Table_1_Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection—A Prospective Pilot Study.DOCX

<p>Background: Blood or tissue culture or histology prove invasive Candida infection, but long time to result, limited feasibility and sensitivity call for new approaches. In this pilot project, we describe the diagnostic potential of quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes in blood of patients with invasive Candida infection.</p><p>Methods: We used flow cytometry quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes from peripheral blood of patients with invasive Candida infection, from patients at risk and healthy volunteers as controls.</p><p>Results: Elevated levels of Candida-reactive lymphocytes were measured in 13 patients with proven invasive Candida infection and in one patient with probable hepatosplenic candidiasis. Results of three candidemia patients were uninterpretable due to autofluorescence of samples. Twelve of 13 patients had Candida identified to species level by conventional methods, and T cell reactivity correctly identified Candida species in 10 of 12 patients. Nine hematological high-risk patients and 14 healthy donors had no elevated Candida-reactive T cell counts.</p><p>Conclusions: This Candida-reactive lymphocyte assay correctly identified the majority of patients with invasive Candida infection and the respective species. Our assay has the potential to support diagnosis of invasive Candida infection to species level and to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.</p

Table_2_Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection—A Prospective Pilot Study.DOCX

<p>Background: Blood or tissue culture or histology prove invasive Candida infection, but long time to result, limited feasibility and sensitivity call for new approaches. In this pilot project, we describe the diagnostic potential of quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes in blood of patients with invasive Candida infection.</p><p>Methods: We used flow cytometry quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes from peripheral blood of patients with invasive Candida infection, from patients at risk and healthy volunteers as controls.</p><p>Results: Elevated levels of Candida-reactive lymphocytes were measured in 13 patients with proven invasive Candida infection and in one patient with probable hepatosplenic candidiasis. Results of three candidemia patients were uninterpretable due to autofluorescence of samples. Twelve of 13 patients had Candida identified to species level by conventional methods, and T cell reactivity correctly identified Candida species in 10 of 12 patients. Nine hematological high-risk patients and 14 healthy donors had no elevated Candida-reactive T cell counts.</p><p>Conclusions: This Candida-reactive lymphocyte assay correctly identified the majority of patients with invasive Candida infection and the respective species. Our assay has the potential to support diagnosis of invasive Candida infection to species level and to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.</p

Primary diseases of 155 investigated immunocompromised patients using the azole resistance PCR assays.

<p>AML, acute myeloid leukemia; ALL, acute lymphatic leukemia; CLL, chronic lymphatic leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; COPD, chronic obstructive pulmonary disease; HIV, human immunodeficiency virus; JMML, juvenile myelomonocytic leukemia, MDS, myelodysplastic syndrome; NHL, non Hodgkin lymphoma; OMF, osteomyelofibrosis.</p

Results of azole PCR based investigation of a multi-azole resistant <i>Aspergillus fumigatus</i> clinical isolate of a patient with heroin addict and aspergilloma, the corresponding clinical isolate of patient 3 suffering from AML and isolates A68 and A71 from immunocompromised patients.

<p>PCR and DNA sequence analysis of all isolates revealed the TR34/L98H alterations.</p

Azole resistance PCR results of 25 investigated blood (PB = peripheral blood), 120 BAL, 17 tissue biopsies and 19 CSF samples.

<p>106 samples including all blood samples were PCR negative in the most sensitive L98H PCR assay. Investigation of 32 samples revealed positive PCR results in all three PCR assays and showed three times the L98H mutation, two times in combination with the TR34. n, number of clinical samples.</p

Characteristics of patients 1–3 and the corresponding clinical samples showing <i>cyp51 A</i> TR34/L98H alterations.

<p>All clinical samples have been tested positive for <i>Aspergillus</i> DNA in our diagnostic nested PCR assay. Pts., patients; AML, acute myeloid leukemia; T-ALL, T-lymphoblastic acute leukemia; COPD, chronic obstructive pulmonary disease.</p

Determination of the improved one-step L98H PCR assay sensitivity.

<p>The determination was performed by Gelstar (Bio-Rad GmbH, Munich, Germany) stained agarose gel electrophoresis using serially diluted <i>A. fumigatus</i> wildtype DNA. The PCR amplicons of the patients were used for DNA sequencing analysis after elution from the agarose gel. BPM = base pair marker; Spiking control = 100 ng human DNA +50 ng <i>A. fumigatus</i> wildtype DNA.</p

DataSheet1.pdf

<p>Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied.</p><p>Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene.</p><p>Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V.</p><p>Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.</p