Resilience and Protection of Health Care and Research Laboratory Workers During the SARS-CoV-2 Pandemic: Analysis and Case Study From an Austrian High Security Laboratory

The SARS-CoV-2 pandemic has highlighted the interdependency of healthcare systems and research organizations on manufacturers and suppliers of personnel protective equipment (PPE) and the need for well-trained personnel who can react quickly to changing working conditions. Reports on challenges faced by research laboratory workers (RLWs) are rare in contrast to the lived experience of hospital health care workers. We report on experiences gained by RLWs (e.g., molecular scientists, pathologists, autopsy assistants) who significantly contributed to combating the pandemic under particularly challenging conditions due to increased workload, sickness and interrupted PPE supply chains. RLWs perform a broad spectrum of work with SARS-CoV-2 such as autopsies, establishment of virus cultures and infection models, development and verification of diagnostics, performance of virus inactivation assays to investigate various antiviral agents including vaccines and evaluation of decontamination technologies in high containment biological laboratories (HCBL). Performance of autopsies and laboratory work increased substantially during the pandemic and thus led to highly demanding working conditions with working shifts of more than eight hours working in PPE that stressed individual limits and also the ergonomic and safety limits of PPE. We provide detailed insights into the challenges of the stressful daily laboratory routine since the pandemic began, lessons learned, and suggest solutions for better safety based on a case study of a newly established HCBL (i.e., BSL-3 laboratory) designed for autopsies and research laboratory work. Reduced personal risk, increased resilience, and stress resistance can be achieved by improved PPE components, better training, redundant safety measures, inculcating a culture of safety, and excellent teamwor

Limiting factors for wearing personal protective equipment (PPE) in a health care environment evaluated in a randomised study.

Pandemics and re-emerging diseases put pressure on the health care system to prepare for patient care and sample logistics requiring enhanced personnel protective equipment (PPE) for health care workers. We generated quantifiable data on ergonomics of PPE applicable in a health care setting by defining error rates and physically limiting factors due to PPE-induced restrictions. Nineteen study volunteers tested randomly allocated head- or full body-ventilated PPE suits equipped with powered-air-purifying-respirators and performed four different tasks (two laboratory tutorials, a timed test of selective attention and a test investigating reaction time, mobility, speed and physical exercise) during 6 working hours at 22°C on one day and 4 working hours at 28°C on another day. Error rates and physical parameters (fluid loss, body temperature, heart rate) were determined and ergonomic-related parameters were assessed hourly using assessment sheets. Depending on the PPE system the most restrictive factors, which however had no negative impact on performance (speed and error rate), were: reduced dexterity due to multiple glove layers, impaired visibility by flexible face shields and back pain related to the respirator of the fully ventilated suit. Heat stress and liquid loss were perceived as restrictive at a working temperature of 28°C but not 22°C

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

BACKGROUND:Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. METHODS:Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. RESULTS:All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. CONCLUSION:PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment

Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, such as storage conditions and duration. It has been discussed that the improper dehydration of tissue during processing influences the quality of NAs and the shelf life of fixed tissue. Here, we report on establishing a method for determining the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin or a non-crosslinking fixative) and its correlation to the performance of NAs in quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The amount of residual water depended primarily on the fixative type and the dehydration protocol and, to a lesser extent, on storage conditions and time. Moreover, we found that these parameters were associated with the qRT-PCR performance of extracted NAs. Besides the cross-linking of NAs and the modification of nucleobases by formalin, the hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research but must also be taken into account in clinical diagnostics where the reanalysis of archived tissue from a primary tumor may be required (e.g., after disease recurrence). We conclude that improving the shelf life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative influence of residual water on NAs

Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.

<p>Human relevant fungi cultivated, treated with PAXgene and formalin and investigated for viability.</p

Investigated strains cover the spectrum of bacterial life conditions.

<p>Specific media were used for bacteria cultivation before and after inactivation treatment.</p

Reverse transcription qPCR: comparison of Cq values of PAXgene and formalin-fixed CMV samples.

<p>RT-qPCR sensitivity assay was performed to detect CMV early-immediate gene <i>TRS1</i> and reference gene <i>GAPDH</i> after fixation of CMV infected MRC-5 cells with PAXgene (<i>TRS1</i>_PF, <i>GAPDH</i>_PF), formalin (<i>TRS1</i>_FF, <i>GAPDH</i>_FF) and not fixed control samples (<i>TRS1</i>_PBS; <i>GAPDH</i>_PBS) in triple biological samples. Low Cq values indicate early detection. Statistical significance p < 0.0001 (***) or p < 0.03 (*).</p

a,b: Results of inactivation experiments of human-relevant fungi by two hours fixation with PBS as positive control, PAXgene Fix, PAXgene Fix and Stab, and formalin.

<p>At least two assays per species (1–3 experiments) were performed. a) Cfu/mL was normalised to 10⁵. The dashed line indicates the threshold for minimum of reduction of 10⁴ used for disinfectants for fungi. b) Bold printed numbers indicate minimal growth after inactivation.</p

a,b,c,d,e,f,g: Results of bacterial inactivation experiments after treatment with PAXgene and formalin.

<p>Bacterial strains show variable viability after treatment with PAXgene Fix, PAXgene Fix and Stab compared to formalin and PBS (viability control) for 30 minutes (a, b, c, e, f, g) and 2 hours (d). The x-axis indicates the amount of experiments, the y-axis cfu/ml. To obtain comparable results all counted cfus were normalised to 10⁶. Columns represent the mean values (and error bars) calculated from the results of a series of different dilutions for one experiment. Dashed lines indicate the reduction limit of 10⁵.</p

Work flow of bacterial and fungal inactivation experiments.

<p>Work flow of bacterial and fungal experiments. Bacteria experiments were performed with four (<i>Pa</i>) and six (<i>Ms</i>) independent experiments, respectively, when no colony was detected after inactivation, six experiments if colonies were detected (<i>Bs</i>, <i>Sa and Mt</i>) and seven experiments with the most variable strain <i>Cs</i>. Two-hour-treatments were performed with fungi (two experiments and all strains listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151383#pone.0151383.t001" target="_blank">Table 1</a>) and additionally with <i>Cs</i> (three experiments).</p