Asian Pacific Journal of Tropical Medicine

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 14  |  Issue : 10  |  Page : 451--455

Prevalence of non-tuberculosis mycobacteria among samples deposited from the National Tuberculous Reference Laboratory of Iran (2011-2018)


Saman Ayoubi1, Parissa Farnia1, Poopak Farnia2, Jafar Aghajani1, Jalaledin Ghanavi1, Ali Akbar Velayati1,  
1 Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Biotechnology, School of Advanced Technology in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Correspondence Address:
Saman Ayoubi
Mycobacteriology Research Center (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran
Iran

Abstract

Objective: To investigate the prevalence of non-tuberculosis mycobacteria (NTM) among the samples deposited from the National Tuberculosis Reference Laboratory of Iran between 2011 and 2018. Methods: The study evaluated the prevalence of NTM among specimens from patients with pulmonary tuberculosis symptoms (n=15 771) deposited at the National Tuberculosis Reference Laboratory of Iran from 2011 to 2018. Detection of Mycobacterium (M.) tuberculosis was based on presence of a 190-bp amplicon from IS6110 insertion sequence using Tb1 and Tb2 primers, and amplicon-negative specimens were tested for NTM and M. tuberculosis (refractory to IS6110 amplification) using restriction fragment length polymorphism PCR of hsp65 amplicon fragment. Results: A total of 7 307 (46.33%) M. tuberculosis and 658 (4.17%) NTM specimens were found, the latter mainly comprising M. abscessus (10.18%), M. avium (2.28%), M. chelonae (8.97%), M. intracellulare (10.49%), M. kansasii (4.71%), and M. simiae (56.08%). Conclusions: As treatment for NTM differs from that for M. tuberculosis, accurate detection of Mycobacterium sp. is of public health significance.



How to cite this article:
Ayoubi S, Farnia P, Farnia P, Aghajani J, Ghanavi J, Velayati AA. Prevalence of non-tuberculosis mycobacteria among samples deposited from the National Tuberculous Reference Laboratory of Iran (2011-2018).Asian Pac J Trop Med 2021;14:451-455


How to cite this URL:
Ayoubi S, Farnia P, Farnia P, Aghajani J, Ghanavi J, Velayati AA. Prevalence of non-tuberculosis mycobacteria among samples deposited from the National Tuberculous Reference Laboratory of Iran (2011-2018). Asian Pac J Trop Med [serial online] 2021 [cited 2021 Dec 5 ];14:451-455
Available from: https://www.apjtm.org/text.asp?2021/14/10/451/329007


Full Text

 1. Introduction



Tuberculosis (TB) is one of the oldest diseases of mankind[1] and still remains a major cause of morbidity and mortality of 10 million and 1.4 million people respectively in 2019 worldwide, with Mycobacterium (M.) tuberculosis the predominant causative agent[2], followed by non-tuberculosis mycobacteria (NTM)[3]. NTM are found in the environment, such as food, soil and water and infections in animals are often zoonotic[4],[5],[6]. NTM infection in humans is on the rise in most countries around the world[3]. Pathogenic NTM species include M. abscessus, M. avium, M. ferrititum, M. intracellularare, M. kansasii, and M. simiae[7],[8],[9],[10],[11]. NTM can infect a variety of human organs, such as lung, skin and soft tissue, particularly among immunocompromised individuals[12],[13],[14],[15],[16],[17],[18].

Given their widespread presence in the environment and global distribution, epidemiological surveillance of NTM is of particular importance[14],[19],[20]. In Iran, prevalence of NTM from 2002 to 2006 was 12%[7]. However, in 2011, NTM prevalence in Isfahan was reported as high as 62%[21],[22], but in Khuzestan Province in 2016, prevalence of only 30% was reported[23]. Thus, accurate identification of Mycobacterium sp. is critical as all patients with acid-fast bacterial infection, especially those with respiratory symptoms, are diagnosed as having TB infection, which requires isolation and/ or long term antibiotic treatment[24],[25],[26]; whereas NTM infection causes a wide range of diseases[27],[28],[29],[30] and NTM are sensitive to a different set of antibiotics used to treat TB[31],[32],[33].

[INLINE:1]

Given the importance of the growing prevalence of diseases caused by NTM worldwide, studies have been conducted to evaluate the prevalence of NTM in Iran[30],[34]. Here, we report NTM prevalence and species in samples deposited from National Tuberculosis Reference Laboratory of Iran from 2011 to 2018 to determine the extent of the disease so that appropriate measures can be carried out to control and eliminate this public health problem in the country.

 2. Materials and methods



2.1. Study method and sample collection

This cross-sectional descriptive study was performed on 15 771 sputum samples obtained from patients presenting with pulmonary tuberculosis symptoms and sent to the Mycobacteriology Research Center, Tehran, Iran from 2011-2018. Samples came from provinces all across the country for mycobacterial tests to confirm presence of M. tuberculosis (Ethical approval: IR.SBMU.NRITLD.REC.1397.569).

2.2. Laboratory procedures

Initial isolation of Mycobacterium spp. was performed on Lowenstein-Jensen media (Bio-Rad, Marnes-la-Coquette, France)[25]. Subsequently, mycobacterial DNA was extracted from samples using a QiAamp DNA kit (QIAGEN, Germantown, MD)[26]. In order to identify M. tuberculosis, a 190 bp fragment of IS6110 insertion sequence was amplified using primers Tb1 (5′-ATCCTGCGAGCGTAGGCGTCGG-3′) and Tb2 (5′-CAGGACCACGATCGCTGATCCGG-3′) in a reaction mixture (50 μL) containing 8 pmol each of Tb1 and Tb2 primers, 10× buffer (QIAGEN, Germantown, MD), 1.5 mM MgCl2, 0.2 mM dNTPs, 2% (v/v) dimethylsulfoxide (DMSO), 1 U Taq polymerase (QIAGEN, Germantown, MD), and 1 μg of DNA[35]. Thermocycling was done as follows: 95 °C for 10 min; 35 cycles of 93 °C for 20 sec, 65 °C for 60 sec and 72 °C for 20 sec; and a last step of 72 °C for 5 min. Amplicons were analyzed by 1.5% agarose gel-electrophoresis and visualized under UV illumination by ethidium bromide staining. M. tuberculosis strain H37Rv[28] was used as reference control. PCR-negative specimens were analyzed for NTM species using restriction fragment length polymorphism (RFLP)-PCR of a hsp65 fragment[36]. Nested-PCR was performed using primers Tb15 (5′-CGTAYGACGAAGAGGCCCGT-3′) and Tb17 (5′-WASGGRTCCTCSAGGACSGC-3′) were employed in the first PCR step in a reaction mixture (50 μL) containing 4 pmol each of primers Tb15 and Tb17, 10× buffer (QIAGEN, Germantown, MD), 1 mM MgCl2, 1 mM dNTPs, 1% (v/v) DMSO, 1 U Taq polymerase (QIAGEN, Germantown, MD), and 1 μg of DNA, with thermocycling conditions as described above, except that 30 cycles and annealing temperature of 60 °C were used. The second PCR step was performed using primer pairs Tb11 (5’-ACCAACGATGGTGTGTCCA-3’) and Tb12 (5’-CTTGTCGAACCGCATACCCT-3’) in a reaction mixture (50 μL) containing 8 pmol each of primers Tb11 and Tb12, 10× buffer (QIAGEN, Germantown, MD), 1.5 mM MgCl2, 0.2 mM dNTPs, 2% (v/v) DMSO, 1 U Taq polymerase (QIAGEN, Germantown, MD), and 1.5 μL of first-step reaction solution. Thermocycling was performed in the above described instrument as follows: 95 °C for 5 min; 30 cycles of 94 °C for 30 sec, 56 °C for 60 sec and 72 °C for 40 sec; with a last step of 72 °C for 10 min. Amplicons (439 bp) were digested with BstE II and with Hae III (Sigma-Aldrich, Darmstadt, Germany) (1.5 U/μL, 60 °C, 5 min) and analyzed by 8% acrylamide gel-electrophoresis and ethidium bromide staining. Fragment sizes were compared with patterns of reference NTM species and M. tuberculosis strain H37Rv [Table 1].{Table 1}

 3. Results



Out of 15 771 sputum specimens sent for examination at the Mycobacteriology Research Center, Tehran, Iran from 2011 to 2018, 6 637 were identified as containing M. tuberculosis (presence of IS6110 190-bp amplicon) while 9 134 were negative, and of the latter samples, RFLP-PCR of hsp65 fragment identified 658 as positive with NTM and 670 with M. tuberculosis, resulting in a total of 42.08% (6 637/15 771) of the samples positive for Mycobacterium spp, consisting of 7 307 (46.33%, 7 307/15 771) M. tuberculosis and the remaining NTM species. The most frequent NTM was M. simiae (56.08%, 369/658) followed by M. intracellulare (10.49%, 69/658), M. abscessus (10.18%, 67/658) and M. chelonae (8.97%, 59/658) [Table 2]. Some representative RFLP-PCR amplicon profiles are shown in [Supplementary Figure 1].{Table 2}

During the period of the survey, the number of NTM species increased over the years, with M. simiae the major NTM species, ranging from 43%-70% of NTM-positive annual samples [Table 2]. It is worth noting M. abscessus was first identified in 2013 (2% of NTM samples) and rose to 28% in 2018.

 4. Discussion



The prevalence of NTM infection in several Asian and European countries have increased over the years, from 8.2 per 100 000 population in 1999 to 16 per 100 000 in 2013[2], a trend observed in the present study. The predominant NTM species varies depending on country. In USA, a survey in 2017 reported M. avium and M. fortuitum as the two major NTM species[37],[38], while in Asian countries (India, Japan, Singapore, and Thailand), M. avium and M. kansasii are predominant[36],[39] and in Turkey, M. fortuitum has the highest prevalence[40],[41]. In the present study, the most major NTM was M. simiae, accounting for 56.08% of the samples examined. However, there are variations in the major NTM species identified in the country depending on location and period of the survey: from 2006 to 2008 in Isfahan, M. fortuitum is the most common NTM sp[38],[39]; an earlier survey in 2010 of samples at the National Tuberculosis Reference Laboratory noted M. simiae as the most common[35],[42]; and in Zahedan in 2014, based on conventional biochemical methods, the most common NTM species reported is M. kansasii[30],[36],[37]. As the samples examined in the present study came from all regions of the country, the data should be more representative of the national NTM prevalence in the human population, at least for bronchial infection. In USA, between 2008 and 2012, the prevalence of NTM has been noted to be on the rise among pulmonary patients[32],[41].

In the present study, the second most abundant NTM species was M. intracellulare, a fast-growing mycobacterium (including other fast growing NTM species) that is found in water source and soil[32],[33],[34],[35]. Simple precautionary measures such as boiling drinking water can prevent from infection and subsequent pulmonary colonization[38],[39].

Given the similarity of NTM species to M. tuberculosis, molecular methods are more accurate, faster and more sensitive than conventional tests for differentiating NTM species from M. tuberculosis[35],[36],[37],[38]. However, absence of PCR amplification of IS6110 sequence does not mean absence of M. tuberculosis; in the present study, RFLP-PCR analysis of hsp65 revealed slightly more samples positive for M. tuberculosis compared to those NTM-positive[38],[40]. As treatment of NTM infection differs from that of M. tuberculosis, their detection and identification are of great importance to physicians because choice of medication is species specific and certain drug combinations can lead to deleterious side effects, which can pose problems due to the lengthy dosage regimen (12-24 months) required for complete cure[40],[41].

In conclusion, the study highlights the usefulness of molecular detection of not only M. tuberculosis but also non-tuberculosis mycobacteria that was present with significant prevalence throughout Iran. Despite the mistaken belief of certain physicians, these mycobacteria pose an important public health problem and their detection and identification should assist in appropriate antibiotic combination therapy and reduce the country’s burden of mycobacterial infection.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

We thank Professor Parissa Farnia for guiding and supporting our project.

Funding

This work was supported by funding from the Mycobacteriology Research Center, National Tuberculosis and Lung Research Institute (NRITLD), Masih Daneshvari Hospital (Grant No. 16597).

Authors’ contributions

Ali Akbar Velayati and Parissa Farnia conceived of the presented idea. Jalaledin Ghanavi developed the theory and performed the computations. Poopak Farnia verified the analytical methods. Saman Ayoubi and Jafar Aghajani carried out the experiment. Saman Ayoubi wrote the manuscript with support from Jafar Aghajani and Parissa Farnia. All authors discussed the results and contributed to the final manuscript.

References

1Joao I, Cristovao P, Antunes L, Nunes B, Jordao L. Identification of nontuberculous mycobacteria by partial gene sequencing and public databases. Int J Mycobacteriol 2014; 3(2): 144-151.
2World Health Organization (WHO). Global tuberculosis report. 2019. [Online]. Available from: https://apps.who.int/iris/bitstream/handle/10665 /329368/9789241565714-eng.pdf. [Accessed on 18 October 2021].
3Larsson LO, Polverino E, Hoefsloot W, Codecasa LR, Diel R, Jenkins SG, et al. Pulmonary disease by non-tuberculous mycobacteria-clinical management, unmet needs and future perspectives. Expert Rev Respir Med 2017; 11: 977-989.
4Donfack VF, Ngando L, Pefura EW, Che DS, Ateba G, Bigna JJ, et al. Comparative study of LoopampTM Mycobacterium tuberculosis complex kit for rapid detection of Mycobacterium tuberculosis complex in Cameroon. BBRJ 2018; 2(1): 46.
5Velayati AA, Farnia P, Mozafari M, Mirsaeidi M. Nontuberculous mycobacteria isolation from clinical and environmental samples in Iran: Twenty years of surveillance. Biomed Res Int 2015; 2015: 254-285.
6Mertaniasih NM, Kusumaningrum D, Koendhori EB, Kusmiati T, Dewi DN. Nontuberculous mycobacterial species and Mycobacterium tuberculosis complex coinfection in patients with pulmonary tuberculosis in Dr. Soetomo Hospital, Surabaya, Indonesia. Int J Mycobacteriol 2017; 6(1): 9-13.
7Ridell M. Mycobacterium abscessus: An environmental mycobacteria being a human pathogen. Int J Mycobacteriol 2015; 4: 41.
8Varahram M, Farnia P, Saif S, Marashian M, Farnia P, Ghanavi J, et al. Identification of different subtypes of rapid growing atypical Mycobacterium from water and soil sources: Using PCR-RFLP using hsp65 and rRNA 16s-23s genes. Int J Mycobacteriol 2016; 5(Suppl 1): S212-S213.
9Farnia P, Masjedi MR, Mohammadi F, Tabarsei P, Farnia P, Mohammadzadeh AR, et al. Colorimetric detection of multidrug-resistant or extensively drug-resistant tuberculosis by use of malachite green indicator dye. J Clin Microbiol 2008; 46: 796-799.
10Akbar Velayati A, Farnia P, Mozafari M, Malekshahian D, Seif S, Rahideh S, et al. Molecular epidemiology of nontuberculous mycobacteria isolates from clinical and environmental sources of a metropolitan city. PloS One 2014; 9: e114428.
11Wu J, Zhang Y, Li J, Lin S, Wang L, Jiang Y, et al. Increase in nontuberculous mycobacteria isolated in Shanghai, China: Results from a population-based study. PLoS One 2014; 9: e109736.
12Mirsaeidi M, Farnia P, Sadikot R, Hsueh PR, Aliberti S. Nontuberculous mycobacteria: Epidemiologic, mycobacteriologic, and clinical aspects. Biomed Res Int 2015; 2015: 523-697.
13Nasiri MJ, Farnia P. Prevalence of rapidly growing mycobacteria (RGM) in Iran: Systematic review and meta-analysis. Int J Mycobacteriol 2015; 4: 145.
14Shah JA, Lindestam Arlehamn CS, Horne DJ, Sette A, Hawn TR. Non-tuberculous mycobacteria and heterologous immunity to tuberculosis. J Infect Dis 2019; 220: 1091-1098.
15Velayati AA, Farnia P, Masjedi MR. The totally drug resistant tuberculosis (TDR-TB). Int J Clin Exp Med 2013; 6: 307-309.
16Garima K, Varma-Basil M, Pathak R, Kumar S, Narang A, Rawat KS, et al. Are we overlooking infections owing to non-tuberculous mycobacteria during routine conventional laboratory investigations? Int J Mycobacteriol 2012; 1(4): 207-211.
17Mirzapour A, Yousefi M, Zaker-Bostanabadi S, Hashemi-Shahraki A, Nazari-Alam A, Ebrahimi SA. Identification of Mycobacterium species isolated from patients using high-performance liquid chromatography in Tehran during 2014-2015. Feyz J Kashan Univ Med Sci 2018; 21: 577-583. [in Persian].
18Farnia P, Mohammadi F, Zarifi Z, Tabatabee DJ, Ganavi J, Ghazisaeedi K, et al. Improving sensitivity of direct microscopy for detection of acid-fast bacilli in sputum: Use of chitin in mucus digestion. J Clin Microbiol 2002; 40: 508-511.
19Farnia P, Mohammadi F, Masjedi MR, Varnerot A, Zarifi AZ, Tabatabee J, et al. Evaluation of tuberculosis transmission in Tehran: Using RFLP and spoloigotyping methods. J Infect 2004; 49: 94-101.
20Velayati AA, Rahideh S, Nezhad ZD, Farnia P, Mirsaeidi M. Nontuberculous mycobacteria in Middle East: Current situation and future challenges. Int J Mycobacteriol 2015; 4: 7-17.
21Merza MA, Farnia P, Tabarsi P, Khazampour M, Masjedi MR, Velayati AA. Anti-tuberculosis drug resistance and associated risk factors in a tertiary level TB center in Iran: A retrospective analysis. J Infect Dev Ctries 2011; 5: 511-519.
22Nezhad ZD, Farnia P, Sheikholslami FM, Karahrudie MA, Mozafari M, Seif S, et al. Prevalence of non-tuberculosis mycobacteria in patients referring to Mycobacteriology Research Center of Iran. Sci J Kurdistan Univ Med Sci 2014; 19: 31-39. [in Persian].
23Tortone CA, Zumarraga MJ, Gioffre A, Oriani DS. Utilization of molecular and conventional methods for the identification of nontuberculous mycobacteria isolated from different water sources. Int J Mycobacteriol 2018; 7: 53-60.
24Rahideh S, Derakhshaninezhad Z, Farnia P, Mozafari M, Seif S, Malekshahian D, et al. Review and meta analysis of nontuberculous mycobacteria in the Middle East. Int J Mycobacteriol 2015; 4: 149.
25Kubica GP. Tuberculosis: Laboratory methods in diagnosis. US Department of Health, Education, and Welfare, Public Health Service; 1960, p. 1-79.
26Ip SC, Lin SW, Lai KM. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA blood mini kit, QIAamp® DNA investigator kit, QIAsymphony® DNA Investigator® kit and DNA IQ™. Sci Justice 2015; 55: 200-208.
27Farnia P, Ghanavi J, Saif S, Farnia P, Velayati AA. Association of interferon-γ receptor-1 gene polymorphism with nontuberculous mycobacterial lung infection among Iranian patients with pulmonary disease. Am J Trop Med Hyg 2017; 97: 57-61.
28Good RC, Snider DE Jr. Isolation of nontuberculous mycobacteria in the United States, 1980. J Infect Dis 1982; 146: 829-833.
29Devana JV, Calambur N, Reddy BR. Pacemaker site infection caused by rapidly growing nontuberculous mycobacteria (RGM). BBRJ 2018; 2(1):82.
30Heydarieh P, Shojaei H, Feyzabadi MM, Havaei A, Hashemi A, Ataei B, et al. Molecular identification and conventional susceptibility testing of Iranian clinical Mycobacterium fortuitum isolates. Iran J Basic Med Sci 2010; 13: 210-215.
31Velayati AA, Farnia P, Hoffner S. Drug-resistant Mycobacterium tuberculosis: Epidemiology and role of morphological alterations. J Glob Antimicrob Resist 2018; 12: 192-196.
32Heidari F, Farnia P, Nowroozi J, Majd A, Masjedi MR, Velayati AA. Evaluating the sensitivity of three primers using PCR-restriction fragment length polymorphism analysis for rapid identification of Mycobacterium simiae isolated from pulmonary tuberculosis patients. Iran J Clin Infect Dis 2010; 5: 30-35.
33Varahram M, Farnia P, Nasiri MJ, Karahrudi MA, Dizagie MK, Velayati AA. Association of Mycobacterium tuberculosis lineages with IFN-γ and TNF-α gene polymorphisms among pulmonary tuberculosis patient. Mediterr J Hematol 2014; 6(1): e2014015.
34Solante MB, Chagan-Yasutan H, Hattori T, Leano S, Garfin AM, Van Soolingen D, et al. High rates of human immunodeficiency virus and drug resistance in tuberculosis patients in Manila, Philippines. Biomed Biotechnol Res J 2017; 1: 157-162.
35Shamsi M, Zolfaghari MR, Farnia P. Evaluation of p2x7 and IFN-γ gene polymorphisms in patients with pulmonary tuberculosis using PCR-RFLP method. Int J Mycobacteriol 2015; 4: 130.
36Mahdaviani SA, Mohajerani SA, Rezaei N, Casanova JL, Mansouri SD, Velayati AA. Pulmonary manifestations of chronic granulomatous disease. Expert Rev Clin Immunol 2013; 9(2): 153-160.
37Simons S, Van Ingen J, Hsueh PR, Van Hung N, Dekhuijzen PR, Boeree MJ, et al. Nontuberculous mycobacteria in respiratory tract infections, Eastern Asia. Emerg Infect Dis 2011; 17: 343-349.
38Baghaei P, Tabarsi P, Farnia P, Marjani M, Sheikholeslami FM, Chitsaz M, et al. Pulmonary disease caused by Mycobacterium simiae in Iran’s national referral center for tuberculosis. J Infect Dev Ctries 2012; 6: 23-28.
39Cirillo DM, Cabibbe AM, De Filippo MR, Trovato A, Simonetti T, Rossolini GM, et al. Use of WGS in Mycobacterium tuberculosis routine diagnosis. Int J Mycobacteriol 2016; 5(1): S252-S253.
40Pokam BD, Guemdjom PW, Yeboah-Manu D, Weledji EP, Enoh JE, Tebid PG. Challenges of bovine tuberculosis control and genetic distribution in Africa. BBRJ 2019; 3(4): 217.
41Joob B, Wiwanitkit V. Common and different lipidomes for lung cancer and tuberculosis: A comparative lipidomics analysis. BBRJ 2019; 3(4): 233.
42Tabarsi P, Baghaei P, Farnia P, Mansouri N, Chitsaz E, Sheikholeslam F, et al. Nontuberculous mycobacteria among patients who are suspected for multidrug-resistant tuberculosis-need for earlier identification of nontuberculosis mycobacteria. Am J Med Sci 2009; 337: 182-184.