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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgement
 ~  References
 ~  Article Tables

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  Table of Contents  
Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 122-125

Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit

Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry-607 402, India

Date of Submission16-Oct-2013
Date of Acceptance05-Jun-2014
Date of Web Publication6-Feb-2015

Correspondence Address:
S Stephen
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry-607 402
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.150912

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 ~ Abstract 

Background: Investigation of extra pulmonary tuberculosis (EPTB) in and around Pondicherry is being carried out since August 2011 in our tertiary care super specialty hospital. Objectives: To compare the rapid Kit SD Bio-Line MPT 64 Ag with conventional and time consuming biochemical tests. Confirmation of Mycobacterium tuberculosis at a reasonable time frame is the main thrust. Materials and Methods: Thirty three Mycobacterium tuberculosis and four Non-Tuberculous Mycobacteria (NTM) grown in MGIT960 system/Lowenstein-Jensen media (LJ) were examined by the rapid MPT 64 antigen detection as well as a battery of conventional tests like niacin, nitrate reduction, paraminobenzoic acid susceptibility and cord formation. Results and Conclusion: . Both the rapid kit and conventional tests correctly identified 33 M.tuberculosis isolates. Keeping conventional identification as reference, sensitivity and specificity for rapid kit was 100%. Rapid kit which takes only 15 minutes is accurate, cost effective, and facilitates early treatment for these EPTB patients, whose clinical specimens are paucibacillary.

Keywords: Extra pulmonary tuberculosis, MPT64 Ag detection, Mycobacterium tuberculosis complex, Mycobacterium tuberculosis complex rapid identification

How to cite this article:
Kandhakumari G, Stephen S. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit. Indian J Med Microbiol 2015;33, Suppl S1:122-5

How to cite this URL:
Kandhakumari G, Stephen S. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit. Indian J Med Microbiol [serial online] 2015 [cited 2020 Apr 9];33, Suppl S1:122-5. Available from:

 ~ Introduction Top

The importance of early diagnosis and correct etiological identification of extra pulmonary tuberculosis (EPTB) need not be over-emphasised, since treatment is different for Mycobacterium tuberculosis and atypical Mycobacteria (non-tuberculous Mycobacteria, NTM). World Health Organization has given guidelines for low and medium income countries for use of liquid culture systems and drug sensitivity testing for tuberculosis work. [1] Automated non-radiometric systems for accelerated isolation of Mycobacterium tuberculosis complex (MTBC), being expensive, are available only in select centres in India and third-world countries. However, most laboratories still depend upon conventional techniques, thus resulting in an extended reporting time of 4-5 weeks. An innovative rapid kit, MPB64-ICA, to detect an established marker of MTBC, the MPT64 antigen, by immunochromatography test (ICT) developed by Japanese scientists [2] found universal acceptance due to its simplicity, accuracy and rapidity .[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] Three commercial kits are now available:

  1. CapiliaTB Neo Assay (TAUNS Laboratories, Numazu Japan).
  2. SD Bioline TBAg MPT 64 Rapid (Standard Diagnostics, Seoul, South Korea).
  3. BD MGIT TBc Identification test (Becton, Dickinson and company, Sparks, MD, USA).

Indian reports on EPTB in general [5],[12],[20] and the use of rapid kits for confirmation of MTBC in particular are few and far between, with recent reports using Capillia TB and SD Bioline kits. [4],[5],[10],[11],[12],[13] Hence our experience with SD Bioline TB Ag MPT64 rapid kit for rapid confirmation of MTBC isolates from EPTB specimens cultured in MGIT960 and Lowenstein-Jensen media (LJ) is presented.

 ~ Materials and Methods Top

Our Institutional Human Ethics Committee scrutinised and approved this research. Patients' informed consent was obtained before collection of specimens. During June 2011 to Oct 2012, 363 single clinical specimens of patients suspected for EPTB were inoculated and incubated in MGIT960 system and LJ media. Ziehl-Neelsen (ZN) smears and blood agar subcultures were made from MGIT tubes flagged positive by automated system. Only those tubes showing acid fast bacilli (AFB) in smears and sterile on blood agar for 48 hours were sub cultured onto LJ media for determining the growth rate, chromogenic property, Niacin production and nitrate reduction as per standard procedures. [21] MGIT tubes showing non-acid fast bacilli and/or fungi were excluded from MPT64 Ag test.

Rapid identification of MTBC from AFB-positive MGIT tubes or LJ media colonies was made using the Rapid kit. Briefly, 100 μl of mixed and vortexed culture fluid from AFB positive MGIT tubes were transferred to sample window of the cassette. Regarding cultures grown on solid medium, two to three colonies from LJ slopes were emulsified in 200 μl of antigen diluent (SD Bioline kit) and 100 μl of this emulsion was used as inoculum. The results of ICT were read within 15 minutes. Positive test had two red to purple bands, one for internal control and the second line for the test. Negative had only one band in internal control slot. Strong or light bands with any intensity were considered to be positive. Reference strains of H37Rv and Mycobacterium fortuitum were included as positive and negative controls, respectively.

Para nitro benzoic acid (PNB) and drug susceptibility test to the first line drugs was performed in MGIT960.

 ~ Results Top

Twenty eight isolates of M. tuberculosis and four non-tuberculous Mycobacteria grown on MGIT960 and five M. tuberculosis isolated on LJ were speciated by conventional techniques. The four NTM isolates included M. scrofulaceum (3) and M. fortuitum (1). All 37 isolates were examined by rapid kit. While 33 MTBC isolates were positive, four NTM were negative for MPT64 antigen [Table 1].
Table 1: Comparison between rapid ICT and conventional tests for confirmation of MTB

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All except one among 33 M. tuberculosis isolates were sensitive to the first line drugs namely Streptomycin, Isoniazid, Rifampicin and Ethambutol. The single isolate was resistant to Streptomycin and Isoniazid in MGIT 960 system.

 ~ Discussion Top

Number of AFB present in clinical specimens from EPTB cases is fewer than in sputum and hence the growth of MTBC was detected between 10-37 days in our MGIT system. Identification and differentiation of M. tuberculosis from NTM by conventional biochemical tests is very expensive, labor-intense and consumes additional 2-5 weeks time. PNB sensitivity test carried out in MGIT 960 system takes 4-13 days to confirm MTBC isolates. In contrast, MPT64 antigen detection results are available within 15 minutes by Rapid kit. This test is easy to perform, do not require any special skill and economical for countries like India where the resources are limited and molecular techniques are not within the easy reach of most of the laboratories. Several reports from different countries recorded specificity of nearly 100% and sensitivity ranging from 95% to 100% for the rapid MPT64 antigen detection kits in comparison to conventional methods for identifying MTB. [2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] We have experienced 100% sensitivity and specificity of this particular kit in comparison to conventional techniques. Our local strains are thus recognised by SD Bioline kit of Korean origin. [5],[10],[11],[12],[13],[14],[15],[16],[17]

In India, SD Bioline kit has been validated by five different researchers: [5],[10],[11],[12],[13] Maurya et al.,[5] from Lucknow reported 99.1% sensitivity and 100% specificity. Kannade et al., [11] from Bombay (Mumbai) examined 165 isolates (125 MTB; 30 NTM; 10 Non-Mycobacterial species) and observed sensitivity of 99.19% and 100% values for specificity, positive predictive value (PPV) and negative predictive value (NPV). Vadwai et al.,[12] from Bombay analysed 394 strains from 280 pulmonary and 114 EPTB samples (388 MTB; 6 NTM) with similar result, i.e. 99.4% sensitivity and 100% specificity. Kumar et al.,[10] from Mysore, Karnataka, analysed 77 isolates (55 MTB; 10 NTM; 12 Non-Mycobacterial species) recorded 100% results for all four parameters. Muddaiah et al.,[13] from Bangalore got identical results with 47 isolates (44 MTB and 3 NTM). Regarding the experience of researchers from Asian continent with this kit, Park et al.,[8] (Seoul, South Korea) recorded 98.6% sensitivity and 100% specificity using 481 isolates (220 MTB; 261 NTM), whereas Ang et al.,[14] from Seoul recorded 97% sensitivity and 100% specificity with 570 isolates (276 MTB; 294 NTM). Akyar et al.,[15] from Turkey and Toihir et al.,[16] from Madagascar examined 394 strains (297 MTB and 97 NTM) and 161 isolates (114 MTB and 97 NTM), respectively. Both studies revealed 100% sensitivity and specificity. Our results from Pondicherry are identical to that of reports form Karnataka [10],[13] , Turkey [15] and Madagascar. [16] A recent article published in 2014 by Japanese workers, Chikamatsu et al.,[17] reports 99.46% sensitivity and 99.4% specificity with 590 strains (500 MTB; 90 NTM).

Till date, only one Indian report by Gomathi et al.,[4] from Chennai has validated Capilia TB kit with 346 isolates (295 MTB; 51 NTM). They reported lower values for sensitivity (76%), specificity (86%), PPV (97%) and NPV (39%), which was attributed to higher rate of contamination in MGIT 960 culture tubes. However, experience from other Asian workers was different. Thus Yin et al.,[7] from China got 100% values for sensitivity, specificity and PPV with 95.2% for NPV. Muyoyeta et al.,[6] from Zambia and South Africa analysed 623 isolates (224 MTB; 399 NTM) and recorded 99.5% sensitivity and 99.25% specificity. A latest report by Chikamatsu et al.,[17] from Japan tested 590 isolates (500 MTB; 90 NTM) with an improved version, Capilia TB- Neo, recorded 99.4% sensitivity and 100% specificity.

TBc Identification kit (BD) has not been evaluated in India so far. Reports from Kenya, China and Taiwan [3],[17],[18],[19] showed sensitivity ranging from 95.2% to 100% and their specificity ranging from 95.96% to100%.

Very faint lines were frequently observed by us when the test was performed with culture fluids from MGIT tubes on the first or sometimes second day of growth flagging by MGIT960, probably due to low levels of MPT64 antigen. SD Bioline Rapid kit requires at least 10 colony forming units (CFU)/ml to detect the MPT64 antigen that is secreted into the medium, as observed by Abe et al. and Park et al. [2],[8]

Gomathi et al.,[4] reported that in high contamination of MGIT 960 setting, MPT 64 is not cost effective. Our contamination rate in MGIT was only 9.6%. AFB smear, Gram  smear and sterility check on blood agar for 48 hours were done to identify the contaminants. Contamination of MGIT tubes with fungi is normally visible within a day or two macroscopically. We have carefully avoided wastage of MPT64 kits by using them only for MGIT positive cultures without contamination and positive ZN smears.

To conclude, globally the prevalence of EPTB is on the increase. Due to prolonged time taken for positive culture and drug susceptibility report by conventional methods in suspected cases of EPTB, the clinicians in developing countries empirically initiate anti-tuberculosis treatment (ATT) with first-line drugs. However, if the etiology happens to be NTM, this would be a burden to the patients and can promote emergence of drug resistance in Mycobacteria. The isolates must be checked for drug sensitivity in this era of increasing drug resistance. Thus rapid isolation of Mycobacterium species using automated MGIT960 system is more beneficial when combined with rapid ICT kit which detects MPT64 Ag in 15 minutes and also differentiates MTBC from NTM isolates.

 ~ Acknowledgement Top

This research project is fully funded by the institution and we express our sincere gratitude to The Chairman, Vice-Chancellor, Research and PG coordinator and Dean, Mahatma Gandhi Medical College and Research Institute, Pondicherry.

We sincerely thank Dr. Vanaja Selvakumar, Scientist F and Deputy Director (Retd), National Institute for Research in Tuberculosis, (NIRT) Chennai, for very kindly providing us H37Rv and other NTM reference strains.

 ~ References Top

World Health Organization. Use of liquid TB culture and drug susceptibility testing (DST) in low and medium income countries: Summary report of the expert group meeting on the use of liquid culture media. Geneva, Switzerland: WHO 2007. Available from: culture_summary_report.pdf [Last accessed on 2013 Mar 28].  Back to cited text no. 1
Abe C, Hirano K, Tomiyama T. Simple and rapid identification of the Mycobacterium tuberculosis complex by immunochromatographic assay using anti-MPB64 monoclonal antibodies. J Clin Microbiol 1999;37:3693-7.  Back to cited text no. 2
Brent AJ, Mugo D, Musyimi R, Mutiso A, Morpeth S, Levin M, et al. Performance of the MGIT TBc identification test and meta-analysis of MPT64 assays for identification of the Mycobacterium tuberculosis complex in liquid culture. J Clin Microbiol 2011;49:4343-6.  Back to cited text no. 3
Gomathi NS, Devi SM, Lakshmi R, Ramachandran R, Wares DF, Kumar V, et al. Capilia test for identification of Mycobacterium tuberculosis in MGIT™-positive cultures. Int J Tuberc Lung Dis 2012;16:788-92.  Back to cited text no. 4
Maurya AK, Nag VL, Kant S, Kushwaha RA, Kumar M, Mishra V, et al. Evaluation of an immunochromatographic test for discrimination between Mycobacterium tuberculosis complex and non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis. Indian J Med Res 2012;135:901-6.  Back to cited text no. 5
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Muyoyeta M, de Haas PE, Mueller DH, van Helden PD, Mwenge L, Schaap A, et al. Evaluation of the Capilia TB assay for culture confirmation of Mycobacterium tuberculosis infections in Zambia and South Africa. J Clin Microbiol 2010;48:3773-5.  Back to cited text no. 6
Yin X, Zheng L, Wu L, Cao N, Zheng F, Hu Y, et al. Comparative evaluation of two rapid methods for differentiating mycobacteria. Tuberculosis (Edinb) 2013;93:227-31.  Back to cited text no. 7
Park MY, Kim YJ, Hwang SH, Kim HH, Lee EY, Jeong SH, et al. Evaluation of an immunochromatographic assay kit for rapid identification of Mycobacterium tuberculosis complex in clinical isolates. J Clin Microbiol 2009;47:481-4.  Back to cited text no. 8
Gaillard T, Fabre M, Martinaud C, Vong R, Brisou P, Soler C. Assessment of the SD Bioline Ag MPT64 Rapid™ and the MGIT™ TBc identification tests for the diagnosis of tuberculosis. Diagn Microbiol Infect Dis 2011;70:154-6.  Back to cited text no. 9
Kumar VG, Urs TA, Ranganath RR. MPT 64 Antigen detection for Rapid confirmation of M. tuberculosis isolates. BMC Res Notes 2011;24:79.  Back to cited text no. 10
Kanade S, Nataraj G, Suryawanshi R, Mehta P. Utility of MPT 64 antigen detection assay for rapid characterization of mycobacteria in a resource constrained setting. Indian J Tuberc 2012;59:92-6.  Back to cited text no. 11
Vadwai V, Sadani M, Sable R, Chavan A, Balan K, Naik A, et al. Immunochromatographic assays for detection of Mycobacterium tuberculosis: What is the perfect time to test? Diagn Microbiol Infect Dis 2012;74:282-7.  Back to cited text no. 12
Muddaiah RK, James PM, Lingegowda RK. Comparative study of Smear Microscopy, Rapid Slide Culture, and Lowenstein-Jensen culture in cases of pulmonary tuberculosis in a tertiary care hospital. J Res Med Sci 2013;18:767-71.  Back to cited text no. 13
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Akyar I, Kocagoz T, Sinik G, Oktem S, Aytekin N, Kocagoz S. Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium. Indian J Med Microbiol 2010;28:308-12.  Back to cited text no. 15
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Chikamatsu K, Aono A, Yamada H, Sugamoto T, Kato T, Kazumi Y, et al. Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex. BMC Infect Dis 2014;14:54.  Back to cited text no. 17
Lu PL, Yang YC, Huang SC, Jenh YS, Lin YC, Huang HH, et al. Evaluation of the Bactec MGIT 960 system in combination with the MGIT TBc identification test for detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol 2011;49:2290-2.  Back to cited text no. 18
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Fraser Wares, Balasubramanian R, Mohan A Sharma SK. Extra pulmonary tuberculosis, management and control. In: Agarwal SP, Chauhan LS, editors. Tuberculosis Control in. India. New Delhi: Elsevier; 2005:95-114.  Back to cited text no. 20
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