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Involvement of methylated HBHA expressed from Mycobacterium smegmatis in an IFN-γ release assay to aid discrimination between latent infection and active tuberculosis in BCG-vaccinated populations

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Abstract

IFN-γ release assays (IGRAs) based on region of difference 1 (RD1) antigens have improved diagnosis of Mycobacterium tuberculosis (M. tb) infection. However, IGRAs with these antigens cannot discriminate between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). M. tb heparin-binding-hemagglutinin (HBHA) induces relatively high IFN-γ responses in LTBI individuals and low responses in ATB patients, but purification of the native methylated HBHA from cultures of M. tb for immunological tests is complex and time-consuming. To overcome these cumbersome procedures, we constructed a recombinant Mycobacterium smegmatis strain that over-expressed HBHA under control of a strong furA promoter. The methylated activity of purified protein was verified by hybridization with anti-methylated Lys antibody, and the methylated HBHA (mHBHA) was further evaluated for antigen-specific IFN-γ responses in BCG-vaccinated Chinese population. A total of 138 individuals including 86 active TB (ATB) patients, 15 latent TB infection (LTBI) cases, and 37 healthy controls (HC) were tested by using an IFN-γ enzyme-linked immunospot (ELISPOT) assay. The results showed that T-cell responses against mHBHA were always lower in ATB patients than in LTBI individuals, regardless of the site of infection or the results of bacteriological tests. This allowed for a good discrimination between these two groups of M. tb-infected individuals, even in the BCG-vaccinated and high TB-incidence setting that is China. Additionally, combination of mHBHA and RD1 antigens in an IFN-γ release assay enhanced diagnostic efficacy for active TB cases. Taken together, inclusion of the immune response to mHBHA can discriminate healthy LTBI cases from ATB patients.

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Authors and Affiliations

  1. School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, 325035, China

    H.-L. Wen, C.-L. Li, G. Li, Y.-H. Lu, J.-X. Lv, S.-H. Lu & X.-Y. Fan

  2. Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of MOE/MOH, Fudan University, Shanghai, 201508, China

    H.-L. Wen, C.-L. Li, G. Li, Y.-H. Lu, H.-C. Li, T. Li, H.-M. Zhao, K. Wu, D. B. Lowrie, S.-H. Lu & X.-Y. Fan

  3. TB Center, Shanghai Emerging and Re-emerging Infectious Disease Institute, Shanghai, 201508, China

    S.-H. Lu & X.-Y. Fan

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  1. H.-L. Wen
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  2. C.-L. Li
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  3. G. Li
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  4. Y.-H. Lu
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  5. H.-C. Li
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  6. T. Li
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  7. H.-M. Zhao
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  8. K. Wu
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  9. D. B. Lowrie
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  10. J.-X. Lv
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  11. S.-H. Lu
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  12. X.-Y. Fan
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Corresponding authors

Correspondence to S.-H. Lu or X.-Y. Fan.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81273328) and Shanghai Science and Technology Commission (17ZR1423900, 15DZ2290200).

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There is no conflict for any of the authors.

Ethical approval

The protocols of this study were approved by the Institutional Review Board of Shanghai Public Health Clinical Center (SPHCC).

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Written informed consent was obtained from all individual participants.

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Wen, HL., Li, CL., Li, G. et al. Involvement of methylated HBHA expressed from Mycobacterium smegmatis in an IFN-γ release assay to aid discrimination between latent infection and active tuberculosis in BCG-vaccinated populations. Eur J Clin Microbiol Infect Dis 36, 1415–1423 (2017). https://doi.org/10.1007/s10096-017-2948-1

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  • DOI: https://doi.org/10.1007/s10096-017-2948-1

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