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

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

Autopsy Study Defines Composition and Dynamics of the HIV-1 Reservoir after Allogeneic Hematopoietic Stem Cell Transplantation with CCR5Δ32/Δ32 Donor Cells.

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

Diagnosis, treatment and supportive management of chronic lymphocytic leukemia: recommendations of the Dutch HOVON CLL working group.

Management of patients with chronic lymphocytic leukemia (CLL) is changing due to considerable advances in the therapeutic armamentarium, and new therapies will possibly continue to emerge in the near future. Therefore, the CLL working group of the Dutch-Belgium Haemato-Oncology Cooperative Group for Adults in the Netherlands (HOVON) necessitated revising the Dutch CLL guidelines. The current guideline is based on the expert opinion of the HOVON CLL working group members and focusses on well-designed clinical trials taking into account efficacy with special emphasis on toxicity, treatment duration and treatment intensity. This article provides recommendations on diagnosis, treatment strategies in front-line and relapsed setting and provides supportive care measurements during novel-based therapies as well as for infectious CLL-related complications. The recommendations presented here are intended to provide guidance for the management of CLL patients in the Netherlands, and take into account the availability of treatment strategies at the time of this publication.

The Role of γδ T Cells as a Line of Defense in Viral Infections after Allogeneic Stem Cell Transplantation: Opportunities and Challenges.

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αß T cells, we describe that by delaying αß T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

HEV infection in stem cell transplant recipients-retrospective study of EBMT Infectious Diseases Working Party.

HEV infection is an emerging cause of acute and chronic hepatitis in stem cell transplant (SCT) recipients. We performed a retrospective observational study among EBMT centers with the aim of describing characteristics, management and outcome of HEV after SCT. There were 34 cases of HEV infection from 12 centers in 6 countries, diagnosed in median 4.5 months after SCT; 20 of acute and 14 of chronic infection. Non-hepatic findings possibly associated with HEV infection were present in 9 (26%). Patients with chronic infection had more characteristics associated with severely immunocompromised status. Ribavirin was provided to 16 patients (47%; 40% with acute and 57% with chronic infection), in median for 75 days. Three (19%) patients discontinued it due to side effects. HEV-RNA clearance occurred in 29 patients (85%; 85% in acute and 86% in chronic infection). HEV was considered a cause of death in 3 (9%), with 2 cases with late diagnosis. Reduction of immunosuppression in those receiving it, and ribavirin treatment in those with chronic infection were associated with shorter time to HEV-RNA clearance. Policy on HEV testing varied between the centers. In conclusion, acute and chronic HEV hepatitis should be promptly diagnosed and managed in SCT recipients.

Vaccine Responses in Adult Hematopoietic Stem Cell Transplant Recipients: A Comprehensive Review.

Consensus on timing of post-hematopoietic stem cell transplantation (HSCT) vaccination is currently lacking and is therefore assessed in this review. PubMed was searched systematically for articles concerning vaccination post-HSCT and included a basis in predefined criteria. To enable comparison, data were extracted and tables were constructed per vaccine, displaying vaccine response as either seroprotection or seroconversion for allogeneic HSCT (alloHSCT) and autologous HSCT (autoHSCT) separately. A total of 33 studies were included with 1914 patients in total: 1654 alloHSCT recipients and 260 autoHSCT recipients. In alloHSCT recipients, influenza vaccine at 7-48 months post-transplant resulted in responses of 10-97%. After 12 months post-transplant, responses were >45%. Pneumococcal vaccination 3-25 months post-transplant resulted in responses of 43-99%, with the response increasing with time. Diphtheria, tetanus, pertussis, poliomyelitis and Haemophilus influenzae type b at 6-17 months post-transplant: 26-100%. Meningococcal vaccination at 12 months post-transplant: 65%. Hepatitis B vaccine at 6-23 months post-transplant: 40-94%. Measles, mumps and rubella at 41-69 months post-transplant: 19-72%. In general, autoHSCT recipients obtained slightly higher responses compared with alloHSCT recipients. Conclusively, responses to childhood immunization vaccines post-HSCT are poor in comparison with healthy individuals. Therefore, evaluation of response might be indicated. Timing of revaccination is essential for optimal response. An individualized approach might be necessary for optimizing vaccine responses.

Predictive factors for vaccine failure to guide vaccination in allogeneic hematopoietic stem cell transplant recipients.

Vaccination after hematopoietic stem cell transplantation (HSCT) is essential to protect high-risk patients against potentially lethal infections. Though multiple studies have evaluated vaccine specific responses, no comprehensive analysis of a complete vaccination schedule post-HSCT has been performed and little is known about predictors for vaccine failure. In this context, allogeneic HSCT (alloHSCT) patients were included and vaccinated starting one year post-transplantation. Antibody responses were measured by Multiplex Immuno Assay for pneumococcal (PCV13), meningococcal C, diphtheria, pertussis, tetanus and Haemophilus influenza type b one month after the last vaccination and correlated to clinical and immunological parameters. Vaccine failure was defined as antibody response above vaccine-specific cut-off values for less than four out of six vaccines. Ninety-six patients were included of which 27.1% was found to have vaccine failure. Only 40.6% of all patients responded adequately to all six vaccines. In multivariate analysis, viral reactivation post-HSCT (OR 6.53; P = 0.03), B-cells <135 per mm3 (OR 7.24; P = 0.00) and NK-cells <170 per mm3 (OR 11.06; P = 0.00) were identified as predictors for vaccine failure for vaccination at one year post-alloHSCT. Measurement of antibody responses and an individualized approach for revaccination guided by clinical status and immune reconstitution of B-cells and NK-cells may improve vaccine responses.

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

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

Comparative in vitro stimulation with lipopolysaccharide to study TNFalpha gene expression in fresh whole blood, fresh and frozen peripheral blood mononuclear cells.

In vitro stimulation with fresh and frozen peripheral blood mononuclear cells (PBMCs) or fresh whole blood has been widely used in animal studies and clinical trials to study the immunological features of a number of human diseases. The objective of this study was to determine the difference in response to stimulation of fresh PBMCs, frozen PBMCs, and fresh whole blood by change of TNFalpha gene expression levels after 4-hour in vitro lipopolysaccharide (LPS) stimulation. Our results demonstrate that TNFalpha gene expression significantly increases in both fresh PBMCs and fresh whole blood when 1 microg/ml or 10 microg/ml LPS was used but only after stimulation with 10 microg/ml LPS in frozen PBMCs. Mean fold change of TNFalpha gene expression levels after 1 microg/ml LPS stimulation was significantly higher using fresh whole blood (51.01+/-7.91) as compared to fresh PBMCs (8.92+/-2.16) or frozen PBMCs (3.04+/-0.55) (p