Antifungal Sensitivity Testing


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Anti fungal Sensitivity Testing

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Antifungal Sensitivity Testing

  1. 1. Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Increase of Fungal Infections Several factors havecontributed to the increasein fungal infections - mostnotably, increasing numberof immunosuppressed casese.g AIDS, cancer or diabetes,the use of broad spectrumantibiotics, cytotoxicchemotherapy, and organtransplantations Dr.T.V.Rao MD 2
  3. 3. Fugal Infections have increased Morbidity and Mortality The increasing incidence of opportunistic severe fungal infections has greatly enhanced the interest in novel methods for in vitro antifungal susceptibility testing, the standardized methodology Dr.T.V.Rao MD 3
  4. 4. Antifungal agents Mode of actionAmphotericin B binds to plasma membrane creating poresAzoles inhibits cytochrome P450 enzymes in the fungal cell5FC converts to 5FU, incorporated into RNA, abnormal proteinsGriseofulvin binds microtubule proteins, inhibit cell wall synthesisTerbinafine is an ergosterol inhibitor useful for systemic mycosisEchinocandins target their action on fungal cell wall Dr.T.V.Rao MD 4
  5. 5. Antifungal agents Griseofulvin Source Penicillium griseofulvum Produced in 1939 Not used until 1958 Spectrum DermatophytesGentles first used orally in guinea pigs prior to its use in humans Anti-inflammatory properties Inhibits keratolytic action Dr.T.V.Rao MD 5
  6. 6. Antifungal agents PolyenesPolyenes are produced from StreptomycesCyclic moleculesNystatinAmphotericin BNatamycinMepartricinBroad spectrum Dr.T.V.Rao MD 6
  7. 7. Formulations of polyenes are ToxicAmB The most widely used antifungal for systemic infections High level of toxicityNystatin Significant nephrotoxicity Has not been developed to treat systemic fungal infections Dr.T.V.Rao MD 7
  8. 8. antifungal agents Amphotericin BYellow powder, water insolubleBile salt allows solubility (weak association)Floats free in the aqueous medium, causes toxic effectsBroad spectrum, binds to sterol in the cell membraneFungicidal activity @ 3 h with 1 g/mlAzole-amphotericin B is never synergisticAmphotericin B and 5FC gives synergyCandida lusitaniae is usually resistant to Amphotericin B Dr.T.V.Rao MD 8
  9. 9. Amphotericin B Toxicity• Early intolerance reaction• Thrombophlebitis• Nephrotoxicity• Hematotoxic effects• The liposomal preparation of Amphotericin B reduces the risk of nephrotoxicity Dr.T.V.Rao MD 9
  10. 10. Antifungal agentsAzole DerivativesA chemical pentacyclic structure with 2 nitrogen atomsWater insoluble except fluconazolePreferentially inhibit cytochrome P450 enzymesFungistatic, Modify cytochrome P450 enzymeFirst generation Imidazoles:Clotrimazole & MiconazoleClotrimazole requires high doses – poorly toleratedParenteral dosages no longer available for Miconazole Dr.T.V.Rao MD 10
  11. 11. antifungal agents Cytochrome P 450 (CYP 450)CYP is a host of enzymes that use iron to oxidize thingsCYP disposes harmful substances by making them water-solubleCYP is something like a hydroxyl groupP450-mediated oxidation is referred to as "Phase I metabolism”CYP in man is found in the liver, small intestineCYP is vital to the formation of cholesterol & steroidsNADPH + H+ + O2 + RH ==> NADP+ + H2O + R-OH Dr.T.V.Rao MD 11
  12. 12. antifungal agents CYP 450 …..Fungal plasma membranes have nonpolar sterol (ergosterol)Amphotericin B binds to ergosterol permitting rapid leakageCytochrome P450 catalyzes synthesis of ergosterolAzole antifungal agents interfere with cytochrome P450 Dr.T.V.Rao MD 12
  13. 13. antifungal agentsKetoconazoleOrally well absorbed imidazole of second generationKetoconazole is the only imidazole for systemic useCSF penetration is very weakHepatotoxicity restricts its useAlso interacts with other molecules Dr.T.V.Rao MD 13
  14. 14. antifungal agents Third generation azoles Triazole derivatives (contain three nitrogen atoms) Fluconazole Itraconazole Voriconazole Posaconazole RevuconazoleSatisfactory tolerability, Suitable for systemic use Dr.T.V.Rao MD 14
  15. 15. antifungal agents Fluconazole & ItraconazoleFluconazole has been extensively used for yeast infectionsUseful for systemic infectionsReadily and completely absorbed by gastrointestinal tractDistributed equally in different organs and tissueCandida krusei Intrinsically resistant to fluconazoleItraconazole is used to treat Aspergillus infectionsEntirely metabolized in the liverEliminated in the feces and urine Dr.T.V.Rao MD 15
  16. 16. antifungal agentsVoriconazole is a modified fluconazoleA broad spectrum antifungal agentRapid absorption after oral administrationDistributes in tissues and body fluidsMetabolized in the liverEliminated in the urine in unchanged formAzoles carry some side effectsHepatotoxicity, gastrointestinal and endocrine toxicitySkin rash, pruritis and other hypersensitivity Dr.T.V.Rao MD 16
  17. 17. antifungal agents EchinocandinsCaspofunginCaspofungin is semisynthetic, synthesized from Glarea lozyensisWhitish powder, water & methanol soluble, fungicidalFungicidal against, Aspergilli, Candida and P. cariniiNo cross resistance amongst strains resistant to Ampho B or azolesNo activity against Cryptococcus neoformans, Fusarium & RhizopusEffective against Pneumocystis cariniiMicafungin and Anidulafungin – are under investigation Dr.T.V.Rao MD 17
  18. 18. antifungal agents TerbinafineTerbinafine belongs to allylamines, synthetic, highly lipophilicOral and topical (cream) formulationsTerbinafine inhibits ergosterol biosynthesisUsed to treat superficial mycosisAlso useful against systemic mycosis (yeast & other fungi)Adverse reactions to terbinafine are in general transient and mild Dr.T.V.Rao MD 18
  19. 19. Newer Methods are EmergingThe establishment of astandardized broth referencemethod for antifungalsusceptibility testing of yeastshas opened the door to anumber of interesting anduseful developments. Also, theavailability of referencemethods provides a usefultouchstone for thedevelopment of commercialproducts that promise to bemore user friendly and tofurther improvement of teststandardization. Dr.T.V.Rao MD 19
  20. 20. Antifungals can be Optimally Used Incorporation of antifungal susceptibility testing methods into the clinical trials of new antifungal agents will facilitate the establishment of clinical correlates and further enhance the clinical utility of antifungal susceptibility testing Dr.T.V.Rao MD 20
  21. 21. Diagnosis of Invasive Fungal Infections Clinical signs and symptoms Rapid tests (issues: sensitivity, specificity) qPCR Galactomannan detection 1-3 β D-glucan detection PNA FISH Smear; histology stains Culture (issues: low % positive, time to positive result) Susceptibility testing Speciation: C. glabrata or another species? Dr.T.V.Rao MD 21Alexander et al. CID. 2006;43:S15–27.
  22. 22. Increased Interest in Antifungal Susceptibility Testing Changing epidemiology of isolated organisms eg, non-albicans Candida on the increase Newer drugs; more choices More immunocompromised patients Antifungal susceptibility testing becoming more commonplace ? Dr.T.V.Rao MD 22Turner et al. Expert Opin Emerg Drugs. 2006;11(2):231–250.Maertens et al. Curr Med Chem-Anti-infective Agents. 2002;1:65–81.
  23. 23. What Are the Current Antifungal Susceptibility Tests? Dr.T.V.Rao MD 23
  24. 24. IntroductionAntifungal susceptibility testing Minimum inhibitory concentration (MIC) suggests the target fungal species is susceptible to antifungal drug MIC values in vitro might not necessarily correlate with the in vivo efficacy noted clinical testing in vivo must be done to confirm any finding in vitro Dr.T.V.Rao MD 24
  25. 25. IntroductionThe National Committee for ClinicalLaboratory Standards (NCCLS) Subcommitteeon Antifungal Susceptibility Tests has provided guidelines to increase the reproducibility of MIC testing of filamentous fungi M27-A broth dilution method Candida spp Cryptococcus neoformans Dr.T.V.Rao MD 25
  26. 26. Diagnosis of Invasive Fungal Infections Clinical signs and symptoms Rapid tests (issues: sensitivity, specificity) qPCR Galactomannan detection 1-3 β D-glucan detection PNA FISH Smear; histology stains Culture (issues: low % positive, time to positive result) Susceptibility testing Speciation: C. glabrata or another species?Alexander et al. CID. 2006;43:S15–27.
  27. 27. Susceptibility Testing Methods Disk diffusion Qualitative results- interpretation only: an isolate is Susceptible or Intermediate or Resistant) MICs (Minimum Inhibitory Concentration) Preferable; quantitative results: a value in g/mL and an interpretation (S,I,R) Other: echinocandin (eg, caspofungin) “susceptibility” tests (NOT for routine laboratories) Inhibition of glucan synthesis (IC50 values) Mutations in FKS gene Dr.T.V.Rao MD 27Pfaller. Curr Drug Targets. 2005;6:929–943.
  28. 28. Antifungal CLSI and EUCAST Guidelines*Yeast-M27-A2 **EUCAST Yeast0.5–2.5 x 103 cfu/mL MethodRPMI1640, pH 7, MOPS 0.5–2.5 x 105Macro/microbroth 35ºC cfu/mL48h (others)–72h (Cryptococcus) RPMI 1640 withInterp: 100% inhibition 2% glucose Amphotericin B (AmB); Micro broth, 35ºC, prominent for others pH 7, * CLSI Method 24h incubation Dr.T.V.Rao MD 28
  29. 29. Antifungal CLSI andEUCAST Guidelines Yeast-M44-A (disk) 1-5 x 106 cfu/mL MHA +2% glucose, 0.5 ug/mL methylene blue Disk diffusion 35ºC 20–24h Interp: measure zone of inhibition; to date for fluconazole and VOR Dr.T.V.Rao MD 29
  30. 30. Reading Disk Diffusion Test Disk with drug Measure diameter of zone of inhibition Lawn of yeast or mould Dr.T.V.Rao MD 30Pfaller. Curr Drug Targets. 2005;6:929–943.
  31. 31. Antimicrobial Gradient Testing E-test® Read plates afterrecommended Incubation Read MIC where elipse intersects scale Dr.T.V.Rao MD 31
  32. 32. Antifungal susceptibility testing in candidemia: current « guidelines »Guideline Recommandation Comment on choice of therapyGermany 2003 None NASpain 2003 AFST (not graded) NoneFrance 2004 Routine E-test (B-II) NoneU.S.A. 2004 NCCLS M27A & FCZ Helpful in case of lack of Not a standard of care clinical response Helpful in deep or Not graded support oral Switch to May hematogenous infections azole (long-term therapies)
  33. 33. Antifungal susceptibility testing (AFST)AFST should beperformed inhematologicalpatients onisolates from bloodor normally sterilesites, in order Dr.T.V.Rao MD 33
  34. 34. Antifungal Susceptible Testing Methods CLSI M27-A3 and M27-S3 method for yeasts: RPMI-1640 medium with MOPS buffer to pH 7.0. • CLSI M44-A and M44-S2 method for disk diffusion testing for yeasts: Mueller-Hinton Agar supplemented with glucose and 0.5 ug/ml methylene blue dye [GMB] medium. • CLSI M38-A2 method for filamentous Fungi RPMI- 1640 medium with MOPS buffer to pH 7.0. Inoculum prepared by spectrophotometer with the spore suspension density adjusted for different species. Dr.T.V.Rao MD 34
  35. 35. Methods for susceptibility testingM38-A referencemethod forfilamentous fungi,published by theClinical LaboratoryStandard Institute(CLSI) Dr.T.V.Rao MD 35
  36. 36. CLSI M38-ACharacteristics CLSI M38A Suitable Conidium-and spore forming fungi Inoculum 0.4x104-5x104 CFU/ml Inoculum Spectrophotometrically Standardization Test medium RPMI 1640 Format Microdilution Temperature 35°C Duration of incubation 48h Endpoint No growth Dr.T.V.Rao MD 36
  37. 37. Limitations of susceptibility testing methods (M38-A, …) size of inoculum the use of growth medium the time of incubation the inoculum preparation method the use of Tween concentrationLack of detection of amphotericin B resistanceNo breakpoints Dr.T.V.Rao MD 37
  38. 38. E-testE-test is a commercially available method for antimicrobial susceptibility testing. This technique is based on a combination of the concepts of dilution and diffusion tests.For Aspergillus spp., good correlations with amphotericin B and Itraconazole Etest and M38-A method have been demonstrated. Dr.T.V.Rao MD 38
  39. 39. Different TestsMTT, XTT, viability testing……………………… and several otherantifungal susceptibility testing methods for moulds have beendevelopedall of these alternative methods correlate more or less with thestandard methodeach also has its own disadvantages: XTT or MTT method is cumbersome E test is relatively expensive Disk diffusion Viability tests are suitable for MFC Ramani 2003; Espinel-Ingroff 1997; Balajee 2002; Lass-Flörl 2001 Dr.T.V.Rao MD 39
  40. 40. Antifungal Drug Sensitivity Needs Special SkillsCharacteristics CLSI M38-A EUCASTSuitability Conidium forming fungi Aspergillus fumigatus Aspergillus spp.Inoculum 0.4-5x104 CFU/ml 1-2.5x105 CFU/mlInoculum Spectrophoto= Haemocytometerstandardization metricallyTest medium RPMI 1640 RPMI 1640 G2%Format Microdilution MicrodiluationTemperature 35°C 35°CDuration of incubation 48h 48hEndpoint No growth No growth
  41. 41. Caspofungin Activity in Aspergillus spp.Different from Routine Tests
  42. 42. Caspofungin Activity in Aspergillus spp. Activity does not fit classic definition of fungicidal No reduction in the number of colony count In Aspergillus the 1,3-β-D-glucan synthase complex is localized in the apical tips of the growing hyphae Inhibition results in profound change in growth, morphology, and cell wall structure of hyphae Structural change decreases ability to invade blood vessels but does not decrease colony count Caspofungin shows in vitro activity against A. fumigatus, A. flavus, A. nidulans, A. niger, A. terreus, and A. candidusBowman et al. Antimicrob Agents Chemother. 2002;46(9):3001–3012.
  43. 43. Colony Forming Unit Quantitation: Yeast vs Aspergillus Candida spp. and Other Yeasts Aspergillus spp. 10 Colony Forming Units 1 Colony Forming Unit 4 Colony Forming Units 1 Colony Forming UnitBowman et al. Antimicrob Agents Chemother. 2002;46(9):3001–3012.
  44. 44. Caspofungin Activity in Aspergillus spp. Colony counts ≠ number of viable cells with filamentous fungi Traditional endpoints like MICs are not useful for interpretation MEC or Minimum Effective Concentration concentration of caspofungin where microscopically swollen, distorted hyphae are observedPfaller MA. Curr Drug Targets. 2005;6:929–943.
  45. 45. Caspofungin Inhibits Aspergillus spp. Growth in Liquid MIC Assays Caspofungin appears static in vitro but demonstrates cidal activity in in vivo studies g/mL 6464 3232 1616 8 µg/ml 8 4 4 2 21 1 0.5 0.25 0.125 0.03 0.03 0.5 0.25 0.13 0.06 0.06 Caspofungin Amphotericin B CLSI M38-A: Caspofungin — prominent inhibition (>50%) at 24 hours AmB — 100% Inhibition at 48 hoursPfaller. Curr Drug Targets. 2005;6:929–943.
  46. 46. Can an In Vitro Susceptibility Test Predict the In Vivo Human Response? MICs are not an absolute measurement MICs can vary based on medium, temperature of incubation, inoculum, etc. The in vitro - in vivo correlation for antifungal drugs is poor “S”* does not predict successful treatment “R”* does not necessarily predict clinical failure Host factors (immune status/underlying disease) play a crucial role in clinical outcome * S= susceptible, R = resistant Dr.T.V.Rao MD 46Rex et al. Clin Microbiol Rev. 2001;14(4):643–658.Rex et al. Clin Infect Dis. 2002;35:982–989.Pfaller. Curr Drug Targets. 2005;6:929–943.
  47. 47. Speciation is Important in Optimal Antifungal Administration Speciation of the infecting fungal pathogen may be more important, ie, is the organism C. glabrata or not C. glabrata? Based on the species of the isolates, the choice of antifungal agent becomes important Dr.T.V.Rao MD 47Rex et al. Clin Microbiol Rev. 2001;14(4):643–658.Rex et al. Clin Infect Dis. 2002;35:982–989.
  48. 48. New antifungal agentsPradimicins-benanomicins bind to cell wall mannoproteins causing osmotic sensitive lysis and cell deathNikkonycins competitive inhibitors of fungal chitin-synthase enzymesAllylamines/thiocarbamates non-competitive inhibitors of squalene epoxidaseSordarins inhibit protein synthesis, i.e. elongation factor 2Cationic peptides bind to ergosterol and cholesterol and lead to cell lysis
  49. 49. Created by Dr.T.V.Rao MD for ‘e’ learning for Medical and Paramedical Students in the Developing World Email Dr.T.V.Rao MD 49
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