MycobacteriologyEfficiency of the TK Culture System in the diagnosis of tuberculosis☆
Introduction
Tuberculosis continues to be one of the major health problems around the world. The most important element in tuberculosis control is to identify patients who have tuberculosis bacilli in their sputum and treat them efficiently, before they transmit the disease to healthy individuals. Culture is the gold standard, the most sensitive and specific method for the diagnosis of tuberculosis. Unfortunately, it takes 3 to 6 weeks to detect mycobacterial growth in classical media like Löwenstein-Jensen (LJ) (de Waard and Robledo, 2007, Manterola et al., 1998, Turkkani et al., 2010). Rapid automated culture systems are not used as widely as classical media, mainly because of their high cost, requirement for expensive instrumentation, and their media being not ready to use. All of the most commonly used rapid mycobacterial culture systems like BACTEC 460, BACTEC MGIT (Becton Dickinson Diagnostic Instrument Systems, Towson, MD, USA) (Huang et al., 2001, Pfyffer et al., 1997, Saitoh and Yamane, 2000, Sorlozano et al., 2009, Telenti et al., 1993, Tortoli et al., 1999, Turkkani et al., 2010), MB/BacT ALERT 3D (bioMérieux, Marcy l'Etoile, France) (Manterola et al., 1998, Saitoh and Yamane, 2000, Sorlozano et al., 2009), and ESP Culture System II (ESP II; Trek Diagnostics, Westlake, OH, USA) (Turkkani et al., 2010) use Middlebrook broth that requires the addition of oleic acid, albumin, dextrose, catalase (OADC), and selective antimicrobials before inoculation of the processed sample. These manipulations require extra time and effort, and increase the risk of contamination.
We have developed a ready-to-use, biphasic, rapid mycobacterial culture medium called TK Medium (SALUBRIS, Woburn, MA, USA) that enables early detection of mycobacterial growth by changing its color (Kocagöz, 2010). TK Medium includes egg and additional nutrients like glutamic acid and iron. Since the production process does not require heating the heat-susceptible vital factors contained in the egg, these probably make the medium richer than classical egg-based media, enabling better mycobacterial growth. The medium contains pH indicators, and the color change basically depends on pH change due to bacterial growth. Mycobacterial growth causes its original red color to turn yellow (Kocagöz et al., 2007, Pai et al., 2006, World Health Organization (WHO), Foundation for Innovative New Diagnostics (FIND), 2006. The color change occurs even before the colonies have grown sufficiently to become visible on the solid part of the medium. TK Medium has the advantage of differentiating mycobacterial growth from the growth of most common contaminants like fungi and Gram-negative bacilli because the growth of those contaminants causes the medium to change to green instead of yellow (Fig. 1). Although some Gram-positive bacteria may change the color of TK Medium to yellow, they rarely survive decontamination, and even if they do, they are inhibited by antimicrobials included in TK SLC, the selective form of the medium. TK Medium is designed as a biphasic medium to enable the visualization and isolation of individual colonies on the solid part and to visualize features like cord factor in the liquid phase (Fig. 2). Pure cultures of mycobacteria can be obtained by subculturing individual mycobacterial colonies if mixed organisms are obtained in the original culture. To lower the rate of contamination, TK Medium was supplemented with selective antimicrobials, polymyxin B, piperacillin, amphotericin B, nalidixic acid, and trimethoprim, to produce TK SLC. To eliminate the possibility that these antimicrobials may inhibit the growth of mycobacteria belonging to different species, we have previously inoculated 98 different species of mycobacteria (culture collection strains obtained from the American Type and Culture Collection [Manassas, VA, USA] or from DSMZ [Germany]) into TK SLC, and all of them have grown well without a sign of inhibition (unpublished data). The TK Anti Tb and PNB kit is used for simultaneous susceptibility testing and for Mycobacterium tuberculosis complex and MOTT (mycobacterium other than tuberculosis) differentiation. It contains a suspension tube, a dilution tube, a TK Medium growth control tube, and TK Media tubes containing isoniazid (INH) (0.2 μg/mL), rifampin (1.0 μg/mL), streptomycin (2.0 μg/mL), ethambutol (7.5 μg/mL), and para-nitro benzoic acid (PNB) (250 μg/mL). TK SLC and the TK Anti Tb and PNB Kit are stored at 4 °C, and their shelf-life is 6 months.
Since the growth in TK Media is detected by color change, it can be easily followed by visual evaluation and can be used in laboratories that have a regular 37 °C incubator. On the other hand, it has a very elaborate automated incubator reader, called Mycolor TK (SALUBRIS-technica) (Fig. 3). Mycolor TK detects and monitors the color changes (from red to yellow or to green). Mycolor TK provides growth curves, and its expert system predicts the type of growing microorganism.
This study was done to investigate the performance and advantages of the TK Rapid Mycobacterial Culture System (TK System) in daily use at the Yedikule Chest Diseases and Chest Surgery Education and Research Hospital, located in Istanbul, which is one of the largest hospitals taking care of tuberculosis patients in Turkey. This is the first large evaluation of the TK System after being introduced to clinical practice.
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Materials and methods
A total of 16,303 clinical samples from patients suspected to have tuberculosis, submitted for microscopic examination and culture, during a 1-year period between May 2009 and May 2010, to the tuberculosis laboratory of Yedikule Chest Diseases and Chest Surgery Education and Research Hospital, Istanbul, Turkey, were evaluated. Sputum samples (91.5%) constituted most of the specimens followed by bronchoalveolar lavage (BAL) (7.3%) and other samples (1.2%) including tracheal aspirate, pleural
Results
The results of microscopy and culture in LJ and TK SLC are summarized in Table 1. Among 16,303 samples included in the study, 1374 (8.42%) were AFB positive by microscopic examination. There was no growth in any type of media from 91 (0.56%) of the samples which were AFB positive by microscopy. In 2150 (13.04%) samples, mycobacteria were isolated in at least 1 type of medium. These belonged to 1571 patients (since for some patients more than 1 sample was positive in culture). While LJ isolated
Discussion
Culture continues to be the gold standard method for the diagnosis of tuberculosis. Additional to its ability to establish a definite diagnosis, culturing mycobacteria also enables the determination of antituberculosis drug susceptibility and allows to perform epidemiologic studies (de Waard and Robledo, 2007, Pai et al., 2006, World Health Organization (WHO), Foundation for Innovative New Diagnostics (FIND), 2006. Since classical media like LJ require 3 to 6 weeks for the detection of growing
Acknowledgments
The authors thank Andrew Ramsay and WHO for providing the mycobacterial collection strains for susceptibility testing and Richard O'Brien and Mark Perkins for their assistance. They also thank David Oliver and Nadi Bakirci for their guidance and help in the preparation of the manuscript.
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2014, Advanced Drug Delivery ReviewsCitation Excerpt :In the presence of anti-TB drugs, a color change from red to yellow indicates DR strains, whereas no change in color indicates drug susceptible strains. A recent study, utilizing 16,303 clinical samples, showed that the TK Rapid Mycobacterial Culture System is a practical and reliable automated system that shortens the time required for both culture and susceptibility results [23]. Although this test is simple, easy to visualize and rapid, further clinical validation is needed.
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2015, Frontiers in Microbiology
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This study was partly supported by the Foundation for Innovative New Diagnostics (FIND). FIND provided financial and scientific support for the development of the TK Culture System. Investigators did not receive any direct support from Salubris, Inc., for this study.