Diagnostic Value of Microbial Cell-free DNA Sequencing for Suspected Invasive Fungal Infections: A Retrospective Multicenter Cohort Study.

Background: An early diagnosis and treatment of invasive fungal disease (IFD) is associated with improved outcome, but the moderate sensitivity of noninvasive diagnostic tests makes this challenging. Invasive diagnostic procedures such as bronchoalveolar lavage (BAL) have a higher yield but are not without risk. The detection and sequencing of microbial cell-free DNA (mcfDNA) may facilitate a noninvasive diagnosis. Materials: In a prospective observational study, we collected plasma in the 120 hours preceding or following a BAL in patients with hematological malignancies suspected for a pulmonary IFD. The EORTC/MSGERC2020 criteria were used for IFD classification. Sequencing was performed by Karius (Redwood City, CA) using their Karius Test (KT) on plasma and a "research use only test" on BAL fluid if available. Cases with a probable/proven IFD were identified based on standard diagnostic tests on serum and BAL (microscopy, polymerase chain reaction, galactomannan, culture) and used to calculate the sensitivity, specificity, and additional diagnostic value of the KT. Results: Of 106 patients enrolled, 39 (37%) had a proven/probable invasive aspergillosis, 7 (7%) a non-Aspergillus IFD, and 4 (4%) a mixed IFD. The KT detected fungal mcfDNA in 29 (28%) patients. Compared with usual diagnostic tests, the sensitivity and specificity were 44.0% (95% confidence interval [CI], 31.2-57.7) and 96.6% (95% CI, 88.5%-99.1%). Sensitivity of the KT was higher in non-Aspergillus IFD (Mucorales:2/3, Pneumocystis jirovecii: 3/5). On BAL, the sensitivity was 72.2% (95% CI, 62.1-96.3), and specificity 83.3% (95% CI, 49.1-87.5). Conclusions: Sequencing of mcfDNA may facilitate a noninvasive diagnosis of IFD in particular non-Aspergillus IFD. However, on plasma and similar to currently available diagnostics, it cannot be used as a "rule-out" test.

Epidemiological characteristics and outcome of viral respiratory tract infections in the first year after allogeneic hematopoietic cell transplantation.

Adverse outcomes of viral respiratory tract infections (RTI) have been reported in recipients of allogeneic hematopoietic cell transplantation. Using a laboratory-developed multiparameter PCR in a consecutive series of 242 patients, we found the highest incidence of viral RTI in the pre-engraftment phase. The occurrence of multiple episodes of viral RTI or viral pneumonia was significantly associated with a higher hazard of non-relapse mortality in the first year after transplantation. We observed a 90-day mortality of 19.7% after viral RTI, which was significantly different between patient groups stratified according to the ISI score.

Baseline chest computed tomography for diagnosis of invasive aspergillosis in patients with acute myeloid leukaemia treated with intensive chemotherapy: A retrospective single-centre cohort study.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a relatively common infection in patients with acute myeloid leukaemia (AML), and is associated with high mortality rates. Optimising early detection is key to reduce the burden of IPA in this population. In this retrospective cohort study, we evaluated the added value of baseline chest CT before start of classical induction chemotherapy. METHODS: Adult patients receiving first-line intensive chemotherapy for AML were included if a baseline chest CT scan was available (±7 days). Data were collected from the electronic health record. IPA was classified using the EORTC/MSGERC 2020 consensus definitions. RESULTS: Between 2015 and 2019, 99 patients were included. During first-line treatment, 29/99 (30%) patients developed a probable IPA. Baseline chest CT was abnormal in 61/99 (62%) and 14/61 (23%) patients had typical radiological signs for IPA. An abnormal scan showed a trend towards higher risk for IPA (hazard ratio (HR): 2.12; 95% CI 0.95-4.84). Ground glass opacities were a strong predictor for developing IPA (HR 3.35: 95% CI 1.61-7.00). No probable/proven IPA was diagnosed at baseline; however, a bronchoalveolar lavage (BAL) at baseline was only performed in seven patients. Twelve-week mortality was higher in patients with IPA (7/26, 27% vs. 5/59, 8%; p = .024). CONCLUSION: Baseline chest CT scan could be an asset in the early diagnosis of IPA and contribute to risk estimation for IPA. In patients with an abnormal baseline CT, performing a BAL should be considered more frequently, and not only in patients with radiological findings typical for IPA.

Retrospective Multicenter Evaluation of the VirClia Galactomannan Antigen Assay for the Diagnosis of Pulmonary Aspergillosis with Bronchoalveolar Lavage Fluid Samples from Patients with Hematological Disease.

Galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid samples has become an essential tool to diagnose invasive pulmonary aspergillosis (IPA) and is part of diagnostic guidelines. Enzyme-linked immunosorbent assays (ELISAs) (enzyme immunoassays [EIAs]) are commonly used, but they have a long turnaround time. In this study, we evaluated the performance of an automated chemiluminescence immunoassay (CLIA) with BAL fluid samples. This was a multicenter retrospective study in the Netherlands and Belgium. BAL fluid samples were collected from patients with underlying hematological diseases with a suspected invasive fungal infection. Diagnosis of IPA was based on the 2020 European Organisation for Research and Treatment of Cancer (EORTC)/Mycoses Study Group Education and Research Consortium (MSGERC) consensus definitions. GM results were reported as optical density index (ODI) values. ODI cutoff values for positive results that were evaluated were 0.5, 0.8, and 1.0 for the EIA and 0.16, 0.18, and 0.20 for the CLIA. Probable IPA cases were compared with two control groups, one with no evidence of IPA and another with no IPA or possible IPA. Qualitative agreement was analyzed using Cohen's κ, and quantitative agreement was analyzed by Spearman's correlation. We analyzed 141 BAL fluid samples from 141 patients; 66 patients (47%) had probable IPA, and 56 cases remained probable IPA when the EIA GM result was excluded as a criterion, because they also had positive culture and/or duplicate positive PCR results. Sixty-three patients (45%) had possible IPA and 12 (8%) had no IPA. The sensitivity and specificity of the two tests were quite comparable, and the overall qualitative agreement between EIA and CLIA results was 81 to 89%. The correlation of the actual CLIA and EIA values was strong at 0.72 (95% confidence interval, 0.63 to 0.80). CLIA has similar performance, compared to the gold-standard EIA, with the benefits of faster turnaround because batching is not required. Therefore, CLIA can be used as an alternative GM assay for BAL fluid samples.

Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

Immunologic Monitoring after Allogeneic Stem Cell Transplantation: T-SPOT.CMV and QuantiFERON-CMV, Are They the Same?

Despite prophylactic and preemptive strategies, cytomegalovirus (CMV) reactivation and disease remains major concerns after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In recent years, immunologic monitoring using CMV commercially available IFN-γ release assays (IGRAs) has gained interest to better risk-stratify immunocompromised patients or to guide prophylactic therapy. CMV-IGRA can quantify CMV cell-mediated immunity by measuring the IFN-γ that is released by CD4+ and CD8+ T lymphocytes in the presence of CMV antigens. However, the 2 most widely used CMV-IGRAs, T-SPOT.CMV and QuantiFERON-CMV, had not yet been compared in the setting of an allo-HSCT. In the present study, we performed a method comparison between T-SPOT.CMV and QuantiFERON-CMV at 28 days and 100 days post-allo-HSCT, and to assess predictive values of both tests for CMV reactivation. Twenty-seven patients were included in a bicentric prospective trial. Samples were obtained on days +28 and +100 post-allo-HSCT, and patients' clinical information was collected up to day +270 post-HSCT. Comparisons of methods were performed using Cohen's κ. On day +28 (n = 26) post-allo-HSCT, T-SPOT.CMV yielded 3 positive test results and QuantiFERON-CMV yielded 2 positive results. On day +100 (n = 24), T-SPOT.CMV produced 7 positive test results, and QuantiFERON-CMV produced 9. One discordant result was obtained at day +28 (n = 26), and 6 discordant results were obtained at day +100 (n = 24). Method comparison showed a strong agreement on day +28 (κ = .780; 95% confidence interval [CI], .366 to 1.000) but only a moderate agreement on day +100 (κ = .442; 95% CI, .070 to .814) and in pooled data from both time points (κ = .578; 95% CI, .300-.856). Four clinically significant CMV infections (CS-CMVi) were observed, all occurring after discontinuation of letermovir prophylaxis. None of those 4 patients had a positive result with either test at day +100 (or day +28). Thus, the negative predictive value (NPV) and sensitivity were very high, at 100% for both tests measured at day +100. Positive predictive values (PPVs) and specificity were considerably lower at day +100 (T-SPOT.CMV: PPV, 23.5%; specificity, 35%; QuantiFERON-CMV: PPV, 26.7%; specificity, 45%). T-SPOT.CMV and QuantiFERON-CMV had only moderate agreement (at day +100) after allo-HSCT. Although these IGRAs are very promising, as shown by their very high NPVs for protection against CS-CMVi, they are not interchangeable. Future research should stipulate which IGRA was used, and future guidelines preferably should be assay-specific. As QuantiFERON-CMV still lacks a large post-allo-HSCT validation study, the moderate agreement with T-SPOT.CMV poses a significant hurdle in the routine implementation of this test.

Infectious diarrhea after allogeneic hematopoietic cell transplantation assessed by a multiplex polymerase chain reaction assay.

OBJECTIVES: To determine the incidence of infectious diarrhea after allogeneic hematopoietic cell transplantation (HCT) using a multiplex polymerase chain reaction assay and assess risk factors for developing infectious diarrhea. METHODS: This was a single-center retrospective study of 140 consecutive allogeneic HCT recipients. Infectious diarrhea was assessed using a laboratory-developed multiplex polymerase chain reaction the first year after transplantation. RESULTS: The incidence rate of infectious diarrhea episodes was 47 per 100 person-years, with the highest rate observed in the pre-engraftment phase. Most episodes were seen as nosocomial infections (38%) and most affected patients (82%) had only one episode of infectious diarrhea. The cumulative incidence of at least one episode of infectious diarrhea was 32% after 1 year. Nonrelapse mortality was higher in transplant recipients with at least one episode of infectious diarrhea (hazard ratio (HR) 2.02, 95% CI = 1.07-3.80). The most frequently observed pathogens were Clostridium difficile, adenovirus, Enteropathogenic Escherichia coli, and Campylobacter jejuni. Patients with acute lower gastrointestinal graft-vs-host disease stage 3 or 4 (HR 3.68, 95% CI = 1.57-8.63) conferred a higher risk for a first infectious diarrhea episode. CONCLUSION: Infectious diarrhea after allogeneic HCT was seen in about one-third of the patients, mostly as nosocomial infection in the pre-engraftment phase.

Blood Mucorales PCR to track down Aspergillus and Mucorales co-infections in at-risk hematology patients: A case-control study.

Introduction: Serum Mucorales PCR can precede the final diagnosis of invasive mucormycosis by several days or weeks and could therefore be useful as a non-invasive screening tool. Methods: We assessed the performance of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics, Maastricht, The Netherlands) on prospectively collected banked sera from hematology patients at risk for invasive mould infections. We evaluated if there is an underestimated incidence of missed Mucorales co-infections in patients with invasive aspergillosis (IA). We tested Mucorales PCR on the sera of all patients with a diagnosis of at least possible IA (EORTC-MSGERC consensus criteria) before the start of any antifungal therapy, and in a control group of similar high-risk hematology patients without IA (in a 1:4 ratio). When a positive Mucorales PCR was observed, at least 5 serum samples taken before and after the positive one were selected. Results: Mucorales PCR was performed in 46 diagnostic serum samples of cases and in 184 controls. Serum Mucorales PCR was positive in 4 cases of IA (8.7%; 12.9% of probable cases) and in 1 control case (0.5%) (p=0.0061, OR=17.43 (1.90-159.96). Post-mortem cultures of the positive control became positive for Rhizopus arrhizus. Mortality of IA cases with and without a positive Mucorales PCR was not significantly different. Only in the PCR positive control case, serial serum samples before and after the diagnostic sample were also positive. Discussion: It is not entirely clear what a positive Mucorales PCR in these cases implies since the 4 Mucorales PCR positive cases were treated with antifungals with activity against Mucorales. In addition, PCR was positive only once. This study does not provide enough evidence to implement Mucorales PCR screening. However, our findings emphasize once more the importance of considering the possibility of dual mould infections, even in patients with a positive galactomannan detection.

Case report: Hemorrhagic fever with renal syndrome presenting as hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis may occur in patients with genetic predisposition and in sporadic cases due to malignancy or infection. We describe a 49-year old man with hemorrhagic fever, type 1 respiratory insufficiency and acute kidney injury. Diagnostic work up showed a hyperinflammatory syndrome, hypertriglyceridemia, hemophagocytosis, very high ferritin and significantly elevated sCD25. The findings were compatible with hemophagocytic lymphohistiocytosis based on the HLH-2004 criteria. Serological testing indentified Puumala virus as the causal pathogen. The patient was successfully treated with pulse corticosteroids, intravenous immunoglobins and supportive therapy.

Performance of the JF5-Based Galactomannoprotein EIA Compared to the Lateral Flow Device and the Galactomannan EIA in Serum and Bronchoalveolar Lavage Fluid.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of "probable" invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4-deviating from the 0.5 threshold recommended by the manufacturer-sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

A unique phenotype of longitudinal extensive transverse myelitis in autoimmune lymphoproliferative syndrome.

A 49-year-old patient with a history of lymphoproliferation and autoimmune cytopenias presented with unexplained longitudinal extensive transverse myelitis. Flow cytometry on peripheral blood showed an elevated level of double negative T lymphocytes, a finding typical for autoimmune lymphoproliferative syndrome (ALPS). Inborn error of immunity (IEI) gene panel demonstrated a heterozygous variant in the FAS gene (c.857G > A, p.(Gly286Glu)), formally confirming the diagnosis. Autoimmune neurological conditions in a context of predisposition for infection and lymphoproliferation should raise suspicion of IEI. Specific testing for ALPS should be considered in patients with a history of non-malignant lymphoproliferation, multilineage cytopenias and unexplained autoimmune (neurological) manifestations.

Early high antibody titre convalescent plasma for hospitalised COVID-19 patients: DAWn-plasma.

BACKGROUND: Several randomised clinical trials have studied convalescent plasma for coronavirus disease 2019 (COVID-19) using different protocols, with different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralising antibody titres, at different time-points and severities of illness. METHODS: In the prospective multicentre DAWn-plasma trial, adult patients hospitalised with COVID-19 were randomised to 4 units of open-label convalescent plasma combined with standard of care (intervention group) or standard of care alone (control group). Plasma from donors with neutralising antibody titres (50% neutralisation titre (NT50)) ≥1/320 was the product of choice for the study. RESULTS: Between 2 May 2020 and 26 January 2021, 320 patients were randomised to convalescent plasma and 163 patients to the control group according to a 2:1 allocation scheme. A median (interquartile range) volume of 884 (806-906) mL) convalescent plasma was administered and 80.68% of the units came from donors with neutralising antibody titres (NT50) ≥1/320. Median time from onset of symptoms to randomisation was 7 days. The proportion of patients alive and free of mechanical ventilation on day 15 was not different between both groups (convalescent plasma 83.74% (n=267) versus control 84.05% (n=137)) (OR 0.99, 95% CI 0.59-1.66; p=0.9772). The intervention did not change the natural course of antibody titres. The number of serious or severe adverse events was similar in both study arms and transfusion-related side-effects were reported in 19 out of 320 patients in the intervention group (5.94%). CONCLUSIONS: Transfusion of 4 units of convalescent plasma with high neutralising antibody titres early in hospitalised COVID-19 patients did not result in a significant improvement of clinical status or reduced mortality.

A Large Retrospective Assessment of Voriconazole Exposure in Patients Treated with Extracorporeal Membrane Oxygenation.

BACKGROUND: Voriconazole is one of the first-line therapies for invasive pulmonary aspergillosis. Drug concentrations might be significantly influenced by the use of extracorporeal membrane oxygenation (ECMO). We aimed to assess the effect of ECMO on voriconazole exposure in a large patient population. METHODS: Critically ill patients from eight centers in four countries treated with voriconazole during ECMO support were included in this retrospective study. Voriconazole concentrations were collected in a period on ECMO and before/after ECMO treatment. Multivariate analyses were performed to evaluate the effect of ECMO on voriconazole exposure and to assess the impact of possible saturation of the circuit's binding sites over time. RESULTS: Sixty-nine patients and 337 samples (190 during and 147 before/after ECMO) were analyzed. Subtherapeutic concentrations (<2 mg/L) were observed in 56% of the samples during ECMO and 39% without ECMO (p = 0.80). The median trough concentration, for a similar daily dose, was 2.4 (1.2-4.7) mg/L under ECMO and 2.5 (1.4-3.9) mg/L without ECMO (p = 0.58). Extensive inter-and intrasubject variability were observed. Neither ECMO nor squared day of ECMO (saturation) were retained as significant covariates on voriconazole exposure. CONCLUSIONS: No significant ECMO-effect was observed on voriconazole exposure. A large proportion of patients had voriconazole subtherapeutic concentrations.

Point of care aspergillus testing in intensive care patients.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is an increasingly recognized complication in intensive care unit (ICU) patients, especially those with influenza, cirrhosis, chronic obstructive pulmonary disease, and other diseases. The diagnosis can be challenging, especially in the ICU, where clinical symptoms as well as imaging are mostly nonspecific. Recently, Aspergillus lateral flow tests were developed to decrease the time to diagnosis of IPA. Several studies have shown promising results in bronchoalveolar lavage fluid (BALf) from hematology patients. We therefore evaluated a new lateral flow test for IPA in ICU patients. METHODS: Using left-over BALf from adult ICU patients in two university hospitals, we studied the performance of the Aspergillus galactomannan lateral flow assay (LFA) by IMMY (Norman, OK, USA). Patients were classified according to the 2008 EORTC-MSG definitions, the AspICU criteria, and the modified AspICU criteria, which incorporate galactomannan results. These internationally recognized consensus definitions for the diagnosis of IPA incorporate patient characteristics, microbiology and radiology. The LFA was read out visually and with a digital reader by researchers blinded to the final clinical diagnosis and IPA classification. RESULTS: We included 178 patients, of which 55 were classified as cases (6 cases of proven and 26 cases of probable IPA according to the EORTC-MSG definitions, and an additional 23 cases according to the modified AspICU criteria). Depending on the definitions used, the sensitivity of the LFA was 0.88-0.94, the specificity was 0.81, and the area under the ROC curve 0.90-0.94, indicating good overall test performance. CONCLUSIONS: In ICU patients, the LFA performed well on BALf and can be used as a rapid screening test while waiting for other microbiological results.

A randomized, multicentre, open-label phase II proof-of-concept trial investigating the clinical efficacy and safety of the addition of convalescent plasma to the standard of care in patients hospitalized with COVID-19: the Donated Antibodies Working against nCoV (DAWn-Plasma) trial.

BACKGROUND: The COVID-19 pandemic has imposed an enormous burden on health care systems around the world. In the past, the administration of convalescent plasma of patients having recovered from SARS and severe influenza to patients actively having the disease showed promising effects on mortality and appeared safe. Whether or not this also holds true for the novel SARS-CoV-2 virus is currently unknown. METHODS: DAWn-Plasma is a multicentre nation-wide, randomized, open-label, phase II proof-of-concept clinical trial, evaluating the clinical efficacy and safety of the addition of convalescent plasma to the standard of care in patients hospitalized with COVID-19 in Belgium. Patients hospitalized with a confirmed diagnosis of COVID-19 are eligible when they are symptomatic (i.e. clinical or radiological signs) and have been diagnosed with COVID-19 in the 72 h before study inclusion through a PCR (nasal/nasopharyngeal swab or bronchoalveolar lavage) or a chest-CT scan showing features compatible with COVID-19 in the absence of an alternative diagnosis. Patients are randomized in a 2:1 ratio to either standard of care and convalescent plasma (active treatment group) or standard of care only. The active treatment group receives 2 units of 200 to 250 mL of convalescent plasma within 12 h after randomization, with a second administration of 2 units 24 to 36 h after ending the first administration. The trial aims to include 483 patients and will recruit from 25 centres across Belgium. The primary endpoint is the proportion of patients that require mechanical ventilation or have died at day 15. The main secondary endpoints are clinical status on day 15 and day 30 after randomization, as defined by the WHO Progression 10-point ordinal scale, and safety of the administration of convalescent plasma. DISCUSSION: This trial will either provide support or discourage the use of convalescent plasma as an early intervention for the treatment of hospitalized patients with COVID-19 infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT04429854 . Registered on 12 June 2020 - Retrospectively registered.

Epidemiology of Pneumocystis jirovecii Pneumonia and (Non-)use of Prophylaxis.

Objectives:Pneumocystis jirovecii pneumonia (PCP) is an AIDS-defining illness. In patients with HIV, the benefit of PCP prophylaxis is well-defined when the CD4 T-cell count decreases below 200 cells/µL. In other immunocompromised patients, the value of PCP prophylaxis is not always as well-established. This study aimed to describe the epidemiology of PCP in recent years and assess how many patients with PCP did or did not receive prophylaxis in the month preceding the infection. Material and Methods: A multicenter retrospective study was performed in 3 tertiary care hospital. A list of patients that underwent broncho-alveolar lavage sampling and Pneumocystis jirovecii (PJ) PCR testing was retrieved from the microbiology laboratories. An in-house PJ quantitative PCR (qPCR) was used in each center. A cycle threshold (Ct) value of ≤ 28.5-30 was considered a probable PCP. For patients with a positive PJ qPCR but above this threshold, a predefined case definition of possible PCP was defined as a qPCR Ct value ≤ 34-35 and both of the following criteria: 1. Clinical and radiological features compatible with PCP and 2. The patient died or received PCP therapy and survived. Patient files from those with a qPCR Ct value ≤ 35 were reviewed to determine whether the patient fulfilled the case definition and if PCP prophylaxis had been used in the weeks preceding the PCP. Disease-specific guidelines, as well as hospital-wide guidelines, were used to evaluate if prophylaxis could be considered indicated. Results: From 2012 to 2018, 482 BAL samples were tested. Two hundred and four had a qPCR Ct value ≤ 35 and were further evaluated: 90 fulfilled the definition of probable and 63 of possible PCP while the remaining 51 were considered colonized. Seventy-four percentages of the patients with PCP were HIV-negative. Only 11 (7%) of the 153 patients had received prophylaxis, despite that in 133 (87%) cases prophylaxis was indicated according to guidelines. Conclusion: In regions where HIV testing and treatment is available without restrictions, PCP is mainly diagnosed in non-HIV immunocompromised patients. More than four out of five patients with PCP had not received prophylaxis. Strategies to improve awareness of antimicrobial prophylaxis guidelines in immunocompromised patients are urgently needed.

Outpatient parenteral antifungal therapy (OPAT) for invasive fungal infections with intermittent dosing of liposomal amphotericin B.

Triazole resistant A. fumigatus has been documented in many parts of the world. In the Netherlands, incidence is now above 10% and results in the need for long-term parenteral therapy with liposomal amphotericin B (LAmB). The long terminal half-life of LAmB suggests that intermittent dosing could be effective, making the application of outpatient antifungal therapy (OPAT) possible. Here, we report our experience with the use of OPAT for Invasive Fungal Infections (IFI). All adult patients treated with LAmB with a 2 or 3 times weekly administration via the outpatient departments in four academic tertiary care centers in the Netherlands and Belgium since January 2010 were included in our analysis. Patient characteristics were collected, as well as information about diagnostics, therapy dose and duration, toxicity, treatment history and outcome of the IFI. In total, 18 patients were included. The most frequently used regimen (67%) was 5 mg/kg 3 times weekly. A partial response to the daily treatment prior to discharge was confirmed by CT-scan in 17 (94%) of patients. A favorable outcome was achieved in 13 (72%) patients. Decrease in renal function occurred in 10 (56%) cases but was reversible in all and was treatment limiting in one patient only. The 100-day mortality and 1-year mortality after initiation of OPAT were 0% and 6%, respectively. In a selected population, and after confirmation of initial response to treatment, our data support the use of OPAT with LAmB for treatment of IFI in an intermittent dosing regimen.