I. Plasmodium and Cryptosporidium: Biology and Biopreservation

Plasmodium and Cryptosporidium are apicomplexan parasites characterized by distinct tissue tropism and host specificities, determining their unique transmissibility and infectivity.Reference Rueckert⁴ Both parasites have adapted to a range of vertebrate hosts, diverging into host-specific species. While the monoxenous life cycle of Cryptosporidium requires only one host, the Plasmodium life cycle is heteroxenous and employs a mosquito vector for transmission between vertebrate hosts.

The life cycle of both parasite genera occurs in the intracellular niche; Cryptosporidium develops in the epithelium of the gastrointestinal tract, while Plasmodium develops in hepatocytes and red blood cells of the vertebrate host and a midgut epithelium of the invertebrate vector. Within the vertebrate host, only a few life stages appear briefly in the extracellular space between events of cell invasion, specifically in the gut lumen for Cryptosporidium or blood plasma for Plasmodium. This distinct tissue tropism dictates unique pathology and symptomology in the vertebrate host, a diarrheal disease in the case of cryptosporidiosis and a systemic multi-organ disease in case of malaria. Related to the distinct tissue tropism is the path by which the pathogen evacuates from the host, which defines the risk of disease transmission and spread within the population. These differences between life cycles of the two parasites contribute to their respective biosecurity profiles in the context of biopreservation.

Infection with Cryptosporidium leads to an acute diarrheal disease in animals and in humans. Most human cryptosporidiosis is caused by the anthroponotic C. hominis and zoonotic C. parvum circulating in cattle. Both species transmit between hosts by the fecal-oral route, including waterborne, foodborne, person-to-person, and animal-to-person modes of transmission. After completion of a life cycle in the intestine, Cryptosporidium evacuates its host with feces in the form of a free-living oocyst. Encapsulated in a thick protective wall, oocysts remain dormant in the environment for several weeks awaiting consumption by the next host. Millions of oocysts can evacuate during a single fecal dischargeReference Chappell⁵ leading to a substantial contamination of the environment, soil, and groundwater. Cryptosporidium is extremely infectious; consumption of as few as 10 oocysts can lead to infection.Reference Okhuysen⁶ As a result of their resistance to chemical disinfectants and their small size allowing for filtration bypass, oocysts persist in the environment, giving rise to frequent outbreaks associated with waterborne transmission via ingestion of recreationalReference Sorvillo⁷ or consumable water.Reference Widerström⁸ Foodborne outbreaks via consumption of fecally contaminated food have also been reported.Reference Quiroz⁹ Additionally, infection among farmed cattle exponentially amplifies the environmental parasite burden and exacerbates the risk of large zoonotic outbreaks.Reference Kenzie¹⁰

The largest cryptosporidiosis outbreak in humans occurred in the United States and was caused by C. parvum. The outbreak was associated with contamination of water by agricultural runoff, leading to an estimated 200,000 infections and a breakdown of the state healthcare system.¹¹ The growing number of US cryptosporidiosis outbreaks in the last decade is concerning as there are limited therapeutics and no vaccines available.¹² Given the potential of oocyst dissemination via water, high morbidity rates, and the need for specific diagnostic capacity, the Centers for Disease Control and Prevention (CDC) designate C. parvum as a bioterrorism agent Category B.Reference Sandrock and Broaddus¹³

Although infectious, the Plasmodium parasite poses a less immediate biosecurity risk as the life cycle does not include a free-living infectious form and instead relies on continuous circulation between various vertebrate hosts and a mosquito vector.Reference Sato¹⁴ Five malaria parasite species are responsible for human disease, putting almost half the world’s population at risk.Reference Patrapuvich¹⁵ Transmission to humans occurs when an infected Anopheles mosquito injects Plasmodium sporozoites into the bloodstream while taking a blood meal.

Chronicity of infection in the host and the abundance of mosquitoes perpetuate the disease burden in several regions.¹⁶ Although rare, human-to-human malaria transmission has also occurred via blood transfusions, sharing contaminated needles, solid organ transplantation, and transplacental transmission.Reference Rosso¹⁷ Approaches to reduce the malaria burden include vector control, implementation of antimalarial drugs, and recently vaccine prevention.Reference Hemingway¹⁸ Due to the parenteral access required for disease transmission and availability of effective treatments and prevention, the biosecurity risk associated with Plasmodium research in a laboratory setting is greatly reduced.

Cryopreservation methods have been established for both Plasmodium and Cryptosporidium parasites. In case of P. falciparum, methods of cryogenic preservation of asexual intracellular blood stages have been widely available for decades and are useful for the preservation of both in vitro cultured parasites and clinical isolates obtained from patient samples.Reference Moll¹⁹ These methods rely on principles of slow cooling in the presence of cryoprotective agents (CPAs). In an effort to improve access to quality-controlled Plasmodium parasites, the National Institute of Allergy and Infectious Disease (NIAID) at the National Institutes of Health (NIH) convened The Malaria Research and Reference Reagent Resource Center (MR4), which is currently responsible for cryobanking and sharing the asexual blood stages of multiple strains of Plasmodium species. Methods for preservation of sexual blood stages and extracellular sporozoites by slow cooling have been previously reported,Reference Keister and Kaslow²⁰ but are not yet widely used in laboratory practice.

In contrast, traditional slow cooling methods have not been successfully applied for the cryopreservation of Cryptosporidium parasites, despite extensive efforts. This failure is likely due to the impermeability of the oocyst wall to CPAs to impart protection to the oocyst and the sensitivity of the parasite to ice crystallization. Only in recent years has cryopreservation of infectious Cryptosporidium been accomplished by application of newer cryopreservation technologies, namely by vitrification.Reference Jaskiewicz²¹ Vitrification is an ice-free approach to cryopreservation where relatively high concentrations of CPAs are combined with rapid cooling rates to achieve formation of amorphous solidification in a glassy state as opposed to crystalline formation of ice. Vitrification of various species of infectious Cryptosporidium parasites by ultra-rapid cooling has been achieved using highly thermally conductive silica microcapillaries.²² Due to the small volume restrictions imposed by microcapillaries, additional technologies utilizing high aspect ratio specimen containers have been developed to successfully cryopreserve larger volumes (~100 μL) of C. hominis and C. parvum.²³ However, cryopreservation of other parasitic stages, namely extracellular sporozoites and intracellular stages, has not yet been achieved.

Advanced technologies for biopreservation have the potential to be a game changer in basic and translational research on pathogens. However, biopreservation of infectious pathogens requires balancing benefits and risks, as well as appropriate governance. Below we discuss cryopreservation of Plasmodium and Cryptosporidium as model pathogens of differing risk to biosecurity, to offer a recommended oversight framework.

II. Benefits of Pathogen Cryopreservation

Cryopreservation is broadly utilized in scientific and clinical research for the purpose of preserving biological materials ranging from cells to tissues and even small organisms. Cryopreservation relieves logistical challenges by allowing researchers to bank biospecimens for dissemination or for later use. In infectious disease research, access to reference organisms is fundamental to studying pathogen biology and host-pathogen interactions, discovering therapeutics and vaccines, evaluating those interventions in preclinical and clinical trials, and providing standards for diagnostics and outbreak investigations.

Pathogen species exist in nature in a multitude of strains, each characterized by distinct genetic markers, antigenic properties, or other unique traits that distinguish them from one another. To standardize a pathogen strain for research purposes, a wild-type isolate is first obtained from an infected host or the environment. This is followed by laboratory adaptation to in vitro and in vivo systems designed to replicate the pathogen and mirror its life cycle in a host. Additionally, standardized pathogens are genetically altered to gain or eliminate function, giving rise to transgenic and mutant varieties. This process generates a considerable inventory of laboratory-adapted pathogen species and strains, each of them unique and valuable to research. Biobanking these pathogens is vital to the continuity of research and collective progress, as it safeguards unique organisms and enables access and sharing with the broader scientific community.

A large number of Plasmodium strains are in existence in laboratories around the world. The Malaria Research and Reference Reagent Resource Center (MR4) alone biobanks 250 unique Plasmodium strains,²⁴ facilitating centralized access to quality-controlled and authenticated materials that are critical within the malaria research community and beyond. P. falciparum blood stages can be maintained in in vitro cultures using red blood cells. Further, the entire parasite lifecycle of P. falciparum and other species can be generated from infection of a species-specific vertebrate host and a mosquito vector. While P. falciparum blood stages can be effectively cultured in vitro, prolonged cultivation may curb progression to sexual differentiation, making use of an animal model and the mosquito vector indispensable for maintenance of parasite strains.Reference Ponnudurai, Taylor and Baker²⁵ However, the vertebrate animal models for cultivation of human species of malaria are expensive and not readily accessible (i.e., non-human primate and humanized mouse models), making maintenance costly and restricted to specialized laboratories.Reference Simwela and Waters²⁶ Further, to mimic a natural infection of the host via a mosquito bite, a live infected mosquito vector is necessary to isolate fresh sporozoite stages. The mosquito vector is typically procured by transport from specialized facilities. Transit of Plasmodium-infected mosquitos is vulnerable to breach of safety standards and therefore carries a risk of accidental release of the pathogen and non-native species of mosquitoes to the environment.

In contrast to Plasmodium, only a limited number of laboratory strains of Cryptosporidium are maintained worldwide, including a single human isolate of C. hominis and a few bovine isolates of C. parvum. However, the number of C. parvum isolates has grown with the emergence of methods for genetic manipulation.Reference Sateriale, Mead and Arrowood²⁷ Maintenance of Cryptosporidium isolates entails serial propagation in a single susceptible animal host, specifically mice and neonatal calves for C. parvum Reference Ananthasubramanian and Ananthan²⁸ and gnotobiotic piglets for C. hominis.Reference Akiyoshi²⁹ Infected animals develop gastrointestinal illness and release oocysts which can be purified from feces. Due to the short shelf life of oocysts, animal passage is scheduled 4–6 times per year, an expensive and time-consuming process. Continuous generation of C. hominis in gnotobiotic piglets is particularly labor-intensive as it involves surgical derivation via Cesarean section and housing of piglets in sterile isolators, and therefore requires access to specialized facilities and trained veterinary personnel.

The rigors of maintaining pathogenic specimens in a laboratory creates a major barrier to research due to technical difficulties and inherent risks. In the case of Plasmodium and Cryptosporidium, the ability to cryogenically bank large quantities of strains can significantly reduce the labor and cost associated with continuous maintenance of reference laboratory organisms. Another benefit to cryopreservation is a reduction in the number of animals used for propagation, thus improving animal welfare under the ethical framework of the Three Rs principle, which promotes replacement, reduction and refinement of animal participation in experimental studies.Reference Hubrecht and Carr³⁰ Additionally, cryopreservation can maintain genetic characteristics that may otherwise be altered as a result of prolonged cultivation or propagation. Streamlined cryopreservation also enables creation of pathogen biorepositories to serve as a centralized source for dissemination of specimens. This enables wider access to biological specimens, improves sharing between laboratories and importantly, extends access to researchers lacking capacity for routine maintenance of host species. Because cryopreservation enables on-demand availability as opposed to continuous culture, this technology also increases the number of clinical isolates and strains that can be studied, thus accelerating potential research. Cryopreservation is also a potential strategy to reduce the risk of accidental release of infectious specimens during transport, as typically a precise protocol for recovery from the frozen state must be executed to preserve infectivity. In the case of Plasmodium sporozoites, transport of live malaria-infected mosquitoes could be entirely avoided with streamlined methods of sporozoite preservation, eliminating the risk of pathogen and vector release to the environment. Further, biobanking of unique pathogen isolates and strains protects from catastrophic specimen loss due to unforeseen events such as pandemics, natural disasters, and associated disruptions to the supply chain.

Although discovered two decades apart, research on Cryptosporidium lags in comparison to that on Plasmodium, and this delay may be partially attributed to many technical limitations, including lacking access to cryobanking. The first method of Cryptosporidium cryopreservation was achieved only recently,³¹ while methods of Plasmodium cryopreservation have been routinely in use for five decades.Reference Diggs, Aikawa and Haynes³² This lag coincides with the delay in scientific discovery as the search for effective drugs and vaccines against cryptosporidiosis continues.Reference Gilbert³³

In drug and vaccine development, pathogen cryopreservation is an important enabling technology with applications in research and clinical studies. For example, performing controlled human infection models (CHIM) or early clinical trials require utilization of a quality-controlled pathogen source across study subjects to ensure uniformity of the infectious dose. However, each passage of a pathogen in an animal or cell culture may alter pathogen characteristics such as infectivity and virulence and give rise to between-batch variation. Therefore, each propagated batch demands optimization, standardization, and in vitro validation prior to use in clinical trials. The ability to cryobank multiple inocula from a single standardized source allows researchers to thaw an individual infectious dose on an as-needed basis, which can eliminate variability and improve the uniformity of infection under trial conditions.Reference Jumani³⁴ In the case of infectious organisms that predominantly affect persons living in low-and middle-income countries or that exhibit seasonal patterns of infection, cryopreservation enables routine access to research specimens globally. The development of biopreservation technologies for infectious organisms can thus advance research to develop therapeutics and vaccines, ultimately yielding health, societal, and economic benefits through the relief of disease burden and improvement of social equity.Reference Luyten and Beutels³⁵

Lastly, cryopreservation is essential for the long-term preservation of eradicated pathogen species (e.g., smallpox and rinderpest viruses or eradicated European P. falciparum strain³⁶). This secures the opportunities for continuation of scientific research, diagnostics, drug and vaccine development in the event of resurgence of these diseases but without the risks related to the maintenance of live cultures. However, special safety considerations should be observed during the cryopreservation of eradicated pathogens to prevent accidental release and potential reemergence.

III. Risks of Pathogen Cryopreservation

While pathogen cryopreservation promises significant benefits, it may also carry risks over time and space. Although handling of both Plasmodium and Cryptosporidium pathogens requires compliance with biosafety level 2 (BSL-2) standards, these seemingly similar pathogens carry different degrees of biological risk. Due to the potential for waterborne transmission, Cryptosporidium poses a threat to public health, hence its classification by CDC as a Category B bioterrorism agent.Reference Hagen, Loderstaedt and Frickmann³⁷ Its release in the environment, whether accidental or intentional, can lead to contamination of drinking or recreational water and result in large outbreaks, potentially affecting thousands of people in a short period of time.³⁸ In contrast, transmission of Plasmodium relies on blood-to-blood contact, whether by injection or mosquito bite, which — in tandem with existing preventative and therapeutic solutions — considerably reduces the risks associated with release of biospecimens to the population in relation to cryopreservation. Consequently, in this section we will focus solely on potential perils associated with Cryptosporidium cryopreservation as an example of a pathogen with high biological risk, defined here as having the potential to cause human harm.³⁹

Biological risk refers to the potential threat or danger posed by biological agents that have the capacity to cause harm to human health, the environment, or other living organisms. Biological agents include microorganisms, toxins, genetically modified organisms, and prions. Risk is inherent to the laboratory handling of biological agents and is therefore rigidly managed. Strategies for mitigating biological risks include implementation of biosafety, biosecurity, and biocontainment procedures to prevent accidental exposures, intentional misuse, or accidental release of biological agents. Although biosafety, biosecurity, and biocontainment are related concepts, they refer to different aspects of biological material handling.⁴⁰ Biosafety refers to the set of practices designed to protect laboratory workers from accidental exposure to biological agents and their further release to the environment. Biosecurity involves measures preventing unauthorized access, theft, loss, or intentional release of hazardous biological materials. Its primary focus is therefore on safeguarding biological materials and associated information from intentional misuse, whether by individuals, groups, or nations. Lastly, biocontainment refers to the physical and procedural barriers implemented to confine biological agents to a controlled space and prevent their escape into the surrounding environment. Together, these measures form a comprehensive framework that ensures responsible and safe practices in the field of biological research and protects the public and the environment from introduction of biological agents.

Standard laboratory handling of Cryptosporidium carries risks relating to biosafety, biosecurity, and biocontainment. Technologies to cryopreserve Cryptosporidium may increase the risk of inadvertent pathogen escape, whether accidental or intentional, under certain conditions. Thus, utilization of advanced technologies for pathogen preservation should proactively identify these elevated risks. Further, implementation of these advanced technologies in pathogen cryopreservation may inherently elevate biosafety risk. For example, the technologies developed for vitrification of Cryptosporidium oocysts involve use of devices and procedures prone to pressurization and aerosolization, and therefore introduce a risk of unintentional release through an exposed laboratory worker.⁴¹ It is therefore critical that both cooling and thawing, events separated in time and likely in space, are performed by skilled and trained personnel in an appropriate BSL environment.

Pathogen cryopreservation carries the potential to undermine biosecurity measures. This could present a potential for misuse, deliberate release, or theft, raising concerns about illicit experimentation, biowarfare, or other malicious activities. Intentional misuse is especially relevant to C. parvum due to its recognition as a bioterrorism agent. A major concern is that biorepositories of pathogens and associated information may be vulnerable to unauthorized access, therefore implementation of robust security measures is essential to mitigate such risk. The security protocols established for live pathogens should be extended to encompass cryopreserved specimens, securing both the physical specimens and the digital information about those specimens. The long-term nature of cryogenic storage introduces challenges in consistently maintaining and enforcing strict biosecurity protocols over extended periods of time. This could be due to employee turnover or poor recordkeeping, which could allow sensitive materials to be accidentally relocated to unsecured facilities.

Pathogen cryopreservation introduces potential biocontainment challenges as well, primarily by altering the dynamics of pathogen storage. Cryogenic storage of pathogens could lead to a risk of inadvertent release or exposure during thawing. Release of Cryptosporidium oocysts, even in limited quantities, poses a significant threat to individuals who come into contact with the contaminated environment. This necessitates stringent biocontainment measures during the handling and storage of such pathogens. The transport of cryopreserved materials further amplifies this vulnerability. A breach in biocontainment during static cryostorage or transport could result from infrastructure failures, equipment malfunction, unexpected environmental conditions, human error, or inadequate adherence to protocols. Maintaining biocontainment becomes especially challenging when utilizing emerging preservation technologies which sometimes rely on specimen containers that are either open to the environment or only partially contained, such as microcapillary,⁴² dropletReference de Vries⁴³ or meshReference Zhan⁴⁴ used for vitrification. To address these concerns, comprehensive risk assessments, rigorous personnel training programs, and careful adherence to established biosafety protocols are essential.

The primary objective of cryopreserving pathogens is to maintain their viability for research or storage. However, the inherent stress imposed by freezing and thawing can induce various changes in the organisms i.e., genetic mutations, epigenetic alterations, and thus adaptation to selective pressures, which has been observed in other microorganisms.Reference Stoycheva⁴⁵ Though not previously reported, these changes could potentially promote the survival of variants tolerant to cryopreservation stresses and result in novel phenotypes exhibiting modified functions in terms of virulence or drug sensitivity. A comprehensive understanding of the molecular mechanisms underlying these alterations should be a future direction for the field to study. This information can help inform the establishment of biosafety, biosecurity, and biocontainment measures to reduce the impact of these variants and prevent their release into the environment.

IV. Existing Oversight Framework in Pathogen Research

The US policy framework on pathogen handling, whether live or biopreserved, involves a combination of laws, regulations, guidelines, and oversight mechanisms at the federal, state, and institutional levels. The primary goal is to ensure the safe and responsible handling of pathogens to protect public health and prevent accidental releases and intentional misuse.

V. Recommendations

Biopreserving pathogens extends their reach and persistence in time and space, emphasizing the need for protective measures. While the US policy framework concerning pathogen handling comprehensively addresses various aspects of biopreservation, research involving the development and application of protocols to preserve, transport, and store pathogens require attention to several issues. These include considering the perils of sharing protocols for the biopreservation of dangerous pathogens, ensuring proper training of personnel handling pathogens, ensuring the security and compliance of facilities housing pathogens, verifying safe transport, developing and monitoring standard operating procedures (SOPs) over time, and building systems to detect and promptly address breaches.

Publishing protocols for cryopreservation of pathogens raises the classic dual-use problem of enabling beneficial research but potentially also empowering laboratories with inadequate biosafety measures as well as malicious actors to cause harm inadvertently or deliberately. A version of this problem arose nearly a decade ago in debate over publishing gain-of-function (GOF) protocols involving pathogens with pandemic potential (P3) in the open scientific literature.Reference Selgelid⁶⁷ Although there are clear differences between protocols that may increase the danger posed by P3 versus protocols to permit pathogen biopreservation, transport, and storage, both may be a source of risk if laboratories failing to take appropriate precautions or malevolent actors intentionally use the pathogens to cause harm. In the GOF context, although the US National Science Advisory Board for Biosecurity (NSABB) initially recommended the publication of these studies in a redacted form, with key findings included but detailed descriptions of materials and methods omitted, they later approved the release of full information.⁶⁸ This decision was influenced by a risk/benefit analysis and a recognition of the challenges in reconciling redacted or classified publication with the norms and practicalities of open science. Cryopreservation of pathogens promises the substantial research benefits outlined above, such as aiding development of treatments and vaccines, thus outweighing potential risks associated with open literature access.

Although the dangers of careless or deliberate misuse of Cryptosporidium (the more dangerous of the two pathogens we review) fall short of P3, its cryopreservation requires safeguards, and the level of prophylaxis required could be greater for more dangerous pathogens. Those safeguards should include, but are not limited to:

• • Training: Appropriate training should be in place for all personnel working within a space that houses cryopreserved infectious specimens. This includes institution-based training but must also involve direct training by the laboratory director in pathogen-specific SOPs for the storage of cryopreserved infectious materials. This includes training regarding PPE, spill procedures, risk assessment, safety reporting procedures, recordkeeping and appropriate handling techniques when accessing the storage tank or thawing specimens. Careful adherence to established protocols is essential for maintenance of biosafety and biocontainment.

• • Biosafety: Biorepositories with infectious materials should be maintained in an appropriate BSL facility accessible only by authorized personnel (i.e., storage of cryopreserved stocks of Cryptosporidium should remain in BSL-2). For pathogens stored in the liquid phase of nitrogen, cross-contamination within a storage unit should be prevented and steps should be taken if a vial containing the pathogen ruptures.Reference Bayers⁶⁹ While pathogens liberated from cryostorage without an appropriate thawing technique may not survive, any materials exiting a tank should be considered potentially infectious, as only a few surviving pathogens could in theory cause infection.

• • Technical handling: Use of advanced technologies for cryopreservation may increase risks to staff. For example, vitrification of Cryptosporidium in glass microcapillaries and high aspect ratio cassettes creates additional risks related to pressurization and exposure to sharps. Detailed protocols should be published for these methods, with stress on the importance of proper use of PPE, engineering controls and practices (e.g., expelling contents directly into liquid to prevent aerosolization or use of blunt-tip and safety needles to reduce opportunity for sharp injury).⁷⁰ The design of specific cryopreservation technologies should also prioritize safety of the end user. For example, the microcapillary for vitrification of Cryptosporidium utilizes a fused silica material, which was selected due to its high resistance to cracking under thermal shock.Reference Deng⁷¹ The risk of sharp injury was further reduced by utilization of microcapillaries coated with polyimide, which increases durability and flexibility of the device.

• • Transport of cryopreserved pathogens: The removal or addition of materials from the cryopreserved inventory must be carefully documented, including full information on transfer of custody. Whether transported by courier or moved across the building, cryopreserved materials should be properly packaged and labeled with biohazard symbols to prevent accidental exposure to others and potential release to environment. Cryptosporidium, for example, is classified by the DOT as a Category B biological hazard, thus shipping should follow UN3373, Class 6.2 regulations.⁷² Given the complexity of these guidelines, institutions require specific training courses that provide up-to-date requirements for certification to ship biological materials.

• • Maintenance of SOPs over time: The prolonged storage of pathogens is vulnerable to a lapse in oversight due to personnel change or facility closures. Such a lapse may pose a risk to biosafety, biocontainment, and biosecurity. Establishment and monitoring the adherence to SOPs for storage of pathogens can guarantee continuity. Particularly critical is the SOP for reporting potential exposures and breaches to oversight authorities for risk assessment and mitigation. As long as pathogens remain in biorepositories, IBCs or relevant bodies should maintain oversight and ensure compliance with policies, which may evolve over time. Additionally, pathogens may lose viability over time, depending on the temperature of long-term storage. Implementation of periodical viability testing is therefore essential to ensure consistency of the inventory and inform the need for renewal of stocks.