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. 2018 Mar 16;18(1):133.
doi: 10.1186/s12879-018-3007-y.

Mycobacterium tuberculosis thymidylate kinase antigen assays for designating incipient, high-risk latent M.tb infection

Misaki Wayengera  1   2 , David P Kateete  3   4 , Benon Asiimwe  4 , Moses L Joloba  3   4
Affiliations

Mycobacterium tuberculosis thymidylate kinase antigen assays for designating incipient, high-risk latent M.tb infection

Misaki Wayengera et al. BMC Infect Dis. .

Abstract

Background: Precise designation of high risk forms of latent Mycobacterium tuberculosis-M.tb infections (LTBI) is impossible. Delineation of high-risk LTBI can, however, allow for chemoprophylaxis and curtail majority cases of active tuberculosis (ATB). There is epidemiological evidence to support the view that LTBI in context of HIV-1 co-infection is high-risk for progression to ATB relative to LTBI among HIV-ve persons. We recently showed that assays of M.tb thymidylate kinase (TMKmt) antigen and host specific IgG can differentiate ATB from LTBI and or no TB (NTB, or healthy controls). In this study, we aimed to expose the differential levels of TMKmt Ag among HIV+ve co-infected LTBI relative to HIV-ve LTBI as a strategy to advance these assays for designating incipient LTBI.

Methods: TMKmt host specific IgM and IgG detection Enzyme Immuno-Assays (EIA) were conducted on 40 TB exposed house-hold contacts (22 LTBI vs. 18 no TB (NTB) by QunatiFERON-TB GOLD®); and TMKmt Ag detection EIA done on 82 LTBI (46 HIV+ve vs 36 HIV-ve) and 9 NTB (American donors). Purified recombinant TMKmt protein was used as positive control for the Ag assays.

Results: IgM levels were found to be equally low across QuantiFERON-TB GOLD® prequalified NTB and TB exposed house-hold contacts. Higher TMKmt host specific IgG trends were found among TB house-hold contacts relative to NTB controls. TMKmt Ag levels among HIV+ve LTBI were 0.2676 ± 0.0197 (95% CI: 0.2279 to 0.3073) relative to 0.1069 ± 0.01628 (95% CI: 0.07385 to 0.14) for HIV-ve LTBI (supporting incipient nature of LTBI in context of HIV-1 co-infection). NTB had TMKmt Ag levels of 0.1013 ± 0.02505 (5% CI: 0.0421 to 0.1606) (intimating that some were indeed LTBI).

Conclusions: TMKmt Ag levels represent a novel surrogate biomarker for high-risk LTBI, while host-specific IgG can be used to designate NTB from LTBI.

Keywords: Latent M.tb infections (LTBI);Thymidylate Kinase; Mycobacterium tuberculois; Serodiagnosis; Tuberculosis.

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Conflict of interest statement

Ethics approval and consent to participate

This protocol was approved by the School of Biomedical Sciences Institutional Review & Ethics Committee (SBS-IREC) at the College of Health Sciences, Makerere University Kampala, Uganda as protocol # SBS 263 titled “Exploration of Mycobacterium tuberculosis thymidylate kinase based culture- and immunodiagnostic- assays towards rapid and easy detection of Tuberculosis”. Since the study used broadly consented serum of TB house-hold contacts and LTBI previously collected by the Makerere University-Case-Western Reserve University (MU-CWRU) TB Research Unit Project, the need for participant consent was waived by the SBS-IREC.

Consent for publication

Not Applicable

Competing interests

“Tehe authors declare that they have no competing interests.” M.W has previously filed a related patent # UG/P/2013/000006 at the Uganda Registration Services Bureau (URBS) and African Regional Intellectaul Property Organization (ARIPO).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Biophysical Profiles of the TMKmt B cell Epitopes in the Immune Epitope Database Analysis Resource and DiscoTope. This figure shows the 5 biophysical profiles of the linear B cell epitope (UG-Peptide 1) within the IEDB-AR (Plates 1 to 5: surface accessibility, antigenicity, beta-turn, flexibility and hydrophilicity) alongside the DiscoTope profile of the non-linear B cell epitope (UG-Peptide 1)
Fig. 2
Fig. 2
Loci of the non-linear B cell epitope on the 3-D Crystal structure of TMKmt. This figure shows the loci of the 22 amino acids ( A:G57, A:E148, A:S150, A:R151, A:G152, A:R153, A:A154, A:Q155, A:R156, A:D157, A:P158, A:G159, A:A160, A:A161, A:R162, A:A163, A:N164, A:E166, A:R167, A:D168, A:A169, A:T179) on the 3-D crystal structure of TMKmt (PdB entry: “1g3u”).
Fig. 3
Fig. 3
TMK host specific IgM levels among the NTB controls and TB exposed house-hold contacts. This figure shows TMKmt host specific IgM levels among the 9 NTB controls (American donors) and 40 TB exposed house-hold contacts. IgM levels were found to be within the same range across both categories. This is consistent with TB epidemiology and immune-pathogenesis wherein exposure occurs early and is followed by M.tb host-specific IgM responses that last only about 1 to 3 months; being replaced by IgG antibodies
Fig. 4
Fig. 4
TMKmt host specific IgG levels among the NTB controls and TB exposed house-hold contacts. This figure shows TMKmt host specific IgG levels among the 9 NTB controls (American donors) and 40 TB exposed house-hold contacts. Higher IgG levels were observed among the TB exposed house-hold contacts relative to the NTB controls
Fig. 5
Fig. 5
Discordance of distribution of TMKmt host specific IgG levels among the TB house-hold contacts. This figure shows the discordance observed in the distribution of TMKmt host specific IgG levels among the TB house-hold contacts after categorization by QuantiFERON TB Gold® Assay as 18 NTB and 22 LTBI. Considering TMKmt host specific IgG levels, some of the NTB are actually LTBI and vice versa
Fig. 6
Fig. 6
TMKmt Ag levels among HIV+ve LTBI and HIV-ve LTBI relative to NTB controls. This figure shows TMKmt Ag levels among 46 HIV+ve LTBI and 36 HIV-ve LTBI relative to 9 NTB controls. Recombinant TMKmt Ag cloned and expressed in E.coli BL21 (DE) was used as a positive control. TMKmt Ag levels among HIV+ve controls where higher than those among HIV-ve LTBI, supporting the high-risk nature of LTBI among PLWHA. Two incidental HIV-ve LTBI had high TMKmt Ag levels, possibly due to false negative HIV-1 test, or another form of immune-deficiency
Fig. 7
Fig. 7
Bar-Graph of TMKmt Ag levels among HIV+ve LTBI and HIV-ve LTBI relative to NTB controls. This figure shows a Bar-Graph of the same TMKmt Ag levels among 46 HIV+ve LTBI and 36 HIV-ve LTBI relative to 9 NTB controls shown in Fig. 6
Fig. 8
Fig. 8
Dot-Graph of TMKmt Ag levels among HIV+ve LTBI and HIV-ve LTBI relative to NTB controls. This figure shows a Dot-Graph of the same TMKmt Ag levels among 46 HIV+ve LTBI and 36 HIV-ve LTBI relative to 9 NTB controls shown in Fig. 6
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