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

Peter D Burbelo,Bassirou Diarra,Craig S Rhodes,Sophia Siddiqui,Sarah K Browne,Ploenchan Chetchotisakd,James S Klimavicz,Joseph A Kovacs,Ahmad Bayat,Jason Keller,Sasisopin Kiertiburanakul,Yupin Suputtamongkol,Steven M Holland,Jason Wagner

doi:10.1186/s12866-015-0545-y

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.

Highlights

There is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB)
Based on the literature [11,12,13,14, 17], a panel of twenty four different Mycobacterium tuberculosis (MTB) proteins was chosen for production as Renilla luciferase fusion proteins
Luciferase Immunoprecipitation Systems (LIPS) testing of MTB antigens generated by synthetic genes As described in the material and methods, nine of the twenty four constructs for expression of MTB proteins utilized synthetic gene synthesis

Summary

Introduction

There is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB). We describe a serological test based on light emitting recombinant proteins for the diagnosis of pulmonary Mycobacterium tuberculosis infection. Mycobacterium tuberculosis (MTB) infects more than one-third of the global population and is one of the world’s leading causes of mortality, resulting in approximately 1.7 million deaths annually [1]. Despite T- and B-cell mediated immunity against MTB, approximately 30 % of individuals develop latent, asymptomatic infection (LTBI) following primary infection. Prophylaxis for patients identified with latent MTB infection can greatly reduce the risk of subsequent active infection [4]. XpertMTB, a nucleic acid amplification test, shows high sensitivity and specificity for the diagnosis of active pulmonary disease including for detecting rifamycin resistance [6]. Tuberculin skin testing is used for detecting latent infection, but it has poor specificity and requires patients to return for evaluation. Interferon-γ release assays, which exploit T cell responses, are highly effective for detecting LTBI, yet these assays are technically complex and require several days to process [8]

Methods

Results

Conclusion