A tick bite patient with fever and meningitis co-infected with Rickettsia raoultii and Tacheng tick virus 1: a case report

Background Increasing numbers of tick-borne pathogens are being discovered, including those that infect humans. However, reports on co-infections caused by two or more tick-borne pathogens are scarce. Case presentation A 38-year-old male farmer was bitten by a hard tick, presented with fever (37.7 °C), severe headache and ejection vomiting. Lumbar puncture was performed in the lateral decubitus. The cerebrospinal fluid (CSF) was clear, and analysis showed severe increased pressure (320 mm H2O), mild leukocytosis (126.0 × 106/L, mononuclear cells accounting for 73%) and elevated total protein concentration (0.92 g/L). Bacterial cultures of CSF and blood were negative. The diagnosis of Rickettsia raoultii and Tacheng tick virus 1 (TcTV-1) co-infection was confirmed by amplifying four rickettsial genetic markers and the partial small (S) RNA segment of TcTV-1 from the patient’s blood. The patient gradually recovered after treatment with levofloxacin and ribavirin. Conclusions This is the first reported co-infection case with fever and meningitis caused by R. raoultii and TcTV-1. It is vital to screen for multiple pathogens in tick-bitten patients, especially in those with severe complex symptoms.

In addition, some emerging bunyaviruses, such as Tacheng tick virus 1 (TcTV-1) and Tacheng tick virus 2 (TcTV-2), are suggested to caused human infections in XUAR [11,12]. Epidemiological investigations showed that (i) 7.7% (26/339) of adult ticks tested positive for TcTV-1, and (ii) syndrome of an index patient infected with TcTV-1 included fever (as high as 39.0 °C), a local skin bulge (about 5 × 5 cm in size), rash, chill, muscle Open Access *Correspondence: wangyuanzhi621@126.com † Yu Zhang, Liang Jiang and Yicheng Yang contributed equally to this work 1 School of Medicine, Shihezi University, Shihezi 832002, Xinjiang Uygur Autonomous Region, China Full list of author information is available at the end of the article soreness and arthralgia. Moreover, the patient showed neurologic signs, such as headache and dizziness [11].

Case presentation
The patient was a 38-year-old previously healthy man, who was engaged in agricultural work. He reported that he was bitten by a tick on his left upper limb, at the tenth intercostal area of the left anterior axillary line on May 23, 2019. When he noticed and removed the tick, it was engorged. Two days later, a painful erythematous mass (about 6 × 6 mm in size) developed around the site of the tick bite. On May 30, he developed persistent fever (37.7 °C ± 0.2 ℃) and headache, and consulted a doctor at a local medical center. He was treated with calcium gluconate (1 g/day intravenously) and amoxicillin (0.5 g thrice daily orally) for 3 days. On June 4, his headache became increasingly severe, and was accompanied with nausea, mild neck stiffness and intense vomiting (more than 20 times per night). On June 5, the patient visited the Manasi County Hospital, XUAR. The patient received the brain MRI examination with DWI, and no obvious abnormality was found. Clinical hemogram, biochemical tests showed that most indexes were within the normal range. However, the white blood cell count (16. Table 1. Blood and CSF samples were negative in BacT/Alert blood culture system. To search for more eventual co-infecting pathogens, the patient' anticoagulated blood samples were collected upon admission (being diagnosed as acute meningitis). The DNA and RNA were extracted by a TIANamp Genomic DNA Kit and an TIANamp Genomic RNA Kit (Tiangen Biotech, Beijing, China), respectively. The complementary DNA (cDNA) was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Transgen Biotech, Beijing, China). Known tick-borne pathogens, including Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp., Francisella spp., Borrelia spp., forest encephalitis virus, severe fever and thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus, TcTV-1 and TcTV-2, were detected using nested PCR (nPCR) or reverse transcription-PCR (RT-PCR).
Four Rickettsia-specific genetic markers, including 543-, 445-, 435-, and 364-bp products of the genes encoding the cell surface antigen 1 (sca1), outer membrane proteins A (ompA), 17 kilodalton antigen (17-kDa), and mitochondrial 16S ribosomal DNA (16SrDNA), were amplified using previously described primers [17]. At the same time, the partial S segment of TcTV-1 (328 bp) was also screened by RT-PCR [11]. The PCR products were purified using the TIAN gel Midi Purification Kit (Tiangen, Beijing, China) and sequenced by Sanger di-deoxy sequencing method (Sangon Biotech, Shanghai, China). Each test was repeated three times. Obtained sequences corresponded to those of R. raoultii and TcTV-1 as The patient was treated with intravenous levofloxacin (0.5 g/day) and ribavirin (0.5 g/day) for 8 days and supportive treatment (potassium chloride oral solution, intravenous 20% mannitol, vitamin C and B6 injections). His mental status and body temperature improved at day 3 during hospitalization. On June 15, the patient was discharged from hospital although the data of mild laboratory abnormalities persisted, including the elevated protein concentration of CSF and gamma-glutamyl transpeptidase levels of serum. The patient was put on continuous oral doxycycline (100 mg, twice daily) and ribavirin (0.45 g, thrice daily) treatment at home for 4 days. After a 14-month follow-up, the tick bite patient recovered with no other complications.

Discussion and conclusions
In this study, we reported a severe acute meningitis case in a tick bite patient co-infected with R. raoultii and TcTV-1. Clinical manifestations included fever (37.7 °C ± 0.2 ℃), severe headache, persistently intense vomiting, mild neck stiffness and raised intracranial pressure (320 mm H 2 O) with mild mononuclear cells and elevated CSF protein levels. It is well known that spotted fever can cause central nervous system infection, presenting as fever, headache, vomiting, increased intracranial pressure and aseptic meningitis [19][20][21]. In similar studies, slightly increased intracranial pressure (200 mm H 2 O) was reported in a case of neurologic abnormalities caused by R. raoultii in northwestern China after tick bite, resulting in right eyelid ptosis, lethargy, fever (38.0-41.0 °C), headache and CSF leukocytosis [9]. Elevated intracranial pressure (235 mm H 2 O) have been reported in a case of Japanese spotted fever with central nervous system involvement and multiple organ failure [22]. In the present case, severe increased intracranial pressure (320 mm H 2 O) was also present, complicated with persistently intense vomiting. This finding means that (i) CSF pressure measurement is necessary in spotted fever patients with neurologic signs, and (ii) some emerging tickborne viruses should also be considered as co-infecting agents in all endemic areas of spotted fever.   Previously, an index patient single-infected with TcTV-1 also showed lymphocytic meningitis similar to that after SFGR infection [9,11,22]. In our study, the patient co-infected with TcTV-1 and R. raoultii presented increased intracranial pressure (320 mm H 2 O), resulting in severe headache and intense vomiting (> 20 times per night), which might account for overlapping clinical signs of the central nervous system between TcTV-1 and R. raoultii infections. In addition, there are major differences in therapeutic measures between infections caused by various tick-borne pathogens, as exemplified by R. raoultii and TcTV-1. Therefore, early diagnosis based on identification of multiple pathogens, is crucial in tickbitten patients with severe clinical syndrome. Public health workers and physicians need to be more aware of the clinical complexity of tick-borne pathogen infections, especially in high-risk areas of tick-borne diseases.
There are several limitations to our study. Firstly, rickettsia and TcTV-1 markers should have also been detected in CSF samples, although cellular and Fig. 1 Phylogenetic tree of patient's R. raoultii strain compared with avilable nucleotide sequences, selected using BLAST (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi). The tree was constructed by maximum-likelihood (ML; 1000 bootstrap replicates) of concatenated sequence data of four genes (17-kDa-sca1-rrs-ompA) using Molecular Evolutionary Genetics Analysis (MEGA, version 7.0; http:// www. megas oftwa re. net/). The concatenated sequence of R. raoultii are indicated by a black triangle (▲). The sequences of Rickettsia felis were used as the outgroup biochemical characteristics of CSF were useful in early diagnosis. Secondly, bacterial and viral metagenomic analysis should have been performed in more clinical samples (e.g., pharyngeal swab, urine, and cerebrospinal fluid) associated with the tick-bite patients. In future cases, these would help us to better understand complex clinical manifestations due to infection of single or multiple tick-borne pathogens.   2 Phylogenetic tree of patient's TcTV1 strain compared with available nucleotide sequences, selected using BLAST (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi). Phylogenetic tree of TcTV1 and related orthonairovirus were constructed based on the partial sequences (328 bp) of nucleoprotein with maximum likelihood method (ML; 1000 bootstrap replicates) using Molecular Evolutionary Genetics Analysis (MEGA, version 7.0; http:// www. megas oftwa re. net/). The TcTV1 sequence obtained in this study is indicated with a black triangle (▲). The sequences of hantaviridae orthohantavirus were used as the outgroup