Growth of recombinant Mycobacterium tuberculosis H37Ra in mouse macrophages

Mycobacterium tuberculosis H37Rv and H37Ra were derived from the same parental strain but differ strikingly in their virulence for experimental animals. Transfer of genetic material between these closely related strains resulted in the isolation of a number of recombinant H37Ra clones bearing the in vivo growth-promoting ivg locus of H37Rv. The recombinant strain was phagocytosed by murine peritoneal macrophages infected in vivo or in vitro and their intracellular growth rates were compared with the vector control. The intracellular growth of the recombinant was significantly faster than the vector control, but substantially slower than the wild-type H37Rv control, regardless of the method used to infect the macrophages. The slower intracellular growth observed for the recombinant strains was not due to a genetically induced metabolic defect, since they grew in synthetic liquid medium at rates equal to those observed for both H37Rv and H37Ra. Peritoneal macrophage monolayers provide a rapid and convenient assay by which to screen H37Ra recombinants for the presence of putative virulence genes.     

Proliferation of distinct human T cell subsets in response to live, killed or soluble extracts of Mycobacterium tuberculosisand Myco. avium

The proliferative responses of distinct cell subsets from healthy, bacille Calmette–Guérin (BCG)-vaccinated blood donors were assessed after in vitro stimulation with live or UV-killed Mycobacterium tuberculosis and Myco. avium or with soluble extracts obtained from either mycobacterial species. Proliferation of cell subsets was evaluated by flow cytometric determination of 5-bromo-2′-deoxy-uridine incorporation into DNA and simultaneous identification of surface phenotypic markers. In the presence of monocytes, the response to whole (live or killed) bacteria was characterized by a predominant proliferation of CD4⁺ αβ⁺ T cells and, to a lesser extent, of CD8⁺ αβ⁺ T cells. Proliferation of CD8⁺ αβ⁺ T cells was primarily elicited by live rather than killed bacilli (P < 0.05). Conversely, when soluble bacterial extracts were used as stimulators, a preferential proliferation of γδ⁺ T cells, expressing predominantly Vγ9⁺ and Vδ2⁺ T cell receptor chains, was recorded. Moreover, when monocyte-depleted cell populations were directly cultured with live bacteria, a marked proportion of CD3⁻CD16⁺ (natural killer (NK)) cells was detected among the responding cells. Although both αβ, γδ and NK cells have been previously shown to react with mycobacteria in vitro, their relative contributions to the response have been difficult to assess. Using a flow cytometric technique which allows direct identification of proliferating cells within complex cell populations, our study demonstrates significant differences in the ability of various mycobacterial antigen preparations to elicit proliferation of distinct cell subsets.     

Comparison of in-house PCR with conventional techniques and Cobas Amplicor M. tuberculosis kit for detection of Mycobacterium tuberculosis

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.     

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.     

IS6110-restriction fragment length polymorphism and spoligotyping analysis of Mycobacterium tuberculosis clinical isolates for investigating epidemiologic distribution in Korea

The Beijing family of Mycobacterium tuberculosis has been emerging in the world. However, there are few nationwide data of genotypic distribution in Korea. This study aimed to identify the genotypic diversity of clinical isolates of M. tuberculosis and to demonstrate the population of Beijing family in Korea. We collected 96 clinical M. tuberculosis isolates from 11 university hospitals nationwide in Korea from 2008 to 2009. We observed 24 clusters in IS6110-RFLP analysis and 19 patterns in spoligotyping. Seventy-five isolates were confirmed to be Beijing family. Two isolates of the K strain and 12 isolates of the K family strain were also found. We found that drug resistance phenotypes were more strongly associated with Beijing family than non-Beijing family (P=0.003). This study gives an overview of the distribution of genotypes of M. tuberculosis in Korea. These findings indicate that we have to pay more attention to control of M. tuberculosis strains associated with the Beijing family.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Drug resistance rates of Mycobacterium tuberculosis at a private referral center in Korea

The goals of this study were to identify first-line drug resistance in new and previously treated tuberculosis (TB) cases and to determine risk factors for multidrug resistant TB (MDR-TB) at a private referral center in Korea. All patients with culture confirmed pulmonary TB over a 2-yr period between July 2002 and June 2004 were prospectively included in this study. In total, 637 patients were included; 512 (80.4%) were new cases, and 125 (19.6%) were previously treated cases. Resistance to at least one first-line drug was identified in 11.7% of new cases and 41.6% of previously treated cases. MDR-TB was detected in 3.9% of new cases and 27.2% of previously treated cases. The proportion of extensively drug-resistant TB among MDR-TB patients was 16.7% (9/54). Factors associated with MDR-TB included age under 45 yr, previous TB treatment, and the presence of cavitation on chest radiography. Rates of first-line drug resistance are high, particularly in previously treated patients, in the private sector in Korea. This underscores the need for an improved control program, coupled with early diagnosis of MDR-TB, to reduce the spread and development of resistance.     

T cell responses to a mixture of Mycobacterium tuberculosis peptides with complementary HLA-DR binding profiles

The T cell response to a mixture of eight peptides derived from sequences of the Mycobacterium tuberculosis 16-, 19- and 38-kD antigens (MTBmix-8) has been studied. The peptides were selected on the basis of complementary binding to nine HLA-DR molecules (HLA-DR1 to DR9). MTBmix-8 at 6.25 and 50 μg/ml gave rise to significant stimulation (P< 0.05) of peripheral blood mononuclear cells (PBMC) from healthy tuberculin-positive and both untreated and treated diseased subjects, but not in any of a control group of healthy tuberculin-negative subjects. MTB-mix-8 stimulated proliferation of PBMC from healthy tuberculin-positive individuals at lower concentrations than the individual component peptides. However, the maximal stimulation achieved was only slightly higher than that achieved with individual peptides. MTBmix-8 also stimulated the production of interferon-gamma (IFN-γ) in vitro. Using the mean ± 2 s.d. of the values for IFN-γ production in the tuberculin-negative population as a cut-off, MTBmix-8 at 6.25 μg/ml was able to detect infection with a sensitivity of 100% in untreated patients, 87% in treated patients, and 82% in tuberculin-positive controls. The corresponding figures for the most potent single peptide (16p91–110) were: 66% in untreated patients, 71% in treated patients and only 42% in controls. Thus, using the IFN-γ-based assay, which has the additional advantages of speed and does not require radioactivity, the mixture of peptides is more sensitive than single peptides in diagnosing infection.     

Age-dependent association of mannose-binding lectin polymorphisms with the development of pulmonary tuberculosis in Viet Nam

Mannose-binding lectin (MBL) binds to pathogens and induces complement-mediated opsonophagocytosis. Although the association between MBL2 polymorphisms and tuberculosis (TB) has been studied in various populations, the results are controversial. We explored the stages of TB associated with MBL2 polymorphisms. X/Y (rs7096206) and A/B (rs1800450) were genotyped in 765 new patients with active pulmonary TB without HIV infection and 556 controls in Hanoi, Viet Nam. The MBL2 nucleotide sequences were further analyzed, and plasma MBL levels were measured in 109 apparently healthy healthcare workers and 65 patients with TB. Latent TB infection (LTBI) was detected by interferon-gamma release assay (IGRA). The YA/YA diplotype, which exhibited high plasma MBL levels, was associated with protection against active TB in younger patients (mean age = 32) ≦ 45 years old (odds ratio, 0.61; 95% confidence interval, 0.46–0.80). The resistant diplotype was less frequently found in the younger patients at diagnosis (P = 0.0021). MBL2 diplotype frequencies and plasma MBL levels were not significantly different between the IGRA-positive and -negative groups. MBL2 YA/YA exhibited a protective role against the development of TB in younger patients, whereas the MBL2 genotype and MBL levels were not associated with LTBI. High MBL levels may protect against the early development of pulmonary TB after infection.     

Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion

Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.     

Adjuvant modulation of the cytokine balance in Mycobacterium tuberculosis subunit vaccines; immunity, pathology and protection

It is known that protection against tuberculosis is mediated primarily by T helper type 1 (Th1) cells but the influence of the Th1/Th2 balance of a vaccination response on the subsequent protection and pathology during infection has not been studied in detail. We designed a panel of Ag85B-ESAT-6 subunit vaccines based on adjuvants with different Th1/Th2-promoting activities and studied cellular responses, bacterial replication and pathology in the lungs of mice infected with Mycobacterium tuberculosis. All vaccines induced cell-mediated and humoral responses but with markedly different interferon-γ : interleukin-5 (IFN-γ : IL-5) and immunoglobulin G1 (IgG1) : IgG2 ratios. The vaccines promoted different levels of control of bacterial replication with the most efficient protection being exerted by cationic liposomes containing monophosphoryl lipid A and low to completely absent immunity with conventional aluminium. The level of protection correlated with the amount of IFN-γ produced in response to the vaccine whereas there was no inverse correlation with the level of IL-5. Characterizing a protective response was an accelerated recruitment of IL-17 and IFN-γ-producing lymphocytes resulting in the early formation of granulomas containing clustered inducible nitric oxide synthase-activated macrophages. In comparison, non-protected mice exhibited a different inflammatory infiltrate rich in neutrophil granulocytes. This study indicates that the adjuvant component of a tuberculosis vaccine may be crucial in determining the kinetics by which effective granulomas, pivotal in controlling bacterial growth, are formed.     

Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules

Despite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT_1721, MT_1694, MT_2462 and MT_3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non-diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculin-positive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.     

Differential fadE28 expression associated with phenotypic virulence of Mycobacterium tuberculosis

Ability to persist in human macrophages is central to the virulence of Mycobacterium tuberculosis and is not invariable among various strains. Differential gene expression that is associated with phenotypic virulence may provide additional information of virulent genes involved in the pathogenesis of M. tuberculosis, which is not fully elucidated. Three hypervirulent strains of M. tuberculosis isolated from patients suffering with tuberculous meningitis were shown to grow more rapidly inside human macrophages in a previous study. In the current investigation, expression of 7 mycobacterial genes (fadE28, mce1A, mymA, acr, sigA, sugC, and Rv3723) of these strains during ex vivo macrophage challenge and in vitro acid shock was quantified by real-time PCR. Using rrs gene as a normalisation gene, fadE28 gene exhibited differential gene expression that is associated with phenotypic virulence, whereas the other 6 genes showed indistinguishable expression patterns. Up-regulation of fadE28 gene in the hypervirulent strains may account for virulence by increasing the efficiency of beta-oxidation, which is important for the persistence in macrophages as M. tuberculosis uses fatty acids preferably inside phagosome of macrophages. The fadE28 gene, together with its adjacent genes may also be critical in the process of lipid modification that could facilitate parasitism in human macrophages.     

Recombinant early secreted antigen target 6 protein as a skin test antigen for the specific detection of Mycobacterium tuberculosis infection

Although the delayed-type hypersensitivity skin test reaction to tuberculin purified protein derivative (PPD) is used worldwide for tuberculosis (TB) detection, it is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacille Calmette-Guérin (BCG) vaccination or infection with non-tuberculous Mycobacteria. As a result, there is an urgent need for a more specific diagnostic tool for TB. This study reports the skin reactions of guinea pigs and human volunteers to recombinant early secreted antigen target 6 (rESAT6), a secretory protein found only in MTB, M. bovis and few other mycobacterial species. These volunteers had varying histories of BCG vaccination and exposure to MTB, allowing us to determine the specificity of their response to TB exposure. Our results show that 1.0 microg of the purified MTB rESAT6 antigen elicited a positive skin response in both animals and humans exposed to MTB, as well as in animals exposed to M. bovis and M. marinum, all species of Mycobacteria that contain the gene for early secreted antigen target 6 (ESAT6). ESAT6 appears to be more specific to MTB infection than PPD, as demonstrated by the fact that we saw no skin responses in the BCG-vaccinated volunteers, nor in the guinea pigs sensitized with BCG vaccine, or with Mycobacteria that do not contain the gene encoding ESAT6. We believe that this is the first report of the use of a rESAT6 protein in a skin test in human volunteers, and that these data support its use in the specific detection of MTB infection.     

Comparison of diagnostic performance of three real-time PCR kits for detecting Mycobacterium species

Purpose

PCR is widely used for rapidly and accurately detecting Mycobacterium Species. The purpose of this study was to assess the diagnostic performance of three real-time PCR kits and evaluate the concordance with two older PCR methods.

Materials and Methods

Using 128 samples, the five PCR methods were assessed, including an in-house PCR protocol, the COBAS Amplicor MTB, the COBAS TaqMan MTB, the AdvanSure TB/NTM real-time PCR, and the Real-Q M. tuberculosis kit. The discrepant results were further examined by DNA sequencing and using the AdvanSure Mycobacteria Genotyping Chip for complete analysis.

Results

For Mycobacterium tuberculosis (MTB) detection, all five kits showed 100% matching results (positive; N = 11 and negative; N = 80). In non-tuberculous mycobacterium (NTM) discrimination, the AdvanSure yielded two true-positive outcomes from M. intracellulare and one false positive outcome, while the Real-Q resulted in one true-positive outcome and one false negative outcome for each case and another false negative result using the provided DNA samples.

Conclusion

Real-time PCR, yielded results that were comparable to those of the older PCR methods for detecting MTB. However, there were disagreements among the applied kits in regard to the sample test results for detecting NTM. Therefore, we recommend that additional confirmatory measures such as DNA sequencing should be implemented in such cases, and further research with using a larger numbers of samples is warranted to improve the detection of NTM.     

Survival of Mycobacterium tuberculosis organisms for 8 days in fresh lung tissue from an exhumed body

Mycobacterium tuberculosis was isolated from the lung tissue of an 86-year-old unembalmed woman who was exhumed for an autopsy 8 days after her death. Autopsy prosectors should consider performing microbiological culture in all cases with a history or gross pathological findings suggestive of an infection even if the postmortem interval is extended. In addition, prosectors should still adhere to biosafety precautions for airborne pathogens, because a long postmortem interval does not necessarily provide assurance that these organisms are not viable.     

Mycobacterium tuberculosis heat-shock protein 70 impairs maturation of dendritic cells from bone marrow precursors, induces interleukin-10 production and inhibits T-cell proliferation in vitro

In different inflammatory disease models, heat-shock proteins (hsp) and hsp-derived peptides have been demonstrated to possess anti-inflammatory properties. While some studies have shown that hsp can directly interact with antigen-presenting cells, others report that bacterial hsp can induce specific T cells with regulatory phenotypes. Effective characterization of the immunomodulatory effects of hsp 70, however, has historically been confounded by lipopolysaccharide (LPS) contamination. In this study, we compared the effects of LPS-free Mycobacterial tuberculosis hsp 70 (TBhsp70) and its possible contaminants on dendritic cells (DC). We demonstrate herein that LPS-free TBhsp70 inhibits murine DC maturation in vitro, while LPS-contaminated TBhsp70 induces DC maturation. Mock recombinant preparations have no effect. In contrast to LPS, TBhsp70 does not induce tumour necrosis factor-alpha production by DC, but interleukin-10. In vivo, only LPS-contaminated TBhsp70 induces up-regulation of CD86 in splenic mature DC. Finally, TBhsp70 inhibited phytohaemagglutinin-induced T-cell proliferation. Our results support the hypothesis that TBhsp70 does not have inflammatory potential, but rather has immunosuppressive properties.     

A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

Purpose

The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guérin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health.

Materials and Methods

A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates.

Results

All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level.

Conclusion

The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.     

Single Nucleotide Polymorphism in Ag85 Genes of Mycobacterium Tuberculosis Complex: Analysis of 178 Clinical Isolates from China and 13 BCG strains

Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. Human T-cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) were evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. However, in our previous studies, proteins MPT64, PstS1, Rv0309 and Rv2945c all harbored higher numbers of amino acid substitutions in their T cell epitopes, which suggests their roles in ongoing immune evasion. Here, we used the same set of 180 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Ag85 complex, and compared the sequences. The results showed that Ag85 were hyperconserved in T/B cell epitopes and the genes were more likely to be under purifying selection. The divergence of host immune selection on different proteins may result from different function of the proteins. In addition, A312G of Ag85A and T418C of Ag85B may represent special mutations in BCG strains, which may be used to differentiate M.bovis and BCG strains from MTB strains. Also, C714A in Ag85B seems to be a valuable phylogenetic marker for Beijing strains.