Serodiagnostic efficacy of Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex, ESAT-6, and CFP-10 in patients with active tuberculosis

Abstract

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.

Fig. 1

Antibody reactivity to various secretory antigens of M. tuberculosis with sera derived from different clinical groups (FTB: fresh untreated, DTB: defaulters, RTB: relapsed, EPTB: extra-pulmonary TB) of tuberculosis patients and controls (HC: healthy controls, LP: leprosy patients). TtTB denotes the treated cases of tuberculosis. Each scatter represents a tested sample of serum, the dotted line denotes the cutoff decided by ROC

Fig. 2

Area under ROC of three significant antigens with sera from tuberculosis patients

Fig. 3

Lanes 1–5 of whole-cell extracts (WCEs) of clinical isolates of M. tuberculosis. a, b Blotting with BCG-vaccinated healthy individual’s sera. c–d Reactivity with individual tuberculosis patient’s sera. e Reactivity pattern of contact with tuberculosis. f 12% SDS–PAGE gel pattern of WCEs of clinical isolates and H37Rv. g Hybridization of polyclonal antibody against ESAT-6 and CFP-10 with whole-cell extracts. h Hybridization of monoclonal antibody (CS-90) against Ag85 complex with WCEs of clinical isolates and H37Rv. Rv: M. tuberculosis laboratory reference strain H37Rv; arrow shows the reactivity pattern of Ag85 complex