UV-C Decontamination: NASA,Prions,and Future Perspectives

Abstract

Ultraviolet (UV) radiation in the UV-C waveband is considered an effective microbicidal method employed in the fight against MRSA (methicillin-resistant Staphylococcus aureus), influenza, and other hospital-acquired infections. Utilized at the peak germicidal wavelength of 254 nm, UV-C is considered an effective microbicide for rapid inactivation of DNA- and RNA-based pathogens (bacteria, viruses, fungi, etc). With regard to pathogenic prion isoforms, where nucleic acid is absent and resistance to decontamination is expressed, UV-C remains totally unexplored in other wavelengths of possible relevance for inactivation by peptide bond photodissociation on contaminated surfaces and surgical instruments. Here it is hypothesized as a viable UV approach to the unusually stable protein-based pathogen, thus aiding in the prevention of Creutzfeldt-Jakob disease by surgical transmission. UV-C is also largely underutilized outside the common context of human health and only passively by NASA (National Aeronautics and Space Administration) in planetary protection protocols, with microbial reduction standards remaining largely unaltered since the Viking robotics program of the 1970s. NASA and the European Space Agency cleanroom environments in particular have experienced positive biological assay detections of bacteria native to cleanroom conditions. Therefore, the goal of this article is to discuss these 2 distinct uses of UV in the context of effective UV-C decontamination, to propose broader applications of UV irradiative practice, and to address future exploration of a newer UV approach applied to prion inactivation by further investigation of wavelengths beyond those considered only germicidal.

The current methods for sterilization for the majority of medical and scientific laboratories and instruments often include a combination of physical and chemical methods, such as autoclaving, steam, and, in the case of NASA (National Aeronautics and Space Administration) spacecraft, dry heat microbial reduction. As of 2013, hospital-acquired infections affect >440,000 patients per year and cost approximately $10 billion annually, according to US News & World Report. ¹ In addition to the common nosocomial infections, prion infections and exposures have been documented. In February 2014, 18 neurosurgery patients at the Forsyth Medical Center in Winston-Salem, North Carolina, were exposed to Creutzfeldt-Jakob disease (CJD) over a 3-week period. ² A rare form of transmissible spongiform encephalopathy, CJD can be transmitted via consumption of infected brain and spinal products, as well as products contaminated by central nervous system material, direct injection or grafting, immunoglobulin, and contaminated surgical instruments. As with all such prion-induced encephalopathies, CJD is invariably fatal. Protection against such prion exposure from contaminated materials remains problematic given that standard sterilization procedures are not effective for CJD and other prion-induced illnesses.

Chemical sterilization—ranging from ozone and hydrogen peroxide to more aggressive approaches, such as bleach and sodium hydroxide—remains the universal standard for the elimination and inactivation of microbes in most laboratories, hospitals, and cleanrooms. While these methods are generally effective, hardy microbes pose not only increased health risks but also possible contamination and confounding of scientific results and data, distinctly affecting NASA astrobiological and planetary exploration aims in the solar system.

Ultraviolet Germicidal Irradiation Systems

A possible approach to sterilization involves the use of ultraviolet germicidal irradiation (UVGI), which has often been used as a viable, low-cost alternative for wastewater treatment, supplemental air purification, aquaria cleaning, and even limited medical and scientific instrument sterilization. As a form of shortwave radiation—particularly at the UV-C portion of the electromagnetic spectrum and usually accomplished at 254 nm—the mutagenic properties of ultraviolet (UV) light are typically utilized for viral and bacterial inactivation by disruption of a pathogen’s DNA and RNA. As UV photons interact with nucleic acid structures, a process of dimerization occurs. Pyrimidine dimers, specifically thymine dimers, are molecular lesions—that is, damage to the structure of a biological molecule that results in reduction or absence of normal function—that form from thymine and cytosine bases in DNA. ³ This photochemical reaction, when left unrepaired (repair not applicable in the case of sterilization), results in the loss of vital cell functions, and the pathogen is killed or otherwise rendered inactive, as infectivity is nullified.

Possible Solutions for Use by NASA

One approach to present concerns is to examine NASA’s microbial reduction methods, which remain the current and only approved methods for spacecraft sterilization. In light of current precautions, note that these measures do not constitute a truly sterile environment with microbial life detected by biological assays under current bioburden constraints. This policy—largely unchanged since the Viking missions of the 1970s, though with more recent additions, such as vapor-phase hydrogen peroxide and gamma irradiation in the case of the Beagle-2 Mars probe parachute ⁴ —has raised several issues regarding sterilization and the prevention of forward and back contamination in various NASA missions, concerning spacecraft and cleanroom procedures. As early as 2007, >100 types of hardy microbes (some previously unknown to science) were discovered at NASA’s Jet Propulsion Laboratory, the Kennedy Space Flight Center, and the Johnson Space Center. With the recent discovery of Tersicoccus phoenicis in NASA and European Space Agency cleanrooms, ⁵ it has become increasingly clear that new methods for sterilization are required as part of NASA’s planetary protection protocol and overall cleanroom procedures.

The chief purpose of planetary protection is the prevention of forward contamination (ie, the seeding of life from Earth on another planet) and back contamination (possible extraterrestrial life proliferation on Earth) with regard to NASA’s current and future missions. Currently most applicable to robotic planetary flight programs, NASA’s cleanroom policy follows federal class 100,000 cleanroom standards, closely aligned with that of ISO class 8, where a cleanroom has at most 3,520,000 particles per cubic meter of air for particles ≥0.5 µm. ⁴ This is achieved with laminar airflow systems to filter contaminants, as well as with sterile instruments, pressurized barriers, and typical cleanroom garmenting determined on a mission-specific basis. Spacecraft and relevant materials receive a general cleaning to reduce initial biological burden before being subjected to high-temperature treatment. Packaged in bioshielding, they are then baked at 111.7ºC for 30 hours, with some variation depending on the treated material.

The possibility of applying UVGI to cleanroom environments to help alleviate the ongoing challenge of damaging sensitive electronic instruments by the above heating measure remains a viable alternative to explore. NASA continues its current exploration of alternatives to accomplish proper decontamination while fulfilling obligations to procedural requirements and mission classification. ⁴ While the planetary protection officer has approved a newer method of vapor-phase hydrogen peroxide, ⁶ UVGI appears to be, for some circumstances, a less complicated and less time-consuming bioburden reduction supplement, with some consideration given to its limitation of surface-only sterilization.

Utilizing UV radiation allows the application of already existing passive methods regarding spacecraft sterilization in an actively applied manner. For example, in the case of Mars, there is some reliance on the UV environment of Mars in microbial reduction. The National Research Council ⁷ task group proceedings concluded that “during the entire Martian year, the UV flux is sufficient to sterilize the Martian surface.” Also noted was that, given microbial decline in certain mission scenarios, the surface of Mars can be considered as sterilizing, ⁸ with some exception made regarding dust layers altering surface exposure. This is the property that, given the overall effectiveness of UV sterilization, could be more widely utilized and integrated procedurally for future NASA missions.

While there are advantages to the adoption of UVGI systems as a matter of decontamination, ⁹ such is not without its relative drawbacks. The mutagenic and probable carcinogenic properties of UV wavelengths are clear, ¹⁰ as the same types of dimerization and damage to microorganisms can also be replicated in human cells by hazardous exposures. Erythema and photokeratitis remain as side effects for employee overexposure to far UV light, ¹¹ as well as eye burning, pain, and swelling/peeling of facial skin. This of course necessitates the need to take proper precautions with proper shielding and safety measures to lessen the likelihood of exposure or overexposure.

Another common problem rests with equipment maintenance and efficacy over time. Given that UV-C is not well suited to subsurface penetration, the germicidal effects of 254 nm can be further inhibited with the addition of dust layers on emitting bulbs, ¹² necessitating weekly cleaning. However, evidence indicates that such buildup may be somewhat mitigated by increased airflow near the bulb surfaces. Such airflow should be used for the additional purpose of germicidal lamp cooling to prevent overheating—though only enough to increase bulb effectiveness at the optimum temperature for output (demonstrated at 77°F-80°F), ¹² which can also be affected by overcooling if the airflow is too great. While issues of humidity also play some role in the effectiveness of germicidal lamps, such would likely be mitigated by the controls and protocols of the cleanroom atmosphere.

Cleanroom Application Recommendations for NASA and Beyond

Concerning cleanroom application, UVGI systems have been utilized by some hospitals as part of disinfection, decontamination, and infection prevention. Muskogee Community Hospital (Muskogee, Oklahoma) was the subject of a case study involving hospital-acquired infections over 21 months. UVGI systems played a partial role in the final results—a total of zero such infections transmitted over the course of that period. ¹³ UV-C light is used here in HVAC systems to disinfect coils and drain pans, ultimately serving as an effective inactivation method for preventing airborne pathogens passing through ventilation systems. Additionally, as part of the lighting systems, UV-C bulbs were installed in surgery and patient rooms and operate 5 days per week at a maximum of 8 hours per day in unoccupied rooms. ¹³ This largely bathes each room in this light and effectively sterilizes the room after each use. Note that these methods not only further demonstrate the viability and effectiveness of these systems for augmentation in NASA cleanrooms, should this option be explored, but also serve as a template for broader applicability and universal adoption in medical environments to lower the fiscal and human costs of medically acquired infections.

Prions and the Problem of Present Sterilization Methods

The sterilization of surgical instruments and otherwise contaminated material is of great interest concerning the control and inactivation of CJD and other prion diseases. As a protein-based pathogen, its constitution is largely that of the typical protein (ie, an amino acid chain connected by peptide bonds). Differing from the typical protein, however, the prion pathogen is a misfolded isoform that acts as the blueprint for other proteins, causing normal proteins in the body to take on the misfolded form. This misfolding eventually leads to brain malformations that resemble holes in healthy brain tissue, with no immune responses or inflammation elicited as a reaction to infection. The resulting illnesses are dubbed transmissible spongiform encephalopathies given their aforesaid effects on the brain. Some symptoms include behavioral changes, cognitive decline and memory deficits, lack of coordination and problems with movement, visual/perceptual decline, trouble swallowing (as the disease progresses), and, invariably, death. Commonly known examples of such encephalopathies include CJD, chronic wasting disease (deer and elk), mad cow disease (bovine), scrapie (sheep), and kuru.

In the case of prion inactivation in CJD, the existence and replication of a prion’s misfolded protein structure does not rely on nucleic acid, as evidenced by the existing literature. ¹⁴ This not only reinforces the well-established view of the prion as a protein-only pathogen but also provides an explanation for current resistance to the mutagenic properties of ionizing radiation and the remarkable stability of prion structure under commonly utilized shortwave UV-C ranges. While the small size of prototypical PrP^Sc (scrapie) and other isoforms plays some role in resistance, the overall amino acid/polypeptide structure disallows the usual approaches of UVGI application given the lack of DNA or RNA. However, the constituent amino acid and peptide bond elements of the prion protein may be the most affected by shortwave UV radiation.

Prion inactivation is often difficult to achieve given its remarkable stability and resistance to traditional approaches. Resistance to protease (a protein-digesting enzyme), heat, formalin, and even common UV radiation wavelengths ¹⁵ is what makes the prevention of prion transmission a significant challenge. More aggressive methods, as mentioned in the introduction, are employed. According to World Health Organization recommendations, contaminated instruments should be subject to treatments such as gravity-displacement autoclaving at 121°C for 30 minutes and immersion in sodium hypochlorate. ¹⁶ Other methods have included bleaching, steam, incineration, formalin, and iodine, as well as the use of alkaline hydrolysis digestion in the case of animal carcass disposal, which degrades the proteins into salts of free amino acids, achieving inactivation by the destruction of peptide bonds. ¹⁷ With the exception of this last example, these harsher treatments, while proven most effective to date, do not guarantee total inactivation.

The aggressiveness of present chemical and physical methods for the sterilization of prions remains a continuing problem. Given the stability of the infectious isoforms and their resistance to normally applied chemical and physical sterilization techniques, the extended use of high temperatures, autoclaving, caustic soda, bleaching, and environ LpH ¹⁸ remains the most effective treatment for inactivation to date. However, even after harsh treatment is applied to exposed instruments, total prion inactivation remains elusive and even reversible (artificially) from partially denatured states. ¹⁹

UV and the Possibility of Mistaken Interpretation

Although the present literature definitively demonstrates that prions are resistant to germicidal wavelengths, ²⁰ this is the area where researchers may be inclined to examine the use of UV-C radiation more carefully. Note that most research on this resistance originates from the 1970s and 1980s, where experiments investigated whether the prion was a “slow virus” containing nucleic acid or it existed as a protein-only pathogen. The most widely cited study is that of Bellinger-Kawahara et al, ¹⁴ indicating purified scrapie protein resistance to inactivation by UV radiation. Based on the typical germicidal wavelength at 254 nm, the findings were later consistent with the now well-established protein-only model of prions. Academic literature searches regarding the subject of UV resistance demonstrate that the aforementioned article is almost invariably incorporated as evidence of the claim. This leads to the possibility of improperly interpreting results beyond the scope of their subject, in essence implying resistance to all radiation after demonstrating resistance only to specifically germicidal wavelengths. All such studies, while illuminating the lack of nucleic acid, merely demonstrate that one cannot approach prions in the same way as the typical pathogen; hence, these studies should no longer be treated as the final interpretation of radiation-prion interaction.

Newer UV Approach to Prions

Noting the importance of peptide bonds in prions is necessary. The absorption spectra of peptide bonds have been shown to be between 190 and 230 nm, indicative of susceptibility to UV light. ²¹ As stated, the research performed on prions has largely been outside this range at nanometer lengths considered “germicidal,”^14,22 with the exception of Alper’s discovery of effective 237-nm inactivation (used only to investigate the nature of the prion agent). Studies that were performed close to the relevant peptide range (or at least in part, including the above) showed greater effectiveness in prion inactivation. ²³ The Johnson study, however, revolved around the use of 185-nm radiation in the production of ozone as a possibly effective agent, discounting any direct effect of shortwave radiation with citation of the aforementioned scrapie study. ¹⁴

Given that the primary results are indicative of negative findings for nucleic acid, a more targeted approach, similar to alkali hydrolysis, is warranted. If the degradation and dissociation of peptide bonds are instrumental in prion inactivation (and what is aimed for chemically), then further research is needed to determine actual UV susceptibility at wavelengths more appropriate to peptide absorption spectra and with further consideration to such radiation methods in amide bond breaking investigated by absorption spectroscopy. ²⁴

Recommendations

To approach a further-reduced biological burden beyond that achieved by current NASA microbial reduction protocols and to address the ongoing difficulties of prion inactivation, the recommendations of this article are summarized thusly:

• Broader application of UVGI systems with specific augmentation for NASA’s Office of Planetary Protection and relevant cleanroom environments

• Exploration into the possibility of applying UV-C radiation within nanometer ranges relevant to peptide bond absorption in prion inactivation by such means other than those previously studied