Utjecaj medija i temperature na tvorbu gliotoksina u sojeva Aspergillus fumigatus

Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 °C resulted in higher gliotoxin production than at 25 °C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL-1 (in YES at 37 °C) and 1.07 mg mL-1 (in CZA at 25 °C). YES broth as a medium containing 4 % sucrose and 2 % of yeast extract is a very rich substrate for the production of gliotoxin in vitro.Gliotoksin je sekundarni metabolit iz skupine epipolitiodioksipiperazina s biološki aktivnim internim disulfidnim mostom u molekuli, koji tvore razne plijesni i gljivica Candida albicans. Plijesan Aspergillus fumigatus vodeći je uzročnik invazivnih aspergiloza i također može tvoriti gliotoksin. Pretpostavlja se da in situ tvorba gliotoksina utječe na patogenezu aspergiloze. U ovom radu ispitali smo in vitro tvorbu gliotoksina u devet sojeva A. fumigatus vrste, izoliranih iz imunokompromitiranih pacijenata. Praćen je utjecaj medija i temperature inkubacije na tvorbu gliotoksina. Gliotoksin je ekstrahiran iz biomase i koncentracija mu je utvrđena polukvantitativno tankoslojnom kromatografijom. Tvorba gliotoksina uočena je već nakon trodnevne inkubacije u koncentracijama 4,06 mg mL-1 (u bujonu s kvaščevim ekstraktom – YES na 37 °C) i 1,07 mg mL-1 (u sintetskom Czapek-Dox bujonu – CZA na 25 °C). Tvorba gliotoksina bila je veća u YES bujonu, za razliku od sintetskog CZA bujona. Viša temperatura inkubacije (37 °C) također utječe na jaču tvorbu gliotoksina nego niža temperatura (25 °C). YES bujon s dodatkom 4 % saharoze i 2 % kvaščeva ekstrakta vrlo je bogat supstrat za in vitro tvorbu gliotoksina, kao jednog od lipofilnih sekundardnih metabolita plijesni vrste A. fumigatus

Evaluacija imunokromatografskog testa za otkrivanje galaktomanana Aspergillus spp. u uzorcima seruma i bronhoalveolarnih lavata – preliminarni rezultati

Background: Detection of biomarkers, such as galactomannan (GM), has proven to be of great significance in early recognition of invasive aspergillosis (IA). The aim of our study was to evaluate the lateral flow assay (LFA) for the detection of GM on serum and bronchoalveolar lavage (BAL) samples previously proven positive by enzyme-linked immunosorbent assay (ELISA). Methods: The study was performed on serum and BAL samples obtained from patients with suspected IA in the period from February 2019 to January 2020, which were previously GM positive by ELISA (Platelia Aspergillus Ag, Biorad, Hercules, USA). Samples were then tested by LFA (Aspergillus Galactomannan LFA, IMMY, Oklahoma, USA) with test line intensity visually read as 1+, 2+, 3+, or 4+. Results: A total of 45 GM ELISA positive serum and/or BAL samples were obtained from 41 patients; 25 (55,6 %) were BAL and 20 (44,4 %) serum samples. LFA showed a positive result in 39 out of 45 (86,7%) GM ELISA positive samples; 22/25 (88.0 %) BAL samples and 17/20 (85.0 %) serum samples tested positive. In BAL samples, low intensity test line of 1+ was significantly more frequent in GM ELISA positive samples with optical density index (ODI) 4.0) had low intensity line of 1+ when tested with LFA. Conclusions: Results obtained by LFA are comparable to GM ELISA. Since low intensity lines were found in serum samples with high ODI, this potentially makes BAL a superior sample for LFA, at least when visual and not automated reading is done.Uvod: Određivanje galaktomanana (GM) igra značajnu ulogu u ranom otkrivanju invazivne aspergiloze (IA). Cilj je ovog istraživanja bila evaluacija imunokromatografskog testa (LFA) za određivanje GM u uzorcima seruma i bronhoalveolarnih lavata (BAL) s pozitivnim rezultatom koji su utvrđeni prethodno napravljenom ELISA metodom. Metode: Istraživanje je napravljeno na uzorcima seruma i BAL-a pacijenata sa sumnjom na IA prikupljenim u razdoblju od veljače 2019. do siječnja 2020. godine. Uzorci s pozitivnim rezultatom utvrđenim ELISA metodom (Platelia Aspergillus Ag, Biorad, Hercules, USA) testirani su s LFA (Aspergillus Galactomannan LFA, IMMY, Norman, Oklahoma, USA) s vizualnim očitavanjem testne linije u rasponu 1+, 2+, 3+, ili 4+. Rezultati: Od 41 bolesnika dobiveno je 45 pozitivnih uzoraka seruma i/ili BAL-a testiranih GM ELISA metodom; 25 (55,6 %) uzoraka BAL-a i 20 (44,4 %) uzoraka seruma. LFA je pokazao pozitivni rezultat kod 39 od 45 (86,7%) uzoraka pozitivnih GM ELISA metodom; 22/25 (88.0 %) uzoraka BAL-a i 17/20 (85.0 %) uzoraka seruma. Kod uzoraka BAL-a, testna linija slabog intenziteta 1+ bila je značajno češća kod pozitivnih uzoraka testiranih GM ELISA metodom s ODI 4.0) imala su pri testiranju s LFA testnu liniju slabog intenziteta 1+. Zaključak: Rezultati dobiveni s LFA usporedivi su s GM ELISA metodom. S obzirom da su testne linije niskog intenziteta utvrđene u uzorcima seruma s visokim ODI, uzorci BAL-a su potencijalno pogodniji uzorci za LFA, barem kad se radi o vizualnom, a ne automatiziranom očitavanju testa

Influence of Media and Temperature on Gliotoxin Production in Aspergillus Fumigatus Strains

Gliotoksin je sekundarni metabolit iz skupine epipolitiodioksipiperazina s biološki aktivnim internim disulfidnim mostom u molekuli, koji tvore razne plijesni i gljivica Candida albicans. Plijesan Aspergillus fumigatus vodeći je uzročnik invazivnih aspergiloza i također može tvoriti gliotoksin. Pretpostavlja se da in situ tvorba gliotoksina utječe na patogenezu aspergiloze. U ovom radu ispitali smo in vitro tvorbu gliotoksina u devet sojeva A. fumigatus vrste, izoliranih iz imunokompromitiranih pacijenata. Praćen je utjecaj medija i temperature inkubacije na tvorbu gliotoksina. Gliotoksin je ekstrahiran iz biomase i koncentracija mu je utvrđena polukvantitativno tankoslojnom kromatografijom. Tvorba gliotoksina uočena je već nakon trodnevne inkubacije u koncentracijama 4,06 mg mL-1 (u bujonu s kvaščevim ekstraktom – YES na 37 °C) i 1,07 mg mL-1 (u sintetskom Czapek-Dox bujonu – CZA na 25 °C). Tvorba gliotoksina bila je veća u YES bujonu, za razliku od sintetskog CZA bujona. Viša temperatura inkubacije (37 °C) također utječe na jaču tvorbu gliotoksina nego niža temperatura (25 °C). YES bujon s dodatkom 4 % saharoze i 2 % kvaščeva ekstrakta vrlo je bogat supstrat za in vitro tvorbu gliotoksina, kao jednog od lipofilnih sekundardnih metabolita plijesni vrste A. fumigatus.Gliotoxin is a secondary metabolite of the epipolythiodioxopiperazine family with biologically active internal disulfide bridge. It is produced by many fungal species, including Aspergillus fumigatus and A. terreus. A. fumigatus, which produces gliotoxin and more than twenty other secondary metabolites, is the leading cause of invasive aspergillosis. Gliotoxin production in situ influence the development of aspergillosis. This study investigated the in vitro production of gliotoxin in nine A. fumigatus isolates from the upper respiratory tract of immunocompromised patients. The effects of media composition and incubation temperature were studied. Gliotoxin was extracted from biomass and its concentration was semi-quantitatively analysed using thin-layer chromatography. Gliotoxin production was higher in the yeast-extract liquid medium (YES) than in the synthetic Czapek-Dox liquid medium (CZA). Incubation at 37 °C resulted in higher gliotoxin production than at 25 °C, probably because higher temperatures favour expansive growth of the mycelium. Gliotoxin could be detected after three days of incubation at concentrations 4.06 mg mL-1 (in YES at 37 °C) and 1.07 mg mL-1 (in CZA at 25 °C). YES broth as a medium containing 4 % sucrose and 2 % of yeast extract is a very rich substrate for the production of gliotoxin in vitro

Epidemiology of Candidemia: Three-Year Results from a Croatian Tertiary Care Hospital

Invasive candidosis is the most common invasive fungal infection in hospitalized patients and is associated with a high mortality rate. This is the first study from a Croatian tertiary care hospital describing epidemiology, risk factors and species distribution in patients with candidemia. A three-year retrospective observational study, from 2018 to 2020, was performed at the University Hospital Centre Zagreb, Zagreb, Croatia. A total of 160 patients with candidemia (n = 170 isolates) were enrolled. Candidemia incidence increased from 0.47 to 0.69 per 1000 admissions in 2018 and 2020, respectively. Ninety-five patients (58.38%) were in the intensive care unit. The main risk factors for candidemia were central venous catheter (CVC) (84.38%), previous surgical procedure (56.88%) and invasive mechanical ventilation (42.50%). Candida albicans was identified in 43.53% of isolates, followed by C. parapsilosis (31.76%) and C. glabrata (12.36%), C. krusei (5.29%), C. tropicalis (2.35%) and C. lusitaniae (2.35%). The study discovered a shift to non-albicansCandida species, particularly C. parapsilosis, and made it possible to determine the main tasks we should focus on to prevent candidemia in the hospital, these being mainly infection control measures directed towards prevention of catheter-related bloodstream infections, specifically comprising hand hygiene and CVC bundles of care. The potential benefit of fluconazole prophylaxis in certain populations of surgical patients could also be considered