A 53-year-old male patient with a one-week history of fever with a maximum body temperature of 39.4℃, shivering with cold, followed by occasional sputum cough and no skin lesions, was admitted to our hospital on March 21, 2019.
The patient had a history of hepatitis B for over twenty years and type 2 diabetes treated with insulin. On October 9, 2008, the patient underwent orthotopic liver transplantation due to hepatitis B cirrhosis. The patient had a regular outpatient visit. Tacrolimus was given for anti-rejection therapy to maintain plasma concentrations at normal levels (reference range: 5-15ng/ml). The patient was on anti-HBV therapy with entecavir (0.5g/day.). In October 2018, the patient underwent a biopsy of the transplanted liver for elevated alanine aminotransferase (118 U/L). Pathology showed chronic inflammatory liver injury (G2-3S3), which was considered as acute rejection. The patient was on oral methylprednisolone (8 mg/day) for over three months.
A computed tomography (CT) scan of the chest on admission showed bilateral infiltrations and scattered multiple nodules (the largest one being 14 mm in diameter) in both lungs (Fig. 1A). The blood tests performed on March 22 showed a white blood cell (WBC) count of 7.37×109/L, an absolute lymphocyte (LYMP) value of 1.14×109/L, a hemoglobin (Hb) level of 146 g/L, platelet count of 44×9/L, aspartate aminotransferase (AST) of 78 IU/L, ALT of 81 IU/L, total bilirubin (TBILI) of 38.1 µmol/L, direct bilirubin (DBILI) of 17.7 µmol/L and serum creatinine of 90 µmol/L. Serum 1,3-beta-D-glucan (BDG) level was below 3.836 pg/ml. Aspergillus galactomannan (GM) was 1.593 µg/L. C-reactive protein (CRP) was 59.6 mg/L, and procalcitonin (PCT) was 0.85 ng/ml. Prothrombin time (PT) was 18.1 sec. Intravenous cefoperazone sulbactam (3.0 g every 12h) and moxifloxacin (0.4 g/day) were started for antimicrobial therapy. Intravenous voriconazole at a loading dose of 400 mg every 12h on day one and 200 mg every 12h after that was started for antifungal therapy after admission. However, the patient still had a fever of up to 39.9°C with chills. Antibacterial therapy was switched to meropenem (1 g every 8h) on March 23. Moreover, antifungal therapy with caspofungin was introduced on March 24, with a loading dose of 70 mg on day one and a maintenance dose of 50 mg daily after that. The dose of tacrolimus was gradually reduced from 1 mg bid to 1 mg q3d. After these treatments, the patient's body temperature gradually returned to normal, and the patient has been fever-free since March 25. Human immunoglobulin for intravenous administration (IVIg) was given at a dose of 5 g daily for 14 days starting March 26. On March 28, blood cultures showed T. marneffei, which was performed on admission. Sputum culture was negative. On April 5, blood tests showed a WBC count of 3.04×109/L, LYMP of 0.38×109/L, Hb level of 103 g/L, platelet count of 52×10E9/L, AST of 24 IU/L, ALT of 18 IU/L, and serum creatinine of 70 µmol/L, TBILI of 20.8 µmol/L, DBILI of 8.8 µmol/L. The plasma tacrolimus concentration was 8.8 ng/ml. On April 6, a chest CT showed that the bilateral lung infiltrations were significantly absorbed, and most of the nodules in both lungs became smaller. A blood culture performed on April 5 was negative. On April 8, intravenous voriconazole, which had been used for two weeks, was switched to oral administration at a dose of 200 mg twice daily, and the patient was discharged from the hospital.
Two days later (on April 10), the patient suddenly developed a high fever of 39.4℃ and was admitted to our hospital again. Blood tests showed a WBC count of 3.6×109/L, LYMP of 0.68×1009/L, Hb level of 108 g/L, and a platelet count of 66×1009/L. The high sensitivity C-reactive protein (hsCRP) was 49.34 mg/L. PCT was 0.179ng/ml. Intravenous imipenem cilastatin (1.0g every 8 hours) and voriconazole (200 mg every 12 hours), and oral oseltamivir (75 mg twice daily) were initiated. Tacrolimus was discontinued. Furthermore, intravenous methylprednisolone (MP) was started at 20 mg/day for four days and then reduced to 10 mg/day for one day. The patient was fever-free from April 12 to April 14. However, on April 15, the patent became febrile again, and oseltamivir was discontinued. On April 16, the chest CT showed infiltrations of both lungs were absorbed, and some nodules in both lungs became smaller. Peripheral blood and sputum samples were collected and sent for mNGS testing by Vision Medicals CO. Ltd (Guangzhou, China) using Illumina NextSeq 550 (USA). IVIg was given at 20 g per day for five days. mNGS detected 11 high-confidence sequence reads for T. marneffei in blood and 20,628 high-confidence sequence reads for Enterococcus faecium in sputum on April 17. The T. marneffei sequence reads mapped to the T. marneffei reference genome (NW_002196661.1), the Enterococcus faecium sequence reads mapped to the Enterococcus faecium reference genome (NC_017960.1) (Fig. 2). The patient had a fever of 39.9°C and was given linezolid (600 mg every 12 h) against Enterococcus faecium; imipenem/cilastatin was switched to cefoperazone/sulbactam (3.0 g every 12 h). Considering the side effects of amphotericin B, caspofungin (50 mg/day) was used synergistically as an antifungal. Although the fever peak was decreased, the patient still had a daily fever with a peak of 38.5°C. The antinuclear antibody (ANA), anti-double-stranded DNA antibody (dsDNA), anti-neutrophil cytoplasmic antibody (ANCA) series, and extractable nuclear antigen (ENA) series were all negative. GM was 5.81µg/L. The BDG was 129.983 pg/ml. The DNA copies of CMV and EBV were less than 500 copies/ml. Hepatitis B and C viruses, and HIV were all tested negative by serology. Syphilis was tested positive by TPPA (119.58) but negative by TRUST. The plasma concentration of tacrolimus was 7.7 ng/ml. Sputum was negative for smear analysis of acid-fast bacilli. The A and B antigens of Mycobacterium tuberculosis were negative according to the T-SPOT blood assay. The flow cytometry of peripheral blood cells showed CD3 + CD4 + T cells/ T lymphocytes was 25.1% (34%-70%), CD3 + CD8 + T cells/T lymphocytes was 53.5% (25%-54%), and CD3 + CD4 + T cells/CD3 + CD8 + T cells was 0.47 (0.68–2.47). The blood cultures performed on April 11 and April 16 were both negative. Sputum culture was also negative. A chest CT on April 24 showed increased and larger nodules (Fig. 1B). Caspofungin was discontinued, and cefoperazone-sulbactam was switched to piperacillin-tazobactam (4.5 g every 8h). Amphotericin B was started via the central venous catheter on April 25. The dose of amphotericin B was started at 1 mg, then 3 mg, then 5 mg, and increased by 5 mg daily until the daily dose reached 35 mg. From May 3, the dose of amphotericin B was 35 mg daily. The MP was given to reduce the side effects of amphotericin B at a dose of 20 mg daily for three days, then 10mg daily. Linezolid was discontinued on April 26. Since April 27, the patient has been fever-free. On May 8, BDG was below 3.836 pg/ml, and voriconazole was discontinued, which was reduced to 200 mg per day on May 2. On May 8, IVIg was given at a dose of 15 mg per day for four days. On May 9, a chest CT showed a decrease in nodules and a smaller size. The creatinine rose to 203 µmol/L on May 13, and amphotericin B was switched to liposomal amphotericin B at a dose of 100 mg for three days on May 14, then 150 mg for seven days, then 100 mg for one day, and discontinued at discharge on May 24. Meanwhile, dexamethasone 2 mg was used daily to reduce its adverse effects. On May 17, piperacillin-tazobactam was switched to cefoperazone-sulbactam (3.0 g every 12h) and discontinued at discharge. The sequence in blood detected by mNGS turned negative on May 17. On May 24, blood tests showed a WBC count of 7.88×109/L, the absolute value of lymphocyte (LYMP) of 0.8×109/L, a hemoglobin (Hb) level of 88 g/L, a platelet count of 53×109/L, an AST of 18 IU/L, an ALT of 39 IU/L, serum creatinine of 120 µmol/L, TBILI of 14.6µmol/L, DBILI of 6.9µmol/L. The patient was discharged without fever or cough and given oral voriconazole at a dose of 200 mg every 12 h. On August 6, the sequence in the blood was detected by mNGS was negative. On August 30, a chest CT showed that the nodules had been absorbed (Fig. 1C), and oral voriconazole was discontinued after 14 weeks. The patient continues to be followed up regularly at the outpatient department of the liver transplant clinic and has not relapsed.