| ObjectiveMPT64 is the secretory proteins of early main ingredients in Mycobacterium tuberculosis (MTB).MPT64 was studied as a early marker of MTB growth and further try to establish an accurate, rapid, simple and universal method of detecting MTB growth in this paper. And its performance was evaluated as a method to differentiate the Mycobacterium tuberculosis Complex from Mycobacteria.Methods1,Experimental study on the rapid detection of growth of Mycobacterium tuberculosis by detecting MPT64 in the culture filtrate 32 cases of 4 strains of MTB suspensions which included 7 dilutions of 10-fold dilution in 1mg/ml pure cultures for each strains, 32 cases of acid-fast stain smear positive samples and 64 cases of non-selective clinical samples were cultured in the BacT/ALERT 3D system. For the suspension samples,the detection of MPT64 in the culture filtrate of the next dilution suspension samples continued once every day when the MPT64 was positive in the certain dilution suspension samples until the positive results showed in the culture or 42 days continuous culture .And for acid-fast stain positive samples,the detection of MPT64 in the culture filtrate continued once every day until the positive results showed in the culture or 42 days continuous culture.For the non-selective clinical samples, the MPT64 detections were performanced once every week, and continued 6 weeks for the negative samples.2,Study on Identification of the Mycobacterium tuberculosis Complex by Detecting Secretory Protein MPT64For samples isolated from clinical patients, 0.1ml of either positive liquid culture medium applied in BacT/ALERT 3D or bacterial sodium suspension , 2h placed with 1 Mcfarland Standard in RT made from L?wenstein-Jensen solid medium cultured colonies, was detected for MPT64 using immunochromatographic assay test(ICA) kit with colloid gold marked anti-MPT64 monoclonal antibody . For reference strains, both positive liquid culture medium and bacterial sodium suspension were applied. Results were compared with that of species identification by the method of gas chromatography analysis of whole-cell fatty acid. Results1,(1)the dilution tests of 1mg/ml pure cultures of 4 strains of MTB showed that the minimum concentration was 10-7～10-6mg/ml for MPT64 and 10-6～10-5mg/ml for the BacT/ALERT 3D system. (2) 31 of 32 acid-fast stain smear positive cases displayed culture positive, including 29 strains of MTB and 3 strains of non-tuberculous mycobacteria(NTM),and culture positive time of 58 strains of MTB was 6～30 days (average 13.7 days). Mpt64 detection was positive in 28 cases of 29stains of MTB during 3～16 days (average 7.8 days) and was negative for 3 strains of NTM. (3) 13 strains were identified as MTB in 15 cases of culture positive samples for non-selective clinical samples, of which the culture positive time was 7～39 days (average 19.2 days). Mpt64 detection was positive in 13 cases of culture positive samples identified as MTB. But 2 of 47 culture negative samples, of which MTB-DNA were positive, showed MPT64 positive during 4 weeks. And MPT 64 tests of others of culture negative samples and 2 cases of NTM were all negative at the end of 6 weeks. (4)The sensitivity and specificity of the method taking MPT64 as a marker to indicate early MTB growth were 97.61% and 96.29% respectively when the BacT/ALERT 3D system culture method was regarded as reference method.2,Totally, 23 reference strains and 133 clinical isolated strains of mycobacteria were detected. (1) In 23 reference strains, M. tuberculosis, M. bovis and M. microti showed both positive result in two kind sample types and the others(including M. kansasii, M. intracellulare, M. avium, M. chelonae, M. abscessus, M. gordonae, M. fortuitum, M. marinum, M. gilvum, M. gilvum, M. aichiense, M. smegmatis, M. parafortuitum, M. terrae, M. nonchromogenicum, M. vaccae, M. phlei, M. scrofulaceum, M. gastri, M. triviale and M. xenopi)presented negative result. The correction rate is 100 %. (2) Among 56 clinical isolated strains from BacT/ALERT 3D, 27 were M. tuberculosis and 25 of them showed MPT64 positive. In the rest 29 non-tuberculosis mycobacterium (NTM) samples, 27 samples showed MPT64 negative. The accuracy of identification was 92.85%. 29 NTM samples were M. gordonae (9 strains), M. chelonae (6 strains), M. abscessus (5 strains), M. avium-intracellulare complex (5 strains), M. fortuitum (1 strains), M. nonchromogenicum (1 strains), M. scrofulaceum (1 strains) and M. kansasii (1 strain).The two false positive results came from 2 strains of M. gordonae. (3) Among 77 clinical isolated strains from L?wenstein-Jensen solid medium, 58 were M. tuberculosis and 55 of them showed MPT64 positive. In the rest 19 NTM samples, all samples showed MPT64 negative. The accuracy of identification was 96.11%. 19 NTM samples were M. avium-intracellulare complex (8 strains), M. chelonae (3 strains),M. gordonae (3 strains), M. abscessus (2 strains), M. fortuitum (1 strains), M. scrofulaceum (1 strains) and M. nonchromogenicum (1 strains) . (4) For all samples, it's sensitivity, specificity and accuracy were 94.31%,97.06%和95.51% respectively. Conclusion1,It is an accurate, rapid, simple and universal method of detecting MTB growth to use MPT64 as a marker to indicate its early growth. But how to determine the optimal time check point remains to be further studied.2,Identifying the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 was a accurate, fast and simple method. Furthermore, it could be used in clinic widely because of no special instrument. |
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