Suitable Disinfectants with Proven Efficacy for Genetically Modified Viruses and Viral Vectors.

Viral disinfection is important for medical facilities, the food industry, and the veterinary field, especially in terms of controlling virus outbreaks. Therefore, standardized methods and activity levels are available for these areas. Usually, disinfectants used in these areas are characterized by their activity against test organisms (i.e., viruses, bacteria, and/or yeasts). This activity is usually determined using a suspension test in which the test organism is incubated with the respective disinfectant in solution to assess its bactericidal, yeasticidal, or virucidal activity. In addition, carrier methods that more closely reflect real-world applications have been developed, in which microorganisms are applied to the surface of a carrier (e.g., stainless steel frosted glass, or polyvinyl chloride (PVC)) and then dried. However, to date, no standardized methods have become available for addressing genetically modified vectors or disinfection-resistant oncolytic viruses such as the H1-parvovirus. Particularly, such non-enveloped viruses, which are highly resistant to disinfectants, are not taken into account in European standards. This article proposes a new activity claim known as "virucidal activity PLUS", summarizes the available methods for evaluating the virucidal activity of chemical disinfectants against genetically modified organisms (GMOs) using current European standards, including the activity against highly resistant parvoviridae such as the adeno-associated virus (AAV), and provides guidance on the selection of disinfectants for pharmaceutical manufacturers, laboratories, and clinical users.

Hygiene and disinfection measures for monkeypox virus infections.

In Germany, recommendations on infection prevention and control of current virus outbreaks are given as communications by the Association for Applied Hygiene e.V. (VAH) together with the joint Disinfectant Commission of the German Association for the Control of Virus Diseases e.V. (DVV) and the Society of Virology* (GfV). The DVV was founded in 1954 in response to the ongoing threat to the population from polio and was given its current name in 1977. The DVV is supported by the Federal Ministry of Health, the Ministries of Health of the Federal States, scientific societies, as well as social foundations and organisations. Private individuals cannot be members of the DVV. The Society of Virology e.V. (GfV) is a scientific society for all virological fields in Germany, Austria and Switzerland, and is thus the largest virological society in Europe. With numerous commissions, guidelines and statements, it is the authoritative contact for research, healthcare and politics. The joint commission "Virus Disinfection" of these scientific societies focuses on the efficacy of chemical disinfection procedures against viruses. The VAH bundles the expertise of scientific societies and experts on infection prevention and is particularly committed to the quality assurance of hygiene measures. With the VAH disinfectant list, the association provides the standard reference for the selection of high-quality disinfection procedures. This disinfectant list has a tradition of more than 60 years in Germany. The original German version of this document was published in August 2022 and has now been made available to the international professional public in English. The document contains recommendations on hygiene and disinfection measures for monkeypox virus infections. Disinfectants against monkeypox must have at least proven efficacy against enveloped viruses (active against enveloped viruses); products with the efficacy ranges "limited virucidal activity" and "virucidal" can also be used. The disinfectant list of the VAH or the disinfectant list of the Robert Koch Institute are available for the selection of products. Especially in the case of contamination with crust or scab material, it should be noted that protein contamination can have a protective or stabilising effect on monkeypox. Therefore, cleaning - before disinfection - should always be carried out in this situation. Preventive measures such as vaccination and hygiene in the vicinity of people with monkeypox must be taken to prevent transmission to small children, pregnant women or people with a pronounced immune deficiency.

Ethanol is indispensable for virucidal hand antisepsis: memorandum from the alcohol-based hand rub (ABHR) Task Force, WHO Collaborating Centre on Patient Safety, and the Commission for Hospital Hygiene and Infection Prevention (KRINKO), Robert Koch Institute, Berlin, Germany.

BACKGROUND: The approval of ethanol by the Biocidal Products Regulation has been under evaluation since 2007. This follows concern over alcohol uptake from ethanol-based hand rubs (EBHR). If ethanol is classified as carcinogenic, mutagenic, or reprotoxic by the European Chemicals Agency (ECHA), then this would affect infection prevention and control practices. AIM: A review was performed to prove that ethanol is toxicological uncritical and indispensable for hand antisepsis because of its unique activity against non-enveloped viruses and thus the resulting lack of alternatives. Therefore, the following main points are analyzed: The effectiveness of ethanol in hand hygiene, the evidence of ethanol at blood/tissue levels through hand hygiene in healthcare, and the evidence of toxicity of different blood/tissue ethanol levels and the non-comparability with alcoholic consumption and industrial exposure. RESULTS: EBHR are essential for preventing infections caused by non-enveloped viruses, especially in healthcare, nursing homes, food industry and other areas. Propanols are effective against enveloped viruses as opposed to non-enveloped viruses but there are no other alternatives for virucidal hand antisepsis. Long-term ingestion of ethanol in the form of alcoholic beverages can cause tumours. However, lifetime exposure to ethanol from occupational exposure < 500 ppm does not significantly contribute to the cancer risk. Mutagenic effects were observed only at doses within the toxic range in animal studies. While reprotoxicity is linked with abuse of alcoholic beverages, there is no epidemiological evidence for this from EBHR use in healthcare facilities or from products containing ethanol in non-healthcare settings. CONCLUSION: The body of evidence shows EBHRs have strong efficacy in killing non-enveloped viruses, whereas 1-propanol and 2-propanol do not kill non-enveloped viruses, that pose significant risk of infection. Ethanol absorbed through the skin during hand hygiene is similar to consumption of beverages with hidden ethanol content (< 0.5% v/v), such as apple juice or kefir. There is no risk of carcinogenicity, mutagenicity or reprotoxicity from repeated use of EBHR. Hence, the WHO Task Force strongly recommend retaining ethanol as an essential constituent in hand rubs for healthcare.

Tolerance of biofilm of a carbapenem-resistant Klebsiella pneumoniae involved in a duodenoscopy-associated outbreak to the disinfectant used in reprocessing.

BACKGROUND: One possible transmission route for nosocomial pathogens is contaminated medical devices. Formation of biofilms can exacerbate the problem. We report on a carbapenemase-producing Klebsiella pneumoniae that had caused an outbreak linked to contaminated duodenoscopes. To determine whether increased tolerance to disinfectants may have contributed to the outbreak, we investigated the susceptibility of the outbreak strain to disinfectants commonly used for duodenoscope reprocessing. Disinfection efficacy was tested on planktonic bacteria and on biofilm. METHODS: Disinfectant efficacy testing was performed for planktonic bacteria according to EN standards 13727 and 14561 and for biofilm using the Bead Assay for Biofilms. Disinfection was defined as ≥ 5log10 reduction in recoverable colony forming units (CFU). RESULTS: The outbreak strain was an OXA-48 carbapenemase-producing K. pneumoniae of sequence type 101. We found a slightly increased tolerance of the outbreak strain in planktonic form to peracetic acid (PAA), but not to other disinfectants tested. Since PAA was the disinfectant used for duodenoscope reprocessing, we investigated the effect of PAA on biofilm of the outbreak strain. Remarkably, disinfection of biofilm of the outbreak strain could not be achieved by the standard PAA concentration used for duodenoscope reprocessing at the time of outbreak. An increased tolerance to PAA was not observed in a K. pneumoniae type strain tested in parallel. CONCLUSIONS: Biofilm of the K. pneumoniae outbreak strain was tolerant to standard disinfection during duodenoscope reprocessing. This study establishes for the first time a direct link between biofilm formation, increased tolerance to disinfectants, reprocessing failure of duodenoscopes and nosocomial transmission of carbapenem-resistant K. pneumoniae.

[Disinfectants during the COVID-19 pandemic: a challenge]. / Desinfektionsmittel in der COVID-19-Pandemie: eine Herausforderung.

Disinfection measures have become more important as a result of the COVID-19 pandemic in Germany. The increased need for disinfectants at the beginning of the pandemic required temporary legal regulations in order to provide a sufficient quantity of products for the necessary disinfection in the medical sector on the one hand and for the additional demand in the population on the other. For this purpose, the Federal Institute for Drugs and Medical Devices (BfArM) and the Federal Institute for Occupational Safety and Health (BAuA) issued a general ruling, which is explained in more detail in this article. The focus was on measures for hygienic hand disinfection. However, other applications such as surface disinfection in relation to pandemic respiratory diseases are also addressed. The experience gained in ensuring the supply of disinfectants that are effective and safe to use should be used to prepare for further pandemics.

The European tiered approach for virucidal efficacy testing - rationale for rapidly selecting disinfectants against emerging and re-emerging viral diseases.

When facing an emerging virus outbreak such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a quick reaction time is key to control the spread. It takes time to develop antivirals and vaccines, and implement vaccination campaigns. Therefore, preventive measures such as rapid isolation of cases and identification and early quarantine of cases' close contacts-as well as masks, physical distancing, hand hygiene, surface disinfection and air control-are crucial to reduce the risk of transmission. In this context, disinfectants and antiseptics with proven efficacy against the outbreak virus should be used. However, biocidal formulations are quite complex and may include auxiliary substances such as surfactants or emollients in addition to active substances. In order to evaluate disinfectants' efficacy objectively, meaningful efficacy data are needed. Therefore, the European Committee for Standardisation technical committee 216 'Chemical disinfectants and antiseptics' Working Group 1 (medical area) has developed standards for efficacy testing. The European tiered approach grades the virucidal efficacy in three levels, with corresponding marker test viruses. In the case of SARS-CoV-2, disinfectants with proven activity against vaccinia virus, the marker virus for the European claim 'active against enveloped viruses', should be used to ensure effective hygiene procedures to control the pandemic.

Decontamination of Personal Protective Equipment.

Exploratory field analyses of the inactivation capacity of disinfectants on contaminated personal protective equipment (PPE) are required to select a suitable surrogate for biohazardous agents like spores of Bacillus anthracis. The objectives of our study were (1) the determination of an appropriate surrogate for the inactivation of spores of B. anthracis with peracetic acid (PAA), and (2) application of optimized inactivation conditions for an effective decontamination of PPE with PAA under field conditions. For inactivation studies, B. anthracis spores from different strains and B. thuringiensis spores were fixed by air drying on carriers prepared from PPE fabric. Time and concentration studies with PAA-based disinfectants revealed that the spores of the B. thuringiensis strain DSM 350 showed an inactivation profile comparable to that of the spores of the B. anthracis strain with the highest stability, implying that B. thuringiensis can serve as an appropriate surrogate. Rapid (3 to 5 minutes) and effective surface decontamination was achieved with 2% PAA/0.2% surfactant. In field studies, PPE contaminated with spores of B. thuringiensis was treated with the disinfectant. Optimizing the decontamination technique revealed that spraying in combination with brushing was effective within 5 minutes of exposure.

The Bead Assay for Biofilms: A Quick, Easy and Robust Method for Testing Disinfectants.

Bacteria live primarily in microbial communities (biofilms), where they exhibit considerably higher biocide tolerance than their planktonic counterparts. Current standardized efficacy testing protocols of disinfectants, however, employ predominantly planktonic bacteria. In order to test the efficacy of biocides on biofilms in a standardized manner, a new assay was developed and optimized for easy-handling, quickness, low running costs, and above all-repeatability. In this assay, 5 mm glass- or polytetrafluoroethylene beads in 24 well microtiter plates served as substrate for Pseudomonas aeruginosa biofilms. After optimizing result-relevant steps, the actual performance of the assay was explored by treating P. aeruginosa biofilms with glutaraldehyde, isopropanol, or peracetic acid in predefined concentrations. The aspired 5 log10 reduction in CFU counts was achieved by glutaraldehyde at 5% (30 min), and by peracetic acid at 0.3% (10 min). In contrast, 80% isopropanol (30 min) failed to meet the reduction goal. However, the main accomplishment of this study was to unveil the potential of the array itself; most noteworthy here, a reliable repeatability of the results. The new bead assay for biofilms is a robust, quick and cost-effective method for assessing the efficacy of biocides against biofilms.

Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy.

Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non-infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation (i.e., the negative staining) and the detection of viruses. To examine the efficiency of FA inactivation, we used Vaccinia virus, Human adenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature-shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy.

Evaluation of a virucidal quantitative carrier test for surface disinfectants.

Surface disinfectants are part of broader preventive strategies preventing the transmission of bacteria, fungi and viruses in medical institutions. To evaluate their virucidal efficacy, these products must be tested with appropriate model viruses with different physico-chemical properties under conditions representing practical application in hospitals. The aim of this study was to evaluate a quantitative carrier assay. Furthermore, different putative model viruses like adenovirus type 5 (AdV-5) and different animal parvoviruses were evaluated with respect to their tenacity and practicability in laboratory handling. To evaluate the robustness of the method, some of the viruses were tested in parallel in different laboratories in a multi-center study. Different biocides, which are common active ingredients of surface disinfectants, were used in the test. After drying on stainless steel discs as the carrier, model viruses were exposed to different concentrations of three alcohols, peracetic acid (PAA) or glutaraldehyde (GDA), with a fixed exposure time of 5 minutes. Residual virus was determined after treatment by endpoint titration. All parvoviruses exhibited a similar stability with respect to GDA, while AdV-5 was more susceptible. For PAA, the porcine parvovirus was more sensitive than the other parvoviruses, and again, AdV-5 presented a higher susceptibility than the parvoviruses. All parvoviruses were resistant to alcohols, while AdV-5 was only stable when treated with 2-propanol. The analysis of the results of the multi-center study showed a high reproducibility of this test system. In conclusion, two viruses with different physico-chemical properties can be recommended as appropriate model viruses for the evaluation of the virucidal efficacy of surface disinfectants: AdV-5, which has a high clinical impact, and murine parvovirus (MVM) with the highest practicability among the parvoviruses tested.

How long do nosocomial pathogens persist on inanimate surfaces? A systematic review.

BACKGROUND: Inanimate surfaces have often been described as the source for outbreaks of nosocomial infections. The aim of this review is to summarize data on the persistence of different nosocomial pathogens on inanimate surfaces. METHODS: The literature was systematically reviewed in MedLine without language restrictions. In addition, cited articles in a report were assessed and standard textbooks on the topic were reviewed. All reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of surface were included. RESULTS: Most gram-positive bacteria, such as Enterococcus spp. (including VRE), Staphylococcus aureus (including MRSA), or Streptococcus pyogenes, survive for months on dry surfaces. Many gram-negative species, such as Acinetobacter spp., Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Serratia marcescens, or Shigella spp., can also survive for months. A few others, such as Bordetella pertussis, Haemophilus influenzae, Proteus vulgaris, or Vibrio cholerae, however, persist only for days. Mycobacteria, including Mycobacterium tuberculosis, and spore-forming bacteria, including Clostridium difficile, can also survive for months on surfaces. Candida albicans as the most important nosocomial fungal pathogen can survive up to 4 months on surfaces. Persistence of other yeasts, such as Torulopsis glabrata, was described to be similar (5 months) or shorter (Candida parapsilosis, 14 days). Most viruses from the respiratory tract, such as corona, coxsackie, influenza, SARS or rhino virus, can persist on surfaces for a few days. Viruses from the gastrointestinal tract, such as astrovirus, HAV, polio- or rota virus, persist for approximately 2 months. Blood-borne viruses, such as HBV or HIV, can persist for more than one week. Herpes viruses, such as CMV or HSV type 1 and 2, have been shown to persist from only a few hours up to 7 days. CONCLUSION: The most common nosocomial pathogens may well survive or persist on surfaces for months and can thereby be a continuous source of transmission if no regular preventive surface disinfection is performed.

Microbicidal efficacy of superheated steam. II. Studies involving E. faecium and spores of B. xerothermodurans and B. coagulans.

The authors' studies on the heat resistance of microorganisms and the superheating of steam (Spicher et al., Zbl. Hyg. Umweltmed. 201, 541-553, 1999) have been continued, using the vegetative bacterium, Enterococcus faecium, and spores of B. xerothermodurans and B. coagulans. The temperature of the saturated steam was 68 degrees C (E. faecium), 105 degrees C (B. xerothermodurans) and 110 degrees C (B. coagulans), respectively. The steam was superheated by 30-40 K, as a maximum. The test organisms had been fixed to fibre glass fleece. The time of exposure to saturated or superheated steam after which 50% of the bioindicators yielded no viable germs was used as a measure of resistance of the organisms. In the discussion, reference has also been made to the findings for B. subtilis and B. stearothermophilus spores obtained in the preceding study. E. faecium exhibited a maximum resistance when superheating the steam by 5 K. The superheated steam required a 74-fold exposure time compared to saturated steam of 68 degrees C. Resistance became gradually reduced as superheating was further increased. Even superheating by 30 K required a 11-fold exposure time. The bacterial spores exhibited maxima of resistance on superheating by 23-30 K. In these cases necessary exposure times were 119 (B. subtilis), 30 (B. xerothermodurans), and 4 times (B. coagulans, B. stearothermophilus) longer than those required in saturated steam. In the superheating range below 10 K, behaviour patterns varied. Thus, heat resistance may initially become reduced with increasing superheating (B. coagulans), remain on almost the same level as under exposure to saturated steam (B. stearothermophilus), reach a stage of weakly enhanced resistance (B. xerothermodurans), or approach a maximum of resistance in an almost linear mode (B. subtilis). It appears that there are differences between strains of one and the same bacterial species.