Helicobacter pylori eradication improves motor fluctuations in advanced Parkinson's disease patients: A prospective cohort study (HP-PD trial).

BACKGROUND: Helicobacter pylori (HP) is a bacterium associated with many gastrointestinal (GI) diseases and has shown a high prevalence in Parkinson's disease (PD). As HP-associated GI dysfunction could affect L-dopa (levodopa) absorption, HP eradication might improve the clinical response and decrease motor fluctuations.
METHODS: A prospective cohort study was conducted on the clinical symptoms of PD patients with motor fluctuations. The 13C-urea breath test was used to diagnose a current HP infection. All patients with HP infection received a 2-week regimen of triple therapy. The changes in the Unified Parkinson's Disease Rating Scale (UPDRS) motor score, L-dopa onset time, wearing-off symptoms, mean daily on-off time, GI symptom scores, and quality of life score were measured at baseline and at a 6-week follow-up.
RESULTS: A total of 163 PD patients were assessed, of whom 40 were enrolled. Fifty-five percent of the enrolled patients (22/40) had a current HP infection, whereas HP eradication was identified in 17 of 22 (77.3%) patients who received eradication therapy. Patients with HP eradication showed a significant decrease in daily 'off' time (4.0 vs. 4.7 h, p = 0.040) and an increase in daily 'on' time (11.8 vs. 10.9 h, p = 0.009). Total wearing-off score (4.4 vs. 6.0, p = 0.001) and the GI symptom score (8.1 vs. 12.8, p = 0.007) were significantly improved. There was no significant improvement in L-dopa onset time, UPDRS motor score, or quality of life score.
CONCLUSIONS: HP eradication leads to significant clinical improvement in the symptoms of PD. Eradication of HP not only increases the total daily 'on' time but also decreases wearing-off symptoms and improves GI symptoms.

Introduction

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders that commonly affect elderly people. Despite enormous advanced knowledge of PD, L-dopa (levodopa) remains the most effective treatment for PD. Almost all PD patients require varying doses of L-dopa to manage their motor symptoms and maintain an acceptable quality of life. However, chronic L-dopa use in PD is associated with motor complications, including motor fluctuations and L-dopa-induced dyskinesia. After 5 years of L-dopa exposure, more than half of PD patients may experience some degree of these problems that significantly affects their functionality and quality of life [1]. Although PD mainly affects the motor system, it has been well established that PD is also associated with several non-motor symptoms (NMS). Gastrointestinal (GI) dysfunction is one of the most problematic NMS in PD and may precede the appearance of motor symptoms by several years. Recent evidence suggested an integral role of the microbiota-gut-brain axis for the early pathogenesis and progression of PD [2-5]. Also, several studies have shown an increased risk of PD in populations with chronic constipation and inflammatory bowel disease [6-8].

Helicobacter pylori (HP) is a Gram-negative bacterium that characteristically causes sustained inflammation of the gastric mucosa, which is associated with many GI diseases such as peptic ulcer, gastroduodenitis and fatal GI diseases especially gastric cancer. A recent population-based study has shown an increased risk of PD in chronic HP infection [9]. Also, meta-analysis has shown a significantly higher prevalence of HP infection in PD patients; furthermore, HP-infected PD patients demonstrated poorer control of motor symptoms than non-infected patients [10]. As GI dysfunction and L-dopa bioavailability play an important role in the pathophysiological changes underlying motor fluctuations, the eradication of HP might positively influence clinical outcome in the treatment of PD. This study aimed to evaluate the therapeutic effects of HP eradication on motor fluctuations and clinical symptoms in patients with advanced PD.

Materials and methods

Study design

A prospective cohort study was conducted between January and December 2019 at the Movement Disorders Clinic, Thammasat University Hospital, Thailand. The study was designed as a single-arm, open-label trial to evaluate the therapeutic effects of HP eradication in PD patients with motor fluctuations. The estimated sample size was determined according to the previous studies [11-14] with a level of significance of 5%, the statistical power of 80%, and the effect size of 0.8. The calculated number of HP infected patients needed in this study was 15 patients. Given the prevalence of HP infection in the general Thai population of 45% and the possibility of a 15% drop out rate, a total of 40 advanced PD patients were recruited. Consecutive PD patients were screened by a movement disorder specialist. Clinical assessment took place at baseline and 4 weeks after the 2-week HP eradication regimen period. Outcome assessors were blinded to the HP results. The primary outcomes were measurable changes in daily on-off time, L-dopa onset and peak time that result from successful HP eradication. The secondary outcomes were changes in clinical symptoms and the quality of life scores. All participants were verbally explained regarding the study before signing the consent form. Written informed consent was obtained from all subjects prior to enrolment. The study protocol was approved by the local ethics committee of Thammasat University (MTU-EC-IM-6-179/61) and registered with the Thai Clinical Trials Registry (TCTR 20190222005).

Participants

Inclusion criteria for the study population were age over 18 years, diagnosis of idiopathic PD according to the UK Parkinson’s Disease Society Brain Bank Diagnostic Criteria (UKPDSBB), current presence of motor fluctuations and receiving treatment with L-dopa. Exclusion criteria were severe cognitive dysfunction or dementia, severe dysphagia, history of gastric lesions or prior gastric surgery, prior HP eradication therapy and allergic to amoxicillin, clarithromycin or omeprazole.

Clinical assessment

Baseline characteristic data, taken from face-to-face interviews and medical records, were collected by a neurologist. Activities of daily living (ADL), NMS, GI symptoms, quality of life, wearing-off symptoms and daily motor fluctuations were assessed by the Schwab and England Activities of Daily Living Scale (SE-ADL), Non-Motor Symptom Questionnaire (NMSQ) application [15], seven-item GI complaint score (GI score), Parkinson’s disease questionnaire-8 (PDQ-8), a Thai version of the 9-item Wearing-off Questionnaire (TWOQ-9) [16], and home PD diary, respectively. Disease duration and L-dopa equivalent dose (LED) were recorded. Motor impairment was examined by a neurologist and a movement disorder specialist during ‘off’ and ‘on’ periods with the Unified Parkinson’s Disease Rating Scale (UPDRS), the Modified Hoehn and Yahr Scale (H&Y) and the Time Up and Go (TUG) test. Patients were asked to withhold all dopaminergic medications and fasting overnight (≥ 8 h) for the morning ‘off’ assessment. Then the patients were given the regular morning dose of L-dopa. Both ‘onset time’ and ‘peak time’ of L-dopa effects were reported subjectively by the patients at the clinic. The ‘on’ motor assessments were done during the reported peak time or at 90 min after oral L-dopa intake, whichever comes earlier.

Evaluation of a current HP infection

All patients underwent the 13C-urea breath test (Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan) during their fasting, before taking L-dopa, and in the upright position. Patients must be free from antibiotics for at least 4 weeks and discontinue H2-receptor antagonist or proton pump inhibitors for at least 2 weeks before the HP assessment. The breath samples were collected using a special breath-collecting bag at baseline and 20 min after swallowing a 100-mg tablet of 13C-labelled urea. The breath samples were measured for isotope-labelled carbon dioxide ratio using infrared spectrophotometry. A cut-off delta value of ≥ 2.5% difference compared with pre- and post-dose breath samples is considered a positive result for current HP infection. The test has a reported sensitivity of 97.7% and a specificity of 98.4% [17].

HP eradication therapy and outcome assessment

All patients continued with their usual dose of dopaminergic medication. The HP positive patients additionally received standard triple therapy with amoxicillin (1 mg twice daily), clarithromycin (500 mg twice daily) and omeprazole (40 mg twice daily) for 2 weeks.

Patients were not allowed to adjust or change their medication regimen. All patients received reminder phone calls for the follow-up date, 4 weeks after the 2-week regimen HP treatment. The Clinical Global Impression Scale (CGI) was assessed. HP eradication was defined as a negative 13C-urea breath test at least 4 weeks after treatment.

Statistical analysis

Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Descriptive statistical values were presented as mean ± standard deviation (SD) and percentage values. The independent t-test, Mann-Whitney test or chi-squared test was used to assess differences in various parameters between HP positive and HP negative groups. The dependent sample t-test or Wilcoxon matched-pairs signed rank test was used to assess the different outcomes between pre- and post-HP eradication in the HP positive group. Correlation analysis and multiple linear regression analysis were used to assess the associated factors of HP infection. All tests were two-sided and a probability value of p < 0.05 was considered to be statistically significant.

Results

Baseline characteristics and comparison

A total of 163 PD patients were consecutively assessed for eligibility. Of these, 123 patients were excluded, and 40 patients were enrolled in the study (Fig 1). The baseline characteristics of the enrolled patients are shown in Table 1. All patients received L-dopa treatment, dopamine agonist (77.5%), catechol-O-methyltransferase (COMT) inhibitor (52.5%) and monoamine oxidase B (MAOB) inhibitor (10.0%). The mean LED was 740.70 ± 310.94 mg/day. Two patients were currently on deep brain stimulation (5.0%).

Fig 1

Flow diagram of patient selection process.

Table 1

General demographic data of the study population.

	Total (n = 40)	HP positive (n = 22)	HP negative (n = 18)	p
Male gender, n (%)	20 (50.0)	12 (60.0)	8 (40.0)	0.525
Age (years)	63.6 ± 9.6	60.2 ± 10.3	67.8 ± 6.8	0.010
Age at onset (years)	55.9 ± 9.6	52.8 ± 10.0	59.7 ± 8.0	0.023
Smoking, n (%)	2 (5.0)	2 (9.1)	0 (0.0)	0.176
Vegetable/fruit (servings/day)	2.3 ± 0.7	2.2 ± 0.8	2.5 ± 0.6	0.173
Disease duration (years)	7.9 ± 5.0	7.5 ± 4.4	8.4 ± 5.8	0.542
SE-ADL	80.0 ± 6.4	77.7 ± 4.3	82.8 ± 7.5	0.018
Hoehn & Yahr stage	2.2 ± 0.7	2.2 ± 0.6	2.2 ± 0.9	0.966
Total LED (mg/day)	740.7 ± 310.9	738.8 ± 289.3	743.1 ± 344.1	0.966
• Dopamine agonist, n (%)	31 (77.5)	19 (86.4)	12 (66.7)	0.144
• COMT inhibitor, n (%)	21 (52.5)	10 (45.5)	11 (61.1)	0.336
• MAO-B inhibitor, n (%)	4 (10.0)	2 (9.1)	2 (11.1)	0.839
• Anticholinergic use, n (%)	8 (20.0)	7 (31.8)	1 (5.6)	0.039
Total TWOQ-9 score	5.3 ± 1.8	5.7 ± 1.5	4.7 ± 2.0	0.092
• Tremor, n (%)	26 (65.0)	18 (81.8)	8 (44.0)	0.014
• Anxiety, n (%)	10 (25.0)	7 (31.9)	3 (16.7)	0.271
• Mood change, n (%)	11 (27.5)	8 (36.4)	3 (16.7)	0.165
• Slowness, n (%)	38 (95.0)	21 (95.5)	17 (94.4)	0.884
• Reduced dexterity, n (%)	33 (82.5)	20 (90.9)	13 (72.2)	0.122
• Stiffness, n (%)	26 (65.0)	15 (68.2)	11 (61.1)	0.641
• Slowness of thinking, n (%)	16 (40.0)	8 (36.4)	8 (44.4)	0.604
• Muscle cramping, n (%)	25 (62.5)	13 (59.1)	12 (66.7)	0.622
• Pain/aching, n (%)	25 (62.5)	16 (72.8)	9 (50.0)	0.140
Total GI symptom score	13.7 ± 7.4	12.4 ± 6.7	15.4 ± 8.1	0.205
Total NMSQ score	35.4 ± 17.1	33.8 ± 13.5	37.9 ± 20.9	0.425
PDQ-8 score	9.9 ± 5.8	10.4 ± 4.9	9.2 ± 6.8	0.526
‘Off’ UPDRS motor score	30.9 ± 12.2	31.7 ± 9.9	29.8 ± 14.8	0.653
‘On’ UPDRS motor score	14.7 ± 9.3	14.3 ± 7.5	15.1 ± 11.3	0.800
Time up and go (sec)	12.0 ± 7.4	11.9 ± 8.3	12.1 ± 6.3	0.942
L-Dopa onset time (min)	19.2 ± 9.5	22.1 ± 8.1	15.4 ± 10.0	0.025
L-Dopa peak time (min)	35.0 ± 9.5	35.4 ± 8.8	34.5 ± 10.5	0.785
Daily ‘on’ time (h)	11.4 ± 2.2	10.9 ± 1.9	12.0 ± 2.4	0.117
Daily ‘off’ time (h)	4.3 ± 1.9	4.6 ± 1.6	3.9 ± 2.2	0.211
Dyskinesia (h)	1.4 ± 2.1	1.3 ± 1.8	1.5 ± 2.3	0.738

HP: Helicobacter pylori; SE-ADL: Schwab and England Activities of Daily Living Scale; LED: Levodopa equivalent dose; COMT: Catechol-O-methyltransferase; MAO, monoamine oxidase; TWOQ-9: Thai version of the 9-item Wearing-off Questionnaire; GI: Gastrointestinal; NMSQ: Non-Motor Symptom Questionnaire; PDQ: Parkinson’s Disease Questionnaire; UPDRS: Unified Parkinson’s Disease Rating Scale; L-Dopa: Levodopa.

Statistically significant p-values are in bold.

Twenty-two patients (55.0%) had a current HP infection. The parameters at baseline between HP positive and HP negative groups are shown in Table 1. There was no statistically significant difference in the mean daily LED between groups. Patients with HP infection had statistically significantly younger age (60.2 vs. 67.8 years, p = 0.010) and age of onset (52.8 vs. 59.7 years, p = 0.023), poorer SE-ADL (77.7 vs. 82.8, p = 0.018), a higher rate of anticholinergic use (31.8 vs. 5.6%, p = 0.039), a higher rate of wearing-off tremor (81.8 vs. 44%, p = 0.014) and delayed L-dopa onset time (22.1 vs. 15.4 min, p = 0.025) than the non-HP-infected patients. Multiple linear regression analysis showed significant association between levodopa onset time (p = 0.014), SE-ADL scale (p = 0.038), and HP infection. The common GI symptoms in both groups were constipation (87.5%), bloating (60.0%) and abdominal pain (37.5%). There was no statistically significant difference in disease duration, dietary history, UPDRS motor scores, H&Y stage, TUG test, total TWOQ-9 scores, daily ‘on’ and ‘off’ times, PDQ-8 score, total NMSQ score or total GI symptom scores between groups.

Analysis of HP eradication effects

HP eradication was identified in 17 patients (77.3%). Patients with confirmed HP eradication showed statistically significantly decreased daily ‘off’ time (4.0 vs. 4.7 h, p = 0.040) and increased total daily ‘on’ time (11.8 vs. 10.9 h, p = 0.009) by approximately 54 min per day from baseline. The success of HP eradication therapy significantly reduced daily ‘off’ time and increased ‘on’ time by 0.7 ± 1.3 and 0.9 ± 1.2 h, respectively, compared with a 0.6 ± 0.4 h increase in ‘off’ time and a 1.6 ± 2.3 h decrease in ‘on’ time with eradication failure (HP eradication effect: −1.3 off-hours, p = 0.041, and +2.5 on-hours, p = 0.004) (Fig 2). In addition, there were significant improvements in total TWOQ-9 score (4.4 vs. 6.0, p = 0.001), TWOQ-9 tremor subscore (0.5 vs. 0.8, p = 0.009), TWOQ-9 mood change subscore (0.1 vs. 0.4, p = 0.029), total GI symptom score (8.1 vs. 12.8, p = 0.007), bloating subscore (1.1 vs. 2.9, p = 0.007) and dysphagia subscore (0.3 vs. 0.7, p = 0.029) following successful eradication (Table 2). Multiple testing correction by Bonferroni adjustment showed a statistically significant improvement only in total TWOQ-9 score (corrected p = 0.029) (S1 Table). Moreover, there were trends toward improvement but not statistically significant in L-dopa onset time, UPDRS motor score, total NMSQ score, and PDQ-8 score. By contrast, patients who failed eradication therapy did not demonstrate any benefit after treatment but showed significantly increased daily ‘off’ time (4.9 vs. 4.3 h, p = 0.033) (Table 3).

Fig 2

Mean change from baseline for motor fluctuations between successful and failed Helicobacter pylori (HP) eradication groups.

Table 2

Clinical symptoms before and after successful Helicobacter pylori (HP) eradication (n = 17).

	Before HP eradication	After HP eradication	p
Total TWOQ-9 score	6.0 ± 1.3	4.4 ± 1.7	0.001
• Tremor	0.8 ± 0.4	0.5 ± 0.5	0.009
• Anxiety	0.4 ± 0.5	0.1 ± 0.3	0.096
• Mood changes	0.4 ± 0.5	0.1 ± 0.2	0.029
• Slowness	0.9 ± 0.2	0.9 ± 0.2	1.000
• Reduced dexterity	0.9 ± 0.3	0.8 ± 0.4	0.163
• Stiffness	0.8 ± 0.4	0.6 ± 0.5	0.188
• Slowness of thinking	0.7 ± 0.5	0.5 ± 0.5	0.431
• Muscle cramping	0.8 ± 0.4	0.7 ± 0.5	0.431
• Pain/aching	0.8 ±0.4	0.7 ± 0.5	0.431
Total GI symptom score	12.8 ± 7.3	8.1 ± 4.5	0.007
• Heartburn	1.2 ± 2.8	0.6 ± 1.4	0.443
• Bloating	2.9 ± 3.2	1.1 ± 1.6	0.007
• Nausea	0.2 ± 0.5	0.2 ± 0.5	1.000
• Vomiting	0.1 ± 0.5	0.1 ± 0.5	1.000
• Abdominal pain	1.8 ± 2.6	0.9 ± 2.2	0.112
• Diarrhea	0.4 ± 1.1	0.0 ± 0.0	0.130
• Constipation	6.2 ± 3.5	5.2 ± 2.8	0.146
Total NMSQ score	33.4 ± 13.3	29.3 ± 14.1	0.258
• Dysphagia	0.7 ± 1.0	0.3 ± 0.6	0.029
PDQ-8 score	10.9 ± 5.3	8.6 ± 5.2	0.080
‘Off’ UPDRS motor score	29.9 ± 9.6	26.0 ± 10.0	0.104
‘On’ UPDRS motor score	12.7 ± 7.0	10.6 ± 5.0	0.185
Time up and go (sec)	11.8 ± 8.5	9.8 ±2.8	0.242
L-Dopa onset time (min)	21.2 ± 6.9	19.5 ± 9.6	0.468
L-Dopa peak time (min)	35.7 ± 7.9	34.9 ± 13.8	0.782
Daily ‘on’ time (h)	10.9 ± 1.9	11.8 ± 2.0	0.009
Daily ‘off’ time (h)	4.7 ± 1.5	4.0 ± 2.1	0.040
Dyskinesia (h)	1.1 ± 1.6	1.3 ± 1.8	0.524

TWOQ-9: Thai version of the 9-item Wearing-off Questionnaire; GI: Gastrointestinal; NMSQ: Non-Motor Symptoms Questionnaire; PDQ: Parkinson’s Disease Questionnaire; UPDRS: Unified Parkinson’s Disease Rating Scale; L-Dopa: Levodopa.

Statistically significant p-values are in bold.

Table 3

Clinical symptoms before and after receiving the eradication therapy in Helicobacter pylori (HP) eradication failure group (n = 5).

	Before HP eradication	After HP eradication	p
Total TWOQ-9 score	4.8 ± 1.6	4.2 ± 2.2	0.529
• Tremor	0.8 ± 0.4	0.4 ± 0.5	0.178
• Mood changes	0.2 ± 0.4	0.4 ± 0.5	0.374
Total GI symptom score	10.8 ± 4.2	11.4 ± 1.3	0.763
• Bloating	1.8 ± 2.2	2.6 ± 2.1	0.338
Total NMSQ score	30.0 ± 14.9	25.2 ± 16.0	0.549
PDQ-8 score	8.6 ± 3.1	9.4 ± 6.4	0.759
‘Off’ UPDRS motor score	33.0 ± 6.2	30.4 ± 8.2	0.610
‘On’ UPDRS motor score	17.2 ± 7.6	12.0 ± 5.0	0.127
Time up and go (sec)	9.1 ± 2.6	8.3 ± 1.0	0.478
L-Dopa onset time (min)	23.7 ± 12.4	22.0 ± 6.7	0.645
L-Dopa peak time (min)	33.6 ± 12.8	28.9 ± 7.3	0.248
Daily ‘on’ time (h)	10.9 ± 2.3	9.3 ± 2.3	0.191
Daily ‘off’ time (h)	4.3 ± 2.3	4.9 ± 2.3	0.033
Dyskinesia (h)	1.6 ± 2.8	1.8 ± 3.3	0.374

TWOQ-9: Thai version of the 9-item Wearing-off Questionnaire; GI: Gastrointestinal; NMSQ: Non-Motor Symptoms Questionnaire; PDQ: Parkinson’s Disease Questionnaire; UPDRS: Unified Parkinson’s Disease Rating Scale; L-Dopa: Levodopa.

Statistically significant p-values are in bold.

Compared with baseline status in HP negative group, the success of HP eradication therapy demonstrated a significant lower total GI symptom score (15.4 vs. 8.1, p = 0.003), heartburn subscore (2.4 vs. 0.6, p = 0.024), bloating subscore (3.3 vs. 1.1, p = 0.007), and dysphagia subscore (1.3 vs. 0.3, p = 0.008). There was no significant difference in motor-related scores, L-dopa onset time, and quality of life between groups (Table 4).

Table 4

Clinical symptoms between patients with Helicobacter pylori (HP) negative and successful HP eradication groups.

	HP negative (n = 18)	HP positive at baseline (n = 17)
		Pre-HP eradication	pa	Post-HP eradication	pa
Total TWOQ-9 score	4.7 ± 2.0	6.0 ± 1.3	0.028	4.4 ± 1.7	0.625
Total GI symptom score	15.4 ± 8.1	12.8 ± 7.3	0.335	8.1 ± 4.5	0.003
• Heartburn	2.4 ± 2.8	1.2 ± 2.8	0.211	0.6 ± 1.4	0.024
• Bloating	3.3 ± 2.7	2.9 ± 3.2	0.740	1.1 ± 1.6	0.007
• Nausea	0.6 ± 1.5	0.2 ± 0.5	0.267	0.2 ± 0.5	0.267
• Vomiting	0.1 ± 0.5	0.1 ± 0.5	0.968	0.1 ± 0.5	0.968
• Abdominal pain	1.6 ± 2.5	1.8 ± 2.6	0.760	0.9 ± 2.2	0.404
• Diarrhea	0.5 ± 1.2	0.4 ± 1.1	0.819	0.0 ± 0.0	0.096
• Constipation	6.9 ± 3.5	6.2 ± 3.5	0.516	5.2 ± 2.8	0.115
Total NMSQ score	37.9 ± 20.9	33.4 ± 13.3	0.544	29.3 ± 14.1	0.173
• Dysphagia	1.3 ± 1.3	0.7 ± 1.0	0.124	0.3 ± 0.6	0.008
PDQ-8 score	9.2 ± 6.8	10.9 ± 5.3	0.409	8.6 ± 5.2	0.759
‘Off’ UPDRS motor score	29.8 ± 14.8	29.9 ± 9.6	0.740	26.0 ± 10.0	0.388
‘On’ UPDRS motor score	15.1 ± 11.3	12.7 ± 7.0	0.618	10.6 ± 5.0	0.146
Time up and go (sec)	12.1 ± 6.3	11.8 ± 8.5	0.817	9.8 ±2.8	0.289
L-Dopa onset time (min)	15.4 ± 10.0	21.2 ± 6.9	0.041	19.5 ± 9.6	0.229
L-Dopa peak time (min)	34.5 ± 10.5	35.7 ± 7.9	0.669	34.9 ± 13.8	0.979
Daily ‘on’ time (h)	12.0 ± 2.4	10.9 ± 1.9	0.140	11.8 ± 2.0	0.781
Daily ‘off’ time (h)	3.9 ± 2.2	4.7 ± 1.5	0.181	4.0 ± 2.1	0.819
Dyskinesia (h)	1.5 ± 2.3	1.1 ± 1.6	0.630	1.3 ± 1.8	0.802

a Compared to HP negative group.

TWOQ-9: Thai version of the 9-item Wearing-off Questionnaire; GI: Gastrointestinal; NMSQ: Non-Motor Symptoms Questionnaire; PDQ: Parkinson’s Disease Questionnaire; UPDRS: Unified Parkinson’s Disease Rating Scale; L-Dopa: Levodopa.

Statistically significant p-values are in bold.

In terms of side-effects related to HP eradication therapy, only one patient in the treatment failure group reported significant side effects (stomach pain, nausea and vomiting) to the treatment. Patients who failed eradication therapy were all male and had a significantly lower intake of vegetables and fruit (1.4 vs. 2.4 servings/day, p < 0.01) as well as a higher rate of smoking (40.0 vs. 0.0%, p < 0.01) compared with the HP eradication group. There was no statistically significant difference in age, age at onset, disease duration, SE-ADL scale, H&Y stage, UPDRS motor score, LED and antiparkinsonian medications between groups (Table 5). Patients with successful HP eradication reported significant clinical benefit on the CGI scale: 60% compared with 20% in the failure group.

Table 5

Demographic and clinical baseline status between Helicobacter pylori (HP) positive patients who succeeded and failed the eradication therapy.

	Successful eradication (n = 17)	Failure eradication (n = 5)	P
Male gender, n (%)	7 (41.2)	5 (100.0)	0.020
Age (years)	61.1 ± 10.6	57.4 ± 9.8	0.495
Age at onset (years)	53.3 ± 10.3	50.4 ± 9.5	0.544
Smoking, n (%)	0 (0.0)	2 (40.0)	0.006
Vegetable/fruit (servings/day)	2.4 ± 0.6	1.4 ± 0.9	0.009
Disease duration (years)	7.6 ± 4.2	7.0 ± 5.5	0.834
SE-ADL	77.1 ± 4.7	80 ± 0.0	0.184
Hoehn & Yahr stage	2.2 ± 0.6	2.2 ± 0.3	0.976
‘Off’ UPDRS motor score	31.3 ± 10.9	33.0 ± 6.2	0.664
‘On’ UPDRS motor score	13.5 ± 7.5	17.2 ± 7.6	0.370
Total LED (mg/day)	774.3 ± 312.7	617.9 ± 156.9	0.152
• Dopamine agonist, n (%)	14 (82.4)	5 (100.0)	0.312
• COMT inhibitor, n (%)	9 (52.9)	1 (20.0)	0.193
• MAO-B inhibitor, n (%)	1 (5.9)	1 (20.0)	0.334
• Anticholinergic use, n (%)	6 (35.3)	1 (20.0)	0.519

SE-ADL: Schwab and England Activities of Daily Living Scale; UPDRS: Unified Parkinson’s Disease Rating Scale; LED: Levodopa equivalent dose; COMT: catechol-O-methyltransferase; MAO: Monoamine oxidase.

Statistically significant p-values are in bold.

Discussion

The reported prevalence of HP infection in PD patients varied between 32% and 70% in different studies, depended on age group, gender, geographical origin, socioeconomic status of the studied population and methods for diagnosis [18]. This study found HP infection in 55% of Thai patients with advanced PD, which is slightly higher than finding prevalence rates of 40–50% among the Thai general population [12]. However, the prevalence in our study is much higher in the subgroup of patients aged 40–60 years than the age-matched general Thai population (78.6% vs. 48.1%) [19]. In contrast to a study by Tan et al. [20], our study demonstrated a statistically significantly younger age and age of onset of the HP positive group than the HP negative group. This could be due to the difference in study population, with our study mainly focusing on patients with advanced PD experiencing motor fluctuations. Also, young PD patients in our study had a significantly higher rate of anticholinergic use, which might be associated with a reduction in volume and acidity of gastric secretion as well as GI motility, resulting in an alteration of the normal flora that could contribute to bacterial colonization, overgrowth, and HP infection.

In terms of GI symptoms, our study did not demonstrate any difference between HP positive and HP negative groups. However, HP eradication significantly alleviated the symptoms of bloating and dysphagia in HP-infected PD patients. This suggested that GI symptoms in PD are not solely the effect of HP infection, but the HP infection could aggravate and induce chronic inflammation in the gastroduodenal mucosa, causing several GI symptoms. Dysphagia typically emerges in PD patients with advanced motor symptoms, and often related to gastroesophageal reflux disease. Eradication of HP in our study did not significantly improve motor scores or reflux symptoms. However, HP positive patients demonstrated significant improvement of dysphagia subscore after eradication therapy. Also, the post-eradication total GI symptoms, bloating, heartburn, and dysphagia scores were significantly less than baseline scores of HP negative patients. This might suggest additional effects of eradication therapy, especially from a gastric acid-lowering agent on gastroesophageal reflux disease that might contribute to dysphagia in PD patients. It should be noted that patients with severe dysphagia were excluded from the study. The significant improvement of dysphagia score in our study was minimal and might not significantly be observed in clinical practice. A multidisciplinary approach and identification of modifiable factors and causes are keys to the success of GI management. Although 10–14 days of triple therapy is recommended as a first-line HP eradication method in Thailand, treatment failure might be observed in approximately 20% [21]. In our study, the HP eradication rate was 77.3%, which is slightly lower than that of previous reports in Thailand [21,22]. This might reflect the increasing rate of antimicrobial resistance of the microorganism, therefore the preferred eradication regimen should vary by region depending on the known or anticipated pattern of antimicrobial resistance [21]. Interestingly, patients who failed the eradication therapy had a significantly lower intake of fruit and vegetables per day and a higher rate of smoking. This might support the interaction between environmental components and the outcome of HP infection therapy.

Regarding motor symptoms, patients with HP infection had longer mean L-dopa onset time and a higher frequency of TWOQ-9 tremor compared to those without infection, which could suggest an association between HP infection and L-dopa bioavailability. Although the L-dopa onset time was not changed after HP eradication, patients with successful HP eradication demonstrated a reduction in total TWOQ-9 score and daily ‘off’ time, as well as an increase in total daily ‘on’ time which consistent with the previous findings [11-14], but contrasts with a post hoc analysis of a recent randomized controlled trial study using a motor fluctuation score detected by wearable sensor [23]. Discrepancies with this report might be explained by the difference of the study population which mainly focus on stable PD without motor fluctuations. There were only 7 PD patients with motor fluctuations in the treatment group evaluated in this post hoc analysis [23]. In our study, HP eradication improved ‘on’ time by approximately 54 min per day which similar to a finding in the previous study of 56 min per day at 6 weeks [14], and comparable to adjunct therapy with 1 mg rasagiline in PD patients with mild motor fluctuations [24]. The prolonged “on” duration without changing in L-dopa onset and peak times in our study were consistent with the previous pharmacokinetic study that demonstrated a significantly higher of the area under plasma L-dopa concentration-time curve without a significant changing in time to maximum concentration after the HP eradication [11]. Therefore, the definite process by which HP may affect L-dopa absorption is still unknown. It was hypothesized that gastroduodenitis induced by HP infection could delay the gastric emptying time, causing delayed L-dopa delivery into the duodenum and impaired L-dopa active transport at the site of L-dopa absorption [11,14,25]. Moreover, HP might indirectly interrupt L-dopa absorption by producing urease, an enzyme that hydrolyses urea to ammonia and carbon dioxide, thus raising the gastric pH and possibly affecting the pH-sensitive L-dopa solubility [26]. Furthermore, HP might play a part in the biosynthesis of neurotoxins that directly affect dopaminergic neurons [27]. Eradication of HP should normalize the gastric pH and ameliorate L-dopa absorption, resulting in improvement of the clinical response [11,14]. Although our study did not demonstrate a significant benefit of HP eradication on L-dopa onset time, there were trends toward the improvement of L-dopa onset time as suggested in previous studies [12-14]. Similar to a recent randomized-control trial study [23], our study did not demonstrate a significant benefit of HP eradication on UPDRS motor score, gait speed, and NMSQ score. These findings suggested that HP eradication should not be used as a symptomatic adjunct therapy in stable PD but might be considered as a treatment for motor fluctuations.

Strengths of this study were the study design that focuses on PD patients with motor fluctuations and the therapeutic effects of HP eradication on various GI symptoms. The outcome assessors were blinded to the patients’ HP status both at baseline and after the eradication treatment. Also, the participants in our study had a high adherence with prescribed treatment. There were no dropouts during the 6-week follow-up.

We acknowledge that our present study has some limitations. First, our study was based on one center and the number of participants was limited. Even though the study population was adequate to estimate the effect of HP eradication on motor fluctuation, it might be too small to identify the definite prevalence and clinical symptoms of HP infection in PD patients. Also, there were multiple comparisons in the statistical analysis that might cause statistical significance occurred by chance. Second, our study was an open-label, single-arm, 6-week clinical trial. Thus, the benefit of HP eradication might contribute to the placebo effect, however this was not demonstrated in the treatment failure group. A multicenter, prospective, randomized controlled study with a larger sample size and long-term follow-up should be considered in the future. Third, some data such as L-dopa onset and peak and daily ‘on’ and ‘off’ times were based on subjective evaluation. An objective motor evaluation with home-based wearable measurements should be considered. Lastly, the benefits seen in our short-term study may not translate into improvements in the longer term. Also, variations in lifestyle factors such as dietary habits, the compositional changes of gut microbiota, and other associated factors might affect the disease progression or interfere with L-dopa absorption.

Conclusions

Successful HP eradication therapy leads to significant clinical improvements in the symptoms of PD patients with motor fluctuations. Eradication of HP not only increases the total daily ‘on’ time but also decreases wearing-off symptoms and improves GI symptoms. HP eradication was well tolerated and should be considered as an additional treatment for motor fluctuations in PD.

TREND statement checklist.

(PDF)

Click here for additional data file.

Clinical symptoms before and after successful Helicobacter pylori (HP) eradication with corrected p-values.

(DOCX)

Click here for additional data file.

Study protocol (Thai).

(PDF)

Click here for additional data file.

Study protocol (English).

(PDF)

Click here for additional data file.

30 Dec 2020

PONE-D-20-33347

Helicobacter pylori eradication improves motor fluctuations in advanced Parkinson's disease patients: A prospective cohort study (HP-PD trial)

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Reviewer #2: Yes

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Reviewer #1: This is an interesting and important study. My comments are as follows:

1. The authors reported the changes of UPDRS motor scores and GI symptoms scores after H. pylori eradication (before and after). It is suggested that the authors may add a table to compare patients receiving eradication therapy versus those not receiving eradication therapy (HP negative group).

2. The case number of patients receiving eradication therapy is small (N=17), whereas the authors conducted multiple comparisons in the statistical analysis. Multiple testing correction might be needed or at least should be addressed in the discussion.

3. Whether the improvement of UPDRS score is directly related to the disappearance of H. pylori or alterations in gut microbiota may be addressed in the discussion section.

Reviewer #2: The article describes clearly about the effect of Helicobacter pylori (HP) eradication on the motor and non-motor performance in Parkinson's disease (PD). The authors concluded that HP eradication may increase "on" time and decrease "off" time and also significantly improved the end-of-dose score and GI symptoms including bloating and dysphagia. The article is well written with useful information. However, there are several questions needed to be clarified.

1.

Is there any difference of all the profiles in patients who failed the eradication therapy? Because PD patients had high placebo effects when receiving the treatment, the data may help clarify the effects of eradication therapy.

2.

Please provide the difference of LED and the percentage of relevant medications of PD patients with successful HP eradication with and without motor fluctuations. Because motor fluctuations may be improved by the adjustment of antiparkinsonian medications, the data may elucidate the effects of the eradication therapy.

3.

The International Movement Disorder Society has verified wearing-off questionnaires as WOQ-19 and WOQ-9, which are frequently used. The end-of-dose questionnaire in the article seemed to be similar to the questions of WOQ-9 or WO1-19. Could the authors explain the reasons why using the end-of-dose questionnaire? Please provide the all the profiles of questionnaires and the reference of the questionnaire.

4.

It’s very interesting and plausible to know the improvement of bloating symptoms in PD patients receiving eradication therapy. The authors mentioned there was no significant difference of motor scores and NMSQ scores, could the authors provide the evidence and explain the possible mechanism of the improvement of dysphagia symptoms?

**********

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20 Jan 2021

I appreciated the constructive feedback of the editor and reviewers. I have addressed each of their concerns as outlined below.

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Response: Thank you for your suggestion. We have corrected the manuscript to meets PLOS ONE’s style requirements, including file naming and affiliations formatting.

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Response: We appreciate your comment. We have added this information in the methods section of the manuscript and ethics statement in the online submission information as the following paragraph.

“All participants were verbally explained regarding the study before signing the consent form. Written informed consent was obtained from all subjects prior to enrolment.”

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"We thank the Digestive Diseases Research Centre (DRC) at Thammasat University

Hospital and also Thai Otsuka Pharmaceutical Co., Ltd, who provided the infrared

spectrophotometer for the study."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

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"This study was supported by a grant from

Thammasat University Research Fund (TU Research Scholar, Contract No. 2/4/2562) and Thai Parkinson’s Disease and Movement Disorder Society (Thai-PDMDS). "

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Response: Thank you for your suggestion. We have updated and removed funding-related text in the acknowledgment, funding statement, and competing interests as follows:

Acknowledgements

We thank the Digestive Diseases Research Centre (DRC) at Thammasat University Hospital for their assistance with the UBT tests. We would also like to show our gratitude to the patients and their families who took part and participated in the study.

Funding Statement

This study was supported by a grant from Thammasat University Research Fund (TU Research Scholar, Contract No. 2/4/2562) and Thai Parkinson’s Disease and Movement Disorder Society (Thai-PDMDS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests

The 13C-urea breath test and infrared spectrophotometer used in the study was provided by Thai Otsuka Pharmaceutical Co., Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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Reviewers' comments:

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: No

Reviewer #2: Yes

Response: Thank you for your suggestion. We have updated tables and data underlying the findings in the manuscript as suggestion.

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Reviewer #2: Yes

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Reviewer #1: This is an interesting and important study. My comments are as follows:

1. The authors reported the changes of UPDRS motor scores and GI symptoms scores after H. pylori eradication (before and after). It is suggested that the authors may add a table to compare patients receiving eradication therapy versus those not receiving eradication therapy (HP negative group).

Response: We appreciate your important suggestion. We have compared general demographic data of the study population between HP positive (all have received eradication therapy) and HP negative group in Table 1. Also, we compared the successful HP eradication group and HP negative group's clinical symptoms in Table 4 as your suggestion.

2. The case number of patients receiving eradication therapy is small (N=17), whereas the authors conducted multiple comparisons in the statistical analysis. Multiple testing correction might be needed or at least should be addressed in the discussion.

Response: We appreciate your important comment, and we are concerned about this issue. We have performed multiple testing correction by Bonferroni adjustment in our statistic data and revealed the statistically significant difference in total TWOQ-9 score before and after eradication therapy. We have mentioned this issue in the results section and also addressed this limitation in the discussion as follows:

Results

“Multiple testing correction by Bonferroni adjustment showed a statistically significant improvement in total TWOQ-9 score.”

Discussion section

“We acknowledge that our present study has some limitations. First, our study was based on one center and the number of participants was limited.……. Also, there were multiple comparisons in the statistical analysis that might cause statistical significance occurred by chance.”

3. Whether the improvement of UPDRS score is directly related to the disappearance of H. pylori or alterations in gut microbiota may be addressed in the discussion section.

Response: We appreciate your important comment. There is emerging evidence to support the hypothesis that gut microbiota alteration may be related to the pathogenesis, clinical symptoms, and PD progression. The compositional changes of microbiota may occur after antibiotic usage, smoking, or dietary modifications. In our study, the alterations in microbiota may be occurred after receiving the HP eradiation therapy and might relate to the clinical improvement or L-dopa absorption. Since our study did not aim or provide the baseline type and number of gut microbiota, we could not answer this question. We have addressed this issue in our limitation in the discussion as follows:

“Lastly, benefits seen in our short-term study may not translate into improvements in the longer term, due to variations in lifestyle factors such as dietary habits, the compositional changes of gut microbiota, and other associated factors that might effect on the disease progression or interfere with L-dopa absorption.”

Reviewer #2: The article describes clearly about the effect of Helicobacter pylori (HP) eradication on the motor and non-motor performance in Parkinson's disease (PD). The authors concluded that HP eradication may increase "on" time and decrease "off" time and also significantly improved the end-of-dose score and GI symptoms including bloating and dysphagia. The article is well written with useful information. However, there are several questions needed to be clarified.

1. Is there any difference of all the profiles in patients who failed the eradication therapy? Because PD patients had high placebo effects when receiving the treatment, the data may help clarify the effects of eradication therapy.

Response: We appreciate your comment. We have added the clinical profiles of patients who fail to eradicate therapy in table 3 as your suggestion.

Besides, we also added the demographic profiles of patients who fail the eradication therapy in Table 5. Patients who failed the eradication therapy were all male and had a significantly lower intake of vegetables and fruit (1.4 vs. 2.4 servings/day, p < 0.01) as well as a higher rate of smoking (40.0 vs. 0.0%, p < 0.01) compared with the HP eradication group. There was no statistically significant difference in age, age at onset, disease duration, SE-ADL scale, H&Y stage, UPDRS motor score, LED and antiparkinsonian medications between groups (Table 5).

2. Please provide the difference of LED and the percentage of relevant medications of PD patients with successful HP eradication with and without motor fluctuations. Because motor fluctuations may be improved by the adjustment of antiparkinsonian medications, the data may elucidate the effects of the eradication therapy.

Response: Thank you for your comment. All patients in the study had motor fluctuations. All patients who received the HP eradication therapy continued their anti-PD medications and were not allowed to adjust or change their PD medication regimen (mentioned in methods: the HP eradication therapy and outcome assessment section). Therefore, there is no change in LED and the percentage of PD medication before and after receiving HP eradication therapy. We believed that the outcomes in our study were mainly the effects of eradication therapy. However, there is no control of the variations in lifestyle factors such as dietary habits or non-parkinsonian medications that might interfere with L-dopa absorption (mentioned in the discussion).

3. The International Movement Disorder Society has verified wearing-off questionnaires as WOQ-19 and WOQ-9, which are frequently used. The end-of-dose questionnaire in the article seemed to be similar to the questions of WOQ-9 or WO1-19. Could the authors explain the reasons why using the end-of-dose questionnaire? Please provide the all the profiles of questionnaires and the reference of the questionnaire.

Response: We appreciate your comment and concern. The end-of-dose (EOD) questionnaire that we used in our article/center is similar to the questions of WOQ-9 in Thai language. The questionnaire was developed by Thai Parkinson’s Disease and Movement Disorder Society (Thai PDMDS) and was published in Thai clinical practice guideline for diagnosis and management of PD in 2012. For clarification and understanding, we had edited the name of the questionnaire in our article from “the end-of-dose questionnaire” to "Thai version of the 9-item Wearing-off Questionnaire (TWOQ-9)". We also provided a questionnaire in the supporting file (S1 Study Protocol Thai), data finding in Table 1 and 2, and the article's reference.

4. It’s very interesting and plausible to know the improvement of bloating symptoms in PD patients receiving eradication therapy. The authors mentioned there was no significant difference of motor scores and NMSQ scores, could the authors provide the evidence and explain the possible mechanism of the improvement of dysphagia symptoms?

Response: Thank you very much for your comment. We have added the possible mechanism in the discussion section as follows:

“Dysphagia typically emerges in PD patients with advanced motor symptoms, and often related to gastroesophageal reflux disease. Eradication of HP in our study did not significantly improve motor scores or reflux symptoms. However, HP positive patients who received eradication treatment demonstrated significant improvement of dysphagia subscore. Also, the post-eradication total GI symptoms, bloating, heartburn, and dysphagia subscores were significantly less than baseline scores of HP negative patients. This might suggest additional effects of eradication therapy, especially from a gastric acid-lowering agent on gastroesophageal reflux disease that might contribute to dysphagia in PD patients. It should be noted that patients with severe dysphagia were excluded from the study. The significant improvement of dysphagia score in our study was minimal and might not significantly be observed in clinical practice.”

6. PLOS authors have the option to publish the peer review history of their article (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 #1: No

Reviewer #2: No

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Response: Thank you very much for your suggestion. We have edited figure files using PACE digital diagnostic tool as your suggestion.

Best regards,

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Mar 2021

PONE-D-20-33347R1

Helicobacter pylori eradication improves motor fluctuations in advanced Parkinson's disease patients: A prospective cohort study (HP-PD trial)

PLOS ONE

Dear Dr. Lolekha,

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Wan-Long Chuang, M.D., Ph.D.

Academic Editor

PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting and important study. The authors have responded to the comments from the reviewer.

Reviewer #2: The great efforts of the authors to answer the question are appreciated. I think their work may help the clinicians and scientists to uncover the complex mechanism between HP and PD.

Reviewer #3: Abstract: There are two percentages mentioned, and it isn't quite clear how these are calculated - 22 patients (55%) and 17 patients (77.3%) - the higher number has the lower percentage, so, to be correct the denominator must be different, but it isn't clear what it is from this text. Could you please clarify, perhaps by stating what the denominator was (22/40 and then 17/22?)?

Study design - pg 3, line 74 - what was the effect size or mean and standard deviation of change in daily on-off time, L-dopa onset or peak time used in the power calculation? What was the difference in groups the study aimed to test? or was it only in those patients who eradicated HP that were of interest? Why were the patients who were HP negative at baseline included? How were the numbers reduced from 163 to 40?

It is unclear how the various groups were recruited from the text, but it is clearer once Figure 1 is examined - could the text be cleared up?

pg 9, line 190 - mention multiple testing correction method with statistical methods (as "p<0.05 was considered to be statistically significant") on page 6 isn't the full story. Include the Bonferroni corrected p-values where they were calculated.

Table 4: It needs to be clear that this is baseline HP negative results and after treatment HP eradication results. For the statistically significant results - it may have been that the results were just different at baseline between these two groups of patients - would it have been of interest to see if there was a difference in the later time point data for the two groups, adjusted for the baseline variables?

Could be 4 columns of results be presented? Pre and post treatment for those who were HP negative at baseline and those who ended up as HP negative after treatment?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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25 Mar 2021

Reviewer #3:

1. Abstract: There are two percentages mentioned, and it isn't quite clear how these are calculated - 22 patients (55%) and 17 patients (77.3%) - the higher number has the lower percentage, so, to be correct the denominator must be different, but it isn't clear what it is from this text. Could you please clarify, perhaps by stating what the denominator was (22/40 and then 17/22?)?

Response: We appreciate your comment. For clarification, we have edited the abstract as follow:

“A total of 163 PD patients were assessed, of whom 40 were enrolled. Fifty-five percent of the enrolled patients (22/40) had a current HP infection, whereas HP eradication was identified in 17 of 22 (77.3 %) patients who received eradication therapy.”

2. Study design - pg 3, line 74 - what was the effect size or mean and standard deviation of change in daily on-off time, L-dopa onset or peak time used in the power calculation? What was the difference in groups the study aimed to test? or was it only in those patients who eradicated HP that were of interest? Why were the patients who were HP negative at baseline included?

How were the numbers reduced from 163 to 40? It is unclear how the various groups were recruited from the text, but it is clearer once Figure 1 is examined - could the text be cleared up?

Response: We appreciate your important comments, and we have addressed each comment as follows:

- What was the effect size or mean and standard deviation of change in daily on-off time, L-dopa onset or peak time used in the power calculation?

The effect size we used in our study was set at 0.8. From previous studies, the mean change in L-dopa onset time and total daily on time after eradication therapy were approximately 13 and 106 minutes, respectively. There was no report of the standard deviation of the mean differences. We have revised the paragraph as below:

“The estimated sample size was determined according to the previous studies [11-14] with a level of significance of 5%, the statistical power of 80%, and the effect size of 0.8. The calculated number of HP infected patients needed in this study was 15 patients. Given the prevalence of HP infection in general Thai population of 45% and the possibility of a 15% drop out rate, a total of 40 advanced PD patients were recruited.”

- What was the difference in groups the study aimed to test? or was it only in those patients who eradicated HP that were of interest?

The study aimed to compare the clinical effects between before and after receiving HP eradication therapy in advanced PD, so it was mainly focused on PD patients with current HP infection.

- Why were the patients who were HP negative at baseline included?

To identify current HP infected patients, we needed to screen the HP status in all advanced PD. We included the HP negative patients in order to identify risk factors or clinical effects that might be associated with HP infection in PD patients. As shown in Table 1, the young age and history of anticholinergic use were risk factors for HP infection. Also, the HP infected patients demonstrated significant delayed L-dopa onset time and tremor during the off period than the non-infected group. These results might support the possible benefit of HP eradication therapy in the later part of our study.

Moreover, there was no significant difference in motor symptoms (UPDRS motor score, daily on-off time, quality of life score) between HP negative patients at baseline and the successful HP eradication therapy group (table 4). These results might suggest that HP eradication is only beneficial in the treatment of motor fluctuations by increased daily on time but not motor symptoms or affects disease progression.

- How were the numbers reduced from 163 to 40? It is unclear how the various groups were recruited from the text, but it is clearer once Figure 1 is examined - could the text be cleared up?

Thank you for your suggestion, we have revised the recruitment process as follow:

“A total of 163 PD patients were consecutively assessed for eligibility. Of these, 123 patients were excluded, and 40 patients were enrolled in the study (Fig 1).”

3. pg 9, line 190 - mention multiple testing correction method with statistical methods (as "p<0.05 was considered to be statistically significant") on page 6 isn't the full story. Include the Bonferroni corrected p-values where they were calculated.

Response: Thank you very much for your comment. We did the corrected p-values according to the reviewer’s suggestion in the revised manuscript, and we found that only the total TWOQ-9 score was statistically significant with the corrected p = 0.029. We have added the corrected p-values as the following paragraph and provide data of corrected p-values in the supporting table (S1 Table).

“Multiple testing correction by Bonferroni adjustment showed a statistically significant improvement only in total TWOQ- 9 score (corrected p = 0.029) (S1 Table).”

4. Table 4: It needs to be clear that this is baseline HP negative results and after treatment HP eradication results. For the statistically significant results - it may have been that the results were just different at baseline between these two groups of patients - would it have been of interest to see if there was a difference in the later time point data for the two groups, adjusted for the baseline variables? Could be 4 columns of results be presented? Pre and post treatment for those who were HP negative at baseline and those who ended up as HP negative after treatment?

Response: Thank you very much for your suggestion. We have revised Table 4 as below. Patients with HP negative were not reassessed at the 6-week follow-up, so we compared pre-post HP eradication with the HP negative at baseline. From table 4, a significant improvement of the GI symptom scores was observed only in the post-HP eradication. Also, the TWOQ-9 score and L-dopa onset time after the eradication were comparable to the HP negative at baseline.

Submitted filename: Response to Reviewers3.docx

Click here for additional data file.

19 Apr 2021

Helicobacter pylori eradication improves motor fluctuations in advanced Parkinson's disease patients: A prospective cohort study (HP-PD trial)

PONE-D-20-33347R2

Dear Dr. Lolekha,

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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Kind regards,

Wan-Long Chuang, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: (No Response)

**********

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

Reviewer #3: (No Response)

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: (No Response)

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: (No Response)

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

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Reviewer #3: No

26 Apr 2021

PONE-D-20-33347R2

Helicobacter pylori eradication improves motor fluctuations in advanced Parkinson's disease patients: A prospective cohort study (HP-PD trial)

Dear Dr. Lolekha:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Wan-Long Chuang

Academic Editor

PLOS ONE