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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 22  |  Issue : 1  |  Page : 12-16

Occurrence and diversity of non-tuberculous mycobacteria among suspected and confirmed cases of pulmonary tuberculosis


1 Department of Microbiology, King George Medical University, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, King George Medical University, Lucknow; Department of Biotechnology, GLA University, Mathura, Uttar Pradesh, India

Date of Submission14-Jun-2019
Date of Acceptance07-Dec-2019
Date of Web Publication13-Aug-2020

Correspondence Address:
Prof. Amita Jain
Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_13_19

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  Abstract 


BACKGROUND: Non-tuberculous mycobacteria (NTM) may cause pulmonary disease that resembles tuberculosis (TB) and it may also coexist with Mycobacterium tuberculosis. Here, we aimed to study the occurrence and diversity of NTM among suspected and confirmed cases of TB.
METHODS: During 2017–2018, we received 11,094 sputum samples, of which 4288 samples were from equal number of presumptive TB patients. The rest of 6866 samples were from known multidrug-resistant TB patients and at different months of treatment follow-up. All samples were subjected to liquid culture and recovered isolates were identified as M. tuberculosis complex (MTBC) or NTM based on microscopy and immunochromatograhic (MPT-64Ag) tests. NTM isolates were further speciated using commercial GenoType® Mycobacterium CM assay.
RESULTS: A total of 2782 culture isolates were recovered, of which 2722 were MTBC and the rest 60 were considered as NTM. NTM was isolated both from presumptive and confirmed TB cases. NTM speciation could be achieved for 42 isolates; Mycobacterium intracellulare (50%) was identified as the most prevalent species, followed by Mycobacterium abscessus (23.8%), Mycobacterium fortuitum (16.7%) and others (9.5%).
CONCLUSION: The proportion of NTM isolation among suspected/confirmed cases of pulmonary TB is low; however, if isolated, patients should be carefully evaluated for possible NTM disease. Molecular speciation of NTM is useful to provide rapid and precise diagnosis.

Keywords: GenoType® Mycobacterium common mycobacteria assay, Mycobacterium tuberculosis complex, non-tuberculous mycobacteria


How to cite this article:
Sinha KK, Singh PK, Singh U, Dixit P, Jain A. Occurrence and diversity of non-tuberculous mycobacteria among suspected and confirmed cases of pulmonary tuberculosis. J Acad Clin Microbiol 2020;22:12-6

How to cite this URL:
Sinha KK, Singh PK, Singh U, Dixit P, Jain A. Occurrence and diversity of non-tuberculous mycobacteria among suspected and confirmed cases of pulmonary tuberculosis. J Acad Clin Microbiol [serial online] 2020 [cited 2020 Sep 27];22:12-6. Available from: http://www.jacmjournal.org/text.asp?2020/22/1/12/291886




  Introduction Top


Non-tuberculous mycobacteria (NTM) are ubiquitous micro-organisms and are increasingly reported as a cause of pulmonary infection from different parts of the world.[1] In India, epidemiological and surveillance data of NTM infections are seriously limited. This scarcity is due to the major attention of researchers and public health programmes towards combating 'pulmonary tuberculosis' (caused by Mycobacterium tuberculosis complex [MTBC]) which contributes the highest burden in the world. In addition to this, less awareness among physicians and lack of routine laboratory practice for appropriate diagnosis of NTM leads to underreporting of NTM in the country.

Many environmental NTM species cause pulmonary disease that is often indistinguishable to pulmonary tuberculosis (PTB). Moreover, NTM infection may falsely be diagnosed as multidrug-resistant (MDR) TB due to positive result in widely used TB test (smear microscopy) and due to usual non-responsiveness of patients to antituberculous medication. NTM has been isolated earlier in variable frequency from suspects of TB and MDR-TB,[2],[3] and interestingly, it may be also co-occurred with M. tuberculosis in considerable proportion.[4] In addition to this, growing evidence suggests that NTM lung disease is a risk factor for developing PTB [5] and vice versa.[6] It is, therefore, imperative to know the NTM epidemiology in region with high TB burden.

Speciation of NTM is important, as infections with different Mycobacterium species often require different management. Biochemical tests were the mainstay for NTM speciation for many years in the past. However, with recent availability of commercial DNA-based line probe assay (LPA) such as GenoType Mycobacterium ® CM/AS (Hain Lifescience, Nehren, Germany), it is increasingly adopted by many clinical laboratories. Here, we aimed to study the occurrence of different NTM species in sputum that are submitted for the diagnosis of TB or for monitoring of ongoing treatment response among MDR-TB cases in Uttar Pradesh, India.


  Methods Top


Study sample

This prospective observational study took place at the Department of Microbiology, King George's Medical University, Lucknow. Our laboratory is recognised as an intermediate reference laboratory under the Revised National Tuberculosis Control Program (RNTCP) of the Government of India. The laboratory has successfully passed proficiency testing by the National Reference Laboratory, Agra, India, as Quality Assurance Programme. Besides it, the laboratory has also accreditation of ISO-15189:2012 for conducting various TB tests. From August 2017 to July 2018, a total of 11,094 clinical sputum samples with request of conducting M. tuberculosis culture were received from different districts (under RNTCP network) as well as from our tertiary hospital. A sample other than sputum was not included in this study. These samples were collected either from patients with presumptive diagnosis of TB (n = 4288) or from confirmed MDR-TB patients for follow-up (n = 6866; multiple samples from single patients at different time intervals) of ongoing treatment.

This study is approved by the institutional ethics committee, Ref. code: 88th ECM 11 B-Thesis/P11.

Culture and identification

Sputum samples were decontaminated and concentrated using standard N-acetyl-L-cysteine–sodium hydroxide method.[7] Subsequently, the processed samples were inoculated into modified Middlebrook 7H9 liquid media of BACTEC MGIT 960 system (BD, Sparks, MD, USA), as per a protocol described by Siddiqi and Rusch-Gerdes.[8] Sample-inoculated media flagged by automated MGIT 960 as 'positive' were further subjected to the Ziehl–Neelsen staining and inoculation over brain–heart infusion (BHI) agar media. Liquid growth showing acid-fast bacilli (AFB) or serpentine cord structure in smear microscopy was considered as mycobacterial growth and was confirmed further for the presence of MTBC using specific MPT-64 antigen-based immunochromatographic test SD Bioline TB Ag MPT 64 Rapid® assay. Growth with positive results in MPT-64-based immunochromatographic test (ICT) was considered as 'MTBC isolates', whereas growth with negative results (along with AFB positive growth) is presumed as NTM isolates and was included for further testing and analysis. Culture isolates with growth on BHI agar media (after 48 h of incubation) and with negative test result both for AFB microscopy and ICT were considered as contaminated. All 'MTBC-confirmed positive growth', 'contaminated growth' and 'negative growth (as flagged by MGIT 960)' were excluded from further analysis.

Non-tuberculous mycobacteria speciation

Speciation of NTM was performed using the reagents provided with Mycobacterium CM ® (Hain Lifescience, Nehren, Germany) LPA kit. For DNA extraction, we used recommended GenoLyse ® DNA extraction kit (Hain Lifescience, Nehren, Germany). Following this, polymerase chain reaction (PCR) amplification, hybridisation of the PCR products to the LPA strips, detection and interpretation of the results were done according to manufacturer's recommended protocols, equipment and facility. We included DNA from Mycobacterium fortuitum reference strain as a positive control, and DNA-negative control (amplification reaction mixture without DNA) was also used to rule out DNA contamination during the setting up of the test. All data were recorded in a Microsoft Office Excel spreadsheet. The frequency of different NTM species was counted in number and presented in percentage.


  Results Top


Of the total 11,094 sputum samples, 2782 were found positive for AFB growth. On subsequent testing of isolates by ICT, 2722 isolates were confirmed as 'MTBC', whereas the remaining 60 isolates, recovered from different patients, were considered as 'NTM' (due to negative test result in ICT). Among these 60 'NTM' isolates, 36 were isolated from presumptive TB cases and the rest of 24 'NTM' cases isolated from patients on-treatment for MDR-TB.

Speciation of NTM isolates by GenoType ® Mycobacterium CM assay could be done for 42 (70%) isolates. The rest of NTM isolates could not be determined by LPA due to unmatched band pattern (14, 23.3%) or invalid test results (4, 6.7%). In our study, Mycobacterium intracellulare was found as the most frequent species (21/60; 35%), followed by Mycobacterium abscessus (10/60; 16.7%), M. fortuitum (7/60; 11.7%), Mycobacterium kansasii (2/60; 3.3%) and Mycobacterium scrofulaceum (1/60; 1.7%). One isolate, recovered from presumptive TB case, was found to have co-occurrence of M. abscessus and Mycobacterium intracellulare strains. The frequency and distribution of NTM species stratified into type of cases tested, i.e., 'presumptive TB case' and 'on-treatment MDR-TB case', are depicted in [Figure 1].
Figure 1: Proportion and diversity of non-tuberculous mycobacteria isolated in the study. NTM: Non-tuberculous mycobacteria; MTBC: Mycobacterium tuberculosis complex; AFB: Acid-fast bacilli; TB: Tuberculosis; MDR: Multidrug resistant

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  Discussion Top


The epidemiology of NTM remains poorly understood in India, the highest TB burden country in the world. This is probably the first report from the country that documented isolation of NTM from on-treatment MDR-TB patients. In this study, we were able to recover 36 and 24 NTM isolates from sputum samples of presumptive TB and on-treatment MDR-TB patients, respectively. Although the proportion of NTM isolation in our study is low (2.2% among culture-positive cases), previous studies from Africa, a country with high TB burden, suggested that a significant proportion of patients with suspected TB or MDR-TB (3.7%–18%) might have NTM pulmonary disease instead.[9],[10],[11]

NTM coinfections with M. tuberculosis disease are generally misdiagnosed owing to similar clinical manifestations.[12] Besides this, there is evidence on colonisation of NTM in PTB patients [13] and can also be co-isolated from active-TB cases.[4],[14],[15] The isolation of NTM from on-treatment MDR-TB patients is surprising to us since NTM resistance to second-line anti-TB drugs is rarely studied. However, it seems possible as NTM known to have intrinsic resistance to a wide spectrum of antibiotics, including most of the TB drugs.[16] Recently, Izadi et al.[17] reported that NTM coinfection may persist for a long time in patients who underwent repeated treatment using antitubercular drugs (both first and second lines). Furthermore, NTM may also acquire drug resistance through genetic mutations during prolonged course of treatment. Acquired resistance to aminoglycosides (used for MDR-TB) has been demonstrated in M. abscessus clinical isolates due to mutations at position 1408 of 16S rRNA (rrs).[18] In addition, an in vitro study by Nessar et al.[19] has also shown that mutations at positions 1406, 1409 and 1491 in M. abscessus could also confer a high level of resistance. These observations indicate the importance of drug resistance profile before initiating treatment for NTM infection, especially if recovered from patients suspected for drug-resistant TB. Macrolides are the key drugs in the treatment of NTM-associated lung disease.[20] The standard treatment regimen (for Mycobacterium avium complex-associated lung diseases) contains rifampicin, ethambutol and a macrolide (clarithromycin or azithromycin) for 18–24 months.[21] Previous reports are available that reported acquired drug resistance against macrolides, rifampicin and ethambutol due to genetic mutations.[22] Therefore, in case of treatment failure/non-responsiveness or laboratory-proven drug resistance, the addition of moxifloxacin to multidrug regimens (including injectable) is considered to improve treatment outcomes.[23]

NTM speciation is crucial for initiating appropriate treatment regimen. The method still relies on conventional method involving a variety of phenotypic tests and enzymatic properties. However, these tests are labour-intensive, time-consuming and are able to identify only common species.[24] Currently, DNA sequencing, targeting different genes (including 16S rDNA, rpo B and hsp 65), is considered as the gold standard method as it has high resolution to discriminate and identify different mycobacterial species.[25] Recently, matrix-assisted laser desorption ionisation–time-of-flight (MALDI-TOF) mass spectrometry was also demonstrated to accurately identify the mycobacterial species. Although the speed, robustness and minimal costs of sample preparation and measurement are key advantages of MALDI-TOF, it suffers due to huge cost for facility establishment and maintenance.[26] Since DNA sequence-based approach is superior to conclude the mycobacterial species, other molecular tests were also developed. Of these, a PCR-based strip hybridisation test (commonly known as LPA) is most promising to identify the clinically relevant mycobacterial species directly from liquid culture medium and within a shortened turnaround time. GenoType Mycobacterium CM and AS (Hain Lifescience, Nehren, Germany) are LPA-based commercial tests, targeting 23S rDNA region, that enable to identify 31 species of NTM.[27] LPA-based method also has limitations such as it requires a high level of expertise and limited number of species can only be identified.

In this study, we used GenoType Mycobacterium CM® version 1.0 assay (Hain Lifescience, Nehren, Germany) which enables the identification of 14 clinically relevant NTM species. As limitation of this kit, some isolates that were not identifiable to the species level but recognised as members of the genus Mycobacterium are hereby referred as unspeciated NTM [Figure 1]. In our study, we could speciate only 70% of total NTM isolates though the sensitivity of the kit was found 93.3% which is in agreement of previous reports that stated sensitivity and specificity as 97.9% and 92.4%, respectively.[27] On analysing the distribution of different NTM species, M. intracellulare was found to be the most prevalent species, followed by M. abscessus, M. fortuitum and other species [Figure 1]. A similar distribution of NTM species was also observed earlier on majority of the pulmonary specimens in an Indian study.[28] In contrast, Maurya et al.[29] reported M. fortuitum as the most frequent species, followed by M. abscessus; however, they had investigated only extrapulmonary specimen, and this could be a possible reason for disparity in distribution.

Species identification was missed in 13 NTM isolates, in which the hybridisation pattern did not match with expected ones, though universal probe was present. This indicates the presence of species more closely related to the Mycobacterium genus. Russo et al.[30] reported that the sensitivity of GenoType Mycobacterium CM ® may be further increased using another strip (GenoType Mycobacterium AS ®); however, this kit was not applied in our study. Four isolates resulted as invalid that may be due to poor amplification or hybridisation. Since repeat testing was not performed, the result for these four isolates remained inconclusive. The kit has also identified the growth of M. abscessus and M. intracellulare in one sample recovered from TB suspect patients. Our study showed that GenoType ® Mycobacterium CM assay is a rapid and reliable identification test of non-tuberculous mycobacterial species, which may provide a prompt and reliable result for initiating appropriate therapy. Conventional methods such as colony morphology, growth characteristics and a panel of different biochemical tests are unable to distinguish many mycobacterial species since they overlap in their biochemical properties.[31],[32] Moreover, biochemical test may be less reliable to identify mixed NTM infection; however, it can be identified by genotypic method.

Our study has some limitations, namely (i) test result could not be correlated with clinical data (radiology and detailed treatment history) due to limited access of patients' information and (ii) repeat NTM isolation culture on the second sample was not performed due to submission of an only single sample. Although a combination of clinical, radiological and microbiological criteria is important to establish NTM disease,[21] our study, nevertheless, demonstrated the presence of NTM species in pulmonary specimens from suspected/confirmed cases of TB. Therefore, patients who have NTM isolated need a careful investigation to confirm NTM disease and then treatment accordingly.


  Conclusion Top


The rate of NTM isolation from suspected/confirmed cases of TB is low in our region with a high TB burden. Molecular speciation of NTM is useful to provide a rapid and precise diagnosis of NTM and thus should be adopted in routine by mycobacterial laboratory. Increased awareness among clinicians about the local epidemiology of NTM is important as it could improve the differential diagnosis of NTM lung disease with PTB and can also reduce unnecessary use of antitubercular treatment.

Acknowledgement

We acknowledge the Revised National Tuberculosis Programme for providing necessary financial, workforce, equipment and consumable support to our laboratory services. The authors would like to thank all staff of TB laboratory for providing routine patient diagnosis care services at our institution.

Financial support and sponsorship

This study was financially supported by the Revised National Tuberculosis Control Programme for workforce, fund, equipment and consumables support.

Conflicts of interest

There are no conflicts of interest.



 
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