LE INFEZIONI FUNGINE INVASIVE: “Recenti …Antifungals MIC mg/L(S/I/R) Amfotericina B 0.064 S...
Transcript of LE INFEZIONI FUNGINE INVASIVE: “Recenti …Antifungals MIC mg/L(S/I/R) Amfotericina B 0.064 S...
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Fabio Arena
Dip. Biotecnologie Mediche
Università di Siena
LE INFEZIONI FUNGINE
INVASIVE: “Recenti
progressi nella
diagnostica
microbiologica”
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Check-Pos Pos-Gram Pos/IDM Pos-ID Pos-ATBM POS-ATB
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Check-Pos Pos-Gram Pos-IDM Pos-ID Pos-ASTM Pos-AST
Tim
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ays)
Arena et al. accepted
Sorveglianza Turn Around Time (Maggio 2015)
• 5 Laboratori italiani medio-grandi membri GLIPaC
• TAT 350 emocolture monomicrobiche, esclusi CoNS
31 Candidemie
78 H
30 H 28 H
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Bassetti M. Intensive Care Med 2014 Bouza E. Int J Antimicrob Agent 2009
• High mortality rates 50-70%
• Difficulties in the diagnosis
The real challenge of candidemia
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• β-D-glucan (mannan, anti-mannan) • Test molecolari PCR, PCR-ESI/MS
• T2 magnetic resonance
Test supplementari esame colturale
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PCR-ESI/MS
S Strenghts W Weaknesses
O Opportunities T Threats
-No step colturale -TAT 6H
-Costo elevato -Apparecchiatura ingombrante -Studi clinici deludenti Sensibilità
-ampio pannello di germi riconosciuti
-Inibitori -basse cariche
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Diagnosis: non cultural methods PCR-ESI/MS
Ecker DJ et al. Nat Rev Microbiol 2008
• More than 750 bacterial and fungal species • Time to results < 6 h (1/2 H hands-on time) • Limit of detection 16 CFU/mL
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PCR-ESI/MS evaluation from positive blood culture
N. campione Standard Iridica
Concordanza Genere Specie Genere Specie
1016 Candida albicans Escherichia coli No
2037 Staphylococcus aureus Negative . Negative PCR
2049 Staphylococcus aureus Escherichia coli No
2052 Streptococcus oralis Finegoldia magna No
3011 Acinetobacter baumannii Escherichia coli No
4026 Enterobacter cloacae Klebsiella pneumoniae No
5040 Escherichia coli Negative . Negative PCR
5050 Enterococcus faecalis Negative . Negative PCR
5063 Clostridium paraputrificium Negative . Negative PCR
6070 Enterococcus faecium Negative . Negative PCR
7010 Streptococcus pyogenes Negative . Negative PCR
8001 Listeria monocytogenes Negative . Negative PCR
8038 Pseudomonas aeruginosa Candida albicans No
Vincent et al. Crit Care Med 2015
19.1% FN
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PCR-ESI/MS evaluation from positive blood culture
19 FN
43% Sensibilità
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T2 Magnetic resonance
S Strenghts W Weaknesses
O Opportunities T Threats
-No step colturale -TAT <5H
-Costo elevato
- dedicato per candidemia
-limitata esperienza clinica -presenza inibitori
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Neely LA et al. Sci Trasl Med 2013
T2 Magnetic resonance/nanoparticle
• 5 Candida species • Time to results < 5 h
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T2 Magnetic resonance/nanoparticle
-2013-2014 -12 centers -Comparison with conventional Blood culture with clinical and spiked samples
Spiked samples sensitivity (n.250): 92.3% C. albicans/C. tropicalis 94.2% C. parapsilosis 88.1% C. krusei/ C. glabrata
Mylonakis et al. CID 2015
Limit of detection: 1 CFU/mL C. tropicalis/C. krusei 2 CFU/mL C. albicans/C. glabrata 3 CFU/mL C. parapsilosis
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12% invalid results...INIBITORS
• 31 discordant cases (1.7% of the total) • 2 positive for BC only • 29 positive for T2 only
T2 Magnetic resonance/nanoparticle
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Spanu T. J Clin Microbiol 2012 Stone NR. J Clin Microbiol 2013 Poritz MA. PlosOne 2011
Yeast identification from positive blood culture bottle
MALDI-TOF PNA-FISH Nested PCR microarray
Molecular diagnosis from positive blood culture
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Specie Fenotipo
C. krusei resistente a fluconazolo
C. rugosa, C. norvegensis, C. lipolytica, C. valida, C. inconspicua, C. zeylanoides
ridotta sensibilità agli azoli
C. guilliermondii, C, metapsilosis, C. parapsilosis, C. fermentati
ridotta sensibilità alle echinocandine
C. lusitaniae ridotta sensibilità all’amfotericina B
A. lentulus, A. pseudofisheri e A. fumigatiaffinis
resistenti agli azoli
A. lentulus e A. fumigatiaffinis, A. terreus
resistenti all’amfotericina B
A. calidioustus resistenza intrinseca agli azoli ed a caspofungina
A. niger può manifestare resistenza acquisita agli azoli
Relevance of fungal identification
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Esame colturale
S Strenghts W Weaknesses
O Opportunities T Threats
-Test di sensibilià -Costo ridotto
-TAT >48 H -bassa sensibilità
- isolato per successivi studi
-problemi intepretativi
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Candida albicans n.32
Candida glabrata n.7
Candida krusei (Issatchenkia
orientalis) n.1
Candida parapsilosis
n.9
Candida tropicalis n.1
Candida species from blood culture 2015
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Micafungin n. isolates MIC Range % Susceptible C. albicans 32 <=0.008 100
C. parapsilosis 8 0.5-2 100 C. glabrata 7 0.015-0.3 100
C. krusei 1 0.12 - C. tropicalis 2 0.015-0.3 -
Fluconazole n. isolates MIC Range % Susceptible C. albicans 31 <=0.12-1 100
C. parapsilosis 9 0.25-1 100 C. glabrata 3 >128 0 C. tropicalis 1 2 -
AST Candida from blood culture 2015
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Eun-Jung Cho et al. J. Antimicrob. Chemother. 2015
Emergence of multiple resistance profiles involving azoles, echinocandins and amphotericin B in Candida glabrata isolates from a neutropenia patient with prolonged fungaemia.
MDR Candida glabrata
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Antifungals MIC mg/L(S/I/R)
Amfotericina B 0.064 S
Anidulafungina 1 R
Caspofungina 0.75 R
Micafungina 0.5 R
Fluconazolo >256 R
- Pz. donna - 76 aa - ricoverata in istituto di
riabilitazione - portatrice CVC
- C. glabrata da sangue periferico
MDR Candida glabrata
Agosto 2016
- Saggio di sensibilità con Gradient diffusion
- Interpretazione EUCAST
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Siopi et al. J. Clin. Microbiol. 2015
AST problemi interpretativi
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Formazione biofilm
• Paired blood cultures • A TTP cut-off of 30 h was 100% sensitive and 51.4%
specific for catheter-related candidaemia
N. Høiby et al. CMI 2014
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Conclusioni
• Candidemia pone probelmi diagnostici e terapeutici
• esame colturale, (1,3)-β-D-glucan, e test di identificazione rapida da emocoltura test consolidati (MALDI-TOF, PNA-FISH e MOLECOLARI rapidi)
• Nuovi diagnostici direttamente da sangue, T2 risonanza magnetica promettente ma poca esperienza clinica
• Resistenze in Candida glabrata
• Test di sensibilità adeguati
• Standardizzazione tecnica per rilevazione biofilm