Clonal Relapse Dynamics in Acute Myeloid Leukemia Following Allogeneic Hematopoietic Cell Transplantation.

Patients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by utilizing error-corrected next-generation sequencing (NGS) in 74 AML patients relapsing after alloHCT evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least one MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 months prior to relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, while 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, while mutations in signaling genes demonstrated a shorter lead-time to relapse. Both DTA and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.

MRD as Biomarker for Response to Donor Lymphocyte Infusion after Allogeneic Hematopoietic Cell Transplantation in Patients with AML.

Donor lymphocyte infusions (DLIs) can directly target leukemic cells through a graft-versus-leukemia effect and play a key role in the prevention and management of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Predictors of response to DLIs are not well established. We evaluated measurable residual disease (MRD) before, 30 and 90 days after DLI treatment as biomarkers of response. MRD was assessed by next-generation sequencing in 76 DLI-treated acute myeloid leukemia patients. MRD status before DLI treatment was independently prognostic for event-free survival (EFS, p < 0.001) and overall survival (OS, p < 0.001). Within 90 days of DLI treatment, 73% of MRD+ patients converted to MRD- and 32% of patients without remission achieved remission. MRD status 90 days after DLI treatment was independently prognostic for the cumulative incidence of relapse (CIR, p = 0.011) and relapse-free survival (RFS, p = 0.001), but not for OS. To evaluate the role of DLI treatment in MRD- patients, 23 MRD- patients who received DLIs were compared with a control cohort of 68 MRD- patients not receiving DLIs. RFS (p = 0.23) and OS (p = 0.48) were similar between the two cohorts. In conclusion, MRD is prognostic before (EFS, OS) and after (CIR, RFS) DLI treatment and may help in the selection of patients who benefit most from DLIs.

Multimodal Treatment of Nasopharyngeal Carcinoma in Children, Adolescents and Young Adults-Extended Follow-Up of the NPC-2003-GPOH Study Cohort and Patients of the Interim Cohort.

Nasopharyngeal carcinoma (NPC) in children and young adults has been treated within two consecutive prospective trials in Germany, the NPC-91 and the NPC-2003 study of the German Society of Pediatric Oncology and Hematology (GPOH). In these studies, multimodal treatment with induction chemotherapy, followed by radio (chemo)therapy and interferon-beta maintenance, yielded promising survival rates even after adapting total radiation doses to tumor response. The outcome of 45 patients in the NPC-2003 study was reassessed after a median follow-up of 85 months. In addition, we analyzed 21 further patients after closure of the NPC-2003 study, recruited between 2011 and 2017, and treated as per the NPC-2003 study protocol. The EFS and OS of 66 patients with locoregionally advanced NPC were 93.6% and 96.7%, respectively, after a median follow-up of 73 months. Seven patients with CR after induction therapy received a reduced radiation dose of 54 Gy; none relapsed. In young patients with advanced locoregional NPC, excellent long-term survival rates can be achieved by multimodal treatment, including interferon-beta. Radiation doses may be reduced in patients with complete remission after induction chemotherapy and may limit radiogenic late effects.

Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT. MRD was detected in 34 patients on day 90 and/or day 180 (25%). The rate of MRD positivity was similar when DTA and non-DTA mutations were considered separately (17.6% vs 19.8%). DTA mutations had no prognostic impact on cumulative incidence of relapse, relapse-free survival, or overall survival in our study and were removed from further analysis. In the remaining 131 patients with at least 1 non-DTA mutation, clinical and transplantation-associated characteristics were similarly distributed between MRD-positive and MRD-negative patients. In multivariate analysis, MRD positivity was an independent adverse predictor of cumulative incidence of relapse, relapse-free survival, and overall survival but not of nonrelapse mortality. The prognostic effect was independent of different cutoffs (above limit of detection, 0.1% and 1% variant allele frequency). MRD log-reduction between diagnosis and post-alloHCT assessment had no prognostic value. MRD status post-alloHCT had the strongest impact in patients who were MRD positive prior to alloHCT. In conclusion, non-DTA mutations are prognostic NGS-MRD markers post-alloHCT, whereas the prognostic role of DTA mutations in the posttransplant setting remains open.

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

Postinfectious Onset of Myasthenia Gravis in a COVID-19 Patient.

Objective: We report the case of a young woman with postinfectious onset of myasthenia gravis after COVID-19 with mild respiratory symptoms and anosmia/ageusia 1 month before admission to our neurological department. Methods: Patient data were derived from medical records of Hannover Medical School, Germany. Written informed consent was obtained from the patient. Results: The 21-year-old female patient presented with subacute, vertically shifted double vision evoked by right sided partial oculomotor paresis and ptosis. About 4 weeks earlier she had suffered from mild respiratory symptoms, aching limbs and head without fever, accompanied by anosmia/ageusia. During the persistence of the latter symptoms for around 10 days the patient had already noticed "tired eyes" and fluctuating double vision. Clinical assessment including a positive test with edrophonium chloride and increased acetylcholine receptor antibodies related the ocular manifestation etiologically to myasthenia gravis. Antibodies (IgA/IgG) against SARS-CoV-2 using three different serological tests (Abbott, DiaSorin, Euroimmun) were detected in serum suggesting this specific coronavirus as previously infectious agent in our patient. The myasthenic syndrome was treated successfully with intravenous immunoglobulins and oral pyridostigmine. Conclusion: This is the first case presentation of postinfectious myasthenia gravis as neurological complication in a COVID-19 patient.

Seropositivity for pathogens associated with chronic infections is a risk factor for all-cause mortality in the elderly: findings from the Memory and Morbidity in Augsburg Elderly (MEMO) Study.

Immunostimulation by chronic infection has been linked to an increased risk for different non-communicable diseases, which in turn are leading causes of death in high- and middle-income countries. Thus, we investigated if a positive serostatus for pathogens responsible for common chronic infections is individually or synergistically related to reduced overall survival in community dwelling elderly. We used data of 365 individuals from the German MEMO (Memory and Morbidity in Augsburg Elderly) cohort study with a median age of 73 years at baseline and a median follow-up of 14 years. We examined the effect of a positive serostatus at baseline for selected pathogens associated with chronic infections (Helicobacter pylori, Borrelia burgdorferi sensu lato, Toxoplasma gondii, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2, and human herpesvirus 6) on all-cause mortality with multivariable parametric survival models. We found a reduced survival time in individuals with a positive serostatus for Helicobacter pylori (accelerated failure time (AFT) - 15.92, 95% CI - 29.96; - 1.88), cytomegalovirus (AFT - 22.81, 95% CI - 36.41; - 9.22) and Borrelia burgdorferi sensu lato (AFT - 25.25, 95% CI - 43.40; - 7.10), after adjusting for potential confounders. The number of infectious agents an individual was seropositive for had a linear effect on all-cause mortality (AFT per additional infection - 12.42 95% CI - 18.55; - 6.30). Our results suggest an effect of seropositivity for Helicobacter pylori, cytomegalovirus, and Borrelia burgdorferi sensu lato on all-cause mortality in older community dwelling individuals. Further research with larger cohorts and additional biomarkers is required, to assess mediators and molecular pathways of this effect.

Leptomeningeal Metastasis: The Role of Cerebrospinal Fluid Diagnostics.

Background: Metastatic spread into the cerebrospinal fluid (CSF) represents a severe complication of malignant disease with poor prognosis. Although early diagnosis is crucial, broad spectrums of clinical manifestations, and pitfalls of magnetic resonance imaging (MRI) and CSF diagnostics can be challenging. Data are limited how CSF parameters and MRI findings relate to each other in patients with leptomeningeal metastasis. Methods: Patients with malignant cells in CSF cytology examination diagnosed between 1998 and 2016 at the Department of Neurology in the Hannover Medical School were included in this study. Clinical records, MRI findings and CSF parameters were retrospectively analyzed. Results: One hundred thirteen patients with leptomeningeal metastasis were identified. Seventy-six patients (67%) suffered from a solid malignancy while a hematological malignancy was found in 37 patients (33%). Cerebral signs and symptoms were most frequently found (78% in solid vs. 49% in hematological malignancies) followed by cranial nerve impairment (26% in solid vs. 46% in hematological malignancies) and spinal symptoms (26% in solid vs. 27% in hematological malignancies). In patients with malignant cells in CSF MRI detected signs of leptomeningeal metastasis in 62% of patients with solid and in only 33% of patients with hematological malignancies. Investigations of standard CSF parameters revealed a normal CSF cell count in 21% of patients with solid malignancies and in 8% of patients with hematological malignancies. Blood-CSF-barrier dysfunction was found in most patients (80% in solid vs. 92% in hematological malignancies). Elevated CSF lactate levels occurred in 68% of patients in solid and in 48% of patients with hematological malignancies. A high number of patients (30% in solid vs. 26% in hematological malignancies) exhibited oligoclonal bands in CSF. Significant correlations between the presence of leptomeningeal enhancement demonstrated by MRI and CSF parameters (cell count, lactate levels, and CSF/Serum albumin quotient) were not found in both malignancy groups. Conclusion: CSF examination is helpful to detect leptomeningeal metastasis since the diagnosis can be challenging especially when MRI is negative. CSF cytological investigation is mandatory whenever leptomeningeal metastasis is suspected, even when CSF cell count is normal.

The Influence of Blood Contamination on Cerebrospinal Fluid Diagnostics.

Background: Blood contamination due to traumatic lumbar puncture presents a diagnostic pitfall in cerebrospinal fluid (CSF) analysis. It is controversially discussed if phagocytosis of erythrocytes which can be found in the CSF after subarachnoid hemorrhage can also develop in vitro in the presence of artificial blood contamination. Furthermore, there is no consensus about the acceptable amount of artificial blood contamination on CSF protein results. Methods: Two measurement series were performed in order to investigate the role of artificial blood contamination on the possible development of erythrophages and siderophages in the CSF: (1) blood contamination was simulated in vitro by adding blood into the CSF. (2) CSF was investigated when blood contamination occurred during a traumatic lumbar puncture. In both types of experiments, CSF including blood was incubated for 24 h and for 72 h at room temperature or at 4°C. In the third measurement series, the effects of artificial blood contamination on CSF protein results were investigated. Blood contamination was simulated in vitro by adding different amounts of blood ending up with five different samples containing erythrocyte counts of 2,500, 5,000, 7,500, 10,000, and 20,000 per µl CSF. Results: Cytological examination revealed no evidence of erythrophages or siderophages in vitro. In contrast, already a low blood contamination (2,500 erythrocytes/µl CSF) led to false pathological results of total protein and albumin. Along with increasing amounts of blood, the frequency of false pathological protein results increased. A blood contamination of 5,000 erythrocytes/µl CSF resulted in a false positive intrathecal IgM production in nearly every fifth patient. In contrast, blood contamination with 5,000 erythrocytes/µl CSF was the acceptable amount of blood which did not lead to a false positive intrathecal synthesis of IgG and IgA. Conclusion: Erythrophages and siderophages do not develop in vitro. An extensive diagnostic work up for the source of blood in the CSF should be performed when erythrophages or siderophages are found in the CSF. The contamination of CSF with increasing volume of blood resulted in falsely elevated CSF protein concentrations. Hence, the amount of blood contamination has to be taken into consideration when interpreting CSF protein measurement results.

Prolonged detection of herpes simplex virus type 2 (HSV-2) DNA in cerebrospinal fluid despite antiviral therapy in a patient with HSV-2-associated radiculitis.

Herpes simplex virus type 2 (HSV-2) can cause radiculo-myelitis as a neurological manifestation. We report a case of ongoing HSV-2 DNA positivity in the cerebrospinal fluid (CSF) of at least eight weeks under antiviral therapy with acyclovir in a highly immunocompromised hemato-oncologic patient with HSV-2-associated radiculitis. Upon admission, the patient presented with pain, leg paresis, and urinary incontinence, as well as pleocytosis in the CSF. Quantitative real-time PCR of the CSF at day 3 after admission revealed HSV-2 with a concentration of 2.0×10(5) copies/ml and treatment with acyclovir intravenously and prednisolone by mouth was started. Clinical symptoms resolved almost completely after approximately 3 weeks of antiviral therapy. However, CSF samples of day 12, 19, 26, 33, 39, 48 and 54 after admission showed a slow decline of HSV-2 DNA concentrations. HSV-2 DNA was still detectable (1.6×10(4) copies/ml) at day 54 after admission. Genotypic resistance testing showed, as far as available, no mutations indicative for acyclovir resistance. Since an increasing specific antibody index for HSV was observed, we speculate that the prolonged detectability of HSV-2 DNA in the CSF might not necessarily indicate ongoing viral replication but neutralized virus. Other hypotheses and the consequences on treatment are discussed. To our knowledge this is the first report about the long-term viral load kinetics of HSV-2 in the CSF of a patient with radiculitis under antiviral therapy, highlighting the need for further studies on HSV DNA kinetics in the CSF and their significance for an appropriate antiviral treatment.

Comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of RT-PCR for the detection of novel 2009 influenza A (H1N1) virus in respiratory specimens.

Although infections with the novel pandemic 2009 influenza A (H1N1) virus (A/H1N1/2009) appeared to be relatively mild during the first summer of circulation ('off season'), there has been significant morbidity and hospitalization and several fatal cases. Thus, rapid detection of A/H1N1/2009 is crucial for efficient treatment and infection control measures. In contrast to seasonal influenza, where point-of-care (POC) rapid antigen tests and direct fluorescent antibody (DFA) staining ensure rapid detection, diagnosis of A/H1N1/2009 has so far been based on RT-PCR. This study retrospectively compared the performance of the Quidel QuickVue POC test, DFA staining and virus isolation with that of RT-PCR for A/H1N1/2009 detection in 526 respiratory specimens collected during the first wave of the outbreak from May to September 2009. A/H1N1/2009 was detected in 9.1% (48/526) of samples. One hundred and thirty-seven of the A/H1N1/2009 PCR-negative samples were additionally tested using a RealAccurate Respiratory RT-PCR panel, revealing other respiratory viruses (mainly entero/rhino- and adenoviruses) in 42.3% (58/137). All methods analysed detected A/H1N1/2009 with excellent specificity but different sensitivities (POC test: 18.2%; DFA staining: 38.7%; virus isolation: 45.7%). Therefore, the POC test was not suitable for diagnosis, detecting A/H1N1/2009 only if present in high concentrations (corresponding median Ct value=19.0; range=16.5-21.4). DFA staining was also able to detect A/H1N1/2009 in specimens with a lower virus concentration (median Ct value=24.0; range=16.5-29.8). Virus isolation, which was positive after a median time of 7.5 days, was too time-consuming. In summary, DFA staining is superior to POC testing and may be appropriate for patients expected to have a rather high level of virus replication. Nevertheless, in DFA-negative specimens, A/H1N1/2009 should be excluded by RT-PCR.

Are meteorological parameters associated with acute respiratory tract infections?

BACKGROUND: Information on the onset of epidemics of acute respiratory tract infections (ARIs) is useful in timing preventive strategies (eg, the passive immunization of high-risk infants against respiratory syncytial virus [RSV]). Aiming at better predictions of the seasonal activity of ARI pathogens, we investigated the influence of climate on hospitalizations for ARIs. METHODS: Samples obtained from 3044 children hospitalized with ARIs in Mainz, Germany, were tested for pathogens with a multiplex reverse-transcriptase polymerase chain reaction enzyme-linked immunosorbent assay from 2001 through 2006. Hospitalizations for ARIs were correlated with meteorological parameters recorded at the University of Mainz. The frequency of hospitalization for RSV infection was predicted on the basis of multiple time series analysis. RESULTS: Influenza A, RSV, and adenovirus were correlated with temperature and rhinovirus to relative humidity. In a time series model that included seasonal and climatic conditions, RSV-associated hospitalizations were predictable. CONCLUSIONS: Seasonality of certain ARI pathogens can be explained by meteorological influences. The model presented herein is a first step toward predicting annual RSV epidemics using weather forecast data.

No temporal association between influenza outbreaks and invasive pneumococcal infections.

OBJECTIVE: To assess whether the influenza peak in populations precedes the annual peak for invasive pneumococcal infections (IPI) in winter. DESIGN: Ecological study. Active surveillance data on influenza A and IPI in children up to 16 years of age collected from 1997 to 2003 were analysed. SETTING: Paediatric hospitals in Germany. PATIENTS: Children under 16 years of age. RESULTS: In all years under study, the influenza A season did not appear to affect the IPI season (p = 0.49). Specifically, the influenza peak never preceded the IPI peak. CONCLUSION: On a population level there was no indication that the annual influenza epidemic triggered the winter increase in the IPI rate or the peak of the IPI distribution in children.

Diagnostic performance of a rapid antigen test for RSV in comparison with a 19-valent multiplex RT-PCR ELISA in children with acute respiratory tract infections.

A rapid and reliable diagnosis of respiratory syncytial virus (RSV) infection in childhood is very important for clinical management. In this study we compared a rapid antigen test (enzyme immunoassay, EIA) for the detection of RSV with a reverse transcriptase polymerase chain reaction (RT-PCR) [19-valent multiplex RT-PCR enzyme-linked immunosorbent assay (ELISA)] to assess the diagnostic performance. Furthermore the diagnostic value of the EIA in terms of age and season relation was analyzed. A total of 400 nasopharyngeal or tracheal secretions from pediatric patients with clinical signs of lower respiratory tract infection were included. The specimen had to be taken in a standardized manner within 72 h after admission. Specimens were tested in parallel with the EIA and the multiplex RT-PCR ELISA. The RT-PCR technique was used as the target assay. The EIA reached a sensitivity of 58% and a specificity of 90% for all samples tested. For patients < [corrected] 1 year the post-test probability for a positive EIA was 91% during the RSV season; a negative test result decreased disease probability from 53 to 25%. For older patients a positive test raised disease probability from 23 to 45% during the RSV season. Negative test results did not markedly change disease probability.

Ten years' experience with year-round active surveillance of up to 19 respiratory pathogens in children.

INTRODUCTION: Surveillance systems for acute respiratory infections (ARI) in children currently are often limited in terms of the panel of pathogens and the age range investigated or are only syndromic and at times only active in the winter season. METHODS: Within PID-ARI.net, a research network for ARI in children in Germany, an active, year-round surveillance system was formed in three regions from north to south for population-based analysis. Children from birth to 16 years of age were included and up to 19 noncolonizing airway pathogens were tested for with multiplex RT-PCR. RESULTS: In the 10-year period from July 1996 to June 2006, a total of 18,899 samples were tested. The positive rate increased with the size of the test panel to up to 72.9%. Picornaviruses (35-39%), paramyxoviruses (23-28%) and orthomyxoviruses (5.8-12.5%) comprised the highest fraction. Reoviruses and Legionella pneumophila were not found at all and Chlamydia pneumoniae and Bordetella parapertussis only rarely. Respiratory syncytial virus and parainfluenza virus (PIV) type 3 were anticyclical in rhythmicity with metapneumovirus and PIV1 and PIV2. The age medians per pathogen depended predominantly upon the attack rate and interepidemic intervals. CONCLUSION: Active surveillance systems for ARI are superior to passive systems. They should be pathogen-specific and comprehensive for viruses and bacteria and age ranges. They should be population-based and multilevel to avoid bias. The impact of atypical bacteria in children was highly overestimated in earlier studies.

Novel human metapneumovirus sublineage.

In a pediatric surveillance network, 287 (5.1%) of 5,580 specimens from patients with acute respiratory infections tested positive for human metapneumovirus (HMPV). Phylogenetic analysis of N- and F-gene sequences of identified HMPV showed that 30% belonged to a novel phylogenetic cluster.

Can respiratory syncytial virus etiology be diagnosed clinically? A hospital-based case-control study in children under two years of age.

An unmatched, hospital-based case-control study was performed, to determine, whether respiratory syncytial virus (RSV) etiology in hospitalized young children can be predicted clinically. Children under 2 years of age admitted with a lower respiratory tract infection in three hospitals in northern Germany were included (one tertiary and two secondary centers). Cases were children tested positive for RSV by multiplex RT-PCR. One control group consisted of children tested negative for RSV in the multiplex-RT-PCR and a second control group consisted of patients in whom no PCR was done. A weighted backward stepwise logistic regression model was applied for multivariate analysis. RSV-etiology could be predicted with a sensitivity of 72.8% and a specificity of 73.2%. Young age, disease entity--pneumonia or bronchiolitis, center, intercostal retractions, absence of an underlying condition, low level of C-reactive protein, short duration of symptoms (all on admission), prematurity and epidemiologic year were predictive; anatomical infiltrates and wheezing were not. Pathogen specific diagnosis is necessary for individual therapy, allocation in observational studies or treatment trials and for surveillance of airway infections in children, since the positive predictive value is too low for an accurate diagnosis and decision making. Multivariate techniques are effective tools in complex clinical research for deconfounding.