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Background To unravel how the integrity of nuclear and mitochondrial circulating cell-free DNA (cfDNA) contributes to its plasma quantity in colorectal cancer (CRC) patients. Methods CfDNA from plasma samples of 80 CRC patients stratified by tumour stage and 50 healthy individuals were extracted. Total cfDNA concentration was determined and equal template concentrations (ETC) were analyzed by quantitative real-time PCR (qPCR) resulting in small and long fragments of KRAS, Alu and MTCO3. The obtained data was also examined relative to the total cfDNA concentration (NTC) and diagnostic accuracy was estimated using receiver operating characteristics. Results Total cfDNA levels were significantly higher in CRC group compared to healthy control and increased with tumour stage. Long nuclear fragment levels were significantly lower in CRC patients in ETC but not NTC condition. The integrity indices of nuclear cfDNA decreased from controls to patients with highly malignant tumor. Mitochondrial cfDNA fragment quantities were strongly reduced in early and late stages of tumor patients and prognostic value was higher in ETC. Predictive models based on either ETC or NTC predictor set showed comparable classification performance. Conclusion Increased blood cfDNA concentration in late UICC stages inversely correlate with nuclear cfDNA integrity index and suggest that necrotic degradation is not a major cause for higher total cfDNA quantity. The diagnostic and prognostic value of MTCO3 is highly significant in early stages of CRC and can be evaluated more comprehensively, using ETC for qPCR analysis. Trial registration The study was registered retrospectively on DRKS, the german register for clinical trials (DRKS00030257, 29/09/2022).

By implementing a focused must-have vaccination strategy (Easy Vaccination in Oncology [EVO]), we aimed to increase rates for high-impact vaccinations (Streptococcus pneumoniae, influenza, herpes zoster and hepatitis B) in the at-risk population of oncological patients.In this German multicentre interventional non-randomised controlled two-arm open trial with repeated cross-sectional data collection, we evaluated the EVO strategy as an easy to implement approach. Vaccination rates were assessed in the outpatient setting and re-assessed after 3 months. A generalised linear mixed model (GLMM) was used to assess the primary endpoint (Streptococcus pneumoniae vaccination rates according to recommendations), taking clustering within clinics into account.Vaccination rates substantially increased in the intervention group; Streptococcus pneumoniae +21.5% (+16.7% according to recommendations), influenza +12.2%, herpes zoster +13.3% (+13.6% age group 50+), and hepatitis B +11%. Vaccination rates in the control group tended to decrease or increase only moderately (-5.8% [-3.8%], +7.4%, +2.1% [1.4%], and -1.7%, respectively). GLMM showed significant effect of the intervention (OR 7.50, 95% CI 2.18-25.80, p = 0.001).This easy-to-implement and resource-saving approach has the potential to increase vaccination rates in oncological patients and to have a considerable impact protecting oncological patients from preventable infectious diseases.The study was registered at the German Resister for Clinical Studies (DRKS) under DRKS00020118.

Standard operating procedures for autologous stem cell transplantation (SCT) aim to guarantee best possible engraftment. Three procedures are routinely used for transplant infusion: regular bag infusion (Procedure 1), injection via syringe (Procedure 2), and combination of regular bag infusion and syringe (Procedure 3). We conducted a retrospective analysis of all autologous stem cell transplants done in the hematology department of the Vivantes Clinic Neukoelln in Berlin, Germany, between January 1, 2016 and March 4, 2017. Of the total of 69 patients, 17 underwent Procedure 1, 32 Procedure 2, and 20 Procedure 3. Although speed of transplant reinfusion differed significantly between procedure types, these differences had no effect on duration of leukopenia. However, duration of leukopenia did correlate with need for blood transfusion and use of antibiotics. Our findings contradict the general perception that very rapid reinfusion is necessary. Nevertheless, considering the limitations of this study (retrospective, single center, small sample size) and that longer duration of aplasia is associated with greater need for intervention, efficient transplant reinfusion is advisable. More research is needed regarding timeliness and type of procedure used.

Background: Vaccinations have the potential to significantly lower the burden of disease for many major infections in the high-risk population of hematological and oncological patients. In this regard Shingrix®, an inactivated Varicella Zoster Virus vaccine, received market approval in the European Union in March 2018, after prior US approval in October 2017, and recommendations specifically state immunocompromised, including oncological, patients. As vaccination rates are considered to be poor in oncological patients, determining the current vaccination rates for Shingrix® two years after market approval is important in defining the need for intervention to bring this potentially high-impact vaccine to the patients. Methods: We analyzed data of the EVO Study to provide data for Herpes zoster vaccination rates in oncological patients. The EVO Study was an interventional study evaluating the potential of increasing vaccination rates of specified must-have vaccinations by an instructional card in the oncological setting. Numbers presented in this publication merged baseline data and follow-up data of the control group; hence data not affected by the intervention. Results: Data of 370 patients were analyzed; 21.1% with hematological malignancies and 78.9% with solid cancer. Only 3.0% were vaccinated with Shingrix®. Patients with hematological malignancy were more likely to be vaccinated than those with solid cancer (7.7 vs. 1.7%). Conclusion: Despite clear recommendations and a pressing need in the high-risk population of hematological and oncological patients, the vast majority of patients are still left without vaccine protection against Herpes zoster by Shingrix®.

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets. Supplementary Information The online version contains supplementary material available at 10.1007/s00277-021-04492-1.

Whether a patient receives general or specialized outpatient palliative cancer care rarely follows clear criteria, leading to undertreatment or overtreatment. Detailed scores exist to predict prognosis, but not treatment requirements, leaving caregivers to follow their intuition. As a phenomenological indicator incorporating possibly important subjective information, intuition may in fact be a helpful tool. In this prospective observational study, a score to estimate three global dimensions of patients' resources was applied: Medical prognosis, feeling of strength and feeling of support. The score results were correlated with the actual amount and effort of care required during the subsequent palliative care time. This phenomenological score correlated well with the performance index and the Hospice and Palliative care Evaluation score. Whilst various individual items correlated significantly with the score or its constituent parameters, there was no uniform coherent pattern, reflecting the complexity of palliative care and the potential value of this predictive tool.

(1) Background: Healthcare workers (HCWs) are prone to intensified exposure to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in the ongoing pandemic. We prospectively analyzed the prevalence of antibodies against SARS-CoV-2 in HCWs at baseline and follow up with regard to clinical signs and symptoms in two university hospitals in Brandenburg, Germany. (2) Methods: Screening for anti-SARS-CoV-2 IgA and IgG antibodies was offered to HCWs at baseline and follow up two months thereafter in two hospitals of Brandenburg Medical School during the first wave of the COVID-19 pandemic in Germany in an ongoing observational cohort study. Medical history and signs and symptoms were recorded by questionnaires and analyzed. (3) Results: Baseline seroprevalence of anti-SARS-CoV-2 IgA was 11.7% and increased to 15% at follow up, whereas IgG seropositivity was 2.1% at baseline and 2.2% at follow up. The rate of asymptomatic seropositive cases was 39.5%. Symptoms were not associated with general seropositivity for anti-SARS-CoV-2, however, class switch from IgA to IgG was associated with increased symptom burden. (4) Conclusion: The seroprevalence of antibodies against SARS-CoV-2 was low in HCWs but higher compared to population data and increased over time. Screening for antibodies detected a significant proportion of seropositive participants cases without symptoms.

The diagnosis of invasive aspergillosis in hematologic patients is a complex composite of clinical preconditions and features, imaging findings, biomarker combinations from appropriate clinical samples and microbiological and/or histological findings.Recent developments in the evolving landscape of diagnostic tests for invasive aspergillosis in adult hematology patients are highlighted.Novel approaches and tools are currently under development. Focusing optimized diagnostic performance, in particular the combination of biomarkers from appropriate clinical samples, improved diagnostic performance distinctly.

Invasive pulmonary aspergillosis (IPA) is one of the major complications in immunocompromised patients. The mainstay of diagnostic imaging is non-enhanced chest-computed-tomography (CT), for which various non-specific signs for IPA have been described. However, contrast-enhanced CT pulmonary angiography (CTPA) has shown promising results, as the vessel occlusion sign (VOS) seems to be more sensitive and specific for IPA in hematologic patients. The aim of this study was to evaluate the diagnostic accuracy of CTPA in a larger cohort including non-hematologic immunocompromised patients. CTPA studies of 78 consecutive immunocompromised patients with proven/probable IPA were analyzed. 45 immunocompromised patients without IPA served as a control group. Diagnostic performance of CTPA-detected VOS and of radiological signs that do not require contrast-media were analyzed. Of 12 evaluable radiological signs, five were found to be significantly associated with IPA. The VOS showed the highest diagnostic performance with a sensitivity of 0.94, specificity of 0.71 and a diagnostic odds-ratio of 36.8. Regression analysis revealed the two strongest independent radiological predictors for IPA to be the VOS and the halo sign. The VOS is highly suggestive for IPA in immunocompromised patients in general. Thus, contrast-enhanced CTPA superior over non-contrast_enhanced chest-CT in patients with suspected IPA.

Aspergillus species, primarily Aspergillus fumigatus, are still the most emerging fungal pathogens. Within recent years, novel molecular methods have been developed to improve the diagnosis of life-threatening invasive aspergillosis in high risk patients. Especially patients with malignant hematological diseases undergoing intensive chemotherapy are at risk and mortality rates are exceptionally high, in part due to difficulties and delays in establishing a microbiologic diagnosis. Early diagnosis and treatment are crucial for an adequate therapeutical management, but, however, are hardly achieved in the clinical setting because most of the current conventional diagnostic tools either lack specificity or acceptable sensitivity at the critical early phase of the infection. Areas covered: To review the clinical value, advantages and problems as well as drawbacks of molecular approaches, especially polymerase chain reaction (PCR)-based assays to detect genomic DNA of Aspergillus species in clinical samples of immunocompromised, especially hematological patients at high risk for IA, a comprehensive review of the literature was performed and expert opinion was expressed. Expert commentary: The results of numerous attempts to diagnose invasive aspergillosis by PCR-based detection of fungal genome in clinical samples highlight the potential of the PCR technique to improve early diagnosis of invasive aspergillosis in patients with hematological malignancies during intensive antineoplastic treatment, combined with imaging surveillance and serologic diagnostic tools. Further comparative validation of reliable assays in prospective multicenter studies is mandatory and urgently needed in order to establish a harmonization and standardization, so that 'gold standard assays' may be incorporated into diagnostic and therapeutic algorithms that improve the prognosis of patients with life-threatening infections caused by Aspergillus species.

Invasive aspergillosis (IA) is a severe complication in immunocompromised patients. Early diagnosis is crucial to decrease its high mortality, yet the diagnostic gold standard (histopathology and culture) is time‐consuming and cannot offer early confirmation of IA. Detection of IA by polymerase chain reaction (PCR) shows promising potential. Various studies have analysed its diagnostic performance in different clinical settings, especially addressing optimal specimen selection. However, direct comparison of different types of specimens in individual patients though essential, is rarely reported. We systematically assessed the diagnostic performance of an Aspergillus‐specific nested PCR by investigating specimens from the site of infection and comparing it with concurrent blood samples in individual patients (pts) with IA. In a retrospective multicenter analysis PCR was performed on clinical specimens (n = 138) of immunocompromised high‐risk pts (n = 133) from the site of infection together with concurrent blood samples. 38 pts were classified as proven/probable, 67 as possible and 28 as no IA according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions. A considerably superior performance of PCR from the site of infection was observed particularly in pts during antifungal prophylaxis (AFP)/antifungal therapy (AFT). Besides a specificity of 85%, sensitivity varied markedly in BAL (64%), CSF (100%), tissue samples (67%) as opposed to concurrent blood samples (8%). Our results further emphasise the need for investigating clinical samples from the site of infection in case of suspected IA to further establish or rule out the diagnosis.

BACKGROUND: Initial results from the ongoing GALLIUM trial have shown that patients with follicular lymphoma have a longer progression-free survival after first-line immunochemotherapy with obinutuzumab than with rituximab. The aim of this secondary analysis was to evaluate the prognostic value of PET-CT responses after first-line immunochemotherapy in the GALLIUM study.METHODS: GALLIUM is an open-label, parallel-group randomised, phase 3 trial, which recruited previously untreated patients with CD20-positive follicular lymphoma (grades 1-3a; disease stage III/IV, or stage II with largest tumour diameter ≥7 cm) who were aged 18 years or older and met the criteria for needing treatment. Eligible patients were randomly assigned in a 1:1 ratio to receive intravenous administration of obinutuzumab (1000 mg on days 1, 8, and 15 of cycle 1, then day 1 of subsequent cycles) or rituximab (375 mg/m2 on day 1 of each cycle), in six 21-day cycles with cyclophosphamide, doxorubicin, vincristine, and prednisone (known as CHOP; oral administration) followed by two 21-day cycles of antibody alone, or eight 21-day cycles cyclophosphamide, vincristine, and prednisone (known as CVP; oral administration), or six 28-day cycles with bendamustine, followed by maintenance antibody every 2 months for up to 2 years. The primary endpoint of the trial, investigator-assessed progression-free survival, has been reported previously. This secondary analysis reports PET and CT-based responses at end-of-induction therapy and explains their relation with progression-free and overall survival outcomes in patients with available scans. As per protocol, during the trial, PET scans (mandatory in the first 170 patients enrolled at sites with available PET facilities, and optional thereafter), acquired at baseline and end of induction (PET population), were assessed prospectively by investigators and an independent review committee (IRC) applying International Harmonisation Project (IHP) 2007 response criteria, and retrospectively by the IRC only applying current Lugano 2014 response criteria. IRC members (but not study investigators) were masked to treatment and clinical outcome when assessing response. The landmark analyses excluded patients who died or progressed (contrast enhanced CT-based assessment of progressive disease, or started next anti-lymphoma treatment) before or at end of induction. GALLIUM is registered at ClinicalTrials.gov, number NCT01332968.FINDINGS: 1202 patients were enrolled in GALLIUM between July 6, 2011, and Feb 4, 2014, of whom 595 were included in the PET population; 533 (IHP 2007; prospective analysis), and 508 (Lugano 2014; retrospective analysis) were analysed for progression-free survival (landmark analysis). At end of induction, 390 of 595 patients (65·5% [95% CI 61·6-69·4]) achieved PET complete response according to IHP 2007 criteria, and 450 (75·6% [95% CI 72·0-79·0]) obtained PET complete metabolic response according to Lugano 2014 criteria. With a median of 43·3 months of observation (IQR 36·2-51·8), 2·5-year progression-free survival from end of induction was 87·8% (95% CI 83·9-90·8) in PET complete responders and 72·0% (63·1-79·0) in non-complete responders according to IRC-assessed IHP 2007 criteria (hazard ratio [HR] 0·4, 95% CI 0·3-0·6, pINTERPRETATION: Our results suggest that PET is a better imaging modality than contrast-enhanced CT for response assessment after first-line immunochemotherapy in patients with follicular lymphoma. PET assessment according to Lugano 2014 response criteria provides a platform for investigation of response-adapted therapeutic approaches. Additional supportive data are welcomed.FUNDING: F Hoffmann-La Roche.

Invasive pulmonary aspergillosis (IPA) is an emerging and life-threatening infectious disease in patients admitted to the intensive care unit (ICU). Most diagnostic studies are conducted in hematological patients and results cannot readily be transferred to ICU patients lacking classical host factors. In a multicenter, prospective clinical trial including 44 ICU patients, hematological (n = 14) and non-hematological patients (n = 30), concurrent serum and bronchoalveolar lavage (BAL) samples were analyzed by conventional culture, galactomannan (GM), 1-3-beta-D-glucan (BDG) as well as an Aspergillus specific nested polymerase chain reaction (PCR). Nine patients (20%) had putative IPA according to AspICU classification. GM and PCR showed superior performance in BAL with sensitivity/specificity of 56%/94% and 44%/94% compared to 33%/97% and 11%/94% in serum. Despite better sensitivity of 89%, BDG showed poor specificity of only 31% (BAL) and 26% (serum). Combination of GM and PCR (BAL) with BDG (serum) resulted in 100% sensitivity, but also reduced specificity to 23%. Whereas mean GM levels were significantly higher in hematological patients BDG and PCR did not differ between hematological and non-hematological patients. Under present clinical conditions test combinations integrating both BAL and blood samples are advantageous. BDG might best serve as possible indicator for ruling out IPA. Trial registration ClinicalTrials.gov Identifier: NCT01695499 . First posted: September 28, 2012, last update posted: May 8, 2017.

Fluoroquinolone (FQ) and fluconazole prophylaxis is recommended for patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). However, due to an uncertain scientific basis and the increasing emergence of resistant germs, this policy should be questioned. Therefore, FQ and fluconazole prophylaxis was omitted in alloHCT at our center. In this retrospective analysis, all consecutive patients (n = 63) who underwent first alloHCT at our institution from September 2010 to September 2013 were included. Patients neither received FQ nor fluconazole prophylaxis. Day 100 mortality, incidence of febrile neutropenia, bacterial infections, and invasive fungal diseases (IFD) were assessed. Sixteen patients who started conditioning under antimicrobial treatment/prophylaxis due to pre-existing neutropenia (3/16), IFD (12/16), or aortic valve replacement (1/16) were excluded from the analysis. Finally, 47 patients were transplanted without prophylaxis as intended. Day 100 mortality was 9 %. Febrile neutropenia occurred in 62 % (29/47); 17/47 patients (36 %) experienced a blood stream infection (BSI) with detection of Gram-positive bacteria in 14 patients, Gram-negative bacteria in five patients, and candida in one patient, respectively. Coagulase-negative staphylococci were the most frequently isolated Gram-positive bacteria; 12/21 isolated Gram-positive and 3/6 Gram-negative bacteria were FQ resistant. In 21 % (10/47) of the patients, IFD (1x proven, 1x probable, and 8x possible) were diagnosed. To conclude, all three criteria, day 100 mortality, the incidence of IFD, and BSI, are in the range of published data for patients transplanted with FQ and fluconazole prophylaxis. These data demonstrate that alloHCT is feasible without FQ and fluconazole prophylaxis.

Wilms' tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1high vs. 63% for WT1low vs. 93% for WT1int; p=0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1high vs. 63% for WT1low vs. 83% for WT1int; p=0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.

Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic EORTC/MSG criteria are often not met. As biomarkers might elucidate the pathogen, we analysed the performance of an Aspergillus PCR assay in blood for diagnosis of IA in immunocompromised paediatric patients with suspected infections. Ninety-five haemato-oncological paediatric patients were included over a period of 3 years, the underlying diseases consisting of acute leukaemia, solid tumours, non-malignant immunocompromising disorders and haematopoietic stem cell transplantation recipients. We retrospectively analysed 253 consecutive episodes of suspected infections. Thirty-eight patients had possible IA, none of the patients fulfilled EORTC/MSG criteria of probable/proven IA. PCR positivity was observed in 97/967 analyses. Sensitivity, specificity, positive and negative predictive value of the PCR per episode were 34%, 78%, 31% and 81% using possible IA as endpoint. Taken together, an undirected blood screening by Aspergillus-specific PCR is of little diagnostic value in a heterogenous paediatric patient cohort. Harnessing PCR for diagnosis of IA should thus be focused on blood analyses of more homogenous high-risk patients and/or analyses of bronchoalveolar lavage, tissue or cerebrospinal fluid specimens.

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in patients receiving chemotherapy or undergoing allogeneic haematopoietic stem cell transplantation for acute leukemia. The existing tools to diagnose IPA lack specificity or sensitivity, or both; the search for improved diagnostic tools for IPA has focused on novel serologic and molecular methods. Aspergillus Galactomannan enzyme-linked immunosorbent assay (GM) analyses showed sensitivity rates in serum samples ranging in a wide span; testing GM in bronchoalveolar lavage (BAL) originated from the primary site of the infection seems to be more sensitive in patients with IPA. Other novel diagnostic markers to detect fungal DNA directly in clinical samples, rapidly, early, sensitively and specifically, are provided by polymerase chain reaction (PCR) based assays; higher sensitivity and specificity rates have been observed for BAL samples in IPA, even under antifungal treatment. The clinical place value of a diagnostic approach combining PCR and GM in BAL is unclear.

High mortality rates of invasive fungal disease (IFD), especially invasive aspergillosis (IA), in immunocompromised haematological patients and current diagnostic limitations require improvement of detection of fungal pathogens by defining the optimal use of biomarkers and clinical samples. Concurrent bronchoalveolar lavage (BAL) and peripheral blood samples of 99 haematological patients with suspected IFD were investigated within a multicentre prospective study. Diagnostic performance of a galactomannan (GM) enzyme immune assay (EIA), a 1,3-β-D-glucan assay (BDG), an Aspergillus PCR, and a multifungal DNA-microarray (Chip) alone or in combination were calculated. IFD were classified as proven (n=3), probable (n=34), possible (n=33), and no IFD (n=29) according to EORTC/MSG criteria. GM, PCR, and Chip showed superior diagnostic performance in BAL than in blood, whereas specificity of BDG in BAL was poor (48% (14/29)). The combination of GM (BAL) with BDG (blood) showed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and DOR (diagnostic odds ratio) of 92% (34/37), 93% (27/29), 94%, 90%, and 153.0, respectively. Combining GM (BAL) with PCR (BAL) showed convincing diagnostic potential for diagnosing IA with sensitivity, specificity, PPV, NPV, and DOR of 85% (17/20), 97% (28/29), 94%, 90%, and 158.7. Addition of the DNA-microarray resulted in further detection of two mucormycetes infections. In 1 out of 15 Aspergillus DNA-positive samples a triazole resistance-mediating Cyp51A mutation was found. Combination of biomarkers is superior to their sole use in diagnosing IFD, particularly IA. Integrating blood and BAL samples into a diagnostic algorithm is an advantageous approach.

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

Objectives Diagnosing invasive pulmonary aspergillosis (IPA) remains a challenge in patients with hematological malignancies. The clinical significance of testing bronchoalveolar lavage (BAL) samples both with polymerase chain reaction (PCR) and Aspergillus galactomannan ELISA (GM) is unclear, and the BAL cutoff for GM has not been clearly evaluated yet. Methods Using a validated nested PCR assay and a GM ELISA, we prospectively examined BAL samples from 87 hematological patients at high risk of IPA. Of 76 (87%) evaluable patients, 29 patients had proven or probable disease. Results The receiver operating characteristic (ROC) analysis of GM optical density (OD) cutoff levels yielded a BAL OD of 0.5 to be optimal. We identified 29 probable or proven cases based on this OD. Sensitivity and specificity for BAL GM were 0.79 (95% CI, 0.62–0.9) and 0.96 (95% CI, 0.86–0.99), respectively. For BAL PCR, sensitivity and specificity were 0.59 (95% CI, 0.41–0.75) and 0.87 (95% CI, 0.75–0.94), respectively. Combining BAL GM and PCR for diagnosis showed a sensitivity and specificity rate of 0.55 (95% CI, 0.38–0.72) and 1.0 (95% CI, 0.93–1.0), respectively, if positivity was defined by positive results for both tests. If either positive BAL GM or positive BAL PCR results defined test positivity, the sensitivity was 0.83 (95% CI, 0.65–0.92), and the specificity was 0.83 (95% CI, 0.70–0.91) Conclusions In terms of optimal sensitivity and specificity, a GM OD cutoff of 0.5 was determined for BAL samples. Positivity for both GM and Aspergillus PCR in BAL makes a pulmonary aspergillosis highly likely.

Myelodysplastic syndromes (MDS) are mainly a disease of the elderly. Commonly, MDS patients are treated in an outpatient setting making hematological/oncological private practices (PP) an important backbone in the management of MDS patients. To gain more insights into the characteristics of patients with MDS treated in hematological/oncological PP and to evaluate the daily diagnostic routines and classification systems used, we performed questionnaire-based analyses. Moreover, to investigate whether characteristics of MDS in PP differ from patients treated in specialized MDS centers in university hospitals (UH), we compared both cohorts of MDS patients. In total, 197 patients in PP and 165 patients in UH were enrolled. Patients in UH were significantly younger as compared to PP. Furthermore, in UH, a greater proportion of patients with international prognostic scoring system (IPSS) higher risk were found, whereas patients with IPSS lower risk were more frequent in PP. In addition, patients in UH had significantly lower hemoglobin levels and platelet counts compared to PP. Our data show that PP and UH are approached by different MDS patient cohorts resulting in different diagnostic workups of MDS patients.

Introduction The incidence of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is increasing, and early diagnosis of the disease and treatment with antifungal drugs is critical for patient survival. Serum biomarker tests for IPA typically give false-negative results in non-neutropenic patients, and galactomannan (GM) detection, the preferred diagnostic test for IPA using bronchoalveolar lavage (BAL), is often not readily available. Novel approaches to IPA detection in ICU patients are needed. In this multicenter study, we evaluated the performance of an Aspergillus lateral-flow device (LFD) test for BAL IPA detection in critically ill patients. Methods A total of 149 BAL samples from 133 ICU patients were included in this semiprospective study. Participating centers were the medical university hospitals of Graz, Vienna and Innsbruck in Austria and the University Hospital of Mannheim, Germany. Fungal infections were classified according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Results Two patients (four BALs) had proven IPA, fourteen patients (sixteen BALs) had probable IPA, twenty patients (twenty-one BALs) had possible IPA and ninety-seven patients (one hundred eight BALs) did not fulfill IPA criteria. Sensitivity, specificity, negative predictive value, positive predictive value and diagnostic odds ratios for diagnosing proven and probable IPA using LFD tests of BAL were 80%, 81%, 96%, 44% and 17.6, respectively. Fungal BAL culture exhibited a sensitivity of 50% and a specificity of 85%. Conclusion LFD tests of BAL showed promising results for IPA diagnosis in ICU patients. Furthermore, the LFD test can be performed easily and provides rapid results. Therefore, it may be a reliable alternative for IPA diagnosis in ICU patients if GM results are not rapidly available. Trial registration ClinicalTrials.gov NCT02058316. Registered 20 January 2014.

The prognosis of patients with CNS involvement of solid tumors is poor. In these patients, systemic chemotherapy has a theoretical advantage of concurrent treatment of systemic disease and reduced risk of neurotoxicity. Here, we report on the efficacy and toxicity of topotecan/ifosfamide (TOPO/IFO) combination chemotherapy in patients treated for CNS involvement of different solid malignancies. Fourteen patients with CNS manifestations (seven with brain metastases, two meningeal carcinomatosis, and five both) of solid tumors (seven with breast cancer, six lung cancer, and one unknown primary cancer) received TOPO/IFO treatment. Eleven patients each were pretreated with 1-6 systemic therapy regimens and whole-brain irradiation. Patients received a total of 34 (median 2) TOPO/IFO cycles. TOPO dosage was 3.6 mg/m(2) (1.2 mg/m(2), days 1-3) and IFO dosage 3,000 mg/m(2) (1,500 mg/m(2), days 1-2) per cycle. Of 12 patients with brain metastases, one patient had partial remission, three stable disease, two progressed, and six had no radiologic CNS response evaluation. Response of meningeal carcinomatosis was found in two and progressive disease in two (three patients not evaluated). Neurologic improvement or stabilization was observed in six of twelve evaluable patients. No systemic tumor response was seen in seven evaluated patients. Grade 3/4 toxicities in eleven evaluable patients were leukopenia (n = 9), infection (n = 6), and thrombopenia (n = 5). Median time to treatment failure was 43 days and median overall survival 107 days. Symptom control was frequently achieved with TOPO/IFO systemic chemotherapy despite a low objective response rate. The feasibility of this treatment is impaired by severe hematotoxicity.

Reliable detection of the JAK2 V617F mutation is a major criterion in the diagnosis of BCR/ABL-negative myeloproliferative neoplasms such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis. A multitude of methods has been applied to both qualitative and quantitative assessment of the mutational status of patients, without defining a gold standard for the daily diagnostic routine so far.We developed a melting point assay to be used on a Rotor-Gene thermal cycler machine, using asymmetric primer concentrations. A human erythroleukemia cell line (HEL) was used as a positive control in a 3-fold lower concentration than the negative control because of the gene amplification of the mutated JAK2 kinase in this cell line. Routine samples from both blood and bone marrow were processed. Additionally, samples were analyzed using an amplification refractory mutation system (ARMS).The sensitivity of the melting point approach was a 5% mutational load. Of 314 bone marrow or blood DNA samples tested, 101 were ARMS positive, and of these, 90 samples tested positive in the melting point assay. Most of the patients had a mutational load between 20% and 50%. No patient had a JAK2 V617F burden higher than 80%. There was no significant difference in the source (bone marrow versus blood), sex, and patient age.We present a reliable and feasible approach for quantitative assessment of the JAK2 V617F status from both blood and bone marrow. A homozygotic mutated cell line or plasmids should be used for dilution standards. We recommend combining this assay with ARMS PCR for result confirmation and higher overall sensitivity.

Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic cells treated with ionizing radiation or chemotherapeutic drugs. They can be quantitatively assessed by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX), where the number of γH2AX foci represents the number of DNA DSB. Real-time assessment of DSB could help tailoring cytotoxic therapies to individual patients regarding both response and adverse events. This would require reliable automated quantification technology not yet routinely available. Here we explore this concept in the context of malignant lymphoma. Methods: To investigate the DSB response to cytotoxic treatment in vitro , peripheral lymphocytes of healthy donors were incubated with bendamustine, an alkylating drug commonly used in lymphoma therapy. To mimic the clinical setting, the drug concentration per number of donor cells was either calculated as a standard dose or based on the body surface area of the individual donor. DNA DSB were quantified by an automatized immunofluorescence γH2AX assay using the AKLIDES NUK ® system. Results: Across all donors, the mean number of γH2AX foci per cell was 1.29, IQR (1.08) after bendamustine treatment as opposed to 0.04, IQR (0.125) in untreated freshly isolated peripheral blood mononuclear cells (PBMCs). The standardized incubation dosage resulted in a mean of 0.89, IQR (0.51) foci per cell, while individualized dose calculation yielded 1.57, IQR (0.5) foci per cell. The difference in γH2AX foci between the two dosage calculations was significant (P=0.036). In addition, we observed a trend towards a negative correlation between the donors’ body surface area and the number of foci per cell. Between donors, no significant correlation of the number of foci in response to a given dose was observed. Dose titrations on the cells of individual donors demonstrated a significant response (P Conclusions: The automatic AKLIDES analysis platform can assess γH2AX foci for routine use. The individual γH2AX foci response to in vitro bendamustine is dose-dependent and can be monitored timely. Calculating individual in vitro dosage from the donor’s body surface area resulted in a broad variation of foci counts between individuals, suggesting that this dosage method does not result in equivalent biological effects among different individuals. Adjusting the dose individually based on biological responses such as DNA DSB could offer a way of personalized medicine with conventional substances, reducing toxicity while increasing therapeutic efficacy.

Invasive aspergillosis (IA) is an important cause of morbidity and mortality in children and adults with haematologic malignancies or undergoing allogeneic haematopoietic stem cell transplantation, and early diagnosis and adequate antifungal treatment improve outcome. However, important differences exist between children and adults regarding epidemiology, underlying disease, and comorbidities, and the value of diagnostic tools to detect IA may also differ between these patient populations. Imaging studies are important to detect IA early, but typical findings of IA in chest computed tomography of adults are not detected in the majority of children. Whereas the value of the serum marker galactomannan seems to be comparable in children and adults, data on the performance of beta-d-glucan in children are too limited for firm conclusions. PCR-based assays are a promising diagnostic approach to rapidly and reliably detect and identify Aspergillus species in various clinical samples. However, as the majority of data on PCR-based approaches has been obtained in adult patients, the value of this method in paediatric patients has not been defined to date. The present review focuses on studies of PCR-based methods to diagnose IA in immunocompromised paediatric patients.

The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection mainly affecting immunocompromised patients. Early diagnosis is critical, but the diagnostic gold standard (histopathology and culture) is time consuming and cannot offer early confirmation of IPA. Fungal biomarkers like galactomannan (GM) are a promising extension to the diagnostic repertoire. However, it still remains under discussion if biomarker analysis from the site of the infection is superior to testing blood samples. We retrospectively evaluated the diagnostic performance of concurrent serum GM and bronchoalveolar lavage (BAL) GM (obtained within 24 h) of immunocompromised patients at high risk of IPA. Twenty-six proven/probable patients and eight patients with no IPA according to the EORTC/MSG 2008 criteria were included in this study. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were for BAL GM: 85%, 88%, 96%, 64% and 38.5, and for serum GM: 23%, 88%, 88%, 26% and 2.1 respectively. BAL GM proved to be significantly more sensitive for the detection of IPA compared to same-day serum GM in patients at high risk of IPA (P < 0.0001). Our data show that BAL GM testing is significantly superior to serum GM implying that diagnostic efforts should focus on specimens from the site of infection.

Infectious complications are a major cause of morbidity and mortality in patients with hemato-oncological diseases. Although disease-related immunosuppression represents one factor, aggressive treatment regimens, such as chemotherapy, stem cell transplantation, or antibody treatment, account for a large proportion of infectious side effects. With the advent of targeted therapies affecting specific kinases in malignant diseases, the outcome of patients has further improved. Nonetheless, dependent on the specific pathway targeted or off-target activity of the kinase inhibitor, therapy-associated infectious complications may occur. We review the most common and approved kinase inhibitors targeting a variety of hemato-oncological malignancies for their immunosuppressive potential and evaluate their risk of infectious side effects based on preclinical evidence and clinical data in order to raise awareness of the potential risks involved.

The effect of mould-active antifungal (AF) therapy/prophylaxis on the performance of the Aspergillus-specific lateral-flow device (LFD) test for diagnosing invasive pulmonary aspergillosis (IPA) was evaluated. This was a retrospective analysis of patients diagnosed with probable or proven IPA (according to revised EORTC/MSG criteria) at the Medical University of Graz (Austria) and the University Hospital of Mannheim (Germany) between February 2011 and December 2014. In total, 60 patients with 63 bronchoalveolar lavage fluid (BALF) samples were included in the analysis. Patient charts were reviewed regarding AF treatment at the time of bronchoscopy, and the influence of AFs on the performance of the LFD and BALF galactomannan (GM) ELISA results was calculated. Overall, 54 patients (57 BALF samples) had probable IPA and 6 patients (6 samples) had proven IPA. In 21/63 samples (33%) (from 19 patients), systemic mould-active AFs had been initiated before bronchoscopy. Of 63 BALF samples, 16 (25%) yielded a false-negative LFD result. The sensitivity of the LFD for probable/proven IPA was significantly lower in those receiving mould-active AFs compared with those without (52% vs. 86%; P=0.006). Similar results were found for BALF GM, with sensitivities decreasing under systemic AFs (71% vs. 95%, P=0.013 with the 0.5 ODI cut-off; 52% vs. 81%, P=0.036 with the 1.0 cut-off). These results suggest that the sensitivity of the BALF LFD and BALF GM assays may be reduced in the presence of mould-active AF treatment. Negative results in patients on AFs should therefore be interpreted with caution.

Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic criteria according to EORTC/MSG guidelines are often not met and have low sensitivity. Hence there is an urgent need to improve diagnostic procedures by developing novel approaches. In the present study, we present a proof of concept experiment for the monitoring of Aspergillus associated protease activity in serum specimens for diagnostic purpose. Synthetic peptides that are selectively cleaved by proteases secreted from Aspergillus species were selected from our own experiments and published data. These so called reporter peptides (RP, n=5) were added to serum specimens from healthy controls (HC, n=101) and patients with proven (IA, n=9) and possible (PIA, n=144) invasive aspergillosis. Spiked samples were incubated ex vivo under strictly standardized conditions. Proteolytic fragments were analyzed using MALDI-TOF mass spectrometry. Spiked specimens of IA patients had highest concentrations of RP-fragments followed by PIA and HC. The median signal intensity was 116.546 (SD, 53.063) for IA and 5.009 (SD, 8.432) for HC. A cut-off >36.910 was chosen that performed with 100% specificity and sensitivity. Patients with PIA had either values above [53% (76/144)] or below [47% (67/144)] this chosen cut-off. The detection of respective reporter peptide fragments can easily be performed by MALDI TOF mass spectrometry. In this proof of concept study we were able to demonstrate that serum specimens of patients with IA have increased proteolytic activity towards selected reporter peptides. However, the diagnostic value of functional protease profiling has to be validated in further prospective studies. It is likely that a combination of existing and new methods will be required to achieve optimal performance for diagnosis of IA in the future.

Background: We performed a prospective, randomized, open-label trial to evaluate the efficacy of low-dose liposomal amphotericin B (L-AmB) to reduce the incidence of invasive fungal infections (IFI) in patients with hematological malignancies and prolonged neutropenia (>10 days) following intensive chemotherapy. Patients and methods: In 219 neutropenic episodes (NE) of 132 patients randomization was performed. Patients received either 50 mg L-AmB every other day (arm A) or no systemic antifungal prophylaxis (arm B). Results: In the first NE of each patient the incidence of proven or probable IFI (primary end point) was five of 75 patients (6.7%) in arm A and 20 of 57 patients (35%) in arm B (P = 0.001). Invasive aspergillosis occurred less frequently in patients receiving L-AmB-prophylaxis (P = 0.0057), whereas the reduction of invasive candidiasis did not reach statistical significance (P = 0.0655). In all NE the incidence of IFI was five of 110 NE (4.6%) in arm A versus 22 of 109 NE (20.2%) in arm B (P

Background: Invasive infections caused by Aspergillus fumigatus as well as Pneumocystis pneumonia are serious and often lethal diseases in severely immunocompromised patients. Case Report: We report on a rare case of a pulmonary coinfection of Pneumocystis jirovecii and Aspergillus fumigatus in a 59-year-old female patient with a history of astrocytoma treated with high doses of corticosteroids and a subsequent CD4 lymphocyte count of 30/µl. Conclusion: A heightened clinical awareness and a stringent and accurate diagnostic work-up encompassing diagnostic tools to detect even coincident fungal infections is crucial for sufficient and early antifungal treatment in immunocompromised patients with lung infiltrates.

Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta- d -glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA.

Central nervous system (CNS) involvement is a severe complication of BCR-ABL-positive leukemia after allogenic stem cell transplantation (alloSCT) associated with fatal outcome. Although second-generation tyrosine-kinase inhibitors (TKI) such as nilotinib have shown activity in systemic BCR-ABL+disease, little data exists on their penetration and efficacy within the CNS. Four patients (3 male, 1 female; age 15–49) with meningeal relapse after alloSCT and subsequent treatment with nilotinib were identified. A total of 17 cerebrospinal fluid (csf) and serum samples were assessed for nilotinib concentration and patient outcome was recorded. Nilotinib concentrations showed a low median csf/plasma ratio of 0.53% (range 0.23–1.5%), yet pronounced clinical efficacy was observed with long-lasting responses (>1 year) in three patients. Comparison with historical data showed a trend towards superior efficacy of nilotinib versus imatinib. Despite poor csf penetration, nilotinib showed significant clinical activity in CNS relapse of BCR-ABL+leukemias. As nilotinib has a high protein-binding affinity, the low-protein concentration in csf could translate into a relatively higher amount of free and therefore active nilotinib in csf as compared to blood, possibly explaining the observed efficacy. Thus, treatment with a 2nd generation TKI warrants further investigation and should be considered in cases of CNS relapse of BCR-ABL-positive leukemia after alloSCT.

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

Although it is a severe complication in immunocompromised patients, diagnosing invasive fungal disease (IFD), especially invasive aspergillosis (IA), remains difficult. In certain clinical scenarios, examining tissue samples for identification of the infectious organism becomes important. As culture-based methods rarely yield results, the performance of an Aspergillus -specific nested PCR in fresh tissue or pleural effusion samples was evaluated. Fresh tissue ( n = 59) and effusion ( n = 47) specimens from 79 immunocompromised patients were subjected to an Aspergillus -specific PCR assay. Twenty-six patients had proven ( n = 20) or probable ( n = 6) IFD, according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, while the remaining patients were classified as having either possible IFD ( n = 30) or no IFD ( n = 23). IA was identified as the underlying IFD in 21/26 proven/probable cases. PCR positivity was observed for 18/21 proven/probable and 6 possible IA cases; cases classified as no IA did not show positive signals. Patients with proven IFD ( n = 5) with cultures positive for non- Aspergillus molds also had negative Aspergillus PCR results. Aspergillus PCR performance analysis yielded sensitivity and specificity values of 86% (95% confidence interval [CI], 65% to 95%) and 100% (95% CI, 86% to 100%), respectively, thus leading to a diagnostic odds ratio of >200. In this analysis, good diagnostic performance of the PCR assay for detection of IA was observed for tissue samples, while effusion samples showed lower sensitivity rates. PCR testing represents a complementary tool; a positive PCR result strengthens the likelihood of IA, whereas IA seems unlikely in cases with negative results but findings could indicate non- Aspergillus IFD. Thus, PCR testing of these specimens enhances the diagnostic capabilities.

Invasive infections caused by Aspergillus fumigatus as well as Pneumocystis pneumonia are serious and often lethal diseases in severely immunocompromised patients.We report on a rare case of a pulmonary coinfection of Pneumocystis jirovecii and Aspergillus fumigatus in a 59-year-old female patient with a history of astrocytoma treated with high doses of corticosteroids and a subsequent CD4 lymphocyte count of 30/µl.A heightened clinical awareness and a stringent and accurate diagnostic work-up encompassing diagnostic tools to detect even coincident fungal infections is crucial for sufficient and early antifungal treatment in immunocompromised patients with lung infiltrates.

Central nervous system (CNS) invasive aspergillosis (IA) is a fatal complication in immunocompromised patients. Confirming the diagnosis is rarely accomplished as invasive procedures are impaired by neutropenia and low platelet count. Cerebrospinal fluid (CSF) cultures or galactomannan (GM) regularly yield negative results thus suggesting the need for improving diagnostic procedures. Therefore the performance of an established Aspergillus-specific nested polymerase chain reaction assay (PCR) in CSF samples of immunocompromised patients with suspicion of CNS IA was evaluated. We identified 113 CSF samples from 55 immunocompromised patients for whom CNS aspergillosis was suspected. Of these patients 8/55 were identified as having proven/probable CNS IA while the remaining 47 patients were classified as having either possible (n = 22) or no CNS IA (n = 25). PCR positivity in CSF was observed for 8/8 proven/probable, in 4/22 possible CNS IA patients and in 2/25 NoIA patients yielding sensitivity and specificity values of 1.0 (95% CI 0.68–1) and 0.93 (95% CI 0.77–0.98) and a positive likelihood ratio of 14 and negative likelihood ratio of 0.0, respectively, thus resulting in a diagnostic odds ratio of ∞. The retrospective analysis of CSF samples from patients with suspected CNS IA yielded a high sensitivity of the nested PCR assay. PCR testing of CSF samples is recommended for patients for whom CNS IA is suspected, especially for those whose clinical condition does not allow invasive procedures as a positive PCR result makes the presence of CNS IA in that patient population highly likely.

In recent years galactomannan antigen testing (GM) and also Aspergillus PCR have become increasingly important for diagnosis of invasive aspergillosis (IA). Whether or not these tests need to be performed with bronchoalveolar lavage fluid (BALF; i.e., primary site of infection), or testing of blood samples is sufficient, remains, however, a matter of debate. We evaluated the diagnostic performance of GM ELISA, and Aspergillus PCR by using BALF samples and blood samples obtained at the same day from a total of 53 immunocompromised patients (16 with probable/proven IA and 37 with no evidence of IA according to the revised EORTC/MSG criteria; 38 patients with hematological malignancies were prospectively enrolled at the Medical University of Graz, Austria, 15 patients with mixed underlying diseases at the Mannheim University Hospital). Patients with possible IA were excluded from this analysis. A total of 34/53 (64%) of all patients and 12/16 (75%) of patients with probable/proven IA received mold-active antifungal prophylaxis/therapy at the time of the BALF procedure. Sensitivities of GM and Aspergillus PCR were 38% and 44% in BALF, and 31% and 0% in blood, respectively. Best sensitivity (75%) for detecting proven/probable IA was achieved when BALF Aspergillus PCR, BALF GM (>1.0 ODI), BALF-culture and serum-GM (>0.5 ODI) were combined (specificity 95%). In conclusion, sensitivities of the evaluated diagnostic tests-when interpreted on their own-were low in BALF and even lower in blood, sensitivities increased markedly when diagnostic tests were combined.

We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leukemia (APL). At 10 years, the overall survival (OS) was 66% in all patients and 75% in patients who achieved a complete remission (CR). Patients in the BAALC(low) group showed an OS of 87% as compared to 60% in the BAALC(high) group (p=0.019). This difference was more pronounced in treatment responders (92% vs. 70%; p=0.035). In multivariate analyses low BAALC expression retained its prognostic relevance. In conclusion, BAALC expression analysis might be useful in further risk stratification in APL patients.

OBJECTIVES Invasive aspergillosis (IA) is a life-threatening infection in severely immunocompromised patients, especially those receiving intensive chemotherapy or undergoing haematopoietic stem cell transplantation. As the clinical diagnosis of IA is mostly based on biomarkers (galactomannan, β-d-glucan, PCR assays) indicating Aspergillus as the underlying pathogen, the effect of antifungal treatment on the performance of these parameters is still controversial. We evaluated the effect of antifungal treatment on the performance of an Aspergillus-specific PCR assay in bronchoalveolar lavage (BAL) samples. PATIENTS AND METHODS Two-hundred-and-twenty-six BAL samples from 226 patients with haematological malignancies at high risk for IA classified according to the 2008 European Organization for the Research and Treatment of Cancer criteria were analysed retrospectively for the diagnostic performance of a nested Aspergillus PCR assay in relation to the number and type of mould-active antifungals received prior to BAL sampling. RESULTS Sensitivity of BAL PCR for patients without antifungal treatment prior to BAL sampling was 0.69, whereas specificity was 0.87. While no significant change in diagnostic performance by the addition of one antifungal was observed, receiving two or more antifungals prior to BAL sampling led to a significant decrease in the diagnostic performance of BAL PCR testing (P

Background Invasive pulmonary aspergillosis (IPA) is a frequent complication in hematologic patients (pts) and proper diagnosis is difficult as culture based methods are time consuming and show only modest sensitivity, especially under the current practice of early mold-active antifungal therapy (AFT) or prophylaxis. Analyzing specimens representing the specific site of infection, i.e. bronchoalveolar lavage (BAL), is supposed to be superior compared to testing blood samples. However, that has not yet been shown in direct comparison in individual patients within a 24h time window. To further elucidate this issue the diagnostic performance of an established Aspergillus specific nested Polymerase Chain Reaction (PCR) assay and of Galactomannan antigen testing (GM ELISA) in BAL was evaluated and compared to results obtained from concurrent blood samples of immunocompromised patients, most of all with hematological malignancies. Methods: We retrospectively identified 125 immunocompromised pts suspected to harbor IPA in whom BAL (n=92) and concurrent serum samples (within 24 hours) had been analyzed with Aspergillus PCR. Of these, 30/125 pts (24%) had proven/probable IPA according to 2008 EORTC/MSG criteria while the remaining 95 were classified as possible (n=65) or no IPA (n=30). Furthermore we also identified pts with same-day BAL and serum samples that had been analyzed with GM ELISA. Among those were another 26 pts that could be classified as proven/ probable IPA. Results PCR positivity in samples directly from the site of infection (BAL) was observed for 19/30 samples yielding cumulative sensitivity/specificity values of 64%/100%. PCR performance of single corresponding blood samples of these pts was significantly inferior with only 3/33 patients with proven/probable IPA resulting in a sensitivity/specificity of 8%/87% (p Conclusions In this retrospective, direct comparison of clinical specimens we observed a significantly superior performance of an Aspergillus PCR as well as GM ELISA assay directly from the site of infection compared to concurrent blood sample testing for pts with suspected IPA during AFT. Thus, PCR analyses or GM ELISA for early diagnosis of IA should be preferably directed at specimens such as BAL when IPA is suspected. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Buchheidt:Astellas: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Gilead: Consultancy, Honoraria.

Norovirus (NV) infections are a frequent cause of gastroenteritis (GE), but data on this disease in immunocompromised patients are limited. We analyzed an NV outbreak, which affected immunosuppressed patients in the context of chemotherapy or HSCT. On recognition, 7 days after admission of the index patient, preventive measures were implemented. Attack rates were only 3% (11/334) and 10% (11/105) among patients and staff members, respectively. The median duration of symptoms was 7 days in patients compared with only 3 days in staff members (P = .02). Three patients died of the NV infection. Commonly used clinical diagnostic criteria (Kaplan-criteria) were unsuitable because they applied to 11 patients with proven NV-GE but also to 15 patients without NV-GE. With respect to the therapeutic management, it is important to differentiate intestinal GVHD from NV-GE. Therefore, we analyzed the histopathologic patterns in duodenal biopsies, which were distinctive in both conditions. Stool specimens in patients remained positive for NV-RNA for a median of 30 days, but no transmission was observed beyond an asymptomatic interval of 48 hours. NV-GE is a major threat to patients with chemotherapy or HSCT, and meticulous measures are warranted to prevent transmission of NV to these patients.

In patients with parenchymal brain masses of uncertain origin responsive to corticosteroids, primary CNS lymphoma (PCNSL) should be considered. PCNSL is a rare but aggressive brain tumor that is highly sensitive to high-dose methotrexate (HDMTX)–based chemotherapy. We report a series of six patients with brain masses without histologic confirmation suspicious for PCNSL based on clinical and radiomorphologic criteria after exclusion of some infectious conditions. All patients were treated with HDMTX. We observed two complete responses, two partial responses, and one stable disease. One patient had progressive disease and received rescue whole-brain irradiation. All patients were alive without disease progression 12–48 months after HDMTX start. No symptoms of late neurotoxicity have occurred so far. The response and survival data in this small series of patients are encouraging and suggest a benefit for patients with suspected PCNSL after initial treatment with HDMTX.

WT1 is a transcription factor involved in differentiation and proliferation of acute myeloid leukemia (AML) blasts and is expressed in 90% of cases, as determined by nested reverse transcription polymerase chain reaction (RT-PCR). It is proposed to be a key molecule in leukemia promotion. To assess the relevance of WT1 expression, we analyzed blood and bone marrow samples from 58 AML patients (37 at diagnosis, 8 in hematological remission, and 13 at relapse) for the level of WT1 expression, using quantitative real-time RT-PCR. In addition, 21 randomly chosen samples were also analyzed for the quantitative expression of the main WT1 splice variants. As expected, samples from patients at the time of diagnosis or relapse showed significantly higher WT1 expression compared to samples from patients in remission or control samples. No striking difference in expression levels was found between various French-American-British (FAB) subtypes. The level of WT1 expression observed in patients at the time of initial diagnosis was similarly high in patients at relapse. Expression of the four main isoforms (E5+/KTS+, E5−/KTS+, E5+/KTS−, and E5−/KTS−) was found in all samples with significantly higher expression levels of the E5+ variants. Together, these findings support the potential of WT1 as a target for novel treatment approaches in AML.

Dear Editor, Cystic fibrosis (CF) is the most common life-limiting autosomal recessive hereditary disorder in Europe; the mean expected lifetime is approximately 35 years and has significantly increased over the last decades because of major advances in supportive treatment, especially treatment of infectious complications [1, 2]. In patients with CF, digestive tract cancers remain the majority of malignancies, whereas the risk for hematological neoplasias is low [5]. We hereby present a case of a patient with CF and newly diagnosed Hodgkin's Lymphoma (HL). Due to infectious issues and pulmonary comorbidity, the curative treatment of HL in this particular clinical setting is a challenge. A 21-year-old male patient with a history of CF was newly diagnosed with HL, Ann Arbor stage IIIb. Sputum samples repeatedly showed colonization with Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus fumigatus, and Mycobacterium abscessus that had already been present prior to the diagnosis of HL. The previous prophylactic antibiotic regimen at that time consisted of inhaled colistin (polymyxin E). To prevent infectious complications caused by A. fumigatus, Mycobacterium abscessus, gram-negative bacteria, or Pneumocystis jirovecii during chemotherapy-induced immunosuppression, the anti-infective prophylaxis was expanded to include voriconazole, clarithromycin, levofloxacinand trimethoprim/sulfamethoxazole, as well as regular prophylactic applications of G-CSF after chemotherapy to prevent neutropenia. Chemotherapy was based on a modified BEACOPPbasis regime without bleomycine due to the patient's impaired lung function. To maximize curative potential, eight cycles of “EACOPPbasis” were initially planned. However, due to recurring infectious complications of increasing length and severity (fever of unknown origin after the first course of chemotherapy preceding primarily lower respiratory tract infections with distinctly increasing pulmonary infiltrates), the cytotoxic treatment had to be delayed for a total of 42 days after only six cycles. Recurrent fever of unknown origin and documented infections (increased lung infiltrates) were treated with adequate antibiotic agents (e.g., imipenem, ceftazidime, cefepime, and aztreonam, intermittently supplemented with clindamycin or vancomycin). Microbiological sampling (blood culture and respiratory specimens) did not yield any additional pathogens. Despite intensive antimycobacterial treatment with clarithromycin, amikacin, imipenem, cefoxitin, moxifloxacin, linezolid, or tigecyclin [4], M. abscessus was repeatedly cultured from respiratory samples [3]. Chest CT scans revealed a moderate increase of preexisting lung infiltrates (Fig. 1). Restaging procedures including positron emission tomography CT showed a persisting complete remission after 17 months of follow-up care. Infectious complications, Stefan Baumann and Friederike Kuchler contributed equally to this work. S. Baumann : F. Kuchler :M. Reinwald :N. Hartel : W.-K. Hofmann :D. Buchheidt (*) Department of Haematology and Oncology, Hospital Mannheim University, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany e-mail: dieter.buchheidt@umm.de