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Year : 2017  |  Volume : 60  |  Issue : 2  |  Page : 243-246
Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex


Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry, India

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Date of Web Publication19-Jun-2017
 

   Abstract 

At present, three rapid kits are available globally for the confirmation of Mycobacterium tuberculosis complex (MTBC) in cultures by MPT64 antigen (MPT64 Ag) detection. These include Capilia TB, SD Bioline, and BD MGIT TBc Identification (TBcID). The third kit is yet to be validated in India. We have tested this kit and compared with SD Bioline using conventional tests as gold standard. Seventy-one MTBC (70 M. tuberculosis and one Mycobacterium bovis) and four nontuberculous mycobacteria (NTM) were isolated from 649 clinical specimens in MGIT 960 and/or Lowenstein–Jensen slants (LJ). MPT64 Ag was detected by both TBcID and SD Bioline kits in all the 71 clinical isolates and the reference strain M. tuberculosis H37Rv. All NTM species tested were negative by the two different kits. Thus, TBcID kit showed 100% concordance in terms of sensitivity and specificity. Rapid kits confirm MTBC cultures within 15 min in contrast to several weeks' time required by conventional techniques.

Keywords: Extrapulmonary tuberculosis, MPT64 antigen, Mycobacterium tuberculosis complex, SD Bioline, TBc Identification

How to cite this article:
Kandhakumari G, Stephen S. Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex. Indian J Pathol Microbiol 2017;60:243-6

How to cite this URL:
Kandhakumari G, Stephen S. Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex. Indian J Pathol Microbiol [serial online] 2017 [cited 2022 Jan 27];60:243-6. Available from: https://www.ijpmonline.org/text.asp?2017/60/2/243/208407



   Introduction Top


India ranks first among high tuberculosis (TB) burden countries and accounts for 23% of the total 9.6 million incident cases reported worldwide in 2014.[1] TB can affect any part of the body leading to extrapulmonary tuberculosis (EPTB) posing diagnostic challenge. EPTB specimens was paucibacillary; hence, smear microscopy and solid cultures are less sensitive. Use of liquid cultures increases the isolation of nontuberculous mycobacteria (NTM) and necessitates differentiation from Mycobacterium tuberculosis complex (MTBC). Molecular techniques such as polymerase chain reaction and recently introduced Xpert MTB/RIF and line probe assay are widely used for diagnosis with limitations,[2] but these tests require expertise and costlier equipment. Simple and rapid test kits which confirm and differentiate MTBC from NTM have become available in the recent past.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15]

MPT64 is a mycobacterial protein secreted by MTBC. BD MGIT TBc Identification test (Becton, Dickinson and company, Sparks, MD, USA) is a rapid immunochromatographic assay for qualitative detection of MPT64 antigen (MPT64 Ag) and differentiation of MTBC with ease from AFB-positive mycobacteria growth indicator tubes (MGITs). Antibodies against MPT64 Ag are conjugated to visualizing particles on the test strip. The antigen and antibody complex then migrates to reaction area where it binds to a second specific MPT64 antibody coated on the membrane and color is developed by the labeled colloidal gold particles and visualized as pink to red line.

Rapid detection of MTBC using MPT64 Ag developed by Abe et al.[3] has shortened the duration compared to conventional techniques. In India, there was only one report of moderate performance of Capilia TB by Gomathi et al.[4] and satisfactory performance of SD Bioline by others.[5],[6],[7] For TBc Identification (TBcID) kit, various researchers from abroad have evaluated and reported sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) ranging from 90.1% to 100%, 95.6 to 100%, 99.2% to 100%, and 80.6% to 98.4%,[8],[9],[10],[11],[12],[13],[14],[15] respectively. The performance assessments of TBcID kit are shown in [Table 1]. BD TBcID is yet to be validated in India. This study evaluates the performance of TBcID and compares with SD Bioline kit, keeping conventional biochemical tests as the “gold standard.”
Table 1: Performance assessment of TBc Identifification kits by various workers

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   Methods Top


This research was approved by our Institutional Human Ethical Committee and the work was carried out in compliance with the principles of the Declaration of Helsinki. Clinical samples from patients were collected after obtaining informed consent. Between June 2011 and October 2014, 649 single clinical samples from EPTB patients were processed. These included 165 pleural fluids, 132 pus, 70 ascitic fluids, 66 LN aspirates/cold abscess, 43 cerebrospinal fluids, 40 synovial fluids, 33 gastric aspirates, 28 biopsies, 27 urine samples, 24 TB spine, 9 peritoneal fluids, 8 pericardial fluids, 2 retrocardial fluids, and one each from bone marrow aspirate and adrenal fluid. After decontamination with NALC-NaOH, the specimens were inoculated into MGIT 960 tubes and Lowenstein–Jensen (LJ) slants and incubated up to 6 and 8 weeks, respectively. ZN smears were made from MGIT tubes flagged positive by the machine or from the colonies grown on LJ media. Identification of MTBC was confirmed by a combination of standard tests such as ZN smear, cord factor, colony morphology, growth rate, niacin production, nitrate reduction, catalase, and aryl sulfatase tests [6] as the gold standard. Seventy-one isolates of MTBC which include seventy strains of M. tuberculosis and one isolate of M. bovis and 20 NTM strains comprising four clinical isolates (three isolates of Mycobacterium scrofulaceum and one isolate of Mycobacterium fortuitum) and 16 stock cultures of NTM were tested for MPT64 Ag by SD Bioline and TBcID kits. Details of MTBC and NTM species are listed in [Table 2]. Reference strains of M. tuberculosis H37Rv, M. bovis BCG, and M. fortuitum were also included as controls for both rapid kits.
Table 2: Details of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species, subjected to two rapid tests

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SD Bioline and BD MGIT TBcID Kits: SD Bioline MPT64 Ag test was performed for isolates recovered in MGIT and LJ as per the manufacturers' instructions and as reported earlier.[6] Since there is no provision in TBcID kit for testing solid media grown cultures, colonies from LJ were emulsified in 200 μl of 10 mM phosphate buffer containing 0.1% Tween 80 as per Abe et al.[3] and 100 μl from the emulsion was added to the sample window and observed for the development of a purple band within 15 min. Even faint lines were considered positive.


   Results Top


MTBC was isolated from 45.8% of TB spine, 30.3% of LN aspirates/cold abscess, 17.9% of biopsies, 14.8% of urine, 12.9% of pus, 6.7% of pleural fluid, and 4.3% of ascitic fluid. Out of 94 strains analyzed for MPT64 Ag detection, all the 71 isolates of MTBC comprising seventy M. tuberculosis and a single isolate of M. bovis were positive in both the kits. Four clinical NTM isolates which include three M. Scrofulaceum and one M. fortuitum and 16 stock cultures of NTM were uniformly negative in SD Bioline and TBcID kits. While the reference strain M. tuberculosis H37Rv was positive for MPT64 Ag, M. bovis BCG and M. fortuitum were negative by both kits. [Figure 1] represents the images of MPT64 Ag detection in TBcID and SD Bioline. All the 71 MTBC isolates were true positive and four NTM isolates tested were true negative by both rapid tests. No false positivity or false negativity was reported from both kits [Table 3]. Regarding the sensitivity, specificity, PPV, and NPV for SD as well as BD, the concordance was 100% against the gold standard conventional biochemical tests. Two microbiologists independently performed the test and assessed the reproducibility of the results and reported as 100%. Even faint bands were considered positive.
Figure 1: MPT64 antigen detection in TBc Identification and SD Bioline. MPT64 antigen positive (both control and test bands present)

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Table 3: Statistical analysis of SD Bioline and TBc Identifification rapid tests against conventional tests as gold standard (n=71)

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   Discussion Top


Different EPTB specimens show culture positivity ranging from 0% to 80%.[2] Confirmation of M. tuberculosis by conventional biochemical tests takes several weeks. The rapid kits, on the other hand, discriminate MTBC from NTM within 15 min. Capilia TB was evaluated in only one Indian study [4] with sensitivity, specificity, PPV, and NPV of 76%, 86%, 97%, and 39%, respectively. This kit can be procured only through FIND (Foundation for Innovative New Diagnostics, Geneva, Switzerland).

SD Bioline kit has been evaluated in India and in several countries such as France, Japan, Madagascar, South Korea, and Turkey, with sensitivity and specificity of 94.5%–100% and 98.3%–100%, respectively.[6]

BD MGIT TBc Identification kit has become available in Indian market from the past 3–4 years only and yet to be validated in India. Our investigation has established that TBcID kit has 100% sensitivity, specificity, PPV, and NPV. Manufactures of TBcID kit recommend positive liquid cultures only to be tested for MPT 64 Ag. However, a few researchers and we have additionally tested solid media grown cultures.[8],[14]

The MPT 64 Ag detection limit for the Capilia TB and SD Bioline kits was set as 105 CFU/ml,[4],[5] whereas for MGIT TBcID kit, it was 5 × 105 CFU/ml according to Yu et al.[11] Inoculum concentration does affect the test performance. In view of this, sufficient amount of LJ growth was scrapped to prepare our inoculum. Regarding the use of TBcID from MGIT tubes, only when the growth units were more than 800 as indicated by MGIT system, ICT was done.

In terms of turnaround time, both SD Bioline and TBcID kits take only about half an hour. Both kits are simple to perform. Regarding the cost, while of SD Bioline costs approximately Rs. 250, TBcID costs Rs. 350 per test.


   Conclusion Top


To conclude, rapid kits detect and confirm MTBC cultures within 15 min when compared to several weeks' time required by conventional methods. These kits are quite relevant today as EPTB due to NTM is on the increase. Empirical treatment of EPTB cases without culture could be hazardous as many NTM strains are resistant to the first-line antituberculous drugs, and thus liable to be misdiagnosed as “multidrug-resistant TB.” In our experience, TBcID kit and SD Bioline kit perform equally well and extremely cost-effective and time saving, thus benefiting resource-poor countries.

Limitations of the study

Quantification of LJ growth was not known as CFU/ml was not done. The number of MTBC isolates was only 71. We could only proceed with this moderate number of isolates. Inclusion of large number of isolates might give a better and clear picture.

Acknowledgment

This research project was fully funded by the institution and we express our sincere gratitude to The Chairman, Vice-chancellor, Dean-Research, PG coordinator, and Dean, Mahatma Gandhi Medical College and Research Institute, Puducherry. We sincerely thank Dr. Vanaja Selvakumar, Scientist F and Deputy Director (Retired), National Institute for Research in Tuberculosis, Chennai, for providing us H37Rv and NTM stock cultures.

This study was fully funded by the institution.

Financial support and sponsorship

This study was fully funded by the institution.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
World Health Organization. Global Tuberculosis Report; 2015. Available from: http://www.apps.who.int/iris/bitstream/ 10665/191102/1/9789241565059_eng.pdf. [Last accessed on 2016 Aug 15].  Back to cited text no. 1
    
2.
Mehta PK, Raj A, Singh N, Khuller GK. Diagnosis of extrapulmonary tuberculosis by PCR. FEMS Immunol Med Microbiol 2012;66:20-36.  Back to cited text no. 2
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3.
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. 3
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4.
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
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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. 5
    
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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;33:122-5.  Back to cited text no. 6
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Nerurkar V, Kattungal S, Bhatia S. Utility of MPT64 antigen test for differentiating mycobacteria: Can correlation with liquid culture smear morphology add further value? Indian J Pathol Microbiol 2016;59:185-7.  Back to cited text no. 7
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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. 8
    
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Said HM, Ismail N, Osman A, Velsman C, Hoosen AA. Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures. J Clin Microbiol 2011;49:1939-42.  Back to cited text no. 9
    
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Martin A, Bombeeck D, Fissette K, de Rijk P, Hernández-Neuta I, Del Portillo P, et al. Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture. J Microbiol Methods 2011;84:255-7.  Back to cited text no. 10
    
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Yu MC, Chen HY, Wu MH, Huang WL, Kuo YM, Yu FL, et al. Evaluation of the rapid MGIT TBc identification test for culture confirmation of Mycobacterium tuberculosis complex strain detection. J Clin Microbiol 2011;49:802-7.  Back to cited text no. 11
    
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Roberts SA, Lowe O, Pandey S, Williamson DA, Newton S, Vaughan R. Comparison of the MGIT TBc immunochromatographic assay with the Accuprobe Gen-Probe TB assay for identification of Mycobacterium tuberculosis complex: Results from a low-burden tuberculosis setting. Diagn Microbiol Infect Dis 2012;74:415-6.  Back to cited text no. 12
    
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Považan A, Vukelic A, Savkovic T, Kurucin T. Use of immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex from liquid culture. Bosn J Basic Med Sci 2012;12:33-6.  Back to cited text no. 13
    
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Machado D, Ramos J, Couto I, Cadir N, Narciso I, Coelho E, et al. Assessment of the BD MGIT TBc identification test for the detection of Mycobacterium tuberculosis complex in a network of mycobacteriology laboratories. Biomed Res Int 2014;2014:398108.  Back to cited text no. 14
    
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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. 15
    

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Correspondence Address:
Selvaraj Stephen
Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondy.Cuddalore Main Road, Puducherry - 607 402
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_695_15

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