Methods to assess antibacterial, antifungal and antiviral surfaces in relation to touch and droplet transfer: a review, gap-analysis and suggested approaches.

To help assess whether a potentially antimicrobial material, surface, or coating provides antimicrobial efficacy, a number of standardised test methods have been developed internationally. Ideally, these methods should generate data that supports the materials efficacy when deployed in the intended end-use application. These methods can be categorised based on their methodological approach such as suspension tests, agar plate/zone diffusion tests, surface inoculation tests, surface growth tests or surface adhesion tests. To support those interested in antimicrobial coating efficacy, this review brings together an exhaustive list of methods (for porous and non-porous materials), exploring the methodological and environmental parameters used to quantify antibacterial, antifungal, or antiviral activity. This analysis demonstrates that antimicrobial efficacy methods that test either fungi or viruses are generally lacking, whilst methods that test bacteria, fungi and viruses are not designed to simulate end-use/lack realistic conditions. As such, a number of applications for antimicrobial activity across medical touch screens, medical textiles and gloves and transport seat textiles are explored as example applications, providing guidance on modifications to existing methods that may better simulate the intended end-use of antimicrobial materials.

Clarifications on the Intended Use of USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.

In the execution of its legislated responsibilities, the United States Food and Drug Administration commonly refers to standard test methods detailed in the United States Pharmacopeia (USP). Microbiological test methods (contained in general chapters) are listed in chapters <51> to <80> with details regarded as enforceable where referenced as a test method. USP <61> "Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests" is a globally harmonized chapter that has been successfully employed for the enumeration of microorganisms recoverable from nonsterile finished drug products. The content of USP <61> is not always scientifically principled nor emphatically understood by all pharmaceutical microbiologists. Consequently, misunderstanding and misapplication of USP <61> may result in analyses and assessments of microbiological quality that are flawed or erroneous. In this article, clarification is provided to assist the pharmaceutical microbiologist in the appropriate and intended use of USP <61>, including provision of details not always commonly known or understood.

Comparison of Medical Tape Performance Using Skin Response Quantitative Measurements on Healthy Volunteers.

BACKGROUND: Medical tapes can lead to skin damage upon removal in susceptible patients with fragile skin and at higher risk of developing tissue injury. PURPOSE: We compared the effect of medical tapes with silicone-based versus acrylate-based adhesives on the back or volar forearm stratum corneum using analytical techniques to assess skin condition and potential damage post product removal on 88 healthy volunteers. METHODS: Two studies were conducted in separate facilities (Study 1: 3M In-house Clinical Facility, St. Paul, Minnesota; Study 2: DermiCo, LLC, Broomall, Pennsylvania). Four commercially available tapes were the same in both studies, two for each type of adhesive. We evaluated adhesion to the skin, total proteins and corneocytes removed by the tapes, changes in transepidermal water loss (TEWL), and induction of the inflammatory cytokine interleukin-1 alpha (IL-1a). RESULTS: One of the silicone tapes displayed the strongest adhesion at 24 hours, and one of the acrylate tapes had the lowest adhesion, showing differences in performance within adhesive categories. The adhesion forces did not correlate with the amount of total protein or corneocytes removed. Silicone adhesives removed less total protein and corneocytes than acrylate adhesives. Silicone adhesives did not alter TEWL, whereas acrylate adhesives significantly raised TEWL. There were no differences in interleukin-1alpha induction. CONCLUSION: The silicone adhesive tapes were less disruptive to the skin barrier than the acrylate adhesive tapes, even in healthy volunteers whose skin is not as fragile as what is observed in typical patients. This type of data could guide clinical product usage decisions.

The Accomplishments of KoCVAM in the Development and Implementation of Alternative Methods in Korea.

The Korean Center for the Validation of Alternative Methods (KoCVAM), which promotes the Three Rs principles and the use of alternative methods in Korea, has been operating within the Toxicological Screening and Testing Division of the Ministry of Food and Drug Safety (MFDS) since 2009. KoCVAM has exchanged opinions and information on the development and validation of non-animal alternative test methods as part of the International Cooperation on Alternative Test Methods (ICATM), and provided input into draft OECD Test Guidelines (TGs). Several Korean laws (e.g. the Cosmetics Act) encourage the use of alternative test methods for chemical testing and assessment. To promote and support the use of alternative test methods in the country, KoCVAM has published information and provided training on the national guidelines, which are based on the OECD TGs. In addition, KoCVAM has held annual training workshops on alternative test methods, to help Korean research institutions (including GLP test facilities) to implement them. In addition, by helping to develop and validate alternative test methods that were adopted in OECD TG 442B, TG 492 and TG 439, KoCVAM has contributed to the enhanced competitiveness of Korean industry on the worldwide stage.

The frictional energy absorber effectiveness and its impact on the pressure ulcer prevention performance of multilayer dressings.

Pressure ulcers including heel ulcers remain a global healthcare concern. This study comprehensively evaluates the biomechanical effectiveness of the market-popular ALLEVYN® LIFE multilayer dressing in preventing heel ulcers. It focuses on the contribution of the frictional sliding occurring between the non-bonded, fully independent layers of this dressing type when the dressing is protecting the body from friction and shear. The layer-on-layer sliding phenomenon, which this dressing design enables, named here the frictional energy absorber effectiveness (FEAE), absorbs approximately 30%-45% of the mechanical energy resulting from the foot weight, friction and shear acting to distort soft tissues in a supine position, thereby reducing the risk of heel ulcers. Introducing the novel theoretical FEAE formulation, new laboratory methods to quantify the FEAE and a review of relevant clinical studies, this research underlines the importance of the FEAE in protecting the heels of at-risk patients. The work builds on a decade of research published by our group in analysing and evaluating dressing designs for pressure ulcer prevention and will be useful for clinicians, manufacturers, regulators and reimbursing bodies in assessing the effectiveness of dressings indicated or considered for prophylactic use.

The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together the authors of this article around the topic: "From bench to validated test guidelines: (pre)val­idation of test methods". Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facil­itate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, large amounts of funding have been invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential reg­ulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer's laboratory. This article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model among those who benefit from validated methods.

Validation activities for methods intended to be used to assess chemical safety have a cost but also bring substantial benefits when the validated methods are established as OECD Test Guidelines, which results in mutual acceptance of data generated by the methods across OECD member and adhering countries. The article discusses some of the challenges faced when method validation is underfunded and unattractive for researchers. Proposals are made to improve the current situation, gain efficiency, and make validation a shared responsibility.

Retrospective evaluation of the eye irritation potential of agrochemical formulations.

Multiple in vitro eye irritation methods have been developed and adopted as OECD health effects test guidelines. However, for predicting the ocular irritation/damage potential of agrochemical formulations there is an applicability domain knowledge gap for most of the methods. To overcome this gap, a retrospective evaluation of 192 agrochemical formulations with in vivo (OECD TG 405) and in vitro (OECD TG 437, 438, and/or 492) data was conducted to determine if the in vitro methods could accurately assign United Nations Globally Harmonized System for Classification and Labelling of Chemicals (GHS) eye irritation hazard classifications. In addition, for each formulation the eye irritation classification was derived from the classification of the contained hazardous ingredients and their respective concentration in the product using the GHS concentration threshold (CT) approach. The results herein suggest that the three in vitro methods and the GHS CT approach were highly predictive of formulations that would not require GHS classification for eye irritation. Given most agrochemical formulations fall into this category, methods that accurately identify non-classified agrochemical formulations could significantly reduce the use of animals for this endpoint.

Biodegradation of plastics in the pelagic environment of the coastal zone - Proposed test method under controlled laboratory conditions.

The increasing quantities of plastic litter accumulated in the oceans, including microplastics, represent a serious environmental threat. Despite the recent legislative actions, the plastic littering problem will not disappear in a short time. It may, however be ameliorated by replacing conventional non-degradable plastics with bio-based materials biodegradable in marine environment (targeting the non-recycled or mismanaged plastic waste). Although priority is set to prevention of plastic litter by means of the circular economy principles, biodegradability is a means of controlling unintentional plastic pollution. In this effort, the development of reliable test methods that would be used along with standard specifications for determining the biodegradability of novel polymeric materials or plastics in marine environments, is a necessary complementary component of the whole strategy to control the marine plastic litter and micro-, nano-plastics threat. The present work focuses on identifying gaps and improving available laboratory test methods for measuring the aerobic biodegradation of plastics in the seawater column within the coastal zone (pelagic environment). The research work followed a methodology that is based on recommendations of ASTM D6691:2017 concerning biodegradation of plastics in the seawater and the similar ISO 23977-1:2020. Three different implementation schemes of the test method were applied using different experimental setups and measuring techniques for monitoring the evolved CO2. The effect of critical parameters affecting nutrient adequacy (concentration in inoculum) and oxygen adequacy (bioreactor size, sample size, frequency of aeration) on the biodegradation of four tested materials was explored, and optimal values are proposed. The results allowed for the refinement of the proposed test method to improve reliability and reproducibility.

Comparison of female rat reproductive effects of pubertal versus adult exposure to known endocrine disruptors.

A prevailing challenge when testing chemicals for their potential to cause female reproductive toxicity is the lack of appropriate toxicological test methods. We hypothesized that starting a 28-day in vivo toxicity study already at weaning, instead of in adulthood, would increase the sensitivity to detect endocrine disruptors due to the possibility of including assessment of pubertal onset. We compared the sensitivity of two rat studies using pubertal or adult exposure. We exposed the rats to two well-known human endocrine disruptors, the estrogen diethylstilbestrol (DES; 0.003, 0.012, 0.048 mg/kg bw/day) and the steroid synthesis inhibitor ketoconazole (KTZ; 3, 12, 48 mg/kg bw/day). Specifically, we addressed the impact on established endocrine-sensitive endpoints including day of vaginal opening (VO), estrous cyclicity, weights of reproductive organs and ovarian histology. After 28 days of exposure, starting either at weaning or at 9 weeks of age, DES exposure altered estrous cyclicity, reduced ovary weight as well as number of antral follicles and corpora lutea. By starting exposure at weaning, we could detect advanced day of VO in DES-exposed animals despite a lower body weight. Some endpoints were affected mainly with adult exposure, as DES increased liver weights in adulthood only. For KTZ, no effects were seen on time of VO, but adrenal and liver weights were increased in both exposure scenarios, and adult KTZ exposure also stimulated ovarian follicle growth. At first glance, this would indicate that a pubertal exposure scenario would be preferrable as timing of VO may serve as sensitive indicator of endocrine disruption by estrogenic mode of action. However, a higher sensitivity for other endocrine targets may be seen starting exposure in adulthood. Overall, starting a 28-day study at weaning with inclusion of VO assessment would mainly be recommended for substances showing estrogenic potential e.g., in vitro, whereas for other substances an adult exposure scenario may be recommended.

Perkinsus olseni in green-lipped mussels Perna canaliculus: diagnostic evaluation, prevalence, and distribution.

Perkinsus olseni (Perkinsidae) is a molluscan parasite notifiable to the World Organisation for Animal Health that is reported in several shellfish hosts in New Zealand, including the native green-lipped mussel Perna canaliculus. Green-lipped mussels comprise over half of New Zealand's aquaculture export value and have historically been considered free of serious diseases based on extensive histology-based surveillance. The discovery of P. olseni in green-lipped mussels has raised questions about future disease threats to green-lipped mussels, particularly under changing ocean climatic conditions. Using mussels collected from farmed (n = 358) and wild (n = 236) populations, we aimed to determine the distribution and prevalence of P. olseni in green-lipped mussels around New Zealand, and assess the performance of diagnostic tests, including real-time PCR, conventional PCR, and culture using Ray's fluid thioglycolate medium (RFTM). Prevalence and diagnostic test performance was evaluated using Bayesian latent class analysis with informative priors. The prevalence of P. olseni was 0-3%, except for 1 wild population from a harbour where prevalence was 22%. Real-time PCR had the highest diagnostic sensitivity (87%) compared to 62 and 21% for conventional PCR and RFTM, respectively. Diagnostic specificity was similar among all methods (96-98%). No mortality was observed during the study. Our results suggest that real-time PCR is the diagnostic test best suited for surveillance of P. olseni in subclinically infected green-lipped mussels under New Zealand conditions.

A roadmap to strengthen standardisation efforts in risk governance of nanotechnology.

A roadmap was developed to strengthen standardisation activities for risk governance of nanotechnology. Its baseline is the available standardised and harmonised methods for nanotechnology developed by the International Organization for Standardization (ISO), the European Committee for Standardization (CEN), and the Organisation for Economic Co-operation and Development (OECD). In order to identify improvements and needs for new themes in standardisation work, an analysis of the state-of-the-art concepts and interpretations of risk governance of nanotechnology was performed. Eleven overall areas of action were identified, each including a subset of specific topics. Themes addressed include physical chemical characterisation, assessment of hazard, exposure, risk and socio-economic factors, as well as education & training and social dialogue. This has been visualised in a standardisation roadmap spanning a timeframe of ten years and including key outcomes and highlights of the analysis. Furthermore, the roadmap indicates potential areas of action for harmonisation and standardisation (H&S) for nanomaterials and nanotechnology. It also includes an evaluation of the current level (limited, moderate, intense) of ongoing H&S activities and indicates the time horizon for the different areas of action. As the identified areas differ in their state of development, the number and type of actions varied widely amongst the different actions towards achieving standardisation. Thus, priority areas were also identified. The overall objective of these actions is to strengthen risk governance towards a safe use of nanomaterials and nano-related products. Though not explicitly addressed, risk-based legislation and policies are supported via the proposed H&S actions.

Uncertainty of Standardized Track Insulation Measurement Methods for Stray Current Assessment.

Stray current is a relevant phenomenon in particular for DC electrified transportation systems, affecting track and infrastructure within the right of way and other structures and installations nearby. It worsens with time and the level of protection depends on timely maintenance, as well as correct design choices. The assessment of track insulation is the starting point for both stray current monitoring systems and at commissioning or upon major changes. Standardized methods (ref. EN 50122-2 or IEC 62128-2) have been almost unchanged in the last 20 years but suffer from accuracy issues and variability due to parameters and conditions not under the operator's control. The uncertainty of test methods is increasingly important now that contractual specifications require a high level of insulation for new systems. A critical discussion and analysis of the sources of variability and practical constraints is proposed, followed by an evaluation of uncertainty, with the objective not only to assess the accuracy of the provided results, but also to foster research on innovative, more flexible and accurate methods.

Russell and Burch's 3Rs then and now: The case of Switzerland.

Since Russell and Burch introduced and defined the 3Rs, i.e., the replacement, reduction, and refinement of animal use in research, in 1959, different definitions have emerged and been implemented in guidelines and policies. Switzerland is known for having some of the most restrictive legislation regarding the use of animals, in which the 3Rs are also defined and implemented. To our knowledge, the purpose and definitions of the 3Rs used in the Swiss Animal Welfare Act, Animal Protection Ordinance, and Animal Experimentation Ordinance have never been compared with Russell and Burch's original purpose and definitions. In this paper we make this comparison with two aims: to reveal ethically relevant departures from the original purpose and definitions, and to provide an ethical evaluation of the current Swiss law regarding the 3Rs. In doing so, we first expose the similarity of purposes. We then identify one risky departure from the original definition of replacement in Swiss law, which shows a problematic focus on species. Finally, we address Swiss law's failure to apply the 3Rs in the most effective way. With respect to this last point, we discuss the need for 3R conflict resolution, the timing of application of the 3Rs, problematic prioritizations and choices of convenience as well as a solution to apply the 3Rs more effectively using Russell and Burch's concept of total sum of distress.

Off to a good start? Review of the predictivity of reactivity methods modelling the molecular initiating event of skin sensitization.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods' predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.

Improving the design and conduct of aquatic toxicity studies with oils based on 20 years of CROSERF experience.

Laboratory toxicity testing is a key tool used in oil spill science, spill effects assessment, and mitigation strategy decisions to minimize environmental impacts. A major consideration in oil toxicity testing is how to replicate real-world spill conditions, oil types, weathering states, receptor organisms, and modifying environmental factors under laboratory conditions. Oils and petroleum-derived products are comprised of thousands of compounds with different physicochemical and toxicological properties, and this leads to challenges in conducting and interpreting oil toxicity studies. Experimental methods used to mix oils with aqueous test media have been shown to influence the aqueous-phase hydrocarbon composition and concentrations, hydrocarbon phase distribution (i.e., dissolved phase versus in oil droplets), and the stability of oil:water solutions which, in turn, influence the bioavailability and toxicity of the oil containing media. Studies have shown that differences in experimental methods can lead to divergent test results. Therefore, it is imperative to standardize the methods used to prepare oil:water solutions in order to improve the realism and comparability of laboratory tests. The CROSERF methodology, originally published in 2005, was developed as a standardized method to prepare oil:water solutions for testing and evaluating dispersants and dispersed oil. However, it was found equally applicable for use in testing oil-derived petroleum substances. The goals of the current effort were to: (1) build upon two decades of experience to update existing CROSERF guidance for conducting aquatic toxicity tests and (2) to improve the design of laboratory toxicity studies for use in hazard evaluation and development of quantitative effects models that can then be applied in spill assessment. Key experimental design considerations discussed include species selection (standard vs field collected), test substance (single compound vs whole oil), exposure regime (static vs flow-through) and duration, exposure metrics, toxicity endpoints, and quality assurance and control.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

OECD workshop consensus report: Ethical considerations with respect to human derived products, specifically human serum, in OECD test guidelines.

The ethical needs and concerns with use and sourcing of human materials, particularly serum, in OECD in vitro test guidelines were explored in a dedicated international workshop held in 2019. The health-related aspects of the donation procedure, including tissue screening, donor health, laboratory work health protection, permission from the donor for commercial use, payment of the donors and the potential for exploitation of low-income populations and data protection of the donors; supply, availability, and competition with clinical needs; traceability of the serum and auditability/GLP needs for the Test Guideline Programme, were examined. Here we provide the recommendations of the workshop with respect to the use of human serum, and potentially other human reagents, specifically with regard to test method development for OECD Test Guideline utility as part of the Mutual Acceptance of Data requirement across all OECD member countries. These include informed donor consent terminology, a checklist of human serum information requirements to be included with the Good Laboratory Practise report, and suitable sources for human serum to ensure waste supplies are used, that can no longer be used for medical purposes, ensuring no competition of supply for essential medical use.

Current testing programs for pesticides adequately capture endocrine activity and adversity for protection of vertebrate wildlife.

The toxicity and ecotoxicity of pesticide active ingredients are evaluated by a number of standardized test methods using vertebrate animals. These standard test methods are required under various regulatory programs for the registration of pesticides. Over the past two decades, additional test methods have been developed with endpoints that are responsive to endocrine activity and subsequent adverse effects. This article examines the available test methods and their endpoints that are relevant to an assessment of endocrine-disrupting properties of pesticides. Furthermore, the article highlights how weight-of-evidence approaches should be applied to determine whether an adverse response in (eco)toxicity tests is caused by an endocrine mechanism of action. The large number of endpoints in the current testing paradigms for pesticides make it unlikely that endocrine activity and adversity is being overlooked. Integr Environ Assess Manag 2023;19:1089-1109. © 2023 Bayer CropScience and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Bioremediation of microplastics in freshwater environments: A systematic review of biofilm culture, degradation mechanisms, and analytical methods.

Microplastics, defined as particles <5 mm in diameter, are emerging environmental pollutants that pose a threat to ecosystems and human health. Biofilm degradation of microplastics may be an ecologically friendly approach. This review systematically summarises the factors affecting biofilm degradation of microplastics and proposes feasible methods to improve the efficiency of microplastic biofilm degradation. Environmentally insensitive microorganisms were screened, optimized, and commercially cultured to facilitate the practical application of this technology. For strain screening, technology should focus on microorganisms/strains that can modify the hydrophobicity of microplastics, degrade the crystalline zone of microplastics, and metabolise additives in microplastics. The biodegradation mechanism is also described; microorganisms secreting extracellular oxidases and hydrolases are key factors for degradation. Measuring the changes in molecular weight distribution (MWD) enables better analysis of the biodegradation behaviour of microplastics. Biofilm degradation of microplastics has relatively few applications because of its low efficiency; however, enrichment of microplastics in freshwater environments and wastewater treatment plant tailwater is currently the most effective method for treating microplastics with biofilms.

India-discovered levonadifloxacin & alalevonadifloxacin: A review on susceptibility testing methods, CLSI quality control and breakpoints along with a brief account of their emerging therapeutic profile as a novel standard-of-care.

BACKGROUND: Levonadifloxacin (intravenous) and alalevonadifloxacin (oral prodrug) are novel antibiotics based on benzoquinolizine subclass of fluoroquinolone, licensed for clinical use in India in 2019. The active moiety, levonadifloxacin, is a broad-spectrum antibiotic with a high potency against methicillin-resistant Staphylococcus. aureus, multi-drug resistant pneumococci and anaerobes. OBJECTIVE: This review, for the first time, critically analyses the antimicrobial susceptibility testing methods, Clinical Laboratory & Standards Institute (CLSI)-quality control of susceptibility testing and breakpoints of levonadifloxacin. Further, the genesis, discovery and developmental aspects as well as therapeutic profile of levonadifloxacin and alalevonadifloxacin are briefly described. CONTENTS: In order to aid the scientific and clinician communities with a single comprehensive overview on all the key aspects of levonadifloxacin and alalevonadifloxacin, the present article covers the reference MIC and disk diffusion methods for levonadifloxacin susceptibility testing that were approved by CLSI and the reference ranges for quality control strains published in the CLSI M100 document. The breakpoints of levonadifloxacin were derived in concordance to US FDA, European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI approaches. Further, the article provides a brief account of challenges encountered during the discovery stages of levonadifloxacin and alalevonadifloxacin, activity spectrum and safety benefits accruing from structural novelty-linked mechanism of action. Further, the review also covers in vitro and in vivo activities, registrational clinical studies and patient-friendly features of levonadifloxacin/alalevonadifloxacin. Cumulatively, levonadifloxacin has a potential to offer a long awaited new standard-of-care treatment for the resistant Gram-positive bacterial infections.