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

ABSTRACT: 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 OD630 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-?, IgG1, and IgG2a, but a higher level of IgG1 than IgG2a.

SUBMITTER: Ren N

PROVIDER: S-EPMC6116745 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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

Ren Ningning N JinLi Jingfang J Chen Yingyu Y Zhou Xia X Wang Jieru J Ge Pan P Khan Farhan Anwar FA Zhang Li L Hu Changmin C Robertson Ian D ID Chen Huanchun H Guo Aizhen A

Microbial biotechnology 20180627 5

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<sub>630</sub> ratio of ...[more]

PMID: 29952084

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Project description:The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bovine tuberculosis in cattle. We have used the advantages of the murine model to identify promising candidates in the host transcriptome post-infection. RNA from BALB/c splenocytes and lung cells after aerogenic Mycobacterium bovis infection were with high-density microarrays to defining biomarkers of disease progression. In antigen-stimulated splenocytes we found statically significant modulation of 1109 genes early after infection and 1134 at later time-point post-infection. 618 of these genes were modulated at both time points. In the lung 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were granzyme A in spleen, and cxcl9, granzyme B, interleukin-22, interluekin-17A receptor, and ccr6 in lungs. The expression of 20 of the most up-regulated genes identified in the murine studies were evaluated using PBMC from uninfected and naturally infected cattle. We could show that the expression of cxcl9, granzyme A and interleukin-22 following in vitro stimulation with PPD was significantly increased in infected cows compared to naïve animals. Thus, we have demonstrated that murine transcriptome analysis can be used to predict responses in cattle. In addition, we have prioritised CXCL9, GnzA and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis. Two groups of 5 BALB/c mice each were infecetd with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested. One group of 5 mice were used as a control. Their splenocytes and lung cells were stimulated in vitro for 3 days with M7 protein pool. Antigen cell culture stimulations in mice were performed using an equal pool of seven secreted, immunogenic recombinant mycobacterial proteins (Rv1886c, Rv3019c, Rv3763, Rv3804c, Rv0251, Rv0287 and Rv0288) common to M. bovis and BCG, referred here as M7 protein cocktail. Each protein was used at final concentration of 2μg/ml in 3-day culture.

Project description:Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography-electrospray ionization-quadruple time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette-Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16?0/0?0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic biomarkers.

Project description:BackgroundNew diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB.MethodsRecombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals.ResultsA robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses.ConclusionThese results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.

Dataset Information

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

Publications

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

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