Improved recovery of Cryptosporidium and Giardia in water by optimizing immunomagnetic separation using acid and heat dissociation Open Access

Cryptosporidium and Giardia are protozoa responsible for waterborne diseases, with infections reported worldwide (Smith et al. 1989; McCuin et al. 2001; Efstratiou et al. 2017). These protozoa pose a significant public health concern as they can survive in the environment for extended periods in the form of (oo)cysts and exhibit high resistance to chlorine disinfection, which is commonly used in water treatment (Korich et al. 1990; Bukhari et al. 1998; Winiecka-Krusnell & Linder 1998). Consequently, several countries, including the United States and the United Kingdom, monitor Cryptosporidium and Giardia to ensure the safety of tap water (Ligda et al. 2020).

The standard method for detecting Cryptosporidium oocysts and Giardia cysts in water can be broadly divided into four steps (USEPA 2012; Ministry of Environment 2021); filtration of a water sample (10 L) using a capsule filter, extraction of (oo)cysts attached to the filter, concentration and separation of (oo)cysts via immunomagnetic separation (IMS), followed by fluorescent staining and microscopic observation. Recovery rates tend to decrease as the process progresses through multiple stages. Since the infectious dose of Cryptosporidium and Giardia is low, enhancing the sensitivity of the detection method is crucial to identifying even small quantities of these protozoa (Schaefer et al. 1991; DuPont et al. 1995; Okhuysen et al. 1999; Connell et al. 2000; Temesgen et al. 2021). Among these steps, IMS plays a critical role in improving protozoan recovery (McCuin et al. 2001; Pinto et al. 2016). This method separates Cryptosporidium and Giardia (oo)cysts by attaching an antibody that binds to the (oo)cysts onto a magnetic body (Fradette et al. 2022). A hydrochloric acid solution (0.1 N HCl) is used to dissociate the organisms from the antibody–bead complex, and a sodium hydroxide solution (1 N NaOH) is employed to neutralize the acid (Fradette et al. 2022). This acid dissociation and neutralization process is the most crucial step in IMS, and even slight deviations in pH from neutral can impact the recovery rate of Cryptosporidium and Giardia (oo)cysts (Connell et al. 2000; Arona et al. 2023). Consequently, the recovery levels of (oo)cysts can be quite low and highly variable (Connell et al. 2000; Hu et al. 2004).

Several studies have reported differences in recovery rates depending on the final pH during the IMS process. These studies have found that when the pH deviates from neutrality, it can affect the stability of antigen–antibody binding, leading to a lower recovery rate of oocysts (Kuhn et al. 2002; Ware et al. 2003). However, to our knowledge, few studies have assessed the effects of the HCl and NaOH used during the IMS step on the recovery rates and have investigated variations in the recovery rates of both Cryptosporidium oocysts and Giardia cysts in relation to pH. Additionally, only a limited number of studies have evaluated the recovery rates of (oo)cysts using heat dissociation, which may serve as an alternative to acid dissociation depending on the pH of the reagent used. Pinto et al. (2016) reported that acid dissociation was more effective than heat dissociation in improving the recovery of Cryptosporidium oocysts and Giardia cysts. However, Ware et al. (2003) suggested that a heat-based IMS separation method improved the recovery of Cryptosporidium oocysts. Because previous studies presented conflicting results, it was difficult to judge the effectiveness of heat dissociation (Ware et al. 2003; Pinto et al. 2016).

Therefore, we evaluated the difference in the recovery rates resulting from pH changes in HCl and NaOH solutions during the IMS step, as well as the cause of these differences in recovery rates of Cryptosporidium oocyst and Giardia cyst. Additionally, heat dissociation was applied as an alternative to acid dissociation and neutralization processes, which are sensitive to pH changes, and the differences in recovery rates of (oo)cysts were compared. Finally, we aimed to propose an IMS method that could enhance the recovery rates of Cryptosporidium and Giardia (oo)cysts.

A total of 86 samples were tested for Cryptosporidium and Giardia (oo)cysts according to the standard method (USEPA 2012; Ministry of Environment 2021). Test samples were prepared by spiking purified water with commercially available Cryptosporidium oocysts and Giardia cysts (EasySeed^™, BTF, Sydney, Australia). The samples (10 L) were filtered using a capsule filter (Envirochek^™, Pall Corporation, Ann Arbor, MI, USA). Particulate materials on filter were eluted and concentrated. The (oo)cysts in the pellets were isolated by IMS using magnetic beads coated with specific antibodies that bind to the surface of the (oo)cysts (Dynabeads^TM GC-Combo, Thermo Fisher Scientific, Vilnius, Lithuania). The (oo)cysts were sequentially stained with fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (EasyStain^™ C&G, BTF, Sidney, Australia) and 4′,6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich, St. Louis, USA), and examined using fluorescence and differential interference contrast (DIC) microscopy (Zeiss^™ Axioskop, Jena, Germany). Of the 86 samples, 40 were analyzed to evaluate differences in the recovery of (oo)cysts based on the pH of 0.1 N HCl and 1 N NaOH (Daejung chemicals & metals, Gyeonggi-do, Republic of Korea). The remaining 46 samples were analyzed to compare the recovery rates between acid and heat dissociation.

To compare dissociation methods for (oo)cysts bound to antibody–bead complex during the IMS step, acid dissociation was applied to 23 samples, while heat dissociation was applied to the remaining 23 samples. For acid dissociation, 0.1 N HCl was used, and for acid neutralization, 1 N NaOH was used. To eliminate the effect of pH variations in 0.1 N HCl and 1 N NaOH on the recovery rate, the pH of each reagent was measured and confirmed to ensure the final pH was within the neutral range. Heat dissociation was performed by treating the samples in a heating block maintained at 80 °C for 10 min (Ware et al. 2003; Pinto et al. 2016).

The results were analyzed using paired Student's t-test, and statistical significance was evaluated at a significance level of 5% (Excel 2016).

IMS is a method that uses beads coated with antibodies to detect and isolate target microorganisms from large volume samples, and is an important step in detecting Cryptosporidium oocysts and Giardia cysts in environmental water (Hsu & Huang 2007; Pinto et al. 2016). While the IMS method can increase the recovery rate of protozoa, it can also be a major cause of (oo)cyst loss (Pinto et al. 2016). Since the IMS process separates (oo)cysts through pH changes via antigen–antibody binding, it has been recognized that the pH of the reagent used affects the recovery rate of (oo)cysts (Kuhn et al. 2002; Ware et al. 2003; Koompapong et al. 2009; Lee et al. 2011; Pinto et al. 2016). In our study, the final pH, achieved through the reaction of 0.1 N HCl and 1 N NaOH, showed the highest recovery rate at pH 7.0–7.1 for both Cryptosporidium oocysts and Giardia cysts, indicating that pH neutralization had a significant effect on the recovery rate (Figure 1). Additionally, deviations from a neutral pH were shown to decrease the FITC signal, ultimately reducing the recovery rate (Figure 2). Since pH changes in the reagent reduced antibody affinity or fluorescent dye signal, thereby decreasing the recovery rate of (oo)cysts, it was found that adjusting the pH to neutral during the IMS process is crucial for improving the recovery rates (Kuhn et al. 2002; Ware et al. 2003; Koompapong et al. 2009; Pinto et al. 2016). If the final pH was maintained at a neutral level, the pH of each HCl and NaOH was also important in improving the recovery rate of (oo)cysts. In particular, the pH of HCl had a greater inpact on the recovery rate of Giardia cysts than on Cryptosporidium oocysts. It was confirmed that using HCl maintained at a pH of 0.9–1.0 could enhance the recovery rate of Giardia cysts (Figure 3). When the pH of NaOH was in the range of 13.0–13.1, the recovery rate of (oo)cysts was highest. Additionally, the pH of NaOH was found to have a greater effect on the recovery rate of Giardia cysts than on Cryptosporidium oocysts (Figure 4). The larger the absolute value of zeta potential, the higher the stability of the colloidal solution (Martins et al. 2025). In general, particles need to have a zeta potential greater than ±30 mV to be stably dispersed in the solution (Martins et al. 2025). The average zeta potentials in waters at pH 7.0 were −38 mV for Cryptosporidium oocysts and −17 mV for Giardia cysts (Hsu & Huang 2002). Since the cysts exist in a more unstable state than oocysts in water, they are more likely to aggregate and are estimated to be more influenced by pH.

Additionally, our results indicated the potential applicability of heat dissociation as an alternative to the acid dissociation method, since there was no statistically significant difference in the recovery rates between the two methods (Figure 5). Unlike acid dissociation, heat dissociation does not require the use of HCl and NaOH solutions. As a result, it is not affected by the pH of these reagents, leading to more stable outcomes. Of the two repeated processes during heat dissociation, the recovery rate of Giardia cysts tended to be higher in the second than in the first. This suggests that cysts may require more time to dissociate than oocysts, as they are unstable and prone to aggregation.

The pH affects the recovery rate of Cryptosporidium oocysts and Giardia cysts during the IMS step, where (oo)cysts bound to antibody-coated beads are separated using 0.1 N HCl and neutralized with 1 N NaOH. Therefore, it is essential to periodically monitor the pH of 0.1 N HCl and 1 N NaOH used in acid dissociation to improve the recovery of (oo)cysts in environmental water. In addition, heat dissociation could serve as an alternative to pH-sensitive acid dissociation, contributing to more stable results. Notably, Giardia cysts are more sensitive to small deviations from neutral pH during acid dissociation and require more time for heat dissociation compared to Cryptosporidium oocysts. Consequently, greater attention should be given to the IMS step to enhance recovery rate.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.