toxicity of amphotericin b deox ycholate-based in...

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Toxicity of Amphotericin B deoxycholate-based induction therapy 1

in patients with HIV-associated cryptococcal meningitis 2

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Tihana Bicanic1♯, Christian Bottomley2, Angela Loyse1, Annemarie E Brouwer3, 4

Conrad Muzoora4, Kabanda Taseera4, Arthur Jackson5, Jacob Phulusa5, Mina C 5

Hosseinipour5, Charles van der Horst5, Direk Limmathurotsakul6, Nicholas J 6

White6,7, Douglas Wilson8, Robin Wood9, Graeme Meintjes10,11, Thomas S 7

Harrison1, Joseph N Jarvis12-14 8

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1. Institute of Infection and Immunity, St. George’s University of London, 10

UK 11

2. Faculty of Epidemiology and Population Health, London School of 12

Hygiene & Tropical Medicine, UK. 13

3. Elisabeth Hospital, Tilburg, the Netherlands 14

4. Mbarara University of Science and Technology, Uganda 15

5. University of North Carolina (UNC) Project, Lilongwe, Malawi 16

6. Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol 17

University, Bangkok, Thailand 18

7. Centre for Tropical Medicine and Global Health, Nuffield Department of 19

Clinical Medicine, University of Oxford 20

8. Edendale Hospital, Pietermaritzburg, South Africa 21

9. Desmond Tutu HIV Centre, Institute of Infectious Disease and Molecular 22

Medicine, University of Cape Town, South Africa 23

AAC Accepted Manuscript Posted Online 8 September 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.01698-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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10. Division of Infectious Diseases and HIV Medicine, Department of 24

Medicine, University of Cape Town, South Africa 25

11. Institute of Infectious Disease and Molecular Medicine, University of 26

Cape Town, South Africa 27

12. Department of Clinical Research, Faculty of Infectious and Tropical 28

Diseases London School of Hygiene and Tropical Medicine, UK 29

13. Botswana-UPenn Partnership, Gaborone, Botswana 30

14. Division of Infectious Diseases, Department of Medicine, Perelman 31

School of Medicine, University of Pennsylvania, USA 32

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Running title: AmB toxicity cryptococcal meningitis HIV 34

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♯ Address correspondence to Tihana Bicanic [email protected] 36

Senior Lecturer and Consultant in Infectious Diseases 37

Institute of Infection and Immunity, St George’s University of London 38

Cranmer Terrace, London, SW17 0RE, UK 39

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Key words: amphotericin B; amphotericin B deoxycholate; cryptococcal 42

meningitis; drug therapy; drug toxicity; antifungal 43

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Word count 2536 45

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Abstract 51

Amphotericin B deoxycholate (AmBd) is the recommended induction treatment 52

for HIV-associated cryptococcal meningitis (CM). Use is hampered by toxicities 53

that include electrolyte abnormalities, nephrotoxicity and anaemia. Protocols to 54

minimise toxicity are applied inconsistently. In a clinical trial cohort of AmBd-55

based CM induction treatment, a standardised protocol of pre-emptive 56

hydration and electrolyte supplementation was applied. Changes in blood 57

counts, electrolytes, and creatinine over 14 days were analysed in relation to 58

AmBd dose, treatment duration (short course, 5-7 days or standard, 14 days), 59

addition of flucytosine, and outcome. In the 368 patients studied, haemoglobin 60

dropped by a mean(95% CI) of 1.5(1.0-1.9) g/dL following 7 days of AmBd, and 61

2.3(1.1-3.6) g/dL after 14 days. Serum creatinine increased by 37(30-45) μmol/L 62

by day 7 and 49(35-64) μmol/L by day 14 of AmBd. Overall 33% of patients 63

developed grade III/IV anaemia, 5.6% developed grade III hypokalaemia, in 64

9.5% creatinine exceeded 220μmol and 6% discontinued AmBd prematurely. 65

Addition of 5FC was associated with a slight increase in anaemia but not 66

neutropenia. Laboratory abnormalities stabilised or reversed during the second 67

week in patients on short course induction. Grade III/IV anaemia(aOR 2.2, 95% 68

CI 1.1-4.3, p= 0.028); and nephrotoxicity(aOR 4.5, 95% CI 1.8-11, p=0.001) were 69

risk factors for 10-week mortality. In summary, routine intravenous saline 70

hydration and pre-emptive electrolyte replacement during AmBd-based 71

induction regimens for HIV-associated CM minimised incidence of 72

hypokalaemia and nephrotoxicity. Anaemia remained a concerning adverse 73


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effect. Addition of flucytosine was not associated with increased neutropenia. 74

Shorter AmBd courses were less toxic, with rapid reversibility. 75

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Introduction 77

Amphotericin B deoxycholate(AmBd) is a polyene antifungal that binds to 78

ergosterol in the fungal cell membrane, resulting in increased permeability and 79

cell death(1), as well as to cholesterol in mammalian cell membranes, partly 80

explaining its adverse effects of nephrotoxicity, electrolyte imbalance and 81

anaemia(1). 82

83

Less toxic lipid formulations of AmB, developed in the 1990s, have now largely 84

supplanted the use of the AmBd preparation for the treatment of systemic 85

mycoses in high-resource settings. AmBd has been used to treat cryptococcal 86

meningitis(CM) since the pre-HIV era(2, 3). AmB exhibits concentration-87

dependent killing(4), and successive randomised controlled trials used 88

progressively increasing doses as induction therapy for HIV-associated CM, 89

+/- flucytosine(5FC) or fluconazole as second agents(5-9). AmBd 1mg/kg/d 90

plus 5FC provided the best fungal clearance and 10-week survival in a recent 91

phase III trial(10) and remains the ‘gold standard’ for treatment of HIV-92

associated CM in IDSA and WHO guidelines(11, 12). 93

94

AmBd nephrotoxicity results from decreased renal blood flow resulting in 95

reduced glomerular filtration rate and direct renal tubular toxicity, causing 96

potassium and magnesium loss(1). Nephrotoxicity is cumulative and dose-97

dependent, though reversible(13, 14), and is reduced by pre-loading with saline, 98

adequate hydration and electrolyte replacement(15-19). Anaemia results 99


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primarily from inhibition of renal erythropoietin production, though 100

haemolysis has been reported(20). 101

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In resource-poor settings, facilities to prevent, monitor and manage AmBd 103

toxicity are lacking. Fear of toxicities deters clinicians from using AmBd to treat 104

CM. Instead, less effective but widely available fluconazole monotherapy is 105

used(21). 106

107

Since 2000, we have implemented a standardised protocol of pre-hydration and 108

pre-emptive electrolyte supplementation in clinical trials of AmBd-based CM 109

induction treatment in Asia and Africa(14, 22-27), which is now included in 110

WHO guidelines(12). We present the toxicity analyses of AmBd dosed at the 111

currently recommended dose range (0.7-1mg/kg/d), for treatment durations of 112

5-14 days, alone or combined with a second antifungal, in a large combined 113

cohort of patients with HIV-associated CM managed in resource-limited 114

settings. Our aims were to characterise the association of AmBd dose and 115

duration, and addition of 5FC, with the development of its common toxicities, 116

and determine the relationship between toxicity and mortality. 117

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Materials and Methods 127

Participants and procedures 128

The cohort consisted of adult HIV-infected, antiretroviral therapy(ART)-naïve 129

patients with first episode of CM enrolled in six phase II clinical trials carried 130

out from 2002-10 in Asia and Africa(14, 23-27)(Supplementary Table 1). 131

In trials 1-4 patients received standard (14-day) courses of AmBd, and in 132

studies 5-6 received shorter courses (< 7 days). Induction therapy differed in 133

terms of AmB dose (0.7 or 1mg/kg/d, dosed according to actual body weight), 134

choice of second+/- third drug and adjunctive interferon-γ. All patients on 5FC 135

received 5FC orally, except for 32 patients in the Thai study who received 136

intravenous 5FC as part of a pharmacokinetic sub-study(28). 137

138

Management of toxicity 139

Laboratory monitoring 140

During the first 14 days, all patients had alternate day measurements of 141

complete blood counts, urea, creatinine and electrolytes (magnesium 142

measurement was only available in South Africa). 143

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Electrolytes and renal impairment 145

Patients received daily pre-hydration with 1L 0.9% (normal) saline with 146

20mmol potassium chloride(KCl) before AmBd administration. Additional IV-147

fluids were at study physicians’ discretion. Nephrotoxic drugs such as 148

aminoglycosides and NSAIDS were avoided (pre-medication with paracetamol 149

or chlorpheniramine was allowed in case of infusion reactions). If creatinine 150


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rose to 220μmol/L (2.5mg/dL), equivalent to grade III nephrotoxicity on 151

Division of AIDS (DAIDS) criteria (creatinine rise 1.9-3.4X ULN), the next 152

AmBd dose was omitted and the patient was given additional IV-fluids. The 153

following day, if creatinine was stable or improving, alternate daily dosing was 154

instituted at the same AmBd dose, with return to daily dosing once creatinine 155

had normalised. If creatinine was still increasing, AmBd was stopped and the 156

patient switched early to fluconazole, adjusting dose according to renal 157

function (50% for creatinine clearance 20-40ml/min, 25% if <20ml/min). 158

Flucytosine dose interval was extended to 12-hourly if creatinine clearance 159

decreased to 20-40ml/min, and once daily if creatinine clearance <20ml/min. In 160

all South African trials, patients received oral electrolyte supplementation with 161

2 tablets twice daily of potassium chloride (Slow-K, 600mg, 8mmol K/tab) and 162

2 tablets once daily of magnesium chloride (Slow-Mag 535mg, 5.33mmol 163

Mg/tab), with additional IV or oral supplementation as required. At other sites, 164

additional IV or oral potassium was administered according to laboratory 165

results. 166

167

Bone marrow toxicity 168

Due to limited blood product availability, there was no haemoglobin threshold 169

for transfusion: this was done at study sites’ discretion. Flucytosine was 170

stopped if platelets dropped to <50x109/ml or neutrophils to <0.5x109/L. 171

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Statistical analyses 173


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We explored changes in haemoglobin(Hb), neutrophil and platelet count, 174

creatinine, potassium(K), and magnesium(Mg) over a 14-day period following 175

start of AmBd-based treatment. For each measure we used LOESS (locally 176

weighted scatterplot smoothing) to estimate the mean value as a function of the 177

number of days post-treatment initiation. 178

179

Linear regression models were constructed examining the effects of AmB dose 180

(0.7 or 1mg/kg/d) and duration (5-7 days or 14 days) on Hb, K and creatinine 181

change from baseline to days 7 and 14, peak creatinine and Hb nadir. Sex and 182

treatment with 5FC were included as potential confounders in the Hb analyses. 183

Baseline imbalances between groups were accounted for by using a ‘change 184

from baseline’ analysis rather than adjusting for baseline (with the exception of 185

peak and nadir values). 186

Logistic regression models examined associations between protocol pre-defined 187

nephrotoxicity, DAIDS grade III hypokalaemia (K+ 2-2.4 mmol/L or mEq/L), 188

grade III (Hb 6.5-7.4 g/dL) and IV anaemia (Hb<6.5g/dL) and mortality at 2 189

and 10 weeks, adjusting for baseline value (Hb, creatinine, K); patient weight in 190

the nephrotoxicity analysis, and established prognostic markers baseline fungal 191

burden, altered mental status and CD4-cell count(29). 192

193

Given the pooling of data across studies, clustering by study in both the linear 194

and logistic regression models was accounted for by including ‘study’ as a 195

random effect term. 196


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Data were analyzed using Stata, v12.0 (StataCorp TX77845 USA). 198


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Results 199

368 (52% male) patients were included; 64 (17%) were Thai, 298 (81%) Black 200

African and 3(<1%) of mixed race. Median (IQR) age was 33 (29-38) years and 201

CD4 count 25 (10-55) cells/μL. Changes from baseline (pre-treatment) are 202

shown in Table 1 and LOESS curves (Figures 1 and 2). 203

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From a mean baseline of 11g/dL, mean overall(%) drop in haemoglobin was 205

1.5g/dL(12%) by day 7 and 2.3g/dL(20%) by day 14. With pre-emptive 206

supplementation, potassium values remained stable over time (Supplementary 207

Figure 1a). Mean(95%CI) creatinine change from baseline was +37(30-45) 208

μmol/L at day 7 and +49(35-64) μmol/L at day 14. 209

210

Incidence of serious toxicity 211

Grade III/IV anaemia (Hb<7.5g/dL) developed in 112 patients(33%). 212

Fifty-nine patients(18%) developed grade IV anaemia (Hb<6.5g/dL). Women 213

had a higher incidence of Grade III (54% vs 14%) and grade IV anaemia (31% vs 214

5%, p<0.001) due to sex differences in baseline, rather than differences in on-215

treatment change (d14 Hb drop 2.14g/dL males vs 2.21g/dL females, p=0.92) 216

(Fig 1c). In the South African trials, which collected transfusion data, 12 of 304 217

patients (4%) received blood transfusions, constituting only 20% of patients 218

with grade IV anaemia. 219

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Sixteen of 284 patients(5.6%) developed grade III hypokalaemia(K+<2.5 221

mmol/L), 5 in the first week of treatment, of whom three patients(1.1% of 222

cohort) developed grade IV hypokalaemia (K+<2mmol/L), two on d11 and one 223

on d12 of treatment. In the South African trials using pre-emptive oral in 224

addition to IV K+ supplementation, potassium change from baseline differed 225

significantly from trials not using oral K (mean(95%CI) d7 K change +0.39(0.24, 226

0.54) vs -0.17(-0.47, +0.12), p<0.001 t-test), with a lower incidence of grade III 227

hypokalaemia (4.2% vs 9.7%, p=0.08). 228

229

Thirty-three patients(9.5%) exceeded the protocol-defined nephrotoxicity 230

threshold(Creat>220μmol/L). In the South African trials using standard-course 231

AmBd(n=234), 15 patients(6.4%) discontinued AmBd early due to 232

nephrotoxicity, at a median(range) of 11(5-13)d. In the short-course trials 233

(n=70), 4 patients(5.7%) stopped early (all in the 7-day study), three of whom 234

omitted the final day only. 235

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Toxicity by AmB dose and duration 237

There was a greater absolute drop in Hb at days 7 and 14 in patients receiving 238

the higher AmBd dose and for those receiving standard vs short-course 239

treatment, though differences were non-significant (supplementary Table 2). 240

Patients receiving shorter courses had a higher nadir Hb (mean 9.5 v 8.3g/dL), 241

even after adjustment for sex and 5FC (p=0.033). 242

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There was less renal impairment in those receiving the 0.7mg/kg AmBd dose: 244

mean creatinine rise 28 vs 41μmol/L at d7 (p=0.02); 42 vs 53μmol/L at d14 245

(p=0.09). Mean creatinine peaked at 127 vs 145μmol/L (p=0.04), remaining 246

significant on multivariable analysis (adjusted mean difference 13μmol/L, 247

p=0.04). Despite similarities in peak creatinine, creatinine change to d14 was 248

less in patients receiving short-course versus standard treatment (mean rise 17 249

vs 49μmol/L, p<0.001, supplementary Table 2). 250

251

Reversibility 252

Hb stabilised and started to increase in the second week in the short-course 253

group after stopping AmBd, whilst continuing to decline in the standard 254

group(mean week 2 change -0.12 vs -0.93 g/dL, p=0.01, Figure 1b). 255

256

Renal impairment in the short-course group resolved in week 2 following 257

cessation of AmBd, with creatinine continuing to rise in the standard group 258

(mean week 2 change: -28 vs +14μmol/L, p<0.001, Figure 2c). 259

260

Potassium 261

There was no significant association of AmB dose or duration with potassium 262

change (data not shown). The Loess curves of potassium change 263

(Supplementary figure 1) show a dip in K+ during days 8-10 in the short-course 264

trials, after stopping AmBd and K+ supplementation, supportive of ongoing 265

potassium wasting(30). 266

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Incidence of grade III/IV hypomagnesemia, neutropenia and 268

thrombocytopenia and associations with use of 5FC are shown in the 269

Supplementary Results. 270

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Association of toxicity with 2 and 10-week outcome 272

Development of nephrotoxicity (Creat>220μmol/L) was associated with 2-week 273

mortality (Table 2), however this was no longer significant in the adjusted 274

model (2-week aOR 2.9, 95% CI 0.6-12.7, p= 0.17). The association between 275

nephrotoxicity and 10-week mortality remained significant following 276

adjustment for baseline creatinine, weight, CD4 count, fungal burden, altered 277

mental status and study (10-week aOR 4.5, 95% CI 1.8-11, p=0.001, Table 2). 278

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Severe anaemia (grade III/IV) was a significant risk factor for 10-week 280

mortality following adjustment for baseline Hb, CD4 count, fungal burden, 281

altered mental status and study (aOR 2.2, 95% CI 1.1-4.3, p= 0.028, Table 3). 282

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Development of grade III hypokalaemia (K+<2.5mmol/L) was not associated 284

with mortality (Table 2). 285

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Discussion 289

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This analysis of 368 patients treated with AmBd-based induction regimens in 291

resource-poor settings represents the largest reported toxicity analysis of HIV-292

associated CM treatment. 293

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Published trials reporting toxicity of AmBd at recommended 0.7-1mg/kg/d 295

doses for HIV-associated CM include 6 trials from high-income settings(6, 8, 31-296

34) and 8 trials from low-income settings in Asia and Africa(9, 10, 35-297

40)(Supplementary Table 3). Six trials reported routine use of saline pre-loading 298

and one reported pre-emptive electrolyte replacement(41). Definitions of 299

nephrotoxicity varied. Incidence of grade III nephrotoxicity (or equivalent) was 300

20-33% in older trials without routine fluid pre-loading(8, 32, 36), compared to 301

2-11% in our data and more recent trials using saline pre-loading(10, 39). 6% of 302

our patients discontinued treatment early, usually 1-2 days before completion, 303

in comparison to up to 53% in published trials. 304

305

AmBd nephroxicity is cumulative(42): in our patients, creatinine rose by a mean 306

of 52% by day 7 and 73% by day 14. Although nephrotoxicity was greater using 307

the 1mg/kg/d dose, this translated into a mean difference in peak creatinine of 308

13μmol/L, which is of minor clinical significance and should not deter 309

clinicians from using the more fungicidal higher dose. 310

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The long terminal half-life of AmBd(up to 2 weeks)(4) provides ongoing 312

fungicidal activity after stopping drug administration(25, 27). These data 313

demonstrate better tolerability of 5-7 day regimens, with less nephrotoxicity 314

and reversibility, with creatinine almost returning to baseline one week after 315

stopping short-course AmBd. Ongoing electrolyte losses following AmBd 316

discontinuation mean supplementation and monitoring are needed for several 317

more days: an important implementation issue, as stable patients are usually 318

discharged following discontinuation of intravenous therapy. Short-course 319

regimens minimise toxicity whilst potentially maintaining efficacy, as well as 320

reducing costs of hospitalisation, intravenous administration and laboratory 321

monitoring. A phase III trial comparing short-course to standard AmBd 322

regimens is underway in Africa (ACTA, ISRCTN 45035509). Administration of 323

fewer, higher doses of liposomal preparations may be an alternative (AmBition-324

CM, ISRCTN 10248064). 325

326

Reported incidence of hypokalaemia (7 studies, variable definitions, 327

Supplementary Table 3) ranges from <1 to 56%. In this cohort, with routine pre-328

emptive supplementation with 20mmol KCl (and supplemental oral potassium 329

in South Africa), potassium levels changed little over 14 days. In the recent 330

large Vietnamese trial(10) 18% developed grade III hypokalaemia, as opposed 331

to only 6% in our cohort and 9% in a Ugandan cohort using pre-emptive 332

replacement(41). These data support WHO recommendations for pre-emptive 333


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daily supplementation with 20mmol KCl IV, with additional oral 334

supplementation(12). 335

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Despite oral supplementation, hypomagnesaemia was much more common 337

than hypokalemia in our cohort. Unfortunately, magnesium was not measured 338

at study sites where routine supplementation was not given, for comparison. 339

Lack of availability of magnesium monitoring or oral replacement preparations 340

(magnesium chloride or glycerophosphate) is a problem. IV magnesium 341

(usually stocked for pre-eclampsia) can be considered for patients with severe 342

deficiency or those remaining hypokalaemic despite potassium replacement(12). 343

344

Rates of anaemia reported in 8 studies (variable definitions, Supplementary 345

Table 3) were 37-44%. Two trials reported an average Hb drop of 1.5-2.5g/dL: 346

in one, 59% of patients were transfused (34, 38). Despite development of grade 347

IV anaemia (Hb<6.5g/dL) in 16% of our cohort, with a mean decrease in 348

haemoglobin of 2.3g/dL over 14 days, only 4% of patients received 349

transfusions, reflecting the scarcity of blood for transfusion in Africa. Severe 350

AmBd-induced anaemia is potentially life-threatening in such settings, given 351

the low baseline Hb and current lack of validated preventative interventions. 352

353

Addition of oral 5FC to AmBd did not result in clinically significant excess 354

toxicity in our cohort, with slightly greater drop in haemoglobin, but no 355


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increase in rates of neutropenia or thrombocytopenia, an important observation 356

given this combination remains the recommended CM induction regimen. 357

358

Development of nephrotoxicity and severe anaemia were independent risk 359

factors for mortality, with odds of dying at 10 weeks increased two-fold in 360

patients with a haemoglobin decrease below 7.5g/dL and more than four-fold 361

in those with a creatinine increase above 220μmol/L. Using these cut-offs, 362

anaemia was more than 3 times more common than nephrotoxicity. The lack of 363

association between hypokalaemia and mortality is likely related to routine pre-364

emptive potassium replacement. 365

366

A limitation of this study is the pooling of data from multiple cohorts treated 367

with different AmBd-based combination regimens from four countries on two 368

continents over an 8-year period. Although toxicity management was uniform 369

across trial sites, there may have been heterogeneities in clinical management. 370

To address this, we adjusted for potential variable-specific and outcome-related 371

confounders in the multivariate analyses, as well as including an additional 372

‘study’ variable to try to account for any residual confounding due to 373

unmeasured effects related to temporal or site-specific differences. 374

375

Findings from this large cohort of patients treated in resource-poor settings 376

demonstrate that, with appropriate laboratory monitoring, it is possible to 377

implement the most fungicidal AmBd-based induction-treatment regimens for 378


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HIV-associated CM using a standardised protocol for pre-emptive toxicity 379

management. Hypokalaemia was minimised and nephrotoxicity occurred in 380

<10% of patients. Anaemia remains a concerning side effect. Shorter courses of 381

AmBd are less toxic, with rapid reversibility. The implementation of 382

standardised protocols for pre-emptive toxicity management is a priority in 383

high HIV prevalence countries to maximise the benefits of a drug that is likely 384

to remain a cornerstone of CM treatment for many years to come. 385


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Acknowledgements 386

Conflicts of interest: TB has attended an Advisory Board and received sponsorship 387

for conference attendance from Gilead Sciences Ltd and Astellas Pharma Inc. JNJ 388

and TSH are principal investigators in an investigator-led trial funded by Gilead. 389

For the remaining authors no conflicts were declared. 390

391

Source of Funding: This work was supported by a Wellcome Trust Intermediate 392

Clinical Fellowship (WT 089966 to TB) and a Wellcome Trust Training Fellowship 393

(WT 081794 to JNJ). 394

Other authors’ funding sources include Medical Research Council (UK) to TSH 395

(G0501476), UNC Center for AIDS Research P30-AI50410 and the NIH Fogarty 396

AIDS International Training and Research Program (DHHS/NIH/FIC 2-D43 397

TW01039 to CvDH, MH). Studies in Thailand were part of the Wellcome Trust 398

Mahidol University Oxford Tropical Medicine Research programme funded by the 399

Wellcome Trust. GM is supported by a Wellcome Trust fellowship (098316) and 400

the National Research Foundation (NRF) of South Africa (UID 85858). GM 401

acknowledges that opinions, findings and conclusions or recommendations 402

expressed in any publication generated by the NRF supported research are that of 403

the author(s), and that the NRF accepts no liability whatsoever in this regard. 404

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545

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Figure legends 550

Figure 1. Individual data points and fitted Loess curves for haemoglobin values 551

over the first 14 days of antifungal therapy 552

1a. All patients receiving 14 days’ AmB-based induction therapy 1b. Plot by AmB 553

duration short-course vs standard 1c. Plot by sex 554

Broken line indicates DAIDS grade IV adverse event threshold of 6.5 g/dL. 555

556

Figure 2. Individual data points and fitted Loess curves for creatinine values over 557

the first 14 days of antifungal therapy 558

2a. All patients receiving 14 days’ AmB-based induction therapy 2b. Plot by AmB 559

dose, 0.7 vs 1mg/kg/d. 2c. Plot by AmB duration short-course vs standard 560

Broken line indicates threshold of 220μmol/L (protocol definition of 561

nephrotoxicity). 562

563

564

565

566


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Table 1. Changes in laboratory parameters from baseline over 1 and 2 weeks’ AmBd treatment

Laboratory parameter Baseline value absolute change to day 7 % change to day 7 absolute change to day 14a % change to day 14a

Haemoglobin (g/dL) 11 (10.4, 11.6) -1.5 (-1.9, -1) -12 (-16, -9) -2.3 (-3.6, -1.1) -20 (-32, -8)

Creatinine (umol/L) 77 (68,87) +37 (30, 45) +52 (43, 62) +49 (35, 64) +73 (53, 93)

Potassium (mmol/L) 3.9 (3.8, 4.0) +0.2 (-0.0, 0.5) +9 (3,15) +0.1 (-0.1, 0.3) +6 (-1, 11)

Magnesium (mmol/L) 0.7 (0.6,, 0.8) -0.07 (-0.12, -0.02) -1(-7, 5) -0.15 (-0.2, -0.1) -10 (-33, -13)

Neutrophils (x10^9/L) 3.5 (2.2, 4.9) -0.1 (-0.4, 0.0.1) +8 (0.9, 14) -0.3 (-0.5, 0.0) +8 (-3, 20)

Values shown are means with 95% CI adjusted for study-level clustering

a. changes from baseline to day 14 are calculated only for patients enrolled in studies of standard course AmBd (1-4).


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Table 2. Association between development of toxicities and 2- and 10-week mortality

Toxicity variable Category 2-wk

mortality

OR (95%CI)

univariable

P aOR (95% CI)

multivariable

P 10-wk

mortality

OR (95%CI)

univariable

P aOR (95% CI)

multivariable

P

2-week mortality

Nephrotoxicitya Creat peak >220umol/L 21% 3.1 (1.2-7.8) 0.018 2.8 (0.6-12.7) 0.17 53% 4.2 (2-8.8) <0.00

1

4.5(1.8-11) 0.001

<220umol/L 8% 1 1 21% 1 1

Anaemia

Grade III/ IV

Hb nadir <7.5g/dL 6% 0.8 (0.33-2) 0.65 - 31% 2.1 (1.2-3.5) 0.008 2.2 (1.1-4.3) 0.028

>7.5g/dL 8% 1 18% 1 1

Hypokalaemia

Grade III

K nadir <2.5 mmol/L 6% 0.77 (0.1-

6.1)

0.81 - 25% 0.99 (0.3-

3.2)

0.99 -

>2.5 mmol/L 8% 1 25% 1

Adjusted for the baseline variable (Hb1, Creat1, K1), baseline fungal burden, mental status, CD4 count and study.

a. Because of the association of weight with nephrotoxicity as well as outcome, weight was additionally adjusted for in the nephrotoxicity analysis


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