Determination of azole resistance in aspergillus fumigatus strains isolated from patient samples

Aspergillus enfeksiyonları özellikle immun baskılanmış hastalarda önemli mortalite ve morbidite nedeni olup, A. fumigatus hava yolu ile bulaşan en önemli enfeksiyon etkenidir. Azoller bu enfeksiyonlardan korunmada ve tedavide en fazla tercih edilen ilaçlardır. Ancak, A. fumigatus izolatları arasında artan oranda azol direncine, çok sayıda ülkede rastlanılmıştır. Azollerin klinikte yaygın kullanımı veya çevresel azol fungisitlere maruz kalma, kazanılmış azol direncinin olası sebeplerindendir. Azol direncinde en sık bildirilen mekanizma, ergosterol sentezinde rol alan 14-α-demetilaz enzimini kodlayan cyp51A genindeki mutasyonlardır. Bu çalışmada da, 1999-2012 yılları arasında çeşitli klinik örneklerden izole edilen A. fumigatus suşlarında azol direncinin sıklığı ve dirence sebep olan cyp51A genindeki mutasyonlar araştırılmıştır. Araştırmaya, 419 hastadan izole edilen 746 A. fumigatus suşu alındı. Tüm suşlar 4mg/L itrakonazol (ITZ) içeren Sabouraud's dekstroz agara ekilerek ITZ direnci açısından tarandı. ITZ içeren agarda üreyen suşların, ITZ'ye, vorikonazole (VOR) ve amfoterisin B'ye (AMB) in vitro duyarlılıkları "Clinical and Laboratory Standards Institute" (CLSI M38A) referans metodu kullanılarak değerlendirildi. PCR amplifikasyonu sonrası, mutasyonları belirlemek için tüm in vitro azol dirençli suşların cyp51A gen ve promotor bölgelerinin dizi analizi yapıldı. Tarama testleri ile A. fumigatus suşlarında ITZ direnci %10,2 (76/746) olarak bulundu. Dirençli suşlara 2000 yılından sonra hemen hemen her yıl (2005 hariç) rastlandı ve en yüksek direnç oranı (%24,6) 2007 yılında görüldü. İn-vitro duyarlılık testleri ile tüm suşlar AMB'ye duyarlı iken, ITZ ve VOR'e dirençli bulundu. cyp51A gen ve promotor bölgenin dizi analizi ile suşların %86,8'inde (66 izolat) TR34/L98H mutasyonu olduğu saptandı. Bu çalışma ile klinik izolatlar arasında azol dirençli A. fumigatus suşlarının bulunduğu ve bu suşlardaki esas direnç mekanizmasının zirai fungisitlerle çapraz reaksiyon sonucu oluştuğu bilenen TR34/L98H mutasyonu olduğu gösterildi. Bu çalışma TR34/L98H mutasyonunun ülkemizde gösterildiği ilk çalışma olup, 2000 yılından bu yana görülmektedir. Bu nedenle, bu bulgunun azol birleşikleri kullanırken akılda bulundurulması gerekmektedir.Aspergillus infections are an important cause of mortality and morbidity especially in immunocompromised hosts, and Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens causing infection. Azoles are the drugs of choice for prophylaxis and treatment of these infections. Recently, an increasing frequency of azole resistant A. fumigatus isolates has been described in different countries. Acquired resistance to azoles is possibly, due to widespread clinical use of azoles or through exposure to azole fungicides in the environment. The most commonly reported mechanism conferring azole resistance is the mutation of cyp51A gene which encodes 14-α-demethylase, the enzyme that has an important role in synthesis of ergosterol. In this study, we investigated the frequency of azole resistance among A. fumigatus isolates collected between 1999 and 2012 from clinical specimens and identified mutations in the cyp51A gene. Seven hundred and forty-six A. fumigatus isolates, collected from 419 patients were investigated. All isolates were screened for resistance to itraconazole (ITZ) by subculturing on Sabouraud's dextrose agar that contained 4 mg/L ITZ. For isolates that grew on ITZ containing agar, the in vitro activities of amphotericin B (AMB), itraconazole and voriconazole (VOR) were determined by using the Clinical and Laboratory Standards Institute (CLSI-M38-A) reference method. After PCR amplification, cyp51A gene and its promoter region of all in-vitro azole resistant isolates were sequenced to determine the mutations. ITZ resistance was found in 10.2% (76/746) of A. fumigatus isolates. From the year 2000 onwards, patients were observed annually with an ITZ-resistant isolate (except 2005), with a maximum prevalence (24.6%) in 2007. According to in vitro susceptibility tests, AMB exhibited a good activity against all of them where as ITZ and VOR were resistant. Sequence analysis of the promoter region and cyp51A gene indicated the presence of TR34/L98H in 86.8% (66 isolates) of isolates. This study revealed the presence of azole resistance in our clinical isolates and the dominance of the TR34/L98H resistance mechanism supported the environmental route of cross-resistance to certain agriculture azole fungicides. This is the first study that shows TR34/L98H mutation in Turkey and azole resistance has emerged since 2000. Thus, it is important to keep these results in mind while using the azole compounds

Development of a Real-Time Polymerase Chain Reaction Method for the Identification of Candida Species

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 mu l of extracted DNA, 2 mu l of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 mu l of MgCl2 (5 mmol), 2 mu l of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 mu l of each primer (0.01 nmol/mu l) and 1 mu l of each probe (0.1 mu mol/mu l) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95 degrees C for 10 mins and 50 cycles of denaturation at 95 degrees C for 10 secs, annealing at 62 degrees C for 10 secs and polymerisation at 72 degrees C for 20 secs. A melting curve was created by cooling the producs at 50 degrees C for 30 secs and then heating to 80 C at a rate of 0.1 degrees C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/mu l were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains

First determination of azole resistance in Aspergillus fumigatus strains carrying the TR34/L98H mutations in Turkey

Aspergillus fumigatus is the most important etiological agent of invasive aspergillosis. Recently, an increasing number of azole-resistant A. fumigatus isolates have been described in various countries. The prevalence of azole resistance was investigated in this study using our culture collection of A. fumigatus isolates collected between 1999 and 2012 from clinical specimens. Seven hundred and forty-six A. fumigatus isolates, collected from 419 patients, were investigated. First, all isolates were screened for resistance to itraconazole by subculturing on Sabouraud dextrose agar that contained 4 mg/L itraconazole. For isolates that grew on the itraconazole containing agar, the in vitro activities of amphotericin B, itraconazole, voriconazole and posaconazole were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A reference method. After PCR amplification, the full sequence of the cyp51A gene and its promoter region was determined for all in vitro azole-resistant isolates. Itraconazole resistance was found in 10.2% of the A. fumigatus isolates. From 2000 onwards, patients were observed annually with an itraconazole-resistant isolate. According to in vitro susceptibility tests, amphotericin B exhibited good activity against all isolates whereas the azoles were resistant. Sequence analysis of the promoter region and CYP51A gene indicated the presence of TR34/L98H in 86.8% (n - 66) of isolates. This initial analysis of the resistance mechanism of A. fumigatus from Turkey revealed a common TR34/L98H mutation in the cyp51A gene. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved

Investigation of methicillin resistant Staphylococcus aureus in neonatal intensive care unit

Methicillin resistant Staphylococcus aureus (MRSA) strains lead to severe infections in immunosupressive patients, geriatric population and premature infants. 27 MRSA strains isolated in the Neonatal Intensive Care Unit was considered as an outbreak and it was aimed to investigate the genetic and epidemiologic relation of the MRSA outbreak. MecA gene was investigated in the S. aureus strains and pulsed field gel electrophoresis (PFGE) was used to investigate the genetic relation between outbreak strains. MecA gene was showed in all isolates. PFGE revealed that there were two different strains and most of the isolates (25/27) were owing to same clone. One of the samples were found closely related with the common strain and the other sample was found genetically unrelated. To terminate the outbreak; liquid baby food was gained to the baby food kitchen, no more new patient was imported to the neonatal unit and none of the patients were exported from neonatal unit to other clinics during outbreak, education about infection control precautions was given to all the staff and nursing bottle dishwasher was obtained. To manage and terminate the outbreak, besides the infection control precautions, tests to determine the genetic relation between outbreak strains which are done in the microbiology laboratory are needed. Molecular analysis of outbreak strains will contribute to prove the epidemiologic and evolution of outbreaks