|Year : 2010 | Volume
| Issue : 4 | Page : 308-312
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
I Akyar1, T Kocagoz1, G Sinik2, S Oktem1, N Aytekin1, S Kocagoz1
1 Department of Microbiology, Acibadem University, Gulsuyu Ward, Fevzi Cakmak Avenue, Divan Street, Nr: 1 Maltepe Istanbul, Post code: 346 62, Turkey
2 Trends in Innovative Biotechnology Organization (TIBO), Idealtepe Ward, Akguvercin Street, Nr: 9 Maltepe, Istanbul, Post code: 34841, Turkey
|Date of Submission||21-May-2010|
|Date of Acceptance||30-Aug-2010|
|Date of Web Publication||20-Oct-2010|
Department of Microbiology, Acibadem University, Gulsuyu Ward, Fevzi Cakmak Avenue, Divan Street, Nr: 1 Maltepe Istanbul, Post code: 346 62
Source of Support: None, Conflict of Interest: None
Background: Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. Aim: We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Lφwenstein-Jensen and TK-selective (SLC) medium. Materials and Methods: The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Lφwenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. Results: The growth of mycobacterial strains was observed in 10-12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. Conclusion: With the additive effect of growth on TK-SLC medium in 10-12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.
Keywords: Atypic mycobacteria, MPB64 protein, Mycobacterium tuberculosis, mycobacterium
|How to cite this article:|
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
|How to cite this URL:|
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 [serial online] 2010 [cited 2020 Jun 7];28:308-12. Available from: http://www.ijmm.org/text.asp?2010/28/4/308/71817
| ~ Introduction|| |
It is estimated that one-third of world population is infected with Mycobacterium tuberculosis. Among these, approximately 8.5 million people develop active tuberculosis and 2.3 million die of this disease annually.  This devastating disease is caused by members of the M. tuberculosis complex, a group of closely related species and subspecies that includes M. tuberculosis, Mycobacterium bovis, the causative agent of bovine tuberculosis, and M. bovis BCG, the live, attenuated tuberculosis vaccine strain.  Although M. tuberculosis is the major pathogen, at least 20 different species of mycobacteria cause infections in humans. In recent years, cases caused by mycobacteria other than tuberculosis (MOTT) have increased prominently parallel to the increase of immunocompromised patients due to diseases like AIDS and cancer. For this reason, it is important to identify the species of mycobacteria when isolated from clinical samples. It is also important to differentiate mycobacteria contaminated from environment in mycobacterial cultures from disease causing mycobacteria, to eliminate misdiagnosis and unnecessary treatment. 
Classical species identification of mycobacteria which depends on growth rate, pigmentation of colonies and biochemical features is very cumbersome, requires long period of time and too much effort. Therefore, rapid molecular methods that identify specific nucleic acid sequences for species identification are used more frequently in routine application.  However, these methods require trained personnel, special laboratory set up, 1 or 2 days in practice, and they are expensive and labour-intensive. 
In developing countries, more than 90% of clinical mycobacterial isolates belong to M. tuberculosis. Therefore, it is important in the first step to differentiate M. tuberculosis from MOTT. In many cases, standard treatment regimen for M. tuberculosis is ineffective in MOTT infections. In terms of starting early treatment for M. tuberculosis infection, it is sufficient to differentiate it from MOTT.
The MPB64 antigen is the secretory protein (24 kDa) that is one of the secreted major antigens from tuberculosis bacteria. This immunogenic protein has been found in unheated culture fluids of M. tuberculosis H37Rv and in some strains of M. bovis BCG. This antigen induced a strong delayed-type hypersensitivity reaction similar to that induced by purified protein derivatives in guinea pigs sensitised with these strains, whereas no reaction to MPB64 was observed with Mycobacterium kansasii or Mycobacterium intracellulare. The MPB64 antigen has been shown to be specific for the M. tuberculosis complex.  Thus, MPB64 could be useful in studies on the pathogenesis and cell-mediated immunology of mycobacteria and in the development of diagnostic tests. 
Although the MPB64 antigen was found to induce a strong delayed-type hypersensitivity reaction in guinea pigs  and human beings,  different results were obtained with MPB64 in blood assays. Johnson et al. reported negative blood test results with MPB64 for persons before and after BCG vaccination and for active TB patients.  In contrast, Roche et al. confirmed that MPB64 was recognised more frequently by TB patients and their contacts than by healthy populations vaccinated with BCG. Furthermore, patients with active TB had significant responses to MPB64 in whole blood gamma interferon assay (WBIA). Differentiation of latent tuberculosis infection from a healthy, unexposed population plays a vital role in the strategy of controlling and eliminating tuberculosis. Both CFP21 and MPB64, antigens encoded by the RD2 region which are restricted in the M. tuberculosis complex, are TB-specific diagnostic candidate antigens.  Also, an enzyme-linked immunosorbent assay (ELISA) has been developed to overcome the disadvantages of skin test. In that study, the antigenic proteins including MPB64 are used. Thus, it would be useful in TB serological analysis. 
SD Bioline Tb Ag MPB64 (Standard Diagnostics Inc., Kyonggi , South Korea), is a lateral flow assay that quickly identifies the presence of M. tuberculosis complex by using anti-MPB64 monoclonal antibodies for rapid discrimination between the M. tuberculosis complex and MOTT. 
Also, a rapid growth in mycobacterial culture media will help to detect M. tuberculosis infections in a short time period. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. TK selective is a medium that contains polypeptides, carbohydrates, salts, dye indicators, vitamins and five different antimicrobials (polymixin B 5 μg/ml, piperacillin 50 μg/ml, amphotericin B μg/ml, nalidixic acid 20 μg/ml and trimethoprim 2 μg/ml) to inhibit the growth of other bacterial species and fungi. Although it is 8 times more expensive than Lφwenstein-Jensen medium and 3 times more expensive than Middlebrook medium, TK-SLC is a cost-effective medium taking its features into consideration.
The original red colour of the medium turns yellow by mycobacterial growth and green by the growth of contaminant organisms. The colour change can be followed visually or in the automated incubator reader Mycolor TK. TK culture system is routinely used in our laboratory along with Lφwenstein-Jensen medium. Average growth detection time of M. tuberculosis with TK culture system is 2 weeks compared to 4 weeks with Lφwenstein-Jensen medium. The medium is a biphasic medium with a slant and liquid at the bottom end of the tube. The liquid part of the medium indicating mycobacterial growth, where cord factor can also be observed, is directly used in this study for immunochromatographic assay (ICA) evaluation. ,,,
This new medium shortens TB detection time to half and its speed and sensitivity is comparable to automated systems.  In a study compared with the other mediums TK-SLC medium was accepted to be a practical and rapid culture system for daily use. 
The purpose of this study was to evaluate the efficiency of Tb Ag MPB64 in rapid differentiation of M. tuberculosis and MOTT grown in Lφwenstein-Jensen and TK-SLC media.
| ~ Materials and Methods|| |
In the present study, SD Bioline Tb Ag MPB64, a newly developed immunochromatographic assay (MPB64-ICA) using anti-MPB64 monoclonal antibodies for rapid differentiation between the M. tuberculosis complex and MOTT bacilli, was evaluated with a total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different species of MOTT, 7 M. bovis BCG substrains and total 256 clinical Mycobacterium isolates between January 2009 and January 2010. The results were compared with those of nucleic acid amplification technique synchronically studied. Polymerase chain reaction (PCR) targeting IS6110 is the method routinely used in our laboratory for identification of M. tuberculosis in clinical samples. IS6110 is usually repeated 5-20 times in M. tuberculosis genome. The presence of multiple copies of this gene increases the sensitivity of PCR assay. Rarely, it is reported that some M. tuberculosis strains are missing IS6110, which we believe would not affect the results of this study.
Mycobacterial grown from clinical samples or reference strains subcultured in Lφwenstein-Jensen and TK-SLC media were studied by SD Bioline Tb Ag MPB64 for rapid differentiation of M. tuberculosis complex and MOTT.
The active ingredients of main components in one strip of SD Bioline Tb Ag MPB64 are: gold conjugate mouse monoclonal anti-MPT64-gold colloid (0.24 ± 0.048 μg); test line: mouse monoclonal anti-MPT64 (0.32 ± 0.064 μg); control line: goat anti-mouse immunoglobulin (0.64 ± 0.128 μg); assay diluent: 100 mM phosphate buffer; sodium azide.
The sensitivity and the specificity of the TB Ag MPT64 rapid test were 98.6, and 100%, respectively.
Growth in TK-SLC was observed generally in 10-12 days for M. tuberculosis strains. All the growths of mycobacteria in TK-SLC medium and Lφwenstein-Jensen agar were confirmed by acid fast bacilli (AFB) staining microscopy.
When colonies were observed on Lφwenstein-Jensen, three to four colonies were suspended in 200 μl extraction buffer provided by the kit and 100 μl was added into the sample well. When TK-SLC indicated growth by turning yellow, 100 μl from the liquid part of the medium was directly added into the sample well.  The line indicating the presence of M. tuberculosis usually appeared within a few minutes [Figure 1].
As instructed in the kit, insert mycobacteria were considered as MOTT unless this line was observed within a maximum of 15 minutes. The species of M. tuberculosis complex strains were confirmed by PCR using IS6110 primers specific for M. tuberculosis complex. All the MOTT strains were standard strains from culture collections ATCC or DSMZ. The strains tested are listed in [Table 1].
|Figure 1 :Identification of the M. tuberculosis complex by MPB64-ICA. If the test is positive in addition to the reddish-purple band appearing in the control (C) window, a clear distinguishable reddish-purple band also appears in the test (T) window|
Click here to view
| ~ Results|| |
The mycobacterium growth was detected approximately in 10-12 days in TK-SLC medium, whereas it was detected in 20-28 days in Lφwenstein-Jensen medium. All M. tuberculosis and MOTT species were identified correctly on ICA slides by Tb Ag MPB64 test. For all the M. tuberculosis strains, a second coloured band was observed, within usally few minutes, next to the control band, indicating these as M. tuberculosis strains. In all MOTT strains, only the control band was observed. There were no false positive and false negative results, and the specificity was 100% for both Lφwenstein-Jensen and TK SLC. Similarly, the sensitivity was also 100% in both the media.
| ~ Discussion|| |
When mycobacteria are isolated from clinical samples, the differentiation of the bacteria belonging to M. tuberculosis complex group and MOTT is very important to identify if the species is pathogenic and to choose the right treatment protocol.  Culture-based methods used for this purpose may require weeks and nucleic acid based methods are cumbersome, costly and require experienced personnel. SD Bioline Tb Ag MPB64, which uses an antibody that recognises specifically the antigen of species belonging to M. tuberculosis complex group, is a rapid tool for differentiation of M. tuberculosis complex and MOTT. The application was very easy and positive results were obtained within usually a few minutes. Also, using TK-SLC medium helps to detect the growth of M. tuberculosis in 10-12 days.
It is important to differentiate M. tuberculosis from MOTT when mycobacteria are grown in culture media from clinical samples. Classical methods based on culture are cumbersome and require long period of time. Nucleic acid tests are rapid and specific. However, they require special laboratory set up and trained personnel.  The MPB64 gene has been well characterised, and the antigen has been widely studied. , This immunogenic protein elicits delayed-type hypersensitivity reactions in sensitised guinea pigs and lymphoproliferative responses in patients with tuberculosis. ,
The MPB64 antigen is found in the culture fluids of the M. tuberculosis complex. The antigen is secreted in significant amounts during the early period of culturing and decreases with longer cultivation. MPB64 is a highly specific protein for the M. tuberculosis complex and has shown potential for diagnostic use. , MPBT64-ICA is a rapid immunochromatographic identification test for the M. tuberculosis complex, which uses anti-MPB64 monoclonal antibodies.  Abe et al. in their studies reported a very high specificity for this test with only one Mycobacterium marinum strain and one Mycobacterium flavescence strain giving a very weak reaction.  In our study, M. marinum strain that we tested did not show any positive reaction.
SD Bioline Tb Ag MPB64 is a rapid lateral flow assay that allows one-step identification of M. tuberculosis complex by using MPB64 antigen. The results of this study indicated that SD Bioline Tb Ag MPB64 can differentiate culture grown mycobacteria as M. tuberculosis complex and MOTT, with a very high sensitivity and specificity. This can safely be stated because of the wide spectrum of MOTT and reference strains studied. The method is very easy to apply and time saving; the results are obtained within a maximum of 15 minutes. It is also a cost-effective method as it is nearly half of the price of the molecular methods. The method does not require any special laboratory set up. The application can be learned very easily and can be applied in any laboratory that can perform mycobacterial culture.
It is very important to identify M. tuberculosis directly in clinical samples. SD Bioline Tb Ag MBP64 should be evaluated for its efficiency of identification of M. tuberculosis directly in clinical samples.
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