Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

BACKGROUND: Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72). CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.

[Prokaryotic expression, purification, and immunogenicity analysis of Mycobacterium tuberculosis specific excretive proteins].

OBJECTIVE: To obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity. METHODS: The Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot. RESULTS: After transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot. CONCLUSION: The prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.

[Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay].

OBJECTIVE: To establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility. METHOD: We used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method. RESULTS: The sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide. CONCLUSIONS: PhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.

[Relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis].

OBJECTIVE: To investigate the relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis. METHODS: Mycobacterium tuberculosis (dormant bacteria) was cultured for 100 days, then diluted into 1 mg/ml concentration with 7H9, and further diluted into 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml. Twelve new tubes added with 5 ml 7H9 and divided into two groups: the first group was added with the resuscitation-promoting factor protein, and the second group as control was added with 7H9. In each group the above diluted solutions were added. The tubes were located at 37 degrees C for culture. Optical density (OD) was detected on day 15, 25, 30, and 35. From each tube 1 microl culture solution was plated on 7H11 medium for colony counting. RESULTS: OD detection showed that bacteria proliferation in each group had positive linear correlation (P < 0.05, P < 0.01), indicating that the resuscitation-promoting factor played a similiar role in solutions with different dilution concentrations. 7H11 results and the OD results show that these two detection methods in each group had linear correlation (P < 0.05, P < 0.01), indicating that these two methods showed consistent test results. CONCLUSION: The resuscitation-promoting factor has no effect on the resuscitation of dormant Mycobacterium tuberculosis and its initial bacteria amount.

[Analysis of differentially expressed genes between resuscitating and active Mycobacterium tuberculosis].

OBJECTIVE: To screen key genes of dormant M. tuberculosis for resuscitation. METHODS: M. tuberculosis H37Rv strain cultured for 20 days in 7H9 liquid medium was used as active bacteria. Dormant bacteria were obtained by cultivating active bacteria hermetically at 37 degrees C using methylene blue as the indicator of oxygen free until the blue medium became colorless. Then resuscitation promoting factors were added to the culture and the bacteria were cultivated for 3 days to be resuscitated. RNA was extracted from active bacteria and resuscitating bacteria, disposed of DNA in RNA with DNase I , and mRNA was purified and then were hybridized using suppression subtractive hybridization (SSH) technique. Differentially expressed genes between resuscitating M. tuberculosis and active M. tuberculosis were identified by PCR, cloning, and sequence alignment. Identification of the differentially expressed genes was performed by real-time quantitative PCR. RESULTS: High or specifically expressed genes as tester had been obtained by SSH in correctitude reaction (active M. tuberculosis as tester) and reverse reaction (dormant M. tuberculosis as tester). These genes were cloned into plasmid PGEM-T Easy, and 78 positive bacteria in correctitude reaction and 46 positive bacteria in reverse reaction were obtained. The positive bacteria were amplified by PCR with T7 and M13 primer, and 66 positive bacteria ( >350 bp) in correctitude reaction and 39 positive bacteria ( > 350 bp) in reverse reaction were obtained. After sequencing, 30 positive sequences in correctitude reaction and 21 positive sequences in reverse reaction were obtained. Twenty and 7 high or specifically expressed genes were finally identified in active and resuscitating M. tuberculosis respectively by searching in Genbank. These genes were classified into 8 categories. Real-time quantitative PCR demonstrated that the quantity of 7 high or specifically expressed genes in resuscitating bacteria was more than 4 times that in active bacteria. CONCLUSION: Differentially expressed genes between resuscitating and active M. tuberculosis were identified using SSH technique and the results may help exploring key genes and mechanisms of dormant M. tuberculosis for resuscitation.

[Screening and analysis of in vivo induced genes of Mycobacterium tuberculosis].

OBJECTIVE: To screen in vivo induced genes of Mycobacterium tuberculosis and search possible molecular targets of new drugs, vaccines, and early diagnostic methods. METHODS: In vivo induced antigen technology (IVIAT) was used in this study. Genomic DNA from M. tuberculosis of the strain H37Rv was extracted. The DNA was partially digested with Sau3A I and the purified fragments were inserted into the pET30a (+), pET30b (+) and pET30c (+) expression vectors to construct a genomic library. The library was induced with IPTG and then was screened with pooled tuberculosis patient sera preabsorbed with in vitro grown M. tuberculosis of the strain H37Rv and Escherichia coli of the strain BL21 (DE3). The inserts of positive clones were sequenced with primer T7 promoter. The sequences were aligned in the genomic database of M. tuberculosis strain H37Rv (http://genolist.pasteur.fr/Tuberculose) to identify the open reading frame (ORF). RESULTS: The genomic expression library included 4.3 x 10(4) clones, and more than eighty percent were recombinant plasmids. The library reached the theoretic requirement. The successive adsorptions significantly decreased the anti-M. tuberculosis antibody titer of sera, and no significant difference was found between the last two adsorption groups, suggesting that the antibodies reactive against the M. tuberculosis H37Rv antigens expressed in vitro were removed. After screening of the genomic expression library and searching in the genome database, 51 ORFs were identified and they were classified into 8 categories according to the classification criterion on the website, including 1 virulence gene, 13 cell wall and cell processes genes, 11 intermediary metabolism and respiration genes, 7 lipid metabolism genes, 2 information pathways genes, 3 PE/PPE genes, 12 conserved hypotheticals, and 2 conserved hypotheticals with an orthologue in M. bovis. CONCLUSION: Genes expressed specially during human M. tuberculosis infections can be identified with in vivo induced antigen technology. Analysis of these genes identified using IVIAT shows that some genes are related to virulence, some are essential genes for M. tuberculosis, and some encoded proteins have strong immunogenicity, suggesting that some of them can be used as molecular targets of anti-tuberculosis drugs, vaccines, and tuberculosis early diagnosis.

[Mutations in the thymidylate synthase gene is a major mechanism in the para-aminosalicylic acid resistance of M. tuberculosis].

OBJECTIVE: The mechanisms of resistance to para-aminosalicylic acid (PAS) are undefined. In this study, we explored the mechanisms of M. tuberculosis PAS resistance in clinical isolates. METHOD: The whole sequence of thymidylate synthase (thyA) gene encoding thyA genes was sequenced in 51 para-aminosalicylic acid (PAS)-sensitive and 44 resistant M. tuberculosis clinical isolates. RESULTS: Sixteen of 44 resistant M. tuberculosis clinical isolates had mutations in the thyA genes, a mutation rate of 36.4% (16/44). No mutations were detected in the sensitive clinical isolates. The mutation types included substitutions, conversions and deletions. CONCLUSIONS: Mutations in the thyA gene is associated with PAS resistance in M. tuberculosis clinical isolates, and mutations in thyA gene probably represent a major mechanism of developing resistance to the drug. Thymidylate synthase is likely to be the target of PAS action.

[The in-vitro interferon-gamma release assay for the diagnosis of tuberculosis and Mycobacterium tuberculosis infections].

OBJECTIVE: To investigate the in-vitro interferon-gamma (IFN-gamma) release assay based on three different Mycobacterium tuberculosis antigens in the diagnosis of tuberculosis and Mycobacterium tuberculosis infections. METHODS: The peripheral blood mononuclear cells (PBMC) were collected from the patients with tuberculosis (tuberculosis group, n = 57), patients with lung cancer (lung cancer group, n = 29), and healthy controls (healthy control group 2, n = 27). The PBMCs were co-cultured for 5 days with different antigens: purified protein derivatives (PPD) of tuberculin, early secretary antigenic target 6,000 protein (ESAT6) and 38,000 antigen. The protein levels of IFN-gamma were detected by ELISA, and the results were compared to those with the tuberculin skin test (TST). RESULTS: (1) For healthy controls, the TST was positively related to the history of BCG vaccination and the closeness of contact with sputum-positive tuberculosis patients (P = 0.047, P = 0.041 respectively). The ESAT6 based IFN-gamma release assay was only significantly related to the closeness of contact with sputum-positive tuberculosis patients (P = 0.005), but not to the history of BCG vaccination. (2) There was no significant difference of the TST results among the three groups (P > 0.05). (3) The receiver operating characteristic (ROC) curve analysis indicated that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the IFN-gamma release assay based on 38,000 antigen were 64.9%, 89.3%, 77.0%, 86.0%, and 71.0% respectively for the diagnosis of tuberculosis. CONCLUSIONS: IFN-gamma release assay based on ESAT6 appears to be better than TST in the diagnosis of infection of Mycobacterium tuberculosis, while IFN-gamma release assay based on 38,000 may be helpful for the diagnosis of tuberculosis.

[Nitric oxide production and expression of cytokines by macrophages infected by M. tuberculosis H(37)R(v)].

OBJECTIVE: To study nitric oxide (NO) production and cytokine expression by macrophages infected by M. tuberculosis H(37)R(v), and to compare the difference between dead and live M. tuberculosis in the induction of immune responses, and thus to show if dead bacteria could be a possible candidate for new vaccines. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and ELISA were used to measure the production of NO and cytokines in macrophages infected by H(37)R(v). RESULTS: Macrophages infected by viable M. tuberculosis produced more NO, IL-1, IL-12, IL-18, TNF-alpha and inducible nitric oxide synthases (iNOS), as compared with macrophages infected by dead bacteria. The number of bacteria was also an important factor determining the production of NO and cytokines. CONCLUSIONS: Viable M. tuberculosis H(37)R(v) can induce the activation of macrophages and the production of more NO and cytokines which play important roles in the host immune response. Heat-killed M. tuberculosis H(37)R(v) failed to induce activation of macrophages and the production of NO and cytokines, which makes it unlikely to be a candidate for vaccine development.