A 50-year-old male with end-stage renal disease received a left kidney transplant from a deceased male donor who died in a motor vehicle accident in March 2013. After renal transplantation, the patient received a maintenance immunosuppressive regimen consisted of tacrolimus (3.5 mg, bid), mycophenolate mofetil (360 mg, bid), and prednisone (4 mg, qd). At 11 months postoperatively, 1+ to 2+ proteinuria was found on a routine urine examination. The proteinuria was relieved after treatment with Tripterygium glycosides tablets (10 mg, bid). At 15 months postoperatively, the patient developed BKV viruria with a urinary viral load of 1.25 x 107 copies/mL (normal range for reference, <5,000 copies/mL). The viral load was undetectable after the dosage of tacrolimus was reduced to 1.5 mg BID and treating with immunoglobulin (infusion). In April 2016, the patient had a chronic rejection reaction and the 24-h urinary protein quantity increased to 1.13 g/24 h. To maintain the allograft function and suppress proteinuria, the corresponding treatment regimen was methylprednisolone (40 mg) combined with cyclophosphamide (0.2 g) intravenous drip for 3 days/month. After three courses of treatment, the 24-h urinary protein quantity decreased to 0.56 g/24 h. The patient has had recurrent symptoms of UTI such as frequent and urgent urination without obvious inducement since June 2016. Regular outpatient review of urinary examination revealed leukocytes fluctuating from 1+ to 3+. E. coli was detected in the midstream urine culture and intravenous cefoperazone sodium sulbactam (1.5 g, 1/12 h) was given for 1 week. In September 2018, the patients came to the hospital because of cough for 1 day. mNGS of the alveolar lavage fluid indicated pneumosporidiosis and blood tested positive for herpes simplex virus. The pulmonary infection resolved after treated with compound sulfamethoxazole tablets (480 mg, bid). Besides, serum creatinine decreased from 305 mumol/L to 245 mumol/L. However, BKV viruria relapsed with the urinary viral load fluctuated from 2.84 x 105 copies/mL to 3.81 x 107 copies/mL. The immunosuppressive regimen was adjusted to tacrolimus (1.5 mg, bid), mycophenolate sodium enteric-coated tablets (180 mg, bid), and prednisone (4 mg, qd). In November 2018, the patient's serum creatinine was 197 mumol/L, and color Doppler examination of the transplanted kidney and renal vessels showed no significant abnormalities. Regular color Doppler ultrasound examinations of the allograft and transplanted kidney vessels were performed every 6 months after transplantation, all showing neither significant abnormalities nor transplanted kidney masses until the current admission. The patient had not undergone an allograft puncture biopsy within 3 years after renal transplantation. His postoperative serum creatinine level was 190 mumol/L. And the postoperative glomerular filtrate rate (GFR) was summarized in Figure 1. The patient was admitted to hospital in May 2019 because of frequent and urgent urination. The clinical course of the patient is summarized in three phases according to the disease progression.
In May 2019, the patient was admitted to hospital for recurrent frequent and urgent urination. On admission, his serum creatinine level was 229 mumol/L. Ultrasound and PET/CT showed solid space-occupying lesions in the upper pole of the transplanted kidney and bladder, prostate and seminal vesicles (Figures 2A1-H1). Biopsy of the graft kidney and bladder lesions revealed cryptococcal granulomas, with cystoscopy results provided in Supplementary Figure 1. Extended-spectrum beta-lactamase positive E. coli was cultured from both renal graft tissue and midstream urine. To rule out systemic cryptococcosis, a lumbar puncture was performed to collect CSF and CSF opening pressure measured. Cell counts and biochemical parameters in CSF, and CSF opening pressure were normal. Ink staining and Cryptococcus antigen detection of CSF were both negative. No significant abnormalities were observed in brain MRI and chest CT scans. Based on the imaging and pathological findings, the patient was diagnosed with cryptococcoma and malacoplakia of the genitourinary system including transplanted kidney, bladder, prostate and seminal vesicles (Figures 2A1-H1 and Figure 3), accompanied by an UTI of E. coli. The patient was treated with meropenem (0.5 g twice a day), and fluconazole (50 mg twice a day) combined with flucytosine (0.5 g twice a day). Meanwhile, immunosuppression was reduced by conversion from tacrolimus (1.5 mg twice a day) to cyclosporine (100 mg twice a day). Additionally, cyclosporine was later adjusted to 75 mg BID after use of the antifungal drug, fluconazole, which can affect the concentration of cyclosporine. The concentrations of immunosuppressants from renal transplantation to transplant nephrectomy were shown in Supplementary Figure 4. After 10 days' treatment, his renal function improved and the serum creatinine decreased from 229 to 185 mumol/L. In July 2019, his chest X-ray showed a soft tissue shadow in the left hilum, which was diagnosed as pulmonary tuberculosis by bronchoscopy; and the patient was thus treated with isoniazid (300 mg, once daily), ethambutol (750 mg, once daily) and levofloxacin (250 mg, once daily).
Follow-up ultrasound and FDG-PET/CT examination in August 2019 showed that although the area of bladder lesion was significantly reduced from 4.7 x 4.1 x 5.5 cm to 3.8 x 2.3 x 3.6 cm, the lesion in the transplanted kidney was enlarged from 4.3 x 4.9 x 4.1 cm to 6.0 x 4.0 x 4.7 cm, with the cryptococcoma and malacoplakia in the upper pole of the transplanted kidney protruding into the adjacent Gerota's fascia (Figures 2A2-H2). Fluconazole was given at 100 mg BID against cryptococcal infection while the anti-Escherichia coli as well as the anti-tuberculosis regimen was maintained, as described in Phase I. The net immunosuppression status of an individual is influenced by the immunosuppression regimen and individual susceptibility and can be assessed by immunosuppressive drug concentrations, peripheral blood leukocyte counts, lymphocyte counts, and viral infection conditions. The peripheral blood leukocyte and lymphocyte counts of this case were lower compared to the average level during uninfected period (Supplementary Figure 5). The presence of pneumosporidiosis, herpes simplex virusemia, and BKV uremia were all suggestive of a low net immunosuppressive status. To stop the disease progression, the patient received a transplant nephrectomy 1 week later.
After transplant nephrectomy, the patient's immunosuppressants were discontinued. He was on dialysis three times a week. The anti-tuberculosis treatment was changed to isoniazid (300 mg, once daily), ethambutol (870 mg, once every 2 days) and levofloxacin (250 mg, once every 2 days), while the anti-Cryptococcus treatment was changed to fluconazole (100 mg, twice a day) and flucytosine (0.5 g, three times a day). After 2 months, a follow-up FDG-PET/CT showed that the lesions in the urogenital system and lung were significantly reduced compared with the last examination (Figures 2A3-H3). Six months post-transplant nephrectomy, the lesions in the genitourinary system were eventually eliminated (Figures 2A4-H4).
Gross inspection of the transplanted kidney showed that the resected transplant volume was 11 x 9.0 x 5.0 cm with its capsule closely adherent to the surrounding fat. A solid yellow mass of 6.0 x 4.0 x 4.7 cm was found in the upper pole renal parenchyma without breaking through the renal capsule. Another 5.0 x 2.5 x 2.5 cm yellow mass was found in the hilar sinus fat (Figure 3F). The pathological changes of the transplanted kidney lesions were consistent with Cryptococcus infection (Figures 3A-E).
Metagenome sequencing revealed that only two and four sequences of C. albidus were respectively detected in the CSF and feces specimens in Phase I. Two sequences of C. albidus were detected in the CSF specimen in Phase II. After transplantation nephrectomy, withdrawal of immunosuppressants and anticryptococcal therapy for 2 months, one sequence of C. albidus was detected in the feces specimen but no sequence in CSF in Phase III. All sequences were typed as [Cryptococcus] albidus var. albidus strain NRRL Y-1402 (Table 1). The type of the E. coli detected in urine and granuloma of the allograft in Phase I and Phase II was E. coli 09-02E (Table 1).
In order to understand the interactions between E. coli and Cryptococcus, we cocultured the two microorganisms in vivo. After an 8-h co-culture of E. coli 09-02E filtrate and Cryptococcus neoformans JEC21 (ATCC@96910) in vitro, the Cryptococcus counts in the control group (without E. coli filtrate) and the experimental group (adding 80, 160, 320, 640, and 1,280 mul E. coli filtrate, respectively) were respectively 2.46 +- 0.52 x 105/mL, 2.30 +- 0.57 x 105/mL, 2.83 +- 0.72 x 105/mL, 3.13 +- 0.76 x 105/mL, 3.09 +- 0.61 x 105/mL, and 2.60 +- 0.63 x 105/mL. Comparison between the control group and the experimental Group III (320 mul E. coli filtrate) and Group IV (640 mul E. coli filtrate) showed a statistically significant difference (P < 0.05) (Supplementary Figure 2), indicating that E. coli at these concentrations may stimulate cryptococcal growth.