Effectiveness of SD Bioline MPT 64 Test for Differentiating Mycobacterium tuberculosis Complex and Non-Tuberculous Mycobacteria from Liquid
Culture Positive Isolates
Shreya khandelwal1,Bharti Malhotra2,Widhi Dubey3,Shipra Bhargava4
1Research scholar, Department of Microbiology, JECRC University, Jaipur.
2*Senior Professor, Head of the Department, Department of Microbiology, SMS Medical College, Jawahar Lal Nehru Marg, Jaipur
3Professor, Director, Faculty of Science,JECRC University, Jaipur.
4Consultant Microbiologist, National Tuberculosis Institute, Bangalore.
ABSTRACT
For proper management of TB, it is critical to distinguish Mycobacterium tuberculosis complex (MTBC) from other mycobacteria. The traditional biochemical methods are slow and error prone, require setting up of multiple tests. To differentiate MTBC from Non Tuberculous Mycobacteria (NTM) a new immunochromatographic test kit that is rapid “SD Bioline TB Ag MPT64”has been evolved by Standard Diagnostics, South Korea. It detects MPT64 antigen specific for MTBC.120 liquid culture positive isolates of mycobacteria were enrolled for the study and tested by SD Bioline TB Ag MPT64 test kits. For positive control H37RV standard strain was used. These were further processed for GenoType. Mycobacterium CM/AS assay for further discrimination to the species level.
Among the 120 isolates initially negative with MPT64 and classified as NTM on further testing by CM/AS, 13(10.83%) were identified as MTBC. The liquid culture tube growth of these 13 isolates after one month was repeated for MPT64, 8/13 (61.53%) gave positive results and 5/13 (38.46%) remained negative. MPT64 test may be negative in 10.83% cases even when the isolate is MTBC on initial testing of Acid fast Bacilli (AFB) smear positive liquid culture. Moreover, 8(6.66%) isolates reported to be positive on prolonged incubation (28 days) indicating that the amount of MPT64 antigen secreted may be low in some isolates. Hence care should be taken to identify MPT64 negative isolates as some MTB may be missed otherwise.
Keywords: SD bioline MPT64 test, Non Tuberculous Mycobacteria, M.TB. Complex
Introduction
Rapid early identification of mycobacterial isolate as Mycobacterium tuberculosis (MTB) or Nontubercular mycobacteria (NTM) is important so as to proceed for drug susceptibility testing for Mycobacterium tuberculosis or ask patient to repeat sample in case of non tubercular Mycobacteria. Traditional methods of differentiating MTBC and NTM are slow, require growth of 3- 4 weeks, cumbersome to perform, error prone, require setting up of multiple tests like , P- Nitro benzoic Acid on lowenstein-Jensen , niacin nitrate and catalase test etc.(Kent & Kubica, 1985). As nowadays liquid culture methods are widely used for rapid diagnosis of TB and are also proposed by Revised National Tuberculosis Control Program (RNTCP) for all National Reference Laboratories (NRLs) and Intermediate Reference Laboratories (IRLs) . It is more sensitive than solid culture for cultivation of Mycobacteria. p-nitrobenzoic acid (PNB) and p- nitro-α acetylamino β hydroxypropiophenone (NAP) test have been used on BACTEC and MGIT respectively to differentiate MTBC and NTM but these take about a week time to give results .Many rapid methods have been developed to differentiate MTBC and NTM like capilla , Capilia TB-Neo (TAUNS, Izunokuni, Japan) (Tomiyama et al.,1997;Hillemann et al.,2005;Wang et al.,2007), SD Bioline TB Ag MPT64 (Abe et al., 1999), Accoprobe assay (Salfinger et al.,
1994), NAA (nucleic acid amplification) test (Cleary et al., 2003) and MGIT™ TBc Identification Test (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) (Yu et al., 2011) etc. which can differentiate mycobacterial growth within few minutes to hours from both solid and liquid media.
SD Bioline TB Ag MPT64, manufactured by Standard Diagnostics, Kyonggi-do, South Korea assay is being used routinely under RNTCP to differentiate MTBC and NTM. It detects MPT64, a 24 kDa secretory protein secreted by TB bacteria, the mouse monoclonal MPT64 antibody is immobilized on nitrocellulose strip and the antigen present in liquid media reacts with it to form a antigen antibody complex (Park et al., 2009).Though this kit is being used routinely under program condition it is important to know the effectiveness of this assay in liquid culture for identifying MTBC in comparison to Genotype CM/AS assay. Genotype CM/AS assay is a line probe assay based on reverse hybridization manufactured by Hain Life Sciences. This kit can identify 31 species of mycobacteria including MTBC. (Lee et al., 2009)
Material and Methods
This research was carried out at the Mycobacteriology lab, in Microbiology Department, SMS Medical College, Jaipur, India. This lab is a culture & Drug susceptibility testing lab established under RNTCP program, wherein diagnosis of MDR-TB suspects is being done. In this study we selected 120 mycobacterium isolates negative by SD Bio line and reported as NTM from Jan 2016 to Jan 2018. All the tests were performed in BSL II+ lab.
All positive tubes were first examined by Ziehl–Neelsen staining to confirm their positivity.
After these all-AFB positive tubes were tested with SD bioline test kit as per manufacturer instructions.
Figure 1. Work profile
SD Bioline test
100µl of liquid culture was added in the well of kit and nurtured for 15 minutes at room temperature. Pink colored control band in “C” area demonstrated its legitimacy. No band in “C”
area shows that the test is not valid. If an additional band was observed in the “t” region than the test was declared positive for M.tb.complex (MTBC).No band in “t” region indicated the test to be negative. For positive control reference strain H37Rv was taken. (Park et al., 2009; Gaillard T, et al., 2011)
Genotype CM/AS
For species identity of NTM Genotype CM/AS assay (Hain Life sciences) was performed on all SD bioline negative isolates. This involved three steps, DNA extractions, Master Mix preparation and hybridization. For DNA extraction genolyse kit (Genolyse ver 1.0 Hain Lifescience) was used in accordance with the manufacturer's instructions (Barnad et al., 2012).
Primer nucleotide mix (PNM) is provided with the kit that contains particular sets of primers intended to amplify a species specific 23S rRNA gene series of Mycobacterium species.
Amplification mixture was made by adding (45 μl) 5 μl extracted DNA, 35 μl primer nucleotide mix, 5 μl 10 × buffer for HotStar Taq (QIAGEN, Hilden, Germany), 0.2 μl HotStar Taq, 2 μl 25 mM MgCl 2 solution and 3 μl water (biology grade water). Amplification was carried out in a thermal cycler, which included 01 denaturation solution (DEN) cycles at 95°C for 15 minutes, annealing of primers at 95°C for 30s, 10 cycles of 2 minutes at 58°C, after that 20 cycles at 95°C for 25 seconds, 40 seconds at 53°C, 40 seconds at 70°C, and 40 seconds at 70°C for the final primer extension, 8 min for 01 cycle. The amplified products were stored at temperatures ranging from +8 to 20°C. This was followed by reverse hybridization in GT Blot 48 (Hain Lifescience) that was done as previously described (Singh et al., 2013). The presence and absence of different bands were used to evaluate and interpret the results, which were compared to the interpretation chart provided with the kit.
Results
On performing Genotype CM/AS test on the 120 liquid culture positive and SD bioline negative isolates, 107 were identified as NTM (57 M. intracellulare, 40 M. abscessus, 5 M. fortutum,2 M.
scrofulacium and 3 were M. simiae) but 13 (10.83%) culture isolates were found to belong to M.
Tb.complex (table 1). On repeating the SD Bioline MPT64 test on initially MPT64 negative liquid culture isolates 8/13 (61.53%) were found to be positive for MTBC but had faint bands in
“t” region (except 3 had dark bands) and 5 (38.46%) remained negative (Table 2).
Table 1-Species identified by genotype CM/AS assay
Species identified BY CM/AS No. (%)
M. intracellulare 57 (47.5%)
M. abscessus 40 (33.33%)
M. fortuitum 5 (4.16%)
M. simiae 3 (2.5%)
M. tuberculosis 13 (10.83%)
Table 2- comparative results of MPT64 test and CM/AS assay
Test performed MPT64
By CM/AS Day 1 30th Day
Total No. of isolates 120
MTBC 0 8 13
NTM 120 107 107
NI 0 5 0
Total 120 120 120
Discussion
Rapid early differentiation of MTBC and NTM is very important so as to process MTB separates for drug susceptibility testing DST and repeat sample from patient in the event of an NTM.SD bioline MPT64 test is an immunochromatographic assay which differentiates M.tb. and NTM rapidly. In this study we have analyzed the effectiveness of SD bioline in liquid culture positive for mycobacteria for rapid differentiation of NTM and MTBC. In present study 13(10.83%) isolates were reported as false negative by SD bioline initially, 8 (61.53%) of them converted positive on repetition of test after one month.
Many researchers have evaluated SD Bioline using known isolates of MTBC and NTM and have reported a sensitivity and specificity of 100 percent for differentiating MTB and NTM (Shenoy et al., 2014; Toihir et al., 2011; Hasegawa et al., 2002; Abe et al., 1999 ; Fabre et al., 2010;
Gaillard et al., 2011; Orikiriza et al., 2017 )
However, on using the test on clinical isolates, it has been reported that in some strains of MTB, MPT64 protein secretion is delayed or is poor which leads to false negative results (Diana et al., 2014; Ochang et al., 2013).
Maurya et al. (2012) observed that SD bioline MPT64 protein concentration can be affected by bacterial load. Park et al. (2009) reported the detection limit of MPT64 test to be about 105
proposed that a Culture must be put to the test by SD Bioline only with GU ≥ 300 to avoid false negative outcomes. While Kumar et al. (2015) reported that culture to be tested on day 3 of incubation in BACTEC machine as on day first faint bands were found.
In present study 5 isolates of MTB complex were missed by SD bioline MPT64 even after repeating the test after 1 month, but identified by Genotype CM/AS assay Many studies have observed that some M.bovis strains also do not produce MPT64 protein and are mislabeled as NTM (Hasegawa et al., 2002; Ismail et al., 2009; Park et al., 2009; Marzouk et al.,2011;
Chikamatsu et al., 2014)). Moreover another reason of false negative results or missing of MTBC can be the mutation in the MPT64 gene as (Hirano et al., 2004) described in his report that 12/381 (3.15%) capilia TB negative separates were positive by accuprobe and DNA-DNA hybridization test .Monde et al., 2013 reported 4/52 (7.69%) capilia negative isolates that were identified as MTBC by LPA . Incomplete protein is produced due to mutation which may not react with antibodies in SD bioline MPT64 test kit and show negative results.
Limitation of our study was that we repeated the SD Bioline after about 30 days and not earlier as this was done only after finding the originally labeled NTM to be MTBC by Genotype CM/AS assay. The repeat SD Bioline should have been done in a systematic manner on day 3, day 7, day 10 etc so as to identify the optimum day when such isolates should be repeated by SD Bioline as it is important to rapidly identify MTB in clinical setting so as to proceed for DST and give appropriate therapy. Moreover, sequencing should also have been done to further identify whether there are mutations in MPT 64 gene and further identification of the species in MTB complex identified in Genotype CM/AS assay but missed by SD Bioline.
Conclusion
SD Bioline can rapidly differentiate MTBC and NTM thus helping in further processing of isolates. However, due to various reasons like, growth being lower than detection limit of 105 CFU/ml, mutation in MPT64 gene, presence of M. bovis and delayed protein secretion in some culture, isolates may be mislabeled. it’s important to repeat the test after few days so as not to miss the MTB as in routine RNTCP labs the NTM are not being followed up for species identification. However, further studies are required to know the optimum day when such isolates should be repeated for confirmation.
Acknowledgements
The authors thank the Foundation for Innovative Newer Diagnostics (FIND), India, RNTCP program, SMS Medical College, Jaipur, India for the kits and technical support. We also acknowledge UGC for Financial assistance to Shreya Khandelwal.
References
1. Abe C. Hirano K. and Tomiyama T. (1999): Simple and rapid identification of the Mycobacterium tuberculosis complex by immunochromatographic assay using anti- MPB64 monoclonal antibodies. J Clin Microbiol. 37, 3693–7.
2. Barnard M. Gey van Pittius NC. and Warren RM. (2012): The Diagnostic Performance of the GenoType MTBDRplus Version 2 Line Probe Assay Is Equivalent to That of the Xpert MTB/RIF Assay. J. Clin. Microbiol. 11, 3712-3716
3. Chikamatsu K. Aono A. Yamada H. Sugamoto T. Kato T. Kazumi Y. Tamai K. Yanagisawa H. and Mitarai S. (2014): Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex. BMC Infect Dis. 14,54.
4. Cleary, TJ., Roudel, G., Casillas, O., & Miller, N. (2003). Rapid and specific detection of Mycobacterium tuberculosis by using the Smart Cycler instrument and a specific fluorogenic probe. J. Clin. Microbiol. 41(10), 4783–4786.
5. Gaillard T, Fabre M, Martinaud C, Vong R, Brisou P, Soler C.( 2011) Assessment of the SD Bioline Ag MPT64 Rapid™ and the MGIT™ TBc identification tests for the diagnosis of tuberculosis. Diagn Microbiol Infect Dis.70,154–6.
6. Hasegawa N. Miura T. Ishii K. Yamaguchi K. Lindner TH. Merritt S. Matthews JD.and Siddiqi SH. (2002): New simple and rapid test for culture confirmation of Mycobacterium tuberculosis complex: a multicenter study. J Clin Microbiol.40, 908–912.
7. Hillemann D. Rüsch-Gerdes S. and Rchter E. (2005): Application of the Capilia TB assay for culture confirmation of Mycobacterium tuberculosis complex isolates.Int J Tuberc Lung Dis, 9, 1409–1411.
8. Hirano K. Aono A. Takahashi M. and Abe C.(2004): Mutations including IS6110 insertion in the gene encoding the MPT64 protein of capilla TB-negative Mycobacterium tuberculosis isolates. J ClinMicrobiol. 42, 390-392
9. Hain Life Science GenoType Mycobacterium CM/AS assay Instruction manual. (2014) 10. Ismail NA. Baba K. Pombo D. and Hoosen AA. (2009): Use of an immunochromatographic
kit for the rapid detection of Mycobacterium tuberculosis from broth cultures. Int J Tuberc Lung Dis, 8,1045-7.
11. Kent PT.and Kubica GP. A Guide for the level III laboratory.Atlanta GA. (1985): US Health Service Centers for Disease Control;.Public health mycobacteriology.Laszlo A, Eidus L.(
1978):Test for differentiation of M.tuberculosis and M bovis from other mycobacteria.Can J Microbiol.24,754–6.
12. Lee AS. Jelfs P. Sintchenko V.and Gilbert GL. (2009): Identification of non-tuberculous mycobacteria: Utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRN A gene sequencing. J Med Microbiol.58,900-4.
13. Marzouk M. Kahla IB. Hannachi N. Ferjeni A. Salma WB. Ghezal S.and Boukadida J.(
2011) Evaluation of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in clinical isolates. Diagn Microbiol Infect Dis. 69,396–399.
14. Maurya AK. Naq VL. Kant S. Kushwaha RA. Kumar M. Mishra V. and Tapan N.
D.(2012).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.135(6),901-906.
15. Muyoyeta M. De Haas PE. Mueller DH, Van Helden PD. Mwenge L. Schaap A. Kruger C.
Gey van Pittius NC. Lawrence K. Beyers N. Godfrey-Faussett P.and Ayles H.( 2010):
Evaluation of the Capilia TB assay for culture confirmation of Mycobacterium tuberculosis infections in Zambia and South Africa. J Clin Microbiol. 43, 773–3775.
16. Orikiriza P. Nyehangane D. Atwine D. and Boum Y. (2017), Evaluation of the SD Bioline TB Ag MPT64 test for identification of Mycobacterium tuberculosis complex from liquid cultures in Southwestern Uganda. Afr J Lab Med.6(2), 383.
17. Park MY. Kim YJ. Hwang SH. Kim HH. Lee EY. Jeong SH. and Chang CL.
(2009):Evaluation of an immunochromatographic assay kit for rapid identification of Mycobacterium tuberculosis complex in clinical isolates. J Clin Microbiol.47,481–4 18. Salfinger M.and Pfyffer GE. (1994): The new diagnostic mycobacteriology laboratory. Eur.
J.Clin. Microbiol. Infect. Dis. 11, 961–979.
19. Shenoy VP.and Mukhopadhyay C. (2014): Rapid Immunochromatographic Test for the Identification and Discrimination of Mycobacterium tuberculosis Complex Isolates from Non-tuberculous Mycobacteria. J Clin Diagn Res. 8(4),13–15.
20. Singh A.K. Maurya A.K. Umrao J. Kant S. Kushwaha R.A. Nag V.L and Dhole T.N.( 2013):
“Role of GenoType Mycobacterium common mycobacteria/additional species assay for rapid differentiation between Mycobacterium tuberculosis complex and different species of non- tuberculous mycobacteria”, J. Lab. Physicians., 5 ,83
21. Tomiyama T. Matsuo K. and Abe C. (1997): Rapid identification of Mycobacterium tuberculosis by an immunochromatography using anti-MPB64 monoclonal antibodies. Int J Tuberc Lung Dis .1, 59.
22. Vadwai V. Sadani M. Sable R. Chavan A. Balan K. Naik A.Kambli P.Dhane V. Patankar M.Shetty A. and Rodrigues C.(2012): Immunochromatographic assays for detection of Mycobacterium tuberculosis: what is the perfect time test?” Diagnostic Microbiology and Infectious Disease, 74, 3, 282–287.
23. Wang JY. Lee LN. Lai HC. Hsu HL. Jan IS. Yu CJ. Hsueh PR. and Yang PC (2007):
Performance assessment of the Capilia TB assay and the BD ProbeTec ET system for rapid culture confirmation of Mycobacterium tuberculosis. Diagn Microbiol Infect Dis 59:395–
399.
24. Yu MC. Chen HY. Wu MH. Huang WL. Kuo YM. Yu FL and Jou R. ( 2011): Evaluation of the rapid MGIT TBc identification test for culture confirmation of Mycobacterium tuberculosis complex strain detection. J Clin Microbiol.49, 802-7.
25. Singh, K., Kumari, R., Tripathi, R. et al. (2019): Mutation in MPT64 gene influencing diagnostic accuracy of SD Bioline assay (capilia). BMC Infect Dis 19, 1048 https://doi.org/10.1186/s12879-019-4671-2
