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

<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

Distribution of <i>Haemophilus</i> spp. isolates by clinical material.

<p>Distribution of <i>Haemophilus</i> spp. isolates by clinical material.</p

A comparison between formic acid extraction (FAE) and direct sample deposition (DSD) spectra for selected study isolates.

<p>Inscribed numbers denote the Biotyper identification score. Scores for <i>H. haemolyticus</i> (marked with an asterisk) have been obtained from comparison with an in-house reference spectrum. Although reproducible spectral differences could be observed for some isolates (highlighted with triangles in the topmost spectrum pair), mean identification scores for both techniques did not differ significantly.</p

Formic acid extraction triplicate spectra from the six <i>H. parainfluenzae</i> study isolates with the highest (top) and lowest (bottom) MALDI Biotyper identification scores.

<p>Inscribed numbers denote the highest score from triplicate measurements. Scores ≥2.0 constitute species-level identification. Lower-scoring spectra show reduced signal intensity and signal-to-noise ratio, resulting in reduced peak detection rates. For non-<i>H. haemolyticus</i> study isolates, failed species-level identification with the Bruker mass spectrometry fingerprinting system could be attributed to suboptimal spectrum quality rather than insufficient database coverage.</p

Spectral similarity between consensus spectra (MSPs) from formic acid extraction triplicate spectra of study isolates.

<p>The Biotyper 2.1 software with default parameter settings has been used for MSP creation and dendrogram calculation. Spectra of the four investigated <i>Haemophilus</i> species formed distinct clusters. Closest similarity was observed between <i>H. influenzae</i> and <i>H. haemolyticus</i> isolates. (a. U.  =  arbitrary units).</p

Combined presentation of misleading or contradictory patterns of diagnostic results from 29 out of 77 clinical <i>Haemophilus</i> spp. isolates, 4 well-characterized <i>H. haemophilus</i> strains (donation from the University of Aarhus) and 1 <i>Haemophilus parahaemolyticus</i> strain from a strain collection.

<p>Sequencing of the 16S rRNA gene of clinical <i>Haemophilus</i> spp. isolates and strains from strain collections was done if the best match for any of the diagnostic procedures performed led to misleading or contradictory results. The well-characterized <i>H. haemolyticus</i> strains that were donated by the University of Aarhus were not sequenced. (“No ID” indicates that the diagnostic procedure did not even allow for identification to genus level).</p><p><b>Bold type</b> indicates misleading or contradictory diagnostic results. For clarity, the table presents only best matches without percentages indicating the reliability of each diagnostic result.</p

Perfect matches, borderline matches, and no matching by FISH.

<p>(a, d, g) Cy3-labeled species-specific probes. (b, e, h) FAM-labeled pan-eubacterial probe staining virtually all bacteria. (c, f, i) Non-intercalating DNA stain DAPI. (a–c) Perfect match of <i>H. haemolyticus</i> with a <i>H. haemolyticus</i> probe (Haha 16S1242). Fluorescence due to the species-specific probe is even higher than fluorescence due to the pan-eubacterial probe (intensity 5+). (d–f) Borderline match of <i>H. influenzae</i> with a <i>H. influenzae</i> probe/competitor probe combination (Hain 16S1253/Hain 16S1253comp). Fluorescence due to the species-specific probe is as high as fluorescence due to the pan-eubacterial probe for only few bacteria and is considerably lower for all other <i>H. influenzae</i> bacteria in a pure culture (intensity 3+). (g–i) No matching of <i>H. parahaemolyticus</i> with a probe with specificity for <i>H. influenzae</i>. Only slight autofluorescence is visible in the Cy3-channel (intensity 1+) compared with adequate fluorescence due to the pan-eubacterial probe.</p

Evaluation of the newly designed <i>Haemophilus</i> probes by FISH.

*<p>Non-<i>Haemophilus</i> negative control strains including isolates from the commensal or pathological flora of the upper respiratory tract including <i>Moraxella catarrhalis</i>, <i>Stenotrophomonas maltophilia</i>, <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, <i>Streptococcus anginosus</i>, <i>S. constellatus</i>, coagulase-negative <i>Staphylococcus</i> spp., <i>S. epidermidis</i>, and <i>Micrococcus luteus</i>. <b>Bold type</b> indicates incorrect binding. †Three out of 50 strains were missed.</p

Identification of <i>Haemophilus</i> spp. by FISH and two MALDI-TOF-MS procedures.

*<p>Non-<i>Haemophilus</i> negative control strains including isolates from the commensal or pathological flora of the upper respiratory tract including <i>Moraxella catarrhalis</i>, <i>Stenotrophomonas maltophilia</i>, <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, <i>Streptococcus anginosus</i>, <i>S. constellatus</i>, coagulase-negative <i>Staphylococcus</i> spp., <i>S. epidermidis</i>, and <i>Micrococcus luteus</i>.</p>†<p>Correct identification was possible after implementation of a newly established <i>H. haemolyticus</i> spectrum based on repeated measurements of the reference strain <i>H. haemolyticus</i> ATCC 33390.</p><p><b>Bold type</b> indicates misidentifications to species level.</p

Opposed spectra of 7 <i>H. influenzae</i> and 7 <i>H. haemolyticus</i> spectra in gel view.

<p>The comparison illustrates high overall spectral similarity between the species, with considerable intraspecies variability.</p