Rapid Susceptibility Testing of Medically Important Zygomycetes by XTT Assay

Abstract

The XTT colorimetric assay quantifies fungal growth by measuring fungal metabolism and has been used successfully for susceptibility testing of Aspergillus species after 24 and 48 h of incubation. In the present study using 14 clinical isolates of Zygomycetes (Rhizopus oryzae Infections caused by Zygomycetes have emerged as an increasingly important cause of morbidity and mortality among severely immunocompromised patients, including those with hematological malignancies and hematopoietic stem cell transplant recipients. The prognosis of zygomycosis in these patients remains extremely poor, despite antifungal treatment (10,16,18,19). For years, amphotericin B has been the drug of choice for these devastating infections. Of the recently introduced second-generation triazoles, voriconazole is not active against the Zygomycetes, while posaconazole has demonstrated activity in vitro, in animal models, and in case reports (4-6, 8, 20-22). As zygomycosis may be rapidly fatal, timely determination of the susceptibilities of clinical isolates to these antifungal agents is important.[A colorimetric method, based on reduction of the tetrazolium salt 2,3-bis{2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) by mitochondrial dehydrogenases, has been recently introduced for in vitro susceptibility testing of filamentous fungi. This method quantifies fungal growth by measuring fungal metabolism, using appropriate concentrations of XTT and an electron transfer agent, such as menadione (1,11,12). For Aspergillus species, MICs determined by the XTT method at 24 and 48 h were comparable with those obtained by the National Committee for Clinical Laboratory Standards (now known as the Clinical and Laboratory Standards Institute [CLSI]) method (12). The sensitivity of the XTT assay may be increased depending on the menadione concentration (11). High menadione concentrations could increase the rate of XTT conversion and theoretically allow the detection of metabolic activity associated with small, early fungal growth that is not yet detected visually or spectrophotometrically. In this case, it is likely that the XTT method might even be useful for early determination of MICs of antifungal agents. This hypothesis has been previously discussed (12) but has not been investigated.In preliminary studies using the XTT assay for zygomycete isolates, high metabolic activity was observed even in the presence of small fungal growth (assessed spectrophotometrically) at 12 or 24 h postinoculation and preceded increases in biomass (C. Antachopoulos, J. Meletiadis, E. Roilides, T. Sein, and T. J. Walsh, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1003, 2005). The present study investigated whether significant metabolic activity with the XTT assay could be demonstrated for the Zygomycetes before hyphal growth is macroscopically or spectrophotometrically detected (at 6 or 8 h postinoculation) and whether changes in this early metabolic activity in the presence of antifungal agents could be used f...

CotH3 mediates fungal invasion of host cells during mucormycosis

“…Data were derived from >700 fungal cells interacting with approximately 200 endothelial cells/group/experiment, with an average of 59% cells being endocytosed in the control. (B) Damage was carried out using the 96-well plate 51 Cr release method. Blocking of CotH3 and CotH2 reduced the ability of R. oryzae to cause endothelial cell damage.…”

“…2 slides per arm were used for each experiment, and the experiment was performed in triplicate on different days. R. oryzae-induced endothelial or CHO cell damage was quantified using a 51 Cr release assay (48). Briefly, endothelial cells or CHO cells grown in 96-well tissue culture plates containing detachable wells were incubated with 1 μCi/well Na2 51 CrO4 (ICN) in M-199 medium (for endothelial cells) or α-MEM (for CHO cells) for 16 hours.…”

“…Briefly, endothelial cells or CHO cells grown in 96-well tissue culture plates containing detachable wells were incubated with 1 μCi/well Na2 51 CrO4 (ICN) in M-199 medium (for endothelial cells) or α-MEM (for CHO cells) for 16 hours. On the day of the experiment, the unincorporated 51 Cr was aspirated, and wells were washed twice with prewarmed HBSS. Cells were infected with fungal germlings (1.5 × 10 5 germinated for 1 hour) suspended in 150 μl RPMI 1640 medium supplemented with glutamine.…”

Diagnosis and treatment of mucormycosis in patients with hematological malignancies: guidelines from the 3rd European Conference on Infections in Leukemia (ECIL 3)

“…While fungicidal activity of posaconazole has been demonstrated against Rhizopus and Mucor spp., AmB was more rapidly fungicidal, with 95% killing noted at as early as 6 h and 99.9% killing at 24 h; for comparison, posaconazole showed less than 70% killing at 6 h and 99.9% killing at 48 h. 67 Similar observations have been made by in vitro studies using the XTT metabolic assay. 68 A few animal studies have been conducted to explore the in vivo efficacy of posaconazole in screening models of disseminated mucormycosis that used survival and/or fungal tissue burden as end points. [69][70][71][72][73] Posaconazole prolonged the survival and reduced tissue burden in neutropenic mice with disseminated Mucor infection and was as effective as standard AmB at the highest dose level.…”

Mitochondrial Associated Yeast Flocculation -The Effect of Acetylsalicylic Acid

“…Since it has been shown that ASA reduces yeast flocculation, presumably by targeting oxylipin production and consequently mitochondria, these results encouraged us to investigate the changes in mitochondrial activity at the start of growth (0 h) and during the flocculent (i.e., 20 h) and less flocculent phases (i.e., 6 h) of S. cerevisiae UOFS Y-2330 35 , using the XTT reduction assay. With this assay, XTT (a tetrazolium salt) is cleaved by various mitochondrial dehydrogenases to produce coloured formazans, which are indicators of fungal metabolic activity 3,40 . We found that in S. cerevisiae UOFS Y-2330, mitochondrial activity was significantly higher (p < 0.001) during the flocculent phase (i.e., after 20 h) of growth than during the less flocculent phase (i.e., after 6 h; Fig.…”