and RT-qPCR. II. Total culturable virus assay. J Vis Exp 2016; 107:52437 [View Article] [PubMed]
     [Google Scholar]
  15. Jiang J, Alderisio KA, Singh A, Xiao L. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Appl Environ Microbiol 2005; 71:1135–1141 [View Article]
     [Google Scholar]
  16. Staggs SE, Beckman EM, Keely SP, Mackwan R, Ware MW et al. The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumerating Cryptosporidium spp. Oocysts in the environment. PLoS One 2013; 8:e66562 [View Article] [PubMed]
     [Google Scholar]
  17. Elwin K, Robinson G, Pérez-Cordón G, Chalmers RM. Development and evaluation of a real-time PCR for genotyping of Cryptosporidium spp. from water monitoring slides. Exp Parasitol 2022; 242:108366 [View Article] [PubMed]
     [Google Scholar]
  18. Deer DM, Lampel KA, González-Escalona N. A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays. Lett Appl Microbiol 2010; 50:366–372 [View Article] [PubMed]
     [Google Scholar]
  19. Altman DG, Machin D, Bryant TN. eds Statistics with Confidence: Confidence Intervals and Statistical Guidelines, Second edition London: BMJ Books; 2000
     [Google Scholar]
  20. Uhlig S, Frost K, Colson B, Simon K, Mäde D et al. Validation of qualitative PCR methods on the basis of mathematical–statistical modelling of the probability of detection. Accred Qual Assur 2015; 20:75–83 [View Article]
     [Google Scholar]
  21. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55:611–622 [View Article] [PubMed]
     [Google Scholar]
  22. Chalmers RM, Katzer F, La Carbona S, Lalle M, Razakandrainibe R et al. A guide to standardise artificial contamination procedures with protozoan parasite oocysts or cysts during method evaluation, using Cryptosporidium and leafy greens as models. Food Control 2022; 134:108678 [View Article]
     [Google Scholar]
  23. Hill VR, Narayanan J, Gallen RR, Ferdinand KL, Cromeans T et al. Development of a nucleic acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water. Pathogens 2015; 4:335–354 [View Article] [PubMed]
     [Google Scholar]
  24. Mayer‐Scholl A, Šoba Šparl B, Deksne G, Ayres H, Woolsey I et al. IMPACT: standardising molecular detection methods to IMprove risk assessment capacity for foodborne protozoan PArasites, using Cryptosporidium in ready‐to‐eat salad as a model. EFS3 2022; 19: [View Article]
     [Google Scholar]
  25. Murphy HR, Almeria S, da Silva AJ. Molecular detection of Cyclospora cayetanensis in fresh produce using real-time PCR. U.S. food and drug administration. In Bacteriological Analytical Manual 2017
     [Google Scholar]
  26. Yang R, Paparini A, Monis P, Ryan U. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples. Int J Parasitol 2014; 44:1105–1113 [View Article] [PubMed]
     [Google Scholar]
  27. Montemayor M, Galofré B, Ribas F, Lucena F. Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water. Cytometry A 2007; 71:163–169 [View Article] [PubMed]
     [Google Scholar]
  28. Luo S, Nguyen KT, Nguyen BTT, Feng S, Shi Y et al. Deep learning-enabled imaging flow cytometry for high-speed Cryptosporidium and Giardia detection. Cytometry A 2021; 99:1123–1133 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001715
Loading
A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water
J. Med. Microbiol. 72, 001715 (2023); https://doi.org/10.1099/jmm.0.001715
/content/journal/jmm/10.1099/jmm.0.001715
/content/journal/jmm/10.1099/jmm.0.001715
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error