Differentiation of BCG-induced lymphadenitis from tuberculosis in lymph node biopsy specimens by molecular analyses of pncA and oxyR

Without culture, differentiation of bacille Calmette-Guérin-induced lymphadenitis (BCG-LA) from tuberculosis (TB) is sometimes difficult by histology, but is important because of different treatment schemes. The purpose of this study was to investigate the feasibility of differentiating BCG-LA from TB in lymph nodes (LNs) by molecular analyses of two recently identified genes, pncA and oxyR. In both genes, a single nucleotide difference exists between Mycobacterium bovis and M. tuberculosis. M. tuberculosis complex (MTC) DNA was first detected in nine of ten formalin-fixed, paraffin-embedded LNs from patients aged under 20 years with suspected mycobacterial infections, using polymerase chain reaction (PCR) for IS6110, an insertion sequence specific for MTC species. PCR, together with direct DNA sequencing and PCR–restriction fragment length polymorphism (RFLP) assay, was then performed to identify polymorphic nucleotide in pncA and oxyR, respectively. For comparison, 37 adult cases of tuberculous lymphadenitis were also analysed by PCR–single strand conformation polymorphism (SSCP) assay for pncA and by PCR–RFLP for oxyR. The results revealed that five of the nine IS6110-positive child cases had a G residue at nucleotide 169 in pncA, and also had a three-band pattern after digesting the amplified oxyR segment with AluI, suggesting BCG-LA. The remaining four child cases, as well as all adult cases with detectable IS6110, showed no motility shift in pncA PCR–SSCP and had the same one-band pattern as M. tuberculosis in oxyR PCR–RFLP, suggesting TB lymphadenitis. The data from molecular analyses showed a good correlation with the vaccination history and clinicopathological findings, except for one case. This study indicates that molecular assay of either oxyR or pncA could be a rapid and useful tool to distinguish BCG-LA from TB. © 1998 John Wiley & Sons, Ltd.     

Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

BackgroundA rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes.ObjectivesA PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013–2015.MethodsEighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR.ResultsThe PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9–100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%).ConclusionsPCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use.     

Differentiation of clinical Mycobacterium tuberculosis complex isolates by their GyrB polymorphism

Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates. Materials and Methods: A primer pair MTUB-f and MTUB-r for M. tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the gyrB gene (gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragment specific for M. tuberculosis (gyrB-PCR2). The DNA polymorphisms in the 1,020-bp gyrB fragment for 7 M. tuberculosis strains confirmed by PCR as well as 2 reference strains; M. tuberculosis H37Rv and M. bovis BCG were analyzed with the restriction enzyme Rsa1. Results: Seventy-seven (97.5%) isolates were positive for gyrB-PCR1 and thus identified as members of M. tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the M. tuberculosis isolates showed the typical M. tuberculosis specific Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from M. bovis BCG. Conclusion: The gyrB PCR-RFLP using the endonuclease Rsa1 can be used to differentiate M. tuberculosis from M. bovis in clinical isolates.     

Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques

Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the hupB gene (Rv2986c in M.tuberculosis, or Mb3010c in M.bovis) to differentiate M. tuberculosis and M. bovis was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol. 42:2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (P < 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of M. tuberculosis and M. bovis in bovine samples. The N-PCR products of M. tuberculosis and M. bovis corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli. M. tuberculosis and/or M. bovis was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with M.tuberculosis or M. bovis by N-PCR and culture reflected the clinical categorizations of the cattle (P of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal.     

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.     

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.     

Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China

We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3′ end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.     

Rapid Detection of Mycobacterium tuberculosis and Pyrazinamide Susceptibility Related to pncA Mutations in Sputum Specimens through an Integrated Gene-to-Protein Function Approach

Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.     

Immune Responses in Cattle Inoculated with Mycobacterium bovis,Mycobacterium tuberculosis,or Mycobacterium kansasii

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti [8]). Despite their ∼99.95% sequence identity (12), M. bovis and M. tuberculosis exhibit distinct differences in virulence and host adaptation. Compared to M. tuberculosis, experimental M. bovis infection of mice or rabbits results in a more severe pathology and shorter mean survival times (9, 17, 18). Mycobacterium tuberculosis is primarily a human pathogen that demonstrates a high level of attenuation in cattle (as reviewed by Francis in 1947 [10]), whereas M. bovis has a wider host range and affects many domesticated and free-ranging mammals as well as humans. Prior to the initiation of control and eradication campaigns in the early to mid-1900s, M. bovis infection accounted for up to 30% of human tuberculosis cases, with M. bovis being transmitted to humans primarily by the consumption of unpasteurized dairy products and contact with infected livestock. Control efforts, including slaughter surveillance and test/cull campaigns, have dramatically reduced the prevalence of M. bovis infection in domestic cattle herds, thereby reducing the spread of M. bovis to humans. However, in developing countries, M. bovis infection of humans persists as a serious and relatively common zoonosis (16).Although Mycobacterium kansasii is not a member of the M. tuberculosis complex, it may cause disease in otherwise healthy humans, albeit infrequently, that is clinically indistinguishable from M. tuberculosis infection (1, 3). As with humans, M. kansasii infection of cattle is uncommon; however, it is occasionally associated with granulomatous lesions within lymph nodes and the respiratory tract of cattle (B. Harris, unpublished observations). Of particular relevance for the diagnosis of tuberculosis, M. kansasii infection/sensitization may elicit responses to antigens generally considered to be tuberculosis specific, such as ESAT-6, CFP-10, and MPB83 (2, 30, 35).With experimental M. bovis infection of cattle, the levels of MPB83-specific antibody correlate with disease severity, bacterial burden, and specific cell-mediated immune responses (15, 33). With this particular scenario, disease severity (i.e., pathology) and bacterial burden are intimately linked; thus, it is difficult to define a potential correlation of a particular immune response to either readout independently. Prior studies have demonstrated that virulent and attenuated strains of M. bovis induce similar delayed-type hypersensitivity responses in cattle; however, only the virulent M. bovis strain induces a persistent gamma interferon (IFN-γ), interleukin-2 (IL-2), and antibody response (34). The objective of the present study was to compare mycobacterium-specific immune responses to the patterns of mycobacterial disease expression in which the mycobacterial burden is uncoupled from pathological changes. Disease expression patterns included persistent colonization with an associated pathology (i.e., M. bovis infection), colonization without an associated pathology (i.e., M. tuberculosis infection), and no colonization or pathology (i.e., M. kansasii infection). Antigen-specific immune responses were evaluated for their correlation to manifestations of disease expression.     

Multiple routes of complement activation by Mycobacterium bovis BCG

Mycobacterium tuberculosis is the leading cause of infectious disease in humans in the world. It evades the host immune system by being phagocytosed by macrophages and residing intracellularly. Complement-dependent opsonisation of extracellular mycobacteria may assist them to enter macrophages. This work examines in detail the mechanisms of complement activation by whole mycobacteria using Mycobacterium bovis BCG as a model organism. M. bovis BCG directly activates the classical, lectin and alternative pathways, resulting in fixation of C3b onto macromolecules of the mycobacterial surface. Investigation into the classical pathway has shown direct binding of human C1q to whole mycobacteria in the absence of antibodies. Most human sera contain IgG and IgM-anti-(M. bovis BCG), and pre-incubation with human immunoglobulin enhances C1q binding to the bacteria. Therefore classical pathway activation is both antibody-independent and dependent. The bacteria also activate the alternative pathway in an antibody-independent manner, but Factor H also binds, suggesting some regulation of amplification by this pathway. For the lectin pathway we have demonstrated direct binding of both MBL and L-ficolin from human serum to whole mycobacteria and subsequent MASP2 activation. H-ficolin binding was not observed. No M. bovis BCG cell surface or secreted protease appears likely to influence complement activation. Together, these data provide a more detailed analysis of the mechanisms by which M. bovis BCG interacts with the complement system.     

Pyrazinamide resistance among South African multidrug-resistant Mycobacterium tuberculosis isolates

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.     

Genomic analysis distinguishes Mycobacterium africanum

Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of “modern” M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of “M. africanum-like” isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.     

Extrapulmonary Dissemination of Mycobacterium bovis but Not Mycobacterium tuberculosis in a Bronchoscopic Rabbit Model of Cavitary Tuberculosis

The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 ΔsigC mutant. M. bovis infections generated cavitary CFU counts of 10⁶ to 10⁹ bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10⁴ to 10⁸ bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.     

Rapid Differentiation of Mycobacterium tuberculosis and M. bovis by High-Resolution Melt Curve Analysis

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.

TABLE 1.

Primers used in this study

Mycobacterium bovis BCG immunization induces protective immunity against nine different Mycobacterium tuberculosis strains in mice

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.     

Differentiation between Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.     

Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis.

Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.     

Differentiation of Known Strains of BCG from Isolates of Mycobacterium bovis and Mycobacterium tuberculosis by Using Mycobacteriophage 33D

The use of mycobacteriophage 33D to differentiate strains of BCG from isolates of Mycobacterium bovis was investigated. The procedure was found to be reproducible and, using the commercially available media described, can be recommended for use in mycobacterial reference laboratories.     

Development of Diagnostic Reagents To Differentiate between Mycobacterium bovis BCG Vaccination and M. bovis Infection in Cattle

In Great Britain a recent independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospect for tuberculosis control in British herds. A sine qua non for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that test-and-slaughter-based control strategies can continue alongside vaccination. In order to assess the feasibility of developing a differential diagnostic test for a live vaccine, we chose M. bovis BCG Pasteur as a model system. Recombinant forms of antigens which are expressed in M. bovis but not, or only at low levels, in BCG Pasteur (ESAT-6, MPB64, MPB70, and MPB83) were produced. These reagents were tested either alone or in combination by using peripheral blood mononuclear cells from M. bovis-infected, BCG-vaccinated, and Mycobacterium avium-sensitized calves. All four antigens induced in vitro proliferation and gamma interferon responses only in M. bovis-infected animals. A cocktail composed of ESAT-6, MPB64, and MPB83 identified infected animals but not those vaccinated with BCG. In addition, promiscuous T-cell epitopes of ESAT-6, MPB64, and MPB83 were formulated into a peptide cocktail. In T-cell assays with this peptide cocktail, infected animals were identified with frequencies similar to those obtained in assays with the protein cocktail, while BCG-vaccinated or M. avium-sensitized animals did not respond. In summary, our results suggest that peptide and protein cocktails can be designed to discriminate between M. bovis infection and BCG vaccination.     

Genetic Diversity and Dynamic Distribution of Mycobacterium tuberculosis Isolates Causing Pulmonary and Extrapulmonary Tuberculosis in Thailand

This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies.