Sistemik klinik örneklerden üretilen aspergillus türlerinin antifungal duyarlılıklarının farklı yöntemlerle araştırılması

Aspergillustürlerinin, bağışıklık sistemi baskılanmış kişilerde ölümcül enfeksiyonlara yolaçması ve son yıllarda antifungallere direnç oranının artması nedeniyleantifungal duyarlılık testlerinin önemi artmıştır. Çalışmamızda Aspergillus türleri için disk difüzyon (DD) ve gradientdifüzyon (GD)yöntemlerini, referans duyarlılık yöntemi olan sıvı mikrodilüsyon (SMD) yöntemiile karşılaştırmayı amaçladık. Ayrıca amfoterisin B (AMB), itrakonazol (İTZ), vorikonazol (VOR) vekaspofungine (KAS) karşı duyarlılık durumunu ve MİK değerlerini belirleyerek ülkemizepidemiyolojik verilerine katkıda bulunmayı ve hastanemizde üreyen Aspergillus türlerinde direnç oranlarınıbelirleyerek ampirik tedaviye yön vermeyi hedefledik.Yöntem: Çalışmada62 A. fumigatus, 28 A.flavus ve 16 A. terreus izolatı (toplam 106 Aspergillus izolatı) yer aldı. Çalışmaya dahil edilen Aspergillus izolatları, gelenekselyöntemler ve Matriks İlişkili Lazer Dezorpsiyon İyonizasyonUçuş Zamanı Kütle Spektrometresi (MALDI-TOF MS) ile tanımlandı. Tüm izolatların DD, GD ve SMD yöntemi ile AMB, VOR, İTZ veKAS’a karşıduyarlılıkları belirlendi. SMD yönteminde RPMI 1640 sıvı besiyeri, DDve GD yönteminde saf Mueller Hinton agar (MHA)kullanıldı.Bulgular: ÇalışmamızdaSMD yöntemi ile en düşük MİK/MEK90 değerleri VOR ve KAS (sırasıyla0.5 µg/ml ve 0.06 µg/ml) ile saptandı. AMB ve İTZ MİK90 değerleriise 2 µg/ml olarak bulundu. Tür düzeyinde incelediğimizde, A. flavus ve A. terreus izolatlarınınAMB MİK90 değerleri (sırasıyla 2 µg/ml ve 4 µg/ml), A. fumigatus izolatlarından (1 µg/ml)yüksek saptandı. A. flavus ve A. terreus izolatlarının İTZ MİK90değerleri 1 µg/ml olup, A. fumigatus izolatlarından(2 µg/ml) daha düşük bulundu. Tüm türlerin İTZ MİK değerlerinin literatürlekarşılaştırdırıldığında yıllar içinde artış gösterdiği saptandı.GD yöntemini SMD yöntemi ilekarşılaştırdığımızda ±2 dilüsyonda temel uyum oranları, AMB,VOR, İTZ ve KAS için sırası ile %91.6, %99.1, %100 ve %38.6 olarak saptandı.KAS ile temel uyum oranları düşük bulunmasına rağmen kategorik uyum oranlarıyüksek bulundu.DD yöntemi SMD yöntem ilekarşılaştırıldığında kategorik uyum oranları AMB, VOR, İTZ ve KAS için sırasıile %65.1, %99.1, %77.3 ve %100 olarak saptandı. GD yöntemini SMD yöntemi ilekarşılaştırıldığında tüm türler bir arada değerlendirildiğinde kategorik uyumoranları AMB, VOR, İTZ ve KAS için sırası ile %79.2, %99.1, %87.8 ve %100olarak saptandı. GD yöntemi kategorik uyum oranları, DD yönteminin AMB ve İTZuyum oranlarından yüksek olup VOR ve KAS için her iki yöntemde aynıbulunmuştur. Sonuç:Saptanan yüksek temel ve kategorik uyum oranlarına göre GD yönteminin, SMDyöntemine iyi bir alternatif olabileceğini düşünmekteyiz. Ayrıca GD yöntemiiçin kullandığımız saf MHA, diğer besiyerlerine göre daha ucuz ve kolay ulaşılabilirolmasıyla avantaj sağlamaktadır. CLSI M51-A DD yöntemini SMD yöntemiylekarşılaştırdığımızda tüm ilaçlar için yüksek uyum oranları saptandı. Fakat AMBve İTZ uyum sonuçları diğer ilaçlara göre daha düşük saptanmıştır. Bu sonuçlaragöre DD yöntemi de SMD yöntemine bir alternatif olarak düşünülebilir fakat AMBve İTZ sonuçları daha dikkatli yorumlanmalıdır.Anahtar kelimeler: Aspergillus, antifungal duyarlılık, sıvı mikrodilüsyon, gradientdifüzyon testi, disk difüzyon, amfoterisin B, itrakonazol, vorikonazol, kaspofunginThe importance of antifungal susceptibilitytests has increased due to the fact that Aspergillusspecies cause fatal infections in immunocompromised individuals and theresistance rate to antifungals has increased in recent years.In our study, weaimed to compare the disc diffusion (DD) and gradient diffusion (GD) methodsfor Aspergillus species with thebroth microdilution (SMD) method, which is the reference susceptibilitymethod.In addition, we aimed to contribute to the epidemiological data of ourcountry by determining the susceptibility status and MIC values foramphotericin B (AMB), itraconazole (ITZ), voriconazole (VOR) and caspofungin(CAS) and to guide empirical treatment by determining the resistance rates in Aspergillus species isolated in ourhospital.Method: Sixty-twoA. fumigatus, 28 A. flavus and 16 A. terreusisolates (a total of 106 Aspergillus isolates)were included in the study. Aspergillusisolates included in the study were identified by conventional methods andMatrix Associated Laser Desorption Ionization Time-of-Flight Mass Spectrometer(MALDI-TOF MS). The susceptibilities of all isolates to AMB, VOR, ITZ and CASwere determined by performing DD, GD and SMD methods. RPMI 1640 broth was usedin the SMD method, and nonsupplemented MuellerHinton agar (MHA) was usedin the DD and GD methods.Results: In ourstudy, the lowest MIC/MEC90 values ​​were determined for VOR and CAS(0.5 µg/ml and 0.06 µg/ml, respectively) with SMD method. AMB and ITZ MIC90 values​​were found to be 2 µg/ml. When we analyzed at the species level, A. flavus and A. terreusisolates had higher AMB MIC90 values ​​(2 µg/ml and 4 µg/ml,respectively) than A. fumigatusisolates (1 µg/ml). ITZ MIC90 values ​​of A. flavus and A. terreusisolates were 1 µg/ml and were found lower than A. fumigatus isolates (2 µg/ml). It was determined that ITZ MICvalues ​​of all species have increased over the years when compared with theliterature. When we compared the GD method with the SMDmethod, the essential agreement rates at ±2 dilution were found to be 91.6%,99.1%, 100% and 38.6% for AMB, VOR, ITZ, and CAS, respectively. Althoughessential agreement rates with CAS were low, categorical agreement rates werehigh.When the DD method was compared with the SMDmethod, categorical agreement rates were found to be 65.1%, 99.1%, 77.3% and100% for AMB, VOR, ITZ, and CAS, respectively.When the GD method was compared with the SMDmethod, the categorical agreement rates were found to be 79.2%, 99.1%, 87.8%and 100% for AMB, VOR, ITZ and CAS, respectively if all species were evaluatedtogether. The categorical agreement rates of the GD method were higher than theAMB and ITZ agreement rates of the DD method while they were found to be thesame in both methods for VOR and CAS.Conclusion:According to the high essential and categorical agreement rates, we think thatGD method may be a good alternative for SMD method. Furthermore,nonsupplemented MHA which we used for GD method provides an advantage as it ischeaper and easily accessible than other media.When we compared CLSI M51-A DD method with SMD method,high agreement rates were found for all drugs. However, AMB and ITZ agreementresults were found to be lower than other drugs. According to these results, DDmethod can also be considered as an alternative to SMD method, but AMB and ITZresults should be interpreted more carefully.Keywords:Aspergillus, antifungalsusceptibility, broth microdilution, gradient diffusion test, disc diffusion,amphotericin B, itraconazole, voriconazole, caspofungin</p

Evaluation of species distribution of yeasts isolated from intensive care units during the four years period Yoǧun bakιm ünitelerinden dört yιllιk dönemde izole edilen mayalarιn tür daǧιlιmιnιn deǧerlendirilmesi

The aim of this study was to evaluate the distributions of yeast species according to the years and to detect the emerging pathogens in intensive care units (ICU). For this purpose, yeast isolation rates were detected retrospectively, in the following time periods: Period I: April-December 2001; period II: January-December 2002; period III: January-December 2003; period IV: January-December 2004. A total of 490 yeast isolates recovered from 462 clinical specimens obtained from 360 different ICU patients were investigated during these periods. Urine (62.1%), blood (13.6%) and tracheal aspirate (8.7%) samples were detected as the most common specimens. Of these isolates, 53.3% were identified as Candida albicans, 14.5% as C.tropicalis, 12.2% as C.glabrata, 6.5% as C.parapsilosis, 4.5% as Trichosporon spp., 3.9% as C.kefyr, 1.6% as C.krusei, 1.4% as Geotrichum candidum and 2.1% as other Candida species. The isolation rates of C.albicans in the periods of I to IV were found as 47.7%, 55.5%, 41.7% and 62.4%, respectively. The decrease between the second and third periods, and increase between third and fourth periods were statistically significant (x2=4.15, p=0.04 and x2=8.32, p=0.004). C.glabrata was the second most common species in the first and second periods (14.8% and 15.5%, respectively), followed by C.tropicalis (12.5% and 10.0%, respectively), however this array has changed in the third and fourth periods (C.tropicalis was the second with the rates of 16.7% and 16.8%, while C.glabrata placed in the third line with the rates of 14.8% and 7.6%, respectively), It was concluded that C.albicans has still been the most frequent species among yeast isolates of ICU's in our hospital; however, the incidence of non-albicans species like C.glabrata and C.tropicalis has increased

Investigation of Aspergillus galactomannan levels in antimicrobial agents

The diagnosis of invasive aspergillosis which is a serious infection of immunocompromized patients,, depends on the detection of Aspergillus galactomannan antigen in the serum by enzyme immunoassay (EIA) in routine laboratories. However, it has been previously reported that false positive results in Aspergillus galactomannan test may be obtained in the sera of patients sera receiving piperacillin-tazobactam (PIP-TAZ). The aim of this study was to investigate the presence and levels of Aspergillus galactomannan antigen in the content of PIP-TAZ and some other antimicrobial agents that are often used for the treatment of infections in immunocompromised patients. The level of galactomannan antigen was determined for PIP-TAZ, ampicillin-sulbactam, ampicillin, penicillin G, ceftriaxone, cefepime, imipenem, clarithromycin, ciprofloxacin, vancomycin, gentamicin, trimethoprim-sulfamethoxazole, ornidazole, fluconazole and amphotericin 6, by a commercial EIA (Platelia Aspergillus EIA, Bio-Rad, France) kit. Galactomannan index (Gl) was estimated with the ratio of absorbance values of antimicrobials to cut-off value and evaluated as positive when GI was found > 0.5. Amongst the 15 antibiotics studied, the only positive result was detected for ampicillin with the highest index value (GI=0.540), followed by PIP-TAZ with a relatively high value (GI=0.235) even though it was not in the range of positivity. GI values have ranged from 0.011 to 0.188 for the other antibiotics. In conclusion, the use of especially ampicillin (and probably PIP-TAZ) therapy should be questioned in patients whose sera are being tested for Aspergillus galactomannan antigen by EIA in order to evaluate the positive results in terms of false positivities due to cross reactivity

Molecular epidemiology of Candida species isolated from urine at an intensive care unit

Candida spp. has been the leading microorganism isolated from the urine specimens of patients hospitalized at the Anesthesiology and Reanimation intensive care unit (ICU) of Dokuz Eylul University Hospital, Izmir, since 1998. This study was undertaken to investigate the clonal relationship of Candida urine isolates in order to find the mode of spread among the patients. Epidemiological surveillance of 38 Candida albicans, 15 Candida tropicalis and 12 Candida glabrata recovered from the urine specimens of patients who were hospitalized in the ICU between June 11, 2000 and October 15, 2001 was carried out by antifungal susceptibility testing and randomly amplified polymorphic DNA (RAPD) analysis. Two short primers [Cnd3 (5'-CCAGATGCAC-3') and Cnd4 (5'-ACGGTACACT-3')] were used for RAPD. None of the isolates had high minimal inhibitory concentration (MIC) values (>1 mug ml(-1)) against amphotericin B with MIC(50)values of 0.5 mug ml(-1), 0.5 mug ml(-1) and 0.125 mug ml(-1) for C. albicans, C. tropicalis and C. glabrata isolates, respectively. However, three C. glabrata isolates were resistant and one C. albicans and five C. glabrata isolates were dose-dependent susceptible (D-DS) to fluconazole. Among C. albicans isolates 19 and 20 patterns were detected with primers Cnd3 and Cnd4, respectively. When primers Cnd3 and Cnd4 were evaluated together, three and four genotypes were identified for C. tropicalis and C. glabrata isolates, respectively. Our results suggest that the source of C. albicans isolates was mostly endogenous. It is difficult to interpret the mode of spread of C. tropicalis and C. glabrata urine isolates as we obtained insufficient banding patterns for these species