Polymerase Chain Reaction (PCR) assays are the primary method to detect rickettsial pathogens, especially for the early detection of infection before the development of detectable antibodies. PCR assays, which selectively amplify a specific region of the organisms DNA to detectable levels, may target a number of different genes to enable a variety of levels of detection, from broad Rickettsia genus- or SFGR-level of detection, to species-specific detection (Rickettsia typhi and Orientia tsutsugamushi for example). Assays which target outer membrane proteins such as 17kDa, ompB and 47kDa, generally confirms the presence or absence of Rickettsia spp, R. typhi or Orientia tsutsugamushi (respectively). With other gene targets, down-stream sequencing of the PCR product will, in most cases, identify the specific species. It is strongly recommended that multiple gene targets are used to gain an accurate identification. Different formats of PCR assays are available, all with varying levels of sensitivity and specificity, including conventional, nested and quantitative (real-time) PCRs. In addition a number of LAMP-assays (loop-mediated isothermal amplification) have been developed, allowing relatively sensitive and specific rapid detection at a constant temperature (minimizing the need for expensive thermocyclers).

The sample type used for a PCR assay is very important. Whole blood or buffy coat is the preferred sample to be used for a number of reasons, including that Rickettsia are intracellular (the cellular component being concentrated in the buffy coat fraction, increasing the sensitivity of detection) and the blood sample being easily collected. Serum or plasma may be used but these samples are less than optimal as there will be fewer patient cells (meaning lower rickettsiae concentration). In addition, serum may have increased concentrations of blood fibrinogen and fibrin materials which can bind to rickettsia DNA decreasing availability of DNA target for PCRs. Eschars are a suitable sample type for PCR and may be sampled as either scrapings, swabs or biopsied specimens.

Diagnosis of rickettsial infections, especially scrub typhus and murine typhus which are common causes of fever in Asia, is difficult since it does not present with any distinctive clinical signs compared to other febrile illnesses in this region, except for a necrotic skin lesion (eschar) in some patients. Laboratory tests are therefore crucial to identify of rickettsial infections. Reliable laboratory tests need significant infrastructure and experienced staff, making access to accurate tests very limited in the low-resource endemic regions. Serological tests measure the amount of specific immunoglobulin IgM and IgG antibodies against the pathogen that is causing the rickettsial illness. Serological tests are often used because they are perceived to be cheaper and easier to perform than molecular testing and they use convenient patient samples which are normally serum or plasma. However, serological tests for the diagnosis of rickettsial illnesses is limited and often there is inconsistency in their application and difficulties with interpretation of the final result.

The gold-standard serological test for rickettsial pathogens is the immunofluorescence assay (IFAs which provides a quantitative measure of antibodies in the patient sample. IFAs are not suited to testing large numbers of samples and require specific skills and equipment to read the result (Blacksell et al., 2007). There is a great deal inconsistency in the application of IFA methodologies especially in the area of the types of diagnostic antigens used, the application of diagnostic cut-offs and which antibody isotype should be tested (Blacksell et al., 2007; Dhawan et al., 2020).

Another type of test which is often used is an Enzyme Linked Immunoassays (ELISAs) which provides a semiquantitative result and is often used for screening purposes although can be used for absolute diagnosis however results should be interpreted with caution. There are commercial and in-house ELISAs available for the detection of scrub typhus antibodies (Saraswati et al., 2019; Phanichkrivalkosil et al., 2018; Elders et al., 2020) and only in house ELISAs for the detection of murine antibodies. As is the case with IFA, it is important that the diagnostic cut off the established in each geographic region based on the endemicity and prevailing background antibodies in the community. Generally, there is strong positive relationship between ELISA and IFA antibody levels for both IgM and IgG.

Point of care lateral flow devices are also available for the rapid diagnosis of scrub typhus using Ab based methodologies. These devices tend to lack accuracy and results should be interpreted with caution (Saraswati et al., 2018).

References
Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NP. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis. 2007;44(3):391-401.

Dhawan S, Robinson MT, Stenos J, Graves SR, Wangrangsimakul T, Newton PN, et al. Selection of Diagnostic Cutoffs for Murine Typhus IgM and IgG Immunofluorescence Assay: A Systematic Review. Am J Trop Med Hyg. 2020.

Elders PND, Dhawan S, Tanganuchitcharnchai A, Phommasone K, Chansamouth V, Day NPJ, et al. Diagnostic accuracy of an in-house Scrub Typhus Enzyme linked immunoassay for the detection of IgM and IgG antibodies in Laos. PLoS Negl Trop Dis. 2020;14(12):e0008858.

Phanichkrivalkosil M, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Laongnualpanich A, Chierakul W, et al. Determination of Optimal Diagnostic Cut-Offs for the Naval Medical Research Center Scrub Typhus IgM ELISA in Chiang Rai, Thailand. Am J Trop Med Hyg. 2019;100(5):1134-40.

Organisms that fall under the classification of rickettsial organisms typically fall within the genera Rickettsia, Orientia and Coxiella are obligate intracellular organisms, that require a host cell for culture. Other organisms that are sometimes similarly classified require similar culture techniques. As such the culture methods are like that of viral culture. In recent times Coxiella has been grown axenically, however this method is not recommended for primary isolation as not all Coxiella group types will amplify.

Rickettsial culture is usually employed for the purposes of diagnostics (live pathogen amplification), maintenance of rickettsial stocks and growth of rickettsial antigens. Culture must be done in a suitable laboratory environment that is able to contain Risk Group 3 (RG3) organisms. In many places, the allowed laboratory classification is a minimum of Biosafety Level 3 (BSL3). However, there has been a push to reduce the laboratory containment requirement to Biosafety Level 2 (BSL2) for low concentration amplification which will include primary cultures for certain rickettsial types/species.

Culture of rickettsial organisms utilizing viral culture techniques is usually conducted in tissue culture flasks with a suitable adherent cell line acting as the host. Antibiotic free media is utilized for these cultures as rickettsial organisms are bacteria and are thus susceptible to antibiotics traditionally used in viral culture. Host cell lines can be any cell line that is vulnerable to rickettsial infection at their preferred growth temperature and can come from a mammal (Vero), amphibian (XTC-2), insect (C6/36) or tick source. Another criterion for host cell selection is their susceptibility to cytopathic effect (CPE) by rickettsial organisms, for easy visualization when the rickettsial culture is ready for harvesting for subsequent processes. However, CPE is not commonly seen in primary isolations and a secondary screening technique will need to be implemented. This can be an immunofluorescence technique to visualize the rickettsiae directly or a PCR to detect their DNA.

Choice of host cell line is also determined by the target rickettsial organism's optimal growth temperature. Example for many members of Rickettsia spp, the optimal growth temperature is 28°C, and suitable cell lines are XTC-2, C6/36 or most tick cell lines. For rickettsial organisms that require a higher temperature, Veros are used for temperatures between 32-35°C. It is thus advisable to attempt isolation of a novel or unknown rickettsial organism from a source material in different host cell lines, to determine their host cell affinity and optimal growth temperature.

Source material can come from a range of different sources. The most common that are used include buffy coat, serum, tick hemolymph and vector/biopsy homogenate. Depending on source, additional steps to “clean” the source material (especially vector and biopsy) maybe needed to prevent contamination of culture. This may include cleaning the material in an appropriate disinfectant or adding an additional step such as filtration. To induce entry of rickettsial organisms into the host monolayer, an additional step of centrifugation at a low speed (10-15min at <500g) is recommended. The rickettsial primary culture should then be incubated in media containing 2-4x the normal concentration of antibiotics (pen/strep and gentamycin) for 24 hours before washing the cell layer with phosphate buffered saline (PBS) and changing the media to one without any antibiotics. The initial “antibiotic shock” in the first 24 hours is optional, as it helps to reduce the likelihood of contamination by unwanted microorganisms at the risk of lowering initial rickettsial inoculation numbers.

Rickettsial organisms have a long doubling time of approximately 8 hours and thus the culture needs to be maintained for at least 6 weeks, with media changes every 7 days. It is also recommended to test for rickettsial presence after 2 weeks of culture by doing an immunofluorescence (IFA) or PCR test of the media on a tiny scraping of the monolayer. Overgrowth of the monolayer is common, and the culture can be passaged as per normal into a fresh flask. Maintaining isolation culture attempts up to 16 weeks is not uncommon.

“Orientia tsutsugamushi is difficult to culture and sequence, and the first complete genomes were not available until 2010. These two genomes of the Boryong and Ikeda strains revealed an extraordinary genome with extensive repeat amplification. More recently, further complete genomes have been obtained by using a combination of long and short-read next-generation sequencing. Long-read sequencing, using the PacBio and Oxford Nanopore Technologies platforms, allows for assembly of a complete sequence despite the extreme repeat content of O. tsutsugamushi. The short-read sequencing is used to correct errors introduced due to the higher error rate of these long-read technologies.

Comparative genomic analysis of the ten complete genomes available confirms widespread amplification of repeat elements, with wide variation in the copy number of different highly amplified genes, and shows extensive chromosomal rearrangements and loss of synteny between strains, with few of the conserved core genes being retained in the same order between strains.

Short-read sequencing has also been successfully used to investigate the genomics of O. tsutsugamushi. While it is difficult to assemble complete sequences using short reads alone, due to the heavy repeat content, the reads can be compared to a reference genome and used to call single nucleotide polymorphisms (SNPs) and short insertions and deletions, allowing for phylogenetic analysis of strains.

When whole genomes are not available, the relationships between strains of O. tsutsugamushi can be determined by MLST (multilocus sequence typing), which looks at the sequence of seven housekeeping genes and systematically assigns a number to each new allele of a gene, and to each combination of alleles. Or the highly variable sequences of the 56 kDa and 47 kDa genes can be compared, as there is enough diversity in this gene to be able to see differences between strains.”

As with all human pathogens, culture of Orientia spp or Rickettsia spp in vitro or in vivo carries the intrinsic hazard of infection of laboratory and ancillary staff (known as laboratory-acquired infections [LAIs]) via parental inoculation by accidental self-inoculation or needle-stick incident, animal bite, or inhalation of infectious aerosols generated during laboratory procedures or incidents. A recent review of scrub typhus and murine typhus LAIs by Blacksell et al (2019a) determined that scrub typhus LAIs were documented in 25 individuals, from 1931 to 2000 with 8 (32%) deaths during the preantibiotic era. There were 35 murine typhus LAI reports and no deaths. Results indicated that the highest-risk activities were working with infectious laboratory animals involving significant aerosol exposures, accidental self-inoculation, or bite-related infections.

Orientia spp or Rickettsia spp are currently classified as risk group (RG) 3 pathogens. There is conjecture however that the basis for this classification is not fully justified and consideration should be given to the risk-group reclassification of selected Rickettsia spp. using a risk-based approach. The recent study by Blacksell et al (2019b) has recommended the 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., heightened containment measure 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. A risk-based biosafety approach for in vitro and in vivo culture of O. tsutsugamushi and R. typhi would require that only high-risk activities (animal work or large culture volumes) be performed in high-containment biosafety level (BSL) 3 laboratories. We argue that relatively low-risk activities including inoculation of cell cultures or the early stages of in vitro growth using low volumes/low concentrations of infectious materials can be performed safely in BSL-2 laboratories within a biological safety cabinet.

Further consultation is underway to fully determine the risks associated with research into rickettsial pathogens such that suitable and sufficient risk mitigation strategies can be implemented in low resource settings where rickettsial pathogens are prevalent.

References
Blacksell SD, Robinson MT, Newton PN, Ruanchaimun S, Salje J, Wangrangsimakul T, 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. 2019;19(1):1044.

Blacksell SD, Robinson MT, Newton PN, Day NPJ. Laboratory-acquired Scrub Typhus and Murine Typhus Infections: The Argument for a Risk-based Approach to Biosafety Requirements for Orientia tsutsugamushi and Rickettsia typhi Laboratory Activities. Clin Infect Dis. 2019;68(8):1413-9.