2.1 Study design
This was a single-center, open-label study, conducted at the University of Fukui Hospital. Patients with a confirmed diagnosis of pulmonary MAC and those who had not yet received standard antimicrobial therapy were treated with 3.0 g of JST extract (Tsumura Co., Tokyo, Japan) three times per day. Their weight, chronic obstructive pulmonary disease assessment test (CAT) score (7–10), NK cell activity, chest computed tomography (CT) results, blood sample results, Self-rating Depression Scale (SDS) scores (11), and State-Trait Anxiety Inventory (STAI) scores (12,13) were measured at the following three points in time: (i) before JST administration, (ii) after 3 months, and (iii) at the end of the study. The treatment was administered for 12 months. Patients were determined to require standard treatment for MAC disease progression, if they had the following conditions: (i) new bronchiectasis, bronchiolitis, or nodules appearing in another lung lobe; (ii) the appearance of new infiltrative or cavitary shadows in the same or another lung lobe; or (iii) the appearance of fresh, blood-stained sputum. These patients were terminated from the study at that time. We evaluated the results of their laboratory tests at the end of the study. The chest CT findings were evaluated by two respiratory physicians using a simplified evaluation scale based on previously reported scales (Table 2) (14,15). At the completion of the study period, the group of patients whose CT scores increased by ≥1 point during the study was defined as the exacerbation group, and the group of patients whose CT scores remained unchanged or decreased during the study was defined as the non-exacerbation group. The primary endpoints were the symptom score (using the CAT), body weight, and NK cell activity before and 3 months after the administration of JST. The secondary endpoints were the SDS score, STAI scores for depression and anxiety, CT score for changes observed on images, exacerbation factors in the exacerbation and non-exacerbation groups, and the safety of JST treatment before and after 3 months of treatment. These parameters were evaluated the same way at the end of the study. Various cytokines and chemokines were also examined.
The study was conducted in accordance with the Declaration of Helsinki, and the protocol and informed consent documents were approved by the Ethics Committee of the Faculty of Medical Sciences of the University of Fukui (Registration No : Fukui 20170186, approval date : 21/02/2018) and they were registered in the UMIN-Clinical Trials Registry (Registration No : UMIN000033590, registration date : 01/08/2018). Written informed consent was obtained from all the patients who participated in this study.
2.2 Study population
The inclusion criteria were as follows: patients that were diagnosed with pulmonary MAC as per the diagnostic criteria (shown in Table 1) (16); those aged between 20 and 80 years; and those that were not using rifampicin, ethambutol, azithromycin, clarithromycin, streptomycin, kanamycin, rifabutin, sitafloxacin, or levofloxacin. However, the use of those drugs for less than 1 week for purposes other than the treatment of pulmonary MAC disease was allowed.
2.3 Assessment of the respiratory symptoms
Patients completed the CAT questionnaire before treatment, 3 months after, and at the end of the study. The CAT is a short, validated questionnaire to be completed by patients; it is used to assess the impact of chronic obstructive pulmonary disease on the health status. It consists of eight items (cough, phlegm, chest tightness, breathlessness going up hills/stairs, activity limitations at home, confidence, sleep, and energy) that are formatted using a 6-point differential scale (7-10). As the items correspond to all chronic respiratory diseases, and the CAT can be used not only for chronic obstructive pulmonary disease but also for a wide range of respiratory diseases, it was used in our study to evaluate the results. The St. George’s Respiratory Questionnaire, which was also developed for chronic obstructive pulmonary disease, is the most commonly used instrument for assessing symptoms of chronic respiratory diseases. However, the St. George’s Respiratory Questionnaire uses redundant and complex algorithms and is not suitable for routine use. Previous studies (17) have shown that the CAT and St. George’s Respiratory Questionnaire are strongly correlated; therefore, the CAT was chosen for this study.
2.4 Psychological tests
The SDS score and STAI score were measured before treatment, 3 months after, and at the end of the study.
The SDS score represents a patient’s self-rated depression based on the answers to a questionnaire consisting of 20 items that are extracted based on a factor analytic study of depression and depressive symptoms reported by Grinker et al (18,19). Zung categorized the SDS scores according to the degree of depression as follows: 20 to 39, normal; 40 to 47, mild depression; 48 to 55, moderate depression; and ≥56, severe depression (11).
The STAI consists of two scales: the State Anxiety Scale, which indicates the susceptibility to anxiety at the time of measurement, and the Trait Anxiety Scale, which indicates susceptibility to anxiety as a personality trait. Both scales have the same format and include 20 items rated using a 4-point scale. The two types of anxiety can be differentiated by providing appropriate instructions to those completing the questionnaires. The mean (± standard deviation [SD]) values for normal adults are 36.6 points (SD, ±8.98 points) for state anxiety and 38.8 points (SD, ±9.68 points) for trait anxiety, corresponding to the 75th percentile or higher; the standard values for high anxiety are ≥42 points for state anxiety and ≥44 points for trait anxiety for males and ≥42 points for state anxiety and ≥45 points for trait anxiety for females (12).
2.5 Assessments of inflammation, nutritional status, NK cell activity, cytokines, and chemokines
The erythrocyte sedimentation rate, white blood cell (WBC) count, and C-reactive protein (CRP) level were measured to assess inflammation. Lymphocyte count and albumin, total cholesterol, and cholinesterase levels were measured to assess the nutritional status. Additionally, the Controlling Nutritional Status (CONUT) values were calculated using the CONUT method, which considers the lymphocytes, albumin, and total cholesterol (Table 3) (20). NK cells are mainly found in the bloodstream and have antitumor activity in a nonsensitized state in addition to regulatory effects on the antibody production system. Morphologically, they are identified as granular lymphocytes. NK cells are present in the peripheral blood as well as in the spleen, tonsil, liver, ascites, pleural fluid, and joint synovial fluid during inflammation. By measuring the cytotoxic activity of NK cells, the biological defense mechanism can be evaluated (21).
Serum cytokine CC chemokine ligand (CCL)17/thymus and activation-regulated chemokine (TARC), interferon-α, interferon-γ, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12/IL-23 p40, IL-13, IL-17/IL-17A, IL-18/IL-1F4, and tumor necrosis factor (TNF)-α were assayed (MILLIPLEX® MAP Human Cytokine/Chemokine/Growth Factor Panel A 48 Plex Premixed Magnetic Bead Panel-Immunology Multiplex Assay; MERCK Millipore Corporation, Billerica, MA, USA). Levels of the granulocyte-macrophage colony-stimulating factor was measured using an enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Minneapolis, MN, USA).
2.6 Assessment of the CT images
Our scoring system (Table 2) was created by modifying previously used scoring systems (15,16). Two independent pulmonologists retrospectively evaluated the chest CT images and completed the scoring sheets. In case of discrepancies in their readings, the final decision was made upon reaching a consensus.
Scores were determined by considering the presence, severity, and extent of the following five categories of parenchymal abnormality: bronchiectasis (maximum score of 6); cellular bronchiolitis (maximum score of 6); cavitation (maximum score of 6); nodules (maximum score of 3), and consolidation (maximum score of 3). Therefore, a maximum total CT score of 24 was used to quantify the parenchymal lesions throughout both the lungs in each patient.
Bronchiectasis was considered to be present when the diameter of the bronchial lumen was greater than that of the adjacent pulmonary artery without tapering. Bronchiolitis (the cellular or inflammatory type) was defined as the presence of centrilobular small nodules (diameter, <10 mm) and a tree-in-bud pattern on high-resolution CT images. Other parenchymal abnormalities, such as cavitation nodules (diameter, 10–30 mm) and airspace consolidation, were also documented (Figure 1).
2.7 Safety evaluation
To determine the safety of administering JST, we observed the patients for the occurrence of any side effects. Pseudohyperaldosteronism is a serious side effect of JST that is indicated by increased blood pressure and sodium levels, fluid retention, edema, hypokalemia, and associated myopathy (weakness, limb cramps, and paralysis). Other side effects, such as anorexia, gastric discomfort, nausea, diarrhea, and other gastrointestinal symptoms, can also be seen with JST.
To ensure patient safety while evaluating pulmonary MAC disease, only patients (i) that were not on the standard treatment of rifampicin, ethambutol, and clarithromycin combination therapy; (ii) those with stable disease; and (iii) those who could participate in follow-up were included. Therefore, if patients experienced disease progression, then they were terminated from the study and consequently, the standard treatment was administered to them.
2.8 Assessment and statistical analysis
We compared the CAT score, body weight, NK cell activity, SDS score, STAI score, CT score, various cytokines, and chemokines before treatment, 3 months after, and at the end of the study for the entire treatment group, the exacerbation group, and the non-exacerbation group using Wilcoxon’s signed rank test.
Additionally, to determine if there were any differences between the non-exacerbation and exacerbation groups before treatment, the pretreatment CAT score, body weight, NK cell activity, SDS score, STAI score, CT score, various cytokines, and chemokines were compared using the Mann–Whitney U test. If significant, then the cutoff value for exacerbation was estimated using the receiver-operating characteristic curve.