Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

Long-Term Observation of SARS-CoV-2 Vaccination Response upon High Efficacy Treatment in Multiple Sclerosis-A Real-World Scenario.

Immunomodulatory and immunosuppressive therapy is needed in people with a chronic neuroinflammatory disease of the central nervous system such as multiple sclerosis (MS). Therefore, MS requires monitoring for and preventing against infectious diseases like SARS-CoV-2. Vaccination and anti-viral treatments are, in particular, recommended for elderly people and people at risk of a severe course of infection and of MS. Here, we asked whether repetitive infection or vaccination influenced responses upon receiving high efficacy treatments, namely sphingosine-1-phosphate receptor modulator (S1P) or anti-CD20 B cell antibody (anti-CD20) treatments. We performed a prospective real-world study of people with MS (pwMS) under S1P or anti-CD20 with repetitive exposure to the SARS-CoV-2 virus or vaccine. The measurement of anti-SARS-CoV-2 antibody titres was performed by two independent immunoassays after initial immunisation and after booster vaccination or infection. Other laboratory and clinical parameters were included in the analysis of influencing factors. As secondary outcomes, lymphocyte and immunoglobulin levels were observed longitudinally under intravenous and subcutaneous anti-CD20 treatment. In a long-term real-world cohort of 201 pwMS, we found that despite lymphopenia upon S1P drugs, the SARS-CoV-2 immunisation response increased both in selective and non-selective S1P (100% and 88% seroconversion, respectively), whereas those under anti-CD20 therapies merely exhibited a slight long-term increase in antibody titres (52% seroconversion). The latter was independent of immunoglobulin or total lymphocyte levels, which mostly remained stable. If the individual was immunised prior to therapy initiation, their levels of SARS-CoV-2 antibodies remained high under treatment. PwMS under non-selective S1P benefit from repetitive vaccination. The risk of an insufficient vaccination response mirrored by lower SARS-CoV-2 antibodies remains in pwMS receiving anti-CD20 treatment, even after repetitive exposure to the vaccine or virus. Due to the compromised vaccination response in CD20-depleting drugs, prompt antiviral treatment might be necessary.

The Cellular and Humoral Immune Response to SARS-CoV-2 Messenger RNA Vaccines Is Significantly Better in Liver Transplant Patients Compared with Kidney Transplant Patients.

Patients after organ transplantation have impaired immune response after vaccination against the SARS-CoV-2 virus. So far, published studies have reported quite different response rates to SARS-CoV-2 vaccination, ranging from 15-79% in liver and kidney transplant recipients. Up to one year after the first vaccine dose, we analyzed the humoral and cellular immune response of 21 liver transplant (LTX) patients after vaccination with mRNA vaccines compared with 28 kidney transplant (KTX) patients. We evaluated IgG against the SARS-CoV-2 spike protein as well as SARS-CoV-2 specific T cells using an ELISpot assay that detected IFN-γ- and/or IL-2-expressing T cells. We found a cellular and/or humoral immune response in 100% of the LTX patients compared with 68% of the KTX patients. Antibody titers against the spike protein of SARS-CoV-2 were significantly higher in the LTX group, and significantly more LTX patients had detectable specific IL-2-producing T cells. The immunosuppression applied in our LTX cohort was lower compared with the KTX cohort (14% triple therapy in LTX patients vs. 79% in KTX patients). One year after the first vaccination, breakthrough infections could be detected in 41% of all organ transplant patients. None of those patients suffered from a severe course of COVID-19 disease, indicating that a partial vaccination response seemed to offer protection to immunosuppressed patients. The better immune response of LTX patients after SARS-CoV-2 vaccination might be due to less intense immunosuppressive therapy compared with KTX patients.

Predictors for insufficient SARS-CoV-2 vaccination response upon treatment in multiple sclerosis.

BACKGROUND: Disease-modifying therapies (DMT) for multiple sclerosis (MS) influence SARS-CoV-2 vaccination response, which might have implications for vaccination regimens in individual patients. Expanding the knowledge of predictors for an insufficient vaccination response as a surrogate for protection against severe disease courses of infection in people with MS (pwMS) under DMT is of great importance in identifying high-risk populations. METHODS: Cross-sectional analysis of vaccination titre and its modifiers, in a prospective real-world cohort of 386 individuals (285 pwMS and 101 healthy controls) by two independent immunoassays between October 2021 and June 2022. FINDINGS: In our cohort, no difference in vaccination antibody level was evident between healthy controls (HC) and untreated pwMS. In pwMS lymphocyte levels, times vaccinated and DMT influence SARS-CoV-2 titre following vaccination. Those treated with selective sphingosine-1-phosphate receptor modulators (S1P) showed comparable vaccination titres to untreated; higher CD8 T cell levels prior to vaccination in B cell-depleted patients resulted in increased anti-spike SARS-CoV2 antibody levels. INTERPRETATION: PwMS under DMT with anti-CD20 treatment, in particular those with decreased CD8 levels before vaccination, as well as non-selective S1P but not selective S1P are at increased risk for insufficient SARS-CoV-2 vaccination response. This argues for a close monitoring of anti-spike antibodies in order to customize individual vaccination regimens within these patients. FUNDING: This work was supported by the German Research Foundation (DFG, CRC-TR-128 to TU, SB, and FZ).

Six-Month Follow-Up after Vaccination with BNT162b2: SARS-CoV-2 Antigen-Specific Cellular and Humoral Immune Responses in Hemodialysis Patients and Kidney Transplant Recipients.

Hemodialysis patients (HDP) and kidney transplant recipients (KTR) have a high risk of infection with SARS-CoV-2 with poor clinical outcomes. Because of this, vaccination of these groups of patients against SARS-CoV-2 is particularly important. However, immune responses may be impaired in immunosuppressed and chronically ill patients. Here, our aim was to compare the efficacy of an mRNA-based vaccine in HDP, KTR, and healthy subjects. DESIGN: In this prospective observational cohort study, the humoral and cellular response of prevalent 192 HDP, 50 KTR, and 28 healthy controls (HC) was assessed 1, 2, and 6 months after the first immunization with the BNT162b2 mRNA vaccine. RESULTS: After 6 months, 97.5% of HDP, 37.9% of KTR, and 100% of HC had an antibody response. Median antibody levels were 1539.7 (±3355.8), 178.5 (±369.5), and 2657.8 (±2965.8) AU/mL in HDP, KTR, and HC, respectively (p ≤ 0.05). A SARS-CoV-2 antigen-specific cell response to vaccination was found in 68.8% of HDP, 64.5% of KTR, and 90% of HC. CONCLUSION: The humoral response rates to mRNA-based vaccination of HDPs are comparable to HCs, but antibody titers are lower. Furthermore, HDPs have weaker T-cell response to vaccination than HCs. KTRs have very low humoral and antigen-specific cellular response rates and antibody titers, which requires other vaccination strategies in addition to booster vaccination.

Longitudinal Study of the Signal Strength in two Serologic Assays for Detection of SARS-CoV-2 Specific Antibodies.

BACKGROUND: In the course of the current SARS-CoV-2 pandemic, antibody assays provide an important means for guidance of public health efforts. Thus, characterization of the course of antibody signals on different widely used assays is needed. METHODS: We selected 25 PCR-confirmed SARS-CoV-2 cases among 3,273 healthcare workers and measured the course of the antibody signal using the Abbott Architect SARS-CoV-2 IgG assay and the Roche Elecsys® Anti-SARS-CoV-2 immunoassay. The signal strength was then modelled using linear mixed models adjusted for age. RESULTS: Since first sampling, the assay signal decreased per day in the Abbott assay (standardized slope (ß) = -0.46, 95% CI = -0.54 to -0.39). In contrast, an increase in the signal was ascertained by the Roche immunoassay per day (ß = 0.25, 95% CI = 0.09 to 0.41). CONCLUSIONS: Roche Elecsys® Anti-SARS-CoV-2 immunoassay may exhibit greater sensitivity in detecting SARS-CoV-2-specific antibodies in individuals in late stages of postinfection.

COVID-19-Importance for Patients on the Waiting List and after Kidney Transplantation-A Single Center Evaluation in 2020-2021.

(1) Background: Dialysis patients and recipients of a kidney allograft are at high risk for infection with SARS-CoV-2. It has been shown that the development of potent neutralizing humoral immunity against SARS CoV-2 leads to an increased probability of survival. However, the question of whether immunocompromised patients develop antibodies has not yet been sufficiently investigated; (2) Methods: SARS-CoV-2 antibodies were examined in hemodialysis patients on the waiting list for kidney transplantation as well as patients after kidney transplantation. Patients were interviewed about symptoms and comorbidities, BMI, and smoking history; (3) Results: SARS-CoV-2 antibodies were found in 16 out of 259 patients (6%). The trend of infections here reflects the general course of infection in Germany with a peak in November/December of 2020. Remarkably, patients on the waiting list experienced only mild disease. In contrast, transplanted patients had to be hospitalized but recovered rapidly from COVID-19. Most interesting is that all immunosuppressed patients developed antibodies against SARS-CoV-2 after infection; (4) Conclusions: Even with extensive hygiene concepts, an above-average number of patients were infected with SARS-CoV-2 during the second wave of infections in Germany. Because SARS-CoV-2 infection triggered the formation of antibodies even in these immunocompromised patients, we expect vaccination to be effective in this group of patients. Thus, dialysis patients and patients after kidney transplantation should be given high priority in vaccination programs.

Prevalence of Severe Acute Respiratory Syndrome Coronavirus-2-specific Antibodies in German Blood Donors during the COVID-19 Pandemic.

BACKGROUND: Coronavirus disease-2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While RT-PCR assays are used routinely to diagnose active COVID-19, serological testing offers a means of identifying individuals who previously experienced asymptomatic infections, as well as those who experienced symptomatic infections but no longer carry the virus. METHODS: The presence of SARS-CoV-2 IgG-positive antibodies in the sera of 673 blood donors residing in south-western Germany before and 3,880 donors after the advent of the COVID-19 pandemic was determined and confirmed using two highly sensitive serological tests. RESULTS: Approximately 0.40% of the donors assessed during the COVID-19 pandemic possessed SARS-CoV-2 IgG-positive antibodies, decidedly fewer than the percentage of SARS-CoV-2-infected individuals determined by real-time RT-PCR nationwide. CONCLUSIONS: These findings confirm the efficacy serological testing in identifying asymptomatic COVID-19 patients.

Containment of a Large SARS-CoV-2 Outbreak Among Healthcare Workers in a Pediatric Intensive Care Unit.

OBJECTIVE: Healthcare workers (HCWs) are particularly exposed SARS-CoV-2 because they are critical in preventing viral transmission and treating COVID-19 patients. Within HCWs, personnel of intensive care units (ICUs) are at the forefront of treating patients with a severe course of COVID-19 infection and therefore represent an extremely vulnerable group. Thus, our objective is to contribute to establish means of infection control protecting HCWs in the frontline of the current pandemic. DESIGN: An outbreak of SARS-CoV-2 was detected and contained in a pediatric ICU (PICU). The first positive case was identified with a point-of-care diagnostic system on site. Real-time PCR-based testing systems from self-collected nasopharyngeal samples swabs were used to test for viral RNA of SARS-CoV-2 in the follow-up. SETTING: PICU within a tertiary university hospital in Germany. PARTICIPANTS: Healthcare workers of the PICU. INTERVENTIONS: Positive HCWs were sent into quarantine. Containment measures were implemented including wearing of surgical-masks, physical distancing and systematic testing. RESULTS: Among 432 HCWs, 91 (25%) were tested. Forty-five percent reported symptoms corresponding to characteristics of COVID-19. Of those, only 19,5% (8 HCWs) were tested positive for SARS-CoV-2. No infection occurred outside the PICU. After the implementation of containment measures, viral transmission was stopped. CONCLUSIONS: In the present study, a large outbreak within a team of healthcare workers of a PICU, affecting almost one fifth of the entire personnel is documented, along with detailed insights about how the outbreak was contained and how operability of the unit was maintained.

Seroprevalence and Seroconversion of Cytomegalovirus in a Large Group of Healthy, German Blood Donors: Potential Contribution to Transfusion Transmitted Infections.

BACKGROUND: Immunocompromised patients are at increased risk of morbidity and mortality due to transfusion transmitted cytomegalovirus (CMV) infections. To avoid or minimize such risk, clinicians working in the field continually monitor the changing epidemiology of CMV infections. MATERIALS AND METHODS: A total of 234,192 blood donations obtained from 44,779 donors were tested. CMV seroprevalence and antibody conversion rates were determined over a 3-year period. RESULTS: A significant percentage (37.5%) of all male and female blood donors tested seropositive. Both age and gender were risk factors for CMV infection. A total of 177 seroconversions (0.4% of donors) were identified. The highest antibody conversion rate occurred among men between 30 and 39 years of age; women did not experience a similar peak in antibody conversion rate. Approximately 10% of infected blood donors were identified by CMV DNA testing prior to seroconversion. CONCLUSIONS: The high rates of seroprevalence and seroconversion and the identification of a significant number of CMV DNA-positive (infected) blood donors prior to seroconversion indicate that the routine testing of blood samples for CMV DNA could reduce the potential risk of CMV transmission to high-risk patients.

Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016.

Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.

The impact of whole blood processing and freezing conditions on the quality of therapeutic plasma prepared from whole blood.

BACKGROUND: The quality of whole blood (WB)-derived plasma preparations has been the subject of several studies, but there has been a lack of robust, comparative data for the different methods of processing and freezing. STUDY DESIGN AND METHODS: Six WB-derived plasma units were pooled and split (n = 16) and frozen within either 8 or 24 hours after WB collection, stored at 4°C or at room temperature (RT), and then frozen either slowly at -20°C or rapidly to below -30°C. Plasma units were tested for fibrinogen, Factor (F)V, FVII, FVIII, FXI, and von Willebrand factor (VWF), protein C (PC), protein S (PS) activity and free PS, prothrombin time, and partial thromboplastin time. RESULTS: FVIII was reduced by 9% to 19% after having been stored for 24 hours irrespective of storage temperature. Slow freezing (SF) reduced FVIII by 17% to 25% compared to rapid freezing (RF) to below -30°C. Storage temperature, but not 24-hour storage, decreased PS activity by 20% to 28%. PS activity was 8% to 17% lower in plasma units frozen slowly compared to RF. Storage and freezing had no influence on free PS. SF caused small losses of FVII and FXI activity. CONCLUSION: Twenty-four-hour hold at RT and SF both reduce FVIII levels below 70 U/dL in many plasma units. PS activity is affected substantially by storage temperature and SF, but free PS is not. With regard to plasma quality, freezing to below -30°C within 1 hour is superior to SF at -20°C.

Reference interval determination for N-terminal-B-type natriuretic peptide (NT-proBNP): a study in blood donors.

We assessed reference values in a group of apparently healthy blood donors. A total of 1980 blood donors was recruited and tested for the presence of NT-proBNP using a newly developed electrochemiluminescence immunoassay (ECLIA) method. NT-proBNP clustered in all blood donors below the age of 50 years and an upper limit of normal (ULN) was found to be 84 pg/ml for males and 146 pg/ml for females. Mean NT-proBNP values increased with increasing age which was due to an increasing number of individuals exceeding the ULN. Age- and gender-appropriate NT-proBNP levels decreased with increasing hemoglobin levels. Hemoglobin but not creatinine levels influenced the NT-proBNP concentration in this cohort. The upper limit of normal can be used in clinical studies to further assess groups of diseased individuals to define clinical cutoffs.

N-terminal pro-brain natriuretic peptide (NT-proBNP) in healthy blood donors and in patients from general practitioners with and without a diagnosis of cardiac disease.

BACKGROUND: The natriuretic peptides and especially the N-terminal pro-brain natriuretic peptide (NT-proBNP) are increased in conditions with cardiac ventricular volume and pressure overload. Early stages of ventricular volume and pressure overload are often without signs and symptoms and therefore difficult to diagnose. On the contrary, a normal level of a natriuretic peptide excludes congestive heart failure as a cause of dyspnea with high probability. In addition, natriuretic peptide levels predict the risk of death and cardiovascular events after adjustment for traditional risk factors. A few studies suggest that age, gender and renal function may influence circulating natriuretic peptide levels. This study was therefore initiated to a) assess reference values for the N-terminal pro-brain natriuretic peptide (NT-proBNP) in a group of blood donors and healthy elderly individuals and to relate these levels to age, sex, and creatinine and b) to measure the levels of NT-proBNP in a population of patients presenting to general practitioners and to check the quality of the diagnoses congestive heart failure and dyspnea of other causes (heart failure patients usually present with breathlessness but the low specificity of dyspnea often leads to misdiagnoses). Finally, the percentage of patients with other diagnoses and elevated NT-proBNP as an indicator of an increased cardiovascular risk or up to now unknown cardiac disease was determined. STUDY DESIGN AND METHODS: N=1981 blood donors, N=283 individuals from general practitioners (GP) without cardiac disease and N=570 consecutive patients from GPs were recruited and tested for the presence of NT-proBNP using a newly developed electrochemiluminescence immunoassay run on an automated analyzer (Elecsys, Roche Diagnostics). RESULTS: NT-proBNP was detected at relatively homogenous levels in all individuals below the age of 50 years. NT-proBNP values increased with increasing age which was due to the increasing number of outliers in that group. Females had higher NT-proBNP levels than males. CONCLUSIONS: Based on the assumption that individuals below the age of 50 years are healthy, reference values based on the 97.5th percentile were established. These values were considered to be normal. The presented data and data from the literature suggest that also in the elderly population a cut-off level of 125 pg/ml is useful either to exclude cardiac dysfunction in symptomatic individuals or to risk stratify elderly individuals in terms of the necessity for intervention.

The quality of plasma collected by automated apheresis and of recovered plasma from leukodepleted whole blood.

BACKGROUND: There exists a current lack of information about the composition of the different types of plasma. No direct comparisons between apheresis plasma (AP) and recovered plasma (RP) derived from in-line-filtered whole blood (WB) have been published to date. STUDY DESIGN AND METHODS: Sixty AP units, 100 RP units from in-line-filtered WB held for 3 hours at 20 degrees C between donation and freezing, and an additional 100 RP units held for 15 hours at 20 degrees C before freezing were analyzed for coagulation factors and inhibitors, total protein, immunoglobulin G (IgG), and hemostasis and proteolysis activation markers. The influence of twice freezing and thawing on clotting factors V, VIII, and XI was also examined. RESULTS: AP contains substantially greater activities of factor (F) V, FVIII, F IX, and FXI than RP frozen within 3 hours after WB donation. Prolonged holding of RP at 20 degrees C for more than 15 hours caused an additional reduction in FVIII, FXI, and protein S activities. Significantly greater levels of prothrombin fragments 1 and 2, platelet factor 4, and neutrophil elastase were found in RP compared with AP. IgG was lower in AP compared with RP. Twice freezing and thawing caused a marked drop in FV, FVIII, and FXI activity. CONCLUSION: Higher FVIII and F IX potencies in AP compared with RP can be expected to result in greater yields when used for purification of these clotting factors. AP is presumably more efficient than RP for treating coagulopathies. RP, however, may contain higher IgG levels than AP.