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1.
Lee HR Kim SY Chang HE Song SH Lee HS Park KU Song J Kim EC 《Journal of clinical microbiology》2010,48(12):4612-4614
We developed a novel multiplex PCR assay using dual-priming oligonucleotide primers targeting the RD1 gene for simultaneous identification of the Mycobacterium tuberculosis complex and M. bovis bacillus Calmette-Guérin (BCG). This assay would be useful both for detection of the M. tuberculosis complex and for differentiation of M. bovis BCG from pathogenic M. tuberculosis complex species. 相似文献
2.
Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle
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Mustafa AS Skeiky YA Al-Attiyah R Alderson MR Hewinson RG Vordermeier HM 《Infection and immunity》2006,74(8):4566-4572
The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine. 相似文献
3.
Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids. 总被引:1,自引:3,他引:1
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M M Floyd V A Silcox W D Jones Jr W R Butler J O Kilburn 《Journal of clinical microbiology》1992,30(5):1327-1330
Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis. 相似文献
4.
5.
Suheir Ereqat Gila Kahila Bar-Gal Abedelmajeed Nasereddin Kifaya Azmi Sharif E. Qaddomi Charles L. Greenblatt Mark Spigelman Ziad Abdeen 《Journal of clinical microbiology》2010,48(11):4269-4272
Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly identified the 7 tested Mycobacterium reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.The Mycobacterium tuberculosis complex (MTC) includes Mycobacterium tuberculosis and Mycobacterium africanum, both of which are considered human pathogens, and Mycobacterium microti and Mycobacterium bovis, which are usually associated with animal infections. Although M. tuberculosis is the main cause of human tuberculosis (TB), it has been estimated that M. bovis is responsible for 10 to 15% of new human TB cases in the developing countries (7). The attenuated tuberculosis vaccine strain M. bovis bacillus Calmette-Guérin (BCG) can also cause human TB, especially in patients diagnosed with cellular immunodeficiency (15) or among neonates and children in regions of endemicity who have been vaccinated (5, 7). In 2003, a Palestinian study described an outbreak of BCG complications in the Gaza strip of the Palestinian territories. It affected 225 infants (average age, 4 months), with a complication rate of 36.61 per 1,000 vaccinations (2). Thus, differentiation and identification of M. tuberculosis from other members of the MTC should improve the clinical and therapeutic management of TB. Moreover, differentiation of Mycobacterium species contributes to the understanding of TB epidemiology. Several molecular methods have been reported for genotyping these two pathogens, i.e., multiplex PCR, PCR restriction analysis, allele-specific PCR, and real-time PCR using fluorescent resonance energy transfer (FRET) probes (3, 8, 10, 11, 13). However, these methods are time-consuming, expensive, and complicated. Comparative genome analysis has shown that M. bovis has numerous single-nucleotide polymorphisms (SNPs) in comparison to M. tuberculosis (4). Of these SNPs, the C-to-T transition at position −215 upstream of the GTG start codon in the promoter region of the narGHJI genes was hypothesized to be responsible for the differing nitrate reductase activities between M. tuberculosis and M. bovis (14). Another polymorphic nucleotide was identified in the oxyR gene (11, 12) and was found to be specific for M. bovis. In this study, we describe the use of high-resolution melt curve analysis (HRM) to differentiate between M. bovis and M. tuberculosis by two stepwise reactions. The first reaction is based on the T-to-C transition at position −215 in the promoter region of the narGHJI genes which differentiates between M. tuberculosis and all other members of the MTC (M. africanum, M. microti, and M. bovis) (11). The second reaction is based on the A-to-G polymorphism in the oxyR gene which is specific to M. bovis and which therefore differentiates between M. bovis and M. africanum or M. microti. Such definitive differentiation is essential, even if the organisms are confined to different restricted geographical areas and to different host species. In addition, all M. bovis strains were further identified by targeting the region of differences 1 (RD1; a 9,650-bp deletion which appears as a specific marker for M. bovis BCG) using the HRM assay. The present study was aimed at developing a rapid assay for differentiation between M. bovis and M. tuberculosis with minimal requirements of cost and time.(This study was submitted by S. Ereqat in partial fulfillment of the requirements for a Ph.D. at Hebrew University under the supervision of Gila Kahila Bar-Gal.)The study included 52 samples: 7 were previously identified as M. tuberculosis (6) and kindly provided by the Austrian Agency for Health and Food Safety, Vienna, Austria; 15 clinical isolates were recovered from sputum samples at the central laboratories of the Palestinian Ministry of Health; and 30 DNA samples were extracted from Ziehl-Neelsen-stained sputum smears (during the years 2005 to 2009). All archival samples and isolates had been previously identified by IS6110-based PCR to the complex level. Purified DNA from the reference strains M. tuberculosis (H37Rv), M. bovis, and M. bovis BCG and from nontuberculosis mycobacteria (NTM) (M. phlei, M. avium, M. intracellulare, and M. kansasii) were generously provided by the Hebrew University-Hadassah Medical School, Jerusalem, Israel.Ten samples from TB-negative sputum smears (confirmed negative by IS6110- based PCR) were included as negative controls in the study.The Ziehl-Neelsen-stained material was scraped off the microscopic slides after the addition of 200 μl of tissue lysis buffer and then processed by proteinase K digestion, followed by extraction according to manufacturer''s instructions (High-Pure PCR template preparation; High-Pure, Mannheim, Germany).A two-reaction approach for differentiating between M. bovis and M. tuberculosis was carried out using two real-time PCR assays followed by HRM analysis. Assay 1 consisted of the identification of the Mycobacterium species based on one SNP, a T-to-C transition, within the narGHJI promoter. Assay 2 consisted of the identification of the Mycobacterium species based on one SNP, an A-to-G transition, of the oxyR gene. The primers used in the two assays were designed to amplify short fragments covering these transitions in both targeted genes. Primer selection was facilitated by Primer3 (http://frodo.wi.mit.edu/). A homology search in GenBank revealed 100% specificity of the primers used for the MTC strains. The primer sequences are shown in Table Table11.
Open in a separate windowaThe product melting temperatures (Tm) are given as means ± standard errors.Each reaction mixture contained 10 μl of 2× Thermo-Start PCR master mix (Thermo Scientific) and 1.5 μM SYTO 9 (Invitrogen); primer mixes were used in final concentrations of 250 μM. DNA from the clinical isolates and controls was added in 2-μl volumes in total reaction mixtures of 20 μl. The amplification reaction for both amplicons (108 and 151 bp) was done as follows: the temperature was held at 95°C for 15 min for the hot start reaction and then 40 cycles of 5 s of denaturation, 10 s of annealing, and an extension at 55°C with a temperature increase of 0.2°C done were for each step. The melt domain was between 75 and 95°C. Reactions were carried out using the Rotor-Gene 6000 real-time thermal analyzer (Corbett Life Science).To obtain sufficient DNA for HRM analysis using 14 DNA extracts from Ziehl-Neelsen-stained sputum smears with a low bacterial load, fragments of 200 and 155 bp from the targeted genes were amplified using the previously published primers (13) (Table (Table1)1) and then 0.5-μl volumes from the PCR products were subjected to the real-time PCR HRM analysis as described above. In all amplification reactions, we used the negative-control samples to rule out the possibility of PCR contamination. Moreover, each PCR assay included DNA from the reference strains M. tuberculosis (H37Rv), M. bovis, and M. bovis BCG as positive controls to validate the characterization of the tested samples. In addition to the reference strains, seven clinical isolates identified as M. tuberculosis were added to the PCR assays for confirmation. All reactions were performed in duplicate.The first step was to optimize the assay, which was carried out on the seven reference samples. The results of the real-time PCR HRM assay based on the narGHJI and oxyR SNPs distinguished between M. tuberculosis isolates and M. bovis, as shown in the normalized melt curves (Fig. 1A and B). The melting temperatures of the PCR product for M. tuberculosis were significantly different from those for M. bovis (P < 0.05) (Table (Table1).1). Confirmation of the amplicon size was carried out by agarose gel electrophoresis (data not shown).Open in a separate windowFIG. 1.Characterization of M. tuberculosis and M. bovis based on real-time PCR with high-resolution melt curve analysis identified M. tuberculosis based on the T-to-C transition in the narGHJI genes (A), M. bovis based on the A-to-G transition in the oxyR gene (B), and M. bovis BCG based on the RD1 region (C). For each HRM graph, the x axis shows the temperature in degrees (°C) and the y axis represents the fluorescence signals.The analytical sensitivity of the HRM assay was clarified by performing 10-fold dilutions using 2 ng of purified DNA for both strains (M. bovis and M. tuberculosis). The melting curve was shown as the DNA level reached 2 pg/reaction. The two reactions showed similar sensitivities. HRM specificity was demonstrated by the absence of melting curves with NTM DNA samples in both reactions.The second step was to use the optimized assay on the 15 clinical isolates. Fourteen isolates were identified as M. tuberculosis and one isolate as M. bovis using the narGHJI and oxyR SNP assays. The M. bovis isolate was further identified as M. bovis BCG by targeting the RD1 region (13) and applying the HRM assay (Fig. (Fig.1C).1C). This isolate was obtained from a 2-year-old female child. The results for the 30 DNA samples extracted from Ziehl-Neelsen-stained sputum smears with unknown Mycobacterium strain identification indicated that 29 samples were M. tuberculosis (their normalized melting curve were identical to those of control strains) and one sample (3%) was M. bovis, which was also obtained from a 3-year-old female child. It is important to note that among the M. tuberculosis strains identified, one was obtained from a nonlocal resident originating from Indonesia, where TB is endemic (imported case), and who was undergoing treatment at the time of sampling (smear positive and culture negative).The real-time PCR HRM results were confirmed by direct sequencing. The two M. bovis sequences obtained in this study were found to be identical to each other and to the published sequences of M. bovis and M. bovis BCG (GenBank accession no. and BX248342.1). The sequences of all samples positive for M. tuberculosis (isolates and reference strains) were also found to be 100% identical to each other and to the published M. tuberculosis sequence (GenBank accession no. AP010918.1). Representative DNA sequences (oxyR gene and narGHJI promoter) from patients were deposited in GenBank. The multiple alignment of these DNA sequences is shown in (Fig. BX842579.12A and B).Open in a separate windowFIG. 2.(A) Multiple alignment of DNA sequences for the oxyR gene. 1 to 3, sequences belonging to M. bovis (Mb); 4 to 6, sequences belonging to M. tuberculosis (Mt). 1, M. bovis; 2, M. bovis BCG; 3, CaseTBA isolate; 4, CaseASA isolate (accession no. ); 5, M. tuberculosis (H37Rv); 6, CaseSAA isolate (accession no. HM135443); 7, CaseNHI isolate (accession no. HM135444). (B) Multiple alignment of DNA sequences for the narGHJI genes. 1 and 2, sequences belonging to M. tuberculosis (Mt); 3 and 4, sequences belonging to M. bovis (Mb). 1, M. tuberculosis H37Rv; 2: CaseSAA isolate; 3, CaseASA isolate (accession no. HM135445); 4, M. bovis BCG.The identification of M. bovis among the clinical cases studied is very important, especially for health management. Given that all M. bovis strains are naturally resistant to pyrazinamide (PZA), a first-line antituberculosis agent ( HM1354421, 7, 9), and that all patients newly diagnosed with TB are placed on a four-drug regimen that includes PZA, definitive differentiation between M. tuberculosis and M. bovis allows for appropriate treatment and reduces the emergence of drug-resistant strains.The results of this study support the use of a real-time PCR HRM assay on DNA extracted directly from positive Ziehl-Neelsen-stained sputum smear slides. The enrichment of the DNA by PCR prior to the real-time PCR HRM assay was found to be crucial for samples with a low bacterial load. Establishment of this method is of great interest, especially in patients with negative cultures or when the patient is on antitubercular treatment. Determination of the exact strain of Mycobacterium prior to confirmation by culture, which can take up to 3 weeks or more, will improve the treatment by applying the most appropriate drugs. In addition, the possibility of cross-reactivity of the designed primers with other NTM strains was excluded, as none of the tested NTM strains showed any melting curve. The real-time PCR HRM assay developed in this study is less complicated than the assay developed by Stermann and colleagues (13) as it does not require flourescence-labeled probes or combined primers and probes. The simplicity of the assay and its low cost make it applicable for diagnostic laboratories. An advantage of performing HRM analysis on a real-time PCR machine with HRM capability is that the PCR amplification and HRM analysis are performed in the same run and the results are available for assessment of amplification of all samples earlier than if HRM analysis done solely as a quality control measure. Therefore, we recommend this method for the rapid differentiation of M. tuberculosis from M. bovis. This differentiation will aid in choosing an efficient and appropriate treatment for TB patients, will reduce the transmission of the disease, and may prevent further outbreaks. 相似文献
TABLE 1.
Primers used in this studyPCR | Reaction | Gene name | Primer | Primer sequence | Product size (bp) | Reference | Product (Tm [°C])a |
---|---|---|---|---|---|---|---|
Preliminary | 1 | narGHJI | LC66 | 5′AACCGACGGTGTGGTTGAC′3 | 155 | Stermann et al. (13) | |
LC67 | 5′ATCTCGATGGATGGGCGTC′3 | ||||||
2 | oxyR | LC90 | 5′CGGGTGCCGCTGACCGCG′3 | 200 | Stermann et al. (13) | ||
LC91 | 5′CCAGCCGGCTTCGCGTGG′3 | ||||||
Real-time | 1 | narGHJI | narF | 5′CGCCGTCAACTTGGTTAGA′3 | M. tuberculosis (84.26 ± 0.09) | ||
narR | 5′GTCCTGCCCGGAAGTTGT′3 | 108 | M. bovis (85.04 ± 0.02) | ||||
2 | oxyR | oxyF | 5′ACACTGATTCCGCAGACC′3 | M. bovis (91.9 ± 0.03) | |||
oxyR | 5′AAAGTCAGCTCTGACAGCGC′3 | 151 | M. bovis (91.27 ± 0.04) |
6.
Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography
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Mohamed AM Bastola DR Morlock GP Cooksey RC Hinrichs SH 《Journal of clinical microbiology》2004,42(3):1016-1023
The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control. 相似文献
7.
Knowledge of mycobacterial glycolipid antigens and the study of their specificity have resulted in their utilization as species markers. We describe a thin-layer chromatography method which could serve as a useful adjunct for the identification of Mycobacterium tuberculosis, M. bovis BCG, M. kansasii, M. gastri and M. marinum. 相似文献
8.
Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle
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Coetsier C Vannuffel P Blondeel N Denef JF Cocito C Gala JL 《Journal of clinical microbiology》2000,38(8):3048-3054
We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex. 相似文献
9.
Identification and characterization of genomic variations between Mycobacterium bovis and M. tuberculosis H37Rv
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Amadio A Romano MI Bigi F Etchechoury I Kubica T Niemann S Cataldi A Caimi K 《Journal of clinical microbiology》2005,43(5):2481-2484
Genetic differences between Mycobacterium bovis and M. tuberculosis were identified. We found (i) a deletion of Rv3479 specific to M. bovis, (ii) that the rpfA gene is shortened to various extents in M. bovis, and (iii) an insertion in Rv0648 and a duplication of lppA common in M. tuberculosis complex isolates. 相似文献
10.
Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. 总被引:35,自引:4,他引:35
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A H Kolk M L Ho P R Klatser T A Eggelte S Kuijper S de Jonge J van Leeuwen 《Clinical and experimental immunology》1984,58(3):511-521
Thirty-two monoclonal antibodies (MoAb) to Mycobacterium tuberculosis H37Rv, M. bovis BCG and M. leprae were produced. The spleen cells of BALB/c mice immunized with sonicated or intact bacilli were fused with Sp2/0-Ag-14 myeloma cells. Many more antibody producing hybridomas were found when M. tuberculosis, rather than M. leprae, was used as the immunogen. The MoAb were characterized by an enzyme immunoassay and immunofluorescence on 16 mycobacterial species. The sodium dodecylsulphate polyacrylamide gel electrophoresis immunoperoxidase assay was used to determine the molecular weight of the antigens detected by the MoAb. Antigens of high, low and intermediate molecular weight were found. Some of the antigens were proteinaceous, others of a glycolipid nature. The immunofluorescence assay proved to be essential for the selection of MoAb since some MoAb reacted only in this assay and not in the enzyme immunoassay. The most specific clones were found in the fusions with spleen cells of mice immunized with intact rather than sonicated bacteria. One MoAb (F29-29) reacted only with M. tuberculosis H37Rv; one (F41-3) only with M. leprae and another (F29-45) reacted with M. tuberculosis and M. gastrii. Several MoAb only reacted with three mycobacterial species: M. tuberculosis, M. kansasii and M. gastrii. Others showed unique patterns of reactivity by enzyme immuno- and immunofluorescence assay. The potential use of the MoAb for the identification of mycobacteria and mycobacterial antigens is discussed. 相似文献
11.
Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. 总被引:15,自引:1,他引:15
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ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51. 相似文献
12.
Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis 总被引:2,自引:0,他引:2
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Bodo R. Eing Andrea Becker Arthur Sohns Ronald Ringelmann 《Journal of clinical microbiology》1998,36(7):2023-2029
The new Roche Cobas Amplicor Mycobacterium tuberculosis assay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas Amplicor M. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas Amplicor M. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested. 相似文献
13.
Use of the hupB gene encoding a histone-like protein of Mycobacterium tuberculosis as a target for detection and differentiation of M. tuberculosis and M. bovis
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Prabhakar S Mishra A Singhal A Katoch VM Thakral SS Tyagi JS Prasad HK 《Journal of clinical microbiology》2004,42(6):2724-2732
The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis. 相似文献
14.
PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis kit. MATERIALS AND METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor kit. CONCLUSION: In-house PCR and Cobas Amplicor kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis. 相似文献
15.
Huys G Rigouts L Chemlal K Portaels F Swings J 《Journal of clinical microbiology》2000,38(10):3675-3680
The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrimination of Mycobacterium bovis (17 strains), M. tuberculosis (15 strains), and M. ulcerans (12 strains) at the inter- and intraspecific level. The AFLP technique is a whole-genome coverage genotypic fingerprinting method based on the selective PCR amplification of modified restriction fragments obtained through a double enzymatic digest and subsequent ligation of double-stranded restriction site-specific adapter oligonucleotides. Selective amplification of ApaI/TaqI templates with primer combination A02-T02 (both having an additional C at their 3' end) generated autoradiographic AFLP fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing clear separation of M. ulcerans (cluster I) from the M. tuberculosis complex members M. bovis and M. tuberculosis (cluster II). Calculation of similarities using the band-based Dice correlation coefficient instead of the Pearson product-moment correlation coefficient revealed a further subgrouping in cluster II. The two resulting subclusters corresponded with the phenotypic identity of M. bovis and M. tuberculosis, respectively, and could also be visually identified by two AFLP marker bands. Because of the relatively low degree of genotypic variation among the AFLP band patterns of the latter two taxa, no correlation could be found with previously reported molecular typing data or with geographical origin. The use of primer combination A02-T01 (the latter having an A as selective base) did not increase the resolving power within the M. tuberculosis complex but resulted in a visual subgrouping of the M. ulcerans strains that was not observed with primer combination A02-T02. Based on the presence or absence of a single AFLP marker band, the M. ulcerans isolates could be unambiguously classified in two continental types corresponding with the African and Australian origin of the strains, respectively. In conclusion, the radioactive AFLP method proved to be a reproducible and reliable taxonomic tool for the differentiation of the three mycobacterial species under study and also demonstrated its potential use for typing of M. ulcerans strains when employing multiple primer combinations. 相似文献
16.
Clinical evaluation of the Roche AMPLICOR PCR Mycobacterium tuberculosis test for detection of M. tuberculosis in respiratory specimens. 总被引:1,自引:3,他引:1
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The reliability of the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB) for the diagnosis of pulmonary tuberculosis was evaluated by testing 956 respiratory specimens from 502 patients and comparing results with results by culture and medical history. Of those 135 specimens that were culture positive for mycobacteria, 61 specimens from 31 patients grew M. tuberculosis. Fifty-two specimens were smear positive for acid-fast bacteria (AFB); M. tuberculosis was isolated from 41 of these specimens. On initial testing, the sensitivity and specificity of the AMPLICOR MTB assay, compared with culture, were 78.7 and 99.3%, respectively. After resolution of discrepancies (by review of medical history), the sensitivity, specificity, and positive and negative predictive values of the AMPLICOR MTB assay were 79.4, 99.6, 92.6, and 98.6%, respectively. Two specimens from two patients with no clinical evidence of tuberculosis were AMPLICOR MTB positive and culture positive for Mycobacterium avium complex. For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 97.6, 100, 100, and 90.9%, respectively. For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 40.0, 99.5, 69.2, and 98.7%, respectively. Our results support the use of AMPLICOR MTB for rapid diagnosis of tuberculosis in patients whose respiratory specimens are AFB smear positive. Further studies are needed to determine the most clinically relevant and cost-effective use of this assay with AFB smear-negative specimens. 相似文献
17.
Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M. bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin 总被引:1,自引:0,他引:1
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Chambers MA Williams A Gavier-Widén D Whelan A Hall G Marsh PD Bloom BR Jacobs WR Hewinson RG 《Infection and immunity》2000,68(12):7094-7099
Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals. 相似文献
18.
Hong-Bin Xu Rui-Hai Jiang Wei Sha Ling Li He-Ping Xiao 《Journal of clinical microbiology》2010,48(10):3635-3640
The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are very slow to give results; and due to the emergence of multidrug-resistant M. tuberculosis and extensively drug-resistant M. tuberculosis, there is an urgent demand for new, rapid, and accurate drug susceptibility testing methods. PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been proposed as a rapid method for the detection of resistance to rifampin, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of PCR-SSCP analysis for the detection of rifampin-resistant tuberculosis. We searched the Medline, Embase, Web of Science, BIOSIS, and LILACS databases and contacted authors if additional information was required. Ten studies met our inclusion criteria for rifampin resistance detection. We applied the summary receiver operating characteristic (SROC) curve to perform the meta-analysis and to summarize diagnostic accuracy. The sensitivity of PCR-SSCP analysis for the rapid detection of rifampin-resistant tuberculosis was 0.79 (95% confidence interval [CI], 0.75 to 0.82), the specificity was 0.96 (95% CI, 0.94 to 0.98), the positive likelihood ratio was 16.10 (95% CI, 5.87 to 44.13), the negative likelihood ratio was 0.20 (95% CI, 0.10 to 0.40), and the diagnostic odds ratio was 100.93 (95% CI, 31.95 to 318.83). PCR-SSCP analysis is a sensitive and specific test for the rapid detection of rifampin-resistant M. tuberculosis. Additional studies in countries with a high prevalence of multidrug-resistant M. tuberculosis and also cost-effectiveness analysis are required in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in M. tuberculosis.Tuberculosis (TB) is a major global health problem. Early detection of drug resistance in patients with tuberculosis allows the use of appropriate treatment regimens for the patient, which has an important impact for the better control of the disease. The development of rapid methods for drug susceptibility testing is very important due to the increasing rates of multidrug-resistant tuberculosis (MDR-TB) worldwide and the recently described extensively drug-resistant tuberculosis (XDR-TB). The World Health Organization (WHO) urgently called for expanded access to culture and drug susceptibility testing in response to the spread of MDR-TB and XDR-TB, declared serious emerging threats to public health (28). This poses significant challenges for TB laboratory capacity and the need for faster drug susceptibility testing methods.Conventional culture methods using egg- or agar-based media are still the most commonly used approaches in many countries. To test for drug resistance, the standard methods using Lowenstein-Jensen (LJ) medium include the proportion method, the absolute concentration method, and the resistant ratio method, which are well standardized with clinical isolates, at least for the major antituberculosis drugs. The proportion method was developed in the 1960s and is still the “gold standard” method used in many laboratories, especially in developing countries, because it is an inexpensive method easily accessible in these settings (1, 2). In recent years, due to the long turnaround times of conventional drug susceptibility testing methods, several new approaches have been proposed for the faster detection of MDR Mycobacterium tuberculosis, including both genotypic and phenotypic methods (23, 24). Among the genotypic methods proposed, PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been an important tool for detecting point mutations underlying genetic diseases since it was established in 1989 (9, 21). Under nondenaturing conditions, single-stranded DNA (ssDNA) has a folded structure that is determined by intramolecular interactions and, therefore, by its sequence. Even a single point mutation can alter the conformation of ssDNA, so that the altered conformation affects the migration of ssDNA, which can be detected as abnormal bands on a nondenaturing gel. Thus, SSCP analysis has a high resolving power to distinguish most conformational changes caused by subtle sequence differences even in a several-hundred-base fragment (9). The PCR-SSCP assay is relatively simple and was initially promising. Recently, the PCR-SSCP assay has been used in some studies for the rapid detection of rifampin-resistant M. tuberculosis (3, 11, 12, 17). However, individually, these trials found that the sensitivity and specificity were statistically inconsistent. The purpose of the present study was to perform a systematic review and meta-analysis to synthesize all available literature on the PCR-SSCP assay for the rapid detection of rifampin-resistant M. tuberculosis and to evaluate the overall accuracy of this method for the detection of rifampin resistance in isolates and in sputum samples. 相似文献
19.
Radiometric selective inhibition tests for differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, and other mycobacteria. 总被引:2,自引:3,他引:2
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In the context of a busy reference laboratory, radiometric selective inhibition tests were evaluated for rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis and of the M. tuberculosis complex from other mycobacteria. p-Nitro-alpha-acetylamino-beta-hydroxypropiophenone at 5 micrograms and hydroxylamine hydrochloride at 62.5 and 125 micrograms per ml of 7H12 medium were used to separate the M. tuberculosis complex from other mycobacteria (MOTT bacilli). Since it is important epidemiologically to distinguish M. tuberculosis from M. bovis, susceptibility to 1 microgram of thiophene-2-carboxylic acid per ml was also determined radiometrically. By using these three agents as selective inhibitors, M. tuberculosis, M. bovis, and MOTT bacilli were differentiated with a high degree of specificity by a BACTEC radiometric procedure. Results of tests performed on clinical isolates submitted on solid medium to our reference laboratory were available within 5 days. 相似文献
20.
Differentiation among members of the Mycobacterium tuberculosis complex by molecular and biochemical features: evidence for two pyrazinamide-susceptible subtypes of M. bovis
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The variations in biochemical as well as molecular characteristics among several members of the Mycobacterium tuberculosis complex that are not M. tuberculosis have been assessed to facilitate an unambiguous species identification. Altogether, 96 M. tuberculosis complex strains including 52 M. bovis isolates and 44 M. africanum isolates were analyzed by spoligotyping. The strains could be clustered into five spoligotype groups. All M. bovis isolates showed the typical absence of the spacers 39 to 43 and typical biochemical properties. However, within these strains we found a group of strains that had a spoligotype pattern which is clearly defined by the additional absence of spacers 3 to 16 and that were uncommonly susceptible to pyrazinamide (PZA). This spoligotype pattern has previously been described as being typical for a caprine genotype because of its predominant isolation from sheep and goats. Due to the clinical importance of PZA resistance, we propose two M. bovis subtypes: M. bovis subtype bovis, which is resistant to PZA, and M. bovis subtype caprae, which is susceptible to PZA. Two additional strains that clustered in group 3 showed biochemical and genetic properties typical for M. bovis and were also sensitive to PZA; thus, they may represent a third PZA-susceptible M. bovis subtype. The M. africanum isolates could be clustered into two spoligotype groups which can be differentiated from M. bovis by hybridization to spacers 39 to 43. These groups correspond to the previously described M. africanum subtypes I and II and can be clearly distinguished from each other by spoligotyping and resistance to thiophen-2-carboxylic acid hydrazide. Our results demonstrate that spoligotyping is a useful tool for differentiation of M. bovis and M. africanum. Moreover, we describe two PZA-susceptible M. bovis subtypes and describe a method that facilitates an unambiguous differentiation of the two M. africanum subtypes. 相似文献