Study population
We conducted a cross-sectional study comparing postmenopausal women with MAC-LD with healthy postmenopausal women at a tertiary care center in Japan. Data and samples from outpatients with MAC-LD were obtained between May 2016 and April 2017 from Keio University Hospital, one of the largest referral centers in Tokyo. The hospital treats approximately 400 outpatients annually with MAC-LD in its infectious disease and pulmonary disease clinics. In total, 193 women with MAC-LD provided written informed consent for study participation. The diagnosis of MAC-LD was based on 2007 American Thoracic Society/Infectious Diseases Society of America criteria [
7]. Meanwhile, data and samples from healthy controls were obtained through annual health examinations of the faculty and staff of Keio University held at the Mita (Tokyo), Hiyoshi (Kanagawa), and Shonan Fujisawa (Kanagawa) campuses between September 2015 and November 2015. In total, 383 healthy female faculty and staff, who were not healthcare workers, provided written informed consent for study participation.
We obtained a 9-mL blood sample and clinical information through questionnaires and medical records from each study participant. To analyze sex steroid hormones, we included postmenopausal participants aged 65 years or younger because the levels of sex steroid hormones, especially E2, fluctuate considerably with the menstrual cycle and decline with aging. Because 65 years of age is the retirement age for faculty and staff at Keio University, all healthy controls were aged 65 years or younger. Consequently, we excluded patients with MAC-LD who were older than 65 years.
Participants were also excluded if their information was incomplete regarding menopause and the use of sex hormone-based medications such as oral contraceptives and if they used sex-hormone based medications. Additionally, healthy controls with histories of MAC-LD or chest X-ray findings compatible with MAC-LD were excluded. To exclude healthy controls with latent MAC infection without clinical symptoms or chest X-ray abnormality, we measured anti-GPL core IgA antibody titers using a Capilia MAC Ab ELISA kit (TAUNS Laboratories, Inc. Izunokuni, Shizuoka, Japan). GPL is a specific antigen that constitutes the cell wall of MAC; therefore, anti-GPL antibodies are specific for MAC infection [
8]. A cutoff value of 0.3 U/mL denoted positivity for anti-GPL core IgA antibodies, [
9] and healthy controls with titers exceeding this cutoff were excluded.
To compare the demographic characteristics between the MAC-LD and control groups, we obtained information on age, body mass index (BMI), age at first menstruation, age at menopause, years after menopause, smoking history, and history of tuberculosis, human immunodeficiency virus (HIV) infection, immunosuppression (immunodeficiency or use of immunosuppressive medication), and chronic diseases such as hypertension, diabetes, dyslipidemia, and osteoporosis. Information on age, BMI, history of tuberculosis, HIV, immunosuppression, and history of chronic diseases was obtained from medical and health examination records. Histories of chronic diseases were compiled using medication histories. The ages at first menstruation and menopause, and smoking histories were obtained using a questionnaire. The number of years after menopause was calculated from current age and age at menopause.
To determine if the sex steroid hormone levels were associated specifically with MAC-LD, but not other chronic lung diseases, we conducted an additional study to compare serum sex steroid hormone levels of MAC-LD patients with plasma sex steroid hormone levels of bronchiectasis (BE) patients without NTM infections (non-NTM BE). Pooled blood samples previously obtained for two clinical studies were used. Samples of female MAC-LD patients above 55 years of age, who were diagnosed with MAC-LD based on the 2007 American Thoracic Society/Infectious Diseases Society of America criteria, [
7] were selected from pooled serum samples obtained for the “NTM Biomarker Study,” which included serum samples of NTM patients of Keio University Hospital. Meanwhile, samples of female non-NTM BE patients were selected from pooled plasma samples obtained for the “NTM Genome-Wide Analysis Study,” which included plasma samples of NTM patients and BE patients of Keio University Hospital. The following characteristics defined non-NTM BE: 1) clinically diagnosed as BE by respiratory physicians based on CT examination, 2) all previous sputum Mycobacteria culture and smear were negative (at least 2 negative culture results were required), 3) anti-GPL core antibody was not detected.
Because of the small number of non-NTM BE patients, we selected age- (difference within a range of 3 years) and BMI- (difference within a range of 3 kg/m2) matched MAC-LD patients and non-NTM BE patients at a ratio of 2:1. Among the matched pairs, a pair of MAC-LD and non-NTM BE patients was excluded if one of the patients was taking oral steroids or sex hormone medication or had a history of ovariectomy or hysterectomy, which might affect sex hormone production.
Statistical methods
For baseline variables, summary statistics were constructed employing frequencies and proportions for categorical data, and medians and interquartile ranges for continuous variables. We compared patient characteristics using Fisher’s exact test for categorical outcomes and the Mann-Whitney U test for continuous variables.
Specifically in the analyses comparing MAC-LD patients with healthy controls, to assess factors independently associated with MAC-LD (i.e., age, BMI, years after menopause, E2, testosterone, and DHEA-S), we additionally performed logistic regression analyses via the simultaneous method. For the analyses, we categorized the serum hormone level of each participant as high or low based on the median hormone level of the study population. Thus, the logistic regression model consisted of three continuous (age, BMI, and years after menopause) and three categorical (low or high E2, testosterone, and DHEA-S) values.
Additionally, to assess the strength of the relationship between MAC-LD and low serum E2 levels, we performed receiver operating characteristic (ROC) curve analysis and calculated the area under the curve (AUC). A p-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS version 25 (IBM, Chicago, IL, USA).