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Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2

Abstract

Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness by infection with Orientia spp.

Using a risk-based approach, this review examines current practice, the evidence base and regulatory requirements regarding matters of biosafety and biosecurity, and presents the case for reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe working practices of risk-based activities during the manipulation of Orientia spp. in the laboratory.

We recommend to reclassify Orientia spp. to Risk Group 2 based on the classification for RG2 pathogens as being moderate individual risk, low community risk. We recommend that low risk activities, can be performed within a biological safety cabinet located in a Biosafety Level (BSL) 2 core laboratory using standard personal protective equipment. But when the risk assessment indicates, such as high concentration and volume, or aerosol generation, then a higher biocontainment level is warranted. For, the majority of animal activities involving Orientia spp., Animal BSL 2 (ABSL2) is recommended however where high risk activities are performed including necropsies, Animal BSL (ABSL3) is recommended.

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Background

Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness in the Asia-Pacific region [1] caused by Orientia tsutsugamushi. Other members of scrub typhus group orientiae [2] include Candidatus Orientia chuto from United Arab Emirates [3] and Kenya [4], and the incompletely characterized orientiae that cause scrub typhus in Chile [5,6,7]. Orientia spp. are members of the family Rickettsiaceae in the order Rickettsiales, are Gram-negative, obligate, intracellular bacteria and are transmitted by arthropod vectors [8, 9].

The laboratory diagnosis of scrub typhus requires the detection of antibodies against and/or nucleic acids of O. tsutsugamushi using serological techniques and PCR, respectively [9, 10]. In vitro culture of Orientia spp. is generally not required for diagnostic purposes, though culture is required to provide the raw materials for diagnostic assays, whole genome sequencing, and the study of growth characteristics. The in vitro or in vivo culture of Orientia spp. has the potential hazard of laboratory-acquired infections (LAIs) of staff by means of parenteral inoculation such as accidental self-inoculation, cut, animal bite or scratch, or exposure to aerosols from laboratory procedures or accidents [11,12,13]. Furthermore, Orientia spp. are sensitive to antibiotics including doxycycline and azithromycin [14, 15].

Using a risk-based approach, this review examines the evidence base, regulatory requirements and current practice, regarding biosafety and biosecurity practises, and argues for the reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe practices of risk-based activities during the manipulation of Orientia spp. in the laboratory.

Risk group classification and biocontainment regulations for Orientia spp. research

Risk Groups (RG) (known as Hazard Groups (HG) in the United Kingdom) are used as biosafety “shorthand” to quantify the degree of hazard associated with a pathogen or toxin. Although there is no universal RG classification there is general agreement amongst countries on RG principles (Table 1). Briefly, RG1 pathogens are those of low individual and community risk. RG2 pathogens represent the largest group and have the characteristics of moderate individual risk and low community risk, and there is general agreement that they are classified as those that “can cause human disease and might be a hazard to workers and effective treatment and preventive measures are available (antibiotics or vaccine) and the risk of spread of diseases in the community caused by these pathogens is low”. RG3 pathogens are considered high individual risk with limited/moderate community risk and “likely to cause serious disease and may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available” (Table 1).

Table 1 Examples of worldwide biological Risk Group classifications

At present, all members of Orientia spp. and the related Rickettsia spp. are classified as RG3 pathogens or equivalent in the United States, United Kingdom, Australia, Singapore, Germany, Switzerland, Belgium, and the European Union [17, 19]. As such, significant biosafety controls have been placed on their manipulation, propagation and storage. Interestingly, in many regions where the disease is endemic, there are few statutory regulations or guidelines regarding biosafety, as well as a lack of clear guidance on the level of biocontainment required for laboratory work on Orientia spp. In the absence of clear local regulations, government regulatory authorities often look to the USA or UK regulations, or elsewhere, to provide the basis for biosafety and biocontainment. There is often an imperative from donors to meet specific biosafety and biocontainment guidelines prior to the disbursement of funds. The following is a short description of the guidelines and regulations as specified by national regulators and a summary is provided in Table 2.

Table 2 Biocontainment and mitigation regulations for Orientia spp.

Physical biocontainment is generally classified as biosafety levels (BSL1–4) although there are differing national standards and regulations. To increase standardisation, the World Health Organization (WHO) has recently revised the WHO Laboratory Biosafety Manual (LBM 4th edition) using a risk-based approach [24] with three levels of biocontainment; 1) Core, 2) Heightened Control Measures (HCM) and 3) Maximum containment.

USA regulations

Orientia tsutsugamushi and Rickettsia spp. (e.g. R. prowazekii, R. typhi, R. rickettsii, Rickettsia conorii, R. akari, R. australis, R. sibirica, and R. japonica) are classified as RG3 organisms and are detailed in the BMBL 5th edition [21] in regards to their control as follows (p197–198). “BSL2 practices, biocontainment equipment, and facilities are recommended for non-propagative laboratory procedures, including serological and fluorescent antibody procedures, and for the staining of impression smears. BSL3 practices, biocontainment equipment, and facilities are recommended for all other manipulations of known or potentially infectious materials, including necropsy of experimentally infected animals and trituration of their tissues, and inoculation, incubation, and harvesting of embryonated eggs or cell cultures. Animal BSL (ABSL) 2 practices, biocontainment equipment, and facilities are recommended for the holding of experimentally infected mammals other than arthropods. ABSL3 practices, biocontainment equipment, and facilities are recommended for animal studies with arthropods naturally or experimentally infected with rickettsial agents of human disease. Several species, including R. montanensis, R. rhipicephali, R. bellii, and R. canadensis, are not known to cause human disease and may be handled under BSL2 conditions.”

UK regulations

In the UK, the Advisory Committee on Dangerous Pathogens (ACDP) advises the Health and Safety Executive (HSE) on aspects of hazards and risks to workers and others from pathogen exposure pathogens. All Rickettsia spp. (R. akari, R. canadensis, R. conorii, R. montanensis, R. prowazekii, R. rickettsii, R. (Orientia) tsutsugamushi, R. sennetsu, R. typhi) are classified as HG 3 pathogens in the Approved List of Biological agents 2013 (ACDP/HSE) [20]. The Control of Substances Hazardous to Health Regulations 2002 (Schedule 3, Part I, Paragraph 3 (4) (b)) state that “the minimum biocontainment level… shall be… level 3 for activities which involve working with a Group 3 biological agent” and so, in the UK, all work with confirmed Orientia spp. must be conducted in a Biocontainment Level (CL = UK BSL equivalent) 3 laboratory. The position is less straightforward when specimens from suspected cases of Orientia spp. infection are handled in a clinical laboratory for diagnostic purposes (Table 2).

Australia

In Australia, laboratories are governed by the Australian/New Zealand Standards AS/NZS which outline the safety principles in laboratories, AS/NZS 2243.3.2010 Safety in Laboratories Part 3: Microbiological Safety and Biocontainment [16]. Clinical specimens with unconfirmed microbial aetiology are regarded as low risk and handled at BSL2. However, once a high-risk pathogen has been isolated from a specimen, it must then be handled at a higher biocontainment level. Although rickettsiae are mentioned as RG3 pathogens there is no mention of Orientia spp. in the Standard. However, Orientia spp. were historically labelled as a Rickettsia spp. and this perhaps indicates the need for this standard to be updated.

Singapore

The legislation that governs the possession, use, import, transhipment, transfer, and transportation of biological agents in Singapore falls under the Biologicals and Toxins Act (BATA), that was originally enacted in 2005 [22]. Rickettsial organisms of the genus Rickettsia are classified within the act as First Schedule biological agent meaning “Risk Group 3 Biological Agents which can cause serious disease which is of high risk to the individual”. Members of the genus Orientia are not specifically mentioned but they are generally viewed as similar to Rickettsia spp. and thus require the same restrictions. Although the legislation does not specify the restrictions of the environment where a biological agent should be worked on, organisms are organised into “Schedules” that share similar restrictions (i.e., First Schedule agents may require BSL3, Second Schedule agents may require BSL4, Third and Fourth Schedule may require BSL2). This allows for the testing of normally accepted RG3 pathogens such as Rickettsia and Orientia spp. from non-amplified diagnostic samples to potentially be performed in a BSL2 environment should the appropriate approval be granted after a review of the planned work environment and the conduct of appropriate risk assessments.

Thailand

The Royal Thai Government enacted the Pathogens and Animal Toxins Act in 1982 that is administered by the Ministry of Public Health and focuses on the regulation and security of pathogens and toxins [23]. It has subsequently been updated in 2001 and 2015 with the latter superseding the previous iterations. Pathogens are designated into 4 risk groups, numbered 1 to 4, corresponding to low, moderate, high, and very high risk, respectively. These pathogens were further defined in the official Announcement of the Ministry of Public Health “List of controlled pathogens under section 18 (of the Act)” 2017 [25]. Group 2 pathogens (moderate risk) can be handled under BSL2 conditions and include R. africae, R. bellii, R. canadensis, R. felis, R. helvetica, R. honei, R. japonica, R. montanensis, R. parkeri, R. quintana, R. rhipicephali, R. sennetsu and R. slovaca. Group 3 pathogens include R. akari, R. australis, R. conorii, R. prowazekii, R. rickettsii, R. sibirica, R. (Orientia) tsutsugamushi, and R. typhi. Routine diagnostic laboratory processing within hospital laboratories can be performed in BSL-2 laboratories with strict adherence to Good Laboratory Practice guidelines. Culturing with the aim of producing large amounts of bacteria should be performed in BSL3 laboratories or equivalent safety level (BSL2 enhanced).

Other countries and regions

Germany, Switzerland, Belgium [17] and the European Union classify Orientia tsutsugamushi as an RG3 pathogen [19]. Canada does not provide a RG classification in its Pathogen Safety Data Sheet resource [18].

Risk-assessment for Orientia spp.

The case for reclassification of Orientia spp. to RG2 using a risk-based justification.

Using the broad classification for RG2 pathogens of moderate individual risk, low community risk that “can cause human disease and might be a hazard to workers and effective treatment and preventive measures are available (antibiotics or vaccine) and the risk of spread of diseases in the community caused by these pathogens is low”, a strong argument can be made to reclassify Orientia spp. to RG2. Certainly, Orientia spp. do not fit the characteristics of RG3 pathogens which are considered high individual risk, limited/moderate community risk with the key difference being the risk of spread of the disease in the community. We therefore recommend reclassification of Orientia spp. to RG2 by all countries.

Recommended risk assessments for Orientia spp. research activities

Risk associated with Orientia spp. individual research activities and laboratory procedures.

Laboratory activities associated with risk of accidental exposure of personnel and the environment to Orientia spp. include (1) the processing of clinical specimens from scrub typhus patients and, (2) in vitro culture of Orientia spp. in mammalian cell cultures or embryonated chicken eggs and associated downstream manipulations including centrifugation, harvesting of cells and supernatants and, carrying cultures to and from incubators to a Biological Safety Cabinet (BSC) [11,12,13].

LOW risk activities for both laboratory staff and environmental contamination in case of an accident would be small-scale processing of suspected scrub typhus patient samples such as initial in vitro inoculation or subsequent passage of patient samples on to cell cultures with a guideline of a total of ≤100 ml (in a single or multiple culture vessels) at a concentration of ≤106 cfu/ml Orientia spp. organisms. This may also include small-scale passaging of reference cultures.

MEDIUM risk activities may include those such as medium-scale passaging of reference cultures or medium-scale passage of clinical Orientia spp. isolates to increase the viable organism concentration thereby increasing the risk to the laboratory staff whilst maintaining a low risk of escape to the environment in case of an accident. Suggested guidelines for medium risk activities would be incubation and manipulation guideline of a total of ≤500 ml (in a single or multiple culture vessels) at a concentration of ≤106 cfu/ml Orientia spp. organisms.

HIGH risk activities involve increased volume and concentration of the Orientia spp. organisms manipulated such as in the case of reagent production which may involve large-scale production of highly concentrated Orientia spp. as well as manipulation with increased inherent risk such centrifugation and concentration techniques, as there is an increased risk to the laboratory staff and of escape to the environment in case of an accident. Suggested guidelines for high risk activities would be incubation and manipulation guideline of a total of > 500 ml (in a single or multiple culture vessels) at a concentration of > 106 cfu/ml Orientia spp. organisms.

The determination of the risk level should be made by a biosafety professional in conjunction with stakeholders including principal investigator and laboratory management as the risk mitigation measures are likely to have significant infrastructure and financial implications.

Risk associated with Orientia spp. during experimental animal activities

Experimental infection of animals (vertebrates and invertebrates) with Orientia spp. presents a different range of infection risks than those of laboratory activities. Vertebrates used for experimental infections are predominantly rodents [26, 27], guinea pigs [28] and non-human primates [29, 30] while chiggers (Leptotrombidium spp.) [29] have been the dominant genus of invertebrates. The risks may be broadly characterised as aerosol and parenteral exposures. Aerosol exposures would normally be associated with necropsy activities, cage and bedding changing (if agents is secreted in urine and faeces), and infectious waste exposures [31]. Parenteral exposures would normally be due to bites, cuts, and scratches from animals or sharp equipment [11, 13, 31]. All such activities if performed on a small-scale would normally be considered MEDIUM risk. However, if the number of animals significantly increase or larger animal species are used, then risk would increase to HIGH. As mentioned above, in the case of increased risk activities, this determination should be made by a biosafety professional in conjunction with stakeholders. For MEDIUM risk procedures where there is minimal aerosol generation then Animal Biosafety Level 2 (ABSL2) is recommended however where HIGH risk activities are performed including necropsies, Animal Biosafety Level 3 (ABSL3) is recommended. Mitigation strategies for medium- and high-risk animal activities based on the US BMBL 5th edition regulations [21] are presented in Table 3.

Table 3 Mitigation strategies for medium- and high-risk animal activities based on the US BMBL 5th edition regulations [21]

Recommendations for risk-based biocontainment levels for Orientia spp. laboratory activities

Risk-based assessment of laboratory activities for Orientia spp., when classified as a RG2 pathogen, do not support the need for BSL3 biocontainment for LOW-risk routine, low-volume culturing of Orientia spp. and downstream activities and most MEDIUM-risk activities. Therefore, these activities can be performed at BSL2 biocontainment level in a BSC with standard Personal Protective Equipment (PPE) which would be consistent with WHO HCM. However, for HIGH-risk activities where high concentrations and large volumes of live organisms are involved such as in the case of reagent production, the risk assessment may suggest performing the activities at a higher level of secondary containment, such as BSL3 containment, and PPE may need to include respirators if primary biocontainment equipment (e.g. BSC) are not available or easily able to be used. Stocks of reference cultures at high concentrations require sufficient levels of physical biosecurity including locked freezers, stored behind locked doors with restricted/vetted personnel access to prevent the theft of pure cultures that may be used for nefarious purposes. Risk-based recommendations for biocontainment and associated mitigation strategies for low- and high-risk Orientia spp. laboratory activities are presented in Table 4.

Table 4 Risk-based recommendations for biocontainment and associated mitigation strategies for low- and high-risk laboratory activities

Discussion

Until now, Orientia spp. and Rickettsia spp. have been classified as RG3 pathogens resulting in substantial controls placed on their growth, handling and storage. This RG3 classification may have evolved from association with the more pathogenic R. rickettsii and R. prowazekii –which were classified by the United States as Biological Select Agents in 2007 due to their biothreat status [21, 32], however, only R. prowazekii retains this status in the current Select Agents list [33]. Orientia spp. are classified as RG3 pathogens by the USA, European Union, Australia, New Zealand, Belgium, Germany, Switzerland and Thailand [19, 25]. While Orientia spp. and many Rickettsia spp. have the potential to cause serious infections, it is difficult to justify their status as RG3 pathogens because they do not spread by person-to-person contact and are amenable to antibiotic treatment.

Further confusion around biosafety requirements for Orientia spp. laboratory investigations is the fallacy that RG status is equivalent to biocontainment level [24]. This misinterpretation has resulted in the incorrect assumption that BSL3 biocontainment is a mandated requirement for all Orientia spp. manipulations, without considering the procedural risks. BSL3 biocontainment laboratories fundamentally differ from BSL2 biocontainment as they provide directional airflow via a negative pressure gradient and HEPA filtered exhaust air that provides protection to the outside environment from aerosol contamination in the event of a catastrophic laboratory accident. A consequence of the need for complex engineering solutions to provide adequate BSL3 biocontainment is that such laboratories are expensive to build, maintain, and manage. Furthermore, such laboratories typically have high electricity costs to maintain heating, ventilation and air-conditioning (HVAC) requirements which further compounded by the regulatory necessity for single pass non-recirculated air. Building and operating a BSL3 laboratory places financial strain on already stretched budgets, and those wishing to maintain regulatory compliance often choose not perform such activities at a lower biocontainment levels for fear of not complying with national laws, funder requirements, or incorrectly perceiving the activities to be too “high risk” to be performed.

It is likely that the requirement for BSL3 biocontainment has had unintended consequence of impeding scrub typhus research and vaccine development [34]. Laboratory and regulatory administrators have taken an overly cautious attitude to the regulatory aspects of research on Orientia spp. and other rickettsial pathogens. This has resulted in the proliferation of high-biocontainment laboratories in both relatively-wealthy and developing countries. The need for high-biocontainment laboratories for Orientia spp. propagation has been overstated and the mistaken requirement to perform all such work in BSL3 laboratory facilities through rules imposed by national governments, or foreign institutions providing donor funds has further compounded the issue.

The evidence presented here demonstrates that work with Orientia spp. and Rickettsia spp. requires a risk-based consideration when selecting biosafety and biocontainment controls. Using a blanket RG3/BSL3 approach is inappropriate and restrictive except when the risks are clearly high and the controls justified. The WHO LBM 4th edition [24], uses a risk-based approach and stipulates that only high-risk activities be performed in HCM laboratories, which may include (but is not restricted to) BSL3 laboratory facilities.

The evidence from scrub typhus LAI reports [13, 31], clearly supports the proposal that low risk activities, can be safely performed within a BSC located in BSL2 core laboratory (i.e., HCM laboratory). When warranted, the requirement for increased secondary containment, PPE and enhanced practices should be maintained. This is supported by the fact that scrub typhus LAIs have not been reported for nearly 20 years [13], and exposure of laboratory staff to infectious materials and their products are ameliorated by rigorous biological safety mitigation strategies through administrative controls including a focus on good microbiological practises and procedures, competency assessment, was well as judicious selection of PPE.

Conclusions

We recommend the reclassification of Orientia spp. to RG2 based on the classification of RG2 pathogens as being moderate individual risk and low community risk. Furthermore, using a risk-based approach, we recommend that low risk activities can be performed within a BSC located in a BSL2 core laboratory (i.e., HCM laboratory) and high-risk activities, such as those involving aerosol generation or high bioburden of bacteria, would require the use of BSL3 laboratory facilities. The majority of animal activities involving Orientia spp. would still require ABSL3 containment. Consideration should also be given to the risk-group reclassification of selected Rickettsia spp. using the same risk-based approach.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Abbreviations

ABSL:

Animal Biological Safety Level

BSC:

Biological Safety Cabinet

BSL:

Biological Safety Level

GMPP:

Good Microbiological Practises and Procedures.

HCM:

Heightened Control Measures

LAI:

Laboratory-acquired infection

LBM:

Laboratory Biosafety Manual

PPE:

Personal Protective equipment

RG:

Risk Group

WHO:

World Health Organization

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Acknowledgments

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Disclaimer

The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.

Funding

Stuart D. Blacksell, Matthew T. Robinson, Paul N. Newton, Tri Wangrangsimakul, Jeanne Salje and Nicholas P.J. Day are funded by the Wellcome Trust of the United Kingdom. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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The manuscript concept was devised by SB, PN and ND. SB contributed the main draft of the manuscript. SB, MR, TW, AR, AB and JeS contributed to the sections on risk assessment. MW contributed to the sections on Animal Biosafety. SR and TW contributed to the sections on regulations in Thailand. MA contributed to the sections on regulations in Singapore. JoS contributed to the sections on regulations in Australia. SB, AR, AB contributed to the sections on regulations in the United Kingdom and the United States. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Stuart D. Blacksell.

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Blacksell, S.D., Robinson, M.T., Newton, P.N. et al. Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2. BMC Infect Dis 19, 1044 (2019). https://doi.org/10.1186/s12879-019-4653-4

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Keywords

  • Orientia
  • Scrub typhus
  • Biosafety
  • Biocontainment
  • Risk group