Molecular Description of a Novel Orientia Species Causing Scrub Typhus in Chile

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%–3.5% for rrs and 11.2%–11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloé Archipelago where the pathogen was identified.

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%-3.5% for rrs and 11.2%-11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloé Archipelago where the pathogen was identified. except the BM2016-I sample, which was only from the buffy coat specimen.

Phylogenetic Analysis
We compared Orientia DNA sequences from the 2016-2019 scrub typhus patients with DNA from the first Orientia scrub typhus patient in Chile from 2006 and that of distinct Orientia species, including O. tsutsugamushi and Candidatus O. chuto, different Rickettsia species, and other microorganisms retrieved from GenBank and aligned using ClustalW (http://www. clustal.org). We used MEGAX software (https:// www.megasoftware.net) to infer phylogenetic analyses by the maximum-likelihood method (18) and to perform the search for the most appropriate model of nucleotide substitution for phylogenetic analysis according to the Bayesian information criterion. For the maximum-likelihood method, we obtained initial trees for the heuristic search automatically by applying neighbor-joining and Bio neighbor-joining algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. We based the support of the topology on a bootstrapping of 1,000 replicates; the positions equivalent to gaps or missing data were deleted.

Comparison of Nucleotide Diversity
We created consensus sequences of the generated amplicons after alignment in BioEdit version 7.0.5.3, which we compared with respective sequences of O. tsutsugamushi and Candidatus O. chuto strains as well as the first Orientia case from Chile, obtained from GenBank.
A sequence identity matrix was constructed in BioEdit version 7.0.5.3. The selected databases and algorithms used for alignment and comparison of sequences were in accordance with current recommendations for the taxonomical characterization of prokaryote strains (19).

Cases
The 18 investigated scrub typhus cases were acquired in 3 regions currently known to be endemic for scrub typhus (15), Biobío, Los Lagos, and Aysen, which span >1,120 km (latitude 38°03′S to 47°47′S) in Chile; 5 of the 18 cases were from Chiloé Island (Los Lagos), where the initial case was reported (Table 1). At the time they sought treatment, all but 1 patient exhibited the 3 clinical signs characteristic of scrub typhus: fever, maculopapular rash, and inoculation eschar. The presence of Orientia genomic DNA was confirmed in all cases by qPCR Orien16S from buffy coat or eschar material ( Table 1). All of the patients recovered from scrub typhus without sequelae, 16 after treatment with doxycycline, 1 after treatment with azithromycin, and 1 without specific antimicrobial therapy. Further epidemiologic and clinical details of some of the patients have been published elsewhere (9,15,20).

DNA Sequences and Phylogenetic Analyses
We successfully amplified fragments of rrs from 18 cases and htrA from17 cases; the primers for tsa failed to produce amplicons. For all assays, we successfully amplified a well-defined Orientia strain (Kawasaki clade) from South Korea as a positive control (6). The lengths of clean reads were 886 nt for rrs and 950 nt for htrA. Sequences of the isolates showed a high nucleotide identity (99.7%-100%) for both genes (Appendix Table 1), with a maximum divergence of 2 nucleotides. We were able to distinguish 3 distinct rrs genotypes (1, 2, and 3) and 2 genotypic variants of htrA (a and b) (Appendix Table 2). HtrA variants, although determined by only 1 nucleotide, led to distinct DNA codons with leucine versus phenylalanine. The genotype 1a samples (n = 10) derived from Los Lagos (continental and Chiloé Island), Biobío, and Aysén regions, whereas genotypes 2b (n = 3) occurred in the continental Los Lagos region, 3a (n = 1) in Chiloé Island, and genotype 3b (n = 3) in the Los Lagos region, both continental and Chiloé Island. For 1 genotype 2 strain, we could not amplify htrA (Appendix Table 2). Phylogenetic analyses of both genes from the DNA specimens from Chile formed a unique cluster separate from the 14 O. tsutsugamushi strains included in the analysis as well as from Candidatus O. chuto (Figures 1, 2). However, the rrs sequence from the 2016-2019 samples grouped together with that from the first scrub typhus case in Chile ( Figure 1) (8).

Species Designation
The rrs sequences we analyzed showed a divergence of ≥3% from known Orientia species, indicating that the isolates from Chile constitute a novel species within the genus Orientia (family Rickettsiaceae, order Rickettsiales, class Alphaproteobacteria). Our designation of the bacteria as a new species was corroborated by the divergence of htrA and our inability to generate amplicons with primers of O. tsutsugamushi type-specific antigen gene tsa. Until a type strain is cultivated and characterized, we propose the designation Candidatus Orientia chiloensis for the novel species, after the Chiloé Archipelago (Los Lagos Region, Chile) where the pathogen was first identified (8,9).

Discussion
Because of new diagnostic tools and increasing clinical awareness, our knowledge of rickettsial infections has increased over recent decades (23,24). For scrub typhus, which has been considered the most important rickettsiosis in Asia and Australasia, the discovery of new endemic regions outside of the traditional tsutsugamushi triangle raises questions about established paradigms (25). Since 2006, multiple patients with scrub typhus have been reported in southern Chile, >12,000 km away from known endemic regions (8,9,15). In addition, a case of scrub typhus caused by a novel species, Candidatus O. chuto, was diagnosed on the Arabian Peninsula (7). These findings, together with serologic and molecular data from sub-Saharan Africa and Europe, suggest that scrub typhus caused by various Orientia species might have a much wider than previously known, possibly global, distribution (4,26,27).
Most clinicoepidemiologic and ecologic aspects of scrub typhus in South America are currently unknown. A recent study on Chiloé Island suggested that trombiculid mites of the genus Herpetacarus, which were found to be infected with Orientia-species bacteria, might serve as vectors (28); preliminary phylogenetic analyses showed that the mite-associated strains were 99%-100% identical to those from patients (29). Clinically, the >40 patients with scrub typhus diagnosed in southern Chile during 2015-2019 sought treatment for conditions similar to those for scrub typhus from the Asia Pacific region-fever, generalized rash, and inoculation eschar-and, similarly, had a rapid response to treatment with tetracycline or azithromycin (30 serologic data suggest that the Orientia species in Chile diverge from those in the Asia-Pacific region (8,9,15), but whether they represent distinct O. tsutsugamushi strains or a new species remained inconclusive. Our phylogenetic analyses of larger DNA segments from 2 conserved genes support the conclusion that the isolates from patients in Chile cluster outside known Orientia species and represent a distinct species. Culture-independent sequencing techniques play an important role in prokaryotic taxonomy, especially for strictly intracellular bacteria (31,32). For the description of new species, sequence analyses of the 16S rRNA gene (rrs) are paramount. A ≥3% divergence of rrs sequences from those in known species is the accepted threshold suggesting a novel species (19), although corrected levels of ≥1.30%-1.35% have been suggested (33,34). Isolates with rrs sequence differences of >5%-6% might belong to a distinct genus, if they display unique phenotypic differences (35). Distinct, lower thresholds have been developed for Rickettsia spp. (31), but this approach remains controversial among rickettsiologists (36). As should be the case for all molecularly defined novel species and genera, we have classified this proposed species as 2152 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 9, September 2020 Candidatus, until type strains can be cultivated and fully described (37). The novel Orientia species presented here fulfills the rrs gene criteria described in the previous sections. Our designation of a novel species was affirmed by a high divergence of another genomic marker, htrA, which diverged >11% from O. tsutsugamushi and of >14% from Candidatus O. chuto (Table 3). This con-served gene diverges only <3.7% among O. tsutsugamushi isolates (38). The O. tsutsugamushi type-specific antigen gene, tsa, which has a much higher diversity than htrA (1), was not amplifiable from isolates from Chile using primers designed for O. tsutsugamushi. This suggests that the Candidatus O. chiloensis tsa is unique, requiring the assessment of additional primers, possibly based on results from a future WGS.  Currently, no Orientia culture isolate from Chile is available. Surprisingly, the rrs and htrA sequences from the 18 Orientia samples from Chile were almost identical, showing a maximum variability of only 2 nucleotides. This genetic homogeneity over a wide geographic range is in sharp contrast to O. tsutsugamushi (38). As a unique characteristic among obligate intracellular pathogens, this species displays a dramatic genomic and phenotypic heterogeneity (1), which might be related to homologous recombination and lateral gene transfer (39). Among O. tsutsugamushi isolates, for example, the divergence of reported rrs sequences are up to 1.5% and for htrA sequences up to 3.6% (1,38), compared with ≤0.3% observed among isolates from Orientia DNA from Chile.
The most frequently applied phenotypic and molecular marker of O. tsutsugamushi strain heterogeneity is the highly variable 56-kDa TSA. This Orientia-specific surface protein is also known to be an important determinant of strain-specific pathogenicity and immunity (1). As we mentioned, we were not able to generate amplicons of strains from Chile with the applied tsa PCR or with tsa qPCR (41) or other commonly used primers (e.g., r56_2057). In a previous report, short tsa sequences were produced from 2 samples, but only after prolonged amplification cycles (9). These findings strongly suggest that tsa of Candidatus O. chiloensis is highly divergent from those of other Orientia species. Becaue the TSA surface protein is the main antigenic determinant, such divergence might explain the low serologic cross-reactivity, which was observed in patients with scrub typhus and in seroprevalence studies in Chile using O. tsutsugamushi whole-cell or recombinant antigens (9,41).
In conclusion, our results indicate that scrub typhus in Chile is caused by a novel Orientia species, suggesting an ancient origin of the disease in South America, rather than recent introduction. However, after obtaining cultured isolates of Candidatus O. chiloensis and larger gene sequences including from WGS, deeper comparative studies of the 3 Orientia species and their vectors are necessary to understand the ecology and evolution of these emerging intracellular pathogens, including the mechanisms responsible for the differences in strain variability and surface proteins.

Acknowledgments
We acknowledge Katia Velasquez and all other physicians who participated in identifying the scrub typhus cases included in this study. We also thank Teresa Azócar and Romina Alarcón for their technical help in processing the clinical samples.   Dermatophytosis is also referred to as ringworm or tinea (Latin for "worm") because it can cause ring-shaped patches that are usually red, itchy, and have worm-like borders. In 1910, Raymond Jacques Adrien Sabouraud, a French dermatologist, was the first to report the morphologic characteristics of dermatophytes. During the decades that followed, taxonomy of dermatophytes has gone through revolutionary changes, mostly due to the advent of molecular diagnosis. Although studies performed in the 21st century have resulted in further classification changes and consolidation of new species, debates regarding the taxonomy of dermatophyte agents persist.