Abstract
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.
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.
TABLE 1.
List of bacterial and fungal strains
| Species (no. of strains) |
|---|
| Bacteria |
| Acinetobacter baumannii (4) |
| Aeromonas caviae (2) |
| Aeromonas hydrophila (3) |
| Aeromonas veronii (2) |
| Aeromonas veronii biovar sobria (1) |
| Alcaligenes faecalis (2) |
| Alcaligenes xylosoxidans (2) |
| Bacillus cereus (2) |
| Bordetella bronchiseptica (2) |
| Branhamella catarrhalis (2) |
| Brevundimonas vesicularis (2) |
| Chryseobacterium indologenes (2) |
| Chryseobacterium meningosepticum (2) |
| Citrobacter freundii (4) |
| Comamonas acidovorans (2) |
| Escherichia coli (2) |
| Enterobacter aerogenes (2) |
| Enterobacter agglomerans (2) |
| Enterobacter cloacae (2) |
| Enterobacter intermedius (2) |
| Enterococcus avium (2) |
| Enterococcus casseliflavus (2) |
| Enterococcus faecalis (2) |
| Enterococcus faecium (2) |
| Enterococcus gallinarum (2) |
| Enterococcus raffinosis (2) |
| Flavimonas oryzihabitans (2) |
| Flavobacterium indologenes (2) |
| Klebsiella ornithinolytica (2) |
| Klebsiella oxytoca (2) |
| Klebsiella ozaenae (2) |
| Klebsiella pneumoniae (2) |
| Kocuria rosea (1) |
| Kocuria varians (2) |
| Leclercia adecarboxylata (2) |
| Morganella morganii (2) |
| Myroides spp. (2) |
| Neisseria gonorrhoeae (2) |
| Ochrobactrum anthropi (2) |
| Pichia anomala (2) |
| Pichia ohmeri (2) |
| Plesiomonas shigelloides (2) |
| Proteus vulgaris (2) |
| Providencia rettgeri (3) |
| Providencia stuartii (2) |
| Pseudomonas aeruginosa (2) |
| Pseudomonas fluorescens (2) |
| Ralstonia pickettii (2) |
| Salmonella group D (1) |
| Salmonella paratyphi A (2) |
| Serratia marcescens (2) |
| Shigella sonnei (2) |
| Sphingobacterium spiritivorum (2) |
| Sphingomonas paucimobilis (2) |
| Staphylococcus aureus (4) |
| Staphylococcus epidermidis (2) |
| Staphylococcus haemolyticus (2) |
| Staphylococcus saprophyticus (2) |
| Staphylococcus, coagulase negative (2) |
| Stenotrophomonas maltophilia (2) |
| Streptococcus agalactiae (2) |
| Streptococcus dysgalactiae (2) |
| Streptococcus mitis (2) |
| Streptococcus pneumoniae (2) |
| Streptococcus pyogenes (2) |
| Streptococcus viridans group (2) |
| Subtotal (137) |
| Fungi |
| Candida albicans (3) |
| Candida dubliniensis (2) |
| Candida glabrata (2) |
| Candida krusei (1) |
| Candida pelliculosa (2) |
| Candida tropicalis (2) |
| Cryptococcus humicolus (2) |
| Cryptococcus neoformans (2) |
| Trichosporon asahii (2) |
| Trichosporon beigelii (2) |
| Subtotal (20) |
| Total (157) |
TABLE 2.
List of reference mycobacterial strains
| Mycobacterium species and strain |
|---|
| M. abscessus |
| ATCC 19977 |
| ATCC 23003 |
| M. acapulcensis KTCC9501 |
| M. agri KTCC9502 |
| M. asiaticum KTCC9503 |
| M. austroafricanum KTCC9504 |
| M. avium ATCC 25291 |
| M. branderi ATCC 51788 |
| M. celatum ATCC 51131 |
| M. chelonae KTCC9505 |
| M. diernhoferi KTCC9506 |
| M. flavescens |
| ATCC 14474 |
| ATCC 23008 |
| M. fortuitum |
| KTCC1122 |
| KTCC9510 |
| M. gastri ATCC 15754 |
| M. gilvum KTCC9512 |
| M. gordonae |
| KTCC3036 |
| KTCC9513 |
| M. interjectum ATCC 51457 |
| M. intermedium ATCC 51846 |
| M. intracellulare |
| KIT41105 |
| KTCC9514 |
| M. kansasii KTCC9515 |
| M. marinum |
| ATCC 11564 |
| ATCC 927 |
| M. malmoense ATCC 29571 |
| M. morikaense KTCC9516 |
| M. mucogenicum |
| KTCC19088 |
| ATCC 49650 |
| M. neoaurum ATCC 25795 |
| M. nonchromogenicum ATCC 19530 |
| M. peregrinum |
| KTCC9615 |
| ATCC 14467 |
| M. phlei KTCC2192 |
| M. porcinum KTCC9517 |
| M. pulveris KTCC9518 |
| M. scrofulaceum |
| KTCC9519 |
| ATCC 19981 |
| M. senegalense ATCC 35796 |
| M. shimoidei ATCC 27962 |
| M. simiae |
| ATCC 15080 |
| ATCC 25275 |
| M. smegmatis KTCC1057 |
| M. sphagni ATCC 33027 |
| M. szulgai KTCC9520 |
| M. terrae |
| KTCC9614 |
| ATCC 15755 |
| M. triviale |
| ATCC 23290 |
| ATCC 23292 |
| M. tuberculosis H37Rv |
| M. vaccae |
| KTCC19087 |
| ATCC 15483 |
| M. xenopi ATCC 19250 |
TABLE 3.
List of mycobacteria isolated from clinical specimens
| Medium (total no. of strains) | Species (no. of strains) |
|---|---|
| Solid (3% Ogawa medium) (200) | M. abscessus (5) |
| M. avium-intracellulare complex (21) | |
| M. fortuitum (2) | |
| M. kansasii (4) | |
| M. szulgai (9) | |
| M. tuberculosis complex (159) | |
| Liquid (MGIT) (70) | M. avium-intracellulare complex (7) |
| M. szulgai (1) | |
| Mycobacteria, unidentifiable (2) | |
| M. tuberculosis complex (60) |
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 × 105 CFU/ml) revealed 10% reaction intensity, and the band intensity gradually weakened with serial dilutions until the 1:1,024 suspension (6.8 × 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.
Acknowledgments
C. L. Chang participated in the development of the MPT64 ICA kit as a scientific consultant.
Footnotes
Published ahead of print on 3 December 2008.
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