A 44-year-old male patient, residing in a village of Yunnan Province, was admitted to Xiangyun County People's Hospital in March 2021. He had a history of working in coal transportation. He visited the local Center for Disease Control and a hospital for recurrent cough and sputum for six years. He was diagnosed with PT, tuberculous bronchial stenosis, and silicosis. He was discharged with a recommendation for anti-tuberculosis treatment and given an anti-tuberculosis HRZE regimen (H: isoniazid, R: rifampin, Z: pyrazinamide, E: ethambutol), but the patient did not take the medication according to the doctor's advice every day. After taking anti-tuberculosis drugs on his own for five days, he was admitted to the hospital because of headache and fever for two days with rash, and a high body temperature up to 39.4℃. Rashes were observed on the face, head, neck, chest and abdomen, and limbs, distributing in patches with size ranging from a pinpoint to a grain of rice, which led to pruritus and a red halo at the base without rupture, separated by normal skin. The lips and mouth were slightly cyanotic, and the breath sounds of both lungs were coarse, with scattered moist rales. The other examination results were unremarkable.
By laboratory examination, normal level of blood cells were observed as follows: white blood cells (7.3×109/L), hemoglobin (155g/L), platelets (202×109/L), neutrophil percentage (90.6%) and lymphocyte count (7.8×109/L). Biochemical tests showed impaired liver function: elevated aspartate aminotransferase(AST)(50U/L), elevated gamma-glutamyl transferase༈GGT༉(64U/L), slightly lower prealbumin (145.5 mg/L), but normal alanine aminotransferase༈ALT༉(21U/L). Inflammatory biomarker tests showed an elevated calcitonin inogen (0.63 ng/mL) and elevated C-reactive protein (59 mg/L). Coagulation analysis showed a prolonged prothrombin time of 13.2s. Plasma fibrinogen was elevated at 4.80g/L. Routine urinalysis showed that the patient was positive (+) for urine protein; positive (+) for blood cell; and positive (++) for urinary ketones, suggesting the presence of impairment of renal function.
No acid-fast bacilli were detected by pooling the patient's nocturnal sputum, immediate sputum and morning sputum for testing. According to the patient's chest computed tomography (CT) scan (bilateral lungs and mediastinum) findings, the scattered nodular and lamellar shadows in both lungs were considered as tuberculosis most likely, and the lesions in the lingual segment of the upper lobe of the left lung and the lower lobe of the left lung were probably infectious lesions (Fig. 1A;Figure 1C). The patient was tested for the antibodies (IgM and IgG) against OHV due to the patient from the national foci of HFRS, but the results were negative.
On the first day after admission, the patient suspended his anti- tuberculosis treatment and was given an acetaminophen oral suspension. Subsequently, his temperature decreased and anti-infective treatment s were provided with methylprednisolone sodium succinate. The blood gas analysis of the patient showed that the pH value was 7.48, the oxygen partial pressure was 53.2 mmHg, the oxygen saturation was 90.1%, and the potassium ion (K+) was 2.8 mmol/L. The presence of electrolyte disorders (hypokalemia, hyponatremia) was treated with sodium chloride and potassium chloride injections. On the second day after admission, the patient's temperature fluctuated between 38.1–38.7 ° C, and he still had headache, cough, expectoration and other uncomfortable symptoms. For the symptoms of headache and rash, he took rotundine and ebastine tablets for symptomatic treatment. Later examination showed that the rash on the patient's face, head, chest and neck, and limbs had subsided, and the patient felt relief from the headache. On the third day after admission, the patient continued with symptomatic treatment for anti-infection and cough suppression. The patient sometimes had fever, discomfort, cough and expectoration with little sputum, which was yellow-white sputum. On the Fourth day after admission, the patient was discharged after the rash and fever subsided at the request of the patient and his family.
In cases where serological and clinical examinations could not identify the specific cause of infection, rash and fever, we performed retrospectively etiological testing of the whole blood and serum from the collected patent previously. Nucleic acid (DNA/RNA) extraction kit (TIANGEN, China) was used to extract viral nucleic acid from the patient whole blood and serum, and the gene sequence was amplified by one-step polymerase chain reaction(PCR)method using universal primers of OHV according to reference[13].PCR amplification was performed using the 56-kDa type specific antigen (TSA) gene of OT according to references [14, 15]. The agarose gel electrophoresis experiment was carried out under the imager (Fig. 1E).The PCR products were purified by gel cutting and sent to a sequencing company (Shanghai Sangon Biotech) for sequencing. The 362bp sequence of OHV༈accession no.OP392989༉and the 172bp DNA sequence of OT were obtained from serum samples. Then primers (ICRA-F2:5 '- CCTCAGTATAATGCCC-3' and ICR8A-R: 5 '- TCCTGCATGACGCTGCAA-3') were designed to obtain 449bp DNA sequence of tsutsugamushi (accession no.OP392990).
Nucleotide sequence similarity searches in the public databases were assessed by the Basic Local Alignment Search Tool, implemented in the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/blast/), using BLASTn, and BLASTn optimized for highly similar sequences (MEGABLAST) and BLASTp, algorithms. In BLAST, it was 92.54% and 97.69% that the highest identity of nucleotide(nt) and amino acid(aa) compared the OHV sequence(OP39298)in this study to SEOV L0199 strain(HQ992814) and SEOV Rn-SHY17 ( ADR32120.1 ). The highest nt and aa identity compared the obtained OT sequence༈OP392990༉to Gilliam genotype of HZ01034 strain (MT258795.1) and Orientia tsutsugamushi str. Gilliam (KJV51889.1) was 96.88%, 93.96% respectively.
Phylogenetic trees were analyzed for the obtained OHV sequences (362bp) and OT sequences (449bp), and the related sequences retrieved in the Genbank database. The each sequence set was aligned by Clustal-X, and phylogenetic relationships were reconstructed using MEGA X for the initial trees obtained by the maximum likelihood neighbor joining method. In the nucleotide substitution models, the K2 + I and T92 + G models were selected for Bootstrap analysis using 1000 replicates to improve the confidence level of the phylogenetic tree, respectively (Fig. 1F-G).The results showed that the patient was infected with SEOV of Orthohantavirus and Gilliam genotype of O. tsutsugamushi.