Comparison of efficacies of different treatments for nontuberculous mycobacterial pulmonary disease in Anhui Province, China

Introduction: Although nontuberculous mycobacterial (NTM) infection is a common cause of pulmonary disease worldwide, few studies have focused on epidemiological and therapeutic factors related to NTM cases in Anhui Province, China. This retrospective study aimed to identify aetiological and clinical factors, and treatment outcomes of patients with NTM pulmonary disease (NTMPD) in Anhui. Methodology: Retrospective clinical data obtained from medical records of NTMPD patients seeking care at Anhui Chest Hospital from July 2019 to June 2022 were analyzed. Treatment outcomes were compared between two patient groups: one receiving a standardised NTM treatment regimen and the other receiving precision treatment regimens. Results: Genotypic analysis of 672 clinical NTMPD-associated isolates revealed that most were Mycobacterium intracellulare , while drug-susceptibility test results demonstrated diverse antibiotic resistance profiles for these isolates. Cough was the most common symptom for 101 NTMPD patients. After patients of both groups received treatment, symptoms improved, sputum culture conversion was observed for some patients, imaging findings stabilised; however, no statistically significant intergroup differences in treatment outcomes were found. Conclusions: In this study, M. intracellulare was the predominant NTM species identified in isolates obtained from NTMPD patients. Drug resistance profiles of our patient isolates were complex, highlighting the need for administration of timely, more effective, standardised treatments for patients with NTMPD in Anhui Province, China.


Introduction
Nontuberculous mycobacteria (NTM) are primarily environmental organisms and typically do not cause disease in humans.Nevertheless, certain NTM species are opportunistic pathogens, capable of illnesses distinct from tuberculosis (TB) and leprosy caused by Mycobacterium tuberculosis (MTB) and Mycobacterium leprae, respectively.
In recent years, more than 30 newly identified NTM species have been discovered, many of which exhibit heightened pathogenicity toward humans.An analysis of publicly available data revealed a strikingly high clinical NTM isolation rate in China.However, this rate likely underestimates the true clinical NTM prevalence rate, since pathogenic NTM are frequently unculturable.In cases where a NTM isolate cannot be cultured, diagnoses of patients presenting with pulmonary illnesses of unknown aetiology must rely on clinical findings by experienced physicians [1][2][3].
As opportunistic pathogens, NTM predominantly infect vulnerable individuals, including those with immunodeficiencies, histories of drug abuse, or exposures to specific environmental factors.Once inside susceptible hosts, NTM organisms can infiltrate various tissues and organs, such as lungs, lymph nodes, skin, and bones [4][5][6].Unlike TB, which is caused by MTB, diseases caused by NTM are particularly difficult to treat, due to their inherent resistance to most antibiotics.Moreover, research studies have shown that even individuals who have been successfully treated for NTM disease remain susceptible to reinfection upon reexposure to these opportunistic pathogens [7,8].
NTM pulmonary disease (NTMPD) is the most prevalent form of NTM-related disease, accounting for approximately 70-80% of reported cases outside of China.However, epidemiological survey data based on a large number of isolates obtained from Chinese NTMPD cases have not yet been reported.Nevertheless, findings of smaller NTMPD-focused Chinese studies have indicated a notable increase in the NTM isolation rate from 4.3% in 1979 to 22.9% in 2010, suggesting growing incidence of NTM disease in the country.Susceptibility to NTM infection is influenced by various host risk factors, including underlying pulmonary conditions, drug-induced or disease-induced immunodeficiencies, and environmental factors such as exposure to tap water [2].
Current guidelines for NTM treatment primarily rely on consensus clinical opinions or case reports rather than well-designed large clinical studies.Consequently, current treatments administered to NTMPD patients tend to mirror treatment regimens used for drug-resistant TB.However, these regimens often consist of multiple antibiotics that can potentially trigger serious adverse events.Furthermore, global NTMPD-associated mortality rates have not been determined, due to a lack of studies analyzing treatment outcomes and disease-related adverse events in NTMPD patients [3,9,10].
This study focuses on Anhui Province, a region in central China with a population of over 63 million people.Before 2018, Anhui reported few cases of NTMPD due to limited economic, technological, and healthcare resources there.Our study aimed to retrospectively analyze clinical characteristics of NTMPD patients in Anhui Province using available resources in that region.Moreover, we compared efficacies of standardised and precision NTMPD treatment regimens to provide insights and to guide the development of improved diagnosis and treatment strategies for use in managing NTMPD patient care in resource-limited areas such as Anhui Province.

Study design and population
In this study, we retrospectively collected clinical data from medical records of NTMPD patients seeking care at Anhui Chest Hospital from July 2019 to June 2022.We also collected sputum, bronchoalveolar lavage fluid (BALF), and pus specimens from the patients whose post-culture yielded a total of 776 clinical isolates.Following completion of microbiological screening and identification assays, 104 isolates were identified as normal flora or environmental contaminants, leaving a total of 672 NTM isolates, including 668 cultured from sputum and BALF samples and four cultured from pus samples.Given the NTMPD focus of this study, we selected 101 NTMPD cases with complete clinical data for further statistical analysis.The patient selection process is outlined in Figure 1.Selected patients were assigned to two treatment groups based on NTM drug resistance profiles determined via drug susceptibility testing (DST): the standardised treatment group (53 cases) and the precision treatment group (48 cases).
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committees of Anhui Chest Hospital(K2021-013). Informed consent was obtained from all subjects involved in the study.

Laboratory testing methods: NTM culture, species identification, and drug susceptibility tests (DST)
All patient specimens, including sputum, BALF, and pus, were pretreated and cultured using the BACTEC MGIT 960 liquid culture method (Becton, Dickinson and Company, Maryland, USA).Isolates testing positive via the colloidal gold method with paranitrobenzoic acid (PNB) medium (Baso Biotechnology Co., Ltd., Zhuhai, China) were tentatively identified as NTM using an MPB64 antigen detection kit (Genesis Bio-detection & Biocontrol Co., Ltd., Hangzhou, China).Isolates preliminarily identified as NTM were then classified at the species level using the MeltPro Myco assay using a pan-mycobacterial primer set (Zeesan Biotech, Xiamen, China).Species assignments were conducted based on proteomic fingerprinting via matrix-assisted laser desorption ionisation−time of flight mass spectrometry (MALDI-TOF MS) and interpreted using the Bruker MALDI-TOF MS identifier (Agena Bioscience, Shanghai, China).
DST was performed following the broth microdilution method reported previously by Wang et al. [11]

Patient selection
Data were retrospectively collected from medical records of 101 NTMPD patients treated at Anhui Chest Hospital between July 2019 to June 2022.The patient cohort consisted of 53 males and 48 females with a male-to-female ratio of 1.1:1.The average age of patients was 59.8 (± 13.2) years, with ages ranging from 21-89 years.All patients received treatment for NTMPD for a minimum period of 9 months.
NTMPD patients were identified based on diagnostic criteria outlined in the British Thoracic Society guidelines for the management of nontuberculous mycobacterial pulmonary disease (NTM-PD) (2017) [12] and the Chinese expert consensus on diagnosis and treatment of NTM diseases (2012) [1].NTMPD diagnosis was based on exclusion of other diseases and relied on observed respiratory symptoms and/or systemic symptoms, along with chest imaging findings showing cavitary shadows, multifocal bronchiectasis, and/or multiple small nodular lesions.During collection of patient sputa, BALF, and pus specimens, precautions were taken to prevent contamination of specimens with exogenous organisms.Additionally, patients met one of the following criteria: (i) two positive sputum cultures for the same NTM species; (ii) BALF yielding one NTM-positive culture result and/or one NTM-positive molecular biological test result, along with a positive acid-fast bacilli smear score of ++ or higher; (iii) sputum yielding one NTMpositive culture result and/or BALF yielding one positive molecular biological test result, along with a positive acid-fast bacilli smear score of ++ or higher.
Patients excluded from the study included pregnant or lactating women, patients infected with both MTB and NTM, immunodeficient patients, and those who discontinued treatment for various reasons.
All patients included in the study also met the following criteria: (i) medical records contained complete clinical data, such as sputum acid-fast bacillar smear, sputum culture, chest computed tomography (CT) scan results, and other findings and test results; (ii) strictly adhered to physician-guided medication administration and refrained from altering or discontinuing treatments without physician approval; (iii) experienced no serious adverse reactions during treatment.

Treatment
The patients were assigned to two groups based on DST results, one receiving the standardised treatment and one receiving precision treatments.In the standardised treatment plan, patients received treatment with at least four different types of drugs following established guidelines and consensus recommendations.In the precision plan, patients initially received antibiotics similar to those in the standardised plan.However, once DST results were available, only drugs showing antimicrobial activities against their specific NTM isolates were continued or added to the regimen, while drugs lacking such activity were discontinued.The updated precision treatment plan included a minimum of five drugs with activity against each patient's specific NTM isolate [1].
For example, for patients with M. intracellulare infection, treatment initially followed the standardised plan.As such, patients received oral azithromycin (300 mg/d) or clarithromycin (500 mg/d), rifampicin (450 mg/d), ethambutol (15 mg/kg-d), and injectable amikacin (400 mg, three times per week).In the precision plan, the initial dosage regimen mirrored the standard treatment, except that clarithromycin was discontinued due to DST-confirmed resistance to macrolides.Additionally, patients received a minimum of five drugs based on DST results showing drug sensitivity, which were selected from the following list: linezolid (600 mg/d), rifampicin (450 mg/d), ethambutol (15 mg/kg-d), amikacin (400 mg, three times per week), rifabutin (300 mg/d), moxifloxacin (400 mg/d), or cefoxitin (1 g every 8 hours).

Therapeutic evaluation
Symptom assessment: Numbers of patients experiencing cough, haemoptysis, chest distress, and/or fever before treatment and after 2, 6, and 9 months of treatment were recorded and compared between the two groups.
Bacterial efficacy: Culture and smear testing was conducted on sputum specimens collected before treatment and after treatment for 2, 6, and 9 months for both groups; then results were statistically analyzed.A positive sputum smear result was defined as the detection of acid-fast bacilli in sputum, while a negative sputum smear result was indicated by three negative sputum smear results for acid-fast bacilli.A negative culture result was defined as one or more negative sputum or BALF culture results.
Imaging manifestations: Chest CT scans were taken after 6 and 9 months of treatment; and lesions on scans were counted and lesion extent was determined by calculating the proportion of lung fields containing lesions.Treatment efficacy was defined based on changes in lesion number and/or pathology, as well as on cavity changes.
Lesion number changes were categorised as follows: (i) significant absorption: absorption of over half of original lesions.(ii) absorption: absorption of less than half of the original lesion.(iii) unchanged: no significant absorption or deterioration of lesions.(iv) worsening: increase in lesion size and/or pathological changes.
Cavity changes were categorised as follows: (i) reduction: maximum cavity diameter reduced by half of the original maximum diameter.(ii) unchanged: maximum cavity diameter decreased or increased by less than half of the original maximum diameter.(iii) increase: maximum cavity diameter increased by more than or equal to half of the original maximum diameter.

Data analysis
All data were statistically analyzed using Statistical Package for the Social Sciences (SPSS) 22.0 software.The mean value was expressed as  � ± S. Statistical analysis was performed using the Chi-square test and a significance level of p < 0.05 considered statistically significant.

DST results of NTM strains
Table 1 presents antibiotic resistance rates for each NTM species present in our collection of NTMPD patient isolates.Notably, 100% of M. avium isolates exhibited resistance to multiple drugs, while 95% of M. abscessus isolates exhibited resistance to most antibiotics.Additionally, 100% of NTM isolates belonged to Mycobacterium species M. chelonae, M. columbia, M. marseillense, M. fortuitum, and M. gordonae, and were resistant to imipenem-cilastatin, along with 95% of NTM isolates of other species.An analysis of antibiotic resistance rates of M. intracellulare and M. abscessus isolates (Table 2) revealed significant differences in resistance rates to rifampin, ethambutol, moxifloxacin, and rifabutin.M. abscessus isolates had high-level drug resistance rates to rifampin, ethambutol, moxifloxacin, and rifabutin of 95.35%, 94.19%, 39.53%, and 69.77%, respectively.In contrast, resistance rates of M. intracellulare isolates to these drugs were 4.26%, 23.12%, 1.22%, and 3.04%, respectively.These rates were statistically significantly different between the two species (p < 0.001).

General patient clinical characteristics
Table 3 provides an overview of the general clinical characteristics of all patients.Notably, more than half of patients were affected by marasmus (57.43%), as indicated by low body mass index value.The majority of patients worked as farmers (54.46%), and the proportion of retirees ranked second.Geographically, the highest numbers of patients lived in Hefei, Lu'an, and Fuyang, accounting for 39.60%, 16.83%, and 12.87% of patients, respectively (Figure 3).Approximately 26.73% of patients were illiterate.
NTM: nontuberculous mycobacterial infection.Therapeutic observation Among the 101 NTMPD patients included in this study, 73 were infected with M. intracellulare and 28 with M. abscessus.Patient symptoms, sputum culture results, and imaging findings are summarised in Tables 4-6.Overall, no significant differences in rates of fever, cough, and haemoptysis symptoms were observed between groups receiving standardised and precision treatments after the initial standardised treatment period.However, after 9 months of treatment, the precision treatment group exhibited a higher symptoms remission rate as compared to that of the standardised treatment group.
Our results also showed a marked decrease in the proportion of patients with positive sputum smear results in both treatment groups after 6 months of antibiotic therapy as compared to baseline results (obtained before treatment initiation).Nevertheless, no statistically significant intergroup differences were observed after 2, 6, or 9 months of treatment.Similarly, although the proportion of positive sputum culture results also decreased with increasing treatment duration, no statistically significant intergroup differences were observed after 9 months of treatment.
Analysis of imaging findings revealed no significant differences in lesion severity or cavity changes between the two groups after 6 months of treatment, although lesions of one patient in the standardised treatment group and two patients in the precision treatment group worsened.After 9 months of treatment, the standardised treatment group exhibited a statistically significant higher lesion absorption rate as compared to that of the precision treatment group.However, no significant difference in cavity changes was observed between the two groups (Figure 4).
During the data collection process, 10 patients were excluded from the treatment efficacy analysis due to serious adverse reactions (Supplementary Table 1).Adverse drug-induced reactions were detected in a small proportion of patients; generally mild, and mainly included drug-induced liver damage.Adverse reactions improved after symptomatic treatment (Supplementary Table 2).

Discussion
The results of this retrospective cohort study provide information regarding the prevalence and clinical characteristics of NTM infections occurring in a representative population of Anhui Province.In particular, we also compared the therapeutic effectiveness of two NTMPD patient treatment plans as guided by DST results in the real-world setting of our hospital.Our results demonstrated that treatment of NTM infections can be challenging, thus suggesting that selection of an appropriate therapeutic schedule is of equal importance to the use of DST results to guide the formulation of a suitable antimicrobial treatment regimen.
It is well known that the distribution of NTMs exhibits significant regional diversity.For example, in Europe and North America, NTM-infected patients are frequently found to be infected with organisms belonging to the Mycobacterium avium Complex (MAC), M. gordonae, and M. bufo species.Meanwhile, in South America, predominant opportunistic Mycobacterium species isolated from NTM-infected patients include MAC, M. kansasii, and M. gordonae, while in Asia isolated NTM species are predominantly MAC, M. abscessus, and other opportunistic Mycobacterium species [13].
Notably, results of the current study suggest that the NTM species in Anhui Province are primarily M. intracellulare and M. abscessus, as consistent with findings obtained in Nanjing by Hu et al. [14].However, these results significantly differ from those obtained from NTM patients in Chongqing, Shanghai, and other regions of China, which collectively suggest that notable regional NTM distribution differences exist across China [15,16].
The results of this study also highlight the complexity of NTM drug resistance profiles in Anhui Province and raises serious concerns.Importantly, all NTM isolates analyzed in this study exhibited extreme high-level resistance to imipenem-cilastatin, a commonly used antibiotic in clinical settings.The mechanism of resistance has not been fully elucidated.
Accumulated evidence indicates that NTM exhibit inherent antibiotic resistance through various mechanisms.This resistance may arise from effects of robust NTM cell walls, and/or surrounding biofilm and granuloma-associated barriers with limited drug permeability that reduce NTM drug exposure and drug uptake.Additionally, NTM may possess capabilities supporting enhanced drug efflux, heightened drug metabolism, and/or decreased drug sequestration, and may express proteins that selectively target common antibiotics.Moreover, mutations of the rpoB gene, a target of rifampicin, are the main cause of acquired drug resistance in both M. avium complex and M. kansasii.Furthermore, rifampicin has been shown to preferentially inhibit transcription of one of the two rpoB promoters, leading to increased transcription of the other promoter that supports the development of strains with enhanced rifampicin resistance [17].Consequently, these drugs should be administered with caution to patients with suspected or confirmed NTM infections.
An analysis of patient clinical characteristics revealed that NTMPD patients in Anhui were predominantly elderly male farmers, which aligns with the results of another study conducted in South Korea [18].These observations may be partly explained by the fact that men tend to have higher numbers of daily contacts with soil than women [19].Furthermore, more than half of NTMPD patients exhibited marasmus, as consistent with observations reported by Blakney et al. [20], while a large proportion of our patients suffered from hypoproteinemia and anaemia.Taken together, these findings suggest a possible relationship between nutritional status and development of NTMPD [21].
While the relationship between NTM drug sensitivity and treatment effects has not been extensively discussed previously [22], the results of this study indicate that DST-guided customisation of treatment did not provide obvious advantages.Nevertheless, recommended best practices dictate that once patients are diagnosed with NTMPD, they should receive standardised therapies according to recommended guidelines as soon as possible in order to avoid increased economic and psychological burdens associated with delayed treatment.

Limitations
There were several limitations in this study.This project was a single-centre retrospective study that generated results that may not be applicable to populations outside of Anhui Province.Additionally, the treatment follow-up period of both of our treatment groups was at most 9 months, shorter than the duration of the standard NTM treatment regimen.Moreover, no assessments of treatment-induced organ damage were conducted for use in evaluating and comparing the safety of NTM treatments.Finally, our analysis did not include additional factors, such as drug combination, treatment durations of the different medications, or patient treatment compliance as potentially influential factors on patient treatment outcomes.Additional prospective, controlled studies are needed to evaluate the effects of these factors on patient prognosis and validate the findings of the current study.

Figure 1 .
Figure 1.The flow chart of selection of NTMPD patients.

Figure 3 .
Figure 3. Geographic distribution of NTMPD prevalence in Anhui Province.

Table 1 .
Drug sensitive test results of NTM.

Table 2 .
Drug sensitive test results of M. intracellulare and M. abscessus.
N: number of resistant strains; Rate: drug resistance rate (%).

Table 3 .
Clinical data analysis of NTMPD in Anhui Province.

Table 6 .
Imaging changes after treatment.