The Colon and Terminal Ileum in Patients with Ankylosing Spondylitis and Controls in Bangladesh. A Macroscopic and Microscopic Study

The Colon and Terminal Ileum in Patients with Ankylosing Spondylitis and Controls in Bangladesh.... Introduction: Little is known about gut lesions in Ankylosing Spondylitis (AS) patients in a developing country like Bangladesh.Methods: Full colonoscopy including the terminal ileum was performed in 60 AS patients and 20 controls, without diarrhea, to study macroscopic and microscopic lesions Results: In the colon in 60 AS patients 17 macroscopic lesions were found, of these 11 in rectum, only 1 in 20 controls. Prevalence of microscopic lesions in the ascending colon, sigmoid and rectum was 51, 44 and 50 in patients, and in controls 13, 9 and 8 respectively. In the terminal ileum macroscopic and microscopic lesions were seen in 21/56 and 43/56 AS patients respectively and in 1/20 and 9/20 of controls. In the AS group macroscopic (38.5 vs 5% p<0.01) and microscopic (76.8 vs 45% p=0.009 ) lesions were more frequent than in controls; No IBD was diagnosed. Findings were comparable in axial AS group (n=25) and mainly peripheral group (n=35). In AS patients marked eosinophilic infiltration was observed in the ascending colon and sigmoid but not in the rectum and this more than in controls. Colonic mucosa in controls was otherwise comparable with western studies. Anaemia was seen in 18/60 cases. No association was found between anaemia or HLA-B27 status with gut lesions. `Conclusions There were equal % of microscopic lesions in the whole gut in AS cases and healthy controls. Previous helminthic invasions may have played a role. Lesions only significantly differ in the ileum between AS and controls, so the ileal lesions may be more disease-related than the colonic ones. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 3 Key words: Microscopic, Macroscopic, Colonoscopy, Large gut, terminal ileum, Ankylosing Spondylitis, Healthy controls Running Title: Macro- and microscopic findings in AS colon and controls in Bangladesh. Introduction: Ankylosing spondylitis (AS) is the prototype of a group of disorders called [1] spondyloarthritides showing familial aggregation, arthritis of sacroiliac- and peripheral joints with enthesopathy, high association with HLA B27, and absence of rheumatoid factor [2, 3] . Associations between inflammatory gut lesions caused by Salmonella, Shigella, Yersina [4–6] and reactive arthritis are well established . The prevalence of spondyloarthritides [7, 8] including AS, in Crohn’s disease and ulcerative colitis is high . The prevalence rates have been described as 10–15% for sacroiliitis and of 7–12% for spondylitis, though the figures [7] are probably higher . Some 10% of patients with inflammatory bowel disease attending a gastroenterology unit fulfilled the criteria for ankylosing spondylitis and an additional 18% of patients had [7] asymptomatic sacroiliitis detected by conventional X-ray . On the other hand, subclinical inflammatory gut lesions were also reported in patients with spondyloarthritides. In Belgian and Scandinavian studies, macroscopic and microscopic changes have been identified in [9–11] patients with spondyloarthritides in up to 50% . One study in Bangladesh looked at the Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 4 colon in patients with ankylosing spondylitis and in normal subjects, with short colonoscopy: the frequency of inflammatory lesions was 50% in AS patients. An additional finding in that study was an eosinophilic infiltration in 85.7% of AS patients and of 80% in controls [12] probably because helminthic infections in our part of the world . [13] Anaemia is a common finding in patients with inflammatory arthritis. The exact [14-15] prevalence of anaemia in patients with AS is unknown but it is very common AS . In this study full colonoscopy was performed in AS patients and in controls to answer the following questions: a) What is the prevalence of gut lesions when using full colonoscopy? b) Does the frequency of gut lesions differ between mainly axial and mainly peripheral AS patients? c) Is anaemia in AS patients related with gut lesions? d) Was the eosinophilic infiltration in the rectum lower after deworming than in our earlier study? Methods: This observational study was carried out in the Departments of Rheumatology, BSMMU, ® ® Modern One stop Arthritis Care (MOAC&RC , Dept. of Gastroenterology and Dept. of Pathology (BSMMU), Dhaka, Bangladesh from July 2011 to Jun 2012. Following the purposive sampling method consecutive AS patients were enrolled fulfilling the revised New York criteria (1984), not taking Disease Modifying Antirheumatic Drugs (DMARDs) or corticosteroids, with no history of diarrhea and dysentery in last one month and no contraindication for colonoscopy. A total of 60 consecutive AS patients and 20 age and sex matched controls could be included in the study after informed consent. The 20 controls consisted of 10 clinically healthy volunteers and 10 persons, visiting the gastroenterologist with upper GI problems planning to evaluate the upper gut by endoscopy and having no lower gut complaints and not using NSAIDs in last month, who were invited to participate in the study as controls. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 5 In all AS patients, X-rays were made of the lumbosacral spine including both SI joints (antero-posterior and lateral view) and X-rays of SI joints with oblique view were done to assess radiological status of the disease. At baseline complete blood count (CBC), c- reactive protein (CRP), human leukocyte antigen B27 (HLA-B27) were done. In this study [17] subjects having haemoglobin levels of ≤10gm/dl were considered as anaemic . [18] All AS patients were assessed using items of the ASAS core set : BASDAI for disease activity and Pain- VAS in the past week The English version was interviewer administered. [19] Enthesitis was assessed using the Maastricht score . The AS patients without peripheral arthritis were classified as the axial group and the others with tender or swollen peripheral joints on examination were classified as the peripheral group. After assessment all AS patients and controls were de-wormed with mabendazole 100 mg 12 hourly for 3 days and after 10 days by albendazole 400 mg for total eradication. This de- worming was done in patients and controls, as in our previous study with short colonoscopy [12] a high eosinophyl count was found. and helminthes are in Bangladesh the most common cause of gut eosinophylia After 14 days subjects were prepared for full colonoscopy. The colon was prepared with 20% mannitol, no premedication was used. Macroscopic lesions were graded as follows: Grade 0—normal, Grade I— redness and edema of mucosa, Grade 2— small ulceration of [12] mucosa, Grade 3—mucosal edema, ulcerations, and hemorrhage . Biopsy specimens were taken from colon and rectum of all subjects. Two specimens were taken from all subjects irrespective of the presence or absence of macroscopically evident lesions and another two specimens were taken from macroscopically evident lesions, if available. One biopsy was taken from the proximal colon and two from the distal colon. The biopsy sites of each subject were recorded. Histological features were graded from 0–3 in the specimens [20] following the Cuvelier et al. grading. The higher grading represents more chronic Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 6 inflammation. G- 0 (Normal); G-1 (Lymphoid hyperplasia, increase in chronic inflammatory cell content in the lamina propria, with or without eosinophilia, but no evidence of cryptitis or epithelial abnormalities.); G-2 (.Diffuse increase of inflammatory cells in the lamina propria with partial villous flattening, crypt distortion and reactive hyperplasia of crypt cell epithelium; , infiltration of crypt cell epithelium with neutrophils , crypt abscesses); G-3. Aphthous ulcerations with or without epithelioid granulomas). Stage I lesions were Comment [hr1]: We copied the original tekst of considered to be a part of the spectrum of normal terminal ileal histology. All measurements Cuvelier and observations were done at ×400 magnifications (×10 ocular and × 40 objectives). For eosinophilic infiltration, cell count was done by calculating the mean from three high power [21] fields of each biopsy specimen . Data analysis: Data were entered and calculated using SPSS 17.1 version for Windows. Frequencies of macroscopic and microscopic lesions were calculated as percentage. Associations between different parameters were analyzed by Chi-square test and Fisher’s exact test. P value <0.05 implies statistical significance. Ethics: The study was approved by the Ethics Committee of Bangabandhu Sheikh Mujib Medical University Shahbagh, Dhaka, Bangladesh. The study was performed following the declaration of Helsinki principles and informed consent was obtained from all participants before enrolment. Disclosures: The authors have no financial or personal relationships or interests regarding this study Results: Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 7 A total of 65 subjects in the AS group and 35 subjects in the control group were invited to participate in this study. Four females and one male in AS group, 9 females and 6 males in control group refused to participate, leaving 60 AS patients and 20 controls in the study. In this study the male to female ratio was 3:1 in the AS group and 7:3 in controls. The mean age of the AS group was 30.4±9.6 and of controls 33.0±12.0 years. Of the AS patients 44 (73.3%) had a family history of low back pain (LBP), enthesitis was seen in 51.67% and only one patient had uveitis . The axial group consisted of 25 cases (male 20, female 5) and the predominantly peripheral group of 35 cases (male 25 female 10). HLAB-27 was tested in 37 cases, of these 20 (59%) were HLAB27 positive (14 male female 6) and 17 were HLAB-27 negative (male 13 female 4). The disease duration of the AS patients (n=60) was <12 months in 14 (23.3%), 12 - 24 months in 11 (18.3%), 24- 48 months in 11 (18.3%) and >48 months in 24 (40%). Further demographics are summarized in table 1. Treatment: Among 60 patients, 43 had no history of DMARDs use; in the past 15 had been on sulfosalazopyrine (SSZ) for some time, one had taken MTX, and one hydroxychloroquine (HCQ); in total 9 patients had been on corticosteroids for some time in the past. None had ever taken a biological. Among mainly peripheral AS patients, 11/35 had taken SSZ in the past and one HCQ and 9/35 took corticosteroids for some time previously. Among the mainly axial AS cases 4/25 had taken SSZ and 1/25 had taken MTX previously. All had stopped these treatments at least 3 months before the start of the study. Baseline clinical and laboratory characteristics: The mean score of the Pain VAS was 45.2, the BASDAI score 3,8 ; Haemoglobin 13.0 (gm/dl), ESR (erythrocyte sedimentation rate) 31.5 (mm first hour), mean corpuscular volume 28(fl), mean corpuscular haemoglobin 32.2 (pg), blood eosinophil count 3.7(%), C-Reactive Protein 22,6 (mg/dl). Details are shown in Table 3. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 8 Colonoscopic findings in the AS group (n=60) and controls (n=20): In four patients colonoscopic evaluation of the terminal ileum has not been performed.. The investigator failed to pass the probe through the ileocoecal junction. In the ascending colon and sigmoid colon macroscopic lesions were unremarkable in both groups, but microscopic lesions were more often observed in the AS group than in the control group although the difference was not statistically significant. (table 4) In the rectum: both macroscopic and microscopic lesions were more frequently seen in the AS group than in the control group but the difference was again not statistically significant (Table 4). The terminal ileum: In the terminal ileum in AS (n=56) macroscopic and microscopic lesions were found in AS 21/56 and 43/56 respectively and in controls 1/20 and 9/20. In the AS group both macroscopic (38.5 vs 5% p<0.01) and microscopic (76.8 vs 45% p=0.009 ) lesions were significantly more frequent than in controls (Table 4). AS axial and peripheral subgroups: The ascending colon, sigmoid colon and rectum were studied in the axial group (n=25) and predominantly peripheral group (n=35): In the ascending colon, sigmoid and rectum , macroscopic lesions were unremakable. Microscopic lesions were seen in both groups similarly (mainly grade 1 and 2 in about 70-80%) in the ascending colon and sigmoid but this difference was not statistically different, (Table 5). The Terminal Ileum: The frequency of macroscopic lesions was similar in the axial and peripheral groups. The microscopic lesions were slightly more frequent in the mainly peripheral group (62.5 %) than the axial group (88.5%) , p=0.05 . No IBD was observed. (Table 5). Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 9 Anaemia present (n=18) and absent (n=42) in the AS group: The frequency of macroscopic lesions did not differ between patients with (59%) and without (68%) anaemia. (Table 6). In the terminal ileum the frequency of macroscopic lesions did not differ between patients with (59%) and without(68%) anaemia The frequency of microscopic lesions was comparable in the anaemia present group (88%) and anaemia absent group (72% ) (Table 6 ). Eosinophil infiltration in different sites of large gut: In our earlier study eosinophilic infiltration in the rectum was found in 85.7% of AS [12] patients and in 80% of the controls . In AS patients even more than in controls a marked eosinophil infiltration was observed in the ileum (8.5 vs 6.5), the ascending colon (Median 12,5 resp 7,5) and sigmoid colon (Median 7,0 vs 5,5) but not in the rectum (Median 5,7 vs 3,0). The differences with controls were not significant.(Table 7) . The figures found in the rectum are much lower than in our previous study. There was a wide range of eosinophil counts in different sites of the gut in both AS patients and controls (0-40/HPF versus 1-22/HPF in ascending colon, 0-24/HPF versus 2-12/HPF in sigmoid colon and 0-13/HPF versus 0-11/HPF in rectum); the mean eosinophil count was higher in AS patients with 12-24 months disease duration. In some of these cases a “massive” or “heavy” eosinophilic infiltration was found, while others showed “patchy infiltration”. The mean BASDAI score, VAS Pain score and eosinophil count showed no correlation with macroscopic or microscopic grading, only in the ascending colon a non- significant tendency to increase along with microscopic grading was observed. For details see table 8 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 10 HLA-B27 was done in 37 AS patients, 20 were positive and 17 negative for the antigen. Macroscopic or microscopic lesions did not differ in HLA B27 pos and neg cases in the ascending colon and sigmoid or rectum. (Table 9). Discussion: In this observational study of patients with Ankylosing Spondylitis (AS) and controls, the full colon was evaluated for macroscopic and microscopic lesions and eosinophil infiltration in the lamina propria. To our knowledge, this is the first study in its kind in the South East Asian. In a developing country like Bangladesh the possibilities to diagnose AS following western criteria are restricted. Most patients cannot afford MRI studies and also testing for HLAB27 is restricted due to the cost. For that reason we could not perform this test in all patients. Infestation of the gut with parasites is very common as shown in our earlier study, in the rectum an eosinophilic infiltration in 85.7% of AS patients and of 80% in controls [12]; was found for that reason we had to de-worm the patients before this study could be done. For religious reasons four women refused colonoscopy as this was done by a male doctor. This is in general not a problem in western countries. In Bangladesh 57% of the adult population are illiterate, so taking informed consent has to be done orally in most cases, but this has never been a problem in any of our research projects. The same holds true for questionnaires like the BASDAI and pain VAS, that have to be clarified and filled in by assistants. [22] The male to female ratio in the present study was 3:1 comparable with other studies . In our series 25 (41.7%) had only axial involvement and 35 (58.3%) had both axial and peripheral (mixed). These findings are comparable with those of previous Belgian studies [23-24] Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 11 In our series the frequency of enthesopathy was 51.7% (31/60), comparable with some [25,26] [27,28] European studies and slightly lower than in other series. . Uveitis is not a frequent presentation in ankylosing spondylitis; only one patient in our series and two in that of [23], Mielants et al 1989 had uveitis. HLAB-27 was tested in 37 of the 60 cases. In these 20/37 (59%) were HLAB27 pos. This % is lower than in most western series. The incidence / prevalence of HLA-B27 in the general population or in SpA patients has not been studied in Bangladesh, to our knowledge. In one study regarding 71 patients with enthesitis , there were 49 AS cases and all were HLA B27 pos , but in this study only patients with HLA-B27 were included so this is not a [29] representative series. In our study in symptom free controls the colonic mucosa in the lower gut did not differ from [20] that found in western studies, except the presence of frequent mild to moderate eosinophilic infiltration in the lamina propria which was found both in patients and in controls. Prior to our study, several groups investigated the relationship between spondyloarthropathies and inflammatory lesions of the gut. There are differences between these investigations regarding sample size, study design, the way they evaluated gut lesions and the method of grading of the lesions. Almost all previous studies included a mixture of all kinds of seronegative spondyloarthropathies as study subjects (i.e. reactive arthritis, ankylosing spondylitis, undifferentiated spondyloarthropathies). Macroscopic lesions: In this series we had enrolled only AS cases and controls with no history of diarrhea and dysentery in the last month. In a prospective study, Mielants et al. 1989 had performed total colonoscopy in 211 patients (75AS, 32 ReA, 104 undifferentiated [23] spondyloarthropathies) and 65 controls . Gut lesions were present in 30% (24/75) of the AS Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 12 patients and in none of the controls. Simenon et al 1990, found macroscopic gut lesions in 64.9%, (37/57of the AS patients) in a retrospective study involving 96 subjects with AS, [26] reactive arthritis, and other spondyloarthropathies ; Altomonte et al 1994, in their prospective study included 38 patients with spondyloarthopathy, but none of them fulfilled [30] the criteria of definite AS; macroscopic lesions were found in 65% . In earlier studies Meuwissen et al 1978 and Costello et al 1980, studied gut mucosa by proctosigmoidoscopy and conventional x-rays. The frequencies of macroscopic lesions were lower (5-10%) than [31-32] those observed in more recent studies using total colonoscopy . Our previous study [12] with short colonoscopy showed macroscopic lesions in 14.28% . In the current series we observed macroscopic lesions in 25% (15/60) among them 3 in the ascending colon, 2 in the sigmoid and 11 in the rectum. This frequency was comparable with the findings of [31-32] Meuwissen et al 1978 and Costello et al 1980 . Microscopic lesions: Multiple biopsies allow detection of higher numbers of abnormalities [12] than as seen by colonoscopy alone . Accordingly, microscopic lesions were more frequent than macroscopic gut lesions. In our current study 51 (85%) had microscopic gut lesions in the ascending colon, 44 (73.3%) in the sigmoid and 50 (83.3%) in the rectum, figures [26] comparable with those of Simenon et al 1990 . In most studies in AS patients microscopic [24,26,34] lesions were mostly mild and the frequency varies from was 56-66.7%% . In a small [33} study only 25% (7/28 ) had microscopic lesions. All 38 spondyloarthropathy patients in [30] the series of Altomonte et al 1994 had microscopic lesions at total colonoscopy .. Microscopic lesions are common in the whole colon, both in AS patients and controls It cannot be excluded that previous helminthic infestation plays a role. The fact that our findings are comparable with other studies does not support that idea. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 13 Axial vs peripheral AS. In our study, microscopic lesions were seen in both groups similarly (mainly grade 1 and 2 in about 70-80%) in the ascending colon and sigmoid. . Slightly more microscopic lesions were seen the ileum in the peripheral group (p=0.05) These findings are [24] comparable with those of De Vos et al 1989 . Inflammatory Bowel Disease (IBD) Symptoms of spondyloarthropathy may precede or coincide with IBD; even articular involvement may be subclinical. De Vlam et al 2000, observed asymptomatic sacroiliitis in 18% of IBD patients; they also observed that [35] frequencies of sacroiliitis increased with the duration of IBD . In a prospective study in 521 IBD patients, Protzer et al observed that symptoms of spondyloarthopathy occurred [36] prior to IBD in 26.8% of all patients and in 14.4% simultaneously with IBD . In our series, none of the gut lesions was diagnostic of IBD. neither macroscopic nor microscopic. Anaemia in AS There are only few studies exploring anaemia in AS patients. In the post hoc analysis of the ASSERT study, nearly 20% of patients had anaemia at baseline. The exact prevalence of anaemia in patients with AS is unknown, but 50% of patients with RA have [46] anaemia . There are multiple potential causes of anaemia in patients with AS, including anaemia of chronic disease. Although chronic inflammation may be an important cause of anaemia, the long-term use of NSAIDs, which is rather common in patients with AS, can lead [47-48] to blood loss in the gastrointestinal tract . In our series 30% (18/60) had mild anaemia. There was no statistical difference in the non- anaemic and anaemic group regarding the macroscopic and microscopic lesions in the ascending colon, sigmoid and rectum. We did not analyze the type and cause of anaemia in this series due to financial constraints. In AS eosinophilia is an unexplained phenomenon. A detailed study of endoscopic material was carried out by DeBrosse and others, who found a gradient of eosinophil density from ascending colon to rectum (20/HPF and 8/HPF, resp.) and a wide range (up to 50/HPF), Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 14 [49] comparable to the findings of Lowichik and Weinberg . The proximal to distal gradient of [50] eosinophil distribution has also been described by Gonsalves . In our study in AS patients even more than in controls also a marked eosinophil infiltration was observed in the ascending colon and sigmoid colon but not in the rectum; the difference with controls was not significant. Geographic variation in eosinophil density in the normal colon has also been reported and there may also be variations in response to seasonal antigenic changes and [51-52] subclinical infections . The mean eosinophil count was higher in AS patients with 12- 24 months disease. In some of these cases a “massive” or “heavy” eosinophilic infiltration was found, while others showed “patchy infiltration” with “varying” numbers of eosinophils [53-56] comparable with other studies . Of thirty-four papers in English reporting “eosinophilic colitis” since 1959, eosinophil density was quantified in only seven. In these, the density [57] [58] ranged from >20/HPF anywhere in the colon to >120/HPF . Although at present there are no accepted criteria for distinguishing colonic eosinophil density at the upper range of normal, from a pathological increase diagnostic of primary eosinophilic colitis, many authors suggest a minimum eosinophil density for diagnosis of eosinophilic colitis: 6/ HPF as [59-61] suggested by Odze et al. and Hwang et al. while Lee and Kim suggested 30/ HPF . In the present series the large number of eosinophils counted in some AS patients may suggest eosinophilic colitis. But we cannot exclude that in some cases parasites still played a role as, for example Strongyloides needs different treatment than which had been prescribed in our series . Further studies are needed to make sure whether eosinophilic colitis could be an AS related disease or rather a separate pathological -physiological entity. Association with HLAB27, BASDAI and VAS pain There was no clear association with HLA-B27 status,. We found no clear association of BASDAI, and VAS pain scores with macroscopic and microscopic grading of lesions in all sites, a finding is comparable with [62-63] other studies Limitations: The study was conducted in a tertiary health care center in Bangladesh, so may Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 15 not be a reflection of the community. In scoring normal rectal histology we could not use the whole spectrum of scoring features as proposed by Rubio and Kock (1981) due to adaptation limitations of the dept. of histopathology. HLA-B27 could not be tested in 23 patients with Ankylosing Spondylitis and in controls for resource constraints. The number of controls was restricted as it was not easy to find more people to undergo a voluntary colonoscopy. Strenghts: this is a large series of AS patients and age and sex matched controls. It is the first study in Bangladesh and one of the first in Asian mainland in AS patients without clinical diarrhea; before the study antihelminthics were applied. Colonoscopy included the whole colon and terminal ileum. Eosinophil count was done by calculating the mean of three high power fields of each biopsy specimen. Conclusions: In Bangladeshi AS patients as well as controls macroscopic lesions are mainly found in the ileum. Microscopic lesions are common in the whole colon, both in AS patients and controls It cannot be excluded that previous helminthic infestation plays a role, but the fact that our findings are comparable with earlier European studies does not support that idea. It is unlikely that these microscopic or macroscopic lesions are the cause of anaemia. In none of the AS patients IBD/ M Crohn was found. Lesions are only significantly different between AS cases and controls in the ileum, so the ileal lesions may be more disease-related than the colonic ones. There was no clear association of HLA-B27 status, BASDAI, or VAS pain with macroscopic and microscopic grading of the lesions in the large gut. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 16 The colonic mucosa of symptom free individuals (controls) is similar to that in western studies, except a frequently found mild to moderate eosinophilic infiltration in both patients and controls. Funding : No funding was received from any source and the expenses met up by corresponding author and co authors as a social work. Acknowledgements: We thank all patients and controls for their willingness to participate in the study Authorship: All authors contributed in the conception and design of the work the analysis and interpretation of the data, drafting and critically revising the data and all approve of the final version. All are accountable for all aspects of the work. The selection of patients and controls and clinical aspects were done by NB and MdNI, the colonoscopies performed by SMI, the pathological studies by SA and MK, supervision by MdNI and JJR Conflict of interest: All authors declare to have no conflicts of interest Data sharing statement: The data collected from the participants were analysed and are shown in the manuscript. No additional unpublished data beyond the article are available. If required the authors are willing to share data with the reviewers References Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 17 1.Tak Yan Yu D, McGonagle D, Marzo-Ortega H, van den Bosch F, Leirisalo-Repo M. Undifferentiated spondylarthritis and reactive arthritis. Chapter 71. In: Firestein GS, Budd RC, Harris JrED, McInnes IB, Ruddy S, Sergent JS (eds) Kelley’s textbook of rheumatology, th 8 ed. Elsevier; 2008. p 1191–1200. 2. 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Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 24 Table 1. Demographics of AS patients (n=60) and controls (n=20) Cases (n=60) Control (n=20) Characters n (%) n (%) Gender Male 45 (75) 14 (70) Female 15 (25) 06 (30) Marital status Married 37 (61.70) 17 (85) Unmarried 23 (38.30) 03 (15) Occupation House wife 12 (20) 06 (30) Business 06 (10) 05 (25) Service 14 (23.30) 05 (25) Others 28 (46.70) 04 (20) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 25 Table 2. Other baseline characteristics of the AS patients (N=60) . Characteristics Total N(%) Previous DMARDs use Yes (%) 17 (28.40) 60 (100) No (%) 43 (71.60) Previous Corticosteroid use Yes (%) 9 (15) 60 (100) No (%) 51 (85) IBS Yes (%) 14 (23.30) 60 (100) No (%) 36 (76.70) HLA B27 Pos (%) 20 (33.30) 60 (100) Neg (%) 17 (28.30) Not done (%) 23 (38.40) Anaemia Yes (%) 18 (30) 60 (100) No (%) 42 (70) Pattern of joint involvement Axial (%) 25 (41.67) 60 (100) Predominantly peripheral (%) 35 (58.33) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 26 Table 3. Baseline clinical and laboratory characteristics of AS patients (N=60) Clinical characteristics Range Mean ±SD Pain last week Visual analog scale (0-100) 0-80 45.20 17.50 BASDAI score 0-8.70 3.80 1.80 Haemoglobin (gm/dl) 9-16.8 13.00 1.80 Erythrocyte Sedimentation Rate 2-97 31.50 24.20 Mean Corpuscular Volume (fl) 58.7-95.8 86.20 7.90 Mean Corpuscular Haemoglobin (pg) 20.2-31.6 28.00 2.60 Mean Corpuscular Haemoglobin Concentration (mg/dl) 24.2-35.8 32.20 1.70 Blood eosinophil count (%) 0-12 3.70 2.70 C-Reactive Protein (mg/dl) 0.9-151 22.60 30.00 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 27 Table 4 Grading of macroscopic and microscopic lesions in the large gut in AS patients and controls Macroscopic Finding Microscopic Finding Patient Control P-value Patient Control P-value (n=60) (n=20) (n=60) (n=20) Ascending n (%) n (%) n (%) n (%) colon Grade -0 56 (93.30) 20 (100) 0.31 9 (15) 7 (35) 0.1 Grade -1 0 (0) 0 (0) - 40 (66.70) 13 (65) 0.55 Grade -2 4 (6.70) 0 (0) 0.57* 9 (15) 0 (0) 0.1 Grade -3 0 (0) 0 (0) - 2 (3.30) 0 (0) 0.50* Sigmoid colon Grade -0 58 (96.70) 20 (100) 1 15 (25) 11 (55) 0.03 Grade -1 1 (1.70) 0 (0) 1* 38 (63.30) 9 (45) 0.19 Grade -2 1 (1.70) 0 (0) 1* 6 (10) 0 (0) 0.32 Grade -3 0 (0) 0 (0) - 1 (1.70) 0 (0) 1.0* Rectum Grade -0 49 (81.70) 19 (95) 0.27 10 (16.70) 12 (60) 0.001 Grade -1 10 (16.70) 1 (5) 0.27 40 (66.70) 8 (40) 0.63 Grade -2 1 (1.70) 0 (0) 1.0* 9 (15) 0 (0) 0.43 Grade -3 0 (0) 0 (0) - 1 (1.70) 0 (0) 0.56* Terminal Ileum (n=56) Grade -0 35 (62.5) 19 (95.0) 0.01 13 (23.2) 11 (55) 0.009 Grade -1 5 (8.9) 0 0.31* 33 (58.9) 9 (45) 0.28 Grade -2 16 (28.6) 1 (5.0) 0.03 6 (10.7) 0 0.33 Grade -3 0 0 - 4 (7.1) 0 0.56* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 28 Table 5 Macroscopic and microscopic lesions in the large gut in the axial (n=35)and in the predominantly peripheral group of AS patients (n=25 ) Macroscopic Finding Microscopic Finding Axial predominantly P-value Axial predominantl P-value group peripheral group y peripheral (n=25) (n=35) (n=25) (n=35) Ascending Number Number (%) Number Number (%) colon (%) (%) Grade -0 24 (96) 32 (91.40) 0.63 5 (20) 4 (11.40) 0.47 Grade -1 0 (0) 0 (0) - 16 (64) 24 (68.60) 0.78 Grade -2 1 (4) 3 (8.60) 0.63* 4 (16) 5 (14.30) 1 Grade -3 0 (0) 0 (0) - 0 (0) 2 (5.70) 0.50* Sigmoid colon Grade -0 24 (96) 34 (97.10) 1 3 (12) 12 (34.30) 0.73 Grade -1 0 (0) 1 (2.90) 1.00* 18 (72) 20 (57.10) 0.28 Grade -2 1 (4) 0 (0) 0.41* 3 (12) 3 (8.60) 0.68* Grade -3 0 (0) 0 (0) - 1 (4) 0 (0) 0.41* Rectum Grade -0 21 (84) 28 (80) 0.74 2 (8) 8 (22.90) 0.17 Grade -1 3 (12) 7 (20) 0.49 17 (68) 23 (65.70) 1 Grade -2 1 (4) 0 (0) 0.41* 5 (20) 4 (11.40) 0.47 Grade -3 0 (0) 0 (0) - 1 (4) 0 (0) 0.41* Terminal Ileum (n=56) Grade -0 15 (62.5) 20 (62.5) 1 9 (37.5) 4 (12.5) 0.05 Grade -1 0 (0) 5 (15.6) 0.06 11 (45.8) 22 (68.8) 0.1 Grade -2 9 (37.7) 7 (21.9) 0.24 2 (8.3) 4 (12.5) 0.69* Grade -3 0 (0) 0 (0) - 2 (8.3) 2 (6.3) 1* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 29 Table 6 In large gut, macroscopic and microscopic lesions with anaemia in patients and controls Macroscopic Finding Microscopic Finding Anaemia Anaemia P-value Anaemia Anaemia P-value present absent present absent (n=18) (n=42) (n=18) (n=42) Ascending n (%) n (%) n (%) n (%) colon Grade -0 16 (88.90) 40 (95.20) 0.34 5 (27.80) 4 (9.50) 0.08 Grade -1 0 (0) 0 (0) - 9 (50) 31 (73.80) 0.07 Grade -2 2 (11.10) 2 (4.80) 0.58* 3 (16.70) 6 (14.30) 0.54 Grade -3 0 (0) 0 (0) - 1 (5.60) 1 (2.40) 0.51* Sigmoid colon Grade -0 18 (100) 40 (95.20) 0.35 6 (33.30) 9 (21.40) 0.32 Grade -1 0 (0) 1 (2.40) 0.70* 10 (55.60) 28 (66.70) 0.41 Grade -2 0 (0) 1 (2.40) 0.70* 2 (11.10) 4 (9.50) 0.59* Grade -3 0 (0) 0 (0) - 0 (0) 1 (2.40) 0.70* Rectum Grade -0 15 (83.30) 34 (81.00) 0.82 5 (27.80) 5 (11.90) 0.13 Grade -1 3 (16.70) 7 (16.70) 0.66 10 (55.60) 30 (71.40) 0.23 Grade -2 0 (0) 1 (2.40) 0.70* 2 (11.10) 7 (16.70) 0.45 Grade -3 0 (0) 0 (0) - 1 (5.60) 0 0.30* Terminal Ileum (n=56) Grade -0 10 (58.8) 25 (68.4) 0.708 2 (11.8) 11 (28.2) 0.16 Grade -1 2 (11.8) 3 (7.7) 0.634* 12 (70.6) 21 (53.8) 0.376 Grade -2 5 (29.4) 11 (28.2) 0.927 2 (11.8) 4 (35) 0.598* Grade -3 0 (0) 0 (0) - 1 (5.9) 3 (7.7) 0.647* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 30 Table 7 In study groups eosinophil infiltration in different sites of large gut in AS patients and controls Patient (n=60) Control (n=20) *P-value (Count Per HPF ) Mean±SD Median Mean±SD Median Ascending colon 18.5±21.5 12.50 11.7±9.9 7.50 0.17 Sigmoid colon 11.4±12.5 7.00 6.6±4.6 5.50 0.09 Rectum 5.7±6.8 4.00 4.8±5.9 3.00 0.61 Terminal ileum 15.6±19.6 8.5 8.8±8 6.5 0.14 *Two mean test High Power Field Table 8 Comparison of AS disease activity (BASDAI score and VAS) and eosinophil count in different macroscopic and microscopic lesions in large gut Microscopic grading Number BASDAI VAS score Eosinophil score count/HPF Ascending colon (N=60) N Mean±SD Mean±SD Mean±SD G-0 9 3.82±1.85 42.2±17.8 13.4±8.4 G-1 40 3.59±1.76 44.0±17.8 20.9±24.0 G-2 9 4.02±1.52 48.9±14.5 9.4±14.6 G-3 2 6.75±2.75 65.0±21.2 35.5±24.7 Sigmoid colon (N=60) G-0 15 3.32±1.27 40.0±11.9 16.9±18.4 G-1 38 4.02±1.98 46.1±19.2 10.5±9.8 G-2 06 3.61±2 48.3±16.0 4.3±3.7 G-3 01 3.20 70 05 Rectum (N=60) G-0 10 4.04±1.93 47.0±13.3 3.5±2.2 G-1 40 3.83±1.92 44.0±19.7 5.8±5.4 G-2 09 3.53±1.30 48.9±11.7 7.4±13.5 G-3 01 2.40 40 05 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 31 Table 9 Frequency of macroscopic and microscopic lesion in large gut (n=60) inHLA- B27 patient groups Macroscopic Finding Microscopic Finding HLA- HLA- HLA- P- HLA- HLA- HLA- P- B27 B27 B27 value B27 B27 B27 value positive negative not positiv negative not (n=20) (n=17) done e (n=17) done (n=23 (n=20) (n=23 ) ) Ascending n (%) n (%) n (%) n (%) colon Grade -0 19 (95) 16 (94.1) 21 0.879 2 (10) 4 (23.5) 3 (13) 0.489 (91.3) Grade -1 0 (0) 0 (0) 0 (0) - 13 (65) 11 (64.7) 16 0.932 (69.6) Grade -2 1 (5) 1 (5.9) 2 (8.7) 0.879 4 (20) 2 (11.8) 3 (13) 0.741 Grade -3 0 (0) 0 (0) 0 (0) - 1 (5) 0 (0) 1 0.66 (4.3) Sigmoid colon Grade -0 19(95) 17 (100) 22 0.66 5 (25) 1 (5.9) 9 0.056 (95.7) (39.1) Grade -1 1 (5) 0 (0) 0 (0) 0.362 14 (70) 13 (76.5) 11 0.133 (47.8) Grade -2 0 (0) 0 (0) 1 (4.3) 0.441 1 (5) 3 (17.6) 2 0.427 (8.7) Grade -3 0 (0) 0 (0) 0 (0) - 0 (0) 0 (0) 1 0.441 (4.3) Rectum Grade -0 14 (70) 14 (82.4) 21 0.197 3 (15) 2 (11.8) 5 0.684 (91.3) (21.7) Grade -1 5 (25) 3 (17.6) 2 (8.7) 0.356 14 (70) 12 (70.6) 14 0.754 (60.9) Grade -2 1 (5) 0 (0) 0 (0) 0.362 3 (15) 2 (11.8) 4 0.886 (17.4) Grade -3 0 (0) 0 (0) 0 (0) - 0 (0) 1 (5.9) 0 (0) 0.276 Terminal Ileum (n=18) (n=15) (n=23 (n=18) (n=15) (n=23 ) ) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 32 Grade -0 9 (50) 11 (73.3) 15 0.364 4 (22.2) 2 (13.3) 7 0.471 (65.2) (30.4) Grade -1 2 (11.1) 1 (6.7) 2 (8.7) 0.904 9 (50) 10 (66.7) 4 0.607 (60.9) Grade -2 7 (38.9) 3 (20) 6 0.461 3 (16.7) 2 (13.3) 1 0.417 (26.1) (4.3) Grade -3 0 (0) 0 (0) 0 (0) - 2 (11.1) 1 (6.7) 1 0.703 (4.3) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rheumatology Advances in Practice Oxford University Press

The Colon and Terminal Ileum in Patients with Ankylosing Spondylitis and Controls in Bangladesh. A Macroscopic and Microscopic Study

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Abstract

Introduction: Little is known about gut lesions in Ankylosing Spondylitis (AS) patients in a developing country like Bangladesh.Methods: Full colonoscopy including the terminal ileum was performed in 60 AS patients and 20 controls, without diarrhea, to study macroscopic and microscopic lesions Results: In the colon in 60 AS patients 17 macroscopic lesions were found, of these 11 in rectum, only 1 in 20 controls. Prevalence of microscopic lesions in the ascending colon, sigmoid and rectum was 51, 44 and 50 in patients, and in controls 13, 9 and 8 respectively. In the terminal ileum macroscopic and microscopic lesions were seen in 21/56 and 43/56 AS patients respectively and in 1/20 and 9/20 of controls. In the AS group macroscopic (38.5 vs 5% p<0.01) and microscopic (76.8 vs 45% p=0.009 ) lesions were more frequent than in controls; No IBD was diagnosed. Findings were comparable in axial AS group (n=25) and mainly peripheral group (n=35). In AS patients marked eosinophilic infiltration was observed in the ascending colon and sigmoid but not in the rectum and this more than in controls. Colonic mucosa in controls was otherwise comparable with western studies. Anaemia was seen in 18/60 cases. No association was found between anaemia or HLA-B27 status with gut lesions. `Conclusions There were equal % of microscopic lesions in the whole gut in AS cases and healthy controls. Previous helminthic invasions may have played a role. Lesions only significantly differ in the ileum between AS and controls, so the ileal lesions may be more disease-related than the colonic ones. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 3 Key words: Microscopic, Macroscopic, Colonoscopy, Large gut, terminal ileum, Ankylosing Spondylitis, Healthy controls Running Title: Macro- and microscopic findings in AS colon and controls in Bangladesh. Introduction: Ankylosing spondylitis (AS) is the prototype of a group of disorders called [1] spondyloarthritides showing familial aggregation, arthritis of sacroiliac- and peripheral joints with enthesopathy, high association with HLA B27, and absence of rheumatoid factor [2, 3] . Associations between inflammatory gut lesions caused by Salmonella, Shigella, Yersina [4–6] and reactive arthritis are well established . The prevalence of spondyloarthritides [7, 8] including AS, in Crohn’s disease and ulcerative colitis is high . The prevalence rates have been described as 10–15% for sacroiliitis and of 7–12% for spondylitis, though the figures [7] are probably higher . Some 10% of patients with inflammatory bowel disease attending a gastroenterology unit fulfilled the criteria for ankylosing spondylitis and an additional 18% of patients had [7] asymptomatic sacroiliitis detected by conventional X-ray . On the other hand, subclinical inflammatory gut lesions were also reported in patients with spondyloarthritides. In Belgian and Scandinavian studies, macroscopic and microscopic changes have been identified in [9–11] patients with spondyloarthritides in up to 50% . One study in Bangladesh looked at the Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 4 colon in patients with ankylosing spondylitis and in normal subjects, with short colonoscopy: the frequency of inflammatory lesions was 50% in AS patients. An additional finding in that study was an eosinophilic infiltration in 85.7% of AS patients and of 80% in controls [12] probably because helminthic infections in our part of the world . [13] Anaemia is a common finding in patients with inflammatory arthritis. The exact [14-15] prevalence of anaemia in patients with AS is unknown but it is very common AS . In this study full colonoscopy was performed in AS patients and in controls to answer the following questions: a) What is the prevalence of gut lesions when using full colonoscopy? b) Does the frequency of gut lesions differ between mainly axial and mainly peripheral AS patients? c) Is anaemia in AS patients related with gut lesions? d) Was the eosinophilic infiltration in the rectum lower after deworming than in our earlier study? Methods: This observational study was carried out in the Departments of Rheumatology, BSMMU, ® ® Modern One stop Arthritis Care (MOAC&RC , Dept. of Gastroenterology and Dept. of Pathology (BSMMU), Dhaka, Bangladesh from July 2011 to Jun 2012. Following the purposive sampling method consecutive AS patients were enrolled fulfilling the revised New York criteria (1984), not taking Disease Modifying Antirheumatic Drugs (DMARDs) or corticosteroids, with no history of diarrhea and dysentery in last one month and no contraindication for colonoscopy. A total of 60 consecutive AS patients and 20 age and sex matched controls could be included in the study after informed consent. The 20 controls consisted of 10 clinically healthy volunteers and 10 persons, visiting the gastroenterologist with upper GI problems planning to evaluate the upper gut by endoscopy and having no lower gut complaints and not using NSAIDs in last month, who were invited to participate in the study as controls. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 5 In all AS patients, X-rays were made of the lumbosacral spine including both SI joints (antero-posterior and lateral view) and X-rays of SI joints with oblique view were done to assess radiological status of the disease. At baseline complete blood count (CBC), c- reactive protein (CRP), human leukocyte antigen B27 (HLA-B27) were done. In this study [17] subjects having haemoglobin levels of ≤10gm/dl were considered as anaemic . [18] All AS patients were assessed using items of the ASAS core set : BASDAI for disease activity and Pain- VAS in the past week The English version was interviewer administered. [19] Enthesitis was assessed using the Maastricht score . The AS patients without peripheral arthritis were classified as the axial group and the others with tender or swollen peripheral joints on examination were classified as the peripheral group. After assessment all AS patients and controls were de-wormed with mabendazole 100 mg 12 hourly for 3 days and after 10 days by albendazole 400 mg for total eradication. This de- worming was done in patients and controls, as in our previous study with short colonoscopy [12] a high eosinophyl count was found. and helminthes are in Bangladesh the most common cause of gut eosinophylia After 14 days subjects were prepared for full colonoscopy. The colon was prepared with 20% mannitol, no premedication was used. Macroscopic lesions were graded as follows: Grade 0—normal, Grade I— redness and edema of mucosa, Grade 2— small ulceration of [12] mucosa, Grade 3—mucosal edema, ulcerations, and hemorrhage . Biopsy specimens were taken from colon and rectum of all subjects. Two specimens were taken from all subjects irrespective of the presence or absence of macroscopically evident lesions and another two specimens were taken from macroscopically evident lesions, if available. One biopsy was taken from the proximal colon and two from the distal colon. The biopsy sites of each subject were recorded. Histological features were graded from 0–3 in the specimens [20] following the Cuvelier et al. grading. The higher grading represents more chronic Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 6 inflammation. G- 0 (Normal); G-1 (Lymphoid hyperplasia, increase in chronic inflammatory cell content in the lamina propria, with or without eosinophilia, but no evidence of cryptitis or epithelial abnormalities.); G-2 (.Diffuse increase of inflammatory cells in the lamina propria with partial villous flattening, crypt distortion and reactive hyperplasia of crypt cell epithelium; , infiltration of crypt cell epithelium with neutrophils , crypt abscesses); G-3. Aphthous ulcerations with or without epithelioid granulomas). Stage I lesions were Comment [hr1]: We copied the original tekst of considered to be a part of the spectrum of normal terminal ileal histology. All measurements Cuvelier and observations were done at ×400 magnifications (×10 ocular and × 40 objectives). For eosinophilic infiltration, cell count was done by calculating the mean from three high power [21] fields of each biopsy specimen . Data analysis: Data were entered and calculated using SPSS 17.1 version for Windows. Frequencies of macroscopic and microscopic lesions were calculated as percentage. Associations between different parameters were analyzed by Chi-square test and Fisher’s exact test. P value <0.05 implies statistical significance. Ethics: The study was approved by the Ethics Committee of Bangabandhu Sheikh Mujib Medical University Shahbagh, Dhaka, Bangladesh. The study was performed following the declaration of Helsinki principles and informed consent was obtained from all participants before enrolment. Disclosures: The authors have no financial or personal relationships or interests regarding this study Results: Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 7 A total of 65 subjects in the AS group and 35 subjects in the control group were invited to participate in this study. Four females and one male in AS group, 9 females and 6 males in control group refused to participate, leaving 60 AS patients and 20 controls in the study. In this study the male to female ratio was 3:1 in the AS group and 7:3 in controls. The mean age of the AS group was 30.4±9.6 and of controls 33.0±12.0 years. Of the AS patients 44 (73.3%) had a family history of low back pain (LBP), enthesitis was seen in 51.67% and only one patient had uveitis . The axial group consisted of 25 cases (male 20, female 5) and the predominantly peripheral group of 35 cases (male 25 female 10). HLAB-27 was tested in 37 cases, of these 20 (59%) were HLAB27 positive (14 male female 6) and 17 were HLAB-27 negative (male 13 female 4). The disease duration of the AS patients (n=60) was <12 months in 14 (23.3%), 12 - 24 months in 11 (18.3%), 24- 48 months in 11 (18.3%) and >48 months in 24 (40%). Further demographics are summarized in table 1. Treatment: Among 60 patients, 43 had no history of DMARDs use; in the past 15 had been on sulfosalazopyrine (SSZ) for some time, one had taken MTX, and one hydroxychloroquine (HCQ); in total 9 patients had been on corticosteroids for some time in the past. None had ever taken a biological. Among mainly peripheral AS patients, 11/35 had taken SSZ in the past and one HCQ and 9/35 took corticosteroids for some time previously. Among the mainly axial AS cases 4/25 had taken SSZ and 1/25 had taken MTX previously. All had stopped these treatments at least 3 months before the start of the study. Baseline clinical and laboratory characteristics: The mean score of the Pain VAS was 45.2, the BASDAI score 3,8 ; Haemoglobin 13.0 (gm/dl), ESR (erythrocyte sedimentation rate) 31.5 (mm first hour), mean corpuscular volume 28(fl), mean corpuscular haemoglobin 32.2 (pg), blood eosinophil count 3.7(%), C-Reactive Protein 22,6 (mg/dl). Details are shown in Table 3. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 8 Colonoscopic findings in the AS group (n=60) and controls (n=20): In four patients colonoscopic evaluation of the terminal ileum has not been performed.. The investigator failed to pass the probe through the ileocoecal junction. In the ascending colon and sigmoid colon macroscopic lesions were unremarkable in both groups, but microscopic lesions were more often observed in the AS group than in the control group although the difference was not statistically significant. (table 4) In the rectum: both macroscopic and microscopic lesions were more frequently seen in the AS group than in the control group but the difference was again not statistically significant (Table 4). The terminal ileum: In the terminal ileum in AS (n=56) macroscopic and microscopic lesions were found in AS 21/56 and 43/56 respectively and in controls 1/20 and 9/20. In the AS group both macroscopic (38.5 vs 5% p<0.01) and microscopic (76.8 vs 45% p=0.009 ) lesions were significantly more frequent than in controls (Table 4). AS axial and peripheral subgroups: The ascending colon, sigmoid colon and rectum were studied in the axial group (n=25) and predominantly peripheral group (n=35): In the ascending colon, sigmoid and rectum , macroscopic lesions were unremakable. Microscopic lesions were seen in both groups similarly (mainly grade 1 and 2 in about 70-80%) in the ascending colon and sigmoid but this difference was not statistically different, (Table 5). The Terminal Ileum: The frequency of macroscopic lesions was similar in the axial and peripheral groups. The microscopic lesions were slightly more frequent in the mainly peripheral group (62.5 %) than the axial group (88.5%) , p=0.05 . No IBD was observed. (Table 5). Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 9 Anaemia present (n=18) and absent (n=42) in the AS group: The frequency of macroscopic lesions did not differ between patients with (59%) and without (68%) anaemia. (Table 6). In the terminal ileum the frequency of macroscopic lesions did not differ between patients with (59%) and without(68%) anaemia The frequency of microscopic lesions was comparable in the anaemia present group (88%) and anaemia absent group (72% ) (Table 6 ). Eosinophil infiltration in different sites of large gut: In our earlier study eosinophilic infiltration in the rectum was found in 85.7% of AS [12] patients and in 80% of the controls . In AS patients even more than in controls a marked eosinophil infiltration was observed in the ileum (8.5 vs 6.5), the ascending colon (Median 12,5 resp 7,5) and sigmoid colon (Median 7,0 vs 5,5) but not in the rectum (Median 5,7 vs 3,0). The differences with controls were not significant.(Table 7) . The figures found in the rectum are much lower than in our previous study. There was a wide range of eosinophil counts in different sites of the gut in both AS patients and controls (0-40/HPF versus 1-22/HPF in ascending colon, 0-24/HPF versus 2-12/HPF in sigmoid colon and 0-13/HPF versus 0-11/HPF in rectum); the mean eosinophil count was higher in AS patients with 12-24 months disease duration. In some of these cases a “massive” or “heavy” eosinophilic infiltration was found, while others showed “patchy infiltration”. The mean BASDAI score, VAS Pain score and eosinophil count showed no correlation with macroscopic or microscopic grading, only in the ascending colon a non- significant tendency to increase along with microscopic grading was observed. For details see table 8 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 10 HLA-B27 was done in 37 AS patients, 20 were positive and 17 negative for the antigen. Macroscopic or microscopic lesions did not differ in HLA B27 pos and neg cases in the ascending colon and sigmoid or rectum. (Table 9). Discussion: In this observational study of patients with Ankylosing Spondylitis (AS) and controls, the full colon was evaluated for macroscopic and microscopic lesions and eosinophil infiltration in the lamina propria. To our knowledge, this is the first study in its kind in the South East Asian. In a developing country like Bangladesh the possibilities to diagnose AS following western criteria are restricted. Most patients cannot afford MRI studies and also testing for HLAB27 is restricted due to the cost. For that reason we could not perform this test in all patients. Infestation of the gut with parasites is very common as shown in our earlier study, in the rectum an eosinophilic infiltration in 85.7% of AS patients and of 80% in controls [12]; was found for that reason we had to de-worm the patients before this study could be done. For religious reasons four women refused colonoscopy as this was done by a male doctor. This is in general not a problem in western countries. In Bangladesh 57% of the adult population are illiterate, so taking informed consent has to be done orally in most cases, but this has never been a problem in any of our research projects. The same holds true for questionnaires like the BASDAI and pain VAS, that have to be clarified and filled in by assistants. [22] The male to female ratio in the present study was 3:1 comparable with other studies . In our series 25 (41.7%) had only axial involvement and 35 (58.3%) had both axial and peripheral (mixed). These findings are comparable with those of previous Belgian studies [23-24] Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 11 In our series the frequency of enthesopathy was 51.7% (31/60), comparable with some [25,26] [27,28] European studies and slightly lower than in other series. . Uveitis is not a frequent presentation in ankylosing spondylitis; only one patient in our series and two in that of [23], Mielants et al 1989 had uveitis. HLAB-27 was tested in 37 of the 60 cases. In these 20/37 (59%) were HLAB27 pos. This % is lower than in most western series. The incidence / prevalence of HLA-B27 in the general population or in SpA patients has not been studied in Bangladesh, to our knowledge. In one study regarding 71 patients with enthesitis , there were 49 AS cases and all were HLA B27 pos , but in this study only patients with HLA-B27 were included so this is not a [29] representative series. In our study in symptom free controls the colonic mucosa in the lower gut did not differ from [20] that found in western studies, except the presence of frequent mild to moderate eosinophilic infiltration in the lamina propria which was found both in patients and in controls. Prior to our study, several groups investigated the relationship between spondyloarthropathies and inflammatory lesions of the gut. There are differences between these investigations regarding sample size, study design, the way they evaluated gut lesions and the method of grading of the lesions. Almost all previous studies included a mixture of all kinds of seronegative spondyloarthropathies as study subjects (i.e. reactive arthritis, ankylosing spondylitis, undifferentiated spondyloarthropathies). Macroscopic lesions: In this series we had enrolled only AS cases and controls with no history of diarrhea and dysentery in the last month. In a prospective study, Mielants et al. 1989 had performed total colonoscopy in 211 patients (75AS, 32 ReA, 104 undifferentiated [23] spondyloarthropathies) and 65 controls . Gut lesions were present in 30% (24/75) of the AS Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 12 patients and in none of the controls. Simenon et al 1990, found macroscopic gut lesions in 64.9%, (37/57of the AS patients) in a retrospective study involving 96 subjects with AS, [26] reactive arthritis, and other spondyloarthropathies ; Altomonte et al 1994, in their prospective study included 38 patients with spondyloarthopathy, but none of them fulfilled [30] the criteria of definite AS; macroscopic lesions were found in 65% . In earlier studies Meuwissen et al 1978 and Costello et al 1980, studied gut mucosa by proctosigmoidoscopy and conventional x-rays. The frequencies of macroscopic lesions were lower (5-10%) than [31-32] those observed in more recent studies using total colonoscopy . Our previous study [12] with short colonoscopy showed macroscopic lesions in 14.28% . In the current series we observed macroscopic lesions in 25% (15/60) among them 3 in the ascending colon, 2 in the sigmoid and 11 in the rectum. This frequency was comparable with the findings of [31-32] Meuwissen et al 1978 and Costello et al 1980 . Microscopic lesions: Multiple biopsies allow detection of higher numbers of abnormalities [12] than as seen by colonoscopy alone . Accordingly, microscopic lesions were more frequent than macroscopic gut lesions. In our current study 51 (85%) had microscopic gut lesions in the ascending colon, 44 (73.3%) in the sigmoid and 50 (83.3%) in the rectum, figures [26] comparable with those of Simenon et al 1990 . In most studies in AS patients microscopic [24,26,34] lesions were mostly mild and the frequency varies from was 56-66.7%% . In a small [33} study only 25% (7/28 ) had microscopic lesions. All 38 spondyloarthropathy patients in [30] the series of Altomonte et al 1994 had microscopic lesions at total colonoscopy .. Microscopic lesions are common in the whole colon, both in AS patients and controls It cannot be excluded that previous helminthic infestation plays a role. The fact that our findings are comparable with other studies does not support that idea. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 13 Axial vs peripheral AS. In our study, microscopic lesions were seen in both groups similarly (mainly grade 1 and 2 in about 70-80%) in the ascending colon and sigmoid. . Slightly more microscopic lesions were seen the ileum in the peripheral group (p=0.05) These findings are [24] comparable with those of De Vos et al 1989 . Inflammatory Bowel Disease (IBD) Symptoms of spondyloarthropathy may precede or coincide with IBD; even articular involvement may be subclinical. De Vlam et al 2000, observed asymptomatic sacroiliitis in 18% of IBD patients; they also observed that [35] frequencies of sacroiliitis increased with the duration of IBD . In a prospective study in 521 IBD patients, Protzer et al observed that symptoms of spondyloarthopathy occurred [36] prior to IBD in 26.8% of all patients and in 14.4% simultaneously with IBD . In our series, none of the gut lesions was diagnostic of IBD. neither macroscopic nor microscopic. Anaemia in AS There are only few studies exploring anaemia in AS patients. In the post hoc analysis of the ASSERT study, nearly 20% of patients had anaemia at baseline. The exact prevalence of anaemia in patients with AS is unknown, but 50% of patients with RA have [46] anaemia . There are multiple potential causes of anaemia in patients with AS, including anaemia of chronic disease. Although chronic inflammation may be an important cause of anaemia, the long-term use of NSAIDs, which is rather common in patients with AS, can lead [47-48] to blood loss in the gastrointestinal tract . In our series 30% (18/60) had mild anaemia. There was no statistical difference in the non- anaemic and anaemic group regarding the macroscopic and microscopic lesions in the ascending colon, sigmoid and rectum. We did not analyze the type and cause of anaemia in this series due to financial constraints. In AS eosinophilia is an unexplained phenomenon. A detailed study of endoscopic material was carried out by DeBrosse and others, who found a gradient of eosinophil density from ascending colon to rectum (20/HPF and 8/HPF, resp.) and a wide range (up to 50/HPF), Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 14 [49] comparable to the findings of Lowichik and Weinberg . The proximal to distal gradient of [50] eosinophil distribution has also been described by Gonsalves . In our study in AS patients even more than in controls also a marked eosinophil infiltration was observed in the ascending colon and sigmoid colon but not in the rectum; the difference with controls was not significant. Geographic variation in eosinophil density in the normal colon has also been reported and there may also be variations in response to seasonal antigenic changes and [51-52] subclinical infections . The mean eosinophil count was higher in AS patients with 12- 24 months disease. In some of these cases a “massive” or “heavy” eosinophilic infiltration was found, while others showed “patchy infiltration” with “varying” numbers of eosinophils [53-56] comparable with other studies . Of thirty-four papers in English reporting “eosinophilic colitis” since 1959, eosinophil density was quantified in only seven. In these, the density [57] [58] ranged from >20/HPF anywhere in the colon to >120/HPF . Although at present there are no accepted criteria for distinguishing colonic eosinophil density at the upper range of normal, from a pathological increase diagnostic of primary eosinophilic colitis, many authors suggest a minimum eosinophil density for diagnosis of eosinophilic colitis: 6/ HPF as [59-61] suggested by Odze et al. and Hwang et al. while Lee and Kim suggested 30/ HPF . In the present series the large number of eosinophils counted in some AS patients may suggest eosinophilic colitis. But we cannot exclude that in some cases parasites still played a role as, for example Strongyloides needs different treatment than which had been prescribed in our series . Further studies are needed to make sure whether eosinophilic colitis could be an AS related disease or rather a separate pathological -physiological entity. Association with HLAB27, BASDAI and VAS pain There was no clear association with HLA-B27 status,. We found no clear association of BASDAI, and VAS pain scores with macroscopic and microscopic grading of lesions in all sites, a finding is comparable with [62-63] other studies Limitations: The study was conducted in a tertiary health care center in Bangladesh, so may Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 15 not be a reflection of the community. In scoring normal rectal histology we could not use the whole spectrum of scoring features as proposed by Rubio and Kock (1981) due to adaptation limitations of the dept. of histopathology. HLA-B27 could not be tested in 23 patients with Ankylosing Spondylitis and in controls for resource constraints. The number of controls was restricted as it was not easy to find more people to undergo a voluntary colonoscopy. Strenghts: this is a large series of AS patients and age and sex matched controls. It is the first study in Bangladesh and one of the first in Asian mainland in AS patients without clinical diarrhea; before the study antihelminthics were applied. Colonoscopy included the whole colon and terminal ileum. Eosinophil count was done by calculating the mean of three high power fields of each biopsy specimen. Conclusions: In Bangladeshi AS patients as well as controls macroscopic lesions are mainly found in the ileum. Microscopic lesions are common in the whole colon, both in AS patients and controls It cannot be excluded that previous helminthic infestation plays a role, but the fact that our findings are comparable with earlier European studies does not support that idea. It is unlikely that these microscopic or macroscopic lesions are the cause of anaemia. In none of the AS patients IBD/ M Crohn was found. Lesions are only significantly different between AS cases and controls in the ileum, so the ileal lesions may be more disease-related than the colonic ones. There was no clear association of HLA-B27 status, BASDAI, or VAS pain with macroscopic and microscopic grading of the lesions in the large gut. Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 16 The colonic mucosa of symptom free individuals (controls) is similar to that in western studies, except a frequently found mild to moderate eosinophilic infiltration in both patients and controls. Funding : No funding was received from any source and the expenses met up by corresponding author and co authors as a social work. Acknowledgements: We thank all patients and controls for their willingness to participate in the study Authorship: All authors contributed in the conception and design of the work the analysis and interpretation of the data, drafting and critically revising the data and all approve of the final version. All are accountable for all aspects of the work. The selection of patients and controls and clinical aspects were done by NB and MdNI, the colonoscopies performed by SMI, the pathological studies by SA and MK, supervision by MdNI and JJR Conflict of interest: All authors declare to have no conflicts of interest Data sharing statement: The data collected from the participants were analysed and are shown in the manuscript. No additional unpublished data beyond the article are available. If required the authors are willing to share data with the reviewers References Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 17 1.Tak Yan Yu D, McGonagle D, Marzo-Ortega H, van den Bosch F, Leirisalo-Repo M. Undifferentiated spondylarthritis and reactive arthritis. Chapter 71. In: Firestein GS, Budd RC, Harris JrED, McInnes IB, Ruddy S, Sergent JS (eds) Kelley’s textbook of rheumatology, th 8 ed. Elsevier; 2008. p 1191–1200. 2. 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Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 24 Table 1. Demographics of AS patients (n=60) and controls (n=20) Cases (n=60) Control (n=20) Characters n (%) n (%) Gender Male 45 (75) 14 (70) Female 15 (25) 06 (30) Marital status Married 37 (61.70) 17 (85) Unmarried 23 (38.30) 03 (15) Occupation House wife 12 (20) 06 (30) Business 06 (10) 05 (25) Service 14 (23.30) 05 (25) Others 28 (46.70) 04 (20) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 25 Table 2. Other baseline characteristics of the AS patients (N=60) . Characteristics Total N(%) Previous DMARDs use Yes (%) 17 (28.40) 60 (100) No (%) 43 (71.60) Previous Corticosteroid use Yes (%) 9 (15) 60 (100) No (%) 51 (85) IBS Yes (%) 14 (23.30) 60 (100) No (%) 36 (76.70) HLA B27 Pos (%) 20 (33.30) 60 (100) Neg (%) 17 (28.30) Not done (%) 23 (38.40) Anaemia Yes (%) 18 (30) 60 (100) No (%) 42 (70) Pattern of joint involvement Axial (%) 25 (41.67) 60 (100) Predominantly peripheral (%) 35 (58.33) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 26 Table 3. Baseline clinical and laboratory characteristics of AS patients (N=60) Clinical characteristics Range Mean ±SD Pain last week Visual analog scale (0-100) 0-80 45.20 17.50 BASDAI score 0-8.70 3.80 1.80 Haemoglobin (gm/dl) 9-16.8 13.00 1.80 Erythrocyte Sedimentation Rate 2-97 31.50 24.20 Mean Corpuscular Volume (fl) 58.7-95.8 86.20 7.90 Mean Corpuscular Haemoglobin (pg) 20.2-31.6 28.00 2.60 Mean Corpuscular Haemoglobin Concentration (mg/dl) 24.2-35.8 32.20 1.70 Blood eosinophil count (%) 0-12 3.70 2.70 C-Reactive Protein (mg/dl) 0.9-151 22.60 30.00 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 27 Table 4 Grading of macroscopic and microscopic lesions in the large gut in AS patients and controls Macroscopic Finding Microscopic Finding Patient Control P-value Patient Control P-value (n=60) (n=20) (n=60) (n=20) Ascending n (%) n (%) n (%) n (%) colon Grade -0 56 (93.30) 20 (100) 0.31 9 (15) 7 (35) 0.1 Grade -1 0 (0) 0 (0) - 40 (66.70) 13 (65) 0.55 Grade -2 4 (6.70) 0 (0) 0.57* 9 (15) 0 (0) 0.1 Grade -3 0 (0) 0 (0) - 2 (3.30) 0 (0) 0.50* Sigmoid colon Grade -0 58 (96.70) 20 (100) 1 15 (25) 11 (55) 0.03 Grade -1 1 (1.70) 0 (0) 1* 38 (63.30) 9 (45) 0.19 Grade -2 1 (1.70) 0 (0) 1* 6 (10) 0 (0) 0.32 Grade -3 0 (0) 0 (0) - 1 (1.70) 0 (0) 1.0* Rectum Grade -0 49 (81.70) 19 (95) 0.27 10 (16.70) 12 (60) 0.001 Grade -1 10 (16.70) 1 (5) 0.27 40 (66.70) 8 (40) 0.63 Grade -2 1 (1.70) 0 (0) 1.0* 9 (15) 0 (0) 0.43 Grade -3 0 (0) 0 (0) - 1 (1.70) 0 (0) 0.56* Terminal Ileum (n=56) Grade -0 35 (62.5) 19 (95.0) 0.01 13 (23.2) 11 (55) 0.009 Grade -1 5 (8.9) 0 0.31* 33 (58.9) 9 (45) 0.28 Grade -2 16 (28.6) 1 (5.0) 0.03 6 (10.7) 0 0.33 Grade -3 0 0 - 4 (7.1) 0 0.56* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 28 Table 5 Macroscopic and microscopic lesions in the large gut in the axial (n=35)and in the predominantly peripheral group of AS patients (n=25 ) Macroscopic Finding Microscopic Finding Axial predominantly P-value Axial predominantl P-value group peripheral group y peripheral (n=25) (n=35) (n=25) (n=35) Ascending Number Number (%) Number Number (%) colon (%) (%) Grade -0 24 (96) 32 (91.40) 0.63 5 (20) 4 (11.40) 0.47 Grade -1 0 (0) 0 (0) - 16 (64) 24 (68.60) 0.78 Grade -2 1 (4) 3 (8.60) 0.63* 4 (16) 5 (14.30) 1 Grade -3 0 (0) 0 (0) - 0 (0) 2 (5.70) 0.50* Sigmoid colon Grade -0 24 (96) 34 (97.10) 1 3 (12) 12 (34.30) 0.73 Grade -1 0 (0) 1 (2.90) 1.00* 18 (72) 20 (57.10) 0.28 Grade -2 1 (4) 0 (0) 0.41* 3 (12) 3 (8.60) 0.68* Grade -3 0 (0) 0 (0) - 1 (4) 0 (0) 0.41* Rectum Grade -0 21 (84) 28 (80) 0.74 2 (8) 8 (22.90) 0.17 Grade -1 3 (12) 7 (20) 0.49 17 (68) 23 (65.70) 1 Grade -2 1 (4) 0 (0) 0.41* 5 (20) 4 (11.40) 0.47 Grade -3 0 (0) 0 (0) - 1 (4) 0 (0) 0.41* Terminal Ileum (n=56) Grade -0 15 (62.5) 20 (62.5) 1 9 (37.5) 4 (12.5) 0.05 Grade -1 0 (0) 5 (15.6) 0.06 11 (45.8) 22 (68.8) 0.1 Grade -2 9 (37.7) 7 (21.9) 0.24 2 (8.3) 4 (12.5) 0.69* Grade -3 0 (0) 0 (0) - 2 (8.3) 2 (6.3) 1* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 29 Table 6 In large gut, macroscopic and microscopic lesions with anaemia in patients and controls Macroscopic Finding Microscopic Finding Anaemia Anaemia P-value Anaemia Anaemia P-value present absent present absent (n=18) (n=42) (n=18) (n=42) Ascending n (%) n (%) n (%) n (%) colon Grade -0 16 (88.90) 40 (95.20) 0.34 5 (27.80) 4 (9.50) 0.08 Grade -1 0 (0) 0 (0) - 9 (50) 31 (73.80) 0.07 Grade -2 2 (11.10) 2 (4.80) 0.58* 3 (16.70) 6 (14.30) 0.54 Grade -3 0 (0) 0 (0) - 1 (5.60) 1 (2.40) 0.51* Sigmoid colon Grade -0 18 (100) 40 (95.20) 0.35 6 (33.30) 9 (21.40) 0.32 Grade -1 0 (0) 1 (2.40) 0.70* 10 (55.60) 28 (66.70) 0.41 Grade -2 0 (0) 1 (2.40) 0.70* 2 (11.10) 4 (9.50) 0.59* Grade -3 0 (0) 0 (0) - 0 (0) 1 (2.40) 0.70* Rectum Grade -0 15 (83.30) 34 (81.00) 0.82 5 (27.80) 5 (11.90) 0.13 Grade -1 3 (16.70) 7 (16.70) 0.66 10 (55.60) 30 (71.40) 0.23 Grade -2 0 (0) 1 (2.40) 0.70* 2 (11.10) 7 (16.70) 0.45 Grade -3 0 (0) 0 (0) - 1 (5.60) 0 0.30* Terminal Ileum (n=56) Grade -0 10 (58.8) 25 (68.4) 0.708 2 (11.8) 11 (28.2) 0.16 Grade -1 2 (11.8) 3 (7.7) 0.634* 12 (70.6) 21 (53.8) 0.376 Grade -2 5 (29.4) 11 (28.2) 0.927 2 (11.8) 4 (35) 0.598* Grade -3 0 (0) 0 (0) - 1 (5.9) 3 (7.7) 0.647* *Fisher’s exact test; Chi-square test Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 30 Table 7 In study groups eosinophil infiltration in different sites of large gut in AS patients and controls Patient (n=60) Control (n=20) *P-value (Count Per HPF ) Mean±SD Median Mean±SD Median Ascending colon 18.5±21.5 12.50 11.7±9.9 7.50 0.17 Sigmoid colon 11.4±12.5 7.00 6.6±4.6 5.50 0.09 Rectum 5.7±6.8 4.00 4.8±5.9 3.00 0.61 Terminal ileum 15.6±19.6 8.5 8.8±8 6.5 0.14 *Two mean test High Power Field Table 8 Comparison of AS disease activity (BASDAI score and VAS) and eosinophil count in different macroscopic and microscopic lesions in large gut Microscopic grading Number BASDAI VAS score Eosinophil score count/HPF Ascending colon (N=60) N Mean±SD Mean±SD Mean±SD G-0 9 3.82±1.85 42.2±17.8 13.4±8.4 G-1 40 3.59±1.76 44.0±17.8 20.9±24.0 G-2 9 4.02±1.52 48.9±14.5 9.4±14.6 G-3 2 6.75±2.75 65.0±21.2 35.5±24.7 Sigmoid colon (N=60) G-0 15 3.32±1.27 40.0±11.9 16.9±18.4 G-1 38 4.02±1.98 46.1±19.2 10.5±9.8 G-2 06 3.61±2 48.3±16.0 4.3±3.7 G-3 01 3.20 70 05 Rectum (N=60) G-0 10 4.04±1.93 47.0±13.3 3.5±2.2 G-1 40 3.83±1.92 44.0±19.7 5.8±5.4 G-2 09 3.53±1.30 48.9±11.7 7.4±13.5 G-3 01 2.40 40 05 Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 31 Table 9 Frequency of macroscopic and microscopic lesion in large gut (n=60) inHLA- B27 patient groups Macroscopic Finding Microscopic Finding HLA- HLA- HLA- P- HLA- HLA- HLA- P- B27 B27 B27 value B27 B27 B27 value positive negative not positiv negative not (n=20) (n=17) done e (n=17) done (n=23 (n=20) (n=23 ) ) Ascending n (%) n (%) n (%) n (%) colon Grade -0 19 (95) 16 (94.1) 21 0.879 2 (10) 4 (23.5) 3 (13) 0.489 (91.3) Grade -1 0 (0) 0 (0) 0 (0) - 13 (65) 11 (64.7) 16 0.932 (69.6) Grade -2 1 (5) 1 (5.9) 2 (8.7) 0.879 4 (20) 2 (11.8) 3 (13) 0.741 Grade -3 0 (0) 0 (0) 0 (0) - 1 (5) 0 (0) 1 0.66 (4.3) Sigmoid colon Grade -0 19(95) 17 (100) 22 0.66 5 (25) 1 (5.9) 9 0.056 (95.7) (39.1) Grade -1 1 (5) 0 (0) 0 (0) 0.362 14 (70) 13 (76.5) 11 0.133 (47.8) Grade -2 0 (0) 0 (0) 1 (4.3) 0.441 1 (5) 3 (17.6) 2 0.427 (8.7) Grade -3 0 (0) 0 (0) 0 (0) - 0 (0) 0 (0) 1 0.441 (4.3) Rectum Grade -0 14 (70) 14 (82.4) 21 0.197 3 (15) 2 (11.8) 5 0.684 (91.3) (21.7) Grade -1 5 (25) 3 (17.6) 2 (8.7) 0.356 14 (70) 12 (70.6) 14 0.754 (60.9) Grade -2 1 (5) 0 (0) 0 (0) 0.362 3 (15) 2 (11.8) 4 0.886 (17.4) Grade -3 0 (0) 0 (0) 0 (0) - 0 (0) 1 (5.9) 0 (0) 0.276 Terminal Ileum (n=18) (n=15) (n=23 (n=18) (n=15) (n=23 ) ) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018 32 Grade -0 9 (50) 11 (73.3) 15 0.364 4 (22.2) 2 (13.3) 7 0.471 (65.2) (30.4) Grade -1 2 (11.1) 1 (6.7) 2 (8.7) 0.904 9 (50) 10 (66.7) 4 0.607 (60.9) Grade -2 7 (38.9) 3 (20) 6 0.461 3 (16.7) 2 (13.3) 1 0.417 (26.1) (4.3) Grade -3 0 (0) 0 (0) 0 (0) - 2 (11.1) 1 (6.7) 1 0.703 (4.3) Downloaded from https://academic.oup.com/rheumap/advance-article-abstract/doi/10.1093/rap/rky016/5025118 by Ed 'DeepDyve' Gillespie user on 12 July 2018

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Rheumatology Advances in PracticeOxford University Press

Published: May 29, 2018

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