Decreased fecal calprotectin levels in Spondyloarthritis patients colonized by Blastocystis spp.

Spondyloarthritis (SpA) is a group of chronic inflammatory systemic diseases mainly characterized by inflammation in the spine and/or peripheral joints. Although a link between SpA-pathogenesis, intestinal inflammation and gut dysbiosis has been proposed, studies have been focused on bacteria-host interactions and very little has been reported regarding intestinal parasites. Here, intestinal parasitic infection of 51 SpA-patients were evaluated and compared to healthy control individuals. No significant differences in the frequency of any parasite between SpA-patients and control individuals were found. Significantly higher levels of fecal calprotectin (FCP) were found in the SpA-patients compared to the control individuals. However, FCP levels were the same when comparing SpA-patients and control individuals, both colonized by Blastocystis spp. On the other hand, when comparing Blastocystis spp. colonized and Blastocystis spp. free SpA-patients, FCP levels were significantly higher in those Blastocystis spp. free. Without ignoring the small sample size as a study limitation, the results showed that in the SpA-patients colonized by Blastocystis spp., the FCP levels were significantly lower than those in the Blastocystis spp. free group and comparable to those in the control group. These findings seem to suggest a relationship between Blastocystis spp. and intestinal inflammation in SpA-patients, but studies intended to explore that interaction specifically should be designed.

www.nature.com/scientificreports/ suggested that intestinal inflammation alters the immune system and intestinal permeability, which allows antigens and activated macrophages to enter into circulation and produce joint inflammation 9,10 . On the other hand, although there is evidence of a relationship between intestinal inflammation and SpA pathogenesis, the role of the microbiome in that interaction is still unclear. In fact, there are very few reports in this regard and most of them explore the relationship between certain types of bacterial gut infections and AS. Although some studies have not found disturbances of the main bacterial groups in the fecal microflora of AS-patients (Bacteroides, Eubacterium-Clostridium and Bifidobacterium) 11 , Costello et al. demonstrated a relationship between disease status and bacterial community composition when comparing AS-patients to control individuals. Therefore, the authors presented the first characterization and identification of intestinal dysbiosis in the AS microbiome 12 . At the same time, the association between the disease and Klebsiella pneumoniae (which was proposed initially as the main microbial trigger and/or perpetuator of AS) has been investigated exhaustively, with contradictory results 13 . The picture is more complex when considering that gut microbiota studies in SpA-patients have focused on bacteria-host interactions and very little has been reported regarding intestinal parasites. There are only a few known case reports that point to Blastocystis spp. 14,15 , Cryptosporidium 16,17 , Dracunculus 18 , Endolimax nana 19 , Entamoeba hartmanni 20 , Entamoeba histolytica 19,21 , Giardia intestinalis [22][23][24] , Strongyloides stercoralis 19,25,26 and Taenia saginata 19,25 as causal agents of ReA. Some reports propose Toxoplasma gondii as a triggering agent of ReA 19 but more recent studies suggest these findings may be coincidental 27 . As far as we know, there are only a few studies on intestinal parasitosis and SpA, and all of them focus on ReA: the aforementioned study on T. gondii 27 , another one on G. intestinalis 28 and another on Giardia in a retrospective and large health insurance cohort 29 .
The aim of this work was to analyze intestinal parasitosis in a group of SpA-patients and to evaluate its relationship with intestinal inflammation. Because fecal calprotectin (FCP) is released during neutrophil activation 30 and reflects the increased migration of these cells into the intestinal lumen and through the inflamed mucosa, FCP levels are high in patients with active inflammatory bowel diseases 31 . Additionally, several studies have shown this protein is elevated in SpA-patients and IBD-patients, and that it correlates well with histological inflammation detected by colonoscopy [31][32][33][34][35][36][37][38] . Therefore, FCP was used as a marker of inflammation.

Results
Population. Fifty-one SpA-patients and 50 control individuals were included in the study. Among the SpApatients, 86.27% (n = 44) were AS-patients, 7.84% (n = 4) uSpA-patients and 5.89% (n = 3) PsA-patients. Regarding clinical expression, 82.35% (n = 42) had axial involvement, 11.76% (n = 6) peripheral and 5.89% (n = 3) mixed. In all, 96.08% (n = 49) were receiving treatment, and ASDAS-CRP and ASDAS-ESR disease activity indices both showed active disease in 90.2% of the cases. The demographic and clinical characteristics of the participants are summarized in Table 1. FCP and clinical characteristics. Elevated FCP levels (≥ 120 ng/ml) were found in 21.57% (n = 11) of the SpA-patients and 14.0% (n = 7) of the control individuals. The median FCP concentration was significantly higher in the SpA-patients compared to the control group (p = 0.0082) ( Table 1). Analyzing the subgroup of SpApatients who had elevated FCP versus the subgroup of SpA-patients with normal FCP (< 120 ng/ml), gastrointestinal symptoms were present in 80.0% of the former group and 81.82% of the latter. On the other hand, according to the limit values established for CRP (C-reactive protein) and ESR (Erythrocyte Sedimentation Rate) (< 3 mg/L and < 20 mm/h, respectively), 11.76% (n = 6) of the SpA-patients had elevated values for CRP, 31.37% (3 men and 13 women) for ESR and 7.84% (n = 4) for both. Among SpA-patients, 52.94% (n = 27) had normal values for FCP, ESR and CRP simultaneously, and none had all three markers elevated at the same time. Finally, Spearman correlation coefficients showed that FCP was positively correlated with ESR, BASDAI and ASDAS-ESR, and had no correlation with CRP, BASFI and ASDAS-CRP (Table 2).

Intestinal parasite infection.
There were no significant differences in the frequency of any parasite between SpA-patients and control individuals and none of the samples were positive for helminths (Table 3. Supplementary Fig. S1). No parasitic species was exclusive to any of the groups, except Iodamoeba bütschlii, which only occurred in one control individual (Supplementary Table S1). The qPCR assay detected five (5) other Blastocystis spp. positive samples in addition to those detected by microscopy. In the case of G. intestinalis, qPCR revealed only one additional infection. Entamoeba histolytica PCR revealed six (6) infections in addition to the three (3) detected for the Entamoeba complex by microscopy (Table 3). For 27.45% of the SpA-patients and 21.57% of the control individuals, E. nana was the only parasite identified. Dual colonization with E. nana and Blastocystis spp. occurred in 51% of the SpA-patients and 56% of the control individuals. The next most frequent combination was E. nana, Blastocystis spp. and Entamoeba coli, which occurred in 7.84% of the SpA-patients and 10% of the control individuals (Supplementary Table S1).
In summary, all the participants were infected with at least one of eight intestinal protists (Table 3). Because E. nana was present in 100% of the population, this parasite was not taken into account in the analyses of association with clinical characteristics. Parasites that were present in a very low proportion, or as co-infection with any other intestinal parasite different from E. nana, were also not included. Therefore, I. bütschlii, G. intestinalis, C. mesnili, Entamoeba coli, E. histolytica and the Entamoeba complex were not included in the analysis (Table 3  and Supplementary Table S1). The only parasite that met the requirements for analysis was Blastocystis spp.
Blastocystis spp. Although it was established that there was a significant difference in FCP levels between the SpA-patients and the control individuals (p = 0.0082), analysis showed no significant difference when Blastocystis spp. colonization was taken into account. To perform this analysis, individuals without Blastocystis spp. www.nature.com/scientificreports/ cant difference in FCP levels was still observed between SpA-patients and the control group in individuals without Blastocystis spp., there was no difference when individuals with Blastocystis spp. were compared (Table 4). Additionally, when comparing median FCP concentrations among the SpA-patients, significantly lower values were observed in those colonized by Blastocystis spp. In the control group, no such difference was observed between Blastocystis spp. colonized and Blastocystis spp. free individuals (Table 5).
Among the SpA-patients colonized by Blastocystis spp. 15.38% had elevated CRP and 34.61% had elevated ESR. In the SpA-patients without Blastocystis spp., these frequencies were similar (14.  Table S2).

Discussion
In this work, clinical characteristics of 51 SpA-patients and the frequency of intestinal parasitosis in them, were evaluated. As noted in the introduction section, research on the associations between SpA and intestinal microbiome has focused mainly on an analysis of prokaryotes, while information on parasites has been limited to a few case reports of ReA [14][15][16][17][18][19][20][21][22][23][24][25][26] .
In this study, 100% of the participants (51 SpA-patients and 50 control individuals) showed infection with some intestinal protist and none of them with helminths (Table 3). There were no significant differences in the frequency of any parasite between the SpA-patients and the control individuals, and the most prevalent parasite in both groups was E. nana, followed by Blastocystis spp.
Although there are some studies that have proposed E. nana as a cause of diarrhea, these findings have been debated for reasons including lack of evidence regarding co-infection with other intestinal microorganisms causing this symptomatology, and even misdiagnosis of E. nana 40 . Similarly, a study that proposed E. nana as a cause of parasitic rheumatism 41 also was refuted because no tests were done to diagnose the reported ReA 40 . We concur with the opinion that E. nana does not cause intestinal inflammation per se. Coupled with the fact that it was present in 100% of the participants, it meant this parasite was not taken into account in the analyses of association with demographic or clinical characteristics. It is relevant to note that there is very little research focused specifically on the study of E. nana and, in general, the reported prevalences correspond to findings in studies focused on other parasites or to general searches for intestinal parasitosis. In fact, a search for "Endolimax nana [title]" in PubMed (on the 15th of July 2022) identified only 15 articles and most of them are relatively old. In Colombia, high frequencies of E. nana colonization were reported when simultaneous diagnosis is made by direct examination and by concentration (for example, 60% in Bermúdez et al. 42 and 77.35% in Hernández et al. 43 ). However, these were local studies on minority groups or schoolchildren from vulnerable communities. Furthermore, when comparing epidemiological data from different studies, not only the diagnostic method and the target population should be considered, but also the period when the surveillance was carried out. For example, a retrospective study in Chile involving 68.142 preschool and school children showed that E. nana decreased from 26.26% in 1980 to 10.14% in 2007 44 . However, a later Chilean study in 103 students from urban and rural schools found an E. nana prevalence of 40.2% 45 . A similar result (41.2%) was obtained among 119 healthy food handlers from Zulia, Venezuela 46 . In Brazil, a study conducted in 3,245 individuals (aged 1 to 93 years) attending the FIOCRUZ Institute identified E. nana as the most common enteric parasite (6.66%) 47 . The same in 595 children and adults living in Brazilian urban slums in whom E. nana was detected with a prevalence of 16% 48 . In the United States, 2,896 patients from throughout the country were tested for intestinal parasites and E. nana was detected in 46 participants (1.6%) 49 . A similar prevalence was found (1.5%) in 2,604 patients from the states of Colorado, Montana, Utah, and New Mexico 50 . In Algeria, E. nana was found in 233 out 2,054 children and adult patients (11.34%) 51 , while the frequency of the parasite was found to be much higher (63.4%) in 93 school attendees in Zambia 52 . In Iran, in 1,025 stool samples collected from 50 villages, E. nana prevalence varied from 18.18% in the youngest (1-10 years) to 34.04% in the group over 50 years of age 53 . In Europe, a study in areas with poor sanitation in Slovakia analyzed 2,760 individuals aged 1 month to 88 years and revealed 16 (0.58%) positive samples for E. nana 54 . Similarly, in a retrospective study in 572 Swiss children attending a hospital, E. nana was the second most frequent parasite (0.5%) 55 . Considering the available background on the epidemiology of E. nana, it is not possible to estimate the frequency of this parasite in the study population (SpA-patients and healthy adults from urban areas with socioeconomic conditions comparable to those of developed countries) and to try to explain the high prevalence found.
Regarding Blastocystis spp., it is also difficult to compare the frequency found in this study to those in earlier reports, since studies have shown that Blastocystis spp. prevalence is highly dependent on the population studied and techniques employed for diagnosis. For example, a literature search on reports of Blastocystis spp. in Brazil between 2000 and 2020 revealed frequencies between 0.5% and 86.6% in the 52 studies ultimately included in the analysis 56 . Similarly, a study in China from 1990 to 2019 found prevalences of Blastocystis spp. in a range of 0.04% to 43.26% 57 . In the present study, the prevalence of Blastocystis spp. was 66.67% in the SpA-patients and 76.47% in the control individuals, which did not represent a significant difference. The high prevalence of Blastocystis spp. in healthy adult groups (even in industrialized countries) has already been reported 58,59 , as well as its higher prevalence in control groups compared to patients with intestinal inflammation [60][61][62] .
As for FCP, its concentration was significantly higher in the SpA-patients compared to the control individuals. Similar findings have been previously reported [34][35][36][37][38] . Simultaneously, in the SpA-patients, a positive and significant correlation was observed between FCP/ESR, FCP/BASDAI and FCP/ASDAS-ESR, but not between FCP/CRP, FCP/BASFI or FCP/ASDAS-CRP (Table 2). No relationship was found between the level of FCP and gastrointestinal symptoms. www.nature.com/scientificreports/ On the other hand, when analyzing FCP in individuals without Blastocystis spp., the result was the same as that found in the general population: FCP concentration was significantly higher in the SpA-patients than in the control individuals. However, when SpA-patients and control individuals, both colonized by Blastocystis spp. were compared, there was no difference in FCP levels (Table 4). Likewise, when comparing Blastocystis spp. colonized and Blastocystis spp. free SpA-patients, FCP levels were significantly lower in those colonized. This difference was not observed in the control group (Table 5). This behavior of the control group differed with the findings of Nieves-Ramirez et al. who enrolled healthy individuals free of gastrointestinal disorders and measured FCP in subgroups of subjects who were positive and negative for Blastocystis spp. They showed that FCP levels in all the individuals were well below levels compatible with clinical inflammation, but were significantly lower in those colonized by Blastocystis spp. 63 . More recently, Ibrahim et al. analyzed stool samples from children and showed that colonization with Blastocystis spp. had no significant effect on FCP levels 64 . In the face of contradictory evidence, we believe these findings may not be comparable due to the diverse aims and approaches of the studies and the differences in experimental designs and sociodemographic characteristics of the study populations. In addition, the existence of indirect factors that have not been contemplated and could influence these findings cannot be ruled out. Therefore, studies aimed at specifically analyzing the effect of Blastocystis spp. colonization on intestinal inflammation in SpA-patients are needed.
In a recent study in which rats were inoculated with Blastocystis cysts (isolated from feces of an asymptomatic human donor) and colitis was induced to monitor the intensity of inflammation compared to a control group, it was found that long-term colonization with Blastocystis spp. was associated with a more severe initial response but a more rapid recovery from colitis 65 . Although it will have to be confirmed that these findings can be translated to humans, they suggest that long-term colonization with Blastocystis spp. may be a protective factor by promoting a more rapid resolution of intestinal inflammation.
With regard to ESR and CRP, no differences in the values of these parameters were observed between SpApatients with and without Blastocystis spp. Similarly, Coskun et al. compared Blastocystis spp. colonized and Blastocystis spp. free ulcerative colitis patients and found no significant differences in ESR and CRP values 66 . In contrast, Javaherizadeh et al. reported significantly higher ESR values in individuals colonized by Blastocystis spp. (11.16 ± 8.67 versus 10.32 ± 5.01. p < 0.01). However, the study included subjects who went to hospitals for routine checkups and does not state or analyze the existence of any underlying disease or clinical condition 67 . The authors also found elevated CRP values in colonized individuals more frequently (1.46%) than in non colonized individuals (0.5%), but did not report median concentration values for each group.
Finally, it is necessary to remark the potential influence of drug therapy on FCP levels. In this study, 68.63% of the SpA-patients received treatment with bDMARDs (Anti IL-17 or Anti TNF-α), 17.65% with NSAIDs and 9.80% with cDMARDs (sulfasalazine or sulfasalazine and methotrexate). As for NSAIDs, their gastrointestinal toxicity has been widely reported. It is known they can increase intestinal permeability within hours of administration and trigger small bowel inflammation days later. In the long term, they can lead to a condition known as NSAID-induced enteropathy, which causes elevation of inflammatory markers such as FCP 68 . Even so, it is unlikely these drugs contribute significantly to the etiology of inflammation in SpA, as NSAID enteropathy is not usually localized in the ileum or colon 4,69 . Along the same line, a study in Spain with 33 SpA-patients found that treatment with NSAIDs did not modify FCP levels 37 . Likewise, a study in Brazil with 85 SpA-patients 31 and another in Croatia with 61 children who were diagnosed with JSpA 70 found that median FCP concentrations were the same between patients who received NSAIDs or DMARs and those who did not. The foregoing leads to the assumption that the observed differences in FCP levels or parasite colonization within the SpA-patients group are unlikely to be the result of treatment. Other authors such as Klingberg et al. showed that FCP levels in AS-patients increased as the frequency of NSAID intake increased, and discontinuation of the drug for three weeks led to a drop in FCP values. However, the authors pointed out that this effect was not due solely to the use of this type of medication, since they found elevated FCP levels in 63% of the patients who did not use NSAIDs or did so only a few times a year 34,36 . Similarly, these authors found significantly lower FCP values in patients receiving methotrexate and infliximab treatment, but not so in those receiving etanercept or sulfasalazine 34,36 . The findings for adalimumab were conflicting 34,36 . Although the number of individuals is not sufficient to make a conclusive comparison, in this study we found no difference in median FCP values between the SpA-patients receiving anti-TNF-(n = 31), NSAIDs (n = 9) or sulfasalazine (n = 3) treatment: 49.70 ng/ml, 49.97 ng/ml and 45.65 ng/ml, respectively.
The study limitations included the small sample size and the fact that we are unable to establish whether Blastocystis spp. can actually influence FCP concentration in SpA-patients, or whether an unknown intestinal context promotes the presence of Blastocystis spp. and reduced inflammation. A study specifically focused on Blastocystis spp. is needed, which also takes into account the intestinal microbiome and the possible influence of therapeutic treatment.
To our knowledge, this is the first study that simultaneously analyzes the parasitic intestinal population and the clinical characteristics of SpA-patients. Without ignoring the aforementioned study limitations, the results showed that in the SpA-patients colonized by Blastocystis spp., the FCP levels were significantly lower than those in the Blastocystis spp. free group and comparable to those in the control group. Although the data seem to suggest a relationship between Blastocystis spp. and intestinal inflammation in SpA-patients, studies intended to explore that interaction specifically should be designed.

Methods
Study population. A group of individuals with diagnosis of SpA (SpA-patients) and a group of healthy individuals (control) were included in the sample. Participants were enrolled between April 2019 and February 2020. The SpA-patients were subjects older than 18 and younger than 65 years of age. They were selected  Table S3) were used to confirm successful DNA extraction by qPCR 73 . Enterococcus faecalis strain ATCC 29212 was used as positive control. The qPCR reactions were performed with TaqMan® Fast Advanced Master Mix (BioRad. Hercules, USA) and run on a CFX96 Touch real-time PCR system (BioRad. Hercules. USA).
Because E. histolytica cannot be morphologically differentiated from E. dispar and E. moshkovskii through microscopic examination, a semi-nested PCR protocol to amplify the 18S rRNA gene was done to identify E. histolytica 74,75 . Both PCR reactions were carried out in a mix containing 0,2 mM dNTPs, 2 mM MgCl2, 0.5 μM of each primer (synthesized by Macrogen. Seoul, Korea) (Supplementary Table S3) and DNA GoTaq® Flexi DNA Polymerase (Promega. Madison, USA). Reactions were performed on a T100 PCR thermal cycler (BioRad. Hercules, USA) as follows: 3 min at 94 °C and 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, with a final step of 7 min at 72 °C. As template, 3 μl of each DNA extraction were used in the first PCR and then 2 μl of the PCR products from the first step were used as templates for the second step. A plasmid containing the product of the first amplification was produced and used as positive control. The identity of the construct was confirmed by sequencing (Macrogen-Seoul, Korea).
Giardia intestinalis and Blastocystis spp. were identified by qPCR using the primers and TaqMan probes reported by Mejia et al. 75 and Stensvold et al. 76 , respectively (synthesized by Macrogen. Seoul, Korea) (Supplementary Table S3). Reactions were performed individually for each parasite with 2 μl of DNA extraction as template and TaqMan® Fast Advanced Master Mix (BioRad. Hercules, USA). Programs were run on a CFX96 Touch Real-Time PCR System (BioRad. Hercules, USA) as in Mejia et al. 75 and Stensvold et al. 76 . DNA of G. intestinalis (strain WB) and Blastocystis spp. (strain BT1) were used as templates in the qPCR reactions and the obtained products were cloned (vector pJET1.2blunt.Thermo Scientific™. Waltham, USA), sequenced (Macrogen. Seoul, Korea) and used as templates in calibration curves (dilutions between 10,000 fg/μL to 0.1 fg/μL).
Statistical Analysis. Descriptive statistics, including mean, median, standard deviation (SD), and interquartile ranges, were used to characterize the study population according to the assumption of normality, as evaluated using the Shapiro-Wilk test. Group comparisons were performed using a mean-difference Z test to continuous variables conforming to a normal distribution; otherwise, a Wilcoxon rank-sum test was applied to other continuous variables. Dichotomic qualitative variables were compared between two groups using a Z test of proportion differences. Correlations between variables were evaluated by Spearman's correlation due to a non-normal distribution in those variables. When it was appropriate, Bonferroni correction was applied to www.nature.com/scientificreports/ account for multiple comparisons. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using Stata V.12.1.

Data availability
The data underlying this article cannot be shared publicly due to the privacy of individuals that participated in the study. The data will be shared on reasonable request, and may be requested via email to romeromaria@ unbosque.edu.co. www.nature.com/scientificreports/