Mutations Including IS 6110 Insertion in the Gene Encoding the MPB64 Protein of Capilia TB-Negative Mycobacterium tuberculosis Isolates

Abstract

A simple immunochromatographic assay, Capilia TB, using anti-MPB64 monoclonal antibodies, is a kit for discriminating between the Mycobacterium tuberculosis complex and mycobacteria other than tubercle bacilli. The sensitivity of the kit was estimated to be 99.2% (381 of 384 samples). The sequencing analysis revealed that all of the Capilia TB-negative isolates had mutations within the mpb64 gene, leading to the production of an incomplete protein as a result of a deletion of the C-terminal region of the protein.Mycobacterium tuberculosis has been known to secrete more than 33 different proteins (3). One of the predominant proteins is MPB64, a 24-kDa protein initially isolated from culture filtrates of Mycobacterium bovis BCG Tokyo (5,11,20). The MPB64 antigen was found in the culture fluid of only strains of the M. tuberculosis complex and some substrains of M. bovis BCG (1, 5, 10, 11) and has shown potential for diagnostic use [1, 12, 13; T. Tomiyama, K. Matsuo, and C. Abe, abstract, Int. J. Tuberc. Lung Dis. 1(Suppl. 1): S59, 1997]. Recently, a simple immunochromatographic assay, Capilia TB (TAUNS, Numazu, Japan), using anti-MPB64 monoclonal antibodies for rapid discrimination between the M. tuberculosis complex and mycobacteria other than tubercle bacilli (MOTT), was developed [1; Tomiyama et al., Int. J. Tuberc. Lung Dis. 1(Suppl. 1): S59, 1997]. The test strip consists of a sample pad, a reagent pad, a nitrocellulose membrane, and an absorbent pad. Because the test sample applied in the sample well flows laterally through the membrane, the antibody-colloidal gold conjugate binds to the MPB64 antigen in the sample. The complex then flows further and binds to the monoclonal antibodies on the solid phase in the test zone, producing a red-purple band within 15 min. Some evaluation studies have demonstrated that the Capilia TB is very useful for rapid confirmation of the M. tuberculosis complex in liquid cultures without any troublesome sample preparation in a laboratory [1, 2, 6, 8; Tomiyama et al., Int. J. Tuberc. Lung Dis. 1(Suppl. 1):S59, 1997]. The kit is commercially available in Japan (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) and is routinely used in most clinical laboratories in hospitals or commercial laboratory centers.More recently, Capilia TB-negative M. tuberculosis strains have been isolated in some districts of Japan; therefore, the sensitivity of the Capilia TB test was reexamined by using a large number of clinical specimens in this study. A total of 13,942 clinical specimens, mostly sputum, were used for the study. Patients were admitted to Fukujuji Hospital (Japan Anti-Tuberculosis Association, Kiyose-shi, Tokyo) with symptoms of pulmonary tuberculosis from September 2001 to October 2002. Of these, 784 (5.6%) were culture positive for Mycobacteria. All mycobacterium-positive cultures were differentiated and identified by the Capilia TB, and the results were compared with those of the Accuprobe-M. tuberculosis complex culture confirmation test (Gen-Probe, San Diego, Calif.) and the DN...

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

“…Several cases of MTB have been reported with negative test results. A possible explanation for the false negative results is that the strain had mutations within the mpt64 gene, which may have led to the production of an incomplete protein (Hirano et al 2004). In this case, conventional techniques for the diagnosis of MTB should be recommended if clinical symptoms and microbiological criteria are suggestive for TB.…”

Detection of Mycobacterium Tuberculosis among Infertile Patients Suspected with Female Genital Tuberculosis

“…MPB64 gene could be appropriate or useful in the detection of M. tuberculosis complex but our results revealed that the role of MPB64 gene is limited to the detection of M. Bovis among patients with FGTB. This may suggest mutations within the MPB64 gene, leading to the production of an incomplete protein as a result of deletion in the C-terminal region of the protein [57]. The results of this study inveterate that TRC4 element might be universally detected, especially among so called south Indian strain of M. tuberculosis [56].…”

Evaluation of the Rapid MGIT TBc Identification Test for Culture Confirmation of Mycobacterium tuberculosis Complex Strain Detection

“…These two isolates were confirmed as true positives using IS6110 real-time PCR and were found to contain a 63-bp deletion at position 196 of the mpb64 gene. Mutations (13,14), deletions (14,23), and IS6110 or CG insertions (13,14,21,23) in the coding region (22,29) of the mpb64 gene that result in false-negative test results have been reported previously.…”

“…Thus, several such tests are commercially available, including the Capilia TB assay (Tauns Laboratories, Inc., Numazu, Japan), the Tibilia rapid test (Hangzhou, China), the SD Bioline TB Ag MPT64 rapid test (Standard Diagnostics, South Korea), and the MGIT TBc ID test. Previous studies have demonstrated that these assays all had outstanding performance, exhibiting 92.4 to 100% sensitivity and 94 to 100% specificity in identifying the M. tuberculosis complex (Table 4) (13,14,21,23,24,27, and M. Warns et al, presented at the 30th Annual Congress of the European Society of Mycobacteriology, Porto, Portugal, July 2009). In this study, we found that the TBc ID test exhibited 98.8% sensitivity and 100% specificity compared to biochemical tests.…”