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Disruptive Activity of Acetic Acid of Salmonella enterica Serovar Typhimurium and Escherichia coli O157 Biofilms Developed on Eggshells and Industrial Surfaces

2 Replies

DOI: 10.31038/MIP.2024511

Abstract

Communities of enteropathogenic microorganisms adhere as biofilms to both natural and artificial surfaces encountered by eggs and chickens during production, constituting a major source of food cross-contamination. Given the rising bacterial resistance to chemical sanitary agents and antibiotics, there is a need to explore alternative approaches, particularly using natural products, to control the proliferation of these microorganisms along the surfaces of the poultry production chain. This study investigates and compares the bactericidal and antibiofilm properties of acetic, citric, and lactic acids against Salmonella enterica serovar Typhimurium and Escherichia coli O157 cells. Biofilms were allowed to develop on eggshells, stainless steel, and polystyrene surfaces at temperatures of 22°C and 37°C, and subsequently exposed to the acids for durations of 2 and 24 hours. The three organic acids exhibited varying degrees of reduction in planktonic, swarmer, and biofilm cells. Notably, acetic acid consistently produced the most promising outcomes, resulting in a reduction between 3 and 6.6 Log10 in the quantities of young and mature biofilm cells adhered to eggshells or stainless steel. Additionally, a decrease of 1 and 2.5 optical density units was observed in biofilms formed on the polystyrene surface. Overall, these findings suggest that acetic acid can effectively act as an anti-biofilm agent, disrupting both newly formed and matured biofilms formed under conditions encountered along the production chain of eggs and broilers.

Keywords

Food-contamination, Bactericidal, Organic acids, Enteropathogenic bacteria, Poultry production

Introduction

Foodborne pathogens, such as Salmonella enterica Serovar Typhimurium (S. Typhimurium) and E. coli O157, linked to poultry production and the food industry, are major concerns in global gastroenteritis outbreaks affecting humans. According to the USA Centers for Disease Control and Prevention, these pathogens contribute to 76 million infections, 325.000 hospitalizations, and 5.000 deaths annually in the USA alone [1]. In Colombia, a South American country, the Colombian National Institute of Health-Sivigila reported a total of 9.781 cases of foodborne illnesses involving 679 outbreaks in 2017 [2]. Despite the inherent protective physical and chemical barriers in eggs, research reveals that S. Typhimurium, E. coli O157, and other enteropathogenic bacteria can contaminate and infect them. Eggs typically become contaminated through three general routes: before oviposition, when the reproductive organs suffer an infection, and secondly, by encountering feces or contaminated surfaces [3,4]. Accumulating evidence illustrates that S. Typhimurium and E. coli O157, through the formation of biofilms, not only colonize eggs but also surfaces throughout the production chain (Figure. 1). This contamination of surfaces may result in the transmission of these pathogens, posing significant risks to public health [4-6].

fig 1

Figure 1: Areas and surfaces of the Colombian poultry production chain at risk for contamination by biofilms formed by enteropathogenic bacteria. Numbers highlight the different steps at which eggs and chickens can be contaminated by enteropathogenic bacteria. Italic letters indicate the places or utensils that may be made of stainless steel or polystyrene from which cross-contamination of eggs and chickens can occur.

Several studies have demonstrated how Salmonella and E. coli strains that are common causes of human gastroenteritis presented firm attachment of their bacterial cells to the eggshell surface and several types of foods and plants of production surfaces, facilitating the formation of biofilms [7-9]. The formation of a biofilm comprises several distinct steps. First, the initial reversible adsorption of cells onto the surface. Second, production of surface polysaccharides or capsular material occurs followed by the formation of an extracellular polymeric matrix. At this stage, biofilm cells form a strong attachment to the surface. In the following steps, the biofilm architecture is developed and matured. The process ends with the liberation of single motile cells that disperse into the environment and initiate again the process [10]. Biofilm formation is known to be influenced by several environmental cues, such as as availability and concentration of nutrients, and the physicochemical parameters of the of the surrounding environment, such as temperature and the material composition of the surface [11]. The surface type can influence microbial interactions among pathogens and promote co-biofilm formation, increases in individual pathogen biomass, and cell activity [12]. By nature, biofilm structure allows microbes to resist chemical or biological sanitizers, while bacterial cells are more vulnerable during the planktonic state and in a short contact time than when sequestered and protected in biofilms. Bacteria cells within biofilms are more resistant to environmental stresses, such as desiccation and UV light exposure, as well as to host-mediated responses, such as phagocytes [13]. Bacterial biofilms are more resistant to antimicrobial agents than are free-living cells, which makes it difficult to eradicate pathogens from surfaces commonly used in the poultry industry [5].

With the rise in the occurrence of foodborne outbreaks associated with poultry production, there is increasing interest in the use of novel biocide applications to prevent or reduce microbial contamination in food industries. The viability of microbes on food contact surfaces varies according to the biofilm state and formation ability, as well as the type of surface. Biofilm formation from the highest to lowest degree follows the order of eggshell > rubber > stainless steel > plastic [14,15]. As reported by Lee [15], rinsing surfaces with water, even extensively, appears to have limited effect on reducing S. Typhimurium biofilm viability. The regular application of cleaning and disinfecting procedures are common strategies employed to control pathogen establishment on industrial equipment [16]. Importantly, Chemical sanitizer efficacy can significantly depend on surface types, bacterial strains, and relative humidity [17]. Therefore, such procedures may not be fully effective in impeding or disrupting biofilms and can induce the formation and persistence of resistant phenotypes [18].

Novel alternatives, such as natural compounds extracted from bacterial cultures or aromatic plants, as well as organic acids, are currently under evaluation for their potential in eradicating biofilms. These compounds may exhibit high lethality against pathogens, efficiently penetrate the structure of a biofilm, and degrade easily in the environment [16]. Organic acids are generally recognized as safe (GRAS) by the USA Federal Drug Administration (FDA) and have been documented to possess antimicrobial activities against different pathogens [5]. In studies involving antibiotic-resistant bacteria, Clary [19] demonstrated how low concentrations (5%) of acetic acid rapidly killed (30 min) planktonic cells of Mycobacterium abscessus. On the other hand, Bradhan [20] demonstrated that lactic acid can decrease viable cell counts of planktonic as well as biofilm-forming cells of multiple carbapenem-hydrolyzing, multi-drug-resistant Klebsiella pneumoniae strains. Acetic acid demonstrated antimicrobial effectiveness on both smooth and rough cell morphotypes. Besides directly affecting bacterial cell viability, organic acids can also influence the electrochemical properties of the attachment surface, leading to an effective antimicrobial outcome [21].

An antimicrobial mechanism of organic acids, such as citric acid, acetic acid, and lactic acid, involves decreasing the environmental pH, creating unfavorable growth conditions for pathogenic bacteria [22]. Weak acids like acetic acid, when at a pH lower than their pKa and in their undissociated form, have shown the ability to reduce biofilm formation by permeating the biofilm structure and inner cell membrane. Kundukad [23] demonstrated that these weak acids, including acetic acid, could effectively eliminate bacteria without harming human cells if the pH remains close to their pKa. Organic acids in their undissociated form possess lipophilic properties, enabling them to diffuse across bacterial cell membranes, thereby disrupting cell function upon reaching the cell interior [5].

Research focusing on evaluating alternative treatments and methods to control S. Typhimurium and E. coli O157 biofilm formation on surfaces along the egg and other animal-derived food production chains is crucial to reduce cross-contamination. Accordingly, the present study aimed to assess the efficacy of organic acids in: 1) controlling biofilm formation by S. Typhimurium and E. coli O157 during the initial stages of development, and 2) disrupting mature biofilms. Eggshells, stainless steel, and polystyrene were utilized to simulate potential soiling surfaces encountered by eggs and broilers throughout the production chain. Two temperatures were assessed as key environmental variables: 22°C, representing the mean environmental temperature of the largest broiler-producing region in Colombia, and 37°C, simulating the optimal growth temperature of these pathogens. Additionally, to track the impact of exposure time and the potential development of resistance, the biofilms were subjected to organic acids for 2 and 24 hours.

Materials and Methods

Bacterial Strains and Growth Conditions

Bacterial strains used in this study were S. Typhimurium ATCC 14028 (American Type Culture Collection, Manassas, VA., USA) and E. coli O157 strain AGROSAVIA_CMSABV_Ec-col-B-001-2007 from the Animal Health collection of the AGROSAVIA Microbial Germplasm Bank (Mosquera, Cundinamarca, Colombia). The bacteria were grown on nutrient agar (Merck, Darmstadt, Germany) or Luria Bertani low salt agar (LBL) (peptone (ThermoFisher, Waltham, Massachusetts, USA) at 10 g.L-1, yeast extract (Merck) at 5 g.L-1, sodium chloride (Merck) at g.L-1, agar (Merck) at g.L-1. When required, LBL agar was acidified to pH 3 with 0.3% (v/v) acetic acid (Merck), 0.2% (v/v) citric acid (Merck), or 0.2% (v/v) lactic acid (Merck). For biofilm assays, LBL broth (LBL without agar) was used.

Growth Curves

S. Typhimurium and E. coli O157 were aerobically grown on LBL agar plates at 37°C for 24 h. The inocula were prepared by scraping the surface of the agar plates following the addition of 10 mL of LBL broth at pH 7 or acidified to pH 3 with acetic (0.3% v/v), citric (0.2% v/v) or lactic acid (0.2% v/v). These cell suspensions were adjusted to an OD at 600 nm of 0.1 (2.2 × 103 colony-forming units (cfu).mL-1) or 1.8 (2.8 × 109 103 cfu.mL-1). Three bacterial suspensions (n=3) per treatment and the control at an initial OD of 0.1 or 1.8 were incubated aerobically at 37°C for 48 h with constant shaking at 140 rpm. Every 2 h, 1-mL aliquots of the bacterial cultures were taken, and 10-fold serial dilutions and plating on LBL agar were made to determine Log10 cfu.mL-1 at each time.

Surface Spreading Assays

S. Typhimurium and E. coli O157 were grown aerobically in 5 mL of LBL broth at 37°C until reaching an optical density (OD) at 600 nm of 1 (16 h). Then, 1 mL of each culture was concentrated 10-fold by centrifugation at 4.400 × g for 5 min at room temperature. The pellets were suspended in 100 µL of LBL broth. The concentration of inocula for S. Typhimurium was 8.25 × 1010 and for E. coli O157 was 1.05 × 1011 cfu.mL-1. Semi-solid agar surface spreading plates were prepared as described by Amaya [24] with 20 mL of LBL broth containing 8% (w/v) of glucose and 0.6% (w/v) of agar and if required acidified with acid acetic (0.3% v/v), citric acid (0.2% v/v), or lactic acid (0.2% v/v). A 5-µL drop of the suspended bacteria was placed in the center of the plates (n=10) and allowed to air-dry for 10 min. The plates were inverted and incubated aerobically for 24 h at 37°C. The areas of the spreading colonies were measured with ImageJ software 1.52a (Wayne Rasband, National Institute of Health, Bethesda, MD, USA) by delimiting the coly area using the shaped and measured tools.

Disruption of Newly Formed Biofilms Developed on Eggshells and Stainless Steel

S. Typhimurium and E. coli O157 were grown aerobically on LBL agar plates at 37°C for 24 h. The inocula of the pathogens were prepared by scraping the cell mass grown from the surface of the plates and washing twice in 2 mL of LBL broth at pH 7 or LBL broth at pH 3, acidified with acid acetic (0.3% v/v), citric acid (0.2% v/v), or lactic acid (0.2% v/v), and centrifugation at 4.400 × g for 5 min at room temperature. Washed cells were suspended in 20 mL of the respective media. The OD of each suspension was adjusted to 1.8 at 600 nm (5 × 109 cfu.mL-1). Then, six 1-cm2 pieces of eggshell or stainless steel for each treatment, which were sterilized by autoclave at 15 lb of pressure and 121°C for 20 min, were weighted and covered with 5 mL of each bacterial suspension in 15-mL Falcon tubes. Negative controls contained each medium without inoculum. Following incubation for 2 or 24 h at 22°C or 37°C, eggshells and stainless-steel pieces were aseptically transferred with sterile forceps to 15-mL Falcon tubes. The eggshells and stainless-steel pieces were rinsed three times with 2 mL of sterile 0.85% NaCl solution to remove unbound cells. To detach the biofilm cells, the eggshells and stainless-steel pieces were sonicated twice in 2 mL of sterile 0.85% NaCl solution for 2 min with a pause of 2 min. Ten-fold serial dilutions were made in sterile 0.85% NaCl solution and plated using drop plate technique on nutrient agar. Plates were incubated aerobically at 37°C for 20 h and the numbers of colony-forming units were counted. The results were expressed as Log10 cfu.g-1 of eggshell or stainless steel.

Disruption of Mature Biofilms Formed on Eggshells and Stainless Steel

Pathogen biofilms were allowed to develop on the surface materials (n=9) for 2 or 24 h at 22 or 37°C in LBL broth (pH 7), following the procedures described above. Once the biofilms were formed, eggshells and stainless-steel pieces were aseptically transferred to LBL broth at pH 7 or acidified with acetic acid (0.3% v/v) to pH 3. The 2-h-old biofilms were incubated aerobically for 2 h and the 24-h-old biofilms were incubated for 24 h, at 22°C or 37°C. After rinsing three times with 2 mL of sterile 0.85% NaCl solution and sonication in 2 mL of sterile 0.85% NaCl solution, 10-fold serial dilutions were made and plated using drop plate technique on nutrient agar. Results were expressed as Log10 cfu.g-1 of eggshell or stainless steel.

Disruption of Biofilms Formed on Polystyrene

S. Typhimurium and E. coli O157 inocula were prepared as described above for the evaluation of biofilm formation on eggshells and stainless steel. To evaluate the disruption of young biofilms by acetic acid, ninety-six-well polystyrene plates (Becton Dickinson, Franklin Lakes, NY, USA) were inoculated with 180 µL of S. Typhimurium or E. coli O157 inoculum adjusted to an OD of 1.8 (approximately 5.12 × 109 cfu.mL-1) in LBL broth pH 7 or broth acidified to a pH of 3 with acetic acid (0.3% v/v), n=24. The multi-well plates were incubated aerobically for 2 or 24 h at 22 or 37°C, without shaking and under humid conditions to prevent evaporation. To evaluate the disruption of matured biofilms, first, the biofilms were allowed to form aerobically in LBL broth at pH 7 for 2 or 24 h. Subsequently, the culture broth was removed and 150 µL of LBL broth at pH 7 or broth acidified to pH 3 with acetic acid (0.3% v/v) was added to the wells, the number of wells used per treatment was of 24. Then the plates were incubated aerobically once more for an additional 2 or 24 h at 22°C or 37°C. Controls consisted of uninoculated broths. At the end of the incubation times, the OD was read at 600 nm using a SunriseTM microtiter plate reader (Tecan Group Ltd, Männedorf, Switzerland). Subsequently, the liquid contents of each well were gently removed, and the biofilms were stained for 1 h with 180 µL of 0.01% (w/v) crystal violet (Sigma-Aldrich, St. Louis, MO., USA) 3. Excess dye was removed, and the wells were rinsed three times with sterile distilled water. The plates were allowed to air-dry at room temperature before adding 180 µL of ethanol: acetone (80: 20) to each well. Crystal violet-stained biofilms were measured at 600 nm using a SunriseTM microtiter plate reader.

Statistical Analysis

At least three biological replicates of each experiment were carried out to ensure the reproducibility of results. Data of surface spread colony areas, cfu.g-1 of eggshell or stainless steel and crystal-violet stained biofilms were Log10 (x + 1) transformed to homogenize variances between treatments. Linear models (LM) were employed for statistical analyses using R v. 3.6.0 (http://www.R-project.org/) with packages lme4, car, and emmeans. Surface spreading data were analyzed using LM and pairwise comparisons were performed for the interaction between all factors. The cfu.g-1 of eggshell or stainless steel and OD data for multi-well plate assays were analyzed with a negative binomial distribution. The negative binomial theta parameter was established with an alternating iteration procedure using the glm.nb function. Pairwise multiple comparisons were carried out using the false discovery rate (FDR) for P-value corrections.

Results

Impact of Acetic Acid on S. Typhimurium and E. coli O157 Planktonic Cells

Growth curves were conducted to monitor the antimicrobial activity of the three organic acids on planktonic cells. Initial low (0.1 OD) and high (1.8 OD) concentrations of cells were employed to simulate the numbers used in young and mature biofilm inoculants, respectively. The results indicated that irrespective of the initial concentration (Figure 2C and 2D), all three organic acids exhibited bactericidal activity against S. Typhimurium and E. coli O157 planktonic cells. In both scenarios, a progressive decrease in colony-forming unit (cfu) numbers was observed over time. Compared to cultures at pH 7 with an initial OD of 0.1, cultures in LBL broth acidified with acetic, citric, and lactic acids exhibited reductions of 7.86 Log10 cfu.mL-1 for S. Typhimurium (Figure 2A) and 8.17 Log10 cfu.mL-1 for E. coli O157 (Figure 3B). When initial cell concentrations were high, cfu.mL-1 numbers also decreased in cultures acidified with the three organic acids. After 48 hours of incubation, viable Log cfu.mL-1 counts of S. Typhimurium and E. coli O157 in acidified cultures revealed reductions of 8.36 and 8.10, respectively.

fig 2

Figure 2: S. Typhimurium and E. coli O157 growth curves for control (pH 7) and acid (pH 3) broth cultures with an initial optical density of 0.1 (A and B, respectively) and 1.8 (C and D, respectively). Error bars indicate standard error of the mean (n=9).

Interference of Organic Acids with Surface Spreading

Bacterial surface motility is known to be involved at different stages of biofilm formation, especially at its initial stages. We evaluated the impact of acetic, citric, and lactic acids on this phenotype. Compared to control conditions, a significant (P < 0.05) decrease in the surface spreading abilities of S. Typhimurium and E. coli O157, ranging between 97 to 98%, was observed on the semi-solid agar plates containing any of the three organic acids (Figure 3).

fig 3

Figure 3: Effect of organic acids on S. Typhimurium (A) and E. coli O157 (C) surface spreading. Error bars indicate the standard error of the means (n=10). Bars with the same letter do not differ significantly (P > 0.05). B and D demonstrate the observed surface spreading patterns of S. Typhimurium and E. coli O157 at 24 h post-inoculation, respectively (bar=1 cm).

Disruption of Newly Formed Biofilms

First, the capacity of acetic, citric, and lactic acid to disrupt biofilms formed at 2 and 24 h post-inoculation (hpi) on eggshells was evaluated. Under the control treatment conditions, the numbers of S. Typhimurium and E. coli O157 attached cells were similar in most comparisons at 2 and 24 hpi (Table 1); although at 24 hpi at 37°C, fewer (P < 0.05) E. coli O157 than S. Typhimurium cells were found to be attached. Of the three acids, acetic acid generated (P < 0.05) higher reductions on newly formed biofilms developed by both pathogens, with an overall 3 Log10 cfu.g-1 of eggshells decrease at both times and temperatures compared to the controls. An exception was at 2 hpi and 37°C with biofilm formation by S. Typhimurium being controlled to a greater extent by lactic acid rather than by acetic and citric acids. Compared to the effect achieved by the other two organic acids, at 2 and 24 hpi, acetic acid also yielded the highest (P < 0.05) reduction of E. coli O157 biofilm formation at both temperatures.

Table 1: Organic acids inhibition of young Salmonella Typhimurium and Escherichia coli O157 biofilms developed on eggshells and stainless steel surfaces.

     

Bacteria (Incubation Temperature)

Surface1

 Treatment Time (h) ST (22°C) ST (37°C) EC (22°C) EC (37°C)
ES Con 2 8.58 ± 0.05aA 8.23 ± 0.12aA 8.31 ± 0.11aA

8.24 ± 0.13aA
24 8.48 ± 0.06aA 8.40 ± 0.18aA 8.33 ± 0.09abA 8.05 ± 0.10bA
AA 2 5.12 ± 0.07bC 7.36 ± 0.08aB 5.03 ± 0.03aC

5.12 ± 0.06bC
24 5.13 ± 0.06aC 5.07 ± 0.04aC 5.03 ± 0.03aC 5.06 ± 0.04aD
LA 2 5.17 ± 0.08cB 5.08 ± 0.05cC 7.33 ± 0.29bB

8.36 ± 0.06aA
24 7.56 ± 0.21aB 7.69 ± 0.07aB 7.65 ± 0.06aB 6.59 ± 0.07bC
CA 2 6.33 ± 0.17bB 8.13 ± 0.13aA 7.52 ± 0.30aB

6.23 ± 0.34bB
24 7.59 ± 0.08cB 8.14 ± 0.10Ba 7.79 ± 0.05cB 8.40 ± 0.11aB
SS Con 2 10.48 ± 0.01bA 10.72 ± 0.01aA 10.31 ± 0.01cA

10.50 ± 0.01bA
24 10.63 ± 0.01bA 10.96 ± 0.00aA 10.33 ± 0.01dA 10.50 ± 0.01cA
AA 2 4.04 ± 0.21aB 4.23 ± 0.01aB 4.20 ± 0.27aB

4.23 ± 0.03aB
24 4.41 ± 0.05bB 4.45 ± 0.03bB 4.61 ± 0.01aB

4.59 ± 0.02aB

1ES: Egg Shell, SS: Stainless Steel, Con: Control medium at pH 7, AC: Acetic acid medium at pH 3, LA: Lactic acid medium at pH 3, CA: Citric acid medium at pH 3, ST: Salmonella Typhimurium, EC: Escherichia coli O157.
abcdMeans (Log10 cfu/g) ± SE (n=9) in rows and with different letters are significantly different (P < 0.05).
ABCDMeans (Log10 cfu/g) ± SE (n=9) in columns, with the same surface material, and the same time, and with different letters are significantly different (P < 0.05).

The disruptive activity that citric and lactic acid caused on newly formed biofilms was found to depend on the time of exposure and the incubation temperature. Citric acid was found to be more effective in disrupting the 2-h-old biofilms formed by S. Typhimurium at 22°C, and by E. coli O157 at 37°C, causing a reduction in the number of cfu.g-1 of eggshell of 2.25 and 2.01 Log10, respectively. On the other hand, lactic acid exerted the higher antibiofilm activity against S. Typhimurium biofilms generating a decrease in the number of cfu attached per gram of eggshell at 22°C of 3.41 Log10 and at 37°C of 3.15 Log10. At the same time and temperatures, E. coli O157 biofilms saw a decrease of 1 and 0 Log10. Biofilms formed during 24 h, treated with this organic acid showed an overall decrease of less than 1 Log10 cfu.cm2-1 of eggshell, in both pathogens. Because acetic acid was observed to be the most effective organic acid in controlling S. Typhimurium and E. coli O157 biofilm formation on eggshells, this organic acid was selected for further studies.

As seen with eggshells, the number of cfu.g-1 of stainless steel attached at 2 and 24 h showed similar numbers by S. Typhimurium and E. coli O157 within control and acetic acid treatments (Table 1). The Attached S. Typhimurium cells to this surface were higher in the control treatment at 2 and 24 hpi (P < 0.05) at 37°C and lower (P < 0.05) for E. coli O157 at 22°C, although the differences were small. At all times and temperatures, acetic acid caused a reduction (P < 0.05) by nearly 6 Log10 of S. Typhimurium and E. coli O157 cfu.g-1 of stainless steel. All counts for acetic acid-treated biofilms were similar (P > 0.05) at 2 hpi; however, at 24 hpi, E. coli O157 counts at both temperatures were slightly higher (P < 0.05) than those for S. Typhimurium.

The formation of biofilms by both pathogens on multi-well polystyrene plates was also found to be influenced by temperature and incubation temperature (P < 0.05, Table 2). Lower (P < 0.05) biofilm OD values were found for S. Typhimurium and E. coli O157 at 22°C than at 37°C at 2 hpi and 24 hpi for the control and acetic acid treatments. Treatment with acetic acid resulted in both pathogens producing less (P < 0.05) biofilm at both temperatures when compared to control OD values at 2 and 24 hpi. However, there were higher decreases in OD values for both acetic acid-treated pathogens at both temperatures at 24 hpi as compared to 2 hpi. While an overall reduction of nearly 1 OD unit was obtained at 2 hpi for both pathogens, a decrease at 24 hpi of 2 OD units and 1.7 OD units was found for S. Typhimurium and E. coli O157, respectively.

Table 2: Acetic acid inhibition of young Salmonella Typhimurium and Escherichia coli O157 biofilms developed on polystyrene surfaces.

     

Bacteria (Incubation Temperature)

Surface1

 Treatment Time (h) ST (22°C) ST (37°C) EC (22°C) EC (37°C)
PS Con 2 2.76 ± 0.04bA 3.14± 0.03aA 2.43 ± 0.07cA

2.98 ± 0.05aA
24 3.04 ± 0.02bA 3.62 ± 0.09aA 3.03 ± 0.03bA 3.12 ± 0.04bA
AA 2 1.68 ± 0.06bB 1.84 ± 0.08abB 1.68 ± 0.05bB

1.91 ± 0.05aB

24

 0.64 ± 0.05cB 1.59 ± 0.10aB 1.22 ± 0.04bB

1.53 ± 0.09aB

1PS: Polystyrene, Con: Control medium at pH 7, AC: Acetic acid medium at pH 3, ST: Salmonella Typhimurium, EC: Escherichia coli O157
abcdMeans (OD) ± SE (n=24) in rows and with different letters are significantly different (P < 0.05).
ABMeans (OD) ± SE (n=24) in columns and with different letters are significantly different (P < 0.05).

Acetic Acid Disruption of Mature Biofilms

Control treatments showed that the number of cfu of S. Typhimurium and E. coli O157 attached per g of eggshell did not significantly increase from 2 to 24 h (P < 0.05) at any of the evaluated temperatures. On the other hand, 2 more hours of incubation were enough to allow higher (P < 0.05) numbers of S. Typhimurium and E. coli O157 cells to be attached to stainless steel than to eggshells for control and acetic acid-treated cultures at both temperatures. As observed in the assays of young biofilms, treatment with acetic acid for 2 and 24 h also generated significant (P < 0.05) reduction on the already formed and mature S. Typhimurium and E. coli O157 biofilms, regardless of the evaluated surface (Tables 3). Compared to control treatments, there was an overall 6.6 Log10 reduction in the number of cfu attached to eggshells and stainless-steel surfaces. Exposure to acetic acid for 2 h was enough to disrupt the already formed biofilms. Interestingly, prolonged exposure to acetic acid for 24 h did not incrementally affect these mature biofilms (Table 3). Furthermore, as observed when evaluating the disruption of young biofilms, the antibiofilm activity of acetic was higher on the biofilms formed on stainless steel than on eggshells.

Table 3: Acetic acid disruption of matured Salmonella Typhimurium and Escherichia coli O157 biofilms developed on eggshells and stainless surfaces.

     

Bacteria (Incubation Temperature)

Surface1

 Treatment Time (h) ST (22°C) ST (37°C) EC (22°C) EC (37°C)
ES Con 2 8.67 ± 0.03aA 8.77 ± 0.03aA 8.31 ± 0.03bA

8.32 ± 0.05bA
24 8.78 ± 0.08bA 8.94 ± 0.01aA 8.39 ± 0.03cA 8.33 ± 0.03cA
AA 2 2.24 ± 0.01aB 2.23 ± 0.01aB 2.21 ± 0.02aB

2.22 ± 0.03aB
24 2.41 ± 0.05bB 2.45 ± 0.03bB 2.59 ± 0.01aB 2.59 ± 0.02aB
SS Con 2 10.54 ± 0.02bA 10.67 ± 0.02aA 10.11 ± 0.01cA

10.68 ± 0.02aA
24 10.76 ± 0.04bA 10.92 ± 0.01aA 10.85 ± 0.01aA 10.92 ± 0.00aA
AA 2 3.46 ± 0.03bB 3.65 ± 0.01aB 3.57 ± 0.03aB

3.62 ± 0.04aB

24

 3.56 ± 0.04cB 3.88 ± 0.02aB 3.55 ± 0.07cB

3.72 ± 0.04bB

1ES: Egg Shell, SS: Stainless Steel, PS: Polystyrene, Con: Control medium at pH 7, AC: Acetic acid medium at pH 3, LA: Lactic acid medium at pH 3, CA: Citric acid medium at pH 3, ST: Salmonella Typhimurium, EC: Escherichia coli O157
abcdMeans (Log10 cfu/g) ± SE (n=9) in rows and with different letters are significantly different (P < 0.05).
ABCDMeans (Log10 cfu/g) ± SE (n=9) in columns, with the same surface material, and with different letters are significantly different (P < 0.05).

Incubation of the mature biofilms formed on the polystyrene surface for an additional 2 and 24 h generated significant differences (P < 0.05) in the OD values for S. Typhimurium and E. coli O157 biofilms. Regardless of the time and temperature of incubation, the OD values of E. coli O157 biofilms were higher than those of S. Typhimurium. Additionally, while S. Typhimurium showed higher OD values at 24 hpi than at 2 hpi, E. coli O157 OD values were reduced over time. An overall reduction on the OD values caused by acetic acid was observed in the matured biofilms formed by the two pathogens, however the antibiofilm activity of the acid varied depending on the time and temperature (P < 0.05). The higher antibiofilm activity exerted by acetic acid on S. Typhimurium matured biofilms was observed at 24 hpi and 22°C (1.08). Similarly, the higher reduction in the OD values in E. coli O157 was found at 22°C; although it was observed at 2 (2.49) and 24 hpi (2.33). Extending the exposure to acetic acid of S. Typhimurium matured biofilms formed at 22°C led to a higher reduction on the OD values at 24 hpi than at 2 hpi. However, this decrease caused by a longer exposure to acetic acid was not observed for the matured biofilms formed at 37°C by S. Typhimurium or by E. coli O157 at any of the evaluated temperatures (Table 4).

Table 4: Acetic acid disruption of matured Salmonella Typhimurium and Escherichia coli O157 biofilms developed polystyrene surfaces.

     

Bacteria (Incubation Temperature)

Surface1

 Treatment Time (h) ST (22°C) ST (37°C) EC (22°C) EC (37°C)
PS Con 2 2.60 ± 0.05bA 1.19 ± 0.08cA 4.40 ± 0.06aA

2.77 ± 0.07bA

24

3.57 ± 0.06bA

 2.09 ± 0.06dA 4.20 ± 0.08aA

2.35 ± 0.08cA

AA

2

 2.20 ± 0.15aB 0.46 ± 0.07bB 1.91 ± 0.1aB

1.75 ± 0.09aB

24

 2.49 ± 0.11aB 1.32 ± 0.14cB 1.87 ± 0.08bB

1.53 ± 0.11abB

1PS: Polystyrene, Con: Control medium at pH 7, AC: Acetic acid medium at pH 3, ST: Salmonella Typhimurium, EC: Escherichia coli O157
abcdMeans (OD) ± SE (n=24) in rows for each surface and with different letters are significantly different (P < 0.05).
ABMeans (OD) ± SE (n=9) in columns, with the same time, and with different letters are significantly different (P < 0.05).

Discussion

Complete removal of enteropathogenic bacteria from the poultry production chain environment is essential to ensure overall food safety. Pathogens like S. Typhymurium and E. coli O157 possess the capability to form biofilms, enabling their survival under unfavorable conditions by adhering to abiotic surfaces such as metals, plastic, or glass while creating a protective barrier [25,26]. Despite the implementation of numerous hygienic measures, concerns persist regarding the efficacy of disinfectants due to the emergence of bacterial resistance [27]. Moreover, several chemical sanitizers previously used for human health purposes are now prohibited, leading to a renewed interest in substituting chemical industrial sanitizers with natural antimicrobial agents. Organic acids, considered safe for food animal and human health, stand out as exceptional alternatives in this regard [28]. They are affordable and known to be safe compounds.

The results from the current study demonstrate the efficacy of acetic acid as an antibiofilm agent against S. Typhimurium and E. coli O157 biofilms. Halstead [29] similarly revealed the bactericidal actions of this organic acid against pathogens such as E. coli, Staphylococcus aureus, and Acinetobacter baumannii. However, in contrast to these findings, other studies have suggested that acetic acid might not be the most efficient biofilm disruptor when compared to other organic acids. For instance, Ban [30] evaluated the antibiofilm activities of propionic acid, acetic acid, lactic acid, malic acid, and citric acid, and found lactic acid to be the most effective in disrupting 6-day-old S. Typhimurium, E. coli O157: H7, and Listeria monocytogenes biofilms. Moreover, Amrutha [5] reported that, when comparing the activity of acetic, lactic, and citric acids at a 2% concentration, lactic acid achieved maximum inhibition of Salmonella sp. and E. coli biofilms formed on cucumber. The degree of antimicrobial effect might be influenced by the concentration of organic acid and the exposure time [28]. According to Beier [31], acetic, butyric, and propionic acids required lower molar amounts than citric, formic, and lactic acids to significantly inhibit enteropathogens. Furthermore, Bardhan [20] indicated that lactic acid was an effective antimicrobial against clinical carbapenem-hydrolyzing, multi-drug-resistant Klebsiella pneumoniae planktonic and biofilm-forming cells. The authors observed cell membrane damage and high rates of bacteriolysis after treatment with lactic acid at concentrations of 0.15% and 0.225%.

The antibacterial activity of organic acids has been associated with their pKa and the optimal pH for dissociation [28]. The pKa values for acetic, citric, and lactic acid are 4.476, 3.86, and 3.13, respectively. Kundukad [23] demonstrated that maintaining their pH close to their pKa enables weak acids like acetic and citric acid to eliminate persistent cells within biofilms of antibiotic-resistant bacteria such as Klebsiella pneumoniae KP1, Pseudomonas putida OUS82, Staphylococcus aureus 15981, Pseudomonas aeruginosa DK1-NH57388A, and P. aeruginosa PA_D25. When provided at a pH lower than their pKa, these compounds can penetrate the biofilm matrix and bacterial cell membranes. While lactic acid is considered a stronger acid than acetic acid based on their pKa values, the efficacy of organic acids also relies on pH levels. The proximity between the pKa value of lactic acid and the pH of 3 used in this study might explain why acetic acid exhibited better performance against biofilm formation and disruption than lactic acid. Further studies comparing the effectiveness of these organic acids at various pH values are necessary to confirm these observations.

In general, it has been suggested that increasing the contact time with disinfectants enhances their antibiofilm activities on various material surfaces [15]. In the current study, it was observed that prolonged exposure of S. Typhimurium and E. coli O157 planktonic cells to the tested organic acids resulted in lower OD values, as depicted by the presented growth curves. However, when mature biofilms of these microbes were exposed to acetic acid on polystyrene surfaces, this time-related effect was not observed. The OD values for mature biofilms did not decrease after exposure to acetic acid for 2 or 24 hours. Similar resistance over time was noted for biofilm cells attached to eggshells and stainless steel when the biofilm formation and contact time with organic acids extended from 2 hours to 24 hours. Amrutha [5] reported that exposure to acetic, citric, and lactic acids did not significantly reduce the production of exopolysaccharides in Salmonella sp. biofilms and resulted in reductions of 10.89%, 6.25%, and 13.42% in E. coli O157: H7 biofilms, respectively. The extracellular matrix developed by biofilm cells acts as a barrier, impeding the penetration or inactivation of antimicrobial compounds [31,32]. Therefore, the limited reduction in S. Typhimurium and E. coli O157 biofilms formed on eggshells, stainless steel, and polystyrene with increased exposure time to acetic acid is likely due to the obstruction presented by the biofilm matrix against the passage of organic acids. Research focusing on disrupting the biofilm matrix using alternative methods before exposure to organic acids could lead to the development of complementary approaches to enhance the antimicrobial activity of organic acids.

In addition to the biofilm matrix defensive shield, it is conceivable that the remaining cells inside the S. Typhimurium and E. coli O157 biofilms would respond to the effects of acetic acid by triggering other protection strategies. Changes in membrane lipids have been described as one of these defensive mechanisms [33]. Additional cell protective strategies would include the release of ammonia [34], the pumping out of protons, and the proton-consuming decarboxylation processes. More recently, Clary [19] demonstrated how the bacterial colony diversification (morphotype) would define the outcome of tolerance to a particular stressor during the process of biofilm formation and its persistence against environmental assaults. Amrutha [5] demonstrated that a reduction of exopolysaccharide (EPS) synthesis, EPS composition and organization, swimming and swarming cell patterns, and a negative impact on quorum sensing play crucial roles in microbial community architecture as well as resistance to toxic substances. Further research is required to identify which of these mechanisms are used by the remaining S. Typhimurium and E. coli O157 biofilm cells attached to eggshells and the industrial surfaces evaluated in the current study.

The antibiofilm activity of organic acids, such as acetic acid, might encounter hindrances due to alterations in the biofilm structure caused by temperature shifts and variations in adhesion surface types. Generally, temperature and surface material have been reported to influence the attachment ability of enteropathogenic and other bacteria, consequently affecting the biofilm structure [35]. In the current study, we did not assess the impact of temperature on the biofilm structure on the tested surfaces. However, our findings indicate that at 22°C, acetic acid exhibited less control only over mature biofilms formed by S. Typhimurium on polystyrene, differing from the conditions at 37°C. Similar temperature-related alterations in biofilm capacity were observed by Andersen [36] when evaluating the biofilm-forming capacity of several E. coli K12 clinical isolates. They reported a higher number of attached cells at 30°C compared to 35°C, observing denser and more evenly distributed biofilms on silicone surfaces at the lower temperature. Andersen [36] suggested that the presence of curli fibers, which facilitate cell adhesion, might have influenced the type and creation of the biofilm structure, particularly at lower temperatures where these cell surface adhesins are produced. Furthermore, another study focused on E. coli O157: H7 biofilms formed at 4°C and 15°C on beef processing surfaces concluded that while a slight decrease in the number of attached cells was noted at 4°C, it did not hinder the overall increase in attached cell numbers over time [37].

Conclusion

The efficacy of compounds utilized for sanitation involves multifaceted events associated not only with the morphology and physiology of the target microbial cells but also with factors such as relative surface hydrophobicity, material surface roughness, and the impact of shear stress [38]. Organic acids can influence the internal chemical equilibrium of microbial cells, leading to alterations in cell membrane integrity or cellular activities, ultimately resulting in cell death. Consequently, organic acids represent an important option for sanitizing purposes and may potentially be combined or incorporated into innovative carrier matrices with other established antimicrobial molecules, such as essential oil components, thereby improving molecule stability and extending their biological activity [39]. The results obtained from this study offer new insights into the effectiveness of acetic acid as an antibiofilm agent, which can be utilized to control S. Typhimurium and E. coli O157 biofilms formed under conditions encountered along the poultry production chain. This newfound information may facilitate the integration of this natural compound into hygiene programs aimed at preventing cross-contamination of eggs, broilers, and broiler meat products.

Acknowledgments

This work supported by the United States Department of Agriculture under grant number 58-3091-7-028-F; and by the Colombian Ministry of Agriculture and Rural Development under grant numbers Tv18 and Tv19. We thank Yessica Muñoz and Xiomara Abella for technical assistance, and Corporación Colombiana de Investigación Agropecuaria – Agrosavia for supporting this research.

Contributions

AGC and CVAG were involved in the experimental and performed biofilm experiments. AGC, MEH, FRV and CVAG participated in data analysis and wrote the manuscript.

Ethics Approval

Not applicable.

Consent to Participate

All authors approved the manuscript.

Consent for Publication

The authors consented for the publication.

Statements and Declarations

Competing Interests

The authors declare no competing interests.

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Attacks on ‘First Responders’ in the United States: Can AI Using Mind Genomics ‘Thinking’ Identify Mindsets and Provide Actionable Insight?

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DOI: 10.31038/JCRM.2024714

Abstract

Using generative AI, the paper investigates the nature of individuals who are likely to attack first responders (e.g., police, fire fighter, medical professionals). AI suggested five different mind-sets, and a variety of factors about these mind-sets, including what they may be thinking, and how they can be recognized. The approach of synthesizing mind-sets provides society with a way to understand negative behaviors, and to protect against them.

Introduction

In today’s society, the traditional feeling towards first responders such as emergency services, law enforcement and firefighters at the scene of an accident or crime, as well as doctors and nurses providing care in clinics, is usually one of respect and gratitude. These individuals are seen as heroes who put their own lives at risk to help others in need. People typically view first responders as dedicated professionals, essential to maintaining order and providing crucial assistance in emergency situations. Often, their work is so stressful that in some cases they end up suffering with PTSD years after their efforts [1-6].

However, violence against first responders, appears to be a growing threat. While underreported, studies suggest a concerning rise. A 2019 report by the National Fire Protection Association (NFPA) highlights that a staggering 69% of EMS personnel experienced some form of violence on the job within a year, with a third being physically assaulted (NFPA 2019).

During the past 30 years, however, the United States has experienced significant changes in societal attitudes and behaviors which end up in the often-unthinkable behavior of attacking first responders, whether these be public servants like police [7] or doctors and nurses in clinics [8-10]. At first glance this behavior seems irrational because the first responders are actively helping the public.

Among the key reasons:

Emotional Intensity and Stress: Emergency situations can be highly emotional and stressful for everyone involved. First responders often encounter distressed individuals, family members, or witnesses. The intensity of these situations can lead to aggression directed at responders [11].

Substance Abuse and Mental Health Issues: People under the influence of drugs or alcohol may act irrationally and become aggressive. Additionally, individuals with mental health conditions might not respond well to assistance. This problem is made worse by the fact that mental health services are underfunded and under supported, which increases the likelihood that first responders may face violent incidents [12].

Vocal And Emotionally Charged Skepticism Towards Government, Law Enforcement, And The Media: Some scholars suggest that this trend owes its growth to the increasingly. The result is a culture where challenging authority is increasingly the norm. Sometimes this erosion is expressed by a simple expression, ‘is nothing sacred anymore?’ [13-15].

Economic Disparities and Social Inequalities in the US; Economics and daily struggle cannot help but create pockets of resentment and frustration within marginalized communities. First responders, often interacting with these communities in times of crisis, end-up becoming targets for the projected frustration and anger emerging from this economically driven sense of powerlessness and injustice. This was recognized more than a half century ago [16]. Also adding to the distrust and antagonism towards first responders is the militarization of police forces and the general increase in the use of force by police officers. When the public views police as tyrannical or hostile, trust in law enforcement may suffer and communities may stop working together to keep the peace [17].

AI Predictions about the Future for First Responders

To understand this topic and to offer recommendations capable of minimizing attacks on these essential workers, we used the AI embedded in the Mind Genomics platform to help us understand the mind-sets of people who attack first responders. The approach asked the AI embedded in Mind Genomics (SCAS, Socrates as a Service) what would happen if the current attitude towards the first responders were to be unchecked. Our specific questions were what would happen in 2026, then in 2030.

SCAS returned with the following ‘prediction’: ‘People will probably still respect and admire first responders in 2026, but they may be worried about their safety due to the rising frequency of assaults on them. Many people may start to be more cautious of the dangers that come with becoming a doctor, nurse, or police officer in light of the increasing number of occurrences targeting these professions. There may be a rising chorus of voices demanding more funding and assistance to shore up the safety nets that now shield first responders. Looking further ahead to 2030, if the trend of attacks on first responders continues unabated, people’s feelings towards these essential workers may become deeply polarized. There may be a growing divide between those who continue to view first responders as heroes deserving of support and admiration, and those who have lost faith in the system and believe that drastic measures are needed to address the root causes of the problem. The traditional feeling of respect and gratitude towards these individuals may be overshadowed by a sense of resentment and anger at the injustices faced by those who dedicate their lives to helping others.

Deeper Understanding of the Problem of Attacking First Responders: Mind-sets and the Contribution of Mind Genomics

Based upon the foregoing ‘prediction’ by AI, we move to a deeper understanding of the minds of people who are described as ‘attacking first responders.’ The approach was based upon the work in Mind Genomics, an emerging branch of psychology dealing with how people respond to the world of the everyday [18,19].

How people respond to stimuli is influenced by their cognitive biases, cultural background, childhood, and life experiences. Studying these individual differences, Mind Genomics zeroes down on the minute details of daily life by classifying individuals according to their thoughts on a subject, their motivations for doing something, and even their barriers to action. Mind genomics achieves this by utilizing a combination of controlled experiments, data analysis, and cognitive psychology principles to identify distinct mind-sets and predict corresponding behaviors [20-22].

Recently, attention has shifted to using artificial intelligence to suggest mind-sets [23]. By using AI, it becomes possible to create a situation where the different mind-sets are identified, along with their possible ‘internal conversation before the attack’, as well as things that can be done immediately as well as long term to discourage these behaviors.

Mind Genomics Empowered by AI, to Explore ‘Who’ Attacks and Why

The rest of the paper is devoted to an exploration of different mind-sets, using AI to drive the creation of the mind-set. The AI is Chat GPT [24], with a series of prompts developed specifically for Mind Genomics. The prompts enable the user to find out specific information about a topic, and later apply AI to further ‘analyze’ the information originally provided by AI. The system is called Socrates as a Service, abbreviated as SCAS. It will be SCAS which allows us to interact with AI.

The exploration begins by presenting SCAS, viz., the embedded AI, with background material, or more correctly with a simple prompting statement. This statement, chosen by the user, is simply the statement: There are six radically different mind-sets of individuals who attack first responders. This statement is presented as fact. (Note that AI will return with only five mind-sets). The rest of the information presented to SCAS is a set of six questions, generated by the user. Table 1 shows the information and request provided to AI.

Table 1: The input information provided by the user and the request for additional information. Note that AI ended up returning only five mind-sets.

TAB 1

The simplicity of the system reduces the anxiety of the user. The user ends up setting the scene for AI by stating the number of mind-sets, and then requests that the AI (viz., SCAS) become a tutor, by answering six questions for each mind-set just synthesized by AI.

Once the user has specified the requested information AI returns quickly with suggestions about the mind-sets. The request has to be made properly. In the effort to create Table 2, it took four iterations to get the request correct, viz., the request shown in Table 1. The iterations are fast, requiring about 15 second each, allowing for a trial-and-error change of instructions so that they end up being clear, and without ambiguity. It is important to emphasize that the ‘errors’ instructing the AI are usually the result of ambiguous instructions, and all-too-often, instructions which contain impossible-to-satisfy requests.

Table 2: The five mind-sets developed by SCAS as a direct response to the request

TAB 2

Table 2 shows the set of five mind-sets ‘synthesized’ by AI. A second iteration might return with some of the same mind-sets, but perhaps with one or two new mind-sets, as well as four of the previous five mind-sets. Note that although the user can request a certain number of mind-sets, the request ends up being a suggestion. Quite often AI returns with fewer mind-sets than requested, but never more than the number requested by the user.

The mind-sets appear with the relevant questions. Whether or not the information is accurate is not as important as the fact that within minutes the user has begun to learn about assaults against first responders. Just the information alone begins to educate, providing insights about what may be going on in the minds of those who do the assaulting, as well as what to say to them in terms of ‘slogans’.

Putting the Ideas into Action after Knowing Mind-sets

AI can predict and prevent attacks on first responders by understanding threat mindsets. By analyzing past incidents, AI can identify patterns and intervene before violence. This knowledge can de-escalate volatile encounters, suggest communication tactics, and prevent violence. With the right tools, first responders can manage unpredictable situations safely.

A short description of each mind-set was given to AI (SCAS), along with the background shown at the top of Table 3. The different mind-sets were provided to give AI a sense of the range of the different ways people might feel about first responders. The request, however, was to come back with a single strategy. The request was given twice, generating two iterations. These are shown in Table 3.

Table 3: Putting the ideas into action – how to prevent or ameliorate the attacks

TAB 3

Strategies Suggested by AI to Minimize Attacks on First Responders

The final activity in this exploration of attacks against first responders comprises the education of professionals. Here let us assume that we are dealing with police officers in a local precinct. The assumption here is that many of the potential attackers are thought to fall into the grouping of ‘Aggressive Defender.’

The strategy is first to create a briefing document for all officers to read (Table 4, and then to create a set of posters showing how the officers should behave towards the Aggressive Defender (Table 5). The briefing document and posters for police officers can enhance their understanding of Aggressive Defender mindsets. The briefing document provides detailed information on their characteristics, behaviors, and motivations, enabling them to anticipate, respond to, and de-escalate situations, thereby improving their safety and effectiveness on the job. In turn, the posters for the precinct teach the police officers how to effectively interact with Aggressive Defenders and potential threats.

It’s important to note that briefing documents and posters are just one method for communicating the information outlined. Multimedia formats for the same information, such as video generated by prompts or text are generally available, and could be used as an adjunct to or substitute for the poster approach outlined below.

Table 4: The briefing document for police officers, focusing on the AGGRESSIVE DEFENDER mind-set

TAB 4

Table 5: Three types of posters for the police precinct, dealing: The briefing document for police officers, focusing on the AGGRESSIVE DEFENDER mind-set.

TAB 5

Who Would be Interested in These AI-based Simulations of Potential Attacker Mind-sets’?

We close the ‘results section’ (viz., the simulations) with a second-level analysis by SCAS. Once the iterations are complete and delivered to the user, the embedded AI reviews the information, and provides deeper analysis of what was presented in the results immediately delivered to the user. This secondary ‘summarization’ of the information occurs some time later, after the project is closed.

Part of the summarization analysis considers WHO would be the audiences. SCAS is pre-programmed to provide three different groups: those who are interested, those who are opposed, and those who think differently and may bring new viewpoints to the problem. These appear in Table 6.

Table 6: AI summarization of three different types of audiences faced with information and simulation of potential attacker mind-sets.

TAB 6

Discussion and Conclusions

Understanding the roots of violence today is critical to safeguarding our first responders. They are continuously exposed to risky circumstances that might develop into violent assaults. The police are often the most visible targets of this assault, but physicians at clinics are also at danger. Individual physicians have been targeted in violent assaults because they are blamed for poor medical results.

Using AI to model mindsets may assist first responders in better understanding and anticipating possible violence. Mind Genomics is a helpful tool for better analyzing and communicating with diverse mindsets. Understanding the mindsets of prospective attackers allows first responders to effectively de-escalate situations and protect themselves and others. This may greatly enhance the safety and efficacy of our first responders in high-risk circumstances.

Imagine a future in which all first responders are educated to comprehend and communicate with diverse mindsets utilizing AI technology. This might transform how our essential front-line workers handle perilous circumstances, shield themselves from injury, and maintain public support. The capacity to detect and avoid violence may be the difference between life and death for the first individuals on the scene.

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  14. Gibbs JC (2013) Targeting blue: Why we should study terrorist attacks on police. In: Examining Political Violence, Routledge (pp. 341-358).
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  20. Ilollari O, Papajorgji P, Gere A, Zemel R, Moskowitz H (2019) Using Mind Genomics to understand the specifics of a customer’s mind.The European Proceedings of Social, Behavioural Sciences EpSBS ISSN: 2357-1330.
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The Financial Incentives Leading to the Overutilization of Cardiac Testing and Invasive Procedures

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DOI: 10.31038/JCRM.2024712

 
 

The overutilization of cardiac testing and unnecessary referrals to invasive coronary angiography are significant clinical and health policy concerns. Inappropriate imaging cardiac stress tests are estimated to cost the U.S. healthcare system $500 million annually and expose many patients to unnecessary radiation. The unjustifiable use of diagnostic tests to screen for cardiac disease in asymptomatic and low-risk chest pain patients may lead to further testing and invasive procedures that are costly and potentially harmful, and have no clear outcome benefits. The principal trend in the treatment strategy for stable ischemic heart disease (SIHD) over the past two decades has been the utilization of percutaneous coronary intervention (PCI) and diminishing utilization of medical treatment and coronary artery bypass surgery (CABG). Despite these long-term changes in strategy, overall mortality has not improved significantly while costs have risen exponentially. One deleterious consequence has been an increasingly greater dependence on testing and interventional volume to maintain the revenue stream of cardiology practices.

Historical Background

The origins of this dependence are related to the original PCI learning curve. PCI quantity became a surrogate for quality: at an early stage, the standard was that “the more you do, the better you are”. This misconception persisted long after it was demonstrated to not be an accurate measure of quality despite the proposal of better metrics. There were several reasons for this tenacity. First, with the high reimbursement for PCI, cardiology sections and departments of medicine had found a “cash cow” in an era of “cost containment” that financed program expansion and higher compensation. Interventional leaders at first rigorously maintained high evidentiary standards of case selection. But then, as fellows were trained and entered outside practice with their newly minted skills, the potential income to physicians and hospitals became apparent. Teaching hospitals suddenly were in competition with previously small community hospitals, including those that previously were established referral sources. More and more interventionists entered practice, and competition expanded further; maintaining high volume meant moderating standards of case selection.

Another factor was an inherent uncertainty and unpredictability with balloon angioplasty. It was accepted that there was a risk of dissection and acute closure requiring urgent CABG, and thus only those who were surgical candidates could be PCI candidates. Some pioneers pushed that envelope with great success in otherwise hopeless cases. With the introduction of stents, the incidence of acute closure requiring CABG became zero. And with this fantastic tool, there was suddenly no contraindication to any patient with a severe lesion, including those with no symptoms at all.

Impact of Financial Incentives

Thereafter, the volume of procedures increased exponentially, and with it, revenue to hospitals, doctors, and programs at a time of diminishing reimbursements for cognitive skills. Hospital administrators, with the bottom line fully in focus, insisted on even more volume. As hospital systems increasingly acquired practices, these non-physicians became physician-leaders, and their bottom line was income generation. Any physician who wanted to see the science that showed evidence that all of these patients were getting benefits were suddenly no longer considered to have high standards, but rather naïve. The cardiology department and cardiac catheterization laboratory directors were expected to increase cath lab volumes.

In parallel, an entire lesion detection infrastructure sprung up with various forms of high-volume, moderately well-reimbursed stress testing being performed on any patient with even the most atypical symptoms. In a patient with a low pretest probability of coronary artery disease, a positive stress test is more likely to be a false positive than a true positive. Cardiologists developed an entire system to detect CAD that was revenue generating, even though the evidence suggesting it saved lives or improved quality of life was lacking. Finding disease to prevent sudden death is an attractive concept and was used to justify the liberalization of testing.

The fact that this testing strategy has led to millions of procedures with no scientific evidence to support it is unwelcome news to many. Science has taken a back seat to dogma in the promotion of procedures designed for a paradigm (obstructive lesion → ischemia → MI → mortality) that is known to be highly simplistic and incorrect. Any suggested harms became controversial and subjects of debate, in particular, whether a “small myocardial infarction” related to microthrombi and embolization during the procedure has long-term prognostic implications.

With academic leaders in interventional cardiology promoting PCI for MI prevention, it should have been no surprise that certain physicians with large practices of SIHD patients were doing unnecessary procedures on non-significant lesions, and sometimes, with no visible stenosis at all. A significant culprit of this time told the media that his 7-figure income was not an influence for placing 30 stents in a day. A few physician reputations were destroyed, but no hospitals went out of business—others, to keep that volume coming in, acquired them. The blame was placed on the “bad apple”, not the tree.

Guidelines

Rather than undertake a serious introspective evaluation at what was transpiring, an indirect evaluation was proposed. The cardiology societies collaborated to develop appropriateness criteria to classify which indications for revascularization were acceptable and which were not. The idea was to self-police and control the destiny of medical practice rather than allow outside agendas, clearly not attuned to the patient, control the procedure. Hospitals became interested in developing and paying for quality assurance programs as a defense against obvious malfeasance. These criteria were most notable for posing a temporary obstacle for clever interventionists to work around rather to assure that the right procedure is done for the right patient.

The flaws in these criteria were clear to many from the outset. Improved survival is not the only benefit a treatment strategy can offer, just the easiest to measure. Most patients prefer improved quality of life to longer survival alone, especially in regard to symptom status, but these are less objective in their assessment. If subjective improvement in symptoms is considered a benefit, then there was no way to generalize classifications, and they could also be subjectively influenced, so they weren’t included. Nearly all interventionists were displeased with a cookbook approach to case selection without reference to the individual patient. And with every new tweak of devices and technique, there was a disregard for prior studies that failed to show a benefit, even when new studies continued to show almost identical results. It is no coincidence that the most important PCI trials of the last 15 years (COURAGE, BARI2D, and ISCHEMIA) were not led by interventional cardiologists.

Contemporary Practice

Today, cardiologists can no longer compensate for declining reimbursement for their services by increasing the number of services they provide. The volume of coronary interventions performed in most institutions and by most interventional cardiologists is declining, just as the number of heart surgeries has been declining for years. Insurance companies require pre-approval for coronary CT angiograms, nuclear imaging, and other procedures. The pressure for interventional cardiologists to do as many cases as possible is motivated by demand from hospital and practice administration to increase revenue, which seems to conflict with the scientific evidence provided by randomized trials and summarized in practice guidelines.

Intervention has devolved to that of a commodity, a service provided on order as if there was no downside risk, with great benefits, and as if no alternative exists. Medical therapy remains the implied least attractive treatment modality, resorted to only when PCI or CABG are not favorably viewed from a technical standpoint. Standard management remains that invasive procedures always yield information that benefits the patient’s outcome. Discordant clinical trials are characterized as flawed in design.

As cardiologists, we see the patients referred to us to consider if a procedure is indicated, then we do the procedures, for which we are compensated; but receive only the fee for office visit if we do not advise the procedure be performed. That is self-referral, and the inherent conflict of interest this business model incorporates has had a substantial influence on modern practice. The pressure to do more cases is constantly applied from the administrative hierarchy: to prove quality, to generate income, to develop new referrals.

The response of third-party payors to the exponential rise in procedures was to suggest non-payment when the physician’s guidelines were abrogated. The physician’s response was to liberalize the criteria, eliminate the term “inappropriate” so that no case could be said to be not scientifically based, and denounce lack of payment for services in a fee-for-service environment. Consequently, the insurance companies now pay decreasing amounts for the procedure, currently at laughably low levels, because they realized that doctors and hospitals have no incentive to become partners in trying to control costs.

The decreased payment per case, of course, adds further pressure to do even more cases and procedures, of even less proven benefit to the patient, to generate more revenue. Hypothermia, ventricular assist devices, multivessel stenting in MI and shock, and specific treatment devices, have been advocated in these guidelines despite no studies showing benefits and even some showing a lack of benefit and even harm. Cycles of increasing indications for procedures following diminishing reimbursement have resulted.

Can This Be Fixed?

As Deming said, “Every system is perfectly designed to get the result that it does”; so to change the outcome, it would be necessary to change the system and its component parts which derive profit from these circumstances. One place to start is how trainees are taught. It’s not just what is said to fellows and housestaff, but how their teachers actually act. If they see their attendings say one thing and do another, with a wink and a nod, they get it. The practice of today has to reflect the values medicine should optimally follow in the future.

Incorporating the results of the ISCHEMIA Trial into practice guidelines is a significant challenge. The finding that SIHD with moderate-to-severe ischemia treated by revascularization had no benefit beyond OMT in preventing major cardiovascular events after 4 years challenges all of our preconceived notions. The premise that severely symptomatic SIHD should be treated invasively to improve mortality is incorrect: since worsening severity of ischemia is associated with increased mortality, logically it would seem to follow that procedures that reduce ischemia should improve survival, but this was not the case. Moreover, the traditional teaching that revascularization does not prevent MI in SIHD may be incorrect: the rate of spontaneous MI during 4-year follow-up was lower in the revascularization subgroup (HR 0.67 (0.53, 0.83), p<0.01), suggesting that perhaps PCI may reduce type I MIs.

For most patients with SIHD but without left main coronary disease or severely reduced left ventricular function, shared decision‐making about revascularization should be based on discussions of symptom relief and quality of life and not about reduction in mortality.

As better evidence is developed, more definitive appropriateness criteria should be implemented to ensure we deliver effective, valuable care — and contain costs.

This change would have immediate repercussions, as the entire medical payment system would have to re-equilibrate after decades of deception on all sides. It will mean less revenue in an environment in which over-utilized procedures are underpaid. Professional societies must take on the hard battles, showing responsibility and leadership. Mechanisms to self-regulate are needed. Those who repeatedly take advantage of the lack of objectivity in testing, without regard to costs to the patient, have to be discouraged, not rewarded, by their practice pattern.

Hospitals and physicians must agree to allow oversight of quality by outside, objective agencies and methods, and welcome it. The alternative is to continue down the current path, where costs are rising, reimbursement diminishing, income is threatened, and procedures are done with modest reference to clinical trials that determine what really helps the patient. The delivery of optimal clinical benefit requires an ongoing self-assessment structure comparing actual results to accepted benchmarks, with timely modification of practices when deficiencies are identified. The critical quality elements include adhering to evidence-driven case selection, ensuring proficient technical performance, and monitoring clinical outcomes [1-4].

References

  1. Klein LW, Dehmer GV, Anderson HV, Rao SV (2020) Overcoming obstacles in developing and sustaining a cardiovascular procedural quality program. Journal of the American College of Cardiology – Cardiovascular Interventions 13(23): 2806-2810. [crossref]
  2. Klein LW, Anderson HV, Rao SV (2019) Proposed framework for the optimal measurement of quality assessment in percutaneous coronary intervention. Journal of the American Medical Association – Cardiology 4(10): 963-964. [crossref]
  3. Klein LW, Anderson HV, Rao SV (2020) Performance metrics to improve quality in contemporary PCI practice. Journal of the American Medical Association – Cardiology 5(8): 859-860. [crossref]
  4. Anderson HV, Shaw RE, Brindis RG, et al. (2005) Relationship between procedure indications and outcomes of percutaneous coronary interventions by American College of Cardiology/American Heart Association Task Force Guidelines. Circulation 112 (18), 2786-2791. [crossref]

Overview of Hard Cyclic Viscoplastic Deformation as a New SPD Method for Modifying the Structure and Properties of Niobium and Tantalum

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DOI: 10.31038/NAMS.2024721

Abstract

In this overview work the changes in the structure and properties of commercially pure niobium and tantalum under conditions of hard cyclic viscoplastic deformation are studied. During linear compression-tension deformation of the sample, wich was carried out in the strain control mode in the range from ε=±0.2% to ε=±3.0% with a frequency f=0.2-2.5 Hz and with a number of cycles in the range from 20 up to 40, respectively. In addition to classical methods of severe plastic deformation, this method can be used to improve and stabilize the microstructure, mechanical, physical and functional properties of single crystalline, coarse-grained, ultrafine and nanocrystalline metallic materials. The experimental results obtained can be used to study the stability and viability of metallic materials, as well as predict their suitability over time in harsh environments such as space and military applications, and thereby expand new understandings and connections in materials science.

Impact Statement

This overview article comprehensively reviews recent advantages of the development of niobium and tantalum structure and properties studyied by severe plastic deformation (SPD) and hard cyclic viscoplastic deformation (HCVD) at room temperature.

Keywords

Severe plastic deformation, Hard cyclic viscoplastic deformation, Microstructure, Mechanical properties, Physical properties, Wear, Tribological properties, Viability, Electrical conductivity, Hydrogen storage, Young´s modulus

Introduction

Written science in the field of SPD first appeared in the late 20th century [1,2], but archaeological research has shown that the process was known and used at least 2,700 years ago [3] in the production of knives and swords. Humanity has been working with metals since the Bronze Age and has created a common and understandable terminology for all communities. For example, the metalworking process with severe plastic deformation (SPD) in turn contains about a hundred technological processes and a large number of terms to describe these processes and their results. The field of SPD is constantly being improved with new methods already developed and partially patented for more than a hundred names. Currently, more than 1000 papers are published annually in the field of SPD. The emergence and application of new processes in materials science also require the development and addition of new terminologies.It is well known, that the SPD methods are popular due to their ability to modify the microstructure [4-6] and mechanical properties [7-10] of various plastic metal materials. At that time, it was well known that the mechanical properties of SPD-processed materials were significantly better compared to their coarse-grained counterparts [11-15]. Experimental results show that it is possible to change the initial microstructure from a coarse-grained (CG) to an ultrafine-grained (UFG) structure with grain sizes in the range from 1000 to 100 nanometers at ECAP and to a nanocrystalline (NC) structure with a crystal size below 100 nanometers [16-21] at HPT. Unfortunately, the scientific works on SPD listed in [1-21] have so far mainly studied only changes in the microstructure and mechanical properties of materials, such as hardness and strength. At the same time, a number of recent scientific articles have shown that changes in microstructure and mechanical properties during SPD also lead to changes in electrical conductivity [22-27], phase transformations [28-31], wear resistance [32-35], cyclic plasticity [36-41], and so on. Unfortunately, in these works relatively little attention was paid to changes in functional properties, which limits the widespread use of these materials in modern industry.

Components and entire systems are characterized by time-varying cyclic loads and often random load sequences that can cause material fatigue and damage. Therefore, understanding the relationship between fatigue damage and random cyclic loading is a necessary prerequisite for reliable sizing of components and structures. However, this design of fatigue components is motivated not only by the desire to avoid damage to products and their repair. Today, issues of materials and energy efficiency are becoming increasingly important, and therefore increasing their sustainability during operation, which requires accurate knowledge of the operating loads of the systems and the corresponding fatigue behavior of the materials. Sustainability in today’s sense means making the most efficient use of available resources, and this goal can only be achieved for many components and structures if the load sequences present in the operation are known and taken into account when optimizing materials, design and production in industry. Information on the latest developments in the field of variable load fatigue, new scientific approaches and industrial applications of materials, components and designs is up to date. The achievements and results of research in recent years, new approaches and the latest processes in various industries are highly appreciated.

Tension-compression amplitudes, as characteristic features of typical workload sequences in various mechanisms, are becoming very important parameters in the design and optimization of components and structures. Material testing methods such as low cycle fatigue (LCF) [42] and high cycle fatigue (HCF) [43], ratchet [44], Bauschinger effect [45-47], Young’s modulus [54], are very important to determine the durability of materials in actual use.

For example, the number of cycles to failure for the LCF test method is typically less than 10,000, and the failure mode is typically ductile failure. During HCF testing, the material or component fails after a large number of cycles, typically greater than 10,000. Thus, HCF is typically associated with very low strain amplitude at tensile, elastic deformation, crack initiation and growth. The difference between these test methods, LCF and HCF, depends on the level of strain under tensile stresses, the ductility of the material and the degree of elastic deformation. Fatigue behavior is characterized by loading frequency, loading history, loading type, ambient temperature, microstructure, defects and residual stresses in the material.

The ratcheting method uses only tensile deformation with controlled tensile strain and a very small number of cycles. The ratcheting method is based on the well-known Bauschinger effect. The method for testing of metallic materials in viscoplastic states is so called as Hard Cylic Viscoplastic Deformation (HCVD) is described in [55-59]. The viscoplastic behavior and hardening/softening of metallic materials allows you to very quickly, simply and cheaply change and study the structure and properties of metallic materials. For example, this method has been used at firstly to study the microstructure, mechanical and functional properties of metallic materials such as coarse grained (CG) copper [60], ultrafine-grained copper alloys [61], pure niobium [62-65], pure tantalum [66-69] with oligocrystalline structure and also Ni-based single-crystal superalloy [70-73], etc.

This overview study on Nb and Ta by uising HCVD technique is based primarily on my own research work in which I have studied materials using various SPD techniques. HCVD principles were first presented in 2004 at the TMS Ultrafine Grained Materials III Annual Meeting, Charlotte, North Carolina, USA [74] and at the 4th DAAAM International Conference on Industrial Engineering – Innovation as a Competitive Advantage for SMEs, Tallinn, Estonia. [75]. Unfortunately, HCVD as a new process in materials science has not yet been widely used in studying the evolution of the structure and properties of metallic materials. At present time the stability and viability of metallic materials, and predicting their suitability over time in harsh environments such as space and military applications is actual. Studying the behavior of metallic materials in viscoplastic states using HCVD method allows us to expand concepts and new connections in materials science.

Experimental Section

Materials

The materials for present experimental work were technically pure niobium (Nb) and tantalum (Ta) ingots, which were produced by electron beam melting (EBM) technique on Neo Performance Materials (NPM) Silmet AS, Estonia. The chemical analysis of NPM Silmet AS showed that the pure Nb ingots, with diameter of 220 mm, contained the following non-metallic elements: N (30 ppm), O (72 ppm), H (˂10 ppm), c (˂20 ppm), and metallic elements: Ta (160 ppm), Si (˂20 ppm), P (˂15 ppm), Mo (˂10 ppm), and W+Mo (˂20 ppm), respectively.

The Ta inots diameter was 120 mm and an oligocrystalline macrostructure. The chemical composition of Ta was: Al, Mg, Pb, Cu, Fe, Mo, Mn, Na, Sn (for all ˂5 ppm), Nb (˂20 ppm), W (˂10 ppm), and Si (˂10 ppm) and non-metallic elements: N (˂ 20 ppm), O (˂ 30 ppm), H (˂10 ppm), S (˂10 ppm), C (˂10 ppm), respectively. These ingots had a oligocrystalline macrostructure up to 15-20 cm in length and approximately 5-6 cm in thickness (Figure 1). The Nb and Ta samples were before for recrystallization heat treated in vacuum furnace at 1100°C for 30 min.

FIG 1

Figure 1: Oligocrystalline macrostructure of Ta after EBM by industrial processing

The IEAP Technique and Test Samples Manufacturing

Using hard-to-deform Nb and Ta samples, the ECAP matrix was modified and a new so-called “indirect extrusion angle pressing” (IEAP) method was developed [34,63,66,67,76]. IEAP format channels do not have the same cross-section. The cross-section of the output channel, taking into account the elastic deformation of the base metal, was reduced to 5-7%, which makes it possible to use the conveyor method when pressing without intermediate processing of the cross-section of the workpiece. This is due to elastic deformation of the matrix and an increase in the cross-section of the sample during pressing. To process workpieces under HCVD, the developed IEAP technology was used. In the work under consideration, the microstructure of the samples under study was modified up to 12 passes of the IEAP along the BC route. The maximum degree of von Mises deformation during one pressing was 1.155, and after 12 passes – up to ~13.86, respectively. As experiments have shown, this IEAP die is convenient for processing high-strength materials, since at higher extrusion passes, when the strength of the materials increases sharply, the friction between the punch and the matrix during pressing also increases, and thus the risk of damage to the matrix or plunger increases. [67]. For comparison, the same metals (Ta and Nb of the oligocrystalline as well as recrystallization structure received at heat treatment) were used so that the results could be compared. Using the IEAP method, samples with dimensions of 12x12x130 mm are produced. The processing steps of IEAP samples are shown in Figure 2.

FIG 2

Figure 2: Diagram of the IEAP stamp and the corresponding stages (a, b, c, d) of sample processing using the so-called conveyor method, with this method in the stamp at the final stage two samples are simultaneously processed, the second sample pushes out the first (d).

The specimens for hard cyclic viscoplastic deformation (HCVD) were manufacturing from EBM and IEAP-treated samples (Figure 3a and 3b). The mechanical cutting and electroerosion tchniques were used. The samples for electrical conduction, hardness, gases content, density, XRD, and microstructure study were cut off from HCVD sample (Figure 3c) and tensile tests minisamples Figure 3f) were cut off by electrical discharge method from HCVD sample (Figure 3d and 3e). The strain amplitude at HCVD testing was measured by extensometer with base length of 10 mm and it was mounted on sample with minimal cross section (Figure 3c). The strain amplitude for other cross sections were calculated. IEAP of the samples was carried out on a hydraulic press with a capacity of 100 tons [68].

FIG 3

Figure 3: Mechanically cut sample from EBM ingot and heat treated at 1100°C for 30 minutes (a), IEAP treated sample (b), test sample for Young’s modulus measure by HCVD with stepped cross section (specimens A1, A2, A3, A4, A5 and A6 (as cast) with 5 mm in length) for measuring microhardness, density, XRD, gases content and electrical conductivity (c), mini-specimens (d) for tensile strength tests (MS1, MS2, MS3, MS4, MS5 and MS6-as cast), cut from the sample after HCVD in diametrical section in three layers 1, 2 and 3 (e), cut out using the electrical discharge method, and a mini-specimen for tensile testing with dimensions in mm is shown in (e).

The HCVD Technique

The HCVD technique was elaborated for materials structure modifing and properties testing [53,58,74,75]. The HCVD as a new process is not yet widespread in the study of metallic materials structure and properties evolution. It is well known that “viscoplasticity” is a response of solids involving time-dependent and irreversible deformations. In this research, we investigate the viscoplasticity of metallic materials and the accompanying changes in microstructure and properties at room temperature. To do this, we use cyclic deformation with a constant strain amplitude at each stage of the experiment. In the HCVD process, the strain amplitude ranges from ε=±0.2% to ±3.0% per cycle. The HCVD was conducted on an Instron-8516 metrials tester, Germany.This method is characterized by the generation of cyclic stress, the magnitude of which depends on the strength properties of the material at a given compression-tension deformation or strain amplitude. Therefore, this research method of metallic materials is called hard cyclic viscoplastic deformation (HCVD). The name of this new process begins with the word “hard”, which means that tensile and compressive deformations are used in the high-amplitude viscoplastic field in both tension and compression. The evolution of the microstructure of mixed metal materials is mainly studied from the deformation rate, the number of cycles, and the deformation stress amplitude of the HCVD method. The effect of HCVD on the improvement of the mechanical and physical properties of test materials has been described in various works [61,69,70,73]. In present overview work these large strain amplitudes of ε1=±0.2%, ε2 ±0.5%, ε3 ±1.0%, ε4 ±1.5%, ε5 ±2.0%, ε6 ±2.5%, and ε7±3.0% are used, respectively. At each degree of deformation, up to 20÷30 cycles are performed. The cycles number depend on material mechanical properties and viability. The frequency of cycling was chosed in the interval of ƒ=0.5 to 2.5 Hz, and were ƒ1=0.5 Hz, ƒ2=1.0 Hz, ƒ3=1.5 Hz, ƒ4=2.0 Hz, and ƒ5=2.5 Hz, respectively. The number of tests of the HCVD method starts from 20 cycles and up to 30 cycles per test for a series with the corresponding constant strain amplitude. The maximal number of cycles was not more then 100 for one sample. The frequency and strain amplitude at HCVD influenced on the strain rate and corresponding changes in the microstructure and properties.

To achieve the required results, the rated voltage, frequency, and a number of cycles are selected based on the test results. Usually, the technical strength is the strength of various constructions in calculations in elastic strain up to ε1=0.2% at tensile deformation, that is, until the beginning of plastic deformation of the material. In the LCF and HCF tests, the amount of elastic deformation is small, less than 0.2% of the deformation. At such a value of deformation, the metal material has an elastic behavior. The HCVD method determines the magnitude of the controlled amplitude of deformation and is controlled by an extensometer using a computer program that controls the process and displays the corresponding results on the computer screen. Typically, the fatigue test of metallic materials checks the tensile strength but does not check the deformation, which develops automatically according to the mechanical properties of the material. Micromechanical multiscale viscoplastic theory has been developed to relate the microscale mechanical responses of amorphous and crystalline subphases to the macroscale mechanical behavior of fibers, including cyclic hardening and stress recovery responses. The HCVD method can be used as a new test method in materials science when it is necessary to determine the behavior of a material under stresses that can exceed the elastic limit and deform under extreme operating conditions. For example, such extremes may occur in aviation, space, or military technology because these devices have a minimum calculated strength or margin of safety compared to other devices. For example, the compressor blades of a turbojet engine for military fighters have a safety margin of no more than 3-5%.

Methods for Other Properties Testing

The microhardness in cross section of samples A1, A2, A3, A4, A5, and A6 was measured using a Mikromet-2001 tester after holding for 12 s at a load of 50 and 100 g. At follows, the mini-samples (MS1, MS2, MS3, MS4, MS5 and MS6-as cast) at tension up to fracture were tested on the MDD MK2 Stand test system manufactured in the UK. The tribological behavior of materials under dry sliding conditions was investigated before and after IEAP, HCVD and heat treatment to provide a comparison over a range of material properties as well as collected strain to understand their influence on the coefficient of friction and on the specific wear rate. Dry sliding wear was studied in a ball-plate system with a tribometer (CETR, Bruker, and UMT2) using an aluminum oxide (Al2O3) ball with a diameter of 3 mm as a counter surface. The coefficient of friction (COF) was obtained automatically. For wear volume calculations, the cross-sectional area of the worn tracks was measured by the Mahr Pertohometer PGK 120 Concept 7.21. The content of metals inclusions (in ppm) were studied according to MBN 58.261-14 (ICP-OES Agilent 730) and gases concentration according to method of MBN 58.266-16 (LECO ONH-836) and S according to method of MBN 58.267-16 (LECO CS-844), respectively. The electrical conductivity (MS/m and/or %IACS) of metal materials was determined with a measurement uncertainty of 1% for different orientations on flat samples by means of the Sigmatest 2.069 (Foerster), accordingly to NLP standards at 60 and 480 kHz on a calibration area of 8 mm in diameter. The electrical conduction was measured at room temperature of 23.0±0.5°C and humidity of 45±5% according to the international annealed copper standard (IACS) in the Estonian national standard laboratory for electrical quantities. To obtain one electrical conductivity data, 30 measurement tests were automatically performed and the result was displayed on the computer screen.

The samples density after IEAP with different pressing number was measured by OHAUS Scout-Portable balances, Italy at room temperature. The dislocation density was calculated by the Rechinger method according to the results of the X-ray investigation by the D5005 AXS (Germany) and Rigaku (Japan) diffractometer. To study the microstructure, the samples were mechanically polished with silicon papers up to 4000, and then with diamond paste on Struers grinder. After the grinding, the samples were etched by an ion polishing/etching facility using precision etching system at 30 kV for 30 min in an argon atmosphere. The microstructure of the samples was studied using an optical microscope Nikon CX, Japan, and electron microscopes Zeiss EVO MA-15 and Gemini Supra-35, Germany, equipped with an EDS apparatus.

Results

Microstructure Evolution of Nb and Ta during HCVD

For example, full-scale diagrams of the HCVD of the Nb manufactured with various processing methods, microstructure, and properties at different strain amplitudes are shown in Figure 4a-4e [63].

FIG 4

Figure 4: HCVD curves of pure Nb for the viscoelastic tension-compression straining at an amplitude of ε = ±0.1% and corresponding deformation amplitude of v ꞊ ± 0.01 mm in the base length of 10 mm (a), viscoelastic tension-compression straining at strain amplitude of ε = ±0.5% and v ꞊ ± 0.05 mm (b) and at strain amplitude of ε = ±2.0% with the corresponding deformation amplitude of v ꞊ ± 0.2 mm (c). The sample E12 HCVD time-deformation (d) and time-stress (e) curves received at ε = ±2% of strain amplitude. The effect of the elastic-plasticity of Nb on the deflection of the curves during the compression (C) and tension (T) cycles is shown by arrows.

As can be seen in Figure 5, high-purity niobium as well as tantalum EBM ingots contain very large millimeter-sized grains connected by a fully wetted triple grain boundary (GB) (Figure 5a). This width of GBs is in nanometers because the metal is of high purity with thin grain boundaries as shown in the figures. Unfortunately, such large grains contain gas pores with dimensions in micrometers (Figure 5b). Under hydrostatic pressure in the shear region of the IEAP die, these pores are compressed and velded to zero. Such pores and GB defects can be completely repaired by hydrostatic compression and simple shear in the IEAP die. These changes take place in sample for 4 passes of IEAP by BC route (c) and for 12 passes of IEAP by BC route (d), respectively. Microstructural evolution in bulk Ta samples during HCVD are presented in Figure 6. As you can see the microstructure of high purity Ta has GB-s on atomic level [63,68].

FIG 5

Figure 5: The triple grain boundary (a) and pores (b) in EBM as-cast Nb, and SEM pictures of microstructure evolution via grains fracture by slip lines (SL is shown by arrows) in the shear region of IEAP at von Mises strain of ƐvM=4.62 by BC route (c) and UFG microstructure formed at ƐvM=13.86 (d), respectively.

FIG 6

Figure 6: Microstructure evolution of pure Ta processed by HCVD at 5 test series (5 x 20 cycles with strain step-by-step increase up to ε5 ± 2.0%) for 100 tension-compression cycles in sum (a) and atomic level GB-s with different orientations of two grains is presented in (b, c).

The microstructure forming in Nb and Ta samples from initial to 8 passes by BC route for Nb and for 12 passes by BC route for Ta is shown in Figure 7a-7d. The relative frequency of grain size in mm was calculated by by ImageJ software and is presented in Figure 7e-7h. As you can see the grain size of Nb was decreased about 3 times and Ta grain size was decreased about 2.5 times, respectively [67].

FIG 7

Figure 7: Distribution of grains sizes of Nb (a, b) for initial (Nb0) and after eight passes (Nb8) and Ta (c, d) for initial (Ta0) and after twelve (Ta12) passes by BC route of IEAP. The corresponding grain size measurements were made by ImageJ software (e, f, g, h), respectively.

The microstructure evolution of IEAP Nb sample during HCVD, with a number of 100 cycles for 5 test series is shown in Figure 8a. For comparison, the IEAP Nb after LCF testing for 100 cycles in Figure 8b. The fatigue cracks are formed during LCF. The TEM images of SB-s with a lowered dislocation density at HCVD in Figure 8c and with high dislocation density after LCF of IEAP sample in Figure 8d, respectively [64].

FIG 8

Figure 8: Optical pictures of double-banded microstructure forming in Nb sample at HCVD for 100 cycles (5 x 20 cycles) (a) [64,69] at increased strain amplitude to ε5 ± 2.0% and cracks initiation during LCF testing (b) for 100 cycles of the ECAP sample [69]. TEM images of SB-s with a lowered dislocation density at HCVD (c) and with high dislocation density after LCF of IEAP sample (d).

Young´s Modulus Evolution of Nb at IEAP and at HCVD

The evolution of physical properties during HCVD depends on the microstructure and properties of the metallic material achieved by IEAP treatment, as well as on the strain rate, which depends on the strain amplitude (measured in mm) during HCVD (Figure 4a-4c). As shown in the diagram (Figure 9a), the increase in tensile strength during HCVD at a strain amplitude ε4=±1.5% for all IEAP samples (E2, E4, E6, E8 and E12) with different accumulated von Mises strains is maximum. With an increase in deformation to ε5=±2.0%, the tensile strength of samples E8 and E12 decreased, since these workpieces had a UFG microstructure obtained in IEAP, with higher tensile strength and hardness. During the HCVD process, at a deformation amplitude ε5=±2.0%, softening occurs, since the GS begins to increase during the coalescence process. In these workpieces, Young’s modulus also decreases as they soften (Figure 9b) during HCVD treatment. The Young’s modulus of IEAP-treated samples decreases during the HCVD process as the dislocation density decreases (Figure 8c and 9) [63,64].

FIG 9

Figure 9: The IEAP samples E2, E4, E6, E8 and E12 tensile strength increases up to strain amplitude ε4=± 1.5%, as well as the strain rate (v=0.3 s-1) increases during HCVD and decrease for E8 and E12 by strain amplitude increase to ε4=±1.5%, (a) and the Yung modulus (b) increases when the von Mises strain increases to ƐvM=11.55 by BC route during IEAP, and the Young’s module decreases in samples E12 by increased von Mises strain up to ƐvM=13.86 by BC route as well by cycles number increase up to 100 cycles during HCVD, respectively.

Ta Physical Properties Evolution at HCVD

The Vickers hardness of Ta was measured, and it was found that with increasing of von Mises strain at IEAP in sample with stepped cross-section, the hardness of Ta increased mainly during the first pressing. By this, the hardness depends on the measurement of orientation to the sample; it is higher in the transverse direction (TD) and lower in cross directions (CS). As shown in Figure 10, a, the Vickers hardness of Ta was increased by increasing the von Mises strain at HCVD. The Vickers microhardness of Ta was increased from 100 HV0.2 to 285 HV0.2, respectively. It should be noted that the methods for measuring hardness according to Martens and Vickers are different. The Marten’s hardness is calculated from the difference between the maximum depth of indentation and after removing the load, when the Vickers hardness is calculated from the length of the diagonal, indentation load and only hardness are measured. The electrical conductivity of Ta (Figure 10b) has similar dependence on strain level and orientation of measure in the heat-treated sample [68].

FIG 10

Figure 10: The evolution of Vickers microhardness (a), and electrical conductivity (b) depends on measuring orientation and heat treatment temperature from 20°C to 165°C with a heating rate of 1°C•min−1. TD: Transverse Direction and CS: Cross-Section.

A well-known fact from the scientific literature is that the Young’s modulus is constant in materials at room temperature. This modulus decreases with increasing temperature and increases with increasing the density of materials. Young’s modulus of Ta is about 186 GPa at room temperature and the maximum value is 193 GPA at 10−6 K. As can be seen in Figure 11, this modulus may also be affected by the amount of strain applied to the material (or the number of passes in IEAP) and the change in equivalent strain-stress amplitude during HCVD. The changes of Young’s module in the Ta samples (S1-initial, S2 – 5 pressings of IEAP, ƐvM=5.77, and S3 – 12 pressings of IEAP, ƐvM=13.86) are shown in Figure 11. Before Young module measure the samples were processed by HCVD at strains of ε2=±0.5%, ε3=±1.0%, and ε4=±1.5% for 20 cycles at one strain level. It should be mentioned that Young’s modulus of each sample was measured for three times in the intervals of tensile strains from 0÷0.06% and from 0÷0.1% to ensure about the reliability of results. Was established, that this modulus depends on von Mises strain, strain rate as well on the interval of strain, at which this parameter was measured. When the material is harder, Young’s modulus is higher at the tension in the interval of the strain of 0-0.1% (S2) and when the material is softer, Young’s modulus is higher for 0÷0.06% strain interval (S1) and lowers at 0÷0.1%, respectively [68].

FIG 11

Figure 11: Change of Young’s modulus in Ta at uniaxial tension (measured after IEAP and HCVD) at strain of 0–0.06% (blue) and of 0–0.1% (red) of samples S1 (a), S2 (b) and S3 (c), respectively.

Changes in electrical conductivity and Vickers microhardness in IEAP samples (S1, S2, and S3) were measured with different orientations (Figure 12). These values vary depending on the strain applied during processing or hardness, as well as on the orientation of the measurement, in cross-section (CS) or transverse (TD) direction. As you can see (Figure 12), when the Vickers microhardness is higher, the conductivity is lower when the heating rate is low. In work is shown, that the electrical conductivity depends on the hardness and strength properties of CuCr-alloys. In the works [39] is shown, that the electrical conductivity and Vickers microhardness of CuCr-alloys increase with temperature increase and revealed maximal values at ~550°C. Accordingly, these parameters depend not only on the microhardness because the density of dislocations was lowered during heat treatment. In the present work, the Ta samples were heat treated at a very low heating rate of 1°C·min−1, and the Vickers microhardness and electrical conductivity increased in sample S2 and decrease in sample S3, respectively. The conductivity is expressed as a percentage of the International Standard Annealed Copper (%IACS), which is 5.80 × 107 Siemens/m at 20°C. Results show, that the electrical conductivity varied in dependence on energy associated with dislocations, grain boundaries state, and vacancy concentration in Ta samples during ECAP and HCVD, respectively (Figures 12 and 13) [68,69].

FIG 12

Figure 12: Influence of processing routes as well as microstructure on microhardness, and dislocation density (a), electrical conductivity and density (b), oxygen and hydrogen contents (c) of pure Niobium. Designations: E12-12 passes of IEAP by BC route, H5-five test series by strain rate increase during HCVD, E12-350°C- heat treatment temperature of sample E 12.

FIG 13

Figure 13: Creep and Relaxation of different materials vs. manufacturing technologies (a). Designations: N1 – By electrical forging (EF) processed Ni-based Fe containing superalloy, N2 – SC Ni-based superalloy, N3 – Cold-drawn pure Cu, N4 – Recrystallized pure Cu, and N5 – ECAP processed nanocrystalline pure Cu [78]. Density evolution of pure Ta processed by IEAP and HCVD (b). Designations: (A4) IEAP processed only, and (A1, A2, and A3) after followed HCVD with different strain amplitude (see Figure 3c) (b).

XRD Investigation of Changes in the Tantalum during EBM, IEAP, and HCVD

Anisotropic deformation during IEAP, and HCVD processes, as well as anisotropic properties in samples, can lead to the formation of anisotropic crystallites and, therefore, anisotropic peak intensities. The X-ray diffraction patterns of the samples: Ta, 2x EBM, Ta, 5x IEAP, Ta, 12x IEAP, and Ta, 5x HCVD are presented in Figure 14. As you can see the X-ray diffraction patterns of the HCVD Ta with compare to EBM and IEAP are differ significantly from other samples. It should be noted that this sample (5x HCVD) had a recrystallized microstructure before HCVD. During HCVD, the microstructure changed, and only one peak appeared in the X-ray diagram at ~ 55.6°. Such an X-ray pattern with a single peak is characteristic of a single-crystal metal and a single-crystal Ni-based superalloy [71]. XRD investigation revealed that at the phase transformation took place at the SPD processing [70,72]. The crystallite size and the dislocation density is possible to determine by X-ray line profile analysis [67,68].

FIG 14

Figure 14: X-ray diffractogram (a) of the Ta samples. Designations: Ta, 2x EBM, initial, Ta, 5x IEAP, Ta, 12x IEAP, and Ta, 5x 5HCVD), respectively.

Influence of HCVD on Wear and Tribological Properties of Nanocrystalline Materials

The specific wear rate (the volume loss per distance per normal load) and coefficient of friction (COF) measurements show their dependence from sample material chemical composition, sample (surface) hardness as well material wear track surface softening/hardening [68,76] during wear testing (Figure 15). Results show that HCV deformed sample in surface was hardened from 77HV0.05 to 90HV0.05 and on the wear track surface from 115 HV0.05 to 126HV0.05, respectively. In this case the surface hardening was induced by cyclic straining and wears track hardening as result of sliding [69].

FIG 15

Figure 15: Influence of the load applied on the COF and wear track cross/sectional area of IEAP-12 Niobium (a) and specific wear rate of pure Niobium for different passes number and temperatures for a load of 50 gr (b).

The SEM investigation of the worn track surface shows (Figure 16a) that the UFG microstructure of Nb was abrased by the alumina ball during the dry sliding testing. In our experiments, the wear debris was not removed from the contact zone during testing, which has an influence on the results [35]. The damaged surface of the worn track has wear debris with size of approximately 100 nm. The test results show that the as-cast sample has the lowest amplitude of COF then UFG Nb. The as-cast material has the lowest COF (0.78) and the lowest specific wear rate (2.1, ×10-2×mm2×g-1) when compared to sample E12 (Figure 15b). The maximal COF was obtained for samples after HCV deformation and samples that were tested in directions that crossed the slip band direction. The specific wear rate was significantly increased after heat treatment at a temperature of 350°C (Figure 16) [69].

FIG 16

Figure 16: SEM picture of wear track surfaces for 15, 50, 100 and 150 g (a) and UFG Nb wear surfaces with debris formed under a load of 100 g (b), and worn surfaces at high magnification of 100000x of samples after 6 passes of IEAP (c).

When comparing our results with the results presented in previous work, the mass loss decreased remarkably as the number of ECAP passes increased, being affected more by the sliding distance than by the applied load under the experimental conditions. From these data, it has been shown that the wear mechanism was observed to be adhesive and delaminating initially, and an abrasive mechanism appeared as the sliding distance increased. In our experiments, the abrasive wear mechanism did not show any dependence on sliding distance.

Discussion

In the papers, a series of experiments were carried out to study the effect of IEAP and HCVD on the microstructure and properties of metallic materials at room temperature. During the subsequent HCVD, we studied the effect of the strain value during tension-compression with a gradual stepwise increase in strain, strain amplitude, and the corresponding strain rate on the microstructure, functional, physical, chemical, and mechanical properties of the studied Nb and Ta. Then, a series of experiments were carried out to study the influence of the number of deformation cycles, the magnitude of axial deformation, the frequency of cycling, and the strain rate during HCVD and followed heat treatment on the microstructure and evolution of the properties of materials in comparison with their initial state. A comparative analysis was carried out, according to the results of which the following conclusions can be drawn: This owerview study evaluated the impact of a new processing method so called as “Hard Cyclic Viscoplastic Deformation” (HCVD) on microstructure, mechanical, physical, chemical, functional, performance, etc. properties of metallic materials such as niobium, and tantalum. To expand the capabilities of the new processing method, the metal materials Nb and Ta with various structures were tested, such as oligocrystalline, coarse-grained, ultrafine-grained, and nanocrystalline. For pre-treatment, multiple methods of severe plastic deformation were used, such as “Indirect Extrusion Angular Pressing” (IEAP). With this new HCVD test method, it is possible to initiate and study the processes occurring in the microstructure of materials before their destruction. HCVD is based on the application of a cyclic tensile/compressive load by controlled strain amplitude on materials at a constant frequency at a given strain level. In this test method, the main parameters are the strain amplitude of compression/tension in the range from 0.2% to 3.0% with the number of cycles from 20 to 40 for one level of deformation and with a frequency of 0.5 to 2.5 Hz. The rest of the process parameters are set automatically depending on the strength properties of the tested metal material in general.

Conclusions

This review study evaluates the effect of a new processing method, the so-called HCVD, on the microstructure microstructure evolution, and mechanical, physical, chemical, functional, tribological properties change, as well as on phase transformations and interatomic interactions, and service life of various metallic materials. To expand the capabilities of the new processing, metallic materials selected for the study, such as Nb and Ta, with different structures: oligocrystalline, coarse-grained, ultrafine-grained and nanocrystalline were tested.

Using this new test method, it is possible to initiate and study the processes occurring in the microstructure and properties of materials during HCVD before their fatigue failure. HCVD is based on the application of cyclic tensile and compressive loads to materials through a controlled strain amplitude at a constant frequency and a specified level of strain. In this test method, the main parameters are the compression-tensile deformation amplitude in the range from ε=±0.2% to ε=±3.0% with a number of cycles from 20 to 40 for one level of deformation amplitude and with a frequency ƒ=from 0, 5 Hz to 2.5 Hz. The rate of deformation depends on the basic strength parameters of the material. The remaining process parameters are set automatically depending on the strength properties of the metal material being tested as a whole. The main outcomes of this overview work can be summarized as follows:

  • The microstructure of Nb and Ta processed by HCVD is significantly different from the microstructure obtained by other SPD methods.
  • During IEAP processing of EBM as-cast Nb or Ta samples, the gases pores and any defect in GB-s at hydrostatic compression pressure concurrently with the simple shear stress are eliminated.
  • The electrical conductivity in SPD processes decreases with increasing hardness, tensile stress and dislocation density and increases when HCVD is combined with heat treatment and lowering of dislocation density.
  • The density of pure Nb increased from 8.27 g/cm3 in the as-cast condition to 8.65 g/cm3 after IEAP and HCVD processing, whit’s is higher than the theoretical (8.55 g/cm3) density.
  • The density of pure Ta incrased from 16.26 g.cm-3 to 16.80 g.cm-3 during HCVD.
  • During the followed HCVD, the nanostructure (20-90 nm) was formed in shear bands.
  • The electrical conductivity during IEAP decreased and during HCVD it increased as a result of the dislocation density decreasing from 5E + 10 cm−2 to 2E + 11 cm−2 since the dislocations are the main obstacles for electrons moving.
  • During IEAP, Young’s modulus of Nb was increased to 105 GPa at the von Mises strain ƐvM = 13.86 and then decreased to 99 GPa during the HCVD. The Young modulus (89 GPa) was minimal for sample E12 after HCVD at strain amplitude (Ɛ5= ±2.0%).
  • The softening of the material is related to the decrease of Young’s modulus at the HCVD with the increase of the strain rate higher than έ (t)  = 0.3 s−1.
  • In turn, the decrease in Young’s modulus indicates a decrease in the attraction of interatomic forces in the metal.
  • The micromechanical properties differ for IEAP and HCVD samples, as well as for SB and in-body metal. For example, after IEAP for 12 passes by BC route, the SB maximal Vickers nano-hardness was NH = 4.78 GPa and the indentation modulus was Er = 177.7 GPa, respectively.
  • During the followed HCVD, these parameters were reduced to NH = 3.29 GPa and Er = 111.4 GPa, respectively.
  • The GB width of pure Nb is so small that it is not possible to measure its micromechanical properties by the nano-indentation method used in the present study.
  • The gas content in Nb depends on the microstructure condition and it is minimal for UFG pure Nb.
  • Compared to the LCF and HCF tests, the HCVD tests require a shorter timeframe.
  • Using the HCVD method, it is also possible to study the viability of different metallic materials during operation in aviation, space, and defense under conditions of high load, and close conditions before failure, when the margin of safety is only a few percent.
  • Accordingly, this overeview article provides a brief overview of the structure and properties of metallic materials that change as a result of HCVD and thereby extending materials science with new relationships.

Acknowledgment

This research was sponsored by the Estonian Research Council (Grant No. PRG1145).

ORCID Lembit Kommel http: //orcid.org/0000-0003-0303-3353.

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  58. Kommel L, Hussainova I, Traksmaa R (2005) Characterization of the viscoplastic behavior of nanocrystalline metals at HCV deformation. Rev Adv Mater Sci 10: 447-453.
  59. Kommel L, Mikli V, Traksmaa R, Saarna M, Pokatilov A, et al. (2011) Influence of the SPD processing features on the nanostructure and properties of a pure niobium. Mater Sci Forum 667-669: 785-790.
  60. Kommel L, Rõzkina A, Vlasieva I (2008) Microstructural features of ultrafine-grained copper under severe deformation. Mater Sci (Medžiagotyra) 14: 206-209.
  61. Kommel L, Hout J, Shahreza BO (Effect of hard cyclic viscoplastic deformation on the microstructure, mechanical properties, and electrical conductivity of Cu-Cr alloy. J Mater Eng Perform.
  62. Kommel L, Saarna M, Traksmaa R, Kommel I (2012) Microstructure, properties and atomic level strain in severely deformed rare metal niobium. Mater Sci (Medžiagotyra) 18;330-335.
  63. Kommel L (2019) Microstructure and properties that change during hard cyclic visco-plastic deformation of bulk high purity niobium. Int J Ref Met Hard Mater 79: 10-17.
  64. Kommel L, Laev N (2008) Mechanism for single crystal refinement in high purity niobium during equal-channel angular pressing. Mater Sci (Medžiagotyra) 14: 319-323.
  65. Kommel L (2008) UFG microstructure processing by ECAP from double electron-beam melted rare metal. Mater Sci Forum 584-586: 349-354.
  66. Kommel L, Shahreza BO, Mikli V (2019) Structuration of refractory metals tantalum and niobium using modified equal channel angular pressing technique. Key Eng Mater 799: 103-108.
  67. Omranpour B, Kommel L, Mikli V, Garcia E, Huot J (2019) Nanostructure development in refractory metals: ECAP processing of Niobium and Tantalum using indirect-extrusion technique. Int J Refr Met Hard Mater 79: 1-9.
  68. Kommel L, Shahreza BO, Mikli V (2019) Microstructure and physical-mechanical properties evolution of pure tantalum processed with hard cyclic viscoplastic deformation. Int J Ref Met Hard Mater 83: 104983.
  69. Kommel L, Kimmari E, Saarna M, Viljus M (2013) Processing and propeties of bulk ultrafine-grained pure niobium. J Mater Sci 48: 4723-4729.
  70. Kommel LA, Straumal BB (2010) Diffusion in SC Ni-base superalloy under viscoplastic deformation. Def Diff Forum.
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  72. Kommel LA, Straumal BB (2010) Diffusion in SC Ni-based superalloy under viscoplastic deformation. Defect and Diff Forum 297-301: 1340-1345.
  73. Kommel L (2015) Effect of hard cyclic viscoplastic deformation on phase’s chemical composition and micromechanical properties evolution in single crystal Ni-based superalloy. Acta Physica Polonica A.
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  77. Omranpour B, Kommel L, Garcia Sanchez E, Ivanisenko J, Huot J (2019) Enhancement of hydrogen storage in metals by using a new technique in severe plastic deformations. Key Eng Mater.
  78. Kommel L (2001) The influence of development of new technology and materials on resource of gas turbine engines.

Evaluation of Skin and Organ Dose of Patients Caused by Computed CT and Comparison with Monte Carlo Simulation Software GEANT4 (GATE)

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DOI: 10.31038/NAMS.2024714

Abstract

Today, the use of CT scan as a type of diagnostic tool has increased dramatically. Therefore, controlled use and in accordance with protective regulations in order to reduce the harmful  effects of radiation, it is necessary. The purpose of this study was to measure the dose received by patients in computed CT scan protocols and compare it with Monte Carlo simulation using GEANT4 software. Radiation parameters were collected from 11 patients referred to Tohid Hospital in Sanandaj to measure DLP quantity in common protocols. In this study, DLP values for Chest Abdomen protocol were measured and compared with simulation values. Our results show Monte Carlo software outputs experimental data well and is a good benchmark for this software. Thus, the simulated and measured doses agreed well.

Keywords

Computed tomography, Chest CT scan, Monte Carlo, Dose during scan, Reference dose limit

Introduction

CT scan is an advanced imaging technique that provides cross- sectional and transverse images of body parts using X-rays using computer algorithms and calculations [1]. Today, use of CT scan as a type of diagnostic tool has increased dramatically. Specific information is required including activity distribution and organ boundaries for patient-specific dosimetry. CT data provides anatomical information which can be used for defining volume of interests specifying internal organs [2,3]. Nevertheless, using CT images for segmentation of anatomic structures of patient body, despite being more accurate, is time consuming. The alternative is using phantoms or Atlas data with already segmented organs and known organ boundaries. The anatomical structures are derived from these databases very easily [4]. In the United Kingdom, CT scans ranged from 250,000 to about 5 million from 1980 to 2013, representing a 20-fold increase, while in the United States, CT scans ranged from 2 million to 85 million. It has been shown to show a growth of approx. 43 [5]. In the United Kingdom and the United States, CT scans account for 11% and 17% of all medical X-ray tests and 67% and 49% of the cumulative effective dose, respectively. Absorption dose in tissues in CT scan is a higher component of the doses received by patients in diagnostic radiology methods [6,7]. Different parameters affect the dose received by patients in CT imaging. One of the most important factors influencing the dose received by patients is the intensity of the current in the tube (current generated in the tube due to the flow of electrons inside it) as a determinant of the amount of X-rays. For dosimetry calculations GATE (GEANT4) application to Tomographic Emission) [8], a Monte Carlo based script interface dedicated to nuclear medicine, was used. Different versions of this free open source toolkit are available on the open GATE collaboration website [9]. For dosimetry applications, GATE is capable to take either patient’s CT or a digital atlas phantom as input [10]. GATE has certain attractive features; some of them are inherited from GEANT4 [11] and some are additionally developed. These include flexible simulation geometry capable of accommodating a large variety of detector and source details and the physical events. In this study we review the evaluation skin and organ dose of patients caused by CT scan and comparison with Monte Carlo simulation software GEANT4 using DLP index.

Methods

Patient Study

This study was performed on 11 patients referred to Tohid Hospital in Sanandaj for chest CT scan. GE Light Speed RT, a third generation standard radiotherapy CT (GE Medical Systems, Milwaukee WI), was used in this study. The scanner has a large bore (80 cm), distance X-ray tube and isocenter 60.6 cm and performs 4-slice helical scanning. The tube voltage 80-140 kV step 20, tube current 10-440 mA step 5, rotation times of 1, 2, 3 and 4 seconds are available. Images were acquired with slice thicknesses of 2.5 mm on 10.0 mm collimation (4 × 2.5 mm) (GE Light Speed RT CT scanner technical evaluation November 2005). This scanner is used routinely for obtaining patient images for radiotherapy treatment planning at the Akdeniz University School of Medicine Department of Radiation Oncology. The regular quality assurance (QA) for image quality, 120-200 kV-mA measurement and mechanical tests based on national and international processes was performed. Three different body regions of the Rando phantom (head, chest and pelvic) were scanned by applying typical clinical protocols. The scan parameters kV, mA, pitch, FOV (field of view), rotation time, slice thickness of the CT examinations which were used in this study are given in Table 1. The scan length for each scanning protocol is also shown in Figure 1.

Table 1: Quality control tests include the accuracy and reproducibility of the parameters of each scan

Protocol

 Mode KVp mAs P T (mm) I(mm)

L (cm)

Breast

 Helical 120 200 1.5 10 10

33.26-1.5

fig 1

Figure 1: A typical transverse slice of CT image of two patients

Monte Carlo Simulation

For both simulations of patient-specific dosimetry with the CT and XCAT phantom, the simulations were performed in GATE Monte Carlo code (version 6.0.0). The data of SPECT, CT and XCAT phantoms were processed to prepare suitable input file formats for GATE. The results of the internal dosimetry for the real activity distribution in the patient body based on the computed CT data were calculated for the CT image and the XCAT phantom in skin as well as in the total body. Photon absorption, Compton and Rayleigh scattering, ionizations, multiple scattering photons were simulated. After completion of simulations, GATE produced two binary files, containing respectively the absolute absorbed dose delivered into the voxels as DLP index (mGy) and the corresponding uncertainties [12]. Dycom photos of each case with VV the 4D slicer software converted into an MHA file or in another way with Mimics Medical 21.0 software converted to 3D STL files. Then, dosimetry separate programs were written for each of these inputs, in MHA and STL formats, and the output of both was almost the same, but in the 2D mode the results were closer to reality.

Dosimetry Calculations

Dose length product (DLP) measured in mGy*cm is a measure of CT tube radiation output/exposure. It is related to volume CT dose index (CTDIvol), but CTDIvol represents the dose through a slice of an appropriate phantom. DLP accounts for the length of radiation output along the z-axis (the long axis of the patient).

DLP = (CTDIvol) * (length of scan, cm)

[units: mGy*cm]

DLP does not take the size of the patient into account and is not a measure of absorbed dose. If the AP and lateral dimensions of the patient are available, then the size specific dose estimate (SSDE) can be used to estimate the absorbed dose.

It is important to remember that the dose length product is not the patient’s effective dose. The effective dose depends on other factors including patient size and the region of the body being scanned. Some multipliers, called k-factors, have been estimated to convert DLPs into effective doses, depending on the body region. If interested, consult reference.

Results

Organ dose simulations were performed using the scan parameters for the chest and abdomen-pelvis CT examinations. The scan range used for the chest CT contained the entire pulmonary area and that used for the abdominal-pelvic CT extended from the diaphragm to the pubic symphysis. In each simulation the obtained results of DLP values for the dedicated GE Light Speed RT CT scanner for organ were about 250 mGy (Table 2). The reported values by manufacturer are 30.16 mGy and 23.9 mGy (GE Report 2005) so Commutated CT is used these values as standards at spreadsheet. The obtained results of DLP values from this study were less then reported values. As a general in the literature, the DLP value for conventional CT scanner is reported to be from 17 to 48 mGy [13-15]. For this dedicated CT scanner, the DLP values were in the range of values from conventional CT. In this study, the organ dose values were obtained by another measurement using the GATE Monte Carlo code (version 6.0.0) calculator and the two methods were compared for each scan protocol. The organs that were in the scanned region are blind listed in Table 3. First result of this study showed that the organ dose is relatively higher in helical mode by using GATE Monte Carlo simulation scanning.

Table 2: Results about dosimetry based on computed CT

Mode

 DLP (mGy-cm) Number
helical 259.9

W .1

helical

 226.5 M .2
helical 248.7

-W .3

helical

 231.6 -M .4
helical 247.3

-M .5

helical

 258.3 -M .6
helical 241.6

-M .7

helical

 230.5 -M .8
helical 259.7

-W .9

helical

 255.9 -M .10
helical 244/3

-M .11

helical

 243/9

-W .12

Table 3: Comparison between dosimetry based on CT and GATE Monte Carlo simulation

(DLP) GATE Monte Carlo code

 DLP (CT Scan) Number
267.4 259.9

1

232.5

 226.5 2
296.1 248.7

3

264.7

 231.6 4
270.8 247.3

5

280.5

 258.3 6
255.2 241.6

7

266.3

 230.5 8
298.5 259.7

9

276.5

 255.9 10
264.9 244.3

11

282.3

 243.9

12

Discussion

We observed similar organ dosimetry results based on phantom with and patient’s CT data (Table 2). The similarity of the whole body dosimetry shows that the phantom and the calculation/simulations are generally acceptable. Variation between the organ boundaries and geometry of organs between patient and phantom may cause the differences and affect the organ dosimetry. In this study we used the GATE Monte Carlo code for calculation of absorbed dose. GATE code is already validated for dosimetry in many clinical situations including brachytherapy, external beam radiotherapy with photons/electrons, systemic radiotherapy, and proton-therapy. One of the main privileges of GATE is the capability to support both imaging and therapy modeling procedures [16]. The method we used has been employed with variations in other studies [17] for example to study mathematical phantom derived from the MIRD-type adult phantom. The use of phantoms is already validated for internal dosimetry purposes. Another reports showed that the dosimetry based on phantom is different from those based on the Zubal phantom as well as different dosimetry estimations obtained from different BMIs. We showed, the calculated doses have a good approximation in the simulated software and the higher percentage of dose in the simulation can be attributed to the use of this approximation that the use of mono energy source in the simulated CT scan. the energy spectrum of the tube is not mono, and in a wide spectrum with a peak of one-third of energy, it sleeps like a rabbit. So in general, the computational results of DLS were similar.

Conclusion

In this study, we showed that the results of dosimetry Similar when the CT phantom is used in place of patient’s CT image and GATE Monte Carlo code simulation. Providing a simulation method could be an option to give less right to CT scams.

References

  1. Grimes J, Celler A, Birkenfeld B, Shcherbinin S, Listewnik MH, et al. (2011) Patient- specific radiation dosimetry of 99mTc-HYNIC-Tyr3-octreotide in neuroendocrine J Nucl Med 52: 1474-81. [crossref]
  2. Kolbert KS, Sgouros G, Scott AM, Bronstein JE, Malane RA, et al. (1997) Implementation and evaluation of patient-specific three-dimensional internal J Nucl Med 38: 301-308. [crossref]
  3. Saeedzadeh E, Sarkar S, Abbaspour Tehrani-Fard A, Ay MR, Khosravi HR, et al. (2012) 3D calculation of absorbed dose for 131I-targeted radiotherapy: A Monte Carlo study. Radiat Prot Dosimetry 150: 298-305. [crossref]
  4. Sgouros G, Kolbert KS, Sheikh A, Pentlow KS, Mun EF, et (2004) Patient-specific dosimetry for 131I thyroid cancer therapy using 124I PET and 3-dimensional- internal dosimetry (3D-ID) software J Nucl Med 45: 1366-72. [crossref]
  5. Tsougos I, Loudos G, Georgoulias P, Theodorou K, Kappas C (2010) Patient-specific internal radionuclide dosimetry. Nucl Med Commun 31: 97-106. [crossref]
  6. Dewaraja YK, Frey EC, Sgouros G, Brill AB, Roberson P, et al. (2012) MIRD pamphlet 23: quantitative SPECT for patient-specific 3-dimensional dosimetry in internal radionuclide therapy. J Nucl Med 2053: 1310-25. [crossref]
  7. Buck AK, Nekolla S, Ziegler S, Beer A, Krause BJ, et (2008) SPECT/CT. J Nucl Med 49: 1305-19.
  8. Segars WP, Sturgeon G, Mendonca S, Grimes J, Tsuen BMW (2010) 4D XCAT phantom for multimodality imaging research. Med Phys 37: 4902-15. [crossref]
  9. Bauman G, Charette M, Reid R, Sathya J. (2005) Radiopharmaceuticals for the palliation of painful bone metastasis-a systemic Radiother Oncol 75: 258-70. [crossref]
  10. Taschereau R, Chow PL, Cho JS, Chatziioannou (2006) A microCT X-ray head model for spectra generation with Monte Carlo simulations. Nucl Instrum Methods Phys Res A 569: 373-377.
  11. Parach AA, Rajabi H (2011) A comparison between GATE4 results and MCNP4B published data for internal radiation dosimetry. Nuklearmedizin 50: 122-133. [crossref]
  12. Díaz-Londoño G, García-Pareja S, Salvat F, Lallena AM (2015) Monte Carlo calculation of specific absorbed fractions: variance reduction Phys Med Biol 60: 2625-44. [crossref]
  13. Fallahpoor M, Abbasi M, Kalantari F, Parach AA, Sen A (2017) Practical Nuclear Medicine and Utility of Phantoms for Internal Dosimetry: XCAT Compared with Radiat Prot Dosimetry 174: 191-197. [crossref]
  14. Fallahpoor M, Abbasi M, Parach AA, Kalantari F (2017) Internal dosimetry for radioembolization therapy with Yttrium-90 J Appl Clin Med Phys 18: 176-180. [crossref]
  15. Fallahpoor M, Abbasi M, Parach AA, Kalantari F (2017) The importance of BMI in dosimetry of 153Sm-EDTMP bone pain palliation therapy: A Monte Carlo study. Appl Radiat Isot 124: 1-6. [crossref]
  16. Parach AA, Rajabi H, Askari MA (2011) Paired organs-should they be treated jointly or separately in internal dosimetry? Med Phys 38: 5509-21. [crossref]
  17. Loevinger R, Budinger TF, Watson EE (1988) MIRD primer for absorbed dose calculations. New York: Society of Nuclear Medicine.

Guidelines to Living with Schizophrenia, Humor

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DOI: 10.31038/PSYJ.2024624

 
 

Schizophrenia is a nasty, vile, pernicious disease that you may hope to survive. Not much good ever came of it. However, with a few handy rules you can learn how to prosper as a mentally ill adult. Be courageous. You may find yourself up against the machinations of hidden enemies, who like Dr Mengele seek to experiment on you for their own amusement. But, having compiled this handy guide to living with schizophrenia, I can teach you how to pass for sane or at the very least fool most people about your aim, goals, and intentions, which are usually just simply a means of escape. Without further introduction, I present my thoughts on How to Understand and Communicate Effectively with Schizophrenia.

  1. Learn to read the world through symbols. Nothing means what it says, not magazines, newspapers, or journals. Beneath the text of a thing lies a meaning that you have to decode. It’s best if you begin to learn to draw inferences early in your psychiatric episode as it may take you many months to perfect your ability to draw conclusions from display cases in department stores and the stacking of fruits and vegetables in the produce aisle.
  2. Further, ignore the surface meaning of all communication. If someone tells you it’s a nice day be sure to figure out what nice means and if this is a cue for someone else to drop nuclear weapons on your town. Meaning lies in patterns that skim the surface. You will see patterns everywhere. It’s your job to learn as quickly as possible how to read those patterns so you know how to respond adequately and shape your actions accordingly. It’s up to you to decode those patterns and make sense of them so they aren’t just random noise or static of sounds and phenomena. This is why you make a great spy. You’re a breaker of codes! A seer of symbols. An oracle of hidden messages.
  3. Regard all means of communication as private signifiers beamed to you and for you only. Listen closely. Even other people having a conversation are words meant just for you to overhear. TV, movies, print, and just plain talk all have something important to impart. Go to movies to receive instructions in the text hidden in the script. Read between the lines of headlines in the newspaper every day so you learn what is really happening in the world. If you learn to read the medium correctly, you’ll soon realize that WWIII is imminent. Isn’t that what authorities are always trying to hide? Either that or aliens rule the planet and always will.
  4. To maintain physical safety, communicate as little as possible with other people. This way you will frustrate their attempts to torture you in small, dimly lit rooms. Other people are problematic. Who knows where their loyalties lie and to whom they report? Nefarious leaders always work through flunkies. Silence is golden and doctors may be CIA plants. Why else would they ask you so many inane questions? Isn’t the CIA after you to exploit your incredibly astute brain power anyway?
  5. If you can, lie! It’s a safety issue. Your safety.
  6. In learning how to understand your world that has just turned upside down and inside out, interpret everything according to rampant paranoia. Assume everyone is against you unless otherwise specified. Be suspicious of all communications. Facts in the paper aren’t real. That war in Ukraine? It’s a plant to help you understand you’re the one at war. Ordinary events are charged with meaning and threat. Even rodents like squirrels are suspect agents looking to gather data on you. The Russians have been known to use Beluga whales as spies. And squirrels are much more agile than whales. We have technology to make cameras pretty tiny now.
  7. Also, learn to interpret random events as a directly threatening action taken by enemies against you! This especially holds true for earthquakes, the rising and setting of the moon, and even the pull of gravity itself. People are hunting you. They are able to utilize the earth’s physical properties in their search. Flee while you still can.
  8. When you see advertising banners at department stores take them really personally. Sure, the sale is over-hyped. But that’s because store means to get your attention. Enter said store and buy lots of unneeded, unnecessary, but not unappealing or unattractive stuff. It’s about the most fun you’re going to have in a while. The more you buy, the more fun you’ll have and to be perfectly honest, you’ve been given permission by the giant banner to go on a shopping spree. Perhaps you already have a pair of sunglasses? But this pair makes you look really cool and maybe even sexy. So what if you drain your bank account? The shadowy organization for whom you work will magically replenish your finances. It’s best to stay away from your bank at this time.
  9. And now, to communicate with your overlords speak quietly in your head. Everyone else can read your mind anyway, though you tend to forget that most of the time. Try to stifle thought around risky individuals so they don’t learn how you plan to finalize your escape. Learn meditating tricks to quiet your brain around others which is churning at breakneck speed through your mental landscape.
  10. Finallly, be sly and crafty with all individuals. It just might save your life. Nurses have it out for you and aides are cranky with you for a reason. Never let them know of your interior life if you can help it.

If you follow this comprehensive plan, you too can take a stab at being a successful psychiatric patient. It won’t really be much fun, except for the shopping, but you will probably help save the planet. France will thank you. Germany will fete you. And England will award you an honorary degree. If you want, the US will in all probability dedicate the Macy’s Thanksgiving Day Parade to you. And that’s all the achievement anyone could ever ask for.

Potato Virus Y-based Tumor In Situ Vaccination

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DOI: 10.31038/NAMS.2024713

Abstract

In situ vaccination (ISV) is an established and growing cancer therapy strategy. Preclinical studies show that ISV antitumor efficacy is achievable by of plant virus nanoparticles (PVNPs), in which PVNPs are directly administered into the tumor. It had previously shown that some PVNPs are potent in inducing long-lasting antitumor immunity when used as an in situ vaccine. Here, we investigate a filamentous plant virus nanoparticle, potato virus Y (PVY), for in situ vaccination treatment of 4T1, the very aggressive and metastatic murine triple-negative breast cancer model. PVY used as ISV does not significantly slow down tumor progression. Data indicate that some PVNPs are more suitable for application as in situ vaccines than others; understanding the intricate differences and underlying mechanisms of immune-activation may set the stage for clinical development of these technologies.

Keywords

Immunotherapy, Cancer, In situ vaccination, Plant virus nanoparticle, Potato virus Y

Introduction

Over 120 years ago Dr. William Coley set the framework for cancer immunotherapy. It was directly administrated live and attenuated bacteria to tumor. This approach led to cancer regression and complete cures in subset of patients [1,2]. The mechanism is now partially understood to involve activating macrophages and lymphocytes, producing immune’s mediator such as cytokines and chemokines [3]. In this approach that known in situ vaccination (ISV), the tumor itself serves as the source of the antigen and what is introduced is an adjuvant [4]. One particular type of ISV involves the use of attenuated mycobacteria known as Bacille Calmette-Guérin (BCG), which has been the established standard of care for superficial bladder cancer for more than four decades [5]. Another type of ISV that has recently received FDA approval is talimogene laherparepvec (T-VEC) developed by Amgen. T-VEC is a therapeutic treatment that utilizes an attenuated herpes simplex virus. T-VEC is injected directly into identified tumors and its therapeutic effects are achieved through the recruitment and activation of immune cells through the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) [6,7]. Recently, plant virus nanoparticles (PVNPs)-based nanotechnologies have provided evidence that can induce a strong immune response against tumors when administered as ISV [3,8,9]. The effectiveness of this approach has been demonstrated in mouse models of melanoma, breast cancer, ovarian cancer, and colon cancer [3,7,10,11]. The data suggests that antitumor effects leading to the development of immune memory and providing protection against tumor recurrence [11]. Investigations have revealed antitumor effects in these models was associated with activation of a broad spectrum of immune cells [3,12,13]. A subset of immune cell such as APCs, neutrophils have been become activated upon engulf PVNPs, resulting in an early inflammatory phase characterized by upregulation of pro-inflammatory cytokines, leukocyte recruitment [7,13]. Toward this end, have recently demonstrated that ISV with various shapes/sizes of PVNPs such as icosahedral cowpea mosaic virus (CPMV) [7], rod-like papaya mosaic virus (PapMV) [14] and tobacco mosaic virus (TMV) [7], bacillus alfalfa mosaic virus (AMV) [15] can induce immune-mediated antitumor responses. It has been demonstrated that filamentous such as potato virus X (PVX) also can exhibit antitumor effectiveness in the context of melanoma [16]. Here, we specifically asked whether other filamentous viruses qualify as in situ vaccine for cancer immunotherapy. In this regard, we employed a library of structurally similar plant virus; specifically, potato virus Y (PVY). PVY virions have a filamentous, flexuous form, with a length of 730 nm and a diameter of 12 nm and a single-stranded, positive sense RNA genome [17,18].

Material and Methods

Preparation and Characterization of PVY Nanoparticles

PVY was propagated within Nicotiana benthamiana plants and purified as previously reported [18]. Transmission electron microscopy (TEM) imaging was conducted to validate the integrity of PVY filaments. The Malvern Zetasizer (Malvern Instruments, Worcestershire, United Kingdom) was utilized to determine the electrostatic surface map and zeta potential measurements.

Determination of Inherent Immunogenicity

For the assessment of canonical pro-inflammatory cytokine IL-6 levels, the inherent immunogenicity was determined by previous method. Summary, purified PVY added in vitro cultures of peripheral human blood mononuclear cells (PBMCs) with a cell density of 106 cell/ml that were purified as previously reported.

Cell Culture and Cell Viability Assay

Cell culture and a cell viability assay were performed using 4T1 cells (ATCC). These cells were cultured in RPMI, supplemented with 10% (v/v) fetal bovine serum (FBS, Atlanta Biologicals) and 1% (v/v) penicillin-streptomycin (penstrep, Life Technologies). The cells were maintained at a temperature of 37 °C and a CO2 concentration of 5%. Upon reaching confluency, the cells were detached with 0.05% (w/v) trypsin-EDTA (Life Technologies), seeded at a density of 2 × 103 cells/100 μL/well in 96-well plates, and incubated overnight at 37 °C and 5% CO2. On the following day, the cells were washed twice with PBS and exposed to PVY at concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, 75, 100 μg/ml for duration of 24 h, in triplicate. Cell viability was assessed using an MTT proliferation assay as suggested by the manufacturer (ATCC, catalog number: 30-1010K™).

Tumor Model

Female Balb/C mice of six weeks of age were acquired from the Pasteur Institute of Iran. Subcutaneous injection of the breast cancer cell line (4T1) was performed at the right side of the abdomen of a mouse with a seeding density of 106 cell/ml. The tumor volume was measured using a digital caliper and calculated using the equation volume=0.5 (length × width2). Mice were then randomly assigned to either the untreated group (PBS) or the group treated with PVY (n=4). In situ vaccination was conducted on day 10 post-tumor induction, where 100 μg of PVY in 50 μL of PBS was intratumorally injected with 72 h intervals. Tumor size was monitored daily, and mice were sacrificed when the tumor volume exceeded 1000 mm3, as per the approved protocol of the institutional animal care and use committee (IACUC) of Tabriz University of Medical Science.

Immunohistochemistry

Immunohistochemistry was performed using antibodies obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The tumors were fixed in PBS and sent to the Sara-Co lab (https://www.sara-co.com) for sectioning and staining. Optic densities (OD) were quantified using Fiji image analysis software.

Statistical Analysis

Data analysis and chart generation were performed using GraphPad Prism 8 software for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using two-way or one-way analysis of variance (ANOVA). A significance level of P≤ 0.05 was considered for comparisons between groups.

Results

Characterization of PVY

In this study, our objective was to investigate whether PVY could be utilized as an in situ vaccine against 4T1 tumors. PVY is filamentous virus with a length of 730 nm and a diameter of 12 nm. The ratios of RNA to protein (A260/280=1.7) indicated that the preparation consisted of uncontaminated and intact PVY (Figure 1A). Figure 1B displays the zeta potential of the PVY particles, which is recorded as-4.4 millivolts (Figure 1B).

fig 1

Figure 1: PVY characteristics A) Transmission electron microscopy confirms the structural stability of PVY particles. B) Dynamic light scattering was used to measure the zeta potential of PVY.

PVY is Immunogenic and Non-cytotoxic for Cancer Cells

4T1 cell line for evaluating the anticancer efficacy of the PVY nanoparticles (100 μg/ml) was used for cytotoxicity assay, the results did not exhibit direct cell toxicity on treated cells (Figure 2A). PVY possesses immunogenic properties and does not exhibit cytotoxic effects on cancer cells. When Human PBMCs were exposed to PVY for 24 hours, there was a significant increase in the expression of interleukin-6 (IL-6), a well-known pro-inflammatory cytokine, compared to the control cells that were left untreated (Figure 2B). This observation suggests that PVY inherently stimulates the immune cells.

fig 2

Figure 2: A) Cell viability of 4T1 cancer cell line exposed to PVY. B) Human PBMCs exposed to PVY produce elevated levels of IL-6 pro-inflammatory cytokine in vitro. C) Therapeutic efficacy of PVY in a breast tumor model. Balb/C mice inoculated with 1 × 106 4T1 cells followed by 100 µg of PVY, once tumors were approximately 100> mm3, 10 days post-inoculation. Tumor growth followed by measuring volume (n=4).

PVY ISV Treatment has not Potent Efficacy in Treating the 4T1 Model

PVY treatment was done 5 times for each mouse in three days intervals starting 10 days after 4T1 cell injection and tumors well established. Control group mice were injected with PBS on the same schedule. The tumor growth (volume) was no significantly delayed in PVY treated groups compared to untreated control (Figures 2C). Inflammatory cytokines were assayed by Immunohistochemistry. Intra-tumor concentrations of pro-inflammatory cytokines, IFN-β, IL-1β, IL-12 andIL-6 don’t significantly increased in PVY-treated animals compared to untreated control animal tumors (Figures 3).

fig 3

Figure 3: Immunohistochemistry don’t reveal dramatic changes in cytokines associated with treatment. Tumor sections stained for INF-β, IL-1β, IL-12 and IL-6. Quantitative analysis was performed using Fiji software to determine relative optical densities of the stained sections (p<0.05), magnification 20.

Discussion

We have elected filamentous plant virus, namely PVY as ISV for the first time. Intratumoral injection of the viral nanoparticles (VNPs) achieved with the combination of virus genome and multivalent capsid proteins (wild virus) with diameters of approximately 12 nm and long 700nm (Figure 1A). In order to assess their inherent immunogenicity, we introduced of the PVYs into in vitro cultures of PBMCs extracted from human. After duration of 24 hours, we measured the level of pro-inflammatory cytokine, IL-6 secreted using ELISA (Figure 2B). We have observed that in the context of dermal 4T1 in mice, the utilization of PVY as an in situ vaccine don’t leads to a suboptimal antitumor immune response against breast tumors. In contrast, it could not be showed with the effectiveness of CPMV, AMV, and PapMV. However, it did observe a relative similarity with PVX. When comparing the formulation of PVY with PVX, no apparent differences in antitumor efficacy were observed [16]. It demonstrated that there was no statistical difference in tumor growth rate or survival time between PVX-DOX complex versus PVX or DOX alone, but PVX+DOX did significantly slow tumor growth rate versus PVX and DOX alone [16]. Furthermore, PVX in mixture with OVA did not prove to be an effective adjuvant. Unexpectedly, no differences in IgG titers between mice immunized with individual OVA and a composition of OVA+PVX were revealed [19]. Furthermore, TMV used as an in situ vaccine elicits a weak antitumor immune response against melanoma [7]. Analysis of cytokines indicated no statistical difference. Prominent cytokine included IL-6, this cytokine also play crucial roles in inducing pro-inflammatory responses to infection and inflammation. In stark contrast, PVY elicits an immune response at in vitro, characterized by the production of pro-inflammatory cytokine, primarily IL-6. It has been observed that IL-6 signaling is associated with attributes that promote tumor growth, such as the ability to control the differentiation of macrophages and neutrophils into their tumor-promoting phenotypes, specifically M2 and N2 [20]. Our results don’t show very significant stimulation of INF-β, IL-1β, IL-12 and IL-6cytokines by PVY in situ vaccination. These cytokines are particularly intriguing due to their significance in eliciting immune responses against tumors through the mediation of T cells [21]. Regardless of their origin, interferons possess the ability to potentially exert antitumor effects either directly or indirectly [22]. The cytokine IL-12 holds great importance in the realm of cancer immunotherapy as it has the capability to activate immune cells with antitumor properties, including those that combat melanoma, by stimulating IFN-γ signaling and production [23]. It can exhibit that PVY can be suitable for epitope presentation. It is demonstrated that the preS1 epitope displayed on PVY (chimeric PVY CP particles) can immunize of mice with a strong anti-preS1 immune response, even in the absence of adjuvants [24].

Conclusion

Plant virus-based materials have emerged as innovative in situ vaccines, potentially initiating an immune response against tumors. These nanoparticles interact with innate immune sensors, reprogramming the immunosuppressive tumor microenvironment to an immune-activated state. This reactivation of the cancer-immunity cycle leads to systemic elimination of cancer cells through the adaptive immune system. It has been documented that CPMV, PapMV, PVX, TMV, and AMV-based ISV can induce anti-tumor responses, reduce tumor growth, and improve survival rates when administered directly to the tumor site. In this study, we present evidences that PVY-based ISV, in compare with them, no efficacy or immune-stimulation in a mouse model. Nevertheless, it can exhibit that PVY can be suitable for epitope presentation. Further research is required to fully elucidate the mechanism of plant viral in situ vaccines.

References

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  2. McCarthy EF (2006) The toxins of William B. Coley and the treatment of bone and soft-tissue sarcomas. The Iowa Orthopaedic Journal 26: 154. [crossref]
  3. Lizotte P, Wen A, Sheen M, Fields J, Rojanasopondist P, et al. (2016) In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer. Nature Nanotechnology 11: 295-303. [crossref]
  4. Shahgolzari M, Fiering S (2022) Emerging potential of plant virus nanoparticles (PVNPs) in anticancer immunotherapies. Journal of Cancer Immunology 4: 22. [crossref]
  5. Kamat AM, Flaig TW, Grossman HB, Konety B, Lamm D, et al. (2015) Consensus statement on best practice management regarding the use of intravesical immunotherapy with BCG for bladder cancer. Nature Reviews Urology 12: 225-35. [crossref].
  6. Johnson DB, Puzanov I, Kelley MC (2015) Talimogene laherparepvec (T-VEC) for the treatment of advanced melanoma. Immunotherapy 7: 611-9. [crossref].
  7. Murray AA, Wang C, Fiering S, Steinmetz NF (2018) In situ vaccination with cowpea vs tobacco mosaic virus against melanoma. Molecular Pharmaceutics 15: 3700-16. [crossref]
  8. Shukla S, Wang C, Beiss V, Cai H, Washington T, et al. (2020) The unique potency of Cowpea mosaic virus (CPMV) in situ cancer vaccine. Biomaterials Science 8: 5489-503. [crossref].
  9. Shahgolzari M, Pazhouhandeh M, Milani M, Yari Khosroushahi A, Fiering S (2020) Plant viral nanoparticles for packaging and in vivo delivery of bioactive cargos. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 12: e1629. [crossref]
  10. Kerstetter-Fogle A, Shukla S, Wang C, Beiss V, Harris PL, et al. (2019) Plant virus-like particle in situ vaccine for intracranial glioma immunotherapy. Cancers 11: 515. [crossref]
  11. Gautam A, Beiss V, Wang C, Wang L, Steinmetz NF (2021) Plant viral nanoparticle conjugated with anti-PD-1 peptide for ovarian cancer immunotherapy. International Journal of Molecular Sciences 22: 9733. [crossref].
  12. Wang C, Fiering SN, Steinmetz NF (2019) Cowpea mosaic virus promotes anti‐tumor activity and immune memory in a mouse ovarian tumor model. Advanced Therapeutics 2: 1900003. [crossref]
  13. Shahgolzari M, Dianat-Moghadam H, Fiering S (2022) Multifunctional plant virus nanoparticles in the next generation of cancer immunotherapies. In: Seminars in Cancer Biology 86: 1076-85. [crossref]
  14. Lebel M-È, Chartrand K, Tarrab E, Savard P, Leclerc D, et al. (2016) Potentiating cancer immunotherapy using papaya mosaic virus-derived nanoparticles. Nano Letters 16: 1826-32.[crossref]
  15. Shahgolzari M, Pazhouhandeh M, Milani M, Fiering S, Khosroushahi AY (2020) Alfalfa mosaic virus nanoparticles-based in situ vaccination induces antitumor immune responses in breast cancer model. Nanomedicine 16: 97-107. [crossref]
  16. Lee KL, Murray AA, Le DH, Sheen MR, Shukla S, et al. (2017) Combination of plant virus nanoparticle-based in situ vaccination with chemotherapy potentiates antitumor response. Nano Letters 17: 4019-28. [crossref]
  17. Della Bartola M, Byrne S, Mullins E (2020) Characterization of potato virus Y isolates and assessment of nanopore sequencing to detect and genotype potato viruses. Viruses 12: 478. [crossref]
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  19. Evtushenko EA, Ryabchevskaya EM, Nikitin NA, Atabekov JG, Karpova OV (2020) Plant virus particles with various shapes as potential adjuvants. Scientific Reports 10: 10365.
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  24. Kalnciema I, Skrastina D, Ose V, Pumpens P, Zeltins A (2012) Potato virus Y-like particles as a new carrier for the presentation of foreign protein stretches. Molecular Biotechnology 52: 129-39. [crossref]

Risky Business: A Comment on Nurse’s Exposure to Infectious Diseases at Work, the Experiences of WW1 Nurses from New Zealand

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DOI: 10.31038/IDT.2024513

 
 

The impact of the COVID 19 pandemic on health professionals around the world highlighted that front line health care staff continue to pay a high price in the provision of care. It has been well documented as to how the lack of appropriate personal protective equipment (PPE), unequitable access to testing, vaccinations as well as the share volume of patients exposed frontline staff to infection[1]. In other smaller events such as Ebola, the early days of AIDS (Acquired immune Deficiency Syndrome) and tuberculosis as other examples where health care workers were unprotected. As the impact of COVID-19 seems to be waning, despite high numbers of infection circulating, it remains to be seen whether the lessons learnt about protecting and supporting frontline health care staff will continue to be upheld in future pandemics.

These modern-day experiences are mirroring what generations of health care professionals, and in particular nurses have experienced in the past. By the nature of their work, nurses are at a greater risk of infection than most other health professionals, as they tend to have more frequent and prolonged contact with patients, are involved in handling bodily fluids, and work close to patients performing any number of duties.

Between 1914-1919 of World War One and the immediate years afterwards, New Zealand send 20% of its nursing population overseas to provide care to the 1000s of wounded and ill soldiers of the Allied nations, amounting to approximately 540 registered nurses. Others such as Voluntary Aid Detachments (VADs) and Red Cross nurses also were sent however these are not accounted for in New Zealand Army service records. On their return to New Zealand, most were then sent straight to work at a new front line, that of the catastrophic influenzas epidemic, brough home by returning soldiers and ravaging through the population[2].

The NZANS left New Zealand fit and healthy, aged between 26-45, and were considered the cream of the crop of their provincial and city hospitals. Prior to departing nurses were inoculated for typhoid and paratyphoid, which caused deadly enteric fever. They were also inoculated against smallpox. The nurses were sent away in batches, starting with the first fifty in April 1915. It was thought at the time, that this would be all that was needed, however by June dozens more were sent and on it went over the next few years [1]. Those early nurses did not return to New Zealand until, after the war ended unless they were “invalided home” (as stated in individual medical files).

The nurses were often working in what could be considered austere environments, in temporary field hospital with poor sanitation. They may also be working in confined environments such as hospital trains dealing with men straight from the front, with infected wounds or infectious diseases such as dysentery. Hygiene measures may be difficult in such confined spaces. In the hotter countries like Egypt and Samoa, flies were also a problem. Another environment which was a breeding ground for infectious diseases was the hospital and troop ships. One of the most well-known New Zealand examples is the Troop Ship Tahiti which had over 1000 men on board, with ten passenger nurses heading to England in 1918. An unwelcome passenger came on board en route, and by the time the ship docked in Southampton over 100 men had died from influenzas, one passenger nurse and at least five others were quarantined for several weeks. Many more soldiers were ill and incapacitated with influenza, and those who died did so from pneumonia, untreatable in the pre antibiotic era[3].

As all the New Zealand war service files from World War one have been digitised, it has been possible to review each of the 550 service files. A data base was created collecting all the available information know about the nurses, age at service, age at death, length of service and locations, along with any details of sicknesses and their amount of time absent with these. This work has been reported elsewhere [1,2], as part of a bigger project. Rather, this commentary is going present the impact of the infectious disease the nurses experienced as well as some of the long-term consequences of their service. Some of these infectious disease ae now virtually non-existent in a well vaccinated population, and of course this can have some complications as the vaccine hesitant may not see the value in vaccinating against a disease they think has been non-existent for generations [3].

In analysing all the files of the nurses who served overseas, three main infectious diseases are prevalent, accounting for over 50% of the recorded sickness. These were influenza, with regular outbreaks through 1916, 1917 and the large global pandemic of 1918. The impact on nurses was at least three weeks off sick in a hospital and then they would have to convalesce in a nursing home to regain their strength before going back to duty. The next most common infectious disease was measles, which remains the most contagious infectious disease currently. The third main infectious disease is tuberculosis, which was often not labelled as such in the notes. For those nurses who ended up in a sanatorium on their return to NZ, their medical boards described firstly a period of chronic cough fatigue and weight loss for weeks and up to three months. They would then receive a diagnosis of “CPDI’, which is chronic pulmonary disease indeterminate. Then after about six months they were usually invalided back to New Zealand and sent to a sanatorium for further treatment. Once they had recovered sufficiently they were then often requested to stay on working in the sanatorium on “light duties”, ostensibly with the idea that they couldn’t get sick again with TB.

Another infectious disease mentioned in several files is diphtheria, a disease long gone from Western countries due to vaccination. This is a potentially fatal disease of the airways causing obstruction and death if the membrane occluding the airways is not removed [4]. Modern treatment of diphtheria is with antibiotics and an antitoxin. Other common infections reported in the files of nurses are skin infections such as boils, which often results in several weeks off sick, or the need for draining of boils. Whilst these nurses may not have been very ill with the skin infections (most likely staphylococcus), it would have been precarious for the soldiers if they received a wound infection.

Whilst the 1918 Spanish infauna pandemic is generally well known and the devastating impact it had on war depleted populations around the world, it was not the first influenza outbreak during world war one. Whilst influenza was known as an illness and that it was caused by a virus and spread via droplet infection, the actual virus was not isolated until 1932 [5]. Until then diagnosis was made by clinical symptoms only; very high temperature, muscle pain and severe debility all appearing suddenly. Often in nurses files the original record would state “PUO”, meaning pyrexia unknown origin followed by “NYD”, not yet diagnosed. Then a few days later the entry would read “influenza” (never “flu”) and then there would be a period of convalescence lasting up to three weeks. Prior to the 1918 influenza outbreak, which claimed the lives of several nurses bother overseas and in New Zealand, the most severe epidemic was in 1916 and is considered the precursor of the 1918 outbreak. The epidemic began in a large British camp in Etaples which had over 100, 00 soldiers and 24 hospitals. The onset of symptoms was rapid and severe, with a high mortality rate [6].

Conclusion

This discussion of the impact of infectious disease on New Zealand army nurses in World War One has highlighted historically the risk nurses have been in, whilst caring for patients. Despite advances in modern health, with improved vaccinations, PPE and understanding of transmission of infectious disease, the experiences of the early days of the COPVID-19 pandemic were eerily similar to historical events, leaving frontline staff under protected and many paying the ultimate price of losing their lives, to what has become essentially preventable through vaccinations and hygiene measures.

[1] https://www.who.int/news/item/03-03-2020-shortage-of-personal-protective-equipment-endangering-health-workers-worldwide

[2] https://nzhistory.govt.nz/culture/influenza-pandemic-1918

[3] https://ww100.govt.nz/influenza-on-the-ss-tahiti

References

  1. Maddocks WA (2023) Broken nurses: an interrogation of the impact of the Great War (1914-1918) on the health of New Zealand nurses who served-a cohort comparison study. BMJ Military Health, p. e002325. [crossref]
  2. Maddocks W (2022) Too Sick For Caring? An Analysis of The Health Impact of The Great War (1914-1918) on The First Cohort of New Zealand Nurses Who Served. Journal of Military and Veterans Health 32(2).
  3. Bullock J, JE Lane, FL Shults (2022) What causes COVID-19 vaccine hesitancy? Ignorance and the lack of bliss in the United Kingdom. Humanities and Social Sciences Communications 9(1): p. 87.
  4. Roddis LH (1957) A Short History of Diphtheria. Military Medicine 120(1): p. 51-54. [crossref]
  5. Potter CW (2001) A history of influenza. Journal of Applied Microbiology 91(4): p. 572-579. [crossref]
  6. Oxford JS, et al. (2002) World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis 2(2): p. 111-4.

SARS-CoV-2 Infection and Multiple Sclerosis: Proactive Approach in a Vulnerable Patient Group through Daily Vitamin D Supplementation?

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DOI: 10.31038/IDT.2024512

 
fig 1

Introduction

An explicit goal of multiple sclerosis (MS) therapy is the “best possible disease control” taking into account the “best possible quality of life” of the patient, with the option of using highly effective therapeutic agents early or as early as possible in response to disease activity [1], but also a to seek proactive therapy by making use of all therapy options [2].

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease with a suspected autoimmune origin. The disease begins earlier than current diagnostic criteria can detect. It affects the entire central nervous system and not just the white matter, as the original term-inflammatory demyelinating disorder-suggests [3]. It is characterized by a very heterogeneous course of the disease, which is represented by relapse-associated neurological deterioration, but also by an increase in disability that is independent of relapse [4]. It is generally accepted that infections in people with MS (PwMS) can have a negative impact on the course of the MS disease. This justifies that all potentially therapeutic and preventive options, especially for COVID-19, should be exploited.

MS is associated with reduced vitamin D status [5]. The molecular mechanisms in the pathogenic effect of vitamin D deficiency in MS are diverse and are orchestrated by encephalitogenic T cells with B cells, microglia, dendritic cells, interleukins (IL-1 beta, IL-6, IL-12, IL-17, TNF alpha, (tumor necrosis factor), MHCII, interferon gamma, among others) [6].

Not only is vit D deficiency associated with MS risk, but s25(OH)D levels are inversely correlated with risk of relapse, CNS lesions, and disability progression. Vitamin D suppl. reduces the number of new Gd+-enhancing or new/enlarged T2 lesions on MRI [7-10]. With MS, which is currently not curable, a high level of activity is required to prevent complications, especially infections of any kind. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an exceptionally transmissible and pathogenic coronavirus that emerged in late 2019, causing a pandemic of acute respiratory illness known as coronavirus infection 2019 (COVID-19). New omicron variants are constantly being discovered [11], for example BA.2.86 (Pirola),JN. New findings show that BA.2.86 efficiently enters lung cells and uses TMPRSS2 for entry into lung cells. The mutations S50L and K356T are for the efficient Lung cell entry of BA.2.86 is responsible. BA.2.86 has a high resistance to therapeutic antibodies and evades the antibodies induced by infection and vaccination [12,13]. COVID-19 can develop into a severe disease associated with immediate and delayed sequelae in various organs, including the central nervous system (CNS) [11].

Over the last 3 years, a complex connection between SARS-CoV-2 infection and MS has emerged [14].

Daily Vitamin D Supplementation is a Prerequisite for the Suppression of Inflammatory Processes

The risk of severe infection from COVID-19 should provide additional motivation for one daily high-dose vitamin D administration [15,16]. As part of prevention, it is worth mentioning that with circulating s25(OH)D values ≥ 55 ng/mL, the SARS-CoV-2 positivity rate was significantly lower than with values below or with deficiency [17]. Current data suggest a protective role for VitD, particularly with a lower risk of intensive care unit admission and a reduced risk of death [18,19]. In addition, the occurrence of Long-Covid is an aspect of implementing this simple, effective, safe and costeffective therapy with a broad therapeutic window for the prevention and treatment of COVID-19 disease [20-22]. Although there is still no indisputable evidence that Vit D supplementation (VitD suppl.) reduces the risk of SARSCov-2 infection in healthy individuals, there is collective evidence that it benefits vulnerable individuals [23]. PwMS with comorbidities, psychiatric illnesses, hypertension, obesity (an increased BMI may correlate with a severe course of Covid-19), age > 50 years, severe disability and methylprednisolone boost therapy as well as some DMTs (disease-modifying therapies) have a higher risk of infection and an increased risk of severe COVID-19 courses [24-26]. Infections (SARS-COV-2) can increase MS symptoms (pseudo-relapses) or cause real relapses [27]. In post-COVID syndrome (Long Covid), one in eight patients presents with symptoms such as fatigue, shortness of breath, cough, joint pain, chest pain, muscle pain, headache and paresthesia in the limbs after at least 3 months. The latter can also occur in PwMS per se [28,29]. if vitamin D administration results in a lower risk of infecton,severity of illness with admission to the intensive care unit or a reduced risk of death in people at risk,Long-Covid occurs less frequently [18,19,21,22,30-33],it is not ethically justifiable to withhold high dose vitamin-D administration from pleople at risk.

Mechanisms of Action of Vitamin D in COVID-19

Barrea et al. list in detail 14 mechanisms as described by Vit D suppl. the risk of COVID-19 infection can be reduced and sufficient Covid 19 vaccination is supported [19,29,32,34-38]. Vit D and its metabolites inactivate viruses (increase in antimicrobial peptide cathelicidin, defensins), lead to reduction of the risk of cytokine storm, reduce matrix metalloproteinase-9 concentration and thereby increase the host’s metabolic tolerance to damage, reduce the risk of pneumonia and myocarditis, lead to the reduction of the concentration of pro-inflammatory cytokines, especially interleukin 6 (IL-6), which promotes the permeability of the BBB, which leads to the potentiation of CNS damage in PwMS, is serious [29,39-42].

Vit D enables neuroprotection by reducing inflammation and oxidative stress. Low 25(OH)D levels were inversely correlated with high IL-6 levels and were independent predictors of COVID-19 severity and mortality [43]. 1,25(OH)2D3 inhibits immunoglobulin synthesis, regulates B cell activity and reduces auto-Ab production. It converts B cells into plasma cells [18]. Vit D reduces the risk of infection with EBV [29].

Current studies show evidence that chronic inflammation in Long Covid-19 Infection with reactivation of the latent Epstein-Barr virus (EBV) can lead to a worsening of the health status in PwMS [44-48]. In MS, there is a high level of molecular mimicry between the EBV transcription factor EBNA-1 and the CNS protein GlialCAM (glial cell adhesion molecule of the central nervous system [49]. Bernal et al. were able to detect EBV reactivation by detecting EBV DNA and antibodies against EBV-lytic genes [50].

In 66.7% of Long Covid patients, EBV reactivation could be demonstrated by a positive titer for EBV EA-D (early antigen-diffuse)-IgG or EBV-VCA (viral capsid-antigen)-IgM) can be provided [45]. Long COVID patients with fatigue and neurocognitive disorders were with serological evidence of recent EBV reactivation (early antigen-D [EA-D] IgG positivity) or high nuclear antigen IgG levels [51].

The triad of inflammatory markers IL-1ß, IL-6 and TNF can be found in both Long Covid and MS [52-54]. Low sun exposure acts synergistically with high EBNA-1 Ab levels and was associated with an increased risk of MS [55]. There is a connection between high EBNA-1 antibody levels and low s25(OH)D levels. On the other hand, a high dose of VitD suppl. the EBNA-1 antibody levels in PwMS [56-58]. Another parallel arises from the increase in GFAP (glial fibrillary acidic protein) as a dysfunction of the astrocytes about 4 months after the start of SARS-Cov2 infection [51]. The concentration of NfL (Neurofilament light chain), GFAP and total tau in CSF in patients with COVID-19 was often elevated with neurological symptoms [59]. Elevated sNfL has already been verified in mild to moderate COVID-19 disease [60]. On the other hand, the risk of mortality increased if sNfL and sGFAP levels were already elevated upon hospital admission [61].

Because there are no effective drugs that block EBV reactivation in Long Covid [62], there are multiple arguments for Vit D suppl. High-dose Vit D-Suppl (14,000 IU/day) for 48 weeks or 20,000 IU/week for 48 weeks selectively reduced anti-EBNA-1 antibody levels in PwMS (RRMS) [58,63].

Several mechanisms are under discussion:

  1. VitD could induce better clearance of EBV infected B cells,
  2. Vit D could directly target and impair viral replication in EBV-infected cells,
  3. Produce better control of inflammation in general,
  4. In an EBV-mediated inflammatory cascade, 1,25(OH)2D3 could suppress the activation of reactive astrocytes
  5. It is likely that at high s25(OH)D levels, the VitD receptor EBNA 2 (Epstein-Barr virus nuclear antigen 2) is displaced upon DNA binding [58,63-68].

Early Start of Therapy is a Crucial Factor

The early start of therapy with VitD suppl. is crucial for influencing influenza and COVID-19 infections [69]. The Corona-19 mortality risk correlates inversely with the VitD status and a mortality rate close to zero could theoretically be achieved at over 50 ng/mL s25(OH)D [70]. The importance of Vit-D metabolism as a potential prophylactic, immunoregulatory and neuroprotective treatment of infections (COVID-19) is increasingly being considered in clinical practice as part of a multitherapeutic approach [34,71,72].

Dosage Suggestions for Vitamin D Supplementation

Currently, there are no consensus guidelines suggesting an appropriate concentration of serum 25(OH)D to prevent COVID-19 or reduce its morbidity and mortality. It is becoming increasingly clear to start with a “loading dose” with high VitD doses over a few days and then continue with a “maintenance dose”, although various variants have been put up for discussion.

For example, one study used a weekly or fortnightly dose totaling 100,000-200,000 IU for 8 weeks (1800 or 3600 IU/day) [73].

To obtain 75 nmol/l s25(OH)D values, the following equation was described:

Dose (IU) = 40 x (75-serum 25(OH)D(3) [nmo/L] x body weight [73].

Over 30 ng/mL s25(OH)D values were also achieved with a single oral dose of 200,000-600,000 IU [38,74].

An s25(OH)D level of 40-60 ng/ml could be achieved by dosing up to 6,000 IU/day over several weeks [75,76]. A daily VitD intake of 10,000 IU/day for 4 weeks would lead to a faster optimal s25(OH)D level in the “status nascendi” of an infection [19].

Another dosage regimen was recommended: cholecalciferol 0.532 mg on day 1 and continued with 0.266 mg on days 3,7,14,21,28 (1 IU vitamin D3 = 0.025 µg vitamin D3 = 65.0 nmol Vitamin D3 [77]. The pharmacokinetic properties of calcifediol allow rapid absorption within hours, facilitating the immediate availability of 25(OH)D2 in target tissues.

This drastically reduced the need for intensive care unit admission and the mortality rate [77]. A key mechanism of 1,25(OH)D2 is its effect on Vit D receptors (VDR) on the adaptive immune system. The activity of TH1 and TH17 cells is reduced and the T regulator (Treg) cells are induced. This results in a reduced production of proinflammatory cytokines (IL-6, IL-8, IL-12, IL17, TNF alpha) and the cytokine storm is weakened [77,78].

The further daily VitD dose will depend on the s25(OH) values. The “maintenance dose” depends on the genetic polymorphism of the enzymes involved in VitD metabolism. Because interindividual differences in the organism’s response to Vit D, particularly in PwMS, are established, one of many explanations for the controversy surrounding the clinical results of Vit D suppl. [6,78,79].

An example of this individual reaction to a Vit D suppl. with 3,200 IU daily for 5 months showed a strong response to peripheral blood mononuclear cells in 60% of healthy individuals, while only a mild to moderate response was recorded in 40% despite reaching 25(OH)D values of 60-90 ng/ML was [80].

Up to Date 2024

Clinical Manifestation of a SARS-Cov-2 Infection in PwMS as Ulcerative Colitis – A Novum

Another challenge in the diagnostic diagnosis of gastroenterological symptoms is exclusively COVID-19-induced colitis (enteropathic infection) without pulmonary manifestation or as the first manifestation of COVID-19 disease [81-87]. In the ileum and colon, there is extensive expression of the angiotensin converting enzyme 2 (ACE2) on the enterocytes, to which the SARS-CoV-2 corona virus binds, penetrates the cells of the intestinal epithelium and causes the inflammation or aggravates existing one chronic inflammatory bowel disease (IBD) [84,87-90]. Molecular mimicry between SARS-CoV-2 and human proteins (enteric epitopes) promotes gut-associated autoimmune diseases [91].

SARS-CoV-2 as an autoimmunogenic virus is seen in association with another 10 autoimmune diseases and multidisciplinary management can be beneficial in long-COVID [92-94]. Vit D deficiency can promote autoimmune dysregulation [95].

PwMS are Predisposed to Comorbid Autoimmune Diseases

PwMS have a tendency to be polyautoimmune [96-98] and hundreds of common genetic susceptibility loci for autoimmune diseases have been identified [99,100]. Up to 18% of PwMS suffer from additional comorbid autoimmune disorders.

Inflammatory bowel diseases (IBD) [ulcerative colitis, Crohn’s disease]) are among the most common autoimmune diseases accompanying MS [101]. About beneficial and adverse effects of DMTs and comorbid autoimmune diseases details in [102]. Knowledge of the tendency towards polyautoimmunity is the key to the precise interpretation of symptoms, even in contrast to treatment-related undesirable side effects of anti-CD20 therapy.

Disease-Modifying Therapies (DMT) Can Increase the Risk of Infection

Long-term observation has shown an increased incidence of respiratory tract infections, urinary tract infections and SARS-CoV-2 during therapy with monoclonal anti-CD20 antibodies in MS (ocrelizumab, ofatumumab, ublituximab, rituximab) [103-105]. The predominant depletion of CD20+ B cells, but also CD20+ T cells and the effect on CD8 T cells by ocrelizumab as well as the additional reduction in immunoglobulins (IgG, IgA, IgM) explains the increased risk of infection [103,106].

Discussion

The connection between Vit D and COVID-19 has been critically examined in over 120 clinical studies, including 41 RCTs, and a strong connection between Vit D and clinical outcomes in Covid-19 has been proven.

Several mechanisms have been discussed:

  1. Affects 1,25(OH)2D3 antimicrobial peptides (cathelicidin), tigth junction proteins and adherenc junction proteins (ZO-1, occludin, claudin-10, ß-catenin, VE-cadherin) [107].
  2. 1,25(OH)2 D3 suppresses the activity of TH 1 and TH 17 cells and induces Treg cells. As a result, there is a reduced production of proinflammatory cytokines (IL-6, IL-8, IL-12, IL-17, TNF alpha) and a weakening of a cytokine storm [79].
  3. Vit D plays an important role in controlling the renin-angiotensin-aldosterone system. Details in [78].

Furthermore, genetic polymorphisms of the Vit D metabolism pathway and nongenetic reasons could explain the controversies surrounding the clinical results of Vit D supplementation [78,79,108]. If the physiological basis for the use of Vit D to improve the health of the general population has already been found with Vit D daily doses of 5000-7000 IU/day [109], it is biologically plausible to use a Vit D suppl. to be carried out preventively in the event of impaired immune homeostasis in PwMS to improve immune function. The daily dose of Vit D is crucial for the therapeutic success of broad gene expression. A daily dose of 10,000 IU leads to genomic changes that were several times higher than with 4000 IU/day [80].

Through the immunomodulatory effect of 25(OH)D and its anti-inflammatory mechanisms, immune-mediated colitis caused by anti-CD20 antibody therapy or ulcerative colitis caused by SARS-CoV-2 could be suppressed or alleviated. This form of manifestation of COVID-19 disease is particularly important in vulnerable people (PwMS) receive attention. [81-94].

Calcitriol may play a supportive role in neuroprotection particularly in PwMS by attenuating neuroinflammation and protecting the endothelial integrity of the blood-brain barrier (BBB) [110,111]. The steep learning curve in assessing clinical symptoms in LONG COVID-19 reveals new manifestations of autoimmune diseases, particularly after severe SARS-CoV-2 infections. In addition to the risk of rheumatic diseases, the occurrence of Crohn’s disease and ulcerative colitis must be taken into account in long-term care [112-116] and is a challenge in the future. Comorbidities affect PwMS more frequently than people without MS and are associated with greater physical and cognitive impairment,lower health-related quality of life,and increased mortality [117].In long-term management,one goal is to potentially avoid comorbidities.Due to the predisposition to polyautoimmunity,thyroid diseases (Hahimoto’s thyroiditis,Graves’disease) are not uncommon as comorbidities [118]. An infection of the endocrine system with SARS-CoV-2 (e.g. thyroid, adrenal gland, pituitary gland, etc.) is possible and the virus has been detected in post-mortem samples [119]. SARS -CoV-2 also mainly penetrates here the main receptor ACE2 and its co-receptor TMPRSS2 into the host cells. ACE2 protein expression was detected in about 87% of deceased COVID-19 patients. Pathological thyroid function tests correlated with the severity of the disease [119,120]. People with one already existing autoimmune disease and Covid-19 were 23% more likely to be diagnosed with another autoimmune disease [112]. In patients with comorbidities, advanced age and SARS-Cov-2, overactivation of T cells, overproduction of proinflammatory cytokines (IL-1 beta, IL-2R, IL-6, IL-8, IL-17, TNF alpha, IFN beta) and a reduction in Treg cells are confirmed [121].  Infections with SARS-CoV-2 and mRNA vaccines can trigger the clinical onset of an autoimmune disease [122]. So it must be during and after the SARS-CoV-2 infection, subacute thyroiditis, Graves disease and Hashimoto’s thyroiditis are expected [122] and the PwMS should be monitored accordingly in the event of clinical symptoms. The immunomodulatory function of vitamin D could be used as part of an early treatment strategy , as vitamin D deficiency increases the risk of autoimmune thyroid diseases [123]. There is a negative relationship between anti-thyroid antibodies (TPO-Ab, TgAb, TSHR Ab) and a sufficient serum 25(OH)D level. A Vit D suppl. led to a decrease in thyroid antibodies and in hypothyroidism, TSH levels decreased. Vit D positively influenced Hashimoto’s thyroiditis and graves disease [124-137].

A Covid-19 cohort showed a significantly higher risk of IBD and celiac disease [138]. Patients with ulcerative colitis were more likely to develop a severe form of Covid-19 than the general population [139]. Despite partly contradictory results of studies on the relationship between vitamin D, Covid-19 and IBD, it can be recognised that 25(OH)D levels above 30ng/mL can exert a protective function [140].

As Covid-19 is not a thing of the past and appears to be here to stay, an easy-to-use and inexpensive vitamin D supplement is needed and should be offered to at-risk groups. The active form of Vit D not only shows a dual effect on SARS-CoV-2 and MS, but also has a versatile spectrum of action on MS.

Summary

People with multiple sclerosis could proactively influence the course of their disease and reduce the risk of infections with possible complications through long-term prophylaxis with daily vitamin D supplementation. The immunomodulatory influence of vitamin D is undisputed and cytokine storms (COVID-19) as well as a severe course of the disease could be prevented. 25(OH)D serum values of over 50 ng/mL should be aimed for through individual daily vitamin D supplementation. The 25(OH)D serum values obtained in studies in the general population with daily doses of 5000-10,000 IU/day cannot be adequately transferred to people with multiple sclerosis and must be titrated individually. Due to the known immunopathological mechanisms of vitamin D and its benefits, it would be desirable to integrate this add-on therapy into standard clinical care.

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Febrile Neutropenia in Pediatric Oncology: Prevalence and Risk-Factors for Bacterial and Fungal Infection

1 Reply

DOI: 10.31038/IDT.2024511

Introduction

Febrile neutropenia (FN) frequently complicates cancer treatment, contributing to overall morbidity and the burden of hospitalization in children with cancer [1-6]. Myelosuppression is a side-effect of cytotoxic chemotherapy, resulting in recurring episodes of neutropenia; fever complicates about 27-34% of neutropenic episodes among children receiving chemotherapy or undergoing hematopoietic stem cell transplant (HSCT) [1-3]. Due to the risk of serious bacterial or fungal infections in children with FN, the difficulty of localizing infections in neutropenic children, and the mortality rate associated with inadequate treatment [4,5] the historical standard in pediatric hematology-oncology was hospitalization with empiric broad-spectrum antibiotics until the fever resolves and the neutropenia improves [6-8]. More recently, however, there is evidence of greater practice variation [9,10] based on practice guidelines emphasizing risk-stratification of children with chemotherapy-induced FN and the benefits of decreasing inpatient hospitalization [9,11-27]. Most FN episodes resolve without diagnosis of a serious infection; bacteremia, the most common infection complicating FN episodes, has a prevalence of 20%-29 [11,28-32]. Invasive fungal infection (IFI) is less common, occurring in less than 5% of FN episodes [32] and data on bacterial infection of sites other than the bloodstream are more limited. There is no single approach to risk-stratification in pediatric FN [26] which necessitates ongoing analysis of risk factors for serious infection, which facilitate risk-stratification and step-down management of children at lower risk [25,27,32,33].

Materials and Methods

Study Design

To evaluate the prevalence and potential predictors of bacterial and fungal infection among pediatric oncology patients with FN at our institution, we conducted a retrospective cohort study containing a nested case-control study. Using hospital billing codes and electronic medical records, we obtained a consecutive 3-year sample of children admitted with FN to the pediatric hematology-oncology teams at UCSF Benioff Children’s Hospital Oakland (Oakland, CA, US), with the end of the sample period preceding the Covid-19 pandemic. Children receiving treatment for cancer were included in the cohort if they had an absolutely neutrophil count (ANC) <500 x109/L or (if no ANC was reported) a total white blood cell count (WBC) <500 x109/L, as well as a single temperature >38.3°C or a sustained temperature >38°C [34]. Participants were excluded if they were receiving or had previously received allogeneic or autologous HSCT or had an underlying syndrome (such as Fanconi anemia) associated with chronic neutropenia. This study was approved by our hospital’s institutional review board and conducted in accordance with the Declaration of Helsinki.

Statistical Methods

Continuous variables, including participants’ ages and days to infection diagnosis, were not normally distributed and are described using median and interquartile range (IQR). Children with multiple episodes of FN during the sample period reentered the cohort for each episode. For the case-control analysis, we randomly sampled one episode per participant. Cases were defined by culture-proven bacteremia, urinary tract infection (UTI), meningitis, cellulitis, osteomyelitis, neutropenic colitis (typhlitis), Clostridium difficile enterocolitis, or invasive fungal infection (IFI). Clinical and radiographic findings were accepted for diagnosis of typhlitis, osteomyelitis, and IFI if cultures were not available [35]. Per institutional standards of care, any positive blood culture from a central venous catheter (CVC) was considered infectious, including coagulase-negative staphylococci. Controls were sampled at a two-to-one ratio with cases. To compare clinical and laboratory findings between the case and control groups, we used rank-sum tests for continuous variables and standard two-by-two tables with Fisher exact tests for categorical variables. Associations were considered significant with an uncorrected p-value <0.05. We also report each association’s relative risk ratio (RR) with a 95% confidence interval. Data analysis was performed using Stata 13 (Statacorp, College Station, TX).

Results

Study Cohort

The cohort (Table 1) consisted of 199 FN episodes among 140 participants, 43% female, with a median age at cohort entry of 6.1 years (3.1-12.3). Most participants (71.4%) were hospitalized once for FN during the study period; among the rest, the number of hospitalizations ranged from 2 to 8. The most common diagnoses were acute leukemia and lymphoma. There were 5 participants (3.6%) with trisomy 21, all of whom had acute leukemia. Nearly all of the participants had a CVC, and 31 (22.1%) had a history of at least one prior infection, including bacteremia (N=21), another bacterial infection (N=7), or IFI (N=4). All participants received empiric intravenous antibiotics with antipseudomonal activity upon the onset of fever. Most of the FN episodes (81.9%) developed in outpatients who were then admitted; 36 FN episodes (18.1%) occurred in children who were already hospitalized, especially those receiving high-intensity chemotherapy for acute myeloid leukemia (AML) or brain tumors. Among participants with acute lymphoblastic leukemia (ALL), 26 FN episodes (13.1% of the total) occurred during the lower-intensity maintenance phase of therapy.

Table 1: Demographic and clinical characteristics

Parameter

N (%)

Sex
 Female

60 (42.9)
 Male

80 (57.1)
Age at onset (years), median (IQR)

6.1 (3.1-12.3)
Trisomy 21

5 (3.6)
Diagnosis
 ALL

61 (43.6)
 Brain tumor

19 (13.6)
 Sarcoma

18 (12.9)
 Lymphoma

13 (9.3)
 AML

7 (5.0)
 Neuroblastoma

7 (5.0)
 Wilms tumor

6 (4.3)
 Hepatoblastoma

5 (3.6)
 Other diagnosis*

4 (2.9)
History of cancer relapse

21 (15.0)
Central venous catheter

127 (90.7)

IQR: Interquartile Range; ALL: Acute Lympoblastic Leukemia; AML: Acute Myelogenous Leukemia; UTI: Urinary Tract Infection.
*Desmoplastic small round cell tumor (N=1), renal carcinoma (N=2), and rhabdoid liver tumor (N=1).

Infectious complications

Of the 199 FN episodes studied, 43 (21.6%) led to a diagnosis of bacterial or fungal infection (Figure 1), with 6 episodes (3%) involving multiple infections. The most common was bacteremia, of which there were 29 cases (14.6%); cultures were positive for Gram-positive organisms in 18 (including 8 with coagulase-negative staphylococci), Gram-negative organisms in 8, and mixed flora in 3. Bacteremia was diagnosed a median of 1 day (1-3) after fever onset. There were 16 cases (8%) of other bacterial infections, which were diagnosed a median of 4 days (2-6) after fever onset and included typhlitis (N=5), Clostridium difficile enterocolitis (N=4), UTI (N=3), and cellulitis (N=3); 5 of these infections occurred along with bacteremia. There were 5 cases of IFI, most commonly pulmonary aspergillosis, diagnosed a median of 5 days (0-9) after fever onset. Overall, the median time from fever onset to diagnosis was 2 days (1-4). Distributive shock requiring intensive care occurred in 4 FN episodes (2%) due to bacteremia or meningitis, and one of these children died.

fig 1

Figure 1: Overview of bacterial and fungal infections in the cohort. For episodes with multiple infections, the left panel categorizes the first diagnosed. UTI, urinary tract infection; C. difficile, Clostridium difficile enterocolitis.

Risk Factors for Bacterial or Fungal Infection

The case-control sample consisted of 40 cases and 80 controls (Table 2). There was not a statistically significant difference in age or sex between the cases and controls, although the case group contained a larger proportion of children <1 year of age and a larger proportion of children who were already hospitalized at fever onset. at the onset of FN. The relative risk of infection was markedly higher in children with trisomy 21 (RR 3.11 [2.39-4.03]) and those with AML (RR 2.11 [1.13-3.95]), although these p-values were >0.05. While cases were slightly more likely to have a temperature ≥39°C, presenting temperature and laboratory values were not significantly different between cases and controls, nor were clinical findings like mucositis and gastrointestinal upset. Cases were more likely to have fever recurrence after >24 hours afebrile and also to have fevers lasting ≥7 days, although these associations were not statistically significant. There was a significantly increased risk of infection (p<0.004) for participants with a prior history of prior bacterial or fungal infection (RR 2.16 [1.34 to 3.48]).

Table 2: Univariate analysis of a nested case-control sample of pediatric patients with febrile neutropenia (FN)

Risk factor, N (%)

 Cases (N=40) Controls (N=80) p

Relative risk (95% CI)
Demographic and historical features
Sex

27 (67.5)

 41 (51.2) 0.118 1.59 (0.91 to 2.77)

Relapsed

 6 (15.0) 12 (15.0) 1.000

1.00 (0.49 to 2.03)
Age at onset (years)*

6.6 (4.2-15.9)

 7.2 (3.1-12.3) 0.432 —

Age <1 year

 3 (7.5) 2 (2.5) 0.332

1.86 (0.87 to 4.00)
Trisomy 21

2 (5.0)

 0 (0.0) 0.109 3.11 (2.39 to 4.03)

Diagnosis of AML

 4 (10.0) 2 (2.5) 0.095

2.11 (1.13 to 3.95)
Prior infection

18 (45.0)

 15 (18.8) 0.004

2.16 (1.34 to 3.48)
Findings at FN onset
Temperature (oC)*

39 (38.6-39.7)

 38.8 (38.4-39.3) 0.052 —

Presenting WBC (x109/L)*

 0.3 (0-0.8) 0.5 (0.2-0.9) 0.106

—
Presenting ANC (x109/L)*

115.5 (0-250)

 86.0 (11-348) 0.538 —

Already admitted

 10 (25.0) 10 (12.5) 0.118

1.67 (0.98 to 2.83)
Rhinitis or rhinorrhea

5 (12.5)

 16 (20.0) 0.445 0.67 (0.30 to 1.51)

Severe mucositis

 7 (17.5) 12 (15.0) 0.793

1.13 (0.59 to 2.16)
Abdominal pain

8 (20.0)

 11 (13.8) 0.430 1.33 (0.73 to 2.42)

Vomiting

 13 (32.5) 15 (18.8)

0.111

1.58 (0.95 to 2.63)
Findings at reevaluation
Fever duration (days)*

2 (1.5-5)

 2 (1-4) 0.346 —

Fever recurrence

 12 (30.0) 13 (16.3) 0.097

1.63 (0.97 to 2.72)
Fever for ≥7 days

7 (17.5)

 8 (10.0) 0.255

1.48 (0.81 to 2.73)

*Reported as median and interquartile range.
P-values are from Fisher exact tests for proportions and rank-sum tests for continuous variables. CI: Confidence Interval; AML: Acute Myeloid Leukemia; WBC: White Blood Cells; ANC: Absolute Neutrophil Count.

Discussion

In this consecutive sample of 199 FN episodes in a typical pediatric oncology population at a United States tertiary-care hospital, 21.6% were complicated by a bacterial or fungal infection, most frequently Gram-positive bacteremia. UTI was more common than expected, likely reflecting our emergency department’s practice of obtaining non-catheterized urine samples from most febrile children. Although undiagnosed UTI would likely be treated by empiric antibiotics, this source of pathology in children with FN warrants further investigation. In a nested case-control analysis, the relative risk of bacterial or fungal infection was higher in children with trisomy 21 and those with AML and considerably higher in those with a prior history of infection. Infections diagnosed during the study period were generally not relapses of prior infection; instead, infection risk may reflect cumulative person-level factors, including duration of chemotherapy, cumulative antibiotic exposure, and differences in the microbiome.

As with any observational retrospective study, these findings are not definitive. Our broadly inclusive definition of infection was designed to reflect clinical decision-making, with emphasis on clinical data that would indicate a change in management or a longer course of inpatient observation. The overall similarity between groups in the case-control analysis, as well as the fact that infections occurred during relatively low-intensity chemotherapy (like maintenance ALL therapy) emphasizes the challenge of risk-stratifying children with FN. Most infections in this cohort, however, were diagnosed within the first 4 days after the onset of fever. For children without trisomy 21, AML, or a prior infection history, who do not have overt signs of infection, there may be less benefit of hospitalization longer than through neutrophil recovery, as long as careful outpatient follow-up can be assured.

Conflict of Interest

The authors have no conflicts of interest or external funding sources to disclose.

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