Candidatus Rickettsia xinyangensis as Cause of Spotted Fever Group Rickettsiosis, Xinyang, China, 2015

In 2015, we evaluated 221 patients with undifferentiated fever and tick bite or animal exposure in Xinyang, China, for Rickettsia infection. Three with mild disease were infected with Candidatus R. xinyangensis, which clustered with R. fournieri and R. vini in phylogenetic analyses. Field investigations suggest Haemaphysalis longicornis ticks might be involved in transmission.

In 2015, we evaluated 221 patients with undifferentiated fever and tick bite or animal exposure in Xinyang, China, for Rickettsia infection. Three with mild disease were infected with Candidatus R. xinyangensis, which clustered with R. fournieri and R. vini in phylogenetic analyses. Field investigations suggest Haemaphysalis longicornis ticks might be involved in transmission.
We collected serum samples from patients during the acute and convalescent phases of illness and tested for IgG against R. rickettsii by using an indirect immunofluorescence assay (Rickettsia IFA IgG; Focus Diagnostics, https://www.focusdx.com). Results showed that 2 patients had seroconverted and 1 had a 4-fold increased IgG titer (Appendix Table  2). In addition, we tested patients for acute infection with Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, and Babesia microti by PCR and indirect immunofluorescence assay (5), and all blood samples were negative for both DNA of and specific IgG against these pathogens.
Two of 3 patients had reported history of tick bite, and 1 had reported animal contact (Table). All 3 patients had fever, asthenia, and anorexia. Two patients had eschar, 1 had lymphadenopathy, and none had rash. None of the 3 patients had any severe complications (i.e., hemorrhagic or neurologic signs or symptoms). Laboratory test results showed that 3 patients had leukopenia; 2 had thrombocytopenia; and 1 had elevated levels of hepatic aminotransferase, lactate dehydrogenase, and creatine kinase when admitted to the hospital (Appendix Figure 3). Clinical signs resolved and laboratory test findings were null (except for 1 patient with elevated hepatic aminotransferase levels) after 4-9 days' hospitalization.
To identify local natural foci of SFGR, we performed a field investigation for infections among ticks captured around the 3 patients' residences. We collected 232 hostseeking Haemaphysalis longicornis ticks and subjected each tick separately to DNA extraction with the DNeasy Blood & Tissue Kit (QIAGEN). Rickettsia sp. XY118 was detected in 2 (0.9%) ticks, and the nucleotide sequences of the gltA (GenBank accession no. KY617774) and ompA (accession no. KY617775) genes from these ticks were identical to those found in our patients. To further describe the genetic characteristics of this new genotype, we amplified the 16S rRNA gene (rrs; accession no. KY617772), 120-kDa genus common antigen gene (ompB; accession no. KY617776), PS120 protein-encoding gene (sca4; accession no. KY617777), and 17-kDa antigen gene (htrA; accession no. KY617773). The nucleotide sequence (1,320 bp) of rrs of XY118 had 99.7% (1,316/1,320) similarity with that of R. japonica (accession no. AP017602) and 99.6% (1,315/1,320) similarity with that of R. heilongjiangesis (accession no. CP002912; Appendix Figure 1). The nucleotide sequences of htrA (99.5%), gltA (99.6%), ompA (96.1%), and ompB (99.8%) from XY118 had the highest identity with the corresponding genes from R. vini. Compared with the partial sca4 sequence of R. fournieri, the corresponding sequence of XY118 contained 5 variable base pair sites and an 18-bp deletion (Appendix Figure 2). A phylogenetic tree that we constructed using the 2,546-bp nucleotide sequence of these 5 genes concatenated showed that Rickettsia sp. XY118, R. fournieri, and R. vini comprise a separate cluster that appears most closely related to R. japonica and R. heilongjiangensis (Figure). According to the gene sequence-based criteria for taxonomic classification of new Rickettsia isolates (6,7), a Candidatus status could be assigned to XY118, so we named this species Candidatus Rickettsia xinyangensis.

Conclusions
We found a novel SFG Rickettsia in human patients and ticks in China and propose the name Candidatus R. xinyangensis for this species. Our phylogenetic analyses involving comparisons with 5 different rickettsial genes showed that this newly identified SFG Rickettsia was most closely related to R. fournieri, a strain first isolated from Argas lagenoplastis ticks in Australia in 2013 (8) that has unknown pathogenicity in humans.
Our finding of Candidatus R. xinyangensis in 0.9% of H. longicornis ticks suggests a natural foci of this bacterium in Xinyang. However, extended field surveys and tick surveillance are required to understand the distribution of this agent and to identify specific tick vectors.
For  in China (9,10), H. longicornis ticks in Japan (11), H. bispinosa ticks in Bangladesh (12), and Apodemus agarius rodents in Korea (13), Candidatus R. xinyangensis could be a tickborne infection of immense clinical relevance in humans. In our study, patients with Candidatus R. xinyangensis infection had similar relatively mild febrile illnesses, as well as leukopenia and elevated hepatic enzyme levels, both of which are features of other SFG rickettsioses. In contrast, unlike patients with many other SFG rickettsioses, including Rocky Mountain spotted fever, our patients had eschars, not rashes (1,14). However, the patients described in this report were few in number and from a single hospital, and the true disease presentation of Candidatus R. xinyangensis infection might be more variable. Future investigations to further assess the disease spectrum of this pathogen and its contribution to clinical cases are needed.