title: Serological diagnosis of pulmonary Mycobacterium tuberculosis infection by LIPS using a multiple antigen mixture.
abstract: BACKGROUND
  There 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.
  
  
  METHODS
  Luciferase 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.
  
  
  RESULTS
  LIPS 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.
  
  
  CONCLUSIONS
  A LIPS immunoassay employing multiple M. tuberculosis antigens shows promise for the rapid and quantitative serological detection of pulmonary TB.
authors.affiliation: Dental Clinical Research Core, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. burbelop@nidcr.nih.gov.|Dental Clinical Research Core, NIDCR, 10 Center Drive, Building 10, Rm. 5 N102/106, Bethesda, MD, 20892, USA. burbelop@nidcr.nih.gov.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Burbelo
authors.affiliation: Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. jmk451@gmail.com.
authors.forename: Jason
authors.initials: J
authors.lastname: Keller
authors.affiliation: Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. wagner.jason.m@gmail.com.
authors.forename: Jason
authors.initials: J
authors.lastname: Wagner
authors.affiliation: Dental Clinical Research Core, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. jamesk1@iastate.edu.
authors.forename: James S
authors.initials: JS
authors.lastname: Klimavicz
authors.affiliation: Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. ahmadbayat@hotmail.com.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Bayat
authors.affiliation: Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. rhodes@dir.nidcr.nih.gov.
authors.forename: Craig S
authors.initials: CS
authors.lastname: Rhodes
authors.affiliation: Project SEREFO, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako, Mali. bdiarra@icermali.org.
authors.forename: Bassirou
authors.initials: B
authors.lastname: Diarra
authors.affiliation: Khon Kaen University, Khon Kaen, Thailand. ploencha@kku.ac.th.
authors.forename: Ploenchan
authors.initials: P
authors.lastname: Chetchotisakd
authors.affiliation: Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. ysuputtamongkol@gmail.com.
authors.forename: Yupin
authors.initials: Y
authors.lastname: Suputtamongkol
authors.affiliation: Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. sasisopin.kie@mahidol.ac.th.
authors.forename: Sasisopin
authors.initials: S
authors.lastname: Kiertiburanakul
authors.affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA. SHOLLAND@niaid.nih.gov.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Holland
authors.affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA. Sarah.Browne@fda.hhs.gov.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Browne
authors.affiliation: Collaborative Clinical Research Branch, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, USA. SSIDDIQUI@niaid.nih.gov.
authors.forename: Sophia
authors.initials: S
authors.lastname: Siddiqui
authors.affiliation: Critical Care Medicine Department, NIH Clinical Center, National Institutes of Health, Bethesda, MD, 20892, USA. jkovacs@mail.nih.gov.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Kovacs
mesh_terms: D000349:Africa; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D015331:Cohort Studies; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008156:Luciferases / Q000032:analysis; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D010865:Pilot Projects; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*; D013785:Thailand; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*; D014481:United States
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D008156:Luciferases