Rickettsia typhi infection in severe fever with thrombocytopenia patients, China

Rickettsia typhi infection in severe fever with thrombocytopenia patients, China Shao-Fei Zhang*, Juan Du*, Xian-Miao Mi*, Qing-Bin Lu, Jie-Ying Bai, Ning Cui, Zhen-Dong Yang, Zhi-Bo Wang, Xiao-Ai Zhang, Pan-He Zhang, Hao Li and Wei Liu a State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing Key Laboratory of Vector Borne and Natural Focus Infectious Diseases, Beijing, People’s Republic of China; School of Public Health, Peking University, Beijing, People’s Republic of China; Laboratory Animal Center, Academy of Military Medical Sciences, Beijing, People’s Republic of China; The 154 Hospital, People’s Liberation Army, Xinyang, People’s Republic of China


Dear Editor,
Severe fever with thrombocytopenia (SFTS) is an emerging infectious disease caused by a novel bunyavirus (SFTSV) that was widely identified in mainland China [1]. Ticks have been recognized as the vectors involved in SFTSV transmission to humans, and several domestic animals are considered potential reservoir host [1]. Therefore people who live in rural areas or have intense contact with insects or animals are at a high-risk of acquiring SFTSV infection. The disease has also been reported in Korea and Japan, indicating the possibility of spreading in the wider geographic range [2,3]. Although infection with SFTSV is associated with a self-limiting illness in the majority of individuals, it can cause severe clinical disease in a proportion of individuals [4][5][6]. Some individuals with severe disease proceed to the critical phase, which is associated with multiorgan dysregulation, haemorrhagic manifestation, neurological symptoms; whereas those with self-limited disease proceed to the recovery phase without developing any of these complications [6]. But at the early phase of the disease, patients have only non-specific febrile illnesses, making it difficult to achieve clinical based diagnosis [4,5]. Furthermore, laboratory capacity to diagnose the causative etiological agents is often lacking in rural areas. Consequently, the determination of the etiological diversity that might be causing SFTS warrants investigation with intense efforts. In recent years, an increased number of vector-borne pathogens other than SFTSV has been detected from the clinically diagnosed SFTS patients, comprising species of spotted fever group rickettsiae (SFGR), Hantaan and Seoul viruses, Orientia tsutsugamushi, and Anaplasma phagocytophilum [7][8][9][10]. Moreover, the previous study found that only 27% of the SFTS like patients reported tick bite history [9], indicating that the patients might be either infested by other vectors or infected with other vector-borne pathogens. Murine typhus is a flea-borne febrile illness caused by Rickettsia typhi, and is hard to be diagnosed due to the non-specific clinical features [11]. In China mainland, R. typhi was only rarely reported to cause human infection. In a field epidemiological survey performed in Tianjin, the antibody positive rates determined by IFA for R. typhi ranged from 5.0% to 58.2% from 2007 to 2009, suggesting a high possibility of acquiring R. typhi infection in people living in rural areas in China [12]. In our sentinel hospital that recruited SFTS like patients in Xinyang city, although SFTSV and SFGR were found to be the most frequently seen tick-borne agents [9,10] the causative agents in a percentage of patients remained to be determined. On the other hand, R. typhi was indeed detected in the captured mice in the same region, with a positive rate of 6.45%, indicating the epidemic status of R. typhi in natural reservoir [13]. This has inspired us to undertake a clinically based surveillance study to explore the presence of R. typhi in SFTS endemic regions. The clinical symptoms and laboratory findings of patients infected with R. typhi were used to differentiate diagnosis with those of SFTSV infection.
This retrospective study was conducted in Xinyang city, Henan Province, where SFGR infection used to be determined from clinical diagnosed SFTS patients [9,10]. The region has a humid subtropical climate with annual precipitation of around 1100 millimetres. The natural landscape of the region is plain and mountain, and the main local agriculture activities include preparing land for cultivation, planting crops, pasturing cattle, and tea-picking activity, etc. From March to November, 2014-2015, patients with suspected vector-borne disease, which was defined as: fever with axillary temperature >38°C, and absence of a focal site of infection (e.g. pneumonia, urinary tract infection), and had a reported history of tick or other vector bite or animal contact, were recruited from the vectorborne disease clinic of the PLA 154 Hospital and the Shangcheng People's Hospital. Paired blood samples were obtained from all consenting patients with the first and second blood collected at least more than two weeks apart. The molecular test of SFTSV, SFG rickettsiae, and other tick-borne agents, including Anaplasma phygocytophilum, Babesia microti, and Borrelia burgdorferi sensu lato, was performed in the acute serum samples by PCR or real-time PCR (Appendix Table A1), and those positive were excluded from the study. The patients with paired serum were tested for R. typhi specific IgG antibody by applying the indirect immunofluorescence assay (IFA) (Focus, USA). Titre of 1:64 of IgG antibody was considered as positive reaction. An acute infection of R. typhi was defined by seroconversion or a four-fold increase in titres of IgG antibodies between paired serum samples.
A total of 1348 hospitalized patients who had illnesses meeting the case definition were recruited in 2014-2015, among whom 767 patients were confirmed with single SFTSV infection, 73 were determined to be with SFGR infections, or infections with other tickborne agents. The other 508 patients who were negative for SFTSV were subject to anti-R. typhi antibody test. Seroconversion was evident for 60 (11.8%) patients. The mean age of the patients was 58.4 ± 11.3 years, lower than that of the SFTSV infection (62.0 ± 12.3) (P = .028, Table 1). Totally 34 (56.7%) were female, comparable with SFTSV infection (450, 58.7%). All patients were farmers and reported recent contact with livestock animals such as cattle, sheep, goats, or dogs, whereas only nine patients additionally provided evidence of insect bites. The 60 patients of R. typhi infection sought medical care from April and August, and the majority of the patients (47, 78.3%) sought care between May-June, which aligned with the seasonal distribution of SFTS.
In SFTS endemic region, fever remains one of the major reasons to seek healthcare but the causes remain ill-defined. Here we provided evidence that R. typhi, a flea-borne rickettsia was prevalent in this region, with clinical syndromes and laboratory abnormalities highly resembling those with SFTSV infection [10], thus pathogen spectrum of the SFTS should be further expanded. The competent vector of R. typhi existed widely in China, especially in regions with warm and humid environment. However, the human cases with R. typhi infection were rarely reported and the clinical data were lacking, contributing to a difficulty clinical diagnosis in most of the endemic region in China. Compared to the data from a systematic review [11], several common clinical features of R. typhi infection, such as headache, chills, and rash, were less frequently seen in our patients. The main reason for this inconsistency might be that our participants were all recruited from SFTS like patients. We believe that due to the lack of typical signs of R. typhi infection, misdiagnosis of R. typhi infection was likely to happen in SFTS endemic regions. The elderly patients with R. typhi infection were more prone to present severe complications, which was consistent with previous studies [11,14,15]. Due to the possibility of severe complications, the recognition of the R. typhi infection and empirical treatment with doxycycline should be stressed, especially for eldly patients in regions with abundant transmission vectors and suitable environment.
The similar clinical manifestations between R. typhi and SFTSV infections could be interpreted from the point of pathogenesis that was common between them. R. typhi proliferates and spreads via the blood stream causing injury to endothelial and vascular smooth muscle cells, composing the pathophysiologic basis for meningoencephalitis and a skin rash [16]. The haemorrhage might also be caused by increased vascular permeability that is related with the infection. Resembling R. typhi infection, pathogenesis of SFTSV infection also involves vascular damage, which leads to an increased vascular permeability and further development of bleeding [17], the damages that underlie the similar complications observed in R. typhi infection [16]. The current finding should also be interpreted from the point of coinfection, which might likely happen. According to our previous knowledge, SFTSV coinfection with serological diagnosed spotted fever rickettsia was associated with aggravated disease [10]. Given the potential synergistic effect of SFTSV and R. typhi infection, the coinfection should also be warned in SFTS endemic regions.
The study was subject to a major limitation. Due to the fact that event of insect bite or animal contact is most often not recalled by the patients [11], we might miss some of the patients with R. typhi infection when using this as one of the inclusion criteria, thus might underestimate the actual infection rate of R. typhi. Despite the current limitation, we have confirmed R. typhi infection in SFTS like patients, described the clinical and laboratory characteristics of R. typhi infection. This information could be helpful for early differentiation between SFTSV and R. typhi infection. Further studies are needed to investigate their coinfection in the endemic regions.

Disclosure statement
No potential conflict of interest was reported by the authors.

Molecular test of SFTSV by real-time PCR
Viral RNA was isolated from serum samples using QIAamp Viral RNA Mini Kit (Qiagen), according to the manufacturer's instructions. The real-time PCR was performed to detect SFTSV RNA with the use of the One step Primer Script RT-PCR Kit (TaKaRa) as previously described [18].

Molecular test of SFG rickettsia
DNA was isolated from blood specimens collected at admission with the use of the QIAamp Blood Mini Kit (Qiagen) according to manufacturer's instructions. Nested PCR assays targeting the outer membrane protein A encoding gene (ompA), citrate synthase gene (gltA), and 17-kDa antigenencoding gene were concurrently performed to detect the presence of SFG rickettsial DNA. Nucleotide sequences of the primers were shown in Appendix Table A1.

Molecular test of Anaplasmataceae
Nested PCR assays targeting the 16S rRNA gene (Eh16S rRNA) was concurrently performed to detect the presence of A. phygocytophilum DNA. Nucleotide sequences of the primers were shown in Appendix Table A1.

Molecular test of Babesia
Nested PCR assays targeting the SSU rRNA gene was concurrently performed to detect the presence of Babesia microti DNA. Nucleotide sequences of the primers were shown in Appendix Table A1.

Molecular test of B. burgdorferi sensu lato
The real-time PCR assays targeting the 16S rRNA gene (Bor16SrRNA) was concurrently performed to detect the presence of B. burgdorferi sensu lato DNA. Nucleotide sequences of the primers were shown in Appendix Table A1.