Evaluation of a microscale quantitative suspension test to determine the bactericidal and yeasticidal activity of glutaral - one step to improve sustainability in disinfectant testing.

Aims: To evaluate a newly developed microscale quantitative suspension test compared to the existing standard suspension test using determination of the bactericidal and yeasticidal activity of glutaral as one step to improve the sustainability of disinfectant testing. Methods: The testing principles of the quantitative suspension test according to VAH method 9 (comparable to EN 13727) was used as a standard suspension test using 8.0 mL product test solution, 1.0 mL organic load and 1.0 mL test suspension. In addition, a micro-scale suspension test was performed in 96-well plates with 160 µL product test solution, 20 µL organic load and 20 µL test suspension. S. aureus ATCC 6538, P. aeruginosa ATCC 15442 and C. albicans ATCC 10231 were test organisms. Glutaral was tested at concentrations of 0.05%, 0.1%, 0.2% and 0.3% with exposure times of 1, 5 and 15 min. Polysorbate 80 (30 g/L), lecithin (9 g/L), L-histidine (1 g/L) and glycine (10 g/L) were used as validated neutralizers. After serial dilution of the disinfectant-neutralizer-mixture, plates were incubated for 48 h at 36°C (bacteria) or 72 hours at 30°C (C. albicans) and colony forming units (cfu) counted. The lg reduction was calculated as the difference between the results of the water control and the disinfectant at the end of the exposure time. All experiments were done in triplicate under clean conditions. Means of lg reduction were compared with the unpaired t-test, p<0.05 was considered to be significant. Results: Sufficient bactericidal activity according the VAH test requirements of at least 5 lg was found with both methods in 16 data sets of 24 data sets in total, and insufficient bactericidal activity of less than 5 lg was found with both methods in 7 data sets. In one data set, the mean lg reduction was above 5 lg with the microscale method and <5 lg with the VAH method, with no significant difference between the data sets (p=0.3096; 0.2% glutaral, 1 min, P. aeruginosa). A sufficient yeasticidal activity of at least 4 lg was found with both methods in one data set, an insufficient yeasticidal activity of less than 4 lg was found with both methods in 8 data sets. With one exception, no significant differences were detected between the two methods below the efficacy threshold. Conclusions: The microscale quantitative suspension test proved to provide results similar to those of VAH method 9 when the bactericidal and yeasticidal activity of glutaralwas evaluated, with 32 out of 33 evaluations yielding consistent results in terms of efficacy. Its suitability should be confirmed with additional bacterial species, additional biocidal active substances and in other laboratories.

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.

Environmental Contamination and Persistence of Clostridioides difficile in Hospital Wastewater Systems.

Clostridioides difficile produces an environmentally resistant dormant spore morphotype that infected patients shed to the hospital environment. C. difficile spores persist in clinical reservoirs that are not targeted by hospital routine cleaning protocols. Transmissions and infections from these reservoirs present a hazard to patient safety. This study aimed to assess the impact of patients acutely suffering from C. difficile-associated diarrhea (CDAD) on C. difficile environmental contamination to identify potential reservoirs. Twenty-three hospital rooms accommodating CDAD inpatients with corresponding soiled workrooms of 14 different wards were studied in a German maximum-care hospital. Additionally, four rooms that never accommodated CDAD patients were examined as negative controls. Stagnant water and biofilms from sinks, toilets, and washer disinfector (WD) traps as well as swabs from cleaned bedpans and high-touch surfaces (HTSs) were sampled. For detection, a culture method was used with selective medium. A latex agglutination assay and a Tox A/B enzyme-linked immunosorbent assay were performed with suspect colonies. Stagnant water and biofilms in hospital traps (29%), WDs (34%), and HTSs (37%) were found to be reservoirs for large amounts of C. difficile during the stay of CDAD inpatients that decreased but could persist 13 ± 6 days after their discharge (13%, 14%, and 9.5%, respectively). Control rooms showed none or only slight contamination restricted to WDs. A short-term cleaning strategy was implemented that reduced C. difficile in stagnant water almost entirely. IMPORTANCE Wastewater pipes are microbial ecosystems. The potential risk of infection emanating from the wastewater for individuals is often neglected, since it is perceived to remain in the pipes. However, sewage systems start with siphons and are thus naturally connected to the outside world. Wastewater pathogens do not only flow unidirectionally to wastewater treatment plants but also retrogradely, e.g., through splashing water from siphons to the hospital environment. This study focused on the pathogen C. difficile, which can cause severe and sometimes fatal diarrheas. This study shows how patients suffering from such diarrheas contaminate the hospital environment with C. difficile and that contamination persists in siphon habitats after patient discharge. This might pose a health risk for hospitalized patients afterward. Since this pathogen's spore morphotype is very environmentally resistant and difficult to disinfect, we show a cleaning measure that can almost entirely eliminate C. difficile from siphons.

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.

Inactivation of Polyomavirus SV40 as Surrogate for Human Papillomaviruses by Chemical Disinfectants.

Human papillomaviruses (HPV) are non-enveloped DNA viruses infecting cutaneous and mucosal squamous epithelia. Sexually transmitted HPV-types that are carcinogenic to humans such as HPV16 can induce cervical and other anogenital cancers. Virus transmission through fomites such as inadequately disinfected gynecological equipment is a further potential transmission route. Since HPV cannot be easily grown in cell culture, polyomavirus SV40 has been used as a surrogate virus when testing the virucidal activity of chemical disinfectants. So far, studies that have compared the virucidal activity of different disinfectants against HPV and SV40 are lacking. Here, we evaluated the susceptibility of HPV16 pseudovirus and SV40 to seven active biocidal substances using quantitative suspension tests. Ethanol, glutaraldehyde (GTA), dodecyldipropylentriamin (DPTA), and ortho-phthalaldehydes (OPA) were able to reduce the infectivity of HPV16 pseudovirus >99.99% after 5 min. In contrast, isopropanol, peracetic acid (PAA), and quaternary ammonium compounds with alkylamines (QAC) only led to a slight or no reduction in infectivity. Concerning SV40, only GTA (60 min contact time), PAA, and OPA had virus-inactivating effects. In conclusion, the virucidal activity of three out of seven disinfectants tested was different for HPV16 pseudovirus and SV40. In this study, SV40 was shown to be a reliable surrogate virus for HPV when testing isopropanol-, GTA-, QAC-, and OPA-based disinfectants.

Air filtration as a tool for the reduction of viral aerosols.

For testing the effectiveness of air purification devices in regard to the reduction of virus-containing aerosols, a test method involving test viruses has been lacking until now. The use of bacteriophages (phiX174 phages) is a method to test the efficiency of air purification devices under experimental conditions. Using air purifiers with a HEPA filter H14, a 4.6-6.1 Log reduction of test viruses can be achieved if bacteriophages are directly aerosolised into the air purifier, which corresponds to a reduction of 99.9974-99.9999%. Due to the complexity and individuality of air flow, an experimental approach was used in which all outside influences were minimised. The experimental setup was practical and chosen to project a scenario of direct transmission by an emitting source to a recipient. The experiments were performed with and without the air purifier at a distance of 0.75 m and 1.5 m each. Using the air purifier at a setting of 1000 m3/h, the concentration of the phiX174 phages in the air could be reduced by 2.86 Log (mean value). Nevertheless, the experiments without the air purifier showed a similar reduction rate of 2.61 Log (mean value) after 35 min. The concentration of phiX174 phages in the air could be additionally reduced up to 1 log step (maximum value) by the use of the air purifier in comparison to the experiments without. Distance was shown to be an important factor for risk reduction.

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.

Test methods for surface disinfection: comparison of the Wiperator ASTM standard E2967-15 and the 4-field test EN 16615.

Aim: Two test methods for surface disinfection (phase 2, step 2) - the Wiperator method (ASTM standard E2967-15) and the 4-field test (EN 16615) - were compared using a disinfectant solution based on quaternary ammonium compounds and a ready-to-use alcohol-based wipe. As test organisms, Staphylococcus aureus and Pseudomonas aeruginosa were used. Results: While the 4-field test is a manual method and better reflects the process in practice, with the Wiperator, the wiping process is better controlled because it is an automated procedure. A comparison of the effects of both methods on the target log10-reduction of S. aureus and P. aeruginosa indicates a statistically significant difference between the two test methods (Mann-Whitney U-Test. S. aureus: 0 (Umin)<4 (U crit ); n 1=8, n 2=8, p=0.001; 2-sided. P. aeruginosa: 24 (Umin)<26 (Ucrit); n 1=11, n 2=10, p=0.025, 2-sided). In addition, the results indicate that the wipe used has a major influence on the success of the disinfection process. Discussion: Both methods are suitable for efficacy studies of surface disinfectants, yet they differ in some aspects. Additionally our data indicate a statistically significant difference between the two test methods. Conclusion: Efficiency testing of surface disinfection is a complex process that depends on many different parameters. Since the 4-field test better reflects the practice, it makes sense to stick to this test procedure, taking into account that the EN 16615 was approved by CEN TC 216 in 2015 after method validation ring trials.

Chemical disinfection in healthcare settings: critical aspects for the development of global strategies.

Chemical disinfection is an indispensable means of preventing infection. This holds true for healthcare settings, but also for all other settings where transmission of pathogens poses a potential health risk to humans and/or animals. Research on how to ensure effectiveness of disinfectants and the process of disinfection, as well as on when, how and where to implement disinfection precautions is an ongoing challenge requiring an interdisciplinary team effort. The valuable resources of active substances used for disinfection must be used wisely and their interaction with the target organisms and the environment should be evaluated and monitored closely, if we are to reliable reap the benefits of disinfection in future generations. In view of the global threat of communicable diseases and emerging and re-emerging pathogens and multidrug-resistant pathogens, the relevance of chemical disinfection is continually increasing. Although this consensus paper pinpoints crucial aspects for strategies of chemical disinfection in terms of the properties of disinfectant agents and disinfection practices in a particularly vulnerable group and setting, i.e., patients in healthcare settings, it takes a comprehensive, holistic approach to do justice to the complexity of the topic of disinfection.

The Washing Machine as a Reservoir for Transmission of Extended-Spectrum-Beta-Lactamase (CTX-M-15)-Producing Klebsiella oxytoca ST201 to Newborns.

During the period from April 2012 to May 2013, 13 newborns (1 to 4 weeks of age) and 1 child in a pediatric hospital ward in Germany were colonized with Klebsiella oxytoca producing an extended-spectrum beta-lactamase (ESBL) (CTX-M-15). A microbiological source-tracking analysis with human and environmental samples was carried out to identify the source and transmission pathways of the K. oxytoca clone. In addition, different hygienic intervention methods were evaluated. K. oxytoca isolates were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine that was used in the same ward to wash laundry for the newborns, as well as in two sinks. These strains were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The environmental findings were compared with those for the human strains and the isolates detected on clothing. The results from both techniques showed that the strains were identical (sequence type 201 and PFGE type 00531, a clone specific to this hospital and not previously isolated in Germany), emphasizing the washing machine as a reservoir and fomite for the transmission of these multidrug-resistant bacteria. After the washing machine was taken out of use, no further colonizations were detected during the subsequent 4-year period.IMPORTANCE Washing machines should be further investigated as possible sites for horizontal gene transfer (ESBL genes) and cross-contamination with clinically important Gram-negative strains. Particularly in the health care sector, the knowledge of possible (re-)contamination of laundry (patients' clothes and staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and to control nosocomial infections. This report describes an outbreak with a single strain of a multidrug-resistant bacterium (Klebsiella oxytoca sequence type 201) in a neonatal intensive care unit that was terminated only when the washing machine was removed. In addition, the study implies that changes in washing machine design and processing are required to prevent accumulation of residual water where microbial growth can occur and contaminate clothes.

Antibiotic resistance: What is so special about multidrug-resistant Gram-negative bacteria?

In the past years infections caused by multidrug-resistant Gram-negative bacteria have dramatically increased in all parts of the world. This consensus paper is based on presentations, subsequent discussions and an appraisal of current literature by a panel of international experts invited by the Rudolf Schülke Stiftung, Hamburg. It deals with the epidemiology and the inherent properties of Gram-negative bacteria, elucidating the patterns of the spread of antibiotic resistance, highlighting reservoirs as well as transmission pathways and risk factors for infection, mortality, treatment and prevention options as well as the consequences of their prevalence in livestock. Following a global, One Health approach and based on the evaluation of the existing knowledge about these pathogens, this paper gives recommendations for prevention and infection control measures as well as proposals for various target groups to tackle the threats posed by Gram-negative bacteria and prevent the spread and emergence of new antibiotic resistances.

Portable UV light as an alternative for decontamination.

We evaluated the capability of a commercially available hand-held device that emits ultraviolet (UV) light to disinfect plain surfaces. Eight bacterial species were tested, including Clostridium difficile ribotype 027 and 3 other spore-forming species. Even bacterial spores could be successfully inactivated within a few seconds of irradiation. UV light may provide an alternative for the decontamination of medical products, such as mobile phones or tablet computers, that cannot be treated otherwise.

The role of surface disinfection in infection prevention.

BACKGROUND: The Rudolf Schuelke Foundation addresses topics related to hygiene, infection prevention and public health. In this context a panel of scientists from various European countries discussed "The Role of Surface Disinfection in Infection Prevention". The most important findings and conclusions of this meeting are summarised in the present consensus paper. AIM: Although the relevance of surface disinfection is increasingly being accepted, there are still a number of issues which remain controversial. In particular, the following topics were addressed: Transferral of microbes from surface to patients as a cause of infection, requirements for surface disinfectants, biocidal resistance and toxicity, future challenges. METHODS AND FINDINGS: After discussion and review of current scientific literature the authors agreed that contaminated surfaces contribute to the transmission of pathogens and may thus pose an infection hazard. Targeted surface disinfection based on a risk profile is seen as an indispensable constituent in a multibarrier approach of universal infection control precautions. Resistance and cross-resistance depend on the disinfectant agent as well as on the microbial species. Prudent implementation of surface disinfection regimens tested to be effective can prevent or minimize adverse effects. CONCLUSIONS: Disinfection must be viewed as a holistic process. There is a need for defining standard principles for cleaning and disinfection, for ensuring compliance with these principles by measures such as written standard operating procedures, adequate training and suitable audit systems. Also, test procedures must be set up in order to demonstrate the efficacy of disinfectants including new application methods such as pre-soaked wipes for surface disinfection.

Long-term effects of disinfectants on the community composition of drinking water biofilms.

Numerous investigations have demonstrated efficiencies of different disinfection methods, but until now only little is known about long-term effects on community compositions of drinking water biofilms. Changes in the community structure, especially regrowth of hygienically relevant microorganisms could be critical for the drinking water quality. In this study the long-term effect of disinfection methods on biofilm communities in drinking water systems was analysed. Old drinking water biofilms grown in silicone tubes were exposed to different preparations of disinfectants (free chlorine, chlorine dioxide, hydrogen peroxide combined with fruit acid, silver and silver with peracetic acid, respectively) and subsequently further exposed in the original drinking water. The comparison of the treated and regrown biofilm populations with untreated ones by the DNA-fingerprinting method denaturing gradient gel electrophoresis (DGGE) revealed a considerable population shift caused by the disinfectants. The disinfection methods induced a selection pressure on the biofilm populations depending on the composition and concentrations. The similarities between the treated and untreated biofilms were generally low. Compared to preparations with peracetic acid the disinfection with hydrogen peroxide and silver resulted in higher similarities of the treated and untreated biofilms, but the microbial diversity increased. It can be concluded that the disinfectants have a major impact on the drinking water biofilm communities and that possibly the intervention selects persisters and microorganisms, which can live on the residuals of the dead biofilm cells. For the evaluation of the efficiency of disinfection methods in drinking water installations it is necessary not only to consider reduction of certain bacteria but also to pay attention to the biofilm community.

Testing and listing disinfectants - instrument and product of quality assurance.

In Germany, the Disinfectants Commission of the Association for Applied Hygiene (VAH) ensures that the user can avail of procedures that meet the stipulated quality requirements. These requirements are based on the tried and tested standard methods of the German Society of Hygiene and Microbiology (DGHM) as well as on European standards. They take account of the different requirements dictated by the various fields of application, reflecting the quality assurance system in a transparent manner. Special emphasis is increasingly placed on retesting the products already available on the market. In multi-center trials the inter- and intralaboratory fluctuations in the test results are ascertained, the quest for suitable standard substances intensified, culture conditions and detection procedures are standardized and the test procedures and potential influence factors analyzed in detail. The aim here is to devise test procedures that will yield a reproducible and reliable result independently of the test location, and meet the requirements of everyday practice. Attention must be paid to, inter alia, the prevailing microbial spectrum in quality and quantity, the prevailing organic and inorganic load as well as material and surface properties. The test procedures are gradually brought into line with the current stock of scientific knowledge, because such a task calls for conscientiousness, tenacity and patience as well as continuous dialog between research, industry and practice. Thanks to these joint efforts, we are increasingly better able to meet the demands made by intelligent and selective prophylactic disinfection.