key: cord-1018831-7hzc0d01 authors: Borkow, Gadi; Salvatori, Rachel; Kanmukhla, Vikram K. title: Drastic Reduction of Bacterial, Fungal and Viral Pathogen Titers by Cuprous Oxide Impregnated Medical Textiles date: 2021-02-01 journal: J Funct Biomater DOI: 10.3390/jfb12010009 sha: 0e9d1671553979059686e30c4741aa18f46262e6 doc_id: 1018831 cord_uid: 7hzc0d01 Hospital patients and personnel are at risk of nosocomial viral infections, as clearly manifested during the COVID-19 pandemic. Transmission of respiratory viral pathogens can occur through contaminated surfaces, including from medical textiles. Copper has potent biocidal properties, and cuprous oxide impregnated medical textiles (CMT) reduce hospital-acquired bacterial infections. In the current study we confirm the antimicrobial properties of CMT and determine their capacity to reduce infectious titres of human coronavirus (HCoV-229E) in an independent laboratory. The antibacterial and antiviral activities of the CMT were determined according to AATCC TM100-2019 and ISO 18184:2019 standards, respectively. The CMT reduced by 4 logs the viable titers of MRSA, Klebsiella pneumoniae, Enterococcus faecalis, and Candida auris after 2 h of incubation. Viable titers of Clostridium difficile were reduced by 2.3, 3, and 4 logs after 2, 6, and 18 h, respectively. Infectious titers of HCoV-229E exposed to CMT for 2 h were reduced by 2.8 and 4 logs (99.85% and 99.99% reductions) as compared to Time-0 control and initial inoculum, respectively. The CMT retain their antibacterial efficacy even after 100 industrial washings. Use of cuprous oxide impregnated textiles in clinical settings may reduce not only hospital acquired infections caused by bacterial and fungal pathogens, but also, and equally important, those caused by coronavirus and other viruses. Infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, can occur through direct exposure to aerosol particles and droplets generated during coughing, sneezing or talking, both by symptomatic and asymptomatic individuals [1, 2] , or indirectly by touching contaminated surfaces [3] . Unfortunately, SARS-CoV-2 can remain infectious on inanimate surfaces from hours to days [4] . On fabrics the virus can remain infectious for up to 2 days [5] . Similarly, other coronaviruses, as well as other enveloped and non-enveloped viruses, can remain infectious for days on inanimate surfaces [6] [7] [8] . Copper has potent biocidal properties [9] . Recently the capacity of copper surfaces to reduce the infectious titers of SARS-CoV-2 has been reported [10, 11] . No viable SARS-CoV-2 was found on metallic copper after 8 h, while on cardboard, plastic, and stainless steel, viable SARS-CoV-2 could be found even after 24, 72 and 72 h, respectively [11] . Similar higher susceptibility to copper than to all other tested surfaces was also found for SARS-CoV-1 [11] . Significantly, it has been shown that cuprous oxide (Cu 2 O), which primarily forms on copper under ambient conditions, and copper ions released from the metallic copper, are the active forms that cause the inactivation of the viruses and bacteria that come in contact with metallic copper surfaces [12] [13] [14] . Furthermore, it has been shown that monovalent copper ions (Cu + ) are much more potent than divalent copper ions (Cu ++ ) in their biocidal action in a wide range of tested conditions [15] . In general, the redox cycling between Cu ++ and Cu + catalyze the production of highly hydroxyl radicals that can damage microbial biomolecules, such as lipids, proteins, and DNA [16] . Under ambient conditions copper oxidizes to cuprous oxide, generating monovalent copper ions [17, 18] . Impregnating fibers, yarns, and woven and non-woven textiles with cuprous oxide microparticles endows these products with wide spectrum biocidal properties [19, 20] . Woven cuprous oxide impregnated medical textiles (CMT), such as sheets, patient gowns, blankets, and towels, already being used in at least 25 hospitals and in different clinical settings, have been found to reduce hospital acquired bacterial infections [21] [22] [23] [24] [25] . In the current study we confirm the capacity of two generations of textiles impregnated with cuprous oxide microparticles, which are currently in use in clinical settings, to reduce bacterial and fungal pathogens and examine their capacity to reduce the infectious titers of human coronavirus (HCoV-229E). HCoV-229E is one of the viruses responsible for the common cold, and its infection is associated with a range of respiratory symptoms, including pneumonia and bronchiolitis. The significance of this study is the demonstration that CMT can also be an additional significant weapon in the arsenal of measures in the battle against hospital-acquired viral infections. The study (Project ID 0620-CHJ-01-1) was performed according to the ISO Standard 18184:2019-Textiles-Determination of antiviral activity of textile products by Microbial Product Test Laboratory, Situ Biosciences LLC, Wheeling, IL, USA (https://www. situbiosciences.com/). The following human pathogens were tested: Methicillin resistant Two medical textile fabrics, used to make hospital medical textiles, were tested-CMT Gen 1 and CMT Gen 2, hereafter referred to as Test Sample 1 and Test Sample 2. Test Sample 1 is a 3.2 Oz/Sy plain woven fabric made of 55% cotton and 45% 150D/48 polyester filament impregnated with 2.6% cuprous oxide microparticles weight/weight (w/w) in the warp (Figure 1 ). The final concentration of the cuprous oxide in Test Sample 1 is 1.17% w/w. Test Sample 2 is a 3.3 Oz/Sy plain woven fabric made of 60% 60:40 CVC (60% Cotton, 40% polyester blend) in the warp and 40% 150D/48F polyester filament impregnated with 2.6% cuprous oxide microparticles in the weft. The total copper oxide of Test Sample 2 is 2.6 × 40% = 1.04% w/w. The production of the CMT has been described elsewhere [26] . Briefly, cuprous oxide microparticles in a polyester master batch were added to the slurry of the polyester to a final concentration of 2.6% w/w. The fibers were then extruded to yield a 150 denier 48 filament yarn. The cuprous oxide microparticles were physically embedded and homogenously distributed throughout the filament yarn. This polyester yarn was then blended with cotton yarns to form the CMT. As a negative untreated test control material, the same woven fabric but without cuprous oxide was used, hereafter referred as Control Fabric. The antimicrobial testing was based on the AATCC TM 100-2019. The test bacteria and candida were propagated at 37 • C ± 1 • C in tryptic soy broth overnight. Dilutions of the bacterial suspensions were created with 0.89% NaCl, 5% D/E nutrient broth, and 0.01% Triton X and for C. auris in 0.89% NaCl, 5% Sabouraud Dextrose Broth (SDB) and 0.01% Triton X to obtain a final microbial concentration between 1-2 × 10 5 colony forming units (CFU)/mL. The Control and Test samples were autoclaved to sterilize them before testing. The samples were then aseptically cut into 1.0 g per piece and laid down onto individual vials. One ml of the microbial test inoculum was pipetted onto the individual fabric samples. Immediately after inoculation, microorganisms from half of the tested samples were recovered by adding 5 mL of D/E neutralizing broth and vortexing for 1 min (Referred to as Time 0). Various dilutions in 0.89% NaCl, 5% D/E nutrient broth, and 0.01% Triton X of the recovered microorganism were plated onto tryptic soy agar in petri dishes. The other half of the tested samples were incubated at 37 • C ± 1 • C and relative humidity of >90% for 2, 6, or 18 h. After the incubation, the microorganisms were recovered from all test samples by adding 5 mL of D/E neutralizing broth and vortexing for 1 min. The recovered microorganisms were plated onto tryptic soy agar in petri dishes. The number of CFU were determined after 48 h of incubation of the plates at 37 • C ± 1 • C and relative humidity of >90%. The calculations of the microbial titre reductions were determined as follows: Reduction = % reduction = 100(C − A)/C, where A = the microbial CFU/mL recovered from the inoculated treated test specimen swatches incubated over the contact period (2, 6, or 18 h), and where C = the microbial CFU/mL recovered from the inoculated test specimen swatches immediately after the inoculation. The antimicrobial efficacy of the fabrics was also tested after 100 washes at a commercial hospital laundry (Handcraft, Richmond, VA, USA). The amount of copper present before and after every 10 washes was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The antiviral test was performed according to the ISO 18184:2019 standard. Vero-E6 cells were cultured in DMEM (EM-1) media with antibiotics and 10% fetal bovine serum (FBS) and were inoculated with −80 • C cryopreserved Human Coronavirus 229E (HCoV-229E). The virus infection was monitored by observable cytotoxic effects (CTE). The virus was harvested once the relative percentage of cells affected by CTE was approximately 90%. The virus stock infectivity titer (TCID 50 /mL) was then determined as described below. The Control and Test samples were autoclaved to sterilize them before testing. The samples were then aseptically cut into 0. In order to control for potential cytotoxicity caused by eluting molecules from the test specimens during vortexing to the neutralizing solution, the following test was conducted: 20 mL of the neutralizing solution was added to non-virus inoculated control and test samples and after vortexing, aliquots from these solutions were tenfold diluted and added to the Vero cells. Cytotoxicity was then monitored. The potential cytotoxicity caused by the neutralizing solution alone at the various dilutions was also examined. End-point dilutions were conducted with the recovered virus inocula using serial log 10 where, the total CTE-is the average of the column logarithm of the number of viable virus, in cells/cm 2 , recovered from the untreated test specimens immediately after inoculation (Time 0). Replicate count per dilution-the number of well replicates inoculated at each dilution. Log dilution factor-is the dilution factor used for each serial dilution (10x = log 10 (10) = 1). The antiviral activity value was calculated as follows: log(Va/Vc) = log(Va) − log(Vc), where, log(Va) = the common logarithm average of 3 infectivity titer values immediately after inoculation of the control sample; log(Vc) = the common logarithm average of 3 infectivity titer values immediately after the 2 h contact time with the tested samples. Replicate data were utilized in the calculation by the Spearman-Karber method; no additional statistical analysis was conducted. Table 1 details the antibacterial and antifungal efficacy of the CMT tested against a panel of human nosocomial pathogens. A 4-log reduction of the viable titers of the tested pathogens was achieved after 2 h of incubation, with the exception of C. difficile, in which a 3 and a 4-log reduction was demonstrated after 18 h of incubation. As can be seen in Table 2 , the antimicrobial efficacy of the Test Samples was maintained even after 100 washes. After 100 washes there was a 1.3% mean loss of copper weight as quantitatively determined by ICP-OES. Table 2 . Antimicrobial efficacy after 100 washes. The initial bacterial titer (CFU) and the titer after 24 h of contact time, and % reduction as compared to the initial inoculum, are detailed. Enterobacter aerogenes 1 0 6.25 × 10 5 2.70 × 10 5 5.65 × 10 5 24 3.00 × 10 2 1.00 × 10 2 1.00 × 10 2 % reduction >99.9 >99.9 >99.9 2 0 6.25 × 10 5 2.70 × 10 5 5.65 × 10 5 24 3.00 × 10 3 1.00 × 10 2 7.50 × 10 2 % reduction 99.52 >99.9 99.87 Table 3 details the CTE caused by the coronavirus alone (initial inoculum), and after being exposed to the Control and Test Samples for 0 and 2 h, and the CTE caused by the neutralizing solution alone or after being retrieved from the non-virus inoculated control and test samples. The infectious titer of the viral inoculum was 3.2 × 10 5 TCID 50 /mL (5.5 log 10 TCID 50 /mL). The recovered viral titers from the inoculated control and test samples at Time 0 were 2.2 × 10 4 TCID 50 /mL (4.3 log 10 TCID 50 /mL). After 2 h of incubation, the infectious titers of the virus were reduced to 3.2 × 10 1 TCID 50 /mL (1.5 log 10 TCID 50 /mL) in both Test Samples, i.e., 2.8 and 4 log 10 reductions (99.853% and 99.99% reductions), as compared to the Time 0 control and initial inoculum, respectively. In contrast, no reduction of the infectious titers occurred in the virus exposed to the Control Sample. No cytotoxicity occurred due to the sample extracted from the fabrics without virus and due to the neutralizer used in all three experiments performed. Hospital patients and personnel are at risk of nosocomial viral infections [8, 27] , as clearly manifested during the current COVID-19 pandemic [28, 29] . Textiles, especially reusable healthcare textiles that are in close contact with patients for extended periods of time, can significantly contribute to bacterial, fungal and viral nosocomial infections [30] [31] [32] . It has been found that indirect cross-contamination through transmission of microorganisms, including viruses, which remain infectious on fomite soft and hard surfaces, can contribute to nosocomial infections [33, 34] . The capacity of metallic copper and copper alloys to readily neutralize coronaviruses has been demonstrated [14] . Similar infectivity reduction findings were recently reported for SARS-CoV-2 [11] . In the Test Samples, the active copper form is already in the oxidized (activated) form (cuprous oxide). This is one significant step closer to releasing the active copper ions that damage viruses. Importantly, cuprous oxide particles release monovalent copper ions (Cu+), which have been found to be much more potent antivirals and antibacterials than divalent ions (Cu++) [35] . Accordingly, N95 respiratory masks, in which the external layers were made with nonwoven polypropylene fabric impregnated with cuprous oxide microparticles, have been shown to reduce the infectious titers of Human Influenza A (H1N1) and Avian Influenza Virus (H9N2) virions that remain on the mask by more than three logs within 30 min of contact [36] . Similarly, copper oxide impregnated nonwoven fibers and fabrics were shown to reduce infectious titers of 12 additional enveloped and non-enveloped pathogenic viruses [37, 38] . The cuprous oxide impregnated woven medical textiles have been in use in medical settings for more than 6 years and 3.4 million patient days, without any adverse effects. The high safety of cuprous oxide impregnated medical and consumer products, such as adult diapers, antimicrobial wound dressings and masks, has been demonstrated in many studies [36, [39] [40] [41] . Cuprous oxide impregnated wound dressings have been cleared by the FDA for the treatment of acute and chronic wounds following extensive biocompatibility studies required for medical devices, demonstrating that they are safe for human tissues. The use of biocidal textiles can significantly reduce nosocomial infections [30] , as demonstrated in several studies in which woven medical textiles, such as sheets, patient gowns, blankets, and towels, impregnated with cuprous oxide microparticles, reduced hospital acquired bacterial infections [21] [22] [23] [24] [25] . Indeed, the potent wide spectrum antibacterial and antifungal efficacy of the tested fabrics used to make medical textiles, was demonstrated in the current study. Further, CMT have proven to be durable; even after 100 washes, more than 98% of their copper remained intact, allowing for their continued antibacterial efficacy. Not surprisingly, the cuprous oxide impregnated medical textiles reduced the infectious titers of the HCoV-229E by more than 99% in two hours of exposure, indicating that the use of such textiles in clinical settings may also contribute to the reduction of hospital acquired viral infections. The use of cuprous oxide impregnated medical textiles may be an important measure in the arsenal of interventions taken to reduce hospital acquired viral infections. This may be of special significance especially during ongoing pandemics, such as that caused by the current coronavirus SARS-CoV-2, responsible for the COVID-19 pandemic. Author Contributions: G.B. and V.K.K. analyzed the data and wrote the article. R.S. performed preliminary experimental laboratory studies in Cupron's microbiology laboratory in preparation for the independent studies conducted by Situ Biosciences lab, which results are described in the current study. The design of the study was performed by the authors in collaboration with the testing lab, Situ Biosciences LLC, in accordance with ISO Standard 18184:2019 requirements and guidelines. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Cupron Inc., Richmond, Virginia. Institutional Review Board Statement: Not applicable. Data Availability Statement: The data and independent study reports used for preparing the manuscript are available from the corresponding author on reasonable request. Seyed Alinaghi, S. Transmission Modes of COVID-19: A Systematic Review Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany The distribution of SARS-CoV-2 contamination on the environmental surfaces during incubation period of COVID-19 patients The role of environmental factors to transmission of SARS-CoV-2 (COVID-19) Stability of SARS-CoV-2 in different environmental conditions Survival of Enveloped and Non-Enveloped Viruses on Inanimate Surfaces Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: The possible role of dry surface contamination Using copper to fight microorganisms A Surface Coating that Rapidly Inactivates SARS-CoV-2 Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1 Pure and Oxidized Copper Materials as Potential Antimicrobial Surfaces for Spaceflight Activities Role of copper oxides in contact killing of bacteria Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials Factors Enhancing the Antibacterial Effect of Monovalent Copper Ions Copper as a biocidal tool Oxidation of polycrystalline copper thin films at ambient conditions Comparison of the early stages of corrosion of copper and iron investigated by in situ TM-AFM Putting copper into action: Copper-impregnated products with potent biocidal activities Endowing textiles with permanent potent biocidal properties by impregnating them with copper oxide Analysis of the role of copper impregnated composite hard surfaces, bed linens and patient gowns in reducing healthcare-associated infection rates Reduced health care-associated infections in an acute care community hospital using a combination of self-disinfecting copper-impregnated composite hard surfaces and linens Effect of copper-impregnated composite bed linens and patient gowns on healthcare-associated infection rates in six hospitals Reduction of healthcare-associated infections in a long-term care brain injury ward by replacing regular linens with biocidal copper oxide impregnated linens Reduction of health care-associated infection indicators by copper oxide-impregnated textiles: Crossover, double-blind controlled study in chronic ventilator-dependent patients Copper oxide impregnated textiles with potent biocidal activities Role of viral bioaerosols in nosocomial infections and measures for prevention and control Transmission of COVID-19 to Health Care Personnel During Exposures to a Hospitalized Patient-Solano County Epidemiology, Pathogenesis, and Control of COVID-19 Biocidal textiles can help fight nosocomial infections The contribution of beds to healthcare-associated infection: The importance of adequate decontamination The role of textiles as fomites in the healthcare environment: A review of the infection control risk A model of the transmission of micro-organisms in a public setting and its correlation to pathogen infection risks Virus hazards from food, water and other contaminated environments Comparison of the antiviral effect of solid-state copper and silver compounds Journal of Hazardous Materials A novel anti-influenza copper oxide containing respiratory face mask Neutralizing viruses in suspensions by copper oxide based filters Deactivation of human immunodeficiency virus type 1 in medium by copper oxide-containing filters Copper oxide impregnated wound dressings: Biocidal and safety studies Safety of using diapers containing copper oxide in chronic care elderly patients Safety of using copper oxide in medical devices and consumer products We thank Don Satchell, who runs the Situ Biosciences lab that conducted the actual lab work. All authors are employees of Cupron Scientific or Cupron Inc., which produces cuprous oxide impregnated products.