Gas Chromatography-Mass Spectrometry Detection of Thymoquinone in Oil and Serum for Clinical Pharmacokinetic Studies.

Thymoquinone (TQ) is the primary component of Nigella sativa L. (NS) oil, which is renowned for its potent hepatoprotective effects attributed to its antioxidant, anti-fibrotic, anti-inflammatory, anti-carcinogenic, and both anti- and pro-apoptotic properties. The aim of this work was to establish a method of measuring TQ in serum in order to investigate the pharmacokinetics of TQ prior to a targeted therapeutic application. In the first step, a gas chromatography-mass spectrometry method for the detection and quantification of TQ in an oily matrix was established and validated according to European Medicines Agency (EMA) criteria. For the assessment of the clinical application, TQ concentrations in 19 oil preparations were determined. Second, two serum samples were spiked with TQ to determine the TQ concentration after deproteinization using toluene. Third, one healthy volunteer ingested 1 g and another one 3 g of a highly concentrated NS oil 30 and 60 min prior to blood sampling for the determination of serum TQ level. After the successful establishment and validation of the measurement method, the highest concentration of TQ (36.56 g/L) was found for a bottled NS oil product (No. 1). Since a capsule is more suitable for oral administration, the product with the third highest TQ concentration (No. 3: 24.39 g/L) was used for all further tests. In the serum samples spiked with TQ, the TQ concentration was reliably detectable in a range between 5 and 10 µg/mL. After oral intake of NS oil (No. 3), however, TQ and/or its derivatives were not detectable in human serum. This discrepancy in detecting TQ after spiking serum or following oral ingestion may be attributed to the instability of TQ in biomatrices as well as its strong protein binding properties. A pharmacokinetics study was therefore not viable. Studies on isotopically labeled TQ in an animal model are necessary to study the pharmacokinetics of TQ using alternative modalities.

Invasive aspergillosis-on-chip: A quantitative treatment study of human Aspergillus fumigatus infection.

Invasive pulmonary aspergillosis is associated with a high mortality rate and poses a direct threat to immunocompromised patients. Here, we present the invasive aspergillosis-on-chip (IAC) model to investigate Aspergillus fumigatus infection in vitro. The model allows the study of the lateral growth and the invasive behaviour of fungal hyphae from the epithelium into the endothelial cell layer in an alveolus-on-chip model. We established an algorithm-based analysis pipeline for three-dimensional confocal microscopy images to visualize and quantify fungal morphology, including hyphal growth and branching. Human macrophages in the IAC model partially inhibited the growth of the fungus, contributed to the release of proinflammatory cytokines (IL-1, IL-6, TNF) and chemokines (IL-8 and MCP-1) associated with an increased number of invasive hyphae. Similar to in vivo, the application of the fungistatic drug caspofungin limited the fungal growth and resulted in morphological changes of the hyphal tree previously described in other studies. The IAC infection model allows the identification and characterization of cellular infection targets and in vitro testing of antifungal drugs in clinically relevant concentrations. It thus represents a promising tool to broaden the understanding of pathogenicity and pathophysiology of invasive aspergillosis.

Managing hematological cancer patients during the COVID-19 pandemic: an ESMO-EHA Interdisciplinary Expert Consensus.

BACKGROUND: The COVID-19 pandemic has created enormous challenges for the clinical management of patients with hematological malignancies (HMs), raising questions about the optimal care of this patient group. METHODS: This consensus manuscript aims at discussing clinical evidence and providing expert advice on statements related to the management of HMs in the COVID-19 pandemic. For this purpose, an international consortium was established including a steering committee, which prepared six working packages addressing significant clinical questions from the COVID-19 diagnosis, treatment, and mitigation strategies to specific HMs management in the pandemic. During a virtual consensus meeting, including global experts and lead by the European Society for Medical Oncology and the European Hematology Association, statements were discussed and voted upon. When a consensus could not be reached, the panel revised statements to develop consensual clinical guidance. RESULTS AND CONCLUSION: The expert panel agreed on 33 statements, reflecting a consensus, which will guide clinical decision making for patients with hematological neoplasms during the COVID-19 pandemic.

COVID-19 in cancer patients: clinical characteristics and outcome-an analysis of the LEOSS registry.

INTRODUCTION: Since the early SARS-CoV-2 pandemic, cancer patients have been assumed to be at higher risk for severe COVID-19. Here, we present an analysis of cancer patients from the LEOSS (Lean European Open Survey on SARS-CoV-2 Infected Patients) registry to determine whether cancer patients are at higher risk. PATIENTS AND METHODS: We retrospectively analyzed a cohort of 435 cancer patients and 2636 non-cancer patients with confirmed SARS-CoV-2 infection, enrolled between March 16 and August 31, 2020. Data on socio-demographics, comorbidities, cancer-related features and infection course were collected. Age-, sex- and comorbidity-adjusted analysis was performed. Primary endpoint was COVID-19-related mortality. RESULTS: In total, 435 cancer patients were included in our analysis. Commonest age category was 76-85 years (36.5%), and 40.5% were female. Solid tumors were seen in 59% and lymphoma and leukemia in 17.5% and 11% of patients. Of these, 54% had an active malignancy, and 22% had recently received anti-cancer treatments. At detection of SARS-CoV-2, the majority (62.5%) presented with mild symptoms. Progression to severe COVID-19 was seen in 55% and ICU admission in 27.5%. COVID-19-related mortality rate was 22.5%. Male sex, advanced age, and active malignancy were associated with higher death rates. Comparing cancer and non-cancer patients, age distribution and comorbidity differed significantly, as did mortality (14% vs 22.5%, p value < 0.001). After adjustments for other risk factors, mortality was comparable. CONCLUSION: Comparing cancer and non-cancer patients, outcome of COVID-19 was comparable after adjusting for age, sex, and comorbidity. However, our results emphasize that cancer patients as a group are at higher risk due to advanced age and pre-existing conditions.

[The German keratomycosis registry : Initial results of a multicenter survey]. / Das Deutsche Pilz-Keratitis-Register : Erste Ergebnisse einer multizentrischen Erhebung.

BACKGROUND AND PURPOSE: Mycotic keratitis is a serious but relatively rare disease. No targeted data collection in Germany existed until the foundation of the German Pilz-Keratitis Register in 2015. PATIENTS AND METHODS: The inclusion of retrospective and prospective patients was carried out. INCLUSION CRITERIA: diagnosis confirmed by the polymerase chain reaction (PCR), culture, histology or confocal microscopy (IVCM). Collected parameters: date of symptom onset, date and method of diagnosis, risk factors, visual acuity and findings at admission and at follow-up, conservative and surgical treatment. RESULTS: By January 2018, a total of 102 eyes from the years 2000-2017 were reported from 16 centers (64.3% female, mean age 52 years, range 18-95 years). The initial diagnosis was made correctly in only 20.6% of cases. The mean time to correct diagnosis was 31.7 ±â€¯46.9 (0-296) days. The diagnosis was confirmed in cultures in 74.5%, histologically in 30.4%, by PCR in 38.2% and IVCM in 27.4%. Fungal species identified were: 36.7% Fusarium spp., 35.8% Candida spp., 6.4% Aspergillus spp. and 21.1% other. The most important risk factor was the use of contact lenses. The most commonly used antifungal agent was voriconazole (64.7%) followed by amphotericin B (37.2%). Penetrating keratoplasty was performed in 65.7% of the cases and 8.8% of the affected eyes had to be enucleated. The visual acuity of the entire study population increased from the initial 0.16 ±â€¯0.25 (0.001-1.0) decimal to 0.28 ±â€¯0.34 (0-1.0) decimal. CONCLUSION: The correct diagnosis of fungal keratitis is often significantly delayed. The treatment can be very difficult and keratoplasty is often necessary. In order to gain a better understanding of this disease, to recognize previously unknown risk factors and, if necessary, a change in the spectrum of pathogens and to identify approaches to treatment optimization, the fungal keratitis registry will be continued.

Management of sepsis in neutropenic cancer patients: 2018 guidelines from the Infectious Diseases Working Party (AGIHO) and Intensive Care Working Party (iCHOP) of the German Society of Hematology and Medical Oncology (DGHO).

Sepsis and septic shock are major causes of mortality during chemotherapy-induced neutropenia for malignancies requiring urgent treatment. Thus, awareness of the presenting characteristics and prompt management is most important. Improved management of sepsis during neutropenia may reduce the mortality of cancer therapies. However, optimal management may differ between neutropenic and non-neutropenic patients. The aim of the current guideline is to give evidence-based recommendations for hematologists, oncologists, and intensive care physicians on how to manage adult patients with neutropenia and sepsis.

Anti-infective vaccination strategies in patients with hematologic malignancies or solid tumors-Guideline of the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Medical Oncology (DGHO).

Infectious complications are a significant cause of morbidity and mortality in patients with malignancies specifically when receiving anticancer treatments. Prevention of infection through vaccines is an important aspect of clinical care of cancer patients. Immunocompromising effects of the underlying disease as well as of antineoplastic therapies need to be considered when devising vaccination strategies. This guideline provides clinical recommendations on vaccine use in cancer patients including autologous stem cell transplant recipients, while allogeneic stem cell transplantation is subject of a separate guideline. The document was prepared by the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Medical Oncology (DGHO) by reviewing currently available data and applying evidence-based medicine criteria.

[Pneumonia in immunocompromised patients]. / Pneumonien bei immunsupprimierten Patienten.

Pneumonia occurs frequently in immunocompromised patients and often shows a complicated course of disease when compared to immunocompetent persons. The type of pathogen involved is directly associated with the type of immunosuppression and includes a wide variety of pathogens. Congenital and primary immunodeficiencies often appear during childhood. Acquired immunodeficiencies are most commonly caused by immunosuppressive medication. The concept of immunosuppression can be extended to patients with COPD or elderly patients because the variety of pathogens and specific features regarding frequency and course of the disease are similar to immunosuppressed patients. Computed tomography can provide an indication of the pathogen and is superior to the chest x­ray in this respect. Blood cultures, antigen and PCR tests are non-invasive diagnostic tools for pathogen diagnostics. Invasive tests include fiberoptic bronchoscopy and complete the diagnostic methods of identifying the causative pathogen.

Diagnosis and empirical treatment of fever of unknown origin (FUO) in adult neutropenic patients: guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO).

Fever may be the only clinical symptom at the onset of infection in neutropenic cancer patients undergoing myelosuppressive chemotherapy. A prompt and evidence-based diagnostic and therapeutic approach is mandatory. A systematic search of current literature was conducted, including only full papers and excluding allogeneic hematopoietic stem cell transplant recipients. Recommendations for diagnosis and therapy were developed by an expert panel and approved after plenary discussion by the AGIHO. Randomized clinical trials were mainly available for therapeutic decisions, and new diagnostic procedures have been introduced into clinical practice in the past decade. Stratification into a high-risk versus low-risk patient population is recommended. In high-risk patients, initial empirical antimicrobial therapy should be active against pathogens most commonly involved in microbiologically documented and most threatening infections, including Pseudomonas aeruginosa, but excluding coagulase-negative staphylococci. In patients whose expected duration of neutropenia is more than 7 days and who do not respond to first-line antibacterial treatment, specifically in the absence of mold-active antifungal prophylaxis, further therapy should be directed also against fungi, in particular Aspergillus species. With regard to antimicrobial stewardship, treatment duration after defervescence in persistently neutropenic patients must be critically reconsidered and the choice of anti-infective agents adjusted to local epidemiology. This guideline updates recommendations for diagnosis and empirical therapy of fever of unknown origin in adult neutropenic cancer patients in light of the challenges of antimicrobial stewardship.

Influenza virus infections in patients with malignancies -- characteristics and outcome of the season 2014/15. A survey conducted by the Infectious Diseases Working Party (AGIHO) of the German Society of Haematology and Medical Oncology (DGHO).

Influenza virus infections (IVI) may pose a vital threat to immunocompromised patients such as those suffering from malignancies, but specific data on epidemiology and outcome in these patients are scarce. In this study, we collected data on patients with active cancer or with a history of cancer, presenting with documented IVI in eight centres in Germany. Two hundred and three patients were identified, suffering from haematological malignancies or solid tumours; 109 (54 %) patients had active malignant disease. Influenza A was detected in 155 (77 %) and Influenza B in 46 (23 %) of patients (genera not determined in two patients). Clinical symptoms were consistent with upper respiratory tract infection in 55/203 (27 %), influenza-like illness in 82/203 (40 %), and pneumonia in 67/203 (33 %). Anti-viral treatment with oseltamivir was received by 116/195 (59 %). Superinfections occurred in 37/203 (18 %), and admission on an intensive care unit was required in 26/203 (13 %). Seventeen patients (9 %) died. Independent risk factors for death were delayed diagnosis of IVI and bacterial or fungal superinfection, but not underlying malignancy or ongoing immunosuppression. In conclusion, patients with IVI show high rates of pneumonia and mortality. Early and rapid diagnosis is essential. The high rate of pneumonia and superinfections should be taken into account when managing IVI in these patients.

Outcome of empirical or targeted antifungal therapy after antifungal prophylaxis in febrile neutropenia.

Azole prophylaxis has been shown to be effective in preventing invasive fungal infections (IFIs) and increasing survival in patients with prolonged neutropenia after myelosuppressive chemotherapy for haematological malignancies. Similarly, empirical antifungal therapy for persistent neutropenic fever has been shown to reduce IFI-related mortality. However, to date, there is little information with regard to the outcome of patients who receive both strategies. Here, we present our retrospective data on three cohorts of patients receiving empirical or targeted antifungal therapy after different antifungal prophylaxis regimens. All records from patients who received myelosuppressive induction chemotherapy for acute myelogenous leukemia (AML) in our centre from 2004-2010 were analysed. From 2004-2006, itraconazole was used as antifungal prophylaxis; for the first 6 months in 2007, local polyenes and from mid-2007 till 2010, posaconazole. Data of 315 courses of chemotherapy in 211 patients were analysed. Antifungal therapy (empirical or targeted, time point and antifungal agent at the physician's discretion) was initiated in 50/174 (29 %), 7/18 (39 %) and 34/123 courses (28 %, p = 0.615) in the itra cohort, the cohort without systemic prophylaxis and the posa cohort, respectively, and was effective in 24/50 (48 %), 5/7 (71 %) and 22/34 courses (65 %, p = 0.221), respectively. IFI occurred in 25/174 (14 %), 4/18 (22 %) and 16/123 (13 %) courses, respectively (p = 0.580). IFI-related survival was not different in the three cohorts. Antifungal treatment in patients with AML who received azole prophylaxis resulted in the expected efficacy-importantly, prior posaconazole prophylaxis did not render subsequent antifungal treatment less effective than prior itraconazole prophylaxis.

Polymorphisms of Toll-like receptors (TLR2 and TLR4) are associated with the risk of infectious complications in acute myeloid leukemia.

Infectious complications continue to be one of the major causes of morbidity and mortality in patients with acute myeloid leukemia (AML). Several single-nucleotide polymorphisms (SNPs) of Toll-like receptors (TLRs) can affect the genetic susceptibility to infections or even sepsis. We sought to investigate the impact of different SNPs on the incidence of developing sepsis and pneumonia in patients with newly diagnosed AML following induction chemotherapy. We analyzed three SNPs in the TLR2 (Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) gene in a cohort of 155 patients with AML who received induction chemotherapy. The risk of developing sepsis and pneumonia was assessed by multiple logistic regression analyses. The presence of the TLR2 Arg753Gln polymorphism was significantly associated with pneumonia in AML patients (odds ratio (OR): 10.78; 95% confidence interval (CI): 2.0-58.23; P=0.006). Furthermore, the cosegregating TLR4 polymorphisms Asp299Gly and Thr399Ile were independent risk factors for the development of both sepsis and pneumonia (OR: 3.55; 95% CI: 1.21-10.4, P=0.021 and OR: 3.57, 95% CI: 1.3-9.86, P=0.014, respectively). To our best knowledge, this study represents the first analysis demonstrating that polymorphisms of TLR2 and TLR4 influence the risk of infectious complications in patients with AML undergoing induction chemotherapy.

Treatment with lenalidomide induces immunoactivating and counter-regulatory immunosuppressive changes in myeloma patients.

Lenalidomide activates the immune system, but the exact immunomodulatory mechanisms of lenalidomide in vivo are poorly defined. In an observational study we assessed the impact of lenalidomide on different populations of immune cells in multiple myeloma patients. Lenalidomide therapy was associated with increased amounts of a CD8(+) T cell subset, phenotypically staged between classical central memory T cells (TCM) and effector memory T cells (TEM), consequently termed TCM/TEM. The moderate expression of perforin/granzyme and phenotypical profile of these cells identifies them as not yet terminally differentiated, which makes them promising candidates for the anti-tumour response. In addition, lenalidomide-treated patients showed higher abundance of CD14(+) myeloid cells co-expressing CD15. This population was able to inhibit both CD4(+) and CD8(+) T cell proliferation in vitro and could thus be defined as a so far undescribed novel myeloid-derived suppressor cell (MDSC) subtype. We observed a striking correlation between levels of TCM/TEM, mature regulatory T cells (T(regs)) and CD14(+) CD15(+) MDSCs. In summary, lenalidomide induces both activating and inhibitory components of the immune system, indicating the existence of potential counter-regulatory mechanisms. These findings provide new insights into the immunomodulatory action of lenalidomide.

Tigecycline in febrile neutropenic patients with haematological malignancies: a retrospective case documentation in four university hospitals.

OBJECTIVES: Tigecycline (TGC) is a first-in-class glycylcycline with an expanded spectrum of activity. Although TGC has not been prospectively studied in febrile neutropenia (FN), we observed that occasionally critically ill neutropenic patients unresponsive to other antibiotics were treated with TGC in our departments. The aim of our study was to analyse effectiveness and toxicity of TGC in FN. METHODS: Data of infectious episodes treated with TGC were retrospectively collected. Baseline data of patients, haematological malignancy, infection and adverse events were documented. Success was defined as defervescence (≥7 days) in the absence of any sign of persistent infection. RESULTS: Data of 35 patients with haematological malignancies and FN were evaluated. Median duration of neutropenia was 25 days (range 6-69 days). The type of infection was pneumonia in 24 patients, four microbiologically documented infections, three clinically documented infections and four with fever of unknown origin. The TGC was administered after a median of two (range 1-5) prior antibiotic regimens. Treatment was successful in 15 (43 %) patients. In patients with prolonged neutropenia (≥28 days), response was significantly lower (13 vs. 79 %; p =0.001). Eight (23 %) patients died during the fever episode. Grade 3-4 toxicity occurred in five (14 %) patients. CONCLUSION: Our results showed promising response rates to TGC and very low toxicity rates compared to the generally low response rate of third-line antibiotic therapies, indicating that TGC may be a successful alternative for salvage treatment of febrile neutropenia, but further study is needed.

Primary prophylaxis of bacterial infections and Pneumocystis jirovecii pneumonia in patients with hematological malignancies and solid tumors : guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO).

Bacterial infections are the most common cause for treatment-related mortality in patients with neutropenia after chemotherapy. Here, we discuss the use of antibacterial prophylaxis against bacteria and Pneumocystis pneumonia (PCP) in neutropenic cancer patients and offer guidance towards the choice of drug. A literature search was performed to screen all articles published between September 2000 and January 2012 on antibiotic prophylaxis in neutropenic cancer patients. The authors assembled original reports and meta-analysis from the literature and drew conclusions, which were discussed and approved in a consensus conference of the Infectious Disease Working Party of the German Society of Hematology and Oncology (AGIHO). Antibacterial prophylaxis has led to a reduction of febrile events and infections. A significant reduction of overall mortality could only be shown in a meta-analysis. Fluoroquinolones are preferred for antibacterial and trimethoprim-sulfamethoxazole for PCP prophylaxis. Due to serious concerns about an increase of resistant pathogens, only patients at high risk of severe infections should be considered for antibiotic prophylaxis. Risk factors of individual patients and local resistance patterns must be taken into account. Risk factors, choice of drug for antibacterial and PCP prophylaxis and concerns regarding the use of prophylactic antibiotics are discussed in the review.

Diagnosis of chronic disseminated candidosis from liver biopsies by a novel PCR in patients with haematological malignancies.

Hepatic Candida infection (HCI; known as chronic disseminated candidosis or CDC) is a distinct form of disseminated Candida infection with predominant involvement of the liver. Diagnosis of HCI is usually made on clinical suspicion together with multiple lesions in liver on ultrasound (US), CT and/or MRI scan. Fungal elements may not always be visible in liver tissue and mycological culture is frequently negative, making the evidence for proven fungal disease difficult. We studied a novel commercially available low-cost and density-array (LCD) chip technique for a molecular diagnosis of HCI. This is a two-step procedure with PCR amplification after DNA extraction followed by hybridization on a small chip provided by the manufacturer (Fungi 2.1, Chipron GmbH). The analysis of DNA from 45 fungal control strains showed an excellent specificity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignancies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, supporting the clinical diagnosis of HCI/CDC. Analysis of liver biopsies from controls was negative for fungal DNA in all samples studied. In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in liver biopsies from patients with haematological malignancies and suspected HCI/CDC but was negative in control biopsies.

Plerixafor with and without chemotherapy in poor mobilizers: results from the German compassionate use program.

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 µg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/µL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

Management of sepsis in neutropenic patients: guidelines from the infectious diseases working party of the German Society of Hematology and Oncology.

Sepsis is a leading cause of mortality in neutropenic cancer patients. Early initiation of effective causative therapy as well as intensive adjunctive therapy is mandatory to improve outcome. We give recommendations for the management of adults with neutropenia and sepsis. The guidelines are written for clinicians involved in care of cancer patients and focus on pathophysiology, diagnosis and treatment of sepsis during neutropenia.

False positivity of the Aspergillus galactomannan Platelia ELISA because of piperacillin/tazobactam treatment: does it represent a clinical problem?

OBJECTIVES: False-positive results of the galactomannan (GM) ELISA caused by concurrent administration of piperacillin/tazobactam have been reported in patients with febrile neutropenia. PATIENTS AND METHODS: This prospective study investigated different sampling times in 30 patients receiving piperacillin/tazobactam for febrile neutropenia. RESULTS: Prior to the first piperacillin/tazobactam infusion, a median GM index of 0.2 [interquartile range (IQR) 0.1-0.3] was noted; in two patients (7%) the index was 0.5. Immediately after piperacillin/tazobactam infusion, the median index increased to 0.3 (IQR 0.2-0.4, P = 0.002) leading to 21% (7/30) false-positive results, if > or = 0.5 is assumed as the cut-off level. GM indices before the next piperacillin/tazobactam infusion were not increased (median 0.2, IQR 0.2-0.35, P > 0.05), but 10% (3/30) were still > or = 0.5. With a cut-off level of > 0.7, no false-positive results were noted at any sampling time point. CONCLUSIONS: We conclude that the clinical relevance of false-positive GM results during piperacillin/tazobactam treatment is small if samples are collected prior to infusion and if a cut-off level of > 0.7 is used.