Exploring challenges in Giardia cyst visualisation by common microscopy methods

For a long time, water, sanitation, and hygiene (WASH) practitioners and regulating bodies have relied heavily on indicator microorganisms for many of their decisions, particularly in developing nations. However, recent studies have indicated that total reliance on faecal indicator bacteria may be misleading, since this group is often less persistent and more easily inactivated during treatment processes than other organisms, including the protozoa Giardia spp. (Mraz et al. 2021).

Giardia duodenalis is one of the commonest intestinal parasites that infects humans worldwide (Yason & Rivera 2007). From 2011 to 2016, 37% of worldwide waterborne protozoa outbreaks were caused by this zoonotic protozoan (Efstratiou et al. 2017). The chlorine-resistance characteristic combined with cysts’ ability to permeate filtration units because they are small (6–15 μm) can explain the occurrence of giardiasis epidemics after consumption of treated drinking water in several countries (Smith 1998; Omarova et al. 2018). This illustrates the importance of considering this pathogen when making decisions regarding water and sanitation, as well as including it in regular monitoring of water systems, either conventional or point-of-use. The latter, particularly, is very important in low-income and self-supplied regions, which are more likely to be contaminated than others (Genter et al. 2021).

Because of the typical cyst morphology that avoids mistaking Giardia for other organisms, optical microscopy is still the main methodology used routinely to detect this organism in water, food, and faecal samples (Adeyemo et al. 2018). In addition, as it is low-cost (compared to molecular-based tools) and relatively easy to run, this sort of technique is the most suitable for developing countries (Tangtrongsup & Scorza 2010).

Brightfield (BF) microscopy associated with Lugol's iodine stain is considered the gold standard methodology for detecting cysts in stool samples (Hooshyar et al. 2019). It enhances the internal structures of cysts, which stand out from the other materials on the slide (Tangtrongsup & Scorza 2010). Nevertheless, in the field of water and sanitation, the standard protocol is direct immunofluorescence assay (IFA). This technique consists of adding fluorescent-labelled antibodies that will link specifically to the cysts’ wall producing a bright fluorescence that can be observed in the microscope (USEPA 2012, 2014). For confirmation, it has been suggested that DAPI (4′,6-diamidino-2-phenylindole) must be applied at the same time as the IFA test (USEPA 2012). The stain highlights the nuclei when it binds to DNA (USEPA 2012).

More recently, Belini et al. (2018) showed that darkfield (DF) microscopy was effective in detecting Giardia spp. in purified suspensions. This illumination technique is based on the formation of a hollow cone of light that encloses the objective lens, causing the light to bypass them. Thus, only diffracted/scattered light from objects in the mounting medium enters the objective lens, forming bright white structures against a dark background (Murphy 2001).

Despite the variety of microscopy techniques for Giardia cyst detection, selecting the most suitable method still poses a challenge, especially for practitioners. Therefore, this study aimed to explore, under controlled conditions, common methods for detecting Giardia spp. cysts, shedding light onto the possibilities for choosing protocols for detecting this parasite.

Purified suspensions of Giardia duodenalis (200 cysts μL⁻¹) and Giardia muris cysts (149 cysts μL⁻¹) (Waterborne, Inc., New Orleans, USA) were used for microscopy. Although G. duodenalis is the only species considered pathogenic to humans, the rodent-exclusive pathogen, G. muris has been used as a model for G. duodenalis since their morphologies are similar (Haas & Kaymak 2003), making it also relevant to environmental, water and sanitation studies.

Aliquots of 5 μL of each suspension were spiked together onto flat glass microscopy slides for three different preparations – see below. The procedure was repeated for each species on an individual slide.

For BF microscopy, one drop of Lugol's iodine was added to the slides. G. duodenalis and G. muris identity were confirmed based on characteristics of cyst size (7–10 μm long × 8–12 μm wide) and internal structures, according to classic literature description (Filice 1952; Jakubowski 1984).

As for fluorescence microscopy, spiked samples were kept dry overnight. Then, as recommended in Method 1623.1 (USEPA 2012), IFA was performed using the Merifluor^® kit (Meridian Bioscience, Inc.), according to the manufacturer's recommendations. When using this kit, Giardia cysts appear as brilliant apple green fluorescence objects under ultraviolet light, with a typical size and an oval to round shape (USEPA 2012).

To visualise the cysts’ nucleus, two drops of Fluoroshield™ with DAPI (Sigma-Aldrich^®) were added and incubated for 10 min at room temperature. This reagent stains the cysts a light blue internally, staining up to four distinct nuclei with a green rim or highlight (USEPA 2012). A second fluorescence microscopy process involved a simpler preparation method using only Fluoroshield^TM with DAPI.

An epifluorescence microscope (BX51, Olympus^®) was used for both BF and fluorescence microscopy. Use of the former involved conventional staining with Lugol's iodine and observation of the organisms’ structures under visible light using a 40× objective lens. The latter involved the use of FITC and DAPI as optical filters (Table 1) for visualising cysts and nuclei, respectively. Physical dimensions determined on Image-Pro^® 6.3 were compared to reported patterns for Giardia cysts, according to USEPA (2012).

DF images were acquired by adapting the standard microscope using an inexpensive, custom-made DF setup developed by Belini et al. (2018). A dry objective lens (Olympus BX41TF) and a charge-coupled device camera (Samsung^® SDC313) were used. Image capture was carried out on PixelView^®. Knowledge of the cysts’ characteristic morphological features improved Giardia detection accuracy (Belini et al. 2018).

Although Lugol's iodine is the most used dye in routine laboratory diagnostics, its use requires an expert to identify organisms and their typical structures (Charakova 2010). Furthermore, internal quality control of the working iodine solution must be performed regularly on known reference organisms to ensure the performance of the stain (Dalynn Biologicals 2005). As staining is not selective, this method hinders cyst detection in environmental samples, which are likely to contain debris and therefore require concentration and purification steps, and deep reliance on the microscopist's subjective analysis.

Despite the high sensitivity and specificity of Merifluor^®, its datasheet indicates that the kit is recommended for use on faecal human samples, in which G. duodenalis is, possibly, the only species present. Therefore, the monoclonal antibodies of the kit are likely to be specific to G. duodenalis antigens. This assumption could explain the difference in fluorescence intensity between the two species but could also be a limiting factor for the kit's use in environmental matrices, since there is no assurance which species might be present in the sample (Edelstein & Edelstein 1989; Alderisio et al. 2017). From a public health perspective, this is particularly important, as differences in fluorescence staining may mislead reported cyst occurrences and concentrations in environmental samples (Alderisio et al. 2017).

It is noted that IFA refers to the current protocol established by USEPA methods 1623.1 and 1693 for analysis of water and disinfected wastewater (2012, 2014), thus it is assumed to be the first resource WASH practitioners would rely on for periodically monitoring quality considering protozoan parasites. In fact, several studies on environmental samples that do not have a standardised detection method include IFA (Olson et al. 1999; Greinert et al. 2004; Graczyk et al. 2008; Grit et al. 2012; Giglio & Sabogal-Paz 2018; Sammarro-Silva & Sabogal-Paz 2020, 2021a, 2021b; Ogura & Sabogal-Paz 2021, 2022). The motive for choosing this protocol is assumed to be its high sensitivity, as older references that consider complex matrices were also immunofluorescence-based, as in Olson et al. (1999), which used IFA to quantify the survival of cysts seeded into soil and cattle waste.

Image results obtained for transmitted DF illumination demonstrated that, despite being unable to differentiate G. duodenalis from G. muris, DF microscopy allowed them to be distinguished by their typical oval shape from other illuminated elements. These interferers are particles that are smaller than the diffraction resolution limit for conventional light microscopy (∼200 nm) (Murphy 2001).

DF imaging can be accomplished in conventional light microscopes equipped with commercially available accessories or, as in the case of Belini et al. (2018), with a low-cost, easy-to-use ring illuminator built with standard light-emitting diodes. By using transmitted DF illumination, the authors observed well-defined cyst walls and intracellular structures, including nuclei and retracted cytoplasm, while axonemes were only imaged in the reflected DF mode. These results encourage further use of transmitted DF microscopy in Giardia spp. visualisation.

DF microscopy is supported mainly by its low-cost and lack of need for reagents, dyes, or specific equipment (Belini et al. 2018). So, it might be an alternative for detecting Giardia spp. cysts in resource-poor settings. However, in a general scenario contemplating environmental samples, scattering of other particles may hinder visualisation, particularly if the slide contains debris, thus purification methods would apply just as they do in all protocols mentioned above. Like Lugol's iodine in BF, DF effectiveness depends on well-trained observers. Due to possible microscopist subjectiveness, it is recommended that at least two professionals analyse samples for quantification.

Table 2 summarises general properties of BF, DF, and fluorescence protocols. These may be either advantages or limitations depending on circumstances – e.g., laboratory settings, available resources and level of urgency for cyst detection/analysis, personnel, matrix quality, etc. On the basis of both the micrographs obtained in this study and experience, the combination of techniques is believed to be an alternative to deal with the constraints inherent in individual methods. If more than one observer is available, that is also important to avoid bias in slide interpretation, as well as human error, as all the procedures cited above are observer-dependent.

Selecting a microscopy imaging method for Giardia cyst identification depends on several factors, including available laboratory infrastructure and human resources, professional expertise, the matrix to be analysed, and time. Still, protocols that require bulky equipment and costly reagents may be challenging.

An exploratory approach was used to demonstrate that BF, DF, and IFA (combined or not with DAPI) enable Giardia spp. cyst detection in purified samples, despite not providing species identification This suggests that simple and inexpensive techniques such as BF and DF microscopy may be useful in low- and middle-income countries when IFA protocols cannot be performed, if validation is carried out.

Ideally, analysis for Giardia cyst detection should include a combination of at least two techniques, so that constraints are compensated for. Additionally, microscopy samples should preferably be analysed by different professionals, with repetitions, for more reliable results. This is important, particularly in environmental analyses, in which interferents and different species of pathogens may be present, nonetheless, it would increase sample processing costs considerably depending on the method.

This work was supported by the São Paulo Research Foundation – FAPESP (Process 12/50522-0), The Royal Society (ICA\R1\201373 – International Collaboration Awards 2020), and National Council for Scientific and Technological Development (CNPq-Brazil, process no. 308070/2021-6). The Coordination for the Improvement of Higher Education Personnel (PNPD/CAPES) awarded a fellowship to M.J.R.d.C.

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

The authors declare there is no conflict.