Effects of Chinese herbal medicine Pugongying for reducing the application of antibiotics in breastfeeding women with acute mastitis: study protocol of a randomized, active-controlled, outcome assessor-blinded trial

• Background : Acute mastitis influences the health condition and quality of life of the infants and mothers 7 during the lactation. Pugongying (a kind of Chinese patent medicine, Herba Taraxaci ) has shown 8 benefits in lactating women with acute mastitis in clinical practice. However, there is no solid evidence to 9 support its effectiveness and safety. • Methods : A three-arm, multicenter, randomized, active-controlled, outcome assessor-blinded clinical 11 trial will be undergoing in three hospitals in Beijing. 306 participants will be randomly assigned into three 12 groups in 1:1:1 ratio with Pugongying alone, cefdinir alone, and combination of Pugongying and cefdinir 13 for 3-day intervention drugs administration. And in combination of Pugongying and cefdinir group, the 14 participants will be administrated with 2-day cefdinir and 3-day Pugongying. The primary outcomes are 15 resolution of fever, visual analogue scale (VAS) scores of breast pain, and the size of the breast mass by 16 palpation. The secondary outcomes are the patency of milk, Traditional Chinese Medicine (TCM) 17 symptoms scores, white blood cell count, the percentage of neutrophil and C-reactive protein, relapse at 18 3 th day of follow up after completion of treatment, and safety assessment including routine blood, liver 19 and renal function and electrocardiography. Besides, the incidence of surgery and the quantity of 20 additional intervention drugs will also be evaluated. • Discussion : The results of this trial are expected to confirm whether Chinese herbal medicine 22 Pugongying could alleviate the symptoms and signs in lactating women with acute mastitis, and they 23 could reduce application of cefdinir in clinical practice. We analyze all available data from the pilot study on breast-pain VAS scores in lactating women with acute mastitis (unpublished) in Third Affiliated Hospital of Beijing University of Chinese Medicine, and the suggestions from the practitioners to calculate sample size. In this trial, sample size formula in the 4 th edition 223 clinical epidemiology is used as reference.[20] In terms of sample size, there is in excess of a 95% power 224 and a (2-side) 10% significance level in detecting treatment differences. The standard deviations are 0.81 in 225 CPM, 0.76 in CPM & ACC, 0.9 in ACC, respectively. The means are 3.55 in CPM, 3.10 in CPM & ACC, 3.34 226 in ACC, respectively.


Background and rationale {6a} 35
Acute mastitis is a common problem in lactating women. [1] It is defined that part of one breast becomes red, 36 painful, swollen and hard, sometimes with common symptoms of fever and malaise. [1] The prevalence rates 37 of acute mastitis in breastfeeding women range from 2% to 33% according to previous mastitis prevalence 38 data. [1] Acute mastitis may produce overwhelming acute symptoms that causes women to consider to stop Page 4 of 23 breastfeeding, or health-care workers will advise women who are administered antibiotics therapy to stop 40 breastfeeding, both of which will result in breastfeeding failure and the infants lose their optimal nutrition. [1][2] 41 What's more, acute mastitis also can bring severe complications such as breast abscess, and occasionally 42 be fatal if inadequately treated. [1] These conditions can lead to a considerable burden of disease and involve 43 substantial costs.
[3] Previous studies have indicated that Gram-positive staphylococci is the main pathogenic 44 bacteria in acute mastitis. [4][5][6][7] With the increasing application of metagenomic sequencing technology in milk 45 microecology field, the research methods of acute mastitis pathogenic bacteria have gradually turned from 46 the isolation and cultivation of pathogens to equilibrium between microorganism. And these studies suggest 47 that two theories may explain the occurrence of acute mastitis. One is that Staphylococcus and 48 Corynebacterium could not be inhibited by commensal bacteria, the other is lower microbial diversity in milk, 49 with increased abundance of conditioned pathogens and depletion of commensal obligate anaerobes. [8][9][10] 50 The pathogenesis of acute mastitis has not been thoroughly described, but most investigators prefer that

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We hypothesize that Pugongying have positive effects on fever-resolution, less breast pain and mass-80 dissipating, and to some extent, it can reduce the application of cefdinir.

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This trial is a Principle Investigator-initiated, three-arm, multicenter, randomized, active-controlled, outcome 83 assessor-blinded, parallel assignment clinical trial in which 306 participants will be assigned to three groups 84 in 1:1:1 ratio with Pugongying alone, cefdinir alone or combination of Pugongying and cefdinir. The 85 investigators plan to allocate a 3-day treatment and 3-day follow-up to participants. Two visits will be 86 scheduled for each participant: baseline, day-3. At the day-6, the investigators will follow the participants up 87 by telephone or Wechat (a social media used in China).     participants should be assured that they can quit the study any time.

Additional consent provisions for collection and use of participant data and 119 biological specimens {26b} 120
The participants in Pugongying group and cefdinir group will sign another ICF which contains the item about 121 milk samples collection for further genetic analysis. Three milk samples will be collected on the baseline day 122 (day-0) and day-3. All samples will not be preserved.

Explanation for the choice of comparators {6b} 125
The recommended pharmacological measure of acute mastitis is mainly antibiotics.

Intervention description {11a} 132
In clinics, participants will be randomly assigned to the Chinese patent medicine group (CPM), combination    co. LTD. The form of the drug is granule, and the participants will take the drug after dissolved. Cefdinir is 141 also named as cefdinir capsule and produced by Astellas Pharma Inc. The form of the drug is capsule, and 142 participants will orally take the capsules. The investigators will follow them up for 3 days after 3-day drug Page 8 of 23 administration.

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All participants will receive education, including dietary, emotional regulation and the knowledge of 145 breastfeeding. The investigators will encourage participants to remove milk effectively.

Criteria for discontinuing or modifying allocated interventions {11b} 147
The participants with body temperature above 41℃ in the 3-day treatment period will be recommended to 148 withdraw from the trial and receive intravenous drip antibiotics therapy. If the participants have preference 149 for particular intervention and would not like to continue receiving assigned intervention, they can withdraw 150 from the trial at any time. In the above situations, the investigators will try their best to obtain the participants' 151 data when they withdraw from the trial.

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During 3-day treatment, if B-ultrasound hints that there is a mammary abscess, the doctor will discuss with 153 the participants and additionally perform the surgery to prevent them from severe complications if necessary.

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The participants in CPM group with body temperature above 39℃ will be administered by twice dose of 155 Pugongying.

Strategies to improve adherence to interventions {11c} 157
Before the beginning of enrollment, the doctors will inform them about high recurrence rate of acute mastitis 158 and the necessity of 3-day drugs administration. And the doctors can assess the disease condition by related 159 laboratory tests and examinations on day-3 to ensure the interests of all enrolled participants. Finally, the 160 investigators will distribute 120% intervention drugs and require the participants to return the drugs at day-3.

Outcomes {12} 169
Primary outcome measures 170 Primary outcome measures are the common chief complaints which have strong relevance to acute mastitis. 171 ·Resolution of fever: Body temperature will be measured by mercury thermometer and recorded on the 172 prepared card by participants. The temperature reduces to 37.2℃ or more below, assessing as the normal 173 temperature. And the normal temperature lasts for at least 24 hours, considered as fever-resolution. To 174 evaluate onset time and the temperature changing from baseline to the end of 3-day treatment, participants 175 will be encouraged to measure body temperature every 4 hours for 3 days, specifically on 2:00 am, 6:00 am,        Table 1 shows the participant timeline (Please see Table 1 at the end of the document).

Sample size {14} 220
Page 11 of 23 We analyze all available data from the pilot study on breast-pain VAS scores in lactating women with acute

Recruitment {15} 233
The costs of intervention drugs (Pugongying and cefdinir), laboratory tests (routine blood, routine urine) and 234 examinations (breast B-ultrasound and electrocardiogram) will be provided by the sponsor.

Concealment mechanism {16b} 239
Page 12 of 23 The randomized number and allocation details will be sealed in opaque envelopes.

Implementation {16c} 241
JHX will generate the allocation sequence. The doctors who in clinics will enroll participants. And the 242 investigators (e.g. JHX and XYJ) will assign participants to interventions.

Who will be blinded {17a} 245
The trial is an outcome assessor-blinded, data collector-blinded and data analyst-blinded study. Treatment 246 allocations will be concealed from the data analysts by group 1, 2 and 3. The data-collectors (e.g. CG) and 247 outcome assessors (e.g. nurses) blinded about intervention allocations will record the data and evaluate the 248 mass size, patency of milk, TCM symptoms and relapse. Participants preference can have minor influence 249 on body temperature, white blood cell count, the percentage of neutrophil and C-reactive protein.

Procedure for unblinding if needed {17b} 251
Not applicable. In this trial, participants and doctors will not be blinded, therefore there is no unblinding 252 procedure.

Plans to promote participant retention and complete follow-up {18b} 263
During intervention period and follow-up period, the investigators will communicate with all enrolled 264 participants by telephone or Wechat to obtain the information of the participants' conditions (e.g. body 265 temperature, breast pain). If the participants discontinue or deviate from the study, the investigators will 266 persuade them to receive laboratory tests and examinations to assess their disease conditions and protect 267 their interests.

Data management {19} 269
All data will be input and checked by two statisticians using EpiData 3.1.

Confidentiality {27} 271
All participants' personal information will be confidential to the extent permitted by Chinese laws. The 272 samples of enrolled participants will be identified by study numbers rather than their name. Unless the 273 permission is obtained, information that identifies individuals will not be disclosed to anyone other than 274 members of the study group. The investigators, the supervisor appointed by CFH, the ethics committee and

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CFDA are allowed to access participants' medical records related to the study to ensure the authenticity and 276 accuracy of the data, but other individual information will not be shared. Case report forms (CRF) will be 277 reserved in cabinet unless investigators allow to open. Electronic data will be input according to the study 278 number and accessed under the permission of investigators. When the results of this study are published, no 279 information about the participants will be disclosed.

Plans for collection, laboratory evaluation and storage of biological specimens for 281 genetic or molecular analysis in this trial/future use {33} 282
Four blood, two urine and three milk samples will be collected on the baseline day (day-0) and day-3. Blood 283 samples will be utilized in laboratory tests, including routine blood test, liver and kidney function test. Blood 284 and urine samples will not be saved. All abnormal values will be evaluated based on reference values.

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Milk samples will be collected by the investigator (XYJ) in sterile conditions. Then they will be storage in -80

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℃ refrigerator and will be transported to Beijing Major Biomedical Technology Co., Ltd in drikold environment Page 14 of 23 for further genetic analysis. All samples will not be preserved.

Statistical methods for primary and secondary outcomes {20a} 290
Statistical analysis will be conducted by Centre for Evidence-Based Chinese Medicine, Beijing University of 291 Chinese Medicine. The statistician (not in authorship) will be blinded from the intervention allocations. SPSS 292 25.0 statistical software packages will be used to analyze the data. Prior to all analyses, a detailed statistical 293 analysis protocol is developed.

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Continuous variables will be expressed as median and standard deviations. Three groups will be compared 295 using Analysis of Variance (ANOVA) or Kruskal Wallis test, as appropriate, based on the data distribution.

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Two groups will be compared using Least Significant Difference (LSD) if there is significant differences 297 between three groups. Dichotomous variables will be expressed as "yes" or "no". Groups will be compared 298 using the chi-square or Fisher's exact test, as appropriate, based on the expected counts. Participants 299 characteristics and past history will be reported and compared between groups. Descriptive statistics will be 300 presented to describe the trial results. A two-sided P < 0.05 will be considered statistically significant.

Interim analyses {21b} 302
Not applicable. The interim analyses are not planned to conduct.

Methods for additional analyses (e.g. subgroup analyses) {20b} 304
Not applicable. Additional analyses are not planned to conduct.

Methods in analysis to handle protocol non-adherence and any statistical methods 306 to handle missing data {20c} 307
The intention-to-treat (ITT) population is defined as the patients who are randomized and receive at least

Composition of the data monitoring committee, its role and reporting structure {21a} 323
Data monitoring committee is not applicable. The data will be monitored by the sponsor.

Adverse event reporting and harms {22} 325
Any serious adverse events will be reported to the principle investigator within 24 hours and recorded in CRF 326 to analyze the relationship between events and intervention. The serious adverse events will be reported to 327 2018 Capital's Funds for Health Improvement and Research following GCP guidelines.

Frequency and plans for auditing trial conduct {23} 329
The sponsor will randomly audit some clinical trials and commission the third party to conduct the audit 330 procedures to avoid interest conflicts.

Dissemination plans {31a} 337
We will put up a poster and Wechat post via public account to disseminate this trial.

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The results of this trial are expected to provide convincing evidence that Pugongying is effective and safety 340 for alleviating manifestations of lactating women with acute mastitis, and they could reduce application of 341 cefdinir in clinical practice.

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As we know, milk is not sterile and has a wide range of microbiome which has important health 343 implications. [22,23] And the relationship of microbiome in milk is in a dynamic equilibrium. The application of 344 antibiotics may induce dysbiosis of milk microbiome. [24] In TCM theory, "vital qi" is a collective designation 345 for all normal function of the human body and the abilities to maintain health, including the abilities to self-346 regulation, adaptation to the environment, resistance against pathogens and self-recovery from illness, and "

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There are some limitations of this trial. First, the outcomes of this trial may not be sufficient to evaluate the 366 effectiveness and safety of Pugongying from all angles in the treatment of acute mastitis. However, the core 367 outcome set of mastitis is not available. We try to assess the manifestations of acute mastitis based on 368 clinical experience. Second, we do not use double-blinded design and this may lead to performance bias.

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But the outcomes measures except the scores of breast pain, are either objective indicators or outcome 370 assessor-evaluation outcomes. The preference of participants and investigators is minimal. Third, the title is