Use of semi-permeable bag materials to facilitate on-site treatment of biological agent-contaminated waste.

Clean up following the wide-area release of a persistent biological agent has the potential to generate significant waste. Waste containing residual levels of biological contaminants may require off-site shipment under the U.S. Department of Transportation's (US DOT) solid waste regulations for Category A infectious agents, which has packaging and size limitations that do not accommodate large quantities. Treating the waste on-site to inactivate the bio-contaminants could alleviate the need for Category A shipping and open the possibility for categorizing the waste as conventional solid waste with similar shipping requirements as municipal garbage. To collect and package waste for on-site treatment, a semi-permeable nonwoven-based fabric was developed. The fabric was designed to contain residual bio-contaminants while providing sufficient permeability for penetration by a gaseous decontamination agent. The nonwoven fabric was tested in two bench-scale experiments. First, decontamination efficacy and gas permeability were evaluated by placing test coupons inoculated with spores of a Bacillus anthracis surrogate inside the nonwoven material. After chlorine dioxide fumigation, the coupons were analyzed for spore viability and results showed a ≥6 Log reduction on all test materials except glass. Second, filters cut from the nonwoven material were tested in parallel with commercially available cellulose acetate filters having a known pore size (0.45 µm) and results demonstrate that the two materials have similar permeability characteristics. Overall, results suggest that the nonwoven material could be used to package waste at the point of generation and then moved to a nearby staging area where it could be fumigated to inactivate bio-contaminants.

Evaluation of disinfection methods for personal protective equipment (PPE) items for reuse during a pandemic.

The COVID-19 pandemic resulted in many supply chain issues, including crippling of essential personal protective equipment (PPE) needed for high-risk occupations such as those in healthcare. As a result of these supply chain issues, unprecedented crisis capacity strategies were implemented to divert PPE items such as filtering facepiece respirators (FFRs, namely N95s) to those who needed them most for protection. Large-scale methods for decontamination were used throughout the world to preserve these items and provided for their extended use. The general public also adopted the use of non-specialized protective equipment such as face coverings. So, the need for cleaning, decontamination, or disinfection of these items in addition to normal clothing items became a necessary reality. Some items could be laundered, but other items were not appropriate for washing/drying. To fill research gaps in small-scale, non-commercial cleaning and disinfection, this bench-scale research was conducted using small coupons (swatches) of multiple PPE/barrier protection materials inoculated with virus (non-pathogenic bacteriophages Phi6 and MS2) and tested against a range of decontamination methods including bleach-, alcohol- and quaternary ammonium compound (QAC)-based liquid sprays, as well as low concentration hydrogen peroxide vapor (LCHPV) and bench-scale laundering. In general, non-porous items were easier to disinfect than porous items, and the enveloped virus Phi6 was overall easier to inactivate than MS2. Multiple disinfection methods were shown to be effective in reducing viral loads from PPE coupons, though only laundering and LCHPV were effective for all materials tested that were inoculated with Phi6. Applications of this and follow-on full-scale research are to provide simple effective cleaning/disinfection methods for use during the current and future pandemics.

Impact of filter material and holding time on spore sampling efficiency in water.

Bacillus anthracis and other environmentally persistent pathogens pose a significant threat to human and environmental health. If contamination is spread over a wide area (e.g. resulting from a bioterrorism or biowarfare incident), readily deployable and scalable sample collection methods will be necessary for rapidly developing and implementing effective remediation strategies. A recent surge in environmental (eDNA) sampling technologies could prove useful for quantifying the extent and levels of contamination from biological agents in environmental and drinking water. In this study, three commonly used membrane filtration materials (cellulose acetate, cellulose nitrate, and nylon) were evaluated for spore filtration efficiency, yielding recoveries from 17%-68% to 25%-117% for high and low titer samples, respectively, where cellulose nitrate filters generated the highest recoveries. A holding time test revealed no statistically significant differences between spore recoveries when analyzed at the specified timepoints, suggesting that eDNA filter sampling techniques can yield and maintain a relatively high recovery of spores for an extended period of time between filtration and analysis without a detrimental impact on spore recoveries. The results shown here indicate that emerging eDNA technologies could be leveraged for sampling following a wide-area contamination incident and for other microbiological water sampling applications.

Determining Viral Disinfection Efficacy of Hot Water Laundering.

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the coronavirus disease 2019 (COVID-19) pandemic, it is intended to be a framework, adaptable to other virus disinfection studies; it demonstrates the steps for preparing the test virus, inoculating the test material, assessing visual and integrity changes to the washed items due to the laundering process, and quantifying the reduction in viral load. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the personal protective equipment (PPE) items after multiple laundering cycles. The representative results presented with the protocol use the Phi6 bacteriophage inoculated onto cotton scrub, denim, and cotton face-covering materials and indicate that the hot water laundering and drying process achieved over a 3-log (99.9%) reduction in viral load for all samples (a 3-log reduction is the disinfectant performance metric in U.S. Environmental Protection Agency's Product Performance Test Guideline 810.2200). The reduction in viral load was uniform across different locations on the PPE items. The results of this viral disinfection efficacy testing protocol should help the scientific community explore the effectiveness of home laundering for other types of test viruses and laundering procedures.

Virucidal efficacy of antimicrobial surface coatings against the enveloped bacteriophage Φ6.

AIMS: Antimicrobial coatings, for use in combination with routine cleaning and disinfection, were evaluated for their effectiveness in reducing virus concentration on stainless steel surfaces. METHODS: Twenty antimicrobial coating products, predominantly composed of organosilane quaternary ammonium compounds, were applied to stainless steel coupons, dried overnight and evaluated for efficacy against Φ6, an enveloped bacteriophage. Additionally, two peel and stick polymer-based films, a copper-based film and three copper alloys were evaluated. Efficacy was determined by comparison of recoveries from uncoated (positive control) and coated (test) surfaces. RESULTS: The results indicated that some of the coating products initially demonstrated >3-log reduction of Φ6; no direct correlation of efficacy was observed with an active ingredient or its concentration. The peel and stick films and copper alloys each demonstrated efficacy in initial testing. However, none of the spray-based products retained efficacy after subjecting the coating to abrasion with either a hypochlorite or quaternary ammonium-based solution applied in accordance with EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings. Of the products tested for this durability, only one peel and stick polymeric film retained efficacy; the copper alloys were not tested for their durability in this study. CONCLUSIONS: These results suggest that while some organosilane quaternary ammonium compound-based products demonstrate antiviral efficacy, more research and development is needed to understand effective formulations with sufficient durability to perform as supplements to routine cleaning and disinfection.

Decontamination of soil contaminated at the surface with Bacillus anthracis spores using dry thermal treatment.

In the event of a large, aerosol release of Bacillus anthracis spores in a major metropolitan area, soils and other outdoor materials may become contaminated with the biological agent. A study was conducted to assess the in-situ remediation of soil using a dry thermal treatment approach to inactivate a B. anthracis spore surrogate inoculated into soil samples. The study was conducted in two phases, using loam, clay and sand-based soils, as well as biological indicators and spore-inoculated stainless-steel coupons. Initial experiments were performed in an environmental test chamber with temperatures controlled between 80 and 110 °C, with and without added humidity, and with contact times ranging from 4 h to 7 weeks. Tests were then scaled up to assess the thermal inactivation of spores in small soil columns, in which a heating plate set to 141 °C was applied to the soil surface. These column tests were conducted to assess time requirements to inactivate spores as a function of soil depth and soil type. Results from the initial phase of testing showed that increasing the temperature and relative humidity reduced the time requirements to achieve samples in which no surrogate spores were detected. For the test at 80 °C with no added humidity, 49 days were required to achieve soil samples with no spores detected in clay and loam. At 110 °C, 24 h were required to achieve samples in which no spores were detected. In the column tests, no spores were detected at the 2.5 cm depth at four days and at the 5.1 cm depth at 21 days, for two of the three soils. The experiments described in the study demonstrate the feasibility of using dry thermal techniques to decontaminate soils that have been surficially contaminated with B. anthracis spores.

Comparison of surface sampling methods for an extended duration outdoor biological contamination study.

Bacillus anthracis, the causative agent for anthrax, is a dangerous pathogen to humans and has a history as a bioterrorism agent. While sampling methods have been developed and evaluated for characterizing and clearing contaminated indoor sites, the performance of these sampling methods is unknown for use in outdoor environments. This paper presents surface sampling data for Bacillus atrophaeus spores, a surrogate for B. anthracis, from a 210-day outdoor study that evaluated the detection and recovery of spores using five different sampling methods as follows: sponge sticks, 37-mm vacuum filter cassettes, residential wet vacuums, robotic floor cleaners, and grab samples of soil, leaves, and grass. The spores were applied by spraying a liquid suspension onto the surfaces. Both asphalt and concrete surfaces were sampled by all the surface sampling methods, excluding grab sampling. Stainless steel coupons placed outdoors were additionally sampled using sponge sticks. Sampling methods differed in their ability to collect detectable spores over the duration of the study. The 37-mm vacuums and sponge sticks consistently detected spores on asphalt through day 37 and robots through day 99. The wet vacuums detected spores on asphalt for days 1 and 4, but not again until day 210. On concrete, all samplers detected spores until day 210 except for sponge stick samplers that detected spores only up until the day 99 time point. For all sampling methods, spore recoveries were higher from concrete than from asphalt surfaces. There was no statistically significant difference in recoveries of sponge sticks and 37-mm vacuums from either asphalt or concrete surfaces. Processing of grab samples was challenging due to non-target background microorganisms resulting in high detection limits for the samples.

Alternative fast analysis method for cellulose sponge surface sampling wipes with low concentrations of Bacillus Spores.

Environmental sampling is a critical component of the post decontamination verification process following a bioterrorism event. The current work was performed to produce a less labor-intensive method for processing cellulose sponge-wipes used for sampling areas potentially contaminated with low concentrations (i.e., post-decontamination) of Bacillus anthracis spores. An alternative fast-analysis processing method was compared to the processing protocol validated by the Centers for Disease Control and Prevention (CDC) for the Laboratory Response Network (LRN). Glazed tile coupons (1102 cm2) were inoculated with 50, 500, or 5000 spores of Bacillus thuringiensis subsp. kurstaki (Btk), then sampled with cellulose sponges. Sampling was limited to a 25- by 25-cm area and performed in the same manner as the CDC sampling method. Samples were then processed using either the alternative "Fast Analysis" method or the "CDC method". Three different analysts repeated the tests at each concentration utilizing each method. Mean recoveries, labor time, and potentially hazardous waste produced were compared for the two methods. The mean percent recoveries and standard errors for the samples processed using the "CDC method" were 39.9 ±â€¯6.7, 43 ±â€¯7.6, and 36.8 ±â€¯10.1 for the 5000, 500, and 50 spore loading levels, respectively; compared to 54.2 ±â€¯12.9, 64.2 ±â€¯21.7, and 45.2 ±â€¯8.6 for the "Fast Analysis" method. At each titer tested the "Fast Analysis" method resulted in a statistically significant higher percent recovery. Furthermore, analysts processed samples utilizing the "Fast Analysis" method in less than half the time and generated half as much potentially hazardous waste compared to the "CDC method".

Expression of ERG Protein and TMRPSS2-ERG Fusion in Prostatic Carcinoma in Egyptian Patients.

AIM: Prostate cancer (PCa) is the second most common cancers in men worldwide. Its incidence can be influenced by several risk factors including genetic susceptibility. Therefore the search for the expression of a certain gene (ERG) and its rearrangement could give us clues for proper identification of PCa. And the study of ERG expression and its comparison to FISH in Egyptian patients can show whether ERG immunophenotype could be used instead of FISH, as it is cheaper. MATERIALS AND METHODS: This study was performed on 85 cases of PCa, showing 30 cases with HGPIN and 30 cases of prostatic hyperplasia. All were immunohistochemistry stained using ERG monoclonal rabbit antihuman antibody was used (clone: EP111). FISH analysis was performed in 38 biopsies of PCa cases to detect TMRPSS2-ERG rearrangement using the FISH ZytoLight TriCheck Probe (SPEC TMRPSS2-ERG). RESULTS: ERG expression was found in 26% of PCa cases and 20% of HGPIN cases. FISH analysis showed fusion of 21 cases of PCa (out of 22 cases showing ERG immunoexpression). CONCLUSION: Our findings emphasise that only malignant and pre-malignant cells and not benign cells from the prostate stain positive. ERG expression may offer a simpler, accurate and less costly alternative for evaluation of ERG fusion status in PCa.

Expression of FGFR3 Protein and Gene Amplification in Urinary Bladder Lesions in Relation to Schistosomiasis.

BACKGROUND: Bladder cancer represents the fifth most common malignancy worldwide and a major cause of cancer-related morbidity and death. Incidence and mortality rates have remained relatively constant over the past four decades. Urothelial bladder cancers have identified multiple risk factors. AIM: We aimed at evaluating the expression of the FGFR3 protein and gene amplification in the urothelial cells of neoplastic and non-neoplastic urothelial lesions of the urinary bladder, and correlation with tumour grade, stage and associated bilharziasis. MATERIAL AND METHODS: One hundred and five different urinary bladder lesions were studied, including 15 cystitis cases (9 bilharzial and 6 non-bilharzial cystitides), 75 urothelial carcinoma cases (18 bilharzial associated and 57 non-bilharzial associated) and 15 squamous cell carcinoma associated with bilharziasis, beside 5 control cases. Data concerning age, sex, tumour grade, stage, and associated bilharziasis were obtained. Each case was studied for FGFR3 expression, and FISH technique was applied on forty malignant cases that show high protein expression. RESULTS: The highest incidence of cystitis was in the fourth decade while of bladder cancer was in the seventh decade. Tumour grade was correlated significantly with tumour stage. FGFR3 correlates significantly with tumour grade, stage and with a bilharzial infestation. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. CONCLUSIONS: FGFR3 overexpression in malignant cases was significantly higher than in chronic cystitis. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. FGFR3 may be further studied as a subject for target therapy of bladder cancer.

The pharmacological approach to reverse portal hypertention and hepatic schistosomal fibrosis in Egypt, control experimental study.

Schistosoma mansoni is the most prevalent cause of liver fibrosis in Egypt. It is characterized by hepatocyte damage, inflammation and chronic parasite egg-induced granuloma formation leading to fibrosis. Its management, particularly fibrosis, has focused primarily on treating and preventing the complications of portal hypertension. Unfortunately, there is no therapy that has been proved to prevent progressive hepatic fibrosis which is associated with a significant morbidity and mortality due to granulomatous hypersensitivity to parasite eggs. However, recent developments in understanding hepatic fibrogenesis confirm that recovery from advanced fibrosis is possible. There is a considerable imperative to develop anti-fibrotic strategies that are applicable to liver fibrosis. It was noted that a marked increase in the amount of different interstitial collagens types are associated with the development of fibrotic liver diseases. Meanwhile, it has been suggested that as long as the relative portions of liver collagen are still within the normal limits, the fibrosis may still be reversible. If it exceeds the normal limits fibrogenesis will proceed to its end stage, even if the etiological agent is removed. Collagen type IV and procollagen type III are two of the most accurate fibrosis markers which allow reliable non-invasive diagnosis. The T lymphocytes and the immuno-regulatory cytokines may be important in the host response to S. mansoni granuloma formation and fibrosis. Chronic parasite egg-induced granuloma formation can lead to fibrosis, which is immunologically characterized by the dominant Th2 response. Corticosteroids and prostaglandins interfere with both efferent and afferent mechanisms of immune function. These data indicate that this adjuvant therapy can be a candidate for therapeutic intervention in hepatic fibrosis through induction of a balance between Th1 and Th2 cells response as will be documented by the fibrosis markers. One hundred S. mansoni infected hamsters (150-250 gm) were obtained from the BRPU-TBRI (5 groups, 20 hamsters each). Treatment was started 10 weeks post infection. First G (20 hamsters) was neither infected nor treated, second G. was infected but untreated, third group infected and PZQ treated, forth G. infected and PZQ and MP treated and fifth group infected and PZQ and PgE1 treated. Samples (liver and blood) were obtained 20 weeks post infection. The serum level of: liver functions, procollagen type III, collagen type IV & Th1 cytokine (IL-2) and Th2 cytokine (IL-10) were performed. Histopathology was performed to study liver fibrosis, measuring the proliferate activity of the hepatocytes using cell image analyzer system and granuloma cells using the indirect immuno-histochemistry by monoclonal antibody proliferating cell nuclear antigen (PCNA). In this study, G.V showed high significant reduction in granuloma size, type and percentage of fibrosis and significant elevation in percentage of degenerated ova compared to Gs.III & IV. The proliferation index measured using PCNA showed high proliferative activity of hepatocytes in non treated group which declined in the treated Gs.III, IV & V. The proliferation activity of hepatocytes and granuloma forming cells decreased significantly in G.V compared to G.IV. There was a significant reduction in liver function tests even tendency for normalization in G.V compared to group III and IV. Procollagen type III and collagen type IV were significantly low in the serum in G.V compared to Gs.III & IV. Th1 (IL-2) level was significantly high in G.V compared to Gs.III, IV and Th2 (IL-10) was significantly low in G.V compared to Gs III & IV indicating the low amount of fibrosis was in the group treated with PZQ PgE1.PgE1 with PZQ to treat S. mansoni infected hamsters can modulate liver fibrosis and improves the liver function tests up to normalization. The balance between Th1 and Th2 cytokines level could be modulated to help reverse or decrease fibrosis in S. mansoni infected hamsters. This may pave the way for clinical application as combined therapy PZQ and PgE1 may by an effective approach to reverse hepatic fibrosis in schistosomiasis by the induction of dominant Th1 response.