INTRODUCTION:

More than 170 species of non-tuberculous mycobacteria have been detected. Non-tuberculous mycobacteria (NTM) are opportunistic environmental pathogens that live naturally in soil and drinking water^1-3. Non-tuberculosis disease has increased globally. It can cause disease and death^4,5.^.A previous systematic review showed that the five-year mortality rates were 27% in Europe, 35% in the US, and 33% in Asia⁶.

The incidence of non-tuberculous mycobacteria (NTM) ranges between 7.2 and 13.6 per 100,000 people in Surakarta, Indonesia⁷. 15% of non-tuberculous mycobacteria (NTM) were NTM strains detected in sputum of suspected tuberculosis (TB) collected from January 2013 to December 2017⁸. In Surabaya, research conducted from January 2014 to September 2015 showed that out of 2440 sputum specimens collected from suspected pulmonary tuberculosis (TB) patients, 141 isolate species (5.78%) were detected as NTM and 459 isolate species (18.81%) were M. tuberculosis species⁹. On the other hand, tuberculosis caused by M tuberculosis, is also an infectious disease that causes death every year^10-13.Latent TB is present in about one-third of the population, and nine million new TB cases are reported annually, creating a major health issue^14-16. Tuberculosis Pulmonary has similarity symptom with NTM-LD(non-tuberculous mycobacteria lung disease). In the past two decades, the prevalence of non-tuberculous mycobacterial lung diseases (NTM-LD) has increased in the United States and several parts of the world. It is possible that Indonesia also faces an increase in NTMLD cases¹⁷.

Identification and differentiation of NTM from M. tuberculosis is a significant diagnostic value. TB is notoriously difficult to diagnose since the symptoms are rarely constant and similarly with NTM infections Because treatment and response rates vary by bacterial species, identifying NTM species is essential. Precise NTM species levels on the prognosis and treatment regimen. NTM is treated with a combination of medications over a long period^18-20. Given this background, this study investigated the specific detection of clinical NTM isolates at a tertiary referral hospital through a sequencing analysis targeting 16S rRNA genes that specific for bacterial identification^21-23.

MATERIALS AND METHODS:

Patients:

This research was carried out at Soetomo General Hospital Surabaya, Indonesia, with 1444 beds. According to the diagnostic microbiological test, the research was a retrospective cohort study involving all patients (n=20) with clinical suspected positive NTM. Patients were admitted to the hospital from January to December 2014.

The medical records of the patients were examined and recorded: these include demographics, comorbidity (Diabetes mellitus), and the patients status of the human immunodeficiency virus (HIV); previous TB: clinical data, i.e., dyspnoea, cough, hemoptysis, and night sweats, and radiographic data (chest X-rays and computed tomography (CT).

Microbiological diagnosis:

Mycobacteria’s microbiological cultures of respiratory samples (sputum, Bronchoalveolar lavage) were performed. According to the ATS guidelines, clinical specimens were stained using the Ziehl Neelsen method. The respiratory specimens were pretreated with semi-alkaline protease NALc-NaOH for digestion and decontamination^24,25. The cultured specimens were treated on a Bactec MGIT 960 (BD Diagnostic Systems). Positive results of the mycobacteria culture on the MGIT were analyzed using TB Ag MPT 64 (TB ID).

The macroscopic colony and microscopic properties of NTM species were used to identify the species and continued with molecular identification by sequencing DNA segments comprising 16S rRNA. The PCR method was used to identify 20 NTM isolates that were subcultured in Lowenstein Jensen Medium. Bacterial colonies were extracted using the QiAmp DNA Kit (Qiagen) following their respective protocol kits. The PCR assay targeted 16S rRNA and used primers such as Mb246 5’ ₈AGA GTT TGA TGG CTC AG₂₈ 3’ and MbR 247 5’ ₆₀₉ TTT CAC GAA CAA CGC GAC AA ₅₉₀ 3'. After amplification, the product's subsequent nested amplification used primers Mb1 5’ ₁₀₅ AGT GGS GAA CGG GTG AGT Aac ₁₂₆ 3’ and MbR7 5’₅₇₃ TTA CGC CCA GTA ATT CCG GAC AA ₅₅₁ 3’. The amplified DNA segments from PCR were separated by electrophoresis in 2% agarose gel. The Mycobacterium genus and species confirmed PCR DNA by DNA sequencing by the ABI 377 System Sequencer Machine, and reference sequences were used to compare all of the sequences using the Gen Bank (http://www.ncbi.nlm.hih.gov/GenBank/) for the identification of species. The Ethics Committee approved the study of Soetomo General Hospital, Surabaya, Indonesia, reference code: 306.

RESULT:

The research population was 20 patients with suspected TB and positive NTM. Twenty of 51 stored NTM isolates growing from 433 respiratory specimens (sputum and bronchoalveolar lavage) were randomly selected and submitted to the TB laboratory of Soetomo General Hospital Surabaya from January to December 2014 to detect chronic TB diagnosis. Identified species by colony appearance were shown in Figure 1.

Figure 1: Culture of NTM colony from respiratory specimens of patients with suspected pulmonary TB in a Lowenstein Jensen medium

This study showed that 80% of the patients were male and 60% had a history of pulmonary TB infection. One patient had bronchiectasis, and seven had underlying non-pulmonary diseases, including diabetes mellitus (Table 1). The nested double amplification PCR was conducted by utilyzing primers Mb246+ Mb reverse 247 to amplify a 590 amplicon (Figure 2).

In this study, M kansasii was found as the highest species (12/20; 60%), followed by M. gordonae (2/ 20;10%), M. paracrofulaceum (2/20; 10%), M. simiae (1/20;5%), M. chelonae (1/20; 5%), and M. terrae (1/20; 5%). Table 2 presents the clinical and radiological profiles of patients who detected positive Mycobacterium kansasii and non-mycobacterium tuberculosis, images of radiological and CT scans of the patients are illustrated in Table 2. Five patients showed cavitation in the right lobe.

Table 1: Patients’ characteristics

Characteristics	Frequency (n:20)
Age, (mean age )	49.7 years (28-71 years)
Male Gender	16
Female	4
Pulmonary abnormal
Previous TB	12
Bronchiectasis	1
Comorbid diseases, number (%)
Diabetes	7 (35%)
HIV AIDS	3 (15%)
Clinical data,
Cough	18
Hemoptysis	7
Dyspnoea	15
Chest pain	3
Sweats	6
Fever	6
Abnormal auscultation	14
Anemia	8
Microbiological data
Mycobacterium kansasii	12 (60%)
Mycobacterium gordonae	2 (10%)
Mycobacterium parascrofulaceum	2 (10%)
Mycobacterium avium	1 (5%)
Mycobacterium simiae	1 (5%)
Mycobacterium chelonae	1 (5%)
Mycobacterium terrae	1 (5%)

Figure 2: PCR results of 16S rRNA on amplification with primers Mb1 and MbR7 (nested) at 469 bp amplicon (M: Marker S Px; Sample, K1: control: Control H37) (a) Electropherogram of sample M: Marker (b)

Table 2: Data of individual patients. Clinical and images findings for non-tuberculous mycobacteria pulmonary infected patients

No	General symptoms	Sex	Age	Clinical specimens	HIV status	Other associated conditions	Chest X-ray	High-resolution computerized tomography of the chest	Species
1	Cough, dyspnea	M	51	Sputum	-	Previous TB, DM	Multiple cavities RUL	Multiple cavities RUL and LUL, multiple blebs RUL and LUL	M. kansasii
2	Cough, dyspnea	F	63	Sputum	-	DM	Infiltrates RUL, Cavitation LUL	ND	M. kansasii
3	Cough, dyspnea	M	56	Sputum	-	Previous TB, DM	ND	ND	M. kansasii
4	Cough, dyspnea, hemoptysis, sweat	M	58	BAL	-	Previous TB	Fibrionfiltat, multiple cavitas LL, pleura effusion bilateral organised.	ND	M. kansasii
5	Cough, hemoptysis dyspnea, swet	M	61	Sputum	-	Previous TB, DM	Fibroinfiltrat LL,RL, multiplecavitas bilateral	ND	M.kansasii
6	Cough, hemoptysis, Dyspnea	M	56	Sputum	-	Previous TB	ND	ND	M. kansasii
7	Cough, dyspnea	M	54	Sputum	HIV	-	Infiltrate Ruland LUL	ND	M. kansasii
8	Cough, hemoptysis	F	63	Sputum	-	-	ND	ND	M. kansasii
9	Cough, hemoptysis, dyspnea	M	57	Sputum	-	Bronchiectasis	Fibroinfiltrat RUL	Bronchiectasis RUL apical segment and superior segment LLL	M. kansasii
10	Cough	M	57	Sputum	-	Previous TB	Fibroinfiltrat LL, RL, emphysema	ND	M. kansasii
11	Cough, dyspnea	M	22	Sputum	-	-	Fluidopneumothorax Left,reticulo granular pattern LL,RL.	ND	M. kansasii
12	Cough, dyspnea, hemoptoe, chest pain	M	54	Sputum	-	DM Previous TB	ND	Single nodul segmen anterior RUL	M. kansasii
13	Dyspnea	M	28	BAL	AIDS	-	Normal	ND	M.avium

14	Cough, dyspneu, sweat	M	36	Sputum	AIDS	-	ND	ND	M.gordonae
15	Cough	F	31	Sputum	-	Previous TB	Pneumothorax Left, Fibroinfiltrat RL; Right Pleural effusion	ND	M.gordonae
16	Cough, dyspneu, sweat	F	65	Sputum	-	-	Cavitation RL,	Fibrosis LUB, cavitation RL, right pneumothorax	M.ghelonae
17	Cough, dyspneu, sweat, hemoptoe	M	57	Sputum	-	Previous TB	Pleural effusion	ND	M.parascrofulaceum
18	Cough	F	67	Sputum	-	Previous TB	ND	ND	M.paracsrofulaceum
19	Cough, dyspneu	M	18	BAL	-	-	Single nodule RUL	ND	M.simiae
20	Cough	M	71	Sputum		Previous Tb, DM	ND	ND	M.terrae

Note: F (female);M(male); AFB (acid fast baccilli); -(negative); +positive: BAL (bronchoalveolarlavage); TB (tuberculosis); DM(Diabetes mellitus.); LUB: left Upper lobe, RUB: Right Upper lobe; LLL; left lower lobe; ND: No Data

DISCUSSION:

The epidemiology of NTM remains poorly understood in Indonesia, the second-highest burden TB country in the world. According to the first report from the tertiary referral hospital²⁶. Twenty NTM isolates from sputum samples of suspected chronic TB patients were detected ^27,28. NTM disease incidence varies greatly depending on NTM species, regional distribution, sex, race/ethnicity, age, and risk factors (such as comorbidities)²⁸. It is known that more than half of NTM detected patients were males. While early reports identified predisposing circumstances in older male patients, approximately 80% of patients were middle-aged or elderly females.

NTM can be found in almost any habitat NTM is more likely to affect patients who already have a lung impairment²⁹. NTM lung infection is associated with chronic obstructive pulmonary diseases, pneumoconiosis, prior pulmonary tuberculosis, and bronchiectasis³⁰. According to the guidelines, diagnosing NTM lung diseases is the initial measure to control disease progressiveness such as TB and fungal infection³¹. Previous research in Africa, which has a high TB prevalence, revealed that individuals with suspected TB or MDR-TB infections (3.7-1.8%) might suffer from NTM pulmonary disease²⁷.

The incidence and prevalence of NTM lung diseases have risen worldwide³². Slow Growth Mycobacteria (SGM), such as M. avium complex (MAC) and M. kansasii, which slowly grow and rapidly grow mycobacteria (RGM), such as M. abscesses complex (MABC), are common causes of lung infection^28,33. This study discovered that (12/20) 60% of patients were positive for M. kansasii. After the M. avium complex, M. kansasii is the second most prevalent cause of NTM lung infection in some areas²⁶. However, M. kansasii was a relatively infrequent cause of NTM lung disease in Korea, but it was likely to be a clinically significant cause of pulmonary infections at the hospital under research²⁸.

In addition, M. kansasii is considered` the most dangerous NTM and the primary cause of NTM lung disease in the UK and Western Europe. Besides the M. avium complex, chronic lung disease, previous TB, cancer, and drinking risk factors for M. kansasii infection³⁴. Although roughly 40% of immunocompetent patients had no concomitant illness, pulmonary disease is the most common clinical^35,36. This current study showed that M. kansasii was detected in one patient with AIDS, half patients (6/12) had comorbid diabetes mellitus and 67% (8/12) with a history of TB infection, and one patient with bronchiectasis³⁷. All of that comorbid are susceptible to NTM infections. Previous research among the Korean population also revealed that bronchiectasis was associated with M. kansasii colony in lung disease patients. This observation showed a higher rate of pre-existing lung disease, ancient tuberculosis in NTM pulmonary infection. Patients with M. kansasii infection showed more cavitation unilateral illness and were at a higher risk of developing right upper lobe disease³⁷. In this current study, three patients showed these conditions as well. Despite similarities in the image analysis of mycobacteriosis and other lung diseases like TB, the middle lobe and lingula are primarily parts of suspected NTM-LD³⁸.

Cough, hemoptysis, weight loss, fatigue and malaise are symptoms of NTM lung diseasesthat are ambiguous and resemble pulmonary TB symptoms, and thus they may represent underlying lung diseases such as bronchiectasis and chronic obstructive pulmonary disease^18,35. The current case series showed that patients had general symptoms such as cough 90% (18/ 20). Hemoptysis was detected in 40% of patients with positive M. kansasii. Many patients with NTM lung disease had hemoptysis, and independent of the volume, a few patients of NTM lung disease required Bronchial Artery embolization (BAE)³⁹. Endobronchial disease and cavitation-induced bronchial artery degradation have been associated with hemoptysis in pulmonary infections, although these were not investigated further hemoptysis in this study³⁴.

One of the M. avium complex (MAC) isolates was found in sputum samples of AIDS patients. Furthermore, MAC is the primary pathogen often encountered among various NTM species²⁵. In his case, a patient did not have clinical symptoms of cough. Moreover, it correlates with a study that showed that M. avium is a producer of a sulfated glycopeptidolipid but not sulfolipid1 (SL-1), which can activate nociceptive neurons and induce cough⁴⁰. Moreover, M. chelonae causes skin, soft tissue, and bone infections due to piercing wounds, contaminated tattoo inks, plastic surgery, or liposuction. It is usually found in hospitals, and its species are highly resistant to nosocomial pathogens, antibiotics, and disinfectants²⁸. In these case series, elderly female patients were infected with M chelonae in the left lobe pulmonary. In Korea, M. parascrofulaceum detected clinically presented hemoptoe and effusion pleural condition. It is similar to the previous study case report⁴¹. Therefore, long-term follow-up and recurrent sputum analysis are required to confirm whether M. chelonae and M. parascrofulaceum caused a temporary infection, disease progression, or colonization.

The identification of NTM is necessary for an initial effective treatment plan. Several phenotypic tests and enzymatic characteristics are labour-intensive, time-consuming, and limited to detect. Common species DNA sequencing targeting various genes 16S rDNA, rpoB, and hsp65 is the standard method for discriminating and identifying different mycobacterial species as it has a high resolution⁴². Positive may imply infection, colonization, or specimen contamination, given the prevalence of NTM in nature. All of the patients in this study had a single positive NTM culture. As opportunistic pathogens, colonization and active mycobacterial diseases were hard to identify when NTM isolated are taken from clinical specimens³³.

Clinical, radiographic, and microbiological data should be combined to diagnose NTM lung disease. However, the diagnosis can be verified by at least two positive cultures in sputum, one positive bronchoscopic lavage, or a transbronchial or another lung biopsy with a positive culture of NTM or histopathological features such as granulomatous inflammation or stainable acid-fast bacilli (AFB), and one positive culture in bronchial lavage or sputum regardless of the mycobacterial strain. As a result, to diagnose NTM lung disease, symptomatic individuals with comparable radiographic results must meet the microbiological criteria²⁵.

This study has three main limitations: a small number of patients, retrospective selection of patients, and unconfirmed significance of NTM infection based on the American Thoracic Society) guidelines that two sputum samples or respiratory specimens are used to diagnose NTM infection challenges in the clinical microbiology laboratory were visible when a positive microbiologic test compatible with NTM was found. The actual clinical significance of the positivity is still in question. To solve this question, clinicians and microbiologists should conduct a comprehensive investigation: Firstly, a detailed assessment of the patient's underlying disease, risk factors, and current clinical status is required. Possible colonization, transient infection, or environmental contamination by species should be excluded. Besides, a microbiological diagnosis test should be done. The risk group should be screened based on the diagnostic criteria of NTMLD and on specific radiological findings that, in some cases, do not meet the radiological abnormalities³⁸. A positive microbiology culture test and other techniques (e.g., PCR) should be performed to detect species^43,44. When radiological images are not specific and cannot support the positivity of microbiological tests, a definitive diagnosis of NTMLD is challenging to perform. Future studies with a larger population are needed to corroborate these current findings.

CONCLUSION:

NTM species identified was dominated by Mycobacterium kansaii. The predominance of NTM species from clinical respiratory can be a reference to determine prudent treatment for patients in high-burden TB countries. Improving clinician awareness of the local epidemiology of NTM is essential to advance the different diagnoses of NTM lung disease with pulmonary TB and to reduce the unnecessary use of antitubercular treatment.

CONFLICT OF INTEREST:

None declared.

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Received on 23.12.2022 Modified on 30.04.2023

Research J. Pharm. and Tech 2023; 16(12):5889-5895.

DOI: 10.52711/0974-360X.2023.00954