Clinical Isolates Of Aspergillus Spp Research Articles

716. Characterization of Azole Susceptibility Profiles of Aspergillus spp. Isolates Obtained From Immunocompromised Patients in Cali, Colombia

Abstract Background Aspergillus spp. are opportunistic filamentous fungi causing a spectrum of diseases described aspergillosis. Aspergillosis is treated with triazole antifungals of second-generation, mostly voriconazole or itraconazole, which inhibit the ergosterol synthesis, an important component of the fungal membrane. However, the efficacy of these drugs has been affected by the presence of resistance found in different Aspergillus spp. species, as a mutations in CYP51 gene We describe the azole susceptibility profiles of Aspergillus spp., isolated from clinical samples in a hospital in Cali, Colombia. Methods A total of 63 Aspergillus spp. clinical isolates were identified at a phenotypic and protein level through matrix-assisted laser ionization (MALDI-TOF-MS), susceptibility profiles against voriconazole and itraconazole were subsequently characterized. Following the guidelines for susceptibility determination issued by EUCAST, 96-well plates were prepared with the azole antifungals itraconazole (ITZ) and voriconazole(VCZ), using concentrations ranging from 0.06 to 32 mg/L. Each well was then inoculated with 100μL of the fungal inoculum previously diluted in distilled water and 20% tween adjusted to a concentration of 0.5 on the Mcfarland scale. The plates were incubated at 37 °C and readings were taken after 48 hours of incubation. Results A total of 63 clinical isolates of Aspergillus spp were collected, of these 44% corresponded to isolates of A. fumigatus, 25% A. flavus/oryzae, 18% A. niger, 5% A. parasiticus, 3% A. terreus and 3% A. versicolor. In vitro characterization of the susceptibility profiles revealed variable phenotypes, with a predominance of susceptible strains for the two antifungals tested, however, of the total isolates, 8% (5/63) were resistant to itraconazole (ITZ), and 6% (4/63) to voriconazole (VRZ). Figure 1. Distribution or Frequency of Aspergillus spp. Species and Their Resistance Profiles Conclusion Azole resistance was low 6-8% . Susceptibility profiles of the strains isolated from clinical samples is important to carry out an accurate identification of each one of the agents involved in infections caused by Aspergillus spp in order to differentiate common species from cryptic ones, since these have increasingly acquired importance at the clinical level,. Disclosures All Authors: No reported disclosures

Antifungal Resistance in Clinical Isolates of Aspergillus spp.: When Local Epidemiology Breaks the Norm.

Aspergillosis is a set of very frequent and widely distributed opportunistic diseases. Azoles are the first choice for most clinical forms. However, the distribution of azole-resistant strains is not well known around the world, especially in developing countries. The aim of our study was to determine the proportion of non-wild type strains among the clinical isolates of Aspergillus spp. To this end, the minimum inhibitory concentration of three azoles and amphotericin B (used occasionally in severe forms) was studied by broth microdilution. Unexpectedly, it was found that 8.1% of the isolates studied have a diminished susceptibility to itraconazole. This value turned out to be similar to the highest azole resistance rate reported in different countries across the world.

Establishment and application of loop-mediated isothermal amplification for detecting clinical isolates of Aspergillus spp

Objective To establish a method for detecting Aspergillus spp.by Loop-mediated isothermal amplification. Methods Aspergillus spp.specific primers were designed from the relative conservation region of the published sequence of 28S rRNA genes of A.fumigatus (GenBank accession number AY660917),A.terreus (GenBank accession number AF454183),A.flavus (GenBank accession number AF454158),and A.niger (GenBank accession number AF454169).Genomic DNA were extracted from Aspergillus standard strains,clinical control strains and clinical samples,and amplified by LAMP.The amplified results could be read with the naked eye by the coloring effect of fluorescent nucleotide dye without the DNA electrophoresis.Approximately 103 each Aspergillus conidia suspension was added to the serum from healthy volunteers,and detected these simulative clinical samples by LAMP assay. Results The LAMP and PCR assays obtained positive results for all four Aspergillus species and 8 simulative clinical samples (double samples for each Aspergillus species) including 103 conidia,but negative in the remaining 15 non-Aspergillus species,human total blood genomic DNA,30 clinical serum samples infected with non-Aspergillus and 10 healthy volunteers.The LAMP assay had a minimum detection of 0.05-0.5pg,by means of detect different levels of 500,50,5,0.5,0.05 pg template in each reaction tube. Conclusion The results confirm that LAMP is a simple,rapid,sensitive and specific method,and can be used for detection of Aspergillus strains in clinical and environmental specimens.（Chin J Lab Med,2014,37:140-143） Key words: Aspergillus; Nucleic acid amplification techniques; Sensitivity and specificity

Antifungal activity of statins againstAspergillusspecies

The cholesterol-lowering agents known as statins have in vitro activities against human pathogenic fungi, such as Candida species, Cryptococcus neoformans, and Zygomycetes. Synergy between statins and azoles against these fungi has also been reported. We evaluated the in vitro activities of two statins, lovastatin and simvastatin, alone and in combination with azoles and amphotericin B, against clinical isolates of Aspergillus spp. A disk diffusion assay showed that both statins were active against Aspergillus spp. The minimal inhibitory concentration (MIC) ranges for lovastatin and simvastatin against Aspergillus spp. were 16 to >256 microg/ml and 4 to >256 microg/ml, respectively. Although both statins were fungicidal for A. fumigatus, the MICs were vastly higher than clinically achievable concentrations. The results of a combined agar dilution-Epsilometer test as well as a disk diffusion assay showed that neither statin had any effect on the in vitro activities of itraconazole, voriconazole, or amphotericin B against Aspergillus spp.

In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest ®: results of a French multicentre study

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest ® and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

Serotonin (5-HT) enhances the activity of amphotericin B against Aspergillus fumigatus in vitro

We investigated the in vitro synergistic antifungal potential of combining serotonin (5-HT) and sertraline with amphotericin B and itraconazole against clinical isolates of Aspergillus spp. Synergy tests were performed using the chequerboard microdilution method. Activity was measured against Aspergillus fumigatus ( n = 7), Aspergillus flavus ( n = 3) and Aspergillus terreus ( n = 2), and compared with that for Candida albicans and Candida parapsilosis. The fractional inhibitory concentration (FIC) indices ranged between 0.25 and 3 for the various isolates tested. 5-HT was shown to enhance the activity of amphotericin B against Aspergillus spp. Combination studies with 5-HT and itraconazole and with sertraline and itraconazole or amphothericin B showed different activities for the various strains, including synergism (FIC < 1.0), additivity (FIC = 1), and indifference (FIC between 1.0 and 2.0). 5-HT and sertraline showed antagonistic activity (FIC > 2) with amphotericin B and itraconazole against C. parapsilosis.

In vitro synergy of caspofungin and itraconazole against Aspergillus spp.: MIC versus minimal effective concentration end points.

Caspofungin and itraconazole were studied alone and in combination against 31 clinical isolates of Aspergillus spp. according to NCCLS M38-P guidelines. MICs and microscopic minimal effective concentrations (MECs) were recorded, and synergy was calculated by using both end points. Synergy or synergy to additivity was found in 30 of 31 isolates by using MIC end points. With MEC end points no synergy was found and indifference was detected in 26 of 31 strains.

Antifungal activity of a new triazole, voriconazole (UK-109496), against clinical isolates of Aspergillus spp.

Voriconazole is a new triazole antifungal agent with potent activity against yeast and molds. We investigated the in-vitro activity of voriconazole compared with that of other antifungal agents against 50 clinical isolates of Aspergillus spp., measured by the National Committee for Clinical Laboratory Standards (NCCLS) reference method described in the M27-A document, and by an alamar blue colorimetric method. Voriconazole was the most potent agent against Aspergillus fumigatus (minimum inhibitory concentration [MIC]90, 0.5 mg/l) and Aspergillus niger (MIC90, 1.0 mg/l). Voriconazole was less active (MIC90, 1.0 mg/l) against Aspergillus flavus than itraconazole (MIC90, 0.5 mg/l). Voriconazole was more active than itraconazole against Aspergillus fumigatus and Aspergillus flavus by the alamar blue indicator method for the measurement of MIC. Based on these results, voriconazole has promising activity against commonly encountered isolates of Aspergillus spp., and its clinical usefulness should be established by further studies.

Discrepancies between MIC and MLC values of amphotericin B against isolates of Aspergillus species.

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains of Aspergillus fumigatus, 20 strains of Aspergillus flavus and 20 strains of Aspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70 Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates of Aspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 microgram/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 micrograms/ml, respectively (p < 0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.