Research tests for the diagnosis of tuberculosis infection

Figures & data

Table 1. Characteristics of routine and new routine tests for TBI diagnosis.

		Characteristics	Advantages	Disadvantages
Routine skin test	TST	Stimulation: intradermal inoculation of PPD; Readout: intradermal induration (48 h-72 h).	Large scale test for screening; Inexpensive; No laboratory infrastructure required.	False positive results in BCG-vaccinated; False negative results in immunocompromised; Second visit needed; Low accuracy to discriminate TB from TBI.
New skin tests	Diaskin test EC-skin test c-Tb	Stimulation: intradermal inoculation of ESAT-6/CFP-10; Readout: intradermal induration (48 h-72 h).	Large scale test for screening; Inexpensive; No laboratory infrastructure required; Not affected by BCG vaccination.	False negative results in immunocompromised; Second visit needed; Low accuracy to discriminate TB from TBI.
Routine IGRA	QFT-TB Gold Plus T-SPOT.TB	Sample: whole blood;Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by ELISA. Sample: PBMC; Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by ELISPOT.	Higher specificity for TBI compared with TST; Not affected by BCG vaccination; No need for a second visit.	False negative results in immunocompromised; Expensive; Laboratory facilities needed; Low accuracy to discriminate TB from TBI.
New IGRA and in vitro tests	STANDARD E TB-Feron AdvanSure TB-IGRA LIOFeron TB/LTBI	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides and TB7.7 protein; Readout: IFN-γ detection by ELISA. Sample: Whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by ELISA. Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides, TB7.7 protein, ADH antigen fusion protein; Readout: IFN-γ detection by ELISA.	Higher specificity for TBI compared with TST; Not affected by BCG vaccination; No need for a second visit.	False negative results in immunocompromised; Expensive; Laboratory facilities needed; Limited data for some tests; Low accuracy to discriminate TB from TBI.
	LIAISON QFT Gold Plus AdvanSure I3 TB-IGRA	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; eadout: IFN-γ detection by chemiluminescence. R	Higher specificity for TBI compared with TST; Not affected by BCG vaccination; Ability to process large volume of samples; Less manual handing than ELISA-based IGRA; Reduce manual hand-on time and increase throughput; No need for a second visit.	Expensive; Laboratory facilities needed; Limited data for some tests; Low accuracy to discriminate TB from TBI.
	VIDAS TB-IGRA	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by ELFA.	Higher specificity for TBI compared with TST; Automated; Not affected by BCG vaccination; No need for a second visit.	Expensive; Laboratory facilities needed; Limited data for some tests; Low accuracy to discriminate TB from TBI.
	STANDARD F TB-FeronFIA QIAreach QuantiFERON-TB ichroma IGRA-TB	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides and TB7.7 protein; Readout: IFN-γ detection by quantitative LFA. Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by qualitative LFA. Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: IFN-γ detection by quantitative LFA.	Higher specificity for TBI compared with TST; Not affected by BCG vaccination; Rapid; Less manual handing than ELISA-based IGRA; No need for a second visit.	Medium cost; Laboratory facilities needed; Limited data for some tests; Low accuracy to discriminate TB from TBI.
	Erythra TB-KIT	Sample: whole blood; Stimulation: PPD peptides; Readout: IFN-γ detection by quantitative LFA.	No need for a second visit; Rapid; Limited data available;	Expensive; Laboratory facilities needed; Limited data available; Low accuracy to discriminate TB from TBI.
	GBTsol Latent TB Test Kit	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: detection of Mtb peptides bound to T-cells.	No need for a second visit; Limited data available.	Expensive; Laboratory facilities needed; Limited data available; Low accuracy to discriminate TB from TBI.
	IP-10 IGRA ELISA IP-10 IGRA LFA	Sample: whole blood; Stimulation: ESAT-6/CFP-10 peptides; Readout: IP-10 detection by quantitative ELISA. Sample: whole blood;Stimulation: ESAT-6/CFP-10 peptides;Readout: IP-10 detection by quantitative LFA.	Higher specificity for TBI compared with TST; Not affected by BCG vaccination; No need for a second visit.	False negative results in immunocompromised; Expensive; Laboratory facilities needed; Low accuracy to discriminate TB from TBI.

Note: Abbreviations: TB: tuberculosis; TBI: tuberculosis infection; TST: tuberculin skin test; PPD: purified protein derivative; ESAT-6: early secretory antigenic target 6; CFP-10: culture filtrate protein 10; ADH: alanine dehydrogenase; BCG: Bacille Calmette – Guérin; IFN-γ: interferon-γ; IP-10: IFN-γ-induced protein-10; IGRA: IFN-γ release assays; ELISA: enzyme-linked immunosorbent assay; ELISPOT: enzyme-linked immunospot; ELFA: enzyme-linked fluorescent assay; LFA: lateral flow assay.

Table 2. New strategies for M. tuberculosis DNA detection.

Clinical Cohorts	HIV status	Sample type	DNA extractionmethod	DNA detection method	Target(s)	Reference
Treated TB, HD	NR	BM-MSC	Qiagen DNeasy Blood & Tissue kit	qPCR	Mtb-specific 12.7 kb fragment	[42]
TBI, HD	NR	LT-pHSC	Lysozime/Proteinase K; chloroform isoamyl alcohol	qPCR	IS6110 MPB64	[44]
TBI	Infected, Uninfected	CD34^− cells CD34^+ cells	CTAB; Lysozime/Proteinase K; chloroform isoamyl alcohol	ddPCR	IS6110 rpoB	[46]
TB, TBI, Non-TB, HD	Uninfected	PBMC	Actiphage test	qPCR	IS6110	[47]
TB, TBI	NR	Plasma	QIAamp Blood DNA Mini Kit	ddPCR or qPCR	IS6110	[48]

Note: Abbreviations: TB: tuberculosis; TBI: tuberculosis infection; HD: healthy donors; BM-MSC: bone marrow-derived mesenchymal stem cells; LT-pHSC: long-term repopulating pluripotent hematopoietic stem cells; PBMC: peripheral blood mononuclear cells; Mtb: Mycobacterium tuberculosis; qPCR: quantitative PCR; ddPCR: droplet digital PCR; NR: Not Reported; CTAB: Cetyltrimethylammonium bromide.

Table 3. New strategies for M. tuberculosis antigens detection.

Clinical Cohorts	Animal model	HIV status	Sample type	Mtb Antigen	Method	Results	Reference
TB, TBI, HD	/	NR	Serum	Rv1860, RV3881c, Rv2031c, Rv3803c	Protein microarray	High sensitivity and specificity in discriminating TB from TBI	[54]
/	Non-human primates	NA	Plasma	CFP-10-derived peptides	MS	Lower detection of CFP-10-derived peptides in TBI compared to TB	[55]
TB, TBI, NTM, Non-TB, HD	/	Infected, Uninfected	Serum	ESAT-6 and CFP-10-derived peptides	MS	Detection of ESAT-6 and CFP-10-derived peptides in 12.9% of TBI	[56]
TB, Non-TB	/	Infected, Uninfected	Serum exosomes	Ag85B, Ag85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64-derived peptides	MS	Detection of Mtb peptides in the circulating exosomes of TBI	[57]
TB, Non-TB	/	NR	Serum	MPT64, MPT32, CFP10, PstS1-derived peptides	MS	Higher detection of these peptides in TB compared to Non-TB individuals	[58]

Note: Abbreviations: TB: tuberculosis; TBI: tuberculosis infection; HD: healthy donors; NTM: nontuberculous mycobacteria; Mtb: Mycobacterium tuberculosis; ESAT-6: early secretory antigenic target 6; CFP-10: culture filtrate protein 10; MS: mass spectrometry; NR: Not Reported; NA: Not Applicable.

Table 4. New strategies for detection of anti-M. tuberculosis antibodies.

Clinical Cohorts	HIV status	Antibodies	Mtb antigen	Results	Reference
TB, Recent TBI, Remote TBI, Non-TB	NR	IgG	ESAT-6, CFP10, Ag85A, MDP1, Acr	Mtb-specific IgGs were significantly higher in TB vs TBI and in Recent TBI vs Remote TBI	[61]
TB, TBI, Non-TB	NR	IgG	PPE17	Mtb-specific IgGs levels do not discriminate TBI from TB	[62]
TB, TBI, Non-TB	Infected, Not infected	IgG	PPE55	Mtb-specific IgGs were detected in 81% of HIV-infected prior TB development	[63]
TB, TBI	Infected, Not infected	IgG, IgA, IgM	209 Mtb antigens	Mtb-specific antigen-specific IgG and IgM levels and Fc receptor-binding characteristics discriminated TB from TBI	[65]
TB, TBI, HD	Not Infected	IgG	PPD	Mtb-specific antibody glycosylation pattern of TBI had less fucose and more galactose and acid sialic compared to TB	[66]
TB, TBI, HD	Not Infected	IgG	PPD, Ag85A, Ag85B, ESAT6, CFP10, GroES, glcB, HspX	Mtb-specific antibody glycosylation pattern differentiated TB from TBI and cured TB; Mtb-specific IgG4 antibodies correlated with TB	[68]

Note: Abbreviations: TB: tuberculosis; TBI: tuberculosis infection; HD: healthy donors; Mtb: Mycobacterium tuberculosis; ESAT-6: early secretory antigenic target 6; CFP-10: culture filtrate protein 10; PPD: purified protein derivative; NR: Not Reported.

Table 5. Host blood transcriptomic signatures for incipient TB diagnosis.

Country	Clinical Cohorts	HIV status	Sample type	Method	Signature name	Number of genes	Reference
South Africa, Gambia	TBI, TB contacts	Uninfected	WB	RNA sequencing, Multiplex qRT-PCR	Zak16	16	[72]
South Africa, Gambia, Ethiopia	TBI, TB contacts	Uninfected	WB	RNA sequencing qRT-PCR	RISK4	4	[73]
South Africa	TB, TBI	Uninfected	WB, PBMC	qRT-PCR	RISK11	11	[74]
South Africa	Recurrent TB, Treated TB, TBI	Infected, Uninfected	WB, PBMC	qRT-PCR	RISK11	11	[75]
South Africa	Past TB, TBI, HD	Uninfected	WB	qRT-PCR	RISK11	11	[76]
South Africa	TB, Subclinical TB, HD	Infected	WB	qRT-PCR	RISK11	11	[77]
South Africa, Gambia, Ethiopia, Brazil, Perù	TBI, TB contacts	Infected, Uninfected	WB	microfluidic qRT-PCR	RISK 6	6	[78]
Bangladesh, Georgia, Lebano, Madagascar	TB, TBI, HD	Uninfected	WB	microfluidic qRT-PCR	RISK 6	6	[79]

Note: Abbreviations: TB: tuberculosis; TBI: tuberculosis infection; HD: healthy donors; qRT-PCR: quantitative reverse transcription PCR; WB: whole blood.

Figure 1. Routine and research tests for TBI diagnosis. Diagnosis of TBI may be based on the detection of either host responses to Mtb antigens or of the pathogen itself. The routine immune-based tests currently used are the TST, new versions of EC-based skin tests, IGRA and its new developments. Other possible strategies are based on the detection of host factors such as the serological approach, for the detection of mycobacterial-specific antibodies, and the transcriptomic approach, which characterizes the host response in unstimulated blood, reflecting changes in gene expression between different disease states. On the other hand, microbiological tests able to identify Mtb itself are based on the detection of Mtb DNA or antigens from blood circulation. EC: ESAT-6/CFP-10; IGRA: interferon (IFN)-γ release assays; Mtb: Mycobacterium tuberculosis; NK: natural killer; TBI: tuberculosis infection; TST: tuberculin skin test. Created with BioRender.com.

Figure 2. Routine and research tests for the identification of TB progressors. TBI individuals are routinely screened by EC-based skin tests or IGRA that reveal host immune responses to Mtb, in absence of clinically and/or microbiological manifestations of TB disease. The 5–10% of TBI individuals can develop TB disease in their life span, and half of those develop the disease within the first 2 years from infection. Progressors from TBI to TB disease may be identified by host-based or pathogen-based diagnostic tests. EC: ESAT-6/CFP-10; IGRA: interferon (IFN)-γ release assays; Mtb: Mycobacterium tuberculosis; TBI: tuberculosis infection; TST: tuberculin skin test. Created with BioRender.com.

Das B, Kashino SS, Pulu I, et al. CD271 + bone marrow mesenchymal stem cells may provide a niche for dormant mycobacterium tuberculosis. Sci Transl Med. 2013;5(170):170ra13. doi: 10.1126/scitranslmed.3004912

 PubMed Web of Science ®Google Scholar

Tornack J, Reece ST, Bauer WM, et al. Human and mouse hematopoietic stem cells are a depot for dormant mycobacterium tuberculosis. Plos One. 2017;12(1):e0169119. doi: 10.1371/journal.pone.0169119

 PubMed Web of Science ®Google Scholar

Belay M, Tulu B, Younis S, et al. Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study. Lancet Microbe. 2021;2(6):e267–e275. doi: 10.1016/S2666-5247(21)00043-4

 PubMedGoogle Scholar

Verma R, Swift BMC, Handley-Hartill W, et al. A novel, high-sensitivity, bacteriophage-based assay identifies low-level mycobacterium tuberculosis bacteremia in immunocompetent patients with active and incipient tuberculosis. Clin Infect Dis Off Publ Infect Dis Soc Am. 2020;70:933–936. doi: 10.1093/cid/ciz548

 PubMed Web of Science ®Google Scholar

Pan S-W, Su W-J, Chan Y-J, et al. Mycobacterium tuberculosis–derived circulating cell-free DNA in patients with pulmonary tuberculosis and persons with latent tuberculosis infection. Plos One. 2021;16(6):e0253879. doi: 10.1371/journal.pone.0253879

 PubMed Web of Science ®Google Scholar

Li J, Wang Y, Yan L, et al. Novel serological biomarker panel using protein microarray can distinguish active TB from latent TB infection. Microbes Infect. 2022;24(8):105002. doi: 10.1016/j.micinf.2022.105002

 PubMed Web of Science ®Google Scholar

Shu Q, Liu S, Alonzi T, et al. Assay design for unambiguous identification and quantification of circulating pathogen-derived peptide biomarkers. Theranostics. 2022;12(6):2948–2962. doi: 10.7150/thno.70373

 PubMed Web of Science ®Google Scholar

Liu C, Zhao Z, Fan J, et al. Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring. Proc Natl Acad Sci U S A. 2017;114(15):3969–3974. doi: 10.1073/pnas.1621360114

 PubMed Web of Science ®Google Scholar

Kruh-Garcia NA, Wolfe LM, Chaisson LH, et al. Detection of mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS. Plos One. 2014;9(7):e103811. doi: 10.1371/journal.pone.0103811

 PubMed Web of Science ®Google Scholar

Chen H, Li S, Zhao W, et al. A peptidomic approach to identify novel antigen biomarkers for the diagnosis of tuberculosis. Infect Drug Resist. 2022;15:4617–4626. doi: 10.2147/IDR.S373652

 PubMed Web of Science ®Google Scholar

Maekura R, Kitada S, Osada-Oka M, et al. Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection. Microbiol Immunol. 2019;63(3–4):130–138. doi: 10.1111/1348-0421.12674

 PubMed Web of Science ®Google Scholar

Abraham PR, Devalraju KP, Jha V, et al. PPE17 (Rv1168c) protein of Mycobacterium tuberculosis detects individuals with latent TB infection. Plos One. 2018;13(11):e0207787. doi: 10.1371/journal.pone.0207787

 PubMed Web of Science ®Google Scholar

Singh KK, Dong Y, Patibandla SA, et al. Immunogenicity of the Mycobacterium tuberculosis PPE55 (Rv3347c) protein during incipient and clinical tuberculosis. Infect Immun. 2005;73(8):5004–5014. doi: 10.1128/IAI.73.8.5004-5014.2005

 PubMed Web of Science ®Google Scholar

Nziza N, Cizmeci D, Davies L, et al. Defining discriminatory antibody fingerprints in active and latent tuberculosis. Front Immunol. 2022;13:856906. doi: 10.3389/fimmu.2022.856906

 PubMed Web of Science ®Google Scholar

Lu LL, Chung AW, Rosebrock TR, et al. A functional role for antibodies in tuberculosis. Cell. 2016;167(2):433–443.e14. doi: 10.1016/j.cell.2016.08.072

 PubMed Web of Science ®Google Scholar

Grace PS, Dolatshahi S, Lu LL, et al. Antibody subclass and glycosylation shift following effective TB treatment. Front Immunol. 2021;12:679973. DOI:10.3389/fimmu.2021.679973

 PubMed Web of Science ®Google Scholar

Zak DE, Penn-Nicholson A, Scriba TJ, et al. A blood RNA signature for tuberculosis disease risk: a prospective cohort study. Lancet Lond Engl. 2016;387:2312–2322.

 PubMed Web of Science ®Google Scholar

Suliman S, Thompson EG, Sutherland J, et al. Four-gene pan-african blood signature predicts progression to tuberculosis. Am J Respir Crit Care Med. 2018;197(9):1198–1208. doi: 10.1164/rccm.201711-2340OC

 PubMed Web of Science ®Google Scholar

Darboe F, Mbandi SK, Thompson EG, et al. Diagnostic performance of an optimized transcriptomic signature of risk of tuberculosis in cryopreserved peripheral blood mononuclear cells. Tuberculosis. 2018;108:124–126. doi: 10.1016/j.tube.2017.11.001

 PubMed Web of Science ®Google Scholar

Darboe F, Mbandi SK, Naidoo K, et al. Detection of tuberculosis recurrence, diagnosis and treatment response by a blood transcriptomic risk signature in HIV-Infected persons on antiretroviral therapy. Front Microbiol. 2019;10:1441. doi: 10.3389/fmicb.2019.01441

 PubMed Web of Science ®Google Scholar

Scriba TJ, Fiore-Gartland A, Penn-Nicholson A, et al. Biomarker-guided tuberculosis preventive therapy (CORTIS): a randomised controlled trial. Lancet Infect Dis. 2021;21(3):354–365. doi: 10.1016/S1473-3099(20)30914-2

 PubMed Web of Science ®Google Scholar

Mendelsohn SC, Fiore-Gartland A, Penn-Nicholson A, et al. Validation of a host blood transcriptomic biomarker for pulmonary tuberculosis in people living with HIV: a prospective diagnostic and prognostic accuracy study. Lancet Glob Health. 2021;9(6):e841–e853. doi: 10.1016/S2214-109X(21)00045-0

 PubMed Web of Science ®Google Scholar

Penn-Nicholson A, Mbandi SK, Thompson E, et al. RISK6, a 6-gene transcriptomic signature of TB disease risk, diagnosis and treatment response. Sci Rep. 2020;10(1):8629. doi: 10.1038/s41598-020-65043-8

 PubMed Web of Science ®Google Scholar

Bayaa R, Ndiaye MDB, Chedid C, et al. Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring. Sci Rep. 2021;11(1):13646. doi: 10.1038/s41598-021-93059-1

 PubMed Web of Science ®Google Scholar