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Accuracy of anti-mycobacterial protein 51 antibodies as a biomarker for latent TB infection in asymptomatic HIV positive individuals: a cross-sectional diagnostic study

BMC Research Notes volume 18, Article number: 73 (2025) Cite this article

Abstract

Objective

To evaluate the diagnostic accuracy of anti-Mycobacterial Protein 51 (MPT51) antibodies in latent Tuberculosis (TB) detection among asymptomatic Human Immune Virus (HIV) positive individuals using Interferon Gamma Release Assay (IGRA) (QuantiFERON-TB Gold Plus) as the gold standard and determine the factors associated with anti-MPT51 antibody positivity among asymptomatic HIV positive individuals.

Results

Considering QuantiFERON-TB Gold Plus as the gold standard, antibody reactivity to MPT51 revealed sensitivity of 32.6% (95% CI 24.5–40.6), specificity of 56.1% (95% CI 47.6–64.6), positive predictive value of 61.7% (95% CI 53.4–70.1) and negative predictive value of 27.7% (95% CI 20-35.4). Among the factors tested, none was independently associated with an increased risk of antibody reactivity against MPT51.

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Introduction

Mycobacterium tuberculosis (M. tb) is an ancient bacterium that causes tuberculosis (TB), a disease that affects millions of people worldwide, accounting for over 4100 deaths in 2022 [1, 2]. Tuberculosis disease probability is heightened in people living with Human Immune-deficiency Virus (PLHIV), with approximately 214,000 deaths in 2022 [3, 4]. Despite the global introduction of a vaccine in 1953 and the discovery of an effective four-drug treatment regimen in the 1960s, M. tb continues to ravage the world [5,6,7]. The TB pandemic is fueled by several factors, including poverty and HIV, but also insufficient understanding of the spectrum of TB pathogenesis [8] that limits diagnosis and appropriate treatment [9,10,11].

Latent tuberculosis Infection (LTBI), defined as infection with viable M. tb for which progression to TB disease is not expected to occur soon without any significant immunological compromise, is increasingly described as a spectrum [12, 13]. Despite the progression from LTBI to active TB being higher in PLHIV [2], it is still diagnosed by proxy immune-based tests, the Tuberculin Skin Test (TST) and Interferon Gamma Release Assay (IGRA) [14,15,16], which have poor prediction of the progression and cannot differentiate between the phenotypes along the TB spectrum [17]. Singh et al. reported antibodies to the Mycobacterial Protein 51 (MPT51) antigen in 90% of asymptomatic patients with incipient and subclinical TB [18, 19], the most concerning phenotypes of the spectrum. Furthermore, studies have reported that these antibodies are elicited during the early stages of TB infection [20, 21] and can aid in early diagnosis and treatment.

However, evaluation against IGRA to map the use of these antibodies along the spectrum of TB pathogenesis for targeted diagnosis is still lacking. Information on factors associated with the positivity of these antibodies among asymptomatic HIV-positive individuals is also limited. This study, therefore, sought to evaluate the diagnostic accuracy of the anti-MPT51 antibodies in asymptomatic HIV-positive individuals against IGRA. Positive results would position anti-MPT51 antibodies as a biomarker to improve LTBI diagnosis [22, 23].

Methods

Ethical approval was obtained from Makerere University School of Biomedical Sciences Research and Ethics Committee. One hundred forty-six adult HIV-positive individuals asymptomatic for TB aged 18 years or more were enrolled in this cross-sectional study carried out between July 2023 and September 2023. Participants were recruited from the Infectious Diseases Institute adult HIV clinic and invited to give written consent to participate after being briefed about the study. Consenting participants gave social demographic information, including whether or not they share a house with a TB patient, experienced any TB signs and symptoms, were previously diagnosed with TB, were previously on any TB treatment, have been on any TB preventive therapy (TPT) and whether they have used any substances (alcohol/smoking). Also, information was collected on the last HIV viral load results, and weight and height were measured. Participants then donated 5 ml of blood, of which 4 ml were used for IGRA testing and 1 ml for MPT51 testing.

ELISAs. The ELISA for MPT51 testing was done according to Wanchu et al. [24]. Briefly, Wells of 96-well microtiter plates (Immulon 2HB; Dynax) were coated with MPT51 antigen at 2 µg/ml (100 µl/ well) and incubated for 16 h at 4 °C. After the incubation period, plates were brought to room temperature and then washed 6 times with 200µL of Phosphate Buffered Saline (PBS) in 0.05% tween 20. The wells were then blocked with 200µL of skimmed milk in 0.1% of tween 20 for one hour at 37 °C. After the incubation period, the plates were washed again 6 times with 200µL of PBS in 0.05% tween 20. 100µL of the sample was then added, and plates were incubated at 37 °C for one hour. After incubation, wells were washed again, and 100µL of antihuman IgA HRP (1:2000; sino biological) was added, after which incubation for an hour at 37 °C followed. 100µL of TMB substrate was then added and incubated for 30 min at 37 °C. 100µL of sulphuric acid was then added to stop the reaction. Optical Densities were read at 405 nm.

Blood for IGRA was collected directly into the IGRA tubes. The IGRA assay, QuantiFERON-TB Gold-plus (Qiagen, Hilden, Germany), was performed according to the manufacturer’s instructions. The results were calculated using QFT-Plus analysis software version 2.71.2.

Statistical analysis

A 2 by-2 table was used to calculate the diagnostic accuracy (sensitivity, specificity, positive and negative predictive values) of anti-MPT51 antibodies against QuantiFERON-TB Gold-plus at 95% CI. In inferential analysis, a random effect logistic regression analysis was used to determine factors associated with antibody positivity against MPT51. Bivariate logistic regression models were used to obtain unadjusted odds ratios for all characteristics potentially associated with antibody positivity against MPT51. Multivariate logistic regression analysis with forward stepwise logit function was done with variables included as a priority based on biological plausibility with antibody positivity against MPT51. Adjusted odds ratios were reported with 95% confidence intervals (95% CI). Data was analyzed using Stata/IC 15.0, Stata Corp LLC Texas USA. For all comparisons, a two-tailed P-value < 0.05 was considered significant.

Results

Demographic characteristics of the study participants

130/146 of the participants enrolled in the study were included in the analysis [25]. Sixteen [16] participants had indeterminate IGRA results; their -anti-MPT51 ELISA was not done.

Of the 130 participants, 82/130 (63.1%) were females, 68/130 (52.3%) were 33 years and below, 85/130 (65.4%) were of normal weight, 18/130 (13.85%) shared a house with a TB patient, 16/130 (12.31%) experienced signs and symptoms of TB, 41/130 (31.54%) were previously diagnosed with TB, 44/130 (33.85%) were previously on TB treatment, 84/130 (64.62) had been on TPT before while 37/130 (28.68%) had used substances (smoking or taking alcohol) before, and 127/130 (97.69%) had suppressed viral load, (Table 1).

Table 1 Socio-demographics and clinical characteristics of the study participants
Full size table

Diagnostic accuracy of antibody reactivity against MPT51

Considering IGRA (QuantiFERON-TB Gold Plus) as the gold standard, antibody reactivity to MPT51 revealed a sensitivity of 32.6% (95% CI 24.5–40.6), specificity of 56.1% (95% CI 47.6–64.6), positive predictive value of 61.7% (95% CI 53.4–70.1) and negative predictive value of 27.7% (95% CI 20-35.4) (Table 2).

Table 2 A 2 by-2 table and antibody reactivity against MPT51 in comparison with QuantiFERON TB Gold Plus as the gold standard*
Full size table

Characteristics

Anti-MPT51 antibody reactivity

Sensitivity (95% CI)

32.6% (24.5–40.6)

Specificity (95% CI)

56.1% (47.6–64.6)

Positive predictive value (95% CI)

61.7% ( 53.4–70.1)

Negative predictive value (95% CI)

27.7% (20-35.4)

  1. *Sensitivity = [29/ (29 + 60)) x100] = 32.6%; Specificity = [23/ (23 + 18)) x 100] = 56.1%; Positive Predictive value (PPV) = [29/ (29 + 18)) x 100] = 61.7%; Negative predictive value (NPV) = [23/ (60 + 23)] x 100 = 27.7%

Factors associated with antibody reactivity against MPT51

At bivariate analysis, among the factors tested, only sex was associated with an increased risk of antibody reactivity against MPT51 with a (Prevalence Ratio (PR) 0.52 95% CI 0.24–1.13). However, at multivariate analysis, none of the factors was independently associated with an increased risk of antibody reactivity against MPT51: sharing vs. not sharing a house with a TB patient (adjusted Prevalence Ratio (aPR): 1.52, 95% CI 0.48–4.77), experiencing vs. not any TB signs and symptoms (aPR: 1.65, 95% CI 0.47–5.77), previously being vs. not being diagnosed with TB (aPR: 1.66, 95% CI 0.13–20.8), previously on vs. not being on TB treatment (aPR: 0.46, 95% CI 0.03–6.12), previously on vs. not being any TPT (aPR: 1.35, 95% CI 0.56–3.26), substance use vs. no substance use (aPR: 1.00, 95% CI 0.43–2.35), having vs. not having a suppressed viral load (aPR: 1.00, 95% CI 0.94-1.00) and being 33 years of age and below vs. being above 33 (aPR: 1.62, 95% CI 0.71–3.12), see Table 3.

Table 3 Logistic analysis for factors associated with antibody reactivity against MPT51
Full size table

Discussion

The study results revealed a sensitivity, specificity, PPV, and NPV of anti-MPT51 of 32.6%, 56.1%, 61.7%, and 27.7%, respectively, for diagnosing LTBI. These results are similar to findings by Wanchu et al. among HIV-positive individuals without TB in India, which revealed a sensitivity of 32% [19, 24]. MPT51 is an adhesin of M. tb, likely expressed during active replication of the bacteria, as suggested by its presence in culture filtrates of M. tb [26,27,28]. It is, therefore, conceivable that antibody reactivity against MPT51 reflects increasing M. tb infection activity [20] and a heightened risk of progressing to active TB. The low sensitivity and specificity of 27.7% and 56.1%, respectively, would not lead to replacing IGRA as an LTBI test; however, a positive MPT51 antibody test is still informative as the presence of these antibodies predicts a higher likelihood of progression to active TB as has been demonstrated in earlier studies [18, 20].

Among the factors tested, none was independently associated with an increased risk of antibody reactivity against MPT51. This could have resulted from most participants having a suppressed viral load as they all were on antiretroviral therapy (ART). A study by Achkar et al., 2010 showed that worsening immunosuppression with concomitantly reduced capacity to control M. tb replication translates into a high risk for progression to TB, increasing the chance of detecting antibodies against MPT51 [20].

The other factors, such as currently or previously being on TPT, having been diagnosed with TB before or having taken any TB treatment, sharing a house with a TB patient, and substance use, had never been evaluated before.

Limitations

Long-term monitoring of our study participants was not done, making it impossible to determine the prognostic value of the antibody responses. Long-term monitoring would allow serial assessments of antibody reactivity over time to determine whether it increases, wanes, or persists.

Even though we intended to have high statistical power, we only managed to collect samples from 146 participants due to financial constraints. This could have affected the study’s results.

Data availability

Data is provided within the manuscript. The datasets generated and analyzed in the current study are available from the corresponding author.

Abbreviations

TB:

Tuberculosis

HIV:

Human Immunodeficiency Virus

AIDS:

Acquired Immune Deficiency Syndrome

ART:

Antiretroviral Therapy

M. tb :

Mycobacterium tuberculosis

MPT51:

Mycobacterial Protein 51

WHO:

World Health Organization

TPT:

Tuberculosis Preventative Therapy

ELISA:

Enzyme-Linked Immunosorbent Assay

IGRA:

Interferon Gamma Release Assay

LTBI:

Latent Tuberculosis Infection.

IDI:

Infectious Disease Institute

PR:

Prevalence Ratio

PLHIV:

People Living with HIV

References

  1. World Health Organisation. 2022. Available from: www.paho.org/en/campaigns/world-tuberculosis-day-2022

  2. UNAIDS W. 2022 [cited 2022 May 15]. Available from: www.unaids.org/en/resources/documents/2022/tb-fact-sheet

  3. WHO. Tuberculosis [Internet]. 2021 [cited 2022 Jul 16]. Available from: https://www.who.int/news-room/fact-sheets/detail/tuberculosis

  4. WHO. Facts sheet. 2021.

  5. Houben RMGJ, Dodd PJ. The Global Burden of Latent Tuberculosis Infection: A Re-estimation Using Mathematical Modelling. Metcalfe JZ, editor. PLOS Med. 2016;13(10):e1002152.

  6. Centers for Disease Control and Prevention. TB and HIV coinfection [Internet]. 2016. Available from: https://www.cdc.gov/tb/topic/basics/tbhivcoinfection.htm

  7. NIH gov. HIV and Opportunistic Infections, Coinfections, and Conditions [Internet]. 2021. Available from: https://hivinfo.nih.gov/understanding-hiv/fact-sheets/hiv-and-tuberculosis-tb

  8. Drain PK, Bajema KL, Dowdy D, Dheda K, Naidoo K, Schumacher SG, et al. Incipient and subclinical tuberculosis: a clinical review of early stages and progression of infection. Clin Microbiol Rev. 2018;31(4):e00021–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Technology Research Education and Technical Assistance for Tuberculosis. Uganda TREAT TB [Internet]. [cited 2022 Jul 16]. Available from: https://treattb.org/trial-sites/uganda/

  10. Heifets LB, Good RC. Current Laboratory Methods for the Diagnosis of Tuberculosis. In: Bloom BR, editor. Tuberculosis [Internet]. Washington, DC, USA: ASM Press; 2014 [cited 2022 May 14]. pp. 85–110. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1128/9781555818357.ch7

  11. Caulfield AJ, Wengenack NL. Diagnosis of active tuberculosis disease: from microscopy to molecular techniques. J Clin Tuberc Mycobact Dis. 2016;4:33–43.

    Google Scholar 

  12. Barry CE, Boshoff HI, Dartois V, Dick T, Ehrt S, Flynn J, et al. The spectrum of latent tuberculosis: rethinking the biology and intervention strategies. Nat Rev Microbiol. 2009;7(12):845–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Achkar JM, Jenny-Avital ER. Incipient and subclinical tuberculosis: defining early Disease States in the context of host Immune Response. J Infect Dis. 2011;204(suppl 4):S1179–86.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Domínguez J, Latorre I, Santin M. Diagnosis and therapeutic approach of latent tuberculosis infection. Enfermedades Infecc Microbiol Clin Engl Ed. 2018;36(5):302–11.

    Article  Google Scholar 

  15. Fukushima K, Akagi K, Kondo A, Kubo T, Sakamoto N, Mukae H. First clinical evaluation of the QIAreachTM QuantiFERON-TB for Tuberculosis infection and active pulmonary disease. Pulmonology. 2022;28(1):6–12.

    Article  CAS  PubMed  Google Scholar 

  16. Biraro IA, Kimuda S, Egesa M, Cose S, Webb EL, Joloba M et al. The Use of Interferon Gamma Inducible Protein 10 as a Potential Biomarker in the Diagnosis of Latent Tuberculosis Infection in Uganda. Delogu G, editor. PLOS ONE. 2016;11(1):e0146098.

  17. Carranza C, Pedraza-Sanchez S, de Oyarzabal-Mendez E, Torres M. Diagnosis for latent tuberculosis infection: New Alternatives. Front Immunol. 2020;11:2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Singh KK, Dong Y, Belisle JT, Harder J, Arora VK, Laal S. Antigens of Mycobacterium tuberculosis recognized by antibodies during Incipient, subclinical tuberculosis. Clin Vaccine Immunol. 2005;12(2):354–8.

    Article  CAS  Google Scholar 

  19. Wanchu A, Dong Y, Sethi S, Myneedu VP, Nadas A, Liu Z, Biomarkers for Clinical and Incipient Tuberculosis: Performance in a TB-Endemic Country. Gold JA,PLoS ONE., Corbett EL, Charalambous S, Moloi VM, Fielding K, Grant AD, Dye C, De Cock KM, Hayes RJ, Williams BG, Churchyard GJ et al. 2004. Human immunodeficiency virus and the prevalence of undiagnosed tuberculosis in African gold miners. Am. J. Resp. Crit. Care Med. 170:673–679.

  20. Achkar JM, Jenny-Avital E, Yu X, Burger S, Leibert E, Bilder PW, et al. Antibodies against Immunodominant Antigens of Mycobacterium tuberculosis in subjects with suspected tuberculosis in the United States compared by HIV Status. Clin Vaccine Immunol. 2010;17(3):384–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Achkar JM, Dong Y, Holzman RS, Belisle J, Kourbeti IS, Sherpa T, et al. Mycobacterium tuberculosis Msynthasenthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis. Clin Vaccine Immunol. 2006;13(11):1291–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. WHO. The End TB strategy [Internet]. 2015 [cited 2022 Jul 16]. Available from: https://www.google.com/url?sa=t%26rct=j%26q=%26esrc=s%26source=web%26cd=%26ved=2ahUKEwi05qry6_v4AhVKyKQKHfXDCksQFnoECBEQAQ%26url=https%3A%2F%2Fapps.who.int%2Firis%2Fbitstream%2Fhandle%2F10665%2F259636%2FTBstrat-eng.pdf%3Fsequence%3D1%26usg=AOvVaw01JU6kPJKcXw9yNaqiaqoT

  23. Ramalingam B, Devi KRU, Raja A. Isotype-specific Anti-38 and 27 kDa (mpt 51) response in Pulmonary Tuberculosis with Human Immunodeficiency Virus Coinfection. Scand J Infect Dis. 2003;35(4):234–9.

    Article  CAS  PubMed  Google Scholar 

  24. Wanchu A, Dong Y, Sethi S, Myneedu VP, Nadas A, Liu Z, et al. Biomarkers for clinical and incipient tuberculosis: performance in a TB-endemic country. PLoS ONE. 2008;3(4):1–8.

    Article  Google Scholar 

  25. Buderer NMF. Statistical methodology: I. incorporating the prevalence of Disease into the sample size calculation for sensitivity and specificity. Acad Emerg Med. 1996;3(9):895–900.

    Article  CAS  PubMed  Google Scholar 

  26. Kinhikar AG, Vargas D, Li H, Mahaffey SB, Hinds L, Belisle JT, et al. Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin. Mol Microbiol. 2006;60(4):999–1013.

    Article  CAS  PubMed  Google Scholar 

  27. Wilson RA, Maughan WN, Kremer L, Besra GS, Fütterer K. The structure of Mycobacterium tuberculosis MPT51 (FbpC1) defines a New Family of non-catalytic α/β hydrolases. J Mol Biol. 2004;335(2):519–30.

    Article  CAS  PubMed  Google Scholar 

  28. Dunn MF, Ramírez-Trujillo JA, Hernández-Lucas I. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology. 2009;155(10):3166–75.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to thank the staff at the Immunology Laboratory under the Department of Immunology and Molecular Biology at Makerere University and the staff at IDI Adult Clinic for the support they offered. May God bless you.

Funding

Funding was received from the Microbiology and Immunology Training for HIV and HIV Related Research in Uganda (MITHU) funded by the NIH Fogarty, Grant No. D43TW010319 (PI: W. Henry Boom) under Case Western Reserve University.

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Author notes

  1. Jonathan Mayito and Obondo James Sande contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Molecular Biology, School of Biomedical Sciences, College of Health Sciences, Makerere University, Kampala, Uganda

    Josephine Victo Namugga, Carol Musubika & Obondo James Sande

  2. College of Natural Sciences, Makerere University, Kampala, Uganda

    Stuart Niwagaba

  3. Infectious Diseases Institute (IDI), Makerere University, Kampala, Uganda

    Jonathan Mayito

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  1. Josephine Victo Namugga
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Contributions

JVN contributed to the conception, proposal development, sample collection and analysis, data collection and interpretation, and drafted the manuscript. CM generated the suitable in-house ELISA for MPT51 and performed sample analysis. SN contributed to the data analysis and interpretation. OJS and JM contributed to the conception, proposal development, and data interpretation and substantively revised the work. All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to Obondo James Sande.

Ethics declarations

Ethical approval and consent to participate

The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice Guidelines. Ethical approval was obtained from the School of Biomedical Sciences Research Ethics Committee (Approval number: SBS-2022-265) under the College of Health Sciences at Makerere University and the IDI REC. Administrative clearance was obtained from the IDI management for conducting the research at the IDI adult HIV clinic. Informed written consent was obtained from all study participants before any study procedure, and their information was treated with confidentiality.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.

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Namugga, J.V., Musubika, C., Niwagaba, S. et al. Accuracy of anti-mycobacterial protein 51 antibodies as a biomarker for latent TB infection in asymptomatic HIV positive individuals: a cross-sectional diagnostic study. BMC Res Notes 18, 73 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-025-07134-1

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Keywords

BMC Research Notes

ISSN: 1756-0500

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