Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues.

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

Detection of Ebola Virus RNA Through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates.

Ebola virus disease is a serious illness of humans and nonhuman primates (NHPs). Direct contact has been shown to be the primary source of Ebola (EBOV) transmission. We used a high-volume air sampler to determine whether EBOV could be detected during 3 independent studies with EBOV-challenged NHPs. Viral RNA was recovered during days 9 and 10 of Study I and days 7 and 8 of Study III. Viral RNA levels were below limits of detection during all other collections. The results demonstrate that the biosafety level 4 (BSL-4) suit protects workers from aerosols in a BSL-4 environment using proper engineering and administrative controls.

Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass spectrometry.

Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current method expands upon this by quantifying eight UGT1A isoforms by nanobore high-performance liquid chromatography (HPLC) coupled with a linear ion trap time-of-flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney, and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8, and 1A10 were below detection limits (<1 pmol/mg protein) in human liver microsome (HLMs). In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9, and 1A10 protein were higher relative to that of liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.

Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans.

Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies conducted entirely in humans. We present a strategy to identify genetic polymorphisms that may underlie susceptibility to adverse drug reactions. First, in a cohort of healthy adults who received the maximum recommended dose of acetaminophen (4 g/d x 7 d), we confirm that about one third of subjects develop elevations in serum alanine aminotransferase, indicative of liver injury. To identify the genetic basis for this susceptibility, a panel of 36 inbred mouse strains was used to model genetic diversity. Mice were treated with 300 mg/kg or a range of additional acetaminophen doses, and the extent of liver injury was quantified. We then employed whole-genome association analysis and targeted sequencing to determine that polymorphisms in Ly86, Cd44, Cd59a, and Capn8 correlate strongly with liver injury and demonstrated that dose-curves vary with background. Finally, we demonstrated that variation in the orthologous human gene, CD44, is associated with susceptibility to acetaminophen in two independent cohorts. Our results indicate a role for CD44 in modulation of susceptibility to acetaminophen hepatotoxicity. These studies demonstrate that a diverse mouse population can be used to understand and predict adverse toxicity in heterogeneous human populations through guided resequencing.

Biological Validation of a Chemical Effluent Decontamination System.

Introduction: Failure of an existing effluent decontamination system (EDS) prompted the consideration of commercial off-the-shelf solutions for decontamination of containment laboratory waste. A bleach-based chemical EDS was purchased to serve as an interim solution. Methods: Studies were conducted in the laboratory to validate inactivation of Bacillus spores with bleach in complex matrices containing organic simulants including fetal bovine serum, humic acid, and animal room sanitation effluent. Results: These studies demonstrated effective decontamination of >106 spores at a free chlorine concentration of ≥5700 parts per million with a 2-hour contact time. Translation of these results to biological validation of the bleach-based chemical EDS required some modifications to the system and its operation. Discussion: The chemical EDS was validated for the treatment of biosafety levels 3 and 4 waste effluent using laboratory-prepared spore packets along with commercial biological indicators; however, several issues and lessons learned identified during the process of onboarding are also discussed, including bleach product source, method of validation, dechlorination, and treated waste disposal.

Stability of SARS-CoV-2 on Produce following a Low-Dose Aerosol Exposure.

We modeled the stability of SARS-CoV-2 on apples, tomatoes, and jalapeño peppers at two temperatures following a low-dose aerosol exposure designed to simulate an airborne transmission event involving droplet nuclei. Infectious virus was not recovered postexposure.

Modeling the Stability of SARS-CoV-2 on Personal Protective Equipment (PPE).

We modeled the stability of SARS-CoV-2 on personal protective equipment (PPE) commonly worn in hospitals when carrying out high-risk airway procedures. Evaluated PPE included the visors and hoods of two brands of commercially available powered air purifying respirators, a disposable face shield, and Tyvek coveralls. Following an exposure to 4.3 log10 plaque-forming units (PFUs) of SARS-CoV-2, all materials displayed a reduction in titer of > 4.2 log10 by 72 hours postexposure, with detectable titers at 72 hours varying by material (1.1-2.3 log10 PFU/mL). Our results highlight the need for proper doffing and disinfection of PPE, or disposal, to reduce the risk of SARS-CoV-2 contact or fomite transmission.

Modeling the stability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on skin, currency, and clothing.

A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. The extent and efficiency of SARS-CoV-2 pandemic is far greater than previous coronaviruses that emerged in the 21st Century. Here, we modeled stability of SARS-CoV-2 on skin, paper currency, and clothing to determine if these surfaces may factor in the fomite transmission dynamics of SARS-CoV-2. Skin, currency, and clothing samples were exposed to SARS-CoV-2 under laboratory conditions and incubated at three different temperatures (4°C± 2°C, 22°C± 2°C, and 37°C ± 2°C). We evaluated stability at 0 hours (h), 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure. SARS-CoV-2 was stable on skin through the duration of the experiment at 4°C (14 days). Virus remained stable on skin for at least 96 h at 22°C and for at least 8h at 37°C. There were minimal differences between the tested currency samples. The virus remained stable on the $1 U.S.A. Bank Note for at least 96 h at 4°C while we did not detect viable virus on the $20 U.S.A. Bank Note samples beyond 72 h. The virus remained stable on both Bank Notes for at least 8 h at 22°C and 4 h at 37°C. Clothing samples were similar in stability to the currency. Viable virus remained for at least 96 h at 4°C and at least 4 h at 22°C. We did not detect viable virus on clothing samples at 37°C after initial exposure. This study confirms the inverse relationship between virus stability and temperature. Furthermore, virus stability on skin demonstrates the need for continued hand hygiene practices to minimize fomite transmission both in the general population as well as in workplaces where close contact is common.

Inactivation of West Nile virus in serum with heat, ionic detergent, and reducing agent for proteomic applications.

Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.

Absolute quantification of human uridine-diphosphate glucuronosyl transferase (UGT) enzyme isoforms 1A1 and 1A6 by tandem LC-MS.

UGT enzymes catalyze the formation of glucuronic acid conjugates. Specifically selected representative stable isotope (C(13), N(15)) labeled peptide internal standards of each enzyme were employed to quantify UGTs 1A1 and 1A6 by LC-MS/MS using isotope dilution techniques. Inter day variability (n=5) for human liver microsomes was