Analysis of the types and characteristics of non-tuberculous mycobacteria in high endemic areas of tuberculosis

doi:10.21203/rs.3.rs-3996092/v1

Objective: To understand the epidemiological, demographic and clinical characteristics of non-tuberculous mycobacterial lung disease (NTM-PD) in areas with high incidence of tuberculosis.

Methods: A prospective surveillance study was conducted for non-tuberculous mycobacterial(NTM) at the Affiliated Hospital of Zunyi Medical University for five consecutive years. A total of 6259 culture-positive specimens grown on Roche medium between January 2017 and December 2021 were collected. After acid-fast staining, colony morphology, growth time, and p-nitrobenzoic acid detection, suspicious NTM strains were screened and identified by gene sequencing, and the dominant groups were subjected to drug sensitivity test.

Results: A total of 107 suspected NTM colonies were screened from 6259 positive cultures, including 51 cases of NTM, 13 cases of MTB, and 43 cases of other bacteria. Among the 51 NTM positive patients, 43.1% were 45-65 years old. All the NTM positive specimens were derived from the respiratory tract. In 2017-2021, the isolation rate of NTM was the highest (1.18%) and the lowest (0.77%), and the main bacterial type was Mycobacterium abscessus (33.33%), followed by Mycobacterium intracellulare (27.45%), Mycobacterium chelates (11.76%), Mycobacterium kansasii (9.80%), Mycobacterium avium (7.84%), Mycobacterium gordonae (3.92%), Mycobacterium smegmatis (3.92%), and Mycobacterium bradyxanthum (1.96%). Other bacteria were dominated by Gordonia spp. The dominant NTM bacteria were Mycobacterium abscessus and Mycobacterium intracellulare, which had a high drug resistance rate. Middle-aged, male, bronchiectasis and serum albumin < 3.5 g/L were risk factors for NTM-PD.

Conclusions: The trend of NTM infection rate is stable in this area, but the drug resistance rate is high. M. abscessus is the most common pathogen of NTM. Middle-aged, male patients with bronchiectasis and serum albumin < 3.5 g/L have a high risk of progressing to NTM-PD. In areas with a high incidence of TB, clinicians should make a differential diagnosis between suspected pulmonary TB and NTM infections.

Non-tuberculous mycobacteria (NTM)

Mycobacterium abscessus

Mycobacterium intracellulare

Resistance to drugs

Non-tuberculosis mycobacteria (NTM) is a general term for mycobacteria other than Mycobacterium tuberculosis (MTB) complex and Mycobacterium leprosy. NTM is widely found in water, soil, dust and other natural environments, and more than 200 species of NTM have been identified to date [1]. NTM is an opportunistic pathogen, and the morbidity and mortality of NTM diseases have been increasing steadily worldwide. Human-to-human transmission of NTM has been reported internationally, and data show that the proportion of immunocompetent children and adults infected with pulmonary NTM is also increasing [2]. NTM infection can invade multiple organs and tissues of the human body, and chronic lung disease is more common. However, the clinical symptoms and imaging examinations of lung disease caused by NTM are similar to those of tuberculosis (TB), which is difficult to distinguish [3]. Moreover, in countries or regions with a high burden of TB, acid-fast staining is the main laboratory examination method. Because acid-fast staining cannot distinguish between MTB and NTM, patients with NTM infections are easily misdiagnosed as TB, especially as multidrug-resistant TB (MDR-TB) [3, 4]. Most NTMs are resistant to anti-TB drugs and antibiotics, further adding to the treatment challenge [5]. China has a high burden of TB, and the isolation rate of NTM is increasing. The incidence and drug resistance of TB in Guizhou province are serious concerns [6-8]. At present, there is no report on the incidence of NTM in this area. Therefore, it is important to study the epidemic types and clinical characteristics of NTM in areas with severe TB epidemic for the diagnosis and treatment of mycobacterial infection.

1.1 Materials

1.1.1 Source of specimens

The Affiliated Hospital of Zunyi Medical University is a tertiary general hospital in Guizhou Province and one of the main medical centers in the healthcare system of Guizhou Province. The hospital was designated as the main medical institution in the regional referral system of MDR-TB in 2014, and specialized in treating MDR-TB patients diagnosed in the hospital and referred from three prefecture-level cities in northern Guizhou province (Bijie, Tongren, and Zunyi). A total of 6259 positive cultures were collected from the Affiliated Hospital of Zunyi Medical University between January 2017 and December 2021, and 6213 were positive by acid-fast staining. According to the colony morphology, growth time and p-nitrobenzoic acid test, 107 suspected NTM were screened for subsequent study. (Figure 1)

1.1.2 Reagent information

Acid-fast staining reagent, acid Roche medium, P-nitrobenzoic acid medium and solid drug sensitive medium were purchased from Zhuhai Bso Biotechnology Co., LTD.

1.2 Methods

1.2.1 NTM traditional bacterial type identification and drug sensitivity test

The laboratory of Respiratory Medicine has been certified by the Chinese Center for Disease Control and Prevention (CDC) to conduct MTB culture and drug susceptibility testing. Sputum samples from patients with clinically suspected pulmonary TB were cultured and tested for drug sensitivity by the respiratory medicine laboratory according to the protocol recommended by the World Health Organization (WHO). Suspected NTM isolates were screened from positive culture colonies of suspected TB patients for testing. The suspected NTM positive cultures were first subjected to acid-fast staining, positive colonies were subjected to PNB test, and then the positive colonies were subjected to sequencing analysis. After sequencing, the dominant NTM species were selected for drug sensitivity test to 14 anti-TB drugs.

1.2.2 Colony DNA extraction and sequencing

One or two bacterial colonies in logarithmic growth phase were scraped into the inner tube of a 1.5 ml EP, after which 200 μl of normal saline was added. After the screw lid was tightened, the bacterial colonies were sealed with sealant and placed in an 85℃ water bath for 15 minutes for inactivation. The samples were packaged and sent to Qingk Biotechnology Co., LTD for sequencing (NTM specific primer sequences were: 16S rRNA-P1: TGGAGAGTTTGATCTGG

CTCAG, 16S rRNA-P2: TACCGCGGCTGCTGGCAC). The sequencing data were submitted to www.ridom-rdna.de database for comparison, and the highest similarity (> 97%) were the result type. If the comparison did not correspond to any known mycobacterial species, it was likely a new mycobacterium or other kinds of bacteria, which were further analyzed on the NCBI (www.ncbi.nlm.nih.gov/BLAST). The raw sequencing data of the 14 bacteria identified by 16sRNA have been uploaded to GeneBank under bioproject BankIt(SUB14273842).

1.2.3 Data analysis

Demographic and clinical factors of suspected NTM infections were tabulated, and conditional logistic regression (binary model) was used to investigate the associated factors of NTM-PD. In the model, NTM was used as the dependent variable (MTB and other bacterium= 0, NTM-PD = 1).

2.1 Population informatics

Among the 51 NTM positive patients, 68.63% were male, 31.37% were female, and 92.16% were Han nationality. The youngest patient was 18 years old, the oldest patient was 79 years old, and the mean age was 48 years. The majority were male patients, between 40 and 65 years old (Figure 2).

2.2 A total of 107 suspected NTM were screened from 6259 positive cultures

Between January 2017 and December 2021, 6259 positive cultures were isolated and cultured from 53114 clinical samples, 6213 were positive for acid-fast staining and 46 were negative. A total of 107 suspected NTM cases were screened from 6213 acid-fast positive cultures by colony morphology and PNB test, and finally 51 cases of NTM, 13 cases of MTB and 43 cases of other bacteria were identified by 16S rRNA (Figure 3).

2.3 Species distribution of the 51 NTM cultures

A total of 51 cases of NTM were identified among the 107 cases. Mycobacterium abscessus 33.33% (17/51), Mycobacterium intracellulare 27.45% (14/51), Mycobacterium chelates11.7%(6/51) ,Mycobacterium kansasii 9.80% (5/51), Mycobacterium avium 7.84% (4/51), Mycobacterium gordonae 3.92% (2/51), Mycobacterium smegmatis 3.92% (2/51), Mycobacterium bradyxanthum 1.96% (1/51), of which Mycobacterium abscessus, Mycobacterium intracellulare and Mycobacterium chelates were the main pathogens. Thirteen cases of MTB complex were identified, including 10 cases of MTB and three cases of Mycobacterium bovis. Other bacteria were also identified in 43 cases. These included Gordonia sp 65.12% (28/43), Nocardia spp. 16.28% (7/43), Corynebacterium 11.63% (5/43), Tsukamurella sp 6.97% (3/43) and Gordonia sp. (Figure 3).

2.4 Changes in isolation rates over five years of NTM

The isolation rates of NTM in the five years from 2017 to 2021 were 1.03%, 1.06%, 1.18%, 1.09%, and 0.77%, respectively, with the highest isolation rate in 2019 and slightly lower in 2021. The isolation rates of MTB were 99.31%, 99.36%, 9.13%, 99.17% and 99.40%, respectively, and increased slightly in 2021. The isolation rates of other bacteria were 0.68%, 0.42%, 0.79%, 0.82% and 0.59%, respectively, with the highest in 2020 and the lowest in 2021 (Figure 4).The overall trend in the past five years decreased slightly, while the Mycobacterium chelonae increased. Among the eight NTM species, the isolation rates of Mycobacterium intracellulare and Mycobacterium abscessus fluctuated widely, while the other species remained relatively stable (Figure 5).

2.5 Drug resistance of NTM dominant bacteria in this area

A total of 35 cases of Mycobacterium intracellulare, Mycobacterium abscessus and Mycobacterium chelonae were selected for drug sensitivity test of 14 anti-TB drugs (rifampicin, isoniazid, streptomycin, ethambutol, moxifloxacin, levofloxacin, p-aminosalicylic acid, amikacin, clarithromycin, capreomycin, Prothionamide, kanamycin, Kam; Reck lung dis; clofazimin). The resistance rates of 14 drugs were 71.43%, 91.43%, 68.57%, 65.71%, 45.71%, 42.86%, 65.7%, 54.29%, 45.71%, 48.57%, 62.86%, 48.57%, 68.57%, 51.43%, respectively.

2.6 Risk factors for NTM infections

Among 51 patients, 16 cases (31.37%) were complicated with bronchiectasis, 14 cases (27.45%) with COPD, four cases (7.84%) with tumor, one case with diabetes, one case with hepatitis, one case with autoimmune disease, and one case with chronic kidney disease. Moreover, 32 cases (62.5%) had cough and expectoration, 47 patients had lower albumin (P<0.05), of which 40 cases were lower than the normal value.Table 1

This is the first report on the prevalence and characteristics of NTM-PD in Zunyi. NTM was isolated in 1.03% of patients with suspected pulmonary TB symptoms, which was lower than that reported in Chongqing (7.1%), Kunming (2.7%), Nanning (7.59) and other places near Zunyi, and lower than that reported in Nanjing, Zhejiang, etc [4, 9, 10]. It was also lower than in the USA and South Korea [11-13]. This difference may be related to the local climate and geographical characteristics, given the regional differences in the prevalence of NTM in China, which shows a gradual upward trend from north to south and from west to east [4]. In addition, the isolation rate of NTM in this area has remained stable for five consecutive years, with a slightly decreasing trend, while other places in China have reported an increasing trend of NTM, which may be related to the decrease of NTM isolation due to the reduction of patients' opportunities to seek medical treatment during the COVID-19 epidemic.

Table 1 Demographic and clinical characteristics associated with nontuberculous mycobacteria pulmonary diseases in Zunyi city.

Characteristics	NTM-PD N (%)	TB+Others N (%)		OR (95% CI)	P value
Gender
Men	35(68.63)	35(62.50)	0.253 ~ 1.133		0.102
Women	16(31.37)	21(37.50)	0.253 ~ 1.133		0.102
Age group(years)
18-40	16(31.37)	18(32.14)	0.967 ~ 1.006		0.180
41-65	22(43.14)	22(39.29)
65-79	13(25.49)	16(28.57)
Ethnicity
Han	47(92.16)	49(87.50)	0.290 ~ 2.443		0.752
Other	4(7.84)	7（12.50）	0.290 ~ 2.443		0.752
Sample
Sputum	49(96.08)	51(91.07)	0.091 ~ 0.901		0.033
Broncho Alveolar Lavage Fluid	2(3.92)	2(3.57)
Pleural effusion	0(0)	4(7.14)
Previous
Yes	11(21.57)	10(17.86)	18.950~ 5.838		0.000
no	40(78.43)	46(82.14)	18.950~ 5.838		0.000
Complications (yes v no)
Bronchiectasia	16(31.37)	10(17.86)	0.037 ~ 2.241		0.623
Diabetes	1(1.96)	3(5.36)	0.106 ~ 6.421		0.235
Hepatitis	1(1.96)	2(3.57)	0.617 ~ 2.972		0.854
COPD	14(27.45)	15(26.79)	0.283 ~ 2.448		0.450
Tumor	4(7.84)	3(5.36)	0.035 ~ 2.172		0.739
Autoimmunity disease	1(1.96)	3(5.36)	0.110 ~ 6.136		0.221
Chronic nephrosis	1(1.96)	1(1.79)	0.037 ~ 2.241		0.848
Clinical symptoms (yes v no)
Cough	32(62.75)	31(55.36)	0.389 ~ 2.679		1.021
Fever	6(11.76)	10(17.86)	0.580 ~ 1.977		1.071
Chest pain	7(13.73)	5(8.93)	0.794 ~ 4.461		1.883
Alb
<35	25(49.02)	24(42.86)	0.964 ~ 0.996		0.017
35-50	22(43.14)	21(37.50)
>50	4(7.84)	11(19.64)

*p-values < 0.05 are shown in bold for comparisons between the NTM-PD and control groups

and Others other bacterium(NTM nontuberculosis mycobacteria) ; Alb albumin; OR odds ratio.

Mycobacterium avium complex is common in Europe and North America, while Mycobacterium abscessus is most common in Asia and Oceania [10, 14]. Even within the same country, the frequency of clinical isolation of NTM varies between cities. In this study, eight NTM strains were isolated from lung specimens, and Mycobacterium abscessus was the most common (33.33%). The most common pathogens were Mycobacterium intracellulare (27.45%), Mycobacterium chelates (11.76%), Mycobacterium kansasii (9.80%), Mycobacterium avium (7.84%), Mycobacterium gordonae (3.92%), Mycobacterium smegmatis (3.92%), Mycobacterium bradyxanthum (1.96%), and the proportion of Mycobacterium abscessus was higher than that in the national multi-center report (23.1%). The number of Mycobacterium intracellulare isolates was lower than that reported in China (52.6%), Zhejiang (66.7%) and Nanjing (70.1%), but avium intracellulare complex (35.29%) was the predominant NTM in respiratory system, which was close to that reported in Beijing (33.7%) [15]. A recent multi-center study in China reported that M. intracellulare accounted for about half of the pulmonary NTM cases [4]. The dominant NTM species in this area are different from those in other areas, which may be related to the epidemic region of NTM. For example, Mycobacterium avium complex (MAC) is common in European countries, and Kansas is more common in Poland and Slovakia [16]. Some studies have reported that the differences in soil, temperature, humidity and daily evapotranspiration levels are related to pulmonary NTM diseases. However, both host susceptibility and environmental factors should be considered in clinical diagnosis and treatment [17]. Studies have reported this discrepancy in relation to differences in the protective efficacy produced by Bacillus Calmette-Guérin (BCG) vaccination against different species of NTM [4].

The common rapid growth mycobacteria(RGM) include Mycobacterium abscessus, Mycobacterium fortuitum group, Mycobacterium chelonae complex, etc. Mycobacterium abscessus complex is the main cause of RGM lung disease. The differences in erm gene between Mycobacterium massiliense and Mycobacterium bolletii may lead to different susceptibilities to macrolides [15]. The common slow-growing mycobacteria, the resistance rate of MAC, Mycobacterium kansasii and Mycobacterium marinum in SGM was higher than that in RGM. Our data showed that common NTM is not sensitive to conventional anti-TB drugs, and the treatment of NTM infections is related to the type, site and severity of infection [18]. Because the results of drug-susceptibility testing for some drugs may be applicable to specific species, the CLSI and most mycobacterial experts strongly recommend species-level identification [19].

The 51 NTM positive patients were mainly males, aged 40-65 years, and the main clinical symptoms of pulmonary disease after NTM infection were cough and expectoration, which was consistent with other reports in China [4, 9, 11, 13, 15, 20]. Patients with bronchiectasis, COPD, tumor and other underlying diseases were susceptible to NTM infection, which was consistent with other reports [4, 17]. These underlying diseases can damage the respiratory mucosa and reduce local immunity, leading to increased risk of infection and disease progression. In addition, our data showed that nearly half of the patients with NTM infections have low serum albumin, which can reflect the host nutritional or immune status, and <3.5 g/dL was one of the poor prognostic factors for death after NTM infection [21-23].

The limitations of this report were that the small sample size may weaken the statistical strength of the conclusions, and the NTM patients were not continuously followed-up after treatment. The clinical treatment of NTM segregation-positive patients will be followed-up in the future.

In summary, the trend of NTM infection rate in this region is stable but the drug resistance rate is high, and Mycobacterium abscessus is the most common NTM pathogen. Middle-aged, male patients with bronchiectasis and serum albumin <3.5 g/L have a high risk of progressing to NTM-PD. In areas with a high incidence of TB, clinicians should make a differential diagnosis between suspected pulmonary TB and NTM infections.

Ethics approval and consent to participate

Ethics approval and consent to participate

The study protocol was approved by the ethics committee of Affiliated Hospital of Zunyi Medical University [Approval No: kll-2019-017]. The need for written informed consent was waived by the Affiliated Hospital of Zunyi Medical University ethics committee due to retrospective nature of the study. and the study was carried out in accordance with the principles of the Declaration of Helsinki.

Consent for publication

Not applicable.

Availability of data and materials

All data in this study are provided by Professor Nana Li, with contact information: E-mail-2525

[email protected]. Add: No.149 Dalian Road,Huichuan district, Zunyi(563000), China. Tel: +86–851-28609237. All data are original data of the laboratory, all materials are owned by the laboratory. The data sets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

All the authors declare that they have no confict of interest.

Funding

This study was supported by The Guizhou Provincial Science and the Technology Foundation (grant number Qiankehe jichu [2022]648) and the Zunyi City Science and the Technology Foundation (Grant number zun shi ke he HZ zi [2019]77).

Authors’ contributions

Conception and design: Nana Li, Ling Chen and Yuanbo Lan. Collection and assembly of data: Nana Li, Si-yu Lou, Yong-hong Ma, Mei Liu, Wan-min Zhang, Xiao-ke Zhang. Data analysis and interpretation: Nana Li, Zhangli Peng, Ling Chen. Manuscript writing: Nana Li. Manuscript revision: Ling Chen and Yuanbo Lan. All authors read and approved the final manuscript. All data were generated inhouse, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. The author(s) read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

Analysis of the types and characteristics of non-tuberculous mycobacteria in high endemic areas of tuberculosis

Status:

Version 1

Abstract

Figures

Introduction

1. Materials and Methods

2. Results

3. Discussion

Declarations

References

Additional Declarations

Status:

Version 1