title: Rapid discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by fluorescence in situ hybridization (FISH) and two matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) platforms.
abstract: BACKGROUND
  Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level.
  
  
  METHODS
  A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction.
  
  
  RESULTS
  Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.
authors: Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Hamburg, Germany. Frickmann@bni-hamburg.de
authors: Hagen
authors: H
authors: Frickmann
authors: Martin
authors: M
authors: Christner
authors: Martina
authors: M
authors: Donat
authors: Anja
authors: A
authors: Berger
authors: Andreas
authors: A
authors: Essig
authors: Andreas
authors: A
authors: Podbielski
authors: Ralf Matthias
authors: RM
authors: Hagen
authors: Sven
authors: S
authors: Poppert
mesh_terms: D006190:Haemophilus / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006192:Haemophilus Infections / Q000175:diagnosis; D006193:Haemophilus influenzae / Q000235:genetics; D044135:Haemophilus parainfluenzae / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D012189:Retrospective Studies; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
references: J Infect Dis. 2007 Jan 1;195(1):81-9
references: 17152011
references: J Clin Microbiol. 2001 Apr;39(4):1553-8
references: 11283086
references: J Clin Microbiol. 2000 Feb;38(2):818-25
references: 10655391
references: J Clin Microbiol. 2007 Nov;45(11):3493-7
references: 17728469
references: J Microbiol Methods. 2000 Jul;41(2):85-112
references: 10991623
references: J Antimicrob Chemother. 2002 Dec;50 Suppl S2:59-73
references: 12556435
references: Environ Microbiol. 2008 Oct;10(10):2894-8
references: 18647333
references: J Clin Microbiol. 2010 Mar;48(3):900-7
references: 20053859
references: J Bacteriol. 1953 Jun;65(6):750-1
references: 13069455
references: Biomed Environ Sci. 2012 Jun;25(3):367-71
references: 22840589
references: J Bacteriol. 2009 Feb;191(3):822-31
references: 19060144
references: J Clin Microbiol. 2005 Aug;43(8):4070-5
references: 16081953
references: J Clin Microbiol. 1988 Aug;26(8):1524-34
references: 3262623
references: Infect Immun. 1995 Nov;63(11):4409-16
references: 7591078
references: Nat Protoc. 2009;4(5):732-42
references: 19390529
references: Auris Nasus Larynx. 2010 Oct;37(5):594-600
references: 20392581
references: Clin Infect Dis. 1997 Jun;24(6):1087-94
references: 9195062
references: J Clin Microbiol. 2011 Mar;49(3):1104-6
references: 21227988
references: J Clin Microbiol. 2008 Aug;46(8):2681-5
references: 18579712
references: Mol Microbiol. 2002 Dec;46(5):1367-80
references: 12453222
references: J Clin Microbiol. 2012 Jul;50(7):2462-5
references: 22573587
references: Eur J Microbiol Immunol (Bp). 2011 Dec;1(4):289-96
references: 24516735
references: J Clin Microbiol. 2007 Dec;45(12):4051-3
references: 17942653
references: J Clin Microbiol. 2007 Oct;45(10):3207-17
references: 17687018
references: Microbiology (Reading). 2006 Dec;152(Pt 12):3683-3690
references: 17159221
references: J Clin Microbiol. 2000 Feb;38(2):830-8
references: 10655393
references: Nucleic Acids Res. 2004 Feb 25;32(4):1363-71
references: 14985472
references: Appl Environ Microbiol. 1990 Jun;56(6):1919-25
references: 2200342
references: Eur Arch Otorhinolaryngol. 2009 Mar;266(3):315-23
references: 18985371
references: Acta Pathol Microbiol Scand B Microbiol Immunol. 1974 Dec;82(6):835-42
references: 4533223
references: Br J Biomed Sci. 2012;69(1):37-9
references: 22558805
references: Mol Biol Evol. 1996 Mar;13(3):451-61
references: 8742634
references: J Infect Dis. 1995 Jul;172(1):132-5
references: 7797903
references: J Mass Spectrom. 1998 Aug;33(8):750-6
references: 9745723
references: J Clin Microbiol. 2010 Mar;48(3):946-8
references: 20071555
references: Int J Med Microbiol. 2011 Jan;301(1):64-8
references: 20728405
references: J Clin Microbiol. 2006 Nov;44(11):4186-8
references: 17088371
references: J Clin Microbiol. 2005 Jul;43(7):3390-7
references: 16000464
references: Braz J Infect Dis. 2001 Apr;5(2):87-97
references: 11493414
references: PLoS One. 2012;7(1):e30150
references: 22291908
references: J Clin Microbiol. 2010 Jul;48(7):2557-9
references: 20463165
references: J Clin Microbiol. 1994 Jan;32(1):187-9
references: 8126177
references: Trop Med Int Health. 2012 Sep;17(9):1117-26
references: 22776353
references: J Med Microbiol. 1984 Aug;18(1):1-16
references: 6146721
references: Mycoses. 2012 May;55(3):e114-23
references: 22126597
references: J Clin Microbiol. 2008 Feb;46(2):406-16
references: 18039799
references: J Clin Microbiol. 2000 Jan;38(1):125-32
references: 10618075
references: Diagn Microbiol Infect Dis. 2009 Aug;64(4):366-73
references: 19446978
references: BMC Bioinformatics. 2006 May 04;7:240
references: 16672074
references: Eur J Clin Microbiol Infect Dis. 2002 Mar;21(3):196-203
references: 11957021