Neues aus der Sepsis-Diagnostik

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1 Neues aus der Sepsis-Diagnostik
> Wiener Intensivmedizinische Tage 2006 < Neues aus der Sepsis-Diagnostik Petra Apfalter Klinische Mikrobiologie AKH Wien

2 Kennen Sie dieses Szenario?
Fiebernder, vortherapierter ICU-Patient Infektion? Sepsis = SIRS durch Infektion Wenn ja, welche? Gefragt wäre rasch ein relevanter Befund Erreger, Empfindlichkeitsprofil

3 Warum ist Sepsis- diagnostik wichtig?
Haupttodesursache auf nichtkardiologischen ICU Direkte Behandlungskosten in D: 1,1 bis 2,45 Milliarden Euro/Jahr Therapie oft empirisch 11% - 46% ICU Patienten initial falsch therapiert 1, 2, 3 Zeitfaktor essentiell für Outcome Letalität hoch (40%) Sepsis ist eine komplexe systemische inflammatorische Wirtsreaktion auf eine Infektion. Es gibt keinen Parameter, der allein zur Diagnose der Sepsis führen kann. Nicht alle septischen Patienten sind gleich krank. Sepsis, schwere Sepsis und septischer Schock definieren ein Krankheitskontinuum, das über eine Kombination aus Vitalparametern, Laborwerten, hämodynamischen Daten und Organfunktionen definiert wird. Die Schwere der Erkrankung korreliert mit der Inzidenz von Organdysfunktionen und Letalität. 1 KOLLEF M et al, Inadequate antimicrobial treatment of infections, Chest 1999, 115: 2 GARNACHO-Montero J et al, Impact of inadequate empirical antibiotic therapy on the outcome of patients admitted to the ICU with Sepsis, Crit Care Med. 2003, 31 (12): 3 Byl B, et al, Impact of Infectious Disease Specialists and Microbiological Data on the Appropriateness of Antimicrobial Therapy for Bacteremia, Clin Infect Dis 1999: 29:60-66 1 Kollef M, Chest, Garnacho-Montero J, CCM, Byl B, CID, 1999

4 Von welchen Infektionen geht eine Sepsis am häufigsten aus?
Lunge % Harnwege % Bauchorgane % Wunden und Weichteile % Katheter % ZNS % Herzklappen % Andere (z.B. Sinus) % Bakterien im Blut (positive Blutkultur) 17.3% (Angus DC, Crit Care Med 2001; 29: )

5 Anteil positiver Blutkulturen AKH Wien 2005
Krankenhaushygiene, AKH Wien

6 Worum es heute geht Konventionelle Diagnostik Diagnostik - Neu
Blutkultur (BK) Diagnostik - Neu PNA-FISH aus BK Light Cycler®SeptiFAST aus EDTA-Blut rDNA-Sequenzanalyse Diagnostik - In Entwicklung Chip-Techniken

7 Alt, aber gut: die BK Blutmenge ist DER entscheidende Faktor!
Bakteriämie: Erwachsene < KBE/ml Kinder > KBE/ml Gesamtvolumen: ml (Kinder 1-5 ml) Standard: 1 aer. + 1anaer. Fl. od aer. Flaschen 3% Ausbeutesteigreung pro ml kultiviertem Blut!

8 Wann BK abnehmen? Möglichst früh bei Symptomen, die auf eine Sepsis hinweisen Vor Behandlungsbeginn, ansonsten am Ende eines Antibiotika – DI 2-3 Kulturen in rascher Folge Bei negativem Ergebnis nach 24h WH! Keine „Überwachungs-BK“ Keine „follow-up“ BK nach Diagnosestellung Nur eine Blutkultur u. U. nicht ausreichend (zu geringe Sensitivität, Relevanz des gezüchteten Keimes u. U. fraglich) Mehr als 3 Kulturen nicht erforderlich (hohe Kosten, iatrogene Anämie)

9 Diagnostischer Zeitrahmen BK
Empirische AB-Therapie Anpassen – De-Eskalieren Tag 1 Tag 2 Tag 3 Tag 4 Tag 5 Tag 6 Tag 7 Tag 28 PILZE Blutkultur ID + AB Gram

10 Worum es heute geht Konventionelle Diagnostik Neu In Entwicklung
Blutkultur (BK) Neu PNA-FISH aus BK Light Cycler® SeptiFAST aus EDTA-Blut rDNA-Sequenzanalyse In Entwicklung Chip-Techniken

11 PNA-FISH Peptide Nucleic Acid Fluorescence In Situ Hybridization
Fluoreszierende PNA Sonde hybridisiert mit ribosomaler RNA Blut-Kultur Identifikation in 2.5 h Für in vitro Diagnostik PNA are DNA mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or of ribonucleic acid [RNA]), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. PNA was originally designed as a ligand for the recognition of double stranded DNA. The concept was to mimic an oligonucleotide binding to double stranded DNA via Hoogsteen base pairing in the major groove. Thus the nucleobases of DNA were retained, but the deoxyribose phosphodiester backbone of DNA was replaced by a pseudo-peptide backbone that according to computer model building was homomorphous with the DNA backbone. In theory a neutral (peptide) backbone should improve the triplex binding capability of the ligand, and we believed that the pseudo peptide backbone was a good chemical scaffold that would allow us to design recognition moieties that went beyond homopurine targets. It was, however, apparent that the PNA designed for triplex formation would also be a mimic of single stranded nucleic acids by default. Although, it was impossible to imagine all the properties and applications that could be developed based on the neutral backbone, it was intriguingto attempt to make a water soluble mimic of an oligonucleotide with a neutral backbone. The potential of the resulting structure as an antisense agent and as a molecular biology tool was obvious although abstract at the point of conception. The pursuit of a neutral backbone drove the design, and the leap to peptide (or amide) chemistry was easy because of the well-established robustness and flexibility of solid phase peptide synthesis (SPPS) technology. During the early stages of the design many structures were considered. However, by applying additional criteria for the structure such as, rigidity, water solubility and not at least chemical accessibility, the structure now known as PNA came into being. The very first experiments conducted with homo-thymine PNAs clearly demonstrated that these bound sequence-specifically to double stranded DNA. It was realized that two homothymine PNAs had formed a triplex with the homoadenine target in the double stranded DNA, while displacing the homothymine strand in the DNA target. Later it was found that PNAs with both purine and pyrimidine bases form very stable duplexes with DNA and RNA, although not with the extremely high stability of the homo-pyrimidine 2PNA/DNA triplexes, but still more stable than the corresponding DNA/DNA and DNA/RNA duplexes. With many properties that set them apart from traditional DNA analogs, PNAs have added a new dimension to synthetic DNA analogs and mimics in molecular biology, diagnostics, and therapeutic.

12 Diagnostischer Zeitrahmen PNA-FISH vs. BK: - 1d
Empirische AB-Therapie Anpassen – De-Eskalieren Tag 1 Tag 2 Tag 3 Tag 4 Tag 5 Tag 6 Tag 7 Tag 28 -> PILZE ! Blutkultur ID + AB Gram

13 PNA-FISH: Target rRNA Hoch konserviert
Kopien/Zelle (natürliche Amplifikation) rRNA Sequenzen = taxonomische Referenz

14 Schnell-ID positiver BK
S. aureus PNA FISH GPCC Gram Fbg. C. albicans PNA FISH Yeast 2005% BK: 35% MRSA von Sau Three AdvanDx kits are registered for use. Your laboratory will first look at gram stain morphology. Our first three kits are for the most frequently isolated gram-positive infections. You choose the S. aureus PNA FISH kit for gram positive cocci in clusters. You choose the C albicans kit for yeast, presumptive Candida You choose the E faecalis kit for gram positive cocci in pairs and chains. We will show you larger images of these results; you can see that positive cells are seen as bright fluorescence. GPCPC E. faecalis PNA FISH

15 Light Cycler® SeptiFAST aus EDTA-Blut
Diagnostics Multiplex Real-time PCR am Light Cycler 2.0 (Roche)

16 The LightCycler SeptiFast Test Hypothesis: DNA in human blood?
Diagnostics PCR Kultur freie DNA lebende Bakterien/Pilze lebende Bakterien/Pilze tote Bakterien/Pilze phagozytierte Bakterien/Pilze lebende, phagozytierte Bakterien/Pilze (?) human DNA

17 Multiple specific HybProbe probes
The LightCycler SeptiFast Test Target Region – Internal Transcribed Spacer Diagnostics Multiple specific HybProbe probes Spacer Region (ITS*) Multiple copies Well investigated Especially suited for Species testing Ribosomal Genes Multiple copies Well investigated Especially suited for Group testing Single Copy Genes No enhancement Less investigated * ITS = internal transcribed spacer

18 Das HybProbe Prinzip 2 Sonden = benachbart hybridisierendes Oligopaar
Signal (FRET) nur wenn beide nebeneinander binden Sequenz-spezifische Detektion Mutationsanalyse in der Hybprobe-Targetsequenz durch Schmelzkurvenanalyse möglich!

19 The LightCycler SeptiFast Test Negative Control / Internal Control
Sample or Negative Control - MagNA Lyser Instrument - Sample Preparation Diagnostics Lysis Internal Control Extraction Purified NA 50 µl eluate each LightCycler Instrument The LightCycler SeptiFast Kit Gram (-) Gram (+) Fungi

20 The LightCycler SeptiFast Test Channel distribution Gram (+)
Diagnostics t [°C] CoNS from SML Staphylococcus aureus S. spp from SML (670) Streptococcus pneumoniae S.pneu S.aureus S.spp S. spp. from SML (610) CoNS Internal Control Enterococcus faecium Enterococcus faecalis IC E.fum E.fis Channel 610 Channel 640 Channel 670 Channel 705

21 SeptiFast Master List Gram (-) Gram (+) Fungi Escherichia coli
Staphylococcus aureus Candida albicans Candida tropicalis Candida parapsilosis Candida glabrata Candida krusei Aspergillus fumigatus Klebsiella (pneumoniae/oxytoca) CoNS 1 Strep. pneumoniae Serratia marcescens Streptococcus spp. 2 Enterobacter (cloacae / aerog.) Enterococcus faecium Enterococcus faecalis Proteus mirabilis Pseudomonas aeruginosa 1 Coagulase Negative Staphylococci (Species which represent the group are listed in the PI). 2 Species which represent the group Streptococcus spp. are listed in the PI. 3 Species referred as A. calcoaceticus- A.baumannii are not detected. Acinetobacter baumannii 3 Stenotrophomonas maltophilia Each bullet point represents a specific melting point, species in brackets are not differentiated

22 Keimspektrum AKH Wien 2005 Sepi 25% Sau 12% 46 21 6 4 5 2 10 15 20 25 30 35 40 45 50 Staphylokokken Enterobakterien Streptokokken Enterokokken Pseudomonas Candida Andere E. coli 9% Klebsiella 5% Enterobacter 3% % Krankenhaushygiene, AKH Wien

23 Tag 1 Tag 2 Tag 3 Tag 4 Tag 5 Tag 6 Tag 7
Empirische AB-Therapie Anpassen – De-Eskalieren Tag 1 Tag 2 Tag 3 Tag 4 Tag 5 Tag 6 Tag 7 Tag 28 -> PILZE ! Blutkultur 6 h ID + AB SeptiFAST Gram

24 rDNA-Sequenzanalyse Real-time PCR + Sequenzierung

25 rDNA-Sequenzanalyse 16S rDNA für Bakterien
18S, 28S rDNA & ITS1 & 2 Region für Pilze Direkt von Patientenprobe Amplifikation mit universellen Primern Sequenzieren der Amplicons Alignment mit Datenbank Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts.

26 Zielgene rDNA Operon 16S rDNA = universelles bakterielles Gen
Real-time PCR-Amplifikation 16S rDNA Universelle Primer 16S rDNA – ca 1600bp……200 bp unsere Fragmente in hypervariablen Region V3 (ca. bei ), 2. Reaktion V6 (ab 900…)…realTime Real-Time PCR V3…forward Sequencing primer, V 6…. reverse sequencing primer -> 2 Reaktionen rDNA Operon 16S rDNA = universelles bakterielles Gen Konservierte Regionen … Primertarget Hypervariable Regionen (zB V3) … Phylogenetische Analyse

27 Überblick Sequenzreaktion
Sequenzreaktion & Aufreinigung MicroSeq Komponenten dRhodamine dye Terminatoren, cycle sequencing A dR6G An automated sequencer runs on the same principle as the Sanger method (dideoxynucleotide chain termination). A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. Where the manual method uses radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs (a different dye for each nucleotide). This makes it possible for all four reactions (dGTP, dATP, dCTP, and dTTP) to be run in one lane, so you can have huge numbers of reactions on one gel. This is a very efficient method. It is important to remember, however, that a computer can make mistakes. Don't trust the computer. Always check your printout for accuracy. You are looking for a good signal, at least in the 100s, and proper spacing, ideally about 12. Look also for big gaps between the bases since the computer can miss bases. It may often miss a G after an A, especially after an AA. T C G dROX dTAMRA dR110

28 Analyse der Daten Editieren der Sequenz

29 Technik Anfordern: Besuchen Sie uns! klinischemikrobiologie

30 Worum es heute geht Konventionelle Diagnostik Neu In Entwicklung
Blutkultur (BK) Neu PNA-FISH aus BK SeptiFAST aus EDTA-Blut rDNA-Sequenzanalyse In Entwicklung Chip-Techniken

31 Microarrays Detektion hunderter Mikroorganismen simultan ( e Gene) Detektion wichtiger Resistenzgene Bestimmung von verschiedenen Genotypen, Mutationen Quantifikation möglich In contrast, the basic idea of microarrays is a different one Theoretically it´s possible to diagnose hundreds of microorganisms simultaneously by detection of thousands of genes, mutations as well as single nucleotide polymorphisms for detection of microbes and their susceptibilties to antimicrobial agents. Independent of the application for which the chip is used, a microarray usually consists of a glass slide (1cm by 1 cm small) containg about to microscopically tiny spots. In microbiology microarrays are predominantely used for gene expression analysis of host cells after infection with a certain microorganism to study host-pathogen interactions.

32 Oligo-shotgun-Chip Austrian Research Center Seibersdorf Cloning of interesting genes in E.coli ( plasmids) genomic 16S rRNA gene- fragments Amplicons(oligos) of all gene fragments spotted onto chip = probes Anyhow, I´d like to show you another kind of chip, in its development our lab currently is involved in : a microbial oligo-shotgun chip, for which we use shorter oligos instead of the targets (consisting of bp) of interesting genes only (which means the whole genome must not have been sequenced)..... This kind of chips is most useful in situations where we have relatively few targets (e.g. few bacteria within tissue or blood), but a large number of oligos we wish to test (library of variable regions of 16S rRNA for bacterial ID and important resistance genes). The basic idea in these chips is similar. The intention is to apply the chip directly on patients blood specimens to keep the time as short as possible until a definite diagnosis can be made,which is extremely valuable in critically ill patients. Such a chip could replace blood culture one day. However, at the moment these chips are for research use only. (The length of the oligos used depend on the application, but they are usually no longer than 25 basesr, thus the density is much higher in these chips. For instance, a chip that is 1cm by 1cm can easily contain 100,000 oligos. The chips are used in a similar manner. They are exposed to a solution containing many copies of the target DNA. Hybridization occurs between oligos and matching DNA and then the chip can be heated in order to repeat the experiment.) Patterns of green spots identify species + resistence present in sample Hybridization DNA* extraction from specimen (*incl. unknown bacterial DNA) -> PCR (whole bacterial DNA tagged with Cy3)

33 Diagnostischer Peptidchip
Anwendung in Körperflüssigkeiten Simultane Detektion verschiedener Erreger & Resistenzen: Protein/Epitop am Chip detektiert spezifische Immunantwort Ziel dieses interdisziplinären Projektes (Medizin, Molekularbiologie, Bioinformatik) ist die Entwicklung eines Prototypen zur Erregerdiagnostik basierend auf einem Peptidchip – ELISA, i.e. dem Nachweis von erregerspezifischen Antikörpern im Serum des Patienten, mit folgender Innovation: Simultane Diagnostik der gängigsten Pathogene (Bakterien, Pilze, und Viren) Rasche Durchführung (1 – 1,5 Stunden) Erweiterbarkeit auf zusätzliche Erreger Einbezug von Resistenz -– Information Information uber den Immunzustand des Patienten

34 Peptidchip - Strategie
Funded by: Co-Operate Vienna Wiener Wirtschaftförderungsfond Peptidchip - Strategie Prediction of Bacterial Antigens in silico Whole genome B cell epitopes Collection of Sera Sera from patients (with bacterial infections) Sera from healthy persons 1.) Erstellen einer systematischen Serumkollektion von Patienten mit mikrobiologisch / klinisch erwiesener Infektion 2.) Identifizierung der relevantesten Peptid-Epitope aus dem von der Firma Intercell AG bereitgestellten Datensatz von Antigenen aus den Organismen S. pneumoniae, S. aureus, S. epidermidis und E. faecalis 3.) Vorhersage von neuen Epitopen von zusätzlichen Pathogenen (P. aeruginosa, C. albicans, CMV) mittels der virtuellen Scoringroutine EpiScore 4.) Experimentelle Bestätigung der identifizierten Peptid - Epitope an Hand Infektions-bestätigter Seren im Peptid-ELISA Ansatz 5.) Erster Test eines Peptidchip Prototyps Bacterial Epitope Identification Peptide Synthesis Peptide ELISA with Sera Validation of Antigens Peptide ELISA Peptide Chip

35 Take home message: Besuchen Sie uns! klinischemikrobiologie