Side-By-Side Evaluation of Three Commercial ELISAs for the Quantification of SARS-CoV-2 IgG Antibodies.

In December 2020, WHO presented the first international standard (WHO IS) for anti-SARS-CoV-2 immunoglobulin. This standard is intended to serve as a reference reagent against which serological tests can be calibrated, thus creating better comparability of results between different tests, laboratories, etc. Here, we have examined three different commercial ELISA kits for the quantification of SARS-CoV-2 IgG antibodies, namely the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) (Euroimmun, Lübeck, Germany), the SERION ELISA agile (Institut Virion Serion, Würzburg, Germany), and the COVID-19 quantitative IgG ELISA (DeMediTec Diagnostics, Kiel, Germany). According to the manufacturers, all are calibrated against the WHO IS and can provide results in either international units (IU) (DeMediTec) or arbitrary antibody units (BAU) per milliliter (Euroimmun, Virion Serion), which are numerically identical, according to the WHO. A total of 50 serum samples from vaccinated individuals were tested side by side and according to the manufacturer's instructions. We compared the test results of all three assays with each other to assess comparability and with a quantitative in-house virus neutralization test (micro-NT). In summary, our data are consistent with other studies published on this topic that tested similar assays from different manufacturers. Overall, the agreement between quantitative ELISAs is variable and cannot be used interchangeably despite calibration against a standard. Therefore, interpretation of results must still be individualized and tailored to each case. More importantly, our results highlight that quantitative ELISAs in their current form cannot replace neutralization tests.

In-Depth Analysis of Bacillus anthracis 16S rRNA Genes and Transcripts Reveals Intra- and Intergenomic Diversity and Facilitates Anthrax Detection.

Analysis of 16S rRNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ, in vitro, and in silico assays to assess the unknown 16S state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing, and bioinformatics, we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis SRA data sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons. IMPORTANCE For severe infectious diseases, precise pathogen detection is crucial for antibiotic therapy and patient survival. Identification of Bacillus anthracis, the causative agent of the zoonosis anthrax, can be challenging when querying specific nucleotide sequences such as in small subunit rRNA (16S rRNA) genes, which are commonly used for typing of bacteria. This study analyzed on a broad genomic scale a cryptic and hitherto underappreciated allelic variant of the bacterium's 16S rRNA genes and their transcripts using a set of in situ, in vitro, and in silico assays and found significant intra- and intergenomic heterogeneity in the distribution of the allele and overall rRNA operon copy numbers. This allelic variation was uniquely species specific, which enabled sensitive pathogen detection on both DNA and transcript levels. The methodology used here is likely also applicable to other pathogens that are otherwise difficult to discriminate from their less harmful relatives.

NOTIFy (non-toxic lyophilized field)-FISH for the identification of biological agents by Fluorescence in situ Hybridization.

The rapid and reliable diagnostics of highly pathogenic bacteria under restricted field conditions poses one of the major challenges to medical biodefense, especially since false positive or false negative reports might have far-reaching consequences. Fluorescence in situ hybridization (FISH) has the potential to represent a powerful microscopy-based addition to the existing molecular-based diagnostic toolbox. In this study, we developed a set of FISH-probes for the fast, matrix independent and simultaneous detection of thirteen highly pathogenic bacteria in different environmental and clinical sample matrices. Furthermore, we substituted formamide, a routinely used chemical that is toxic and volatile, by non-toxic urea. This will facilitate the application of FISH under resource limited field laboratory conditions. We demonstrate that hybridizations performed with urea show the same specificity and comparable signal intensities for the FISH-probes used in this study. To further simplify the use of FISH in the field, we lyophilized the reagents needed for FISH. The signal intensities obtained with these lyophilized reagents are comparable to freshly prepared reagents even after storage for a month at room temperature. Finally, we show that by the use of non-toxic lyophilized field (NOTIFy)-FISH, specific detection of microorganisms with simple and easily transportable equipment is possible in the field.