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 Table of Contents  
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 14-18

Recent methods for diagnosis of nontuberculous mycobacteria infections: Relevance in clinical practice

1 Department of Microbiology, All Institute of Medical Sciences, Jodhpur, Rajasthan, India
2 Department of Pulmonary Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India

Date of Web Publication24-Jul-2017

Correspondence Address:
Anand Kumar Maurya
Department of Microbiology, All India Institute of Medical Sciences, Jodhpur - 342 005, Rajasthan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_18_17

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Nontuberculous mycobacteria (NTM) infections are ever more important in recent years for leading causes of morbidity and mortality worldwide. Clinical appearance of Mycobacterium tuberculosis (TB) complex and NTM is same, but the treatment regimen is always different. NTM is challenging for both diagnostic and therapeutic with reason that it mimic pathological, microbiological, immunological, and radiological findings of TB. Newer molecular diagnostic methods allow for a better identification of NTM infections in patients not responding to antitubercular treatment and falsely categorized as drug-resistant TB. This article will explore the recent methods for the diagnosis and identification of NTM infections in clinical practice. In the future, the molecular-based diagnosis will significantly reduce the turnaround time of the diagnosis and thereby improving patient outcome.

Keywords: Biomarkers, Mycobacterium tuberculosis complex, nontuberculous mycobacteria, tuberculosis

How to cite this article:
Maurya AK, Nag VL, Kant S, Sharma A, Gadepalli RS, Kushwaha RA. Recent methods for diagnosis of nontuberculous mycobacteria infections: Relevance in clinical practice. Biomed Biotechnol Res J 2017;1:14-8

How to cite this URL:
Maurya AK, Nag VL, Kant S, Sharma A, Gadepalli RS, Kushwaha RA. Recent methods for diagnosis of nontuberculous mycobacteria infections: Relevance in clinical practice. Biomed Biotechnol Res J [serial online] 2017 [cited 2023 May 30];1:14-8. Available from: https://www.bmbtrj.org/text.asp?2017/1/1/14/211406

  Introduction Top

Tuberculosis (TB) is most important infectious diseases cause of death worldwide. Nontuberculous mycobacteria (NTM) another name as mycobacteria other than TB are acid-fast bacilli, but they are also differ from Mycobacterium TB complex (MTBC). These mycobacteria are widely present in our environment, and they are found in natural recourses of water, biofilm, damp walls, and also found in tap water.[1] NTM is lacking the evidence of human to human transmission, but it infects to human from environmental sources.[1]

Mycobacterium avium complex was reported as the most frequently isolates in all these countries, including India as per the International Union against TB and Lung Diseases reports.[2] Mycobacterium fortuitum was the most frequently reported in Belgium (2.1%), Italy (2.5%), Denmark (5.3%), United Kingdom (6.0%), France (6.5%), Finland (6.7%), Spain (10.8%), Germany (12.2%), Portugal (16.5%), Czech Republic (17.5%), Switzerland (17.5%), and Turkey (33.9%). The study suggested that environment is the main source of NTM infection[2],[3] and reported prevalence in worldwide with varying frequencies,[4],[5],[6],[7],[8] isolation rates were between 0.7% and 34% in India.[3],[9]

NTM treatment are different according to the species involvement, disease site, and susceptibility pattern.[1] Clinical appearance and progresses are often slow in NTM infection. Population-based studies data showed that NTM disease is increasing in the United States.[2],[5] American Thoracic Society and Infectious Diseases Society of America were working together to develop recommended guidelines for NTM disease.[2],[10] Studies reported from USA region showed that NTM lung disease is most commonly due to M. avium complex and Mycobacterium kansasii being second.[5] M. kansasii is the pathogen most commonly seen in in England and Wales, whereas M. malmoense is the most commonly in Scotland. Mycobacterium xenopi is common in Southeast England.[11] M. avium complex and M. kansasii are also most common in Japan.[6] NTM prevalence reported from India, 17.4% of fibrocavitary disease and 7.4% of other clinical specimens among M. fortuitum. The most frequently NTM species isolated from Delhi and Kasauli was M. avium intracellulare (8.6%) from sputum specimens.[8],[12],[13],[14],[15]

  Laboratory Diagnostics of Nontuberculous Mycobacteria Top

Laboratory methods are important role in the NTM diagnosis as well as monitoring of treatment and prevention.


Various staining methods are available for staining as Ziehl-Neelsen (ZN) stain and modified Kinyoun stain are indicate the presence of mycobacteria. Corbol fuschin is generally used in modified Kinyoun and ZN stain and is directly visible by light microscopy. Fluorescence microscopy uses auramine and rhodamine which both fluoresces at short wavelength. Studies reported that sensitivity rates of the ZN and auramine stain are higher than Kinyoun stain,[16],[17] but when compared to culture, it is only 60% and 90%.[16],[17],[18] In histopathological examinations, the sensitivity of fluorescence microscopy and ZN staining is low due to negatively influence by formalin fixation.[19],[20] On the basis of staining, it is not very easy to discriminate between MTBC and NTM. Smear sensitivity is lower in extra-pulmonary TB patients, and persons with the disease due to NTM Infection. However, it is very clear that ZN microscopy is an important method to the detection of mycobacteria, but it unable to help in identification of NTM, and should be pursued by culture.


Lowenstein-Jensen (LJ) is conventional and an excellent medium for the growth of Mycobacterium tuberculosis, but are generally inferior to Middlebrook agar as an all-purpose medium for both M. tuberculosis and NTM.[2] Guidelines and Recommendation for culture of NTM as per ATS (American Thoracic Society) include two types of medium, one solid medium (LJ or Middlebrook medium) and one liquid medium culture system BACTEC (Becton-Dickenson Diagnostics), MB Redox (Heipha Diagnostika), BacT/ALERT® MP (bioMerieux, France), MGIT (BD Diagnostics), and Septi-check (BD Diagnostics).[21],[22],[23],[24] Most media require additives OADC enrichment (mixture of Bovine albumin, Dextrose, Catalase, and Oleic acid) to increase the growth rate and PANTA antibiotic (mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) are often added to inhibit the growth of contaminants.[22],[23],[24],[25] A conventional method for the growth of mycobacteria is time-consuming (6–8 weeks), but they are considered “gold standard.”[2] Liquid-based culture is high sensitivity because the growth of M. tuberculosis can be detected within 1–2 weeks. However, they always use in combination with the conventional LJ method for NTM culture. Moreover, the final report can be sent after 6 weeks if no growth detected in the liquid-based culture and 8 weeks of incubation on the LJ slants.

Biochemical identification of nontuberculous mycobacteria species identification

Various biochemical test for NTM species differentiation includes reduction of nitrate, niacin secretion, tween eighty hydrolysis, growth on MacConkey agar, reduction of tellurite, urease activity, without crystal violet, catalase activity, caratogenesis, at 68°C, semi-quantitative catalase activity, growth activity with arylsulfatase, pyrazinamidase, and ß-glucocidase.[24] Data from various paper showed that specific growth inhibitors as nitrobenzoic acid (PNB), and nitro-alpha-acetylamino-beta-hydroxypropiophenone inhibit to the M. tuberculosis- complex and it may be useful for the differentiation of NTM. The rate of growth, pigmentation of colonies, and various biochemical reactions are used for phenotypic identification of NTM species.[24]

  Molecular Methods Top

Traditional methods are available for identification of NTM by solid media, biochemical tests, and antimicrobial sensitivity testing, but they need more time to detection and less sensitive to molecular testing. Mycobacterium identification by molecular testing continues transforming the diagnosis of TB worldwide. Molecular testing is based on the principle of nucleic acid amplification which allows a speedy and precise identification of the Mycobacterium species <24 h.[26],[27] DNA hybridization and DNA sequencing are very common and essential in nowadays laboratory setup for identification and differentiation of mycobacteria.[26],[28] Various molecular methods are commercially available like DNA probes are Gen-Probe Amplified M. tuberculosis Direct Test (Gen-Probe, San Diego, California, USA) and AMPLICOR nucleic acid amplification test (USA) and its application are used for identification and differentiation of mycobacterial species, including M. tuberculosis complex, M. intracellulare, M. Avium, M. kansasii, M. avium complex, M chelonae, M fortutium, and M. gordonae.[28],[29] Molecular studies reported the sensitivity of nucleic acid amplification testing to detect more greater than 95% M. tuberculosis nucleic acid in positive acid-fast bacillus sputum smears cases[28],[29] and negative results show strong association of an NTM species.[30],[31]

Line probe assays

Line probe assays are based on nucleic acid amplification and reverse hybridization to detect NTM and its speciation of NTM to various species from clinical and culture isolates by DNA hybridization based GenoType® Mycobacterium common mycobacteria/additional species (CM/AS) assay (Hain Lifescience, Nehren Germany). This is commercial kit detect to the identification and differentiates different species of NTM from cultures. This method based on nucleic acid amplification targeting the 23S ribosomal RNA gene region, followed by reverse hybridization on nitrocellulose membrane strips. There are two kits available-the CM, recognize 15 Mycobacterium species, including M. tuberculosis complex whereas the AS denotes 16 additional less common NTM species available. The GenoType® Mycobacterium CM/AS assay is very precise, rapid, and consistent test for the identification of mycobacterial species, which dedicate set up and qualified laboratory staff.[9] Polymerase chain reaction (PCR)-based sequencing, genes coding for the 32 kDa protein,[32],[33] 16S ribosomal RNA, 65 kDa heat shock protein,[34] and 16S-23S ribosomal RNA internal transcribed spacer are other molecular methods available in laboratories.[32]

  Immunodiagnostic Test Top

Interferon-gamma based determinations

Various studies revealed that immunodominat antigens like as 6-kDa Early Secretory Antigenic Target (ESAT-6) and Culture Filtrate Protein (CFP-10) may act as potential makers for TB diagnosis.[35],[36],[37],[38] Based on these studies, Interferon-Gamma Release Assays (IGRAs) based detection are important developments in the diagnostic for TB. The advantage of IGRA to detect MTBC-specific antigens encoded in region of difference (RD) 1 and increased specificity towards detection of MTBC infection. A recent assessment showed that IFN-γ assays using MTB RD1 antigens, including ESAT-6 and CFP-10, may have advantages over tuberculin skin testing.[36],[37],[38],[39] Various INF-γ commercial kits available are as commercial: enzyme-linked immunospot T SPOT-TB assay (United Kingdom) and QuantiFERON-TB, and its enhanced versions QuantiFERON-TB Gold and QuantiFERON-TB Gold in-Tube assays (Australia). In India, Government of India has banned the use of blood-based serodiagnostic kits for TB and has discouraged the use of tests such as TB Gold in 2012.[40]

Immunochromatographic test

Rapid and precise detection of mycobacteria could help in additional patients from needless treatment in cases of NTM. A new, simple, and rapid assays Immunochromatographic test (standard deviation [SD] MPT64 TB Ag Kit) developed by SD Bioline, South Korea which facilitates rapid detection and differentiation of MPT 64 antigen in M. tuberculosis isolates and NTM.[41],[42],[43],[44] MPT 64 TB Ag kit is highly sensitive and speedy identification of MTBC, together with M. tuberculosis, Mycobacterium africanum, Mycobacterium bovis, and substrains of M. bovis BCG.[41],[42],[45],[46] The study reported the sensitivity was found to 99% and 100% specificity from MPT64 TB Ag test.[47],[48] The advantage of MPT64 TB Ag test are easily and direct culture positive specimens does not require any extraordinary equipment and easily distinguish between MTBC and NTM.[9],[48]

  Molecular Typing Methods Top

Molecular genotyping studies on mycobacterial species provide insights into epidemiological behavior, evolution, and transmission of the mycobacteria. Reported typing studies[49],[50] and taxonomical relations have been established between species and subspecies.[51],[52] These methods are available for molecular typing, i.e. pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD). RAPD is easy to perform and often applied in the investigation of, for instance, pseudo-outbreaks. Fluorescent amplified fragment length polymorphism is based on fluorophore-labeled PCR primers, which building the amplified fragments detectable to an automated DNA sequences.[53] Other genome segments are used in genotyping are repetitive elements like insertion sequences (IS).

IS6110 is especially members of the MTBC, and it can be used as a significant useful diagnostic marker in the identification of MTBC. It has been widely used as epidemiological, typing, and identification of MTBC.[54] Various IS reported for NTM, IS900, and IS901 used as biomarkers as identification of Mycobacterium paratuberculosis and M. avium strains, IS1245 is using for typing of M. avium,[55] identification of Mycobacterium celatum by IS1407, IS1395 M. xenopi and both IS2404 and IS2606 for Mycobacterium ulcerans, IS1245 for M. avium for genotyping and identification of NTM.[56]

IS6110-RFLP is using as “gold standard” typing for M. tuberculosis. The advantage of IS6110-RFLP are simple do, less time consuming, and need a little quantity of genomic DNA from clinical specimens and/or culture isolates from NTM.[57]

Mycobacterial intergenic repetitive units-variable number of tandem repeats (MIRUs-VNTR) is a powerful technique for epidemiology, phylogeny, and group of genetic elements used for genotyping of TB.[58] The principle of this technique is based on DNA segments have “tandem repeated” sequences at loci dispersed in the region of the M. tuberculosis genome.[58] This tool is applicable for MTBC and NTM (M. tuberculosis, Mycobacterium leprae, M. ulcerans and M. avium).[58] They are very short repetitive sequences which are readily incorporated or deleted by DNA polymerase and therefore highly variable in length between strains.[59],[60],[61] Various studies showed that 15 MIRU's found in M. tuberculosis, they were also able to differentiate between phylogenetic lineages[62] and 22 MIRU's were identified in M. avium.[63] The advantage of MIRU-VNTR typing is easy to perform, inexpensive, adaptable technique, and high discriminatory and reproducibility.[57]

Multilocus sequence typing is a typing method for strain classification that indexed variation in multiple housekeeping genes and also based on DNA sequencing-based method which shows nucleotide variations presence in sets of genetic loci.[64] This could help in finding out the source of the epidemic and new infection.[64] However, it acceptable the purpose of the unpredictability between subspecies and strains of M. avium, enhanced genetic divergence and established to be precious in investigations of NTM.[65],[66]

  Conclusion Top

NTM infection is a major concern for both diagnose and treatment worldwide. Molecular methods are a valuable tool in reduces the possibility of an inadequate treatment of NTM infections. This may gradually replace conventional methods for the identification and differentiation of NTM. Molecular typing methods may also facilitate the analysis of the outbreaks and track transmission patterns in the community.

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Conflicts of interest

There are no conflicts of interest.

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