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Original Article

An Overview of Pulmonary due to Rapidly Growing Mycobacteria in South Asia and Impressions from a Subtropical Region

Kamal Shrivastava1, Chanchal Kumar1, Anupriya Singh1, Anshika Narang1, Astha Giri1, Naresh Kumar Sharma1, Shraddha Gupta1, Varsha Chauhan1, Jayanthi Gunasekaran1, Viswesvaran Balasubramanian2, Anil Chaudhry3, Rupak Singla4, Rajendra Prasad2, Mandira Varma‑Basil1 Departments of 1Microbiology and 2Pulmonary Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, 3Department of Pulmonary Medicine, Rajan Babu Institute of Pulmonary Medicine and , 4Department of TB and Respiratory Diseases, National Institute of Tuberculosis and Respiratory Diseases, Delhi, India

Abstract

Background: Rapidly growing mycobacteria (RGM) comprise nearly half of the validated of nontuberculous mycobacteria (NTM) and have been reported to have a higher incidence in Asia as compared to Europe and America. There is limited information on RGM infections from South Asia. Hence, the present study aimed to ascertain the incidence of pulmonary infections due to RGM in Delhi and to review the status of available information on the prevalence of RGM in South Asia, a region endemic for tuberculosis. Methods: We analyzed 933 mycobacterial isolates obtained from pulmonary samples in Delhi and performed species identification by polymerase chain reaction (PCR)‑restriction analysis (restriction fragment length polymorphism) and line probe assay. Drug susceptibility testing (DST) was performed by broth microdilution method. We also reviewed reports available on pulmonary infections in South Asia, attributed to RGM. Results: Of the 933 mycobacterial isolates studied, NTM were identified in 152 (16.3%). Of these, 65/152 (42.8%) were RGM comprising fortuitum (34/65; 52.3%), (25/65; 38.5%), (3/65; 4.61%), Mycobacterium mucogenicum (2/65; 3.1%), and (1/65; 1.5%). On applying the American Thoracic Society/Infectious Diseases Society of America guidelines, 11/25 (44%) M. abscessus, 3/3 (100%) M. chelonae, and both isolates of M. mucogenicum were found to be clinically relevant. DST revealed that maximum susceptibility of the RGM was seen to , , and . Conclusions: Of the RGM isolated in the present study, 16/65 (24.6%) were found to be clinically relevant. Hence, it is important to recognize these organisms as potential to identify patients with RGM disease to initiate appropriate therapy.

Keywords: India, nontuberculous mycobacteria, rapidly growing mycobacteria, rapidly growing mycobacteria pulmonary infections, South Asia

Submitted: 15-Nov-2019 Accepted: 23-Nov-2019 Published: 06-Mar-2020

Introduction favors the formation of biofilms, accounting for their resistance to and commonly used .[5] Dispersal Rapidly growing mycobacteria (RGM) are ubiquitous of the organisms from biofilms may also be a source of organisms isolated from soil, dust, rocks, and water and are characterized by visible growth on solid media within Address for correspondence: Dr. Mandira Varma‑Basil, 7 days.[1] Although generally of low , RGM Department of , Vallabhbhai Patel Chest Institute, especially, Mycobacterium abscessus, Mycobacterium University of Delhi, Delhi ‑ 110 007, India. E‑mail: [email protected] fortuitum, Mycobacterium chelonae, and Mycobacterium mucogenicum, are being increasingly seen to cause a wide ORCID: spectrum of diseases including pulmonary, skin, soft tissue, and https://orcid.org/0000-0001-5562-015X disseminated infections.[1‑4] Moreover, the high hydrophobicity

of RGM, and other nontuberculous mycobacteria (NTM), This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to Access this article online remix, tweak, and build upon the work non‑commercially, as long as appropriate credit Quick Response Code: is given and the new creations are licensed under the identical terms. Website: For reprints contact: [email protected] www.ijmyco.org

How to cite this article: Shrivastava K, Kumar C, Singh A, Narang A, DOI: Giri A, Sharma NK, et al. An overview of pulmonary infections due to 10.4103/ijmy.ijmy_179_19 rapidly growing mycobacteria in South Asia and impressions from a subtropical region. Int J Mycobacteriol 2020;9:62-70.

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Shrivastava, et al.: RGM infections in South Asia

nosocomial in patients through water pipes or other articles, we extracted the following data using a data devices.[1] In fact, it has been reported that hospital‑acquired extraction sheet: research setting, study period, population infections due to NTM are most commonly associated with tested and numbers, NTM species isolated, method for NTM RGM.[6] However, due to their ubiquitous nature, of identification, prevalence of pulmonary NTM isolation/disease, RGM from clinical specimens does not necessarily indicate HIV coinfection rate, and risk factors for NTM acquisition. At NTM disease and was until recently, often ignored. The recent least two authors reviewed each article. progress in the development of rapid molecular methods Data analysis in clinical microbiology has led to increased isolation and In estimating country‑level and overall prevalence of NTM identification of these organisms from clinical specimens.[3,4,7] in South Asia, a pooled estimate was computed based on Consequently, a number of new species have been identified the reported prevalence. We checked all retrieved articles and some species previously considered to be contaminants, for application of the American Thoracic Society (ATS) are now recognized as pathogens.[8] With increasing awareness diagnostic criteria for clinically relevant RGM and recorded of the importance of RGM, clinicians are facing challenges the proportion of patients meeting these criteria and NTM while treating patients with RGM infection, especially if the species responsible for infection. RGM has been recently identified as a . Reporting the rapidly growing mycobacteria associated Species‑level identification of RGM is recommended as the with pulmonary infections in Delhi, India susceptibility pattern varies among different species.[8] Since conventional biochemical tests are time‑consuming and Clinical specimens and mycobacterial isolates cumbersome, laboratorians rely on molecular methods of A total of 933 isolates were obtained from patients suspected identification such as polymerase chain reaction restriction of suffering from pulmonary mycobacterial disease between analysis (PRA), line probe assay (LPA), and sequencing of January 2014 and April 2019 at the Department of Microbiology, hsp65 or16s rRNA. Furthermore, most RGM are resistant to Vallabhbhai Patel Chest Institute, Delhi, India, after approval first‑line antituberculous drugs. In fact, M. abscessus is the from the Institutional ethical committee. The patients had most difficult to treat. Hence, drug susceptibility has been reported to the outpatient unit of Vallabhbhai Patel Chest Institute, recommended for all RGM found to be clinically relevant.[2,9] Rajan Babu Institute of Pulmonary Medicine and TB of Delhi, or National Institute of TB and Respiratory Diseases, Delhi, India. Although reports on the incidence of RGM infection are The clinical isolates obtained were characterized by their colony increasing, most of the reports come from industrialized morphology on Lowenstein–Jensen medium and were subjected nations of the world.[2,10] There is still a paucity of data on RGM to biochemical identification by niacin, nitrate reduction, and infections from South Asia, a region that is also endemic for semi‑quantitative catalase tests.[14] Further characterization of tuberculosis (TB).[11,12] Here, we report our experience with the isolates was performed by PRA of the hsp65 gene using the RGM associated with pulmonary infections in Delhi, India, and enzymes NruI and BamHIas previously described.[15] present an overview of clinically significant RGM identified in South Asia, to understand the extent of awareness of these Species identification by line probe assay pathogens in this region. Species identification of the isolates identified as NTM was performed by GenoType Mycobacterium CM/AS (Hain Lifescience GmbH, Germany). Methods Literature search Sanger sequencing Species identification of a subset of the clinical isolates A review of the reports on RGM in South Asia was conducted in was confirmed by Sanger sequencing of heat‑shock accordance with PRISMA guidelines.[13] The overall aim of this protein‑65 (hsp65) gene in an Applied Biosystems Automated review was to determine the prevalence of clinically significant Sequencer (Ocimum Biosolutions, Bengaluru, India). RGM in patients with pulmonary infection in South Asia. Sequences were identified by similarity using Blastn We included Afghanistan, Bangladesh, Bhutan, Nepal, India, available at National Center for Biotechnology Information Pakistan, Sri Lanka, and the Maldives in the South Asian region. (NCBI) (www.blast.ncbi.nlm.nih.gov/blast.cgi). Species We searched PubMed, Scopus, EMBASE, and Copernicus identification was confirmed if 97% match was obtained with for publications on RGM involved in pulmonary infection a sequence deposited in the database, according to the criteria in South Asia from January 2002 to June 2018 using various proposed by McNabb et al.[16] combinations of the search terms NTM, atypical mycobacteria, RGM, South Asia, Afghanistan, Bangladesh, Bhutan, Nepal, Clinical relevance of rapidly growing mycobacteria India, Pakistan, Sri Lanka, Maldives, and pulmonary infections. Clinical records of patients were reviewed to assess the clinical relevance of the NTM isolated according to ATS guidelines.[2] Selection process and data abstraction The titles and abstracts of all the articles obtained in the Minimum inhibitory concentration database search were screened and full‑text copies of those Minimum inhibitory concentrations (MICs) of the clinically found to be relevant to our search obtained. For all relevant relevant RGM were performed by broth microdilution

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method described by Li et al. in U‑bottomed microtiter Table 1: Antimicrobials tested against rapidly growing plates (Falcon, New York) using streptomycin (STR), mycobacteria in the present study (INH), ethambutol (EMB), ciprofloxacin (CIP), clarithromycin (CLR), amikacin (AMK), cefoxitin (FOX), Antimicrobials Solvent Range tested (µg/ml) linezolid (LZD), doxycycline (DOX), imipenem (IPM), STR Water 0.5‑64 sulfamethoxazole (SFX), levofloxacin (LVX), tetracycline, INH Water 0.5‑64 and clofazimine (Sigma Aldrich, St. Louis, MO, USA) and RIF DMSO 0.5‑64 (RIF) (MP Biomedicals, Santa Ana, CA, USA).[17] EMB Water 0.5‑64 Various concentrations of the drugs [Table 1], dissolved in AMK Water 0.5‑64 an appropriate solvent, were added to microtiter plates in FOX Water 0.0625‑8 triplicates, followed by the addition of 100 µl of the test CLR Acetone 0.25‑32 inoculum that had been adjusted to 0.5 McFarland’s standard. CIP 0.1 N HCl 0.5‑64 The microtiter plates were sealed with parafilm and incubated LZD DMSO 0.968‑124 DOX DMSO 0.25‑512 at 37°C for 72 h. Freshly prepared 30 µl of Alamar Blue IPM Water 1‑128 reagent (0.2 mg/ml) was added to the wells after 3 days. The SFX Acetone 2‑256 plates were reincubated at 37°C for 24 h, and the color change LVX DMSO 0.5‑64 in all wells, from blue to pink indicating the growth of , CLO Water 0.0625‑8 was recorded. MIC was recorded as the lowest concentration STR: Streptomycin, INH: Isoniazid, RIF: Rifampicin, EMB: Ethambutol, of drug preventing color change. AMK: Amikacin, FOX: Cefoxitin, CIP: Ciprofloxacin, CLR: Clarithromycin, LZD: Linezolid, DOX: Doxycycline, Ethics statement IPM: Imipenem, SFX: Sulfamethoxazole, LVX: Levofloxacin, Written informed consent and detailed history of contact CLO: Clofazimine, DMSO: Dimethyl sulphoxide were taken from each patient prior to the collection of samples, following approval of the study by the Institutional M. chelonae (173/852; 20.3%). Of the 470 RGM isolated Ethics Committee of Vallabhbhai Patel Chest Institute. All from pulmonary NTM disease, M. fortuitum (181/470; 38.5%) experiments were performed in accordance with the ethical was the most common, followed by M. abscessus (146/470; standards of the Declaration of Helsinki. 31%). M. abscessus pulmonary disease was reported in India, though the report from Pakistan reported the occurrence of Results M. chelonae–M. abscessus. In Pakistan, Mycobacterium Description of included studies smegmatis (11/64; 17.19%) and M. mucogenicum (9/64; 14.06%), were the most common RGM found in pulmonary We conducted a review of the literature focusing on samples after M. fortuitum (16/64; 25%) [Figure 1]. RGM reported in South Asia. With the search terms nontuberculous mycobacteria, atypical mycobacteria, rapidly Clinical relevance of nontuberculous mycobacteria growing mycobacteria, South Asia, Afghanistan, Bangladesh, For the analysis, we excluded articles where specific criteria for Bhutan, Nepal, India, Pakistan, Sri Lanka, Maldives, identifying clinical relevance of NTM (viz., ATS guidelines) had pulmonary infections, we identified 81 reports from South not been followed and nine articles were selected.[11,18‑20,25,26,28,29,31] Asia and excluded case reports, reviews, and editorials. However, clinically relevant RGM were identified in only eight We also excluded publications where the isolates had not studies. The ninth study isolated only one RGM, Mycobacterium been clearly differentiated as pulmonary or extrapulmonary, flavescens, which was not clinically relevant.[29] Out of the eight identification had not been done up to species level, where studies included in the final analysis, maximum number of the RGM described were reported only from extrapulmonary reports was from India (6/8; 75%). Pakistan and Sri Lanka had sites or from the environment. Thus, 17 articles were one study each (1/8; 12.5%) [Table 2]. selected [Table S1a and b], of which the maximum number Of the 238 clinically significant NTM isolated from pulmonary of reports was from India (14/17; 82.3%) [Table 2].[12,15,18‑29] specimens, 122 (51.26%) were rapid growers. The most Pakistan, Sri Lanka, and Nepal had one study each (1/17; common RGM was M. chelonae (46/122; 37.7%), followed 5.8%) [Table 2].[11,30,31] The most common method used by M. fortuitum (45/122; 36.89%) and M. abscessus (18/122; for species identification was the difference in growth and biochemical characteristics observed in 15/17 (88.2%) articles. 14.75%) [Figure 2]. Of these, 8/15 (53%) also used a molecular assay. Investigators Drug susceptibility profile of the clinically relevant rapidly used the MPT64 antigen immunochromatography test in growing mycobacteria in the included studies 2/17 (11.7%) studies to differentiate between Mycobacterium The eight articles that identified clinically relevant RGM were tuberculosis complex (MTBC) and NTM [Table 2]. also screened for reports of drug susceptibility profile of the Of the 1324 NTM identified, 852 (64.35%) were rapid RGM and five articles reporting drug susceptibility testing (DST) growers. The most common RGM was M. fortuitum (333/852; were selected for analysis.[11,18,19,26,31] Of these, 3/5 (60%) studies 39.08%), followed by M. abscessus (250/852; 29.34%) and were from India and 1/5 (20%) each were from Pakistan and Sri

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Figure 1: Rapidly growing mycobacteria causing pulmonary infections in South Asia.[11,12,15,18‑31] Highlighted box shows the rapidly growing mycobacteria isolated from pulmonary infections in the present study

Figure 2: Clinically relevant rapidly growing mycobacteria isolated from pulmonary specimens in South Asia.[11,18‑20,25,26,28,29,31] Highlighted box shows the clinically relevant rapidly growing mycobacteria isolated from pulmonary infections in the present study

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Table 2: Description of the studies included from South Asia Country Author Method of species identification Number of rapid growers isolated Clinical guidelines DST (reference) from pulmonary samples followed (yes/no) (yes/no) India Jesudason Growth characteristics 15 Yes Yes et al., 2005 Biochemical tests Khatter et al., Growth characteristics 5 Yes Yes 2008 Biochemical tests Shennai et al., Growth characteristics 30 Yes No 2010 PNB, RLBH assay, PRA Gayathri et al., Biochemical tests, RFLP 72 No Yes 2010 Sequencing Garima et al., Biochemical tests 10 No No 2012 PRA, sequencing Anilkumar Tetraplex‑PCR, RFLP, sequencing 11 No No et al., 2012 Myneedu Growth characteristics 8 No No et al., 2013 Biochemical tests Varma‑Basil Biochemical tests 6 No No et al., 2013 PRA, sequencing Jain et al., Growth characteristics biochemical tests 2 Yes No 2014 Immunochromatography (MPT64) assay PCR for mycobacterium , MTC (ESAT6) MAC specific gene Raveendran Growth characteristics 10 Yes Yes et al., 2015 AccuProbe assay GenoType mycobacterium CM/AS Umrao et al., Biochemical tests 124 No No 2016 MPT64 Ag assay GenoType mycobacterium CM/AS Goswami Growth characteristics 34 No Yes et al., 2016 Biochemical tests Verma et al., Biochemical tests 72 Yes No 2018 PCR‑RFLP Sharma et al., TB‑ID 1 Yes No 2018 PRA Pakistan Ahmed et al., Growth characteristics 64 Yes Yes 2013 Biochemical tests Sri Lanka Keerthirathane Growth characteristics 3 Yes Yes et al., 2016 Biochemical tests Nepal Dhungana Growth characteristics 3 No No et al., 2008 Biochemical tests PCR: Polymerase chain reaction, PRA: PCR restriction analysis, MTC: Mycobacterium tuberculosis complex, DST: Drug susceptibility test

Lanka. Studies that did not report species‑specific susceptibility NTM. Of these, 152/933 (16.3%) isolates were identified pattern were not included in the final analysis. as NTM. The DST pattern revealed that no uniform methodology was used Speciation of the isolated nontuberculous mycobacteria in the five studies that reported the DST. Two studies each (2/5; and establishment of clinical relevance of the rapidly 40%) used broth microdilution assay and disc diffusion assay growing mycobacteria and one (1/5; 20%) used Etest. A varying resistance pattern was Of all the NTM isolated, RGM were identified in observed to all the antibiotics tested [Table 3]. 65/152 (42.8%) isolates and the remaining were slow Rapidly growing mycobacteria associated with pulmonary growers. Majority of the RGM (34/65; 52.3%) were infections in Delhi, India M. fortuitum, followed by M. abscessus (25/65; 38.5%), Culture identification M. chelonae (3/65; 4.6%), M. mucogenicum (2/65; 3%), and Clinical isolates (n = 933) were subjected to primary M. smegmatis (1/65; 1.5%). Mycobacterium intracellulare screening by PRA to differentiate between MTBC and was the most common (45/87; 51.7%) slow‑growing NTM

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Table 3: Drug resistance profile of the clinically relevant rapidly growing mycobacteria isolated from pulmonary samples in the studies included from South Asia Author (reference) Methods of DST Number Number of resistant isolates, n (%) of isolates CIP CLR AMK LZD FOX IPM SFX AZM DOX Raveendran et al., 2015 Etest 16 3 (18.8) 3 (18.8) 0 ‑ ‑ 7 (43.8) 9 (56.3) 10 (62.5) Khatter et al., 2008 Broth microdilution 2 0 2 (100%) 2 (100%) Jesudason et al., 2005 Kirby‑Bauer 47 37 (78.7) ‑ 1 (2.1) ‑ ‑ ‑ ‑ ‑ Ahmed et al., 2013 Broth microdilution 6 2 (33.3) ‑ 0 1 (16.7) ‑ 5 (83.3) ‑ 5 (83.3) Mycobacterium abscessus Raveendran et al., 2015 Etest 14 13 (92.9) 0 1 (7.1) ‑ ‑ 13 (92.9) 14 (100) 13 (92.9) Mycobacterium chelonae Raveendran et al., 2015 Etest 6 3 (50) 0 0 ‑ ‑ 5 (83.3) 4 (66.7) 5 (83.3) Mycobacterium chelonae‑abscessus complex Keerthiratane et al., Agar proportion 3 3 (100) 1 (33.3) 0 ‑ ‑ ‑ ‑ ‑ 2016 and disc diffusion CIP: Ciprofloxacin, CLR: Clarithromycin, AMK: Amikacin, LZD: Linezolid, FOX: Cefoxitin, IPM: Imipenem, SFX: Sulfamethoxazole, AZM: Azithromycin, DOX: Doxycycline, DST: Drug susceptibility test

Table 4: Drug resistance profile of the rapidly growing mycobacteria isolated from pulmonary samples in the present study Antimicrobial RGM number (percentage of resistant) Mycobacterium abscessus (4), n (%) Mycobacterium mucogenicum (1), n (%) Mycobacterium chelonae (2), n (%) STR 4 (100) 1 (100) 2 (100) INH 4 (100) 1 (100) 2 (100) RIF 4 (100) 1 (100) 2 (100) EMB 4 (100) 1 (100) 2 (100) AMK 1 (25) 0 0 FOX 1 (25) 0 0 (100) CLR 0 0 1 (50) CIP 3 (75) 0 1 (50) LZD 0 0 0 DOX 4 (100) 1 (100) 2 (100) IPM ‑‑ 1 (100) ‑‑ SFX 3 (75) 1 (100) 2 (100) LVX 3 (75) 0 1 (50) CLO 2 (50) 1 (100) 2 (100) STR: Streptomycin, INH: Isoniazid, RIF: Rifampicin, EMB: Ethambutol, AMK: Amikacin, FOX: Cefoxitin, CIP: Ciprofloxacin, CLR: Clarithromycin, LZD: Linezolid, DOX: Doxycycline, IPM: Imipenem, SFX: Sulfamethoxazole, LVX: Levofloxacin, CLO: Clofazimine, RGM: Rapidly growing mycobacteria

isolated, followed by Mycobacterium kansasii (24/87; 27.6%), Antimicrobial susceptibility testing (15/87; 17.2%), Mycobacterium The isolates were categorized into sensitive, intermediate, and celatum (1/87; 1.1%), and (1/87; resistant according to the breakpoint described previously.[32] 1.1%). One slow‑growing NTM could not be speciated. Antimicrobial susceptibility testing was performed for the Of the 65 isolates of RGM, clinical relevance was attributed clinically relevant RGM. Since multiple isolates were obtained to 16/65 (24.6%) isolates on the basis of ATS guidelines.[2] Of from patients, a single isolate from each patient was taken up for the 25 M. abscessus identified, 11/25 (44%) were clinically DST. One isolate of M. abscessus was lost during subculture, relevant and were recovered repeatedly from five patients. Two thus antimicrobial susceptibility testing was performed for M. mucogenicum isolates were obtained from repeated samples four isolates of M. abscessus, two isolates of M. chelonae, of a single patient with symptoms of prolonged cough, fever, and one isolate of M. mucogenicum. All the four isolates of M. and loss of appetite. Two isolates of M. chelonae were also abscessus were resistant to STR, INH, RIF, EMB, DOX, and repeatedly isolated from a single patient with tracheostomy. SFX [Table 4]. None of the isolates were resistant to CLR and The third isolate of M. chelonae was a repeat isolate obtained LZD. M. mucogenicum (n = 1) and M. chelonae (n = 2) were from a patient with complaints of cough and weight loss. also resistant to STR, INH, RIF, EMB, and SFX [Table 4].

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M. mucogenicum was susceptible to AMK, FOX, CLR, CIP, an in‑house reverse line blot hybridization assay,[20] while two LZD, and LVX, while M. chelonae showed susceptibility only used the genotype CM/AS assay.[26,27] to AMK, CLR, and LZD. Of the 1324 NTM identified in the 17 articles retrieved from South Asia, 852 (64.35%) were RGM. Although Discussion RGM comprise about 50% of the currently validated NTM RGM comprise nearly half of the validated mycobacterial species encountered in clinical settings, in various surveys species known till date and have been grouped into six conducted previously, RGM were found to vary from 3% to major taxonomic groups on the basis of their phenotypic and 14% of the clinically relevant NTM isolated from pulmonary genotypic characters, namely, M. fortuitum, M. chelonae/M. samples.[33,34] However, in their review of NTM isolation from abscessus complex, M. smegmatis, M. mucogenicum, Asia, Simons et al. reported that RGM were responsible for Mycobacterium mageritense/, 14% of pulmonary NTM infections, except in India, Taiwan, and the pigmented RGM.[33] Although ubiquitous in nature, South Korea, where they were responsible for >30% of RGM can lead to a wide spectrum of diseases ranging from infections. Simons et al. hypothesized that the fact that RGM asymptomatic illness to soft‑tissue infections to chronic were frequently found in pulmonary samples in Asia, could pulmonary infections. In fact, RGM were first implicated reflect ethnic susceptibility, laboratory practices, or higher in human disease in 1938 when da Costa Cruz attributed a environmental exposure to RGM in Asia.[34] postinoculation abscess to an RGM that he described and [1] The most common RGM isolated from pulmonary named M. fortuitum. Since then, RGM have increasingly specimens, identified in the 17 articles included in this been implicated as pathogens and reports have further review, was M. fortuitum (181/470; 38.5%), followed by increased with the advent of molecular assays. M. abscessus (146/470; 31%) [Figure 1]. The rare RGM, Like other NTM, the distribution of RGM also varies by region. M. thermoresistibile was isolated in one study from Pakistan.[35] Although geographic diversity has been studied by some However, the source of these isolates was not mentioned, hence investigators, there are very little data from South Asia. Hence, the study was not included in the final analysis. Although we reviewed the available literature from eight countries in M. thermoresistibile has been isolated earlier in Iran, no cases South Asia, namely, Afghanistan, Bangladesh, Bhutan, Nepal, have yet been reported from South Asia, other than Pakistan.[36] India, Pakistan, Sri Lanka, and the Maldives. It may be noted, that of the 17 studies included, only nine studies had followed the clinical guidelines (namely, ATS We obtained 81 reports from this region, of which we analyzed guidelines) to identify clinically relevant RGM. 17 reports that had given details of the source of samples collected. No data were available from Afghanistan, Bhutan, Drug susceptibility profile was reported in five studies that Bangladesh, or Maldives. A maximum number of reports were demonstrated isolation of clinically relevant RGM. It was from India, followed by Pakistan [Table 2]. Of the 17 articles observed that no uniform method of DST was used in these retrieved, the most common method used for identification studies. Two studies each (2/5; 40%) used broth microdilution was difference in growth and biochemical characteristics assay and disc diffusion assay and one (1/5; 20%) used E test. observed in 15/17 (88.2%) articles, of which 8/15 (53%) used Although a varying resistance pattern was observed to all the a molecular assay in addition to conventional techniques. The antibiotics tested, maximum susceptibility was observed to number of articles reporting use of molecular assays alone or AMK [Table 3]. in conjunction with an immunochromatographic technique In our own investigation, we identified RGM in 65 (42.8%) of was lower (2/17; 11.7%) than the studies using conventional the 152 NTM isolated from clinical specimens. On applying the techniques alone (7/17; 41.18) (Fisher’s exact test >P = 0.05). ATS guidelines for clinical relevance, 11 of the M. abscessus were Given the improvement in molecular diagnostics in the last clinically relevant, as they were isolated from multiple samples decade and the availability of rapid results, we expected a of five patients. Two isolates ofM. mucogenicum were isolated greater number of investigators using molecular diagnostic repeatedly from the respiratory secretions of a single patient. tools. However, the popularity of the conventional methods of M. mucogenicum has rarely been reported from South Asia. diagnostics was probably due to the higher cost of molecular One study in Pakistan isolated nine isolates of M. mucogenicum tests which are not easily accessible in several regions of from pulmonary samples and three from extrapulmonary South Asia. samples.[11] In India, M. mucogenicum was reported to be The most common molecular tests used were PRA (7/17; clinically relevant in a sample from pleural tap in one study. 41.18%) and line probe hybridization assays (3/17; 17.65%). [24] Sankar et al. reported the drug susceptibility of a single Sequencing was used only in four studies (23.53%) [Table 2]. clinical isolate of M. mucogenicum.[37] However, the source of Sequencing is not easily accessible to several laboratories, this isolate was not mentioned. The ATS guidelines recommend while PRA is a sensitive and specific technique which is routine susceptibility testing for both taxonomic identification more cost‑effective than the currently available commercial and treatment of M. fortuitum, M abscessus, and M. chelonae assays, which may account for it being used more frequently with AMK, DOX, fluorinated quinolones, a sulfonamide or than sequencing. Of the studies using LPAs, one study used trimethoprim‑SFX, FOX, CLR, and LZD.[2] Testing against IPM

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is recommended only for M. fortuitum and M. mucogenicum, as mycobacteria in the human environment. J Appl Microbiol it is reproducible. Hence, in the present study, we tested IPM 2009;107:356‑67. 6. Wallace RJ Jr., Brown BA, Griffith DE. Nosocomial outbreaks/ only against M. mucogenicum (n = 1) and found it to be resistant. pseudo‑outbreaks caused by nontuberculous mycobacteria. Annu Rev Microbiol 1998;52:453‑90. The investigations conducted in our laboratory revealed 7. Gharbi R, Mhenni B, Ben Fraj S, Mardassi H. Nontuberculous high susceptibility of M. abscessus to LZD, CLR, and mycobacteria isolated from specimens of pulmonary AMK [Table 4]. Similarly, high susceptibility of M. abscessus tuberculosis suspects, Northern Tunisia: 2002‑2016. BMC Infect Dis to CLR and AMK has been reported in other studies also.[12,26] 2019;19:819. 8. Colombo RE, Olivier KN. Diagnosis and treatment of infections The two isolates of M. chelonae were susceptible to AMK, caused by rapidly growing mycobacteria. Semin Respir Crit Care Med CLR, and LZD, consistent with the report of Goswami et al., 2008;29:577‑88. 9. Woods GL, Bergmann JS, Witebsky FG, Fahle GA, Wanger A, though the latter had not reported the susceptibility of M. Boulet B, et al. Multisite reproducibility of results obtained by the chelonae to LZD.[12] broth microdilution method for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. The Clinical and Laboratory Standards Institute recommends J Clin Microbiol 1999;37:1676‑82. the broth microdilution method for DST of RGM. Agar‑based 10. van Ingen J, Boeree MJ, Dekhuijzen PN, van Soolingen D. methods and E test are not recommended due to inconsistent Environmental sources of rapid growing nontuberculous mycobacteria causing disease in humans. Clin Microbiol Infect 2009;15:888‑93. results. However, a number of articles we reviewed had used 11. Ahmed I, Jabeen K, Hasan R. Identification of non‑tuberculous [18,21,26] the Etest or Kirby–Bauer method for DST of RGM. The mycobacteria isolated from clinical specimens at a tertiary care hospital: variation in the drug susceptibility patterns in various studies A cross‑sectional study. BMC Infect Dis 2013;13:493. highlights the importance of using a uniform technique for 12. Goswami B, Narang P, Mishra PS, Narang R, Narang U, Mendiratta DK. Drug susceptibility of rapid and slow growing non‑tuberculous drug susceptibility assays of RGM. mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India. Indian J Med Microbiol 2016;34:442‑7. 13. Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred Conclusions reporting items for systematic reviews and meta‑analyses: The PRISMA There is a large gap in our knowledge of RGM in South Asia. statement. PLoS Med 2009;6:e1000097. In fact, there are no reports from a number of regions. With 14. Kent PT, Kubica GP. A Guide for the Level III Laboratory. Atlanta, GA: Centers for Disease Control; 1985. the improvement in molecular diagnostic techniques and better 15. Varma‑Basil M, Garima K, Pathak R, Dwivedi SK, Narang A, awareness of the importance of RGM as potential pathogens, Bhatnagar A, et al. Development of a novel PCR restriction analysis it is very important to generate data on the occurrence of these of the hsp65 gene as a rapid method to screen for the Mycobacterium pathogens to be able to identify patients harboring these agents. tuberculosis complex and nontuberculous mycobacteria in high‑burden countries. J Clin Microbiol 2013;51:1165‑70. It is also necessary to follow uniform methodology for DST 16. McNabb A, Eisler D, Adie K, Amos M, Rodrigues M, Stephens G, to be able to treat these patients. et al. Assessment of partial sequencing of the 65‑kilodalton heat shock protein gene (hsp65) for routine identification of Acknowledgment Mycobacterium species isolated from clinical sources. J Clin KS and CK would like to acknowledge the Department of Microbiol 2004;42:3000‑11. 17. Li G, Lian LL, Wan L, Zhang J, Zhao X, Jiang Y, et al. Antimicrobial Science and Technology and Indian Council of Medical susceptibility of standard strains of nontuberculous mycobacteria by Research, respectively, for providing fellowship. microplate alamar blue assay. PLoS One 2013;8:e84065. 18. Jesudason MV, Gladstone P. Non tuberculous mycobacteria isolated Financial support and sponsorship from clinical specimens at a tertiary care hospital in South India. Indian This work was supported by the Indian Council of Medical J Med Microbiol 2005;23:172‑5. Research, India (No. 5/8/5/14/2013‑ECD1). 19. Khatter S, Singh UB, Arora J, Rana T, Seth P. Mycobacterial infections in human immuno‑deficiency virus seropositive patients: Role of Conflicts of interest non‑tuberculous mycobacteria. Indian J Tuberc 2008;55:28‑33. 20. Shenai S, Rodrigues C, Mehta A. Time to identify and define There are no conflicts of interest. non‑tuberculous mycobacteria in a tuberculosis‑endemic region. Int J Tuberc Lung Dis 2010;14:1001‑8. References 21. Gayathri R, Therese KL, Deepa P, Mangai S, Madhavan HN. susceptibility pattern of rapidly growing mycobacteria. J Postgrad Med 1. De Groote MA, Huitt G. Infections due to rapidly growing mycobacteria. 2010;56:76‑8. Clin Infect Dis 2006;42:1756‑63. 22. Garima K, Varma‑Basil M, Pathak R, Kumar S, Narang A, Rawat KS, 2. Griffith DE, Aksamit T, Brown‑Elliott BA, Catanzaro A, Daley C, et al. Are we overlooking infections owing to non‑tuberculous Gordin F, et al. An official ATS/IDSA statement: Diagnosis, treatment, mycobacteria during routine conventional laboratory investigations? 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25. Jain S, Sankar MM, Sharma N, Singh S, Chugh TD. High prevalence Sooriyapathirana SS. Real time PCR for the rapid identification and of non‑tuberculous mycobacterial disease among non‑HIV infected drug susceptibility of mycobacteria present in Bronchial washings. individuals in a TB endemic country‑experience from a tertiary center BMC Infect Dis 2016;16:607. in Delhi, India. Pathog Glob Health 2014;108:118‑22. 32. Pang H, Li G, Zhao X, Liu H, Wan K, Yu P. Drug susceptibility 26. Raveendran R, Oberoi JK, Wattal C. Multidrug‑resistant pulmonary and testing of 31 antimicrobial agents on rapidly growing mycobacteria extrapulmonary tuberculosis: A 13 years retrospective hospital‑based isolates from China. Biomed Res Int 2015;2015:419392.doi: analysis. Indian J Med Res 2015;142:575‑82. 10.1155/2015/419392. 27. Umrao J, Singh D, Zia A, Saxena S, Sarsaiya S, Singh S, et al. 33. Brown‑Elliott BA, Philley JV. Rapidly growing mycobacteria. Microbiol Prevalence and species spectrum of both pulmonary and extrapulmonary Spectr 2017;5:10.1128/microbiolspec.TNMI7-0027-2016.doi:10.1128/ nontuberculous mycobacteria isolates at a tertiary care center. Int J microbiolspec.TNMI7-0027-2016. Mycobacteriol 2016;5:288‑93. 34. Simons S, van Ingen J, Hsueh PR, Van Hung N, Dekhuijzen PN, 28. Verma AK, Sarin R, Arora VK, Kumar G, Arora J, Singh P, et al. Boeree MJ, et al. Nontuberculous mycobacteria in respiratory tract Amplification of Hsp 65 gene and usage of restriction endonuclease for infections, Eastern Asia. Emerg Infect Dis 2011;17:343‑9. identification of non tuberculous rapid growerMycobacterium . Indian J 35. Khanum T, Rasool SA, Ajaz M, Khan AI. Isolation‑drug resistance Tuberc 2018;65:57‑62. profile and molecular characterization of indigenous typical and atypical 29. Sharma P, Singh D, Sharma K, Verma S, Mahajan S, Kanga A. mycobacteria. Pak J Pharm Sci 2011;24:527‑32. Are we neglecting nontuberculous mycobacteria just as 36. Velayati AA, Farnia P, Mozafari M, Mirsaeidi M. Nontuberculous laboratory contaminants? Time to reevaluate things. J Pathog mycobacteria isolation from clinical and environmental samples in Iran: 2018;2018:8907629. Twenty years of surveillance. Biomed Res Int 2015;2015:254285. doi: 30. Dhungana GP, Ghimire P, Sharma S, Rijal BP. Characterization of https://doi.org/10.1155/2015/254285. mycobacteria in HIV/AIDS patients of Nepal. JNMA J Nepal Med 37. Sankar MM, Gopinath K, Singla R, Singh S. In vitro antimycobacterial Assoc 2008;47:18‑23. drug susceptibility testing of non‑tubercular mycobacteria by tetrazolium 31. Keerthirathne TP, Magana‑Arachchi DN, Madegedara D, microplate assay. Ann Clin Microbiol Antimicrob 2008;7:15.

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Table S1a: Details of the studies included from South Asia that reported Rapidly growing mycobacteria S. No. Author and Year Reference Country Duration of No. of NTM Total rapid No. of rapid growers from No. Study isolated growers pulmonary samples 1 Verma AK et al., 2018 28 India 2013‑2014 121 121 72 2 Sharma et al., 2018 29 India 2013‑2014 26 5 1 3 Goswami B et al., 2016 12 India 2007‑2009; 65 34 34 2012‑2013 4 Umrao J et al., 2016 27 India 2013‑2015 263 165 124 5 Raveendran R et al., 2015 26 India 2000‑2012 154 96 10 6 Jain S et al., 2014 25 India 2011‑2012 13 4 2 7 Myneedu VP et al., 2013 24 India 2009‑2011 60 18 8 8 Varma‑Basil M et al., 2013 15 India 2007‑2010 15 6 6 9 Garima K et al., 2012 22 India 2007‑2010 44 12 10 10 Anilkumar AK et al., 2012 23 India 2004‑2009 24 11 11 11 Shennai S et al., 2010 20 India 2005‑2008 127 46 30 12 Gayatri R et al., 2010 21 India 2000‑2008 148 148 72 13 Khatter S et al., 2008 19 India 18 months 8 5 5 14 Jesudason MV et al., 2005 18 India 1999‑2004 115 103 15 15 Ahmed I et al., 2013 11 Pakistan 2010‑2011 104 72 64 16 Keerthirathane et al., 2016 31 Sri 1anka 2014‑2015 21 3 3 17 Dhungana GP et al., 2008 30 Nepal 2004‑2005 16 3 3 Total 1324 852 470 [Downloaded freefromhttp://www.ijmyco.orgonFriday,January29,2021,IP:197.46.112.30]

Table S1b: Details of Rapidly growing mycobacteria isolated in the studies included from South Asia S. No. Reference M M. M. M. M. M. M. M. M. M. M. M. Unidentified No. abscessus fortuitum chelonae chelonae‑abscessus mucogenicum flavescens vaccae phlei smegmatis immunogenum senegalense peregrinum RGM 1 28 5 28 35 0 0 0 0 0 0 3 0 1 0 2 29 0 0 0 0 0 1 0 0 0 0 0 0 0 3 12 3 21 3 0 0 7 0 0 0 0 0 0 0 4 27 65 43 16 0 0 0 0 0 0 0 0 0 0 5 26 4 4 2 0 0 0 0 0 0 0 0 0 0 6 25 0 1 1 0 0 0 0 0 0 0 0 0 0 7 24 0 2 3 0 0 1 0 2 0 0 0 0 0 8 15 3 3 0 0 0 0 0 0 0 0 0 0 0 9 22 0 10 0 0 0 0 0 0 0 0 0 0 0 10 23 7 0 1 0 0 0 0 0 2 0 1 0 0 11 20 20 10 0 0 0 0 0 0 0 0 0 0 0 12 21 39 33 0 0 0 0 0 0 0 0 0 0 0 13 19 0 2 0 0 0 0 2 1 0 0 0 0 0 14 18 0 6 8 0 0 0 0 0 1 0 0 0 0 15 11 0 16 0 6 9 1 4 0 11 0 0 0 17 16 31 0 0 0 3 0 0 0 0 0 0 0 0 0 17 30 0 2 1 0 0 0 0 0 0 0 0 0 0 Total 146 181 70 9 9 10 6 3 14 3 1 1 17