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Journal of Clinical Microbiology, February 2009, p. 481-484, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01253-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates

Mi Young Park,^1* Young Jin Kim,¹ Sang Hyun Hwang,¹ Hyoung Hoi Kim,¹ Eun Yup Lee,¹ Seok Hoon Jeong,² and Chulhun L. Chang^1,3

Department of Laboratory Medicine, School of Medicine,¹ Medical Research Institute, Pusan National University, Busan,³ Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea²

Received 2 July 2008/ Returned for modification 16 August 2008/ Accepted 22 November 2008

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We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (99%) and specificity (100%) and an appropriate detection limit (10⁵ CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories.

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Tuberculosis is a global problem and the single most common cause of death from any bacterial agent (15, 22). Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) are different clinically, so prompt detection, isolation, and discrimination are essential for appropriate management (3, 7).

The MPT64 protein is highly specific for M. tuberculosis complex, including M. tuberculosis, Mycobacterium africanum, Mycobacterium bovis, and some, although not all, substrains of M. bovis BCG (1, 6, 16, 18, 23), and can be detected in culture isolates and biopsy samples (1, 7, 17, 18, 20). Recently, Standard Diagnostics (SD, Yongin, Korea) developed a simple and rapid assay using a mouse monoclonal anti-MPT64 antibody to discriminate between M. tuberculosis complex and NTM by immunochromatography. Mouse monoclonal anti-MPT64 antibodies (SD Bioline TB Ag MPT64; SD) are immobilized on a nitrocellulose membrane as the capture material. Another antibody, which recognizes another epitope of MPT64 and has been conjugated with colloidal gold particles, is used for antigen capture and detection in a sandwich-type assay.

We evaluated the clinical usefulness of the kit using mycobacteria and other organisms. To determine specificity, 137 bacterial isolates (68 species), 20 fungal isolates (10 species), 53 reference mycobacterial isolates (40 species), and 51 NTM isolates from clinical samples were tested (Tables 1 to 3). To determine sensitivity, 159 M. tuberculosis complex strains grown on 3% Ogawa medium (isolated at Pusan National University Hospital), 60 strains from Bactec MGIT 960 culture tubes (isolated at Kosin University Gospel Hospital), and one reference strain, M. tuberculosis H37Rv, were tested. All bacterial, fungal, and mycobacterial isolates were stock cultures that had been kept in a –4°C refrigerator or a –72°C deep freezer for as long as 18 months. The cultured mycobacteria were identified by acid-fast bacillus stain, nucleic acid amplification, and DNA microarray (10, 14). Finally, to determine the detection limit, a series of diluted suspensions of M. tuberculosis H37Rv were inoculated onto Middlebrook 7H10 agar and the resulting colonies were counted (19). One hundred microliters of sample taken from liquid medium was applied directly to the sample well without preparation. Three or four colonies were scraped from the solid medium and suspended in 300 µl of extraction buffer (SD); then, 100 µl of the suspension was added to the sample well. If there was condensation fluid in egg-based medium, 100 µl of the fluid was applied directly to the sample well, instead of using extraction buffer. Tests were interpreted 15 min after sample application. The presence of a control band alone indicates a negative result, whereas the presence of two color bands (control and test bands), no matter which band appears first, indicates a positive result. A color band of any intensity was read as a positive reaction (Fig. 1). If the control band was not visible after 15 min, the result was considered invalid, and the sample was retested.

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TABLE 1. List of bacterial and fungal strains

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TABLE 2. List of reference mycobacterial strains

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TABLE 3. List of mycobacteria isolated from clinical specimens

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FIG. 1. Identification of the M. tuberculosis complex by the MPT64 ICA kit. Top, strong positive; middle, weak positive; bottom, negative.

All bacterial, fungal, and NTM isolates, including reference strains, were negative by the immunochromatographic assay (ICA) (specificity, 100%). One hundred fifty-eight of 159 M. tuberculosis complex strains grown on solid medium and 59 of 60 strains from liquid medium were positive by the ICA (overall sensitivity, 98.6%). The 1:128-diluted suspension (5.5 x 10⁵ CFU/ml) revealed 10% reaction intensity, and the band intensity gradually weakened with serial dilutions until the 1:1,024 suspension (6.8 x 10⁴ CFU/ml) was negative. The detection limit thus was determined to approximate 10⁵ CFU/ml.

Although most culture-positive mycobacteria are M. tuberculosis in regions where tuberculosis is highly prevalent, NTM isolates have been increasing gradually, such that now 20 to 30% of mycobacteria found in clinical specimens in Korea are NTM (13). These organisms trigger diseases and true infections and thus can be important clinically (21). Because of the complexity of test methods, many small hospital laboratories do not discriminate between M. tuberculosis and NTM (4, 11, 12), meaning that NTM are inappropriately managed with first-line antituberculosis drugs (12, 24), worsening the patient's condition and raising the risk of drug resistance. Thus, exact and rapid identification of mycobacteria is important, and a simple, sensitive, and specific identification method is necessary. Direct staining of a colony is simple and fast but does not discriminate between M. tuberculosis and NTM, and traditional biochemical tests not only can produce equivocal results but also take a long time (2). Chemiluminescent DNA probes, nucleic acid amplification, high-performance liquid chromatography, and sequencing of 16S rRNA genes are more sophisticated methods that require expensive equipment (2, 5, 10, 14). Although our study was conducted with culture specimens and needs further direct testing with clinical specimens, the ICA was shown to be rapid and easy and to have high sensitivity and specificity.

In this study, one isolate of M. tuberculosis complex from solid medium was negative. This organism was subcultured twice on 3% Ogawa medium with failure of growth, suggesting that the culture was kept too long in a slant culture format in a refrigerator before testing and lost its viability. The negative isolate that was grown in liquid medium was subcultured on 3% Ogawa medium, and a repeat test also was negative. According to a recent study, MPT64, once secreted into the medium, is stable, as the test remains positive even if performed 1 year after the detection of growth in either solid or liquid medium (7). This suggests another reason for the negative test results besides storage. Some M. bovis BCG substrains lack MPT64 production (1, 6, 16, 18), and this could have been a nonproducing strain. Another possible explanation for the negative test results is that the strain had mutations within the mpt64 gene, leading to the production of an incomplete protein. By sequencing, Hirano et al. identified several such mutations, including deletion of nucleotides, point mutations, and an IS6110 insertion mutation at nucleotide position 501 (8, 9).

The ICA is rapid and easy, is applicable for specimens from both liquid and solid media, and does not require any special equipment. It showed excellent sensitivity (99%) and specificity (100%) and an appropriate detection limit (10⁵ CFU/ml). It can simplify the identification of M. tuberculosis complex strains, avoiding the technical complexity of PCR and similar identification techniques in clinical laboratories.

 
C. L. Chang participated in the development of the MPT64 ICA kit as a scientific consultant.

 
* Corresponding author. Mailing address: Department of Laboratory Medicine, School of Medicine, Pusan National University, Busan 602-739, Korea. Phone: 82-51-240-7417. Fax: 82-51-247-6560. E-mail: CCHL{at}pusan.ac.kr

Published ahead of print on 3 December 2008.

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Journal of Clinical Microbiology, February 2009, p. 481-484, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01253-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Park, M. Y. Articles by Chang, C. L. Search for Related Content PubMed PubMed Citation Articles by Park, M. Y. Articles by Chang, C. L.