NOVOSTI V MIKROBIOLOŠKI DIAGNOSTIKI INVAZIVNIH GLIVIČNIH BOLEZNI
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1 NOVOSTI V MIKROBIOLOŠKI DIAGNOSTIKI INVAZIVNIH GLIVIČNIH BOLEZNI Saša Simčič, UL MF Inštitut za mikrobiologijo in imunologijo
2 Mikrobiološka ka diagnostika invazivnih glivičnih bolezni 1. Pregledovanje direktnih mikroskopskih preparatov iz kliničnih nih vzorcev 2. Histopatološko dokazovanje invazije povzročiteljev v tkiva 3. Osamitev in identifikacija 4. Določanje antigenov gliv v serumu in drugih telesnih tekočinah in določanje protiteles v serumu 5. Dokazovanje nukleinskih kislin v kliničnih nih vzorcih
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5 Omejitve pri dokazovanju nukleinskih kislin v kliničnih nih vzorcih 1. Nezadostno poznavanje bioloških dejavnikov (sproščanje DNA iz gliv na mestu okužbe, kinetika sproščanja in prehod DNA v krvni obtok nekroza tkiva??, vpliv antimikotikov). 2. Metoda PCR še e ni standardizirana!!! Kakšen vzorec je primeren? Katera metoda za izolacijo DNA je primerna? Katere tarčne sekvence pomnoževati? Kateri način pomnoževanja uporabiti (RT-PRC Roche LC, Corbett Rotor-Gene ali Applied Biosystems TaqMan)? 3. Medlaboratorijska primerjava rezultatov izbranih metod PCR kaže na preveliko variabilnost rezultatov (problem analitske občutljivosti PCR metode, problem lažno negativnih rezultatov pri bolnikih z IA in lažno pozitivnih rezultatov pri bolnikih brez kliničnih nih znakov IA). 4. Določitev aspergilusne DNA v vzorcih BAL ne ločuje med kontaminacijo s konidiji in invazijo v tkivo (boljši i izbor je test na GM, ker se GM večinoma sprošča a iz hif med rastjo aspergilusa, in manj iz konidijev).
6 Mikrobiološka ka diagnostika invazivnih glivičnih bolezni - najbolj pogosto uporabljeni testi na antigene gliv 1. Aspergiloza: ELISA na galaktomanan (BioRad, Platelia Aspergillus Antigen) 2. Pan-fungal kolorimetrični test na 1,3-beta beta-d-glukan (Associates of Cape Cod, Fungitell; Wako; Seikegaku Kogyo) 3. Kandidoza: ELISA na manan (BioRad, Platelia Candida Antigen) 4. Kriptokokoza: ELISA na kapsularni polisaharidni antigen (Meridian, Premier Cryptococcus) 5. Histoplasmoza: ELISA na polisaharidni antigen (MiraVista, H.capsulatum Antigen EIA)
7 CID 2004: 39; 199 Conclusion: highly sensitive and specific in detecting early IFIs, including candidiasis, fusariosis, trichosporonosis, and aspergillosis CID 2005: 41; 654 Conclusion: reproducible assay results with high specificity and high PPV
8 Med Mycol 2006: 44; SI85. Conclusions: the G-test was developed in 1992 by Obayashi et al. and has found wide application in Japan in the fields of hematological diseases and HSCT, in the diagnosis of deep mycosis BG was discussed as a surrogate marker for a presumptive therapy of patients with fever not responding to broad-spectrum antibiotics
9 Lancet Infect Dis 2004: 4; 349 Conclusions: may has a significant variation in performance the causes of this variability are multifactorial (biological and epidemiological) understanding the biology of GM release by Aspergillus will greatly enhance our understanding of the kinetics of this and other surrogate markers CID 2006: 42; 1417 Conclusions: the accuracy of the test is variable among different patient populations the test is more useful in patients who have hematological malignancy or who have undergone HSCT than in solid-organ transplant recipients further studies with attention to the impact of antifungal therapy and rigorous assessment of false-positive test results are needed
10 CID 2006: 43 (Suppl 1); S15 Conclusion: the combination of the 2 tests (GM and BG) improved specificity (to 100 %) and PPV (to 100 %) for the diagnosis of IA, without affecting sensitivity and NPV
11 CID 2007: 44; 402 Conclusions: experience with use of beta-glucan assay in HSCT recipients is limited although valuable as diagnostic adjuncts to support a diagnosis of a probable IFI in patients with compatible host factors and radiological findings as defined in the EORTC/MSG criteria, the value of these laboratory markers as screening tools for IFIs is controversial, and more research is required it is a high priority to validate the application of these tests to antifungal algorithms
12 Lažno pozitivni (5 % odrasli -83 % novorojenci) in lažno negativni rezultati na GM v serumu 1. GM je stranski produkt fermentacije pri pridobivanju beta-laktamskih antibiotikov (Tazocin; kljub ukinitvi terapije, lažno pozitivni rezultati lahko ostanejo več dni pri bolnikih z okvaro ledvic). 2. Poškodbe črevesne stene ali nezrela črevesna stena pri otrocih pospešijo absorpcijo GM iz hrane in vode. 3. Infuzija z Racol-om, om, ki vsebuje sojine proteine; prisotnost paraproteinov IgM, CIC in avtoprotiteles v serumu, ki se vežejo ejo s protitelesi v testu (bolniki s kronično no GVHD po alogenski HSCT). 4. Bombažni brisi. 5. Gastrointestinalna kolonizacija z Bifidobacterium spp. (lipoteihoična kislina in lipoglikan) pri novorojenčkih in otrocih, ki ji sledi translokacija.
13 Lažno pozitivni rezultati (od 5% pri odraslih do 83% pri novorojencih) in lažno negativni rezultati na GM v serumu 6. Kriptokokoza (galaktoksilomanan ima epitope, ki so navzkrižno no reaktivni z GM), navzkrižna reaktivnost s Penicillium spp. 7. Vzroki za lažno negativne rezultate pri IA: zmanjšano ano sproščanje GM v kri na mestu okužbe, zaradi dejavnikov iz okolja (ph in omejena količina ina hranil na mestu okužbe vplivata na sproščanje GM v kri), in abscesi. 8. Antimikotična na terapija zmanjša a občutljivost testiranja (AmB in vitro omeji rast micelija). 9. Drugi vzroki za variabnilnost rezultatov: sproščanje GM iz hif, prehod iz mesta okužbe v kri, vezava GM s sestavinami v krvi, dejavniki v bolniku (mesto in obseg bolezni, antimikotiki, starost, metodološki dejavniki prag, definicija pozitivnega izvida), razvrščanje anje IGB.
14 Lažno pozitivni rezultati na GM v drugih kliničnih nih vzorcih 1. BAL: uporaba tekočine za izpiranje Plasmalyte (Baxter), ki vsebuje Na-glukonat. 2. BAL: pogosto pri bolnikih po presaditvi pljuč,, zaradi kolonizacije dihal z aspergilusom; testiranje BAL se kombinira s slikovno diagnosti gnostično metodo hr-ct. 3. Urin: križna reaktivnost protiteles EB-A2 s ciklofosfamidom.
15 Lažno pozitivni rezultati na BG v serumu 1. Gram-pozitivna bakteriemija. 2. Kontaminacija z BG pri izvedbi testa. Lažno negativni rezultati na BG v serumu 1. Velika koncentracija bilirubina in trigliceridov v krvi. 3. Hemoliza vzorca. 4. Terapija s frakcijami plazemskih proteinov ali koagulacijskimi faktorji in nekaterimi zdravili. 5. Hemodializa z uporabo celuloznih membran in stik z bombažnimi gazami. 6. Kardiopulmonarni obtok. 7. Vročinska kap, in nepoznani razlogi. 8. BG v antibiotikih (npr. amoksicilin-klavulanska klavulanska k.)
16 CID 2004: 39; 1467 CID 2006: 43 (Suppl 1); S15 Conclusions: GM can be detected in BAL, CSF and urine from patients with IA with higher sensitivity than is the case with culture, as well as early in the course of infection PPV and NPV are 100 % when BAL GM EIA testing is combined with hr-ct scanning GM EIA and qpcr assay add to the sensitivity of BAL for diagnosing IPA in high risk patients
17 Clin Microbiol Rev 2004: 17; 281. Conclusions: Platelia Candida Mannan EIA is highly specific; there remains however a lack of the sensitivity (40-75%; probably due to a rapid clearance of mannan), necessary for early detection of systemic candidiasis) Test strategy works for C.albicans, C.glabrata and C.tropicalis but does not work as well with C.parapsilosis, C.kefyr, or C.krusei (EBCA1 mab used in the mannan assay recognize a mannose epitope present to a much lesser extent in mannan from the last three species
18 J Clin Microbiol 2005: 43; 2181 Conclusions: The usefulness of antibody detection may be limited when the patients under investigation are immunosuppressed and/or heavily colonized but uninfected The specificities for two antigen detection assays (ELISA and latex agglutination) and PCR method are high, although the Candida PCR method has enhanced sensitivity over both antigen assays Both PCR and ELISA techniques should be used in unison to aid the detection of invasive Candida infections
19 Zaključki ki Vsaka metoda ima omejitve, zato bi bilo najbolje uporabiti serijsko sko testiranje večih vzorcev z večimi metodami (glivična bolezen je dinamičen proces). Slikovno diagnostično metodo hr-ct in zanesljive laboratorijske teste na analite iz gliv bi mogli uporabiti kot markerje za uvedbo antimikotične ne terapije ali kot kriterij za spremembo že e obstoječega ega antimikotičnega nega režima. V prihodnosti si več obetamo od standardiziranih molekularnih metod, ki bodo vključene v mikrobiološko ko diagnostiko IGB.
Lewis White Department of Medical Microbiology and NPHS Cardiff, University Hospital of Wales, Cardiff, UK Friday, February 24, 2006, 10:05-10:25 am
show slides PCR PLATFORMS, STRENGTHS AND WEAKNESSES Lewis White Department of Medical Microbiology and NPHS Cardiff, University Hospital of Wales, Cardiff, UK Friday, February 24, 2006, 10:05-10:25 am
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