Virucidal efficacy of glutaraldehyde for instrument disinfection.

Aim: Glutaraldehyde (GDA) is an active ingredient in many instrument disinfectants and is effective against a broad spectrum of microorganisms. In the past, the virus-inactivating properties of these products were mainly claimed based on quantitative suspension tests with different test viruses. Recently, however, a European Norm EN 17111:2018 has been published which allows examination of instrument disinfectants in a surface carrier test, simulating practical conditions. Therefore, it is of interest to evaluate GDA for the ability to inactivate the viruses used in this European Norm as test viruses. Methods: The virucidal efficacy of GDA as the active ingredient in instrument disinfectants was evaluated with 4 different test viruses in a method simulating practical conditions (EN 17111:2018). Results: With a fixed exposure time of five minutes at 20°C, 100 ppm GDA were necessary to inactivate vaccinia virus, classifying it as a limited spectrum virucidal activity for pre-cleaning products. For adenovirus, 125 ppm GDA were required, whereas for murine norovirus as a surrogate for human norovirus, 4,000 ppm GDA were required for a significant reduction of viral titres. Both non-enveloped viruses must be tested to prove virucidal activity in EN 17111:2018. But even 4,000 ppm were not enough to yield a 4 log10 reduction of the murine parvovirus at 20°C. This virus is only required as a test virus using this method if temperatures ≥40°C are used. Conclusion: GDA, as the active ingredient of many instrument disinfectants, shows virucidal efficacy at 20°C. The necessary concentrations are strongly dependent on the stability of the test viruses. Due to the high stability of murine norovirus, GDA levels of 4,000 ppm were required to inactivate this virus within the 5-minute exposure time.

Evaluation of the virucidal efficacy of disinfectant wipes with a test method simulating practical conditions.

Background: The use of disinfectant wipes in hospitals is increasing over the last years. These wipes should be able to inactivate microorganisms including viruses on environmental surfaces and to prevent their transfer to clean areas.The European norm (EN) 16615:2015 describes a wiping process over four fields starting on the contaminated field 1 followed by fields 2-4 and back to the starting point (4-field test). This test method exclusively describes killing and transfer of vegetative bacteria and fungi by disinfectant wipes without measuring virucidal activities. Therefore, it was the aim of this study to use the existing test methodology additionally to evaluate virus inactivation by wipes. Methods: The 4-field test was performed with four commercially available disinfectant wipes including the examination of the active solutions of these wipes with a reference wipe. Murine norovirus (MNV) as surrogate of human noroviruses, adenovirus (AdV) type 5 and polyomavirus SV40 (SV40) were chosen as test viruses. Results: The per acetic acid (PAA)-based wipe (wipe A) was able to inactivate all three test viruses resulting in a four log10 reduction on test field 1, whereas the quaternary ammonium compound (QAC)-based products (wipes B and C) failed to reach such reduction. Both QAC-based wipes were able to inactivate SV40 and only the active solution of wipe B was effective against MNV. Another wipe with 2-propanol as active ingredient (wipe D) was not able to show a sufficient efficacy against all three test viruses. There was a good agreement between the results of the wipes and the corresponding fluids showing no influence of the material of wipes.Tests with the 2-propanol-based wipe D showed a transfer of all test viruses to the non-contaminated test fields 2-4. SV40 was additionally transferred by the QAC-based wipe C with 0.78% active ingredients to these additional fields. In all other cases no virus transfer to test fields 2-4 was observed. Finally, no virus could be detected in the PAA-based wipe A after usage in the 4-field test in contrast to the other wipes examined. Conclusions: The successful performance of a 4-field test with viruses demonstrated that the existing wiping method with bacteria and fungi can be used in addition for measuring virucidal efficacy. The virus-inactivating properties of surface disinfectants could be evaluated therefore with a test simulating practical conditions with mechanical action resulting in more reliable data than the existing quantitative suspension tests and/or a carrier test without any mechanical action.

Virucidal efficacy of peracetic acid for instrument disinfection.

BACKGROUND: Various peracetic-acid (PAA)-based products for processing flexible endoscopes on the market are often based on a two-component system including a cleaning step before the addition of PAA as disinfectant. The peracetic acid concentrations in these formulations from different manufacturers are ranging from 400 to 1500 ppm (part per million). These products are used at temperatures between 20 °C and 37 °C. Since information on the virus-inactivating properties of peracetic acid at different concentrations and temperature is missing, it was the aim of the study to evaluate peracetic acid solutions against test viruses using the quantitative suspension test, EN 14476. In addition, further studies were performed with the recently established European pre norm (prEN 17111:2017) describing a carrier assay for simulating practical conditions using frosted glass. METHODS: In the first step of examination, different PAA solutions between 400 and 1500 ppm were tested at 20 °C, 25 °C, and 35 °C with three test viruses (adenovirus, murine norovirus and poliovirus) necessary for creating a virucidal action according to the European Norm, EN 14476. A second step for simulating practical conditions based on prEN 17111:2017 followed by spreading a test virus together with soil load onto a glass carrier which was immerged into a peracetic acid solution. A fixed exposure time of five minutes was used in all experiments. RESULTS: In the quantitative suspension test 1500 ppm PAA solution was needed at 35 °C for five minutes for the inactivation of poliovirus, whereas only 400 ppm at 20 °C for adeno- and murine norovirus were necessary. In the carrier assay 400 ppm peracetic acid at 20 °C were sufficient for adenovirus inactivation, whereas 600 ppm PAA were needed at 25 °C and 35 °C and 1000 ppm at 20 °C for murine norovirus. A PAA solution with 1000 ppm at 35 °C was required for complete inactivation of poliovirus. However, a dramatically decrease of titer after the drying and immerging could be observed. In consequence, a four log reduction of poliovirus titer could not be achieved in the carrier test. CONCLUSION: In summary, 1500 ppm PAA at 35 °C was necessary for a virucidal action in the quantitative suspension test. After passing the requirements of the suspension test, additional examinations with adeno- and murine norovirus on glass carriers based on prEN 17111:2017 will not additionally contribute to the final claim of an instrument disinfectant for virucidal efficacy. This is due to the great stability of poliovirus in the preceded quantitative suspension test and the fact that poliovirus could not serve as test virus in the following carrier assay.

Virucidal efficacy of a sonicated hydrogen peroxide system (trophon® EPR) following European and German test methods.

Aim: The virucidal efficacy of an automated ultrasound probe disinfector (trophon® EPR) was evaluated in a three step procedure according to European and German test methods. This system uses sonicated hydrogen peroxide mist (35%) at elevated temperature (50°C) in a closed chamber with control of all parameters within a 7 minute cycle. Methods: In the first step of examination, the peroxide solution was tested in a quantitative suspension assay according to the Guideline of Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) e.V. and Robert Koch-Institute (RKI) and in parallel with the European Norm EN 14476 with all test viruses creating a virucidal claim. In the second step, the virucidal efficacy of the hydrogen peroxide solution was evaluated in a hard surface carrier test according to the Guideline of DVV with adenovirus, murine norovirus and parvovirus simulating practical conditions. Finally, the efficacy was evaluated by the automated system using stainless steel carriers inoculated with test virus and positioned at different levels inside the chamber. Results: A ≥4 log10 reduction of virus titre was demonstrated with all methods including carrier tests with murine norovirus, adenovirus, and parvovirus using the automated device. Conclusion: The automated device is able to inactivate test viruses of German and European norms and can therefore claim efficacy against human pathogenic enveloped and non-enveloped viruses. This includes human papillomaviruses which form part of the complete virucidal claim.

Development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid.

BACKGROUND: Hand disinfectants are important for the prevention of virus transmission in the health care system and environment. The development of broad antiviral spectrum hand disinfectants with activity against enveloped and non-enveloped viruses is limited due to a small number of permissible active ingredients able to inactivate viruses. METHODS: A new hand disinfectant was developed based upon 69.39 % w/w ethanol and 3.69 % w/w 2-propanol. Different amounts of citric acid and urea were added in order to create a virucidal claim against poliovirus (PV), adenovirus type 5 (AdV) and polyomavirus SV40 (SV40) as non-enveloped test viruses in the presence of fetal calf serum (FCS) as soil load. The exposure time was fixed to 60 s. RESULTS: With the addition of 2.0 % citric acid and 2.0 % urea an activity against the three test viruses was achieved demonstrating a four log10 reduction of viral titers. Furthermore, this formulation was able to inactivate PV, AdV, SV40 and murine norovirus (MNV) in quantitative suspension assays according to German and European Guidelines within 60 s creating a virucidal claim. For inactivation of vaccinia virus and bovine viral diarrhea virus 15 s exposure time were needed to demonstrate a 4 log10 reduction resulting in a claim against enveloped viruses. Additionally, it is the first hand disinfectant passing a carrier test with AdV and MNV. CONCLUSIONS: In conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in current guidelines and thereby contributing as valuable addition to the hand disinfection portfolio.

Virucidal activity of Formulation I of the World Health Organization's alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.

BACKGROUND: A recently modified World Health Organization (WHO) Formulation I was examined as 80% and 97% solutions against poliovirus type 1, adenovirus type 5 and murine norovirus according to the new European Norm prEN 14476:2011. In a previous study the unmodified WHO Formulation I had failed to demonstrate a sufficient activity against poliovirus type 1 according to the European Norm EN 14476-2007 whereas a sufficient activity was seen with adeno- and norovirus. FINDINGS: The modified WHO Formulation I demonstrated a virucidal activity against all 3 test viruses of the European Norm prEN 14476:2011 under clean conditions. This was achieved as 80% solution against adeno- and norovirus within 30 seconds and as 97% solution against poliovirus within 60 seconds. Testing the unmodified WHO Formulation I against poliovirus type 1 in the 97% assay of the European Norm prEN 14476:2011 an identical activity could be demonstrated. When comparing the 80% and the 97% assay of the European Norm prEN 14476:2011 the modified WHO Formulation I as 80% solution was active against adenovirus type 5 within 30 seconds whereas the 97% solution failed within 2 minutes exposure time. CONCLUSIONS: The technical possibility in the new European Norm prEN 14476:2011 allows testing a ready-to-use disinfectant as 97% solution and is responsible for the new virucidal claim of the modified WHO Formulation I. In contrast to the improvements with poliovirus type 1 the activity against adenovirus type 5 decreased when increasing the test concentration from 80% to 97%.

Exploring the function of alcohol dehydrogenases during the endophytic life of Azoarcus Sp. strain BH72.

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.

The virucidal activity of 2 hand rubs proposed by the World Health Organization was studied in a quantitative suspension test for chemical disinfectants and antiseptics in human medicine (EN 14476). These formulations are recommended if no hand rubs with declared microbiological activity are available in health care settings. Formulation I, based on ethanol, inactivated bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV), adenovirus, and murine norovirus as a surrogate for human norovirus. Formulation II, based on isopropyl alcohol, was active only against adenovirus and enveloped viruses, such as BVDV and HCV. Both formulations failed to inactivate poliovirus by 4 log(10) steps within 300 seconds.

Inactivation of murine norovirus by chemical biocides on stainless steel.

BACKGROUND: Human norovirus (NoV) causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV) as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces. METHODS: The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA) or glutaraldehyde (GDA) for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells. RESULTS: PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v)] and 1-propanol [30% (v/v)] were able to inactivate MNV under clean conditions (0.03% BSA) on the carriers by > or = 4 log10 within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v). Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes). CONCLUSION: Products based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.