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Recent studies have revealed an important role for Ltbp-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/−) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.
Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.
Introduction: As the immunosuppressive potency of 15-deoxyspergualin (DSG) has been shown in the therapy of renal transplant rejection and Wegener's granulomatosis, the intention of this study was to evaluate the safety of DSG in the therapy of lupus nephritis (LN). Methods: Patients with histologically proven active LN after prior treatment with at least one immunosuppressant were treated with 0.5 mg/kg normal body weight/day DSG, injected subcutaneously for 14 days, followed by a break of one week. These cycles were repeated to a maximum of 9 times. Doses of oral corticosteroids were gradually reduced to 7.5 mg/day or lower by cycle 4. Response was measured according to a predefined decision pattern. The dose of DSG was adjusted depending on the efficacy and side effects. Results: 21 patients were included in this phase-I/II study. After the first DSG injection, one patient was excluded from the study due to renal failure. 5 patients dropped out due to adverse events or serious adverse events including fever, leukopenia, oral candidiasis, herpes zoster or pneumonia. 11/20 patients achieved partial (4) or complete responses (7), 8 were judged as treatment failures and one patient was not assessable. 12 patients completed all 9 cycles; in those patients, proteinuria decreased from 5.88g/day to 3.37g/day (P = 0.028), Selena-SLEDAI decreased from 17.6 to 11.7. In 13/20 patients, proteinuria decreased by at least 50%; in 7 patients to less than 1g/day. Conclusions: Although the number of patients was small, we could demonstrate that DSG provides a tolerably safe treatment for LN. The improvement in proteinuria encourages larger controlled trials.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.
Treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia (Ph+ ALL) has been revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Most patients with CML achieve long-term survival similar to individuals without CML due to treatment with TKIs not only in frontline but also in further lines of therapy. The third-generation TKI ponatinib has demonstrated efficacy in patients with refractory CML and Ph+ ALL. Ponatinib is currently the most potent TKI in this setting demonstrating activity against T315I mutant clones. However, ponatinib’s safety data revealed a dose-dependent, increased risk of serious cardiovascular (CV) events. Guidance is needed to evaluate the benefit–risk profile of TKIs, such as ponatinib, and safety measures to prevent treatment-associated CV events. An expert panel of German hematologists and cardiologists summarize current evidence regarding ponatinib’s efficacy and CV safety profile. We propose CV management strategies for patients who are candidates for ponatinib.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile cellular infection model that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The model is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression (A549-AT). Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV-2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected A549-AT cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel model cell line allows rapid and sensitive monitoring of SARS-CoV-2 infection and the screening for host factors essential for viral replication. HIGHLIGHTS: Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection; The versatile model cell line A549-AT is suitable for rapid and sensitive high-throughput assays; Additional gene specific expression cassettes allow the screening for compounds and cellular factors limiting SARS-CoV-2 replication.
Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany).
Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021.
In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.
Reduced neutralization of SARS-CoV-2 Omicron variant by vaccine sera and monoclonal antibodies
(2021)
Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.
Reduced neutralization of SARS-CoV-2 Omicron variant by vaccine sera and monoclonal antibodies
(2021)
Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab, genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat COVID-19 due to Omicron and other emerging variants of concern.
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections.
We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R.
We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.5-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab.
In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which however, might be circumvented by a combination therapy with casirivimab together.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time.
In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022.
In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities.
The NVX-CoV2373-vaccine has recently been licensed, although data on vaccine-induced humoral and cellular immunity towards the parental strain and variants of concern (VOCs) in comparison to dual-dose mRNA-regimens are limited. In this observational study including 66 participants, we show that NVX-CoV2373-induced IgG-levels were lower than after vaccination with BNT162b2 or mRNA-1273 (n=22 each, p=0.006). Regardless of the vaccine and despite different IgG-levels, neutralizing activity towards VOCs was highest for Delta, followed by BA.2 and BA.1. Interestingly, spike-specific CD8 T-cell levels after NVX-CoV2373-vaccination were significantly lower and were detectable in 3/22 (14%) individuals only. In contrast, spike-specific CD4 T-cells were induced in 18/22 (82%) individuals. However, CD4 T-cell levels were lower (p<0.001), had lower CTLA-4 expression (p<0.0001) and comprised less multifunctional cells co-expressing IFNγ, TNFαα and IL-2 (p=0.0007) as compared to mRNA-vaccinated individuals. Unlike neutralizing antibodies, NVX-CoV2373-induced CD4 T cells cross-reacted to all tested VOCs from Alpha to Omicron, which may hold promise to protect from severe disease.
Ziele: Das Ziel dieser offiziellen Leitlinie, die von der Deutschen Gesellschaft für Gynäkologie und Geburtshilfe (DGGG) und der Deutschen Krebsgesellschaft (DKG) publiziert und koordiniert wurde, ist es, die Früherkennung, Diagnostik, Therapie und Nachsorge des Mammakarzinoms zu optimieren.
Methoden: Der Aktualisierungsprozess der S3-Leitlinie aus 2012 basierte zum einen auf der Adaptation identifizierter Quellleitlinien und zum anderen auf Evidenzübersichten, die nach Entwicklung von PICO-(Patients/Interventions/Control/Outcome-)Fragen, systematischer Recherche in Literaturdatenbanken sowie Selektion und Bewertung der gefundenen Literatur angefertigt wurden. In den interdisziplinären Arbeitsgruppen wurden auf dieser Grundlage Vorschläge für Empfehlungen und Statements erarbeitet, die im Rahmen von strukturierten Konsensusverfahren modifiziert und graduiert wurden.
Empfehlungen: Der Teil 1 dieser Kurzversion der Leitlinie zeigt Empfehlungen zur Früherkennung, Diagnostik und Nachsorge des Mammakarzinoms: Der Stellenwert des Mammografie-Screenings wird in der aktualisierten Leitlinienversion bestätigt und bildet damit die Grundlage der Früherkennung. Neben den konventionellen Methoden der Karzinomdiagnostik wird die Computertomografie (CT) zum Staging bei höherem Rückfallrisiko empfohlen. Die Nachsorgekonzepte beinhalten Untersuchungsintervalle für die körperliche Untersuchung, Ultraschall und Mammografie, während weiterführende Gerätediagnostik und Tumormarkerbestimmungen bei der metastasierten Erkrankung Anwendung finden.
Purpose: The aim of this official guideline coordinated and published by the German Society for Gynecology and Obstetrics (DGGG) and the German Cancer Society (DKG) was to optimize the screening, diagnosis, therapy and follow-up care of breast cancer.
Methods: The process of updating the S3 guideline dating from 2012 was based on the adaptation of identified source guidelines which were combined with reviews of evidence compiled using PICO (Patients/Interventions/Control/Outcome) questions and the results of a systematic search of literature databases and the selection and evaluation of the identified literature. The interdisciplinary working groups took the identified materials as their starting point to develop recommendations and statements which were modified and graded in a structured consensus procedure.
Recommendations: Part 1 of this short version of the guideline presents recommendations for the screening, diagnosis and follow-up care of breast cancer. The importance of mammography for screening is confirmed in this updated version of the guideline and forms the basis for all screening. In addition to the conventional methods used to diagnose breast cancer, computed tomography (CT) is recommended for staging in women with a higher risk of recurrence. The follow-up concept includes suggested intervals between physical, ultrasound and mammography examinations, additional high-tech diagnostic procedures, and the determination of tumor markers for the evaluation of metastatic disease.
Hematotoxicity is one of the major side effects of chemotherapy. The aim of this study was to examine the association between single nucleotide polymorphisms (SNPs) and hematotoxicity in breast cancer patients in a subset of patients of the SUCCESS prospective phase III chemotherapy study. All patients (n = 1678) received three cycles of 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) followed by three cycles of docetaxel or docetaxel/gemcitabine, depending on randomization. Germline DNA was genotyped for 246 SNPs selected from a previous genome-wide association study (GWAS) in a panel of lymphoblastoid cell lines, with gemcitabine toxicity as the phenotype. All SNPs were tested for their value in predicting grade 3 or 4 neutropenic or leukopenic events (NLEs). Their prognostic value in relation to overall survival and disease-free survival was also tested.
None of the SNPs was found to be predictive for NLEs during treatment with docetaxel/gemcitabine. Two SNPs in and close to the PIGB gene significantly improved the prediction of NLEs after FEC, in addition to the factors of age and body surface area. The top SNP (rs12050587) had an odds ratio of 1.38 per minor allele (95% confidence interval, 1.17 to 1.62). No associations were identified for predicting disease-free or overall survival.
Genetic variance in the PIGB gene may play a role in determining interindividual differences in relation to hematotoxicity after FEC chemotherapy.
Background: In recent months, Omicron variants of SARS-CoV-2 have become dominant in many regions of the world, and case numbers with Omicron subvariants BA.1 and BA.2 continue to increase. Due to numerous mutations in the spike protein, the efficacy of currently available vaccines, which are based on Wuhan-Hu 1 isolate of SARS-CoV-2, is reduced, leading to breakthrough infections. Efficacy of monoclonal antibody therapy is also likely impaired.
Methods: In our in vitro study using A549-AT cells constitutively expressing ACE2 and TMPRSS2, we determined and compared the neutralizing capacity of vaccine-elicited sera, convalescent sera and monoclonal antibodies against authentic SARS-CoV-2 Omicron BA.1 and BA.2 compared with Delta.
Findings: Almost no neutralisation of Omicron BA.1 and BA.2 was observed using sera from individuals vaccinated with two doses 6 months earlier, regardless of the type of vaccine taken. Shortly after the booster dose, most sera from triple BNT162b2-vaccinated individuals were able to neutralise both Omicron variants. In line with waning antibody levels three months after the booster, only weak residual neutralisation was observed for BA.1 (26%, n = 34, 0 median NT50) and BA.2 (44%, n = 34, 0 median NT50). In addition, BA.1 but not BA.2 was resistant to the neutralising monoclonal antibodies casirivimab/imdevimab, while BA.2 exhibited almost a complete evasion from the neutralisation induced by sotrovimab.
Interpretation: Both SARS-CoV-2 Omicron subvariants BA.1 and BA.2 escape antibody-mediated neutralisation elicited by vaccination, previous infection with SARS-CoV-2, and monoclonal antibodies. Waning immunity renders the majority of tested sera obtained three months after booster vaccination negative in BA.1 and BA.2 neutralisation. Omicron subvariant specific resistance to the monoclonal antibodies casirivimab/imdevimab and sotrovimab emphasizes the importance of genotype-surveillance and guided application.
Funding: This study was supported in part by the Goethe-Corona-Fund of the Goethe University Frankfurt (M.W.) and the Federal Ministry of Education and Research (COVIDready; grant 02WRS1621C (M.W.).
The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections.
We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R.
We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 5.71 and 3.64, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.6-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab.
In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which however, might be circumvented by a combination therapy with casirivimab together.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time.
In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022.
In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities.