title: Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.
abstract: BACKGROUND
  A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group.
  
  
  METHODS
  Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory.
  
  
  RESULTS
  Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods.
  
  
  CONCLUSIONS
  The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.
authors.affiliation: a Department of Bacteriology and Immunology , Norwegian Institute of Public Health , Oslo , Norway ;
authors.forename: Audun
authors.initials: A
authors.lastname: Aase
authors.affiliation: b Institute of Parasitology, Biology Centre, Czech Academy of Sciences , Ceske Budejovice , Czech Republic ;
authors.forename: Ondrej
authors.initials: O
authors.lastname: Hajdusek
authors.affiliation: c Section for Virology , Norwegian Veterinary Institute , Oslo , Norway ;
authors.forename: Øivind
authors.initials: Ø
authors.lastname: Øines
authors.affiliation: d Department of Medical Microbiology , Sørlandet Hospital Health Enterprise , Kristiansand , Norway ;
authors.forename: Hanne
authors.initials: H
authors.lastname: Quarsten
authors.affiliation: e Department of Clinical and Experimental Medicine, Division of Medical Microbiology , Linköping University , Linköping , Sweden ;
authors.forename: Peter
authors.initials: P
authors.lastname: Wilhelmsson
authors.affiliation: a Department of Bacteriology and Immunology , Norwegian Institute of Public Health , Oslo , Norway ;
authors.forename: Tove K
authors.initials: TK
authors.lastname: Herstad
authors.affiliation: f Department of Engineering and Science , University of Agder , Kristiansand , Norway ;|g Research Unit , Sørlandet Hospital Health Enterprise , Kristiansand , Norway ;
authors.forename: Vivian
authors.initials: V
authors.lastname: Kjelland
authors.affiliation: b Institute of Parasitology, Biology Centre, Czech Academy of Sciences , Ceske Budejovice , Czech Republic ;
authors.forename: Radek
authors.initials: R
authors.lastname: Sima
authors.affiliation: b Institute of Parasitology, Biology Centre, Czech Academy of Sciences , Ceske Budejovice , Czech Republic ;
authors.forename: Marie
authors.initials: M
authors.lastname: Jalovecka
authors.affiliation: e Department of Clinical and Experimental Medicine, Division of Medical Microbiology , Linköping University , Linköping , Sweden ;|h Medical Services , County Hospital Ryhov , Jönköping , Sweden.
authors.forename: Per-Eric
authors.initials: PE
authors.lastname: Lindgren
authors.affiliation: a Department of Bacteriology and Immunology , Norwegian Institute of Public Health , Oslo , Norway ;
authors.forename: Ingeborg S
authors.initials: IS
authors.lastname: Aaberge
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000097:blood* / Q000175:diagnosis / Q000469:parasitology; D001898:Borrelia / Q000235:genetics / Q000302:isolation & purification*; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D006801:Humans; D007223:Infant; D008193:Lyme Disease / Q000097:blood* / Q000175:diagnosis / Q000382:microbiology; D008853:Microscopy / Q000379:methods* / Q000592:standards; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D016054:DNA, Protozoan
keywords: Babesia spp.; Borrelia burgdorferi sensu lato; Lyme borreliosis; Lyme disease; PCR; babesiosis; microscopy