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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.    Methodology  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.    Principal Findings  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.

plos one

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