PMC8635719_01
The mNGS and bone marrow result detected in Case 1. (A) The genome coverage of the detected reads of Leishmania shedunii. (B) The abundance of detected microbe and their reads numbers. A total of 5074 specific reads (98.87%) of Leishmania were detected in the blood sample. (C) The examination of a bone marrow aspirate revealed amastigotes.
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F1
PACE-seq metagenomic next-generation sequencing (mNGS) (Hugobiotech, Beijing, China) detected 5,074 specific reads of Leishmania in the blood sample, also indicating leishmaniasis (Figure 1).
The dose of liposomal amphotericin B (L-AmB) and changes of temperature in Case 1. The initial dosage of L-AmB in the first patient was 10mg/day and the patient's temperature decreased as the dose of L-AmB was escalated. Dose escalation was performed as follows: 10 mg on day 1, 30 mg on day 2, 80 mg on day 3, and 120 mg on day 4. However, the appearance of hypotension on day 4 of the treatment protocol forced the dose reduction on day 5 (80 mg/d). There was mild renal impairment on day 6. Therefore, treatment was interrupted on days 6-11, and his renal function gradually recovered during treatment cessation. Treatment was restarted at a dose of 10 mg/kg from day 12, and the dose was gradually increased at a rate of 10 mg/(kg day). The dose was increased to 40 mg on day 15. Since the patient's renal function was still slightly abnormal at day 15, the dose of L-AmB was not increased, but maintained at 40 mg during the next 12 days (day 16-27). Finally, we stopped the medication until the accumulating dose reached 890 mg(12.6 mg/kg) on the day 27. Polymerase chain reaction of Leishmania in a bone marrow smear was negative on day 17.
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F2
Then dose escalation of the L-AmB for the patient is presented in Figure 2. The patient's temperature and L-AmB dose were presented in Figure 2, and his basic information was summarized in Table 1.
PMC8635719_02
Liposomal amphotericin B (L-AmB) administration protocol and temperature changes in Cases 2-4. TAD, total accumulated dosage.   Normalization of body temperature.   Negative PCR result for bone marrow aspirate. To avoid complications associated with adverse drug reactions, the dosing regimens of L-AmB in case 2-4 were similar to those used for case 1, starting with a low dose that was gradually increased until the maximum tolerated dose was reached. This dosage was used for maintenance therapy. The initial dose for case 2 was 5 mg (~0.1 mg/kg) on day 1. The dose was increased to 10 mg on day 2-3, and no complications occurred. Therefore, the patient received L-AmB in 10 mg dose increments up to a dose of 60 mg (~1.2 mg/kg) on day 8. This dosage (60mg) was used for maintenance therapy until the cumulative dose of L-AmB reached 765 mg (13.9 mg/kg) on day 17. Her body temperature returned to normal on day 4. Polymerase chain reaction (PCR) for Leishmania in bone marrow aspirate was negative on day 12, when the dose of L-AmB was 405 mg. The trapezoidal regimen of L-AmB in case 3 was initiated at a dose of 5 mg (~0.1 mg/kg). The dosage of L-AmB was increased by 5 mg every 2 days as tolerated from day 1 to day 8 and increased by 10 mg every 2 days from day 9 to day 13 to 50 mg (~1.0 mg/kg), which was administrated as maintenance therapy on days 14-22. Treatment was stopped when the cumulative dose of L-AmB reached 740 mg(14.8 mg/kg) on day 22 of protocol. The patient's body temperature had returned to normal on day 4. PCR for Leishmania in bone marrow aspirate was negative on day 15, when the cumulative dose of L-AmB was 400 mg. In case 4, L-AmB treatment was initiated at a dose of 10 mg and increased in increments of 10 mg (~0.14 mg/kg) daily, reaching 70 mg (~1.0mg/kg) on day 7 of the protocol. To avoid complications, instead of further increasing the L-AmB dose, we used a dose of 70 mg as maintenance therapy until the cumulative dose reached 910 mg (13 mg/kg) on day 16. His body temperature returned to normal on day 7. PCR for Leishmania in bone marrow aspirate was negative on day 9, when the cumulative dose of L-AmB was 420 mg.
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F3
The L-AmB protocol followed that presented in Figure 3.
PMC8635719_03
Liposomal amphotericin B (L-AmB) administration protocol and temperature changes in Cases 2-4. TAD, total accumulated dosage.   Normalization of body temperature.   Negative PCR result for bone marrow aspirate. To avoid complications associated with adverse drug reactions, the dosing regimens of L-AmB in case 2-4 were similar to those used for case 1, starting with a low dose that was gradually increased until the maximum tolerated dose was reached. This dosage was used for maintenance therapy. The initial dose for case 2 was 5 mg (~0.1 mg/kg) on day 1. The dose was increased to 10 mg on day 2-3, and no complications occurred. Therefore, the patient received L-AmB in 10 mg dose increments up to a dose of 60 mg (~1.2 mg/kg) on day 8. This dosage (60mg) was used for maintenance therapy until the cumulative dose of L-AmB reached 765 mg (13.9 mg/kg) on day 17. Her body temperature returned to normal on day 4. Polymerase chain reaction (PCR) for Leishmania in bone marrow aspirate was negative on day 12, when the dose of L-AmB was 405 mg. The trapezoidal regimen of L-AmB in case 3 was initiated at a dose of 5 mg (~0.1 mg/kg). The dosage of L-AmB was increased by 5 mg every 2 days as tolerated from day 1 to day 8 and increased by 10 mg every 2 days from day 9 to day 13 to 50 mg (~1.0 mg/kg), which was administrated as maintenance therapy on days 14-22. Treatment was stopped when the cumulative dose of L-AmB reached 740 mg(14.8 mg/kg) on day 22 of protocol. The patient's body temperature had returned to normal on day 4. PCR for Leishmania in bone marrow aspirate was negative on day 15, when the cumulative dose of L-AmB was 400 mg. In case 4, L-AmB treatment was initiated at a dose of 10 mg and increased in increments of 10 mg (~0.14 mg/kg) daily, reaching 70 mg (~1.0mg/kg) on day 7 of the protocol. To avoid complications, instead of further increasing the L-AmB dose, we used a dose of 70 mg as maintenance therapy until the cumulative dose reached 910 mg (13 mg/kg) on day 16. His body temperature returned to normal on day 7. PCR for Leishmania in bone marrow aspirate was negative on day 9, when the cumulative dose of L-AmB was 420 mg.
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F3
The L-AmB protocol followed that presented in Figure 3.
PMC8635719_04
Liposomal amphotericin B (L-AmB) administration protocol and temperature changes in Cases 2-4. TAD, total accumulated dosage.   Normalization of body temperature.   Negative PCR result for bone marrow aspirate. To avoid complications associated with adverse drug reactions, the dosing regimens of L-AmB in case 2-4 were similar to those used for case 1, starting with a low dose that was gradually increased until the maximum tolerated dose was reached. This dosage was used for maintenance therapy. The initial dose for case 2 was 5 mg (~0.1 mg/kg) on day 1. The dose was increased to 10 mg on day 2-3, and no complications occurred. Therefore, the patient received L-AmB in 10 mg dose increments up to a dose of 60 mg (~1.2 mg/kg) on day 8. This dosage (60mg) was used for maintenance therapy until the cumulative dose of L-AmB reached 765 mg (13.9 mg/kg) on day 17. Her body temperature returned to normal on day 4. Polymerase chain reaction (PCR) for Leishmania in bone marrow aspirate was negative on day 12, when the dose of L-AmB was 405 mg. The trapezoidal regimen of L-AmB in case 3 was initiated at a dose of 5 mg (~0.1 mg/kg). The dosage of L-AmB was increased by 5 mg every 2 days as tolerated from day 1 to day 8 and increased by 10 mg every 2 days from day 9 to day 13 to 50 mg (~1.0 mg/kg), which was administrated as maintenance therapy on days 14-22. Treatment was stopped when the cumulative dose of L-AmB reached 740 mg(14.8 mg/kg) on day 22 of protocol. The patient's body temperature had returned to normal on day 4. PCR for Leishmania in bone marrow aspirate was negative on day 15, when the cumulative dose of L-AmB was 400 mg. In case 4, L-AmB treatment was initiated at a dose of 10 mg and increased in increments of 10 mg (~0.14 mg/kg) daily, reaching 70 mg (~1.0mg/kg) on day 7 of the protocol. To avoid complications, instead of further increasing the L-AmB dose, we used a dose of 70 mg as maintenance therapy until the cumulative dose reached 910 mg (13 mg/kg) on day 16. His body temperature returned to normal on day 7. PCR for Leishmania in bone marrow aspirate was negative on day 9, when the cumulative dose of L-AmB was 420 mg.
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F3
The L-AmB protocol followed that presented in Figure 3.