Evaluation of Inoculum Preparation for Etest and EUCAST Broth Dilution to Detect Anidulafungin Polyresistance in Candida glabrata.

The influence of inoculum preparation in EUCAST broth dilution and Etest to detect the coexistence of resistant and susceptible Candida subpopulations (defined as polyresistance [PR]) was evaluated. Cocultures of two echinocandin-resistant and susceptible clinical C. glabrata strains were used to simulate the occurrence of mixed populations in clinical samples, and antifungal susceptibility testing was performed with standard and modified approaches of inoculum preparation. Polyresistant results manifested as microcolonies or double ellipses in Etest and in single reduced optical density (OD) values (dip in OD) in microdilution. The strict inclusion of five distinct colonies of 1:5 and 1:10 resistant and susceptible cocultures led to higher rates of PR and R results compared to including one to two colonies in inoculum preparation (30% and 26% for Etest and broth dilution, respectively). Modifying the inoculum preparation by increasing the turbidity from a 2 to a 4 McFarland standard before redilution to a 0.5 McFarland standard reliably enabled the detection of resistance, with better identification of PR by Etest than by broth dilution (82% versus 32%, respectively) and of resistant minimum inhibitory concentration (MIC) values in 18% of Etests and 67% of microdilutions. The highest identification of PR succeeded with Etest and a modified 3 McFarland standard approach of inoculum preparation. Our data demonstrate that inoculum preparation as recommended and practiced does not reliably identify resistant subpopulations in polyresistant Candida cultures. By increasing the inoculum size for Etest assays from a 2 to a 4 McFarland standard with subsequent redilution, we propose a simple adaptation to increase reliability.

COVID-19 Associated Pulmonary Aspergillosis: Diagnostic Performance, Fungal Epidemiology and Antifungal Susceptibility.

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) raises concerns as to whether it contributes to an increased mortality. The incidence of CAPA varies widely within hospitals and countries, partly because of difficulties in obtaining a reliable diagnosis. We implemented a routine screening of respiratory specimens in COVID-19 ICU patients for Aspergillus species using culture and galactomannan (GM) detection from serum and/or bronchoalveolar lavages (BAL). Out of 329 ICU patients treated during March 2020 and April 2021, 23 (7%) suffered from CAPA, 13 of probable, and 10 of possible. In the majority of cases, culture, microscopy, and GM testing were in accordance with CAPA definition. However, we saw that the current definitions underscore to pay attention for fungal microscopy and GM detection in BALs, categorizing definitive CAPA diagnosis based on culture positive samples only. The spectrum of Aspergillus species involved Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans. We noticed changes in fungal epidemiology, but antifungal resistance was not an issue in our cohort. The study highlights that the diagnosis and incidence of CAPA is influenced by the application of laboratory-based diagnostic tests. Culture positivity as a single microbiological marker for probable definitions may overestimate CAPA cases and thus may trigger unnecessary antifungal treatment.

Multiple colony antifungal susceptibility testing detects polyresistance in clinical Candida cultures: a European Confederation of Medical Mycology excellence centers study.

OBJECTIVES: Many factors influence the outcome of in vitro antifungal susceptibility testing (AFST), including endpoint definition, inoculum sizes, time and temperature of incubation, and growth medium used. This European Confederation of Medical Mycology (ECMM) Excellence center driven study investigated multiple colony testing (MCT) of five separate colonies to investigate the prevalence of polyresistance (PR), defined as heterogeneous MICs from a same-species Candida culture irrespective of the underlying resistance mechanism. METHODS: Candida spp. MCT for fluconazole and anidulafungin was performed by Etest prospectively comprising 405 clinical samples. MCT results were compared to the real-life routine MIC data and PR was assessed. Candida colonies displaying strong PR were selected for genotyping using multilocus sequence typing and random amplified polymorphic DNA assays for C. lusitaniae. RESULTS: Candida PR was observed in 33 of 405 samples (8.1%), with higher rates for non-albicans species (26/186, 14%) than for C. albicans (7/219, 3.2%), and for fluconazole than for anidulafungin. MCT detected acquired resistance more often than routine AFST (18/405, 4.5%) and 9 of the 161 investigated blood cultures showed PR (5.6%). Multilocus sequence typing and random amplified polymorphic DNA did not reveal a uniform genetic correlate in strains studied. CONCLUSIONS: This study shows that Candida single MIC-values obtained in routine diagnostics may be incidental, as they fail to detect PR and resistant subpopulations reliably. The reasons for PR seem to be manifold and should be regarded as a phenotypical expression of genomic variability irrespective of the underlying resistance mechanism, which may help to interpret ambiguous and non-reproducible AFST results.

Serology anno 2021-fungal infections: from invasive to chronic.

BACKGROUND: Diagnosing invasive or chronic fungal infections is a challenge, particularly in the immunocompromised host. Microscopy and culture remain the reference standard, but are insensitive. The use of non-culture-based techniques is recommended in conjunction with conventional methods to improve the diagnostic yield. OBJECTIVES: The aim was to provide an updated 2021 inventory of fungal antigen and serology tests for diagnosing invasive and chronic fungal infections, the key focus was set on Aspergillus, Candida and Cryptococcus species. SOURCES: Pubmed search for publications with the key words fungal antigen tests, laboratory-based diagnosis of invasive pulmonary aspergillosis, chronic pulmonary aspergillosis, invasive candidiasis, invasive fungal infections and cryptococcal infections published from 2017 to 2020. CONTENT: Antigen assays such as the galactomannan (GM) and ß-d-glucan detection systems are frequently used, but these tests vary in sensitivity and specificity, depending on the patient population involved, specimens inspected, cut-offs defined, test strategy applied and inclusion or exclusion of possible fungal case definitions. Multiple different detection systems are available, with recently introduced new point-of-care tests such as the lateral flow device and the lateral flow assay. Despite a wide heterogeneity in populations evaluated, studies indicate a better diagnostic performance of bronchoalveolar lavage GM in comparison with serum GM, and a suboptimal specificity of GM bronchoalveolar lavages (cut-off ≥1) and serum ß-d-glucan in non-neutropenic individuals. Point-of-care cryptococcal antigen tests show excellent performance. IMPLICATIONS: There are fungal antigen detection tests available with excellent to reasonable clinical performance to diagnose invasive fungal infections. Only a few assays are useful to monitor therapeutic response. There are multiple marketed IgG antibody tests to detect Aspergillus fumigatus antibodies, the titres vary widely and the performance differs significantly. In general, diagnostic tests are vulnerable to being affected by the host, the microbe and laboratory setting.