Clinical Forms of Japanese Spotted Fever from Case-Series Study, Zigui County, Hubei Province, China, 2021

We report a case-series study of 5 patients with Japanese spotted fever from the Three Gorges Area in China, including 1 fatal case. Seroprevalence of Rickettsia japonica was ≈21% among the local population. Our report highlights the emerging potential threat to human health of Japanese spotted fever in the area.

USA) and 2 mM glutamine (Sigma, USA) two days before infection. The blood clot homogenate was inoculated into each well (50µL/well), and then the plate was centrifuged in bucket rotors at 700×g for 30 minutes. The plate was incubated at 32 o C and 5% CO2 for 3-5 days. When the culture medium was renewed every 4 days, the cells in each well were scratched manually with a streaking loop. The obtained cells were smeared on a slide and stained by Gimenez staining (Solarbio, China).

Isolation of DNA
Genomic DNA of isolated Rickettsia strain was prepared as previously described (11).
Mainly, rickettsiae was first released from infected Vero cells by a tissue grinder. Host cell debris was removed by centrifugation at 270 rcf for 5 minutes, and the supernatant was filtered through a 2.0 μm filter unit (Jinteng, Tianjin, China). Rickettsiae were recovered from the filtrate by centrifugation (17,000 rcf, 15 min 4°C) and resuspended in D-PBS with calcium and magnesium (Beyotime, Shanghai, China). The free host DNA were digested by treating with DNase I (15 µg/ml; from bovine pancreas Type II-S, Sigma-Aldrich) for 30 min at room temperature. After DNase I treatment rickettsiae were centrifuged again (17,000 rcf, 15 min 4°C) and genomic DNA was extracted using QIAamp Tissue kit (QIAGEN, USA) and quantified using a Qubit fluorometer (Life Technologies, Paisley, UK). The DNA sample was aliquoted and stocked at -80 o C until use. The whole genome of R. japonica str. YC21 was sequenced using MinION Nanopore (Oxford Nanopore Technologies, Oxford, UK) and Illumina Hiseq (Illumina, San Diego, CA, USA) methods.

MinION Sequencing
The library was prepared for MinION Nanopore sequencing using a Genomic DNA

Genome Assembly
Base calling of the fast5 files was performed using GUPPY (version 1.4.3-1; Oxford Nanopore Technologies). Reads were then BLAST searched against the NCBI nucleotide (nt) database (13). All long reads related to the genus Rickettsia were mapped to the reference Rickettsia japonica str. YH genome sequence (GenBank accession number NC_016050) using Minialign 0.5.3 (14) and coverage plots were visualized using Geneious v11.1 (15). To improve the accuracy of our assembly, the whole-genome Illumina short reads were mapped to the Oxford Nanopore long reads using BWA-MEM and errors were corrected (16). Rawdata was submitted to National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa/browse/CRA006321).

Medication of Patients
Case 1 first visited the village infirmary, complaining of a high fever, where she received an intravenous infusion of cefuroxime (1500 mg bid.), levofloxacin (400 mg qd), and ribavirin (500 mg qd). For the next two days, the dose of ribavirin was increased to 600-700 mg qd. She was then referred to the emergency ward of our hospital and treated with an intravenous infusion of ceftazidime (2000 mg q12h) and gastric administration of minocycline (100 mg qd). The patient died of multiple organ failure 2 days after admission.

Determination of Cutoff Values for IFA for JSF Diagnosis
R. japonica str. YC21 was co-cultivated in Vero cell culture to prepare antigen using T75 vented flasks and DMEM supplemented with 2% FBS. R. japonica str. YC21 co-cultured in Vero cells was grown in vented tissue culture flasks at 32 o C in an incubator with 5% CO2.
Briefly, when gross cytopathic effect was evident, we harvested infected cells by 0.25% trypsin digestion. The cell concentration was adjusted to 1.2-1.8×10 5 /mL with DMEM medium containing 2% FBS. Twenty microliters of the cell suspension was added to each well of 12-well slides, and the slides were then air-dried, fixed in acetone and stored at -20°C until they were used.
Sera from 30 anonymous healthy donors from Beijing (no JSF case reported), China and 15 JSF patients (collected by our team and diagnosed via PCR) were used to determine the cutoff values for IFA. The cut-off value was determined following the previous reports (20).