Identification of new diagnostic biomarkers for Mycobacterium tuberculosis and the potential application in the serodiagnosis of human tuberculosis

Abstract

Summary Mycobacterium tuberculosis (M. tuberculosis) regions of difference (RD) encode proteins which are potentially useful as diagnostic reagents for tuberculosis (TB). In this study, 75 genes from M. tuberculosis RD1‐RD16 were successfully cloned from which 68 proteins were expressed and purified. Three serum pools from patients with pulmonary TB (PTB), extra‐pulmonary tuberculosis (EPTB) and healthy controls (HC) were used to preliminarily screen individual RD proteins. The OD 630 ratio of the PTB or EPTB to the HC group ≥ 2‐fold was positive. As a result, 29 proteins were obtained. The serological response to the identified antigens was further verified using 58 PTB samples with 38 sera from smear‐positive PTB (PTB‐SP) patients and 20 sera from smear‐negative PTB (PTB‐SN) patients, 16 EPTB samples, 42 latent M. tuberculosis infection samples and 40 HCs by indirect ELISA. With respect to the PTB diagnosis, receiver operating characteristic analysis showed that Rv0222 [area under the curve (AUC), 0.8129; 95% confidence interval (CI), 0.7280–0.8979] and Rv3403c (AUC, 0.8537; 95% CI, 0.7779–0.9294) performed better than ESAT6/CFP10 (AUC, 0.7435; 95% CI, 0.6465–0.8406). Rv0222 and Rv3403c demonstrated the highest diagnostic ability in the PTB‐SP group (sensitivity, 86.8%; specificity, 80%), while Rv3403c demonstrated the highest diagnostic ability in the PTB‐SN group (sensitivity, 70%; specificity, 80%). With respect to the EPTB diagnosis, Rv0222 exhibited the highest diagnostic value (AUC, 0.7523; sensitivity, 68.8%; specificity, 87.5%). In addition, the combination of Rv0222 and Rv3403c improved the test for PTB‐SN. These results indicate that Rv0222 and Rv3403c would be potential diagnostic biomarkers for active TB serodiagnosis. Mouse experiments demonstrated that Rv0222 and Rv3403c elicited specific cellular and humoral responses which were characterized by production of IFN‐γ, IgG1, and IgG2a, but a higher level of IgG1 than IgG2a.

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection

“…Several individuals in the recent LTBI group had high antibody titers against Ag85A or Acr. The antibody titers to DosS, HBHA, HrpA, and Ag85B were under the detection limits in most participants in this study; however, other useful biomarkers for detecting LTBI, such as Mtb proteins Rv0222 and Rv3403c, Mtb thymidylate kinase antigen, and LAM, have been reported . ESTA6 and Ag85A are expressed in growing bacilli, and Acr and MDP1 are upregulated in the stationary to dormant phases.…”

“…The antibody titers to DosS, HBHA, HrpA, and Ag85B were under the detection limits in most participants in this study; however, other useful biomarkers for detecting LTBI, such as Mtb proteins Rv0222 and Rv3403c, Mtb thymidylate kinase antigen, and LAM, have been reported. [12][13][14][15] ESTA6 and Ag85A are expressed in growing bacilli, and Acr and MDP1 are upregulated in the stationary to dormant phases. The antibody responses against these antigens, which are associated with different growth states and correlated with bacterial burden, [16][17][18] were strongest in patients with active TB, followed by those with recent LTBI (Figure 2).…”

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep

“…The accuracy of each test was determined on the basis of AUC. The cut-off values were generated using the point of maximum of combined specificity and sensitivity for each ROC curve (Ren et al, 2018). The validation parameters were calculated as described by de Oliveira et al (2011).…”

Biomarcadores da tuberculose: uma revisão da literatura.

“…A pesquisa de anticorpos se mostrou pouco interessante também nos esforços de Ren et al (2018). Na pesquisa da resposta humoral aos antígenos Rv0222c e Rv3403c descreveu dinâmicas similares as citadas anteriormente em pacientes humanos (sensibilidade e especificidade no intervalo de 70% a 80% individualmente, melhorando com a união dos analitos, porém com progresso pouco expressivo).…”