Impact on the German asymptomatic screening strategy based on actual user data from SARS-CoV-2 test centers.

Since March 2021, Germany has been providing cost-free severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen tests, and many day-to-day activities following the lockdown have required negative test results. Yet it remains unclear how tests have been used and whether there are patterns connected to mitigation measures. We analyzed over 50,000 anonymized records from eight test centers in a typical medium-sized city, with one of them remaining open continuously from March until December 2021. The centers exhibit distinct patterns of visitor types, with the majority tested only once in the investigated period. Individuals who underwent repeated testing tended to favor the same location. A preference for spontaneous testing grew in proportion to the availability of spare tests. Visitors aged 18 to 30 years were distinctly overrepresented compared to the local demographic. A negative binominal model showed that implemented mitigation measures had an impact on the number of tests conducted. Cost-free testing in private facilities was implemented into the German complementary screening strategy, aiming to achieve weekly population-wide testing. This study demonstrates these facilities were rarely used for regular testing but rather for meeting requirements of certified tests. The results should aid authorities in making future decisions regarding infection control.

Free PoC Testing for SARS-CoV-2 in Germany: Factors Expanding Access to Various Communities in a Medium-Sized City.

During the third wave of the COVID-19 (coronavirus disease 2019) pandemic in Germany, free SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) point-of-care (PoC) antigen tests were offered to citizens at least once a week to prevent spreading by asymptomatic infected individuals. This study investigated user groups, timing, frequency, and test center locations in a typical medium-sized European city. We analyzed 27,369 pseudonymized datasets from eight centers over 12 weeks. Those were evaluated according to age, residence, appointment, and potential repeated test occurrence. The centers were visited by different groups; some centers were preferred by a predominantly younger demographic, whereas a mobile option attracted an older age group by reaching districts with few other testing possibilities. Elderly individuals were tested more spontaneously than younger individuals, and a test center at a 'park and ride' had more spontaneous visitors from outside of the city compared to other test locations. Only a small proportion of less than 4% came for testing more than five times. To preferably address many people for voluntary antigen testing, it is crucial to offer different test opportunities accounting for individual behavioral patterns, despite this requiring more complex and costly design than conventional forms.

Sample Preparation for qPCR Detection of Listeria from Food.

Quantitative PCR, if performed properly, is a highly sensitive and robust tool. Nevertheless, its application to the particular case of pathogen detection from foodstuffs necessitates special requirements for reliable results. Firstly, a robust analytical chain, involving sample preparation and DNA isolation with purification, is necessary to ensure optimal performance. Secondly, for reliable quantification of Listeria monocytogenes from food, reproducible controls for all steps of the analytical chain are needed, which can give quantitative information about the performance of each individual step of the detection chain. Ideally, each individual sample should include a so-called internal sample process control (ISPC) which passes through all steps of the analytical chain and is phenotypically similar to the target organism (in this case L. monocytogenes).This chapter describes the modular and rapid (3 h) sample preparation method "matrix lysis" for the quantification of L. monocytogenes from food and gives detailed information regarding the application of an ISPC based on the example of the L. monocytogenes Δ-prfA/+IAC strain.

qPCR Validation on the Basis of the Listeria monocytogenes prfA Assay.

Quantitative real-time polymerase chain reaction (qPCR) is one of the most used molecular methods. There are numerous qPCR assays on the market, some of them for pathogen detection, and the development of new assays still continues. However, what methods are suitable for assay performance validation and which information do they provide? For conclusions based on qPCR data, it is essential to know which capacities and limitations an assay has. This chapter gives an overview of methods for qPCR assay performance validation and the respective insights and how to combine them. Most of those validation methods have been published in connection with the prfA assay, which specifically detects Listeria monocytogenes. Thereby, it could be demonstrated that this assay reliably quantifies even a single copy of the prfA gene and is thus suitable for detection of Listeria monocytogenes.

Reduced bacterial contamination rates detected on silicone tourniquets compared to conventional tourniquets in clinical routine.

BACKGROUND: Tourniquets used for peripheral venous vascular access such as blood sampling are regularly contaminated in clinical routine. Although most contaminations are harmless, some pose a possible risk for infection. To improve peripheral venous access infection control standards, tourniquets with no or as few as possible bacterial burden should be used. Conventional tourniquets can be reprocessed by autoclaving or by incubating in disinfectants. However, both methods are time-consuming and not suitable for immediate use between patients. In contrast, silicone tourniquets can be quickly and simply reprocessed with wipe disinfection. In vitro studies from the manufacturer have demonstrated reduced bacterial contamination on silicone tourniquets after usage compared to conventional tourniquets. This study aims to independently investigate the bacterial load on both types of tourniquets in clinical routine. METHODS: In a first trial, new conventional and silicon tourniquets were used for blood sampling in one facility with strict guidelines for reprocessing (after each patient or not at all) for 1 day and tested for bacterial contamination. In a second trial, new tourniquets were used in four facilities while the mode and frequency of tourniquets' reprocessing was defined individually by each facility. The number of treated patients, mode and frequency of reprocessing and other relevant handling measures were documented. RESULTS: Under controlled conditions, with strictly specified reprocessing, slightly fewer bacteria were found on silicone than on conventional tourniquets. In routine clinical practice the reprocessing frequency was not higher for silicone tourniquets in practice. Yet, in all four facilities, there were significantly fewer bacteria found on silicone than on conventional tourniquets. CONCLUSION: Although tourniquets are classified as non-critical medical devices, results show - together with benefits of faster and easier reprocessing - that silicone tourniquets can improve infection control of venous vascular access.

Perception of Agrobacterium tumefaciens flagellin by FLS2XL confers resistance to crown gall disease.

Bacterial flagella are perceived by the innate immune systems of plants1 and animals2 alike, triggering resistance. Common to higher plants is the immunoreceptor FLAGELLIN-SENSING 2 (FLS2)3, which detects flagellin via its most conserved epitope, flg22. Agrobacterium tumefaciens, which causes crown gall disease in many crop plants, has a highly diverged flg22 epitope and evades immunodetection by plants so far studied. We asked whether, as a next step in this game of 'hide and seek', there are plant species that have evolved immunoreceptors with specificity for the camouflaged flg22Atum of A. tumefaciens. In the wild grape species Vitis riparia, we discovered FLS2XL, a previously unknown form of FLS2, that provides exquisite sensitivity to typical flg22 and to flg22Atum. As exemplified by ectopic expression in tobacco, FLS2XL can limit crown gall disease caused by A. tumefaciens.

A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens.

Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC-MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.

A Novel Method for Sampling and Long-Term Monitoring of Microbes That Uses Stickers of Plain Paper.

Detection of pathogens is crucial in food production areas. While it is well established, swabbing as a state-of-the-art sampling method offers several drawbacks with respect to yield, standardization, overall handling, and long-term monitoring. This led us to develop and evaluate a method that is easier to use at a lower cost and that should be at least as sensitive. After evaluating sundry promising materials, we tested text-marking paper stickers for their suitability to take up and release Listeria monocytogenes with their nonsticky paper side over a 14-day time period using quantitative PCR. The recovery rate was similar to that in previous studies using conventional swabs, and we also confirmed the feasibility of pooling besides resilience to cleansing and disinfection. In a proof-of-concept experiment that sampled several locations, such as door handles, the occurrences of L. monocytogenes and Escherichia coli were determined. The results suggest that the presented sticker system might offer a promising cost-effective alternative sampling system with improved handling characteristics.IMPORTANCE As a ubiquitous bacterium, Listeria monocytogenes has a propensity to enter food production areas inadvertently via fomites such as door handles and switches. While the bacterium might not be in direct contact with the food products, knowing the microbial status of the surroundings is essential for risk assessment. Our investigation into a novel quantitative PCR (qPCR)-based sampling system with the highest sensitivity and ability to monitor over long periods of time, yet based on paper, proved to be cost-effective and reasonably convenient to handle.

Don't Shut the Stable Door after the Phage Has Bolted-The Importance of Bacteriophage Inactivation in Food Environments.

In recent years, a new potential measure against foodborne pathogenic bacteria was rediscovered-bacteriophages. However, despite all their advantages, in connection to their widespread application in the food industry, negative consequences such as an uncontrolled phage spread as well as a development of phage resistant bacteria can occur. These problems are mostly a result of long-term persistence of phages in the food production environment. As this topic has been neglected so far, this article reviews the current knowledge regarding the effectiveness of disinfectant strategies for phage inactivation and removal. For this purpose, the main commercial phage products, as well as their application fields are first discussed in terms of applicable inactivation strategies and legal regulations. Secondly, an overview of the effectiveness of disinfectants for bacteriophage inactivation in general and commercial phages in particular is given. Finally, this review outlines a possible strategy for users of commercial phage products in order to improve the effectiveness of phage inactivation and removal after application.

Essential role of polymerases for assay performance - Impact of polymerase replacement in a well-established assay.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

Induction of the viable but non-culturable state in bacterial pathogens by household cleaners and inorganic salts.

Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1 h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.

PCR-Stop analysis as a new tool for qPCR assay validation.

Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both with suitable calibration curves and efficiencies. We demonstrated thereby the benefits of PCR-Stop analysis revealing quantitative and qualitative resolution of both assays, the limits of one of those assays and thus avoiding misinterpretations in qPCR analysis. Furthermore, data displayed that a well performing assay starts indeed with its average efficiency.

Virucidal or Not Virucidal? That Is the Question-Predictability of Ionic Liquid's Virucidal Potential in Biological Test Systems.

For three decades now, ionic liquids (ILs), organic salts comprising only ions, have emerged as a new class of pharmaceuticals. Although recognition of the antimicrobial effects of ILs is growing rapidly, there is almost nothing known about their possible virucidal activities. This probably reflects the paucity of understanding virus inactivation. In this study, we performed a systematic analysis to determine the effect of specific structural motifs of ILs on three different biological test systems (viruses, bacteria and enzymes). Overall, the effects of 27 different ILs on two non-enveloped and one enveloped virus (P100, MS2 and Phi6), two Gram negative and one Gram positive bacteria (E. coli, P. syringae and L. monocytogenes) and one enzyme (Taq DNA polymerase) were investigated. Results show that while some ILs were virucidal, no clear structure activity relationships (SARs) could be identified for the non-enveloped viruses P100 and MS2. However, for the first time, a correlation has been demonstrated between the effects of ILs on enveloped viruses, bacteria and enzyme inhibition. These identified SARs serve as a sound starting point for further studies.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.