Candida属における微量液体希釈法とE
Contents
1. 4 Candida albicans non albicans Candida CT 113 0023 2 4 1 TEL 03 3811 0401 FAX 03 3811 0439 E mail 10ms214 hst u bunkyo ac jp 8 Vol 22 No 2 2012 3 fluconazole FLCZ OC albicans amp FLCZ Candida glabrata Candida krusei Candida 3 Clinical and Laboratory Standards In stitute CLSI2. 2 H E test RPMI 1640 DF Cgzgg2a E test Candida CLSI M27 A3 E test amphotericin B AMPH B flucytosine 5 FC itraconazole ITCZ ELCZ 4 MIC
3. AMPH B McFarland No 0 5 E test 3 2 E teet 35 1C 24 48 MIC 4 1 CLSI AMPH B MIC 0 MIC 2 50 IC50 50 IC50 2 2 E test AMPH B 5 FC 90
4. Candida E test MIC 5 FC ITCZ FLCZ 5 FC 3 E test MIC E test CLSI MIC lg x RR 2005 1 14 Vol 22 No 2 2012 2 4 5 6 7 8 9 10 11 12 13 14 AMIE k 0 ONES fe Candida KIEO in vitro 79 1 20 24 Edwards J E Jr 2005 Candida Species p 2938 In Principles and Practice of Infectious Diseases 6 ed Mandell G L J E Bennett R Dolin ed Elsevier Churc
5. E test 48 MIC E test 2 Vol 22 No 2 2012 13 118 FLCZ ITCZ C glabrata Z ITCZ ELCZ C tropicalis ITCZ ITCZ C gla ef 85 7 ELCZ S DD 42 9 C tropicalis D ITCZ S DD 61 1 CLSI 5 EC E test C albicans 3 48 TTCZ FLCZ MIC
6. E test MIC E test Table 3 AMPH B 77 96 3 E test 2 3 38 8 RPMI C albicans 21 MIC E test 3 1 1 95 2 Cgzgg2a Table 2 Comparison of MICs of four antifungal agents determined by broth microdilution and E test among 80 isolates of Candida species MIC ug ml Species No of isolates Agent Method Range 50 90 C albicans 39 AMPH B MD 0 5 2 1 1 E test 0 032 16 0 064 0 125 5 FC MD 0 125 64 0 125 0 5 E test 0 064 64 0 125 2 ITCZ MD 0 032 16 0 064 1 E test 0 032 16 0 064 0 5 FLCZ MD 0 125 64 0 25 64 E test 0 125 64 0 125 C glabrata 21 AMPH B MD 1 2 1 2 E test 0 125 0 5 0 125 0 25 5 FC MD 0 125 0 5 0 125 0 125 E test 0 125 1 0 125 0 125 ITCZ MD 0 064 16 1 16 E test 0 032 4 1 4 FLCZ MD 1 32 8 16 E test 0 5 16 4 16 C tropicalis 18 AMPH B MD 1 2 1 1 E test 0 064 0 5 0 125 0 25 5 FC MD 0 125 0 125 0 125 E test 0 064 0 125 0 125 0 12
7. AMPH B 96 3 E test 2 ITCZ 27 5 FLCZ 25 0 C glabrata C tropicalis 5 FC C tropicalis IZ FLCZ E test MIC Key words Candida CLSI broth microdilution E test MIC O HIV
8. ITTCZ FLCZ L 80 MIC LIS 1 E test 48 AMPH B 5 FC ITCZ FLCZ 4 MIC E test Table 1 2 Candida MIC Table 2 AMPH B E test MICs MIC 2 C albicans D MIC MIC 0 5 pg ml 1 i Vol 22 No 2 2012 9 114 Table 1 Comparison of broth microdilution MICs and E test MICs for four antifungal agents against five reference strains
9. 1 E test C gla pgfg S DD 42 9 E test T 9 5 z 72 Vol 22 No 2 2012 E test MIC C albicans ATCC 90028 AMPH B E test MIC MIC 3 MIC CLSI M27 A3 E test BEA GNI AMPH B MIC E test Favel NOBRE b 0 25 2 ng ml E test C albicans C glabrata CS0 06 1 ug ml C tropicalis Z 0 125 0 5 pg ml range Cgzgg2a R Table 3 MIC
10. Broth microdilution ug ml E test ug ml Organism Agent Reference range MIC Reference range MIC C albicans ATCC 90028 AMPH B 0 5 2 1 0 125 0 5 0 125 5 FC 0 5 2 0 5 0 5 2 2 ITCZ NA 0 125 0 064 0 25 0 064 FLCZ 0 25 1 0 5 0 125 0 5 0 5 C albicans ATCC 24433 AMPH B 0 25 1 1 NA 0 064 5 FC 1 4 1 NA gt 32 ITCZ NA 0 032 NA 0 064 FLCZ 0 25 1 0 25 NA 0 5 C tropicalis ATCC 750 AMPH B 0 5 2 1 NA 0 125 5 FC lt 0 125 0 25 lt 0 125 NA 0 125 ITCZ NA 0 25 NA 0 25 FLCZ 1 4 1 NA 2 C parapsilosis ATCC 90018 AMPH B 0 5 2 1 NA 0 25 5 FC lt 0 125 0 25 lt 0 125 NA 0 125 ITCZ NA 0 125 NA 0 125 FLCZ 0 25 1 1 NA 1 C krusei ATCC 6258 AMPH B 0 25 2 2 0 5 2 0 5 5 FC 4 16 4 NA gt 32 ITCZ 0 125 0 5 0 125 0 25 1 0 5 FLCZ 16 64 16 NA 64 AMPH B amphotericin B 5 FC flucytosine ITCZ itraconazole FLCZ fluconazole NA not available C albicans D 5 FC 4 E test MIC 2 C albicans FLCZ MIC C glabrata TTCZ MICoo C tropicalis D ITCZ tz Bt S MICs MIC E test 2 AMPH B MICs k MIC FLCZ MIC E test 2 3 E test MIC 4
11. 1 20094F 11 12 C albicans 20 13 7 ITCZ 18 C glabrata 20 8 11 1 Candida tropicalis 15 7 8 ITCZ ELCZ 2 C albicans 7 TIMM 3136 TIMM 3164 TIMM 3165 TIMM 3166 TIMM 3209 TIMM 3309 TIMM 3960 4 4 ITCZ FLCZ 3 C albi cans 9 IFM 46910 IFM 54354 IFM 57376 IFM 57378 IFM 57388 IFM 57389 IFM 57408 IFM 57409 IFM 57410 C tropicalis 2 KYTE 1 KYTE 2 73 C albicans ATCC 66027 24433 90028 C glabrata ATCC 90030 C tropicalis ATCC 750 Candi da parapsilosis ATCC 90018 C krusei ATCC 6258 7 2 AMPH B 5 FC ITCZ FLCZ 4 Ro
12. CLST AMPH B ITCZ 0 032 16 ug ml 5 FC FLCZ 0 125 64 ug ml 3 1 CLSI M27 A3 FP ORT McFarland No 0 5 10 FP 0 5 pl F 35 1C 24 48 2 E test E test MIC 1 mol L NaOH pH 7 0 0 1 2 0 165 mol L MOPS RPMI 1640 Sigma Aldrich C albicans 21 RPMI 1640 GIBCO 0 165 mol L MOPS RPMI 1640
13. S DD 4 2 E test 5 1 E test 2 S DD C glabrata Vol 22 No 2 2012 11 116 broth microdilution ug ml AMPH B broth microdilution ug ml E test ug ml ITCZ broth microdilution ug ml 5 FC broth microdilution ug ml E test ug ml FLCZ Fig 1 Correlation between broth microdilution MICs and E test MICs of four antifungal agents in all strains The vertical dashed line shows within 2 fold dilution The shaded frame shows beyond 2 fold dilution AMPH B amphotericin B 5 FC flucytosine ITCZ itraconazole FLCZ fluconazole S DD 2 1 E test 1 C popfcg7s S DD 6e1 1 11 1 E test KS l HAMEL Ko FLCZ C gpicgs 5 3 E test S DD 2
14. 1ng7ml 0 125 pg ml E test MICs 1 pg ml 0 25 ug ml E test C tropicalis MIC L ug ml 0 25 ug ml E test MIC 2 ug ml 0 5 ug ml E test 2 E test 1 2 2 RPMI E test E test RPMI 1640 C albi cans AMPH B 3 AMPHL B CLSI M27 A3 YEYE X E test 5 FC
15. distribution of broth microdilution and E test for 80 isolates Agent 5 4 3 sg j 0 1 2 3 4 5 gt 5 No of isolates AMPH B 80 1 1 3 6 7 5 22 27 5 31 38 8 17 21 3 2 2 5 1 1 3 5 FC 80 4 5 0 66 82 5 4 5 0 2 2 5 1 1 3 3 3 8 ITCZ 80 3 3 8 7 8 8 12 15 0 18 22 5 22 27 5 12 15 0 4 5 0 1 1 3 1 1 3 FLCZ 80 3 3 8 3 3 8 14 17 5 26 32 5 23 28 8 9 11 3 1 1 3 1 1 3 Total 320 7 2 2 6 1 9 21 6 6 41 12 8 43 13 4 50 15 6 112 35 0 25 7 8 7 2 2 1 0 3 2 0 6 5 1 6 The values are the percent of the strains exhibit the difference between E test MIC and microdilution MIC 0 E test MICs are equal to microdilution MICs 1 E test MICs were 1 dilution 2 fold higher than microdilution MICs 1 E test MICs were 1 dilution 2 fold lower than microdilution MICs 2 E test MICs were 2 dilutions 4 fold higher than microdilution MICs 2 E test MICs were 2 dilutions 4 fold lower than microdilution MICs AMPH B amphotericin B 5 FC flucytosine ITCZ itraconazole FLCZ fluconazole Table 4 Distribution of 80 organisms tested according to species and susceptibility profile No of isolates Method arid species AMPH B 5 FC ITCZ FLCZ No of isolates S RF S i R S S DD R S S DD R C albicans 39 MD 36 92 3 3 7 7 38 97 4 E test 39 100 35 89 7 C glabrata 21 MD 19 90 5 2 9
16. 112 2012 Cgzgggz E test D 23 12 1 24 2 10 fluconazole FLCZ IIE Candida albicans IE MIC Candida CLSI M27 A3 E test MIC LTC albicans 36 Candida glabrata 20 Candida tropicalis 17 7 80 amphotericin B AMPH B flucytosine 5 FC itraconazole ITCZ ELCZ 4 1 MIC 5 FC 92 5 T FLCZ 72 6 ITCZ 65 0
17. 5 21 100 1 2 6 30 76 9 4 10 3 5 12 8 32 82 1 2 5 1 5 12 8 4 10 3 31 79 5 4 10 3 4 10 3 35 89 7 1 2 6 3 7 7 1 4 8 2 9 5 18 85 7 12 57 1 9 42 9 E test 21 100 21 100 2 9 5 2 9 5 17 81 0 19 90 5 2 9 5 C tropicalis 18 MD 16 88 9 2 11 1 18 100 7 38 9 11 61 1 18 100 E test 18 100 18 100 16 88 9 2 11 1 18 100 Total 80 MD 72 90 0 8 10 0 79 98 8 E test 80 100 75 93 8 1 1 3 40 50 0 17 21 3 23 28 8 63 788 12 15 0 5 6 3 5 6 3 50 62 5 9 11 3 21 26 3 73 91 3 3 3 8 4 5 0 S susceptible S DD susceptible dose dependent I intermediate R resistant Values were as determined by the CLSI M27 A3 broth microdilution reference method MD broth microdilution 5AMPH B amphotericin B 5 FC flucytosine ITCZ itraconazole FLCZ fluconazole R MIC 22 ug ml There is no official breakpoint in AMPH B MIC I intermediate interpretive category for flucytosine only MIC RPMI 1640 RPMI 1640 JEG AD RPMI 1640 Bourgeois C albicans MIC
18. 5 ITCZ MD 0 032 0 5 0 25 0 5 E test 0 032 0 25 0 064 0 125 FLCZ MD 0 25 2 0 5 1 E test 0 125 2 0 25 0 5 Total 80 AMPH B MD 0 5 2 1 1 E test 0 032 16 0 125 0 25 5 FC MD 0 125 64 0 125 0 25 E test 0 064 64 0 125 0 5 ITCZ MD 0 032 16 0 125 2 E test 0 032 16 0 064 2 FLCZ MD 0 125 64 1 16 E test 0 125 64 0 25 8 AMPH B amphotericin B 5 FC flucytosine ITCZ itraconazole FLCZ fluconazole MD broth microdilution 5 FC 92 5 82 5 ITCZ ELCZ 1 65 0 ITCZ 72 6 FLCZ E test 2 27 5 ITCZ 25 0 FLCZ 48 Table 4 AMPH B 2 ug ml 8 E test 5 FC 4 E test ITCZ C albicans
19. 8 E test Eig 1 AMPH B range 0 5 2 ng ml E test 0 032 0 5 ug ml 1 3 3 8 96 3 E test 2 C albicans 21 EE test range 0 25 0 5 pg ml 5 FC 6 E test 2 70 Vol 22 No 2 2012 4 4 ug ml E test C264 ng ml 0 125 ug ml TITCZ 80 22 27 6 FLCZ 20 25 1 E test 2 C albicans 3 ITCZ ELCZ 16 ug ml ITCZ 264 ug ml FLCZ E test 0 25 pg ml 4
20. E test C albicans MIC MIC 4 MIC AIDS E teet 24 ITCZ 0 06 pg ml 0 125 ug ml FLCZ 0 25 pg ml E teet Barry Matar M 24 MIC E test O 48 MIC ITCZ FLCZ C albicans C glabrata E test MIC 2 S DD S DD 5 FC 3 E test
21. hill Livingstone Philadelphia Pennsylvania Clark T A A Hajjeh R 2002 Recent trends in the epidemiology of invasive mycoses Curr Opin In fect Dis 15 569 574 2009 NCCLS M27 A2 mi crodilution 58 1 15 2i 2009 No 201 55 12 309 320 Fleck R A Dietz H Hof 2007 In vitro susceptibil ity of Candida species to five antifungal agents in a German university hospital assessed by the reference broth microdilution method and E test J Antimi crob Chemother 59 4 767 771 2003 MIC 51 8 470 476 2009 2 36 569 574 FF 2009 1 E 36 536 543 Clinical and Laboratory Standards Institute Refer ence method for broth dilution antifungal suscepti bility testing of yeasts Approved standard third edi t
22. ion M27 A3 CLSI Wayne PA USA 2008 AB Biodisk 2007 Antifungal susceptibility testing for in vitro diagnostic use AB Biodisk Solna Swe den Favel A A Michel Nguyen A Datry et al 2004 Susceptibility of clinical isolates of Candida lusitani ae to five systemic antifungal agents J Antimicrob Chemother 53 3 526 529 Bourgeois N L Dehandschoewercker S Bertout et al 2010 Antifungal susceptibility of 205 Candida spp isolated primarily during invasive Candidiasis and comparison of the Vitek 2 system with the CLSI broth microdilution and E test methods J Clin Mi crobiol 48 1 154 161 Koga Ito C Y J P Lyon M A Resende 2008 Candida 119 15 16 Comparison between E test and CLSI broth micro dilution method for antifungal susceptibility testing of Candida albicans oral isolates Rev Inst Med Trop Sao Paulo 50 1 7 10 Park J Y J H Shin Y Uh et al 2008 In vitro am photericin B susceptibility of korean bloodstream yeast isolates assessed by the CLSI broth microdilu tion method E test and Minimum fungicidal con centration test Korean J Lab Med 28 5 346 352 Park B J B A Arthington Skaggs R A Hajjeh et al 2006 Evaluation of amphotericin B interpretive breakpoints for Candida bloodstream isolates by correlation with therapeutic outcome Antimicrob 18 19 20 Pfaller M A S A Messer A Karlss
23. on et al 1998 Evaluation of the E test method for determining flu conazole susceptibilities of 402 clinical yeast isolates by using three different agar media J Clin Microbi ol 36 9 2586 2589 Barry A L M A Pfaller R P Rennie et al 2002 Precision and accuracy of fluconazole susceptibility testing by broth microdilution E test and disk diffu sion methods Antimicrob Agents Chemother 46 6 1781 1784 Matar M J L Ostrosky Zeichner V L Paetznick et al 2003 Correlation between E test disk diffusion and microdilution methods for antifungal suscepti bility testing of fluconazole and voriconazole Anti microb Agents Chemother 47 5 1647 1651 Agents Chemother 50 4 1287 1292 17 2010 56 6 119 138 Comparison of Broth Microdilution Method with the E Test for Candida spp Susceptibility to Antifungal Agents Hiromi Morishige Yoko Mano Mieko Goto Toyoko Oguri Nobuhiko Furuya 1 Graduate School of Health Care Science Bunkyo Gakuin University Faculty of Health Science Technology Bunkyo Gakuin University 3 The University of Tokyo Hospital Clinical Laboratory Kameda Medical Center Recently the increase in incidence of deep mycoses and the occurrence of fluconazole FLCZ resistant Candida albi cans require the accurate determination of MIC against fungi We compa
24. red and evaluated the MICs of antifungal agents by broth microdilution based on CLSI M27 A3 and E test In this study we tested isolates from each of the fol lowing species reference isolates 7 isolates C albicans 36 isolates Candida glabrata 20 isolates and Candida tropi calis 17 isolates These methods were used to determine the MICs of amphotericin B AMPH B flucytosine 5 FC itraconazole ITCZ FLCZ for all 80 isolates The rates of MIC agreement within 2 fold dilution between broth microdi lution and E test methods were as follows 5 FC 92 5 FLCZ 72 6 ITCZ 65 0 On the other hand MICs of E test were lower more than 4 fold dilution than those of broth microdilution in AMPH B 96 3 ITCZ 27 5 and FLCZ 25 0 Several isolates showed discrepancies of susceptibility according to methods strains and or agents except for C glabrata and C tropicalis for 5 FC and C tropicalis for FLCZ In this study the MICs of E test tended to be lower than broth microdilution method against many isolates tested Therefore these results suggest that interpretation of suscepti bility required the special attention Vol 22 No 2 2012 15
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