Sensitivity, specificity, PPV and NPV for <i>Streptococcus agalactiae</i> and <i>S. pneumoniae</i>.

<p>The study sample on which the calculation is based was represented by <i>Streptococcus</i> spp. only.</p

Classification accuracy and marker peak detection rates.

<p>Classification accuracy and marker peak detection rates.</p

NOREC marker peaks detected by A.B.O.S. analysis.

<p>NOREC marker peaks detected by A.B.O.S. analysis.</p

Agreement between FISH and 16S rDNA sequencing (SEQ, used as gold standard) in the experiments carried out with the Pan-Flavo (Flavo285) probe and the combination of two <i>F. psychrophilum</i> (FlavoP77, FlavoP477) probes.

<p>SE: sensitivity; SP: specificity; PPV: positive predictive value, NPV: negative predictive value.</p

Fluorescent <em>In Situ</em> Hybridization: A New Tool for the Direct Identification and Detection of <em>F. psychrophilum</em>

<div><p><em>F. psychrophilum</em> is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent <em>in situ</em> hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect <em>F. psychrophilum</em> in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of <em>Flavobacterium</em> species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of <em>F. psychrophilum</em> infections in fish. We constructed a generic probe for the genus <em>Flavobacterium</em> (“Pan-Flavo”) and two specific probes targeting <em>F. psychrophilum</em> based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different <em>Flavobacterium</em> and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the <em>F. psychrophilum</em> probe, respectively). FISH was able to detect <em>F. psychrophilum</em> in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of <em>Flavobacterium</em> spp. and <em>F. psychrophilum</em>.</p> </div

Genera of bacteria studied in the first validation step.

*<p>includes <i>Aerococcus</i> (1), <i>Aeromonas</i> (3), <i>Alcaligenes</i> (2), <i>Bacillus</i> (1), <i>Chryseobacterium</i> (2), <i>Corynebacterium</i> (3), <i>Delftia</i> (1), <i>Hafnia</i> (3), <i>Micrococcus</i> (2), <i>Pasteurella</i> (3), <i>Providencia</i> (2), <i>Raoultella</i> (1), <i>Shewanella</i> (1), <i>Vibrio</i> (1).</p

ROC curves for cell suspension of pure strains; area under the curve (AUC) for FISH: 0.89, for culture method: 0.79.

<p>(A). ROC curves for spiked spleens; AUC for FISH: 0.84, for culture method: 0.6 (B).</p

Probes used, target microorganisms and DNA target regions. [Cyanine dye (CY3); Carboxyfluorescein (FAM)].

*<p><i>E.coli</i>, GenBank sequence HM371196.</p

FISH assays of pure cultures.

<p>DAPI staining (A, B, C); Pan-Flavo probe (D, E, F); <i>F. psychrophilum</i> probes (G, H, I) (100x). <i>F. psychrophilum</i> (DSM3660) (A, D, G); <i>Flavobacterium</i> spp. (B, E, H); <i>Chryseobacterium</i> spp. (C, F, I).</p

Identification of Shiga-Toxigenic <i>Escherichia coli</i> outbreak isolates by a novel data analysis tool after matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

<div><p>The fast and reliable characterization of bacterial and fungal pathogens plays an important role in infectious disease control and tracking of outbreak agents. DNA based methods are the gold standard for epidemiological investigations, but they are still comparatively expensive and time-consuming. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable and cost-effective technique now routinely used to identify clinically relevant human pathogens. It has been used for subspecies differentiation and typing, but its use for epidemiological tasks, e. g. for outbreak investigations, is often hampered by the complexity of data analysis. We have analysed publicly available MALDI-TOF mass spectra from a large outbreak of Shiga-Toxigenic <i>Escherichia coli</i> in northern Germany using a general purpose software tool for the analysis of complex biological data. The software was challenged with depauperate spectra and reduced learning group sizes to mimic poor spectrum quality and scarcity of reference spectra at the onset of an outbreak. With high quality formic acid extraction spectra, the software’s built in classifier accurately identified outbreak related strains using as few as 10 reference spectra (99.8% sensitivity, 98.0% specificity). Selective variation of processing parameters showed impaired marker peak detection and reduced classification accuracy in samples with high background noise or artificially reduced peak counts. However, the software consistently identified mass signals suitable for a highly reliable marker peak based classification approach (100% sensitivity, 99.5% specificity) even from low quality direct deposition spectra. The study demonstrates that general purpose data analysis tools can effectively be used for the analysis of bacterial mass spectra.</p></div