Influence of suspended particles and dissolved organic matters on virus enrichment in reclaimed water by two-step tangential flow ultrafiltration: Phenomena and mechanisms.

Enteric virus concentration in large-volume water samples is crucial for detection and essential for assessing water safety. Certain dissolution and suspension components can affect the enrichment process. In this study, tangential flow ultrafiltration (TFUF) was used as an enrichment method for recovering enteric virus in water samples. Interestingly, the bacteriophage MS2 recovery in reclaimed water and the reclaimed water without particles were higher than that in ultrapure water. The simulated reclaimed water experiments showed that humic acid (HA) (92.16% ± 4.32%) and tryptophan (Try) (81.50 ± 7.71%) enhanced MS2 recovery, while the presence of kaolin (Kaolin) inhibited MS2 recovery with an efficiency of 63.13% ± 11.17%. Furthermore, Atomic force microscopy (AFM) revealed that the MS2-HA cluster and the MS2-Try cluster had larger roughness values on the membrane surface, making it difficult to be eluted, whereas MS2-Kaolin cluster had compact surfaces making it difficult to be eluted. Additionally, the MS2-HA cluster is bound to the membrane by single hydrogen bond with SO, whereas both the MS2-Try cluster and the MS2-Kaolin cluster are bound to the membrane by two hydrogen bonds, making eluting MS2 challenging. These findings have potential implications for validating standardized methods for virus enrichment in water samples.

Microbial reductions and physical characterization of chitosan flocs when using chitosan acetate as a cloth filter aid in water treatment.

The World Health Organization (WHO) estimates 2.1 billion people lack access to safely managed water. Cloth filtration is often employed in rural and developing communities of South Asia for point-of-use water treatment, but bacteria and viruses are too small for efficient removal by this filtration method. Chitosan is a biodegradable, cationic, organic polymer derived from the chemical treatment of chitin that acts as a coagulant and flocculant of contaminant of microbes and other particles in water, thereby facilitating filtration of microbes. This research 1) evaluated the use of chitosan acetate as a pre-treatment coagulation-flocculation process followed by cloth filtration for microbial reductions and 2) assessed floc particle size under three stirring conditions. E. coli KO11 bacteria and MS2 coliphage virus removals were quantified using culture-based methods. Chitosan acetate coagulation-flocculation pre-treatment of water, followed by cloth filtration, met or exceeded the protective (2-star) WHO performance levels for bacteria (2 log10 reduction) and viruses (3 log10 reduction), and filtrate turbidity was consistently reduced to < 1 NTU, meeting United States Environmental Protection Agency (EPA) and WHO targets.

Optimizing the synthesis and purification of MS2 virus like particles.

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents' delivery.

Effectiveness of the Nanosilver/TiO2-Chitosan Antiviral Filter on the Removal of Viral Aerosols.

Background: The precaution of airborne transmission of viruses, such as influenza, SARS, MERS, and COVID-19, is essential for reducing infection. In this study, we applied a zero-valent nanosilver/titania-chitosan (nano-Ag0/TiO2-CS) filter bed, whose broad-spectrum antimicrobial efficacy has been proven previously, for the removal of viral aerosols to minimize the risk of airborne transmission. Methods: The photochemical deposition method was used to synthesize the nano-Ag0/TiO2-CS antiviral material. The surface morphology, elemental composition, and microstructure of the nano-Ag0/TiO2-CS were analyzed by a scanning electron microscopy/energy dispersive X-ray spectroscopy and a transmission electron microscopy, respectively. The MS2 bacteriophages were used as surrogate viral aerosols. The antiviral efficacy of nano-Ag0/TiO2-CS was evaluated by the MS2 plaque reduction assay (PRA) and filtration experiments. In the filtration experiments, the MS2 aerosols passed through the nano-Ag0/TiO2-CS filter, and the MS2 aerosol removal efficiency was evaluated by an optical particle counter and culture method. Results and Conclusions: In the MS2 PRA, 3 g of nano-Ag0/TiO2-CS inactivated 97% of MS2 bacteriophages in 20 mL liquid culture (2 ± 0.5 × 1016 PFU/mL) within 2 hours. The removal efficiency of nano-Ag0/TiO2-CS filter (thickness: 6 cm) for MS2 aerosols reached up to 93%. Over 95% of MS2 bacteriophages on the surface of the nano-Ag0/TiO2-CS filter were inactivated within 20 minutes. The Wells-Riley model predicted that when the nano-Ag0/TiO2-CS filter was used in the ventilation system, airborne infection probability would reduce from 99% to 34.6%. The nano-Ag0/TiO2-CS filter could remain at 50% of its original antiviral efficiency after continuous operation for 1 week, indicating its feasibility for the control of the airborne transmission.

Sediment re-suspension as a potential mechanism for viral and bacterial contaminants.

Pathogenic enteric viruses and bacteria tend to occur in higher concentrations and survive longer in aquatic sediments than suspended in the water column. Re-suspension of these organisms can result in a significant degradation of overlying water quality. Additionally, the re-suspension of microbial pathogens in artificial irrigation canals could endanger the consumption of fresh and ready-to-eat produce. Irrigation water has been implicated in numerous fresh produce outbreaks over the last 30 years. This study aimed to quantify the proportions of bacterial and viral re-suspension from sediment in a recirculating flume with varying velocities. MS2 coliphage and Escherichia coli were found to re-suspend at rates that were not significantly different, despite organism size differences. However, E. coli re-suspension rates from sand and clay were significantly different. This suggests that likely sediment-associated particles were recovered with the organisms attached. Similar re-suspension rates are hypothesized to be due to the dynamics of sediment transport, rather than that of the organisms themselves. This study also indicated that the re-suspension of sediment at very low velocities (e.g., less than 10 cm/s), could impact the microbiological quality of the overlaying water. Results from this study conclude that sediment could be a viable mechanism for irrigation water contamination.

Next-generation sequencing as a screening tool for foodborne pathogens in fresh produce.

Next generation sequencing (NGS) approaches are increasingly applied to tracing microbial contaminants entering the food chain due to NGS' untargeted nature and ability to investigate non-culturable (and/or difficult to culture) organisms while yielding genomic information about the microbiota. So far, a plethora of microbes has been shown to be associated with fresh produce, but few studies have utilised NGS to identify contamination with human pathogens. This study aims to establish the limit of detection (LoD) for Salmonella and phage MS2 (a Norovirus surrogate) contamination of fresh produce employing NGS approaches on the Illumina MiSeq: 16S amplicon-sequencing, and RNA-seq, using ScriptSeq (Illumina) and NEBNext (New England BioLabs) kits. ScriptSeq proved the most sensitive approach; delivering an LoD of 104 CFU reaction-1 (Colony Forming Units) for Salmonella and 105 PFU reaction-1 (Plaque Forming Units) for phage MS2. Use of the NEBNext kit resulted in detection of Salmonella at 106 CFU reaction-1 and phage MS2 at 107 PFU reaction-1. 16S amplicon-sequencing yielded a similar LoD of 105 CFU reaction-1 for Salmonella but could not detect MS2. The tested NGS methodologies, in combination with bioinformatics approaches applied, proved less sensitive than conventional microbial detection approaches.

Effect of polymer and glass physicochemical properties on MS2 recovery from food contact surfaces.

Viruses are transmissible via their interaction with contact surfaces of food containers or tools. This study evaluated the recoveries of MS2 coliphage, a virus surrogate, from polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), and glass (borosilicate and soda lime), as influenced by the surface chemistry and topography. MS2 (5-6 logs) in PBS with 1% TSB was inoculated onto each of 9 different surfaces, 24-h cold-incubated, and recovery was quantified by infectivity. The order of MS2 recovery efficiency from smooth surfaces was PP > PE ≥ soda lime glass, which classified into 3 ANOVA groups, p = 0.05. The MS2 recovery ratios of smooth vs. rough surfaces were 1.4-1.5. Atomic force microscopy revealed 21-nm diam pinholes (<28-nm of MS2 size) in the borosilicate glass. The lowest and highest MS2 recoveries among the 9 surfaces were demonstrated by the hole-bearing borosilicate glass (34 ±â€¯8%) and smooth PP (69 ±â€¯14%) respectively. Generally greater MS2 recovery was obtained from smooth PP and PE surfaces compared to glass, but topographic alterations (pinholes or increased roughness) decreased recovery possibly by trapping the viruses.

The use of hollow fiber dialysis filters operated in axial flow mode for recovery of microorganisms in large volume water samples with high loadings of particulate matter.

Ultrafiltration concentration of microorganisms in large volume water samples containing high levels of particulate matter was evaluated in a proof of concept study. The organisms tested were Bacillus atrophaeus subspecies globigii spores and MS2 bacteriophage. To produce the large volume samples, fresh water sediment of a known particle size was added to 51 l of tap water. Five different concentrations of particulate matter were studied: 0, 50, 100, 150 and 750 mg solids/l. The concentration procedure used a dialysis filter as the ultrafilter configured for axial flow, either with or without recirculation. The target number of organisms spiked was 1 × 105 of either spores or bacteriophage per 51 l. After concentration, the filters were dissected to retrieve the fibers which were then washed using surfactant solution which was then analyzed for the target organisms. Two washes of the filter fibers were carried out sequentially. For axial flow with recirculation, the first wash produced statistically greater recovery of B. globigii spores (26-40% of spike) compared to the second wash (8-13% of spike). Total recovery (the sum of the recoveries for the first and second washes) ranged from 35 to 53%. Recovery increased as the solids level increased from 0 to 150 mg solids/l. Recovery at the 100 and 150 mg solids/L loadings was statistically higher at the P < .05 level than recovery at 0 mg/L solids. At 150 mg solids/L, axial flow without recirculation (dead end) yielded lower recovery than axial flow with recirculation, however the difference was not significant at the P < .05 level. Recovery of B. globigii at 750 mg solids/L averaged 38% using dead end axial flow. The average recovery of MS2 bacteriophage was 45% at a solids concentration of 150 mg/L using axial flow with recirculation. PhiX174 and Phi8 were also studied, however these bacteriophage appeared to be inactivated in the matrix of concentrated wash water. One hundred liters of water containing 750 mg solids/L was concentrated using dead end axial flow, and only minimal problems with filter clogging were observed. Results described herein suggest axial flow ultrafiltration is an effective concentration method for microorganisms in water containing high levels of particulate matter.

Virus deposition onto polyelectrolyte-coated surfaces: A study with bacteriophage MS2.

HYPOTHESES: By selecting constituent polyelectrolytes and controlling conditions of their deposition, the resulting polyelectrolyte multilayers can be designed as surface coatings with controlled adhesive properties with respect to viruses. Charge and hydrophilicity of the polyelectrolyte multilayers govern virus adhesion. EXPERIMENTS: Four surfaces of different charges and hydrophobicities were designed using a layer-by-layer assembly of poly(styrene-4-sulfonate) and poly(dimethyl diallyl ammonium chloride). Contact angle measurements gave an estimate of MS2 hydrophilicity in terms of free energy of interfacial interaction in water. Experimental results on MS2 adhesion obtained using quartz crystal microbalance with dissipation monitoring were compared with predictions by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. FINDINGS: MS2 deposition onto polyelectrolyte multilayers occurred in two phases: an early phase defined by virus-surface interactions and a later phase with virus-virus interactions controlling deposition kinetics. Principal component analysis showed that the deposition rates in the two phases were independent one of another and that each was correlated to the depth of the secondary minimum of the corresponding XDLVO energy profile. Hydrophobic and electrostatic interactions governed the deposition process: short range hydrophilic repulsion prevented deposition into the primary minimum while electrostatic interactions defined the dependence of the deposition kinetics on the ionic strength. Different surfaces showed distinct kinetics of and capacities for MS2 deposition pointing to the potential of polyelectrolyte multilayers as easy-to-apply coatings for regulating virus adsorption, inactivating viruses via the virucidal action of cationic polyelectrolytes and reducing human exposure to viruses.

Rapid and selective concentration of bacteria, viruses, and proteins using alternating current signal superimposition on two coplanar electrodes.

Dielectrophoresis (DEP) is usually effective close to the electrode surface. Several techniques have been developed to overcome its drawbacks and to enhance dielectrophoretic particle capture. Here we present a simple technique of superimposing alternating current DEP (high-frequency signals) and electroosmosis (EO; low-frequency signals) between two coplanar electrodes (gap: 25 µm) using a lab-made voltage adder for rapid and selective concentration of bacteria, viruses, and proteins, where we controlled the voltages and frequencies of DEP and EO separately. This signal superimposition technique enhanced bacterial capture (Escherichia coli K-12 against 1-µm-diameter polystyrene beads) more selectively (>99%) and rapidly (~30 s) at lower DEP (5 Vpp) and EO (1.2 Vpp) potentials than those used in the conventional DEP capture studies. Nanometer-sized MS2 viruses and troponin I antibody proteins were also concentrated using the superimposed signals, and significantly more MS2 and cTnI-Ab were captured using the superimposed signals than the DEP (10 Vpp) or EO (2 Vpp) signals alone (p < 0.035) between the two coplanar electrodes and at a short exposure time (1 min). This technique has several advantages, such as simplicity and low cost of electrode fabrication, rapid and large collection without electrolysis.

Transport of bacteriophage MS2 and PRD1 in saturated dune sand under suboxic conditions.

Soil passage of (pretreated) surface water to remove pathogenic microorganisms is a highly efficient process under oxic conditions, reducing microorganism concentrations about 8 log10 within tens of meters. However, under anoxic conditions, it has been shown that removal of microorganisms can be limited very much. Setback distances for adequate protection of natural groundwater may, therefore, be too short if anoxic conditions apply. Because removal of microorganisms under suboxic conditions is unknown, this research investigated removal of bacteriophage MS2 and PRD1 by soil passage under suboxic conditions at field scale. At the field location (dune area), one injection well and six monitoring wells were installed at different depths along three suboxic flow lines, where oxygen concentrations ranged from 0.4 to 1.7 mg/l and nitrate concentrations ranged from 13 to 16 mg/L. PRD1 and MS2 were injected directly at the corresponding depths and their removal in each flow line was determined. The highest bacteriophage removal was observed in the top layer, with about 9 log removal of MS2, and 7 log removal of PRD1 after 16 meters of aquifer transport. Less removal was observed at 12 m below surface, probably due to a higher groundwater velocity in this coarser grained layer. MS2 was removed more effectively than PRD1 under all conditions. Due to short travel times, inactivation of the phages was limited and the reported log removal was mainly associated with attachment of phages to the aquifer matrix. This study shows that attachment of MS2 and PRD1 is similar for oxic and suboxic sandy aquifers, and, therefore, setback distances used for sandy aquifers under oxic and suboxic conditions provide a similar level of safety. Sticking efficiency and the attachment rate coefficient, as measures for virus attachment, were evaluated as a function of the physico-chemical conditions.

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity.

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log10 was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log10 loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log10, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.

Evaluation of hospital-grade disinfectants on viral deposition on surfaces after toilet flushing.

BACKGROUND: Past studies have shown that infectious aerosols created during toilet flushing result in surface contamination of the restroom. The goals of this study were to quantify viral contamination of surfaces in restrooms after flushing and the impact of disinfectants added to the toilet bowl prior to flushing on reducing surface contamination. METHODS: The degree of contamination of surfaces in the restroom was assessed with and without the addition of coliphage MS2 to the toilet bowl before flushing. The bowl water and various surfaces in the restroom were subsequently tested for the presence of the virus. RESULTS: The toilet bowl rim, toilet seat top, and toilet seat underside were contaminated in all trials without a disinfectant added to the bowl water before flushing. All disinfectants significantly reduced concentrations on surfaces when the contact time was ≥15 minutes. Hydrogen peroxide resulted in very little reduction of virus in the toilet bowl (<1 log10). Peracetic acid and quaternary ammonium had the greatest log reductions on virus in the organic matter in the toilet. CONCLUSIONS: Toilet flushing resulted in extensive contamination of surfaces within the restroom. Addition of disinfectant to the toilet bowl prior to flushing reduced the level of contamination in the bowl and fomites after flushing.

Evaluation of the suitability of a plant virus, pepper mild mottle virus, as a surrogate of human enteric viruses for assessment of the efficacy of coagulation-rapid sand filtration to remove those viruses.

Here, we evaluated the removal of three representative human enteric viruses - adenovirus (AdV) type 40, coxsackievirus (CV) B5, and hepatitis A virus (HAV) IB - and one surrogate of human caliciviruses - murine norovirus (MNV) type 1 - by coagulation-rapid sand filtration, using water samples from eight water sources for drinking water treatment plants in Japan. The removal ratios of a plant virus (pepper mild mottle virus; PMMoV) and two bacteriophages (MS2 and φX174) were compared with the removal ratios of human enteric viruses to assess the suitability of PMMoV, MS2, and φX174 as surrogates for human enteric viruses. The removal ratios of AdV, CV, HAV, and MNV, evaluated via the real-time polymerase chain reaction (PCR) method, were 0.8-2.5-log10 when commercially available polyaluminum chloride (PACl, basicity 1.5) and virgin silica sand were used as the coagulant and filter medium, respectively. The type of coagulant affected the virus removal efficiency, but the age of silica sand used in the rapid sand filtration did not. Coagulation-rapid sand filtration with non-sulfated, high-basicity PACls (basicity 2.1 or 2.5) removed viruses more efficiently than the other aluminum-based coagulants. The removal ratios of MS2 were sometimes higher than those of the three human enteric viruses and MNV, whereas the removal ratios of φX174 tended to be smaller than those of the three human enteric viruses and MNV. In contrast, the removal ratios of PMMoV were similar to and strongly correlated with those of the three human enteric viruses and MNV. Thus, PMMoV appears to be a suitable surrogate for human enteric viruses for the assessment of the efficacy of coagulation-rapid sand filtration to remove viruses.

Development of an efficient viral aerosol collector for higher sampling flow rate.

Viral aerosol infection through cough generates large amounts of viral aerosol and can result in many adverse health effects such as influenza flu and severe acute respiratory syndrome (SARS). To characterize the coughed viral aerosol, the sampler needs to sample at higher flow rate and possess high physical collection efficiency as well as high viral preservation. However, most current inertia-based high flow bioaerosol samplers are not suited for viral aerosol sampling since the viability will be lost doing the sampling process. Current condensation growth methods only have good physical collection efficiency and viral preservation at low flow rate (< 10 LPM). In this study, we developed a viral aerosol sampling system using a cooler and steam-jet aerosol collector (SJAC) for bioaerosol collection for the first time. The system is based on mixing condensation growth method and has high viral preservation at a higher flow rate (12.5 LPM). We control the inlet aerosol flow temperature and the SJAC mixing reservoir temperature to improve the physical collection efficiency and viability preservation of the viral aerosol. Results indicate that the physical collection efficiency is 70-99% for aerosol 30-100 nm when the aerosol flow and mixing reservoir temperature was 19 and 50 °C, respectively. In addition, the system was 7 and 22 times more efficient for viability preservation of MS2 bacteriophage than the commonly used All Glass Impinger 30 (AGI-30) and BioSampler®, respectively. Finally, the system can be applied to sample at a lower concentration (105 PFU/m3), and results shows the system was 4.7 times more efficient for viability preservation than using AGI-30 alone. The developed viral collection system will improve our understanding of the characteristics of coughed aerosol and can be used for future evaluation of respiratory protective equipment and environmental sampling.

Removal of model viruses, E. coli and Cryptosporidium oocysts from surface water by zirconium and chitosan coagulants.

The present work evaluates the effect of contact filtration, preceded by coagulation with zirconium (Zr) and chitosan coagulants, on model microorganisms and waterborne pathogens. River water intended for potable water production was spiked with MS2 and Salmonella Typhimurium 28B bacteriophages, Escherichia coli, and Cryptosporidium parvum oocysts prior to coagulation. The hygienic performance demonstrated by Zr comprised 3.0-4.0 log10 removal of viruses and 5.0-6.0 log10 removal of E. coli and C. parvum oocysts. Treatment with chitosan resulted in a removal of 2.5-3.0 log10 of viruses and parasites, and 4.5-5.0 log10 of bacteria. A reference coagulant, polyaluminium chloride (PACl), gave a 2.5-3.0 log10 removal of viruses and 4.5 log10 of E. coli. These results indicate that both Zr and chitosan enable adequate removal of microorganisms from surface water. The present study also attempts to assess removal rates of the selected microorganisms with regard to their size and surface properties. The isoelectric point of the Salmonella Typhimurium 28B bacteriophage is reported for the first time. The retention of the selected microorganisms in the filter bed appeared to have some correlation with their size, but the effect of the charge remained unclear.

A carrier state is established in Pseudomonas aeruginosa by phage LeviOr01, a newly isolated ssRNA levivirus.

ssRNA bacteriophages are very abundant but poorly studied, particularly in relation to their effect on bacterial evolution. We isolated a new Pseudomonas aeruginosa levivirus, vB_PaeL_PcyII-10_LeviOr01, from hospital waste water. Its genome comprises 3669 nucleotides and encodes four putative proteins. Following bacterial infection, a carrier state is established in a fraction of the cells, conferring superinfection immunity. Such cells also resist other phages that use type IV pili as a receptor. The carrier population is composed of a mixture of cells producing phage, and susceptible cells that are non-carriers. Carrier cells accumulate phage until they burst, releasing large quantities of virions. The continuous presence of phage favours the emergence of host variants bearing mutations in genes involved in type IV pilus biogenesis, but also in genes affecting lipopolysaccharide (LPS) synthesis. The establishment of a carrier state in which phage particles are continuously released was previously reported for some dsRNA phages, but has not previously been described for a levivirus. The present results highlight the importance of the carrier state, an association that benefits both phages and bacteria and plays a role in bacterial evolution.

Assessment of Healthcare Worker Protocol Deviations and Self-Contamination During Personal Protective Equipment Donning and Doffing.

OBJECTIVE To evaluate healthcare worker (HCW) risk of self-contamination when donning and doffing personal protective equipment (PPE) using fluorescence and MS2 bacteriophage. DESIGN Prospective pilot study. SETTING Tertiary-care hospital. PARTICIPANTS A total of 36 HCWs were included in this study: 18 donned/doffed contact precaution (CP) PPE and 18 donned/doffed Ebola virus disease (EVD) PPE. INTERVENTIONS HCWs donned PPE according to standard protocols. Fluorescent liquid and MS2 bacteriophage were applied to HCWs. HCWs then doffed their PPE. After doffing, HCWs were scanned for fluorescence and swabbed for MS2. MS2 detection was performed using reverse transcriptase PCR. The donning and doffing processes were videotaped, and protocol deviations were recorded. RESULTS Overall, 27% of EVD PPE HCWs and 50% of CP PPE HCWs made ≥1 protocol deviation while donning, and 100% of EVD PPE HCWs and 67% of CP PPE HCWs made ≥1 protocol deviation while doffing (P=.02). The median number of doffing protocol deviations among EVD PPE HCWs was 4, versus 1 among CP PPE HCWs. Also, 15 EVD PPE protocol deviations were committed by doffing assistants and/or trained observers. Fluorescence was detected on 8 EVD PPE HCWs (44%) and 5 CP PPE HCWs (28%), most commonly on hands. MS2 was recovered from 2 EVD PPE HCWs (11%) and 3 CP PPE HCWs (17%). CONCLUSIONS Protocol deviations were common during both EVD and CP PPE doffing, and some deviations during EVD PPE doffing were committed by the HCW doffing assistant and/or the trained observer. Self-contamination was common. PPE donning/doffing are complex and deserve additional study. Infect Control Hosp Epidemiol 2017;38:1077-1083.

A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.

The detection of aerosolized viruses can serve as an important surveillance and control tool in agriculture, human health, and environmental settings. Here, we adapted an anion exchange resin-based method, initially developed to concentrate negatively charged viruses from water, to liquid impingement-based bioaerosol sampling. In this method, aerosolized viruses are collected in a 20ml liquid sample contained within widely used impingers, BioSamplers (SKC Inc., Eighty Four, PA), and further concentrated via adsorption to an anion exchange resin that is suspended within this liquid. Viral nucleic acids are then extracted from the resin to facilitate molecular analyses through a reduction in the effective sample volume. For this study, various quantities of two negatively charged viruses, type A and type B influenza viruses (FluMist Quadrivalent vaccine) and the male-specific (F+) RNA coliphage MS2 (MS2), were nebulized into a custom-built bioaerosolization chamber, and sampled using BioSamplers with and without anion exchange resin. Compared to direct testing of the BioSampler liquid, detection was improved by 6.77× and 3.33× for type A and type B influenza viruses, respectively, by using the anion exchange resin. For MS2, the anion exchange resin method allowed for an average improvement in detection of 8.26×.

Comparison of internal process control viruses for detection of food and waterborne viruses.

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.