Comparative Assessment of the Efficacy of Commercial Hand Sanitizers Against Human Norovirus Evaluated by an in vivo Fingerpad Method.

Human noroviruses (hNoV) are the leading cause of acute non-bacterial gastroenteritis worldwide and contaminated hands play a significant role in the spread of disease. Some hand sanitizers claim to interrupt hNoV transmission, but their antiviral efficacy on human hands is poorly characterized. The purpose of this work was to characterize the efficacy of representative commercial hand sanitizers against hNoV using an in vivo fingerpad method (ASTM E1838-17). Eight products [seven ethanol-based and one benzalkonium chloride (BAK)-based], and a benchmark 60% ethanol solution, were each evaluated on 10 human volunteers using the epidemic GII.4 hNoV strain. Virus titers before and after treatment were evaluated by RT-qPCR preceded by RNase treatment; product efficacy was characterized by log10 reduction (LR) in hNoV genome equivalent copies after treatment. The benchmark treatment produced a 1.7 ± 0.5 LR, compared with Product A (containing 85% ethanol) which produced a 3.3 ± 0.3 LR and was the most efficacious (p < 0.05). Product B (containing 70% ethanol), while less efficacious than Product A (p < 0.05), performed better than the benchmark with a LR of 2.4 ± 0.4. Five of the other ethanol-based products (labeled ethanol concentration ranges of 62-80%) showed similar efficacy to the 60% ethanol benchmark with LR ranging from 1.3 to 2.0 (p > 0.05). Product H (0.1% BAK) was less effective than the benchmark with a LR of 0.3 ± 0.2 (p < 0.05). None of the products screened were able to completely eliminate hNoV (maximum assay resolution 5.0 LR). Product performance was variable and appears driven by overall formulation. There remains a need for more hand sanitizer formulations having greater activity against hNoV, a virus that is comparatively recalcitrant relative to other pathogens of concern in community, healthcare, and food preparation environments.

Efficacy of an alcohol-based surface disinfectant formulation against human norovirus.

AIM: To evaluate the anti-noroviral efficacy of PURELL® surface sanitizer and disinfectant spray (PSS, an alcohol-based formulation) using human norovirus GII.4 Sydney [hNoV, by RT-qPCR and human intestinal enteroid (HIE) infectivity assay] and its cultivable surrogate, Tulane virus (TuV, infectivity assay), compared to sodium hypochlorite (NaOCl) solutions. METHODS AND RESULTS: PSS efficacy was evaluated in suspension and on surfaces [stainless steel (SS)] using ASTM methods. Results were expressed as log10 reduction (LR) of genome equivalent copy number (GEC, for hNoV, assayed by RT-qPCR) and plaque forming units (PFU, for TuV, per infectivity assay). In suspension, PSS achieved a 2.9 ± 0.04 LR hNoV GEC irrespective of contact time (30 or 60 s) and soil load (2.5% or 5%). Under all treatment conditions, infectious TuV could not be recovered following exposure to PSS, corresponding to the assay limit of detection (3.1-5.2 log10 PFU). Infectious hNoV could not be detected in the HIE model after exposure to PSS. On SS and 2.5% soil, PSS produced a 3.1 ± 0.1 LR hNoV GEC, comparable to 500 ppm NaOCl for 60 s. With 5.0% soil, PSS produced a 2.5 ± 0.2 LR hNoV GEC, which was similar to 1000-5000 ppm NaOCl for 60 s. CONCLUSIONS: PSS showed high anti-hNoV efficacy by RT-qPCR and in in vitro (TuV) and ex vivo (HIE) infectivity assays and performed similar to 1000-5000 ppm NaOCl for a 60-s contact time on SS with added soil. SIGNIFICANCE AND IMPACT OF STUDY: hNoV remains a significant cause of morbidity globally, partly due to its resistance to numerous surface disinfectants. RT-qPCR results from this study indicate PSS efficacy against hNoV is comparable to NaOCl efficacy. Infectivity assays leveraging TuV and the HIE model for hNoV support and confirm loss of virus infectivity. Collectively, these results indicate the product's ability to inactivate hNoV quickly, which could be beneficial in settings having elevated risk for hNoV transmission.

Use of DNA aptamer for sandwich type detection of Listeria monocytogenes.

A single stranded (ss) DNA aptamer, specific to members of Listeria genus, was used to develop a two-site binding sandwich assay for capture and detection of L. monocytogenes. Antibody-immobilized immunomagnetic beads were used to capture L. monocytogenes, followed by their exposure to the aptamer detector. Detection was achieved by amplification of cell-bound aptamers by qPCR. The lower limit of detection for the combined assay was 2.5 CFU L. monocytogenes in 500 µl buffer. This is juxtaposed to a detection limit of 2.4 log10 CFU in 500 µl buffer for immunomagnetic separation coupled with qPCR detection of L. monocytogenes targeting the hly gene. When applied to turkey deli meat, subjected to 24 h of non-selective enrichment, the two-site binding sandwich assay showed positive results at initial inoculum levels of 1-2 log10 CFU per 25 g sample. Because of its lower limit of detection, the assay reported here could be useful for detection of L. monocytogenes in foods and environmental samples.

An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding.

Nucleic acid aptamers are a class of alternative ligands increasingly growing in importance in the face of contemporary detection challenges. Aptamers offer multiple advantages over traditional ligands like antibodies; however, their ability to specifically bind target molecules must first be confirmed after their generation. Use of a plate-based enzyme-linked aptamer sorbent assay (ELASA) is a generally rapid way to screen and characterize aptamer binding to protein targets. ELASA involves directly plating a protein target onto a nonspecific (polystyrene) surface and assessing binding of functionalized (biotinylated) aptamers to those plated proteins using an enzyme conjugate that recognizes the aptamers. Here, we describe an ELASA that was designed and used to evaluate and compare binding of ssDNA aptamers against the capsids of different strains of human norovirus.

Evaluation of a Surface Sampling Method for Recovery of Human Noroviruses Prior to Detection Using Reverse Transcription Quantitative PCR.

Human noroviruses are the most common cause of acute viral gastroenteritis, and the environmental persistence of these viruses contributes to their transmissibility. Environmental sampling is thus an important tool for investigating norovirus outbreaks and for assessing the effectiveness of cleaning and decontamination regimens. The purpose of this study was to evaluate a sampling material (wipes) for their efficacy at recovering human norovirus from hard surfaces and foods. Dilutions of a human norovirus GII.4 stool specimen derived from an outbreak were applied to hard surfaces (stainless steel and ceramic) and the surfaces of representative foods (green pepper, apple, tomato, and cheese). The viruses were recovered at various times postinoculation using the wipes, followed by RNA extraction and reverse transcription quantitative PCR. Recovery efficiency ranged from 74% to almost 100% for all artificially inoculated hard surfaces and for most fresh produce surfaces. Less efficient recovery was observed for cheese. Viral RNA could be recovered from select surfaces for up to 7 days postinoculation, with a <1 log reduction in genome copy number. In field tests, 24 (11%) of 210 environmental samples collected during winter 2012 from restrooms in North Carolina were presumptively positive for human norovirus, and six of these samples were confirmed as GII.4 by sequencing. These wipes may be a valuable tool for investigations of norovirus outbreaks and studies of norovirus prevalence.

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain.

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target-the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

Selection, characterization and application of nucleic acid aptamers for the capture and detection of human norovirus strains.

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5-36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations.

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).