title: Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test.
abstract: BACKGROUND
  As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.
  
  
  RESULTS
  Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.
  
  
  CONCLUSIONS
  Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Beissner
authors.affiliation: Komfo Anokye Teaching Hospital (KATH), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana.
authors.forename: Richard Odame
authors.initials: RO
authors.lastname: Phillips
authors.affiliation: Battke Scientia GmbH, Taufkirchen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Battke
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Malkin
authors.initials: M
authors.lastname: Bauer
authors.affiliation: Institut National d'Hygiène (INH), Ministère de la Santé, Lomé, Togo.
authors.forename: Kossi
authors.initials: K
authors.lastname: Badziklou
authors.affiliation: Komfo Anokye Teaching Hospital (KATH), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana.
authors.forename: Fred Stephen
authors.initials: FS
authors.lastname: Sarfo
authors.affiliation: Institut National d'Hygiène (INH), Ministère de la Santé, Lomé, Togo.
authors.forename: Issaka
authors.initials: I
authors.lastname: Maman
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Agata
authors.initials: A
authors.lastname: Rhomberg
authors.affiliation: Centre Hospitalier Régional Maritime (CHR-Maritime), Tsévié, Togo.
authors.forename: Ebekalisai
authors.initials: E
authors.lastname: Piten
authors.affiliation: Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Komfo Anokye Teaching Hospital (KATH), Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana.
authors.forename: Michael
authors.initials: M
authors.lastname: Frimpong
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Kristina Lydia
authors.initials: KL
authors.lastname: Huber
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Symank
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Jansson
authors.affiliation: German Leprosy and Tuberculosis Relief Association, Togo office (DAHW-T), Lomé, Togo.
authors.forename: Franz Xaver
authors.initials: FX
authors.lastname: Wiedemann
authors.affiliation: Institut National d'Hygiène (INH), Ministère de la Santé, Lomé, Togo.
authors.forename: Abiba
authors.initials: A
authors.lastname: Banla Kere
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Herbinger
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Löscher
authors.affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Gisela
authors.initials: G
authors.lastname: Bretzel
mesh_terms: D000293:Adolescent; D000328:Adult; D054312:Buruli Ulcer / Q000175:diagnosis*; D002648:Child; D002675:Child, Preschool; D003430:Cross-Sectional Studies; D005260:Female; D005612:Freeze Drying; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019095:Point-of-Care Systems; D012680:Sensitivity and Specificity; D055815:Young Adult