Sensitivity testing of filamentous fungi to newer antifungals: clinical relevance

The continuing increase of systemic and invasive mycoses due to filamentous fungi (moulds) such as species of Aspergillus, zygomycetes of the order Mucorales (Rhizopus, Mucor, θιπ.) and rare hyalohyphomycetes such as Fusarium and Scedosporium, and their difficult management because of their resistance in most antifungals, needed the development and standardization of a method to determine susceptibility to these drugs. The present dissertation aimed to the development of an antifungal susceptibility testing method which would be reliable and reproducible, in order to be used as a standard method. This method should be applicable to all filamentous fungi,The strains used for this study were isolates from cases with systemic mycoses with known antifungal susceptibility and outcome, as well as from a prospective registry study. The experiments of the first phase of the study aimed to verify the optimal conditions for the preparation of the inoculum of conidia to be tested for antifungal susceptibility with the broth microdilution method. The main conclusions of this phase were that the haemocytometer counting of conidia was a more reliable method of inoculum preparation than the spectrophotometric adjustment, and that Tween 20, a nonionic surfactant, should be used as a dispersing agent for the optimal suspension of hydrophobic conidia. The ideal inoculum size was 1.0 × 106 -5.0 × 106 CFU/ml. In the second phase, this new method was evaluated in three independent laboratories and its reliability and reproducibility were confirmed, with interlaboratory agreement of 89.2% and a narrow 95% CI (2.20 -2.65). In the third phase of the study the method was used for the susceptibility testing of strains from a prospective registry study of rare invasive mycoses and performed very well. The results of this study have been adopted by the EUCAST and constitute the basis of the guidelines for the determination of the MICs of antifungals against conidia forming moulds (EUCAST DEFINITIVE DOCUMENT E.DEF 9.1)

Analysis of the Influence of Tween Concentration, Inoculum Size, Assay Medium, and Reading Time on Susceptibility Testing of Aspergillus spp.

The influence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinafine. Inoculum size (10(4) CFU/ml versus 10(5) CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 10(5) CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major increases (two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher concentrations of Tween (P < 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 μg/ml, respectively) to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 μg/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of 10(5) CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P > 0.01)

Correlation of patient preferences to treatment outcomes in patients with rheumatoid arthritis treated with tumour necrosis factor inhibitors in Greece

Objectives: To investigate possible associations between rheumatoid arthritis (RA) patient-expressed preferences over anti-tumour necrosis factor (anti-TNF) treatment and clinical and patient-reported outcomes. Methods: PANORAMA was a non-interventional, prospective, multicentre, cohort study of 12 months duration, in patients with moderate-to-severe RA who initiated or switched to anti-TNF treatment. After initiation of anti-TNF, patients completed a preferences questionnaire on attributes related to anti-TNF treatment. Satisfaction with treatment was assessed with the Treatment Satisfaction Questionnaire for Medication (TSQM); compliance and persistence to treatment were recorded via a patient diary. Univariate and multivariate analyses were applied to assess correlations between patients’ preferences over treatment with clinical and patient-reported outcomes. Results: A total of 254 patients were enrolled; 66.1% (168/254) had highly active disease (DAS28-ESR &gt; 5.1), while 65.4% (166/254) were biological-naïve. The 12-month drug-survival rate was 72.3%, while the respective rates of good EULAR response and remission (DAS28-ESR &lt; 2.6) were 56.5% and 40.8%, respectively. By univariate analysis, fulfilment of patient preferences over treatment was associated with increased probability of remaining on therapy (p = 0.019), good EULAR response (p &lt; 0.001) and satisfaction with treatment (p &lt; 0.001). By multivariate analysis, fulfilment of patient preferences was the most important predictor for good EULAR response (OR 5.56, p &lt; 0.001; finding confirmed and after propensity scoring matching), while seropositivity (HR 1.18, p = 0.047) and a high ESR (&gt; 35 mm/h, HR 1.16, p = 0.071) predicted drug survival. Conclusions: In anti-TNF-treated RA patients, fulfilment of treatment preferences was independently associated with a good EULAR response and correlated with drug persistence at 12 months, emphasising the importance of patient preferences in treatment outcomes.Key Points• In anti-TNF treated RA patients, fulfilment of patients’ treatment preferences is associated with a good clinical response at 12 months.• A shared decision-making process can maximise treatment’s outcome in anti-TNF treated patients. © 2020, International League of Associations for Rheumatology (ILAR)

Interlaboratory Evaluation of Hematocytometer Method of Inoculum Preparation for Testing Antifungal Susceptibilities of Filamentous Fungi

A three-laboratory study was performed to evaluate conidium counting in a hematocytometer as a technique of inoculum preparation for susceptibility testing of Aspergillus spp. In addition, inocula were quantified by colony counting and optical density determination. The agreement and correlation coefficient between conidium and colony quantifications were 89.2% and 0.73 (P < 0.01). Correlations with optical density determination were not significant

Inoculum Standardization for Antifungal Susceptibility Testing of Filamentous Fungi Pathogenic for Humans

Two methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately