[Application of recombinant 38000 protein antigen of Mycobacterium tuberculosis in screening Mycobacterium tuberculosis infection].

OBJECTIVE: To evaluate the potential of recombinant 38000 protein of Mycobacterium tuberculosis (38000 protein) as a tuberculosis-specific tuberculin for screening M. tuberculosis infection. METHODS: A total of 1342 subjects (706 men and 636 women, age 18-60 years) from several communities in Kazuo County and Xidaziying Town, Chaoyang, Liaoning Province, and Hongdong County, Linfen, Shanxi Province were enrolled from September 2004 to February 2005. The skin tests were performed with double-blinded and the intradermal injections were administered on both forearms with 0.1 ml solution of PPD and 38000 protein at the right side and the left side, respectively. The vertical and transverse diameters of induration or erythema were measured following 24 h for 38000 protein and 48 h for PPD, respectively. The diameters of the the skin test reactions were defined as the means of the vertical and transverse diameters, and positive skin reactions were identified when the diameter was greater than or equal to 5 mm. The comparison of the positive rate was performed via chi(2) test and the consistency of positive skin test reactions between 38000 protein and TB-PPD was analyzed through calculating Kappa coefficients. RESULTS: The positive rate was 55.1% (740/1342) and 28.6% (384/1342) for PPD and 38000 protein, respectively; the difference being significant (chi(2) = 190.6, P < 0.01). The consistency of positive skin test reactions between 38000 protein and PPD was low due to a negative Kappa coefficient. The positive rates induced by PPD and 38000 protein tended to increase with age except for the 33-37 year group. For a given age group, the positive rate of PPD was much higher than that of 38000 protein. The subjects without BCG scar had a lower positive rate for 38000 protein (24.3%, 137/566) than those with BCG scar (31.9%, 247/776) (chi(2) = 4.7, P < 0.05). The subjects with tuberculosis contact history had a higher positive rate for 38000 protein (74.4%, 32/43) than those without tuberculosis contact history (27.1%, 352/1299). Subjects without tuberculosis contact history had a lower positive rate for 38000 protein (27.1%, 352/1299) than that of PPD (54.0%, 702/1299), all of which showed significant difference (chi(2) test values changed from 4.7 to 192.1, P < 0.05 or P < 0.01). For those with a tuberculosis contact history, no significant difference in the positive rate was found between 38000 protein (74.4%, 32/43) and PPD (88.4%, 38/43) (chi(2) = 1.9, P > 0.05). Nor was significant difference found in the positive rate of 38000 protein between male subjects (28.5%, 201/706) and female subjects (28.8%, 183/636). The diameter of the positive reactions induced by 38000 protein and PPD ranged from 5 - 9 mm and from 5 - 14 mm, respectively. CONCLUSION: Our findings show that 38000 protein may be useful as a tuberculosis-specific skin test antigen for screening Mycobacterium tuberculosis infection.

[Identification of differential genomic genes of Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra by suppression subtractive hybridization].

To study the virulence-related genes in Mycobacterium tuberculosis, we used suppression subtractive hybridization to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra. All of 54 different genes were cloned, sequenced and analyzed by Southern-blotting. Two different DNA fragments in H37Ra are new genes so far, and get the new Genbank number AY534505 and AY560011. Eight different DNA fragments in H37Rv were obtained. One is the fragment of a gene coding virulence factor mce; one fragment belongs to the gene coding for purC synthenzyme; one for PE family protein; the other 4 fragments for putative gene; and the last one is a non-coding fragment. PCR analysis indicated that 2 of the different genes were present exclusively in the clinical virulent strain and in H37Rv, but not in the clinical avirulent strain and in H37Ra. The novel differential genes may provide an important clue for studying the mechanism of M. tuberculosis pathogenesis.

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra.

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.

[Application of multiplex polymerase chain reaction in rapid identification of Mycobacterium bovis BCG].

OBJECTIVE: To establish a method for the rapid identification of Mycobacterium bovis BCG. METHODS: A genomic region designated RD1 was found to be deleted from BCG strains, but present in other strains of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) including Mycobacterium tuberculosis, Mycobacterium africanum, and Mycobacterium microti. With this information, a multiplex PCR method, developed to detect the deletion of RD1, was used to differentiate BCG strains from other strains of Mycobacterium bovis and other members of MTC. RESULTS: RD1 was shown to be absent in 5 BCG vaccine strains and 2 BCG strains isolated from an infant who died of systemic disseminated infection induced by BCG vaccination, but it was present in 3 Mycobacterium bovis standard strains, 6 Mycobacterium bovis strains isolated from diseased cows, deer or patients with pulmonary tuberculosis and other MTC strains including Mycobacterium tuberculosis H(37)Rv and H(37)Ra strains, 48 Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis, and 3 Mycobacterium africanum standard strains. CONCLUSION: The multiplex PCR method is simple, rapid, and specific for the identification of BCG among strains of MTC, and is applicable in clinical laboratories.

Clinical evaluation of the recombinant 38 kDa protein of Mycobacterium tuberculosis.

The diagnosis of tuberculosis remains among public health concerns due to shortcomings of the purified protein derivative (PPD). Recombinant truncated 38 kDa protein (rTPA38) of Mycobacterium tuberculosis was evaluated to screen new tuberculosis-specific tuberculin. 539 patients, 1133 healthy controls, and 55 guinea pigs were recruited to assess their sensitivity and specificity to rTPA38; 221 healthy controls, with negative responses to rTPA38 and PPD, were vaccinated with M. bovis BCG to determine their cross-reactions with M. bovis BCG. The Mantoux technique was adopted to perform skin tests. No difference in the sensitivity of skin tests was detected between rTPA38 and PPD (78.2% vs 83.4%), but there was a significant difference in the specificity of skin tests between rTPA38 and PPD (75.2% vs 47.0%). Compared to PPD, rTPA38 elicited low positive responses for those recruitments vaccinated with M. bovis BCG. The rTPA38 had significant skin reactions in M. tuberculosis-sensitized guinea pigs, and the opposite was true for both M. fortuitum- and M. kansasii-sensitized guinea pigs. These findings indicate that rTPA38 may have potential as a tuberculosis-specific skin test antigen.