Surgical resection is sufficient for incidentally discovered solitary pulmonary nodule caused by nontuberculous mycobacteria in asymptomatic patients.

Incidentally discovered solitary pulmonary nodules (SPN) caused by nontuberculous mycobacteria (NTM) is uncommon, and its optimal treatment strategy remains uncertain. This cohort study determined the clinical characteristics and outcome of asymptomatic patients with NTM-SPN after surgical resection. Resected SPNs with culture-positive for NTM in six hospitals in Taiwan during January, 2010 to January, 2017 were identified. Asymptomatic patients without a history of NTM-pulmonary disease (PD) or same NTM species isolated from the respiratory samples were selected. All were followed until May 1, 2019. A total of 43 patients with NTM-SPN were enrolled. Mycobacterium avium complex (60%) and M. kansasii (19%) were the most common species. The mean age was 61.7 ± 13.4. Of them, 60% were female and 4% had history of pulmonary tuberculosis. The NTM-SPN was removed by wedge resection in 38 (88%), lobectomy in 3 (7%) and segmentectomy in 2 (5%). Caseating granuloma was the most common histologic feature (58%), while chronic inflammation accounts for 23%. Mean duration of the follow-up was 5.2 ± 2.8 years (median: 4.2 years [2.5-7.0]), there were no mycobacteriology recurrence or NTM-PD development. In conclusion, surgical resection is likely to curative for incidentally discovered NTM-SPN in asymptomatic patients without culture evidence of the same NTM species from respiratory specimens, and routine mycobacterium culture for resected SPN might be necessary for differentiating pulmonary tuberculosis and NTM because further treatment differs.

Introduction

With advances in and the widespread use of chest computerised tomography (CT), the detection rate of solitary pulmonary nodules (SPNs) has increased in recent decades, and the detection rate exceeded 50% among asymptomatic patients in some series [1, 2]. The etiology of SPN varies substantially among various studies. A positive correlation between nodule size and the likelihood of malignancy has been demonstrated. According to the Fleischner Society guideline and Mayo Clinic CT Screening Trial, the likelihood of malignancy is less than 1% for nodules less than 6 mm in diameter but up to 50% for those larger than 20 mm [3, 4]. A population-based study conducted in the United States and United Kingdom revealed that the vast majority of SPNs are benign, even in smokers [5].

Granuloma due to infection is the most common aetiology of benign SPN [6], with the exact proportion depending on the geographic location. In a study including 201 SPNs in South Korea, an endemic area for tuberculosis (TB), tuberculoma accounted for 60% of benign SPNs, but none of them yielded nontuberculous mycobacteria (NTM) [7]. In a study in Japan, among 25 benign lesions in 103 surgically removed SPNs, the responsible microorganism was Mycobacterium tuberculosis in 1 (4%) case and Mycobacterium avium complex (MAC) in 4 (16%) cases [8]. In a study conducted in the United States, only 1 (5%) of the 20 resected SPNs was culture-positive for M. tuberculosis, whereas 14 (70%) were positive for NTM [9]. In NTM-SPNs, MAC is the most commonly isolated species (70%–89%) [2, 10–14].

Limited data are available on the treatment of NTM-SPN. The current guidelines suggest that surgical resection without postoperative antibiotics may be sufficient for treating SPN caused by MAC if there is no other radiographic abnormality (poor quality of evidence, Grade D) [15, 16]. The only reported evidence is from a retrospective study conducted in 126 patients with chronic obstructive pulmonary disease (COPD) who underwent lung volume reduction surgery. Among the 142 lung specimens, 14 exhibited caseating granuloma, implying mycobacterial infection. Only three specimens were culture-positive for NTM. In addition, no follow-up imaging and mycobacteriology studies were performed [17].

Because of the globally increasing prevalence of NTM disease [16, 18], the increasing use of low-dose chest CT for routine health examination in Taiwan [19], and the high availability and safety of video-assisted thoracic surgery for SPN resection [20], physicians will encounter incidentally discovered NTM-SPNs more frequently in the future. Reliable evidence confirming the most appropriate treatment strategy for NTM-SPN is required. We therefore conducted this multicentre, retrospective, longitudinal follow-up study of asymptomatic patients with incidentally discovered NTM-SPN to determine their clinical characteristics and outcomes after surgical resection.

Material and methods

Study population

This retrospective, longitudinal study was conducted at two medical centres—the National Taiwan University Hospital (NTUH) and Kaohsiung Medical University Hospital (KMUH)—and their four branch hospitals between January 2010 and January 2017. The study was approved by the institutional review boards (IRB) of the two centres (NTUH REC 201508017RIND and KMUH IRB-SV[I]-2015200266), and IRB waived the need for informed consent because data utilized in this retrospective study have been de-identified.

Lung tissues obtained after surgical resection were retrieved from mycobacteriology databases. Patients whose resected tissues were culture-positive for NTM were identified. Among them, those with multiple pulmonary nodules or consolidation confirmed by chest CT were excluded and only patients with SPNs were selected. For all respiratory specimens, acid-fast smear (AFS), mycobacterial culture, and species identification were performed using previously reported methods [18]. SPN was defined as a small (≤3 cm), well-defined lesion completely surrounded by aerate lung parenchyma on chest CT [3].

Patients were further excluded if (1) they were symptomatic and their NTM-SPNs were not incidentally discovered; (2) PD caused by NTM had been diagnosed previously or was diagnosed at present according to the 2007 guidelines of the American Thoracic Society and Infectious Disease Society of America [15]; (3) same sputum sample was culture-positive for the NTM species isolated from the SPN before resection; (4) they had active pulmonary TB or malignancy concomitantly before pulmonary resection; (5) demographic data were missing; and (6) the duration of follow-up was less than 24 months after resection.

Data collection

For each patient, the following data were collected: age, sex, body mass index (BMI), smoking status, underlying diseases, initial manifestations, SPN size and location on chest CT, preoperative lung function, laboratory data, surgical methods (segmentectomy, wedge resection, or lobectomy), mycobacteriological and histopathological findings of the lung specimen, results of follow-up sputum, latest chest image (CT preferred) before the end of follow-up, treatment course, and survival. Sputum AFS and mycobacterial culture were considered negative if patients failed to expectorate any sputum even after sputum induction [21]. The effective standard anti-NTM treatment was based on current NTM guidelines [15, 16]. Chest images were interpreted by two pulmonologists blinded to the clinical data. Discrepancies were resolved through consensus. All patients were followed up until May 1, 2019 or death. Duration of follow-up was calculated by three different ways: 1) operation to last date of chest CT follow-up; 2) operation to last date of chest x-ray follow-up; and 3) operation to last date of hospital visit.

Outcomes

The primary outcome was the incidence of NTM-PD during the follow-up after resection of SPN. The secondary outcome was the incidence of new lesion on follow-up chest images and bacteriological recurrence in follow-up sputum mycobacterial studies.

Statistical analysis

All statistical analyses were performed using SPSS (version 22.0; SPSS Inc., Chicago, IL, USA). Intergroup differences were determined using the independent sample t test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables, as appropriate. Two-side p values of <0.05 were considered statistically significant.

Results

Selection of study participants

Fig 1 shows the flowchart of patient selection and the enrolment criteria. Between 2010 and 2017, 248 lung specimens yielding NTM from 214 cases were retrieved from the mycobacteriology databases of six hospitals. After applying a serial of exclusion criteria mentioned above, a total of 43 resected NTM-SPNs from 43 cases were finally selected for analysis. The follow-up duration of each patient was at least two years since resection, with the mean of 5.2 ± 2.8 years.

Fig 1

Case selection process.

(CT, computerised tomography; NTM, nontuberculous mycobacteria; PD, pulmonary disease; SPN, solitary pulmonary nodule) a Including echo-guided biopsy, bronchoscopic biopsy, and computerised tomography-guided biopsy. b Including 131 specimens obtained by wedge resection, 40 by lobectomy, and 9 by segmentectomy. c Including 66 specimens from 48 cases of multiple lung nodules, 14 specimens from 10 cases of multiple consolidations, and 23 specimens from 18 cases of other radiographic patterns, such as nodular infiltrations, bronchiectasis, ground-glass opacities, and atelectasis. d The symptom was productive cough in four and hemoptysis in the other three.

Clinical characteristics of patients

The clinical characteristics of the 43 patients with NTM-SPN are summarised in Table 1. The mean age was 61.7 ± 13.4 years, with a female/male ratio of 1.5. Among them, 88% had never smoked and 3 (7%) were malnourished (BMI < 18.5 kg/m2). The most common underlying diseases were the complete remission of malignancy after treatment (n = 11, 26%), including 5 patients with early stage lung cancer, 3 with colorectal cancer, 2 with breast cancer and 1 with prostate cancer. Six (14%) patients had the underlying disease of diabetes mellitus (DM, 14%). None received serology test for Human Immunodeficiency Virus (HIV) infection.

Table 1

Clinical characteristics of the 43 patients with nontuberculous mycobacteria (NTM)-solitary pulmonary nodule.

Variables	N = 43
Age (year)	61.7 ± 13.4
Female sex	26 (60%)
Body-mass index (kg/m2)	22.6 ± 3.5
<18.5	3 (7%)
Smoking status
Current	3 (7%)
Former	2 (5%)
Underlying diseases
History of pulmonary tuberculosis	2 (5%)
Bronchiectasis	1 (2%)
Chronic obstructive pulmonary disease	1 (2%)
Asthma	2 (5%)a
Lung cancer under complete remission	6 (14%)b
Other cancer under complete remission	5 (12%)c
Diabetes mellitus	6 (14%)
Rheumatoid arthritis	2 (4%)d
Baseline laboratory data
Leukocyte (K/uL)	6.1 ± 1.7
Hemoglobin (g/dL)	13.0 ± 1.6
Platelet count (K/uL)	222.8 ± 55.1
Aspartate transaminase (U/L)	23.2 ± 6.9
Alanine transaminase (U/L)	19.4 ± 9.9
Total bilirubin (mg/dL)	0.6 ± 0.2
Creatinine (mg/dL)	0.9 ± 0.3
Albumin (g/dL) (n = 48)	4.3 ± 0.4
CEA (n = 42)	1.9 ± 1.7
Baseline lung function (n = 46)
FEV1 (% of predicted)	110.5 ± 22.1
FEV1/FVC	81.3 ± 4.9
Obstructive defect	1 (2%)
Restrictive defect	4 (9%)

Abbreviation: CEA, carcinoembryonic antigen; FEV1, forced expiratory volume in one second; FVC, forced vital capacity.

Data are either number (%) or mean ± standard deviation.

a One patient had severe asthma under long-term steroid therapy. The other patient received prednisolone at 15 mg per day for 6 months and used long-term inhaled corticosteroids.

b Histological reports of resected lung tissue demonstrated lung adenocarcinoma in five patients and another one is squamous cell carcinoma.

c Three patient had colorectal cancer, one patient had breast cancer, and one patient had prostate cancer.

d Both patients received long-term disease-modifying antirheumatic drugs and steroids.

All patients were asymptomatic, and their SPNs were discovered incidentally during a health examination or routine work-up for underlying comorbidities. Five patients (10%) had impaired lung function. The laboratory data were within normal ranges.

Radiographic features and characteristics of SPN

Table 2 summarises the characteristics of NTM-SPNs. The most common locations of resected SPNs were the right upper lobe (32%) and left upper lobe (16%). The mean diameter of SPNs was 1.8 ± 1.0 cm, nine (21%) of the SPNs were cavitary lesions and two of them had the calcification (5%). Positron emission tomography (PET) was performed in 16 patients, with the maximal standardised uptake value (SUVmax) being 6.4 ± 7.2 (median: 4.2; IQR: 2.0–5.9).

Table 2

Operation methods, results of tissue histopathology, and mycobacteriology studies of the 43 patients with nontuberculous mycobacteria (NTM) solitary pulmonary nodule.

Variables	N = 43
Preoperative radiographic features
Location: right side	29 (67%)
Right upper lobe	14 (32%)
Right middle lobe	7 (16%)
Right lower lobe	8 (19%)
Location: left side	14 (33%)
Left upper lobe	7 (16%)
Left lower lobe	7 (14%)
Cavitation	9 (21%)
Calcification	2 (5%)
Size (cm)	1.8 ± 1.0
PET-CT, SUVmax (n = 16)	6.4 ± 7.2
Operation method
Wedge resection	38 (88%)
Lobectomy	3 (7%)
Segmentectomy	2 (5%)
Histopathology
Caseating granulomatous inflammation	25 (58%)
Non-caseating granulomatous inflammation	8 (19%)
Chronic inflammation	10 (23%)
Tissue mycobacteriology study
No. of AFS-positive case
Grade 1 or 2	7 (16%)
Grade 3 or 4	2 (5%)
Culture
MAC	26 (60%)
M. kansasii	8 (19%)
M. abscessus	3 (7%)
M. fortuitum	2 (5%)
M. chelonae	1 (2%)
Unidentified NTM	3 (7%)a

Abbreviations: AFS, acid-fast smear; MAC, Mycobacterium avium complex; PET-CT, positron emission tomography–computed tomography; SUVmax, maximal standardised uptake value.

Data are represented as either number (%) or mean ± standard deviation.

a The histological reports of the 3 resected SPNs all indicated granulomatous inflammation. Tissue mycobacterial culture yielded photochromogens in 2 and nonphotochromogens in the remaining one. However, species identification was not possible under routine laboratory procedures.

The surgical method of SPN removal was wedge resection in 38 (88%), lobectomy in 3 (7%), and segmentectomy in the remaining 2 patients (5%). The result of lung tissue AFS was positive in 9 (21%) patients. The most common mycobacterial isolate of the 43 SPNs was MAC (n = 26, 60%), followed by M. kansasii (8, 19%). No surgical complications occurred. Among patients with SPN, 33 (77%) had histological findings suggestive of mycobacterial infection, including 25 (58%) and 8 (19%) showed caseating granulomatous inflammation and noncaseating granulomatous inflammation, respectively.

Sputum mycobacteriology study

Mycobacteriological study of either sputum or bronchoalveolar lavage was performed before surgery in 36 (84%) of the 43 patients with NTM-SPN (Table 3). Of these samples, the culture results from bronchoscopic specimens were positive for MAC in one case with M. kansasii-SPN and M. gordonae in another with MAC-SPN.

Table 3

Preoperative and follow-up sputum mycobacteriology study and preoperative radiological features of the 43 patients with nontuberculous mycobacteria (NTM)-solitary pulmonary nodule (SPN).

Variables		N = 43
Pre-operative mycobacteriology study from respiratory tract
Bronchoscopic specimens (n = 12)		12
Number of expectorated sputum samples per case (n = 24)		1.8 ± 1.6
Number of cases with positive sputum AFS		0
Culture-positive for NTM other than that isolated from SPN		2 (5)a
Follow-up mycobacteriology study from respiratory tract (n = 19)b
Bronchoscopic specimens		6
Number of expectorated sputum samples per case		1.9 ± 0.7
Timing of follow-up (years after operation)		3.3 (1.2)
No. of AFS-positive case		0
Culture-positive for NTM		2 (5)
M. gordonae		2
Same species as the tissue culture		0

Abbreviation: AFS, acid-fast smear; MAC, Mycobacterium avium complex.

Data are number (%), mean ± standard deviation or median (IQR).

a The culture results from bronchoscopic specimen were positive for MAC in one case with M.kansasii-SPN and M. gordonae in another with MAC-SPN.

b 24 patients failed to produce any sputum even upon sputum induction using hypertonic saline solution and were considered as sputum culture-negative for NTM.

During follow-up, 24 (56%) patients had no or scanty sputum even upon sputum induction and 6 of them only ever received bronchoscopic samplings thereafter. None of the follow-up respiratory samples yielded the same NTM species as the initial tissue culture. Two isolates of M. gordonae were identified from bronchoscpic specimens in two patients.

Outcome status

A total of four, three with MAC-SPN and one with M. kansasii-SPN ever received standard anti-TB treatment after surgery, with a treatment duration of 160 ± 44.6 days (Table 4). None of the patients received effective anti-NTM treatment for more than 6 months. During the mean duration of follow-up 5.2 ± 2.8 years (median: 4.2 years [2.5–7.0]), there were no mortalities, and NTM-PD was not diagnosed in any patient because of the lack of suggestive radiographic and microbiologic evidence.

Table 4

Treatment outcomes of the 43 patients with nontuberculous mycobacteria (NTM)-solitary pulmonary nodule (SPN).

Variables	N = 43
Ever received any anti-mycobacterial drug	4 (9) a
Received effective chemotherapy against NTM-PD >6 months	0
Follow-up duration (years after operation)
To last date of chest CT follow-up (n = 35)	2.5 ± 1.9
To last date of chest x-ray follow-up (n = 43)	3.8 ± 2.1
To last date of hospital visit (n = 43)	5.2 ± 2.8
Outcome
Mortality	0
New pulmonary lesion	1 (2)
Mycobacteriology recurrence	0
Fulfilling the diagnosis of NTM-PD	0

Abbreviation: PD, pulmonary disease.

Data are expressed as either number (%) or mean ± standard deviation.

a Three cases had Mycobacterium avium complex–SPN, and the remaining one had M. kansasii-SPN. The primary care physicians decided to complete a full course of antituberculosis treatment (160 ± 44.6 days) based on the histological finding of granulomatous inflammation and positive acid-fast smear of resected SPNs. All the 4 patients were clinically asymptomatic.

In one patient who underwent wedge resection of the right upper lobe for MAC-SPN, a new lesion with nodular infiltration was noted in the same lobe but different location as previous one on chest CT at 9 months after the operation. She remained asymptomatic during this period (Fig 2). The subsequent mycobacterial cultures from 3 expectorated sputum samples and 1 bronchoscopic sample were all negative for NTM during a 2-year follow-up.

Fig 2

A) Chest computerised tomography (CT) of a 79-year-old asymptomatic woman before wedge resection shows a 1.7 × 1.0 cm2 lobulated nodule over the right upper lobe (RUL). The tissue culture yielded Mycobacterium avium complex. She remained asymptomatic and did not receive medical treatment after resection. B) CT performed 9 months later shows new nodular infiltrates over the RUL. In the subsequent 2 years, the patient remained asymptomatic. The subsequent mycobacterial cultures from 3 expectorated sputum samples and 1 bronchoscopic sample were all negative for NTM.

Discussion

This is the first retrospective study to investigate the long-term outcomes of asymptomatic patients with incidentally discovered NTM-SPN after surgical resection. Two main findings were obtained. First, surgical resection is curative for NTM-SPN in asymptomatic patients without positive culture of the same NTM species from respiratory specimens and a history of NTM-PD. Further medical treatment for NTM will probably not be necessary. Second, MAC and M. kansasii accounted for 79% of the resected SPNs.

The detection rate of SPN is increasing because of the increasing use of chest CT in current clinical practice and the performance of routine health check-ups. In one study, granuloma was discovered to be the most common cause of benign SPNs and was usually, if not always, assumed to be TB related [6]. NTM pulmonary infection has been demonstrated to present as SPN [11, 15, 22], with an incidence of 2% in a study conducted in South Korea [22]. The true incidence and outcome of NTM-SPN remain unclear because bacteriological confirmation of resected lung tissue has been lacking in most studies [9, 17, 22–25], and sometimes both patients with SPN and those with other radiographic patterns of lung lesions with multiple involvement have been enrolled [22, 26]. According to a literature review, we found only 74 cases of culture-confirmed and pure NTM-SPN, which were identified in six studies (Table 5) [9, 17, 22, 25–27]. Although the duration of follow-up in these studies varied from 0 to 10 years, the overall mean duration was less than 2 years. In addition, crucial information was frequently missing. Medical treatment was administered to only 19 (26%) patients, usually a regimen against TB rather than NTM [9, 26]. Nevertheless, in only 2 of the 74 cases (one of them received anti-NTM treatment after resection), recurrent nodular infiltration was discovered at the other lobe, and no bacteriological evidence of NTM recurrence was obtained [22]. Because of the heterogeneity in patient selection criteria and clinical evaluation, small sample size, and relative short follow-up duration of the above-mentioned studies, reliable recommendations for the management of NTM-SPN remain to be determined.

Table 5

Literature review of the clinical characteristics and postoperative outcomes of patients with solitary pulmonary nodule (SPN) caused by nontuberculous mycobacteria (NTM).

Case No.Ref	Underlying disease	Male sex	Agea(year)	No symptom	Histology	SPN size (cm)	Main NTM spp. (%)	Same NTM spp. in sputum	Medical Txa (%)	FU duration (year)	Recurrent case (%)
14 [9]	n.a.	4	64 (53–70)	100%	GR	n.a.	MAC (86%)	1	10 (71%)b	1.7 (1.0–10)	0
24 [25]	Old TB: 2; Cancer: 2	8	64 (49–77)	96%	GR	0.7–3.0	MAC (100%)	0	0%	Mean: 0.7	0
2 [17]	COPD	n.a.	n.a.	0	CG	n.a.	M. xenopi (100%)	0	n.a.	n.a.	n.a.
28 [26]	n.a.	12	59 (33–79)	89%	n.a.	n.a.	MAC (96%)	2	9 (32%)	Mean: 1.8	2 (7%)c
5 [22]	n.a.	n.a.	n.a.	n.a.	GR	1.3–3.4	n.a.	3	0	0.5–6	0
1 [27]	0	1	46	100%	GR	2.5	M. kansasii	0	0	0.5	0

Abbreviations: CG, caseating granuloma; CI, chronic inflammation; COPD, chronic obstructive pulmonary disease; FU, follow-up; GR, granuloma; n.a., not available; OP, operation; Tx, treatment.

a Data are expressed as median (range) unless otherwise mentioned.

b The 10 patients received anti-tuberculosis treatment.

c Both had recurrent nodule at different lobes from the previous surgical site, but none underwent mycobacterial culture for the recurrent nodule. One received medical treatment for NTM after operation.

Anti-TB treatment lasting 6–12 months is routinely administered after surgery for pulmonary granuloma caused by M. tuberculosis [28]. Because of the global trend of aging, the high risk of toxicity due to “empiric anti-TB treatment” [29] should be carefully balanced with potential benefit of the treatment. In a retrospective study, 67 patients with lung nodule(s) and histopathological findings suggestive of TB but lacking microbiologic confirmation were followed up, and they were not immediately administered anti-TB treatment [30]. Within 3.7 ± 0.7 years, only one, a cancer patient under regular chemotherapy and target therapy, developed active TB, corresponding to an incidence rate of 4 cases/1000 person-years. Although NTM is less virulent than M. tuberculosis, its multidrug treatment is even more toxic and lengthy [31, 32]. Therefore, determining the necessity of postoperative anti-NTM treatment for patients with completely resected NTM-SPN is of practical importance. In this study, which can be considered the largest longitudinal study in NTM-SPN, none of the 43 patients with NTM-SPN developed NTM-PD within 5.2 ± 2.8 years, suggesting that conservative management with regular follow-up, rather than immediate chemotherapy, may be sufficient for treating NTM-SPN.

In this study, MAC was the predominant causative organism of NTM-SPN (60%), which is in compatible with previous reports [9, 11, 22, 25, 26, 33]. Interestingly, M. kansasii was more common in this study cohort than in those of previous reports [26, 27], accounting for 19% of the SPNs. This might be related to increasing M. kansasii isolation in Taiwan [18, 34].

During follow-up, 2 patients had subsequent different NTM isolates from sputum as initial tissue culture. The single NTM isolate from respiratory tract should be interpreted with caution under consideration of the colonization rather than true pathogen for patients with NTM-SPN [15, 26].

This study has some limitations. First, this retrospective study lacked a standardised treatment and follow-up protocol. Second, a resected SPN was sent for mycobacterial culture at the discretion of the surgeon. This might have resulted in underestimation of the incidence of NTM-SPN. Third, 4 patients received the anti-TB medication after surgical resection, which may be a potential confounder to alter the subsequent outcome. Lastly, because serology test for HIV infection was not checked and few patients received steroids or immunomodulating agents, the impact of immunosuppression, either disease-associated or iatrogenic, on the risk and clinical course of NTM-SPN remains to be studies.

In conclusion, regular follow-up rather than immediate administration of toxic and lengthy chemotherapy for incidentally discovered NTM-SPN after surgical resection should be the optimal management strategy for asymptomatic patients without culture evidence of the same NTM species from respiratory specimens. Considering the increasing prevalence of NTM-related disease worldwide and the improving detection of SPNs, routine mycobacterial culture for resected SPN could be considered for differentiating TB and NTM because their treatment differs.

20 Aug 2019

PONE-D-19-16108

Surgical resection is sufficient for incidentally discovered solitary pulmonary nodule caused by nontuberculous mycobacteria in asymptomatic patients

Dear Dr Inn-Wen Chong

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Adriano Gianmaria Duse, MD

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Comments to the Author

Many thanks for the submission of this manuscript. The manuscript is technically sound and well-written. Current practice for patients with asymptomatic solitary pulmonary nodules caused by NTMs is generally to consider that resection is sufficient treatment, but there are limited data to support this practice. While this is a retrospective study, it is unlikely RCTs will be performed given the relative rarity of this condition, and the data do add to current knowledge.

We kindly request you to correct the grammatical errors listed below as well as to address the comments of the reviewers.

Grammatical / typographical errors:

Introduction

"tuberculoma accounted for 60% of benign SPNs, but none of them yielding nontuberculous mycobacteria (NTM)". Suggest "...none of them yielded..."

Methods:

"and their four branch hospitals between January 2010 and Jnauary 2017."

Correct spelling

Results:

"During follow-up, 24 (56%) patients had no or scanty sputum even upon sputum

induction and 6 of them ever received bronchoscopic samplings thereafter"

Not clear what "...ever received bronchoscopic samplings" means. Maybe "...6 of them only ever received bronchoscopic sampling thereafter"?

Discussion:

Further medical treatment for NTM will probably not necessary. Suggest "..not be necessary".

This might be resulted in underestimation of the incidence of NTM-SPN. Suggest "...might have resulted in..."

A few minor clarifications are required:

1) The method of case ascertainment was not entirely clear. Based on the figure it seems that the authors examined a database containing all positive cultures for NTM, and then looked for patients with resected solitary lung nodules. Additional detail regarding the exact methods used to identify potential cases would be helpful.

One of the exclusion criteria was the same sputum sample being culture-positive for the NTM species isolated from the SPN before resection. What do you mean by "same sputum sample" - is this a sputum sample taken at the same time as the surgery? Did you define a time period relative to the surgery when the sputum sample could/should have been taken?

2) As this was a retrospective cohort study, follow-up methods were not standardized for all patients, which may have led to under-ascertainment of recurrent nodules. The authors should specify whether patients were censored in follow-up based on the last CT scan obtained, the last plain chest radiograph obtained, or some other criterion. Ideally, follow-up would only be counted based on the last CT (which would be the "gold standard" test here). There is a footnote to Table 4 that provides some information, but it would be helpful to provide person-years of follow-up based on CT only.

3) Fig 1: Some of the numbers do not add up:

2nd step to 3rd step: starting with 154 cases of surgical lung specimens, and excluding 76 with multiple nodules, leaves 78 cases not 77. 4th step to 5th step: starting with 63 SPNs, and excluding 13 plus 3 plus 7 leaves 40, not 43.

4) It would be helpful to include data on initial PET/CT findings, if any patients had this test performed.

5) 3 patients had "unidentified NTM" in the nodules. Did these patients have granulomatous inflammation and/or acid-fast bacilli noted on histopathology? If not, this raises concern that the NTM were lab contaminants.

6) Table 2: PET-CT and SUV are listed in the legend but not in the table.

7) Table 3 – legend – medium should be median

8) 4 patients received TB treatment – why? Surely this implies they were symptomatic in some way? Or was TB isolated from other specimens, even though they were asymptomatic?

9) Table 4 states that patients received no NTM therapy for > 6months. The text implies received no NTM treatment at all. Please clarify - did some receive NTM therapy for <6 months?

10) Discussion: You state "4 patients received the transient anti-TB medication after surgical resection, which may be a potential confounder to alter the subsequent outcome." Leave out "the transient" - mean TB treatment was 160 days.

11) Was HIV status tested in any of the patients? If not, this is something to mention in the results as the conclusions may not be generalisable to an HIV-infected population. While some of the patients included were on steroids, most were not on any active immune modulating treatment - so the conclusions in relation to any form of immunosuppression may also be questioned.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

26 Aug 2019

Responses to Editor

Q1. We kindly request you to correct the grammatical errors listed below as well as to address the comments of the reviewers.

Ans: We apologize for the errors and have corrected the grammatical errors in the revised manuscript. We have also addressed the reviewers’ comments, item-by-item. Please see the Responses to Reviewers. Thank you.

Responses to Reviewers

1) The method of case ascertainment was not entirely clear. Based on the figure it seems that the authors examined a database containing all positive cultures for NTM, and then looked for patients with resected solitary lung nodules. Additional detail regarding the exact methods used to identify potential cases would be helpful.

Ans: Thank you for the comment. We have revised the step-by-step description in Figure 1 in the revised manuscript. The descriptions on case selection process in the main text have also been revised (2nd and 3rd paragraphs of the methods section).

2) As this was a retrospective cohort study, follow-up methods were not standardized for all patients, which may have led to under-ascertainment of recurrent nodules. The authors should specify whether patients were censored in follow-up based on the last CT scan obtained, the last plain chest radiograph obtained, or some other criterion. Ideally, follow-up would only be counted based on the last CT (which would be the "gold standard" test here). There is a footnote to Table 4 that provides some information, but it would be helpful to provide person-years of follow-up based on CT only.

Ans: We thank for the instructive comment. The definition of follow-up duration was added in the methodology (Page 8, Line 1-4). The durations of chest CT follow-up, chest X-ray follow-up and clinical follow-up were also added in the Table 4 of the revised manuscript.

3) Fig 1: Some of the numbers do not add up: 2nd step to 3rd step: starting with 154 cases of surgical lung specimens, and excluding 76 with multiple nodules, leaves 78 cases not 77. 4th step to 5th step: starting with 63 SPNs, and excluding 13 plus 3 plus 7 leaves 40, not 43.

Ans: Thank you for the reminder and we apologize for the confused numbers in this figure. We repeatedly counted the 3 cancer patients in the group with pulmonary symptoms. We had corrected the numbers of Figure 1 in the revised manuscript.

4) It would be helpful to include data on initial PET/CT findings, if any patients had this test performed.

Ans: Thank you for the nice suggestion. Positron emission tomography (PET) was performed in 16 patients, with the maximal standardised uptake value (SUVmax) being 6.4 ± 7.2 (median: 4.2; IQR: 2.0–5.9). We have provided the information in the Results section (Page 12, Line 16-18) and Table 2.

5) 3 patients had "unidentified NTM" in the nodules. Did these patients have granulomatous inflammation and/or acid-fast bacilli noted on histopathology? If not, this raises concern that the NTM were lab contaminants.

Ans: We apologize for the misleading. The histological reports of the 3 resected SPNs all indicated granulomatous inflammation. Tissue mycobacterial culture yielded photochromogens in 2 and nonphotochromogens in the remaining one. However, species identification was not possible under routine laboratory procedures. We have footnoted this information in Table 2 of the revised manuscript. Thank you for the comment.

6) Table 2: PET-CT and SUV are listed in the legend but not in the table.

Ans: Thank you for the reminder. As suggested by the reviewer in the 4th comment, we have added the information on PET-CT in Table 2 of the revised manuscript.

7) Table 3 – legend – medium should be median

Ans: We apologize for the typo and have corrected it in the revised manuscript. Thank you.

8) 4 patients received TB treatment – why? Surely this implies they were symptomatic in some way? Or was TB isolated from other specimens, even though they were asymptomatic?

Ans: Thank you for the thought-provoking comment. For the 4 patients, the primary care physicians decided to complete a full course of anti-TB treatment based on the histological finding of granulomatous inflammation and positive acid-fast smear of resected SPNs, even knowing that patients were asymptomatic and culture of SPNs yielded NTM. We have footnoted this information in Table 4 of the revised manuscript.

9) Table 4 states that patients received no NTM therapy for > 6months. The text implies received no NTM treatment at all. Please clarify - did some receive NTM therapy for <6 months?

Ans: We apologize for the confusing statement. We have revised the descriptions in Table 4 and in the Results section (Page 17, Para 1) as “None of the patients received effective anti-NTM treatment for more than 6 months”.

10) Discussion: You state "4 patients received the transient anti-TB medication after surgical resection, which may be a potential confounder to alter the subsequent outcome." Leave out "the transient" - mean TB treatment was 160 days.

Ans: Thank you for the comment. We have deleted the incorrect wording “transient”.

11) Was HIV status tested in any of the patients? If not, this is something to mention in the results as the conclusions may not be generalisable to an HIV-infected population. While some of the patients included were on steroids, most were not on any active immune modulating treatment - so the conclusions in relation to any form of immunosuppression may also be questioned.

Ans: Thank you for the instructive comments. In this retrospective study, none of the 43 patients received serology test for HIV infection (mentioned in Page 10, Line 5-6). We totally agree with you that the impact of immunosuppression, either disease-associated or iatrogenic, on the risk and clinical course of NTM-SPN remains to be studied. We have added the limitation in the Discussion section (Page 24, Line 12-15).

Submitted filename: 20190825_Responses.docx

Click here for additional data file.

29 Aug 2019

Surgical resection is sufficient for incidentally discovered solitary pulmonary nodule caused by nontuberculous mycobacteria in asymptomatic patients

PONE-D-19-16108R1

Dear Dr. Chong

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Adriano Gianmaria Duse, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All the recommended changes have been addressed adequately in the revised document.

Reviewers' comments:

4 Sep 2019

PONE-D-19-16108R1

Surgical resection is sufficient for incidentally discovered solitary pulmonary nodule caused by nontuberculous mycobacteria in asymptomatic patients

Dear Dr. Chong:

I am 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.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Adriano Gianmaria Duse

Academic Editor

PLOS ONE