title: Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.
abstract: BACKGROUND
  Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings.
  
  
  METHODS
  The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs.
  
  
  RESULTS
  Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas.
  
  
  CONCLUSIONS
  Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands; Armauer Hansen Research Institute, Addis Ababa, Ethiopia.
authors.forename: Kidist
authors.initials: K
authors.lastname: Bobosha
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Elisa M
authors.initials: EM
authors.lastname: Tjon Kon Fat
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Susan J F
authors.initials: SJ
authors.lastname: van den Eeden
authors.affiliation: Armauer Hansen Research Institute, Addis Ababa, Ethiopia.
authors.forename: Yonas
authors.initials: Y
authors.lastname: Bekele
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jolien J
authors.initials: JJ
authors.lastname: van der Ploeg-van Schip
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Claudia J
authors.initials: CJ
authors.lastname: de Dood
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Karin
authors.initials: K
authors.lastname: Dijkman
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Kees L M C
authors.initials: KL
authors.lastname: Franken
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Wilson
authors.affiliation: Armauer Hansen Research Institute, Addis Ababa, Ethiopia.
authors.forename: Abraham
authors.initials: A
authors.lastname: Aseffa
authors.affiliation: Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: John S
authors.initials: JS
authors.lastname: Spencer
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Tom H M
authors.initials: TH
authors.lastname: Ottenhoff
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Paul L A M
authors.initials: PL
authors.lastname: Corstjens
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Annemieke
authors.initials: A
authors.lastname: Geluk
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology*; D016207:Cytokines / Q000097:blood* / Q000378:metabolism; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D007700:Kinetics; D007918:Leprosy / Q000175:diagnosis* / Q000276:immunology*; D009166:Mycobacterium leprae / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D016207:Cytokines