Serological diagnosis of pulmonary Mycobacterium tuberculosis infection by LIPS using a multiple antigen mixture

Abstract

BackgroundThere is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB). Here we describe a serological test based on light emitting recombinant proteins for the diagnosis of pulmonary Mycobacterium tuberculosis infection.MethodsLuciferase Immunoprecipitation Systems (LIPS), a fluid-phase immunoassay, was used to examine antibody responses against a panel of 24 different M. tuberculosis proteins. Three different strategies were used for generating the constructs expressing the recombinant fusion M. tuberculosis proteins with luciferase: synthetic gene synthesis, Gateway recombination cloning, and custom PCR synthesis. A pilot cohort of African pulmonary TB patients was used for initial antibody screening and confirmatory studies with selected antigens were performed with a cohort from Thailand and healthy US blood donors. In addition to testing M. tuberculosis antigens separately, a mixture that tested seven antigens simultaneously was evaluated for diagnostic performance.ResultsLIPS testing of a pilot set of serum samples from African pulmonary TB patients identified a potential subset of diagnostically useful M. tuberculosis antigens. Evaluation of a second independent cohort from Thailand validated highly significant antibody responses against seven antigens (PstS1, Rv0831c, FbpA, EspB, bfrB, HspX and ssb), which often showed robust antibody levels up to 50- to 1000-fold higher than local community controls. Marked heterogeneity of antibody responses was observed in the patients and the combined results demonstrated 73.5 % sensitivity and 100 % specificity for detection of pulmonary TB. A LIPS test simultaneously employing the seven M. tuberculosis antigen as a mixture matched the combined diagnostic performance of the separate tests, but showed an even higher diagnostic sensitivity (90 %) when a cut-off based on healthy US blood donors was used.ConclusionA LIPS immunoassay employing multiple M. tuberculosis antigens shows promise for the rapid and quantitative serological detection of pulmonary TB.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0545-y) contains supplementary material, which is available to authorized users.

Evaluation of antibody responses to panels of M. tuberculosis antigens as a screening tool for active tuberculosis in Uganda

“…In contrast, our results are largely in agreement with prior studies that have evaluated multiplex serologic testing[ 10 , 25 , 26 ]. Antibody responses in a Brazilian cohort using a panel of 7 antigens (including Rv0934 which also had high variable importance in our study) demonstrated high sensitivity (93%) and specificity (87%) in detecting active TB when compared to TB negative controls from a non-endemic environment[ 26 ].…”

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays

“…Consistent with other studies, we observed a high level of heterogeneity in Ab responses to Mtb proteins, including those with biomarker value (6,7,9,10,31). Hence, our data are consistent with the notion that a single biomarker will not be sufficient for distinguishing TB status in various patient groups (6,32).…”

“…The cellular location for two of the proteins (Rv3405c and Rv3544c) has not been identi- fied. All of the four CFPs (Rv0054 (9, 10, 41), Rv0831c (9, 42), Rv2031c (9,10,43,44) and Rv0222 (45,46)), have already been reported in TB serology studies. Rv2031c (hspX) was also previously identified using a Mtb proteomic level microarray with printing of unpurified Mtb proteins generated in E. coli lysate (6).…”

“…Because of the potential to turn Ab detection assays into simple dipstick formats, TB serology, despite its known limitations, remains a field of study that is worthwhile pursuing further and new biomarker targets need to be identified. The simultaneous use of multiple more recently identified Mtb proteins in form of multiplex microbead immunoassays has already shown promising improved accuracy for TB serodiagnosis in regional case-control studies (9,10). Although the World Health Organization recognizes the limitations of currently available serologic tests and in fact cautions against using them (11), it vigorously encourages further research to meet the need for reliable, simple tests for TB in endemic regions.…”

Identification of Novel Antigens Recognized by Serum Antibodies in Bovine Tuberculosis

“…a High nonspecific binding was found with the following fusion proteins (which were excluded from the data analyses): Acr1-MPT83 (Rv2031-Rv2873), DID64 (Rv2031-Rv0934-Rv387), DID82 (Rv1980c-Rv3619-Rv38814), DID88 (Rv2873-Rv1980c-Rv3881), DID90 (Rv2873-Rv0934-Rv2032), and DID99 (Rv1980c-Rv2108-Rv3881 Advances in genomic and proteomic research have made it possible to provide a large-scale characterization of TB biomarkers eliciting antibody responses in humans and nonhuman primates (21)(22)(23). The studies defined the M. tuberculosis immunoproteome to be approximately 10% of the bacterial proteome enriched for membraneassociated and extracellular proteins, which can lead to the development of more accurate serodiagnostic tools (24). Interestingly, the set of 13 serological targets found to be predominantly associated with active TB in humans (22) included only 2 proteins, Rv1980c and Rv2873, identified in the present study among the top 12 antigens recognized by antibodies in M. bovis-infected cattle.…”