Short-course High-dose Liposomal Amphotericin B for Human Immunodeficiency Virus–associated Cryptococcal Meningitis: A Phase 2 Randomized Controlled Trial

Short-course High-dose Liposomal Amphotericin B for Human Immunodeficiency Virus–associated... Abstract Background We performed a phase 2 noninferiority trial examining the early fungicidal activity (EFA) of 3 short-course, high-dose liposomal amphotericin B (L-AmB) regimens for cryptococcal meningitis (CM) in Tanzania and Botswana. Methods Human immunodeficiency virus (HIV)-infected adults with CM were randomized to (i) L-AmB 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); or (iv) L-AmB 3 mg/kg/day for 14 days (control). All patients also received oral fluconazole 1200 mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid cryptococcal infection (EFA). Noninferiority was defined as an upper limit of the 2-sided 95% confidence interval (CI) of difference in EFA between intervention and control <0.2 log10 colony-forming units (CFU)/mL/day. Results Eighty participants were enrolled. EFA for daily L-AmB was –0.41 log10 CFU/mL/day (standard deviation, 0.11; n = 17). Difference in mean EFA from control was –0.11 (95% CI, –.29 to .07) log10 CFU/mL/day faster with single dose (n = 16); –0.05 (95% CI, –.20 to .10) log10 CFU/mL/day faster with 2 doses (n = 18); and –0.13 (95% CI, –.35 to .09) log10 CFU/mL/day faster with 3 doses (n = 18). EFA in all short-course arms was noninferior to control. Ten-week mortality was 29% (n = 23) with no statistical difference between arms. All arms were well tolerated. Conclusions Single-dose 10 mg/kg L-AmB was well tolerated and led to noninferior EFA compared to 14 days of 3 mg/kg/day L-AmB in HIV-associated CM. Induction based on a single 10 mg/kg L-AmB dose is being taken forward to a phase 3 clinical endpoint trial. Clinical Trials Registration ISRCTN 10248064. cryptococcal meningitis, HIV, AmBisome, amphotericin, randomized clinical trial Early mortality in human immunodeficiency virus (HIV) treatment programs in low-resource settings is considerably higher than in high-income countries [1–4]. Up to 20% of these deaths are directly attributable to cryptococcal meningitis (CM) [2, 5, 6], which was estimated to cause 181100 deaths globally in 2014 [6]. The poor outcomes reported using currently available antifungal therapy are a critical driver of this high CM-related mortality. Mortality using amphotericin B deoxycholate (AmB-d)–based therapy in low-resource settings, even in clinical trials, remains in the region of 35%–45% at 10 weeks [7–10]. Recommended AmB-d–based therapy requires hospitalization for at least 14 days, and its toxicity profile requires costly laboratory monitoring [11]. In most resource-limited settings, the lack of access to reliable laboratory monitoring, limited nursing capacity, and inadequate funding mean that AmB-d is not routinely available. As a consequence, oral fluconazole monotherapy is widely used but, even at a high dose of up to 1200 mg/day, it is much less rapidly fungicidal than amphotericin B and mortality at 10 weeks is around 60% [12, 13]. New treatment strategies are urgently needed. Liposomal amphotericin B (L-AmB) has lower rates of drug-induced toxicities than AmB-d [14]. Although L-AmB is recommended as treatment for HIV-associated CM in several national guidelines [15, 16], optimal regimens are unknown. The long tissue half-life and effective penetration into the brain tissue suggest it may be possible to deliver effective treatment with very short courses of high-dose L-AmB [17, 18]. Pharmacokinetic data from animal models and humans suggest that increasing L-AmB dosing from the currently recommended 3–4 mg/kg may lead to improved outcomes and that very short-course regimens may be as effective as daily therapy [17, 19]. The concept of single- or intermittent-dose L-AmB therapy has been tested in prophylaxis for hematology patients, with single doses of up to 15 mg/kg given without significant toxicities [20–22], and is established in treatment of visceral leishmaniasis where single doses of 10 mg/kg are routinely given and have been shown to be efficacious [23]. The strategy of short-course, high dosing of L-AmB for HIV-associated CM has not been previously tested in a clinical trial. We performed an open-label phase 2 randomized noninferiority trial to compare alternative short-course L-AmB regimens for the treatment of HIV-associated CM. Our aim was to determine which, if any, of the 3 alternative schedules of intermittent high-dose L-AmB could be adopted for the development of a phase 3 randomized controlled clinical endpoint trial. We measured the effects on early fungicidal activity, which is associated closely with all-cause mortality [9, 24, 25]. MATERIALS AND METHODS The trial protocol for the Ambition-cm Trial (Ambisome Induction Optimization for Cryptococcal Meningitis) has previously been published in full [26]. The study was carried out at Princess Marina Hospital (Gaborone, Botswana) and Bugando Medical Centre and Sekou Toure Hospital (Mwanza, Tanzania). The study was approved by the research ethics committees of the London School of Hygiene and Tropical Medicine, the University of Pennsylvania, the Botswana Ministry of Health, and the National Institute of Medical Research Tanzania. The study was conducted in accordance with the principles of International Conference of Harmonisation good clinical practices and was prospectively registered on the International Standard Randomized Controlled Trial Register (ISRCTN 10248064). Participants and Procedures Between October 2014 and September 2016, sequential HIV-infected adults aged ≥18 years with a first episode of CM, diagnosed by cerebrospinal fluid (CSF) India ink or cryptococcal antigen (CrAg) lateral flow assay (IMMY, Norman, Oklahoma), were screened for enrollment in the trial. Pregnant or lactating patients, patients with a previous serious reaction to study drugs, or patients on antifungal treatment for >48 hours were excluded. Patients who were both antiretroviral therapy (ART) naive and ART exposed were recruited. Written informed consent was obtained from participants or, in the case of mental obtundation, from a guardian or person with legal responsibility. Patients with mental obtundation were reconsented on recovery. Patients were block randomized individually to 1 of 4 treatment groups by means of random computer-generated lists with an allocation ratio of 1:1:1:1 and block sizes of 8. Randomization lists were created by an independent statistician who prepared sealed envelopes in advance that were sent to the sites. Trial pharmacists were responsible for randomization at each site. Randomization was stratified by abnormal mental status (Glasgow Coma Scale [GCS] score of 15 or <15) and ART status on admission at each site. The patients and clinical trial team were not blinded. Laboratory staff performing quantitative fungal cultures were blinded to treatment allocation. The 4 treatment arms were (i) L-AmB (AmBisome, Gilead Sciences) 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); and (iv) L-AmB 3 mg/kg/day for 14 days (control). L-AmB was given by intravenous infusion over 2 hours. All patients also received 1200 mg/day oral fluconazole (Diflucan [Pfizer] or fluconazole [Medopharm]) for the first 2 weeks. Unless contraindicated, all patients received 1 L of 0.9% normal saline with 20 mmol of potassium chloride (KCl) prior to L-AmB to minimize nephrotoxicity and were routinely given oral potassium (16 mmol KCl twice daily) and magnesium (11 mmol Mg2+ once daily) supplementation and daily trimethoprim-sulfamethoxazole prophylaxis. After the 2-week induction phase, patients received fluconazole 800 mg/day until 10 weeks and 200 mg/day thereafter. ART consisting of tenofovir, emtricitabine, and efavirenz was commenced 4–6 weeks after initiation of antifungal therapy in individuals not already on ART. Evaluations and Outcomes At baseline patients underwent a lumbar puncture (LP) for opening pressure, cell count and differential, protein, glucose, India ink, CrAg, quantitative fungal culture, and routine bacterial culture. LPs for opening pressure measurements and CSF samples for quantitative fungal culture were repeated on treatment days 3, 7, and 14. Patients with a CSF opening pressure >30 cm H2O or symptoms of raised intracranial pressure underwent daily LPs to remove CSF in accordance with guidelines [15]. Quantitative cryptococcal cultures were plated in serial 10-fold dilution and the dilution with the least colonies, but at least 30 colony-forming units (CFUs)/200 μL, was used to calculate CFU/mL quantitative cryptococcal culture results, as previously described [24]. A linear regression of log10 CFU/mL against time was calculated for each patient. All data points were analyzed except sterile cultures in the second week if these values lessened the slope, as sterility would have been achieved before that day’s LP and using the second week’s value would therefore underestimate the true slope [9, 24]. All participants had baseline blood tests including full blood count, urea, creatinine, electrolytes, alanine aminotransferase (ALT), HIV test (if status unknown), and CD4 cell count. During the 2-week induction phase, patients underwent alternate-day renal function and electrolyte assessment and twice-weekly monitoring of full blood count and ALT. Clinical and laboratory adverse events (AEs) were graded using the National Institutes of Health Division of AIDS toxicity table [27]. Clinical response was monitored daily for the first 2 weeks or until discharge (whichever was later), then in a follow-up clinic 3, 4, 6, and 10 weeks after starting therapy. The primary outcome measure was the mean rate of decrease in CSF cryptococcal CFU, also known as early fungicidal activity (EFA) of each L-AmB treatment arm. Secondary outcome measures were mortality at 2 and 10 weeks; proportion of patients in each treatment arm suffering clinical and laboratory-defined grade 3/4 AEs; and median percentage change from baseline in laboratory-defined parameters. Statistical Analysis Using a noninferiority design, assuming an EFA of 0.50 log10 CFU/mL/day with a standard deviation of 0.25 log10 CFU/mL/day in the standard daily dosing arm, with a prespecified acceptable Δ of 0.2 log10 CFU/mL/day, 1-sided α of .025, and 90% power, gave a sample size of 33 patients per arm. The prespecified Δ of 0.2 log10 CFU/mL/day was selected on the basis of prior evidence showing increased mortality once EFA falls below 0.3 log10 CFU/mL/day (ie, the projected 0.50 log10 CFU/mL/day in the control arm minus prespecified Δ of 0.2 log10 CFU/mL/day) [26]. A sample size of 40 patients per arm (160 patients in total) was planned to allow for patients who died prior to obtaining EFA measurement. An interim analysis was planned after 80 participants were randomized in the study. The primary analysis was based on the intention-to-treat population. Patients who died before having a repeat LP on day 3 or those with a negative baseline culture could not have an EFA calculated and were therefore not included the EFA analysis, but were analyzed for secondary endpoints. Linear regression models were used, with the mean rate of decrease in log10 CSF cryptococcal CFU (EFA) being the dependent variable and the treatment groups (using the control group as a comparator) the primary independent variables. The short-course L-AmB groups were compared to the control arm for noninferiority using the prespecified Δ of 0.2 log10 CFU/mL/day. Statistical significance was defined as P ≤ .05. Following an unadjusted EFA analysis, adjusted analysis was performed including covariates that may determine outcomes (baseline fungal burden, CD4 cell count, abnormal mental status, sex, age, and ART status) giving summary differences with 95% confidence intervals (CIs). Grade 3 and 4 AEs were tabulated by study arm, and the overall numbers of AEs compared using the χ2 test. The proportion of patients experiencing grade 3 and 4 anaemia, renal impairment, and hypokalemia during 2-week induction treatment was compared across study arms using the χ2 test. Mean change in hemoglobin and percentage change in creatinine during 2-week induction therapy were compared across study arms using analysis of variance analysis and χ2 testing, respectively. Mortality was compared across groups using χ2 testing. Data were analyzed using Stata version 13 software (StataCorp, College Station, Texas). RESULTS The study was stopped on the recommendation of the independent data monitoring committee at the preplanned interim analysis as the primary objective had been achieved, with noninferiority achieved in all 3 study arms at both the predefined 95% confidence level and the stringent 99% confidence level, and no safety concerns with short-course treatment, with the recommendation that the trial proceed onto the clinical endpoint phase 3 trial. At the time of stopping, 134 patients had been screened for the trial. Fifty-four patients were excluded (Figure 1), and 80 patients were enrolled and randomized to 1 of the 4 treatment groups: 18 to single dose, 20 to 2 doses, 21 to 3 doses, and 21 to control. One patient was excluded after randomization after it emerged they had been treated for a previous episode of CM; thus 79 patients completed the study, with no loss to follow-up during the initial 2-week induction phase. All participants received the treatment as per the randomization arm. One patient was lost to follow-up between 2 and 10 weeks. Baseline clinical and laboratory characteristics were well balanced between treatment groups (Table 1). Twenty-five patients (32%) were on ART at presentation with CM, the median baseline CD4 count was 32 cells/µL, and 28% (22) had a GCS score <15. Figure 1. View largeDownload slide Consolidated Standards of Reporting Trials (CONSORT) diagram. *All patients received their allocated intervention. No patients were lost to follow-up during the initial 2-week follow-up. †The single patient lost to follow-up had full follow-up data until hospital discharge at 2 weeks that were used in toxicity and mortality analyses. Abbreviations: CM, cryptococcal meningitis; EFA, early fungicidal activity; LP, lumbar puncture. Figure 1. View largeDownload slide Consolidated Standards of Reporting Trials (CONSORT) diagram. *All patients received their allocated intervention. No patients were lost to follow-up during the initial 2-week follow-up. †The single patient lost to follow-up had full follow-up data until hospital discharge at 2 weeks that were used in toxicity and mortality analyses. Abbreviations: CM, cryptococcal meningitis; EFA, early fungicidal activity; LP, lumbar puncture. Table 1. Baseline Characteristics of Trial Participants Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) All patients were of black African ethnicity. Abbreviations: ART, antiretroviral therapy; CFU, colony-forming units; CSF, cerebrospinal fluid; IQR, interquartile range; L-AmB, liposomal amphotericin B; TB, tuberculosis; WBC, white blood cell. aFive patients were missing baseline CD4 cell count, 5 patients were missing CSF WBC count, and a single individual was missing baseline quantitative cryptococcal culture. All other data were complete for all participants. View Large Table 1. Baseline Characteristics of Trial Participants Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) All patients were of black African ethnicity. Abbreviations: ART, antiretroviral therapy; CFU, colony-forming units; CSF, cerebrospinal fluid; IQR, interquartile range; L-AmB, liposomal amphotericin B; TB, tuberculosis; WBC, white blood cell. aFive patients were missing baseline CD4 cell count, 5 patients were missing CSF WBC count, and a single individual was missing baseline quantitative cryptococcal culture. All other data were complete for all participants. View Large Primary Outcome EFA was calculated for 69 patients (17 in the control group, 16 in the single-dose group, 18 in the 2-dose group, and 18 in the 3-dose group). Five patients died prior to follow-up LP and 5 patients had negative baseline cultures precluding EFA calculation. All the short-course, high-dose arms of L-AmB were noninferior in terms of EFA to 14 days of standard-dose L-AmB at the predefined noninferiority margin of 0.2 log10 CFU/mL/day (Figure 2A). The mean (standard deviation) EFA was –0.41 (0.11) log10 CFU/mL/day with standard treatment (control), –0.52 (0.35) log10 CFU/mL/day with single-dose L-AmB, –0.47 (0.29) log10 CFU/mL/day with 2 doses, and –0.54 (0.44) log10 CFU/mL/day with 3 doses. The difference in mean EFA between single dose and control was –0.11 (95% CI, –.29 to .07) log10 CFU/mL/day; between 2 doses and control was –0.05 (95% CI, –.20 to .10) log10 CFU/mL/day; and between 3 doses and control was –0.13 (95% CI, –.35 to .09) log10 CFU/mL/day. There was no evidence for any dose-response effect with additional L-AmB doses, suggesting maximal fungicidal activity was achieved with a single 10 mg/kg dose. This remained the case when the analysis was adjusted for factors that have previously been shown to affect EFA (CSF fungal burden and CD4 cell count), abnormal mental status, and also sex, age, and ART status (Figure 2C). Figure 2. View largeDownload slide Early fungicidal activity (EFA) by treatment group. A, Difference in mean EFA between intervention arms and control. All 3 short-course treatment arms were noninferior to control. B, Individual patient slopes over the initial 14 days of treatment. The mean slope (standard deviation) is given below each plot. Sterile cultures in the second week that lessened the slope and were excluded from EFA calculation (as sterility would have been achieved before that day’s lumbar punctures) are shown in the dotted gray line. C, Adjusted difference in mean EFA between intervention arms and control. All 3 short-course treatment arms remained noninferior to control when adjusted for (i) baseline fungal burden (quantitative cryptococcal culture [QCC]); (ii) baseline CD4 cell count; (iii) baseline mental status; (iv) QCC and CD4 cell count; (v) QCC, CD4 cell count, and mental status; and (vi) QCC, CD4 cell count, mental status, sex, age, and antiretroviral therapy status. Abbreviations: CFU, colony-forming units; CI, confidence interval; SD, standard deviation. Figure 2. View largeDownload slide Early fungicidal activity (EFA) by treatment group. A, Difference in mean EFA between intervention arms and control. All 3 short-course treatment arms were noninferior to control. B, Individual patient slopes over the initial 14 days of treatment. The mean slope (standard deviation) is given below each plot. Sterile cultures in the second week that lessened the slope and were excluded from EFA calculation (as sterility would have been achieved before that day’s lumbar punctures) are shown in the dotted gray line. C, Adjusted difference in mean EFA between intervention arms and control. All 3 short-course treatment arms remained noninferior to control when adjusted for (i) baseline fungal burden (quantitative cryptococcal culture [QCC]); (ii) baseline CD4 cell count; (iii) baseline mental status; (iv) QCC and CD4 cell count; (v) QCC, CD4 cell count, and mental status; and (vi) QCC, CD4 cell count, mental status, sex, age, and antiretroviral therapy status. Abbreviations: CFU, colony-forming units; CI, confidence interval; SD, standard deviation. Mortality Overall all-cause mortality rates were 15% (12/79) at 2 weeks and 29% (23/79) at 10 weeks, with no significant difference between treatment arms. Two-week mortality was 10% (2/21) in the control arm, 11% (2/18) in the single-dose arm, 15% (3/20) in the 2-dose arm, and 25% (5/20) in the 3-dose arm (P = .52). At 10 weeks, mortality was 29% (6/21) in the control arm, 22% (4/18) in the single-dose arm, 15% (3/20) in the 2-dose arm, and 50% (10/20) in the 3-dose arm (P = .09; Table 2). Mortality at 10 weeks was associated with abnormal mental status at baseline in univariable analysis (odds ratio, 3.75 [95% CI, 1.3–10.7]), but not with baseline fungal burden, baseline CD4 cell count, or ART status. The mortality difference between the single-dose and control arms was 6.4% (95% CI, –21% to 34%). Table 2. Primary and Key Secondary Outcomes Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Abbreviations: AE, adverse event; CFU, colony-forming units; CI, confidence interval; EFA, early fungicidal activity; L-AmB, liposomal amphotericin B. aIndividuals who die prior to the day 3 lumbar puncture (LP) or who were culture negative at baseline do not have an EFA value. Overall, 5 patients died prior to follow-up LP (1 control, 1 single dose, 1 two dose, 2 three dose) and 5 patients had negative baseline cultures (3 controls, 1 single dose, 1 two dose). bAll 3 study arms were noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. The respective difference in mean EFA between single dose and control was –0.11 (99% CI, –.35 to .14) log10 CFU/mL/day; between 2 doses and control was –0.05 (99% CI, –.26 to .16) log10 CFU/mL/day; and between 3 doses and control was –0.13 (99% CI, –.42 to .17) log10 CFU/mL/day. Using this more stringent cutoff, all 3 study arms remained noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. View Large Table 2. Primary and Key Secondary Outcomes Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Abbreviations: AE, adverse event; CFU, colony-forming units; CI, confidence interval; EFA, early fungicidal activity; L-AmB, liposomal amphotericin B. aIndividuals who die prior to the day 3 lumbar puncture (LP) or who were culture negative at baseline do not have an EFA value. Overall, 5 patients died prior to follow-up LP (1 control, 1 single dose, 1 two dose, 2 three dose) and 5 patients had negative baseline cultures (3 controls, 1 single dose, 1 two dose). bAll 3 study arms were noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. The respective difference in mean EFA between single dose and control was –0.11 (99% CI, –.35 to .14) log10 CFU/mL/day; between 2 doses and control was –0.05 (99% CI, –.26 to .16) log10 CFU/mL/day; and between 3 doses and control was –0.13 (99% CI, –.42 to .17) log10 CFU/mL/day. Using this more stringent cutoff, all 3 study arms remained noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. View Large Safety There were no safety concerns with short-course treatment in terms of fungal clearance, and no patients receiving short-course L-AmB required additional “rescue” L-AmB therapy. The 3 high-dose short-course L-AmB regimens were all well tolerated. Eighty-eight grade 3 and higher AEs occurred in 47 patients: 45 grade 3 and 43 grade 4/5 AEs, with no significant differences observed between treatment arms (Table 3). Of these, 49 were clinical, and 39 laboratory AEs. There were 10 grade 3 and 2 grade 4 AEs that were attributed to treatment with L-AmB, all of which were expected L-AmB–related side effects (3 grade 3 hypokalemia, 1 grade 4 hypokalemia, 1 grade 3 hypomagnasemia, 4 grade 3 creatinine rises, 1 grade 3 and 1 grade 4 anemia, 1 grade 4 hyponatremia). Both grade 4 L-AmB related events occurred in the control group. During induction therapy, grade 3 and 4 anemia occurred in 6% (n = 5) and 1% (n = 1) overall, renal impairment in 5% (n = 4) and 1% (n = 1) overall, and hypokalemia in 1% (n = 1) and 1% (n = 1) overall, with no significant differences between treatment arms (Table 2). Table 3. Adverse Events and Readmissions During 10-Week Follow-up Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Data are presented as No. unless otherwise indicated. Abbreviations: AE, adverse event; ALT, alanine aminotransferase; CM, cryptococcal meningitis; ICP, intra-cranial pressure; IRIS, immune reconstitution inflammatory syndrome; L-AmB, liposomal amphotericin B. aForty-seven patients had at least 1 AE: 2 patients had 5 AEs, 2 patients had 4 AEs, 5 patients had 3 AEs, 7 patients had 2 AEs, 6 patients had 1 AE, and 33 had no AEs. Twenty-eight patients had grade 3 AEs: 4 in the control arm, 11 in the single-dose arm, 6 in the 2-dose arm, and 7 in the 3-dose arm. Fifteen patients had grade 4 AEs: 5 in the control arm, 5 in the single-dose arm, 2 in the 2-dose arm, and 3 in the 3-dose arm. bRelated includes all AEs classified as possibly, probably, or definitely related to study drug. View Large Table 3. Adverse Events and Readmissions During 10-Week Follow-up Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Data are presented as No. unless otherwise indicated. Abbreviations: AE, adverse event; ALT, alanine aminotransferase; CM, cryptococcal meningitis; ICP, intra-cranial pressure; IRIS, immune reconstitution inflammatory syndrome; L-AmB, liposomal amphotericin B. aForty-seven patients had at least 1 AE: 2 patients had 5 AEs, 2 patients had 4 AEs, 5 patients had 3 AEs, 7 patients had 2 AEs, 6 patients had 1 AE, and 33 had no AEs. Twenty-eight patients had grade 3 AEs: 4 in the control arm, 11 in the single-dose arm, 6 in the 2-dose arm, and 7 in the 3-dose arm. Fifteen patients had grade 4 AEs: 5 in the control arm, 5 in the single-dose arm, 2 in the 2-dose arm, and 3 in the 3-dose arm. bRelated includes all AEs classified as possibly, probably, or definitely related to study drug. View Large Eleven trial participants were readmitted to hospital during the 10-week follow-up period, at a median of 41 (interquartile range, 25–55) days, including 4 in the control arm, 4 in the single-dose arm, none in the 2-dose arm, and 3 in the 3-dose arm. Cryptococcal immune reconstitution inflammatory syndrome (IRIS) was suspected or diagnosed in 5 of the 45 (11%) patients initiating ART, 2 of whom died, with no significant differences between study arms. DISCUSSION The use of a single 10 mg/kg dose of L-AmB was noninferior to standard 3 mg/kg daily dosing for 14 days in reducing CSF cryptococcal burden in patients with a first episode of HIV-associated CM. These findings are consistent with previous human and animal studies demonstrating that shorter courses of amphotericin-based treatment may be better tolerated and as effective as conventional 14-day courses [13, 17, 28–30]. High dosages of L-AmB were well tolerated, and the safety profile of all L-AmB regimens tested compared favorably to data from prior clinical trials using conventional AmB-d in similar patient populations, both in terms of mortality at 10 weeks and drug-induced toxicities [9, 11]. Overall rates of AEs associated with L-AmB were very low, with just 1 patient (1%) developing grade 4 anemia during induction therapy (in the control arm), compared with 18% of a historic cohort of 368 CM patients receiving AmB-d treatment and an identical prehydration and electrolyte supplementation regimen to that used in the current trial [11]. The median fall in hemoglobin during the first 2 weeks of treatment was 0.9 g/dL, compared with 2.3 g/dL in the previous cohort of AmB-d–treated patients [11], and there was a median increase in creatinine of 14% over the initial 2 weeks, compared to 73% in the AmB-d–treated cohort [11]. There were no grade 4 AEs attributed to high-dose L-AmB during the trial. Rates of recurrence of CM symptoms and IRIS were low, with suspected IRIS events occurring in 11% of individuals initiated on ART during the trial. Based on these phase 2 results, single-dose 10 mg/kg L-AmB is being taken forward to a phase 3 clinical endpoint trial (ISRCTN 72509687). Given the correlation between EFA and clinical outcome [9, 25], the rapid EFA seen with single 10 mg/kg doses of L-AmB should result in a clinically efficacious alternative treatment for CM. The 10-week mortality rate of 22% with the single-dose 10 mg/kg L-AmB selected for study in the phase 3 trial, and the overall mortality rate of 29% in the trial, compare favorably with mortality rates of approximately 40% seen in recent large clinical trials of 2-week AmB-d–based treatment [7–9]. Notably, these mortality rates were in the context of fluconazole as a second antifungal agent. The addition to high dose L-AmB of a more efficacious agent such as flucytosine, which has been proven to be superior to fluconazole in the recent Advancing CM Treatment in Africa (ACTA) trial [30], may enable a further reduction in mortality rates. The current phase 2 study was not powered to detect a mortality difference, as shown by the wide 95% CIs around the mortality difference, and as expected, no significant difference in mortality between the 4 L-AmB treatment arms was seen. The higher mortality rate in the 3-dose arm was likely due to chance alone, with 40% (n = 4) of the deaths occurring prior to receipt of the third dose of L-AmB. In conclusion, we have demonstrated that a single 10 mg/kg dose of liposomal amphotericin B given in combination with high-dose fluconazole is noninferior to daily dosed L-AmB at the standard dose of 3 mg/kg plus high-dose fluconazole in terms of rate of fungal clearance in patients with HIV-associated CM. This short-course treatment strategy is now being tested against AmB-d in a clinical endpoint trial. If confirmed to be effective, single high dosages of L-AmB given with an optimized oral antifungal medication backbone would provide a feasible, well-tolerated, and sustainable treatment regimen for HIV-associated CM in resource-limited settings where the safe administration of AmB-d treatment is not possible. Reductions in the need for toxicity monitoring, fewer drug-related AEs, and the potential for shorter periods of hospitalization are likely to mean that a single high-dose L-AmB treatment strategy is cost effective, and a highly favorable alternative to the current standard of care. Notes Author contributions. J. N. J. and T. S. H. conceptualized and designed the study, supervised implementation, analyzed the data, and drafted the final manuscript. T. B. L., A. A. C., G. B., M. M., R. K. K. P., and M. W. T. implemented the study. K. T. and N. L. were the research nurses, C. M. implemented the laboratory aspects of the trial, and N. M. was the study pharmacist. J. K. and J. C. supervised implementation. D. L. drafted the initial manuscript. W. H. assisted with conceptualization and design of the study and critically reviewed the manuscript. S. M. was the trial manager, assisted with study design, supervised implementation and data management, and helped draft the final manuscript. All authors reviewed and approved the final manuscript. The authors had full access to all study data and had final responsibility for the decision to submit for publication. Acknowledgments. We thank the management and staff of Princess Marina Hospital, Bugando Medical Centre, and Sekou Toure Hospital for their assistance and support; Drs Mosepele Mosepele, Miriam Haverkamp, Elizabeth Williams, William Hurt, and Hannah Mitchell for their assistance with patient care; John Bradley for preparing the randomization codes; and the members of the Data Monitoring Committee (Andrew Nunn, Rob Peck, and Mina Hosseinipour) and the Trial Steering Committee (David Boulware, Saidi Kapiga, and Mary Kasule) for their oversight of the trial. Disclaimer. The funding source and drug manufacturers had no involvement in the study design; collection, analysis, and interpretation of data; preparation of manuscripts; or the decision to submit this manuscript for publication. Financial support. Gilead Sciences funded the trial through an Investigator-Initiated award (IN-EU-131-D036) and provided the L-AmB for the trial. The work was made possible through core support from the Penn Center for AIDS Research, an National Institutes of Health (NIH)-funded program (grant number P30 AI 045008). Potential conflicts of interest. J. N. J. and T. H. were recipients of a Gilead Investigator award. T. H. declares consultancy fees from Viamet, lecture fees from Pfizer and Gilead Sciences, and payments from Immuno-Mycologics. W. H. holds or has recently held research grants with F2G, AiCuris, Astellas Pharma, Spero Therapeutics, Matinas Biosciences, Antabio, Amplyx, Allecra, Auspherix, and Pfizer; holds awards from the NIH, the Medical Research Council, the National Institute for Health Research, and the European Commission (FP7 and IMI); has received personal fees in his capacity as a consultant for F2G, Amplyx, Ausperix, Spero Therapeutics, The Medicines Company, Gilead, and Basilea; and is Medical Guideline Director for the European Society of Clinical Microbiology and Infectious Diseases, and an Ordinary Council Member for the British Society of Antimicrobial Chemotherapy. G. B. declares consultancy fees from Pfizer, grants and travel expenses from NIH, and lecture payments from ViralEd. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Braitstein P , Brinkhof MW , Dabis F , et al. ; Antiretroviral Therapy in Lower Income Countries (ART-LINC) Collaboration; ART Cohort Collaboration (ART-CC) groups . Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries . Lancet 2006 ; 367 : 817 – 24 . Google Scholar Crossref Search ADS PubMed 2. Lawn SD , Harries AD , Anglaret X , Myer L , Wood R . Early mortality among adults accessing antiretroviral treatment programmes in sub-Saharan Africa . AIDS 2008 ; 22 : 1897 – 908 . Google Scholar Crossref Search ADS PubMed 3. Amuron B , Namara G , Birungi J , et al. Mortality and loss-to-follow-up during the pre-treatment period in an antiretroviral therapy programme under normal health service conditions in Uganda . BMC Public Health 2009 ; 9 : 290 . Google Scholar Crossref Search ADS PubMed 4. Gupta A , Nadkarni G , Yang WT , et al. Early mortality in adults initiating antiretroviral therapy (ART) in low- and middle-income countries (LMIC): a systematic review and meta-analysis . PLoS One 2011 ; 6 : e28691 . Google Scholar Crossref Search ADS PubMed 5. Park BJ , Wannemuehler KA , Marston BJ , Govender N , Pappas PG , Chiller TM . Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS . AIDS 2009 ; 23 : 525 – 30 . Google Scholar Crossref Search ADS PubMed 6. Rajasingham R , Smith RM , Park BJ , et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis . Lancet Infect Dis 2017 ; 17 : 873 – 81 . Google Scholar Crossref Search ADS PubMed 7. Day JN , Chau TTH , Wolbers M , et al. Combination antifungal therapy for cryptococcal meningitis . N Engl J Med 2013 ; 368 : 1291 – 302 . Google Scholar Crossref Search ADS PubMed 8. Beardsley J , Wolbers M , Kibengo FM , et al. ; CryptoDex Investigators . Adjunctive dexamethasone in HIV-associated cryptococcal meningitis . N Engl J Med 2016 ; 374 : 542 – 54 . Google Scholar Crossref Search ADS PubMed 9. Jarvis JN , Bicanic T , Loyse A , et al. Determinants of mortality in a combined cohort of 501 patients with HIV-associated cryptococcal meningitis: implications for improving outcomes . Clin Infect Dis 2014 ; 58 : 736 – 45 . Google Scholar Crossref Search ADS PubMed 10. Boulware DR , Meya DB , Muzoora C , et al. ; COAT Trial Team . Timing of antiretroviral therapy after diagnosis of cryptococcal meningitis . N Engl J Med 2014 ; 370 : 2487 – 98 . Google Scholar Crossref Search ADS PubMed 11. Bicanic T , Bottomley C , Loyse A , et al. Toxicity of amphotericin B deoxycholate-based induction therapy in patients with HIV-associated cryptococcal meningitis . Antimicrob Agents Chemother 2015 ; 59 : 7224 – 31 . Google Scholar Crossref Search ADS PubMed 12. Longley N , Muzoora C , Taseera K , et al. Dose response effect of high-dose fluconazole for HIV-associated cryptococcal meningitis in southwestern Uganda . Clin Infect Dis 2008 ; 47 : 1556 – 61 . Google Scholar Crossref Search ADS PubMed 13. Jackson AT , Nussbaum JC , Phulusa J , et al. A phase II randomized controlled trial adding oral flucytosine to high-dose fluconazole, with short-course amphotericin B, for cryptococcal meningitis . AIDS 2012 ; 26 : 1363 – 70 . Google Scholar Crossref Search ADS PubMed 14. Hamill RJ , Sobel JD , El-Sadr W , et al. Comparison of 2 doses of liposomal amphotericin B and conventional amphotericin B deoxycholate for treatment of AIDS-associated acute cryptococcal meningitis: a randomized, double-blind clinical trial of efficacy and safety . Clin Infect Dis 2010 ; 51 : 225 – 32 . Google Scholar Crossref Search ADS PubMed 15. Perfect JR , Dismukes WE , Dromer F , et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America . Clin Infect Dis 2010 ; 50 : 291 – 322 . Google Scholar Crossref Search ADS PubMed 16. Nelson M , Dockrell D , Edwards S , et al. ; BHIVA Guidelines Subcommittee . British HIV Association and British infection association guidelines for the treatment of opportunistic infection in HIV-seropositive individuals 2011 . HIV Med 2011 ; 12 ( Suppl 2 ): 1 – 140 . Google Scholar Crossref Search ADS PubMed 17. Lestner J , McEntee L , Johnson A , et al. Experimental models of short courses of liposomal amphotericin B for induction therapy for cryptococcal meningitis . Antimicrob Agents Chemother 2017 ; 61 . doi: https://doi.org/10.1128/AAC.00090-17 . 18. Vogelsinger H , Weiler S , Djanani A , et al. Amphotericin B tissue distribution in autopsy material after treatment with liposomal amphotericin B and amphotericin B colloidal dispersion . J Antimicrob Chemother 2006 ; 57 : 1153 – 60 . Google Scholar Crossref Search ADS PubMed 19. O’Connor L , Livermore J , Sharp AD , et al. Pharmacodynamics of liposomal amphotericin B and flucytosine for cryptococcal meningoencephalitis: safe and effective regimens for immunocompromised patients . J Infect Dis 2013 ; 208 : 351 – 61 . Google Scholar Crossref Search ADS PubMed 20. Gubbins PO , Amsden JR , McConnell SA , Anaissie EJ . Pharmacokinetics and buccal mucosal concentrations of a 15 milligram per kilogram of body weight total dose of liposomal amphotericin B administered as a single dose (15 mg/kg), weekly dose (7.5 mg/kg), or daily dose (1 mg/kg) in peripheral stem cell transplant patients . Antimicrob Agents Chemother 2009 ; 53 : 3664 – 74 . Google Scholar Crossref Search ADS PubMed 21. Mehta P , Vinks A , Filipovich A , et al. High-dose weekly AmBisome antifungal prophylaxis in pediatric patients undergoing hematopoietic stem cell transplantation: a pharmacokinetic study . Biol Blood Marrow Transplant 2006 ; 12 : 235 – 40 . Google Scholar Crossref Search ADS PubMed 22. Cornely OA , Maertens J , Bresnik M , et al. ; AmBiLoad Trial Study Group . Liposomal amphotericin B as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (AmBiLoad trial) . Clin Infect Dis 2007 ; 44 : 1289 – 97 . Google Scholar Crossref Search ADS PubMed 23. Sundar S , Chakravarty J , Agarwal D , Rai M , Murray HW . Single-dose liposomal amphotericin B for visceral leishmaniasis in India . N Engl J Med 2010 ; 362 : 504 – 12 . Google Scholar Crossref Search ADS PubMed 24. Brouwer AE , Rajanuwong A , Chierakul W , et al. Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial . Lancet 2004 ; 363 : 1764 – 7 . Google Scholar Crossref Search ADS PubMed 25. Bicanic T , Muzoora C , Brouwer AE , et al. Independent association between rate of clearance of infection and clinical outcome of HIV-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients . Clin Infect Dis 2009 ; 49 : 702 – 9 . Google Scholar Crossref Search ADS PubMed 26. Molefi M , Chofle AA , Molloy SF , et al. Ambition-cm: intermittent high dose AmBisome on a high dose fluconazole backbone for cryptococcal meningitis induction therapy in sub-Saharan Africa: study protocol for a randomized controlled trial . Trials 2015 ; 16 : 276 . Google Scholar Crossref Search ADS PubMed 27. National Institute of Allergy and Infectious Diseases, Division of AIDS . Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse events, version 2.1 . Bethesda, MD : NIAID , 2017 . 28. Muzoora CK , Kabanda T , Ortu G , et al. Short course amphotericin B with high dose fluconazole for HIV-associated cryptococcal meningitis . J Infect 2012 ; 64 : 76 – 81 . Google Scholar Crossref Search ADS PubMed 29. Livermore J , Howard SJ , Sharp AD , et al. Efficacy of an abbreviated induction regimen of amphotericin B deoxycholate for cryptococcal meningoencephalitis: 3 days of therapy is equivalent to 14 days . MBio 2013 ; 5 : e00725 – 13 . 30. Molloy SF , Kanyama C , Heyderman RS , et al. ; ACTA Trial Study Team . Antifungal combinations for treatment of cryptococcal meningitis in Africa . N Engl J Med 2018 ; 378 : 1004 – 17 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Infectious Diseases Oxford University Press

Loading next page...
 
/lp/oxford-university-press/short-course-high-dose-liposomal-amphotericin-b-for-human-XU9jXB5NIn
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
ISSN
1058-4838
eISSN
1537-6591
D.O.I.
10.1093/cid/ciy515
Publisher site
See Article on Publisher Site

Abstract

Abstract Background We performed a phase 2 noninferiority trial examining the early fungicidal activity (EFA) of 3 short-course, high-dose liposomal amphotericin B (L-AmB) regimens for cryptococcal meningitis (CM) in Tanzania and Botswana. Methods Human immunodeficiency virus (HIV)-infected adults with CM were randomized to (i) L-AmB 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); or (iv) L-AmB 3 mg/kg/day for 14 days (control). All patients also received oral fluconazole 1200 mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid cryptococcal infection (EFA). Noninferiority was defined as an upper limit of the 2-sided 95% confidence interval (CI) of difference in EFA between intervention and control <0.2 log10 colony-forming units (CFU)/mL/day. Results Eighty participants were enrolled. EFA for daily L-AmB was –0.41 log10 CFU/mL/day (standard deviation, 0.11; n = 17). Difference in mean EFA from control was –0.11 (95% CI, –.29 to .07) log10 CFU/mL/day faster with single dose (n = 16); –0.05 (95% CI, –.20 to .10) log10 CFU/mL/day faster with 2 doses (n = 18); and –0.13 (95% CI, –.35 to .09) log10 CFU/mL/day faster with 3 doses (n = 18). EFA in all short-course arms was noninferior to control. Ten-week mortality was 29% (n = 23) with no statistical difference between arms. All arms were well tolerated. Conclusions Single-dose 10 mg/kg L-AmB was well tolerated and led to noninferior EFA compared to 14 days of 3 mg/kg/day L-AmB in HIV-associated CM. Induction based on a single 10 mg/kg L-AmB dose is being taken forward to a phase 3 clinical endpoint trial. Clinical Trials Registration ISRCTN 10248064. cryptococcal meningitis, HIV, AmBisome, amphotericin, randomized clinical trial Early mortality in human immunodeficiency virus (HIV) treatment programs in low-resource settings is considerably higher than in high-income countries [1–4]. Up to 20% of these deaths are directly attributable to cryptococcal meningitis (CM) [2, 5, 6], which was estimated to cause 181100 deaths globally in 2014 [6]. The poor outcomes reported using currently available antifungal therapy are a critical driver of this high CM-related mortality. Mortality using amphotericin B deoxycholate (AmB-d)–based therapy in low-resource settings, even in clinical trials, remains in the region of 35%–45% at 10 weeks [7–10]. Recommended AmB-d–based therapy requires hospitalization for at least 14 days, and its toxicity profile requires costly laboratory monitoring [11]. In most resource-limited settings, the lack of access to reliable laboratory monitoring, limited nursing capacity, and inadequate funding mean that AmB-d is not routinely available. As a consequence, oral fluconazole monotherapy is widely used but, even at a high dose of up to 1200 mg/day, it is much less rapidly fungicidal than amphotericin B and mortality at 10 weeks is around 60% [12, 13]. New treatment strategies are urgently needed. Liposomal amphotericin B (L-AmB) has lower rates of drug-induced toxicities than AmB-d [14]. Although L-AmB is recommended as treatment for HIV-associated CM in several national guidelines [15, 16], optimal regimens are unknown. The long tissue half-life and effective penetration into the brain tissue suggest it may be possible to deliver effective treatment with very short courses of high-dose L-AmB [17, 18]. Pharmacokinetic data from animal models and humans suggest that increasing L-AmB dosing from the currently recommended 3–4 mg/kg may lead to improved outcomes and that very short-course regimens may be as effective as daily therapy [17, 19]. The concept of single- or intermittent-dose L-AmB therapy has been tested in prophylaxis for hematology patients, with single doses of up to 15 mg/kg given without significant toxicities [20–22], and is established in treatment of visceral leishmaniasis where single doses of 10 mg/kg are routinely given and have been shown to be efficacious [23]. The strategy of short-course, high dosing of L-AmB for HIV-associated CM has not been previously tested in a clinical trial. We performed an open-label phase 2 randomized noninferiority trial to compare alternative short-course L-AmB regimens for the treatment of HIV-associated CM. Our aim was to determine which, if any, of the 3 alternative schedules of intermittent high-dose L-AmB could be adopted for the development of a phase 3 randomized controlled clinical endpoint trial. We measured the effects on early fungicidal activity, which is associated closely with all-cause mortality [9, 24, 25]. MATERIALS AND METHODS The trial protocol for the Ambition-cm Trial (Ambisome Induction Optimization for Cryptococcal Meningitis) has previously been published in full [26]. The study was carried out at Princess Marina Hospital (Gaborone, Botswana) and Bugando Medical Centre and Sekou Toure Hospital (Mwanza, Tanzania). The study was approved by the research ethics committees of the London School of Hygiene and Tropical Medicine, the University of Pennsylvania, the Botswana Ministry of Health, and the National Institute of Medical Research Tanzania. The study was conducted in accordance with the principles of International Conference of Harmonisation good clinical practices and was prospectively registered on the International Standard Randomized Controlled Trial Register (ISRCTN 10248064). Participants and Procedures Between October 2014 and September 2016, sequential HIV-infected adults aged ≥18 years with a first episode of CM, diagnosed by cerebrospinal fluid (CSF) India ink or cryptococcal antigen (CrAg) lateral flow assay (IMMY, Norman, Oklahoma), were screened for enrollment in the trial. Pregnant or lactating patients, patients with a previous serious reaction to study drugs, or patients on antifungal treatment for >48 hours were excluded. Patients who were both antiretroviral therapy (ART) naive and ART exposed were recruited. Written informed consent was obtained from participants or, in the case of mental obtundation, from a guardian or person with legal responsibility. Patients with mental obtundation were reconsented on recovery. Patients were block randomized individually to 1 of 4 treatment groups by means of random computer-generated lists with an allocation ratio of 1:1:1:1 and block sizes of 8. Randomization lists were created by an independent statistician who prepared sealed envelopes in advance that were sent to the sites. Trial pharmacists were responsible for randomization at each site. Randomization was stratified by abnormal mental status (Glasgow Coma Scale [GCS] score of 15 or <15) and ART status on admission at each site. The patients and clinical trial team were not blinded. Laboratory staff performing quantitative fungal cultures were blinded to treatment allocation. The 4 treatment arms were (i) L-AmB (AmBisome, Gilead Sciences) 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); and (iv) L-AmB 3 mg/kg/day for 14 days (control). L-AmB was given by intravenous infusion over 2 hours. All patients also received 1200 mg/day oral fluconazole (Diflucan [Pfizer] or fluconazole [Medopharm]) for the first 2 weeks. Unless contraindicated, all patients received 1 L of 0.9% normal saline with 20 mmol of potassium chloride (KCl) prior to L-AmB to minimize nephrotoxicity and were routinely given oral potassium (16 mmol KCl twice daily) and magnesium (11 mmol Mg2+ once daily) supplementation and daily trimethoprim-sulfamethoxazole prophylaxis. After the 2-week induction phase, patients received fluconazole 800 mg/day until 10 weeks and 200 mg/day thereafter. ART consisting of tenofovir, emtricitabine, and efavirenz was commenced 4–6 weeks after initiation of antifungal therapy in individuals not already on ART. Evaluations and Outcomes At baseline patients underwent a lumbar puncture (LP) for opening pressure, cell count and differential, protein, glucose, India ink, CrAg, quantitative fungal culture, and routine bacterial culture. LPs for opening pressure measurements and CSF samples for quantitative fungal culture were repeated on treatment days 3, 7, and 14. Patients with a CSF opening pressure >30 cm H2O or symptoms of raised intracranial pressure underwent daily LPs to remove CSF in accordance with guidelines [15]. Quantitative cryptococcal cultures were plated in serial 10-fold dilution and the dilution with the least colonies, but at least 30 colony-forming units (CFUs)/200 μL, was used to calculate CFU/mL quantitative cryptococcal culture results, as previously described [24]. A linear regression of log10 CFU/mL against time was calculated for each patient. All data points were analyzed except sterile cultures in the second week if these values lessened the slope, as sterility would have been achieved before that day’s LP and using the second week’s value would therefore underestimate the true slope [9, 24]. All participants had baseline blood tests including full blood count, urea, creatinine, electrolytes, alanine aminotransferase (ALT), HIV test (if status unknown), and CD4 cell count. During the 2-week induction phase, patients underwent alternate-day renal function and electrolyte assessment and twice-weekly monitoring of full blood count and ALT. Clinical and laboratory adverse events (AEs) were graded using the National Institutes of Health Division of AIDS toxicity table [27]. Clinical response was monitored daily for the first 2 weeks or until discharge (whichever was later), then in a follow-up clinic 3, 4, 6, and 10 weeks after starting therapy. The primary outcome measure was the mean rate of decrease in CSF cryptococcal CFU, also known as early fungicidal activity (EFA) of each L-AmB treatment arm. Secondary outcome measures were mortality at 2 and 10 weeks; proportion of patients in each treatment arm suffering clinical and laboratory-defined grade 3/4 AEs; and median percentage change from baseline in laboratory-defined parameters. Statistical Analysis Using a noninferiority design, assuming an EFA of 0.50 log10 CFU/mL/day with a standard deviation of 0.25 log10 CFU/mL/day in the standard daily dosing arm, with a prespecified acceptable Δ of 0.2 log10 CFU/mL/day, 1-sided α of .025, and 90% power, gave a sample size of 33 patients per arm. The prespecified Δ of 0.2 log10 CFU/mL/day was selected on the basis of prior evidence showing increased mortality once EFA falls below 0.3 log10 CFU/mL/day (ie, the projected 0.50 log10 CFU/mL/day in the control arm minus prespecified Δ of 0.2 log10 CFU/mL/day) [26]. A sample size of 40 patients per arm (160 patients in total) was planned to allow for patients who died prior to obtaining EFA measurement. An interim analysis was planned after 80 participants were randomized in the study. The primary analysis was based on the intention-to-treat population. Patients who died before having a repeat LP on day 3 or those with a negative baseline culture could not have an EFA calculated and were therefore not included the EFA analysis, but were analyzed for secondary endpoints. Linear regression models were used, with the mean rate of decrease in log10 CSF cryptococcal CFU (EFA) being the dependent variable and the treatment groups (using the control group as a comparator) the primary independent variables. The short-course L-AmB groups were compared to the control arm for noninferiority using the prespecified Δ of 0.2 log10 CFU/mL/day. Statistical significance was defined as P ≤ .05. Following an unadjusted EFA analysis, adjusted analysis was performed including covariates that may determine outcomes (baseline fungal burden, CD4 cell count, abnormal mental status, sex, age, and ART status) giving summary differences with 95% confidence intervals (CIs). Grade 3 and 4 AEs were tabulated by study arm, and the overall numbers of AEs compared using the χ2 test. The proportion of patients experiencing grade 3 and 4 anaemia, renal impairment, and hypokalemia during 2-week induction treatment was compared across study arms using the χ2 test. Mean change in hemoglobin and percentage change in creatinine during 2-week induction therapy were compared across study arms using analysis of variance analysis and χ2 testing, respectively. Mortality was compared across groups using χ2 testing. Data were analyzed using Stata version 13 software (StataCorp, College Station, Texas). RESULTS The study was stopped on the recommendation of the independent data monitoring committee at the preplanned interim analysis as the primary objective had been achieved, with noninferiority achieved in all 3 study arms at both the predefined 95% confidence level and the stringent 99% confidence level, and no safety concerns with short-course treatment, with the recommendation that the trial proceed onto the clinical endpoint phase 3 trial. At the time of stopping, 134 patients had been screened for the trial. Fifty-four patients were excluded (Figure 1), and 80 patients were enrolled and randomized to 1 of the 4 treatment groups: 18 to single dose, 20 to 2 doses, 21 to 3 doses, and 21 to control. One patient was excluded after randomization after it emerged they had been treated for a previous episode of CM; thus 79 patients completed the study, with no loss to follow-up during the initial 2-week induction phase. All participants received the treatment as per the randomization arm. One patient was lost to follow-up between 2 and 10 weeks. Baseline clinical and laboratory characteristics were well balanced between treatment groups (Table 1). Twenty-five patients (32%) were on ART at presentation with CM, the median baseline CD4 count was 32 cells/µL, and 28% (22) had a GCS score <15. Figure 1. View largeDownload slide Consolidated Standards of Reporting Trials (CONSORT) diagram. *All patients received their allocated intervention. No patients were lost to follow-up during the initial 2-week follow-up. †The single patient lost to follow-up had full follow-up data until hospital discharge at 2 weeks that were used in toxicity and mortality analyses. Abbreviations: CM, cryptococcal meningitis; EFA, early fungicidal activity; LP, lumbar puncture. Figure 1. View largeDownload slide Consolidated Standards of Reporting Trials (CONSORT) diagram. *All patients received their allocated intervention. No patients were lost to follow-up during the initial 2-week follow-up. †The single patient lost to follow-up had full follow-up data until hospital discharge at 2 weeks that were used in toxicity and mortality analyses. Abbreviations: CM, cryptococcal meningitis; EFA, early fungicidal activity; LP, lumbar puncture. Table 1. Baseline Characteristics of Trial Participants Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) All patients were of black African ethnicity. Abbreviations: ART, antiretroviral therapy; CFU, colony-forming units; CSF, cerebrospinal fluid; IQR, interquartile range; L-AmB, liposomal amphotericin B; TB, tuberculosis; WBC, white blood cell. aFive patients were missing baseline CD4 cell count, 5 patients were missing CSF WBC count, and a single individual was missing baseline quantitative cryptococcal culture. All other data were complete for all participants. View Large Table 1. Baseline Characteristics of Trial Participants Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) Characteristic All (n = 79) Control (n = 21) Single-Dose L-AmB (n = 18) 2-Dose L-AmB (n = 20) 3-Dose L-AmB (n = 20) Age, y, median (IQR) 38 (32–43) 39 (34–46) 37 (32–40) 37 (30–43) 38 (33–43) Sex, male, % (No.) 54% (43) 57% (12) 67% (12) 50% (10) 45% (9) Weight, kg, median (IQR) 52 (45–61) 52 (48–65) 52 (43–65) 52 (45–55) 56 (45–68) On ART at presentation, % (No.) 32% (25) 38% (8) 22% (4) 35% (7) 30% (6) Currently on TB treatment, % (No.) 11% (9) 14% (3) 11% (2) 15% (3) 5% (1) CD4 count, cells/µL, median (IQR)a 32 (8–58) 24 (5–69) 31 (12–51) 32 (10–50) 32 (16–84) Symptom duration, d, median (IQR) 14 (7–16) 14 (4–16) 14 (7–21) 9 (7–21) 8 (7–14) Glasgow Coma Scale score <15, % (No.) 28% (22) 29% (6) 28% (5) 25% (5) 30% (6) CSF opening pressure, cm H2O, median (IQR) 25 (16–36) 22 (17–31) 22 (16–29) 32 (13–38) 27 (18–55) CSF WBC count, cells/µL, median (IQR)a 12 (5–64) 10 (5–138) 15 (4–40) 15 (5–64) 13 (3–70) CSF fungal burden, log10 CFU/mL, median (IQR)a 5.0 (3.7–5.8) 4.9 (2.7–5.6) 5.2 (3.2–6.0) 5.3 (4.2–5.5) 5.0 (3.9–5.9) Hemoglobin, g/dL, median (IQR) 11 (9.5–12.6) 11.2 (9.5–12.5) 10.6 (9.5–12) 10.4(9.6–13.5) 11.7 (10.1–12.3) Creatinine, μmol/L, median (IQR) 63 (58–89) 73 (59–103) 69 (59–89) 62 (57–75) 62 (55–95) All patients were of black African ethnicity. Abbreviations: ART, antiretroviral therapy; CFU, colony-forming units; CSF, cerebrospinal fluid; IQR, interquartile range; L-AmB, liposomal amphotericin B; TB, tuberculosis; WBC, white blood cell. aFive patients were missing baseline CD4 cell count, 5 patients were missing CSF WBC count, and a single individual was missing baseline quantitative cryptococcal culture. All other data were complete for all participants. View Large Primary Outcome EFA was calculated for 69 patients (17 in the control group, 16 in the single-dose group, 18 in the 2-dose group, and 18 in the 3-dose group). Five patients died prior to follow-up LP and 5 patients had negative baseline cultures precluding EFA calculation. All the short-course, high-dose arms of L-AmB were noninferior in terms of EFA to 14 days of standard-dose L-AmB at the predefined noninferiority margin of 0.2 log10 CFU/mL/day (Figure 2A). The mean (standard deviation) EFA was –0.41 (0.11) log10 CFU/mL/day with standard treatment (control), –0.52 (0.35) log10 CFU/mL/day with single-dose L-AmB, –0.47 (0.29) log10 CFU/mL/day with 2 doses, and –0.54 (0.44) log10 CFU/mL/day with 3 doses. The difference in mean EFA between single dose and control was –0.11 (95% CI, –.29 to .07) log10 CFU/mL/day; between 2 doses and control was –0.05 (95% CI, –.20 to .10) log10 CFU/mL/day; and between 3 doses and control was –0.13 (95% CI, –.35 to .09) log10 CFU/mL/day. There was no evidence for any dose-response effect with additional L-AmB doses, suggesting maximal fungicidal activity was achieved with a single 10 mg/kg dose. This remained the case when the analysis was adjusted for factors that have previously been shown to affect EFA (CSF fungal burden and CD4 cell count), abnormal mental status, and also sex, age, and ART status (Figure 2C). Figure 2. View largeDownload slide Early fungicidal activity (EFA) by treatment group. A, Difference in mean EFA between intervention arms and control. All 3 short-course treatment arms were noninferior to control. B, Individual patient slopes over the initial 14 days of treatment. The mean slope (standard deviation) is given below each plot. Sterile cultures in the second week that lessened the slope and were excluded from EFA calculation (as sterility would have been achieved before that day’s lumbar punctures) are shown in the dotted gray line. C, Adjusted difference in mean EFA between intervention arms and control. All 3 short-course treatment arms remained noninferior to control when adjusted for (i) baseline fungal burden (quantitative cryptococcal culture [QCC]); (ii) baseline CD4 cell count; (iii) baseline mental status; (iv) QCC and CD4 cell count; (v) QCC, CD4 cell count, and mental status; and (vi) QCC, CD4 cell count, mental status, sex, age, and antiretroviral therapy status. Abbreviations: CFU, colony-forming units; CI, confidence interval; SD, standard deviation. Figure 2. View largeDownload slide Early fungicidal activity (EFA) by treatment group. A, Difference in mean EFA between intervention arms and control. All 3 short-course treatment arms were noninferior to control. B, Individual patient slopes over the initial 14 days of treatment. The mean slope (standard deviation) is given below each plot. Sterile cultures in the second week that lessened the slope and were excluded from EFA calculation (as sterility would have been achieved before that day’s lumbar punctures) are shown in the dotted gray line. C, Adjusted difference in mean EFA between intervention arms and control. All 3 short-course treatment arms remained noninferior to control when adjusted for (i) baseline fungal burden (quantitative cryptococcal culture [QCC]); (ii) baseline CD4 cell count; (iii) baseline mental status; (iv) QCC and CD4 cell count; (v) QCC, CD4 cell count, and mental status; and (vi) QCC, CD4 cell count, mental status, sex, age, and antiretroviral therapy status. Abbreviations: CFU, colony-forming units; CI, confidence interval; SD, standard deviation. Mortality Overall all-cause mortality rates were 15% (12/79) at 2 weeks and 29% (23/79) at 10 weeks, with no significant difference between treatment arms. Two-week mortality was 10% (2/21) in the control arm, 11% (2/18) in the single-dose arm, 15% (3/20) in the 2-dose arm, and 25% (5/20) in the 3-dose arm (P = .52). At 10 weeks, mortality was 29% (6/21) in the control arm, 22% (4/18) in the single-dose arm, 15% (3/20) in the 2-dose arm, and 50% (10/20) in the 3-dose arm (P = .09; Table 2). Mortality at 10 weeks was associated with abnormal mental status at baseline in univariable analysis (odds ratio, 3.75 [95% CI, 1.3–10.7]), but not with baseline fungal burden, baseline CD4 cell count, or ART status. The mortality difference between the single-dose and control arms was 6.4% (95% CI, –21% to 34%). Table 2. Primary and Key Secondary Outcomes Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Abbreviations: AE, adverse event; CFU, colony-forming units; CI, confidence interval; EFA, early fungicidal activity; L-AmB, liposomal amphotericin B. aIndividuals who die prior to the day 3 lumbar puncture (LP) or who were culture negative at baseline do not have an EFA value. Overall, 5 patients died prior to follow-up LP (1 control, 1 single dose, 1 two dose, 2 three dose) and 5 patients had negative baseline cultures (3 controls, 1 single dose, 1 two dose). bAll 3 study arms were noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. The respective difference in mean EFA between single dose and control was –0.11 (99% CI, –.35 to .14) log10 CFU/mL/day; between 2 doses and control was –0.05 (99% CI, –.26 to .16) log10 CFU/mL/day; and between 3 doses and control was –0.13 (99% CI, –.42 to .17) log10 CFU/mL/day. Using this more stringent cutoff, all 3 study arms remained noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. View Large Table 2. Primary and Key Secondary Outcomes Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Outcome All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P Value EFA, log10 CFU/mL/d, mean (95% CI) –0.49 (–.56 to –.41), n = 69a –0.41 (–.47 to –.36), n = 17 –0.52 (–.71 to –.33), n = 16 –0.47 (–.6 to –.32), n = 18 –0.54 (–.76 to –.33), n = 18 .64 Mean difference in EFA vs control, log10 CFU/mL/d, mean (95% CI) … … –0.11 (–.29 to .07) –0.05 (–.20 to .10) –0.13 (–.35 to .09) b 2-week mortality, % (No.) 15 (12/79) 10 (2/21) 11 (2/18) 15 (3/20) 25 (5/20) .52 10-week mortality, % (No.) 29 (23/79) 29 (6/21) 22 (4/18) 15 (3/20) 50 (10/20) .09 Grade 3 AEs during induction therapy (days 1–14), % (No.)  Anemia 6 (5) 0 (0) 11 (2) 15 (3) 0 (0) .11  Renal impairment 5 (4) 0 (0) 6 (1) 0 (0) 15 (3) .10  Hypokalemia 1 (1) 0 (0) 0 (0) 5 (1) 0 (0) .39 Grade 4 AEs during induction therapy (days 1–14), % (No.)  Anemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42  Renal impairment 1 (1) 0 (0) 0 (0) 0 (0) 5 (1) .39  Hypokalemia 1 (1) 5 (1) 0 (0) 0 (0) 0 (0) .42 Mean change from baseline to day 14  Hemoglobin, g/dL, mean (95% CI) 0.9 (.5–1.4) 1.2 (.1–2.3) 0.8 (–.1 to 1.7) 0.3 (–.6 to 1.3) 1.4 (.5–2.2) .39  Creatinine, %, mean (95% CI) 14 (3–24) 17 (–9 to 43) 13 (–9 to 35) 24 (6–42) –2 (–22 to 18) .29 Abbreviations: AE, adverse event; CFU, colony-forming units; CI, confidence interval; EFA, early fungicidal activity; L-AmB, liposomal amphotericin B. aIndividuals who die prior to the day 3 lumbar puncture (LP) or who were culture negative at baseline do not have an EFA value. Overall, 5 patients died prior to follow-up LP (1 control, 1 single dose, 1 two dose, 2 three dose) and 5 patients had negative baseline cultures (3 controls, 1 single dose, 1 two dose). bAll 3 study arms were noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. The respective difference in mean EFA between single dose and control was –0.11 (99% CI, –.35 to .14) log10 CFU/mL/day; between 2 doses and control was –0.05 (99% CI, –.26 to .16) log10 CFU/mL/day; and between 3 doses and control was –0.13 (99% CI, –.42 to .17) log10 CFU/mL/day. Using this more stringent cutoff, all 3 study arms remained noninferior to control at the predefined noninferiority margin of 0.2 log10 CFU/mL/day. View Large Safety There were no safety concerns with short-course treatment in terms of fungal clearance, and no patients receiving short-course L-AmB required additional “rescue” L-AmB therapy. The 3 high-dose short-course L-AmB regimens were all well tolerated. Eighty-eight grade 3 and higher AEs occurred in 47 patients: 45 grade 3 and 43 grade 4/5 AEs, with no significant differences observed between treatment arms (Table 3). Of these, 49 were clinical, and 39 laboratory AEs. There were 10 grade 3 and 2 grade 4 AEs that were attributed to treatment with L-AmB, all of which were expected L-AmB–related side effects (3 grade 3 hypokalemia, 1 grade 4 hypokalemia, 1 grade 3 hypomagnasemia, 4 grade 3 creatinine rises, 1 grade 3 and 1 grade 4 anemia, 1 grade 4 hyponatremia). Both grade 4 L-AmB related events occurred in the control group. During induction therapy, grade 3 and 4 anemia occurred in 6% (n = 5) and 1% (n = 1) overall, renal impairment in 5% (n = 4) and 1% (n = 1) overall, and hypokalemia in 1% (n = 1) and 1% (n = 1) overall, with no significant differences between treatment arms (Table 2). Table 3. Adverse Events and Readmissions During 10-Week Follow-up Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Data are presented as No. unless otherwise indicated. Abbreviations: AE, adverse event; ALT, alanine aminotransferase; CM, cryptococcal meningitis; ICP, intra-cranial pressure; IRIS, immune reconstitution inflammatory syndrome; L-AmB, liposomal amphotericin B. aForty-seven patients had at least 1 AE: 2 patients had 5 AEs, 2 patients had 4 AEs, 5 patients had 3 AEs, 7 patients had 2 AEs, 6 patients had 1 AE, and 33 had no AEs. Twenty-eight patients had grade 3 AEs: 4 in the control arm, 11 in the single-dose arm, 6 in the 2-dose arm, and 7 in the 3-dose arm. Fifteen patients had grade 4 AEs: 5 in the control arm, 5 in the single-dose arm, 2 in the 2-dose arm, and 3 in the 3-dose arm. bRelated includes all AEs classified as possibly, probably, or definitely related to study drug. View Large Table 3. Adverse Events and Readmissions During 10-Week Follow-up Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Adverse Event All Control Single-Dose L-AmB 2-Dose L-AmB 3-Dose L-AmB P  Value Overall  All AEs 88a 19 29 14 26 .50  Grade 3 AEs 45 7 18 9 11 …   Elevated creatinine 0 2 1 3   Hypokalemia 1 0 1 0   Hypomagnesemia 1 0 0 0   Hyponatremia 2 4 1 0   Elevated ALT 0 2 1 2   Anemia 0 2 1 0   Neutropenia 1 2 2 0   Prolonged initial hospitalization 2 3 1 5   Persistently raised ICP 0 1 1 0   Other 0 Co-trimoxazole allergy, confusion 0 Pneumonia  Grade 4 AEs 20 6 7 2 5 …   Elevated creatinine 1 1 0 1   Hypokalemia 1 0 0 0   Hyponatremia 2 0 0 1   Hypernatremia 0 1 0 0   Elevated ALT 0 0 1 0   Anemia 1 0 0 0   Neutropenia 0 1 1 0   Prolonged initial hospitalization 0 1 0 1   Recurrence of CM symptoms 1 1 0 2   Other 0 Recurrent seizures, persistently raised ICP 0 0  Grade 5 AEs (deaths) 23 6 4 3 10 … AEs related to L-AmB therapyb  Grade 3 AEs 10 3 2 2 3 …  Grade 4 AEs 2 2 0 0 0 … Readmissions and IRIS  Readmissions 11 4 4 0 3 .48  Possible IRIS 5 1 1 0 3 Data are presented as No. unless otherwise indicated. Abbreviations: AE, adverse event; ALT, alanine aminotransferase; CM, cryptococcal meningitis; ICP, intra-cranial pressure; IRIS, immune reconstitution inflammatory syndrome; L-AmB, liposomal amphotericin B. aForty-seven patients had at least 1 AE: 2 patients had 5 AEs, 2 patients had 4 AEs, 5 patients had 3 AEs, 7 patients had 2 AEs, 6 patients had 1 AE, and 33 had no AEs. Twenty-eight patients had grade 3 AEs: 4 in the control arm, 11 in the single-dose arm, 6 in the 2-dose arm, and 7 in the 3-dose arm. Fifteen patients had grade 4 AEs: 5 in the control arm, 5 in the single-dose arm, 2 in the 2-dose arm, and 3 in the 3-dose arm. bRelated includes all AEs classified as possibly, probably, or definitely related to study drug. View Large Eleven trial participants were readmitted to hospital during the 10-week follow-up period, at a median of 41 (interquartile range, 25–55) days, including 4 in the control arm, 4 in the single-dose arm, none in the 2-dose arm, and 3 in the 3-dose arm. Cryptococcal immune reconstitution inflammatory syndrome (IRIS) was suspected or diagnosed in 5 of the 45 (11%) patients initiating ART, 2 of whom died, with no significant differences between study arms. DISCUSSION The use of a single 10 mg/kg dose of L-AmB was noninferior to standard 3 mg/kg daily dosing for 14 days in reducing CSF cryptococcal burden in patients with a first episode of HIV-associated CM. These findings are consistent with previous human and animal studies demonstrating that shorter courses of amphotericin-based treatment may be better tolerated and as effective as conventional 14-day courses [13, 17, 28–30]. High dosages of L-AmB were well tolerated, and the safety profile of all L-AmB regimens tested compared favorably to data from prior clinical trials using conventional AmB-d in similar patient populations, both in terms of mortality at 10 weeks and drug-induced toxicities [9, 11]. Overall rates of AEs associated with L-AmB were very low, with just 1 patient (1%) developing grade 4 anemia during induction therapy (in the control arm), compared with 18% of a historic cohort of 368 CM patients receiving AmB-d treatment and an identical prehydration and electrolyte supplementation regimen to that used in the current trial [11]. The median fall in hemoglobin during the first 2 weeks of treatment was 0.9 g/dL, compared with 2.3 g/dL in the previous cohort of AmB-d–treated patients [11], and there was a median increase in creatinine of 14% over the initial 2 weeks, compared to 73% in the AmB-d–treated cohort [11]. There were no grade 4 AEs attributed to high-dose L-AmB during the trial. Rates of recurrence of CM symptoms and IRIS were low, with suspected IRIS events occurring in 11% of individuals initiated on ART during the trial. Based on these phase 2 results, single-dose 10 mg/kg L-AmB is being taken forward to a phase 3 clinical endpoint trial (ISRCTN 72509687). Given the correlation between EFA and clinical outcome [9, 25], the rapid EFA seen with single 10 mg/kg doses of L-AmB should result in a clinically efficacious alternative treatment for CM. The 10-week mortality rate of 22% with the single-dose 10 mg/kg L-AmB selected for study in the phase 3 trial, and the overall mortality rate of 29% in the trial, compare favorably with mortality rates of approximately 40% seen in recent large clinical trials of 2-week AmB-d–based treatment [7–9]. Notably, these mortality rates were in the context of fluconazole as a second antifungal agent. The addition to high dose L-AmB of a more efficacious agent such as flucytosine, which has been proven to be superior to fluconazole in the recent Advancing CM Treatment in Africa (ACTA) trial [30], may enable a further reduction in mortality rates. The current phase 2 study was not powered to detect a mortality difference, as shown by the wide 95% CIs around the mortality difference, and as expected, no significant difference in mortality between the 4 L-AmB treatment arms was seen. The higher mortality rate in the 3-dose arm was likely due to chance alone, with 40% (n = 4) of the deaths occurring prior to receipt of the third dose of L-AmB. In conclusion, we have demonstrated that a single 10 mg/kg dose of liposomal amphotericin B given in combination with high-dose fluconazole is noninferior to daily dosed L-AmB at the standard dose of 3 mg/kg plus high-dose fluconazole in terms of rate of fungal clearance in patients with HIV-associated CM. This short-course treatment strategy is now being tested against AmB-d in a clinical endpoint trial. If confirmed to be effective, single high dosages of L-AmB given with an optimized oral antifungal medication backbone would provide a feasible, well-tolerated, and sustainable treatment regimen for HIV-associated CM in resource-limited settings where the safe administration of AmB-d treatment is not possible. Reductions in the need for toxicity monitoring, fewer drug-related AEs, and the potential for shorter periods of hospitalization are likely to mean that a single high-dose L-AmB treatment strategy is cost effective, and a highly favorable alternative to the current standard of care. Notes Author contributions. J. N. J. and T. S. H. conceptualized and designed the study, supervised implementation, analyzed the data, and drafted the final manuscript. T. B. L., A. A. C., G. B., M. M., R. K. K. P., and M. W. T. implemented the study. K. T. and N. L. were the research nurses, C. M. implemented the laboratory aspects of the trial, and N. M. was the study pharmacist. J. K. and J. C. supervised implementation. D. L. drafted the initial manuscript. W. H. assisted with conceptualization and design of the study and critically reviewed the manuscript. S. M. was the trial manager, assisted with study design, supervised implementation and data management, and helped draft the final manuscript. All authors reviewed and approved the final manuscript. The authors had full access to all study data and had final responsibility for the decision to submit for publication. Acknowledgments. We thank the management and staff of Princess Marina Hospital, Bugando Medical Centre, and Sekou Toure Hospital for their assistance and support; Drs Mosepele Mosepele, Miriam Haverkamp, Elizabeth Williams, William Hurt, and Hannah Mitchell for their assistance with patient care; John Bradley for preparing the randomization codes; and the members of the Data Monitoring Committee (Andrew Nunn, Rob Peck, and Mina Hosseinipour) and the Trial Steering Committee (David Boulware, Saidi Kapiga, and Mary Kasule) for their oversight of the trial. Disclaimer. The funding source and drug manufacturers had no involvement in the study design; collection, analysis, and interpretation of data; preparation of manuscripts; or the decision to submit this manuscript for publication. Financial support. Gilead Sciences funded the trial through an Investigator-Initiated award (IN-EU-131-D036) and provided the L-AmB for the trial. The work was made possible through core support from the Penn Center for AIDS Research, an National Institutes of Health (NIH)-funded program (grant number P30 AI 045008). Potential conflicts of interest. J. N. J. and T. H. were recipients of a Gilead Investigator award. T. H. declares consultancy fees from Viamet, lecture fees from Pfizer and Gilead Sciences, and payments from Immuno-Mycologics. W. H. holds or has recently held research grants with F2G, AiCuris, Astellas Pharma, Spero Therapeutics, Matinas Biosciences, Antabio, Amplyx, Allecra, Auspherix, and Pfizer; holds awards from the NIH, the Medical Research Council, the National Institute for Health Research, and the European Commission (FP7 and IMI); has received personal fees in his capacity as a consultant for F2G, Amplyx, Ausperix, Spero Therapeutics, The Medicines Company, Gilead, and Basilea; and is Medical Guideline Director for the European Society of Clinical Microbiology and Infectious Diseases, and an Ordinary Council Member for the British Society of Antimicrobial Chemotherapy. G. B. declares consultancy fees from Pfizer, grants and travel expenses from NIH, and lecture payments from ViralEd. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Braitstein P , Brinkhof MW , Dabis F , et al. ; Antiretroviral Therapy in Lower Income Countries (ART-LINC) Collaboration; ART Cohort Collaboration (ART-CC) groups . Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries . Lancet 2006 ; 367 : 817 – 24 . Google Scholar Crossref Search ADS PubMed 2. Lawn SD , Harries AD , Anglaret X , Myer L , Wood R . Early mortality among adults accessing antiretroviral treatment programmes in sub-Saharan Africa . AIDS 2008 ; 22 : 1897 – 908 . Google Scholar Crossref Search ADS PubMed 3. Amuron B , Namara G , Birungi J , et al. Mortality and loss-to-follow-up during the pre-treatment period in an antiretroviral therapy programme under normal health service conditions in Uganda . BMC Public Health 2009 ; 9 : 290 . Google Scholar Crossref Search ADS PubMed 4. Gupta A , Nadkarni G , Yang WT , et al. Early mortality in adults initiating antiretroviral therapy (ART) in low- and middle-income countries (LMIC): a systematic review and meta-analysis . PLoS One 2011 ; 6 : e28691 . Google Scholar Crossref Search ADS PubMed 5. Park BJ , Wannemuehler KA , Marston BJ , Govender N , Pappas PG , Chiller TM . Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS . AIDS 2009 ; 23 : 525 – 30 . Google Scholar Crossref Search ADS PubMed 6. Rajasingham R , Smith RM , Park BJ , et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis . Lancet Infect Dis 2017 ; 17 : 873 – 81 . Google Scholar Crossref Search ADS PubMed 7. Day JN , Chau TTH , Wolbers M , et al. Combination antifungal therapy for cryptococcal meningitis . N Engl J Med 2013 ; 368 : 1291 – 302 . Google Scholar Crossref Search ADS PubMed 8. Beardsley J , Wolbers M , Kibengo FM , et al. ; CryptoDex Investigators . Adjunctive dexamethasone in HIV-associated cryptococcal meningitis . N Engl J Med 2016 ; 374 : 542 – 54 . Google Scholar Crossref Search ADS PubMed 9. Jarvis JN , Bicanic T , Loyse A , et al. Determinants of mortality in a combined cohort of 501 patients with HIV-associated cryptococcal meningitis: implications for improving outcomes . Clin Infect Dis 2014 ; 58 : 736 – 45 . Google Scholar Crossref Search ADS PubMed 10. Boulware DR , Meya DB , Muzoora C , et al. ; COAT Trial Team . Timing of antiretroviral therapy after diagnosis of cryptococcal meningitis . N Engl J Med 2014 ; 370 : 2487 – 98 . Google Scholar Crossref Search ADS PubMed 11. Bicanic T , Bottomley C , Loyse A , et al. Toxicity of amphotericin B deoxycholate-based induction therapy in patients with HIV-associated cryptococcal meningitis . Antimicrob Agents Chemother 2015 ; 59 : 7224 – 31 . Google Scholar Crossref Search ADS PubMed 12. Longley N , Muzoora C , Taseera K , et al. Dose response effect of high-dose fluconazole for HIV-associated cryptococcal meningitis in southwestern Uganda . Clin Infect Dis 2008 ; 47 : 1556 – 61 . Google Scholar Crossref Search ADS PubMed 13. Jackson AT , Nussbaum JC , Phulusa J , et al. A phase II randomized controlled trial adding oral flucytosine to high-dose fluconazole, with short-course amphotericin B, for cryptococcal meningitis . AIDS 2012 ; 26 : 1363 – 70 . Google Scholar Crossref Search ADS PubMed 14. Hamill RJ , Sobel JD , El-Sadr W , et al. Comparison of 2 doses of liposomal amphotericin B and conventional amphotericin B deoxycholate for treatment of AIDS-associated acute cryptococcal meningitis: a randomized, double-blind clinical trial of efficacy and safety . Clin Infect Dis 2010 ; 51 : 225 – 32 . Google Scholar Crossref Search ADS PubMed 15. Perfect JR , Dismukes WE , Dromer F , et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America . Clin Infect Dis 2010 ; 50 : 291 – 322 . Google Scholar Crossref Search ADS PubMed 16. Nelson M , Dockrell D , Edwards S , et al. ; BHIVA Guidelines Subcommittee . British HIV Association and British infection association guidelines for the treatment of opportunistic infection in HIV-seropositive individuals 2011 . HIV Med 2011 ; 12 ( Suppl 2 ): 1 – 140 . Google Scholar Crossref Search ADS PubMed 17. Lestner J , McEntee L , Johnson A , et al. Experimental models of short courses of liposomal amphotericin B for induction therapy for cryptococcal meningitis . Antimicrob Agents Chemother 2017 ; 61 . doi: https://doi.org/10.1128/AAC.00090-17 . 18. Vogelsinger H , Weiler S , Djanani A , et al. Amphotericin B tissue distribution in autopsy material after treatment with liposomal amphotericin B and amphotericin B colloidal dispersion . J Antimicrob Chemother 2006 ; 57 : 1153 – 60 . Google Scholar Crossref Search ADS PubMed 19. O’Connor L , Livermore J , Sharp AD , et al. Pharmacodynamics of liposomal amphotericin B and flucytosine for cryptococcal meningoencephalitis: safe and effective regimens for immunocompromised patients . J Infect Dis 2013 ; 208 : 351 – 61 . Google Scholar Crossref Search ADS PubMed 20. Gubbins PO , Amsden JR , McConnell SA , Anaissie EJ . Pharmacokinetics and buccal mucosal concentrations of a 15 milligram per kilogram of body weight total dose of liposomal amphotericin B administered as a single dose (15 mg/kg), weekly dose (7.5 mg/kg), or daily dose (1 mg/kg) in peripheral stem cell transplant patients . Antimicrob Agents Chemother 2009 ; 53 : 3664 – 74 . Google Scholar Crossref Search ADS PubMed 21. Mehta P , Vinks A , Filipovich A , et al. High-dose weekly AmBisome antifungal prophylaxis in pediatric patients undergoing hematopoietic stem cell transplantation: a pharmacokinetic study . Biol Blood Marrow Transplant 2006 ; 12 : 235 – 40 . Google Scholar Crossref Search ADS PubMed 22. Cornely OA , Maertens J , Bresnik M , et al. ; AmBiLoad Trial Study Group . Liposomal amphotericin B as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (AmBiLoad trial) . Clin Infect Dis 2007 ; 44 : 1289 – 97 . Google Scholar Crossref Search ADS PubMed 23. Sundar S , Chakravarty J , Agarwal D , Rai M , Murray HW . Single-dose liposomal amphotericin B for visceral leishmaniasis in India . N Engl J Med 2010 ; 362 : 504 – 12 . Google Scholar Crossref Search ADS PubMed 24. Brouwer AE , Rajanuwong A , Chierakul W , et al. Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial . Lancet 2004 ; 363 : 1764 – 7 . Google Scholar Crossref Search ADS PubMed 25. Bicanic T , Muzoora C , Brouwer AE , et al. Independent association between rate of clearance of infection and clinical outcome of HIV-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients . Clin Infect Dis 2009 ; 49 : 702 – 9 . Google Scholar Crossref Search ADS PubMed 26. Molefi M , Chofle AA , Molloy SF , et al. Ambition-cm: intermittent high dose AmBisome on a high dose fluconazole backbone for cryptococcal meningitis induction therapy in sub-Saharan Africa: study protocol for a randomized controlled trial . Trials 2015 ; 16 : 276 . Google Scholar Crossref Search ADS PubMed 27. National Institute of Allergy and Infectious Diseases, Division of AIDS . Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse events, version 2.1 . Bethesda, MD : NIAID , 2017 . 28. Muzoora CK , Kabanda T , Ortu G , et al. Short course amphotericin B with high dose fluconazole for HIV-associated cryptococcal meningitis . J Infect 2012 ; 64 : 76 – 81 . Google Scholar Crossref Search ADS PubMed 29. Livermore J , Howard SJ , Sharp AD , et al. Efficacy of an abbreviated induction regimen of amphotericin B deoxycholate for cryptococcal meningoencephalitis: 3 days of therapy is equivalent to 14 days . MBio 2013 ; 5 : e00725 – 13 . 30. Molloy SF , Kanyama C , Heyderman RS , et al. ; ACTA Trial Study Team . Antifungal combinations for treatment of cryptococcal meningitis in Africa . N Engl J Med 2018 ; 378 : 1004 – 17 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Clinical Infectious DiseasesOxford University Press

Published: Jan 18, 2019

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off