74 research outputs found
Framing Reformulation
No full textGreen Open Access added to TU Delft Institutional Repository 'You share, we take care!' - Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Theory, Territories & Transition
Comparisson of two molecular methods for detection of Aspergillus spp. from lower respiratory tract samples
No full textComparison of extraction methods for the identification of yeast fungi by mass spectrometry
No full textCryptococcus neoformans sensu lato on different tree species and in soil samples
No full textCryptococcus neoformans sensu lato so glive kvasovke, ki se nahajajo v okolju in lahko povzročajo okužbe pri ljudeh. V zadnjih letih se kot njihova ekološka niša omenjajo različne drevesne vrste po vsem svetu. Njihov obstoj v okolju je pogojen s količino padavin in s temperaturo. V sredozemskem okolju tako zasledimo predvsem vrsto C. neoformans, medtem ko zmerno celinsko podnebje ugaja vrsti C. deneofromans. Namen naloge je bil odkriti in opisati prisotnost Cryptococcus neoformans sensu lato na različnih drevesnih vrstah v Sloveniji. Vzorčenje je potekalo v petindvajsetih krajih v šestih različnih regijah, s čimer smo pokrili celotno površino Slovenije. Skupaj smo zbrali tristo dvaintrideset vzorcev iz različnih drevesnih vrst in vinskih trt. Vzorčili smo liste, plodove, prst v bližini drevesa, ptičje iztrebke na deblu dreves, lubje, razpoke med skorjo in votle dele v deblu. Pridobljene vzorce smo gojili na agarju z izvlečkom semen abesinske gizotije. Kremasto ali rjavo obarvane kolonije, sumljive za kriptokoke, smo izolirali in identificirali z MALDI TOF. Skupno smo pridobili štiri izolate C. neoformans sensu stricto, ki smo jih osamili iz pinij (Pinus pinea) ter oljke (Olea europea). Vsi štirje izolati iz dreves so bili pridobljeni z brisanjem razpok med skorjo in iz votlih delov debel, poleg tega so bili vsi dobljeni v Obalno-kraški regiji. Z metodo mikrodilucije smo za vse pridobljene izolate testirali občutljivost za antimikotike. Izolati so pokazali največjo občutljivost za itrakonazol ter posakonazol.Cryptococcus neoformans sensu lato are basidiomycetous yeast that are found in the environment and can cause infections in humans. In recent years, various tree species around the world have been described as their ecological niche. Their presence in the environment is conditioned with the amount of preciptation and temperature. In the Mediterranean the prevalent species is C. neoformans sensu stricto, while C. deneoformans prefers continental climate. The aim of our study was to determine the presence of Cryptococcus neoformans sensu lato on various tree species in Slovenia. Sampeling was performed in six different statistical regions in the country, covering the whole area of Slovenia. Three hundred thirty-two samples from trees and grapevine were collected. The source of samples were leaves, fruits, soil under the trees, bird excreta on trunk, bark, fissures and inner trunk hollows. Samples were cultured on niger seed agar medium. Brownish or creamy colonies, resembling for Cryptococcus spp. were identified with MALDI TOF MS. All four recovered isolates were identified as C. neoformans sensu stricto, obtained from Italian stone pine (Pinus pinea) and olive tree (Olea europea). In addition, all isolates were recovered from fissure and inner trunk hollow swabs in the Costal-Karst statistical region. The antifungal susceptibility testing was performed by broth microdilution method for all cryptococcal isolates. According to antifungal susceptibility testing results, the isolates are most susceptible to itraconazole and posaconazole
Assessment of adequacy of haemoculture bottles for fungi detection in primary sterile bodily fluids
No full textInvazivne mikoze so glivne okužbe med katere uvrščamo širok nabor stanj in bolezni, katerih razplet je odvisen od vrste povzročitelja ter od mesta okužbe. Diagnostika glivnih patogenov v primarno sterilnih kužninah z metodami kultivacije, je ključnega pomena, v postopku zdravljenja bolnika, občutljivost ter čas do detekcije pa sta ključna indikatorja uspešnosti testa. Kultivacija krvi septičnega bolnika v hemokulturnih stekleničkah ob sumu na invazivno mikozo je priporočljiva iz strani EFISG (angl. ESCMID Fungal Infection Study Group). Za potrditev invazivne kandidoze predstavlja detekcija gliv z uporabo hemokulturnih stekleničk zlati standard. V naši raziskavi smo s simuliranimi vzorci kužnin ugotavljali, kako učinkovite (občutljivost in čas do detekcije gliv) so hemokulturne stekleničke za detekcijo glivnih patogenov iz drugih sterilnih kužnin. Ugotovili smo, da je občutljivost metode za detekcijo patogenih gliv s hemokulturno stekleničko BD BACTEC-Mycosis IC/F iz simuliranih primarno sterilnih kužnin je 100 %, občutljivost s stekleničko BD BACTEC Plus Aerobic/F znaša 95,3 %. Ugotovili smo, da je povprečni čas do detekcije vseh skupin gliv, razen pri nekaterih vrstah plesni, najhitrejši pri uporabi hemokulutrne stekleničke BD BACTEC-Mycosis IC/F. Statistično značilno hitrejši čas do detekcije v primerjavi z uporabo hemokulturne stekleničke BD BACTEC Plus Aerobic/F, smo dokazali, pri detekciji gliv iz rodu Cryptococcus pri uporabi koncentracije začetnega inokuluma 10 CFU/mL in 100 CFU/mL (p = 0,002/0,001) in pri detekciji gliv iz rodu Aspergillus, pri uporabi koncentracije začetnega inokuluma 100 CFU mL (p = 0,027). Ugotovili smo, da je uporaba hemokulturne stekleničke BD BACTEC Anaerobic Plus/F neprimerna za detekcijo patogenih gliv v primarno sterilnih kužninah zaradi izjemno nizke občutljivosti (11,6 % - 13,95 %).Invasive mycoses are fungal infections that can cause a broad spectrum of clinical symptoms and diseases, which are dependant on the site of infection and the causative agent. Diagnostics of fungal pathogens in primary sterile bodily fluids is a key factor in treatment of patient. Sensitivity and time to detection are key factors of successfulness of test. Cultivation of primary sterile bodily fluids in hemoculture bottles is recommended, when there is a suspicion of invasive mycosis, by EFISG. For confirmation of invasive candidiasis, detection of pathogen with hemoculture method is golden standard. In our research we used simulated primary sterile bodily fluids, to assess sensitivity and time to detection of blood culture bottles for diagnostics of fungal pathogens. We found that sensitivity of BD BACTEC-Mycosis IC/F bottle for detection of pathogenic fungi in simulated primary sterile bodily fluids was 100 % and that sensitivity of BD BACTEC Plus Aerobic/F bottle was 95,3 %.We found that average time to detection of all groups of fungi, except some genera of moulds, is fastest when we used BD BACTEC-Mycosis IC/F bottle. Statistical difference in time to detection, in comparison with usage of BD BACTEC Plus Aerobic/F bottle, was noticed, when we detected Cryptococcus species at concentration of inoculum 10 CFU/mL and 100 CFU/mL (p = 0,002/0,001) and when we detected Aspergillus species at concentration of inoculum 100 CFU/mL (p = 0,027). We found out that usage of BD BACTEC Anaerobic Plus/F isn\u27t suitable for detection of pathogenic fungi in primary sterile bodily fluids, for it\u27s poor sensitivity (11,6 % - 13,95 %)
Comparison of methods for susceptibility testing of invasive yeasts isolates for antimycotics
No full textKandidoza je glivna okužba, ki jo povzročajo vrste iz rodu Candida pri imunsko oslabelih bolnikih. Zgodnja uvedba ustreznega protiglivnega zdravljenja je povezana z zmanjšano smrtnostjo. Izbor ustreznega zdravila je odvisen od testiranja občutljivosti kvasovk, ki se večinoma izvaja s komercialno dostopnimi testi. Najboljšo primerljivost in natančnost zagotavljajo referenčne dilucijske metode, kot je mikrodilucija po standardu EUCAST. Rezultat mikrodilucije je minimalna inhibitorna koncentracija (MIK) antimikotika, na podlagi katere lahko opredelimo izolat kot občutljiv, mejno občutljiv ali odporen. V raziskavi smo uporabili komercialno mikrotitrsko ploščico (Merlin Diagnostika, Nemčija) z liozifiranimi antimikotiki. Občutljivost smo določili z različicama mikrodilucije, prilagojenima za EUCAST. MIK smo odčitali vizualno z in brez dodanega barvnega indikatorja ter spektofotometrično pri valovnih dolžinah 450 nm in 492 nm. Rezultate smo primerjali z referenčnimi vrednostmi MIK. Ugotovili smo, da je primernost posamezne metode določanja MIK odvisna od kombinacije izolata kvasovke in antimikotika. Pri določanju MIK ehinokandinov smo dobili prenizke vrednosti v primerjavi s kontrolnimi sevi, ki bi jih lahko popravili z uporabo korekcijskega faktorja 2. Pri antimikotikih, ki delujejo fungistatično, smo dobili z vizualnim odčitavanjem kategorično neprimerljive rezultate z rezultati referenčnega laboratorija. Zaključili smo, da prilagojene metode za EUCAST niso primerne za uvedbo v rutinsko delo v mikoloških laboratorijih, saj se nezadostno ujemajo z referenčnimi vrednostmi. Izjema je amfotericin B.Candidosis is a fungal infection caused by Candida spp. in immunocompromised patients. Early antifungal treatment has been shown to significantly reduce mortality and morbidity. Choice of appropriate therapy depends on antifungal susceptibility testing of yeasts, which is mostly performed with commercially avaible tests. The best interlaboratory comparability is provided by reference dilution methods, such as EUCAST microdilution. The result of microdilution are the minimal inhibitory concentrations (MICs) of antimycotics. On the basis of MIC, the isolates can be interpreted as sensitive, intermediate or resistant. In our study a commercial microtiter plate (Merlin Diagnostika, Germany) with lyophilized antimycotics was used. Susceptibility was determined by two microdilution methods modified for EUCAST. MIC was determined visually with and without added colour indicator and spectrophotometrically at wavelengths of 450 nm and 492 nm. We compared our MIC values with reference values. The most suitable method of determining MIC depends on the combination of yeast isolate and antimycotic. We have determed that low echinocandin MICs could be corrected using correction factor 2. The categorical agreement between visual determination of azole MICs and reference values was low. Modified EUCAST methods are not suitable for routine mycology laboratories due to insufficient agreement of results compared to the reference laboratory values. The exception is amphotericin B
Distribution of minimal inhibitory concentrations of antifungal compounds for medically relevant fungi
No full textGlivne okužbe so na našem geografskem območju oportunistične narave in se najpogosteje pojavljajo pri imunokomprimitiranih posameznikih. Z razvojem novih antimikotikov in vedno večjega interesa ter potreb po teh zdravilih se razvija tudi testiranje občutljivosti gliv za antimikotike. Poznamo referenčne metode in več oblik komercialno dostopnih metod za testiranje občutljivosti. V magistrski nalogi smo želeli opredeliti razporeditev minimalnih inhibitornih koncentracij (MIK) antimikotikov za medicinsko pomembne glive, ki so bile izolirane iz kliničnih vzorcev v Laboratoriju za glivične infekcije na Inštitutu za mikrobiologijo in imunologijo Medicinske fakultete Univerze v Ljubljani (IMI MF UL). Te smo primerjali s porazdelitvami MIK iz podatkovne baze Evropske komisije za testiranje antimikrobne občutljivosti (EUCAST), ki so bili pridobljeni z referenčno mikrodilucijsko metodo. V analizo smo vključili 27 različnih medicinsko pomembnih gliv. Podatke o MIK smo pridobili z metodo gradient difuzije, ki so bili v veliki večini že v bazi informacijskega sistema IMI (MBL), deloma pa smo opravili še dodatna testiranja za novejše antimikotike ali redkejše izolate. Rezultate MIK smo primerjali s podatki, pridobljenimi z referenčno metodo mikrodilucije, opredeljeno s strani EUCAST. Kjer je bilo možno, smo opredelili zgornjo mejo divjega tipa (angl. wild-type upper limit, wtUL). Ugotovili smo, da se vrednosti MIK z obema metodama za Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus flavus, Aspergillus fumigatus in Aspergillus niger v več kot 95 % skladata, če upoštevamo razpon dveh redčitvenih stopenj. Ugotovili smo, da imajo vse redkeje osamljene glive kvasovke wtUL za flukonazol višjo (80 %) ali enako (20 %) epidemiološki razmejitveni vrednosti (angl. epidemiological cut-off value, ECOFF) Candida albicans. Vrednosti wtUL za izavukonazol za najpogosteje izolirane glive iz skupine Mucorales in ECOFF A. fumigatus so bile skladne, medtem ko je bil wtUL za posakonazol dve in štiri redčitvene stopnje višji za za Mucor spp. in Rhizopus spp.Fungal infections are opportunistic in our geographic area and most often occur in immunocompromised individuals. With the development of new antifungals and the ever-increasing interest and need for these drugs, antifungal sensitivity testing is also developing. We are familiar with reference methods and several forms of commercially available sensitivity testing methods. In this Master\u27s thesis we wanted to define the distribution of minimum inhibitory concentrations (MIC) of antifungals for medically important fungi, which were isolated from clinical samples in the Laboratory for Fungal Infections at the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana (IMI MF UL). These were compared with MIC distributions from the European Commission Antimicrobial Susceptibility Testing (EUCAST) database, which were obtained using the reference microdilution method. We included 27 different medically important fungi in the analysis. We obtained data on MIC using the gradient diffusion method, the vast majority of which were already in the database of the IMI information system (MBL), if there was no data available, we performed additional testing for newer antifungals or rarer isolates. MIC results were compared with data obtained using the reference microdilution method defined by EUCAST. Where possible, we defined the wild-type upper limit (wtUL). We found that the MIC values with both methods for Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger agree in more than 95 % if we consider a range of two two-fold dilutions. We found that all of the less common yeast isolates had fluconazole wtUL higher (80 %) or equal (20 %) to the epidemiological cut-off value (ECOFF) of Candida albicans. The wtUL values for isavuconazole for the most frequently isolated fungi from the Mucorales group and ECOFF of A. fumigatus were consistent, while the wtUL for posaconazole was two and four two-fold dilutions higher than for Mucor spp. and Rhizopus spp.
Interaction of human microbiota and aerobiome in karst caves
No full textV disertaciji smo raziskovali antropogene vplive na populacije mikroorganizmov v zraku v slovenskih turističnih jamah in speleoterapevtski jami, s poudarkom na ekoloških posledicah jamskega turizma in speleoterapije. Aerosoli v jamah zagotavljajo vpogled v atmosferske razmere in spremembe, ki jih povzroča človek. Študija je vključevala vzorčenje zraka v Postojnski jami, Škocjanskih jamah in v Bolnišnični jami Sežana pred in po prisotnosti ljudi, da bi ocenili vpliv na mikrobne združbe v zraku. Uporabljene so bile metode, kot so gojenje na gojiščih, pretočna citometrija, detekcija ß-D-glukana in metagenomski pristop. Prisotnost ljudi (turistov in bolnikov) je v 91,7 % primerov povzročila povečanje koncentracije mikroorganizmov v zraku, medtem ko ravni ß-D-glukana in CO2 niso pokazale takšnega trenda in so bile bolj pod vplivom sezonskih variacij. V nobeni jami nismo zaznali primarno patogenih mikroorganizmov, zaznali smo le oportunistične vrste, kot so Aspergillus spp., Paecilomyces spp. in Alternaria spp. med glivami ter Staphylococcus spp. in Pseudomonas spp. med bakterijami. Študija je poudarila učinkovitost naravnega in umetnega prezračevanja pri zmanjševanju mikrobne obremenitve. MALDI-TOF MS je pokazal večjo natančnost pri identifikaciji mikrobov, zlasti na ravni rodu, v primerjavi s sistemom OmniLog ID in identificiral mikrobe, povezane s človekom, kot kazalce antropizacije jam. Prevladovali so prokariontski debla Actinomycetota, Bacillota in Pseudomonadota, kar so potrdile tako metode, ki temeljijo na kulturi, kot tudi metagenomski pristopi. Metagenomski pristop je razkril večjo vrstno raznolikost in zaznal približno 100-krat več taksonov kot konvencionalne metode, kar poudarja pomembnost uporabe več metod za celovito analizo. Kot zanesljive indikatorje antropizacije jam smo prepoznali: Micrococcus luteus, Kocuria rosea, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus epidermidis, kompleks Streptococcus mitis in Moraxella spp. Metagenomski pristop je poleg omenjenih bakterijskih vrst v vzorcih zraka identificiral tudi bakterijo Cutibacterium acnes, ki predstavlja del stalne mikrobiote na koži ljudi in jo je težko vzgojiti v standardnih laboratorijskih pogojih.This dissertation investigates the anthropogenic influences on the airborne microbial populations in Slovenian show caves and a speleotherapeutic cave, emphasizing the ecological implications of cave tourism and speleotherapy. Aerosols in caves provide insights into atmospheric conditions and human-induced changes. As part of the study, air samples were taken from Postojnska jama, Škocjanske jame and Bolnišnična jama Sežana before and after the presence of humans to assess the impact on airborne microbial communities. Methods such as culture-dependent techniques, flow cytometry, β-D-glucan detection and shotgun metagenomics were used. Results showed that human activity increased airborne microbial concentrations in ⡤ 91.7% of cases, with seasonal variation in β-D-glucan and CO2 concentrations. No primary pathogens were detected in either cave, only opportunistic species such as Aspergillus spp., Paecilomyces spp. and Alternaria spp. among the fungi and Staphylococcus spp. and Pseudomonas spp. among the bacteria. The study emphasises the effectiveness of natural and artificial ventilation in reducing the microbial load. MALDI-TOF MS showed higher accuracy in the microbial identification, especially at the genus level, compared to the OmniLog ID system and identified human-associated microbes as indicators of cave anthropisation. The predominant prokaryotic phyla identified were Actinomycetota, Bacillota and Pseudomonadota, which was confirmed by both culture-based and metagenomics-based approaches. The metagenomics-based approach revealed greater species diversity and detected about 100 times more taxa than conventional culture-based methods, emphasising the importance of using multiple methods for a comprehensive analysis. Micrococcus luteus, Kocuria rosea, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus epidermidis, Streptococcus mitis complex and Moraxella spp. were identified as reliable indicators of cave anthropisation. The metagenomics-based approach also identified Cutibacterium acnes, a core member of the human skin microbiota that is difficult to cultivate under standard laboratory conditions, as an additional indicator organism
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