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NASA NTRS2022

Dermatitis during Spaceflight Associated with HSV-1 Reactivation

Human alpha herpesviruses herpes simplex virus (HSV-1) and varicella zoster virus (VZV) establish latency in various cranial nerve ganglia and often reactivate in response to stress-associated immune system dysregulation. Reactivation of Epstein Barr virus (EBV), VZV, HSV-1, and cytomegalovirus (CMV) is typically asymptomatic during spaceflight, though live/infectious virus has been recovered and the shedding rate increases with mission duration. The risk of clinical disease, therefore, may increase for astronauts assigned to extended missions (>180 days). Here, we report, for the first time, a case of HSV-1 skin rash (dermatitis) occurring during long-duration spaceflight. The astronaut reported persistent dermatitis during flight, which was treated onboard with oral antihistamines and topical/oral steroids. No HSV-1 DNA was detected in 6-month pre-mission saliva samples, but on flight day 82, a saliva and rash swab both yielded 4.8 copies/ng DNA and 5.3 × 104 copies/ng DNA, respectively. Post-mission saliva samples continued to have a high infectious HSV-1 load (1.67 × 107 copies/ng DNA). HSV-1 from both rash and saliva samples had 99.9% genotype homology. Additional physiological monitoring, including stress biomarkers (cortisol, dehydroepiandrosterone (DHEA), and salivary amylase), immune markers (adaptive regulatory and inflammatory plasma cytokines), and biochemical profile markers, including vitamin/mineral status and bone metabolism, are also presented for this case. These data highlight an atypical presentation of HSV-1 during spaceflight and underscore the importance of viral screening during clinical evaluations of in-flight dermatitis to determine viral etiology and guide treatment.

Satish K. Mehta↗
NASA NTRSDate not supplied

The BioMole Facility: Advancement of In Situ Microbiome Analysis for the International Space Station

Characterization of the International Space Station (ISS) microbiome has been enabled by sample return and Earth-based analysis. As human exploration pushes beyond low-Earth orbit, microbial-related crew health, planetary protection, and space research requires in situ capabilities. Steps toward reducing Earth-dependence for complex sample analysis began in 2016 with the amplification of DNA within the miniPCR thermal cycler and DNA sequencing with the MinION sequencer onboard the ISS; for both, samples were prepared on Earth. In 2017, these platforms synergistically enabled the in-situ identification of unknown bacteria collected and cultured from ISS surfaces, thereby shifting the paradigm that microbial cultures had to be returned to Earth. The following year, a culture-independent, swab-to-sequencer method further advanced spaceflight microbiology, demonstrating that culturing could be excluded and provided enhanced insight into the bacterial profile of ISS surfaces. Based on the success of these payloads in confirming the ability to meet crew health identification requirements and the benefits accompanying a culture-independent method, the BioMole Facility was established by the medical operations Crew Health Care Systems team. BioMole is the set of hardware, consumables, and procedures required to support sample preparation and nanopore sequencing onboard the ISS. BioMole goals include expanding sample sources, comparing data to previous methods, demonstrating onboard data analytics, and validating new hardware. To date, comparative surface analysis, molecular- and culture-based, has been completed. Additionally, the demonstration of a sample-to-answer process was achieved when BioMole data was processed onboard using the IBM Open Data and AI Edge software platform installed on the ISS-residing Spaceborne Computer-2. The taxonomic profiles generated from the edge analysis were as expected and paralleled that of the downlinked processed data. Future BioMole efforts involve microbial profiling of the ISS water system, ISS validation of the MinION Mk1C, and an expansion to a research facility available to investigators.

Sarah L. Castro-Wallace↗
NASA NTRSDate not supplied

Dermatitis During Spaceflight Associated With HSV-1 Reactivation- A Case Study

Human alpha herpesviruses herpes simplex virus (HSV-1 or -2) and varicella zoster virus (VZV) establish latency in various cranial nerve ganglia, and often reactivate in response to stress-associated immune system dysregulation. Reactivation of Epstein Barr Virus (EBV), VZV, HSV-1 and Cytomegalovirus (CMV) is typically asymptomatic during spaceflight, though live/infectious virus has been recovered and the shedding rate increases with mission duration. The risk of clinical disease, therefore, may increase for astronauts assigned to extended missions (>180 days). Here, we report for the first time, a case of HSV-1 skin rash (dermatitis) occurring during a long duration spaceflight. The astronaut reported persistent dermatitis during flight, which was treated onboard with oral antihistamines and topical/oral steroids. No HSV-1 DNA was detected in 6-month pre-mission saliva samples, but on flight day 82, a saliva and rash swab both yielded 4.8 copies/ng DNA and 5.3×104 copies/ng DNA, respectively. Post-mission saliva samples continued to have high infectious HSV-1 load (1.67×107 copies/ng DNA). HSV-1 from both rash and saliva samples had 99.4% genotype homology. Additional physiological monitoring, including stress biomarkers (cortisol, dehydroepiandrosterone (DHEA), and salivary amylase), immune markers (adaptive regulatory and inflammatory plasma cytokines) and biochemical profile markers including vitamin/mineral status and bone metabolism are also presented for this case. These data highlight an atypical presentation of HSV-1 during spaceflight and underscore the importance of viral screening during clinical evaluations of in-flight dermatitis, to determine viral etiology and guide treatment.

Herpes, Viral Reactivation, Spaceflight, Dermatiti↗
NASA NTRS2022

Unlocking the Spacecraft and Human Habitat Microbiome to Enable the Next Generation of Space Exploration

Planetary protection is the discipline that prevents harmful contamination of the solar system during exploration activities. The current international guidelines and NASA policy addressing biological contamination on spacecraft surfaces contains prescriptive guidelines of spore requirements (e.g., 300 spores/m2, 5×105 spores per spacecraft) applicable to spacecraft bound for Mars. To verify these requirements spacecraft engineers sample spacecraft surfaces throughout the assembly, test and launch operations phase of the mission using damp water cotton swabs and polyester wipes. After sampling, the potential biological contamination is enumerated using a series of traditional microbiology techniques to include sonication, heat shocking at 80°C for 15min to select for spores, and growth on tryptic soy agar at 32°C for 72 hours. To enable crewed missions to Mars and robotic exploration of the Ocean Worlds a risk informed decision making / performance-based approach to assess biological contamination offers a promising solution in the trade space. Recognizing the need for a performance-based approach, NASA’s new Planetary Protection policies now incorporate the agility for missions to be able to leverage a performance or prescriptive approach. One of top contenders in the option space is a coupled quantitative, descriptive and functional based approach to be able to assess the quantity, types and capabilities of the biological contamination present on spacecraft surfaces. A tailored, mission by mission assurance case could then be formulated by building an argument around the target body, projected capabilities surrounding the types of organisms their potential for survival and proliferation, and ability to be transported on the target body to contaminate an area of biological interest. A performance-based requirement would then be used to demonstrate the mission’s compliance in protecting the planetary environment safety objectives. This symposium talk will showcase the background and need case for NASA to develop such a capability as well as provide an update on the efforts underway in developing a transparent and responsible performance-based approach to biological contamination assessments on spacecraft surfaces.

Habitat Microbiome↗
NASA NTRSDate not supplied

Nanopore Sequencing in Space: The Advancement of In Situ Microbiome Analysis for the International Space Station and Beyond

Introduction: Routine assessment of the International Space Station (ISS) microbiome has been performed through in-flight culture and Earth-based analysis. Through utilization of the ISS, significant progress toward in situ microbial identification has been achieved. In 2016, the molecular space age began when DNA-based, Earth-prepared samples were amplified within miniPCR (Genes in Space-1), and subsequent samples were sequenced with the MinION (Biomolecule Sequencer). The following year, these platforms were used collectively to yield the first off-Earth identification of unknown bacteria collected and cultured onboard the ISS (Genes in Space-3). Further advancement occurred in 2018 when a culture-independent, direct swab-to-sequencer method revealed a more thorough depiction of the ISS surface microbiome (BEST). As NASA looks towards Artemis and extended exploration missions, it is critical to continue to harness the ISS to expand in situ nanopore sequencing-based analysis in support of microbial-related crew health, planetary protection, and space research initiatives.

Christian G. Mena↗
NASA NTRSDate not supplied

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗
NASA NTRS2024

Herpesviruses in Saliva and Their Clinical Significance

Saliva has been used as a source of biological markers for a wide spectrum of normal and disease states for a long time. It is a non-invasive, easily accessible, and self-collected body fluid that contains a variety of measurable biological substances. While mostly water, saliva also contains ions, carbohydrates, proteins and peptides, exfoliated cells, nucleic acids, and microorganisms. Saliva can reflect tissue levels of some natural substances and a large variety of molecules introduced for therapeutic use, emotional status; hormonal status, immunological status, neurological effects, and nutritional and metabolic status. It can also be used to monitor a variety of drugs including marijuana, cocaine, and alcohol. It is the most cost-effective approach for screening large population in community mass screening programs and for longitudinal sampling of hospitalized individuals aimed at monitoring viral load dynamics and treatment response. During the COVID-19 pandemic, scientific evidence emerged indicating that molecular tests performed on saliva have diagnostic sensitivity and specificity comparable to those observed with nasopharyngeal swabs for SARS-CoV-2 RNA detection. The presence of IgA and IgG antibodies at the mucosal level has been demonstrated to influence the progression of viral infection and the severity of clinical manifestation. As saliva contains both respiratory secretions and immunological components, it has wide applications, ranging from clinical diagnostics to post-vaccine disease burden and immunity surveillance.

Douglass Diak↗
NASA NTRS1992

Habitat automation

A habitat, on either the surface of the Moon or Mars, will be designed and built with the proven technologies of that day. These technologies will be mature and readily available to the habitat designer. We believe an acceleration of the normal pace of automation would allow a habitat to be safer and more easily maintained than would be the case otherwise. This document examines the operation of a habitat and describes elements of that operation which may benefit from an increased use of automation. Research topics within the automation realm are then defined and discussed with respect to the role they can have in the design of the habitat. Problems associated with the integration of advanced technologies into real-world projects at NASA are also addressed.

Swab, Rodney E.↗
NASA NTRS2017

Microstructure Based Material-Sand Particulate Interactions and Assessment of Coatings for High Temperature Turbine Blades

Gas turbine engines for military/commercial fixed-wing and rotary wing aircraft use thermal barrier coatings in the high-temperature sections of the engine for improved efficiency and power. The desire to further make improvements in gas turbine engine efficiency and high power-density is driving the research and development of thermal barrier coatings and the effort of improving their tolerance to fine foreign particulates that may be contained in the intake air. Both commercial and military aircraft engines often are required to operate over sandy regions such as in the Middle-East nations, as well as over volcanic zones. For rotorcraft gas turbine engines, the sand ingestion is adverse during take-off, hovering near ground, and landing conditions. Although, most of the rotorcraft gas turbine engines are fitted with inlet particle separators, they are not 100 percent efficient in filtering fine sand particles of size 75 microns or below. The presence of these fine solid particles in the working fluid medium has an adverse effect on the durability of turbine blade thermal barrier coatings and overall performance of the engine. Typical turbine blade damages include blade coating wear, sand glazing, Calcia-Magnesia-Alumina-Silicate (CMAS) attack, oxidation, plugged cooling holes, all of which can cause rapid performance deterioration including loss of aircraft. The objective of this research is to understand the fine particle interactions with typical ceramic coatings of turbine blades at the microstructure level. A finite-element based microstructure modeling and analysis has been performed to investigate particle-surface interactions, and restitution characteristics. Experimentally, a set of tailored thermal barrier coatings and surface treatments were down-selected through hot burner rig tests and then applied to first stage nozzle vanes of the Gas Generator Turbine of a typical rotorcraft gas turbine engine. Laser Doppler velocity measurements were performed during hot burner rig testing to determine sand particle incoming velocities and their rebound characteristics upon impact on coated material targets. Further, engine sand ingestion tests were carried out to test the CMAS tolerance of the coated nozzle vanes. The findings from this on-going collaborative research to develop the next-gen sand tolerant coatings for turbine blades are presented in this paper.

Engine Tests↗