Correlation of drug exposure and bacterial susceptibility with treatment response for Mycobacterium avium complex lung disease: protocol for a prospective observational cohort study

Abstract

Introduction

The burden of Mycobacterium avium complex (MAC) lung disease is increasing globally and treatment outcome is in general poor. Therapeutic drug monitoring has the potential to improve treatment outcome by ensuring adequate drug exposure. However, very limited population-based studies exist for MAC lung disease. This study aims to describe the distribution of drug exposure for key antimycobacterial drugs at population level, and to analyse them in relationship to treatment outcome in patients with MAC lung disease.

Methods and analysis

A prospective cohort aiming to include 100 adult patients diagnosed with and treated for MAC lung disease will be conducted in Shanghai Pulmonary Hospital, China. Blood samples will be collected after 1 month MAC treatment for measurement of macrolides, rifamycin, ethambutol, amikacin and/or fluoroquinolones, using a validated liquid-chromatography tandem mass spectrometry method. Respiratory samples will be collected at inclusion and once every 3 months for mycobacterial culture until treatment completion. Minimum inhibitory concentration (MIC) determination will be performed using a commercial broth microdilution plate. In addition to mycobacterial culture, disease severity and clinical improvement will be assessed from the perspective of lung function, radiological presentation and self-reported quality of life. Whole genome sequencing will be performed for any longitudinal isolates with significant change of MIC to explore the emergence of drug resistance-conferring mutations. The relationship between drug exposure and treatment outcome will be analysed and potential confounders will be considered for adjustment in multivariable models. Meanwhile, the associations between drug exposure in relation to MIC and markers of treatment response will be explored using Cox proportional hazards or binary logistic regression models, as appropriate.

Ethics and dissemination

This study has been approved by the ethics committee of Shanghai Pulmonary Hospital (No. K22-149Z). Written and oral informed consent will be obtained from all participants. The study results will be submitted to a peer-reviewed journal.

Trial registeration number

NCT05824988.

Correspondence to Professor Wei Sha; [email protected]

STRENGTHS AND LIMITATIONS OF THIS STUDY

• We used a comprehensive approach, taking into account the drug exposure, population pharmacokinetic modelling and bacterial minimum inhibitory concentration (MIC), as well as microbiological, clinical and self-reported measures for treatment response.

• Bacterial culture conversion is an imperfect surrogate marker of the final treatment outcome due to its low sensitivity, thus lung function, radiological presentation and self-reported well-being will be assessed in this study, to fully understand the improvement of patients during Mycobacterium avium complex treatment.

• A limited sampling strategy with population pharmacokinetic models for estimation of drug exposure will be adopted as a supplement to rich blood sampling to increase the study feasibility and sample size.

• Inherent variations exist for microdilution method used for MIC determination, thus the relationship between drug exposure/MIC and treatment outcome will only be investigated exploratorily.

Introduction

Non-tuberculous mycobacteria (NTM) are opportunistic bacterial pathogens commonly seen in populations with underlying lung diseases, immunodeficiency and old age.¹ Along with a growing ageing population, the incidence and prevalence of NTM infections has gradually increased over the years in both developed and developing countries.^2–6 A national surveillance study in 2021 showed that Mycobacterium avium complex (MAC) was the most prevalent NTM specie in China, accounting for nearly 61% of NTM lung disease.⁷ In addition to the rapid increase of disease burden, treatment of MAC is a challenge for both patients and healthcare due to the long treatment duration, frequent drug-induced adverse events, lack of treatment alternatives, poor treatment outcome and high recurrence rate.^{8 9} Despite that treatment of MAC lung disease is recommended for at least 12 months after culture conversion,¹⁰ only 61.4% of treated individuals achieved treatment success according to a systematic review in 2018.⁹

The evidence regarding the association between minimum inhibitory concentration (MIC) and treatment outcome is still limited in MAC lung disease, as highlighted in the latest guidelines released by the ATS/ERS/ESCMID/IDSA.¹⁰ MIC data exists for drugs such as macrolides and amikacin,^{11 12} whereas data is scarce for alternatives such as rifamycin and ethambutol.¹³ The difficulties in explaining the clinical value of MIC testing might partially be attributed to the lack of in vivo drug exposure data, which cannot be accurately predicted by the dose administered due to between-patient pharmacokinetic (PK) variability.¹⁴ A retrospective study found that the suboptimal plasma concentrations of clarithromycin (56%), azithromycin (35%) and ethambutol (48%) were common despite a recommended standard dose.¹⁵ For clarithromycin, another study from Korea found a high proportion of subtherapeutic concentrations (97%, 96/99), defined as peak plasma concentration below 2 mg/L.¹⁶ The drug–drug interaction is one of the causes for subtherapeutic clarithromycin exposure, where rifampicin significantly lowers the drug exposure of clarithromycin by 68% due to liver enzyme upregulation.¹⁵ As for the association between drug exposure of macrolides and treatment response in MAC disease, the results from different studies are conflicting.^{16 17}

In infectious diseases, therapeutic drug monitoring (TDM) is a strategy to guide and personalise treatment by measuring both plasma drug concentrations and pathogen susceptibility. TDM is recommended for antimycobacterial treatment, including patients with NTM with poor treatment response¹⁰ and some categories of patients with tuberculosis.^18–20 Previous cohort studies on tuberculosis have highlighted the clinical benefits of TDM,^{21 22} but no studies are yet published for MAC lung disease. Based on in vitro hollow fibre infection models, a series of studies have been performed to identify the pharmacokinetic/pharmacodynamic (PK/PD) targets for ethambutol, moxifloxacin, linezolid and terizidone to improve dose guidance for treatment of MAC.^23–26 Although the hollow-fibre studies have hypothesised that the treatment failure rate seen in real life is caused by the low probability of target attainment and low kill rate under current doses, this needs to be confirmed in prospective studies. To the best of our knowledge, there is only one study measuring both drug exposure and bacterial MIC in patients with MAC lung disease, although there was no comparison of the PK/PD indices, that is, drug exposure/level of bacteria resistance, with treatment response.¹⁵ The limited number of studies might be attributed to the inconvenience of multiple, so-called rich blood sampling required to estimate area under drug concentration-time curve (AUC). Therefore, a limited sampling strategy, derived from the population PK model, has been proposed as a useful tool to facilitate PK studies and TDM in clinical routine.¹⁸

A more holistic approach is needed for evaluation of response to MAC treatment. The definition of treatment outcome for NTM is based on mycobacterial culture from respiratory samples,²⁷ which is insensitive especially at the late stage of treatment and among patients without symptom of persistent cough. There is some evidence to support that 6-month culture conversion is predictive of final treatment outcome and may be considered as an interim endpoint of efficacy.^{28 29} Other important markers of improvement include lung function tests, as a substantial decline in lung function has been reported to be associated with treatment failure of NTM lung disease.³⁰ Moreover, CT scan can visualise the resolution or deterioration of pulmonary lesions. Considering that MAC treatment is long and cumbersome, the overall health, daily life and perceived well-being are also important aspects for assessment.

In this protocol, we describe a comprehensive approach, taking into account drug exposure, population PK modelling, bacterial MIC as well as a variety of measures for treatment response in patients with MAC lung disease. The primary aim of the study is to describe the distribution of drug exposure for key antimycobacterial drugs at a population level, and to analyse them in relation to treatment outcome. A secondary aim is to explore associations between drug exposure or PK/PD indices and multiple markers of clinical improvement and safety.

Methods and analysis

Study design

A prospective cohort study of drug exposure in relation to bacterial MIC and treatment outcome in patients with MAC lung disease will be conducted in Shanghai, China. This project is led by the Tongji University affiliated Shanghai Pulmonary Hospital, with the guidance and support from Fudan University, The University of Sydney, Karolinska University Hospital, Karolinska Institutet and Shanghai Municipal Center for Disease Control and Prevention. The results of this study will be reported following the Strengthening the Reporting of Observational Studies in Epidemiology Statement guideline for cohort studies.

Study setting

The study will be carried out in Shanghai, a large metropolitan city located in the eastern coastal region of China. Since there is no mandatory requirement for reporting NTM infection, the main source of patients with NTM lung disease in China is patients investigated for tuberculosis. As a national surveillance study showed, the proportion of NTM lung disease in patients examined for tuberculosis is 7–8% in eastern China,⁷ whereas the corresponding number in Shanghai is nearly 10% according to local statistics. The study hospital is one of the largest designated tuberculosis hospitals in China, a teaching hospital with five specialised mycobacteria wards. Recruitment of patients will be performed by the study hospital and a screening log will be kept. The study opened for recruitment on 14 April 2023 and is expected to be completed in October 2026 (ClinicalTrials.gov, NCT05824988). The recruitment will last for 1 year and may be prolonged depending on the number of enrolled patients.

Study participants

Statistics in the study hospital indicate that the annual number of patients with MAC lung disease is around 200. Approximately, half of them are believed to be eligible for enrolment according to the criteria below, which is equivalent to a sample size of 100. Considering that the study aim is descriptive and explorative, no pre-study calculation of sample size was performed. Post hoc power analysis will be performed for the primary outcome when necessary to verify power. Rich blood sampling (six samples over 8 hours) will be performed for the first 30 patients aged <65 years to enable the development of population PK models. A limited sampling strategy will be used for study participants ≥65 years, as the collection of multiple blood samples in older patients is regarded as a sensitive issue in the context of Chinese traditional culture. After population PK models have been developed, limited sampling strategy will be applied for all study participants regardless of age.

Inclusion criteria

• Adults aged ≥18 years.

• Culture-positive MAC lung disease.

• MAC treatment at Shanghai Pulmonary Hospital.

• A regimen composed of at least three core drugs, that is, macrolides, rifamycin and ethambutol, in doses not lower than recommended according to the ATS/ERS/ESCMID/IDSA and Chinese national guidelines.^{1 10}

• Written informed consent.

Exclusion criteria

• Pregnancy.

• Confirmed mixed infection with mycobacterial species, including Mycobacterium tuberculosis and other NTM species.

• Ongoing antimycobacterial treatment for more than 1 month, including tuberculosis and NTM.

• Patients admitted to the intensive care unit.

• Off-label use for any study drugs, such as inhalation of amikacin.

Study outline

The overall study outline is shown in figure 1. The diagnosis and treatment of MAC lung disease will adhere to the ATS/ERS/ESCMID/IDSA guidelines.¹⁰ In brief, two positive culture results from separate expectorated sputum samples or one positive culture from bronchoalveolar lavage fluid (BALF) samples will be considered to meet microbiological criteria for diagnosis. Patients treated with a regimen composed of macrolides, rifamycin and ethambutol at minimum will be screened for eligibility, including those with an addition of other drugs, such as amikacin, fluoroquinolones or linezolid. The recommended doses for study drugs are summarised in table 1. Patients prescribed with lower than recommended doses will not be considered for this study.

Table 1

Summary of pharmacokinetic/pharmacodynamic (PK/PD) characteristics and recommended doses for the most frequently used drugs in study hospital^42–46

Drug	Dose	Administration	Optimal PK/PD indices	Normal Tmax (hours)
Clarithromycin	≥50 kg: 1000 mg BID <50 kg: 750 mg TID	Oral	AUC/MIC	2
Azithromycin	≥50 kg: 500 mg QD <50 kg: 250 mg QD	Oral	AUC/MIC	2–3
Rifampicin	≥50 kg: 600 mg QD <50 kg: 450 mg QD	Oral	AUC/MIC	2
Rifapentine	≥50 kg: 600 mg BIW <50 kg: 450 mg BIW	Oral	AUC/MIC	5–6
Ethambutol	15 mg/kg QD	Oral	AUC/MIC; Cmax/MIC	2–3
Amikacin	10–15 mg/kg QD	Intramuscular	Cmax/MIC; AUC/MIC	0.5–1.5
Levofloxacin	500 mg QD	Oral	AUC/MIC	1–2
Moxifloxacin	400 mg QD	Oral	AUC/MIC	1–2
Linezolid	600 mg QD	Oral	AUC/MIC	1.5

AUC, area under drug concentration-time curve; BID, two times a day; BIW, two times a week; MIC, minimum inhibitory concentration; QD, one time a day; TID, three times a day; T_max, time to peak drug concentration.

Figure 1. Study outline. SGRQ, St. George’s Respiratory Questionnaire. * Additional pharmacokinetic sampling will be performed for patients with poor treatment response at 6 months using a limited sampling strategy (2 and 6 hours).

Baseline is defined as the date when MAC treatment is started in the study hospital, or in other hospitals if a standard regimen was given during the past month. After informed consent, a baseline questionnaire will be used to collect demographic, behaviour and clinical information by a designated study nurse. Meanwhile, lung function test and the St. George’s Respiratory Questionnaire (SGRQ) will be given to obtain baseline levels. Study participants will be followed-up after 1-month treatment, and then every 3 months until treatment completion. A comprehensive assessment will be performed at 6 months of treatment and patients with persistent positive culture or resolution of pulmonary lesions by less than 30% are considered as having a poor treatment response. Post-treatment visits will be given at 6 and 12 months after treatment completion to assess the recurrence of MAC lung disease.

The safety of the study patients will be closely monitored at each clinical visit by liver and renal function tests, routine blood tests and vital signs. On these basis, adverse events will be recorded. Study participants with serious drug-induced adverse events will be admitted for inpatient treatment. Meanwhile, a CT scan is routinely performed once every 3 months in China until completion of MAC treatment, and is required by Chinese NTM treatment guidelines regardless of inclusion or exclusion for this study.¹ In the study hospital, a low-dose CT scan is applied to minimise radiation exposure levels.

Drug concentrations will be measured for all study patients at 1 month after treatment initiation to enable attainment of steady state. Rich blood sampling (0, 1, 2, 4, 6 and 8 hours) will be implemented for the first 30 patients aged <65 years, and limited sampling (2 and 6 hours) for the rest patients. Additional PK sampling will be performed for patients with poor treatment response at 6 months using a limited sampling strategy (2 and 6 hours). If key study drugs or doses were adjusted 1 week prior to the planned day, blood sampling will be delayed by at least 1 week. At the day of blood sampling, detailed drug and food intake will be noted by the study nurse in a record card to assess delayed absorption and possible interactions. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) will be used to measure drug concentrations in plasma.

Respiratory samples (sputum and/or BALF) will be collected at inclusion and once every 3 months for mycobacterial culture using BACTEC MGIT 960 during MAC treatment.³¹ Time to mycobacterial culture positivity will be recorded to estimate the bacterial load as an alternative for colony forming units count.³² MIC determination and whole-genome sequencing (WGS) will be performed for baseline, 6-month and/or the last available positive culture during treatment, to assess the development of acquired drug resistance and reinfection.

Apart from microbiological work, the disease severity will be evaluated and monitored in following aspects. Since patients with underlying disease are at higher risk of MAC infection, the Charlson Comorbidity Index will be used to evaluate comorbid conditions, as it has been translated and validated in Mandarin.³³ Our early work showed that bronchiectasis was the most frequent comorbidity in patients with MAC lung disease, close to 80%.³⁴ Hence, the easy-to-use modified Reiff score will be applied to score the radiological extension of bronchiectasis based on lobe involvement (calculating lingula as a separate lobe) and the degree of dilatation (tubular=1, varicose=2, cystic=3).³⁵ The total score ranges from 0 to 18, with 0 representing no bronchiectasis with CT scan. Lung function test will be performed by a spirometer to monitor the impact of MAC on lung function. The overall health, daily life and perceived well-being will be measured by the SGRQ in a self-reported way. All questionnaires will be translated into a Mandarin version by our research group (online supplemental table 1).

Outcome measure

Primary outcome measures are peak concentrations (C_max) and AUC for key antimycobacterial drugs in patients with MAC lung disease. C_max and AUC will be depicted and their association with treatment response will be investigated, with and without relation to MIC.

Secondary outcome measures include multiple markers of clinical improvement and safety. The final treatment outcome will be evaluated at the end of MAC treatment and at follow-up after treatment completion. The definition will refer to the Nontuberculous Mycobacteria Network European Trialsgroup (NTM-NET) consensus statement.²⁷ In brief, the latter consists of microbiological or clinical cure, cure (both microbiological and clinical), treatment failure, death, unknown outcome (including lost to follow-up and transfer out), treatment halted as well as recurrence. From the microbiology perspective, 6-month culture conversion and time to culture conversion will be recorded. Culture conversion is defined as at least three consecutive negative mycobacterial cultures from respiratory samples, collected at least 4 weeks apart.²⁷ Other secondary outcome measures include acquisition of drug resistance during MAC treatment; resolution of pulmonary lesions or cavitation on CT scan; change of lung function with forced expiratory volume in 1 s and forced vital capacity as the main parameters; improvement of life quality by SGRQ; and occurrence of grade 3 or 4 adverse events defined in accordance with the Division of AIDS guidelines.³⁶

Laboratory methods

Drug concentration measurement

Assays for measurement of clarithromycin, azithromycin, rifampicin, rifapentine, ethambutol, amikacin, moxifloxacin, levofloxacin and linezolid are under development on the basis of an LC-MS/MS system, in specific AB SCIEX Triple Quad 5500MD. The optimised assays will be validated according to the 2018 US Food and Drug Administration guideline for bioanalytical method validation.³⁷ The collected venous blood samples will be centrifuged at 3500 rpm for 10 min within 1 hour from sampling. Aliquots of plasma will be then frozen at −80°C awaiting analysis.

Microbiological work

All microbiological work will be done at a biosafety level 2 laboratory in the Shanghai Pulmonary Hospital, apart from the WGS analysis which will be performed at the Shanghai Municipal Center for Disease Control and Prevention.

Mycobacterial species identification

MeltPro Mycobacteria Identification Kit (Zeesan Biotech, Xiamen, China), a licensed domestic commercial kit, will be used for identification of 19 common Mycobacterium species, based on the probe-based melting curve analysis.³⁸ After positive culture of MAC in the BACTEC MGIT 960 system, 1 mL of liquid culture will be centrifuged at 12 000 rpm for 5 min. After removing the supernatant, a volume of 300 µL lysis buffer will be added. The detailed procedure is referred to the manufacturer’s instructions.

Minimum inhibitory concentration

Baseline, 6-month and/or the last positive mycobacterial isolates during MAC treatment will be collected for MIC determination. A commercially available high-throughput broth microdilution plate, that is, Sensititre SLOMYCO2 Susceptibility Testing Plate (Trek Diagnostic System, Thermo Fisher, USA), is routinely used in study hospital to test the susceptibility of MAC isolates to 12 antibiotics simultaneously. The specific test drug and concentration range is shown in figure 2. The reference isolate Mycobacterium peregrinum ATCC 700686 or Staphylococcus aureus ATCC 29213 will be included in each test run and compared with previously published quality control target ranges for each drug. Result reading will be performed with Sensititre Vizion after 7–14 days, depending on growth.

Figure 2. Commercially available Sensititre SLOMYCO2 Susceptibility Testing Plate used in study hospital. The colours of green, yellow and orange represent susceptible, intermediate and resistant, respectively, as recommended by the CLSI guidelines. The grey indicates there are no recommended breakpoints up to yet. AMI, amikacin; CFZ, clofazimine; CIP, ciprofloxacin; CLA, clarithromycin; CLSI, Clinical and Laboratory Standards Institute; DOX, doxycycline; LZD, linezolid; MIN, minocycline; MFX, moxifloxacin; POS, positive control; RIF, rifampicin; RFB, rifabutin; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole.

Whole genome sequencing

MIC results for baseline, 6-month and/or the last positive isolates during treatment will be compared. Those with at least 2 two-fold dilutions change of MIC, considering the inherent assay variation in the MIC test,³⁹ will be sent for WGS analysis to detect new resistance mutations. In brief, Pathogen Lysis Tubes S Kit and QIAamp DNA Mini Kit (Qiagen, Hilden, GER) will be used for genomic DNA extraction. Libraries will be constructed by Illumina DNA Prep (Illumina, San Diego, California, USA), and paired-end 150 bp DNA sequencing will be performed on a NovaSeq 6000 platform (Illumina, San Diego, California, USA) with an expected coverage of 100×. Pairwise single nucleotide polymorphism (SNP) distances between isolate genomes will be calculated after removing the recombination in Snp-dists (V.0.8.2). The resistance genes will be referred to those of M. tuberculosis, including rifampicin (rpoB), clarithromycin (rrl), ethambutol (embB) and amikacin (rrs).

Data collection and analysis plan

The study hospital has developed a specific database for mycobacterial disease, which extracts data from digital medical charts, examination reports as well as laboratory records on a weekly basis. Examination and laboratory results, including CT scan, lung function, routine blood tests, liver and renal function tests as well as microbiological results, can be exported from it. Study-specific case report forms will be used to collect data on treatment regimens, adherence, self-reported well-beings and other information that is not contained in the mycobacteria specific database. Monthly reports will be prepared to update the project progress within the research group and virtual meetings will be held when discussion is necessary.

Population PK modelling will be performed in the patients assigned with rich blood sampling strategy. The model will be built using a non-linear mixed-effect modelling in Phoenix NLME (V.8.0; Certara, Princeton, New Jersey, USA). The selection of structural model will be based on visual inspections of plasma concentration versus time and a review of existing literatures. The impact of covariates, such as age, bodyweight, body mass index and comorbidities, on PK parameters will be evaluated using a stepwise method with forward inclusion and backward elimination. Visual predictive checks will be performed to assess the performance of developed population PK models.

Developed population PK models will be used to estimate AUC values for study drugs. The distribution of C_max, AUC, MIC, percentage of time that concentration persisted above MIC (%T_>MIC), C_max/MIC and AUC/MIC will be presented and visualised in graphs. The relationship between drug exposure and treatment outcome will be analysed and potential confounders, such as treatment interruption and regimen change after PK sampling, will be considered for adjustment in multivariable models. Meanwhile, the associations between PK/PD indices and markers of treatment response will be explored using Cox proportional hazards or binary logistic regression models, as appropriate. Drug–drug interactions will be explored using population PK models or multivariate adaptive regression splines.

Patient and public involvement

Patients were not involved in the design, recruitment or conduct of the study. The results of the study can be obtained in Mandarin on request at Shanghai Pulmonary Hospital.

Ethics and dissemination

The study will be performed in accordance with Good Clinical Practice and the Declaration of Helsinki. Ethical approval has been obtained from the ethics committee of Shanghai Pulmonary Hospital (No. K22-149Z).

Prior to study initiation, a training workshop on the study protocol and ethical considerations was held by the main study investigators from Shanghai Pulmonary Hospital. All clinicians, nurses and laboratory staff who participated in this study attended the workshop. Patients will be informed about the study orally and in writing by study investigators. They will be ensured that neither study participation nor study termination, regardless of what the reason is, will result in any changes in their treatment. Informed consent will be prepared in duplicate, and be signed by both patient and study investigators. In case of illiteracy, fingerprint is allowed under observation by a witness after being fully informed about the study as confirmation of understanding of the study. Blood samples will be measured within 2 weeks after sampling and the results will be fed back to clinicians on time. Considering the nature of an observational study, the adjustment of treatment regimen is not mandatory but on the decision made by clinicians. Standard of care will be given to all patients at the study hospital and regular monitoring will be performed to ensure their safety.

We aim to present our data in international and domestic conferences and to publish the results in a peer-reviewed journal. Any significant amendments to study protocol will be reported to the ethical boards in Shanghai Pulmonary Hospital.

Discussion

In this prospective observational cohort study regarding TDM for MAC lung disease, we present a comprehensive approach taking into account drug exposure, population PK modelling, bacterial MIC as well as an assessment of treatment response. The findings from our study are likely to be of benefit in future trials on TDM in MAC lung disease. Rich blood sampling will enable the development of population PK models for accurate calculation of PK indices, specifically in this patient population. Since mycobacterial culture has the inherent limitation of insensitivity for treatment response, lung function test, CT scan and well-being will be applied in this study as supplementary measures. The SGRQ will be used to quantify well-being, as it has been reported sensitive to treatment response of Mycobacterium abscessus lung disease.^{40 41} Together with bacterial MIC and clinical data, the C_max/MIC and AUC/MIC for antimycobacterial drugs will be explored to deepen our understanding regarding the correlation of PK and/or PD indices with treatment response and may thus guide the development of new dosing strategies.

There is a large knowledge gap between PK/PD indices and treatment response in MAC lung disease. A milestone study was conducted 10 years ago, with retrospective measurement of both drug exposure and bacterial MIC, and yielded important information on pharmacological interactions and low concentrations of key drugs including macrolides, but did not correlate PK/PD indices with treatment responses.¹⁵ Compared with rare population-based studies, in vitro studies using hollow fibre infection models to suggest tentative targets for antimycobacterial drugs are relatively prosperous,^23–26 including a C_max/MIC>1.23 for ethambutol, an AUC/MIC>17.12 for moxifloxacin and an AUC/MIC>7.82 for linezolid. It is with regret that none of them have been validated by population-based clinical studies.

A limitation of this study protocol is the use of two blood sampling strategies, including a rich and a limited sampling strategy. The main consideration for this design is the sensitive issue of rich blood sampling in China, especially in elder people. To enable the estimation of drug AUC for those with limited sampling, population PK models will be developed in this study. We believe this design can assist in increasing the size and feasibility of our study. Second, despite the fact that the efficacy of antimycobacterial drugs is affected by both drug exposure and bacterial MIC, the primary aim of this study is to analyse drug exposure in relation to treatment outcome. One of the main reasons is the low correlation between MIC and treatment outcome for the drugs used to compose MAC treatment regimen, apart from macrolides and amikacin.¹⁰ In addition, there is innate variability of MICs measured by current commercial MIC plates,³⁹ despite automatic pipette and results reading applied in this study. A variability of up to ±1 two-fold MIC dilution step will result in great changes of PK/PD indices. Third, bacterial culture conversion is an imperfect surrogate marker of final treatment outcome due to its low sensitivity, especially in patients with NTM who have been reported with lower rates of persistent cough compared with patients with tuberculosis. Nevertheless, culture conversion is recognised as the standard measure to define treatment outcome. In this study, lung function, CT scan and perceived well-being will be used as a comprehensive assessment of treatment responses.

In conclusion, the findings from this study will provide useful insights into the distribution of drug exposure among patients with MAC lung disease and its relationship with treatment outcome. PK/PD indices and their association with markers of clinical improvement will be explored. Hopefully, the present study will inspire future studies on TDM for both MAC and other NTM lung diseases. The development of assays for drug concentration analysis and population PK models for core drugs are expected to facilitate TDM studies, thus enabling the generation of population-based evidence to support precise and individualised treatment for MAC lung disease.

Ethics statements

Patient consent for publication

Not applicable.

Footnote

XZ and LW contributed equally.

Contributors XBZ and LW designed the study and wrote the first draft of the manuscript. LDF, YDL, JB, YH, JWA, WS and BX provided critical suggestions on study design, manuscript writing and revision. YYZ, YHC and XJL wrote the laboratory part for this manuscript. All authors read and approved the final manuscript.

Funding This work was supported by the National Natural Science Foundation of China (grant number 82204107), the Shanghai Clinical Research Center for Infectious Disease (Tuberculosis) (grant number 19MC1910800), the Shanghai Key Clinical Specialty Construction Project (Tuberculosis) (grant number shslezdzk03001) and the Shanghai 2020 ‘Science and Technology Innovation Action Plan’ Technological Innovation Fund (grant number 20Z11900500). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests None declared.

Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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