Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples.

Hygiene of drinking water is periodically controlled by cultivation and enumeration of indicator bacteria. Rapid and comprehensive measurements of emerging pathogens are of increasing interest to improve drinking water safety. In this study, the feasibility to detect bacteriophage PhiX174 as a potential indicator for virus contamination in large volumes of water is demonstrated. Three consecutive concentration methods (continuous ultrafiltration, monolithic adsorption filtration, and centrifugal ultrafiltration) were combined to concentrate phages stepwise from 1250 L drinking water into 1 mL. Heterogeneous asymmetric recombinase polymerase amplification (haRPA) is applied as rapid detection method. Field measurements were conducted to test the developed system for hygiene online monitoring under realistic conditions. We could show that this system allows the detection of artificial contaminations of bacteriophage PhiX174 in drinking water pipelines.

On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/µL, 1 GU/µL, and 5 × 10(3) GU/µL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

Probing effects of pressure release on virus capture during virus filtration using confocal microscopy.

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure.

Effects of a pressure release on virus retention with the Ultipor DV20 membrane.

Recent data have shown that a temporary release in the transmembrane pressure can cause a significant increase in virus transmission through a number of commercial virus filters. The objective of this work was to study the effect of a pressure release on retention of the bacteriophage ΦX174 by the Ultipor DV20 membrane, with phage capture within the membrane visualized by confocal microscopy. Phage challenge tests showed a significant transient increase in phage transmission (typically by an order of magnitude) immediately after a pressure release. This transient increase was not due to any damage to the filter or to the presence of a back-flow (from permeate to feed). Confocal images demonstrated that the pressure release caused the migration of previously captured bacteriophage further into the depth of the filter. Data were analyzed using a modified internal polarization model, with the results providing important insights into the factors controlling virus retention during virus filtration.

Transport and removal of bacteriophages MS2 and PhiX174 in steel slag-amended soils: column experiments and transport model analyses.

The aim of this study was to investigate the removal of bacteriophages MS2 and PhiX174 in soils amended with converter furnace steel slag. Column experiments were performed to examine the bacteriophage removal in slag-amended (slag content: 0%, 25%, and 50%) loam soils. For comparison, column experiments were also conducted with Escherichia coli. In addition, chloride (Cl) was used as a conservative tracer to determine transport characteristics. Results showed mass recoveries of Cl of 98.6 +/- 3.5%, indicating that the experiments were conducted successfully. The mass recovery of MS2 was 86.7% in no slag (100% soil), decreasing to 0% in slag contents of 25% and 50%. The mass recovery of PhiX174 decreased from 87.8% to 51.5% with increasing slag content from 0% to 50%. In the case of E. coli, the mass recoveries decreased from 47.0% to 10.5% with increasing slag content from 0% to 50%. In the transport models analyses, the HYDRUS-1D code was used to quantify the sorption parameters from breakthrough curves. For the 100% soil column, a one-site kinetic sorption model was fitted to the data, whereas a two-site kinetic sorption model was fitted for slag-amended (25% and 50% slag) soil data. Results demonstrate that the addition of steel slag to soil enhances the removal of bacteriophages due to the presence of FeO in the steel slag. However, CaO could not contribute to the bacteriophage removal in our experimental conditions because the effluent pH (7.7-8.9) in slag-amended (25% and 50% slag) soils was not high enough to promote the bacteriophage inactivation.

Improved virus removal in ceramic depth filters modified with MgO.

Ceramic filters, working on the depth filtration principle, are known to improve drinking water quality by removing human pathogenic microorganisms from contaminated water. However, these microfilters show no sufficient barrier for viruses having diameters down to 20 nm. Recently, it was shown that the addition of positively charged materials, for example, iron oxyhydroxide, can improve virus removal by adsorption mechanisms. In this work, we modified a common ceramic filter based on diatomaceous earth by introducing a novel virus adsorbent material, magnesium oxyhydroxide, into the filter matrix. Such filters showed an improved removal of about 4-log in regard to bacteriophages MS2 and PhiX174. This is explained with the electrostatic enhanced adsorption approach that is the favorable adsorption of negatively charged viruses onto positively charged patches in an otherwise negatively charged filter matrix. Furthermore, we provide theoretical evidence applying calculations according to Derjaguin-Landau-Verwey-Overbeek theory to strengthen our experimental results. However, modified filters showed a significant variance in virus removal efficiency over the course of long-term filtration experiments with virus removal increasing with filter operation time (or filter aging). This is explained by transformational changes of MgO in the filter upon contact with water. It also demonstrates that filter history is of great concern when filters working on the adsorption principles are evaluated in regard to their retention performance as their surface characteristics may alter with use.

Hygienic quality of artificial greywater subjected to aerobic treatment: a comparison of three filter media at increasing organic loading rates.

With a growing world population, the lack of reliable water sources is becoming an increasing problem. Reusing greywater could alleviate this problem. When reusing greywater for crop irrigation it is paramount to ensure the removal of pathogenic organisms. This study compared the pathogen removal efficiency of pine bark and activated charcoal filters with that of conventional sand filters at three organic loading rates. The removal efficiency of Escherichia coli O157:H7 decreased drastically when the organic loading rate increased fivefold in the charcoal and sand filters, but increased by 2 log10 in the bark filters. The reduction in the virus model organism coliphage phiX174 remained unchanged with increasing organic loading in the charcoal and sand filters, but increased by 2 log10 in the bark filters. Thus, bark was demonstrated to be the most promising material for greywater treatment in terms of pathogen removal.

Mg/Al layered double hydroxide for bacteriophage removal in aqueous solution.

The objective of this study was to investigate the removal of bacteriophages in Mg/Al layered double hydroxide (LDH). Batch experiments were performed with bacteriophage MS2 in a powder form of Mg/Al LDH under various LDH doses. Column experiments were also performed under flow-through condition with bacteriophages MS2 and phiX174 in Mg/Al LDH immobilized on sand surfaces. Batch tests demonstrated that the powder form of Mg/Al LDH was effective in removing MS2 with the removal capacity of 2.2 × 10(8) plaque forming unit (pfu)/g under the given experimental conditions (LDH dose = 2 g/L; initial MS2 concentration = 4.61 × 10(5) pfu/mL). Column experiments showed that the log removal of phiX174 was 4.40 in columns containing 100% Mg/Al LDH-coated sand while it was 0.05 in 100% quartz sand. These findings indicated that Mg/Al LDH-coated sand was effective in removing bacteriophages compared with sand. A more than 4 log removal (=5.44) of MS2 was achieved in 100% Mg/Al LDH-coated sand. This study demonstrates the potential application of Mg/Al LDH for virus removal in water treatment.

Adenovirus, MS2 and PhiX174 interactions with drinking water biofilms developed on PVC, cement and cast iron.

Biofilms colonizing pipe surfaces of drinking water distribution systems could provide habitat and shelter for pathogenic viruses present in the water phase. This study aims (i) to develop a method to detect viral particles present in a drinking water biofilm and (ii) to study viral interactions with drinking water biofilms. A pilot scale system was used to develop drinking water biofilms on 3 materials (7 cm(2) discs): PVC, cast iron and cement. Biofilms were inoculated with viral model including MS2, PhiX174 or adenovirus. Five techniques were tested to recover virus from biofilms. The most efficient uses beef extract and glycine at pH = 9. After sonication and centrifugation, the pH of the supernatant is neutralized prior to viral analysis. The calculated recovery rates varied from 29.3 to 74.6% depending on the virus (MS2 or PhiX174) and the material. Applying this protocol, the interactions of virus models (MS2 and adenovirus) with drinking water biofilms were compared. Our results show that adsorption of viruses to biofilms depends on their isoelectric points, the disc material and the hydrodynamic conditions. Applying hydrodynamic conditions similar to those existing in drinking water networks resulted in a viral adsorption corresponding to less than 1% of the initial viral load.

Ozone efficacy for the control of airborne viruses: Bacteriophage and norovirus models.

This study was designed to test the efficacy of an air treatment using ozone and relative humidity (RH) for the inactivation of airborne viruses. Four phages (φX174, PR772, MS2 and φ6) and one eukaryotic virus (murine norovirus MNV-1) were exposed to low ozone concentrations (1.23 ppm for phages and 0.23 ppm for MNV-1) and various levels of RH for 10 to 70 minutes. The inactivation of these viruses was then assessed to determine which of the tested conditions provided the greatest reduction in virus infectivity. An inactivation of at least two orders of magnitude for φX174, MS2 and MNV-1 was achieved with an ozone exposure of 40 minutes at 85% RH. For PR772 and φ6, exposure to the reference condition at 20% RH for 10 minutes yielded the same results. These findings suggest that ozone used at a low concentration is a powerful disinfectant for airborne viruses when combined with a high RH. Air treatment could therefore be implemented inside hospital rooms ventilated naturally.

Design of salt-tolerant membrane adsorbers for viral clearance.

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50-150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage PhiX174 (pI approximately 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand-binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance-processing step compared to traditional strong anion exchange membrane adsorbers.

Ceramic media amended with metal oxide for the capture of viruses in drinking water.

Ceramic materials that can adsorb and/or inactivate viruses in water may find widespread application in low-tech drinking-water treatment technologies in developing countries, where porous ceramic filters and ceramic granular media filters are increasingly promoted for that purpose. We examined the adsorption and subsequent inactivation of bacteriophages MS2 and (phiX-174 on five ceramic media in batch adsorption studies to determine media suitability for use in a ceramic water filter application. The media examined were a kaolinitic ceramic medium and four kaolinitic ceramic media amended with iron or aluminium oxides that had been incorporated into the kaolinitic clays before firing. Batch adsorption tests indicate increased sorption and inactivation of surrogate viruses by media amended with Fe and Al oxide, with FeOOH-amended ceramic inactivating all bacteriophages up to 8 log10. Unmodified ceramic was a poor adsorbent of bacteriophages at less than 1 log10 adsorption-inactivation and high recovery of sorbed phages. These studies suggest that contact with ceramic media, modified with electropositive Fe or Al oxides, can reduce bacteriophages in waters to a greater extent than unmodified ceramic.

Comparison of the Filter Efficiency of Medical Nonwoven Fabrics against Three Different Microbe Aerosols.

　Exact evaluation of the performance of surgical masks and biohazard protective clothing materials against pathogens is important because it can provide helpful information that healthcare workers can use to select suitable materials to reduce infection risk. Currently, to evaluate the protective performance of nonwoven fabrics used in surgical masks against viral aerosols, a non-standardized test method using phi-X174 phage aerosols is widely performed because actual respiratory viruses pose an infection risk during testing and the phage is a safe virus to humans. This method of using a phage is simply modified from a standard method for evaluation of filter performance against bacterial aerosols using Staphylococcus aureus, which is larger than virus particles. However, it is necessary to perform such evaluations based on the size of the actual pathogen particles. Thus, we developed a new method that can be performed safely using inactivated viral particles and can quantitate the influenza virus in aerosols by antigen-capture ELISA (Shimasaki et al., 2016a) . In this study, we used three different microbial aerosols of phi-X174 phage, influenza virus, and S. aureus and tested the filter efficiency by capturing microbial aerosols for two medical nonwoven fabrics. We compared the filter efficiency against each airborne microbe to analyze the dependency of filter efficiency on the microbial particle size. Our results showed that against the three types of spherical microbe particles, the filter efficiencies against influenza virus particles were the lowest and those against phi-X174 phages were the highest for both types of nonwoven fabrics. The experimental results mostly corresponded with theoretical calculations. We conclude that the filter efficiency test using the phi-X174 phage aerosol may overestimate the protective performance of nonwoven fabrics with filter structure compared to that against real pathogens such as the influenza virus.

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.

Evaluation of the efficiency of medical masks and the creation of new medical masks.

The effectiveness of medical masks in preventing respiratory infection was investigated by testing bacterial leakage, filtration efficiency, respiratory resistance and oxygen concentration of the enclosed space. Polypropylene (PP) fibres were treated with dimethyldioctadecylammonium bromide to impart a positive electrical charge capable of attracting bacteria. The fluffed PP fibres were used to make a polypropylene mask and to edge standard surgical and N-95 respirators to prevent leakage. A PP napkin was created by melting and blowing PP. The PP edging seal dramatically reduced bacterial leakage of standard masks and was more effective than adhesive paper tape edging in reducing respiratory resistance. Bacterial or viral filtration efficiency was almost 100% for the PP mask and the PP napkin. The specially designed PP mask with a synthetic adhesive at the edge of the mask may be more effective than the standard surgical mask and the N-95 respirator. The PP napkin is an important tool in preventing the spread of pathogens.

Investigation of enteric adenovirus and poliovirus removal by coagulation processes and suitability of bacteriophages MS2 and φX174 as surrogates for those viruses.

We evaluated the removal of enteric adenovirus (AdV) type 40 and poliovirus (PV) type 1 by coagulation, using water samples from 13 water sources for drinking water treatment plants in Japan. The behaviors of two widely accepted enteric virus surrogates, bacteriophages MS2 and φX174, were compared with the behaviors of AdV and PV. Coagulation with polyaluminum chloride (PACl, basicity 1.5) removed AdV and PV from virus-spiked source waters: the infectious AdV and PV removal ratios evaluated by means of a plaque-forming-unit method were 0.1-1.4-log10 and 0.5-2.4-log10, respectively. A nonsulfated high-basicity PACl (basicity 2.1) removed infectious AdV and PV more efficiently than did other commercially available PACls (basicity 1.5-2.1), alum, and ferric chloride. The MS2 removal ratios tended to be larger than those of AdV and PV, partly because of differences in the hydrophobicities of the virus particles and the sensitivity of the virus to the virucidal activity of PACl; the differences in removal ratios were not due to differences in the surface charges of the virus particles. MS2, which was more hydrophobic than the other viruses, was inactivated during coagulation with PACl. Therefore, MS2 does not appear to be an appropriate surrogate for AdV and PV during coagulation. In contrast, because φX174, like AdV and PV, was not inactivated during coagulation, and because the hydrophobicity of φX174 was similar to or somewhat lower than the hydrophobicities of AdV and PV, the φX174 removal ratios tended to be similar to or somewhat smaller than those of the enteric viruses. Therefore, φX174 is a potential conservative surrogate for AdV and PV during coagulation. In summary, the surface hydrophobicity of virus particles and the sensitivity of the virus to the virucidal activity of the coagulant are probably important determinants of the efficiency of virus removal during coagulation.

The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices.

The collection of waterborne pathogen occurrence data often requires the concentration of microbes from large volumes of water due to the low number of microorganisms that are typically present in environmental and drinking waters. Hollow-fiber ultrafiltration (HFUF) has shown promise in the recovery of various microorganisms. This study has demonstrated that the HFUF primary concentration method is effective at recovering bacteriophage φX174, poliovirus, enterovirus 70, echovirus 7, coxsackievirus B4 and adenovirus 41 from large volumes of tap and river water with an average recovery of all viruses of 73.4% and 81.0%, respectively. This study also evaluated an effective secondary concentration method using celite for the recovery of bacteriophage and enteric viruses tested from HFUF concentrates of both matrices. Overall, the complete concentration method (HFUF primary concentration plus celite secondary concentration) resulted in a concentration factor of 3333 and average recoveries for all viruses from tap and river waters of 60.6% and 60.0%, respectively.

Rapid and reproducible infectivity end-point titration of virulent phage in a microplate system.

The standard method for measuring the number of infectious phages in solution has traditionally been the plaque forming assay. An alternative method is described where the number of lytic, infectious phages is determined in an endpoint titration assay adapted for a microplate system. In this model system, susceptible Escherichia coli B6 at a density of 4 x 10(7) cells/ml, were mixed with an equal volume (100 microl) of PhiX174 diluted serially in a microtest plate. After 3h of incubation on a microplate shaker the endpoint was determined spectrophotometrically and calculated according to the method of Reed and Muench. A well was considered positive for infection if the OD630-value was < or = 10% compared to the OD630-value of the negative control of uninfected cells. ID50-titers were 2.5x higher than the PFU-titers (CV 15%) and the intra assay reproducibility revealed a CV of 9%. The method has several advantages as compared with the conventional PFU-titration. It is less time and material consuming with the possibility to assess several samples at the same time.

Performance of a membrane adsorber for trace impurity removal in biotechnology manufacturing.

Membrane adsorbers provide an attractive alternative to traditional bead-based chromatography columns used to remove trace impurities in downstream applications. A linearly scalable novel membrane adsorber family designed for the efficient removal of trace impurities from biotherapeutics, are capable of reproducibly achieving greater than 4 log removal of mammalian viruses, 3 log removal of endotoxin and DNA, and greater than 1 log removal of host cell protein. Single use, disposable membrane adsorbers eliminate the need for costly and time consuming column packing and cleaning validation associated with bead-based chromatography systems, and minimize the required number and volume of buffers. A membrane adsorber step reduces process time, floor space, buffer usage, labor cost, and improves manufacturing flexibility. This "process compression" effect is commonly associated with reducing the number of processing steps. The rigid microporous structure of the membrane layers allows for high process flux operation and uniform bed consistency at all processing scales.

Enteric Viral Surrogate Reduction by Chitosan.

Enteric viruses are a major problem in the food industry, especially as human noroviruses are the leading cause of nonbacterial gastroenteritis. Chitosan is known to be effective against some enteric viral surrogates, but more detailed studies are needed to determine the precise application variables. The main objective of this work was to determine the effect of increasing chitosan concentration (0.7-1.5% w/v) on the cultivable enteric viral surrogates, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and bacteriophages (MS2 and phiX174) at 37 °C. Two chitosans (53 and 222 kDa) were dissolved in water (53 kDa) or 1% acetic acid (222 KDa) at 0.7-1.5%, and were then mixed with each virus to obtain a titer of ~5 log plaque-forming units (PFU)/mL. These mixtures were incubated for 3 h at 37 °C. Controls included untreated viruses in phosphate-buffered saline and viruses were enumerated by plaque assays. The 53 kDa chitosan at the concentrations tested reduced FCV-F9, MNV-1, MS2, and phi X174 by 2.6-2.9, 0.1-0.4, 2.6-2.8, and 0.7-0.9 log PFU/mL, respectively, while reduction by 222 kDa chitosan was 2.2-2.4, 0.8-1.0, 2.6-5.2, and 0.5-0.8 log PFU/mL, respectively. The 222 kDa chitosan at 1 and 0.7% w/v in acetic acid (pH 4.5) caused the greatest reductions of MS2 by 5.2 logs and 2.6 logs, respectively. Overall, chitosan treatments showed the greatest reduction of MS2, followed by FCV-F9, phi X174, and MNV-1. These two chitosans may contribute to the reduction of enteric viruses at the concentrations tested but would require use of other hurdles to eliminate food borne viruses.