Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Akira Madarame; Masakatsu Fukuzawa; Kumiko Uchida; Tadashi Ichimiya; Sakiko Naito; Yoshiya Yamauchi; Takashi Morise; Yasuyuki Kagawa; Takahiro Muramatsu; Takao Itoi

doi:10.1371/journal.pone.0334969

. 2025 Oct 30;20(10):e0334969. doi: 10.1371/journal.pone.0334969

Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Akira Madarame ^1,^*,^#, Masakatsu Fukuzawa ^1,^#, Kumiko Uchida ¹, Tadashi Ichimiya ¹, Sakiko Naito ¹, Yoshiya Yamauchi ¹, Takashi Morise ¹, Yasuyuki Kagawa ¹, Takahiro Muramatsu ¹, Takao Itoi ¹

Editor: Mohammed Misbah Ul Haq²

PMCID: PMC12574853 PMID: 41166300

Abstract

This study aimed to determine if the drug-induced lymphocyte stimulation test (DLST) for various oral mesalamines can diagnose mesalamine intolerance and predict the success of retreatment in patients with adverse events (AEs) due to the first oral 5-aminosalicylate (5-ASA) formulations administered. Data from patients with ulcerative colitis who experienced AEs after administration of the first oral 5-ASA and underwent DLSTs for two or more types of mesalamine, including the first oral 5-ASA were retrospectively analyzed. Mesalamine intolerance was defined as AEs within 6 months of starting the first oral 5-ASA and the inability to take oral mesalamine. Clinical characteristics, symptoms, type of first oral 5-ASA, DLST results, and the efficacy of retreatment with oral mesalamine were compared. The DLST for the first oral 5-ASA (F-DLST), highest DLST among the different oral mesalamine types (H-DLST), and mean DLST (M-DLST) were analyzed. Twenty-eight patients (median age 39 years, 57.1% male) were eligible; six patients were tolerant to oral mesalamine and22 were intolerant. Positive F-DLST (odds ratio [OR], 2.300; p = 0.002), positive M-DLST (OR, 2.667; p = 0.007), and older age at diagnosis (median 24.5 vs. 41.5; p = 0.006) were associated with mesalamine intolerance. Fourteen of the 28 patients underwent mesalamine retreatment. Higher F-DLST (median 88.0 vs. 174.0; p = 0.026), M-DLST (median 118.5 vs. 170.3; p = 0.040), and older age at diagnosis (median 24.5 vs. 39.0; p = 0.033) were associated with retreatment failure for oral mesalamine. DLSTs for various oral mesalamine formulations may be useful in predicting mesalamine intolerance and retreatment outcomes. However, their clinical utility should be interpreted with caution due to the risk of false-positive and false-negative results.

Introduction

Formulations of 5-aminosalicylate (5-ASA) are the first-line treatment for patients with mild to moderate ulcerative colitis (UC); 5-ASAs, such as mesalamine, induce and maintain UC remission and prevent colorectal cancer [1,2]. Salazosulfapyridine (SASP) is a sulfonamide-based prodrug that can be used to treat UC. It delivers 5-ASA to the colon through bacterial azo-reduction; however, it contains a sulfapyridine moiety, so SASP differs chemically and pharmacokinetically from pure mesalazine formulations and may cause additional adverse effects, including headache, skin rash, and male infertility. Therefore, mesalamine, a 5-ASA without the sulfapyridine group, was developed. Mesalamine is tolerated better than SASP [3,4]. The efficacy of 5-ASA depends on the concentration in the colonic mucosa. Therefore, 5-ASA formulations require drug delivery systems to deliver the drug to the colon efficiently. In Japan, the approved mesalamine-based 5-ASA formulations include Pentasa® with a time-dependent release system, Asacol® with a pH-dependent colon-targeted oral drug delivery system, and Lialda® with a multimatrix system.

Mesalamine-based 5-ASA formulations are well tolerated but can cause symptoms similar to UC exacerbations, including fever, abdominal pain, diarrhea, and hematochezia [5–7]. Mesalamine intolerance may also cause organ damage such as pericarditis and pneumonia [8–11]. Hiraoka et al. reported that the incidence of adverse events (AEs) related to 5-ASAs more than tripled from 5.3% in 2007–2010–16.2% in 2014–2016 [12]. According to a study by Hibiya et al., a higher percentage of patients with mesalamine intolerance underwent colectomies compared with the percentage of patients tolerating mesalamine who underwent colectomies (Hazard ratio: 4.92; 95% confidence interval: 2.58–9.38) [13]. Patients who experience 5-ASA-induced AEs can be retreated after desensitization therapy or treated with another mesalamine-based ASA [14–16]; however, the AEs may recur [17]. Although mesalamine intolerance sometimes causes serious AEs, methods for diagnosing mesalamine intolerance have not been established.

The drug-induced lymphocyte stimulation test (DLST) measures the uptake of ³H-thymidine by proliferating lymphocytes after stimulation with the target drug [18]. The accuracy of DLST in diagnosing drug allergies varies widely depending on the target drug [19,20], and the low sensitivity of DLST for detecting AEs related to 5-ASA is problematic [21,22]. The accuracy of the DLST for mesalamine affects decisions concerning retreatment with mesalamine. False positives may develop due to an allergy to the excipients of the mesalamine [23], and the opportunity for retreatment with mesalamine may be lost. Several studies have highlighted the high success rate of retreatment with mesalamine, even with a positive DLST for the suspect drug [24,25]. However, Shimizu et al. reported that five patients with positive DLSTs for 5-ASA were rechallenged with 5-ASA, and all patients failed [26]. In previous studies, DLST was performed only for the suspected 5-ASA agent, and the association between DLST results for various oral mesalamine formulations and mesalamine intolerance remains unclear. We hypothesized that testing various mesalamine formulations using DLST would improve the diagnostic sensitivity of mesalamine intolerance. Therefore, this study aimed to evaluate the relationship between DLST results for various oral mesalamine, mesalamine intolerance, and retreatment outcomes.

Materials and methods

Patients

Consecutive UC patients treated from 2014 to 2021 at the Department of Gastroenterology and Hepatology, Tokyo Medical University (Tokyo, Japan) were enrolled in the study. All patients were at least 18 years old and were diagnosed with UC based on standard clinical, endoscopic, and historical criteria according to the Japanese Research Committee on Inflammatory Bowel Disease [27]. Patients were prescribed Pentasa® (Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan), Asacol® (Zeria Pharmaceutical Co., Ltd., Tokyo, Japan), Lialda ® (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan), or Salazopyrin® (Pfizer Co., Ltd., Tokyo, Japan). Patients who experienced AEs after the first oral 5-ASA and underwent DLSTs for two or more types of mesalamine, including the first oral 5-ASA, were identified. Patients with unknown disease extent or type of oral mesalamine at diagnosis, patients who started oral SASP at diagnosis, patients receiving topical mesalamine before oral mesalamine, and patients with a history of hospital visits of less than 6 months were excluded from the study. Patients with a history of using topical 5-ASA formulations were excluded because this study specifically aimed to evaluate mesalamine intolerance and retreatment outcomes associated with oral formulations. The inclusion of patients who experienced AEs only with topical 5-ASA preparations may introduce heterogeneity because their pharmacokinetics and routes of exposure are different from those of oral formulations. Therefore, patients who had prior topical 5-ASA use were excluded to maintain the consistency and validity of the study cohort. A retrospective chart review was performed by two gastroenterologists (AM and MF). Any discrepancies were resolved by discussion with the senior author.

Data collection and definitions

Electronic medical records and the UC database were reviewed to ensure the inclusion of all patients. The clinical characteristics of patients with AEs to the first oral 5-ASA were extracted from medical records, including age at diagnosis, extent of disease at diagnosis of UC, type of first oral 5-ASA, clinical symptoms of AEs due to first oral 5-ASA, and duration of AEs due to first oral 5-ASA.

AEs due to the first oral 5-ASA were defined as symptoms occurring after receiving the first oral 5-ASA that resolved after discontinuing the drug, with or without corticosteroid treatment. Mesalamine intolerance was defined as (1) the occurrence of AEs within 6 months of starting the first oral 5-ASA and the inability to continue taking oral mesalamines owing to intolerance upon retreatment or (2) patients whose initial AEs were severe or involved significant organ toxicity and therefore did not receive mesalamine retreatment. Mesalamine tolerance was defined as patients tolerating at least one oral mesalamine [13]. Patients who experienced only mild AEs from the first oral 5-ASA and had no organ damage received mesalamine retreatment after consultation with their attending physician. Retreatment success was defined as the ability to continue taking at least one oral mesalamine formulation for >6 months after starting the first oral 5-ASA without AEs, whereas retreatment failure was defined as discontinuation of retreatment with mesalamine owing to AEs. Patients who achieved successful retreatment were ultimately assigned to the mesalamine-tolerant group, while those in whom retreatment failed were assigned to the mesalamine-intolerant group.

DLST was performed by SRL Inc. (Hachioji, Japan). For each DLST performed per drug, 5 mL of whole blood was collected and transferred to a heparinized blood collection tube. Lymphocytes were isolated from the whole blood through density gradient centrifugation and suspended in the RPMI1640 medium (DS Pharma Biomedical, Osaka, Japan). The cells were cultured with mesalamine for 72 h. ³H-Thymidine was then added, and incubation was continued for an additional 16 h. The uptake of ³H-thymidine, which was used as a control, in lymphocytes not exposed to mesalamine was measured. The stimulation index (SI) was defined as the ratio of ³H-thymidine uptake between the mesalamine-treated and control samples [18]. Positive DLST was defined as SI > 180%. The SI of the first oral 5-ASA was defined as F-DLST, the highest SI among the various types of oral mesalamines was defined as H-DLST, and the M-DLST was defined as the mean SI for the different mesalamines.

Study design and statistical analyses

The study was a single-center retrospective cohort study. Patients with UC who experienced AEs to the first oral 5-ASA were divided into two groups: (a) mesalamine-tolerant group: patients who were tolerant to another oral mesalamine and (b) mesalamine-intolerant group: patients who were intolerant to one or more oral mesalamines and discontinued mesalamine treatment. Patient characteristics and the medical course were compared using the χ2, Fisher’s exact, or Mann–Whitney U-tests. DLST was selected as a quantitative variable, and missing DLST data were removed. Univariate analyses were performed to identify factors associated with tolerance to retreatment with mesalamine. The cutoff values for DLST in predicting mesalamine intolerance and the efficacy of retreatment with oral mesalamine were examined using receiver operating characteristic (ROC) analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. P values <0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics version 29.0.1.0 (IBM Corp., Armonk, N.Y., USA).

Ethical considerations

The Ethics Committee of Tokyo Medical University School of Medicine approved this study (approval number: T2021-0352) on February 25, 2022, and the study was conducted in accordance with the tenets of the Declaration of Helsinki. Medical records and UC database were accessed for research purposes between February 25, 2022 and September 1, 2022. The authors had access to identifiable participant information during data collection; however, all data were anonymized before analysis. Moreover, this study was conducted using an opt-out approach approved by the institutional ethics committee, and the requirement for written informed consent was waived in accordance with relevant ethical guidelines. Furthermore, information about this study, including the opt-out process, was publicly posted at Tokyo Medical University Hospital, and no patients objected to the anonymized use of their data.

Results

Patient characteristics

Between 2014 and 2021, 695 patients with UC visited our hospital. Seventy-four patients were excluded due to the lack of detailed information, and 13 were excluded owing to prior use of topical mesalamine preparations before initiating oral 5-ASA. Of the remaining patients, 564 (92.8%) had no AEs due to the first oral 5-ASA and 44 (7.2%) had AEs. Moreover, 28 of the 44 patients underwent DLSTs for two or more types of mesalamine, including the first oral 5-ASA (Fig 1). None of the patients had a history of hospital visits of <6 months. Of the 28 patients who underwent DLST, 6 were classified into the mesalamine-tolerant group and 22 into the mesalamine-intolerant group. Table 1 shows the clinical characteristics of the patients with AEs due to the first oral 5-ASA. The median age at diagnosis of UC was 39 years (interquartile range [IQR]: 27.5–50.5), and 16 (57.1%) of the patients were male. At the time of UC diagnosis, 18 patients had extensive colitis, five patients had left-sided colitis, and five patients had proctitis. The median first oral 5-ASA dose was 4,000 mg (IQR: 3,600–4,800). The first oral 5-ASAs were time-dependent in eight cases, pH-dependent in six cases, and multimatrix in 14 cases. The time to the onset of AEs due to the first oral 5-ASA was 13 days (IQR: 9–22.5). The most common symptoms were fever (n = 17, 60.7%), diarrhea/bloody stools (n = 13, 46.4%), and abdominal pain (n = 7, 25.0%). Three patients were affected: one had pneumonia, another experienced myelosuppression, and the third suffered from both pancreatitis and liver dysfunction.

Table 1. Clinical characteristics of patients with adverse events due to the first oral 5-ASA.

Total number	28
Sex (female/male)	12/16
Age at diagnosis of UC (years); IQR	39 (27.5–50.5)
Extent of UC at the time of diagnosis (%)
Extensive	18 (64.3)
Left-sided	5 (17.9)
Rectum	5 (17.9)
First oral mesalamine (%)
Time-dependent release	8 (28.6)
pH-dependent release	6 (21.4)
Multimatrix	14 (50.0)
Median first oral 5-ASA dose (mg); IQR	4,000 (3,600–4,800)
Symptoms of adverse events due to the first oral 5-ASA (%)
Fever	17 (60.7)
Diarrhea and bloody stools	13 (46.4)
Abdominal pain	7 (25.0)
Joint pain	4 (14.3)
Nausea	2 (7.1)
Headache	1 (3.6)
Fatigue	1 (3.6)
Skin rash	1 (3.6)
Pancreatitis	1 (3.6)
Pneumonia	1 (3.6)
Bone marrow suppression	1 (3.6)
Liver dysfunction	1 (3.6)
Time to the onset of adverse events due to the first oral 5-ASA (days); IQR	13 (9–22.5)

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Values are presented as medians or numbers. 5-ASA, 5-aminosalicylate; IQR, interquartile range; UC, ulcerative colitis.

Comparison of the mesalamine-intolerant and tolerant groups

Table 2 shows the comparison of clinical backgrounds between the mesalamine-intolerant and tolerant groups. The mesalamine-intolerant group was younger than the tolerant group by age at diagnosis of UC (23.5 vs 41.5 years, p = 0.006). No significant differences in sex, extent of disease at diagnosis of UC, or type of first oral 5-ASA were detected between the two groups. In addition, no significant differences in the AE symptoms due to the first oral 5-ASA (diarrheal bloody stools, fever up, abdominal pain, nausea, headache, fatigue, skin rash, joint pain, pancreatitis, pneumonia, bone marrow suppression, liver dysfunction) or the time to onset of AEs due to the first oral 5-ASA were detected between the groups.

Table 2. Comparison of the clinical characteristics of patients in the mesalamine-tolerant and mesalamine-intolerant groups.

	Mesalamine-tolerant group (n = 6)	Mesalamine-intolerant group (n = 22)	Odds ratio	95% CI	p value
Sex (female/male)	3/3	9/13	1.444	0.236–8.844	1.000
Age at diagnosis (years): IQR	24.5 (21.0–31.0)	41.5 (32.0–58.0)			0.006
Disease location (%)
Extensive	3 (50.0)	15 (68.2)	2.143	0.342–13.420	0.634
Left-sided	2 (33.3)	3 (13.6)	0.316	0.039–2.550	0.285
Rectum	1 (16.7)	4 (18.2)	1.111	0.100–12.308	1.000
First oral mesalamine (%)
Time-dependent release	1 (16.7)	7 (31.8)	2.333	0.228–23.908	0.640
pH-dependent release	0 (0)	6 (27.3)	1.375	1.065–1.776	0.289
Multimatrix	5 (83.3)	9 (40.9)	0.138	0.014–1.394	0.165
Symptoms of adverse events for the first oral mesalamine (%)
Fever	4 (66.7)	13 (59.1)	0.722	0.108–4.820	1.000
Diarrhea and bloody stools	1 (16.7)	12 (54.5)	6.000	0.598–60.158	0.173
Abdominal pain	2 (33.3)	5 (22.7)	0.588	0.082–4.212	0.622
Joint pain	1 (16.7)	3 (13.6)	0.789	0.067–9.317	1.000
Nausea	1 (16.7)	1 (4.5)	0.238	0.013–4.497	0.389
Headache	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Fatigue	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Skin rash	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Pancreatitis	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Pneumonia	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Bone marrow suppression	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Liver dysfunction	0 (0)	1 (4.5)	1.048	0.956–1.148	1.000
Duration to the onset of adverse events for first oral mesalamine (days); IQR	12.0 (10.0–27.0)	13.0 (8.0–20.0)			0.955

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Values are presented as medians or numbers. 5-ASA, 5-aminosalicylate; IQR, interquartile range.

Relationship between DLST and mesalamine intolerance

In the 28 patients who underwent F-DLSTs, 15 (53.6%) tests were negative and 13 (46.4%) tests were positive. All 13 patients with positive F-DLSTs were mesalamine-intolerant group (p = 0.018, OR 2.444, 95% CI 1.479–4.039) (Table 3). The median F-DLST was higher in the mesalamine-intolerant group than in the mesalamine-tolerant group (250.5% vs. 83.5%, p = 0.002).

Table 3. Comparison of patients who underwent DLST in the mesalamine-tolerant and mesalamine-intolerant groups.

	Mesalamine-tolerant group	Mesalamine-intolerant group	Odds ratio	95% CI	p value
	(n = 6)	(n = 22)
Positive F-DLST (%)	0 (0)	13 (59.1)	2.444	1.479–4.039	0.018
F-DLST (IQR)	83.5 (74.0–90.0)	250.5 (159.0–391.0)			0.002
Positive H-DLST (%)	2 (33.3)	18 (81.8)	9.000	1.201–67.417	0.038
H-DLST (IQR)	153.5 (90.0–226.0)	411.0 (222.0–562.0)			0.005
Positive M-DLST (%)	0 (0)	15 (68.1)	3.143	1.705–5.794	0.005
M-DLST (IQR)	110.4 (84.3–118.75)	311.1 (166.5–384.3)			0.001

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Values are presented as medians or numbers. DLST, drug-induced lymphocyte stimulation test; F-DLST, DLST for the first oral 5-aminosalicylate; H-DLST, highest DLST among various types of oral mesalamine preparations; IQR, interquartile range; M-DLST, mean DLST among various types of oral mesalamine preparations.

DLSTs for various oral mesalamines, including the first oral 5-ASA, were performed in 28 patients. Four patients were tested for one type of oral mesalamine, four patients were tested for two types, 11 patients were tested for three types, and 13 patients were tested for four types. Eight cases (28.6%) were negative and 20 cases (71.4%) were positive for the H-DLST. Eighteen of the 20 patients with positive H-DLSTs were intolerant to oral mesalamines, and the association was significant (p = 0.038, OR 9.000, 95% CI 1.201–67.417). The median H-DLST was higher in the mesalamine-intolerant group than the median H-DLST in the mesalamine-tolerant group (411.0% vs. 153.5%, p = 0.005).

M-DLSTs were positive in 15 patients and negative in 13 patients. The 15 patients with positive M-DLSTs were all intolerant to oral mesalamines (p = 0.005, OR 3.143, 95% CI 1.705–5.794). The M-DLSTs were higher in the mesalamine-intolerant group than the M-DLSTs in the mesalamine-tolerant group (311.1% vs. 110.4%, p = 0.001).

The sensitivity and specificity of a positive F-DLST for predicting mesalamine intolerance were 0.591 and 1.00; the positive and negative predictive values were 1.00 and 0.400. Positive H-DLSTs had a sensitivity of 0.818, a specificity of 0.667, a positive predictive value of 0.900, and a negative predictive value of 0.500. Positive M-DLSTs had a sensitivity of 0.682, a specificity of 1.00, a positive predictive value of 1.00, and a negative predictive value of 0.462.

Retreatment with oral mesalamine after AEs due to the first oral 5-ASA

Fourteen patients underwent retreatment with oral mesalamines after AEs to the first 5-ASA (Fig 2), of whom six achieved retreatment success and were classified into the mesalamine-tolerant group, whereas eight experienced retreatment failure and were classified into the mesalamine-intolerant group. Second, third, and fourth-line therapies with oral mesalamine achieved success rates of 21.4% (three of 14), 60.0% (three of five), and 0% (none of one), respectively. For the second-line therapy, eight patients were switched to a different type of oral 5-ASA preparation. Four patients underwent desensitization therapy with a time-dependent release type of 5-ASA that differed from their initial formulation. One patient underwent desensitization therapy using the same type of time-dependent release 5-ASA as initially administered, and one patient was switched to SASP. Among the second-line therapy, the three successful cases consisted of one patient who was switched to a different oral 5-ASA preparation, one patient who underwent desensitization therapy with a time-dependent release type of 5-ASA that differed from their initial formulation, and one patient who underwent desensitization therapy using the same type of time-dependent release 5-ASA as initially administered. For the third-line therapy, four patients were switched to SASP, and one patient underwent desensitization therapy with a time-dependent release type of 5-ASA that differed from the initial formulation. Among the third-line therapy, the three successful cases consisted of two patients who were switched to SASP and one patient who underwent desensitization therapy with a time-dependent release type of 5-ASA that differed from the initial formulation. Furthermore, for the fourth-line therapy, one patient was switched to a different type of oral 5-ASA preparation. Desensitization therapy with a time-dependent release type of 5-ASA was initiated at 5 mg/day. Treatment with a different type of oral 5-ASA preparation was started at a median dose of 2,400 (IQR: 800–2,400) mg/day, whereas SASP therapy was started at a median dose of 500 (IQR: 375–1,250) mg/day. All treatment regimens were titrated upward to reach at least 2,000 mg/day. Six patients who achieved successful retreatment maintained oral mesalamine therapy at a minimum dose of 2,000 mg/day for at least 6 months, and none discontinued treatment thereafter. No significant correlation was found between positive DLSTs and retreatment outcomes. Higher F-DLSTs (median 83.5 vs. 168.5, p = 0.033) and M-DLSTs (median 110.4 vs. 172.75, p = 0.010) were associated with failure of oral mesalamine retreatment (Table 4). Additionally, older age at diagnosis was associated with retreatment failure (median 24.5 vs. 39.0, p = 0.033). No significant differences in sex, disease location, symptoms of AEs for the first oral 5-ASA, or time to onset of AEs for the first oral 5-ASA were detected between the retreatment success and failure groups (Table 5).

Table 4. Comparison of DLST and failed retreatment with oral mesalamine therapy.

	Success	Failure	Odds ratio	95% CI	p value
	n = 6	n = 8
Positive F-DLST (%)	0 (0)	3 (37.5)	1.600	0.935–2.737	0.209
F-DLST; IQR	83.5 (74.0–90.0)	168.5 (127.0–605.5)			0.033
Positive H-DLST (%)	2 (33.3)	5 (62.5)	3.333	0.362–30.701	0.592
H-DLST; IQR	153.5 (90.0–226.0)	259.5 (168.5–689.5)			0.071
Positive M-DLST (%)	0 (0)	3 (37.5)	1.600	0.935–2.737	0.209
M-DLST; IQR	110.4 (84.3–118.75)	172.75 (136.9–518.8)			0.010

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Table 5. Demographic characteristics of patients who received retreatment with oral mesalamine after developing adverse events due to the first mesalamine therapy.

	Success	Failure	Odds ratio	95% CI	p value
	(n = 6)	(n = 8)
Sex (female/male)	3/3	2/6	3.000	0.312–28.841	0.580
Age at diagnosis (years): IQR	24.5 (21.0–31.0)	39.0 (31.0–45.5)			0.033
Disease location (%)
Extensive	3 (50.0)	6 (75.0)	3.000	0.312–28.841	0.580
Left-sided	2 (33.3)	1 (12.5)	0.286	0.019–4.237	0.538
Rectum	1 (16.7)	1 (12.5)	0.714	0.036–14.347	1.000
Symptoms of adverse events for the first oral mesalamine (%)
Fever	4 (66.7)	5 (62.5)	0.833	0.090–7.675	1.000
Diarrhea and bloody stools	1 (16.7)	4 (50.0)	5.000	0.388–64.387	0.301
Abdominal pain	2 (33.3)	2 (25.0)	0.667	0.065–6.871	1.000
Nausea	1 (16.7)	1 (12.5)	0.714	0.036–14.347	1.000
Headache	0 (0)	1 (12.5)	1.143	0.880–1.485	1.000
Fatigue	0 (0)	1 (12.5)	1.143	0.880–1.485	1.000
Joint pain	1 (16.7)	3 (37.5)	3.000	0.227–39.608	0.580
Time to the onset of adverse events for the first oral mesalamine (days); IQR	12.0 (10.0–27.0)	16.0 (4.5–22.0)			0.948

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Values are presented as medians or numbers. IQR, interquartile range.

The relationship between the number of positive DLSTs for various types of mesalamine and the failure of retreatment with mesalamine is shown in Fig 3. Retreatment failed in 42.9% (three of seven) of patients with no positive DLSTs, in 33.3% (one of three) of patients with one positive DLST, and in 100% (all four) of patients with two or three positive DLSTs.

Relationship between mesalamine intolerance and DLST cutoff values

The diagnostic potential of the DLST for predicting mesalamine intolerance was evaluated by plotting ROC curves (Fig 4a). A cutoff value of 96.0 for F-DLST yielded a sensitivity of 95.5%, a specificity of 83.3%, and a maximum Youden index (0.788) with an area under the curve (AUC) of 0.928 (p < 0.001). A cutoff value of 288.5 for H-DLST resulted in a sensitivity of 68.2%, a specificity of 100%, and a maximum Youden index (0.682) with an AUC of 0.879 (p < 0.001). The cutoff value of 120.2 for M-DLST exhibited a sensitivity of 95.5%, a specificity of 83.3%, and a maximum Youden index (0.788) with an AUC of 0.939 (p < 0.001).

Fig 4b shows the ROC curves for the relationship between DLST and mesalamine retreatment failure. A cutoff value for F-DLST of 98.5 yielded a sensitivity of 87.5%, a specificity of 83.3%, and the maximum Youden index (0.708) was obtained, with an AUC of 0.844 (p = 0.003). The sensitivity and specificity of H-DLST at a cutoff value of 140.0 were 100% and 50.0%, respectively, with a maximal Youden index (0.500) and AUC of 0.792 (p = 0.020). A cutoff value of 120.2 for M-DLST yielded a sensitivity of 100%, a specificity of 83.3%, a maximum Youden index (0.833), and an AUC of 0.917 (P < 0.0001).

Discussion

In this retrospective single-center cohort study, the incidence of AEs in patients receiving the first oral 5-ASA was 7.2%. Positive F-DLST, H-DLST, and M-DLST were significantly more frequent in the mesalamine-intolerant group compared with positive tests in the mesalamine-tolerant group. The F-DLST, H-DLST, and M-DLST in the mesalamine-intolerant group were higher than the test values in the mesalamine-tolerant group, confirming the usefulness of the DLST in diagnosing mesalamine intolerance. In patients retreated with mesalamine, higher F-DLST, and M-DLST were associated with retreatment failure. The AUCs for the ROC curves of F-DLST, H-DLST, and M-DLST were high, confirming the usefulness of the DLST in diagnosing mesalamine intolerance and predicting retreatment failure with mesalamine. To the best of our knowledge, this is the first study to evaluate the use of DLST for various oral mesalamine formulations for diagnosing mesalamine intolerance and failure of mesalamine retreatment in patients with UC.

Mesalamine intolerance is still not clearly defined [28]. In this study, we used the definition of mesalamine intolerance from a multicenter study reported in Japan [13]. Mesalamine intolerance is often defined as the presence of AEs due to at least one mesalamine formulation, independent of whether oral mesalamine can be continued [12,24–26]. Patients with mesalamine intolerance often use advanced therapies such as immunomodulators and biologics [13], which also carry the risk of AEs [29,30] and malignancy [31]. Therefore, continuing mesalamine therapy whenever possible is important in avoiding unnecessary advanced therapies and reducing the risk of colectomy [4]. However, mesalamine retreatment can cause major AEs; thus, it should be used with caution [16,26]. The precise mechanisms underlying mesalamine intolerance remain unclear; however, immune-mediated reactions to 5-ASA, sulfapyridine, or formulation excipients were suggested [28]. A genome-wide association study revealed a significant genetic predisposition to mesalamine intolerance. Moreover, it identified rs144384547 as a genome-wide significant susceptibility single-nucleotide polymorphism associated with mesalamine-induced fever and diarrhea [32]. The mesalamine-intolerant group had higher abundance levels of Faecalibacterium, Streptococcus, and Clostridium than the mesalamine-tolerant group. Based on this finding, dysbiosis may contribute to the development of mesalamine intolerance [33]. DLST, which is commonly used to assess T-cell sensitization to drugs, can evaluate lymphocyte proliferation by determining the incorporation of ³H-thymidine into the DNA of proliferating lymphocytes. This assay is based on the principle that antigen-specific T cells undergo clonal expansion upon antigen recognition [34]. Accordingly, if mesalamine intolerance involves an immunologically mediated hypersensitivity reaction, a high SI would be expected. The mesalamine retreatment rate ranges from 22.6% to 63.9% [12,24–26], and our retreatment rate (50.0%) was comparable to these values. This high retreatment rate indicates that our hospital has an aggressive retreatment policy, considering the degree of side effects and DLST results.

Significant differences in positive F-DLST, H-DLST, and M-DLST and high F-DLST, H-DLST, and M-DLST were identified. An association between mesalamine intolerance as defined in this study and DLST, indicating the usefulness of DLST, has not been previously reported. However, predictors of mesalamine intolerance have been previously reported. In addition to the DLST results, an older age at diagnosis was significantly associated with mesalamine intolerance and failed retreatment with mesalamine. To the best of our knowledge, previous studies have not reported a similar association between mesalamine intolerance and age at diagnosis. Hiraoka et al. reported that successful retreatment with mesalamine was associated with the lack of fever and abdominal pain (odds ratio = 4.64; 95% confidence interval, 0.85–25.3, p = 0.052) [12]. Suzuki et al. reported that fever and diarrhea caused by mesalamine allergy were associated with rs144384547 located upstream of RGS17. The frequency of mesalamine-induced fever and diarrhea was higher in patients heterozygous for rs144384547 compared with patients without the risk allele for this SNP (22.0% vs. 2.34%) [32]. In our study, fever was also the most common AE of 5-ASA but was not a predictor of mesalamine intolerance. The incidence of fever was low (22.0%) in the Suzuki study, even in patients with the gene mutation. Shuji et al. reported that pancolitis tended to be associated with mesalamine intolerance (p = 0.059) [13]. In our study, no significant differences in disease location were detected between patients with and without mesalamine intolerance. Mesalamine intolerance can occur in any disease location.

The mesalamine retreatment success rate in patients who experienced an AE due to the first oral 5-ASA was 42.9% in our study. Significant differences in the success of mesalamine retreatment were detected between patients with high and low mesalamine F-DLST and M-DLST. Previous studies reported mesalamine retreatment success rates of 0%–85.7% [12,24–26], and some reports indicated that positive DLST results to the suspect drug did not affect retreatment [24,25]. Desensitization therapy with time-dependent 5-ASA is often successful, even in patients with positive DLSTs [25]. However, Hirotaka et al. reported that retreatment of five DLST-positive patients with mesalamine resulted in AEs in all patients [26]. The differences in results may be due to allergies, the retreatment starting dose, the influence of excipients, and the involvement of the rs144384547 gene. No association between positive DLSTs and retreatment was detected, but F-DLST and M-DLST were higher in the retreatment failure group compared with the success group, suggesting that the cutoff values for retreatment may be different. All patients with positive DLSTs for more than one mesalamine failed retreatment. Positive DLSTs to more than one mesalamine may be due to allergy to mesalamine and should be carefully considered before retreatment.

DLST cutoff values for mesalamine intolerance or retreatment have not been reported. The cutoff values of the DLST vary for different drugs [21,35]. The reported sensitivity of DLST when a 5-ASA caused AEs is 0.24, the specificity is 0.81, the false positive rate is 0.195, and the false negative rate is 0.76 [21]. In the present study, F-DLST, H-DLST, and M-DLST had high AUCs for predicting mesalamine intolerance; the cutoff values for F-DLST and M-DLST were less than 180%, and the cutoff value for H-DLST was 288.5%. The cutoff values for F-DLST, H-DLST, and M-DLST for predicting retreatment failure were below 180% and correlated well. Importantly, all patients with two or more positive DLSTs failed retreatment, indicating a high likelihood of true mesalamine allergy. In contrast, patients with negative or only one positive DLST often tolerated retreatment, suggesting that intolerance may be formulation-specific rather than related to mesalamine itself. These findings provide a preliminary framework: DLST should be performed after the initial onset of intolerance symptoms following the first oral 5-ASA, and it is advisable to test all available oral mesalamine formulations. Retreatment should be avoided when multiple DLSTs are positive, whereas cautious reintroduction may be considered in cases with negative or single-positive results.

This study has several limitations. First, this was a single-center, retrospective cohort study with a limited and uneven sample size, which restricts the statistical power and limits the external validity and generalizability of the findings. Moreover, only univariate comparisons were performed, and potential confounders such as age, disease extent, type of first 5-ASA, 5-ASA dose, severity of AE, and corticosteroid use were not adjusted for. Although a multivariable logistic regression would be essential to clarify independent predictors of mesalamine intolerance and retreatment outcomes, this was not feasible owing to the small number of patients. Future studies with larger sample sizes are needed to address these potential confounders in a more robust manner. Second, treatment decisions, including mesalamine retreatment and the performance of DLSTs, were made at the discretion of the attending physicians, which may have introduced variability. Additionally, although the DLST showed significant associations with mesalamine intolerance, its clinical applicability remains uncertain due to limited sensitivity and specificity as well as the risk of both false positives and false negatives. Our findings suggest a preliminary framework for clinical decision-making; however, DLST should not yet be applied in routine practice, and further validation in larger, multicenter prospective cohorts is required. Third, the ROC-derived cutoff values, odds ratios, and AUCs were derived and evaluated within the same small dataset, raising concerns about statistical overfitting and overly optimistic estimates. Therefore, external validation in larger, multicenter prospective cohorts will be necessary to confirm the diagnostic performance of the DLST and the generalizability of these thresholds. Finally, to ensure diagnostic clarity and sufficient follow-up, patients with incomplete data, those who had used topical mesalamine preparations before oral mesalamine formulations, and those with a short history of hospital visits were excluded from the analysis. However, these exclusion criteria might have introduced selection bias. In conclusion, future multicenter prospective cohort studies are essential to validate the diagnostic value of the DLST and the clinical management of mesalamine intolerance.

Conclusions

DLSTs for various oral mesalamine formulations may have a predictive value in diagnosing mesalamine intolerance and assessing the likelihood of successful retreatment. Nevertheless, their clinical application requires caution owing to potential false-positive and false-negative results. To improve diagnostic accuracy, DLSTs should be interpreted along with clinical symptoms and, if available, other emerging diagnostic modalities such as genetic testing, once clinically validated.

Acknowledgments

The authors thank Enago (www.enago.jp) for the English language review.

Data Availability

The minimal dataset required to replicate the study’s findings is fully contained within the paper.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0334969.r001

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Reviewer #1: Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

This is an interesting topic.

However, the clinical usefulness and reliability of DLST testing for mesalazine is very low. For this reason, DLST testing is being performed less and less. As a result, it is difficult to find meaning in research discussing the usefulness of DLST testing. There have already been several papers reporting that mesalazine intolerance has a poor prognosis, so this study lacks novelty. It is also unfortunate that the number of studies is small.

Reviewer #2: 1.As a single-center retrospective study, the limited sample size (n=28) may result in insufficient statistical power. Expanding the cohort or conducting multicenter collaborations would improve the reliability of the findings. 2.The exclusion of certain cases (e.g., patients receiving topical mesalazine) during screening could introduce selection bias. The potential impact of this limitation should be explicitly addressed in the Discussion section. 3.The manuscript lacks detailed methodological descriptions of the drug lymphocyte stimulation test (DLST), including critical parameters such as drug concentrations and incubation periods. Supplementary procedural details are recommended to ensure experimental reproducibility. 4.The immunological mechanisms of DLST positivity and mesalazine intolerance are not discussed in depth (e.g., whether it is an allergic reaction or non-immunotoxic). It is recommended that additional mechanistic hypotheses be added to the existing literature. 5. The conclusions emphasise the predictive value of DLST, but there may be a problem of false positives/negatives in practical application. Discussion is needed on how to improve diagnostic accuracy in combination with clinical symptoms or other tests (e.g., genetic testing).

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PLoS One. 2025 Oct 30;20(10):e0334969. doi: 10.1371/journal.pone.0334969.r002

Author response to Decision Letter 1

29 May 2025

Manuscript ID: PONE-D-25-17000

Title: Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Dear Editors and Reviewers,

We would like to thank you for the opportunity to revise our manuscript and the insightful comments. We have carefully addressed each point raised by the reviewers and have revised the manuscript accordingly. Our detailed responses to each comment are provided below.

Reviewer #1

Comment: The clinical usefulness and reliability of DLST testing for mesalazine is very low. For this reason, DLST testing is being performed less and less. As a result, it is difficult to find meaning in research discussing the usefulness of DLST testing. There have already been several papers reporting that mesalazine intolerance has a poor prognosis, so this study lacks novelty.

Response:

Given its relatively low sensitivity and specificity, the use of DLST in clinical practice is limited. Further, its application in other settings has decreased. However, in Japan, DLST is still used as a supportive tool for evaluating suspected drug-induced hypersensitivity reactions, such as mesalamine intolerance.

This study differs from previous ones because previous studies commonly assessed the DLST results only for mesalamine preparation that were initially suspected. Moreover, the present study examined the DLST results for various types of oral mesalamine in each patient, thereby allowing the evaluation of whether a higher M-DLST across various formulations is associated with clinical intolerance and retreatment failure.

In addition, this study explored an optimal SI cutoff value for predicting intolerance, which has not been clearly addressed in previous studies. We believe that our findings offer novel contributions to the field, particularly in using DLST more effectively as part of a multifaceted diagnostic approach. These points have been added to the Introduction and Discussion of the revised manuscript.

Comment: The number of cases is small.

Response:

We acknowledge that this single-center retrospective study was limited by the small sample size. This has been explicitly discussed in the revised Discussion.

Reviewer #2

Comment 1: The limited sample size (n=28) may result in insufficient statistical power. Expanding the cohort or conducting multicenter collaborations would improve the reliability of the findings.

Response:

We agree with this limitation, and this has been clearly stated in the Discussion. We also recommended the need to conduct multicenter prospective studies to validate our findings.

Comment 2: The exclusion of certain cases (e.g., patients receiving topical mesalazine) during screening could introduce selection bias. The potential impact of this limitation should be explicitly addressed in the Discussion section.

Response:

Thank you for this valuable suggestion. A statement acknowledging the possibility of selection bias due to our exclusion criteria and its potential effect on the generalizability of the results has been added to the Discussion.

Comment 3: The manuscript lacks detailed methodological descriptions of the DLST, including drug concentrations and incubation periods. Supplementary procedural details are recommended.

Response:

The Methods was revised to include data on drug concentrations, incubation conditions, and control settings used in DLST.

Comment 4: The immunological mechanisms of DLST positivity and mesalamine intolerance are not discussed in depth (e.g., whether it is an allergic reaction or non-immunotoxic).

Response:

Thank you for your valuable comment. As suggested, the Discussion has been revised. In particular, data on the possible immunological mechanisms underlying mesalamine intolerance and DLST positivity were added. Moreover, information on immune-mediated reactions to 5-ASA, sulfapyridine, or formulation excipients, a genome-wide significant SNP (rs144384547) associated with mesalamine-induced symptoms, and possible involvement of dysbiosis was included. Furthermore, an explanation of the DLST principle was added, thereby emphasizing its relevance in detecting T-cell-mediated hypersensitivity, which may be reflected by a high stimulation index in mesalamine-intolerant cases.

Comment 5: The conclusions emphasise the predictive value of DLST, but there may be a problem of false positives/negatives in practical application. Discussion is needed on how to improve diagnostic accuracy.

Response:

The Conclusion and Discussion were revised to support our claims regarding predictive utility. The risks of false-positive and false-negative results were explicitly discussed. Furthermore, we proposed interpreting the DLST findings in consideration of clinical symptoms and, if available, genetic or immunologic markers.

Additional Revisions

We appreciate the editorial request regarding data sharing. As described in the manuscript, the data were anonymized before analysis in compliance with ethical guidelines and were collected using an opt-out procedure approved by our institutional ethics committee. The requirement for informed consent was waived owing to the retrospective nature of the study and the use of anonymized data.

However, after re-examining the dataset in light of journal publication standards, certain combinations of clinical variables (e.g., age, sex, disease characteristics, and treatment responses) could potentially allow the reidentification of individuals if shared externally. Furthermore, participants did not provide explicit consent for external data sharing. According to Japanese law, disclosing potentially identifiable clinical information to parties outside the institution without consent may constitute a legal violation. Therefore, we have removed the Supporting Information and clarified this limitation in the revised Data availability section.

The revised manuscript with track changes, a clean version of the manuscript, and a response letter were uploaded separately.

We confirmed that our protocols do not require registration in protocols.io, as they do not involve novel laboratory methods.

We hope that the revisions and clarifications satisfactorily address the concerns raised. We greatly appreciate the reviewers’ constructive feedback and your consideration of our revised manuscript.

Sincerely,

Akira Madarame, MD

On behalf of all co-authors

Attachment

Submitted filename: Response to Reviewers.docx

pone.0334969.s001.docx^{(21.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334969.r003

Decision Letter 1

Mohammed Misbah Ul Haq

4 Jul 2025

Dear Dr. Madarame,

Please submit your revised manuscript by Aug 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Partly

Reviewer #4: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: No

Reviewer #4: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

Reviewer #3: This manuscript suggests that DLSTs are useful for retreatment mesalamine in cases with side effects for first 5-ASA.

However, it requires some revisions as listed below.

1. The authors divided the 28 patients who experienced AEs from the first 5-ASA treatment into two groups: a tolerable group of six and an intolerable group of 22. Was this determined by retreatment after discontinuing the first 5-ASA to assess tolerability or intolerance (Page 6, line 107-111)? The Results section states 14 patients underwent retreatment. The study flow and Methods section needs to be clearly described.

2. The definitions of success and failure for 5-ASA retreatment should be indicated. Fig 2 shows that mesalamine retreatment was undergone multiple times. However, it is unclear at what point the retreatment was deemed a success or failure.

3. The authors should provide information on mesalamine retreatment details that could affect outcomes, such as dosage and the presence or absence of desensitization therapy.

4. The spelling of mesalamine should be consistent throughout the text and figures.

Reviewer #4: (No Response)

**********

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Reviewer #3: No

Reviewer #4: No

**********

Attachment

Submitted filename: review-PONE.docx

pone.0334969.s002.docx^{(14.7KB, docx)}

PLoS One. 2025 Oct 30;20(10):e0334969. doi: 10.1371/journal.pone.0334969.r004

Author response to Decision Letter 2

20 Jul 2025

Manuscript ID: PONE-D-25-17000

Title: Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Dear Editors and Reviewers:

We would like to thank you for the opportunity to revise our manuscript as well as for the insightful comments. We have carefully addressed each point raised by the reviewers and have revised the manuscript accordingly. Our detailed responses to each comment are provided below.

Reviewer #3

Comment 1: The authors divided the 28 patients who experienced AEs from the first 5-ASA treatment into two groups: a tolerable group of six and an intolerable group of 22. Was this determined by retreatment after discontinuing the first 5-ASA to assess tolerability or intolerance (Page 6, line 107-111)? The Results section states 14 patients underwent retreatment. The study flow and Methods section needs to be clearly described.

Response:

Thank you for your valuable comment. We apologize for the lack of clarity in the description regarding the patient grouping. In the revised manuscript, we have clarified that mesalamine intolerance was defined as either (1) the occurrence of adverse events (AEs) within 6 months of the first oral 5-ASA and the inability to continue taking oral mesalamines owing to intolerance upon retreatment or (2) patients whose initial AEs were severe or involved significant organ toxicity, and therefore, did not receive mesalamine retreatment. We have added these definitions and clarifications to the Methods section, under the heading Data collection and definitions (Page 6, lines 119–124).

Comment 2: The definitions of success and failure for 5-ASA retreatment should be indicated. Fig 2 shows that mesalamine retreatment was undergone multiple times. However, it is unclear at what point the retreatment was deemed a success or failure.

Response:

Thank you for bringing this to our attention. We have added clear definitions of “retreatment success” and “failure” to the Methods section, under the heading Data collection and definitions. Retreatment success was defined as the ability to continue taking at least one oral mesalamine formulation for >6 months after starting the first oral 5-ASA without AEs, and retreatment failure was defined as discontinuation of mesalamine retreatment owing to AEs (Page 6, lines 126–129).

To make this clearer, we have revised the description of Fig. 2 as well.

Comment 3: The authors should provide information on mesalamine retreatment details that could affect outcomes, such as dosage and the presence or absence of desensitization therapy.

Response:

Thank you for your insightful comment. We have added detailed information in the Results section on the methods of mesalamine retreatment that could affect outcomes. Specifically, we describe the three approaches used, which are as follows: (1) switching to a different type of oral 5-ASA, (2) performing desensitization therapy, and (3) switching to SASP.

Additionally, the initial dose at retreatment was specified, and all treatment regimens were titrated upward to reach at least 2,000 mg/day (Page 14, lines 248–261).

Comment 4: The spelling of mesalamine should be consistent throughout the text and figures.

Response:

Thank you for bringing this to our attention. Accordingly, we have carefully checked the entire manuscript, including the text and figures, to ensure that the spelling of mesalamine is consistent throughout.

Major comments

Materials and methods

Comment 1: Why were patients excluded with previous topical 5-ASA drugs? It is a potential confounder, and excluding them results in selection bias.

Response: Thank you for your insightful comment. We acknowledge the reports regarding mesalamine intolerance caused by topical 5-ASA drugs. However, this study specifically aimed to investigate mesalamine intolerance and retreatment outcomes in patients who experienced AEs from their first dose of oral 5-ASA formulation. The inclusion of patients who only experienced intolerance to topical 5-ASA preparations may introduce heterogeneity as their pharmacokinetics and routes of exposure are different from those of oral formulations. Therefore, patients who previously used topical 5-ASA were excluded to maintain the consistency and validity of the study cohort. For clarity, we have added this rationale to the Materials and Methods section (Page 5, lines 102–108).

Comment 2: It should be emphasized that reintroduction of a different type of 5-ASA formulation is only acceptable and ethical in cases of mild intolerance. In cases of severe adverse events, such as severe renal failure or pancreatitis, re-exposure should be strictly avoided. This important distinction should be clearly stated.

Response: Thank you for this important comment. We agree that retreatment with a different type of 5-ASA formulation should only be considered in cases of mild intolerance without major organ damage.

To clarify this ethical consideration, we have added the following sentence to the Materials and Methods section: “Patients who experienced only mild AEs from the first oral 5-ASA and had no organ damage received mesalamine retreatment after consultation with their attending physician.” (Page 6, lines 124–126)

Results section

Comment 1: Very small and highly imbalanced sample. The comparison between only six and twenty-two patients—set against an already limited total sample size—substantially restricts statistical power and calls into question the external validity and generalizability of the study’s conclusions.

Insufficient statistical modelling. Only univariate comparisons were performed; potential confounders (age, disease extent, 5-ASA dose, AE severity, corticosteroid use) were not adjusted for. A multivariable logistic regression is essential to clarify independent predictors, however, due to the small sample size, it may not be possible.

Response: Thank you for pointing out this important limitation. We completely agree that the small and uneven sample size restricts the statistical power and limits the external validity and generalizability of our study findings.

In addition, only univariate analyses were performed, and adjusting for potential confounders such as age, disease extent, 5-ASA dose, AE severity, and corticosteroid use would have strengthened the conclusions. However, considering the limited number of patients enrolled in this retrospective single-center cohort, conducting a robust multivariable logistic regression analysis was not feasible.

To address this concern, we have clearly discussed these limitations in the Discussion section and have emphasized that future studies should validate our findings in larger, multicenter prospective cohorts, with adequate sample sizes, to allow for multivariable adjustment (Page 21–22, lines 396–416).

Comment 2: Why was 5-ASA reinducted in case of myelosuppression? It should be stated, as current ECCO guidelines advise against 5-ASA re-challenge after serious organ toxicity. Was the pancreatitis mild?

Response: Thank you for raising this important point. We agree that mesalamine reintroduction should be strictly avoided in patients with significant organ toxicity, such as myelosuppression or severe pancreatitis, in line with current ECCO guidelines.

In this study, no patients with serious organ toxicity received mesalamine retreatment. We believe that this misunderstanding may have arisen from the original wording of our definition of mesalamine intolerance.

To clarify this ethical consideration, we have revised the definition in the Materials and Methods section to explicitly state that retreatment was only performed in cases without significant organ damage (Page 6, lines 124–126).

Comment 3: Although both F-DLST and M-DLST showed statistically significant associations with mesalamine intolerance, their limited sensitivity and specificity substantially restrict their clinical utility.

The ROC-derived cut-offs yield apparently excellent AUC values; however, because they were optimised and evaluated on the same, very small dataset (n = 28, with only six tolerant cases), the results are likely over-optimistic and may not generalise. External validation in a larger cohort is essential before these thresholds can be recommended for clinical use.

Response: Thank you for this valuable comment. We agree that despite the significant associations of F-DLST and M-DLST with mesalamine intolerance, their limited sensitivity and specificity substantially restrict their current clinical utility.

We also acknowledge that the ROC-derived cutoff values and AUCs may appear overly optimistic because they were derived and evaluated within the same small cohort (n = 28), which included only six tolerant cases.

To address this, we have further discussed these limitations in the Discussion section and have emphasized that external validation using larger, multicenter prospective cohorts is essential before these cutoff values can be recommended for routine clinical practice (Page 22, lines 405–410).

Minor comments

Introduction

Comment 1: The phrase ‘Salazosulfapyridine (SASP), one of the first 5-ASAs’ is misleading. SASP is a sulfonamide-based prodrug that delivers 5-ASA after bacterial azo-reduction in the colon. Because of its sulfapyridine moiety, SASP differs chemically, pharmacokinetically, and in its adverse-event profile from the later, pure mesalazine formulations. Please rephrase it.

Response: Thank you for raising this point. We agree that the original phrasing may have been misleading. As suggested, we have revised the sentence to clarify that SASP is a sulfonamide-based prodrug that delivers 5-ASA to the colon via bacterial azo-reduction and that it differs chemically, pharmacokinetically, and in its adverse event profile from the later, pure mesalamine formulations (Page 3, lines 45–49).

Materials and methods

Comment 1: As Salazopyrin is not a classical 5-ASA and , I would not include it in the study as it can increase bias. Both its pharmacokinetics and immunologic toxicity differ substantially from those of single-agent 5-ASA preparations. However, as I can see it in the methods section, no patients received Salazopyrin among the observed 28 patients.

Response: Thank you for your comment. We agree that the inclusion of patients who received SASP could introduce bias because SASP has substantially different pharmacokinetics and immunologic toxicity from single-agent 5-ASA formulations.

To avoid this potential bias, we have explicitly stated in the Materials and Methods that patients who initiated oral SASP at diagnosis were excluded from the study (Page 5, line 99–100).

The revised manuscript with tracked changes, a clean version of the manuscript, and a response letter were uploaded separately.

We confirmed that our protocols do not require registration in protocols.io because they do not involve novel laboratory methods.

We hope that the revisions and clarifications satisfactorily address the concerns of the reviewers. We greatly appreciate the constructive feedback of the reviewers and your consideration of our revised manuscript. We hope that the revised manuscript will be acceptable for publication in PLOS ONE.

Sincerely,

Akira Madarame, MD

On behalf of all co-authors

Attachment

Submitted filename: Response_to_Reviewers_auresp_2.docx

pone.0334969.s003.docx^{(25.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334969.r005

Decision Letter 2

Mohammed Misbah Ul Haq

1 Sep 2025

Dear Dr. Madarame,

Please submit your revised manuscript by Oct 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comment:

Reviewer #3:

Reviewer #5:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Partly

Reviewer #5: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: No

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

Reviewer #5: Yes

**********

Reviewer #3: Comments to the Author

The authors has responded properly to the reviewer's comments, so I have no additional comments.

Reviewer #5: This manuscript investigates the diagnostic and predictive value of the drug-induced lymphocyte stimulation test (DLST) in patients with ulcerative colitis who experienced mesalamine intolerance. The topic is clinically relevant, and the authors provide novel insights by analyzing DLST results for multiple mesalamine formulations. The study is clearly written and addresses an important clinical problem. However, there are some methodological and interpretative issues that need further clarification.

Substantial concern is that sample size is too small.

Statistical limitations:The study is based on a small, single-center cohort (n=28), which limits statistical power. The cutoff values derived from ROC analysis and the odds ratios may be subject to overfitting. The authors should acknowledge this more explicitly and emphasize the need for validation in larger, multicenter prospective studies.

Clinical applicability of DLST:While the results suggest that DLST could be useful in predicting intolerance and retreatment outcomes, its clinical application remains unclear due to potential false positives and negatives. The discussion would be strengthened by presenting specific clinical scenarios (e.g., when DLST should be considered in practice, or how to act upon multiple positive results) and by proposing a preliminary decision-making algorithm.

Adjustment for confounding factors:The analysis relies only on univariate comparisons. Potential confounders, such as age, type of first 5-ASA, severity of adverse events, and corticosteroid use, may influence the results. Although the limited sample size may preclude multivariable analysis, the authors should discuss this limitation and outline how future research could address it.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #3: No

Reviewer #5: Yes: Osamu Handa

**********

PLoS One. 2025 Oct 30;20(10):e0334969. doi: 10.1371/journal.pone.0334969.r006

Author response to Decision Letter 3

3 Sep 2025

Manuscript ID: PONE-D-25-17000R2

Title: Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Dear Editors and Reviewers:

We sincerely thank the Academic Editor and Reviewers for their thoughtful and constructive comments, which have been very helpful in improving our manuscript. We have carefully revised the manuscript in accordance with the suggestions, and the specific responses to each comment are detailed below. Page and line numbers refer to the revised version of the manuscript.

Reviewer #3

Comment: The authors have responded properly to the reviewer's comments, so I have no additional comments.

Response:

We thank the reviewer for the positive evaluation and for acknowledging our previous revisions. We are grateful for the constructive feedback provided during the review process.

Reviewer #5

Comment (Statistical limitations):

The study is based on a small, single-center cohort (n=28), which limits statistical power. The cutoff values derived from ROC analysis and the odds ratios may be subject to overfitting. The authors should acknowledge this more explicitly and emphasize the need for validation in larger, multicenter prospective studies.

Response:

We thank the reviewer for raising this important point. We fully agree that the small, single-center cohort limits the statistical power and may increase the risk of overfitting for ROC-derived cutoff values and odds ratios. In the revised Discussion, we have explicitly acknowledged these limitations and emphasized the need for validation in larger, multicenter prospective studies to confirm the diagnostic performance and clinical applicability of DLST (page 22, lines 413–417).

Comment (Clinical applicability of DLST):

While the results suggest that DLST could be useful in predicting intolerance and retreatment outcomes, its clinical application remains unclear due to potential false positives and negatives. The discussion would be strengthened by presenting specific clinical scenarios (e.g., when DLST should be considered in practice, or how to act upon multiple positive results) and by proposing a preliminary decision-making algorithm.

Response:

We thank the reviewer for this constructive suggestion. In the revised Discussion, we have added specific descriptions of how DLST may be applied in clinical practice. Based on our findings, we suggest that DLST should be performed after the first onset of intolerance symptoms to oral 5-ASA and ideally include all available mesalamine formulations. We further describe a preliminary framework in which retreatment should be avoided when two or more DLSTs are positive, whereas cautious reintroduction may be considered in patients with negative or single-positive results (page 21, lines 388–396). We have also emphasized that this framework is preliminary and requires validation in larger, multicenter prospective studies before being implemented in routine clinical practice (page 22, lines 410–413).

Comment (Adjustment for confounding factors):

The analysis relies only on univariate comparisons. Potential confounders, such as age, type of first 5-ASA, severity of adverse events, and corticosteroid use, may influence the results. Although the limited sample size may preclude multivariable analysis, the authors should discuss this limitation and outline how future research could address it.

Response:

We thank the reviewer for highlighting this important issue. As suggested, we have added a statement in the Discussion acknowledging that our analysis was limited to univariate comparisons and did not adjust for potential confounders, including age, type of first 5-ASA, severity of adverse events, and corticosteroid use. We have also noted that, although multivariable analysis was not feasible due to the small sample size, future studies with larger cohorts will be necessary to incorporate these factors and clarify independent predictors of mesalamine intolerance and retreatment outcomes (page 21-22, lines 397–405).

We carefully reviewed the reviewer comments and confirm that no additional references were specifically recommended in this revision round. Accordingly, no new citations were added.

The revised manuscript with tracked changes, a clean version of the manuscript, and a response letter were uploaded separately.

We confirmed that our protocols do not require registration in protocols.io because they do not involve novel laboratory methods.

Sincerely,

Akira Madarame, MD

On behalf of all co-authors

Attachment

Submitted filename: Response_to_Reviewers_auresp_3.docx

pone.0334969.s004.docx^{(20.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334969.r007

Decision Letter 3

Mohammed Misbah Ul Haq

24 Sep 2025

Dear Dr. Madarame,

Please submit your revised manuscript by Nov 08 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Dr. Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #6: All comments have been addressed

Reviewer #7: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #6: (No Response)

Reviewer #7: No

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #6: Yes

Reviewer #7: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #6: Yes

Reviewer #7: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #6: Yes

Reviewer #7: Yes

**********

Reviewer #6: Thank you for your thoughtful revisions to the manuscript. Although this remains a study with a relatively small sample size, I appreciate that you have carefully addressed the points raised by the previous reviewers. I would kindly ask you to consider the following additional questions and clarifications, which may help to further strengthen the clarity of the manuscript.

Comment 1

According to the Methods, retreatment success was defined as the ability to continue oral mesalamine for more than 6 months. However, in the Results and Fig. 2, some patients classified into the intolerant group appear to have achieved ‘success’ with second- or third-line mesalamine therapy. The subsequent clinical course of these patients (e.g., whether they later discontinued mesalamine due to AEs) is not clearly described. Please clarify how such cases were classified into the final tolerant vs. intolerant groups.

Comment 2

In Results (lines 185) and Figure 2, the phrase “28 patients with AEs” is mentioned. Should this actually refer to the 28 patients who underwent DLST?

Comment 3

In the sentence “All 13 patients with positive F-DLSTs were mesalamine intolerant (p = 0.018, OR 2.444, 95% CI 1.479–4.039) (Table 3),”

it would be clearer to state “mesalamine intolerant group” rather than simply “mesalamine intolerant.”

Comment 4

There is an inconsistency between the study period reported in the Materials and Methods section and that in the Results section. Please revise accordingly.

Reviewer #7: This research paper examines 5-ASA intolerance, a topic that has gained attention in recent years. The content is intriguing. However, as Reviewer 5 noted, unfortunately, the number of patients examined in this study is small. Also, were there no patients among the 22 with mesalamine intolerance who used SASP? Unless the authors evaluated the efficacy of SASP, these patients cannot be considered actual cases of mesalamine intolerance. Please reconsider.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #6: No

Reviewer #7: No

**********

PLoS One. 2025 Oct 30;20(10):e0334969. doi: 10.1371/journal.pone.0334969.r008

Author response to Decision Letter 4

24 Sep 2025

Manuscript ID: PONE-D-25-17000R3

Title: Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Dear Editors and Reviewers:

Reviewer # 6, Comment 1:

Response:

We sincerely thank the reviewer for this helpful comment. To address the concern:

1. We revised the Results sentence for clarity. The original wording “Fourteen patients underwent retreatment with oral mesalamines after experiencing AEs in response to the first 5-ASA (Fig 2), including six patients who were tolerant to oral mesalamines and eight patients who were intolerant to oral mesalamines” has been replaced with “Fourteen patients underwent retreatment with oral mesalamines after AEs to the first 5-ASA (Fig 2), of whom six achieved retreatment success and were classified into the mesalamine-tolerant group, whereas eight experienced retreatment failure and were classified into the mesalamine-intolerant group.” (page 13, lines 245–248)

2. We added details of the clinical course for patients with successful retreatment: “Six patients who achieved successful retreatment maintained oral mesalamine therapy at a minimum dose of 2,000 mg/day for at least 6 months, and none discontinued treatment thereafter.” (page 14-15, lines 270–273)

3. To avoid ambiguity, we also added a statement in the Methods explicitly clarifying that patients with successful retreatment were classified into the mesalamine-tolerant group, whereas those with retreatment failure were classified into the mesalamine-intolerant group. (page 6, lines 129–131)

We believe these revisions clearly address the reviewer’s concern and eliminate any potential misunderstanding regarding the classification of patients.

Comment 2:

In Results (line 185) and Figure 2, the phrase “28 patients with AEs” is mentioned. Should this actually refer to the 28 patients who underwent DLST?

Response:

We thank the reviewer for this careful observation. The phrase “28 patients with AEs” was indeed misleading. What we intended to describe was the 28 patients who underwent DLST. We have revised the Results section (page 9, line 186) and the corresponding label in Figure 2 to read “Patients who underwent DLST” to avoid confusion.

Comment 3:

In the sentence “All 13 patients with positive F-DLSTs were mesalamine intolerant (p = 0.018, OR 2.444, 95% CI 1.479–4.039) (Table 3),” it would be clearer to state “mesalamine intolerant group” rather than simply “mesalamine intolerant.”

Response:

We appreciate the reviewer’s helpful suggestion. We agree that “mesalamine-intolerant group” is clearer and avoids potential ambiguity. We have revised the sentence in the Results section (page 12, line 219) to read: “All 13 patients with positive F-DLSTs were in the mesalamine-intolerant group (p = 0.018, OR 2.444, 95% CI 1.479–4.039) (Table 3).”

Comment 4:

There is an inconsistency between the study period reported in the Materials and Methods section and that in the Results section. Please revise accordingly.

Response:

We thank the reviewer for pointing out this inconsistency. We have corrected the discrepancy by unifying the study period throughout the manuscript. The study period is now consistently reported as between 2014 and 2021 in both the Materials and Methods and Results sections. (page 9, line 180)

Reviewer #7:

This research paper examines 5-ASA intolerance, a topic that has gained attention in recent years. The content is intriguing. However, as Reviewer 5 noted, unfortunately, the number of patients examined in this study is small. Also, were there no patients among the 22 with mesalamine intolerance who used SASP? Unless the authors evaluated the efficacy of SASP, these patients cannot be considered actual cases of mesalamine intolerance. Please reconsider.

Response:

We thank the reviewer for these constructive comments.

1. We fully acknowledge the limitation of the relatively small sample size. As noted by Reviewer 5, we have already addressed this point in the Limitations section, emphasizing the need for confirmation in larger, multicenter prospective cohorts.

2. With regard to SASP, we confirm that SASP retreatment was performed and evaluated in this study. In fact, SASP was included among the successful retreatment cases. Specifically, two patients who failed initial 5-ASA therapy subsequently tolerated SASP and maintained treatment without recurrence of adverse events. These patients were therefore classified into the mesalamine-tolerant group. Conversely, three patients experienced retreatment failure with SASP (one in the second-line therapy and two in the third-line therapy). We have revised the Results section (page 14, lines 255–259, 262–265) to make these details explicit, thereby clarifying that the efficacy of SASP was fully assessed in our cohort.

We believe these revisions adequately address the reviewer’s concern.

We carefully reviewed the reviewer comments and confirm that no additional references were specifically recommended in this revision round. Accordingly, no new citations were added.

The revised manuscript with tracked changes, a clean version of the manuscript, and a response letter were uploaded separately.

We confirmed that our protocols do not require registration in protocols.io because they do not involve novel laboratory methods.

Sincerely,

Akira Madarame, MD

On behalf of all co-authors

Attachment

Submitted filename: Response_to_Reviewers_auresp_4.docx

pone.0334969.s005.docx^{(22.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0334969.r009

Decision Letter 4

Mohammed Misbah Ul Haq

6 Oct 2025

Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

PONE-D-25-17000R4

Dear Dr. Madarame,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #8: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

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Reviewer #6: Thank you for your clarifications regarding the classification of mesalamine-tolerant and intolerant patients. However, in Fig. 2 the designation of “(–): N=6” is not clearly explained. According to the Methods, patients achieving retreatment success (≥6 months of mesalamine continuation without AEs) should be classified into the tolerant group. Could you please clarify whether the “(–): N=6” patients in Fig. 2 correspond to the mesalamine-tolerant group?

To avoid further confusion, please revise the figure or legend to explicitly indicate which subgroup in Fig. 2 constitutes the final mesalamine-tolerant group (n=6), and how these patients relate to the intermediate “success” branches shown at the second- and third-line retreatments. This will help ensure consistency between the Methods, the Results, and Fig. 2.

Reviewer #8: This paper is potentially interesting and worthy of eventual publication. Your revised paper is well written and illustrated because all comments are taking into account.

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Reviewer #6: No

Reviewer #8: No

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PLoS One. doi: 10.1371/journal.pone.0334969.r010

Acceptance letter

Mohammed Misbah Ul Haq

PONE-D-25-17000R4

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Submitted filename: Response to Reviewers.docx

pone.0334969.s001.docx^{(21.7KB, docx)}

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pone.0334969.s002.docx^{(14.7KB, docx)}

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pone.0334969.s003.docx^{(25.2KB, docx)}

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pone.0334969.s004.docx^{(20.3KB, docx)}

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pone.0334969.s005.docx^{(22.5KB, docx)}

Data Availability Statement

The minimal dataset required to replicate the study’s findings is fully contained within the paper.

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PERMALINK

Significance of the drug-induced lymphocyte stimulation test for various oral mesalamines in ulcerative colitis with mesalamine intolerance

Akira Madarame

Masakatsu Fukuzawa

Kumiko Uchida

Tadashi Ichimiya

Sakiko Naito

Yoshiya Yamauchi

Takashi Morise

Yasuyuki Kagawa

Takahiro Muramatsu

Takao Itoi

Roles

Abstract

Introduction

Materials and methods

Patients

Data collection and definitions

Study design and statistical analyses

Ethical considerations

Results

Patient characteristics

Fig 1. Flowchart of eligible patients.

Table 1. Clinical characteristics of patients with adverse events due to the first oral 5-ASA.

Comparison of the mesalamine-intolerant and tolerant groups

Table 2. Comparison of the clinical characteristics of patients in the mesalamine-tolerant and mesalamine-intolerant groups.

Relationship between DLST and mesalamine intolerance

Table 3. Comparison of patients who underwent DLST in the mesalamine-tolerant and mesalamine-intolerant groups.

Retreatment with oral mesalamine after AEs due to the first oral 5-ASA

Fig 2. Flowchart of the resumption of oral mesalamine therapy after adverse events caused by the first oral 5-aminosalicylate.

Table 4. Comparison of DLST and failed retreatment with oral mesalamine therapy.

Table 5. Demographic characteristics of patients who received retreatment with oral mesalamine after developing adverse events due to the first mesalamine therapy.

Fig 3. Relationship between the number of positive DLSTs and mesalamine retreatment failure.

Relationship between mesalamine intolerance and DLST cutoff values

Fig 4. Receiver operating characteristic curves for DLST.

Discussion

Conclusions

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Mohammed Misbah Ul Haq

Roles

Author response to Decision Letter 1

Decision Letter 1

Mohammed Misbah Ul Haq

Roles

Author response to Decision Letter 2

Decision Letter 2

Mohammed Misbah Ul Haq

Roles

Author response to Decision Letter 3

Decision Letter 3

Mohammed Misbah Ul Haq

Roles

Author response to Decision Letter 4

Decision Letter 4

Mohammed Misbah Ul Haq

Roles

Acceptance letter

Mohammed Misbah Ul Haq

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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