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A Mycobacterium tuberculosis Dormancy Antigen Differentiates Latently Infected Bacillus Calmette-Guérin-vaccinated Individuals.

Peña D, Rovetta AI, Hernández Del Pino RE, Amiano NO, Pasquinelli V, Pellegrini JM, Tateosian NL, Rolandelli A, Gutierrez M, Musella RM, Palmero DJ, Gherardi MM, Iovanna J, Chuluyan HE, García VE - EBioMedicine (2015)

Bottom Line: LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD.Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients.ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química Biológica, Facultad de Ciencias Exactas y Naturales (IQUIBICEN), UBA (Universidad de Buenos Aires)-CONICET, Intendente Güiraldes 2160, Pabellón II, 4°piso, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina ; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Intendente Güiraldes 2160, Pabellón II, 4°piso, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina.

ABSTRACT
IFN-γ release assays (IGRAs) are better indicators of Mycobacterium tuberculosis infection than the tuberculin skin test (TST) in Bacillus Calmette-Guérin (BCG)-vaccinated populations. However, IGRAs do not discriminate active and latent infections (LTBI) and no gold standard for LTBI diagnosis is available. Thus, since improved tests to diagnose M. tuberculosis infection are required, we assessed the efficacy of several M. tuberculosis latency antigens. BCG-vaccinated healthy donors (HD) and tuberculosis (TB) patients were recruited. QuantiFERON-TB Gold In-Tube, TST and clinical data were used to differentiate LTBI. IFN-γ production against CFP-10, ESAT-6, Rv2624c, Rv2626c and Rv2628 antigens was tested in peripheral blood mononuclear cells. LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD. Additionally, Rv2626c peptide pools to which only LTBI responded were identified, and their cumulative IFN-γ response improved LTBI discrimination. Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients. ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients. Therefore, since only LTBI recognizes specific epitopes from Rv2626c, this antigen could improve LTBI diagnosis, even in BCG-vaccinated people.

No MeSH data available.


Related in: MedlinePlus

IFN-γ production against M. tuberculosis antigens in PBMCs and whole blood from non-TB individuals and TB patients.(A) Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; N = 45), patients with tuberculosis (TB; N = 56) and latently M. tuberculosis infected individuals (LTBI; N = 56) were cultured with Rv2626c or CFP-10 + ESAT-6 for 5 days. Then, cell free supernatants were recovered and IFN-γ production was evaluated by ELISA. (B) Whole blood from HD (N = 28), TB patients (N = 20) and LTBI individuals (N = 25) was cultured with Rv2626c or CFP-10 + ESAT-6 for 24 h. Afterwards, plasma samples were collected and IFN-γ production was evaluated by ELISA. (A–B) Bars represent the Mean ± SEM. Mann–Whitney test for unpaired samples. (C–D) ROC curve analyses for evaluation of the predictive value of whole blood IFN-γ levels produced in response to (C) Rv2626c or (D) CFP-10 + ESAT-6, for differentiating (C) LTBI individuals from non-LTBI individuals (HD or TB), or (D) infected individuals (LTBI or active TB) from HD.Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve.
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f0010: IFN-γ production against M. tuberculosis antigens in PBMCs and whole blood from non-TB individuals and TB patients.(A) Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; N = 45), patients with tuberculosis (TB; N = 56) and latently M. tuberculosis infected individuals (LTBI; N = 56) were cultured with Rv2626c or CFP-10 + ESAT-6 for 5 days. Then, cell free supernatants were recovered and IFN-γ production was evaluated by ELISA. (B) Whole blood from HD (N = 28), TB patients (N = 20) and LTBI individuals (N = 25) was cultured with Rv2626c or CFP-10 + ESAT-6 for 24 h. Afterwards, plasma samples were collected and IFN-γ production was evaluated by ELISA. (A–B) Bars represent the Mean ± SEM. Mann–Whitney test for unpaired samples. (C–D) ROC curve analyses for evaluation of the predictive value of whole blood IFN-γ levels produced in response to (C) Rv2626c or (D) CFP-10 + ESAT-6, for differentiating (C) LTBI individuals from non-LTBI individuals (HD or TB), or (D) infected individuals (LTBI or active TB) from HD.Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve.

Mentions: Since we classified our population using QFT-GIT assay, and considering that this test uses whole blood for more practical and faster testing, we also analyzed the response to Rv2626c stimulating whole blood. Given that both current IGRAs discriminate M. tuberculosis infection (active or latent) from non-infected individuals, we also examined the response of active TB patients (Table 4). We first investigated the response of PBMCs from TB patients, LTBI and HD to Rv2626c and CFP-10 + ESAT-6 (Fig. 2A). We found that, as expected, both active and latently infected individuals (TB patients and LTBI) secreted significantly higher IFN-γ amounts in response to CFP-10 + ESAT-6 as compared to HD. Interestingly, we observed that stimulation with Rv2626c allowed the discrimination between active and latent infections (Fig. 2A) since TB patients did not secrete IFN-γ against this antigen (Fig. 2A), in sharp contrast to the results obtained with CFP-10 + ESAT-6. When we performed our experiments in whole blood, we obtained similar results to those observed in PBMCs: LTBI subjects secreted significantly higher amounts of IFN-γ in response to Rv2626c than HD. In addition, like in PBMCs (Fig. 2A), we observed that TB patients did not produce IFN-γ against Rv2626c (Fig. 2B), in sharp contrast to the results obtained with CFP-10 + ESAT-6 (Fig. 2B). Furthermore, the ROC analysis for IFN-γ responses to Rv2626c in whole blood reinforced the potential of Rv2626c antigen for discriminating LTBI individuals from non-LTBI individuals (patients with active TB or HD; Fig. 2C; AUC, 0.86; p < 0.001; 95% CI: 0.78–0.93). Additionally, by using the data obtained in whole blood and the Youden index (Youden, 1950, Perkins and Schisterman, 2006), we were able to establish an optimal cut off point of > 63.35 pg/ml of IFN-γ, with 78.26% sensitivity and 89.36% specificity. For comparison, Fig. 2D also shows the ROC curve obtained after analyzing the IFN-γ response to CFP-10 + ESAT-6 for the discrimination of M. tuberculosis infected individuals (LTBI + TB patients) versus HD. In conclusion, our findings show that the response to Rv2626c might be useful to differentiate between HD and LTBI, similar to ESAT-6 and CFP-10, antigens employed in IGRAs like QFT-GIT. However, in contrast to QFT-GIT assay, stimulation with Rv2626c would also allow the discrimination between active and latent infections. Therefore, our results indicate that Rv2626c might be useful not only to diagnose LTBI, but also, to differentiate latent from active M. tuberculosis infection.


A Mycobacterium tuberculosis Dormancy Antigen Differentiates Latently Infected Bacillus Calmette-Guérin-vaccinated Individuals.

Peña D, Rovetta AI, Hernández Del Pino RE, Amiano NO, Pasquinelli V, Pellegrini JM, Tateosian NL, Rolandelli A, Gutierrez M, Musella RM, Palmero DJ, Gherardi MM, Iovanna J, Chuluyan HE, García VE - EBioMedicine (2015)

IFN-γ production against M. tuberculosis antigens in PBMCs and whole blood from non-TB individuals and TB patients.(A) Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; N = 45), patients with tuberculosis (TB; N = 56) and latently M. tuberculosis infected individuals (LTBI; N = 56) were cultured with Rv2626c or CFP-10 + ESAT-6 for 5 days. Then, cell free supernatants were recovered and IFN-γ production was evaluated by ELISA. (B) Whole blood from HD (N = 28), TB patients (N = 20) and LTBI individuals (N = 25) was cultured with Rv2626c or CFP-10 + ESAT-6 for 24 h. Afterwards, plasma samples were collected and IFN-γ production was evaluated by ELISA. (A–B) Bars represent the Mean ± SEM. Mann–Whitney test for unpaired samples. (C–D) ROC curve analyses for evaluation of the predictive value of whole blood IFN-γ levels produced in response to (C) Rv2626c or (D) CFP-10 + ESAT-6, for differentiating (C) LTBI individuals from non-LTBI individuals (HD or TB), or (D) infected individuals (LTBI or active TB) from HD.Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve.
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f0010: IFN-γ production against M. tuberculosis antigens in PBMCs and whole blood from non-TB individuals and TB patients.(A) Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD; N = 45), patients with tuberculosis (TB; N = 56) and latently M. tuberculosis infected individuals (LTBI; N = 56) were cultured with Rv2626c or CFP-10 + ESAT-6 for 5 days. Then, cell free supernatants were recovered and IFN-γ production was evaluated by ELISA. (B) Whole blood from HD (N = 28), TB patients (N = 20) and LTBI individuals (N = 25) was cultured with Rv2626c or CFP-10 + ESAT-6 for 24 h. Afterwards, plasma samples were collected and IFN-γ production was evaluated by ELISA. (A–B) Bars represent the Mean ± SEM. Mann–Whitney test for unpaired samples. (C–D) ROC curve analyses for evaluation of the predictive value of whole blood IFN-γ levels produced in response to (C) Rv2626c or (D) CFP-10 + ESAT-6, for differentiating (C) LTBI individuals from non-LTBI individuals (HD or TB), or (D) infected individuals (LTBI or active TB) from HD.Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve.
Mentions: Since we classified our population using QFT-GIT assay, and considering that this test uses whole blood for more practical and faster testing, we also analyzed the response to Rv2626c stimulating whole blood. Given that both current IGRAs discriminate M. tuberculosis infection (active or latent) from non-infected individuals, we also examined the response of active TB patients (Table 4). We first investigated the response of PBMCs from TB patients, LTBI and HD to Rv2626c and CFP-10 + ESAT-6 (Fig. 2A). We found that, as expected, both active and latently infected individuals (TB patients and LTBI) secreted significantly higher IFN-γ amounts in response to CFP-10 + ESAT-6 as compared to HD. Interestingly, we observed that stimulation with Rv2626c allowed the discrimination between active and latent infections (Fig. 2A) since TB patients did not secrete IFN-γ against this antigen (Fig. 2A), in sharp contrast to the results obtained with CFP-10 + ESAT-6. When we performed our experiments in whole blood, we obtained similar results to those observed in PBMCs: LTBI subjects secreted significantly higher amounts of IFN-γ in response to Rv2626c than HD. In addition, like in PBMCs (Fig. 2A), we observed that TB patients did not produce IFN-γ against Rv2626c (Fig. 2B), in sharp contrast to the results obtained with CFP-10 + ESAT-6 (Fig. 2B). Furthermore, the ROC analysis for IFN-γ responses to Rv2626c in whole blood reinforced the potential of Rv2626c antigen for discriminating LTBI individuals from non-LTBI individuals (patients with active TB or HD; Fig. 2C; AUC, 0.86; p < 0.001; 95% CI: 0.78–0.93). Additionally, by using the data obtained in whole blood and the Youden index (Youden, 1950, Perkins and Schisterman, 2006), we were able to establish an optimal cut off point of > 63.35 pg/ml of IFN-γ, with 78.26% sensitivity and 89.36% specificity. For comparison, Fig. 2D also shows the ROC curve obtained after analyzing the IFN-γ response to CFP-10 + ESAT-6 for the discrimination of M. tuberculosis infected individuals (LTBI + TB patients) versus HD. In conclusion, our findings show that the response to Rv2626c might be useful to differentiate between HD and LTBI, similar to ESAT-6 and CFP-10, antigens employed in IGRAs like QFT-GIT. However, in contrast to QFT-GIT assay, stimulation with Rv2626c would also allow the discrimination between active and latent infections. Therefore, our results indicate that Rv2626c might be useful not only to diagnose LTBI, but also, to differentiate latent from active M. tuberculosis infection.

Bottom Line: LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD.Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients.ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química Biológica, Facultad de Ciencias Exactas y Naturales (IQUIBICEN), UBA (Universidad de Buenos Aires)-CONICET, Intendente Güiraldes 2160, Pabellón II, 4°piso, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina ; Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Intendente Güiraldes 2160, Pabellón II, 4°piso, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina.

ABSTRACT
IFN-γ release assays (IGRAs) are better indicators of Mycobacterium tuberculosis infection than the tuberculin skin test (TST) in Bacillus Calmette-Guérin (BCG)-vaccinated populations. However, IGRAs do not discriminate active and latent infections (LTBI) and no gold standard for LTBI diagnosis is available. Thus, since improved tests to diagnose M. tuberculosis infection are required, we assessed the efficacy of several M. tuberculosis latency antigens. BCG-vaccinated healthy donors (HD) and tuberculosis (TB) patients were recruited. QuantiFERON-TB Gold In-Tube, TST and clinical data were used to differentiate LTBI. IFN-γ production against CFP-10, ESAT-6, Rv2624c, Rv2626c and Rv2628 antigens was tested in peripheral blood mononuclear cells. LTBI subjects secreted significantly higher IFN-γ levels against Rv2626c than HD. Additionally, Rv2626c peptide pools to which only LTBI responded were identified, and their cumulative IFN-γ response improved LTBI discrimination. Interestingly, whole blood stimulation with Rv2626c allowed the discrimination between active and latent infections, since TB patients did not secrete IFN-γ against Rv2626c, in contrast to CFP-10 + ESAT-6 stimulation that induced IFN-γ response from both LTBI and TB patients. ROC analysis confirmed that Rv2626c discriminated LTBI from HD and TB patients. Therefore, since only LTBI recognizes specific epitopes from Rv2626c, this antigen could improve LTBI diagnosis, even in BCG-vaccinated people.

No MeSH data available.


Related in: MedlinePlus