Profile of Nontuberculous Mycobacteria in Patients Suspected of Tuberculosis and Drug-Resistant Tuberculosis
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Published online: 2020-11-23 THIEME Original Article 203 Profile of Nontuberculous Mycobacteria in Patients Suspected of Tuberculosis and Drug-Resistant Tuberculosis Megha Sharma1 Bharti Malhotra1 Jitendra Tiwari2 Shipra Bhargava1 1Department of Microbiology, SMS Medical College, Jaipur, Address for correspondence Bharti Malhotra, MBBS, MD, Rajasthan, India Department of Microbiology, SMS Medical College, Jaipur, 2Department of Microbiology, Government Medical College, Rajasthan, 302004 India (e-mail: [email protected]). Bharatpur, Rajasthan, India J Lab Physicians:2020;12:203–211 Abstract Objective Infections due to nontuberculous mycobacteria (NTM) is increasing glob- ally and may present as drug-resistant tuberculosis (DRTB). In India, data on NTM prevalence and species diversity is limited. Present study was conducted to detect the prevalence and profile of NTM among patients suspected of DRTB using paraffin slide culture (PSC)and mycobacteria growth indicator tube (MGIT) culture methods for isolation of NTM. Material and Method A total of 2,938 samples suspected of TB/DRTB were cultured on PSC and MGIT960. Species identification of mycobacterial isolate was done by sequencing of 16s ribosomal RNA gene. Result Among 2938 samples, 35 (1.19%) were found positive for NTM by PSC and 9 (0.30%) were found positive by MGIT. The diversity of NTM species was high (13 species). Out of 35 NTM isolates by PSC, maximum 34.29% (12) isolates were found to be Mycobacterium fortuitum, followed by 11.43% (4) Mycobacterium abscessus and Mycobacterium chelonae, and 42.85% (15) were other species viz. 8.57% (3) were Mycobacterium intracellulare and Mycobacterium kansasii, 5.71% (2) were Mycobacterium peregrinum, and 2.85% (1) were Mycobacterium flavescens, Mycobacterium farcinogenes, Mycobacterium moriokanese, Mycobacterium wolinskyi, Mycobacterium simiae, Mycobacterium goodii, and Mycobacterium terrae each. Coinfection of Mycobacterium tuberculosis(MTB) and NTM was found in 60% (21) samples. Keywords Conclusion Prevalence of NTM was low among multidrug resistant tuberculosis/TB ► nontuberculous suspected patients, similar to other studies done in India. PSC was found better than mycobacteria (NTM) MGIT for the isolation of NTM, though poor separation of NTM and MTB on subculture ► paraffin slide may have led to false negativity in cases of coinfection. About 13 species were iso- culture (PSC) lated; M. fortuitum was the most common of all. Since coinfection of NTM and TB can ► mycobacterium also occur, samples of patients suspected of NTM should be cultured on PSC even if tuberculosis (MTB) positive for MTB. DOI https://doi.org/ © 2020. The Indian Association of Laboratory Physicians. 10.1055/s-0040-1721160 This is an open access article published by Thieme under the terms of the Creative ISSN 0974-2727 . Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/) Thieme Medical and Scientific Publishers Pvt. Ltd., A-12, 2nd Floor, Sector 2, Noida-201301 UP, India 204 NTM Detection and Profile Sharma et al. Introduction presumptive DRTB and TB suspected patients were included in the study. This study was approved by the Institutional Nontuberculous mycobacteria (NTM) are opportunistic Ethical Committee. pathogens that cause surgical-site infections, postinjection abscesses, osteomyelitis, catheter-related bloodstream infec- Inclusion Criteria tions, and central nervous system infections, mostly affect- Pulmonary samples with more than 5 mL volume and ing patients with the pre-existing pulmonary disease such extrapulmonary samples with more than 2 mL volume were as chronic obstructive pulmonary disease (COPD) or tuber- included in the study. culosis (TB), or immunocompromised patients with HIV All samples received in laboratory having adequate volume infection, leukemia, or those patients on immunosuppres- were included in the study. Among 2,938 samples enrolled 1,2 sive drugs. The exact burden of NTM disease is not known in the study, 1,612 were from DRTB suspect and1,326 were because it is not a reportable disease worldwide including from pulmonary and extrapulmonary TB suspects. All clin- India, but now NTM infections are showing increasing trend ical samples, pulmonary (sputum, bronchoalveolar lavage) 3 in India. The clinic radiological signs and symptoms of and extrapulmonary (gastric aspirate, pleural fluid, pus and both NTM and Mycobacterium tuberculosis (MTB) are quite endometrial biopsy), from males and females of all age groups similar; thus, NTM is easily misdiagnosed as MTB, or as were included in this study. Handling of MGIT cultures and multidrug-resistant tuberculosis (MDRTB) and extensively PSC cultures were done in biosafety level 3 (BSL3) laborato- drug-resistant tuberculosis (XDRTB) that pose challenge for ry,while rest all processes were done in BSL2 laboratory as 3 effective treatment. NTM isolation rates range from 0.5 to recommended for MTB. 8.6% in India.4 A recent study from India reported increase in prevalence of NTM from 1.0% in 2005 to 3.5% in 2008; Statistical Analysis 5 88.6% of these NTM were clinically relevant. The isolation All data were entered in Microsoft Excel version 10 and ana- rates of NTM vary widely depending on population and lyzed by SPSS version 21. Chi-squared test was applied to find 6 geographic location. Infections with pathogenic NTM such statistical significance. as Mycobacterium marinum, Mycobacterium fortuitum, and 7 Mycobacterium chelonae have been reported from India. Sample Collection Paraffin baiting has been used for isolating NTM and Samples were collected in sterile, screw cap containers from 8,9 Nocardia as NTM utilizes paraffin wax as the sole source of various districts of Rajasthan, stored in refrigerator at the carbon, while MTB is not able to utilize paraffin wax as the districts for 1 to 5 days and transported to the laboratory by sole source of carbon and does not grow in the paraffin slide courier or hand delivered maintaining cold chain. On receipt culture (PSC). This property helps in isolating NTM from clin- of samples at laboratory, they were processed immediately or ical specimens as this property of paraffin metabolism is not after refrigeration at 4°C overnight.15 For PSC, the deposits of 9 found among other human pathogens. processed samples were stored in–20°C and inoculated once It is important to isolate and identify NTM because of its in 7 to 10 days; rest all work was done daily as per Revised pathogenic potential and higher resistance to antitubercu- National Tuberculosis Control Program (RNTCP) guidelines. losis drugs.10 Phenotypic identification of NTM species is 11 based on a panel of biochemical tests, which have many Sample Processing and Culture drawbacks such as slow turnaround time, difficult to inter- All the samples were subjected to smear microscopy by pret, low specificity, and limited accuracy; laboratories are Ziehl–Neelsen staining method, decontamination byN-ace- now using alternative rapid methods for species identi- tyl-L-cysteine-sodium hydroxide method (with final NaOH fication such as DNA probes (AccuProbe, Gen-Probe, San concentration of 4%) as per RNTCP guidelines.10 The concen- Diego, California, United States),polymerase chain reaction trated sediment (1–1.5mL) was resuspended in 1 to 2 mL of 12 (PCR)-restriction enzyme analysis of the hsp65 gene, or phosphate buffer and processed for MGIT culture (0.5mL) and 13 sequencing of the 16S ribosomal DNA (rDNA). Use of 16S line probe assay (LPA) (0.1mL) as per RNTCP guidelines.16,17 Rest ribosomal RNA (rRNA) gene has now become a “gold stan- of deposit was stored at–20°C; 0.5 mL of the stored deposit of 14 dard” for the identification of NTM. all samples was inoculated in PSC.9 Smear-positive DRTB sus- Hence, present study was conducted to investigate the pected samples were processed directly for LPA from sample, prevalence and profile of NTM among DRTB suspected MTB growth from MGIT was processed for LPA if direct LPA/ patients using mycobacteria growth indicator tube (MGIT) GeneXpert results were not available.17,18 Samples (1mL) of TB and PSC methods for isolation and 16S rRNA gene sequencing suspects were processed directly for GeneXpert as per proto- for species identification. col.18 All the processing done in this study is shown in ►Fig. 1. Procedure PSC Briefly, 0.5 mL of processed sample was inoculated Study Setting into 4.5 mL sterile Czapek broth medium (Czapek broth Study was performed in Mycobacteriology Laboratory, constituents: NaNO3—3.0 g, K2HPO4—1.0, MgSO4H2O—0.5 g, Department of Microbiology, SMS Medical College, Jaipur, KCl—0.5 g, FeSO4—0.01 g) with added BACTEC PANTA Plus Rajasthan from August 2016 to August 2017. Samples of (Becton Dickinson) (polymyxin B, amphotericin B, nalidixic Journal of Laboratory Physicians Vol. 12 No. 3/2020 © 2020. The Indian Association of Laboratory Physicians. NTM Detection and Profile Sharma et al. 205 Fig. 1 Workflow chart (STROBE Guideline). AFB, acid-fast bacilli; DRTB, drug-resistant tuberculosis; LJ, Lowenstein-Jensen medium; LPA, line probe assay; MGIT, mycobacteria growth indicator tube; MTB, Mycobacterium tuberculosis; NTM, nontuberculous mycobacteria; PSC, paraffin slide culture; STROBE, Strengthening The Reporting of Observational studies in Epidemiology. acid, trimethoprim, and