Gaps and inconsistencies in the current knowledge and implementation of biosafety and biosecurity practices for rickettsial pathogens

Scrub typhus, typhus group and spotted fever group rickettsiosis have similar signs and symptoms, including fever, headache, rash, and muscle pain [7]. An eschar, a key pathognomonic sign of STG and SFG rickettsiosis, may sometimes be present as a dark scab-like lesion and can be found at the site of the mite or tick bite [7].

Rickettsia and Orientia are naturally transmitted to humans through the bites or infectious secretions of ectoparasites [5, 7, 9], and human-to-human transmission does not occur naturally [10]. The SFG rickettsiae are transmitted by ticks [5, 11], and TG members, R. prowazekii (epidemic typhus) and R. typhi (murine or endemic typhus) are transmitted by the body louse (Pediculus humanus humanus) and rat flea (Xenopsylla cheopis), respectively [5]. Orientia tsutsugamushi is transmitted by the bite of larval stage Leptotrombidium mites, also known as chiggers, in the primary endemic areas [12, 13].. Candidatus Orientia chiloensis [16] has also been detected in trombiculid mites of Herpetacarus, Quadraseta, and Paratrombicula spp.in Chile [14]. Transitional group members, R. australis, R. akari and R. felis, are transmitted by ticks, mites and fleas, respectively [7].

Rickettsial diseases can be effectively treated with the tetracycline class of antibiotics. Doxycycline is the first choice due to its high efficacy [5, 15]. However, in the late 1990s, there was suspicion of doxycycline-resistant strains of O. tsutsugamushi following the observation of increased fever clearance times in a small number of scrub typhus patients in Chiangrai, northern Thailand [16]. Subsequently, studies have demonstrated that doxycycline-resistant scrub typhus is a misconception, with treatment outcomes likely to be determined by other bacterial, host, and pharmacological factors [17]. Azithromycin may be considered an alternative for scrub typhus treatment [15] but not murine typhus [18].

Orientia tsutsugamushi and most of Rickettsia spp. are classified as risk group (RG) 3 pathogens in the United States, United Kingdom, Australia, Singapore, Germany, Switzerland, Belgium, and the European Union [19, 20]. However, there are inconsistencies in the designation of RGs depending on the pathogen and the country. Table 1 summarizes global RG classifications for O. tsutsugamushi and selected Rickettsia spp.

The infectious dose required to cause human infection for Rickettsia and Orientia pathogens remains insufficiently understood; however, for some rickettsiae, there are results from animal and in vitro models that can provide useful information. Using modelling, the estimated infectious dose for R. rickettsii is 23 ID₅₀ (50% infectious dose), with a 95% confidence interval of 1 to 89 organisms [21] having used results from studies in non-human primates [22‐24], and humans [25] (summarized study results presented in Table ;2). In vitro and in vivo studies have also been performed for other Rickettsia spp. in animal and in vitro models also using results from previous studies for R. prowazekii [26] (Cairo 3 strain), R.typhi [26], R. canada (now known as R. canadensis) [27], R. rickettsii [28], R. conorii [28] and R. sibirica [29] (Table ;2). Infectious dose results demonstrated differences in the susceptibility of the animal species [30], with guinea pigs (GP) generally more susceptible than mice (M), with minimum ID₅₀ of 393 and 15 (M) and 1 and 8 (GP) for R. prowazekii (Breinl and Cairo 3 strains); 2 (M) and 5 (GP) for R. typhi; 9.7 × 10⁴ (M) and 1.1 × 10⁴ (GP) for R. canada; 1.1 × 10⁴ (M) and 126 (GP) for R. rickettsii (Sheila Smith) and 1.6 × 10⁴ (M) and 21(GP) for R. rickettsii (R strain) [30] (Table ;2). The infectious dose for Orientia spp. has not been determined, however, mouse [31] and hybrid models with non-human primates [32] have been developed for vaccine assessment purposes. It has been shown that there are differences in the severity of illness caused by various strains of O. tsutsugamushi in animals [33, 34]..

When assessing the level of risk involved in handling rickettsial pathogens, it is imperative to take into account both the nature of the pathogen and the type of procedure that will be performed. Factors such as pathogenicity, virulence, and the likelihood of aerosolization must be considered. Additionally, the specific activity to be performed (e.g., serology, inoculation of specimens into culture, animal experiments, initial in vitro growth of an isolate, or large-scale production) must also be taken into consideration as the risk will vary depending on the activity. Guidance is provided on these matters by the US [35], Canada [36], UK [37], Australia [38], Singapore [39], Switzerland [40] and Thailand [41] regarding recommendations for biocontainment requirements and special practices and procedures when working with rickettsial pathogens (see Table 3 for full details). In these jurisdictions, the guidance recommends that non-propagative work with clinical specimens be performed in a Biosafety Level (BSL) 2 facility using BSL2 practices for NAATs and serology testing. Canada, the UK, Australia, and Singapore require all in vitro propagation of the rickettsial pathogens to be performed at BSL3 containment. In the US, procedures involving the in vitro propagation of rickettsial pathogens are mandated to use “BSL3 practices and containment equipment… for activities involving culture propagation or specimen preparation and propagation of clinical isolates known to contain or potentially containing Rickettsia spp. pathogenic to humans.”. However, somewhat confusingly, the US regulations also state, “BSL2 facilities with BSL3 practices are recommended for all manipulations of known or potentially infectious materials, including… and inoculation, incubation, and harvesting of embryonated eggs or cell cultures.” The US has specific guidance for working with Orientia spp. pathogens [35] in the BMBL 6th edition; however, locations such as Australia, where scrub typhus is endemic (northern Queensland, Northern Territory) does not have specific guidance [38]. In vivo animal work with rickettsial pathogens is normally confined to BSL-3. However, recently, a mouse model for acute lethal rickettsial disease, using R. parkeri Atlantic Rainforest strain and C3H/HeN mice, has been described with the advantage that this model can be studied in an animal BSL2 containment level [42].

In the US, R. rickettsii and R. prowazekii are classified as Select Agents under the Code of Federal Regulations (42 CFR Part 73), which defines specific regulations regarding the possession, storage, use, and transfer of pathogens [35]. When Select Agent laboratory studies are funded by a US agency, outside of the US, the foreign entity must meet the Select Agent requirements, which often requires review by US government regulators.

Post-exposure prophylaxis (PEP) following a laboratory or clinical exposure to a rickettsial pathogen is potentially lifesaving, depending on the pathogen and the route of inoculation.

Guidance is provided regarding PEP following a laboratory incident, although it is confined to RMSF exposures. The Canadian guidance [10] specifies 100 mg of doxycycline taken twice daily for 5–7 days and until the patient is afebrile for at least 2–3 days (Table S1). The US guidance provided by CDC/NIH [35] detailed in Table S1 does not specify the anti-rickettsial chemotherapy required; however, it also recommends infrastructure development such as availability of an experienced medical officer, signs and symptoms of disease training, non-punitive, anonymous reporting system for accidents; and the reporting of all febrile illnesses.

There are numerous reports of laboratory-acquired infections (LAIs) caused by rickettsial pathogens. RMSF has been acquired through needlesticks [43, 44] (Table S1)and aerosol exposures [45], centrifugation errors, equipment failure, the blending of infected ticks, and direct tick bites when working in the field. Before the introduction of biosafety practices (i.e., biological safety cabinets and PPE) and the use of antibiotics for PEP (i.e., before 1940), there were 63 laboratory-related infections recorded, including 11 deaths [10, 46]. Whilst there are some discrepancies in the absolute numbers, many of these LAIs were summarised by Pike [47], where RMSF (n = 13), scrub typhus (n = 7) and epidemic typhus (R. prowazekii) (n = 3) fatalities were described. In the same article, the infections and subsequent deaths [47] of Howard Ricketts and S. J. M. von Prowazek due to infections acquired during research activities were eponymously honoured due to their dedication and sacrifice. Orientia tsutsugamushi and R. typhi LAIs were often the result of aerosol exposures and needlesticks, often without any risk mitigation for aerosol exposure or animal handling [48]. A study aggregating LAI infections in O. tsutsugamushi and R. typhi reported that between 1931 and 2000, 25 scrub typhus LAIs caused eight deaths and 35 murine typhus infections with no deaths [48], with the cause being largely aerosol and self-inoculation-related [48]. Notably, all O. tsutsugamushi LAIs fatalities occurred exclusively during the pre-antibiotic era [48], demonstrating the effectiveness of the tetracycline group PEP. Following the introduction of antibiotic treatments, later reviews recorded ten reports of LAI R. typhi between 1941 and 1995, resulting in 35 laboratory-borne infections and no fatal cases distributed in countries such as the US, UK, South Korea, Malaysia, and Switzerland [48].

Inactivation of rickettsial pathogens normally relies on the use of chemical disinfectants. However, there is often a lack of clear evidence; several reports and guidance documents recommend using chemicals for the empirical inactivation of rickettsial pathogens. Chemical inactivation includes alcohols (ethanol, isopropanol), aldehydes (formaldehyde, paraformaldehyde, glutaraldehyde), alkalis (sodium hydroxide, ammonium hydroxide), halogens (sodium hypochlorite, iodine), peroxygens (accelerated hydrogen peroxide, e.g., Rescue®), potassium peroxymonosulfate (Virkon-S®), peroxyacetic acid, (Oxy-Sept® 333), phenolic compounds (Lysol®, O-Syl®, Amphyl®, TekTrol®, Pheno-Tek II®), sodium dodecyl sulphate (Qiagen ATL); however, specific concentrations and durations of exposure vary and in many cases have not been validated (Table S1). Limited studies have been conducted to evaluate the efficacy of irradiation for inactivating rickettsiae; however, some methodologies are reportedly successful (Table S1). Combinations of chemicals and radiation have been used to inactivate O. tsutsugamushi; however, the justification for this combination was mandated by the requirement for rickettsial inactivation in blood products. Thermal treatment is effective for inactivating rickettsial pathogens. Exposure to dry heat at a temperature of 56 °C (Table S1) or humid heat at 121 °C was effective in inactivating most rickettsial pathogens, although there are variations in contact time depending on the pathogen (Table S1).