Skip to main content

Working toward improved monitoring of Cryptosporidium and Giardia (oo)cysts in water samples: testing alternatives to elution and immunomagnetic separation from USEPA Method 1623.1

Abstract

Objective

This study was designed to find a method to enhance the recovery of Cryptosporidium spp. and Giardia spp. parasites from water samples for research purposes compared to the results that can be achieved with USEPA Method 1623.1. Four different approaches were used to test water samples that were artificially spiked with parasites. The approaches were: (i) Method 1623.1 itself, (ii) elution of Method 1623.1 combined with microfiltration, (iii) an elution technique based on grinding the filter membrane in a blender before the eluent was concentrated by immunomagnetic separation, and (iv) the blender elution followed by microfiltration. Fluorescence microscopy was used to determine which approach led to the highest parasite recovery rates.

Results

Method 1623.1 gave the best results for Giardia, while all four approaches were statistically equivalent for Cryptosporidium. We evaluated the costs and laboratory time requirements for each protocol to give readers a complete comparison of the methods tested. Elution of Method 1623.1 combined with microfiltration resulted in lower costs and less laboratory work time without compromising the recovery of the parasites.

Introduction

Cryptosporidium spp. and Giardia spp. are parasitic protozoa responsible for gastrointestinal illness in several animal species as well as in humans [1, 2]. One of the predominant means of dissemination of these parasites is the transmission of cysts, robust egg-like structures, through environmental water sources where they are consumed by new hosts [2]. To ensure the distribution of safe water for consumption, Canada mandates that water treatment plants reduce or inactivate 99.9% of Cryptosporidium spp. and Giardia spp. parasites in the water [3]. The United States requires 99% reduction of Cryptosporidium and 99.9% reduction of Giardia [4, 5]. Monitoring the presence of either of these organisms is crucial to validate the efficiency of the water treatment and to determinate the parasitic load of the source water entering the treatment plant. LeChevallier and collaborators analyzed 66 surface waters of the United States and of Canada, and found average concentrations of 0.04–66 Giardia cysts of per 100 L compared to 0.07–484 Cryptosporidium oocysts per 100 L [6]. In a document of 2009, World Health Organization stated that environmental waters worldwide can contain concentrations from 0.01 to 100 oocysts of Cryptosporidium per Litre [7].

Although several biomolecular protocols have been developed since the 1990s to detect these protozoa in environmental samples [8], the only actual standardized method to do it is United States Environmental Protection Agency (USEPA) Method 1623.1. Briefly, this method consists of filtering from 10 to 50 L of raw water with an EnviroChek HV filtration cartridge with a porosity of 1 µm. The biological material is eluted from the filter and parasites are concentrated by immunomagnetic separation (IMS). Then, their cells are enumerated by differential interference microscopy and fluorescence microscopy (with fluorescein isothiocyanate and 4′,6-diamidino-2-phenylindole to stain the cells). Although validated multiple times, this method has several weaknesses, such as common recovery rates for parasites of 30–50% and even lower [9,10,11], a laboratory-work time of several days, for a cost of about 1000 Canadian dollars (CAD) per sample analyzed (Ministère de l'Environnement et de la Lutte contre les changements climatiques, pers. comm.). Alternative elution techniques presented in previous studies gave variable recovery results from 9 to 58% for Cryptosporidium and 2 to 74% for Giardia. [12,13,14,15,16,17,18]. Therefore, we undertook experiments to test an alternative elution protocol combined either with IMS or microfiltration, to try to increase recovery rates of these parasites over the USEPA Method 1623.1 standardized steps. We also sought to shorten the procedures and reduce the costs associated with the analyses. Although none of the approaches tested in this study gave higher recoveries than the standardized method, some alternative protocols yielded statistically similar recovery rates, while reducing the time and costs required.

Main text

Methods

(Oo)cyst preparation and filtration

The spiking process was adapted from the matrix spike control described in USEPA Method 1623.1. To prepare artificially contaminated suspensions of oocysts of Cryptosporidium and cysts of Giardia, ColorSeeds (BioPoint Inc., Australia) containing 98–102 (oo)cysts per vial, which were inactivated by gamma ray exposure and permanently dyed with Texas Red stain, were used. Suspensions were prepared by adding a vial of ColorSeeds to 10 L of ultrapure water in a Cubitainer, plus the equivalent of 500 000 non-pathogenic bacterial cells belonging to the species Bacillus cereus, Pseudomonas putida, Cupriavidus sp., and Escherichia coli, to mimic the microbial content of environmental water samples. The number of cells was determined by adjusting the optic density of the suspension with a standard of known concentration. The artificially contaminated samples were filtered with EnviroChek HV filtration cartridges (Pall Corporation, United States) with a peristaltic pump. Once the 10 L volume was done filtering, an additional 1 L of ultrapure water was added to the Cubitainer and shaken to collect potentially remaining adhered (oo)cysts. This volume was also filtered on the same cartridge. Cartridges were kept at 4 °C for a maximum of 24 h before processing.

Elution and centrifugation

Samples were attributed to a combination of elution and concentration techniques, as illustrated in Fig. 1. Briefly, the twelve cartridges were split between two elution methods: standard USEPA Method 1623.1 [19], and an alternative elution protocol. The latter consists of opening the EnviroChek cartridge inside a laminar flow-hood cabinet with a pipe cutter, slicing the filtration membrane with a scalpel blade, and putting the fragments of the membrane in a commercial blender (Hamilton Beach, United States). The result of the cartridge opening can be seen at figure S1 (Additional file 1). The inside of the blender was coated with liquid silicone (SigmaCote, Sigma-Aldrich, Canada) to prevent the adherence of the (oo)cysts to the surfaces, as both parasites have hydrophilic surfaces [20]. Then, 250 mL of an elution solution containing Laureth-12 10%, Tris 1 M pH 7.4, EDTA 0.5 M pH 8.0, ultrapure water, and Antifoam A was added to the blender. The mixture was blended for 1 min. Then, liquid (with as few membrane pieces as possible) was collected in a centrifugation bottle. Its content was centrifuged for 30 min at 5855×g to collect the parasites in the pellet. This speed and duration were determined to be more efficient in pelleting parasites during preliminary tests with approximately 97% of oocysts pelleting.

Fig. 1
figure 1

Summary of approaches tested and repartition of samples. EPA: elution protocol as described in U.S. EPA Method 1623.1; Blender: Alternative elution protocol consisting of opening the EnviroChek filtration cartridge, slicing the membrane surface and blending the pieces in a commercial blender; IMS: concentration by immunomagnetic separation according to the USEPA Method 1623.1; Microfiltration: concentration by filtrating the eluate with a Sterivex.™ filter of 0.45 µm of pore size

Concentration of (oo)cysts

Products either from the USEPA 1623.1 standardized elution technique or from the alternative elution protocol were submitted to a concentration of the (oo)cysts into a smaller volume. To do so, two concentration techniques were compared to determine the efficiency of each, namely the IMS and an alternative microfiltration protocol. The IMS was performed according to USEPA 1623 protocol and using the Dynabeads™ GC-Combo kit (ThermoFisher Scientific, United States). The microfiltration was done by manually pumping the elution liquid into a sterile syringe and by pushing the solution into a Sterivex™ sterilizing filter of porosity of 0.45 µm. Then, biological material from the Sterivex™ was recovered following the extraction protocol described in [21], which includes opening the cartridge and cutting out the membrane. The membrane pieces were resuspended in 200 µL of PBS 1X buffer and vortexed one minute to allow the cells to be collected in the liquid.

Detection and enumeration of (oo)cysts

Concentrates either from the IMS or the microfiltration were then fixed on a microscope slide and stained with either EasyStain kit (BioPoint Inc., Australia) or Aqua-Glo™ G/C kit (Waterborne Inc., United States) following the manufacturer's instructions as required by the USEPA Method 1623.1. The slides were analyzed by fluorescence microscopy with a Zeiss Axio Observer Z1 microscope connected with an Axiocam MRm camera (Carl Zeiss, North York, ON, Canada) to enumerate the number of (oo)cysts collected. The techniques were compared by calculating the percentage of (oo)cysts collected by each one.

Statistical analysis

Each combination of steps was done in biological triplicate. Statistical analyses were made with the R package RCommandr version 3.5.0. The normality of the recovery values for each condition was determined by a Shapiro–Wilk test. Averages obtained for each condition and each parasite were then compared by the Kruskal–Wallis non-parametric test with a threshold of 5%. The data for the conditions applying to a normal distribution were compared together with an ANOVA test with a threshold of 5%.

Results

Figure 2 presents the results obtained with the various combinations of techniques. Although none of these alternative approaches gave significantly superior recovery rates to USEPA Method 1623.1, equivalent results were obtained in the case of Cryptosporidium results. It was determined that the medians of the data generated with the four approaches were not statistically different for this parasite. For Giardia, the USEPA Method 1623.1 produced recovery rates with a median statistically significantly higher than the three other approaches tested. Even if the distribution of data could look otherwise, averages of the data obtained for Cryptosporidium and Giardia with the USEPA Method 1623.1 were not statistically different. But this global behavior of higher recoveries for Giardia than for Cryptosporidium has been seen in previous studies as well [17, 22, 23]. Only the comparison of data from the USEPA protocol for Giardia and the blender elution followed by IMS gave statistically significantly different averages, with the USEPA method giving higher values.

Fig. 2
figure 2

Recovery of Cryptosporidium and Giardia according to the combination of techniques applied. Condition 1 consists of the complete protocol according to USEPA Method 1623.1. Condition 2 is the combination of the elution according to the USEPA with the concentration by microfiltration. Condition 3 is the alternative elution protocol combined with the concentration by IMS. Condition 4 is the alternative elution protocol paired with the concentration by microfiltration. The symbol ■ states that the averages are statistically significantly different with a threshold of 5%. The symbol □ indicates that the averages are non-statistically significantly different with a threshold of 5%. The symbol means that the medians are non-statistically significantly different with a threshold of 5%

The costs and the laboratory work time required for each approach are summarized in Table 1 and detailed. Some of the alternative approaches allow a gain of time of a few hours of laboratory work. The costs can be significantly reduced from 100 to 650 CAD depending on the method chosen compared to the USEPA Method 1623.1. It could be advantageous especially in the case of Cryptosporidium where all techniques generated non-statistically significant different results. Despite the decrease in costs, the alternative elution paired with the microfiltration does not seem to be the best approach to adopt, given the low recoveries obtained. However, USEPA elution with microfiltration appears to be the most advantageous combination of alternative approaches regarding the time gain and the decrease of costs for a similar recovery. This is particularly promising in a research context.

Table 1 Costs and time required per sample for each combination of techniques tested in this study

Limitations

Some limitations in the approaches tested in this study are worth mentioning. First, our inexperience with the USEPA Method 1623.1 protocol likely may have contributed to the relatively low recovery rates. However, low recovery rates were also reported in the past by interlaboratory validation assays led by the USEPA [11], and our recovery rates align with those. With experience and practice, higher recoveries might be expected. Typically, only well-trained personnel adept at obtaining higher recoveries are allowed to analyse samples from clients in environmental analysis laboratories, while the present study was done in an academic research context.

Second, with the alternative elution protocol tested during this study, it was not possible to physically retrieve the entire filtration membrane from the EnviroChek HV cartridge following its opening with a pipe cutter. Approximately 30% of the filtration surface remained covered by the polycarbonate housing despite all our efforts to cut the cartridge closer to the extremity (Additional file 1, Figure S1). Therefore, the parasites stuck to that portion of the filtration surface could not be collected with this approach.

Third, although the concentration of parasites by IMS is expected to remove all other cells that do not belong to either Cryptosporidium or Giardia genera, our experiments showed that does not always occur. Many bacteria cells could also be seen (Additional file 1, Figure S2). Our experiments were done by artificially spiking 10 L of water with 500 000 bacterial cells to mimic contaminated environmental water. However, some raw water samples may contain even higher concentrations of bacteria. For example, the River Ruhr in Germany, which was studied by Strathmann et al. [24], contained about 3.4 × 106 total cells per mL (more than 50 000 times more bacteria than in our own samples). We conclude that the problem of interfering bacteria carried over during the IMS could be quite cumbersome for the analysis of some environmental samples and could interfere with the analysis. IMS beads can also confuse Cryptosporidium oocyst detection if they are carried over in the sample until the microscopic examination (see Additional file 1, figure S3 for comparison).

Finally, centrifugation is a major issue to consider in this protocol to improve parasite recovery. The USEPA Method 1623.1’s centrifugation step is at 1500 × g for 15 min. In a previous study, this centrifugation caused a loss of 8 to 14% of cells when compared with the same sample composition submitted only to IMS and fluorescence microscopy [25]. Lechevallier et al. [26] found that higher centrifugation speed helped to recover more (oo)cysts; therefore, to increase the proportion of (oo)cysts collected, we chose a centrifugation speed of 5855 × g for 30 min following our alternative elution technique. Since Lechevallier et al. (26) demonstrated that even a speed of 17 300 × g would not disrupt them, it is unlikely that the centrifugation applied here would have broken them. We did not evaluate the loss of parasites at each step in the present study.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on a reasonable request.

Abbreviations

IMS:

Concentration by immunomagnetic separation

USEPA:

United States Environmental Protection Agency

References

  1. Leitch GJ, He Q. Cryptosporidiosis-an overview. J Biomed Res. 2011;25(1):1–16.

    Article  Google Scholar 

  2. Yaoyu F, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24(1):110–40.

    Article  Google Scholar 

  3. Health Canada. Guidelines for Canadian Drinking Water Quality. Guideline Technical Document. Enteric Protozoa: Giardia and Cryptosporidium [Internet]. Federal-Provincial-Territorial Committee on Health and the Environment. 2019. 121 p. www.canada.ca/en/health-canada/services/environmental-workplace-health/reports-

  4. United States Environmental Protection Agency. Cryptosporidium : Drinking Water Health Advisory. 2001.

  5. United States Environmental Protection Agency. Giardia : Drinking Water Health Advisory. 1999.

  6. LeChevallier MW, Norton WD, Lee RG. Occurrence of Giardia and Cryptosporidium spp surface water supplies. Appl Environ Microbiol. 1991;57(9):2610–6.

    CAS  Article  Google Scholar 

  7. World Health Organization. Risk Assessment of Cryptosporidium in Drinking Water. Public Health and Environment Water, Sanitation, Hygiene & Health. 2009

  8. Adeyemo FE, Singh G, Reddy P, Stenström TA. Methods for the detection of Cryptosporidium and Giardia: From microscopy to nucleic acid based tools in clinical and environmental regimes. Acta Trop. 2018;184:15–28. https://doi.org/10.1016/j.actatropica.2018.01.011.

    CAS  Article  PubMed  Google Scholar 

  9. MDDELCC. Bilan de la Qualité de l’Eau Potable au Québec : 2010–2014 [Internet]. 2016 [cited 2017 Jun 6]. p. 72. http://www.mddelcc.gouv.qc.ca/eau/potable/bilans/bilan-qualite2010-2014.pdf

  10. Hu J, Feng Y, Ong SL, Ng WJ, Song L, Tan X, et al. Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623. J Microbiol Methods. 2004;58(3):321–5.

    CAS  Article  Google Scholar 

  11. Connell K, Rodgers CC, Shank-givens HL, Scheller J, Pope ML, Miller K. Building a better protozoa data set. J Am Water Works Assoc. 2000;92(10):30–43.

    CAS  Article  Google Scholar 

  12. LeChevallier MW, Norton WD, Lee RG. Occurrence of Giardia and Cryptosporidium spp surface water supplies. Appl Environ Microbiol. 1991;57(9):2610–6.

    CAS  Article  Google Scholar 

  13. LeChevallier MW, Trok TM, Burns MO, Lee RG. Comparison of the zinc sulfate and immunofluorescence techniques for detecting Giardia and Cryptosporidium. J Am Water Works Assoc. 1990;82(9):75–82.

    Article  Google Scholar 

  14. Musial CE, Arrowood MJ, Sterling CR, Gerba CP. Detection of Cryptosporidium in water by using polypropylene cartridge filters. Appl Environ Microbiol. 1987;53(4):687–92.

    CAS  Article  Google Scholar 

  15. Nieminski EC, Schaefer FW, Ongerth JE. Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water. Appl Environ Microbiol. 1995;61(5):1714–9.

    CAS  Article  Google Scholar 

  16. Swales C, Wright S. Evaluation of a continuous flow centrifuge for recovery of Cryptosporidium oocysts from large volume water samples. Water Res. 2000;34(6):1962–6.

    CAS  Article  Google Scholar 

  17. Hsu BM, Huang C, Hsu YF, Jiang GY, Hsu CLL. Evaluation of two concentration methods for detecting Giardia and Cryptosporidium in water. Water Res. 2001;35(2):419–24.

    CAS  Article  Google Scholar 

  18. Watt PM, Johnson DC, Gerba CP. Improved method for concentration of Giardia, Cryptosporidium, and poliovirus from water. J Environ Sci Heal Part A Toxic/Hazardous Subst Environ Eng. 2002;37(3):321–30.

    Google Scholar 

  19. United States Environmental Protection Agency. Method 1623.1: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. 2012.

  20. Hsu BM, Huang C. Influence of ionic strength and pH on hydrophobicity and zeta potential of Giardia and Cryptosporidium. Colloids Surfaces A Physicochem Eng Asp. 2002;201(1–3):201–6.

    CAS  Article  Google Scholar 

  21. Cruaud P, Vigneron A, Fradette MS, Charette SJ, Rodriguez MJ, Dorea CC, et al. Open the Sterivex TM casing: an easy and effective way to improve DNA extraction yields. Limnol Oceanogr Methods. 2017;15:1015–20. https://doi.org/10.1002/lom3.10221.

    CAS  Article  Google Scholar 

  22. Karim H, Sylvain S, Laurence L, Lucien H, Henry-Michel C. Comparison of three methods to concentrate Giardia cysts and Cryptosporidium oocysts from surface and drinking waters. Water Sci Technol. 2010;62(1):196–201.

    CAS  Article  Google Scholar 

  23. Ongerth JE. The concentration of Cryptosporidium and Giardia in water—The role and importance of recovery efficiency. Water Res. 2013;47(7):2479–88. https://doi.org/10.1016/j.watres.2013.02.015.

    CAS  Article  PubMed  Google Scholar 

  24. Strathmann M, Horstkott M, Koch C, Gayer U, Wingender J. The River Ruhr—an urban river under particular interest for recreational use and as a raw water source for drinking water: the collaborative research project “Safe Ruhr”—microbiological aspects. Int J Hyg Environ Health. 2016;219(7):643–61. https://doi.org/10.1016/j.ijheh.2016.07.005.

    CAS  Article  PubMed  Google Scholar 

  25. Kim KJ, Jung HH, Lee K. Evaluation of cyst loss in standard procedural steps for detecting of Giardia lamblia and Cryptosporidium parvum in water. Biotechnol Bioprocess Eng. 2006;11(4):368–71.

    CAS  Article  Google Scholar 

  26. Lechevallier MW, Norton WD, Siegel JE, Abbaszadegan M. Evaluation of the Immunofluorescence Procedure for Detection of Giardia Cysts and Cryptosporidium Oocysts in Water. Appl Environ Microbiol. 1995;61(2):690–7.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Manuela Villion, Étienne Walker-Lamontagne and Daniel Verreault from the Ministère de l'Environnement et de la Lutte contre les changements climatiques of Quebec, Canada, for the technical support, the training to the USEPA Method 1623.1, and for the revisions made to the manuscript.

Funding

This research project was supported by scholarships to MSF from the Natural Sciences and Engineering Research Council of Canada (NSERC) [ESD3-547356-2020], the Fonds de recherche du Québec—Nature et technologies [B2X-270698-2019-2020], and the Drinking Water Chair of Laval University (CREPUL).

Author information

Authors and Affiliations

Authors

Contributions

MSF and SJC designed the study. MSF performed the laboratory work and statistical analyses and wrote the first version of the manuscript. SJC made revisions to the article and provided technical support for the laboratory work. Both the authors read and approved the final manuscript.

Corresponding author

Correspondence to Marie-Stéphanie Fradette.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

. Figure S1. Photograph of the EnviroChek HV filtration cartridge following opening with a pipecutter during the alternative elution protocol. Figure S2. Picture of bacteria carried over by the immunomagnetic separation. Figure S3.. Comparison of a Cryptosporidium oocyst (A) with beads from the IMS beads (B).Table S1. Detailed evaluation of the costs associated with the techniques presented in this article.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fradette, MS., Charette, S.J. Working toward improved monitoring of Cryptosporidium and Giardia (oo)cysts in water samples: testing alternatives to elution and immunomagnetic separation from USEPA Method 1623.1. BMC Res Notes 15, 254 (2022). https://doi.org/10.1186/s13104-022-06118-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-022-06118-9

Keywords

  • Cryptosporidium
  • Giardia
  • Recovery
  • Water samples
  • USEPA Method 1623.1