The Investigation Of The Etiology And Antituberculous Therapy For Non-puerperal Mastitis

BackgroundMastitis is defined as inflammatory diseases of female breasts,which encompasses a large spectrum of disorders ranging from acute infectious diseases to chronic disease.Acute infectious mastitis usually affects females in lactation period,and with the improvement of sanitary conditions in recent years,lactation mastitis incidence decreased.Meanwhile,non-puerperal mastitis(NPM)incidence increased year by year.Periductal mastitis(PDM)and granulomatous lobular mastitis(GLM)is the most common types of NPM;and PDM constitutes 1-2%of symptomatic breast diseases,while GLM is rare chronic inflammatory lesion of the breast.NPM can affect females in all age groups,especially women of childbearing age.The onset of NPM can be acute or slow,and symptoms are not typical,sometimes only palpable breast lumps but no obvious signs of inflammation.The common clinical manifestations are nipple retraction,breast lumps,nipple discharge and breast abscesses.Because of similar clinical and imaging features,sometimes it is difficult to identify NPM with breast cancer,leading to misdiagnosis and mistreatment.Till now,there is no standard treatment for NPM,and some patients underwent mastectomy which brought great suffering to the patients.Thus far,the etiology of PDM is still obscure.Several factors have been associated with an increased risk for this disease,including smoking,obesity,and diabetes mellitus.However,all of these stated risks are identified based on small case series reports and have not been confirmed epidemiologically.PDM is generally seen in those who smoke heavily,moreover recurrence is also associated with smoking,especially heavy smoking.However,the smoking rate of female in China is only 2.4%,which is significantly lower than that in western countries,so the relationship of smoking and PDM is unclear.There are many disputes about the relationship between the fertility related factors and the disease.Literatures also indicate conflicting information on whether age at first birth,parity,and duration of breastfeeding are potential risk factors for PDM.Etiologically,the exact role of bacterial infection in PDM has been debated for many years.Bacterial infection was thought to be a possible etiological factor of PDM.But studies suggest that a specific pathogenic bacterium may not exist for the infection of PDM.And the investigation of consequent immune response following bacterial infection is still few.Granulomatous lobular mastitis is considered as an autoimmune disorder,which is mainly associated with lactation,hyperprolactinemia and infection.GLM is usually detected in women with a history of breast-feeding or birth recently.Pregnancy,lactation,and hyperprolactinemia have also been put forward as a possible predisposing factor of GLM.Some other factors including obesity and diabetes mellitus are considered as risk factors for GLM,but smoking is not related to this disorder in general studies.Bacteria,especially Corynebacteria,were reported to be detected in GLM;however there are few studies on the pathogenesis of the disease.There are many disputes about the pathogeny of GLM,and no definite conclusion on the role of bacteria and consequent immune response.The treatament of NPM is a thorny problem in the clinical work of breast surgery.Surgical treatment is still the main clinical treatment measures,but nonetheless,it is not the standard treatment for NPM in expert consensus.The reason is that if the operation range is too small,the recurrence rate is very high;but mastectomy will destroy the appearance of the breast and cause large trauma on the psychology of women.For PDM,antituberculous therapy and Chinese medicinal therapy are reported as effective treatment,and for GLM,corticosteroids and antituberculosis drugs are considered as effective alternative treatment.In this study,we collected the information of PDM and GLM patients who received antituberculous therapy,and provided long term follow-up to analyze the clinical application of this approach in NPM.Based on the above findings,we hypothesized that many clinical factor played a role in the pathogenesis of NPM,and the main cause of this disease may be the consequent immune response following bacterial infection instead of the infection itself.We conducted a retrospective non-matched case control study to analyze the risk factors of NPM,and investigated the role of bacterial infection and the consequent immune response in the pathogenesis of this disease.And we evaluated the clinical usage of antituberculous therapy for PDM and GLM.PART I:The Investigation of Risk Factors of NPMObjectivePDM can affect women of all age groups,yet GLM mainly occurs in female of childbearing age,especially in those within 5 years after pregnancy.Our previous research showed that nipple retraction and passive smoking were risk factors of NPM.In this part,we conducted retrospective non-matched case control study.By analyzing the related factors,we could get epidemiological characteristics of PDM and GLM,and analyze the relationship between these factors and NPM.Furthermore,we could make clear the difference between PDM and GLM in clinical manifestations and risk factors.Research Methods1.The collecting of study subjectsThis was a retrospective non-matched case control study.The study was conducted from January 2011 to March 2015.Eighty-seven patients with PDM and 26 patients with GLM were recruited from the Department of Breast Surgery at the Second Hospital of Shandong University.Pathological results were reviewed by two pathologists and any other possible inflammatory breast diseases.Eighty-seven healthy controls were collected from the Physical Examination Center of the Second Hospital of Shandong University from June 2015 to August 2015.2.Data CollectionThe questionnaire contents include two parts:1)Basic questionnaire demographic characteristics,female physiology related factors,diseases history and family history,lifestyle and habits.2)Clinical breast examination table including the apparent diagnosis of breast and palpation,primary diagnosis relevant suggestion which filled out by breast surgeons and breast ultrasound and X-ray mammography report,conventional pathology results.3.Statistical analysisSPSS 17.0 software was used to analyze all data.Statistical methods,including t-test,x2 test,univariate and multivariate unconditional logistic regression analyses,were used to identify the risk factors for PDM and GLM.The odds ratios(OR)with 95%confidence intervals were also calculated.Results1.The results of epidemiological investigationTo investigate the risk factors for PDM and GLM,87 patients with PDM,26 patients with GLM and 87 healthy control subjects were enrolled in this study.The median age of PDM patients at presentation was 34(range 20-62)years,the median age of GLM patients was 29(range 22-44)years,and the median age of controls was 34(range 26-45)years.Overweight/obesity,the number of full-term pregnancy,age at first birth,age of menarche,menopause,nipple retraction and duration of breastfeeding are related with PDM(P<0.05).Miscarriages,galactostasis,history of benign breast disease,chronic disease history,history of allergies,alcohol consumption and passive smoking are not related with PDM(P>0.05),Age of menarche,nipple retraction and body mass index(BMI)are related with GLM(P<0.05).Overweight/obesity,duration of breastfeeding,menopause,history of benign breast disease,chronic disease history,history of allergies,alcohol consumption and passive smoking,miscarriages,galactostasis,and the number of full-term pregnancy are not related with GLM(P>0.05).2.The logistic regression results of risk factors for PDM2.1 Univariate analysisThe results of the univariate analysis indicate that overweight/obesity,age of menarche,the number of full-term pregnancy,history of benign breast disease and nipple retraction are related with PDM risk,and age at first birth is a protective factor(P<0.05).Miscarriages,galactostasis,history of allergies and passive smoking are not related with PDM risk(P>0.05).2.2 Multivariate analysisMultivariate Logistic regression analysis indicates that overweight/obesity(OR,1.358;95%CI,1.083-1.704;P=0.008)and age of menarche(OR,2.408;95%CI,1.378-4.208;P=0.002)are independent predictors of PDM.In contrast,late age at first birth has a protective effect against PDM(OR,0.184;95%CI,0.034-0.983;P=0.048).2.3 Subgroup AnalysisCases and controls are divided into two groups according to the age of 35 years.For patients under the age of 35,age of menarche and nipple retraction are independent predictors of PDM.But for patients over age of 35,age of menarche and the number of full-term pregnancy increased the risk of PDM.3.The logistic regression results of risk factors for GLM3.1 Univariate analysisThe results of the univariate analysis indicate that overweight/obesity,age of menarche and nipple retraction are related with GLM risk(P<0.05).Miscarriages,the number of full-term pregnancy,galactostasis,history of benign breast disease,history of allergies and passive smoking are not related with GLM risk(P>0.05).3.2 Multivariate analysisMultivariate Logistic regression analysis indicates that overweight/obesity(OR,2.934;95%CI,1.264-6.808;P=0.012),age of menarche(OR,16.280;95%CI,3.268-81.093;P=0.001)and the number of full-term pregnancy(OR,9.450;95%CI,1.394-64.055;P=0.021)are independent predictors of GLM.4.The comparation of PDM and GLM in risk factors and clinicopathologic featuresThere is significant difference in age between GLM and PDM,however there are no significant differences in BMI,the number of full-term pregnancy,age at first birth,galactostasis,menopause,duration of breastfeeding,chronic disease history and passive smoking between the two groups(P>0.05).In the clinicopathologic features,the two groups patients have different performance in the onset time,the time of last birth to the onset,mass size,local skin erythroswelling and fistula/sinus formation(P<0.05).Meanwhile,there are no differences in affected side,quadrants,nipple retraction and subareolar region lesions(P>0.05).The infiltration of neutrophils,multinuclear giant cells and foam cells are different in these two groups,but the infiltration of lymphocytes is similar.Conclusion1.Overweight/obesity and age of menarche are independent predictors of PDM.In contrast,late age at first birth has a protective effect against PDM.The risk factors were different after stratified by age.2.Overweight/obesity,age of menarche and the number of full-term pregnancy are independent predictors of GLM.3.There are many differences in clinical manifestations and clinicopathological features between PDM and GLM.PART Ⅱ The Investigation of Microorganism Infection and Th1/Th2/Th17 Cytokines Expression in NPMObjectiveAccording to our previous study and literature reports,bacterial infection,especially mixed bacterial flora,may be one of the initiating factors of NPM and the consequent immune response following bacterial infection may be the key point in the pathogenesis process.To make clear the role of bacterial infection and the expression of main Th1/Th2/Th17 cytokines in NPM patients,we collected the tissues of patients and detected the expression of IL-4,IL-10,IL-12A,IL-17A and IFN-y.By this study,we could have a preliminary understanding of the pathogenesis of PDM and GLM.Research Methods1.Bacterial detectionThirty-three specimens from 31 patients were collected in this study.Normal tissues adjacent to benign breast pathology(including fibroadenoma,mastalgia,and intraductal papilloma of the breast)were used as control specimens(n = 12).Breast tissue samples were collected under aseptic conditions at the time of operation.Genomic DNA was prepared using the bacterial DNA extraction kit and analyzed by 16S rDNA gene sequencing.Nucleotide sequences were analyzed using the National Center for Biotechnology Information BLAST software.2.Th1/Th2/Th17 cytokines expression studyParaffin sections of 31 cases mentioned above and another 26 cases of GLM were used for immunohistochemistry analyses to investigate the expression status of Th1/Th2/Th17 cytokines in PDM and GLM.Normal tissues adjacent to benign breast pathology(including fibroadenoma,mastalgia,and intraductal papilloma of breast)were used as control specimens(n = 22).The stained slides were reviewed and scored independently and blindly by two pathologists.And the expression of Thl/Th2/Thl7 cytokines was graded according to immunohistochemical scores.3.Statistical analysisDifferences of cytokines expression between NPM and controls were evaluated by Chi-square test.P<0.05 was considered significant,and all tests were 2-sided.All statistical analyses were performed by SPSS 17.0.Results1.Microorganism Infection in PDM.All cases were confirmed by pathological examination after operation,and there were no GLM patients in the study group.Thirty-three specimens from 31 patients and 12 cases of normal tissues were collected in this study.1.1 PDM was related to bacterial infectionAfter extraction of bacterial genomic DNA gel electrophoresis,in the case group,the bands were amplified by 16S rDNA,while the control group had no obvious bands,indicating the presence of bacterial infection in patients,but not in normal breast tissue from controls.1.2 Mixed bacterial infection was the most common in PDMIn the case group,a mixture of different bacterial infection was most often observed(n=12),followed by single bacterial infection of Pseudomonas spp.(n=9).Enterococcus faecium,Corynebacterium kroppenstedtii,Bacillus firmus;Sporosarcina,and Staphylococcus aureus were also found in some samples.This result validated the assumption that bacterial infection might be an etiological factor for PDM.2.The expression of Th1/Th2/Th17 cytokines in PDM and GLMThe immunohistochemical result showed that cytokines were mainly expressed in cytoplasm of normal epithelial cells and stromal inflammatory cells.2.1 The expression of Thl cytokines was up-regulated in PDMAfter staining and taking photographs,a ’scoring evaluation of the density and extent of staining for each cytokine was performed.The expressions of IFN-y and IL-12A in stromal inflammatory cells of the breast were increased in PDM compared to normal breast tissues(P<0.05).However,there were no significant differences in the expression of IL-17A,IL-4 and IL-10 between these two groups(P>0.05).2.2 The expression of Thl cytokines was up-regulated in GLMThe expressions of Thl cytokines,IFN-y and IL-12A,in stromal inflammatory cells of GLM patients were increased compared to normal breast tissues(P<0.05).However,there were no significant differences in the expression of Th2 and Th17 cytokines between these two groups(P>0.05).2.3 The expression of Th17 cytokine was higher in PDMThe expressions of Th17 cytokine,IL-17A,in stromal inflammatory cells of PDM patients were increased compared to GLM patients(P<0.05).However,there were no significant differences in the expression of Thl and Th2 cytokines between these two groups(P>0.05).Conclusion1.Bacteria infection,especially mixed bacterial flora,is an important etiological factor for PDM.2.Thl cytokines,IFN-γ and IL-12A,were up-regulated in PDM patients.3.The expressions of IFN-γ and IL-12A were increased in GLM compared with controls.4.Compared with GLM,the expression of IL-17A was higher in PDM.PART Ⅲ The Clinical Usage of Antituberculous Therapy in NPMObjectiveThe treatment of NPM is a difficult problem in the clinical work of breast surgery.Surgical treatment is still the main clinical treatment measures,but nonetheless,it is not the standard treatment for NPM in expert consensus.If the operation range is too small,the recurrence rate is very high;but mastectomy will destroy the appearance of the breast and cause large trauma on the psychology of women.Antituberculous therapy has been reported to be an effective alternative treatment for both PDM and GLM.In this study,we collected the information of PDM and GLM patients who received antituberculous therapy,and provided long term follow-up to analyze the clinical application of this approach in NPM.Research Methods1.The collecting of study subjectsThis is a retrospective single-arm clinical assessment study of consecutive patients referred to the Second Hospital of Shandong University,China,with a diagnosis of PDM or GLM and received antituberculous treatments from January 2011 to January 2015.There is no control group in this study.Patients with any history of malignancy were excluded.Vacuum-assisted biopsy or incisional biopsy was performed depending on the clinical findings at the time of admission.2.Data Collection and follow-upThe demographic,clinical,and histopathologic data of patients were collected,including presenting complaint,size of lesion,and histology findings.The imaging findings were assessed in accordance with the Breast Imaging Reporting and Data System(BI-RADS)criteria.Patients were followed monthly for symptoms(e.g.,pain and macroscopic appearance of the breast)and side effects(e.g.,nausea,vomiting,and liver function damage)of antituberculous treatment.Patients received physical examination and ultrasonography in the same hospital.The changes of mass size,abscess and fistulae were recorded in detail.Side effects were also recorded in the reexaminations and blood examinations were performed to monitor of liver and renal functions monthly.After the end of treatment,patients were required to take review of physical examination and ultrasonography every three months.3.Criteria of efficacyComplete remission was defined as the absence of breast mass or signs of breast inflammation after finishing medical treatment and did not recur after 6 months’ follow-up.Patients in whom the breast mass did not resolve completely were considered as having partial remission.If there was no change in the breast mass or inflammation signs at the last clinical visit,the patient was defined as a non-responder.4.Statistical analysisSPSS 17.0 software was used to analyze all data.Statistical methods,including t-test,χ2 test were used in the process.Results1.The clinical features of patients with antituberculous treatmentFrom 2011 to 2015,64 patients with PDM received antituberculous treatment,and the median age was 31(range 22-58)years.Abscess incision and drainage was performed in 9 PDM patients,and vacuum-assisted biopsy was performed in the remaining 55 patients with Mammotone Biopsy System.Forty patients had local skin redness,swelling and pain,while 38 patients had breast abscesses,and sinus was formed in 15 patients.For GLM,22 patients received triple anti-tuberculosis medicine treatment and median age of 29(range 23-44)years.After admission,abscess incision and drainage was performed in 1 patient,and small pieces of tissue were taken in this process for histopathological examination.Vacuum-assisted biopsy was performed in the remaining 21 patients with Mammotone Biopsy System.Sixteen patients had local skin redness,swelling and pain,while 12 patients had breast abscesses,and sinus was formed in 8 patients.2.Antituberculous therapy was effective in the treatment of PDMDuring the median follow-up period of 26(10-60)months after withdrawal,16 patients were lost to follow-up.Because of side effects,8 patients discontinued the antituberculous treatment.For the remaining 40 patients,35 patients got clinical complete remission and no recurrence was observed.The success rate of antituberculous therapy was 87.50%.3.GLM patients were sensitive to antituberculous therapyDuring the median follow-up period of 29(11-42)months after withdrawal,3 patients were lost to follow-up.For the remaining 19 patients,18 patients got clinical complete remission and no recurrence was observed-The success rate of anti-tuberculosis was over than 90%.Conclusions1.Antituberculous therapy is effective in PDM patients with inflammatory skin changes,abscess formation and sinus.2.GLM patients were sensitive to antituberculous therapy,and this can be an effective alternative therapy in GLM patients.