A 30-year-old male Sanya local farmer with symptoms of emaciation (unintentional loss of 3 kg in 1 month), left lower abdominal pain of unknown cause associated with posture and aggravated by turning, went to the nearby health service center for medical treatment with no apparent improvement. Five days later, on 7th July 2021 (Day 1), he was admitted to Sanya People's Hospital with fever of unknown origin (Tmax 38 C) and complications of hypoproteinemia, electrolyte imbalance and hyperglycemia (Table 1). Medical history: denied hepatitis, tuberculosis, hypertension, diabetes, heart disease, cancer, etc. On 8th July (Day 2), the patient underwent a computed tomography (CT) plain scan of the upper abdomen, which revealed the enlarged spleen with low-density from the splenic and left psoas major, showing a possible abscess with inflammatory exudate (Figure 1). Spleen puncture was not performed due to the poor location of the abscess and the poor patient compliance. The patient was started on symptomatic treatment for sepsis and hyperglycemia. However, the patient deteriorated rapidly and remained febrile with worsening abdominal pain and tendency to ketoacidosis. Cefoperazone-tazobactam was promptly administered to the patient based on the detection of serum anti- Hcp1 antibodies to B. pseudomallei, which were determined to be strongly positive for IgG strip (Figure 2). After intravenous antibiotic and supportive therapy, the relief of symptoms on 16th July (Day 10) provided the window of opportunity for the ultrasound-guided puncture aspiration of the left psoas major abscess. The lesion recovered well on the second day after the abscess was aspirated. The diagnosis of splenic and psoas major abscess due to melioidosis was finally confirmed on 20th July (Day 14) by pus culture as shown in Figure 3 (blood cultures taken prior to antibiotics were negative), and anti-infective therapy was continued. On 27th July (Day 21) the patient gradually improved from sepsis with stable blood glucose. Inflammatory indicators returned to normal, lesion exudation decreased, and the patient was discharged from hospital on oral cefixime eradication therapy for TMP-SMZ resistance. Follow-up imaging with contrast-enhanced abdominal CT on 20th October 2021 (3 months after discharge) showed abscess resolution.  
The antigen reagents for the serological detection, including the recombinant Hcp1 were prepared in our laboratory. Hcp-1 enzyme-linked immunosorbent assay (ELISA), and Hcp-1 gold immunochromatography assay (GICA) were performed to detect IgG and IgM directed against B. pseudomallei in the patient's serum. The sera were collected at five different time points during the days of hospitalization (Figure 2B). Two replicates of the negative control, two replicates of the positive control, one blank control and two replicates of samples were included in each ELISA. After the reaction, the optical density (OD) was measured at a single wavelength of 450 nm. The antibody titres were determined by ELISA method (the maximum dilution multiple of the antibody with OD value above the cut-off). The OD value of the patient's first serum IgG was 1.471 (cut-off 0.105) at a titre of 1:3200; the OD value of the fifth sample was 2.761 at a titre of 1:25,600. The OD value of the patient's first serum IgM was 0.2915 (cut-off 0.29) at a titre of 1:100; the OD value of the fifth sample was 0.23 at a titre of 1:20. For GICA, the diluted serum was added to the strip and read after 15 minutes, only IgG was positive (Figure 2A). According to the Hcp1 ELISA, the patient showed a more than 4-fold increase in IgG titre, while the IgM remained low.
The patient's blood was collected and inoculated into both aerobic and anaerobic blood culture bottles and placed in BACT/ALERT  3D (BioMerieux, France), which showed no growth after 5 days. The aspirated pus from the puncture was thick yellow fluid. Direct smear and Gram stain showed tiny numbers of Gram-negative rods with WBC (Figure 3A). The pus was inoculated onto Columbia blood agar, non-inhibitor chocolate agar, and China blue agar and cultured in a constant temperature incubator in a CO2 enhanced atmosphere at 35 C. After 24 hours of culture, small, smooth, creamy white, neatly edged, milky and slightly sticky colonies were seen on the blood plates. 72 hours later, pale white-yellow, subcircular, dry, wrinkled, and opaque "daisy-like" colonies were observed (Figure 3B-D). No growth was observed on other culture media including anaerobic, mycobacterial, and fungal cultures. The selected single colony stained was showed as Gram--negative bacilli and acid-fast staining was negative. The isolated strain was then subjected to BD Phoenix-100 Automated Microbiology System (BD Biosciences, Franklin Lakes, NJ), and identified as B. pseudomallei. In addition, genomic DNA extracted from the isolated strain using a Bacterial DNA Kit (TIANGEN, Beijing), and amplified using a 16S rDNA Bacterial Identification PCR Kit (Sangon, Shanghai), was sent to BGI company for sequencing. Direct sequencing and phylogenetic analysis of the PCR amplification products showed that the isolate (named BP-HS) was unequivocally clustered with B. pseudomallei (Figure 3E). The scale bar of the evolutionary distance divergence is 0.001.
The strains isolated from this case were also subjected to antimicrobial susceptibility testing using the microbroth dilution method according to CLSI guidelines. The CLSI M45 susceptible/resistant breakpoints were used. Briefly, the isolate was cultured in Columbia blood plate for 24 hours at 35  C in 5% CO2, and the turbidity was adjusted to 0.5 McFarland, then the bacterial suspension was diluted with Mueller Hinton broth. Ceftazidime, imipenem, cefoperazone-sulbactam (CFP-S) and trimethoprim-sulfamethoxazole (TMP-SMZ) were then applied. After incubation in a 5% CO2 incubator at 35 C for 16-24 hours, the clinical isolate of B. pseudomallei showed a susceptibility and resistance profile with susceptibility to ceftazidime (MIC=2mug/mL), cefoperazone-sulbactam (MIC<=16/8mug/mL), imipenem (MIC<=1mug/mL), and resistance to trimethoprim- sulfamethoxazole (MIC=4/76mug/mL).