Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broadrange fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples

Lymphocyte predominant cells detect Moraxella catarrhalis-derived antigens in nodular lymphocyte-predominant Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta' (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD <sup>+</sup> LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD <sup>+</sup> B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD <sup>+</sup> DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD <sup>+</sup> BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies

Oxygen-Independent Stabilization of Hypoxia Inducible Factor (HIF)-1 during RSV Infection

BACKGROUND: Hypoxia-inducible factor 1 (HIF)-1alpha is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1alpha as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1alpha as transcriptional regulator during infections with RSV. METHODOLOGY, PRINCIPAL FINDINGS: We gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1alpha. These studies revealed that RSV-positive cells also stained for HIF-1alpha, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1alpha protein 24 h after RSV infection. In contrast, HIF-1alpha activation was abolished utilizing UV-treated RSV. Moreover, HIF-alpha-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1alpha dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1alpha. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1alpha activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-alpha-activation in a murine in vivo model. CONCLUSIONS/SIGNIFICANCE: Taking together, these studies suggest hypoxia-independent activation of HIF-1alpha during infection with RSV in vitro and in vivo

Evaluation of fluorescence in situ hybridisation (FISH) for the detection of fungi directly from blood cultures and cerebrospinal fluid from patients with suspected invasive mycoses

The aim of this study was to evaluate the diagnostic performance of in-house FISH (fluorescence in situ hybridisation) procedures for the direct identification of invasive fungal infections in blood cultures and cerebrospinal fluid (CSF) samples and to compare these FISH results with those obtained using traditional microbiological techniques and PCR targeting of the ITS1 region of the rRNA gene. In total, 112 CSF samples and 30 positive blood cultures were investigated by microscopic examination, culture, PCR-RFLP and FISH. The sensitivity of FISH for fungal infections in CSF proved to be slightly better than that of conventional microscopy (India ink) under the experimental conditions, detecting 48 (instead of 46) infections in 112 samples. The discriminatory powers of traditional microbiology, PCR-RFLP and FISH for fungal bloodstream infections were equivalent, with the detection of 14 fungal infections in 30 samples. However, the mean times to diagnosis after the detection of microbial growth by automated blood culture systems were 5 hours, 20 hours and 6 days for FISH, PCR-RFLP and traditional microbiology, respectively. The results demonstrate that FISH is a valuable tool for the identification of invasive mycoses that can be implemented in the diagnostic routine of hospital laboratories. © 2015 Da Silva et al

Anti-tubercular activity of a natural stilbene and its synthetic derivatives

Objectives: Tuberculosis (TB) and multidrug- and extensively drug-resistant TB in particular are remaining a major global health challenge and efficient new drugs against TB are needed. This study evaluated the anti-tubercular activity of a natural stilbene and its synthetic derivatives against M. tuberculosis .Methods: Isopropylstilbene and its synthetic derivatives were analyzed for their anti-tubercular activity against M. tuberculosis ATCC 27294 as well as multidrug- and extensively drug-resistant M. tuberculosis clinical isolates by using MGIT 960 instrumentation and EpiCenter software equipped with TB eXiST module. Cytotoxic effects of drug candidates were determined by a MTT dye reduction assay using A549 adenocarcinomic human alveolar basal epithelial cells.Results: Growth of M. tuberculosis ATCC 27294 was suppressed by the natural isopropylstilbene HB64 as well as synthetic derivatives DB56 and DB55 at 25 µg/ml. Growth of clinical isolates MDR and XDR M. tuberculosis was suppressed by HB64 at 100 µg/ml as well as by synthetic derivatives DB56 and DB55 at 50 µg/ml and 25 µg/ml, respectively. No anti-tubercular activity was demonstrated for synthetic derivatives DB53, EB251, and RB57 at 100 µg/ml. Toxicity in terms of IC50 values of HB64, DB55 and DB56 were 7.92 µg/ml, 12.15 µg/ml and 16.01 µg/ml, respectively.Conclusions: Synthetical derivatives of stilbene might be effective candidates as anti-tubercular drugs. However, toxicity of these substances as determined by IC50 values might limit therapeutic success in vivo . Further investigations should address lowering the toxicity for parenteral administration by remodeling stilbene derivatives

Prävalenz multiresistenter Organismen bei Patienten mit Flüchtlingsanamnese an einem Deutschen Universitätsklinikum in Abhängigkeit von deren Aufenthaltsdauer in Deutschland

Background: Refugees have a significant risk of carrying multidrug-resistant organisms (MDRO), including multidrug-resistant gram-negative organisms (MDRGN) and methicillin-resistant Staphylococcus aureus (MRSA). Since the duration of MDRGN colonization has been shown to last for several months, we hypothesize that the prevalence of MDRO in refugees gradually declines during their stay in Germany to the level of MDRO prevalence in non-refugee patients. Knowledge about the dynamics of refugees' MDRO prevalence might provide the basis for appropriate infection control measures for refugees in hospitals as well as refugees' MDRO epidemiology in general.Material and methods: MDRO prevalence in 109 refugees admitted to the University Hospital Frankfurt, Germany, were compared to 819 adult controls and 224 pediatric patients admitted to the intensive care unit between June 2016 and May 2017.Results: 41.3% (95% confidence interval=31.9-51.1) of the refugees, 5.7% (4.2-7.6) of the adult controls and 8.9% (5.5-13.5) of the pediatric controls were positive for at least one MDRGN. The highest MDRGN prevalence was found in refugees who recently arrived (18 Monate unterscheidet sich nicht mehr signifikant von der der Kontrollgruppen (14,9%; 1,8-42,8).Schlussfolgerung: Diese Studie untersucht den Zusammenhang zwischen der Aufenthaltsdauer der Flüchtlinge in Deutschland und deren MRGN-Prävalenz. Es zeigt sich, dass die MRGN-Prävalenz bei Flüchtlingen umso niedriger ist, je länger sie sich in Deutschland aufhielten. Bei Flüchtlingen mit einer Aufenthaltsdauer von mindestens 18 Monaten findet sich im Vergleich zu den hier untersuchten Kontrollgruppen kein statistisch signifikanter Unterschied mehr, wenngleich auch nach dieser Zeitspanne die MRGN-Prävalenz bei Flüchtlingen noch über der Prävalenz der einheimischen kritisch-kranken Kontrollkohorte liegt. Hinsichtlich MRSA zeigt sich bei der Gruppe der Flüchtlinge ebenfalls eine Abnahme der Prävalenz über Zeit. Da allerdings die MRSA-Prävalenz in dieser Patientengruppe auch 18 Monate nach Einreise in Deutschland die der einheimischen Bevölkerung nahezu achtfach übersteigt, sollten risikoadaptierte Hygienemaßnahmen zur Vermeidung einer MRSA-Übertragung auch nach dieser Zeit strikt eingehalten werden

Einfluss der Flächendesinfektion mit Wasserstoffperoxid auf die Prävalenz Vancomycin-resistenter Enterokokken (VRE) auf Krankenstationen

Objective: Vancomycin-resistant enterococci (VRE) are of major concern in infection control. Although broad infection control actions to check VRE have been implemented, VRE remain part of daily infection prevention in clinical settings. Cleaning procedures in the inanimate ward environment might play a key role in controlling VRE. In order to optimize infection control management at University Hospital Frankfurt, Germany (UHF), this study evaluates the impact of H2O2-containing cleaning wipes compared to Glucoprotamin containing wipes on VRE prevalence in intensive care wards.Methods: Retrospective analyses were conducted of the VRE prevalence on environmental materials obtained from three intensive care units (ICU) at UHF for 17 months prior to (T1) and during the 25 months after (T2) the implementation of H2O2-containing cleaning wipes from January 2016 to June 2019. The bactericidal power of the two disinfectants against VRE was compared using the 4-field test according to EN 16615 (2015).Results: At T1 and T2, n=666 and n=710 environmental samples, respectively, were obtained. At T1, 24.2% (n=161/666; 95% confidence interval: 21.0-27.6) and at T2, 6.9% (n=49/710; 5.1-9.0) samples were positive for VRE. In vitro disinfectant testing did not reveal any superiority of H2O2 over glucoprotamin. No effect on the VRE prevalence in patients' rectal screening materials was observed.Conclusion: Though Glucoprotamin and H2O2 were in vitro equally effective against VRE, the prevalence of VRE in ICU environment at UHF decreased after implementation of H2O2-containig wipes. This might be due to multiple factors, of which we consider the impact of the Hawthorne effect to be the strongest. Success of infection control strategies might depend on the compliance of the persons critically involved. Transparent information on infection control strategies is suggested to increase compliance and should therefore be considered both in daily infection control and outbreak management.Hintergrund: Vancomycin-resistente Enterokokken (VRE) stellen zunehmend eine krankenhaushygienische Herausforderung dar. Wenngleich für VRE bereits umfangreiche Empfehlungen zur Infektionsprävention implementiert wurden, bleibt VRE weiterhin ein fester Bestandteil der täglichen Infektionsprävention im klinischen Setting. Reinigungs- und Desinfektionsmaßnahmen der unbelebten Stationsumgebung könnten dabei eine Schlüsselrolle bei der Eindämmung von VRE darstellen. Mit dem Ziel, eine optimierte krankenhaushygienische Herangehensweise zur Eindämmung von VRE am Universitätsklinikum Frankfurt (UKF) zu schaffen, untersucht diese Arbeit den Einfluss von H2O2-enthaltenden im Vergleich zu Glucoprotamin-enthaltenden Reinigungstüchern auf die VRE-Prävalenz in Umweltmaterialien am UKF.Methodik: Retrospektive Analyse der VRE-Prävalenz in Umweltmaterialien dreier Intensivstationen (ITS) am UKF 17 Monate vor (T1) und in den ersten 25 Monate nach (T2) Umstellung auf H2O2-enthaltende Wischtücher zur Oberflächendesinfektion (Zeitraum: Januar 2016 bis Juni 2019). Mittels 4-Felder-Tests (EN 16615; 2015) Vergleich von H2O2 und Glucoprotamin bezüglich ihrer Wirksamkeit auf VRE. Ergebnis: In T1 und T2 wurden n=666 bzw. n=710 Umweltmaterialien untersucht. In T1 wurde VRE in 24,2% der Materialien (n=161/666; 95%-Konfidenzintervall: 21,0-27,6) und in T2 in 6,9% (n=49/710; 5, -9,0) nachgewiesen. Die Desinfektionsmitteltestung in vitro erbrachte keine Überlegenheit von H2O2 gegenüber Glucoprotamin. Die VRE-Prävalenz in Screeningmaterialien von am UKF behandelten Patienten derselben Zeiträume blieb unverändert. Schlussfolgerung: Die VRE-Prävalenz in Umweltmaterialien der ITS am UKF nahm seit Umstellung auf H2O2-getränkte Wischtücher zur Oberflächendesinfektion signifikant ab. Hinsichtlich der antimikrobiellen Wirksamkeit zeigten sich im 4-Felder-Test äquivalente Ergebnisse für Glucoprotamin und H2O2. Die dargestellte Dynamik scheint daher auf multifaktorielle Aspekte zurückzuführen zu sein, wovon wir den Einfluss des Hawthorne Effect am stärksten einschätzen. Der Erfolg von Strategien zur Infektionsprävention hängt nach unserer Erfahrung essenziell von der Compliance der involvierten Personen ab. Diese kann durch eine transparente Informationspolitk im täglichen krankenhausyhgienischen Geschäft wie auch im Ausbruchsfalle gestärkt werden

Anamnestische Risikofaktor-Analyse von Patienten mit Nachweis von Carbapenem-resistenten Enterobacterales und/oder Acinetobacter baumannii - Einfluss auf die krankenhaushygienische Strategie an einem deutschen Universitätsklinikum

Background: Carbapenem-resistant Enterobacterales and Acinetobacter baumannii are of major concern in terms of infection prevention and control. This study evaluated factors that may increase the frequency of Enterobacterales and A. baumannii with carbapenem resistance (CR) in patients admitted to a German University Hospital for implementation of optimized infection control management.Methods: A five-year-retrospective epidemiological cohort analysis was conducted on anamnestic risk factors for carrying Enterobacterales and/or A. baumannii with CR in patients who were first tested positive for these species at University Hospital Frankfurt (UHF) between January 2013 and June 2018. Results: 364 patients were tested positive for Enterobacterales and/or A. baumannii with CR, resulting in n=400 bacterial isolates in total, with Klebsiella pneumoniae being the most frequently detected species (n=146/400; 36.5%; 95% confidence interval: 31.8-41.4). In patients who were tested positive for Enterobacterales and/or A. baumannii with CR, any hospital stay within the previous 12 months was the most frequently reported common factor (n=275/364; 75.5%; 70.8-79.9).Conclusion: A hospital stay within the previous 12 months, including hospitals in Germany and abroad, is a frequent characteristic of patients who tested positive for Enterobacterales and/or A. baumannii with CR. Upon admission, any previous hospital stay of the given patient within the previous 12 months should be determined. Infection control strategies such as screening measures need to be adapted to these patient groups in hospital settings. In order to reflect the varying determinants in "nosocomial" cases in greater detail, the existing criteria used to characterize "nosocomial detection" of gram-negative bacteria with CR should be reviewed.Hintergrund: Enterobacterales und/oder Acinetobacter baumannii mit Carbapenem-Resistenz (CR) stellen in Kliniken eine enorme medizinische Herausforderung dar. Die vorliegende Studie untersucht anamnestische Charakteristika von Patienten des Universitätsklinikums Frankfurt am Main (UKF), die zum Risiko einer Trägerschaft von Enterobacterales und/oder A. baumannii mit CR beitragen. Ziel der Untersuchung ist die Etablierung einer zielgerichteten Strategie zur Infektionskontrolle.Methode: Retrospektive epidemiologischen Kohortenanalyse bei Patienten, die zwischen Januar 2013 und Juni 2018 am UKF positiv auf mindestens eine Spezies Enterobacterales und/oder A. baumannii mit CR getestet wurden. Ergebnisse: Innerhalb des Untersuchungszeitraums wurden bei 364 Patienten 400 Isolate von Enterobacterales und/oder A. baumannii mit CR nachgewiesen. Hiervon am häufigsten vertreten war Klebsiella pneumoniae (n=146/400; 36,5%; 95%-Konfidenzintervall: 31,8-41,4). Die anamnestische Angabe mindestens eines Krankenhausaufenthalts "innerhalb der letzten 12 Monate" wurde am häufigsten berichtet (n=275/364; 75,5%; 70,8-79,9).Zusammenfassung: Da sich ein Krankenhausaufenthalt innerhalb der letzten 12 Monate mit Nachweis von Enterobacterales und/oder A. baumannii mit CR als das am häufigsten berichtete Risikomerkmal herausstellte, sollte das bei Aufnahme eines Patienten sorgfältig abgefragt werden. Krankenhaushygienische Strategien zur Infektionsprävention, z.B. Implementierung von Screeningprotokollen für Patienten mit diesem Risikofaktor, können hilfreich sein und sollten situationsadaptiert eingesetzt werden. Um die derzeit als "nosokomiale Nachweise" zu bewertenden Fälle richtig darstellen zu können, ist eine Überarbeitung der derzeitigen Definitionskriterien notwendig