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Received: 26 February 2022    Revised: 2 June 2022    Accepted: 26 June 2022

DOI: 10.1111/1756-185X.14388

ORIGINAL ARTICLE

Fecal calprotectin as a biomarker of microscopic bowel

inflammation in patients with spondyloarthritis

Júlia Faria Campos1  | Gustavo Gomes Resende2 | Alfredo José Afonso Barbosa3 |

Silas Castro de Carvalho4 | Junia Aguiar Lage5 | Pedro Ferrari Sales Cunha5 |
Stela Cristina Silva de Souza5 | Maria de Lourdes Abreu Ferrari6

1
Instituto Alfa de Gastroenterologia,
Hospital das Clínicas, Universidade Abstract
Federal de Minas Gerais, Belo Horizonte,
Aim: Microscopic bowel inflammation is present in up to 60% of all patients with
Brazil
2
Departamento de Reumatologia, Hospital
spondyloarthritis (SpA) and appears to be associated with more severe joint disease
das Clínicas, Universidade Federal de and a higher risk of developing inflammatory bowel disease (IBD). This study aimed to
Minas Gerais, Belo Horizonte, Brazil
3
determine the utility of fecal calprotectin (fCAL) in evaluating endoscopic and histo-
Departamento de Patologia Clínica,
Faculdade de Medicina, Universidade logical bowel inflammation in SpA patients.
Federal de Minas Gerais, Belo Horizonte, Methods: Ileocolonoscopies with biopsies and fCAL measurements were performed
Brazil
4 in 65 patients with SpA.
Departamento de Endoscopia Digestiva,
Universidade Federal de Minas Gerais, Results: In 47 (72.3%) patients, the fCAL levels were higher than 50 μg/g, whereas
Belo Horizonte, Brazil
5
in 20 (30.7%), these levels were greater than 250 μg/g. A total of 38 (58.5%) patients
Faculdade de Medicina, Universidade
Federal de Minas Gerais, Belo Horizonte, presented with microscopic bowel inflammation, and 13 (20%) presented with signs of
Brazil endoscopic inflammation. fCAL levels were significantly higher in patients with micro-
6
Departamento de Clínica Médica,
scopic bowel inflammation than in those without inflammatory findings (P < .001); ad-
Faculdade de Medicina, Universidade
Federal de Minas Gerais, Belo Horizonte, ditionally, these levels were slightly higher in patients with endoscopic signs of bowel
Brazil
inflammation (P = .053). A fCAL cutoff value of 96 μg/g predicted histological bowel
Correspondence inflammation with 73% sensitivity and 67% specificity. No statistically significant dif-
Júlia Faria Campos Hospital das Clínicas,
ference was observed in the fCAL levels between patients who had been treated or
Universidade Federal de Minas Gerais,
Avenida Professor Alfredo Balena, 110, not treated with nonsteroidal anti-­inflammatory drugs (NSAIDs).
Santa Efigênia, Belo Horizonte, Minas
Conclusion: Our findings confirm a high prevalence of microscopic bowel inflamma-
Gerais, 30130-­100 Brazil.
Email: j.fariacampos@gmail.com tion in SpA patients, regardless of the use of NSAIDs. The evaluation of fCAL levels
proved to be useful in the identification of microscopic inflammation and could help
in the more judicious indication of ileocolonoscopy. These results support the use of
fCAL for the evaluation of microscopic bowel inflammation in SpA patients.

KEYWORDS
ankylosing spondylitis, biomarker, fecal calprotectin, inflammatory bowel disease,
spondyloarthritis

Clinical Trial Registration Number: UFMG Research Ethics Committee (Ethical approval number 2.510.480).

© 2022 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

Int J Rheum Dis. 2022;00:1–9.  |

wileyonlinelibrary.com/journal/apl     1
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2      CAMPOS et al.
1  |  I NTRO D U C TI O N
Inflammatory bowel disease (IBD) has demonstrated a prevalence
of 0.1%-­0 .2%1 in the general population. Approximately 20% of
IBD patients develop spondyloarthritis (SpA), whereas approxi-
2
mately 10% of those patients with ankylosing spondylitis (AS)
present with a diagnosis of IBD throughout life, thus indicating
an association of the 2 diseases that is 10-­ to 100-­fold higher
in these patients than in the general population. It is estimated
that up to 60% of patients with SpA present with some degree of
bowel inflammation, 3 the majority of which are oligosymptomatic.
The bowel is the interface between the environmental stimulus
and the immunological mechanisms that theoretically participate
4
in the onset of joint inflammation in patients with SpA. Much has
been investigated about the genetic susceptibility, the increase in
bowel permeability, the similarities regarding the types of inflam-
matory responses, and the dysbiosis of the intestinal microbiome
presented in SpA and IBD, which are probable physiopathologi-
cal mechanisms in both conditions. 5 However, the precise role of
bowel inflammation in those patients and its outcomes are still
uncertain.
Microscopic bowel inflammation appears to be a marker of SpA
severity. The condition most often occurs in young patients with a
more severe and active joint disease.3 The more expressive bone
edema in the sacroiliac joints6 is associated with a higher risk of evo-
lution to AS and IBD.7 Studies have shown that patients with SpA
and bowel inflammation more often require biological therapy and
present with better response rates to this medication when com-
pared to those with the same severity of disease but without bowel
inflammation.8
The use of ileocolonoscopy as a standard method of screen-
ing for bowel inflammation presents some limitations; due to the
fact that it is an invasive exam, it has limited availability in certain
scenarios, and it has no routine recommendation in the evaluation
of a patient with SpA without intestinal symptoms.9 Fecal calpro-
tectin (fCAL), which is a protein that comprises 60% of the cytosol
of neutrophils, monocytes, and macrophages,10 is a useful, simple,
and noninvasive biomarker. Its presence in the stool is related to
the migration of these inflammatory cells to the mucosa of the gas-
trointestinal tract, thus making it more specific than other serum
markers,11 such as the C-­reactive protein (CRP) and the erythrocyte
sedimentation rate. It also shows a good correlation with clinical and
endoscopic scores in the evaluation of bowel inflammation activ-
ity12 in patients with IBD. Recent studies have suggested that fCAL
can be an even more useful marker, as it increases in the early stages
of IBD, even before the emergence of endoscopic, clinical, or sys-
temic manifestations.13
In order to enhance the knowledge of the association between
SpA and bowel inflammation, this study was developed to investi-
gate the association between fCAL levels and endoscopic and his-
tological findings of bowel inflammation (even if only in the initial
stages) in patients with SpA.
2  |  M ATE R I A L S A N D M E TH O DS
2.1  |  Evaluated patients and variables
This work was a cross-­sectional study, including 65 patients with
SpA, of which 51 patients had axial SpA (44 ankylosing spondyli-
tis [AS] and 7 nonradiographic axial spondyloarthritis [nr-­A x SpA]
patients), classified according to the criteria of the Assessment Of
Spondyloarthritis International Society (ASAS),14,15 and 14 patients
had psoriatic arthritis (PsA), classified according to the Caspar
Criteria.16 All of the patients were over 18 years of age and received
medical care at the Outpatient Clinic of Spondyloarthritis at Hospital
das Clínicas da Universidade Federal de Minas Gerais (HC-­UFMG).
All of the patients who had previously been diagnosed with IBD,
Crohn’s disease (DC), or ulcerative colitis and known chronic dis-
eases of the gastrointestinal tract (microscopic colitis, eosinophilic
colitis, and known and active gastric and duodenal ulcers), as well
as high-­risk patients for ileocolonoscopies (American Society of
Anesthesiology classification -­ ASA III or IV), those patients using
anticoagulants, and pregnant women were excluded from the study.
All of the patients signed the informed consent form, and the study
was approved by the UFMG Research Ethics Committee (Ethical ap-
proval number 2.510.480).
Clinical data (including human leukocyte antigen [HLA]-­B27 sta-
tus) and information about the use of medications were recorded,
and the level of CRP was measured.
Disease activity was measured via the Bath Ankylosing
Spondylitis Disease Activity Index (BASDAI) and Ankylosing
Spondylitis Disease Activity Score (ASDAS CRP)17,18 in 52 and 50
patients with axial disease, respectively, and the Disease Activity
in Psoriatic Arthritis (DAPSA)19 scale was used for 11 PsA patients
with only peripheral symptoms. Patients were also asked about
the history of psoriasis, uveitis, and erythema nodosum, as well as
about the presence of current gastric (heartburn and epigastralgia)
or bowel (abdominal pain, diarrhea, or blood in the stool) symptoms.
2.2  |  fCAL measurement
fCAL was measured in 65 patients who had their first stool sam-
ples collected in the morning 2 days before beginning their prepara-
tion for the ileocolonoscopy exam. The Quantum Blue® rapid test
(BÜHLMANN Laboratories AG®), which has a lower limit of 30 μg/g
and an upper limit of 1000  μg/g (enzyme-­linked immunosorbent
assay correlation coefficient of 0.8920) was used.
2.3  |  Ileocolonoscopic and histological evaluations
The ileocolonoscopy was performed by the same experienced
endoscopist, who aimed to evaluate enanthema, edema, loss of
submucosal vascular pattern, and, most importantly, the presence
CAMPOS et al. |
      3

TA B L E 1  Overall characteristics of the 65 evaluated patients

of erosions and ulcerations of the colonic and ileal mucosa. Two
fragments of each bowel segment were collected (terminal ileum, N = 65 Mean
right, transverse, and left portions of the colon, and rectum). The Characteristics (%) (SD†) Median (IQR‡)

exam was performed in a 15-­day maximum interval from the last Gender
rheumatologic examination in which the disease activity was -­ Male 35 (53.8)
evaluated. Non-­smokers 58 (89.3)
Histology was performed by a single pathologist (A.J.A.B), who Age 49 (±11.8)
classified the findings as being without inflammatory changes and Disease duration in y 18.5 (10–­20)
with signs of acute or chronic inflammation, according to previously
SpA subtype
described classifications.3,9,21
-­AS 44 (67.7)
Briefly, acute inflammation was defined by findings that were
-­PsA 14 (21.5)
similar to those found in bacterial bowel infections, such as the
-­nr-­A xSpA 7 (10.8)
preservation of the mucosal architecture, the infiltration of crypts
Involvement subtype
and villi by neutrophils, the formation of a crypt abscess, and the
presence of polymorphonuclears in the lamina propria. In contrast, -­Exclusively axial 29 (44.6)

chronic inflammation was defined by findings that were similar to -­Exclusively 6 (9.2)
peripheral
those of CD, characterized by a change in the mucosal architecture,
with edema and distortion of the villi and crypts, as well as by mono- -­Axial and peripheral 30 (46.1)

nuclear cell infiltrates in the lamina propria. HLA-­B27 status

-­Positive 36 (55.4)
-­Negative 10 (15.4)
2.4  |  Statistical analysis -­Not evaluated 19 (29.2)
Disease activity
The data were analyzed by using PASS 11 and SPSS software for DAPSA 4.5 (1.2–­20)
Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). The sample ASDAS CRP 2.4 (± 1.2)
size calculation was performed by estimating a statistical power
BASDAI 2.5 (1.15–­4.85)
of 80% to detect microscopic inflammation based on the preva-
CRP, mg/L 5.95 (4.4–­12.92)
lence of microscopic inflammation that was found in a previous
Extra-­joint
similar study, 21 including at least 52 patients with SpA. The nu-
manifestations
merical variables regarding normality were evaluated by using
-­Psoriasis 15 (23,0)
the Shapiro–­W ilk test, and the categorical variables are pre-
-­Uveitis 37 (56.9)
sented as percentages. The numerical variables were compared
-­Erythema nodosum 1 (1.5)
by using Student’s t test or the Mann–­W hitney test, whereas the
-­Current gastric 21 (32.3)
Chi-­s quared test or Fisher’s exact test was used to compare the
symptoms
categorical variables, when appropriate. To compare the medi-
-­Current bowel 28 (43)
ans of more than 2 groups, the Kruskal–­Wallis test was used.
symptoms
The correlation analysis between 2 numerical variables was per-
Treatment
formed by applying the Spearman rank order correlation test.
-­NSAIDs 47 (72.3)
The significance level that was adopted for the P value was less
-­NSAID intake score 50 (±29)
than .05. Moreover, a multivariable logistic regression was per-
-­Corticosteroid 8 (12.3)
formed by using the Poisson regression model, which included
variables that presented a P value <.05 in the final evaluation. -­Methotrexate 9 (13.8)

The fCAL cutoff values for endoscopic and microscopic inflam- -­Immunobiologics§ 16 (24.6)

mation were obtained by using the receiver operating characteristic †

(ROC) curve, which allowed for the selection of those values that
presented the highest sum value of sensitivity and specificity.
3  |   R E S U LT S
The overall characteristics of the 65 patients are summarized in
Table 1.
Standard deviation.
‡
Interquartile range standard deviation.
§
Immunobiologics: anti-­factor of tumor necrosis (infliximab,
adalimumab, and golimumab) and interleukin-­17 inhibitor
(secukinumab).
Abbreviations: CRP, C-­reactive protein; AS, ankylosing spondylitis;
ASDAS, Ankylosing Spondylitis Disease Activity Score; BASDAI, Bath
Ankylosing Spondylitis Disease Activity Index; DAPSA, Disease Activity
in Psoriatic Arthritis; nr-­A xSpA, nonradiographic axial spondyloarthritis;
NSAIDs, nonsteroidal anti-­inflammatory drugs; PsA, psoriatic arthritis.
 |
4      CAMPOS et al.

3.1  |  fCAL
The median value of fCAL was 144  μg/g (interquartile range
[IQR]: 45–­284.50 μg/g), and the mean value was 250.23 μg/g (SD:
± 291.32 μg/g). Forty-­seven (72.3%) patients exhibited levels above
50  μg/g, when considering the cutoff point defined as the refer-
ence for normality, whereas 20 (30.76%) patients exhibited values of
above 250 μg/g, which is a possible reference value for the inflam-
mation observed in IBD.13,22–­24
No statistically significant difference was found between the
values of fCAL that were observed in patients with SpA treated with
NSAIDs (median: 150 μg/g; IQR: 45–­309 μg/g) and those not treated
with NSAIDs (median: 116.50 μg/g; IQR: 40,7–­231 μg/g; P = .623).
The mean ASAS NSAID intake score, which estimates the current
NSAID exposure, was 50 (±29). Upon evaluation of the SpA sub-
types, the patients with AS exhibited a higher median fCAL value
(165.5 μg/g; IQR: 55–­378,75 μg/g) than patients with PsA (82.5 μg/g;
IQR: 39–­228 μg/g) and nr-­A xSpA (88 μg/g; IQR: 41–­183 μg/g), but
the difference was not statistically significant (P  =  .209). Higher
fCAL levels were also observed in patients with axial SpA (AS and
nr-­A xSpA) than in those with peripheral disease (PsA) (150 μg/g vs
83 μg/g, respectively), but the difference was not statistically signif-
icant (P = .150).
3.2  |  Ileocolonoscopy and histology
Among the 65 patients who were evaluated in this study, 13 (20.0%)
patients presented with endoscopic signs of bowel inflammation,
12 of whom presented with ulcerations (8 exclusively observed
in the distal ileum, 3 exclusively observed in the colon, and 1 ob-
served in the ileum and colon), as well as one enanthema observed
in the colon. The histopathological analysis of the intestinal mucosa
showed inflammatory alterations in 38 (58.5%) patients, 34 (52.3%)
of whom had chronic microscopic inflammation, and in 4 (6.1%) pa-
tients who had acute microscopic inflammation (Figure 1B–­D).
3.3  |  Associations between fCAL and bowel
inflammation
A strong association was found between microscopic bowel in-
flammation and fCAL levels (P < .001). Patients with microscopic
inflammation showed a median fCAL level of 235.5 μg/g (IQR: 88–­
656 μg/g), whereas those patients with no inflammation showed a
median fCAL level of 56 μg/g (IQR: 30–­154 μg/g) (Figure 2). No sta-
tistically significant difference was observed between the subtypes
of microscopic inflammation (acute and chronic) and the fCAL levels.
When regarding the endoscopic evaluation, a trend of an association
was found between the fCAL levels and endoscopic inflammation
(P  =  .053), due to the fact that the median fCAL level in patients
with no inflammation when viewed via ileocolonoscopy was 98 μg/g
(IQR: 42–­258 μg/g), whereas in those patients with endoscopic in-
flammation, the median fCAL level increased to 183 μg/g (IQR: 106–­
661 μg/g) (Figure 3).
No association between endoscopic inflammation and micro-
scopic inflammation was observed (P = .179).
No association between treatment with NSAIDs and bowel in-
flammation was observed, either endoscopically (P = .739) or micro-
scopically (P = .087).

F I G U R E 1  Microphotographs of colon biopsies. (A) Hematoxylin and eosin (H&E) staining, increase of 20×. Colon mucosa with no
relevant histological alterations. Evidence of glands and crypts with preserved architectures. (B) H&E staining, increase of 30×. Colon
mucosa with acute inflammatory alterations. (C) H&E staining, increase of 45.3×. Colon mucosa with acute inflammatory alterations. In
B and C, the evidence of polymorphonuclear inflammatory infiltrates, mucosal edema, and preserved crypts. (D) H&E staining, increase
of 26.9×. Colon mucosa with chronic inflammatory alterations. The evidence of mild granulomononuclear inflammatory infiltrates and
distortion of the crypts
CAMPOS et al.
F I G U R E 2  Association between fecal calprotectin (fCAL) levels
in μg/g and the presence of microscopic bowel inflammation
F I G U R E 3  Association between fecal calprotectin (fCAL) levels
in μg/g and the presence of endoscopic bowel inflammation
F I G U R E 4  The receiver operating characteristic (ROC) analysis
of fecal calprotectin (fCAL) levels and microscopic inflammation
The multivariable logistic regression using microscopic inflam-
mation as the outcome and other clinical risk factors as predictors
(such as fCAL levels, endoscopic inflammation, SpA subtype, gen-
der, the use of immunobiologic agents, NSAID index, and CRP level)
|
      5
showed a significant association between microscopic inflammation
and fCAL levels (P < .001) and between microscopic inflammation
and the use of immunobiological agents (P = .010).
When applying the ROC analysis, it was found that fCAL values
demonstrated the best accuracy for the evaluation of microscopic
inflammation at 96 μg/g, with a sensitivity of 73% and a specificity of
69% (P = .0015), an area under the curve of 0.78, a positive predic-
tive value of 67.2%, a negative predictive value of 74.6%, and a like-
lihood ratio for the positive result of 2.4 (Figure 4). For endoscopic
inflammation, the cutoff value was 117.5 μg/g, with a sensitivity of
77%, a specificity of 55% (P = .054), and an area under the curve of
0.67.
Table 2 summarizes the main findings related to the fCAL levels
and the presence of microscopic and endoscopic inflammation.
Disease activity (as evaluated by the BASDAI, ASDAS, or DAPSA)
proved to not be associated with the increase in fCAL levels or with
the presence of endoscopic or microscopic inflammation. Similarly,
no extra-­articular manifestations, gastrointestinal symptoms, CRP
levels, presence of HLA-­B27, SpA treatments, or other medications
used in this study (acetylsalicylic acid or proton pump inhibitors)
demonstrated a significant association with the increase in fCAL
levels or with the presence of endoscopic and microscopic bowel
inflammation.
The IBD diagnosis is defined by a combination of clinical, endo-
scopic, histological, and laboratory findings. In the present study, 6
(9.2%) patients presented with endoscopic inflammation associated
with histological inflammation and a fCAL level of ≥250 μg/g, a level
compatible with active IBD.13,22–­24 Upon the addition of the symp-
toms of abdominal pain and diarrhea that were reported in the ques-
tionnaire, 2 of the 6 patients (3% of the total sample) fulfilled the
criteria, which indicated a probable IBD diagnosis.
4  |  D I S C U S S I O N
The present study aimed to reproduce the routine medical care pro-
vided at outpatient rheumatology clinics. For this reason, this study
included patients with PsA and nr-­A xSpA and not solely those pa-
tients with AS, which is the subgroup that has been more frequently
studied in similar studies. It also included patients undergoing treat-
ment with NSAIDs, due to the fact that this is the first-­line medi-
cation that is most used to treat axial SpA. The suspension of this
medication could lead to the reactivation or worsening of the axial
symptoms.
A high prevalence of microscopic bowel inflammation was
observed in 38 patients (58.4%), thus corroborating the findings
previously described by Mielant et al7 and other researchers. 3
Liesbet et al3 evaluated 65 patients with SpA and found micro-
scopic bowel inflammation in 46% of these patients, with 17%
of the acute subtypes and 29% of the chronic subtypes. In the
present study, 52.3% of the patients presented with the chronic
inflammation subtype, and 6.1% of the patients presented with
the acute subtype.
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6      CAMPOS et al.
TA B L E 2  Association of fCAL and microscopic and endoscopic inflammation
Microscopic
Microscopic inflammation present inflammation absent
Median fCAL, 235.5 (88–­656) 56 (30–­154)
μg/g IQR
Endoscopic inflammation present Endoscopic
inflammation
absent
Median fCAL, 183 (106–­661) 98 (42–­258)
μg/g IQR
Abbreviations: fCAL, fecal calprotectin; IQR, interquartile range. ROC, receiver operating characteristic;
AUC, area under the curve; Sens, sensitivity; Spec, specificity.
When regarding the ileocolonoscopy aspects, 20% of the studied
patients presented with signs of inflammation, with a predominance
of ulceration, especially in the terminal ileum. Among similar studies
from the previous literature, few studies have described the ileoco-
21
lonoscopy aspects. Cypers et al demonstrated macroscopic alter-
ations in 31% of 125 patients who underwent an ileocolonoscopy
exam. However, the authors make no reference to the type of endo-
scopic lesion, or to its localization. Recent studies, which have com-
plemented the study of the small intestine through the endoscopic
capsule, have reinforced the idea that the distal ileum is actually the
segment of the digestive tract that is more often compromised by
macroscopic inflammation. In a study in which 30 patients were se-
lected, 15 of whom had fCAL >100 μg/g and another 15 patients
25
with levels <50 μg/g, Ostgard et al performed the complete study
of the small and large intestine by using the endoscopic capsule (EC).
In the group with high fCAL levels, ulcerated lesions or erosions
were observed in the small intestine in 12 of the 15 patients, com-
pared to colonic lesions in only 3 patients. All patients with colonic
lesions also exhibited erosions or ulcerations in the small intestine.
In contrast, Kopylov et al26 evaluated bowel inflammation in 64 pa-
tients with SpA. Of these patients, 27 presented with suggestive CD
lesions, according to the Lewis score calculated after the EC exam,
whereas 7 patients presented with CD-­suggestive alterations di-
agnosed by the ileocolonoscopy exam. Among the 7 patients with
colonic lesions, 6 patients were also identified via the EC exam. In
contrast, among the patients with lesions in the small intestine, only
6 of the 27 patients were diagnosed with bowel inflammation in the
ileocolonoscopy exam. These results highlight the importance of a
detailed evaluation of the small intestine of patients with SpA and in-
dicate a possible limitation of the endoscopic evaluation solely con-
ducted via an ileocolonoscopy exam, as was observed in this study.
Emerging evidence suggests that subclinical bowel inflammation
in patients with SpA may not be the consequence of the systemic
inflammatory process but rather an important pathophysiological
event that is actively participating in the pathogenesis of the dis-
ease. 27 It also appears to be a marker of SpA severity, as it most
often occurs in young patients with more severe and active joint
disease.3 A large body of data suggests that the presence of micro-
scopic bowel inflammation may be associated with a higher risk of
developing IBD. Studies from the Ghent Group have shown that
ROC cutoff
P value in μg/g AUC Sens Spec
<0.001 96 0.78 0.76 0.69
P Value ROC cutoff AUC Sens Spec
in μg/g
0.053 117.5 0.67 07.7 0.55
AS patients with chronic microscopic bowel inflammation and per-
sistent peripheral arthritis may be at the greatest risk of developing
IBD.7,28,29 In a prospective study on 123 patients with SpA, Mielant
et al showed that 8 (6.5%) patients developed Crohn’s disease at
2–­9 years after the appearance of rheumatic symptoms.7 In another
prospective study, Klingberg et al proposed that fCAL levels could
be a potential biomarker to identify patients with AS who are at risk
of developing IBD. In addition, they showed that CD developed in
1.5% of the patients with AS in 5 years, and a high fCAL level was the
main predictor of this effect.30
Despite the fact that bowel inflammation seems to be an import-
ant prognostic factor in SpA, there is no formal recommendation
of screening clinically silent IBD by means of routine colonoscopy.9
Additionally, it is not recommended to change or escalate SpA treat-
ment guided solely by the finding of subclinical microscopic bowel
inflammation.
In this scenario, fCAL levels emerge as a fast, simple, and nonin-
vasive biomarker that could help patients and professionals to better
understand the association between SpA and bowel inflammation,
as well as the risk of developing IBD. The evaluation of fCAL levels
could play a role in overall patient stratification. Additionally, it could
help in the judicious indication of colonoscopy in SpA patients, thus
enhancing the indication of this endoscopic exam in cases in which
fCAL levels are high or postponing it for those patients with fCAL
levels that are persistently normal.
In this study, the fCAL values proved to be above the reference
value (>50 μg/g) in more than 70% of the patients, approximately
30% of whom demonstrated levels compatible with those found
in patients with active IBD (>250 μg/g).13,22–­24 This increase in
fCAL was highly associated with microscopic bowel inflammation
(P < .001); additionally, among all of the evaluated variables, only
these variables showed a statistically significant association. There
was a trend to associate fCAL levels with endoscopic inflammation
(P = .053), although the association was not statistically significant.
However, the endoscopic evaluation was only conducted via the
ileocolonoscopy exam, with a limited study of the small intestine,
which may well justify (even if only partially) the absence of an as-
sociation between endoscopic bowel inflammation and fCAL levels.
This study set the best accuracy value of fCAL at 96 μg/g for the
presence of microscopic bowel inflammation, with a sensitivity of
CAMPOS et al.
73% and a specificity of 69%, which is very similar to the value de-
fined in the findings reported by Cypers et al, which found a refer-
ence value of 85 μg/g with a sensitivity of 64.3% and a specificity of
21
73.3%. The choice of this cutoff point determined a positive pre-
dictive value of 67.2% and a negative predictive value of 74.6%, with
a likelihood ratio for a positive result of 2.4. Thus, the 2 studies note
that fCAL values near 100 μg/g indicate that a detailed evaluation
of bowel inflammation via an ileocolonoscopy exam could be useful.
Although the presence of microscopic bowel inflammation and
3
high fCAl levels can indicate a greater joint disease activity, the lit-
erature is inconsistent regarding the results about the association
between the BASDAI, ASDAS CPR, and DAPSA scores and bowel
inflammation and fCAL levels. Similar to the results from the present
21 31
study, Cypers and Matzkies observed no association between
the evaluated scores and the fCAL levels. In contrast, Klingberg30
and Duran32 showed that the biomarker is actually associated with
the highest scores of axial and peripheral joint disease.
The use of NSAIDs showed no significant influence on the fCAL
levels or on the microscopic and endoscopic bowel inflammation re-
sults. In this case, it is believed that an important factor to be con-
sidered may well be the possibility of a type II error; specifically, the
patient sample may be insufficient for the evaluation of the desired
effect.
The data about the real impact of the use of NSAIDs in the
fCAL values have been shown to be variable. In a study published
33
by Demir et al, the authors conducted a retrospective analysis
of 5943 patients without IBD, with the aim of striving to recognize
possible factors that could justify the false-­positive results of this
protein. Only patients with fCAL values between 50 and 150 μg/g
were included. The authors demonstrated there was no association
between the fCAL values and the use of NSAIDs, and bowel inflam-
mation was the sole factor related to the levels of this biomarker.
However, other publications have demonstrated different results.
34
One study conducted by Tibble et al compared the fCAL levels to
the excretion rates of indium-­111 (which is a standard marker for the
evaluation of bowel permeability) in 47 patients undergoing treat-
ment with NSAIDs. A strong correlation was observed between the
2 methods, thus reinforcing the utility of fCAL in the evaluation of
increased bowel permeability. In a more objective manner, Rendek
et al35 attempted to quantify the impact of the use of NSAIDs on
the increase in fCAL. To achieve this effect, these authors evaluated
the fCAL levels in serial measurements in 30 healthy patients who
took diclofenac at 50 mg 3 times per day for 14 days. An increase
in the fCAL levels above 50 μg/g was observed in 27% of the pa-
tients, and posterior normalization was observed up to 14 days after
the suspension of the medication. In addition, the study suggested
a possibility of the appearance of mechanisms of bowel adaptation
for the possible lesions provoked by NSAIDs, given that the fCAL
levels significantly increased only in the first 7 days of the use of the
medications but not in the subsequent 7 days. This observation is
relevant because, in the present study, the patients with SpA who
were treated with NSAIDs had taken the medicine chronically for
more than 15 days. A bowel adaptation mechanism can justify (even
|
      7
if only theoretically) the absence of an association between fCAL
levels and the use of NSAIDs. Moreover, the different NSAIDs could
have different effects on the fCAL level. For example, Meling et al36
observed an increase in fCAL levels in patients taking indomethacin
or naproxen for 14 days but not in those patients taking lornoxicam,
which is a selective cyclooxygenase 2 inhibitor.
Thus, despite the well-­known concept that NSAIDs interfere
with fCAL levels and can provoke the appearance of varied types of
lesions in the gastrointestinal tract, there are still doubts about the
magnitude of the impact of its use, as well as about the differences in
its effects. These effects can depend on the type of NSAIDs, the time
of administration, the pattern of use, and the appearance of bowel
adaptive mechanisms. It is important to highlight that the patients
in the present study had been taking NSAIDs for more than 15 days
and, for medical reasons, could not stop taking the medication.
The type II error can also justify the absence of the association
between the fCAL levels and the use of medications with action
in the reduction of bowel inflammation, such as corticosteroids,
methotrexate, and immunobiological drugs. It was hoped that the
biomarker values would be lower, especially in patients taking immu-
nobiological drugs, as demonstrated by Klingberg et al.30 However,
in this study, 16 (24.6%) of the patients were treated with biological
medications; among these patients, the fCAL values showed no dif-
ference from the other patients.
Finally, even if 6 (9.2%) of the patients fulfilled the endoscopic,
histological, and laboratory criteria (fCAL >250 μg/g) that are sug-
gestive of IBD and 2 (3%) of these patients still reported compat-
ible symptoms, the actual diagnosis would only be possible after
a gastroenterological follow-­up and a continued evaluation of the
patients.
The major limitations of this study are represented by the small
sample size, which limits the ability of the authors to adjust for con-
founders, especially SpA therapy, the sole endoscopic evaluation
of the colons and terminal ileum, the histological evaluation being
performed by 1 single pathologist, and the non-­suspension of the
NSAIDs. However, it is important to note that, due to the aim of con-
ducting a study that was as close as possible to real life situations,
the non-­suspension of NSAIDs produces a more applicable outcome
in clinical practice. We believe that prospective studies are needed
to better evaluate the impact of oligosymptomatic microscopic
bowel inflammation, high fCAL levels, and the possible outcomes of
the association between SpA and IBD and its implications in clinical
practice.
5  |  CO N C LU S I O N
A high prevalence of microscopic bowel inflammation was observed
in this study, thus corroborating the previously described findings.3,7
The importance of this strong association between subclinical mi-
croscopic bowel inflammation and SpA has yet to be elucidated,
but it appears to be related to the pathogenesis of the articular dis-
ease and to its course. fCAL is a useful and reliable biomarker of
 |
8      CAMPOS et al.
11. Vilela EG, da Gama Torres HO, Martin FP, de Lourdes de Abreu
microscopic inflammatory activity in patients with SpA. Moreover,
values above 95 μg/g can be used for the judicious recommenda-
tion of the ileocolonoscopy exam to evaluate bowel inflammation in
12. Bertani L, Mumolo MG, Tapete G, et al. Fecal calprotectin: current
these patients and can also play a role in their overall stratification.
However, fCAL is still of unknown value for predicting future IBD
or for guiding therapy choices. These questions require further pro-
spective studies for them to be answered.
AU T H O R C O N T R I B U T I O N S
J.F.C. conceived and designed the analysis, collected the data, per-
formed the analysis, wrote the paper; G.G.R. conceived and de-
signed the analysis, contributed data and analysis tools, performed
the analysis; A.J.A.B. performed the histopathological evaluation
of the bowel biopsies; S.C.C. performed the ileocolonoscopies;
J.A.L., P.F.S.C., S.C.S.S. collected the data; M.L.A.F. conceived and
designed the analysis, contributed data and analysis tools, wrote
the paper
AC K N OW L E D G E M E N T
The investigators would like to thank BÜHLMANN Laboratories
AG® for the donation of The Quantum Blue® rapid test used in this
research.
C O N FL I C T O F I N T E R E S T S
The authors declare they have no conflicts of interest.
ORCID
Júlia Faria Campos  https://orcid.org/0000-0001-7983-534X
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