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. 2021 Jun 30;10(7):828.
doi: 10.3390/pathogens10070828.

Use of Rv0222-Rv2657c-Rv1509 Fusion Protein to Improve the Accuracy of an Antibody ELISA for Extra-Pulmonary Tuberculosis in Humans

Yingyu Chen  1   2   3 , Pan Ge  1   2 , Kailun Zhang  1   2   3 , Jie Xiang  4 , Li Zhang  4 , Ian D Robertson  3   5 , Aizhen Guo  1   2   3
Affiliations

Use of Rv0222-Rv2657c-Rv1509 Fusion Protein to Improve the Accuracy of an Antibody ELISA for Extra-Pulmonary Tuberculosis in Humans

Yingyu Chen et al. Pathogens. .

Abstract

(1) Background: Tuberculosis (TB) in humans is a serious chronic epidemic disease caused by Mycobacterium tuberculosis (M. tb). The diagnosis of TB, especially extra-pulmonary TB (EPTB), is difficult. Isolation of M. tb from culture has a low sensitivity in patients with TB and an even lower sensitivity in cases of EPTB. Although Xpert MTB/RIF assays and serological tests are more sensitive than the above tests, they still lack sensitivity for EPTB diagnosis. (2) Methods: To improve the accuracy of TB diagnosis, a Rv0222-Rv2657c-Rv1509 fusion protein based iELISA was constructed, the diagnosis of TB, pulmonary TB (PTB) and EPTB was then evaluated. Sera of 40 TB patients including 14 with PTB, 14 with EPTB and 12 with no information about the form of TB, and five pneumonia patients were investigated. (3) Results: The sensitivity of the ELISA in TB, PTB and EPTB patients was 80% (95% CI: 64.4, 90.9%), 85.7% (95% CI: 57.2, 98.2%) and 92.8% (95% CI: 66.1, 99.8%), respectively, with a specificity of 70% (95% CI: 53.5, 83.4%). Both the sensitivity and specificity with this fusion protein were higher than for CFP10/ESAT6 (used as reference antigen) fusion protein (71.4%; 95% CI: 41.9, 91.6%, and 67.5%; 95% CI: 50.9, 81.4%), respectively, in cases of EPTB. All pneumonia patients' sera tested negative in both ELISAs. (4) Conclusion: use of these new fusion proteins as antigens in serological assays has the potential to improve the diagnosis of all forms of TB in humans, especially EPTB.

Keywords: diagnosis; extra-pulmonary tuberculosis; fusion protein; pulmonary tuberculosis; tuberculosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Purification of recombinant Rv0222-Rv2657c-Rv1509 protein. The arrow showed the protein Rv0222-Rv2657c-Rv1509. M: Protein Ladder; Lane 1: protein expression before induction; Lane 2: protein expression after induction; Lane 3: protein expressed in supernatant; Lane 4: protein expressed in precipitation; Lane 5: Protein in effluent; Lane 6: protein in binding buffer; Lane 7: protein in elution buffer; Lane 8: purified Rv0222-Rv2657c-Rv1509 protein after ultrafiltration.
Figure 2
Figure 2
ROC and scatter plots for antibody detection using sera from TB and HC against Rv0222-Rv2657c-Rv1509 and CE. (A) ROC of Rv0222-Rv2657c-Rv1509 for TB and HC, (B) Rv0222-Rv2657c-Rv1509 antibody detection of TB and HC, (C) ROC of CE for TB and HC, (D) CE antibody detection of TB and HC. Symbol “***” presents p < 0.001.
Figure 3
Figure 3
ROC and scatter plots for antibody detection using sera from PTB and HC against Rv0222-Rv2657c-Rv1509 and CE. (A) ROC of Rv0222-Rv2657c-Rv1509 for PTB and HC, (B) Rv0222-Rv2657c-Rv1509 antibody detection of PTB and HC, (C) ROC of CE for PTB and HC, (D) CE antibody detection of PTB and HC. Symbol “**” presents p < 0.01.
Figure 4
Figure 4
ROC and scatter plots for antibody detection using sera from EPTB and HC against Rv0222-Rv2657c-Rv1509 and CE. (A) ROC of Rv0222-Rv2657c-Rv1509 for EPTB and HC, (B) Rv0222-Rv2657c-Rv1509 antibody detection of EPTB and HC, (C) ROC of CE for ETB and HC, (D) CE antibody detection of PTB and HC. Symbol “**” presents p < 0.01.
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