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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2016  |  Volume : 59  |  Issue : 2  |  Page : 185-187
Utility of MPT64 antigen test for differentiating mycobacteria: Can correlation with liquid culture smear morphology add further value?


Department of Microbiology, SRL Limited, Mumbai, Maharashtra, India

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Date of Web Publication9-May-2016
 

   Abstract 

Context: Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) infections may or may not be the same, but the treatment is always different. Hence accurate differentiation between MTBC and NTM is of utmost importance.
Aims: To assess in parallel, the utility of MPT64 antigen immunochromatography assay (MPT64 ICT) and bacillary morphology on liquid culture smear, for rapid differentiation between MTBC and NTM in clinical isolates.
Settings and Designs: Private sector reference laboratory, prospective.
Subjects and Methods: Thousand and ninety-three mycobacterial isolates, recovered using Mycobacteria Growth Indicator Tube 960 liquid culture system (BD, USA), were subjected to MPT64 ICT (Standard Diagnostics Inc., Korea), para amino nitrobenzoicacid (PNB), niacin, and nitrate reduction tests. Smears prepared from culture vials were subjected to Ziehl-Neelsen staining and observed microscopically for typical patterns (chords, single cells, etc.,). PNB, nitrate and niacin tests served as the reference method for MTBC identification.
Results: Thousand and fourteen and 79 isolates were identified as MTBC and NTM, respectively. MPT64 ICT correctly identified 955/1014 MTBC and all NTM isolates, yielding sensitivity and specificity of 94.2% and 100%, respectively. 936/1014 (92.3%) MTBC isolates revealed characteristic serpentine chording on culture smear including 56/59 MPT64 ICT negative isolates. Sensitivity and specificity of liquid culture smear were 98.1% and 82.3%, respectively.
Conclusion: Correlation of MPT64 ICT results with liquid culture smear was useful, especially in MPT64 ICT negative isolates, where the latter could help to determine need and/or type of additional confirmatory testing. Liquid culture smear, however, lacked specificity and cannot be used as a stand alone test.

Keywords: Chording, identification, MPT64, Mycobacterium tuberculosis complex, non-tuberculous mycobacteria

How to cite this article:
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

How to cite this URL:
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 [serial online] 2016 [cited 2019 Dec 12];59:185-7. Available from: http://www.ijpmonline.org/text.asp?2016/59/2/185/182009



   Introduction Top


Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) infections may be similar,[1] but treatment is always different.[2] NTM are inherently resistant to conventional anti-tuberculosis drugs, hence often misdiagnosed as multidrug-resistant tuberculosis.[1] With the increasing number of clinical NTM infections, accurate differentiation between MTBC and NTM is crucial.[1] Conventional phenotypic identification methods are cumbersome, while genotypic methods require expertise and substantial monetary and infrastructural investment. Hence, TBMPT64 antigen-based immunochromatography assays (MPT64 ICT) offer promise in rapid and cost-effective differentiation.[1],[2],[3],[4],[5],[6] Another key differentiating factor between MTBC and NTM is the distinctive chording that MTBC exhibit during growth in liquid culture.[7],[8],[9] The aim of this study was to assess in parallel, the utility of a commercial MPT64 ICT and bacillary morphology on liquid culture smear, for rapid differentiation between MTBC and NTM in clinical isolates.


   Subjects and Methods Top


The study was prospectively carried out at a private sector reference laboratory in Mumbai. The Mycobacteriology section is a biosafety level three laboratory, accredited by NABL, CAP, and RNTCP for liquid culture and susceptibility testing for first-and second-line anti-tuberculousdrugs. The study duration was 5 months.

Study duration: Five months During the study period, 5082 consecutive clinical specimens were processed for mycobacterial culture using Mycobacteria Growth Indicator Tube (MGIT) 960 liquid culture system (Ms. BDLtd., USA). All specimens were digested and decontaminated by the N-acetyl-L-cysteine-NaOH method. A volume of 0.5 ml of the processed specimen was inoculated into MGIT 960 vials, supplemented as described by the manufacturer,[10] and 0.5 ml onto Lowenstein-Jensen (LJ) medium slants. All inoculated MGIT vials were incubated in the MGIT 960 instrument either until they flagged positive on the instrument or for a maximum of 6 weeks. L. J medium slants were examined biweekly, for 8 weeks, for the visible appearance of colonies.

Of the total specimens processed, all MGIT vials that flagged positive were subjected to smear preparation and Ziehl-Neelsen (ZN) staining. Smears were observed microscopically for the presence of mycobacteria and morphological patterns (serpentine chords, single cells, etc.,).

Once mycobacterial growth was confirmed on ZN stained culture smear, each isolate was subjected to the following tests:

  1. SD Bioline TBAgMPT64(Standard Diagnostics Inc., Korea) (MPT64 ICT)
  2. Paraamino nitrobenzoicacid (PNB) susceptibility: Tested on MGIT 960 system. The reagent was prepared inhouse
  3. Niacin production: Using commercial strips-BBL™ Taxo™ TB Strip (Ms. BD Ltd, USA)
  4. Nitrate reduction: Prepared in-house from commercial Nitrate broth (Ms. Hi-Media Ltd, India).


All tests were performed as per the respective manufacturer instructions.[10],[11],[12] The interpretative criteria used for above four tests and quality control are shown in [Table 1].
Table 1: Test interpretative criteria and quality control

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Statistical analysis

PNB, nitrate, and niacin tests were considered as the gold standard for MTBC identification. The sensitivity and specificity of MPT64 ICT and liquid culture smear morphology were calculated using two-by-two tables.


   Results Top


One thousand ninety-three mycobacterial isolates were recovered, of which 886 were from pulmonary specimens and 207 from extrapulmonary sources.

Using PNB, nitrate, and niacin methods, 1014 isolates were identified as MTBC and 79 as NTM.

As seen in [Table 2], MPT64 ICTcorrectly identified 955/1014 MTBC and all (79) NTM isolates, yielding a sensitivity and specificity of 94.2% and 100%, respectively.
Table 2: Performance of MPT 64 antigen immunochromatography assay

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[Table 3] shows the correlation between Liquid culture smear and MPT64 ICT, vis-a-vis reference phenotypic method. Sensitivity and specificity of chording as a presumptive marker of MTBC and bunches/singles as a marker of NTM, was found to be 98.1%, 82.3%, respectively.
Table 3: Correlation of culture smear with MPT 64 antigen immunochromatography assay and reference phenotypic method

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   Discussion Top


MPT64 ICT

Mycobacterium tuberculosis MTBC protein 64 (MPT-64) or Rv1980c antigen is a 24kDa secretary protein secreted by MTBC.[1],[2],[5] It is found only in viable and actively dividing cells.[1],[2],[5] Once secreted into the culture medium, it is stable for at least one year.[1] MPT64 ICT was found to be reliable in this study to differentiate between MTBC and NTM. The test was easy to perform, relatively inexpensive, and rapid.

As shown in [Table 2], 59/1014 MTBC isolates had negative MPT64 ICT results. Since tests were performed on fresh isolates, i.e., 24–48 h of MGIT culture flagging positive, false negativity could not be attributed to nonviability or older cultures. Likely causes of the false negativity could be (1) mutations within the mpt64 gene, leading to the production of an incomplete protein, (2) low-level expression of the antigen, and (3) lack of MPT64 production in some Mycobacterium bovis substrains.[1],[4],[5],[6] Studies report MPT64 ICT specificity of 100% with sensitivities ranging from 96 to 100%.[1],[2],[3],[5] Specificity of MPT64 ICT in this study was comparable to other studies, but sensitivity was slightly lower. No particular reason could be identified for the same.

Morphology on liquid culture smear

MTBC grow as tight, rope-like aggregates of acid-fast Bacilli with the bacterial long axes parallel to the cord long axes, commonly described as chording. This effect is caused by the mycolic acid-containing molecules and is implicated in mechanisms responsible for rendering Mycobacterium tuberculosis complex (MTC) virulent. On the other hand, NTM is scattered or dispersed without any orientation.[13],[14]

Several studies document that MTBC yields serpentine cording in liquid medium with a sensitivity of 23–95% and a specificity of 95–100%.[7],[8],[9],[13],[14],[15]

As seen in [Table 3], 92.3% MTBC isolates displayed chording on culture smear including 56/59 MPT64 ICT negative isolates. Similarly, 94.9% of NTM isolates were seen arranged singly or in bunches on smear.

In this study, the high sensitivity of chording allowed early presumptive identification of MTBC. Culture smear morphology, however, lacked sufficient specificity. Likely causes for MTC appearing in singles or bunches could be (1) paucity of AFB that may be misread as the absence of cording [13] and (2) too early time-to-detection of MGIT culture to form serpentine cording.[7] NTM species that can form chords include Mycobacterium avium-intracellulare, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium phlei, Mycobacterium terrae, and Mycobacterium kansasii.[9],[14] Incomplete cords produced by NTM may sometimes be read as true cords due to interpersonal subjectivity.[13],[14] Hence, culture smear morphology, inspite of high sensitivity, cannot be used as a sole test for MTBC/NTM differentiation, a finding expressed by other researchers.[8]


   Conclusion Top


This study points out that additional phenotypic/molecular testing for MTBC/NTM differentiation is required for some percentage of MPT64 ICT negative isolates. It will be incorrect to label all MPT64 ICT negative isolates as NTM, as it will have an adverse effect on patient treatment. Correlation of MPT64 ICT results with bacillary morphology on liquid culture smear is thus found to be useful in routine laboratory practice. The presence of chording in MPT64 ICT positive isolates obviates the need for any further testing to confirm identity. Furthermore, in MPT64 ICT negative isolates, bacillary morphology can help to determine the need of additional phenotypic/molecular testing and guide on the type of nucleic probe to be used for confirmatory identification, thereby improving reporting accuracy in a cost effective manner.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
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. 1
    
2.
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 & non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis. Indian J Med Res 2012;135:901-6.  Back to cited text no. 2
[PUBMED]  Medknow Journal  
3.
Martin A, Bombeeck D, Mulders W, Fissette K, De Rijk P, Palomino JC. Evaluation of the TB Ag MPT64 Rapid test for the identification of Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis 2011;15:703-5.  Back to cited text no. 3
    
4.
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. 4
    
5.
Shenoy VP, Mukhopadhyay C. Rapid immunochromatographic test for the identification and discrimination of Mycobacterium tuberculosis complex isolates from non-tuberculous mycobacteria. J Clin Diagn Res 2014;8:DC13-5.  Back to cited text no. 5
    
6.
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. 6
    
7.
Tu HZ, Chang SH, Huaug TS, Huaug WK, Liu YC, Lee SS. Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex and Mycobacterium kansasii. Ann Clin Lab Sci 2003;33:179-83.  Back to cited text no. 7
    
8.
Morris AJ, Reller LB. Reliability of cord formation in BACTEC media for presumptive identification of mycobacteria. J Clin Microbiol 1993;31:2533-4.  Back to cited text no. 8
    
9.
Yagupsky PV, Kaminski DA, Palmer KM, Nolte FS. Cord formation in BACTEC 7H12 medium for rapid, presumptive identification of Mycobacterium tuberculosis complex. J Clin Microbiol 1990;28:1451-3.  Back to cited text no. 9
    
10.
Siddiqi S, Rüsch-Gerdes S. MGIT Procedure Manual; 2006. Available from: . [Last accessed on 2015 Feb 09].  Back to cited text no. 10
    
11.
SD Bioline TB Ag MPT64 Kit Insert. Available from: . [Last accessed on 2015 Feb 09].  Back to cited text no. 11
    
12.
BBL™ Taxo™ TB Strip Kit Insert. Available from: . [Last accessed on 2015 Feb 09].  Back to cited text no. 12
    
13.
Singhal R, Arora J, Bhalla M, Lal P, Reza S, Behera D, et al. Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium. Indian J Med Microbiol 2012;30:218-21.  Back to cited text no. 13
[PUBMED]  Medknow Journal  
14.
Kadam M, Govekar A, Shenai S, Sadani M, Salvi A, Shetty A, et al. Can cord formation in BACTEC MGIT 960 medium be used as a presumptive method for identification of M. tuberculosis complex? Indian J Tuberc 2010;57:75-9.  Back to cited text no. 14
    
15.
Attorri S, Dunbar S, Clarridge JE 3rd. Assessment of morphology for rapid presumptive identification of Mycobacterium tuberculosis and Mycobacterium kansasii. J Clin Microbiol 2000;38:1426-9.  Back to cited text no. 15
    

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Correspondence Address:
Vidya Nerurkar
Department of Microbiology, SRL Limited, Prime Square Building, Gaiwadi Industrial Estate, S.V. Road, Goregaon (W), Mumbai - 400 062, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.182009

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    Tables

  [Table 1], [Table 2], [Table 3]

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