Conditions: Cryptococcal Meningitis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 48
Healthy volunteers: no

Brief summary:
Cryptococcal meningitis (CM) is a fungal infection that causes a severe syndrome of meningitis that is 100% fatal without antifungal therapy. Even with antifungal therapy, mortality rates remain high, especially in Africa where the ongoing HIV/AIDS pandemic leads to higher prevalence of cryptococcosis. Combination of amphotericin and flucytosine (5-FC) is the mainstay of therapy for the initial management of CM. Indeed, it has even been shown that effective delivery of these therapies in Africa can lower mortality rates by 90%.

This is a prospective open-label trial to compare the efficacy and safety of lower doses of 5FC during induction therapy to historical controls with standard 5FC dosing. Participants in the trial will receive 60mg/kg/day of 5-FC in 3 divided doses for 10 days. Single-dose liposomal amphotericin (10mg/kg) is preferred, if available. Amphotericin B 0.7-1.0 mg/kg/day may be used if needed. Historical controls drawn from the AMBITION trial will be used as a comparison group, selected weighted by inclusion/exclusion criteria, baseline characteristics and therapies received. Induction therapy for control group participants followed the 2018 WHO cryptococcal guidelines with 7 days of 5-FC 100mg/kg/day and 7 days of IV Amphotericin deoxycholate followed by 1200mg fluconazole/day for 7 days. The intervention group received single- dose liposomal amphotericin plus 5-FC and fluconazole 1200 mg/day.

All participants will receive fluconazole 1200mg/day during consolidation therapy from day 1 to 14 then 800mg/day from day 15 to 10 weeks, and 200mg/day after 10 weeks. All participants will receive lumbar punctures at diagnosis, day 3, day 5-7, day 10-14, and additionally as required for control of intracranial pressure and documentation of CSF sterilization. Controls from Ambition will be matched for the same LP windows. Therapeutic LPs conducted during the first week have a \~70% relative survival benefit.

Detailed description:

Arms:
arm 1: Historical controls drawn from the AMBITION trial will be used as a comparison group, selected weighted by inclusion/exclusion criteria, baseline characteristics and therapies received. arm 2: HIV-infected persons in Uganda with cryptococcal meningitis

Interventions: intervention 1: AMBITION trial control intervention 2: Flucytosine
Intervention descriptions: intervention 1: Induction therapy for control group participants followed the 2018 WHO cryptococcal guidelines with 7 days of 5-FC 100mg/kg/day and 7 days of IV Amphotericin deoxycholate followed by 1200mg fluconazole/day for 7 days. intervention 2: Participants in the trial will receive 60mg/kg/day of 5-FC in 3 divided doses for 10 days. Single-dose liposomal amphotericin (10mg/kg) is preferred, if available. Amphotericin B 0.7-1.0 mg/kg/day may be used if needed.

Protocol document:
Prot_SAP_000.pdf: 
FLOOR Trial Protocol 
22 March 2024 
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Optimizing the Dose of Flucytosine for the Treatment of 
Cryptococcal Meningitis. 
Abbreviation: FLOOR 
  
Principal Investigators:  David B Meya, David R Boulware 
Protocol Champion: Thomas McHale 
 
ClinicalTrials.gov Registration number:  
IDI IRB number: IDI-REC-2022-32 
University of Minnesota IRB number: STUDY00017800 
Uganda National Drug Authority (NDA) number:  
U.S. National Institutes of Health: R01NS086312 
 
  
Version Number: 1.2 
Version Date: 22 March 2024 
  
 
 
   
 


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Protocol Summary 
Title: 
Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal 
Meningitis. 
Abbreviation: 
FLOOR 
Phase: 
II 
Population: 
Phase II: HIV-infected persons in Uganda with cryptococcal meningitis 
Rationale: 
Cryptococcal meningitis (CM) is a fungal infection that causes a severe 
syndrome of meningitis that is 100% fatal without antifungal therapy. Even 
with antifungal therapy, mortality rates remain high, especially in sub-
Saharan Africa where the ongoing HIV/AIDS pandemic leads to higher 
prevalence of cryptococcosis.  
Combination of amphotericin and flucytosine (5-FC) is the mainstay of 
therapy for the initial management of CM. Indeed, it has even been shown 
that effective delivery of these therapies in sub-Saharan Africa can lower 
mortality rates by 90%. However, even while 5-FC is included on the WHO 
list of essential medicines, its availability world-wide is limited. Since 5-FC 
has few clinical indications, it is not manufactured widely, leading to high 
costs for the medication, and limited distribution in sub-Saharan Africa.  
While early studies of 5-FC in CM were done in the pre-AIDS era, the dose of 
150 mg/kg/day was selected and resulted in significant toxicities, including 
leukopenia and thrombocytopenia requiring dose reduction or 
discontinuation of 5-FC. Later retrospective studies of dosing in the AIDS era 
were inconclusive about the ideal dose to benefit efficacy and toxicity. In 
1997, van der Horst performed the first major clinical trial for AIDS related 
CM demonstrating efficacy of combination of 5-FC with amphotericin B. The 
selected dose was 100 mg/kg/day, which has been used as standard dosing 
ever since. Optimizing dose necessary for good clinical outcomes could 
reduce the daily dose and increase the total number of patients able to be 
treated at the same cost and supply. In our own studies of Cryptococcus in 
Uganda, the MIC50 is approximately 4-8 mg/L. Preliminary models of dosing 
based on AMBITION pharmacokinetics of 5-FC has shown that lower doses 
may still be acceptable at targets of 40% or 60% time over MIC.  


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Description of 
Study 
Intervention: 
Standard dosing for 5-FC is 100 mg/kg/day divided in 4 doses over waking 
hours for 7-14 days during the period of induction therapy. We will deliver 
doses of: 
1. 60mg/kg/day in 3 divided doses for 10 days 
 
This most commonly will be 2 500mg tablets three times per day 
Prelim Data 
Molloy et al. demonstrated in the ACTA trial published in 2018 that 1 week 
of IV Amphotericin B 1 mg/kg plus oral 5-FC 100 mg/kg is the optimal 
management for the first week of CM. These are shown to have benefits in 
mortality and similar rates of fungal clearance from the CSF to other 
combination regimens.  
Phase II Study 
Design 
Prospective, open-label trial to compare the efficacy and safety of lower 
doses of 5FC during induction therapy to historical controls with standard 
5FC dosing.    
1. Participants in the trial will receive 60mg/kg/day of 5-FC in 3 
divided doses for 10 days, n=36 
2. Single-dose liposomal amphotericin (10mg/kg) is preferred, if 
available.  
3. Amphotericin B 0.7-1.0 mg/kg/day may be used if needed. 
4. Historical controls drawn from the AMBITION trial will be used as a 
comparison group, selected weighted by inclusion/exclusion criteria, 
baseline characteristics and therapies received. Induction therapy for 
control group participants followed the 2018 WHO cryptococcal 
guidelines with 7 days of 5-FC 100mg/kg/day and 7 days of IV 
Amphotericin deoxycholate followed by 1200mg fluconazole/day for 
7 days. The intervention group received single-dose liposomal 
amphotericin plus 5-FC and fluconazole 1200 mg/day. 


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All participants will receive fluconazole 1200mg/day during consolidation 
therapy from day 1 to 14 then 800mg/day from day 15 to 10 weeks, and 
200mg/day after 10 weeks. 
All participants will receive lumbar punctures at diagnosis, day 3, day 5-7, 
day 10-14, and additionally as required for control of intracranial pressure 
and documentation of CSF sterilization. Controls from Ambition will be 
matched for the same LP windows. Therapeutic LPs conducted during the 
first week have a ~70% relative survival benefit.  
Phase II Study 
Endpoints 
Primary:  
1. Rate of CSF Cryptococcus clearance (Early Fungicidal Activity, or 
EFA), quantified by the change of log10 Cryptococcus CFU/mL CSF/day 
as measured by serial quantitative CSF fungal cultures over ~2 
weeks 
Secondary:  
1. CSF culture sterility cumulative incidence over 18 weeks 
2. 18-week survival time 
Key Secondary:  
1. Desirability of Outcome Response (DOOR) as ordinal ranked 
maximum score tested by Win Ratio. 
a. 18-week Survival with CSF sterility by 2-weeks 
b. 18-week survival with CSF culture positivity beyond 2 weeks 
c. Grade 3 hematological adverse event by 2 weeks 
d. Grade 4 hematological adverse event by 2 weeks 
e. Lost to follow up before 18-weeks 
f. 
Serious Adverse Event through 18 weeks (e.g. all-cause re-
hospitalization, permanent neurologic deficit)  
g. Death by 18-weeks 
Safety & Exploratory: 
1. Incidence of Grade 3-5 Laboratory abnormalities over 2 weeks 
2. Incidence of Clinical Grade 3-5 Adverse Events or Serious Adverse 
Events (SAEs) over 2 weeks 
3. Pharmacokinetics of 5FC in blood, CSF, and bodily fluids / tissues. 
Phase II 
Duration: 
18 weeks 
Phase II 
Number of 
Participants 
36 CSF culture positive participants with an EFA estimate  
With adaptive sample size up to 50 CSF CrAg+ participants consented 
Phase II 
Inclusion 
Criteria 
● CSF cryptococcal antigen (CrAg) positive meningitis 
● Ability and willingness to provide informed consent 
● Willing to receive protocol-specified lumbar punctures  
 


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Phase II x 
Criteria 
● 
Age < 18 years 
● 
Inability to take enteral (oral or nasogastric) medicine 
● 
Cannot or unlikely to attend regular clinic visits 
● 
Receiving chemotherapy or corticosteroids 
● 
Suspected Paradoxical immune reconstitution inflammatory syndrome 
(IRIS) 
● 
Pregnancy or breastfeeding 
● 
CrCl < 20 mL/minute 
● 
Absolute neutrophil count < 500 x106 cells/L 
● 
Thrombocytopenia < 50,000 x 106 cells/L 
● 
Patients with prior 5-flucytosine exposure >3 days in the 12 months 
prior to enrollment 
● 
Any condition for which participation would not be in the best interest 
of the participant or that could limit protocol specified assessments.  
Phase II Study 
Drug and 
Dosing 
● Participants will receive 5-FC 60mg/kg/day for 10 days. 
● Participants will receive IV AMB 0.7-1.0 mg/kg/day for 7 days. 
● Liposomal Amphotericin B single IV dose 10 mg/kg is preferred, when 
available. 
● All participants will receive fluconazole 1200 mg/day during 
consolidation therapy from day 1 to 14 then 800 mg/day from day 15 
to 10 weeks, and 200mg/day after 10 weeks. 
Phase II 
Monitoring 
● 
All participants will have CSF quantitative fungal cultures performed on 
Days 1, 2-4, 5-7, and 10-14 of antifungal treatment. 
● 
3x weekly CBC, chemistries, and kidney function tests for duration of 
induction therapy during hospitalization 
Phase II Safety 
Analysis 
The Data Monitoring Committee (DMC) will conduct interim safety analysis 
after 10 participants are enrolled with >2 weeks of accrued data and 
calculable EFA. 
EFA and culture positive Cryptococcus relapse will be monitored by the trial 
investigators, and if statistical evidence shows that EFA <0.2 log10 
CFU/mL/day, then an early DMC will be called.  
Phase II 
Statistical 
Design 
Rationale 
Early fungicidal activity (EFA) is a quantitative measure of antimicrobial 
activity for an antifungal regimen in human patients with cryptococcal 
meningitis. In numerous trials, EFA correlates well with 10-week mortality. 
Specifically, EFAs worse than 0.20 log10 CFU/mL/day are associated with 
higher mortality (>50%) and EFA better than 0.3 log10 CFU/mL/day is 
associated with lower mortality (30-40%). 
EFA is calculated by measuring the slope of the clearance rate of yeast from 
CSF, which is calculated from longitudinal quantitative CSF cultures collected 
over two weeks.  
By intent to treat analysis, all participants who are enrolled in this study will 
be included for analysis.  
Clinical Trial 
Sites 
1. Mulago Hospital, Kampala, Uganda 
2. Kiruddu Hospital, Kampala, Uganda 
3. Mbarara Regional Referral Hospital, Mbarara, Uganda 


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Estimated Time 
to Complete 
Enrollment: 
Up to 12 months 
 
 
 
 
 


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Table of Contents 
1. KEY ROLES 
1 
2. BACKGROUND AND SCIENTIFIC RATIONALE 
2 
2.1 Background Summary and Study Hypotheses 
2 
2.2 Burden of Cryptococcal Meningitis 
2 
2.3 Treatment of Cryptococcal Meningitis 
4 
2.4 Rationale for Optimizing 5FC Dose 
6 
2.5 Early Fungicidal Activity 
7 
2.6 Potential Risks and Benefits 
10 
3. OBJECTIVES 
12 
3.1 Study Objectives 
12 
3.2 Study Outcome Measures 
12 
4. STUDY DESIGN 
13 
4.1 Phase II Study Design 
13 
4.2 Pharmacokinetic Studies 
16 
5. STUDY ENROLLMENT AND WITHDRAWAL 
 
16 
5.1 Inclusion Criteria 
16 
5.2 Treatment Procedures 
17 
5.4 Blinding 
20 
5.6 Study Withdrawal 
21 
6. MEDICATIONS TO BE USED IN THE TRIAL 
21 
6.1 Cryptococcal Meningitis Management 
21 
6.2 Other Antifungals Used in Cryptococcal Treatment 
22 
6.3 Accountability Procedures for the Study Intervention 
26 
6.4 Assessment of Subject Adherence to Study Interventions 
26 
7. PHASE II TRIAL STUDY SCHEDULE 
26 
7.1 Schedule of Events 
26 
7.2 Enrollment 
27 
7.3 Hospital Follow-up Visits 
29 
7.4 Clinic Follow-up Outpatient Visits 
31 
7.5 Termination of Study 
35 
8. STUDY PROCEDURES/EVALUATIONS 
36 
8.1 Procedures 
36 
8.2 Lumbar Puncture and CSF Analysis 
37 
8.3 Laboratory Evaluations 
39 
8.4 Specimen Storage 
40 


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8.5 Biohazard Containment 
40 
9. ASSESSMENT OF SAFETY 
40 
9.1 Adverse Event (AE) Definitions 
40 
9.2 Assessment of Adverse Events by the Investigator 
41 
9.3 Adverse Events of Special Interests 
43 
9.4 Serious Adverse Event 
43 
9.5 Serious Adverse Event Reporting – Procedures for Investigators 44 
9.6 Recording of Adverse Events 
44 
9.7 Reporting Period for Adverse Events 
44 
9.8 Study Medication Discontinuation 
45 
9.9 Pre-existing Conditions 
45 
9.10 Pregnancy Reporting 
46 
9.11 Expedited Reporting 
46 
10. CLINICAL MONITORING 
47 
10.1 Site Monitoring Plan 
47 
10.2 Medical Monitor 
47 
10.3 Data and Safety Monitoring Board (DSMB) Safety Reviews 
47 
11. STATISTICAL CONSIDERATIONS 
48 
11.1 Statistical Analysis Plan 
48 
11.2 Timing of Analyses 
48 
11.4 Primary Endpoint: 
49 
11.5 Secondary Endpoints 
50 
12. ETHICS/PROTECTION OF HUMAN SUBJECTS 
51 
12.1 Ethical Standard 
51 
12.2 Institutional Review Board 
51 
12.3 Informed Consent Process 
52 
12.4 Subject Confidentiality 
52 
12.5 Study Discontinuation 
53 
12.6 Future Use of Stored Specimens 
53 
13. DATA HANDLING AND RECORD KEEPING 
53 
13.1 Source Documents 
54 
13.2 Data Management Responsibilities 
55 
13.3 Data Capture Methods 
55 
13.4 Types of Data 
55 
13.5 Study Records Retention 
55 
13.6 Protocol Violations and Deviations 
56 
13.7 Quality Control and Quality Assurance 
56 
14. ADMINISTRATIVE 
57 


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14.1 Publication of Research Findings 
57 
15. LITERATURE REFERENCES 
58 
Appendix A: Adverse Events observed with IV amphotericin + fluconazole therapy
 
66 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Document History 
 
Document 
Version Date 
Summary of Changes 
Original Protocol 30 March 2023 
Original Protocol 
14 August 2023 
Response to Mulago IRB and UNCST comments. Clarifications of 
the therapies to be used. Additional evidence added to the 
background/justification. Minor additions to the protocol listed 
in the cover letters. 
Original Protocol 
22 March 2024 
Updated the schedule of events and exclusion criteria for better 
efficiency and to align with the schedule that was used for the 
comparator group. Response to Mulago REC comments.  
 
 
 
 
 
 


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Abbreviations 
ACTA  
Advancing cryptococcal meningitis treatment for Africa 
AE 
 
Adverse event 
AMB 
 
amphotericin B deoxycholate 
ART 
 
antiretroviral therapy 
CBC 
 
complete blood count 
CDC 
 
Centers for Disease Control and Prevention 
CFU  
 
colony forming units 
CHAI  
Clinton Health Access Initiative 
CM  
  
cryptococcal meningitis 
COAT  
Cryptococcal Optimal ART Timing 
CrAg 
 
cryptococcal antigen 
CRP 
 
C-reactive protein 
CSF 
 
cerebrospinal fluid 
CTCAE  
Common Terminology Criteria for Adverse Events 
DAIDS  
Division of AIDS (NIH, NIAID) 
DSMB  
Data & Safety Monitoring Board 
EFA 
 
Early Fungicidal Activity 
FDA 
 
Food and Drug Administration 
GCP 
 
Good Clinical Practice 
GCS 
 
Glasgow Coma Scale 
GI 
 
Gastrointestinal 
GRAS  
generally recognized as safe 
ICP 
 
intracranial pressure 
IDSA 
 
Infectious Diseases Society of America 
IRB 
 
Institutional Review Board 
IRIS  
  
immune reconstitution inflammatory syndrome 
IQR 
 
interquartile range 
IV 
 
intravenous 
LFT 
 
liver function tests 
LP 
 
lumbar puncture 
MOP 
 
manual of operations 
NIAID  
National Institute of Allergy and Infectious Diseases 
NIH 
 
National Institutes of Health 
NG 
 
nasogastric 
OI  
 
opportunistic infection 
PHQ-9  
patient health questionnaire – 9  
PO  
 
per os (by mouth) 
SAE 
 
Serious adverse event 
SOC 
 
 
Standard of Care 
SoE  
 
Schedule of Events 
SOPs  
Standard Operating Procedures 
SRA 
 
Stringent Regulatory Authority 
TEAEs               Treatment emergent adverse events


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1. 
KEY ROLES 
Uganda Principal Investigator: 
David B Meya MBChB, MMed, PhD 
Infectious Diseases Institute 
P.O. Box 22418 
College of Health Sciences,  
Makerere University 
Kampala, Uganda 
 
U.S. Principal Investigator: 
David R Boulware MD, MPH, CTropMed 
University of Minnesota 
689 23rd Ave SE 
Minneapolis, MN 55455 USA 
 
 
Protocol Champion: 
Thomas McHale, MD MSc 
University of Minnesota 
420 Delaware St SE MMC 250 
Minneapolis, MN 55455 
 
Investigators: 
Darlisha Williams, MPH 
University of Minnesota 
689 23rd Ave SE, MRF 4-157 
Minneapolis, MN 55455 USA 
 
Mucunguzi Atukunda, MBChB, MPH 
Infectious Diseases Institute 
P.O. Box 22418 
Kampala, Uganda 
 
Conrad Muzoora MBChB, MMed 
Mbarara University  
P.O. Box 1410, 
Mbarara, Uganda 
 
Abdu K Musubire, MBChB MMed 
Infectious Diseases Institute 
P.O. Box 22418 
Kampala, Uganda 
 
Caleb Skipper, MD 
University of Minnesota 
Mayo Memorial Building, MMC 250, 420 
Delaware Street S.E., Minneapolis, MN 
55455, USA 
 
Pharmacologists: 
 
Katharine Stott, PhD, MRCP, DTM&H, 
MSc, MRes 
University of Liverpool 
Liverpool L69 3BX, United Kingdom 
 
Melanie Nicol, PharmD, PhD 
University of Minnesota 
Experimental & Clinical Pharmacology 
2001 6th St SE 
Minneapolis, MN 55455 
 
Biostatisticians: 
Biyue Dai, PhD 
Nicole Engen, MS 
Abdul Wele, MS 
Division of Biostatistics and Health Data 
Science, University of Minnesota 
Coordinating Centers for Biometric Res. 
2221 University Ave SE, Suite 200 
Minneapolis, MN  55414 USA 


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2. 
BACKGROUND AND SCIENTIFIC RATIONALE 
2.1 
Background Summary and Study Hypotheses 
Cryptococcal meningitis (CM) has emerged as one of the most frequent and deadly opportunistic 
infections in HIV patients, with a global burden estimated at nearly 1 million cases annually.1 Early 
mortality from HIV-associated cryptococcal meningitis remains unacceptably high, in large part 
due to the high cost, toxicity, and relatively limited repertoire of effective antifungals. The essential 
role of 5-FC in reducing fungal burden in the first week of therapy has now been firmly 
established.2–4 Importantly, access to the important medication 5-FC remains limited in settings 
where CM incidence and mortality is highest. 
The Advancing Cryptococcal Meningitis treatment for Africa trial (ACTA) recently demonstrated 
in a high quality, randomized controlled trial that Amphotericin B (AMB) in combination with 5-FC 
had a 38% reduced mortality at 10-week follow-up when compared to fluconazole used in 
combination with AMB.2 A cost-effectiveness analysis based on these data showed that the cost 
of saving 1 life from CM would be only $973.5  
While there are 3 stringent regulatory authority (SRA) approved manufacturers of 5-FC, for 
decades these companies have not been registered in Africa.6,7 Consequently, this low-cost, off-
patent, life-saving medication has been widely unavailable in the region of the world where it is 
needed most. Vitaris, one of the SRA approved manufacturers of 5-FC received WHO pre-
qualification to produce 5-FC tablets in 2018.8 This led to registration in South Africa and 
development of an access program that provided 5-FC free of charge to tertiary centers 
throughout the country.9 In this project, after 5-FC was provided free of charge, these centers saw 
an 11% mortality reduction compared to a period before 5-FC was freely available.9 
While funding and production remain limiting factors on distribution of 5-FC in these critical areas, 
another aspect in need of attention is the optimal dosing of 5-FC. Early studies of 5-FC in CM 
used doses as high as 150 mg/kg/day.10,11 In these studies, leukopenia and thrombocytopenia 
were common, seen in up to 30% of patients receiving 5-FC.  Diarrhea and rash were also seen 
but less frequent.10,11 Subsequent studies used a lower dose of 5-FC and 100mg/kg/day and 
found no difference in toxicity compared to groups treated without 5-FC.12 While these studies 
show that 5-FC can be used safely and effectively at 100 mg/kg/day, no study has yet shown 
whether lower doses might yet be effective. This crucial point could greatly affect access to 5-FC 
by reducing the cost and supplies needed for any given patient with CM.  
2.2 
Burden of Cryptococcal Meningitis 
Cryptococcal meningitis has become the most common cause of adult meningitis in many parts of 
Africa,13 where cryptococcosis now rivals tuberculosis in all-cause mortality.14  While long-term 
survival has improved with widespread use of antiretroviral therapy in high-income countries, early 
mortality remains high.15  Furthermore, expanding access to antiretroviral therapy in resource-limited 


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settings has not yet led to compelling improvements in mortality, with 10-week mortality rates 
between 24% and 37%, even under optimal research conditions.16,17  Early mortality rates are often 
~70% in routine practice where access to diagnostics or medications is limited or unavailable, 
intracranial pressure is uncontrolled, or in settings where other barriers to the management of 
cryptococcal meningitis exist.18–20 Intravenous administration of amphotericin B deoxycholate is not 
often possible in resource-limited settings, even when it is available.  
Even with amphotericin-based induction therapy, survival is not optimal. Figure 2.2 displays the 
survival in Uganda from 2013-2017 in the setting of a study using IV amphotericin + fluconazole 
induction therapy and control of intracranial pressure.21,22 Approximately, half of the mortality 
occurs during the first two weeks and the other half during the next 18 weeks, particularly in the 
first 6 weeks (Figure 2.2).  This 18-week time period captures 95% of the 5-year mortality in 
Uganda.23 
 
Figure 2.2: Survival After Cryptococcal Meningitis by Glasgow Coma Scale (GCS)  
in Uganda 
 
 
GCS < 15 = altered mental status, GCS = 15 = intact mental status, responsive to voice 
 
The efficacy and safety of induction therapy for cryptococcal meningitis can be evaluated 
effectively with all-cause mortality as an early clinical outcome. This is because approximately 
half of the mortality related to CM occurs during the first two weeks.24 18-week outcomes will 
also be assessed for further evaluation of primary and secondary endpoints.  
 
Table 2.2: The 2-week mortality of Ugandans on Amphotericin + Flucytosine with GCS=15 
Study 
Number of 
Patients 
Number Died 
2-week 
Mortality 
95% Confidence 
Interval 
EnACT Stage 1-3 
25 
1 
4.00% 
-3.7%, 11.7% 


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Jarvis et al. NEJM 2022; 
Uganda GCS=15 data 
197 
17 
8.60% 
4.7%, 12.6% 
Average 
222 
18 
8.10% 
4.5%, 11.7% 
2.3 
Treatment of Cryptococcal Meningitis 
Standard cryptococcal meningitis treatment is divided into three phases: a 2 week induction 
phase, followed by an 8-week consolidation phase, and an extended maintenance phase 
thereafter for secondary prophylaxis.24 Table 2.3 outlines the treatment recommendations for 
cryptococcal meningitis in HIV-infected individuals.  Historically, international treatment 
guidelines have been informed primarily by large multisite clinical trials with endpoints of 
treatment success and survival at 2 weeks and 10 weeks. Ongoing mortality occurs between 
10-18 weeks. After 18 weeks, approximately 90-95% will be alive at 5 years.23,25 
Table 2.3: 2018, 2022 WHO Antifungal Treatment Recommendations for Cryptococcal 
Meningitis 
Medication and Dose 
Week 1 
Week 2 
Week 3-10 
Week >10 
Amphotericin B (1.0 
mg/kg/day) + flucytosine 
100mg/kg/day  
  
 
 
 
Fluconazole 1200mg daily 
 
 
 
 
Fluconazole 800mg daily 
 
 
 
 
Fluconazole 200mg daily 
 
 
 
Through 12 
months 
 
 
 
 
 
Treatment Phase 
Induction 
Induction 
Consolidation 
Secondary 
Prophylaxis 
These recommendations are based on a published 2018 phase III randomized trial.20  This remains the 
2022 recommended regimen for locations without access to liposomal amphotericin B (Ambisome®).21 
In the published ACTA trial, one week of IV amphotericin B deoxycholate was non-inferior to two 
weeks of IV AMB (Figure 2.3).2 The best therapy in the ACTA trial was 1 week of IV AMB + 
flucytosine with 24% (27/113) 10-week mortality.  
Cryptococcal mortality can be reduced by the use of high-dose amphotericin B (0.7-1.0 
mg/kg/day), alongside aggressive inpatient management of raised intracranial pressure.12 In 


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most resource-limited settings, standard induction therapy for cryptococcosis includes the 
alternate regimen of IV AMB for 14 days combined with high-dose fluconazole (800-
1200mg/day).    
The ability of amphotericin to rapidly and consistently sterilize the cerebrospinal fluid (CSF) of 
cryptococcal patients suggests that amphotericin should be central to any induction strategy.26  
 
 
 
Figure 2.3: ACTA trial, comparison of induction therapies 
Substantial barriers exist in adhering to the accepted guidelines in resource-limited areas, 
where cryptococcal meningitis is most 
prevalent. These include costs and availability 
of drugs, intravenous (IV) drug administration 
which requires hospitalization, co-
administration of IV fluids and supplemental 
electrolytes, and the severe and potentially 
life-threatening toxicity of amphotericin which 
necessitates rapid and reliable laboratory 
monitoring.27,28 For these reasons, 
amphotericin is generally reserved for life-
threatening fungal infections, and in the case 
of cryptococcosis, limited to the induction 
period.  Many centers in low and middle 
income countries consequently find it difficult 
to adhere to recommended guidelines for cryptococcal induction therapy.  Identifying more 
effective and less toxic antifungal therapy is necessary to allow for more rapid fungal clearance 
during the induction phase, which could lead to less dependence on completing long courses of 
IV medications with increased risk of toxicity. 
The combination of amphotericin and flucytosine appears to be rapidly fungicidal and has better 
survival than alternative 14-day regimens.2,29–31 Flucytosine can also be associated with severe 
side effects including hepatotoxicity, bone-marrow depression, and renal toxicity when co-
administered with other nephrotoxic drugs such as amphotericin.32 Furthermore, flucytosine is 
currently not registered or available in most low and middle income countries. Given the 
apparent advantages of flucytosine over fluconazole as combination therapy, substantial 
advocacy efforts are currently underway to secure availability and affordability of flucytosine 
where Cryptococcus is prevalent. In 2019, UNITAID was seeking to make flucytosine available. 
In contrast to amphotericin, fluconazole is generally well-tolerated and readily penetrates the 
central nervous system (CNS).33 Consolidation and subsequent maintenance therapy therefore 
relies on extended courses of oral fluconazole (Table 2.3).  At typical doses (≤400 mg/day), 


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6 
 
however, fluconazole is fungistatic rather than fungicidal, confirmed in a well-designed clinical 
trial demonstrating no change in fungal growth by serial quantitative cultures over 2 weeks with 
400 mg/day of fluconazole.26 Even when used in higher, more fungicidal doses (800-1200 
mg/day)19 as is currently being recommended for induction therapy of CM, fluconazole adds a 
marginal contribution to overall fungal clearance when used together with amphotericin.  
Fluconazole monotherapy, which is still the standard of care in much of the world where 
amphotericin use is limited or impossible to administer, leads to suboptimal clearance, 
resistance and symptomatic relapse.34,35 More efficacious and less toxic oral antifungals for the 
treatment of cryptococcosis are urgently needed. 
2.4 
Rationale for Optimizing 5FC Dose 
Dosing for 5-FC was a matter of debate in early trials that established the efficacy of 5-FC in the 
management of CM. In the era before AIDS, Bennett et al. used a lower dose of amphotericin B 
(0.3 mg/kg/day) in combination with higher dose of 5-FC (150 mg/kg/day) to treat CM in patients 
receiving immunosuppressive medications 10. While the addition of 5-FC proved to have 
superior clinical efficacy, the rate of cytopenias was high (30%).10 In a subsequent retrospective 
study of patients with AIDS, average dose of 5-FC was 75-100 mg/kg/day, but was not 
associated with survival benefit and was associated with 53% of patients having cytopenias 
necessitating discontinuation of the drug.36 Finally, van der Horst et al. performed a high quality 
randomized clinical trial using a higher dose of amphotericin (0.7mg/kg/day) with or without 5-
FC (100 mg/kg/day)12. This trial demonstrated that the addition of 5-FC resulted in higher rates 
of CSF sterilization and lower mortality rates and no difference in toxicity between groups. 
However, the dose of 5-FC was not selected based on a systematic analysis, but rather as an 
attempt to mitigate the toxicity and maximize therapeutic effects.  
While there are no studies to date that establish time over MIC as the critical pharmacodynamic 
property for efficacy of 5-FC, we can extrapolate that time over MIC is an important factor from 
work on 5-FC and Candida spp.37 Preliminary modelling based on AMBITION pharmacokinetics 
by Stott et al. demonstrated that 5-FC may reach CSF concentrations above MIC for 
Cryptococcus even at lower and less frequent dosing (Figure 2.4.1). In our own analyses of 
MIC for 5-FC against Cryptococcus the MIC50 has been approximately 4 mg/L and MIC90 8 mg/L 
(Table 2.4). In this hypothetical model displayed in Figure 2.4.1, the proportion of patients 
receiving 5-FC dosing of 50 mg/kg/day still achieve target time over MIC more than 80% of the 
time even in the strictest target. 
 
 
 
 
 
 
 
 
 


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Figure 2.4.1: Pharmacokinetic modelling of flucytosine CSF levels at lower doses and 
frequencies. 
 
 
Recently, Jezewski et al. endeavored to better understand the mechanisms of how flucytosine 
functions in the in vivo environment.66 For years, 5-FC has been understood to have a 
synergistic effect when used in combination with amphotericin or fluconazole.67-69 However, this 
synergism has been poorly understood and difficult to replicate in vitro. One of the conditions 
that has been thought to potentiate the flucytosine effects is the elevated level of CO2 in the 
body compared to ambient atmospheric levels. In the Jezewski et al. study, isolates grown at 
elevated CO2 concentrations had a 2 to 64-fold reduction in MIC, depending on the CO2 
concentration. We have also found, in our own studies that the ratio of 5FC CSF concentrations 
to MIC is quite high, with a median ratio of 2.98 (Figure 2.4.2). These data indicate that in 
addition to the strong evidence by Stott et al above that lower doses of flucytosine will still 
achieve levels above MIC, the target MIC might be even lower than we have identified in our 
isolates (Table 2.4).  
 
During phase 2 of the AMBITION-cm trial, which established the effectiveness of single high 
dose liposomal Amphotericin 10 mg/kg for induction treatment of CM. Amphotericin was paired 
with high-dose fluconazole without 5-FC.38 In this phase 2 trial, single dose liposomal 
Amphotericin B 10 mg/kg with fluconazole 1200 mg/day for 14 days demonstrated an EFA of -
0.52 log10 CFU/mL/day. This was found to be non-inferior to the standard regimen of liposomal 
Amphotericin 3 mg/kg/day for 14 days with the same dose of fluconazole. These data suggest 
that the dose of 5-FC can be safely adjusted for optimization while maintaining the mortality 
benefit of CM induction therapy. 
 
 
 
 
 
 
 
 


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Table 2: MIC for flucytosine in Cryptococcus isolates from participants in the EnACT clinical trial. 
The top section are the MIC values for the initial screening CSF, prior to significant exposure to 
5-FC. The second section is the last CSF with positive Cryptococcus growth during treatment. 
The 𝜒𝜒2 p-value tests for a statistical difference in MIC between the control and oral 
amphotericin groups. 
 
 
 
 
 
 
MIC 
(µg/mL) 
 
Control 
(n, %) 
Oral Amphotericin 
(n, %) 
 
Total 
(n, %) 
𝜒𝜒2 p-value 
Screening Lumbar Puncture 
≤2 
2 (14.3%) 
7 (21.9%) 
9 (19.6%) 
0.451 
4 
9 (64.3%) 
15 (46.9%) 
24 (52.2%) 
8 
3 (21.4%) 
6 (18.8%) 
9 (19.6%) 
16 
0 (0%) 
4 (12.5%) 
4 (8.7%) 
Last Positive Lumbar Puncture 
≤2 
0 (0%) 
3 (10.0%) 
3 (7.3%) 
0.302 
4 
7 (63.6%) 
12 (40.0%) 
19 (46.3%) 
8 
4 (36.4%) 
15 (50.0%) 
19 (46.3%) 


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Figure 2.4.2: Scatterplot of the ratio of average 5-FC CSF level to MIC, with high-end outliers 
removed. The median 5-FC / MIC ratio was 2.98 and mean was 4.72, indicating the 5-FC levels 
were markedly higher than MIC in the overall population. Points below the red line indicate 5-FC 
levels below MIC at any point during treatment (n=6 participants with ratio <1). There is no 
relationship between EFA and the average 5-FC CSF level to MIC ratio. The blue trendline is 
based on a linear regression formula that is displayed in the top right of the chart. 
 
 
 
 
2.5 
Early Fungicidal Activity 
Rate of fungal clearance, or early fungicidal activity (EFA), provides a measure for evaluating 
the efficacy of induction therapy for CM. Bicanic et al have demonstrated in a pooled series of 
Phase II clinical trials that the rate of cerebrospinal fluid sterilization correlates with clinical 
outcomes.40  
Quantitative CSF culture yeast clearance is generally logarithmic over the first 2 weeks; thus, 
the clearance can be estimated by linear regression of log10 colony-forming units (CFU) per mL 


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of CSF per day of therapy as a unit of measure (i.e., change in log10 CFU/mL CSF/day). This 
approach has allowed for small innovative phase II clinical trials to estimate the early fungicidal 
activity of newer induction regimens with dramatically smaller sample sizes, using early 
fungicidal activity as a surrogate marker.  
Similarly in Uganda, the relationship of EFA to 10-week and 26-week survival in patients with 
CM has been investigated in several clinical trials In a series of three NIH-sponsored clinical 
trials from 2010 to 2018,41 an EFA worse than 0.20 log10 CFU/ml/day was strongly associated 
with increased mortality (P=0.001). Survival was comparable among AMB regimens with EFAs 
greater than 0.20 log10 CFU/ml/day. 
 
Figure 2.5: Early Fungicidal Activity versus 26-week Survival with IV AMB + Fluconazole 
 
Below are examples of EFA spaghetti plots (Figure 2.5.A), where each line is an individual 
research participant, and each dot is a CSF quantitative culture value in log10 CFU/mL CSF 
units. In general, CSF yeast clearance is approximately linear (on a log10 scale).  With IV AMB, 
the rate of clearance improves.  With IV AMB + 5-FC, the rate of clearance improves further 
over IV AMB + fluconazole alone.  Increases in the first 1 day of therapy are common, but after 
Day 2 the majority have a steadily decreasing CFU/mL count over 14 days of induction therapy. 
The occasional outlier patient (~5%) might have increased CSF culture values over time (even 
with IV AMB + 5FC).  
 
Figure 2.5.A: Example of Serial Quantitative CSF Cultures to calculate EFA 
Fluconazole 400mg/day in 
Cape Town 26 
Fluconazole 
1200mg/day in 
Mbarara, Uganda 19 
IV AMB x 5day with 
fluconazole 1200mg in 
Mbarara, Uganda 42 
IV AMB + 5FC x 14 days 
in Cape Town 17 


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Table 2.5: Trials comparing CSF Cryptococcus clearance Early Fungicidal Activity 
(EFA) by regimen. 


F L O O R Tri al Pr ot o c ol  
2 2 M ar c h 2 0 2 4  
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _  
1 2  
 
I n d u cti o n R e gi m e n 
E F A  
± S D  
N  
R ef  
Si n gl e -d os e A m p h ot erici n + 5 F C + fl uc o n a z ol e ( 1 2 0 0 m g / d a y) 
0. 4 0  
0. 1 3  
4 0 7  
4 3  
A m p h ot erici n  
0. 3 1  
0. 1 5  
9 9  
 
A m p h ot erici n + 5 F C  
0. 4 2  
0. 1 0  
1 0 0  
3 0  
A m p h ot erici n + fl uc o n a z ol e ( 8 0 0 m g / d a y)  
0. 3 2  
0. 1 0  
9 9  
 
A m p h ot erici n + 5 F C  
0. 4 1  
0. 2 2  
2 1  
4 4  
A m p h ot erici n + fl uc o n a z ol e ( 8 0 0 m g / d a y)  
0. 3 8  
0. 1 8  
2 2  
A m p h ot erici n + fl uc o n a z ol e ( 1 2 0 0 m g / d a y)  
0. 4 1  
0. 3 5  
2 3  
A m p h ot erici n + v oric o n a z ol e  
0. 4 4  
0. 2 0  
1 3  
A m p h ot erici n ( 5 d a ys) + fl uc o n a z ol e ( 1 2 0 0 m g / d a y) 
0. 3 0  
0. 1 1  
3 0  
4 2  
A m p h ot erici n + 5 F C  
0. 4 9  
N A  
3 0  
4 5  
A m p h ot erici n + 5 F C + I N F - 
0. 6 4  
N A  
6 0  
A m p h ot erici n ( 7 d a ys) + fl uc o n a z ol e ( 1 2 0 0 m g / d a y)  
0. 3 8  
0. 2 0  
1 9  
4 6  
A m p h ot erici n ( 7 d a ys) + fl uc o n a z ol e ( 1 2 0 0 m g / d a y) + 5 F C  
0. 5 0  
0. 1 5  
1 8  
Fl uc o n a z ol e ( 1 2 0 0 m g / d a y)  
0. 1 8  
0. 1 1  
3 0  
1 9  
Fl uc o n a z ol e ( 8 0 0 m g / d a y)  
0. 0 7  
0. 1 7  
3 0  
A m p h ot erici n ( 1 m g / k g / d a y)  
0. 4 8  
0. 2 8  
4 9  
2 6  
Fl uc o n a z ol e ( 4 0 0 m g / d a y)  
0. 0 2  
0. 0 5  
5  
A m p h ot erici n + fl uc o n a z ol e ( 8 0 0 m g / d a y)  
0. 3 6  
0. 2 5  
2 2 3  
2 5  
A m p h ot erici n + fl uc o n a z ol e ( 8 0 0 m g / d a y) + s ertr ali n e  
0. 4 3  
0. 3 9  
1 2 8  
2 1  
Fl uc o n a z ol e ( 1 2 0 0 m g / d a y)  
0. 1 1  
0. 0 9  
2 0  
4 7  
Fl uc o n a z ol e ( 1 2 0 0 m g / d a y) + fl uc yt osi n e  
0. 2 8  
0. 1 7  
2 1  
A m p h ot erici n 1 w e e k ( eit h er 5 F C or fl uc o n a z ol e)  
0. 4 0  
0. 2 4  
1 7 9  
2  
A m p h ot erici n 2 w e e k ( eit h er 5 F C or fl uc o n a z ol e)  
0. 4 2  
0. 2 5  
1 8 2  
Fl uc o n a z ol e ( 1 2 0 0 m g / d a y) + fl uc yt osi n e  
0. 2 6  
0. 1 8  
1 8 2  
A m p h ot erici n + 5 F C  
0. 4 6  
0. 2 5  
1 8 6  
A m p h ot erici n + fl uc o n a z ol e ( 1 2 0 0 m g / d a y) 
0. 3 6  
0. 2 3  
1 7 5  
 
 
Si mil arl y, a s e p ar at el y p u bli s h e d a n al y si s f o u n d a c orr el ati o n b et w e e n E F A a n d 1 0 -w e e k 
m ort alit y ( P = 0. 0 4) u si n g a l ar g er c oll e cti o n of st u di e s ( Fi g ur e 2. 5 . B).4 8 T hi s a n al y si s u s e d t h e 
r e p ort e d E F A fr o m t h e p u bli c ati o n, w hi c h diff er e d sli g htl y a cr o s s p u bli c ati o n s a n d di d n ot h a v e 
pri m ar y d at a, t h u s w a s u n a bl e t o a c c o u nt f or diff eri n g d e m o gr a p hi c s a cr o s s  hi g hl y di v er s e HI V -
p o siti v e a n d HI V -n e g ati v e c o h ort s. T h e i n cl u d e d st u di e s c a m e fr o m: t h e U S, C a n a d a, I n di a, 
T h ail a n d, I n d o n e si a, L a o s, Vi et n a m, U g a n d a, S o ut h Afri c a, M al a wi, B ot s w a n a, Zi m b a b w e a n d 
N et h erl a n d s. T hi s a n al y si s o nl y i n cl u d e d r a n d o mi z e d tri al s, a n d e x cl u d e d t w o fl u c o n a z ol e 
m o n ot h er a p y st u di e s, 1 9, 2 6  w hi c h h a d p o or E F A s < 0. 2 0 l o g 1 0  C F U/ ml/ d a y a n d hi g h m ort alit y o n 
t h e b a si s t h at t h e s e ar e cl e arl y i nf eri or t h er a pi e s. T hi s e x cl u si o n mi s s e s t h e p oi nt t h at E F A i s a 
q u a ntit ati v e m ar k er of mi cr o bi ol o gi c a cti vit y i n h u m a n s.   


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Shortcomings in EFA:  EFA is not a perfect surrogate marker for all-cause mortality on an 
individual patient level basis. Thus, EFA has been criticized.48 There are two problems with 
EFA: 1) Persons who rapidly die after diagnosis may not have a computed EFA (as an EFA 
requires at least two time points); 2) Death in persons with advanced AIDS could be from other 
opportunistic infections and/or AIDS-related 
cancers; 3) Death after cryptococcal 
meningitis is influenced also by the immune 
system. As we have reported, cryptococcal 
meningitis follows the “damage-response 
framework for microbial pathogenesis”49—
whereby those with the worst immune response 
(e.g. lowest CD4 T cell count) and highest 
pathogen burden have high mortality—but 
surprisingly those with highest CD4 counts and 
lowest fungal burden (i.e. sterile CSF cultures), 
actually have relatively higher mortality as well 
(Figure 2.5.C).50 Similarly, over abundant immune 
responses, e.g. unmasking cryptococcal IRIS 
occurring early after HIV therapy initiation, is highly detrimental.51 Excess dysregulated 
inflammation in the brain is often fatal.  
Thus, EFA cannot capture all factors associated with 
mortality, but EFA can assess the microbiologic activity of an 
antifungal regimen.  At present, there is no immune directed 
therapy for cryptococcosis, thus the only therapeutics we 
have (and that are FDA-approved) are focused on antifungal 
therapy.  Thus, while the immune system contributes to 
mortality on an individual patient basis, the group EFA of an 
antifungal regimen has been associated with overall mortality 
as displayed in Figures 2.4.B.   
In this trial, the primary endpoint is EFA as the clearance rate 
of Cryptococcus yeast in CSF. EFA is a direct quantitative 
measure of the antimicrobial activity of 5-FC used in combination with AMB therapy in humans 
with cryptococcal meningitis.  
2.6 
 Potential Risks and Benefits 
2.6.1 
Potential Risks 
First, the EFA, which as described above, indicates the rate of fungal clearance and correlates 
with mortality due to CM will be followed by the study team. If EFA is consistently <0.2 log10 
CFU/mL/day after 10 enrolled patients, then the trial will be suspended until an interim review 


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can be completed. In addition, all patients will be receiving IV AMB which is also a gold standard 
therapy and reduces mortality related to CM, in addition to fluconazole.  
As discussed above 5-FC is a critical aspect of the induction phase of treatment for CM, 
decreasing the fungal burden in the CNS and improving mortality. Given this, there is a risk that 
decreasing the dose of 5-FC will lead to insufficient CSF concentrations to achieve fungal 
clearance and reduced mortality. For this reason, we will implement several safety measures. 
As discussed above, our trial will use the induction therapy regimen established in the 
AMBITION-cm trial and now recommended by the updated WHO guidelines.4 During phase 2 of 
that trial, high dose liposomal Amphotericin B with fluconazole achieved an EFA of -0.52 log10 
CFU/mL/day.38 These data provide strong evidence that optimizing the dose of 5-FC can be 
safely studied in the same context without significantly reducing mortality.  
The risks associated with the traditional IV AMB include nephrotoxicity, life-threatening 
hypokalemia (common), life-threatening hypomagnesemia (common), severe anemia 
(common), nausea (common), vomiting (common), diarrhea (common), severe infusion 
reactions including rigors, local inflammation, and thrombophlebitis (common), and many other 
less common side effects. 
Amphotericin B is an FDA pregnancy category B (no evidence of risk in humans); however, all 
participants will also receive flucytosine and fluconazole, which are both potential teratogens at 
high doses. Therefore, subjects must use effective forms of contraception to avoid becoming 
pregnant. Pregnant women will be excluded. 
 
2.6.2 
Potential Benefits 
The primary benefit for this study will be to improve access to 5-FC for patients in greatest need 
of this therapy. It is hoped that lower doses of 5-FC will be equally effective compared to the 
traditional dosing, which will reduce the cost of therapy for individual patients and eventually HIV 
care programs. A secondary benefit by participating in the study is that the lower doses may be 
associated with lower toxicities hence providing data to recommend decreasing costs of safety 
monitoring in the long term. High doses of 5-FC are associated with leukopenia, 
thrombocytopenia and acute renal injury, especially when used in conjunction with IV AMB. 
Lower doses of 5-FC will likely have reduced burden of side effects on individual patients.  
Participants will receive scheduled lumbar punctures at baseline, Day 3 ±1, Day 7 ±2, and Day 
14 ±2 and additionally as required for control of intracranial pressure and documentation of CSF 
sterilization.  Receipt during the first week of at least one therapeutic lumbar puncture with 
control of intracranial pressure has a 69% relative reduction in 10 day mortality in Uganda.52 
Thus participants will likely benefit from study participation through receipt of scheduled lumbar 
punctures with control of intracranial pressure. 
There is the potential benefit of access to single high dose liposomal AMB, which has recently 
been shown to be as effective as the 7-day course of IV AMB deoxycholate. When available to 
the study team, liposomal AMB will be used which will also reduce incidence of side effects 
related to AMB deoxycholate.  


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3. 
OBJECTIVES 
3.1 
Study Objectives 
3.1.1 
Phase II Trial Objective 
To demonstrate the reduced dose of 5-FC, when combined with IV AMB for 
induction therapy of CM is non-inferior compared to traditional dosing of 5-FC in 
reducing fungal burden of Cryptococcus 
3.1.2 
Phase II Secondary Objective 
● To determine if there remains a mortality benefit to 5-FC for induction therapy at 
reduced dosing. 
● To assess the overall safety and tolerability of 5-FC at reduced dosing.  
3.2 
Study Outcome Measures 
 
Primary Endpoint 
1. CSF culture clearance at 2 weeks (Early Fungicidal Activity, or EFA: change of log10 
Cryptococcus CFU/mL CSF/day as measured by serial quantitative CSF fungal 
cultures)  
Key Secondary Endpoint  
1. Desirability of Outcome Response (DOOR) as ordinal ranked maximum score tested 
by Win Ratio. 
a. 18-week Survival with CSF sterility by 2-weeks 
b. 18-week survival with CSF culture positivity beyond 2 weeks 
c. Grade 3 hematological adverse event by 2 weeks 
d. Grade 4 hematological adverse event by 2 weeks 
e. Lost to follow up before 18-weeks 
f. 
Serious Adverse Event through 18 weeks (e.g. all-cause re-hospitalization, 
permanent neurologic deficit)  
g. Death by 18-weeks 
 
Secondary Endpoints 
1. CSF culture sterility cumulative incidence over 18 weeks 
2. 18-week survival time 
Safety & Exploratory Endpoints 
1. Incidence of Grade 3-5 abnormal laboratory test results (as per the NIAID DAIDS toxicity 
grading scale) through Week 2 of the study 
2. Incidence of clinical Adverse Events (Grade 3-5) or SAEs through week 2 
3. Pharmacokinetics of 5-FC concentrations in blood, CSF, and other tissues. 


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4. 
STUDY DESIGN 
4.1 
 Phase II Study Design 
 
Figure 4.1 
 
 
This is a single-arm trial with historical controls to compare the efficacy and safety of reduced 
dosing of 5-FC induction therapy for the treatment of cryptococcal meningitis in combination 
with IV AMB to standard of care, with EFA as the primary endpoint.  


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Participants will be enrolled into an investigational cohort at study entry.  
1. The investigational cohort will receive 60mg/kg/day of 5-FC divided into 3 doses per day 
for 10 days, n~50 total enrolled to achieve n=36 CSF culture positive.  
2. The control group will consist of historical participants in the AMBITION trial 
3. All participants will receive liposomal Amphotericin B 10 mg/kg, single-dose infusion, if 
available.  
4. If liposomal Amphotericin is unavailable, participants will receive IV amphotericin 
deoxycholate 0.7-1.0 mg/kg/day for 7 days. 
5. All participants will receive fluconazole 1200 mg/day during induction therapy from day 1 
to 14 then consolidation therapy 800 mg/day from day 15 to 10 weeks, and maintenance 
therapy of 200 mg/day after 10 weeks. 
6. All participants will receive lumbar punctures at diagnosis, day 3, day 5-7, day 10-14, 
and additionally as required for control of intracranial pressure and documentation of 
CSF sterilization. Therapeutic LPs conducted during the first week have a ~70% relative 
survival benefit. 
Investigational cohort will receive: 
● IV Amphotericin B deoxycholate 1 mg/kg/day for 7 days or IV Liposomal Amphotericin B 
10mg/kg single dose. 
● Oral flucytosine (5FC) 60mg/kg/day for 10 days in 3 divided doses 
● Oral fluconazole 1200mg/day from Day 1 to 14 
● Oral fluconazole 800mg/day from Day 15 to Week 10 
● Oral fluconazole 200mg/day thereafter 
 
The control cohort will be drawn from the AMBITION trial that was conducted partly in Uganda.43 
This was a randomized clinical trial that evaluated single high-dose amphotericin B (10 mg/kg) 
with fluconazole (1200 mg/day) and flucytosine (100 mg/kg/day) for 2-week induction therapy of 
cryptococcal meningitis compared to standard amphotericin deoxycholate (1 mg/kg/day) with 
flucytosine (100 mg/kg/day). The trial enrolled participants who were HIV positive with a first 
episode of cryptococcal meningitis diagnosed by cryptococcal antigen or India ink staining. 
Participants were excluded if they were pregnant, had already taken amphotericin or 
fluconazole for >48 hours, had a previous serious reaction to the study medications or were 
taking a medication that would be contraindicated with the study medications. In Uganda, the 
trial enrolled 163 participants in the control arm and 164 in the intervention arm for a total 
enrollment of 327 Ugandan participants. The control cohort will be drawn from Ugandan 
participants. If IV liposomal amphotericin B is used in the investigational cohort then they will be 
drawn from the intervention arm. If IV deoxycholate amphotericin B is used in the investigational 
cohort then they will be drawn from the control arm. We will compare the EFA of all the control 
participants to the interventional participants using multivariate mixed linear regression, 
adjusting for confounding factors such as CD4+ count, GCS<15 or not, prior exposure to ART or 
not and other important clinical factors. We will also compare 10-week survival in the 


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interventional and control cohorts, using multivariate Cox regression, adjusting for the same 
clinical factors as the mixed regression model. 
 
This 2018 and 2022 WHO recommended regimen is based on a phase III randomized clinical 
trial demonstrating that: 
i) 
1 week of IV AMB had equivalent survival as 2 weeks of IV AMB;2 
ii) 
1 week of IV AMB combination therapy was less toxic than 2 weeks of IV AMB;2 
and  
iii) 
Adjunctive flucytosine had superior survival to adjunctive fluconazole.2 
 
A DSMB review by the full committee will occur after 10 participants have been enrolled with >2 
weeks of accrued data. A local Ugandan community member will be invited to attend open 
DSMB meetings. The DSMB safety reports will be submitted to the IRBs and regulatory 
authorities. 
 
Timing of ART Initiation or Switch.  
ART will be initiated for ART-naïve participants ~6 weeks after CM diagnosis, per current 
standard of care.25  Persons enrolled with a failing ART regimen will be switched to 2nd line HIV 
therapy at ~6 weeks while persons who have initiated ART in the preceding 2 weeks will have 
their ART interrupted and re-initiated ~6 weeks after CM diagnosis, in accordance with 
consensus best practices.53 ART management decisions for persons on ART longer than 2 
weeks will be at investigators’ discretion. Outpatient visits will occur every 2-4 weeks at the 
study site outpatient HIV clinic until the conclusion of the study at 18 weeks. Patients can elect 
to continue to receive routine HIV care at the site after study completion. If persons have active 
cryptococcal-related issues (e.g. IRIS or relapse) ongoing at 18 weeks, the participant will 
continue to be followed by the study team until their condition has stabilized.  
Prior published outcome data suggest no overall difference in 18-week survival between those 
who present ART-naïve vs. ART-experienced on a failing regimen.51 
4.2 
 Pharmacokinetic Studies 
PK studies will be conducted on CSF from each lumbar puncture performed during the study 
and on selected plasma specimens. Timing of blood and CSF collections will be recorded. For a 
subset of participants, we will do a more intensive plasma sampling regimen by collecting 
multiple peripheral blood samples after the first dose of 5-FC that day.   
In Kampala, cell pellets of CSF white cells and peripheral blood mononuclear cells (PBMCs) will 
be collected for potential immunologic studies. 
 
4.3 
Antifungal Susceptibility Testing 


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Minimum inhibitory concentration (MIC) testing will be performed on all participants in 
the investigational cohort with Cryptococcal growth on CSF culture. Broth microdilution, 
as recommended by the European Committee on Antimicrobial Susceptibility Testing 
will be used to determine the MIC. 
 
5. 
STUDY ENROLLMENT AND WITHDRAWAL 
5.1 
Inclusion Criteria 
● CSF cryptococcal antigen (CrAg) positive meningitis 
● Ability and willingness to provide informed consent 
● Willing to receive protocol-specified lumbar punctures 
 
Exclusion Criteria 
● Age < 18 years 
● Inability to take enteral (oral or nasogastric) medicine 
● Cannot or unlikely to attend regular clinic visits 
● Receiving chemotherapy or corticosteroids 
● Suspected paradoxical immune reconstitution inflammatory syndrome (IRIS) 
● Pregnancy or breastfeeding (tested on screening) 
● CrCl < 20 mL/minute* 
● Absolute neutrophil count < 500 x 106 cells/L* 
● Thrombocytopenia < 50,000 x 106 cells/L* 
● Patients with prior 5-flucytosine exposure >3 days in the 12 months prior to enrollment 
● Any condition for which participation would not be in the best interest of the participant or 
that could limit protocol specified assessments.  
Rationale for Phase II Trial Criteria 
The inclusion and exclusion criteria of the Phase II trial are meant to be broad to enroll a 
representative sample of persons in resource-limited areas, and thereby be broadly 
generalizable. Flucytosine and fluconazole are known teratogens in the first trimester. 
Amphotericin is the preferred drug of choice during pregnancy. Since therapy includes 
flucytosine or fluconazole, pregnant and breastfeeding women are excluded. 
Other exclusion criteria are intended to minimize bias and maximize likelihood of completing the 
study or need to customize therapy (e.g., pregnant women). 
Note on subjects with positive CSF CrAg but subsequent negative CSF cultures: 
Since CSF CrAg is more sensitive than CSF culture in very early disease with a low fungal 
burden,18,54 CSF culture-negative patients with a positive CSF CrAg will be assumed to have 
early CM and be eligible for the study and included in the analyses. Approximately 10-15% of 
participants are estimated to be culture negative. CSF cultures take 7-14 days for results, thus 
are not a realistic criterion for enrollment as therapy is initiated based on CSF CrAg results. CSF 


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20 
 
CrAg LFA is markedly more sensitive than India ink microscopy.54 Although these participants 
will not contribute to the quantitative CSF clearance data, they will contribute to all other 
endpoints for survival, safety, tolerability, and toxicity – all of which are important outcomes.  
This trial will use adaptive sample size, requiring 36 CSF culture positive participants and 
enrolling up to 50 total participants with cryptococcal meningitis.  
Note on subjects with previous history of cryptococcal meningitis 
Subjects with a previous history of prior, known cryptococcal meningitis will be included in the 
trial as long as they did not receive >2 days of 5-FC during their previous treatment. The 
rationale for this is that most patients in Uganda would have previously been treated with 
fluconazole during induction therapy and relapse is highly associated with fluconazole 
resistance.34 These patients may still benefit from an induction regimen that includes 5-FC and 
would be ideal candidates for this therapy when it becomes more widely available.  
 
*Note on laboratory exclusion criteria 
Subjects can be enrolled into the study before the laboratory data is available. Labs should be 
sent on enrollment and should be available within 72 hours. Subjects meeting laboratory 
exclusion criteria will be withdrawn from the study (See section 5.6). 
5.2 
Treatment Procedures 
5.2.1 
Selection of Study Population 
For the phase II trial, potential research participants with active cryptococcal meningitis will be 
identified by active surveillance during hospitalization by the on-site study teams and consented 
for screening. Secondary surveillance will be via the site microbiology laboratories with study 
team notification of new positive culture or CrAg results. 
 
5.2.2 
Timing of Study Enrollment 
For the phase II trial, potential participants would be determined as eligible after lumbar 
puncture results.  The diagnostic lumbar puncture is considered a routine medical procedure; 
with meningitis considered a medical emergency. Participants can be enrolled anytime in the 
first 2 days of 5-FC therapy. This allows time for informed consent without any undue, coercive 
time pressure. Receipt of >2 days of 5-FC therapy is an exclusion.  
 
5.3 
Informed Consent Process 
5.3.1 
Study Screening and Storage Consent 
Since serum and CSF will be stored, consent for specimen storage and data collection will be 
obtained at the time of the initial diagnostic lumbar puncture (when the cryptococcal meningitis 
diagnosis is unconfirmed) prior to obtaining consent for the main overall trial.  A screening and 
storage consent (or surrogate consent from caregiver/next of kin) will be required for study 
personnel to perform a lumbar puncture (LP) screening for CM diagnosis to verify an inclusion 
criterion. Persons with altered mental status are eligible for screening diagnostic lumbar 


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21 
 
puncture for meningitis diagnosis with surrogate consent, even if they are not eligible for the 
overall study (as it would be unethical to withhold appropriate diagnostics from critically ill 
patients). This screening and storage consent requests to “record information about their current 
illness,” including presenting symptoms (assessed prior to LP), final diagnosis (based on CSF 
results and physician diagnosis), and vital status at hospital discharge.  
This screening /storage consent may be via this or another research study (e.g., EnACT) in 
order to make the diagnosis of cryptococcal meningitis and assess for this trial’s eligibility 
criteria. If persons are referred from outside health care facilities with documentation of CM per 
study inclusion criteria, they will be eligible; however, these persons must have an LP with a full 
diagnostic CSF analysis performed after study screening consent and prior to entry into the 
study to confirm the diagnosis and exclude alternative diagnoses.  
As meningitis is a medical emergency, after the diagnosis of cryptococcal meningitis is 
confirmed, persons start on standard IV AMB and 5-FC therapy.  The first dose of 5-FC is study 
day 1. 
5.3.2 
Informed Consent   
Informed consent is a process that is initiated prior to the individual agreeing to participate in the 
study and continuing throughout the individual’s study participation.  After CM diagnosis and 
eligibility criteria are confirmed, informed consent will be obtained by a study investigator who 
will provide potential subjects with an IRB approved consent form in English or Luganda 
(Kampala) or Runyankole (Mbarara).  Informed consent must occur prior to participants 
receiving 12 or more doses of 5-FC. There is a window of time to allow for informed consent 
process without any undue, coercive time pressure or unethical withholding of antifungal 
therapy until consent is provided. Participants may have received fluconazole and remain 
eligible. However, potential participants will be given appropriate time to consider the study.  At 
the time of informed consent, a complete history and physical examination will be performed to 
verify inclusion/exclusion criteria. 
During the Informed Consent process, the investigator will describe the purpose, risks, and 
benefits related to the study.  Each aspect of the forms will be explained in detail with the 
potential subject, and the potential subject will have the opportunity to ask any questions 
regarding the study. The investigator obtaining informed consent will ask questions to assess 
the subject’s understanding.  The investigator will state that participation is voluntary and that 
subjects may refuse participation or withdraw at any time without prejudice to their clinical care. 
Persons who decline participation will continue the same CM induction therapy according to 
current standards of care. 
Since this study may require additional blood draws beyond the standard of care, additional 
consent will be required from subjects receiving blood draws for stored samples for future study 
purposes.  In this case, samples will be stored indefinitely, unless the subject has a change of 
mind and asks for them to be destroyed. 
Specimens do need to be collected as pharmacokinetics is a key component of the study. 


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If, in the opinion of the investigator, potential participants who do not have appropriate 
comprehension, the investigator must re-explain the study or determine whether the 
participant’s current mental capacity is diminished due to the CM and pursue surrogate consent.  
Consent for Lumbar Punctures:  Lumbar punctures will be explained in detail in the meningitis 
screening sample storage consent and main trial informed consent form.  All follow up 
therapeutic LPs are deemed standard of care to control intracranial pressure and document 
clearance of infection. Participants retain the right to refuse a follow up LP, although this is not 
recommended as therapeutic LPs have a strong survival benefit. One follow up therapeutic LP 
is associated with a 69% relative survival benefit in the first ~10 days.52 A second therapeutic 
LP in the second week has a 50% reduction in 30-day mortality (33% vs 22%) in Uganda.55  
Refer to Section 8.2 for the recommended LP schedule. 
Storage Consent:  Informed consent for long-term storage of samples for future research 
testing is necessary by Ugandan regulations. This will be completed according to the COAST 
protocol which is a parallel study into which all FLOOR participants will be enrolled. Refer to 
Section 12.6 for further details. 
5.3.3 
Capacity to Consent 
Assessment of the capacity to consent is required for all participants in the study that have a 
GCS of 15. The UCSD Brief Assessment of Capacity to Consent (UBACC) will be administered 
to all subjects.  The UBACC is an adapted, validated tool consisting of 10 questions worth 3 
points each for assessing the capacity to consent in clinical trials.56 If GCS is 15, then UBACC 
will be administered to assess capacity to consent.  If the subject scores 15 or better on the 
assessment, then they can self-consent.  A GCS of <15 or UBACC assessment score of <15 
means that the individual does not have the capacity to consent.  Documentation that the 
assessment was administered and completed will be included in the study file.  
5.3.4 
Surrogate/Proxy Consent 
Subjects who are unable to give informed consent due the lack of capacity to consent or other 
barrier may enroll in the study with surrogate consent.   
5.3.5 
Literacy 
A person who speaks and understands the language of the informed consent document, but 
does not read and write, can be enrolled in a study by “making their mark” via a thumbprint on 
the informed consent document. 
In this event, an impartial, literate third party must witness the entire consent process and sign 
the informed consent document.  The witness’s name, signature, and relationship must be 
recorded on the informed consent document. A member of the study team is not an impartial 
third party. 
5.3.6 
Waiver of Informed Consent 
CrAg LFA screening by fingerstick, serum, and/or plasma is to be performed as routine 
diagnostic care (without written informed consent) due to the high prevalence of CrAg+ 
individuals hospitalized.  At Mulago Hospital in Kampala, Uganda in 2010, the prevalence of 


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23 
 
CrAg+ individuals with CD4<100 was 21% on the infectious disease ward.57 In Uganda, this 
point-of-care CrAg testing has been standard of care to rapidly identify persons with 
disseminated cryptococcal infection. 
5.4 
 Blinding 
This study will be a single arm phase II trial. Study medications will be dispensed by a study 
pharmacist of record. 
 
5.5 
Premature Discontinuation of the Intervention 
A study subject can discontinue the study intervention (low-dose 5-FC) in the event of any of the 
following: 
● Any clinical adverse event (AE), laboratory abnormality, concurrent illness, or 
other medical condition or situation occurs such that continued participation with 
an intervention would not be in the best interest of the subject 
● Decompensation of the cryptococcal meningitis 
● Subject wishes to voluntarily discontinue therapy 
Subjects are free to discontinue an intervention at any time upon request or in the opinion of the 
investigator in the event of decompensation and disease progression.  Examples of progression 
would be development of altered mental status, increasing CSF quantitative culture CFU counts 
by >1 log10 CFU/mL, other medical events leading to an inability to take oral medicines, etc. 
Subjects discontinuing low-dose 5-FC would then be given standard dose 5-FC, as clinically 
necessary. 
Every effort will be made to undertake protocol-specific study procedures.  If voluntary 
discontinuation occurs, the subject will be asked to continue scheduled evaluations, complete 
an end-of-study evaluation, and be given appropriate care under medical supervision until the 
symptoms of any AE resolve or the subject’s condition becomes stable. The patient will be 
offered ongoing follow-up care at the site outpatient HIV clinic or referred to a clinic of their 
choice if withdrawn from the study.  
Alterations in dose will be considered a protocol deviation, since the goal of the trial is to assess 
the effect of dose-reduction. Patients may remain in the study but dose-adjustments will be 
documented and reported to the IRB. 
Subjects discontinuing the intervention will receive standard of care therapy through 18 weeks. 
5.6 
Study Withdrawal 
By consenting to the trial, participants are consenting to receiving the study treatment, study 
follow-up and study data collection. However, a study subject may discontinue from further 
ongoing study participation at any time if: 
● Subject wishes to voluntarily withdraw. 
● Fulfillment of early withdrawal criterion: absolute neutrophil count < 500 x 106 
cells/L, thrombocytopenia < 50,000 x106 cells/L, or renal creatinine clearance < 20 
mL/minute 


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● Any unacceptable toxicity or adverse event (ie. Development of severe renal 
dysfunction or leukopenia on treatment).  
 
Subjects are free to withdraw from participating in the study at any time upon request.  If 
voluntary withdrawal occurs, the subject will be asked to complete an end-of-study evaluation 
and be given appropriate care under medical supervision until the symptoms of any AE resolve 
or the subject’s condition becomes stable. The patient will be offered ongoing follow-up care at 
the site outpatient HIV clinic or referred to a clinic of their choice if withdrawn from the study.  
Participants who withdraw from Mulago hospital will not be contacted after withdrawal of 
consent.  
Subjects may discontinue the study intervention (as per Section 5.5) and remain in the research 
study for safety monitoring and assessment of endpoints, receiving standard of care therapy.  
Subjects withdrawing from the study will be recommended to receive standard of care therapy. 
 
6. 
MEDICATIONS TO BE USED IN THE TRIAL 
With the exception of the intervention of low-dose 5-FC, all medications received in this study 
are considered standard of care and will be provided by the study. HIV antiretroviral medicines 
will not be provided by the study but are freely available, regardless of study population, via the 
public sector HIV clinics.  
6.1 
Cryptococcal Meningitis Management 
Medication management is summarized in Table 6.2, as per 2018 and 2022 
cryptococcal WHO guidelines,4,58 which exceed the local standard of care. 
 
Table 6.2: 2018 and 2022 WHO Treatment Regimen for Cryptococcal Meningitis 
Medication and Dose 
Week 1 
Week 2 
Week 3-10 
Week >10 
Amphotericin B (1.0 
mg/kg/day)    + flucytosine 
100mg/kg/day a 
  
 
 
 
Fluconazole 1200mg daily 
 
 
 
 
Fluconazole 800mg daily 
 
 
 
 
Fluconazole 200mg daily 
 
 
 
Through 12 
months 
 
 
 
 
 
Treatment Phase 
Induction 
Induction 
Consolidation 
Secondary 
Prophylaxis 
Notes: a Flucytosine (5FC) 100 mg/kg/day preferred where available, otherwise fluconazole at 800–1200mg/day in divided doses 
with two weeks of amphotericin B. KCI 40–60 mEq/day should be given with amphotericin. 


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Individual patient factors often alter patient care and fluconazole dosage on an individual basis 
at physician discretion, such as acute renal failure, duration of cryptococcal culture positivity, 
CM relapse, medication intolerance, and hepatic P450 drug-drug interactions (e.g. rifamycin). 
As well, some individuals will remain CSF culture positive after 14 days of amphotericin, thus 
higher dose fluconazole with fungicidal activity (1200 mg/day) may be used until outpatient clinic 
enrollment at Study Week 4. For persons with persistently positive cultures, refer to Section 7.4 
on outpatient clinical management of CM. Induction therapy using a combination of 
amphotericin and either high-dose fluconazole or flucytosine is recommended as the 
microbiologic activity is greater than that of amphotericin alone. Subjects who remain CSF 
culture positive should continue on at least induction-dose fluconazole until culture negative.  
6.2 
Other Antifungals Used in Cryptococcal Treatment  
6.2.1 
Amphotericin B 
Amphotericin B is an antifungal polyene. Amphotericin is well known for its frequent and 
potentially severe side effects. For this reason, it is used only for the treatment of life-
threatening fungal disease, such as CM, where it may be the only effective treatment available.  
In all cases, possible life-saving benefits of amphotericin must be balanced against its untoward 
and dangerous side effects.  
 
6.2.1.1 
Amphotericin B Deoxycholate 
Amphotericin B deoxycholate is available in single vials.  Each vial contains a sterile, 
non-pyrogenic, lyophilized cake providing 50 mg amphotericin B and 41 mg sodium 
deoxycholate buffered with sodium phosphates.  Amphotericin B is solubilized by the 
addition of sodium deoxycholate to form a mixture, which provides a colloidal dispersion 
for intravenous infusion following reconstitution.  
Typical AMB deoxycholate dosing for CM is 0.7-1 mg/kg daily.  Amphotericin should be 
administered by slow intravenous infusion over a period of approximately 4 hours.  The 
recommended concentration for intravenous infusion is 0.1 mg/mL (1 mg/10 mL). 
Acquisition:  Any nationally registered, GMP-manufactured, and/or FDA-approved 
formulation is acceptable.  
Product Storage and Stability:  Prior to reconstitution, AMB deoxycholate should be 
stored under refrigeration 2° to 8°C (36° to 46°F) and protected against exposure to 
light. 
Study administration for all treatment arms:  
Subjects will receive the WHO standard 7-day course of IV amphotericin B deoxycholate 
0.7-1.0mg/kg/day.  


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Amphotericin is well known for its frequent and often severe side effects, as outlined in 
Appendix A. Significant infusion reactions must be anticipated with a tendency toward 
IV-associated phlebitis.  To help prevent this, IV lines will be flushed after amphotericin 
is administered and peripheral IV sites generally rotated at least every 3 days.  With IV 
AMB, provide 1–2 L saline/day, along with careful monitoring for renal dysfunction and 
particularly electrolyte abnormalities (K and Mg).  Electrolyte management with 
amphotericin is essential, and will follow WHO treatment guidelines including 
supplemental potassium and magnesium.53 
Guidelines for use of IV AMB and management of common toxicities associated with 
amphotericin B are included in the MOP and may be consulted, at the site investigator’s 
discretion. Participants who permanently and prematurely discontinue IV AMB because 
of an AE will be followed closely until resolution of the AE can be documented. Those 
who require additional IV AMB in any arm will be tracked, as additional IV AMB is a 
secondary endpoint.  
When not receiving IV AMB, additional IV fluids will be given as clinically indicated.  
 
6.2.1.2 
Liposomal Amphotericin B 
Liposomal amphotericin B (AmBisome®), Gilead, Inc. is dosed at 10 mg/kg/dose 
in a single dose.  
Acquisition:  Liposomal preparations of amphotericin B are rarely available in 
Africa, but may be more so in the future. AmBisome® may be substituted for 
AMB deoxycholate depending on availability. 
Product Storage and Stability:  
Liposomal amphotericin B may be stored at temperatures up to 25°C. 
Study Administration: 
Subjects will receive the WHO revised standard single high dose liposomal 
Amphotericin B 10 mg/kg/dose.4 Liposomal amphotericin will be the preferred 
regimen when available to the study team. If liposomal Amphotericin is not 
available then Amphotericin deoxycholate will be used.  
The same guidelines for use of IV AMB and management of its toxicities as 
described above in Section 6.2.1.1. will be followed. 


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6.2.2 
Flucytosine 
Flucytosine (5-fluorocytosine) is a fluorinated pyrimidine that is available as 250mg 
and 500mg oral capsules. Flucytosine is a white crystalline powder and the 
following inactive ingredients: cornstarch, lactose, and talc.  
Typical dosing is 100mg/kg/day divided in 6-hour intervals. Flucytosine is rapidly 
and virtually completely absorbed following oral administration. 
Flucytosine is taken up by fungal organisms via the enzyme cytosine permease. Inside the 
fungal cell, flucytosine is rapidly converted to fluorouracil (5-FU) by the enzyme cytosine 
deaminase. Fluorouracil exerts its antifungal activity through the subsequent conversion into 
several active metabolites, which inhibit protein synthesis by being falsely incorporated into 
fungal RNA or interfere with the biosynthesis of fungal DNA through the inhibition of the enzyme 
thymidylate synthetase. 
Acquisition:  Any GMP-manufactured Flucytosine is acceptable.  
Product Storage: Flucytosine should be stored at room temperature between 15-30⁰C. 
Study administration:  
Investigational cohort: Subjects enrolled will receive 10 days of flucytosine at 60mg/kg/day in 3 
divided doses.  Three divided doses are a general recommendation. Based on the number of 
tablets or GI tolerability, the number of doses can be altered. Table 6.2.2 provides an example 
approximate dosing schedule.  
Table 6.2.2 Example Daily Dosing Regimen by Weight 
Weight 
Total 
500mg Tablets Per Day 
Pills per Dose 
Mg/Kg Dose 
Range 
<30 
Weight based dosing 
Respective pills every 
8 hours 
 
30-38 
4 
2, 1, 1 
54.1 - 66.7 
38-44 
5 
2, 2, 1 
56.8-65.8 
45-53 
6 
2, 2, 2 
55.6-66.7 
54-62 
7 
3, 2, 2 
56.2 - 64.8 
63-70 
8 
3, 3, 2 
57.5 - 63.5 
71-79 
9 
3, 3, 3 
57.0 - 63.4 
80-87 
10 
4, 3, 3 
57.5 - 62.5 


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88-94 
11 
4, 4, 3 
58.5 - 62.5 
95-103 
12 
4, 4, 4 
58.3 - 63.2 
>104 
Weight based dosing 
 
 
Estimated weight is acceptable to use (e.g. in comatose or confused persons unable to stand on 
a scale).   Dose should be adjusted once a measured weight is available. There is a general 
preference to round up when between weight bands or when in doubt.  
Cautions. Flucytosine must be given with extreme caution to patients with impaired renal 
function or bone marrow depression. Since flucytosine is excreted primarily by the kidneys, 
renal impairment may lead to accumulation of the drug. Flucytosine serum concentrations 
should be monitored to determine the adequacy of renal excretion in such patients. Dosage 
adjustments should be made in patients with renal insufficiency to prevent progressive 
accumulation of active drug. Patients may be more prone to depression of bone marrow 
function if they: 1) have a hematologic disease, 2) are being treated with radiation or drugs 
which depress bone marrow, or 3) have a history of treatment with such drugs or radiation. 
Bone marrow toxicity can be irreversible and can lead to death. Frequent monitoring of hepatic 
function and of hematopoietic system is indicated during therapy. 
Side Effects. Other common side effects include headache, rash, nausea, vomiting, diarrhea, 
and abdominal pain. The potential side effects will be discussed with patients at study entry, 
with close monitoring for adverse events. 
6.2.3 
Fluconazole 
Fluconazole is a synthetic triazole antifungal agent that is available in 200 mg oral tablets.  
Fluconazole is a white crystalline solid which is slightly soluble in water and saline.  Diflucan 
tablets contain 200mg of fluconazole and the following inactive ingredients: microcrystalline 
cellulose, dibasic calcium phosphate anhydrous, povidone, croscarmellose sodium, FD&C Red 
No. 40 aluminum lake dye, and magnesium stearate.   
Typical dosing is 200-1200 mg daily.  Duration of dosage depends on severity of infection.  The 
oral bioavailability of fluconazole is >90%. 
Fluconazole is a highly selective inhibitor of fungal cytochrome P450 dependent enzymes.  It is 
a potent CYP2C9 inhibitor and moderate CYP3A4 inhibitor.  Thus, patients who are on 
fluconazole and other drugs metabolized through CYP2C9 and CYP3A4 should be monitored. 
Acquisition:  When available, fluconazole will be obtained from the Ministry of Health via the 
Pfizer Diflucan Donation Programme (as per standard clinical practice). Any GMP-manufactured 
fluconazole is acceptable. 
Product Storage and Stability:  Fluconazole needs to be stored below 86°F (30°C). 


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Study administration:  Fluconazole is an oral medication, used for the treatment of many fungal 
infections including CM. After induction therapy, 800mg/day will be given until sterilization of 
CSF can be documented and effective ART is initiated, as outlined in Table 6.2.  If there is 
fluconazole toxicity, the dose can be reduced to 400 mg/day for consolidation therapy after 
negative cultures for Cryptococcus have been documented.  It can be further reduced to 200 
mg/day after 10 weeks for secondary prophylaxis, assuming there is no reason to continue high 
dose fluconazole (history of CM relapse, CM-IRIS, concurrent fungal infection, or other reasons 
per medical officer clinical judgment). 
The justification for the increased dose is based on recent studies that have shown that 
fluconazole is more potent and safely tolerated at higher doses.19,42,44,46 In a study from 
Mbarara, for example, Longley et al showed that fluconazole was more rapidly fungicidal when 
administered at a dosage of 1200 mg per day versus 800 mg per day.19  The current 2018 and 
2022 WHO cryptococcal guidelines recommend 800 mg/day for 8 weeks during consolidation 
therapy (Week 3 to Week 10).4,58 
Possible side effects of the medication are rare, but include headache, rash, nausea, vomiting, 
diarrhea, elevated alkaline phosphatase, and abdominal pain.  Patients will be warned of 
potential side effects at study entry, and closely monitored for adverse events.  Fluconazole 
doses of 800 mg or above will be divided into at least twice daily to avoid side effects. 
 
6.3 
Accountability Procedures for the Study Intervention 
Clinical personnel involved in the dispensation and administration of study drugs to be used in 
the study will adhere to GCP practices. Pharmacies utilized in the study will maintain the supply 
and record keeping of all study drug and antifungal dispensing.  
6.4 
Assessment of Subject Adherence to Study Interventions 
While subjects are hospitalized, study personnel (nurse or medical officer) will directly oversee 
administration of study medication to ensure compliance and record time of administration. 
Administration of antifungals will be by directly observed therapy while subjects are hospitalized 
during the first 1-2 weeks. Upon hospital discharge during outpatient management, self-reported 
adherence collected via a patient diary and pharmacy records will be used to assess study drug 
adherence. Patient diaries will include at minimum, the date the dose was taken, amount of 
dose administered, and number of doses taken in one day. 
 
 


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7. 
PHASE II TRIAL STUDY SCHEDULE 
7.1 
Schedule of Events 
 
 
Procedures 
Sc
ree
nin
g 
Enr
oll
me
nt 
Da
y 3 
D
ay 
5-
7 
D
ay 
1
0-
1
4 
Wee
k 4 
W
ee
k 6 
W
e
e
k 
8 
W
e
e
k 
1
0 
W
e
e
k 
1
2 
W
e
ek 
1
8 
Pre
mat
ure 
Disc
onti
nuat
ion 
Signed Consent Form 
 
X 
 
 
 
 
 
 
 
 
 
 
Assess Eligibility Criteria 
X 
X 
 
 
 
 
 
 
 
 
 
 
Review of Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
 
Enrollment 
 
X 
 
 
 
 
 
 
 
 
 
 
Assess Clinical Status 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Assess Adverse Event 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
X 
Cli
nic
al 
La
bs 
Na, K, creatinine 
X 
X 
 
X 
X 
 
X 
 
 
 
 
 
CBC 
X 
X 
 
X 
X 
 
X 
 
 
 
 
 
CD4 
 
X 
 
 
 
 
 
 
 
 
 
 
ALT, T-bilirubin 
 
X 
 
 
 
 
X 
 
 
 
 
 
CSF Collection & Storage 
X 
 
X 
X 
+1 
+2 
 
 
 
 
 
 
Urine Pregnancy Test 
X 
 
 
 
 
 
 
 
 
 
 
 
Re
se
arc
h 
La
bs 
Storage of Blood for PK and 
research studies 
 
X3 
 
 
 
 
 
 
 
 
 
 
ART Initiation/Change 
 
 
 
 
 
 
X 
 
 
 
 
 
Quantitative Neurocognitive 
 
 
 
 
 
 
 
 
 
X 
 
 
1 Day 14 CSF can be collected between days 10-14. If CSF cultures are sterile from >7 days prior (i.e. culture 
obtained on day 3 is sterile on day 10), lumbar punctures may be deferred at Day 14. Participants may be 
discharged from the hospital after 7 days, if ambulatory and clinically stable. 
2 If CSF culture is positive at Day 14 then an LP must be performed at Week 4. For those who remain culture 
positive thereafter, follow up LPs should be conducted as per best practices to document sterility. 
3Optional: Peripheral blood mononuclear cells (PBMCs) – Kampala only (optional) 
All study visits have an allowable visit window of ±1 day for Day 3-7 visit, ±2 days from 
Day 7-14 visits, and 4 Weeks and beyond have ±1 week visit window. 
7.2 
Enrollment 
At the time of initial hospital presentation, participants presenting with suspected CM will be 
evaluated by the study team. An LP will be performed by the study team per standard of 
care for evaluation of a patient with signs of meningitis (Section 8.2). For women of 
childbearing potential, a urine pregnancy test will be administered.    


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Study staff will verbally screen for inclusion/exclusion criteria when introducing the patient 
information sheet at the start of the informed consent process. Diagnostic CSF analysis 
includes: 
● CSF white cell count 
● CSF protein 
● Quantitative fungal culture59 
● Cryptococcal antigen (CrAg) by lateral flow assay54 
● CSF storage for possible PK studies or future immunology assays 
Documented cryptococcal meningitis will be defined as either CSF fungal culture positive for 
Cryptococcus species and/or CSF CrAg positive. As trial enrollment will be within the first 48 
hours after diagnosis, CSF CrAg results will be used as the inclusion criteria, as the CSF 
fungal culture takes >48 hours. CSF analysis performed at the local approved microbiology 
laboratory.  The purpose of the first lumbar puncture is to make the etiologic diagnosis of 
meningitis (an inclusion criterion) and collect baseline CSF parameters and CSF specimens 
prior to antifungal therapy.  As with every LP in this trial, CSF will be sent for quantitative 
cryptococcal cultures. 
If the participant meets all inclusion/exclusion criteria and completes the informed consent 
process by signing the informed consent document, they will be considered eligible for 
enrollment into the study.   
A physician assessment at this time will include: 
● Complete medical history 
● Vital Signs 
● Complete physical examination  
At enrollment, current signs/symptoms occurring must be recorded. All signs/symptoms 
must be recorded on CRFs at time of enrollment, so that new AEs may be correctly 
assessed and documented on an ongoing basis.  Pre-existing Grade 3-4 events are 
commonly expected among participants at study entry and will likely include: fatigue, weight 
loss, anorexia, nausea, vomiting and neurologic (behavior/altered mental 
status/cognitive/headache) abnormalities.  
Within + 72 hours of enrollment, baseline laboratories will be collected for:  
● Complete blood count (CBC) 
● Chemistry panel (e.g., Na, K, creatinine at minimum, with calculated creatinine 
clearance) 
● CD4 T cell count, as possible 
● HIV RNA (viral load) 
● Liver function tests (ALT, T-bilirubin)  
● Specimen Storage 
● Peripheral blood mononuclear cells (PBMCs) – Kampala only (optional) 
We expect that most patients that undergo screening will already have an HIV diagnosis.  In 
the event of unknown HIV-status, HIV screening will be the rapid test per the current 
hospital protocol of universal HIV testing of all hospitalized patients (i.e. opt-out).  Additional 


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screening for AIDS-related OIs may occur as per standard medical practice, particularly for 
tuberculosis.  
Study days are numbered starting from the first day of diagnosis (Day 1 = the day 
the subject receives first dose of IV AMB therapy).  The format of the visit code will be 
‘xx.y’, where ‘xx’ is the study week, and ‘y’ is the study day of the corresponding week.  
For example, day 1 of the study is the day the subject received the first dose of IV AMB 
and will be denoted ’00.1”.  Day 10 of the study (1 week and 3 days) would therefore be 
denoted ‘01.3’. 
 
Participants may receive up to 2 days of standard dose 5-FC and remain eligible, so as to 
allow participants adequate time for their decision on whether to participate in the research 
project as well as to allow for weekends or holidays. The number of 5-FC doses should 
start with the first investigational dose received, and the schedule of events adjusted 
accordingly.   
7.3 
Hospital Follow-up Visits 
Hospital follow up visits have a visit window, as defined below, to accommodate weekends 
and/or holidays when the local laboratories are closed. Participants will generally be seen 
by a study investigator daily during hospitalization recording administration of study 
medicine, antifungal medicines, and additional IV fluids.  
 
Follow up Visits 
Day ~3  
A study medical officer will assess between day 2-4:  
● Interval history occurring since the prior study visit 
● Vital signs 
● Focused physical and neurologic examination, directed by symptoms  
● Medications and adherence (as appropriate) 
● Adverse events (AEs) 
Laboratory monitoring will include, at a minimum: 
● Serum creatinine 
● Serum chemistry (Na, K) 
● Lumbar puncture with quantitative fungal culture 
● Storage of plasma, serum, and CSF. 
Day 5-7  
A study medical officer will assess during day 5-7:  
● Interval history 
● Vital signs 
● Focused physical and neurologic examination, directed by symptoms  
● Medications and adherence (as appropriate) 


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● Adverse events (AEs) 
Laboratory monitoring will include, at a minimum: 
● CBC with autodiff 
● Lumbar puncture with CSF quantitative culture 
● Storage of plasma, serum, and CSF. 
● Optional: Peripheral blood mononuclear cells (PBMCs) – Kampala only (optional) 
● Optional: Intensive PK sampling of peripheral blood with samples taken 1, 2, 4, 7, 12, 
and 23 hours after the first dose of 5-FC that day 
Participants can be discharged from the hospital after day 7 if they are fully ambulatory and their 
clinical condition has been deemed stable by the study medical officer, but return for a Day 14 
outpatient study visit. 
 
Day 10 - 14 
A study medical officer will assess between day 8-14:  
● Interval history 
● Vital signs 
● Focused physical and neurologic examination, directed by symptoms  
● Medications and adherence (as appropriate) 
● Adverse events (AEs) 
Laboratory monitoring will include, at a minimum: 
● Serum creatinine 
● Serum chemistry (Na, K) 
● CBC with autodiff 
● ± Lumbar puncture with CSF quantitative culture (at physician discretion, see below*) 
● Storage of plasma, serum, and CSF. 
 
* A lumbar puncture at Day 10-14 is at physician discretion, based on earlier quantitative culture 
results. If participants have sterile cultures from >7 days prior (i.e. Day 3 or Day 5-7 CSF 
culture, respectively) AND have an absence of ongoing symptoms of elevated intracranial 
pressure (e.g. headache), then physicians may opt to not perform the lumbar puncture.    
If life-threatening (Grade 4) lab abnormalities are present or develop during the course of the 
study, laboratory monitoring shall occur with greater frequency for subject safety. Example: 
● Creatinine: if values > 3 mg/dL (>265 mmol/L), Creatinine will be repeated at least daily 
until stable 
● Potassium: if values ≤ 2.5 mEq/L or ≥ 6.5 mEq/L potassium will be repeated STAT and 
at least daily until improving 
In the event of a prolonged hospitalization, the study team will continue to follow the patient 
daily in the hospital, with assessments, evaluation, and monitoring undertaken according to 
expected routine standards of care.  If any subject is hospitalized at the time of their expected 
clinic follow-up visit (Week 4, 6, 8, or 10), the study visit will occur while hospitalized.   


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7.4 
Clinic Follow-up Outpatient Visits 
7.4.1 
Clinic Registration, Orientation, and ART counseling 
All subjects will be scheduled to return for outpatient clinic registration in ~1-2 weeks after 
hospital discharge for clinic orientation and ART counseling, as necessary.  After discharge from 
the hospital, patients will be connected with their local HIV clinic for consideration of ART 
initiation. ART counseling and initiation will occur per standard of care at 4-6 weeks per WHO 
guidelines.4,25A study nurse will call the patient to verify plans for attending clinic. If subjects are 
physically unable to attend clinic by ~ 4 weeks, a home visit will occur by study personnel, or a 
driver will be sent to retrieve the subject if necessary. If there are health concerns (and 
specifically if the subject needs to be retrieved due to poor functional status and/or inability to 
ambulate), a study physician visit will occur as clinically needed for a ‘sick visit’, in addition to 
the scheduled events, as described in detail below. 
At the first outpatient visit, the subject’s general health, hydration status, and need for re-
hospitalization will be assessed.  Locator information including a street geographical mapping to 
the home or latitude/longitude coordinates of the home and up to 3 telephone contacts will be 
obtained to minimize lost to follow up. 
Outpatient visits have a ±1 week visit window, but substantial deviation with scheduling is 
discouraged. Visits beyond week 6 can be done by phone.  
Week 4 Outpatient Study Visit 
The Week 4 visit is optional unless the day 10-14 CSF was not sterile. A medical officer will 
assess: 
● Interval history 
● Review of medications and adherence 
● Document any adverse events 
● Focused physical and neurologic exam, directed by symptoms 
● Laboratory evaluation, for all participants: 
▪ Lumbar puncture if Day 14 is not sterile 
● Additional Research labs 
▪ ± Whole blood for PBMC isolation - immunology studies in Kampala 
▪ Storage of blood for pharmacokinetic / immunology studies 
● Consider ART initiation (or switch to second line ART) if clinically stable 
In the event of a prolonged hospitalization or re-hospitalization, the schedule of 
events will continue per schedule with lab monitoring and procedures conducted 
in the hospital.  
A lumbar puncture will be repeated only if the most recent CSF analysis is culture 
positive at time of visit.  
Pharmacy will reconcile previous study medicines and dispense another 2-week 
supply.  


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Week 6 Outpatient Study Visit 
At the Week 6 visit, a medical officer will assess: 
● Interval history 
● Review of medications and adherence 
● Focused physical and neurologic exam, directed by symptoms 
● Laboratory evaluation: 
▪ Complete blood count (CBC) 
▪ Serum sodium, potassium, creatinine  
▪ ALT, AST, T-bilirubin, alkaline phosphatase  
▪ Lumbar puncture if Week 4 is not sterile 
▪ Storage of leftover plasma and serum for pharmacokinetic / 
immunology studies 
● Consider ART initiation (or switch to second line ART) if clinically stable 
● Week 6 is the end of the collection period for expedited SAE reporting as all 
care afterward is the observational standard of care therapy.   
● A lumbar puncture will be repeated only if the most recent CSF analysis is 
culture positive at time of visit.  
Week 8 Outpatient Study Visit 
 
 
Routine HIV care is provided. 
● Any clinically significant (i.e. symptomatic) laboratory abnormality should be 
considered for follow up. 
● A lumbar puncture will be repeated only if the most recent CSF analysis is 
culture positive at time of visit.  
Week 10 Outpatient Study Visit 
At the Week 10 visit, a medical officer will assess: 
● Interval history 
● Review of medications and adherence  
● Focused physical and neurologic exam, directed by symptoms 
● Laboratory evaluation follow up of unresolved AEs 
● Lumbar puncture will be repeated if no prior sterile culture has occurred. 
● Change to Fluconazole 200mg/day secondary prophylaxis; unless there is a 
reason to continue higher dose fluconazole (history of CM relapse, CM-IRIS, 
concurrent fungal infection, or other reasons per medical officer clinical 
judgment), according to current guidelines for long-term secondary 
prophylaxis.4  
 
 


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Week 12 Outpatient Study Visit 
At the Week 12 visit, a medical officer will assess: 
● Interval history 
● Review of medications and adherence 
● Focused physical and neurologic exam, directed by symptoms 
● Evaluation of quantitative neurocognitive function at 12 weeks.60 
Week 18 Termination Visit 
Week 18 is the final scheduled visit of the study. This visit can be conducted by phone or 
in-person. The patient will be offered ongoing HIV follow-up care at the site HIV clinic or 
referred to a clinic of their choice. 
18 weeks signifies completion of the study though subjects may require additional 
evaluation and follow-up in circumstances of ongoing acute complications.  Fluconazole 
will be continued at maintenance dose (200 mg/daily) according to current guidelines for 
long-term secondary prophylaxis. 
7.4.2 
Sick Visits 
In all subjects with suspected culture-positive relapse, paradoxical IRIS, or other health 
concerns, an urgent physician’s visit will be made. Patients will be given the mobile phone 
number of the site study team to make urgent visits on their appointment card. At sick visits, 
study physicians will perform: 
● Interval History, specifically emphasizing review of systems and medication 
adherence 
● Vital Signs 
● Complete Physical Exam 
● LP (if CNS symptoms) 
● Radiologic investigation (as clinically indicated) 
● Blood draw, as clinically directed  
If there is clinical suspicion for meningitis (e.g., therapeutic failure, culture-positive relapse, or 
paradoxical IRIS), an LP must be performed (with subject verbal consent).  The clinical 
presentation of a culture positive meningitis relapse and IRIS is clinically indistinguishable, and 
an LP with complete CSF analysis is necessary to differentiate IRIS from culture-positive 
relapse. MIC will be performed on all relapse meningitis with culture positive CSF. Clinically, 
paradoxical IRIS has a classic phenotype with the basic triad of:  
1. Treated cryptococcal meningitis with a clinical response, 
2. Recently started effective ART, and  
3. Recurrent, aseptic (culture negative) meningitis. 
The primary diagnostic consideration is in excluding cryptococcal relapse in which the CSF 
culture would be positive.  
After a prior response to CM treatment with resolution of symptoms, evidence suggesting 
clinical worsening or new signs/symptoms would commonly include increased headache with or 
Commented [1]: Should move to week 12 to align with 
coast 


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without fever or neurological deficits associated with increased intracranial pressure, increased 
lymphocytic CSF pleocytosis and/or development of cerebral cryptococcal abscess by contrast 
CT scan. 
For persons with seizures, loss of consciousness, or focal neurologic deficit (other than cranial 
nerve VI palsy), a head CT will be performed to exclude other intracranial pathology or contra-
indication to lumbar puncture (if available). If a CT is not available, LP should be performed at 
physician discretion.  Cranial nerve VI palsy is common (20%) with increased intracranial 
pressure >200 mm H2O. The purpose of repeat LP will be to differentiate CM relapse as well as 
to therapeutically relieve elevated intracranial pressure when present. Diagnosis of non-CNS 
cryptococcal IRIS will be based on clinical signs (e.g., lymphadenopathy), imaging, and/or 
biopsy of the relevant site with histopathology and/or culture to exclude alternative etiologies 
(e.g., AFB). 
All other non-IRIS or non-relapse clinical events will be in accordance with current guidelines 
and existing clinic practices.  Refer to Section 10 on AEs and for additional information. 
Any CSF obtained during sick visits will have complete CSF analysis (Section 8.2.1).  
Remaining CSF will be stored for PK studies and future immunology studies. 
7.5 
Termination of Study 
At study termination, the reason for study termination will be documented. Reasons for study 
termination are study completion (after 18 weeks), withdrawal of consent, death, transfer of 
care, or loss-to-follow-up.  Analysis is by intention to treat.  At the study termination visit, the 
following will be documented: 
● Interval history 
● Vital status 
● AEs, updates / final outcomes 
7.5.1 
Early Termination 
In case of early termination, a reason for study termination will be documented.  Reasons for 
early termination include withdrawal of consent, death, transfer of care, or loss-to-follow-up.  An 
attempt will be made to schedule a termination visit.  Assessment and laboratory evaluations for 
an early termination visit will be the same as the Week 18 visit. 
If subjects withdraw from the study, they remain eligible for ART per national guidelines.  
Persons withdrawing will be encouraged to continue to receive ART from the outpatient clinic or 
referred to a clinic of their choice for ongoing HIV care and management of CM. 
Subjects enrolled in the study but choosing to leave the hospital early against medical advice 
will continue to participate in the study if they wish.  Additional phone calls by study personnel 
will encourage the subject to seek follow up HIV care and to rejoin the trial per the ongoing 
schedule of events.  Assessment of vital status will continue via telephone calls at a minimum, 
unless consent is completely withdrawn.  


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All analysis is by intention to treat, thus anyone voluntarily discontinuing study participation must 
have a specific request made to them of whether continued collection of vital status (dead / 
alive) via an 18-week visit or a phone call at a minimum is allowable. 
For persons involuntarily defaulting from study participation due to circumstances beyond their 
individual control (e.g., moving residence due to financial reasons, etc.), appropriate referral to 
local HIV-care will be arranged and a request made for a Week 18 visit.  Actual transport 
expenses may be reimbursed in these rare occasions with prior approval of the site PI. 
Alternatively, phone visits may be conducted as outpatient to assess interval history and vital 
status. 
 
8. 
STUDY PROCEDURES/EVALUATIONS 
8.1 
Procedures 
8.1.1 
Clinical History 
8.1.1.1 
Complete History  
A complete medical history will review prior HIV history, TB history, other past medical 
history, review of systems, medications, and allergies.   
8.1.1.2 
Interval History 
At each subject contact, investigators will seek information on adverse events by open 
ended questioning and, as appropriate, by examination.  This will include structured 
assessment of the: interval history of the present illness, review of systems, adverse 
events, new medications, medication adherence.   
All clearly related signs, symptoms, and abnormal diagnostic results will be 
recorded and grouped under one diagnosis. After entry, all signs/symptoms 
consistent Grade 1-5 clinical adverse events must be recorded at each study visit 
(refer to Section 9.1). Laboratory abnormalities do not need to be individually 
documented on an AE form unless accompanied by a clinical event. 
8.1.2 
Physical Exam 
8.1.2.1 
Complete Physical Exam 
A complete physical exam includes: HEENT (head, eyes, ears, nose, and throat), neck, 
chest, cardiovascular, abdomen, extremities, skin, and neurologic exam, as well as GU 
exam as culturally appropriate.   
 
8.1.2.2 
Focused Physical and Neurologic Exam 
A focused physical and neurologic exam will be directed at current symptoms and 
complaints with a targeted physical exam by study physicians at a minimum. 


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8.1.2.3 
Quantitative Neurocognitive Performance Score (QNPZ-8) 
Neurocognitive function testing may occur at week 12 visit (+2 weeks) using a 
quantitative neurocognitive performance (QNPZ-8) score in Kampala.  
QNPZ-8 is derived from a test battery which includes: 
● 
Grooved Pegboard test 
● 
Color Trails 1 test 
● 
Color Trails 2 test 
● 
WAIS-III Digit Symbol test 
● 
Finger Tapping test 
● 
Hopkins Verbal Learning test – revised (HVLT-R), Learning and Delayed 
Recall 
● 
Semantic Verbal Fluency test (category fluency) 
8.1.2.4 
Depression Screening 
Prevalence of Depression as per the Patient Health Questionnaire (PHQ-9) will be 
collected at 12 weeks. PHQ-9 Total Score Depression Severity Scoring is: 
1-4  
Minimal depression 
5-9  
Mild depression 
10-14 Moderate depression 
15-19 Moderate severe depression 
20-27 Severe depression 
8.2 
Lumbar Puncture and CSF Analysis 
8.2.1 
 Diagnostic Lumbar Puncture (LP) with Complete CSF Analysis 
All patients with suspected meningitis will be screened for cryptococcosis by CrAg lateral 
flow assay (LFA).  A CrAg LFA will initially be evaluated on whole blood obtained via 
fingerstick, with the results (return time = 10 minutes) directing subsequent work-up of 
CSF obtained by diagnostic LP.  The screening informed consent process will be started 
after the fingerstick CrAg is collected.  If the fingerstick CrAg is positive, a diagnosis of 
cryptococcal meningitis will be entertained and CSF studies will be directed to 
confirm/exclude this diagnosis.  To evaluate for CM, a CrAg LFA will be performed on CSF 
obtained at the bedside during the LP procedure. 
In cases of CM confirmed by bedside CrAg LFA, additional CSF studies will be obtained. 
Refer to the most current CSF Lab SOP on meningitis diagnostics. All LPs will be 
performed with manometers and have opening intracranial pressure (ICP) and closing 
pressure recorded.  Quantitative fungal cultures will be performed.  Remaining CSF 
samples will be stored at the site laboratory for future pharmacokinetic and 
pathophysiology studies. 


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If the screening fingerstick CrAg is negative, a diagnostic LP will be performed and non-
specific CSF studies will be obtained to ascertain alternative diagnoses, refer to CSF Lab 
SOP for current details. 
8.2.2 
Therapeutic LPs with Quantitative Cultures 
After cryptococcal diagnosis is known, follow up LPs will be performed to reduce ICP and 
document quantitative fungal culture to determine if the CSF is sterile.  When an elevated 
opening CSF pressure is present (>20 cm H2O), the therapeutic reduction of pressure is 
clinically indicated.62,63 We have previously demonstrated that a therapeutic LP conducted 
during the first week of therapy has a 70% relative reduction in mortality.46   
All follow up ‘therapeutic LPs’ will be analyzed for quantitative fungal culture.  Should a 
persistent headache recur anytime during the study, a therapeutic LP must be 
recommended to decrease the intracranial pressure.  These follow up therapeutic LPs 
require verbal consent of the participant per clinical practice. 
8.2.3 
Quantitative Fungal Cultures 
Fungal cultures will be performed per the SOP: “Protocol for quantitative CSF microbiology 
cultures for Cryptococcus neoformans” as provided by Drs. Tom Harrison and Tihana 
Bicanic.59,61 The quantitative culture SOP includes an undiluted, standard CSF fungal culture, 
the result of which will be used for CM management. Cryptococcal isolates will be frozen at ≤ - 
20ºC in cryovials with 1mL of 25% glycerol.  The purpose of storing isolates is to enable 
external quality assurance and enable future susceptibility testing as needed. Cryptococcus 
isolates will be stored indefinitely. 
8.2.4 
Frequency of LPs 
An initial LP is required for CM diagnosis at the time of enrollment with written informed 
consent.  Therapeutic LPs will be performed at Day 3 ±1 and Day 7 ±2, and whenever 
clinically indicated for the reduction of elevated ICP as recommended by IDSA and DHHS 
guidelines.62,63  Subjects with a positive CSF culture at Day 14 (estimated ~30%) will be 
recommended to have a follow up LP by the Week 4 outpatient visit to document whether 
ongoing culture positive cryptococcosis is continuing. Additionally, subjects with a positive 
CSF culture at Week 4 (estimated ~10%) will be recommended to have a follow up LP by 
the week 6 outpatient visit to document whether ongoing culture positive cryptococcosis is 
continuing. 
The majority of individuals being treated for CM are expected to have sterile CSF cultures 
by Week 4. Those with ongoing CSF culture positivity should have follow up LPs to 
document clearance of the cryptococcal infection, prior to decreasing the fluconazole 
maintenance dosing.  Until CSF is known to be culture negative, fluconazole should 
remain dosed at 800 mg/day.  
Serial LPs are recommended as the standard of care for controlling elevated ICP in CM.  
According to the current standard of care, if a subject has elevated intracranial pressure 


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(>20 cm H2O), an LP should be performed therapeutically to reduce elevated intracranial 
pressure.18,64 Therapeutic LPs conducted within the first one week of therapy have a 
~70% relative reduction in mortality.52 
All follow-up LPs must have verbal consent from the subject prior to performing the LP per 
clinical practice.  As LPs are invasive procedures, subjects will need to give consent prior 
to performing follow up LPs. The initial diagnostic LP at screening visit requires written 
informed consent.  Follow up LPs require verbal consent.  Subjects may decline follow-up 
LPs at their own risk, but at minimum will be offered therapeutic LPs at Day 3 ±1 and Day 
7 ±2.   
Once subjects are CSF culture negative (given at least 7 days of culture growth) and have 
a normal CSF opening pressure (< 20 cm H2O), they should not have a routine follow up 
LP, unless new symptoms develop.  If new symptoms develop concerning for CM-IRIS or 
CM relapse, an LP will be recommended for the subject for diagnosis to guide clinical 
management. 
8.3 
Laboratory Evaluations 
All routine clinical tests as specified on the schedule of events will be performed at the 
local site lab. Print out of lab results will be placed into research charts.  
8.3.1 
Hematology 
CBC monitoring will be encouraged as part of routine care at the start of induction 
antifungal therapy, initial evaluation for ART, and whenever clinically indicated. Scheduled 
draws will occur at enrollment, Day 5-7, Day 14, and Week 6.  
8.3.2 
Chemistry 
Serum creatinine, sodium, potassium, and ALT, total-bilirubin will be monitored at the local 
approved site laboratory as part of routine care at the start of CM therapy, initial evaluation 
for ART, and whenever clinically indicated. Serum aliquots of 0.5-1mL will be frozen at -
80ºC.  An estimated creatinine clearance can be obtained using the Modification of Diet in 
Renal Disease (MDRD) Study equation:65 
GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.212 for 
African) 
8.3.3 
CD4 Profile 
CD4 profiles will be encouraged on initial HIV diagnosis as part of routine care and at 10 
weeks. 
8.3.4 
Stored Specimens 


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Any extra serum and plasma collected for clinical testing (e.g., creatinine and potassium) 
will be stored for later potential cytokine analysis (Bio-Rad, Hercules, CA) via a Luminex 
Magpix instrument to investigate biomarkers which predict risk of mortality and/or IRIS 
pathogenesis. The volume of stored blood should always be < 30mL for all pathogenesis 
studies. 
The CSF supernatant will be frozen for pharmacokinetic and pathogenesis studies. 
If a subject later withdraws storage consent (after initial consent), their study chart should 
be marked for ‘No Storage,’ and the PIs notified in order to destroy any stored specimens. 
8.4 
Specimen Storage 
Specimens processed at the study site will be per the laboratory protocols for the site lab 
for clinical tests.  Extra serum, plasma, and CSF must be stored at -80⁰C. 
8.5 
Biohazard Containment 
Appropriate blood and secretion precautions will be employed by all personnel during 
blood draws, lumbar punctures, and shipping and handling of all specimens for this study, 
as currently recommended by the CDC, the NIH, and national guidelines. 
 
9. 
ASSESSMENT OF SAFETY 
This is a clinical trial investigating whether a lower dose of flucytosine is safe and effective 
compared to standard dosing. There is evidence that lower doses reach adequate levels above 
MIC for Cryptococcus.  
Participants will also receive fluconazole and/or Amphotericin B as well as HIV therapy and 
cotrimoxazole.  HIV therapy will be provided via the public health system. This study will provide 
antifungal therapy while hospitalized, if otherwise unavailable.  Outpatient standard of care 
fluconazole will be provided by this research study.  
9.1 
Adverse Event (AE) Definitions 
The intervention during this trial is the reduced dose of flucytosine for induction therapy.  
Adverse Event (AE) 
An AE is any untoward medical occurrence in a clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have to have a causal relationship with 
this treatment.  An AE can therefore be any unfavorable or unintended sign (including abnormal 
laboratory findings), symptom, or disease temporally associated with the use of a medicinal 
product. Any symptom, sign, illness or experience that develops or worsens in severity during 
the course of the study are considered AEs.  


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Any medical condition already present at the time of screening should not be reported as an 
adverse event unless the medical condition or signs or symptoms present at baseline changes 
in severity or seriousness at any time during the study. In this case, it should be reported as an 
adverse event. 
As a diagnosis may have multiple signs or symptoms associated with the AE (e.g., sepsis 
presenting with fever, hypotension, confusion), a single diagnosis for the AE is preferred over 
reporting each individual symptom.  
Clinically significant abnormal laboratory or other examination (e.g., ECG) findings that are 
detected during the study or are present at the time of screening and significantly worsen during 
the study are defined and should be reported as adverse events. The Investigator will exercise 
his or her medical and scientific judgment in deciding whether an abnormal laboratory finding, or 
other abnormal assessment is clinically significant. Clinically significant abnormal laboratory 
values occurring during the clinical study will be followed until repeat tests return to normal, 
stabilize, or are no longer clinically significant. Any abnormal test that is determined to be an 
error does not require reporting as an adverse event. Laboratory abnormalities will be 
separately categorized from clinical AEs.  
Worsening of the index indication (cryptococcal meningitis) due to insufficient therapeutic effect 
of study drug is captured as an efficacy measure (i.e., death, poor early fungicidal activity) and 
in general will not be considered an adverse event but are captured in other individual trial 
endpoints. As participants enter the trial with Grade 4 cryptococcal meningitis, worsening is not 
possible, unless death occurs.  
9.2 
 Assessment of Adverse Events by the Investigator 
The Investigator will assess the severity (intensity) of each adverse event as mild, moderate, 
severe, or life threatening.  
9.2.1 
Severity of Adverse Events 
The term severity is defined as the intensity grade or level for a specific event, i.e., mild, 
moderate, severe, or life-threatening. Importantly, severity is not the same as seriousness, 
which is based on participant/event outcome or action criteria usually associated with events 
that pose a threat to a subject’s life or functioning (ICH E2A). The Division of AIDS (DAIDS) 
Table for Grading the Severity of Adult and Pediatric Adverse Events (v2.1, July 2017) will be 
used.  Laboratory AEs will be defined using the statistical database by DAIDS Table grading 
thresholds.  
The Investigator will assess the severity (intensity) of each adverse event as mild, 
moderate, severe, or life threatening and will also categorize each adverse event as to its 
potential relationship to study drug using the categories of yes or no. 
Assessment of severity: 
The severity of all adverse events should be graded as per The Division of AIDS (DAIDS) 
Table for Grading the Severity of Adult and Pediatric Adverse Events (v2.1, July 2017 or 


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most current).  For those adverse events not listed, the following grading system should be 
used: 
● Mild (Grade 1): Transient symptoms, awareness of sign/symptom, but easily 
tolerated and no interference with patient’s daily activities. 
● Moderate (Grade 2): Marked signs/symptoms that interfere with a patient's 
usual activities, but still acceptable. 
● Severe (Grade 3): Incapacitating signs/symptoms which cause considerable 
interference with the patient’s daily activities, unacceptable. 
● Life-threatening (Grade 4): Life-threatening or disabling adverse event. 
● Death (Grade 5): Death-related adverse event.  
o Death from pre-existing cryptococcal infection is part of the survival endpoint, which 
is expected to be up to 20% of enrolled subjects. Cryptococcal meningitis deaths 
without other AEs should not be additionally reported as a Grade 5 AE. The 
objective is to discern Grade 5 deaths due to adverse events versus deaths from 
the progression of the pre-existing cryptococcal meningitis.  Overall survival will be 
separately analyzed as the secondary endpoint.   
9.2.2 
Causality of Adverse Events 
The probable causality of the investigational study drug being related to the AE will be 
collected.   
Definitely related 
There is a certainty that the AE is related to the study drug. 
Probably related 
There is a high likelihood that the AE is related to the study 
drug. 
Possibly related  
There is a likelihood that the study drug is the cause of the AE, 
but other causes cannot be ruled out. 
Unlikely to be related  It is not likely that the AE is related to the study drug, and other 
more likely causes are present. 
Unrelated 
Evidence exists that the AE is related to something other than 
the study drug. 
 
AEs will use pre-defined causality for the common, expected medical conditions 
associated with HIV/AID (e.g., TB, new opportunistic infections), other infections (e.g., 
malaria, pneumonia) and cryptococcal meningitis complications. Refer to Manual of 
Operations for details. 
Therapeutic failure of the antifungal therapy alone is not deemed as causal for an AE 
(i.e. it is unrelated).  


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The most common disease-related complications and/or standard of care treatment-
related side effects include: 
● 
Death due to pre-existing cryptococcosis (up to 20% of enrolled subjects); 
● 
Meningitis complications: e.g., seizures, cranial nerve palsies, increased 
intracranial pressure 
● 
Transient renal insufficiency in the first 14 days after study entry due to IV 
amphotericin (20%); 
● 
Cryptococcal IRIS events (~20%); 
● 
AIDS-related opportunistic infections; 
● 
Hospitalization or death due to immune reconstitution inflammatory 
syndrome (IRIS), cryptococcal culture-positive relapse, or AIDS opportunistic 
infection (20-25%);18,19,26 
● 
Common ART or flucytosine side effects (e.g., anemia, leukopenia) 
● 
Common IV amphotericin side effects (e.g., anemia, hypokalemia, renal 
failure) 
● 
Hospital nosocomial infections and other complications (e.g., aspiration 
pneumonia) 
9.3 
Adverse Events of Special Interests 
AEs of special interest will be queried of participants while hospitalized on a daily basis. 
9.4 
Serious Adverse Event 
AEs are classified as serious or non-serious.  A Serious adverse event is:  
● fatal 
● life-threatening 
● requires re-hospitalization (after hospital discharge for the initial, pre-existing 
meningitis) 
● prolonged hospitalization (due to a new AE; not due to pre-existing meningitis or 
HIV/AIDS) 
● results in persistent or significant disability or incapacity 
● results in congenital anomaly or birth defect 
● an important medical event that may jeopardize the subject or may require 
immediate intervention to prevent one of the other outcomes listed in the definition 
above (e.g., anaphylaxis) 
All AEs that do not meet any of the criteria for serious will be classified as non-serious adverse 
events. A standard medical problem, e.g., malaria, which in theory if untreated could result in a 
fatal or life-threatening event, is not by itself a SAE.  If the AE is of sufficient severity to result in 
hospitalization, then the AE is a SAE.   
Routine outpatient medical therapy for a common, expected illness in the study population (e.g., 
malaria, TB) is not a SAE. 
All SAEs will be: 


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● recorded on the appropriate AE CRF 
● followed through resolution by a study clinician 
● reviewed and evaluated by a study clinician 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, 
they may jeopardize the patient and may require medical or surgical intervention to prevent one 
of the outcomes listed above. Examples of such medical events include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalizations, or the development of drug 
dependency. 
Death due to pre-existing cryptococcal meningitis is not a SAE. Death due to a complication of 
meningitis (e.g., new seizure) would be a SAE.  
Prolongation of hospitalization must be due to a new AE, not the pre-existing cryptococcal 
meningitis or pre-existing HIV/AIDS and related conditions.  A new AE must cause the 
prolonged hospitalization for this to be considered a SAE. Hospitalizations due to cryptococcal 
meningitis commonly may last 2-4 weeks.   
9.5 
Serious Adverse Event Reporting – Procedures for Investigators 
Initial reports 
All SAEs occurring from the time of informed consent through 2 weeks within 72 hours of the 
knowledge of the occurrence (this refers to any adverse event that meets any of the 
aforementioned serious criteria).  
Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or until the 
condition becomes chronic in nature, stabilizes (in the case of persistent impairment), or 
the patient dies. 
Within 24 hours of receipt of follow-up information, the investigator must update the SAE 
form for the study and submit any supporting documentation (e.g., patient discharge 
summary or autopsy reports) to the IRB via e-mail. If it is not possible to access the CRF 
system, refer to the procedures outlined above for initial reporting of SAEs. 
9.6 
Recording of Adverse Events 
Clinical AEs (Grades 3-5) occurring during the first 2 weeks of the research study will be 
recorded in the CRF, and the Investigator will give his or her opinion as to the relationship 
of the AE to the study drug treatment (i.e., whether the event is related or unrelated to study 
drug administration). 
All AEs will be documented. A description of the event, including its date of onset and 
resolution, whether it constitutes an SAE or not, any action taken (e.g., changes to study 
treatment), and its outcome should be provided, along with the Investigator’s assessment 


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of causality (i.e., the relationship to the study treatment). For an AE to be a suspected 
treatment-related event, there should be at least a reasonable possibility of a causal 
relationship between the protocol treatment and the AE. Adverse events will be graded 
according to The Division of AIDS (DAIDS) Table for Grading the Severity of Adult and 
Pediatric Adverse Events (v2.1, July 2017).   
If the AE is serious, it should be reported immediately to the IRB. Other untoward events 
occurring in the framework of a clinical study after enrollment are to be recorded as AEs. 
Laboratory test results do not require individual additional reporting on CRFs as these will be 
summarized from the statistical database for Grade 1 lab AEs and above.  
9.7 
Reporting Period for Adverse Events 
All AEs regardless of seriousness or relationship to study treatment, spanning from the 
signing of the informed consent form until 10 weeks are to be recorded on the CRF. The 
AEs occurring from the signing of the ICF until the first dosing of the study drug will be the 
pre-treatment AEs. The AEs occurring at or after the first dosing until completion of 
treatment will be the treatment-emergent AEs (TEAEs). The study will continue with a 
follow-up period after ~30 calendar days post completion of study drug through 10 weeks.   
All AEs resulting in discontinuation from the study should be followed until resolution or 
stabilization. All new AEs occurring during this period must be reported and followed until 
resolution unless, in the opinion of the Investigator, the AE or laboratory abnormality/ies 
is/are not likely to improve because of the underlying disease. In this case, the Investigator 
must record his or her reasoning for this decision in the patient’s medical record. 
 
9.7.1 
Laboratory AEs 
As laboratory anomalies are common in this population with advanced HIV/AIDS and 
causality difficult to discern, all laboratory values will be graded but not assigned 
causality individually.  
All lab results shall be reported on ‘Blood Results CRF.’ Lab abnormalities will be 
summarized from the statistical database and graded for severity according the DAIDS 
Toxicity Table. All grades 1-5 will be summarized.  These laboratory abnormalities will 
be summarized separately from clinical AEs. 
9.8 
Study Medication Discontinuation 
This study medicine can be halted or interrupted if the investigator of record has concern of 
harm by the study drug. If the participant wishes to dose reduce due to intolerance of GI side 
effects (e.g. nausea), this is allowable.  This is not a protocol deviation, but tolerability is an 
exploratory endpoint.  The participant may discontinue the study medicine and remain in the 
study. Any dose reduction or discontinuation should be recorded on the ‘Med CRF’ for the study 
medicine. Before discontinuing the medication, the investigator should consider dividing the 
scheduled daily dose into smaller doses or dose reduction.  


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9.9 
Pre-existing Conditions 
A pre-existing condition is one that is present at study entry.  All persons in the trial have life-
threatening (Grade 4) cryptococcosis and HIV/AIDS. At study entry, any clinically significant 
abnormality should be recorded on the study CRF as a pre-existing condition and graded 1-4. 
Cryptococcosis has a variety of symptoms associated with this disease state, and these 
symptoms will be expected to wax and wane.  Each symptom of cryptococcal meningitis (and 
the disseminated cryptococcosis present thereof) is not an AE; however, new complications of 
cryptococcosis would be a new AE. Examples of complications would include: new seizures, 
new aspiration pneumonia, new obstructive hydrocephalus, etc. 
All pre-existing conditions must be clearly documented at study enrollment as new Grade 3-5 
AEs diagnosis are a study endpoint. As a diagnostic evaluation may be underway at time of 
enrollment (e.g. tuberculosis), the investigator should document if the condition was present 
prior to enrollment, even if the final diagnosis was made after enrollment. If a participant enters 
the study with a diagnosis and then the frequency, intensity, or the character of the condition 
worsens by Grade +1 level during the 2-week reporting period, the event should be defined as a 
new AE.  
At the end of 2 weeks, any new clinically significant findings/abnormalities that meet the 
definition of an AE in retrospective comparison to the pre-existing conditions must also be 
recorded and documented as an AE(s).  
9.10 Pregnancy Reporting 
If the patient or partner of a patient participating in the study becomes pregnant during the 18-
week study, the Investigator should report the pregnancy to the IRB within 24 hours of being 
notified. The IRB will then forward the exposure in-utero form to the investigator for completion. 
If a patient becomes pregnant while in the study, their medical care will be customized in 
discussion with the trial PIs.  Safety and monitoring procedures will continue to be performed. 
The major concern is the known teratogenic effect of high-dose fluconazole in the first trimester.  
The patient should be followed by the investigator until completion of the pregnancy. If the 
pregnancy ends for any reason before the anticipated date, the investigator should notify the 
IRB. At the completion of the pregnancy, the investigator will document the outcome of the 
pregnancy. If the outcome of the pregnancy meets the criteria for immediate classification as an 
SAE (i.e., postpartum complication, spontaneous abortion, stillbirth, neonatal death, or 
congenital anomaly), the investigator should follow the procedures for reporting an SAE.  
9.11 Expedited Reporting 
During the AE reporting period of 2 weeks to comply with national regulations, reports of all 
suspected unexpected serious adverse reactions (SUSARs) will be submitted to the IRB with 
oversight within 7 calendar days of the study site awareness of the AE. Relevant follow-up 
information will subsequently be communicated within 8 days. 
Reporting will be via the ‘Adverse Event Reporting CRF.’  Copies of each report and 
documentation of IRB notification and receipt will be kept in the Clinical Investigator’s binder. 


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All other suspected unexpected serious adverse reactions will be reported to the FDA, 
applicable competent authorities and Ethics Committees as required as soon as possible but 
within a maximum of 15 days of first knowledge by the Sponsor. Relevant follow-up information 
will subsequently be communicated within 15 days. 
The Sponsor will also inform all investigators as required. 
Listings of SAEs related to 5FC will be included in the Development Safety Update Report. 
At the time of the initial report, the following information will be provided: 
● Study identifier 
● Study Site 
● Subject number 
● Date of event  
● A description of the event 
● Medical treatments given/discontinued 
● Working diagnosis 
● Current vital status 
Within the 7 calendar days following the event, the investigator will provide further information in 
the form of a written narrative.  This will be documented along with any other diagnostic 
information that will assist the understanding of the event.  Significant new information on 
ongoing SAEs will be provided promptly to the study sponsor and local IRB of record.  
Any health authority safety reports will also be submitted to the Uganda National Drug Authority, 
both expedited and on an annual basis. 
 
10. CLINICAL MONITORING 
10.1 Site Monitoring Plan 
Site monitoring will be conducted to ensure that the human subject protection, study 
procedures, laboratory, study intervention administration, and data collection processes are of 
high quality and meet the sponsor, ICH E6 and regulatory guidelines.  The objectives of a 
monitoring visit are to:  
● Verify the existence of signed informed consent documents and documentation of 
the Informed Consent Form process for each monitored subject;  
● Verify the prompt and accurate recording of all monitored data points, and prompt 
reporting of all SAEs;  
● Compare data abstracts with individual participants’ records and source documents 
(laboratory analyses and test results, and any other relevant original subject 
information);  
● Help ensure investigators are in compliance with the protocol. 
10.2 Medical Monitor 
The medical monitor will review the overall clinical progress of the study and provide protocol 
guidance and clarifications as needed.  The medical monitor will review AE CRFs for the 
appropriateness of: working/final diagnoses, SAE designation, AE severity grade, 


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expectedness, and relatedness to study medication.  The medical monitor will provide feedback 
to the study investigators, requesting further documentation, as needed.  
10.3 Data and Safety Monitoring Board (DSMB) Safety 
Reviews 
Refer to the DSMB Charter for full details.  
● 
DSMB safety reviews will occur after 10 participants have been enrolled and have 
accrued approximately 2 weeks of follow-up data.  
The DSMB committee will consist of Dr. Peter Williamson as the chairperson, Dr. Agnes 
Kiragga, Dr. Felix Bongomin, Dr.Emilio Letang, and Dr. Kenneth Ssebambulidde.  The trial PIs 
and statistician will present to the DSMB in an open session before the DSMB enters into a 
closed, private session.  A quorum of three members will be required to meet, including the 
DSMB statistician. A local Ugandan community member will be invited to attend open DSMB 
meetings. The PhD study biostatistician will prepare summary progress reports on tolerability, 
safety, and efficacy within approximately 30 days of 10 participants being enrolled and accruing 
2 weeks of follow up data. At time of the safety reviews, all available data will be utilized, 
particularly with respect to control subjects.  
Data are unblinded, thus the PI, on-site investigators, and other team members will additionally 
monitor for any unexpected concern.  If concerns are raised, this will prompt an unscheduled 
DSMB safety evaluation.  
If there are unexpected safety concerns of serious and potentially related AEs, these AEs will be 
reviewed by the DSMB, and reported to the relevant regulatory authorities with oversight.  
Unexpected safety concerns may result in suspension of further trial interventions or closure to 
further enrollment.  The study PIs, DSMB chair, and sponsor each retain the authority to 
suspend additional enrollment and study interventions for the entire study; however, consensus 
agreement will be sought.  Examples of findings that might trigger a safety review are the 
number of SAEs overall, the number of occurrences of a particular type of SAE, severe 
AEs/reactions, or increased frequency of events. In particular, the investigators will be 
monitoring for culture positive Cryptococcal relapse. This has been rare in previous trials and a 
small number of cases would be cause for an early safety review. The biostatistician will 
perform a formal analysis as per the statistical plan.   
Reporting to the IRB of record will occur with annual reporting, and after each DSMB report. We 
will report all deaths, including cryptococcal related deaths to the IRB.  
 
11. STATISTICAL CONSIDERATIONS 
11.1 Statistical Analysis Plan 
Descriptive statistical methods will be used to summarize the data from this study, with 
hypothesis testing performed for the primary efficacy endpoint. Unless stated otherwise, the 
term “descriptive statistics” refers to number of subjects (N), mean, median, standard deviation, 


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standard error, minimum, and maximum for continuous data and frequencies and percentages 
for categorical data. All data collected during the study will be included in data listings. Unless 
otherwise noted, the data will be sorted first by treatment assignment, subject number, and then 
by date. Unless specified otherwise, all statistical testing will be two-sided and will be performed 
using a significance (alpha) level of 0.05. The statistical analyses will be conducted with the 
SAS® software package version 9.4 or higher or R Studio® 2022.07.1 or higher. All analyses will 
be subject to formal verification procedures. Analysis will be by intent-to-treat principle.  
11.2 Timing of Analyses 
The primary analysis of the primary and key secondary endpoints will be conducted once the 
last patient completes 2 weeks of the study or discontinues prematurely, and the resulting 
database is cleaned and locked. 
A follow-up analysis will be conducted including all data through Week 18 once the last patient 
completes 18 weeks on study or discontinues prematurely. 
11.3 Statistical Hypothesis 
We hypothesize that a regimen with lower-dose 5-FC will be well tolerated, have reduced 
toxicity compared to a regimen with standard dose 5-FC, will have a superior rate of CSF fungal 
clearance compared to historical fluconazole monotherapy, and will have a statistically non-
inferior rate of CSF fungal clearance compared to the historical standard-dose 5-FC. Full details 
of statistical considerations for making comparisons with historical controls will be provided in 
Statistical Analysis Plan. 
11.4 Primary Endpoint:  
The primary endpoint is CSF early fungicidal activity (EFA) during induction treatment: as 
measured in the change in log10 CFU/mL of CSF/day and as assessed with general linear 
models. EFA from the low-dose 5-FC group will be compared to historical controls of standard-
dose IV amphotericin + standard dose flucytosine and fluconazole. 
11.4.1 Sample Size & Statistical Power 
Similar to phase II TB trials, rate of CSF yeast clearance has been used in moderate size phase 
II cryptococcal trials to demonstrate microbiologic efficacy as proof of concept. When the EFA is 
better than 0.20 CFU/mL/day, there is reasonably good survival after cryptococcal meningitis 
(Figure 2.5). Once the EFA is better than approximately 0.30 log10 CFU/mL/day, there is less 
obvious incremental gains in survival beyond this threshold.61  
Overall, the EFA is a quantitative measure of microbiologic activity in humans of an antifungal 
regimen. 
For the EFA on a lower-dose 5-FC, we will have 36 participants with adequate data to calculate 
the EFA (i.e. non-sterile CSF culture at baseline and at least 1 follow up LP) and a standard 
deviation of approximately 0.20.   


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The CSF EFA will be interpreted in the context of other antifungal regimens published EFAs and 
the concurrent controls (Table 2.5). For example, if the lower-dose 5-FC arm has an EFA >0.30 
log10 CFU/mL/day and standard deviation of +0.20, then the low dose 5-FC regimen will 
establish superiority compared to fluconazole 1200 mg regimen and non-inferiority with the all 
oral 5FC + fluconazole regimen.  For a low-dose 5-FC arm EFA of 0.35, non-inferiority 
compared to the concurrent controls will be established with a margin of 0.17.  For an EFA 
better than 0.30 log10 CFU/mL/day, survival is relatively similar across antifungal regimens, as 
per section 2.5 and Figure 2.5. 
 
A subsequent statistical analysis of the ASTRO clinical trial has demonstrated that 30 to 40 
patients with EFA is sufficient to determine a significant difference in EFA. This was based on a 
bootstrap analysis of the participants of the ASTRO trial that is documented in table 11.4.1 
below.  
 
Table 11.4.1: EFA Calculated with the ASTRO Study (n=378) 
 
Replicate 
Size 
500 Bootstrap EFAs from 
randomly selected replicates  
N  
Mean (95%CI) 
10 
-0.454 (-0.768, -0.219) 
15 
-0.450 (-0.688, -0.257) 
20 
-0.449 (-0.664, -0.269) 
30 
-0.445 (-0.608, -0.292) 
40 
-0.445 (-0.593, -0.308) 
50 
-0.446 (-0.569, -0.316) 
378 Actual 
-0.444 (-0.489, -0.399) 
 
 
11.5 Secondary Endpoints 
Key Secondary Endpoint 
DOOR as ordinal ranked maximum score tested by Win Ratio. 
a. 18-week Survival with CSF sterility by 2-weeks 
b. 18-week survival with CSF culture positivity beyond 2 weeks 
c. Grade 3 hematological adverse event by 2 weeks 
d. Grade 4 hematological adverse event by 2 weeks 
e. Lost to follow up before 18-weeks 
f. 
Serious Adverse Event through 18 weeks (e.g. all-cause re-hospitalization, permanent 
neurologic deficit)  
g. Death by 18-weeks 


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Secondary Endpoints 
1) CSF culture sterility cumulative incidence over 18 weeks using Gray’s method. All 
persons who are non-sterile at baseline will be included with all CSF culture data 
considered.   
a) CSF culture sterility at 2 weeks, 6 weeks, 10, and 18 weeks will be summarized.  
All persons who are non-sterile at baseline will be included with all CSF culture 
data considered through the relevant study visit window. 
2) 18-week survival time 
 
11.6 Safety and Exploratory Endpoints 
1) Laboratory anomalies (Grade 3-5 AEs) as per the NIAID DAIDS toxicity grading table will 
be summarized for the incidence over 2 weeks of having at least one event and the 
mean number of events with cumulative incidence and linear regression models, 
respectively. If the cumulative incidence of a laboratory AE for any given anomaly (e.g. 
anemia, acute kidney injury) is >10%, the lab measurement will be individually 
summarized as a continuous variable. This is the key safety endpoint. 
a) Grade 1-2 AEs will also be collected and summarized but are not the protocol 
scientific endpoint. 
2) Clinical adverse events (Grade 3-5 AEs or SAEs) will be summarized via cumulative 
incidence function. 
a) Note: Clinical AEs are listed separately from laboratory anomalies as lab AEs 
occur in the majority of this critically ill population with advanced AIDS and 
receiving IV AMB. 
3) Tolerability will be assessed as the percent of received and tolerated doses versus total 
daily doses as a continuous variable for the experimental low-dose 5-FC and control 
standard dose 5-FC groups over the first two weeks. 
a) Dropouts and study withdrawals will be summarized. 
4) Descriptive analyses for the lower-dose 5-FC on pharmacokinetics of 5-FC 
concentrations in blood, CSF, and other bodily fluids/tissues; both antemortem and 
possibly postmortem. 
11.7 Statistical Power for Secondary Outcomes 
Secondary endpoints are primarily descriptive in nature without statistical power for individual 
dose comparison unless the effect size is large.  
 
12. ETHICS/PROTECTION OF HUMAN SUBJECTS 
12.1 Ethical Standard 
This study is to be conducted according to US and international standards of Good Clinical 
Practice (International Conference on Harmonization guidelines), Declaration of Helsinki, and 


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International Ethical Guidelines for Biomedical Research Involving Human Subjects, applicable 
government regulations for Uganda, U.S., international and Institutional research policies and 
procedures. All investigators must have received human subject protection and Good Clinical 
Practice training prior to human subject involvement.  
12.2 
Institutional Review Board 
Prior to the initiation of the study at the clinical research site, the protocol, all informed consent 
forms and the participant Information materials will be submitted to and approved by the local 
IRB of record.  Likewise, any future amendments to the study protocol will be approved by each 
site’s IRB.   
This protocol and any amendments will undergo review and approval by the local site IRB in 
Kampala, Uganda and the Uganda National Council of Science and Technology (UNCST) under 
FWA00001293, University of Minnesota (FWA00000312), and all other relevant local/national 
IRBs for any clinical site. 
12.3 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the research study and continuing throughout the individual’s study participation.  Extensive 
discussion of risks and possible benefits of this therapy will be provided to the subjects and their 
families.  Consent forms describing in detail the study interventions/products, study procedures, 
and risks are given to the subject and written documentation of informed consent is required 
prior to enrollment and starting the study product.  Consent forms will be IRB-approved, and the 
subject will be asked to read and review the document.  Upon reviewing the document, the 
investigator will explain the research study to the subject and answer any questions that may 
arise.  The subjects will sign the informed consent document prior to any procedures being done 
specifically for the study.  Procedures being performed for standard medical care (e.g., lumbar 
puncture, CSF diagnostic testing) may be performed prior to informed consent. Meningitis is a 
medical emergency, and routine medical care should not be withheld, endangering potential 
participants.  
The subjects should have the opportunity to discuss the study with their surrogates or think 
about it prior to agreeing to participate.  The subjects may withdraw consent at any time 
throughout the course of the trial.  A copy of the informed consent document will be given to the 
subjects for their records.  The rights and welfare of the subjects will be protected by 
emphasizing to them that the quality of their medical care will not be adversely affected if they 
decline to participate in this study. 
12.4 
Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participating subjects. 


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The study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study, or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  For research uses, all 
data will be de-identified and coded with the clinic medical record number. 
Protected health information will only be shared through a secure network and will not be used 
or sent through WhatsApp, Zoom, or email.  
Any authorized representatives of the sponsor may inspect all documents and records required 
to be maintained by the investigator, including but not limited to, medical records (office, clinic, 
or hospital) and pharmacy records for the subjects in this study. The clinical study site will 
permit access to such records. 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participating subjects. 
The study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study, or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  For research uses, all 
data will be de-identified and coded with the clinic medical record number. 
Any authorized representatives of the sponsor may inspect all documents and records required 
to be maintained by the investigator, including but not limited to, medical records (office, clinic, 
or hospital) and pharmacy records for the subjects in this study. The clinical study site will 
permit access to such records. 
12.5 
Study Discontinuation 
In the event that the study is discontinued, subjects remain eligible for ART according to 
national guidelines and as provided by PEPFAR, Global Fund, or Ministry of Health sources.  
Participants will be encouraged to continue to receive ART from the outpatient clinic or referred 
to a clinic of their choice for ongoing HIV care and management of CM.  
12.6 
Future Use of Stored Specimens 
According to Ugandan regulations, a separate informed consent for long term storage of 
samples for future research testing is necessary. The “SCREENING STORAGE CONSENT 
FORM FOR MENINGITIS” asks for consent for long term storage of samples. Subjects have the 
option to consent for long term storage of samples for this study only and/or for future research. 
If a subject declines storage consent, this will be noted on their lab order entry form and in their 
study chart, and specimens will be discarded after appropriate clinical testing is completed. The 
purpose of the long-term storage is to enable: 1) future diagnostic testing for assays which are 
not currently commercially available (e.g., multi-band semi-quantitative CrAg LFA; new TB 
tests); 2) pathogenesis studies to better understand immunology and microbiology of 
cryptococcal meningitis in relation to clinical outcome; 3) pharmacokinetic studies of antifungal 
medications; 4) antifungal susceptibility testing of Cryptococcus isolates. 


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There will not be a separate storage informed consent form for the FLOOR study. However, all 
FLOOR participants will be co-enrolled in the ongoing COAST observational study. This study 
includes a separate storage informed consent form. 
 
13. DATA HANDLING AND RECORD KEEPING 
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness 
of the data reported.  All source documents should be completed in a neat, legible manner to 
ensure accurate interpretation of data. Black ink is required to ensure clarity of reproduced 
copies.  When making changes or corrections, cross out the original entry with a single line, and 
initial and date the change. DO NOT ERASE, OVERWRITE, OR USE CORRECTION FLUID 
OR TAPE ON THE ORIGINAL. 
Copies of the CRF will be provided for use as source documents and maintained for recording 
data for each subject enrolled in the study.  Data reported in the CRF derived from source 
documents should be consistent with the source documents or the discrepancies should be 
explained. 
13.1 
Source Documents 
Source data are all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source 
data are contained in source documents.   Source data are all information, original records of 
clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial. Examples of these original documents and data 
records include, but are not limited to, research charts, laboratory notes, memoranda, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions certified 
after verification as being accurate and complete, media, x-rays, and subject files and records 
kept at the pharmacy, at the laboratories, and medico-technical departments involved in the 
clinical trial.  
The research team are the primary healthcare providers for participants after they enter into the 
trial and will manage all routine care for the participants. The research team will work together 
with the hospital team for complete hospital management and the hospital clinical team will be 
involved for emergent and urgent needs of the participant when the research team is not 
available or actively providing care on the wards. 
The Daily Clinical Review form will serve as the source for clinical observations obtained by the 
study team, as this is the first location where data are recorded. During outpatient visits, CRFs 
are completed first with information then transcribed into the electronic medical record. 
Study data forms will be digitally scanned for permanent record keeping and for enabling rapid 
resolution of any discrepancies. 


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The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects. 
Information Type 
Source Document 
Rationale / Record Location 
Lab Values 
Paper Lab Report 
In Research chart 
Microbiology Values 
Paper Lab Report 
In Research chart 
CSF CrAg Bedside Test 
Screening CRF 
CrAg testing is performed at bedside by trained study 
staff and the results are recorded on a CRF   
Pregnancy Test 
CRF 
Point-of-care pregnancy test is performed by study 
staff in the hospital, recorded on the CRF  
Medical History 
Screening CRF and Meningitis 
CRF 
Past medical history is obtained directly from the 
patient and recorded on the Screening and Meningitis 
CRFs. Hospital Records are not used. 
Physical Exam, or 
Clinical Observations 
Daily Clinical Review Form 
Clinical observations are recorded directly in the Daily 
Clinical Review Form. Hospital records are not used 
and are not the source document. 
Study Medicine 
Dispensing 
Pharmacy Dispensing Log 
Research Pharmacy in locked file cabinet 
Study Medication 
Administration 
Daily Clinical Review Form 
and Nurses Notes; Participant 
Adherence Diary 
Research staff record study medication administration 
on daily clinical review forms and nurses notes while 
participant is in hospital; Participants self-report study 
medication administration after hospital discharge in a 
patient adherence diary. 
Concomitant 
Medications 
Med CRF 
Research team prescribes all medications. Medications 
are recorded on Med CRF by the prescribing provider. 
Clinical Adverse Events AE Log 
Research staff record AEs on the AE Log. 
Participants contact research study team for all their 
health care needs. (Given 24/7 contact phone numbers 
and reimbursed for transport costs for scheduled or 
sick visits). All healthcare once enrolled is provided 
without cost (within reasonable relatedness to HIV, 
meningitis, or any AE). 
Lab Adverse Events 
Paper Lab Report 
In Research chart 
 


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13.2 
Data Management Responsibilities  
All source documents and laboratory reports must be reviewed by the clinical team and data 
entry staff, who will ensure that they are accurate and complete. Adverse events must be 
graded, assessed for severity and causality, and reviewed by the site PI or designee. 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site PI. During the study, the investigator must maintain complete and accurate 
documentation for the study. 
13.3 
Data Capture Methods 
Data entry will occur via computer database using DataFax system. 
13.4 
Types of Data 
Data for this study will include safety, laboratory (clinical, immunologic and microbiological), and 
outcome measures (e.g., survival, hospital duration, performance status). 
13.5 
Study Records Retention 
The investigator will retain study essential source documents for 15 years after the completion 
of the study or at least 2 years after market authorization or formal discontinuation of the 
product whichever is longest. Digital images of the source documents will be retained for an 
indefinite period.   
13.6 
Protocol Violations and Deviations 
Protocol Violation: Any change, divergence, or departure from the study procedures in an IRB-
approved research protocol that has a major impact on the subject’s rights, safety, or well-being 
and/or the completeness, accuracy or reliability of the study data. 
Protocol Deviation: Any change, divergence, or departure from the IRB approved study 
procedures in a research protocol that does not have a major impact on the subject's rights, 
safety or well-being, or the completeness, accuracy and reliability of the study data.  The 
noncompliance may be either on the part of the subject, the investigator, or the study site staff. 
As a result of deviations, corrective actions will be developed and implemented promptly.  
These practices are consistent with ICH E6: 
4.5   Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1   Quality Assurance and Quality Control, section 5.1.1 
5.20 Noncompliance, sections 5.20.1, and 5.20.2. 
It is the responsibility of the site to use continuous vigilance to identify and report protocol 
deviations within 5 working days of identification, or within 5 working days of the scheduled 
protocol-required activity.   


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13.7 
Quality Control and Quality Assurance 
A QC/QA Quality Management plan will be implemented centrally via the DataFax system which 
incorporates quality control for complete record keeping. 
This study may be subject to a quality assurance audit by Matinas or its designee, as well as 
inspection by appropriate regulatory authorities.  
 
14. ADMINISTRATIVE 
14.1 
Publication of Research Findings 
The Principal Investigators are expected to publish the results of this clinical trial in a scientific 
journal.  Publication of the results of this trial will be governed by the co-PIs: Drs Meya and 
Boulware in accordance with standard academic practices with the freedom to publish without 
restriction. The investigators agree to submit all manuscripts or abstracts to the Sponsor before 
submission. This allows the Sponsor to protect proprietary information and to provide optional 
comments.  
The clinical study plan and the results of the study will be published on www.ClinicalTrials.gov in 
accordance with 21 CFR 50.25(c).  
All investigators funded by the NIH must submit or have submitted for them to the National 
Library of Medicine’s PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/) an electronic version 
of their final, peer-reviewed manuscripts upon acceptance for publication.  The NIH Public 
Access Policy ensures the public has access to the published results of NIH funded research. 
 
 
 
 
 
 
 
 
 
 
 


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Outcomes. Clin Infect Dis. 2014;58(5):736-745. doi:10.1093/cid/cit794 
41. Pullen MF, Hullsiek KH, Rhein J, et al. Cerebrospinal Fluid Early Fungicidal Activity as a 


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Publ Infect Dis Soc Am. 2020;71(7):e45-e49. doi:10.1093/cid/ciaa016 
42. 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(1):76-81. 
doi:10.1016/j.jinf.2011.10.014 
43. Jarvis JN, Lawrence DS, Meya DB, et al. Single-Dose Liposomal Amphotericin B Treatment 
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44. Loyse A, Wilson D, Meintjes G, et al. Comparison of the early fungicidal activity of high-dose 
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45. Jarvis JN, Meintjes G, Rebe K, et al. Adjunctive interferon-γ immunotherapy for the 
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46. Jackson AT, Nussbaum JC, Phulusa J, et al. A phase II randomized controlled trial adding oral 
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47. Nussbaum JC, Jackson A, Namarika D, et al. Combination Flucytosine and High‐Dose 
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66.  Jezewski AJ, Ristow LC, Krysan DJ. 2023. Carbon Dioxide Potentiates Flucytosine 
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67.  Schwarz P, Janbon G, Dromer F, Lortholary O, Dannaoui E. 2007. Combination of 
Amphotericin B with Flucytosine Is Active In Vitro against Flucytosine-Resistant Isolates of 
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68.  Yamamoto Y, Maesaki S, Kakeya H, Yanagihara K, Ohno H, Ogawa K, Hirakata Y, Tomono K, 
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Antimicrob Agents Chemother 47:3361–3364. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Appendix A: Adverse Events observed 
with IV amphotericin + fluconazole 
therapy 
Cryptococcal Optimal ART Timing (COAT) Trial in Uganda used amphotericin and 
fluconazole with a randomized timing of ART initiation. Data below are as published in the 
manuscript’s supplemental appendix.18  In total, 436 Grade 3-5 adverse events occurred 
in 177 subjects with cryptococcal meningitis. The majority were asymptomatic lab 
abnormalities, related to AIDS, cryptococcosis, or amphotericin B deoxycholate therapy.  
 
 
 


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Figure S3a displays the cumulative incidence of time to a first Grade 3-5 adverse event 
by trial arm as defined by the NIAID DAIDS Toxicity Grading Scale, 2009.  The AE 
distribution by body system was: 
Distribution by Body System 
Earlier ART 
Deferred ART 
Hematology 
99 (47.8%) 
128 (55.9%) 
Chemistries 
65 (31.4%) 
53 (23.1%) 
Infection 
13 (6.3%) 
26 (11.4%) 
Neurological 
11 (5.3%) 
5 (2.2%) 
Gastrointestinal 
6 (2.9%) 
8 (3.5%) 
Cardiovascular 
6 (2.9%) 
5 (2.2%) 
Systemic 
4 (1.9%) 
2 (0.9%) 
Skin, dermatological 
2 (1.0%) 
1 (0.4%) 
Respiratory 
1 (0.5%) 
1 (0.4%) 
Overall (Number of events) 
207 (100%) 
229 (100%) 
The most commons adverse events included anemia (n=88), neutropenia (n=54), 
leukopenia (n=24), hyponatremia (n=21), hypokalemia (n=23), and elevated 
creatinine (n=18). 
Grade 5 events occurred in 14 (15.9%) persons randomized to earlier ART and 
11 (12.3%) randomized to deferred ART. Grade 5 events did not include deaths 
from Cryptococcus. 
 
The line listing of AEs was: 
COAT Trial: Adverse Event Line Listing 
Grade 3-5 Adverse Events  
Grade 4 and 5 Adverse Events 
Body System 
MedDRA Term 
Earlier ART Deferred 
ART 
 
Earlier ART 
Deferred ART 
Cardiovascular 
Cardiac arrhythmias 
1 
0 
 
1 
0 
DVT 
4 
1 
 
1 
0 
Pulmonary embolus 
0 
1 
 
0 
1 
Tachycardia 
0 
1 
 
0 
0 
 
Chemistries 
Alanine aminotransferase (ALT) 
increased 
2 
1 
 
0 
0 
Aspartate aminotransferase (AST) 
increased 
5 
4 
 
2 
1 
Blood alkaline phosphatase increased 
2 
0 
 
1 
0 
Blood bicarbonate decreased 
6 
5 
 
1 
2 
Blood bilirubin increased 
1 
0 
 
0 
0 
Blood sodium decreased 
14 
7 
 
8 
3 


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Blood sodium increased 
0 
2 
 
0 
1 
Hyperkalemia 
2 
0 
 
2 
0 
Potassium serum decreased 
15 
8 
 
4 
1 
Serum creatinine increased 
8 
10 
 
2 
2 
 
Gastrointestinal 
Decreased appetite 
1 
1 
 
0 
0 
Diarrhea 
3 
2 
 
1 
1 
Nausea and vomiting 
2 
2 
 
1 
1 
 
Hematology 
Anemia 
41 
47 
 
23 
31 
Leukopenia 
11 
13 
 
6 
3 
Neutropenia 
23 
31 
 
12 
12 
Thrombocytopenia 
4 
13 
 
1 
3 
 
Infection 
Abdominal pain localized 
0 
1 
 
0 
0 
Acinetobacter bacteremia 
0 
1 
 
0 
1 
Acute pneumonia 
1 
0 
 
0 
0 
Bronchopneumonia 
1 
0 
 
0 
0 
Burkholderia cepacia complex 
0 
1 
 
0 
0 
Cystitis, Klebsiella 
1 
0 
 
1 
0 
Disseminated tuberculosis 
0 
2 
 
0 
2 
Klebsiella sepsis 
1 
1 
 
1 
1 
Pulmonary tuberculosis 
0 
1 
 
0 
0 
Salmonella bacteremia 
0 
1 
 
0 
0 
Salmonella sepsis 
0 
1 
 
0 
0 
Sepsis 
1 
4 
 
1 
3 
Septicemia due to pseudomonas 
1 
1 
 
1 
0 
Staphylococcus aureus septicemia 
1 
0 
 
1 
0 
Tuberculosis 
0 
1 
 
0 
1 
 
Neurological 
Acute mental status changes 
5 
3 
 
4 
0 
Intracranial pressure increased 
3 
0 
 
0 
0 
Muscle weakness right-sided 
2 
0 
 
2 
0 
Sensory neuropathy 
1 
0 
 
0 
0 
 


FLOOR Trial Protocol 
22 March 2024 
________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ 
69 
 
 
Respiratory 
Dyspnea 
1 
1 
1 
 
Dermatological 
Erythroderma 
1 
0 
0 
  
Rash - morbilliform 
0 
1 
0 
Systemic 
HIV wasting syndrome 
0 
1 
0 
 
 
 
 
 
 
 
 
Pyrexia 
1 
0 
0 
 
 
 

ICF_001.pdf: 
Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 1 of 6 
 
Study Title: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal 
Meningitis. 
The lead investigators of this research study are Dr. David Meya of the Infectious Diseases 
Institute (IDI) of Makerere University; Dr. Thomas McHale and Dr. David Boulware of the 
University of Minnesota USA.   
Study Purpose 
Cryptococcal meningitis or “Crypto” is a life-threatening fungal infection around the brain. 
Fortunately, this infection is very treatable with the correct medicine. However, flucytosine, one of 
the medicines currently used to treat cryptococcal meningitis, has serious side effects that can 
affect your kidneys and blood. In Uganda and other countries in Africa, this drug is not widely 
available and supplies are often low, making it difficult to consistently provide good treatment for 
crypto. For this reason, researchers have been searching for ways to improve access to 
flucytosine for patients and reduce side effects.  
Researchers have done studies to determine the best dose for flucytosine. We are able to 
measure the levels of flucytosine that reach the brain. Based on these levels, we believe that 
lower doses of flucytosine may still be effective in treating crypto. We will also be giving you 2 
additional medications to treat crypto meningitis that will be at standard recommended doses and 
have been shown to be effective even without flucytosine. Lower doses will allow more people to 
have access to the medicine and also may have fewer side effects.  
The purpose of this study is to know whether this lower dose of flucytosine is a safe and effective 
treatment for people with cryptococcal meningitis. We hope to enroll around 50 people in this 
study in Uganda. In order to show that this medicine treats crypto at the dose under study, we will 
measure how quickly the crypto is cleared from your around brain. Researchers have used this 
measurement for decades to study how well medicines work for crypto.  
Your participation in this study is voluntary. If you decide to participate in the study, you will 
receive a dose of 60 mg/kg of the medicine flucytosine, which is an experimental dose that is 
lower than the dose of 100 mg/kg that is currently recommended by international guidelines. If you 
decide not to participate in the study, care will still be provided according to the best standards of 
care available in the hospital.  You confirm that the following has been explained to you, and you 
have had a chance to ask questions: 
1. If you decide to participate in the study, you will receive the lower dose of the medicine 
flucytosine  
2. It is unknown for sure whether lower doses of flucytosine will be effective in treating 
cryptococcal meningitis.  
3. It is unknown for sure whether a lower dose of flucytosine will be safer than standard 
dosing.   
4. You will also receive a second standard medicine for your infection, called IV amphotericin 
for either 1 dose (if liposomal) or 7 days (if deoxycholate). This is a standard medicine and 
is not experimental. You will receive a third standard medicine for your infection, called 
fluconazole. This is a standard medicine and is not experimental.  
5. The IV amphotericin is a strong medicine with many side effects including fever, rigors and 


Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 2 of 6 
 
chills. We will need to do blood tests to monitor this medicine’s side effects.  IV 
amphotericin is a standard, recommended medicine and is not experimental. 
6. You will be required to take all of your medications faithfully.  
7. The doctors will take fluid surrounding your spinal cord through a needle in your back, 
which is called a lumbar puncture. You will be required to receive at least 3 lumbar 
punctures, which we strongly recommend regardless of whether you decide to enrol in this 
study. These are important to check the pressure around the brain and to make sure the 
infection goes away.  This is part of the best standard therapy. 
8. After leaving the hospital you will be required to return for clinic visits every 2 to 4 weeks 
for 18 weeks. 18 weeks is the duration of the study. If you are unable to attend clinic 
because you are sick, let us know and we will help you get to clinic or come see you at 
home. If you miss your scheduled visit, a nurse will continue to call until we have reached 
you and a new visit can be scheduled. After 18 weeks, clinic will return to normal frequency 
as decided by you and your doctor. 
9. At the end of 18 weeks, you will have the option to receive ongoing care through our clinic, 
continue receiving care through your own clinic, or establish care at a new clinic near your 
home.  Study staff will assist in ensuring ongoing care at the end of the study period. 
10. You may be contacted by phone or text message as a reminder of clinic appointments, to 
check how you are feeling, or how you are taking your medicines. 
11. You will benefit from this study because you will receive more intensive medical care 
through a group of doctors and nurses that are experienced in treating meningitis. The test 
results will be used by doctors to make decisions for your health.  
12. If you agree, some samples will be collected and stored for future research tests that are 
separate from this study. This may benefit future Ugandans and others around the world, 
but you will not benefit directly.  
13. You can choose not to have your samples stored and still participate in this study. Tests 
will continue to be performed to assist doctors in taking care of you, but will not be stored. 
14. Study doctors or a designated representative of the sponsor will review your medical 
records and tests from this hospital stay.  
15. You will not be paid for participating in this study, but you will be given money to refund 
transport expenses up to 30,000 Uganda Shillings for each study related visit. You may 
also be compensated up to 50,000 Uganda Shillings if extra blood is drawn. 
16. Any information that is collected about you shall be kept private, and your data will be used 
without your name, picture, or identity. 
17. Agreeing to participate in this study is voluntary and you can withdraw from the study at 
any time if you so wish by telling the study doctors. If you withdraw from the study, you can 
continue to receive your crypto and HIV treatment at the hospital or clinic, at no cost. 
18. You will not be giving up any of your legal rights by signing this consent form. 
19. You are going to be given a copy of this form. 
It is recommended by the national guidelines and your doctors that you continue to take 


Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 3 of 6 
 
medicines for cryptococcal meningitis and to receive lumbar punctures while in hospital.  
Risks 
The study has the following risks:   
Until this study is completed, there is no way of knowing for sure how effective lower dose 
flucytosine is for treating cryptococcal meningitis. For this reason, there is a risk that you could 
receive a medicine that is less effective than the standard dose of flucytosine. Lower doses of 
flucytosine have not been used in a lot of people. Side effects can occur, though we think they are 
less than those with standard doses of flucytosine. Common side effects are expected to include 
nausea, diarrhea, or vomiting. Most of the side effects are expected to be mild and go away with 
stopping the medicine. Severe unforeseen adverse reactions could occur but are expected to be 
less likely with this lower dose 
A lumbar puncture (LP) is when a needle is inserted into the back to collect spinal fluid from a 
person with meningitis.  It is the method doctors use to collect spinal fluid for laboratory analysis or 
to relieve headache that results from increased pressure around the brain.  An LP is a part of the 
standard medical treatment for persons with cryptococcal meningitis, and is part of the therapy for 
this infection. Risks of lumbar puncture include possible bleeding around the puncture site, and 
rarely infection or fluid leak. Having an LP has the same risks for you whether you have the LP in 
this study or as part of standard clinical care. Repeated LPs are strongly recommended whether 
you choose to participate in the study or not. 
A lumbar puncture is required for diagnosis, and it is recommended that you receive a total of at 
least 2 more LPs to relieve pressure around your brain, to help decrease the infection, and to 
ensure that the infection is gone at the end of therapy. These LPs will occur approximately every 2 
days for the first week and at the completion of flucytosine therapy. If the pressure around your 
brain is high enough to be causing a headache, you will be recommended to have additional LPs 
to lower the pressure, which also makes your headaches better.  This is the standard of care for 
cryptococcal meningitis and will be recommended regardless of whether or not you enrol in this 
study. We recognize that these LPs can be unpleasant, but the LPs are very important for your 
medical care to give you the best chance of surviving this serious infection. 
This study requires that a small amount of blood be taken through a needle from a vein in your 
arm at several times during the study. Your doctors will do some tests on the blood to make sure 
your liver and kidney are working well, and to know if any severe problems happen. If there are 
problems found, your medicine may be decreased or stopped. This is about 30mL (6 teaspoons) 
more blood than would have been taken over the next 3 months than if you do not participate.  
This small amount of extra blood taken should have no effect on you, since your body will have 
time to make new blood. It is possible that there may be a small amount of bleeding or bruising 
around the needle site. Infection at the needle site is also a very small possibility.  
Pregnancy: 
Pregnant and breastfeeding women cannot enrol into the study.  Pregnancy should be avoided 
when taking the recommended drugs for cryptococcal meningitis due to risks to the unborn child 
(fetus). This risk is not unique to the study. If you are sexually active, we recommend that you use 
at least two methods to prevent getting pregnant.  If you later want to become pregnant or think 


Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 4 of 6 
 
you may be pregnant, please notify the study doctor right away, so that some of your medicines 
may be changed. 
Research Related Injury 
If you experience problems related to crypto infection of the brain, HIV, or a new infection, 
treatment will be available, and you will not have to pay for it.  If you experience other health 
problems, a referral to specialized care will be made, but this study cannot pay for health care, 
which is unrelated to the crypto infection. If you suffer bodily injury as a consequence of 
flucytosine therapy, there is insurance to compensate you through Lion Assurance Company, Ltd. 
P.O. Box 7658, Kampala. 
Sample Storage 
If you agree to participate in this study, we will collect some samples of your blood, urine, stool 
and spinal fluid for your medical care during the study.  We would like to store these samples 
temporarily. These samples will be identified by a unique number, not your name or other personal 
information, so that others will not be able to identify you.  Testing will be for research related to 
meningitis, the immune system, and to measure how much of the medicine gets into your blood.  
Some of the tests will be done locally in Uganda, and some will be done in the United States.   
Extra Sample Collection 
With your permission, we would also like to collect a small volume of extra blood (approximately 4 
teaspoons or 20 mL) along with your other routine blood draws for research testing. This may 
require an additional blood collection that would not normally occur. This may be used to test the 
levels of flucytosine in your blood or may be used for research that studies the types of cells in 
your blood that fight infection. You will be compensated 50,000 Uganda shillings if extra blood is 
drawn. If you prefer not to have any extra blood draws, you may still participate in the study. Many 
research tests will be performed later and will not change your treatment, and thus you will not 
receive the test results.  
Sample ownership and duration of storage 
If you are willing to donate samples for future research, it means you are giving them to us. The 
samples will be under the responsibility of the researchers and may be shared with other 
researchers to assist with testing. You will not personally receive direct benefit from this future 
testing, but your contribution may help others in the future. They will not be sold. There is no limit 
on how long your samples will be stored. However, you can change your mind at any time without 
penalty and tell us to destroy the samples. If you prefer that we do not keep your samples after the 
study is completed, you may still participate in the study. 
Confidentiality 
Your study information will be kept private, but will be included in your medical records if relevant. 
Your name, picture of your face, or identity will not be shared or disclosed in any publications or 
presentations. Your records for the study may be reviewed by representatives of the University, 
and other government regulatory authorities to assure the accuracy and quality of the records and 
the correct conduct of the research study.   
 
Study approval  


Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 5 of 6 
 
Approval to conduct this research study has been granted by the Uganda National Council for 
Science and Technology (UNCST),Infectious Disease Institute Research and Ethics Committee, 
and University of Minnesota. The research study is funded by the National Institute of Health in the 
United States. 
Contacts and Questions 
You may ask any questions you have now. If you have questions later or any urgent health 
concerns, you are encouraged to contact:  
● Dr. David Meya in Kampala on phone number XXXXXXXXX 
● Dr. Conrad Muzoora in Mbarara on phone number XXXXXXXXXXX 
 
You will be given a clinic appointment card with the doctors’ phone numbers before you leave 
hospital. 
In case of any questions regarding: 
● Your welfare and rights as a research participant,  
● Any questions or complaints not being answered by your study doctors 
● You want to talk to someone besides the research team. 
you should contact: 
● Dr. David Patrick Kateete, the Chairman of the Infectious Disease Institute Research 
Ethics Committee (IDIREC) on telephone 0704879922 or 0393193144 
● Uganda National Council for Science and Technology (UNCST), Plot 3 Kimera Road; 
Ntinda, Kampala on telephone 0414-705-513. 
If you have any questions about this research study, you can ask them now or contact the above 
doctors later. The study doctors will see you while in hospital. 
 
For More Information 
A description of this clinical trial will be available on www.ClinicalTrials.gov, as required by United 
States Law. This Web site will not include information that can identify you. At most, the Web site 
will include a summary of the results. You can search this Web site at any time.


Informed Consent: Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis (FLOOR) 
English. Version 1.2 February 2024 
Page 6 of 6 
 
Sample Storage 
I consent to the collection, testing and storage of my samples (blood, spinal fluid, stool and 
urine) for the purposes of this research.  Samples will be stored indefinitely, unless I 
change my mind and ask for my samples to be destroyed.  I also consent to have extra 
blood draws specifically for study purposes. 
I consent to the collection, testing and storage of my samples (blood, spinal fluid, stool and 
urine) for the purposes of this research only.  This does not require additional blood draws, 
and all samples will be destroyed when the study is over. 
 
 
Informed consent 
I hereby consent to participate in this research study called: 
 “Optimizing the Dose of Flucytosine for the Treatment of Cryptococcal Meningitis.”  
 
 
 
 
 
Name of Impartial witness 
(If participant thumb print used) 
 
 
 
 
 
Signature 
 
Date 
Name of Participant 
 
Signature / thumbprint 
(with witness) 
 
 
Date: 
 
 
Name of Surrogate 
 
 
Signature / thumbprint 
of surrogate 
 
 
Date: 
Name of person obtaining consent 
 
 
 
Signature 
 
Date 
The Participant signed with a thumb print. As a witness, I confirmed that all the information 
about the study as given and the participant consented to taking part.  
 
 
 
 
 
 
 
Consent Reviewer (PI or Designee) 
 
Signature 
 
Date