The opportunistic protist, Giardia intestinalis, occurs in gut-healthy humans in a high-income country

ABSTRACT Giardia intestinalis, a cosmopolitan gastrointestinal protist, is detected mainly in patients with clinical giardiasis in high-income countries. In contrast, there is very little information on the presence of Giardia in asymptomatic individuals. Therefore, the aim of this study was to determine the presence and prevalence of Giardia in gut-healthy volunteers in the Czech Republic and to perform a comparative evaluation of different diagnostic methods, since Giardia diagnostics is complicated. Our results confirmed that the qPCR method is the most sensitive method for detecting Giardia and revealed a prevalence of 7% (22/296) in asymptomatic individuals. In most cases, the colonization intensity ranged from 10−1–101. A conventional PCR protocol targeting the TPI gene was used to identify the assemblages. However, this protocol had limited sensitivity for Giardia amplification, effectively detecting colonization above an intensity of 104. In addition, Giardia was detected in 19% of the animals, which were closely associated with the study participants. However, due to methodological limitations, zoonotic transmission could not be clearly confirmed. Notably, contact with animals proved to be the only factor that had a significant impact on the incidence of Giardia in gut-healthy humans.

Giardia is a complex of organisms exhibit a high degree of genetic diversity and colonizes the small intestine of humans and other mammals [2].Eight distinct assemblages, referred to as assemblages A-H, have been documented [2].While assemblages A and B have been found mainly in humans, they display broad host specificity, posing a zoonotic potential [10].The remaining six assemblages (C-H) are restricted to non-human hosts [11], although sporadic detections in humans have also been reported [12,13].Currently, it is considered that Giardia assemblages represent distinct species [14].
Various methodological approaches have been employed to detect G. intestinalis [15].Commonly used diagnostic methods still include coproscopic techniques (e.g.flotation or sedimentation), but their predictive value may be affected by the intermittent shedding of cysts in the host excrements and the expertise of the diagnostician [15].Molecular methods, such as conventional PCR (cPCR) and real-time PCR (qPCR) [15,16], are also used for detection and assemblage identification.However, the identification of assemblages is impeded by the limited sensitivity of existing molecular genotyping tools, particularly in individuals with weak colonization [17].Consequently, there exists a knowledge gap regarding the occurrence of assemblages in such samples, and the assessment of zoonotic potential often remains inconclusive [12,18].
The prevalence of G. intestinalis in high-income countries is reported to be between 2% and 7% in high-income countries, while in low-income countries the prevalence is higher and usually ranges from 20% to 30% [19].Because Giardia diagnostic efforts typically target clinical giardiasis cases based on characteristic symptoms, the occurrence of asymptomatic Giardia colonization in healthy individuals in high-income countries remains understudied [20].With increasing interest in gut microbiome research, it has been recognized that asymptomatic Giardia colonization is quite common within the human population [3][4][5][6][7][8][9].It is important to note that the available studies on asymptomatic individuals have focused on children under the age of 16 [5,8].Here, we sought to address this knowledge by extending our investigation to include a more diverse age range of healthy individuals, from infants to individuals over the age 60.
Given the urgent need to advance our understanding of G. intestinalis, its epidemiology, and public health implications pertaining to asymptomatic colonization in the human population, our study aimed to determine the prevalence of this protist among gut-healthy individuals in the Czech Republic (i.e.those without intestinal disease).We further assessed the fecal Giardia load in positive samples using qPCR and identified assamblages in selected isolates.Furthermore, we sought to investigate the potential zoonotic transmission of Giardia between study participants and their companion animals.To achieve our research objectives, we employed two different diagnostic methodologies such as qPCR targeting SSU RNA, and cPCR assays focusing on three genes, specifically triosephosphate isomerase, beta-giardin, and small ribosomal subunit.One of the sub-objectives was to evaluate and compare the specificity and sensitivity of the above-mentioned cPCR protocols to identify the most robust approach.Finally, we conducted a sensitivity comparison between cPCR and qPCR for the detection of Giardia.

Sample collection
This study employed samples obtained from our previous investigation focused on another intestinal protist, Blastocystis sp [21].Stool samples origin from volunteers who met the criteria of being gut-healthy (i.e.without diarrhoea, abdominal pain, flatulence, etc.).Samples were collected between 2017 and 2019 in the Czech Republic.The participants also completed a questionnaire providing details regarding gender, age, living location, travelling, animal contact [21].Additionally, fecal samples were also collected from the animals closely associated with volunteers (for more details see Lhotská et al. [21]).

DNA isolation and PCR diagnostics
Total genomic DNA was extracted from fecal samples (200 mg) using the commercial kit PSP Spin Stool DNA Kit (Stratec, Germany), according to the manufacturer's protocol (see Lhotská et al. [21]).
Initially, we assessed three distinct cPCR protocols, which targeted different genesspecifically, triosephosphate isomerase (TPI), beta-giardin (BG), and the small ribosomal subunit (SSU rRNA)for their effectiveness in Giardia detection.Detailed information on the primers and PCR conditions is provided in Table 1.Each PCR reaction (20 µl) comprised 2×concentrated Master Mix AccuPower® Taq PCR PreMix (Bioneer, Republic of Korea) and was conducted using the T100 TM Thermal Cycler (Hercules, California, USA).Positive control DNA was obtained from a trophozoite culture (WB ATCC 30957, human isolate).Visualization of cPCR products (15 µl) was achieved using an electrophoresis system (Thermo Fisher Scientific Inc., USA) with 1.5% agarose gel containing ethidium bromide (0.002 mg/ml).Amplicons were purified using the GenElute TM Gel Extraction Kit (Sigma-Aldrich, MO, USA) and sequencing was outsourced to Eurofins GATC Biotech (Germany).
We also applied the qPCR diagnostic protocol [22] utilizing specific primers and a Taqman probe (for details see Table 1).This protocol was optimized for a LightCycler LC 480 I (Roche, Basel, Switzerland) and adapted to our conditions (Master Mix 5x HOT FIREPol® Probe qPCR Mix Plus ROX, Solis BioDyne, Estonia).Each sample was subjected to triplicate analysis (20 µl per qPCR reaction) in a 96-well block, with the positive control identical to that used in cPCR.To estimate the fecal Giardia load via qPCR, we established a quantification curve using trophozoite cultures (further details available in Supplementary Data 1).

Testing DNA inhibition for qPCR
We examined all negative samples for potential qPCR inhibition.This was achieved by adding foreign DNA (from experimental rat tissue) and employing a specific qPCR protocol (commercial primers and Taqman probe for detection of the rat beta-2-microglobulin; ThermoFisher Scientific, Waltham, MA, USA) according to study Šloufová et al. [21]).
We employed a Bayesian generalized linear model with a binomial distribution and logit link function to investigate the impact of several variables on Giardia occurrence as detected by qPCR.The variables considered were age (continuous; years), gender (binary; male/female), living location (binary; city/village), travelling (categorical; non-travel/Europe/outside Europe), and contact with animals (categorical; no contact/contact with pets/contact with pets and livestock) (for more details, see Supplementary data 2).

Prevalence of Giardia intestinalis
In this study, we examine a total of 431 fecal samples, consisting of 296 samples from asymptomatic humans and 135 animal samples collected within 14 regions of the Czech Republic (for more details, see Lhotská et al. [21]).
Initially, we utilized cPCR targeting the TPI gene (530 bp) to analyze Giardia-positive samples, as it proved to be the most efficient protocol when compared to alternative protocols for BG and SSU rRNA.The latter two protocols exhibited low specificity and sensitivity (see Supplementary Data 3 for details).
Additionally, we verified the presence of Giardia in the qPCR amplicons using Sanger sequencing.However, it is important to emphasize that this sequencing did not provide information about specific assemblages.This limitation arises from the conservative nature of the SSU rRNA gene and the very short length of the amplicon (62 bp).
For all 47 positive samples, we identified the fecal Giardia load based on the correlations between obtained Ct values and quantification (10 −1 -10 5 cells per qPCR reaction; Table 2 and Table 4; Supplementary data 1).In most cases (81%), the fecal load corresponded to values of 10 −1 -10 1 cells per one qPCR reaction.
Internal inhibition was not detected in any of the samples.Additionally, all 431 samples underwent testing using Sheather's flotation method, and only two samples tested positive (human sample no.B441/20 and rabbit sample no.B151/18).Interestingly, these samples exhibited higher Giardia loads, specifically 10 4 and 10 6 .

Sensitivity comparison of molecular diagnostics methods
To evaluate the difference in sensitivity between cPCR and qPCR, we compared the 142 samples that were tested by both methods.While cPCR detected G. intestinalis in only seven cases (7/142), qPCR detected Giardia in 24 cases (24/142) (Table 5), indicating that qPCR is the more sensitive method for detecting Giardia in fecal samples (McNemar test, χ 2 = 18.01; p < 0.01; Table 5).

Factors influencing Giardia intestinalis occurrence
The impact of specific factors (Figure 1) on G. intestinalis prevalence was exclusively investigated within a subset of human samples (N = 296).Supplementary Data 5 presents information concerning asymptomatic Giardia-positive individuals.

Discussion
The intestinal protist Giardia intestinalis is considered an opportunistic pathogen with a cosmopolitan distribution.Higher prevalence rates, typically ranging from 20% to 64%, are frequently documented in low-income countries most likely attributed to low hygiene standards [24,25].In contrast, high-income countries report the presence of Giardia primarily in symptomatic cases [20].
To date, there exists a limited number of studies investigating the prevalence of G. intestinalis in guthealthy individuals.Moreover, the majority of these studies have exclusively focused on children below the age of 16 [5,8].Therefore, the principal objective of our study was to address this knowledge gap by assessing the occurrence of Giardia in a diverse age group of gut-healthy individuals (0 to >60 years) within a high-income country, the Czech Republic.We employed qPCR as the main detection method.
Given that epidemiological studies on G. intestinalis encompass a variety of diagnostic methods to identify positive individuals, each with varying sensitivity and specificity, we opted to compare our data solely with studies that utilized conventional PCR (cPCR) and qPCR.Contrary to expectations, our study revealed a Giardia prevalence of 7% in 296 gut-healthy individuals.However, this prevalence is notably lower when compared to the results of a few currently available studies focusing on asymptomatic subjects.For instance, recent surveys conducted in Spain reported asymptomatic Giardia prevalence of 17% and 18% [5,8].Conversely, low-income countries such as Brazil, Ethiopia, Argentina, and Mozambique exhibit considerably higher asymptomatic prevalence of Giardia ranging from 18% to 64% [6,[26][27][28].The low prevalence observed in our study could be attributed to the broad age range of the subjects (0 to >60 years).It should be emphasized that the studies mentioned above were mainly conducted on asymptomatic children under 16 years of age.Hence, additional crosssectional studies are needed to clarify the presence of G. intestinalis in the healthy population, employing qPCR as a diagnostic method, with particular focus on the adult population.Our results demonstrating the occurrence of Giardia in an asymptomatic population are consistent with the observations of Messa et al. [26], who found its higher incidence among asymptomatic individuals (32%) compared to individuals with diarrhea (20%).
For the accurate detection of intestinal protists and the assessment of their prevalence in gut-healthy individuals, the utilization of a precise and highly sensitive molecular method is imperative e.g.[29,30].This choice becomes particularly crucial in the case of Giardia, given the sensitivity and specificity challenges associated with existing molecular targeting protocols [18].In our study, we initially conducted a specificity comparison for cPCR across three different genes [31][32][33], with the TPI gene protocol demonstrating the highest specificity [33].
Subsequently, we compared the sensitivity between TPI cPCR and qPCR for Giardia detection.This comparative analysis aimed to identify the most sensitive method, particularly in the context of our study focus on gut-healthy individuals with low colonization.Our findings corroborate the limited sensitivity of cPCR, especially for detecting weak Giardia colonizations (i.e.below 10 4 ), as qPCR identified an additional 17 positive samples.Conventional coproscopical methods (e.g.Sheather flotations) only detected two positive samples.
In addition to assessing qPCR sensitivity, we quantified the intensity of Giardia colonization in positive samples by constructing a quantification curve.This curve was generated using a dilution series derived from a Giardia culture (range of 10 −1 -10 5 cells per sample).We chose to use trophozoites from the culture instead of cysts to generate the quantification Table 6.Outputs of a Bayesian generalized linear model with binomial distribution and logit link explaining a variation in Giardia occurrence in humans detected using qPCR method.HPD intervals not including zero suggest a statistical significance and a Bayes factor greater than 1 can be interpreted as evidence against the null, a Bayes factor greater than 3 can be considered as substantial evidence against the null.curve because we did not have Giardia-positive samples that contained enough cysts, even from experimental animals [34].A major limitation of our study was the insufficient number of cysts available to construct the curve.
A surprising finding in our study is the remarkable sensitivity of qPCR in detecting of very low colonization intensities (from 10 −1 cells per 1 qPCR reaction), the so-called fecal protist load (according to Šloufová et al. [29]).In contrast, cPCR only identified samples with moderate to high fecal Giardia load, especially those with more than 10 4 cells.This contrast in sensitivity among different molecular protocols underscores the potential for biased information about the presence of Giardia in gut-healthy individuals when employing varying diagnostic approaches.
Our results indicate that asymptomatic cases are characterized by low fecal loads, typically falling within the range of 10 −1 -10 1 , which can only be detected by qPCR and remain undetectable by less sensitive methods.Additionally, our study underscores the difficulty in obtaining sequences for assemblage determination using cPCR, specifically for the TPI gene, in qPCR-positive samples with fecal Giardia loads below 10 3 .Consequently, the identification of assemblages was feasible in only seven out of 47 positive samples, all of which had fecal loads between 10 4 and 10 6 .These identified assemblages included BIV in five rabbits, AII in one dog, and BIII in one human.Our findings align with those of Belkessa et al. [17], who also reported similar challenges related to the sensitivity of qPCR and the sequence obtaining via cPCR.
Another objective of our study was to investigate the presence of Giardia in animals closely associated with volunteers, focusing on possible zoonotic transmission.Our results revealed the Giardia positivity rate of 19% in these animals.However, there is a lack of comprehensive studies examining the presence of Giardia across a range of animal species by PCR.Nevertheless, our results appeared to be among the range of positivity rates reported in different animal species: 29% in dogs [35], 8% in cats [36], 4% in rabbits [37], 33% in pigs [38], 28% in cattle [39], 4% in guinea pigs [40], and 17% in horses [41].
Regarding the identification of assemblages, as mentioned above, we were able to confirm only five in rabbits and one in a dog based on an adequate colonization intensity for cPCR (> 10 4 ).In one case, we detected Giardia by qPCR in both the owner and his pet (a rabbit).Unfortunately, we were unable to obtain a sequence from the owner's sample, primarily because of the low fecal Giardia load (10 1 ).Consequently, zoonotic transmission could not be confirmed in this case.
The distribution of intestinal protists is influenced by various epidemiological factors such as living locality, contact with animals, travel history, age, and gender [21,30].Among these factors, only contact with animals significantly impacted Giardia occurrence in gut-healthy individuals in our study.Individuals in regular contact with animals, whether pets or farm animals, exhibited a higher prevalence, suggesting that close animal contact might play a pivotal role in zoonotic transmission [42,43].This aligns with a previous study in Brazil that identified shared sub-assemblages (BIV) between children and pet dogs, suggesting potential Giardia transmission [44].However, our data cannot definitively confirm zoonotic transmission due to limited sequencing information as previously mentioned.
Interestingly, lifestyle factors, such as travel history and living locality, did not influence Giardia occurrence in the Czech gut-healthy individuals.While travel is often considered a significant predisposing factor [45,46], our study did not reveal a higher prevalence among individuals who travelled within or outside of Europe compared to non-travellers.Additionally, rural residents, who are typically at a risk of exposure to potential sources of Giardia colonization [43,47], did not show here a significantly higher Giardia prevalence compared to urban residents.This is consistent with a Colombian study reporting similar prevalence in both urban and rural areas [48].
Giardia intestinalis is most frequently found in children, particularly those under five years of age [43,47].A study by Muadica et al. [5] focused on healthy asymptomatic children aged 1-16 years, revealed a 17% prevalence (263/1512).Here, we did not observe any significant age-related effects.Regarding gender, we found no difference in occurrence between women (9%) and men (5%), which is consistent with a previous study [24].

Conclusions
In conclusion, our study contributes significantly to filling the knowledge gap regarding the occurrence of G. intestinalis in gut-healthy individuals in a highincome region.However, we found a lower prevalence (7%) compared with previous studies, which may be due to the wider age range of the individuals included in our study.Our results highlight the importance of using accurate and highly sensitive molecular methods such as qPCR for precise Giardia detection.Traditional and conventional methods commonly employed in epidemiological studies often exhibit limited sensitivity and specificity.
Quantitative PCR has demonstrated exceptional sensitivity in detecting even weak Giardia colonization.However, the challenge of obtaining sequences by cPCR from qPCR-positive samples with low fecal loads remains an issue for future studies.In addition, our results highlight the need for further research using advanced tools to explore the genetic diversity of Giardia in animals and its potential transmission to humans.
In our study, close contact with animals was identified as a significant factor associated with a higher prevalence of Giardia in gut-healthy individuals, suggesting a possible role in zoonotic transmission.However, the ability to obtain sequences for assemblage identification is limited, preventing definitive confirmation in this regard.

Figure 1 .
Figure 1.Prevalence of Giardia intestinalis in human samples according to the specific categories such as lifestyle (village life/city life, travelling) and contact with animals (pets and farm animals).(Samples Nnumber of samples obtained in each category out of the total number of samples) * Statistically significant differences.

Figure 2 .
Figure 2. Effect of contact with animals on the probability of Giardia infection in humans.We present the entire posterior distribution of the three animal contact categories with their median (vertical line) generated from posterior draws of a Bayesian model.

Table 2 .
Summary of qPCR-positive human and animal samples (N = 47) for Giardia intestinalis with Ct values and comparison with cPCR results.

Table 3 .
List of human and animal species included in this study subjected to qPCR.

Table 4 .
Evaluation of the fecal load of Giardia intestinalis in human samples based on the quantification curve (set in the range of 10 −1-10 5 trophozoites per qPCR reaction).

Table 5 .
Comparison of the results of two diagnostic methods (cPCR and qPCR) in detection of Giardia intestinalis in 142 samples using McNemar's test (χ 2 = 18.01; p < 0.01).