title: Development of a dry-reagent-based qPCR to facilitate the diagnosis of Mycobacterium ulcerans infection in endemic countries.
abstract: BACKGROUND
  Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.
  
  
  RESULTS
  We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix.
  
  
  CONCLUSIONS
  Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.
authors.affiliation: ATOMycA, Inserm Avenir Team, CRCNA, Inserm U892, 6299 CNRS and LUNAM, CHU and Université d'Angers, Angers, France.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Babonneau
authors.affiliation: SR2B, Avrillé, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Bernard
authors.affiliation: Centre de diagnostic et de traitement de l'ulcère de Buruli, Pobè, Bénin.
authors.forename: Estelle
authors.initials: E
authors.lastname: Marion
authors.affiliation: Centre de diagnostic et de traitement de l'ulcère de Buruli, Pobè, Bénin.
authors.forename: Annick
authors.initials: A
authors.lastname: Chauty
authors.affiliation: Laboratoire de Bactériologie, Institut de Biologie en Santé-PBH, CHU d'Angers, Angers, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Kempf
authors.affiliation: SR2B, Avrillé, France.
authors.forename: Raymond
authors.initials: R
authors.lastname: Robert
authors.affiliation: ATOMycA, Inserm Avenir Team, CRCNA, Inserm U892, 6299 CNRS and LUNAM, CHU and Université d'Angers, Angers, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Marsollier
mesh_terms: D054312:Buruli Ulcer / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000302:isolation & purification; D005260:Female; D006801:Humans; D019911:Mycobacterium ulcerans / Q000235:genetics*; D058069:Neglected Diseases / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
references.citation: J Clin Microbiol. 2006 Nov;44(11):4273-5
references.pmid: 16928966
references.citation: Clin Infect Dis. 2007 Jul 1;45(1):68-75
references.pmid: 17554703
references.citation: Antimicrob Agents Chemother. 2007 Nov;51(11):4029-35
references.pmid: 17526760
references.citation: PLoS Negl Trop Dis. 2010;4(7):e731
references.pmid: 20625552
references.citation: PLoS Med. 2005 Apr;2(4):e108
references.pmid: 15839744
references.citation: Antimicrob Agents Chemother. 2005 Aug;49(8):3182-6
references.pmid: 16048922
references.citation: J Clin Microbiol. 2014 Dec;52(12):4398-400
references.pmid: 25320228
references.citation: J Clin Microbiol. 2003 Sep;41(9):4231-7
references.pmid: 12958250
references.citation: J Clin Microbiol. 1997 May;35(5):1097-100
references.pmid: 9114387
references.citation: J Clin Microbiol. 2005 Jan;43(1):271-6
references.pmid: 15634982
references.citation: Lancet Glob Health. 2014 Jul;2(7):e422-30
references.pmid: 25103396
references.citation: J Clin Microbiol. 2010 Jun;48(6):2263-4
references.pmid: 20375229
references.citation: Clin Infect Dis. 2011 Jan 1;52(1):94-6
references.pmid: 21148526