Rapid Discrimination of <i>Haemophilus influenzae</i>, <i>H. parainfluenzae</i>, and <i>H. haemolyticus</i> by Fluorescence <i>In Situ</i> Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

Abstract

<div><p>Background</p><p>Due to considerable differences in pathogenicity, <i>Haemophilus influenzae</i>, <i>H. parainfluenzae</i> and <i>H. haemolyticus</i> have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal <i>H. haemolyticus</i> as <i>H. influenzae</i>. In a multi-center approach, we assessed the suitability of fluorescence <i>in situ</i> hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of <i>H. influenzae</i>, <i>H. parainfluenzae</i> and <i>H. haemolyticus</i> to species level.</p><p>Methodology</p><p>A strain collection of 84 <i>Haemophilus</i> spp. comprising 50 <i>H. influenzae</i>, 25 <i>H. parainfluenzae</i>, 7 <i>H. haemolyticus</i>, and 2 <i>H. parahaemolyticus</i> 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.</p><p>Principal Findings</p><p>Among the 84 <i>Haemophilus</i> 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 <i>H. haemolyticus</i> 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.</p></div