The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance

Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Predictors for prolonged hospital stay solely to complete intravenous antifungal treatment in patients with candidemia: Results from the ECMM candida III multinational European observational cohort study

Background To date, azoles represent the only viable option for oral treatment of invasive Candida infections, while rates of azole resistance among non-albicans Candida spp. continue to increase. The objective of this sub-analysis of the European multicenter observational cohort study Candida III was to describe demographical and clinical characteristics of the cohort requiring prolonged hospitalization solely to complete intravenous (iv) antifungal treatment (AF Tx). Methods Each participating hospital (number of eligible hospitals per country determined by population size) included the first ~ 10 blood culture proven adult candidemia cases occurring consecutively after July 1st, 2018, and treating physicians answered the question on whether hospital stay was prolonged only for completion of intravenous antifungal therapy. Descriptive analyses as well as binary logistic regression was used to assess for predictors of prolonged hospitalization solely to complete iv AF Tx. Findings Hospital stay was prolonged solely for the completion of iv AF Tx in 16% (100/621) of candidemia cases by a median of 16 days (IQR 8 – 28). In the multivariable model, initial echinocandin treatment was a positive predictor for prolonged hospitalization to complete iv AF Tx (aOR 2.87, 95% CI 1.55 – 5.32, p < 0.001), while (i) neutropenia, (ii) intensive care unit admission, (iii) catheter related candidemia, (iv) total parenteral nutrition, and (v) C. parapsilosis as causative pathogen were found to be negative predictors (aOR 0.22 – 0.45; p < 0.03). Interpretation Hospital stays were prolonged due to need of iv AF Tx in 16% of patients with candidemia. Those patients were more likely to receive echinocandins as initial treatment and were less severely ill and less likely infected with C. parapsilosis

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance. A nationwide survey and rationale for the DB-MSG 002 study protocol

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The presence of FLT3-ITD and mutated NPM1 in ALDH^bright CD34+CD38− progenitor populations.

<p>(<b>A</b>) In CD34-negative AML cases, the ALDH^bright population of AML cells consists, besides CD34+CD38− stem cells of CD38dim and CD38+ progenitors (<b>A</b>, middle panel, AML-464) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078897#pone.0078897.s005" target="_blank">Table S3</a>). The complete ALDH^bright compartment in CD34-negative AML has a normal phenotype as proven by the absence of FLT3-ITD in the CD34+CD38− (<b>A</b>, right upper panel) as well as the CD38dim (<b>A</b>, right middle panel) and CD38+ (data not shown) population of cells. The ALDH^low compartment contains the FLT3-ITD (arrow in right lower panel), indicating leukemic cells. (<b>B</b>) In CD34-positive AML cases, the ALDH^bright population contains, apart from normal CD34+CD38− HSC, CD34+CD38+ progenitors and CD34− cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078897#pone-0078897-g005" target="_blank">Figure 5B</a> middle panel)(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078897#pone.0078897.s005" target="_blank">Table S3</a>). The ALDH^bright CD34+CD38− stem cells lack molecular aberrancies (<b>B</b>, right upper two panels) and are therefore normal. The CD34+CD38+ progenitor compartment (<b>B,</b> right middle two panels) in this case has very small FLT3-ITD and NPM1 mutant peaks likely originating from purification of ALDH^low cells within the ALDH^bright compartment. The ALDH^low compartment is largely neoplastic (<b>B</b>, right lower 2 panels).</p

ALDH^bright CD34+CD38– cells are devoid of both the FLT3 and NPM1 mutation.

<p>AML-808 contains both FLT3-ITD and mutated NPM1 and has no immunophenotypic aberrancy present. However, AML-808 can be segregated into a normal and a leukemic CD34+CD38– compartment by measuring ALDH activity (<b>A–C</b>). The CD34+CD38– stem cells (<b>A</b>, orange) segregate into two separate ALDH compartments (<b>B</b>). The CD34+CD38–ALDH^bright cells of AML-808 do neither contain FLT3-ITD nor mutated NPM1 (<b>C</b>, upper panels), while the CD34+CD38-ALDH^low cells (blue) do (<b>C</b>, middle panel). The ALDH^low cells have both mutations (<b>C</b>, lower panel). AML-575 contains both FLT3-ITD and mutated NPM1. The CD34+CD38– cells (red) segregate into two separate ALDH compartments (<b>D</b>, middle panel). The ALDH^bright (red) cells within AML-575 lack the immunophenotypic aberrancy CD33 and are low in SSC compared to the ALDH^low cells (blue)(<b>F</b>). The CD34+CD38-ALDH^bright cells do neither contain FLT3-ITD nor mutated NPM1 (<b>G</b>, upper panels), while the CD34+CD38-ALDH^low cells do (<b>G</b>, middle panel). The ALDH^low cells have both mutations (<b>G</b>, lower panel). (<b>H</b>) CD34+CD38– HSC, CD34+CD38– LSC (purification based on CLL1+, CD34 expression and scatter properties) from AML-598, and CD34+CD38– HSC from normal BM were purified. RNA was isolated and RNA sequencing was performed. The most abundant ALDH mRNA expressed within both normal HSC fractions (AML and normal BM) was the ALDH1A1 enzyme. LSC had hardly any expression of the ALDH1A1 form. Numbers indicate the amount of ALDH1A1 reads within that fraction.</p

Presence of FLT3-ITD and mutated NPM1 in ALDH^bright and ALDH^low compartments in CD34-positive AML.

<p>Detection of molecular aberrancies in CD34+CD38– ALDH^bright and ALDH^low compartments of CD34-positive AML. The FLT3-ITD percentage is determined in the total leukemic blast population (data not shown), the CD34– cell population and the CD34+CD38-ALDH^low and CD34+CD38-ALDH^bright compartments. The NPM1 mutation analysis is not quantative. # the ALDH^bright compartment contained in one case a small mutant peak (AML-945), likely caused by relatively poor separation of ALDH^bright and ALDH^low populations due to overlap in the boundary region.</p

CD34+CD38– HSC have higher ALDH activity than co-existing CD34+CD38– LSC.

<p>(<b>A–C</b>) CD34+CD38– HSC within normal bone marrow are ALDH^bright and the level of ALDH activity decreases upon differentiation to CD38+ progenitors. Representative flow cytometric ALDH activity patterns (ALDH versus SSC) are shown for (<b>A</b>) total CD45dim normal bone marrow cells treated with or without DEAB and (<b>B</b>) CD45dimCD34+ cells (middle panel) and both CD45dimCD34+ cells (red) and CD45dimCD34– cells (blue)(right panel). In C, the ALDH activity versus SCC of CD34+CD38– stem cells (panel 1 in green and panel 3 in bleu) and CD34+CD38+ progenitor cells (red in panel 2 and panel 3) from the normal BM is shown. (<b>D–I</b>) In CD34-positive AML, the ALDH activity of CD34+CD38– HSC is higher than that of the CD34+CD38– LSC. The aldefluor assay was performed on cells of a CD34-positive AML case (AML-951) and cells were subsequently labeled with anti-CD45 PERCP, anti-CD34 PC7, anti-CD38 APC and anti-CLL1 PE. The CD34+CD38– stem cells (<b>D</b>, purple) showed to be partly CLL-1+ and partly CLL-1– (<b>E</b>). The CLL-1+ stem cells (green) are FSC/SSC^high as compared to the CLL-1– cells (purple)(<b>F</b>). Investigation of the ALDH activity of the CD34+CD38– compartment showed that the CD34+CD38– stem cell population (<b>D</b>) segregates into an ALDH^bright (red) and an ALDH^low (blue) population (<b>G</b>). The ALDH^bright cells (red) are CLL-1 negative (H) and contain only wild type FLT3 kinase (<b>I,</b> upper panel). The ALDH^low cells (blue) are largely CLL-1 positive (<b>H</b>) and contain FLT3-ITD+ cells (<b>I</b>, lower panel). The arrow indicates the FLT3-ITD.</p

CD34+CD38–stem cell frequencies and their marker expression in the ALDH^bright and ALDH^low compartment in CD34-positive AML.

<p>Detection of immunophenotypic aberrancies in CD34+CD38– ALDH^bright and ALDH^low compartments of CD34-positive AML.</p>*<p>The percentage of CD34+CD38– cells in the total ALDH^bright or ALDH^low compartments is indicated.</p>&<p>in the ALDH^bright CD34+CD38– compartment the aberrant marker expression, both CLL-1 and lineage markers, is indicative for the neoplastic character of the cells.</p>#<p>Percentage expression does not quantitatively correlate with % of neoplastic cells: a shift in fluorescence of a total cell population may result e.g. in 50% marker expression based on normalization by isotype controls of negative cell populations.</p>‡<p>data in between parenthesis represent number of cells in cases where there is <25 cells positive for a marker.</p