Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

Although the isolation and counting of small extracellular vesicles (sEVs) are essential steps in sEV research, an integrated method with scalability and efficiency has not been developed. Here, we present a scalable and ready-to-use extracellular vesicle (EV) isolation and counting system (EVics) that simultaneously allows isolation and counting in one system. This novel system consists of (i) EVi, a simultaneous tandem tangential flow filtration (TFF)-based EV isolation component by applying two different pore-size TFF filters, and (ii) EVc, an EV counting component using light scattering that captures a large field-of-view (FOV). EVi efficiently isolated 50-200 nm-size sEVs from 15 µL to 2 L samples, outperforming the current state-of-the-art devices in purity and speed. EVc with a large FOV efficiently counted isolated sEVs. EVics enabled early observations of sEV secretion in various cell lines and reduced the cost of evaluating the inhibitory effect of sEV inhibitors by 20-fold. Using EVics, sEVs concentrations and sEV PD-L1 were monitored in a 23-day cancer mouse model, and 160 clinical samples were prepared and successfully applied to diagnosis. These results demonstrate that EVics could become an innovative system for novel findings in basic and applied studies in sEV research.

Effect of in-line filtration in newborns: study protocol of the Intravenous Neonatal Central Access Safety (INCAS) randomized controlled trial.

BACKGROUND: Particulate contamination due to infusion therapy (administration of parenteral nutrition and medications) carries a potential health risk for infants in neonatal intensive care units (NICUs). This particulate consists of metals, drug crystals, glass fragments, or cotton fibers and can be generated by drug packaging, incomplete reconstitution, and chemical incompatibilities. In-line filters have been shown to remove micro-organisms, endotoxin, air, and particles in critically ill adults and older infants, but its benefits in newborn remain to be demonstrated. Moreover, 50% of inflammatory episodes in the setting of NICUs are blood culture-negative. These episodes could be partly related to the presence of particles in the infusion lines. METHODS: A multicenter randomized single-blind controlled trial was designed. All infants admitted to NICUs for which prolonged infusion therapy is expected will be enrolled in the study and randomized to the Filter or Control arm. All patients will be monitored until discharge, and data will be analyzed according to a "full analysis set." The primary outcome is the frequency of patients with at least one sepsis-like event, defined by any association of suspected sepsis symptoms with a level of c-reactive protein (CRP) > 5 mg/L in a negative-culture contest. The frequency of sepsis, phlebitis, luminal obstruction, and the duration of mechanical ventilation and of catheter days will be evaluated as secondary outcomes. The sample size was calculated at 368 patients per arm. DISCUSSION: This is the first multicenter randomized control trial that compares in-line filtration of parenteral nutrition and other intravenous drugs to infusion without filters. Sepsis-like events are commonly diagnosed in clinical practice and are more frequent than sepsis in a positive culture contest. The risk of these episodes in the target population is estimated at 30-35%, but this data is not confirmed in the literature. If the use of in-line filters results in a significant decrease in sepsis-like events and/or in any other complications, the use of in-line filters in all intravenous administration systems may be recommended in NICUs. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05537389, registered on 12 September 2022 ( https://classic. CLINICALTRIALS: gov/ct2/show/results/NCT05537389?view=results ).

Commercial reverse osmosis point-of-use systems in Egypt failed to purify tap water.

This study addresses the heightened global reliance on point-of-use (PoU) systems driven by water quality concerns, ageing infrastructure, and urbanization. While widely used in Egypt, there is a lack of comprehensive evaluation of these systems. We assessed 10 reverse osmosis point-of-use systems, examining physicochemical, bacteriological, and protozoological aspects of tap water (inlets) and filtered water (outlets), adhering to standard methods for the examination of water and wastewater. Results showed significant reductions in total dissolved solids across most systems, with a decrease from 210 ± 23.6 mg/L in tap water to 21 ± 2.8 mg/L in filtered water for PoU-10. Ammonia nitrogen levels in tap water decreased from 0.05 ± 0.04 to 2.28 ± 1.47 mg/L to 0.02 ± 0.04 to 0.69 ± 0.64 mg/L in filtered water. Despite this, bacterial indicators showed no significant changes, with some systems even increasing coliform levels. Protozoological analysis identified prevalent Acanthamoeba (42.5%), less frequent Naegleria (2.5%), Vermamoeba vermiformis (5%), and potentially pathogenic Acanthamoeba genotypes. Elevated bacterial indicators in filtered water of point-of-use systems, combined with essential mineral removal, indicate non-compliance with water quality standards, posing a public health concern. Further research on the long-term health implications of these filtration systems is essential.

Simultaneous SERS-decoding detection of multiple pathogens in drinking water with home-made portable double-layer filtration and concentration device.

The engineering of a home-made portable double-layer filtration and concentration device with the common syringe for rapid analysis of water samples is reported. The core elements of the device were two installed filtration membranes with different pore sizes for respective functions. The upper filtration membrane was used for preliminary intercepting large interfering impurities (interception membrane), while the lower filtration membrane was used for collecting multiple target pathogens (enrichment membrane) for determination. This combination can make the contaminated environmental water, exemplified by surface water, filtrated quickly through the device and just retained the target bacteria of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes on the lower enrichment membrane. Integrating with surface-enhanced Raman spectra (SERS) platform to decode the SERS-Tags (SERS-TagCVa, SERS-TagR6G, and SERS-TagMB) already labeled on each of the enriched bacteria based the antibody-mediated immuno-recognition effect, fast separation, concentration, and detection of multiple pathogenic bacteria from the bulk of contaminated environmental water were realized. Results show that within 30 min, all target bacteria in the lake water can be simultaneously and accurately measured in the range from 101 to 106 CFU mL-1 with detection limit of 10.0 CFU mL-1 without any pre-culture procedures. This work highlights the simplicity, rapidness, cheapness, selectivity, and the robustness of the constructed method for simultaneous detecting multiple pathogens in aqueous samples. This protocol opens a new avenue for facilitating the development of versatile analytical tools for drinking water and food safety monitoring in underdeveloped or developing countries.

Method for fabricating a mesh ripple filter for charged-particle therapy.

Objective.A simple, low-cost ripple filter consisting of multiple mesh sheets (mRiFi) was previously developed to reproducibly widen the Bragg peak of heavy-ion beams. To fabricate the mRiFi, the mRiFi parameters such as the wire material, wire diameter, wire spacing, and number of mesh sheets had to be determined. However, it was unclear how these parameters contribute to shifting and widening of the Bragg peak as well as to lateral spreading of the beam passing through the mRiFi. The purposes of this study were to clarify the contributions and to propose a recipe for fabricating a mRiFi with the desired performance values.Approach. We established an analytical calculation method to estimate shifting and widening of the Bragg peak and lateral spreading of heavy-ion beams passing through the mRiFi for given mRiFi parameter values. We also performed Monte Carlo simulations to validate the analytical calculation method. The recipe for fabricating the mRiFi with desired performances was established based on the analytical calculation method. Using the recipe, we fabricated the mRiFi for multi-ion therapy and evaluated its performance through demonstration experiments with helium-, carbon-, oxygen-, and neon-ion beams.Main results.The difference between the results of the Monte Carlo simulation and the analytical calculation was less than 0.4 mm for the peak shift, 0.15 mm for the peak width, and less than 0.11 mm for the lateral beam size which validated the analytical calculation method. The experimentally observed shift and width of the Bragg peak were consistent with the analytical calculations.Significance.We proposed a method to determine mRiFi parameters for fabricating a mRiFi with a desired performance, i.e. adequate widening of the Bragg peak with an acceptable peak shift and lateral beam spread. The proposed method allows anyone to fabricate a simple and low-cost mRiFi satisfying desired specifications.

Implications of the operation of continuous granular activated carbon filters on the effluent quality.

Granular activated carbon (GAC) filtration is a commonly used method for advanced wastewater treatment. Filters can be operated continuously or discontinuously, with continuous operation not requiring feed flow interruption for backwashing and circulation (B/C). This study investigated the influence of B/C on the effluent quality of continuous filters. Two continuous GAC filters were operated for 1.5 years, with analysis of dissolved substances and particulate matter in the influent and effluent. The results indicated that various B/C modes had no impact on the removal of dissolved organic carbon and organic micropollutants (OMP), achieving an OMP removal of over 70% after 5,600 treated bed volumes (m3 treated wastewater per m3 GAC). However, it was evident that continuous B/C over 2-4 h resulted in increased turbidity, total suspended solids over 30 mg/L and total phosphorus concentrations of 1.3 mg/L in the filter effluent. Additionally, the study demonstrated that longer and more intensive B/C processes resulted in GAC size degradation with AC concentrations of up to 6.9 mg/L in the filter effluent, along with a change in GAC particle size. Furthermore, the importance of pre-filtration in reducing particulate matter in the filter influent and decreasing hydraulic head loss could be demonstrated.

Lifestraw Family water filters in low- and middle-income countries: a systematic review and meta-analysis to define longer-term public health impact against childhood diarrhoea and inform scale-up.

Background: Diarrhoeal disease disproportionately affects children <5 years in low- and middle-income countries (LMICs). The pathogens responsible for diarrhoea are commonly transmitted through faecally-contaminated drinking water. Lifestraw Family point-of-use water filters have been the subject of intervention studies for over a decade and were the first filters evaluated by the World Health Organization in its water treatment evaluation scheme to provide comprehensive protection against many diarrhoea-causing pathogens. This systematic review aimed to: 1) report on aspects related to physical environment and implementation and 2) conduct an updated meta-analysis on Lifestraw Family filter effectiveness against childhood diarrhoea based on studies with ≥12 months of follow-up. Methods: We conducted a literature search in November 2022 using MEDLINE, Embase, Cochrane, and CINAHL databases. Inclusion criteria were: 1) RCTs, cluster-RCTs, quasi-experimental, or matched cohort studies on 2) Lifestraw Family 1.0 or 2.0 filters 3) conducted in LMICs 4) that evaluated filter effectiveness against diarrhoea in children <5 and 5) analysed ≥12 months of follow-up data on clinical effectiveness against diarrhoea and were 6) published from 2010 with 7) full-text availability in English. A modified Newcastle-Ottawa Scale was used to assess risk of bias. Relative risk (RR) and 95% confidence intervals (CIs) were extracted and analysed using a random-effects meta-analysis. Results: We included 6 studies in LMICs involving 4740 children <5. Of the four clinically-effective interventions, common characteristics were access to improved water sources (75%), the 2.0 version of the filter or the 1.0 version with additional water storage (100%), use of behaviour change theory, community engagement, and health messaging (75%), local filter repair-and-replace mechanisms (75%), and specially-trained local interventionists (100%). The meta-analysis showed a 30% reduction in diarrhoea risk in the intervention group (RR = 0.69; 95% CI = 0.52-0.91, P = 0.01). Conclusions: Lifestraw Family water filters can be effective interventions to reduce diarrhoea in vulnerable paediatric populations for at least one year, though certain aspects related to physical environment and implementation may increase their public health impact. The findings of this study suggest considerations for scale-up that can be applied in settings in need of longer-term interim solutions until universal access to safe drinking water is achieved.

Residual risk of Pseudomonas aeruginosa waterborne contamination in an intensive care unit despite the presence of filters at all water points-of-use.

OBJECTIVE: To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa from a water network colonized by a single genotype [sequence type (ST) 299] despite the presence of antimicrobial filters in a medical intensive care unit (ICU). METHODS: During the first 19-month period since the ICU opened, contamination of the water network was assessed monthly by collecting water upstream of the filters. Downstream water was also sampled to assess the efficiency of the filters. P. aeruginosa isolates from patients were collected and compared with the waterborne ST299 P. aeruginosa by multiplex-rep polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events by other genotypes of P. aeruginosa were also assessed. RESULTS: Overall, 1.3% of 449 samples of filtered water were positive for P. aeruginosa in inoculum, varying between 1 and 104 colony-forming units/100 mL according to the tap. All P. aeruginosa hydric isolates belonged to ST299 and displayed fewer than two single nucleotide polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in five patients showed identical profiles to the hydric ST299 clone on both multiplex-rep PCR and PFGE, and differed by an average of fewer than five SNPs, confirming the water network reservoir as the source of contamination by P. aeruginosa for 4.09% of patients. Cross-transmission events by other genotypes of P. aeruginosa were responsible for the contamination of 1.75% of patients. DISCUSSION/CONCLUSION: Antimicrobial filters are not sufficient to protect patients from waterborne pathogens when the water network is highly contaminated. A microbiological survey of filtered water may be needed in units hosting patients at risk of P. aeruginosa infections, even when all water points-of-use are fitted with filters.

A self-cleaning micro-fluidic chip biospired by the filtering system of manta rays.

Size-based particle filtration has become indispensable in numerous biomedical and environmental applications. In this study, bioinspired by the filter-feeding mechanism (lobe filtration) of manta rays, we designed a U-shaped biomimetic gill rake filter that combined lobe filtration and Dean flow to filter monodisperse suspensions, bi-disperse suspensions and yeast cells. Compared with other equipment using the inertial focusing technology, our equipment can perform high-throughput (up to 8 mL min-1) and high-efficiency filtration of particles (maximum filtration efficiencies of 96.08% and 97.14% for 10 and 15 µm monodisperse suspensions at the optimum flow rate of 6 mL min-1). The complex velocity field of the micro-fluidic flow within the filter is numerically simulated, and in combination with experiments, a threshold for the flow rate is identified. When the inlet flow rate exceeds the threshold value, the efficiency of particle filtration is increased rapidly. Afterwards, by analysing the filtration mechanism, we develop three novel filtration processes. The equilibrium positions of the particles and yeast cells in the main channel are close to the outer wall at high flow rate, which diminishes the likelihood of particles and yeast cells entering the side channel. This configuration establishes a self-cleaning mechanism, ensuring prolonged and efficient operation of the filter with high-throughput processing. Furthermore, the influence of the filter lobe angle and channel width on the filtration efficiency and outlet flow rate ratio are explored, and an optimisation plan is prepared.

A mica filter enables bacterial enrichment from large volumes of natural water for sensitive monitoring of pathogens by nanopore sequencing.

Nanopore sequencing is extremely promising for the high-throughput detection of pathogenic bacteria in natural water; these bacteria may be transmitted to humans and cause waterborne infectious diseases. However, the concentration of pathogenic bacteria in natural water is too low to be detected directly by nanopore sequencing. Herein, we developed a mica filter to enrich over 85% of bacteria from > 10 L of natural water in 100 min, which led to a 102-fold improvement in the assay limits of the MinION sequencer for assessing pathogenic bacteria. Correspondingly, the sequencing time of S. Typhi detection at a concentration as low as 105 CFU/L was reduced from traditional 48 h to 3 h. The bacterial adsorption followed pseudo-first-order kinetics and the successful adsorption of bacteria to the mica filter was confirmed by scanning electron microscopy and Fourier infrared spectroscopy et al. The mica filter remained applicable to a range of water samples whose quality parameters were within the EPA standard limits for freshwater water. The mica filter is thus an effective tool for the sensitive and rapid monitoring of pathogenic bacteria by nanopore sequencing, which can provide timely alerts for waterborne transmission events.

Versatile filter membrane for effective sampling and real-time quantitative detection of airborne pathogens.

Construction of air filter membranes bearing prominent collecting and transferring capability is highly desirable for detecting airborne pathogens but remains challenging. Here, a hyaluronic acid air filter membrane (HAFM) with tunable heterogeneous micro-nano porous structures is straightforwardly constructed through the ethanol-induced phase separation strategy. Airborne pathogens can be trapped and collected by HAFM with high performance due to the ideal trade-off between removal efficiency and pressure drop. By exempting the sample elution and extraction processes, the HAFM after filtration sampling can not only directly disperse on the agar plate for colony culture but also turn to an aqueous solution for centrifugal enrichment, which significantly reduces the damage and losses of the captured microorganisms. The following combination with ATP bioluminescence endows the HAFM with a real-time quantitative detection function for the captured airborne pathogens. Benefiting from high-efficiency sampling and non-traumatic transfer of airborne pathogens, the real-world bioaerosol concentration can be facilely evaluated by the HAFM-based ATP assay. This work thus not only provides a feasible strategy to fabricate air filter membranes for efficient microbial collection and enrichment but also sheds light on designing advanced protocols for real-time detection of bioaerosols in the field.

Fiber-in-Tube Electrifiable Structure for Virus Filtration Self-Generated Static Electricity by Vibration/Sound.

Fiber has been considered as an ideal material for virus insulation due to the readily available electrostatic adsorption. However, restricted by the electrostatic attenuation and filtration performance decline, their long-lasting applications are unable to satisfy the requirements of medical protective equipment for major medical and health emergencies such as global epidemics, which results in both a waste of resources and environmental pollution. We overcame these issues by constructing a fiber-in-tube structure, achieving the robust reusability of fibrous membranes. Core fibers within the hollow could form generators with tube walls of shell fibers to provide persistent, renewable static electricity via piezoelectricity and triboelectricity. The PM0.3 insulation efficiency achieved 98% even after 72 h of humidity and heat aging, through beating and acoustic waves, which is greatly improved compared with that of traditional nonwoven fabric (∼10% insulation). A mask spun with our fiber also has a low breathing resistance (differential pressure <24.4 Pa/cm2). We offer an approach to enrich multifunctional fiber for developing electrifiable filters, which make the fiber-in-tube filtration membrane able to durably maintain a higher level of protective performance to reduce the replacement and provide a new train of thought for the preparation of other high-performance protective products.

A Novel 3D-Printed Gravity-Independent Air-Eliminating Filter for Rapid Intravenous Infusions.

INTRODUCTION: The absence of a consistent downward G vector can make separation of gases from liquids challenging, such as in field medicine without stable upright equipment or during spaceflight. This limits the use of medical equipment and procedures like administration of intravenous (IV) fluids in microgravity and can make field medicine hazardous. Administering IV fluids and medications in microgravity requires a technique to separate air from the liquid phase. Current commercial filters for separation of gases are incompatible with high flow and blood. We present a novel filter designed to provide adequate air clearance without a consistent downward G vector.METHODS: Inline air-eliminating filters were designed for use with IV fluid tubing in microgravity using computer-aided design software and printed using nylon 12 on an EOS Selective Laser Sintering 3D printer. A 0.2-µm membrane filter was adhered around a central, hollow pillar with external spiral baffles allowing separation and venting of air from the fluid. Results were compared against commercially available inline air-eliminating filters.RESULTS: The 3D-printed filters outperformed the commercial filters in both percentage of air removed and flow rates. The centrifugal, baffled filter had flow rates that far exceeded the commercial filters during rapid transfusion.DISCUSSION: IV fluid administration is an often underappreciated and a necessary basic requirement for medical treatment. An air-eliminating filter compatible with blood and rapid transfusion was developed and validated with crystalloid solutions to allow the successful administration of IV fluid and medication without a consistent downward G vector.Formanek A, Townsend J, Ottensmeyer MP, Kamine TH. A novel 3D-printed gravity-independent air-eliminating filter for rapid intravenous infusions. Aerosp Med Hum Perform. 2024; 95(6):327-332.

Description of a 'plankton filtration bias' in sequencing-based bacterial community analysis and of an Arduino microcontroller-based flowmeter device that can help to resolve it.

Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias.

An Immiscible-Phase Filtration Device for Isolation, Amplification, and Detection of Nucleic Acids for Clinical Diagnostics.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

Basics of Sterile Compounding: Sterilization Methods in Sterile Product Manufacturing.

Sterilization methods to produce sterile preparations include heat, gas, radiation, and filtration. This article focuses on heat, gas, and radiation sterilization, plus a brief introduction to bright-light sterilization. Microbiology basics and microbial death kinetics, key to understanding why these sterilization methods work, will also be briefly discussed. Filtration sterilization will be covered in a separate article.

Advancement of membrane separation technology for organic pollutant removal.

In the face of growing global freshwater scarcity, the imperative to recycle and reuse water becomes increasingly apparent across industrial, agricultural, and domestic sectors. Eliminating a range of organic pollutants in wastewater, from pesticides to industrial byproducts, presents a formidable challenge. Among the potential solutions, membrane technologies emerge as promising contenders for treating diverse organic contaminants from industrial, agricultural, and household origins. This paper explores cutting-edge membrane-based approaches, including reverse osmosis, nanofiltration, ultrafiltration, microfiltration, gas separation membranes, and pervaporation. Each technology's efficacy in removing distinct organic pollutants while producing purified water is scrutinized. This review delves into membrane fouling, discussing its influencing factors and preventative strategies. It sheds light on the merits, limitations, and prospects of these various membrane techniques, contributing to the advancement of wastewater treatment. It advocates for future research in membrane technology with a focus on fouling control and the development of energy-efficient devices. Interdisciplinary collaboration among researchers, engineers, policymakers, and industry players is vital for shaping water purification innovation. Ongoing research and collaboration position us to fulfill the promise of accessible, clean water for all.

Loss of micropollutants on syringe filters during sample filtration: Machine learning approach for selecting appropriate filters.

Prefiltration before chromatographic analysis is critical in the monitoring of environmental micropollutants (MPs). However, in an aqueous matrix, such monitoring often leads to out-of-specification results owing to the loss of MPs on syringe filters. Therefore, this study investigated the loss of seventy MPs on eight different syringe filters by employing Random Forest, a machine learning algorithm. The results indicate that the loss of MPs during filtration is filter specific, with glass microfiber and polytetrafluoroethylene filters being the most effective (<20%) compared with nylon (>90%) and others (regenerated-cellulose, polyethersulfone, polyvinylidene difluoride, cellulose acetate, and polypropylene). The Random Forest classifier showed outstanding performance (accuracy range 0.81-0.95) for determining whether the loss of MPs on filters exceeded 20%. Important factors in this classification were analyzed using the SHapley Additive exPlanation value and Kruskal-Wallis test. The results show that the physicochemical properties (LogKow/LogD, pKa, functional groups, and charges) of MPs are more important than the operational parameters (sample volume, filter pore size, diameter, and flow rate) in determining the loss of most MPs on syringe filters. However, other important factors such as the implications of the roles of pH for nylon and pre-rinsing for PTFE syringe filters should not be ignored. Overall, this study provides a systematic framework for understanding the behavior of various MP classes and their potential losses on syringe filters.

Standard addition method for rapid, cultivation-independent quantification of Legionella pneumophila cells by qPCR in biotrickling filters.

Cultivation-independent molecular biological methods are essential to rapidly quantify pathogens like Legionella pneumophila (L. pneumophila) which is important to control aerosol-generating engineered water systems. A standard addition method was established to quantify L. pneumophila in the very complex matrix of process water and air of exhaust air purification systems in animal husbandry. Therefore, cryopreserved standards of viable L. pneumophila were spiked in air and water samples to calibrate the total bioanalytical process which includes cell lysis, DNA extraction, and qPCR. A standard addition algorithm was employed for qPCR to determine the initial concentration of L. pneumophila. In mineral water, the recovery rate of this approach (73%-134% within the concentration range of 100-5000 Legionella per mL) was in good agreement with numbers obtained from conventional genomic unit (GU) calibration with DNA standards. In air samples of biotrickling filters, in contrast, the conventional DNA standard approach resulted in a significant overestimation of up to 729%, whereas our standard addition gave a more realistic recovery of 131%. With this proof-of-principle study, we were able to show that the molecular biology-based standard addition approach is a suitable method to determine realistic concentrations of L. pneumophila in air and process water samples of biotrickling filter systems. Moreover, this quantification strategy is generally a promising method to quantify pathogens in challenging samples containing a complex microbiota and the classical GU approach used for qPCR leads to unreliable results.

Comparison of Plenum and Cage-level Filter Exhaust Dust PCR Testing to Soiled Bedding Sentinel Mice (Mus musculus) on an IVC Rack.

The use of soiled-bedded sentinels (SBSs) has historically been the standard for colony health surveillance monitoring at our institution. With the advent of newer technologies in which dust collected from filters is tested by PCR, we compared traditional SBS with PCR testing of both exhaust air dust collected from a filter in the downstream vertical plenum (exhaust dust test [EDT]) and the SBS cage-level exhaust filter (SCEF). Our hypothesis was that both methods of filter testing would identify more pathogens than SBS testing. Twenty-five individually ventilated mouse racks that used disposable caging were sanitized and placed into rotation. Rack plenums were tested by PCR to verify negative results before the study start. Exhaust dust collection media were placed in the exhaust plenum (n = 25). SBS cages were placed on each side of the rack with 2 mice per cage (n = 42 mice), with the remaining cage slots occupied by research animals. At each triweekly cage change, the exhaust air filters were carefully removed from the cage top, placed in sterile 50-mL conical tubes, and pooled for submission. After 3mo, the SBS mice were tested via serology for bacterial and viral agents and by PCR for Helicobacter species, pinworms, and ectoparasites. In addition, the EDT filter and SCEF were collected for PCR to evaluate for the same agents. Our results indicate that the SCEF consistently detected agents more frequently than the EDT filter placed in the plenum and that the EDT filter media detected agents more frequently than did the SBS mice. Our data suggest that both PCR methods of detection are superior to SBS for individually ventilated disposable rodent cages and that the SCEF is superior to EDT. These data supported our movement of institution toward environmental monitoring as a method of rodent colony health surveillance.