烟曲霉再鉴定、标准化CSP分型及体外药物敏感性

摘要/Abstract

摘要： 目的了解烟曲霉复合体临床株菌种分布,经典烟曲霉临床株CSP基因型及对常见抗真菌药物敏感性状况。方法菌株来源:北京大学真菌和真菌病研究中心保存分离自125名患者的162株烟曲霉复合体菌株。通过形态学,最高生长温度及分子生物学测序分步鉴定;对CSP基因进行扩增、测序,采用国际化命名体系进行CSP分型;采用微量液基稀释法测定经典烟曲霉对伊曲康唑(ITC)、两性霉素B(AMB)、伏立康唑(VRC)及卡泊芬净(CAS)的敏感性。结果所有烟曲霉复合体菌株均为经典烟曲霉;共分为16个CSP基因型,最常见为t04A、t03和t01;分离自4名患者的13株菌对ITC的MICs≥4μg/mL,其中2株菌AMB和VRC的MICs分别为4μg/mL和16μg/mL。CAS的MECs最高为4μg/mL,仅1株。结论未检出烟曲霉相关新种;经典烟曲霉临床株共16个CSP基因型,分布与国际研究结果基本一致,其中5个为新型。我国经典烟曲霉临床株ITC耐药率为3.2%,个别菌株AMB,VRC和CAS耐药。

关键词: 烟曲霉复合体, 经典烟曲霉, 鉴定, CSP分型, 体外药敏试验

Abstract: Objective To investigate the strain distribution of Aspergillus fumigatus complex,the CSP genotypes and in vitro susceptibility of classic A.fumigatus strains.Methods One hundred and sixty-two strains of A.fumigatus complex isolated from 125 patients were re-identified as Aspergillus section Fumigati via macro-and micro-morphological characteristics,and classical A.fumigatus via maximum grow temperature.Atypical strains were identified based on internal transcribed spacer of the rDNA gene(rDNA ITS) sequencing.Classic A.fumigatus was typed by CSP gene sequencing and subsequently analyzed with the international nomenclature.In vitro antifungal susceptibility to itraconazole(ITC),amphotericin B(AMB),voriconazole(VRC) and caspofungin(CAS) were determined by broth microdilution susceptibility test.Results All strains were identified as classic A.fumigatus complex with 16 genotypes.Type t04A,t03 and t01 were most common in our study.MICs of ITC were ≥4 μg/mL in 13 strains isolated from 4 patients(3.2%),two strains of which have MICs against AMB and VRC at 4 μg/mL and 16 μg/mL,respectively.The highest MEC against CAS was 4 μg/mL in 1 strain.Conclusions No newly proposed A.fumigatus associated specie was found.All clinical isolates of classic A.fumigatus in clinical were divided into 16 CSP genotypes,five of which had not been reported previously.Genotype distribution was similar with literature.ITC resistance rate was 3.2%in classical A.fumigatus in China.Resistances to AMB,VRC and CAS exist in some isolates.

Key words: Aspergillus fumigatus complex, classic Aspergillus fumigatus, identification, CSP typing, susceptibility

中图分类号:

R519.8

高露娟, 余进, 李若瑜. 烟曲霉再鉴定、标准化CSP分型及体外药物敏感性[J]. 中国真菌学杂志, 2011, 6(6): 328-331,336.

GAO Lu-juan, YU Jin, LI Ruo-yu. Re-identification,standard CSP typing and in vitro susceptibility of Aspergillus fumigatus complex[J]. Chinese Journal of Mycology, 2011, 6(6): 328-331,336.

参考文献

[1] Messer SA,Jones RN,Fritsche TR.International surveillance of
Candida spp.and Aspergillus spp.:report from the SENTRY
Antimicrobial Surveillance Program(2003)[J]. J Clin Microbi-ol,2006,44(5):1782-1787.
[2] Balajee SA,Houbraken J,Verweij PE.Aspergillus spe-cies identification in the clinical setting[J]. Stud Mycol,2007,
59:39-46.
[3] Hong SB,Shin HD,Hong J.New taxa of Neosartorya and
Aspergillus in Aspergillus section Fumigati[J]. Antonie Van
Leeuwenhoek,2008,93(1-2):87-98.
[4] Balajee SA,Kano R,Baddley JW.Molecular identifica-tion of Aspergillus species collected for the transplant-associated
infection surveillance network[J]. J Clin Microbiol,2009,47
(10):3138-3141.
[5] 高露娟,余进,李若瑜.中国大陆地区曲霉病流行现状分析
[J]. 中国真菌学杂志,2010(4):247-251.
[6] Balajee SA,Tay ST,Lasker BA.Characterization of a no-vel gene for strain typing reveals substructuring of Aspergillus
fumigatus across North America[J]. Eukaryot Cell,2007,6
(8):1392-1399.
[7] Hurst SF,Kidd SE,Morrissey CO.Interlaboratory repro-ducibility of a single-locus sequence-based method for strain typ-ing of Aspergillus fumigatus[J]. J Clin Microbiol,2009,47
(5):1562-1564.
[8] Bain JM,Tavanti A,Davidson AD.Multilocus sequence
typing of the pathogenic fungus Aspergillus fumigatus[J]. J
Clin Microbiol,2007,45(5):1469-1477.
[9] Kidd SE,Nik ZN,Slavin MA.Utility of a proposed CSP
typing nomenclature for Australian Aspergillus fumigatus iso-lates:Identification of additional CSP types and suggested modi-fications[J]. J Microbiol Methods,2009,78(2):223-226.
[10] Howard SJ,Arendrup MC.Acquired antifungal drug resistance
in Aspergillus fumigatus:epidemiology and detection[J]. Med
Mycol,2011,49 Suppl 1:S90-S95.
[11] Howard SJ,Cerar D,Anderson MJ.Frequency and evo-lution of azole resistance in Aspergillus fumigatus associated
with treatment failure[J]. Emerg Infect Dis,2009,15(7):
1068-1076.
[12] Arendrup MC,Garcia EG,Buzina W.Breakthrough
Aspergillus fumigatus and Candida albicans double infection
during caspofungin treatment:laboratory characteristics and im-plication for susceptibility testing[J]. Antimicrob Agents Che-mother,2009,53(3):1185-1193.
[13] Madureira A,Bergeron A,Lacroix C.Breakthrough inva-sive aspergillosis in allogeneic haematopoietic stem cell trans-plant recipients treated with caspofungin[J]. Int J Antimicrob
Agents,2007,30(6):551-554.[6] Vemuganti GK,Garg P,Gopinathan U.Evaluation of agent
and host factors in progression of mycotic keratitis:a histologic
and microbiologic study of 167 corneal buttons[J]. Ophthalmolo-gy,2002,109(8):1538-1546.
[7] Cuenca-Estrella M,Gomez-Lopez A,Mellado E.Head-to-head comparison of the activities of currently available antifungal
agents against 3,378 Spanish clinical isolates of yeasts and fila-mentous fungi[J]. Antimicrob Agents Chemother,2006,50
(3):917-921.
[8] 孙淑娟,娄红祥,苏乐群,等.真菌感染与抗真菌药物[J]. 食
品与药品,2005,7(2):1-4.[14] Arendrup MC,Perkhofer S,Howard SJ.Establishing in
vitro-in vivo correlations for Aspergillus fumigatus:the chal-lenge of azoles versus echinocandins[J]. Antimicrob Agents
Chemother,2008,52(10):3504-3511.
[15] Glass NL,Donaldson GC.Development of primer sets designed
for use with the PCR to amplify conserved genes from filamentous
ascomycetes[J]. Appl Environ Microbiol,1995,61(4):1323-1330.
[16] Klaassen CH,de Valk HA,Balajee SA.Utility of CSP
typing to sub-type clinical Aspergillus fumigatus isolates and pro-posal for a new CSP type nomenclature[J]. J Microbiol Meth-ods,2009,77(3):292-296.
[17] Espinel-Ingroff A,Arthington-Skaggs B,Iqbal N.Multi-center evaluation of a new disk agar diffusion method for suscep-tibility testing of filamentous fungi with voriconazole,posacon-azole,itraconazole,amphotericin B,and caspofungin[J]. J
Clin Microbiol,2007,45(6):1811-1820.
[18] Yaguchi T,Horie Y,Tanaka R.Molecular phylogenetics
of multiple genes on Aspergillus section Fumigati isolated from
clinical specimens in Japan[J]. Nippon Ishinkin Gakkai
Zasshi,2007,48(1):37-46.
[19] Balajee SA,Nickle D,Varga J.Molecular studies reveal
frequent misidentification of Aspergillus fumigatus by morphotyp-ing[J]. Eukaryot Cell,2006,5(10):1705-1712.
[20] Vinh DC,Shea YR,Sugui JA.Invasive aspergillosis due
to Neosartorya udagawae[J]. Clin Infect Dis,2009,49(1):
102-111.
[21] Alcazar FL,Mellado E,Alastruey LA.Aspergillus section
Fumigati:antifungal susceptibility patterns and sequence-based
identification[J]. Antimicrob Agents Chemother,2008,52(4):
1244-1251.
[22] Vinh DC,Shea YR,Jones PA.Chronic invasive aspergil-sp.nov.,a new sibling species of A.fumigatus[J]. Eukaryot
Cell,2005,4(3):625-632.
24] Montenegro G,Sanchez PS,Jewtuchowicz VM.Phenotyp-ic and genotypic characterization of Aspergillus lentulus and
Aspergillus fumigatus isolates in a patient with probable invasive
aspergillosis[J]. J Med Microbiol,2009,58(Pt 3):391-395.
25] Alhambra A,Catalan M,Moragues MD.Isolation of
Aspergillus lentulus in Spain from a critically ill patient with
chronic obstructive pulmonary disease[J]. Rev Iberoam Micol,
2008,25(4):246-249.
26] Levdansky E,Romano J,Shadkchan Y.Coding tandem
repeats generate diversity in Aspergillus fumigatus genes[J].
Eukaryot Cell,2007,6(8):1380-1391.
27] Alvarez-Perez S,Garcia ME,Bouza E.Characterization of
multiple isolates of Aspergillus fumigatus from patients:geno-type,mating type and invasiveness[J]. Med Mycol,2009,47
(6):601-608.
28] Snelders E,van der Lee HA,Kuijpers J.Emergence of
azole resistance in Aspergillus fumigatus and spread of a single
resistance mechanism[J]. PLoS Med,2008,5(11):e219.
29] Lionakis MS,Lewis RE,Chamilos G.Aspergillus suscep-tibility testing in patients with cancer and invasive aspergillosis:
difficulties in establishing correlation between in vitro suscepti-bility data and the outcome of initial amphotericin B therapy[J].
Pharmacotherapy,2005,25(9):1174-1180.