Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic preparedness.

Throughout the SARS-CoV-2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost-effective platform that can be employed in a high-throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS-CoV-2 diagnostics in an academic environment. The strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable with RT-qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

Induction of Long-Lasting Regulatory B Lymphocytes by Modified Immune Cells in Kidney Transplant Recipients.

BACKGROUND: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls. METHODS: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080. RESULTS: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness. CONCLUSIONS: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220.

Molecular epidemiology and disease severity of influenza virus infection in patients with haematological disorders.

Influenza virus infection is a common cause of self-limiting respiratory tract infection (RTI), however immunocompromised patients are at an increased risk for a severe course of disease or fatal outcome. We therefore aimed to gain a better understanding of the molecular epidemiology of influenza viruses from patients with haematological disorders and their impact on the clinical course of disease. Molecular analysis using polymerase chain reaction (PCR) of nasopharyngeal swabs was performed for influenza virus in haematological patients at the Heidelberg University Hospital. Clinical data was evaluated to identify associated risk factors. For phylogenetic analysis, the hemagglutinin (HA) gene was sequenced. Out of 159 influenza positive patients, 117 patients developed upper RTI (influenza A: n = 73; influenza B: n = 44). Lower RTI was observed in n = 42 patients (26%), n = 22/42 patients developed severe disease and n = 16/159 (10.1%) patients died. Risk factors for lower RTI were nosocomial infection (p = 0.02), viral shedding for ≥14 days (p = 0.018), IgG levels <6 g/dL (p = 0.046), bacterial/fungal co-infections (p < 0.001). Risk factors for fatal outcome were age ≥65 years (p = 0.032), bacterial/fungal (p≤0.001) co-infections and high viral load (p = 0.026). Sequencing of the HA gene (n = 115) revealed subtype A(H3N2) (n = 46), A(H1N1)pdm09 (n = 24), B/Victoria (n = 34), B/Yamagata (n = 11). There was no correlation between influenza (sub)type and lower RTI. Influenza infection in haematological patients is associated with significant morbidity and mortality, the risk for aggravating co-infections, prolonged viral shedding and nosocomial transmission emphasizing the need for infection control.

RSV-CLASS -Clinical Assessment Severity Score: An easy-to-use clinical disease severity score for respiratory syncytial virus infection in hospitalized children.

Respiratory syncytial virus (RSV) is the most common cause of acute respiratory tract infection in infants and young children often leading to severe disease requiring hospitalization. However, validated tools for systematic assessment of disease severity are lacking. This study aimed at creating and validating a standardized, simple-to-use disease severity score for RSV infection in children-the RSV-CLASS (Clinical Assessment Severity Score). Therefore, data from over 700 RSV-infected children over six winter seasons (2014-2020) was analyzed using univariate and multiple regression analyses for the prediction of lower respiratory tract infection (LRTI) as a proxy for a severe course of the disease. Testing a broad range of respiratory symptoms, they eventually yielded seven items. Performing stepwise selection, these were reduced to the final four items: cough, tachypnea, rales, and wheezing, each receiving one point in the proposed score named RSV-CLASS. The score was calculated for children in two cohorts A and B, one for development and one for validation, with an area under the curve of 0.90 and 0.87, respectively. With a score value of 3 or 4, 97.8% and 100% of the children, respectively, were admitted with LRTI and classified correctly. The RSV-CLASS is a disease severity score based on a neutral, analytical approach using prospective data from a large study cohort. It will contribute to systematically assessing the disease severity of RSV infection and can be used for evidence-based clinical decision-making as well as for research settings.

Live-virus serum neutralization after bivalent SARS-CoV-2 mRNA vaccination in hemodialysis patients.

The development of bivalent booster vaccines addresses the ongoing evolution of the emerging B.1.1.529 (omicron) variant subtypes that are known to escape vaccine-induced neutralizing antibody response. Little is known about the immunogenicity and reactogenicity of bivalent mRNA vaccines in hemodialysis patients with impaired vaccine response. In this prospective, observational cohort study, we analyzed SARS-CoV-2 anti-S1 IgG, surrogate neutralizing antibodies (SNA), and live-virus neutralization against the SARS-CoV-2 wildtype and the BA.5 variant in 42 hemodialysis patients with and without prior SARS-CoV-2 infection before and after an additional fifth bivalent vaccine dose. Anti-S1 IgG and SNA were significantly higher in hemodialysis patients with prior infection than in patients without infection (p < 0.001 and p < 0.01, respectively). In patients without prior infection, both antibody levels increased, and live-virus neutralizing antibodies against the wildtype and the BA.5 variant were correspondingly significantly higher after bivalent booster vaccination (p < 0.001 for both). Conversely, in patients with prior infection, anti-S1 IgG and SNA did not alter significantly, and bivalent booster vaccination did not induce additional humoral immune response against the SARS-CoV-2 wildtype and the BA.5 variant. Thus, bivalent mRNA vaccines might increase humoral responses in hemodialysis patients without prior infection. Larger clinical trials are needed to help guide vaccination strategies in these immunocompromised individuals.

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.

Severe Dysbiosis and Specific Haemophilus and Neisseria Signatures as Hallmarks of the Oropharyngeal Microbiome in Critically Ill Coronavirus Disease 2019 (COVID-19) Patients.

BACKGROUND: At the entry site of respiratory virus infections, the oropharyngeal microbiome has been proposed as a major hub integrating viral and host immune signals. Early studies suggested that infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with changes of the upper and lower airway microbiome, and that specific microbial signatures may predict coronavirus disease 2019 (COVID-19) illness. However, the results are not conclusive, as critical illness can drastically alter a patient's microbiome through multiple confounders. METHODS: To study oropharyngeal microbiome profiles in SARS-CoV-2 infection, clinical confounders, and prediction models in COVID-19, we performed a multicenter, cross-sectional clinical study analyzing oropharyngeal microbial metagenomes in healthy adults, patients with non-SARS-CoV-2 infections, or with mild, moderate, and severe COVID-19 (n = 322 participants). RESULTS: In contrast to mild infections, patients admitted to a hospital with moderate or severe COVID-19 showed dysbiotic microbial configurations, which were significantly pronounced in patients treated with broad-spectrum antibiotics, receiving invasive mechanical ventilation, or when sampling was performed during prolonged hospitalization. In contrast, specimens collected early after admission allowed us to segregate microbiome features predictive of hospital COVID-19 mortality utilizing machine learning models. Taxonomic signatures were found to perform better than models utilizing clinical variables with Neisseria and Haemophilus species abundances as most important features. CONCLUSIONS: In addition to the infection per se, several factors shape the oropharyngeal microbiome of severely affected COVID-19 patients and deserve consideration in the interpretation of the role of the microbiome in severe COVID-19. Nevertheless, we were able to extract microbial features that can help to predict clinical outcomes.

Neutralizing antibody response against the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants after a third mRNA SARS-CoV-2 vaccine dose in kidney transplant recipients.

Seroconversion after COVID-19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age-matched healthy controls. In addition, vaccine-induced neutralization of SARS-CoV-2 wild-type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live-virus assay. After a third vaccine dose, anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls (p < 0.001). Vaccine-induced cross-neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti-S1 IgG, surrogate neutralizing, and/or anti-RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls (p < .001 for all).

Rapid comparative evaluation of SARS-CoV-2 rapid point-of-care antigen tests.

PURPOSE: The objective of this study was to develop a scalable approach for direct comparison of the analytical sensitivities of commercially available SARS-CoV-2 antigen point-of-care tests (AgPOCTs) to rapidly identify poor-performing products. METHODS: We present a methodology for quick assessment of the sensitivity of SARS-CoV-2 AgPOCTs suitable for quality evaluation of many different products. We established reference samples with high, medium, and low SARS-CoV-2 viral loads along with a SARS-CoV-2 negative control sample. Test samples were used to semi-quantitatively assess the analytical sensitivities of 32 different commercial AgPOCTs in a head-to-head comparison. RESULTS: Among 32 SARS-CoV-2 AgPOCTs tested, we observe sensitivity differences across a broad range of viral loads (9.8 × 108 to 1.8 × 105 SARS-CoV-2 genome copies per ml). 23 AgPOCTs detected the Ct25 test sample (1.6 × 106 copies/ml), while only five tests detected the Ct28 test sample (1.8 × 105 copies/ml). In the low-range of analytical sensitivity, we found three saliva spit tests only delivering positive results for the Ct21 sample (2.7 × 107 copies/ml). Comparison with published data supports our AgPOCT ranking. Importantly, we identified an AgPOCT widely offered, which did not reliably recognize the sample with the highest viral load (Ct16 test sample with 9.8 × 108 copies/ml) leading to serious doubts about its usefulness in SARS-CoV-2 diagnostics. CONCLUSION: The results show that the rapid sensitivity assessment procedure presented here provides useful estimations on the analytical sensitivities of 32 AgPOCTs and identified a widely-spread AgPOCT with concerningly low sensitivity.

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2.

PURPOSE: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. METHODS: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. RESULTS: The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2-87.5%). Specificity was 99.3% (CI 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. CONCLUSION: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. TRIAL REGISTRATION NUMBER AND REGISTRATION DATE: DRKS00021220 and 01.04.2020.

Identification and Cloning of a New Western Epstein-Barr Virus Strain That Efficiently Replicates in Primary B Cells.

The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to B95-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases.IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property.

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity.

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors.IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.

Molecular epidemiology of respiratory syncytial virus in children in sub-Saharan Africa.

OBJECTIVES: This study investigated the molecular epidemiology of respiratory syncytial virus (RSV) among febrile children with acute respiratory tract infection in Ghana, Gabon, Tanzania and Burkina Faso between 2014 and 2017 as well as the evolution and diversification of RSV strains from other sub-Saharan countries. METHODS: Pharyngeal swabs were collected at four study sites (Agogo, Ghana: n = 490; Lambaréné, Gabon: n = 182; Mbeya, Tanzania: n = 293; Nouna, Burkina Faso: n = 115) and analysed for RSV and other respiratory viruses using rtPCR. For RSV-positive samples, sequence analysis of the second hypervariable region of the G gene was performed. A dataset of RSV strains from sub-Saharan Africa (2011-2017) currently available in GenBank was compiled. Phylogenetic analysis was conducted to identify the diversity of circulating RSV genotypes. RESULTS: In total, 46 samples were tested RSV positive (Ghana n = 31 (6.3%), Gabon n = 4 (2.2%), Tanzania n = 9 (3.1%) and Burkina Faso n = 2 (1.7%)). The most common RSV co-infection was with rhinovirus. All RSV A strains clustered with genotype ON1 strains with a 72-nucleotide duplication and all RSV B strains belonged to genotype BAIX. Phylogenetic analysis of amino acid sequences from sub-Saharan Africa revealed the diversification into 11 different ON1 and 22 different BAIX lineages and differentiation of ON1 and BAIX strains into potential new sub-genotypes, provisionally named ON1-NGR, BAIX-KEN1, BAIX-KEN2 and BAIX-KEN3. CONCLUSION: The study contributes to an improved understanding of the molecular epidemiology of RSV infection in sub-Saharan Africa. It provides the first phylogenetic data for RSV from Tanzania, Gabon and Burkina Faso and combines it with RSV strains from all other sub-Saharan countries currently available in GenBank.

Letermovir prophylaxis is effective in preventing cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: single-center real-world data.

Morbidity and mortality after allogeneic hematopoietic cell transplantation (alloHCT) are still essentially affected by reactivation of cytomegalovirus (CMV). We evaluated 80 seropositive patients transplanted consecutively between March 2018 and March 2019 who received letermovir (LET) prophylaxis from engraftment until day +100 and retrospectively compared them with 80 patients without LET allografted between January 2017 and March 2018. The primary endpoint of this study was the cumulative incidence (CI) of clinically significant CMV infection (CS-CMVi) defined as CMV reactivation demanding preemptive treatment or CMV disease. With 14% CI of CS-CMVi at day +100 (11 events) was significantly lower in the LET cohort when compared to the control group (33 events, 41%; HR 0.29; p < 0.001). Whereas therapy with foscarnet could be completely avoided in the LET group, 7 out of 80 patients in the control cohort received foscarnet, resulting in 151 extra in-patient days for foscarnet administration (p = 0.002). One-year overall survival was 72% in the control arm vs 84% in the LET arm (HR 0.75 [95%CI 0.43-1.30]; p < 0.306). This study confirms efficacy and safety of LET for prophylaxis of CS-CMVi after alloHCT in a real-world setting, resulting in a significant patient benefit by reducing hospitalization needs and exposure to potentially toxic antiviral drugs for treatment of CMV reactivation.

Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test.

In 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0-94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5-99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2-92.3%) and 88.9% (40/45; CI 76.5-95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1-99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7-99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing.Clinical Trial DRKS00021220.

Interpretation of myocardial injury subtypes in COVID-19 disease per fourth version of Universal Definition of Myocardial Infarction.

BACKGROUND: Application of the 4th version of Universal Definition of Myocardial Infarction (UDMI) to characterize rates and prognostic relevance of myocardial injury in COVID-19 disease. METHODS: This retrospective, single-centre observational study enrolled 104 patients hospitalized with SARS-CoV-2 infection. Kaplan-Meier analysis and multivariate Cox regression were used to identify influence of acute or chronic myocardial injury on a composite primary (mortality, incident acute respiratory distress syndrome, incident mechanical ventilation) and secondary endpoint (mortality, incident acute myocardial injury during hospitalization, incident venous thrombosis, pulmonary embolism or stroke). RESULTS: A total of 27 (26.0%) patients presented with chronic myocardial injury, and 19 (18.3%) with acute myocardial injury. 42 patients(40.4%) developed an incident myocardial injury during hospitalization. The presence of acute or chronic myocardial injury on admission and incident myocardial injury during hospitalization were associated with higher rates of endpoints. Independent predictors for the primary endpoint were higher severity stages according to Siddiqi et al. classification system and history of dyslipidaemia. Maximal hs-cTnT and D-dimer concentrations during hospitalization showed an association (r = 0.61). CONCLUSIONS: Objective description of myocardial injury according to the 4th UDMI in the current COVID-19 pandemic is crucial in order to discriminate patients with acute myocardial infarction and acute, chronic or incident myocardial injury.

Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19 for incorporation into Multiplex Serology.

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen's kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology.

Validation of Spiked Postmortem Blood Samples from Cornea Donors on the Abbott ARCHITECT and m2000 Systems for Viral Infections.

BACKGROUND: Transplantation of human corneal tissue is associated with the potential risk of transmittance of viral infections. In accordance with European directives and federal laws, in Germany each tissue donor has to be tested for infectious diseases such as hepatitis B and C virus (HBV and HCV) and human immunodeficiency virus (HIV) infection. However, most of the currently available CE-marked serologic and nucleic acid screening systems are only validated for antemortem blood. METHODS: Twenty related and paired ante- and postmortem blood samples from cornea donors were obtained and subsequently analyzed for hepatitis B surface antigen (HBsAg), hepatitis B antibody (anti-HBc), anti-HCV, HCV RNA, anti-HIV-1/2, and HIV p24 Ag using Abbott test systems. The sera were also spiked with reference materials in concentrations giving low and high positivity for HBV, HCV, and HIV markers. RESULTS: The spiked ante- and postmortem sera from related donors showed similar results for HBsAg, anti-HBc, anti-HCV, HCV RNA, anti-HIV, and HIV p24 Ag, indicating a high stability of viral markers in cadaveric specimens. Three cornea donors had a medical history of HBV infection and revealed anti-HBc at similar levels in the ante- and postmortem sera. In addition, there was a single postmortem sample demonstrating a weak signal of anti-HIV-1 and HIV-1 p24 Ag. False-positive or false-negative results were not detected. The results obtained with the Abbott ARCHITECT analyzer and Abbott RealTime HCV PCR showed no significant differences. CONCLUSION: The analyzed screening assays are suitable for the detection of infectious markers of HBV, HCV, and HIV at similar levels in spiked ante- and postmortem sera from cornea donors.

The burden of hepatitis E among patients with haematological malignancies: A retrospective European cohort study.

BACKGROUND & AIMS: The burden of hepatitis E virus (HEV) infection among patients with haematological malignancy has only been scarcely reported. Therefore, we aimed to describe this burden in patients with haematological malignancies, including those receiving allogeneic haematopoietic stem cell transplantation. METHODS: We conducted a retrospective, multicentre cohort study across 11 European centres and collected clinical characteristics of 50 patients with haematological malignancy and RNA-positive, clinically overt hepatitis E between April 2014 and March 2017. The primary endpoint was HEV-associated mortality; the secondary endpoint was HEV-associated liver-related morbidity. RESULTS: The most frequent underlying haematological malignancies were aggressive non-Hodgkin lymphoma (NHL) (34%), indolent NHL (iNHL) (24%), and acute leukaemia (36%). Twenty-one (42%) patients had received allogeneic haematopoietic stem cell transplantation (alloHSCT). Death with ongoing hepatitis E occurred in 8 (16%) patients, including 1 patient with iNHL and 1 patient >100 days after alloHSCT in complete remission, and was associated with male sex (p = 0.040), cirrhosis (p = 0.006) and alloHSCT (p = 0.056). Blood-borne transmission of hepatitis E was demonstrated in 5 (10%) patients, and associated with liver-related mortality in 2 patients. Hepatitis E progressed to chronic hepatitis in 17 (34%) patients overall, and in 10 (47.6%) and 6 (50%) alloHSCT and iNHL patients, respectively. Hepatitis E was associated with acute or acute-on-chronic liver failure in 4 (8%) patients with 75% mortality. Ribavirin was administered to 24 (48%) patients, with an HEV clearance rate of 79.2%. Ribavirin treatment was associated with lower mortality (p = 0.037) and by trend with lower rates of chronicity (p = 0.407) when initiated <24 and <12 weeks after diagnosis of hepatitis E, respectively. Immunosuppressive treatment reductions were associated with mortality in 2 patients (28.6%). CONCLUSION: Hepatitis E is associated with mortality and liver-related morbidity in patients with haematological malignancy. Blood-borne transmission contributes to the burden. Ribavirin should be initiated early, whereas reduction of immunosuppressive treatment requires caution. LAY SUMMARY: Little is known about the burden of hepatitis E among patients with haematological malignancy. We conducted a retrospective European cohort study among 50 patients with haematological malignancy, including haematopoietic stem cell transplant recipients, with clinically significant HEV infection and found that hepatitis E is associated with hepatic and extrahepatic mortality, including among patients with indolent disease or among stem cell transplant recipients in complete remission. Hepatitis E virus infection evolved to chronic hepatitis in 5 (45.5%) patients exposed to a rituximab-containing regimen and 10 (47.6%) stem cell transplant recipients. Reducing immunosuppressive therapy because of hepatitis E was associated with mortality, while early ribavirin treatment was safe and effective.