Clinical trial NCT03002012
Conditions: Cryptococcosis Cryptococcal Infections AIDS-Related Opportunistic Infections
Keywords: cryptococcal antigen preemptive treatment CRAG+ sertraline fluconazole randomized clinical trial HIV
Sponsor: University of Minnesota
Start date: 2017-11-15; Completion date: 2018-03-13
Enrollment: 22 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Cryptococcal meningitis or "Crypto" is a life threatening fungal infection around the brain that requires hospitalization for treatment for 14 days and then continued therapy. Crypto causes 15-20% of HIV/AIDS-related deaths worldwide. However, this infection can be detected before one develops symptoms and becomes ill. People can be screened for infection by a blood test to detect "cryptococcal antigen," (called CrAg), which is part of the fungus, in blood. The World Health Organization and over 22 countries worldwide recommend CrAg screening of all persons with advanced AIDS entering or re-entering into HIV care.

However, it is not known how best to treat people with cryptococcal antigen in their blood, who don't otherwise yet have symptoms of infection around their brain. If no treatment is given, almost all people will develop infection of the brain and/or die. International guidelines suggest using both HIV medicines and an anti-fungal medicine, called fluconazole, to treat this early infection. However, despite this treatment approximately 1 in 4 people may get sick and/or die.

Researchers have recently discovered another medicine that may work against the Cryptococcus fungus. This medicine is called Sertraline, and it is actually a medicine that has been used for more than 25 years to treat depression (sadness). Sertraline is one of the most commonly used medicines worldwide.

The purpose of this research clinical trial is to determine if standard fluconazole antifungal therapy plus a high dose of Sertraline, will be better than standard fluconazole therapy alone for treating early disseminated cryptococcal infection in persons who are asymptomatic and do not yet have infection of the brain (i.e. meningitis).

This study seeks to test if Sertraline will improve survival through 6-months. Prior studies have shown that \>90% of those who survive 6-months will survive \>5 years.

Detailed description:
This is a double-blind, randomized placebo-controlled clinical trial testing sertraline as an antifungal medicine in combination with fluconazole for treatment of HIV-infected persons with AIDS and asymptomatic cryptococcal antigenemia (CrAg+).

Arms: arm 1: Fluconazole Standard of Care + Sertraline arm 2: Fluconazole Standard of Care + Placebo Oral Tablet
Interventions: intervention 1: Sertraline intervention 2: Placebo Oral Tablet intervention 3: Fluconazole
Intervention descriptions: intervention 1: sertraline 400mg/day intervention 2: matched placebo tablet intervention 3: Standard of Care Fluconazole per World Health Organization and Ugandan Guidelines (800mg/day x 2 weeks, 400mg/day x10 weeks, 200mg through 6 months).

Protocol documents:
Prot_000.pdf: 
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1. TITLE PAGE 
 
Short title: Cryptococcal Antigen Screening plus Sertraline 
(C-ASSERT)
Long Title: Randomized Clinical Trial evaluating sertraline plus fluconazole versus 
fluconazole alone for the preemptive treatment of asymptomatic cryptococcal 
antigenemia in HIV-infected persons living with AIDS 
 
 
 
Clinical Trial Phase: II / III 
 
Local Protocol Number(s): 
UNCST Number: HS 2204 
National Drug Authority: 584/NDA/DID/08/17 
NIH NIAID Funding Mechanism: U01AI125003 
Principal Investigator (PI): David Meya / David Boulware 
IND Sponsor: University of Minnesota 
FDA IND Number: 133772 
Protocol Chair: Radha Rajasingham 
NIAID DAIDS Medical Officer: Melanie Bacon  
 
DAIDS-ES ID: 33238  
ClinicalTrials.gov Identifier: NCT03002012 
Version Number: 2.0 
Version Date: 10th October 2017 


C-ASSERT Protocol  
 
 
 
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SIGNATURE PAGE 
I agree to conduct the study in accordance with the relevant, current protocol and 
will not deviate from the protocol without permission of DAIDS, except when 
necessary to protect the safety, rights, or welfare of study participants. 
I agree to personally conduct or supervise this study. 
I will ensure that the requirements relating to obtaining informed consent and 
Ethics Committee (EC) or Institutional Review Board (IRB) review and approval (45 
CFR 46, ICH/GCP, etc.) are met. 
I agree to report to the sponsor adverse experiences as per the protocol that occur 
during the course of this study. 
I agree to maintain adequate and accurate study records and to make those 
records available for inspection by DAIDS, DAIDS’ authorized representatives, and/or 
other applicable regulatory entities. 
I will ensure that an EC or IRB that complies with the requirements of 45 CFR Part 
46 will complete initial and continuing review and approval of the study. I also agree 
to promptly report to the EC/IRB: 1) all changes to the study; 2) safety updates DSMB 
summaries and other information required by the funder, sponsor and EC/IRB in 
compliance; 3)  all unanticipated problems involving risks to human subjects or others. 
Additionally, I will not make any changes to the study without DAIDS and EC/IRB 
approval, except where necessary to eliminate apparent immediate hazards to study 
participants. All protocol changes should be reviewed and approved by the PI, DAIDs, 
and regulatory authorities. 
I agree to ensure that all staff members involved in the conduct of this study are 
informed about their obligations in meeting the above commitments. 
 
 
Principal Investigator:  ______________________________ 
 
 
 
Signed: _____________________________Date: ___________________ 
 
 
Name/Title 


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2. TABLE OF CONTENTS 
 
1. 
TITLE PAGE 
1 
SIGNATURE PAGE 
2 
2. 
TABLE OF CONTENTS 
4 
2.1. 
KEY ROLES 
7 
2.2. 
LIST OF ABBREVIATIONS 
8 
3. 
PROTOCOL SUMMARY 
9 
3.1. 
PARTICIPATING SITES 
12 
4. 
INTRODUCTION 
14 
4.1. 
BACKGROUND ON CRYPTOCOCCAL ANTIGEN SCREENING 
14 
4.2. 
PROBLEM 
16 
4.2.1.  STANDARD OF CARE CRAG SCREENING IN UGANDA 
17 
4.2.2.  EVIDENCE GAPS IN MANAGEMENT OF ASYMPTOMATIC CRAG+ 
17 
4.3. 
STUDY HYPOTHESIS 
19 
4.4. 
RATIONALE: ANTIFUNGAL EFFECT OF SERTRALINE 
19 
4.5. 
VALIDATION: ANTIFUNGAL EFFECT OF SERTRALINE 
21 
4.6 
EXPERIENCE IN HIGHER DOSE SERTRALINE 
23 
5. 
OBJECTIVES 
25 
5.1. 
PRIMARY OBJECTIVE(S) 
25 
5.2. 
SECONDARY OBJECTIVE(S) 
25 
6. 
STUDY DESIGN 
26 
7. 
STUDY POPULATION 
28 
7.1. 
PARTICIPANT ELIGIBILITY CRITERIA 
28 
7.1.1.  INCLUSION CRITERIA 
28 
7.1.2.  EXCLUSION CRITERIA 
28 
7.2. 
RECRUITMENT PROCESS 
30 
8. 
INTERVENTIONS (STUDY PRODUCT) 
32 
8.1. 
BIOMEDICAL INTERVENTIONS 
32 
8.1.1.  REGIMEN (DOSE, SCHEDULE, ROUTE, ADMINISTRATION) 
35 
8.1.2.  RATIONALE FOR SERTRALINE DOSE SELECTION 
35 
8.1.3.  STUDY PRODUCT FORMULATION AND PREPARATION 
38 
8.1.4.  STUDY PRODUCT SUPPLY AND ACCOUNTABILITY 
38 
8.1.5.  SERTRALINE SIDE EFFECT PROFILE 
39 
8.1.6.  SERTRALINE PRECAUTIONS 
40 
8.1.7.  ELECTROCARDIOGRAM QT INTERVAL SAFETY – PUBLISHED DATA 
41 
8.1.8.  SERTRALINE SAFETY IN OVERDOSE 
43 
8.2. 
FLUCONAZOLE 
45 
8.3. 
ASSESSMENT OF PARTICIPANT ADHERENCE 
46 
8.4. 
PROHIBITED MEDICATIONS 
46 
8.5. 
CONCOMITANT MEDICATIONS AND PROCEDURES 
46 
8.5.1.  RESCUE MEDICATIONS 
48 


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8.6. 
PHARMACOKINETIC INTERACTIONS BETWEEN SERTRALINE, ANTIRETROVIRALS, AND FLUCONAZOLE. 
49 
9. 
STUDY PROCEDURES / EVALUATIONS 
50 
9.1. 
SCHEDULE OF EVENTS 
50 
9.2. 
CRAG SCREENING 
50 
9.3. 
PRE‐ENTRY VISIT 
51 
9.4. 
ENROLLMENT VISIT (WEEK 0) 
52 
9.5. 
FOLLOW‐UP DETAILED STUDY VISITS 
52 
9.5.1.  WEEK 2 VISIT: ART INITIATION 
53 
9.5.2.  WEEK 4 AND 8 VISITS 
53 
9.5.3.  WEEK 12 VISIT 
54 
9.5.4.  WEEK 16 VISIT 
54 
9.5.5.  WEEK 20 VISIT 
55 
9.5.6.  OTHER ROUTINE / UNSCHEDULED CLINIC VISITS 
55 
9.5.7.  PREGNANCY VISIT 
55 
9.5.8.  OTHER SICK VISITS 
55 
9.5.9.  STUDY TERMINATION VISIT 
56 
9.6. 
LABORATORY EVALUATIONS 
56 
9.6.1.  CRYPTOCOCCAL ANTIGEN LATERAL FLOW ASSAY (CRAG LFA) 
56 
9.6.2.  ROUTINE LABORATORY MONITORING 
57 
9.6.3.  ADDITIONAL LABORATORY MONITORING 
57 
9.6.4.  SPECIMEN PREPARATION, HANDLING AND SHIPPING 
57 
9.7. 
EKG MONITORING 
58 
10. 
ASSESSMENT OF SAFETY 
59 
10.1.  SAFETY ASSESSMENT OVERVIEW 
59 
10.2.  GRADING ADVERSE EVENT SEVERITY 
60 
10.3.  RECORDING OF SERIOUS AES 
60 
10.3.1.  LABORATORY AES 
61 
10.4.  ADVERSE EVENT PROCEDURES AND REPORTING REQUIREMENTS 
61 
10.5.  EXPEDITED AE REPORTING REQUIREMENTS FOR THE STUDY TO NIH 
61 
10.6.  FDA IND SAFETY REPORTING 
62 
11. 
CLINICAL MANAGEMENT 
64 
11.1.  CLINICAL MANAGEMENT OF ADVERSE EVENTS 
64 
11.2.  SEROTONIN SYNDROME 
64 
11.3.  HEPATIC DYSFUNCTION 
65 
11.4.  EKG QTC ABNORMALITIES 
66 
11.5.  PREGNANCY 
66 
11.6.  FAILURE OF PREEMPTIVE TREATMENT 
68 
11.7.  CRITERIA FOR DISCONTINUATION OF TRIAL PARTICIPATION 
68 
11.7.1.  CRITERIA FOR PREMATURE STUDY MEDICINE DISCONTINUATION 
68 
11.7.2.  CRITERIA FOR STUDY MEDICINE DOSE REDUCTION/RESUMPTION 
68 
12. 
STATISTICAL CONSIDERATIONS 
70 
12.1.  STUDY ENDPOINTS 
70 
12.2.  ANALYSIS PLAN 
70 
12.3.  ANALYSIS PLAN SECONDARY AND OTHER ENDPOINTS 
70 
12.4.  SAMPLE SIZE CONSIDERATIONS 
73 
12.5.  RANDOMIZATION / STRATIFICATION 
73 
12.6.  BLINDING PROCEDURES/ UNBLINDING PROCEDURES 
73 


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12.7.  DATA AND SAFETY MONITORING 
74 
12.7.1.  PLANNED INTERIM ANALYSES AND STOPPING GUIDELINES 
75 
12.7.2.  INTERIM MONITORING AND SAFETY REVIEW 
76 
12.7.3.  QUALITY CONTROL AND QUALITY ASSURANCE 
76 
12.7.4.  TRIAL SAFETY COMMITTEE 
77 
12.7.5.  EARLY SAFETY INDEPENDENT REVIEW (PHASE II ACTIVITY) 
77 
13. 
STUDY MONITORING, AUDITING, AND INSPECTING 
78 
13.1.  STUDY MONITORING PLAN 
78 
13.2.  AUDITING AND INSPECTING 
79 
13.3.  LABORATORY QUALITY CONTROL 
79 
14. 
DATA HANDLING AND RECORDKEEPING 
80 
14.1.  DATA MANAGEMENT RESPONSIBILITIES 
80 
15. 
HUMAN SUBJECTS PROTECTIONS 
81 
15.1.  INSTITUTIONAL REVIEW BOARD/ETHICS COMMITTEE 
81 
15.2.  VULNERABLE PARTICIPANTS 
81 
15.3.  INFORMED CONSENT 
81 
15.3.1.  DOCUMENTATION OF INFORMED CONSENT 
82 
15.3.2.  LITERACY / PROXY CONSENT 
83 
15.4.  WAIVER OF INFORMED CONSENT 
83 
15.5.  STORED SAMPLES AND ASSOCIATED DATA CONSIDERATIONS 
83 
15.6.  RISKS 
84 
15.7.  BENEFITS 
84 
15.8.  COMPENSATION 
84 
15.9.  PARTICIPANT PRIVACY AND CONFIDENTIALITY 
85 
15.10. NEW / INCIDENTAL FINDINGS 
85 
15.11. STUDY DISCONTINUATION 
85 
15.12. POST‐TRIAL ACCESS 
85 
15.13. COMMUNITY ADVISORY BOARD AND OTHER RELEVANT STAKEHOLDERS 
85 
16. 
ADMINISTRATIVE PROCEDURES 
86 
16.1.  PROTOCOL REGISTRATION 
86 
16.2.  REGULATORY OVERSIGHT 
86 
16.3.  CLINICALTRIALS.GOV 
86 
16.4.  CONFLICT OF INTEREST 
86 
17. 
REFERENCES 
88 
18. 
APPENDICES 
99 
APPENDIX A: INFORMED CONSENT 
99 
SAMPLE STORAGE INFORMED CONSENT FORM 
103 
APPENDIX B: STUDY STATISTICAL PLAN 
106 
APPENDIX C: CURRENT STUDY SITE LIST AND PROCESS OVERVIEW 
119 
APPENDIX D. ADVERSE EVENTS OBSERVED IN COAT AND ASTRO‐CM SERTRALINE TRIAL. 
124 
 


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2.1. 
Key Roles 
David R Boulware MD, MPH, CTropMed. Dr. Boulware affiliated with the University of 
Minnesota is the U.S.-chair who will lead protocol development and senior advisor to the 
overall conduct of the project.  He will work with the team to evaluate data in writing 
abstracts and manuscripts related to the data collected.   
David B Meya MBChB MMed, PhD, Dr. Meya is the Ugandan-chair who led the protocol 
development, and will be responsible for supervision of study staff, overseeing study 
implementation, and ensuring that the implementation of the protocol is conducted with 
integrity based on NIH and local regulatory bodies.   
Radha Rajasingham MD. Dr. Rajasingham is the U.S. protocol champion, who will 
develop the protocol, present the protocol to ethical bodies for review and approval.  She 
will serve as an advisor for protocol implementation, and will work with the team to 
analyze data, write abstracts, and manuscripts related to the data collected.    
Elizabeth Nalintya MBChB. Elizabeth is the study coordinator for the C-ASSERT trial, and 
will provide day-to-day supervision of the study staff and supervise project 
implementation in Uganda. 
Conrad Muzoora MBChB, MMed is the site PI in Mbarara, Uganda who will provide input 
for protocol development and led protocol implementation in Mbarara, Uganda.  
Freddie Mukasa Kibengo MMed is the site PI in Masaka, Uganda who will implement the 
trial in Masaka. 
Melanie Bacon RH MPH, is the NIH NIAID DAIDS Medical Officer and representative of 
the funder. 
Biostatistician: 
Kathy Huppler Hullsiek PhD.  Dr. Hullsiek is a biostatistician affiliated with the Division of 
Biostatistics at the University of Minnesota. She will provide statistical support during 
protocol development, and will be primarily responsible for data management, data 
analysis, and dissemination of results.   
 
Additional medical officers will report to the Site PI of record. 
 
The University of Minnesota is the regulatory sponsor of the C-ASSERT trial.


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2.2. 
List of Abbreviations 
AIDS  
acquired immunodeficiency syndrome 
ALT 
 
alanine aminotransferase 
ART 
 
antiretroviral therapy 
AUC 
 
area under the curve 
AZT 
 
zidovudine 
CBC 
 
complete blood count 
CFU   
colony forming units 
COAT  
Cryptococcal Optimal ART Timing 
CrAg  
cryptococcal antigen 
CRP 
 
C-reactive protein 
CSF 
 
cerebrospinal fluid 
DAIDS  
Division of AIDS (NIH, NIAID) 
DSMB  
Data and Safety Monitoring Board 
EKG 
 
electrocardiogram 
EFA 
 
early fungicidal activity 
FDA 
 
Food and Drug Administration 
GCP 
 
Good Clinical Practice 
GCS 
 
Glasgow Coma Scale 
Hgb 
 
Hemoglobin 
HIV 
 
human immunodeficiency virus 
IDI 
 
Infectious Disease Institute 
IDSA  
Infectious Diseases Society of America 
IND 
 
Investigational New Drug 
IRB 
 
Institutional Review Board 
IRIS    
immune reconstitution inflammatory syndrome 
IQR 
 
interquartile range 
IV 
 
intravenous 
JCRC  
Joint Clinical Research Centre 
LFA 
 
lateral flow assay 
LP 
 
lumbar puncture 
MOP  
manual of operations 
NIAID  
National Institute of Allergy and Infectious Diseases 
NIH 
 
National Institutes of Health 
OI  
 
opportunistic infection 
OHRP  
Office of Human Research Protections 
PEPFAR 
President’s Emergency Plan for AIDS Relief 
PO  
 
per os (by mouth) 
SOC 
 
Standard of Care 
SOPs  
Standard Operating Procedures 
QTc 
 
corrected QT interval 
UK MRC 
United Kingdom Medical Research Council 
UNCST 
Ugandan National Council for Science & Technology 
 


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3. PROTOCOL SUMMARY 
Short Title 
Cryptococcal Antigen Screening plus Sertraline 
 (C-ASSERT)  
U01 Award Title 
Operational Research for Cryptococcal Antigen Screening 
(ORCAS) of HIV Patients 
Study Abbreviation 
C-ASSERT 
Intervention 
Asymptomatic CrAg+ persons will be randomized to 
fluconazole (current standard of care) vs. fluconazole plus 
sertraline 
Study Hypothesis 
Cryptococcal-free survival will be improved by an enhanced 
package of CrAg+ preemptive care with the addition of 
adjunctive sertraline 
Sample Size 
600 
Study Population  
HIV-infected persons with cryptococcal antigenemia in blood 
without symptomatic meningitis, predominantly with CD4<100 
Study Design 
Randomized parallel design, double blind trial 
Study Phase 
Integrated Phase II / III 
Participant Duration 
6 months 
Standard of Care 
 (per WHO guidelines) 
Fluconazole 800mg/day x 2 weeks, 400mg/day for 10 weeks, 
then 200mg/day through 6-months 
Expected Mortality 
The expected mortality is 25-30% with standard of care 
therapy; untreated 100% mortality occurs within 6 months 
Intervention 
Sertraline vs. Placebo for 16 weeks 
Escalating doses up to 400mg/day 
Week1 : 100mg/day Week2: 200mg/day Week3: 300mg/day  
Weeks 4-12: 400 mg/day 
Weeks 13-15: Taper 300, 200, then 100mg/day 
Rationale for 
Sertraline Dose 
 Efavirenz induces hepatic P450 metabolism of sertraline 
 Usual 200mg antidepressant sertraline dose has ~50% 
lower levels in Ugandans receiving HIV therapy 
 Higher 400mg/day dose is needed to compensate for this 
metabolism, otherwise sub-therapeutic levels will occur. 
 At 400mg, 95% of participants are projected to achieve 
therapeutic concentrations of sertraline in brain tissue 
based on Ugandan pharmacokinetic data (n=140) and 
Cryptococcus susceptibilities of <4 g/mL for sertraline. 


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Primary Objective 
To determine if adjunctive sertraline provides a cryptococcal-
free survival benefit in persons with asymptomatic 
cryptococcal antigenemia who have a 25-30% risk of death 
with current standard of care.  
Secondary Objective 
To identify incidence and risk factors for meningitis or death 
in asymptomatic CrAg+ persons  
To evaluate safety of sertraline in 300 HIV-infected persons 
Primary Endpoint 
6 month meningitis-free survival  
Secondary Endpoints  6-month Survival Time 
 Incidence of Symptomatic Cryptococcal Meningitis  
 Incidence of Clinical Grade 3,4,5, and/or Serious 
Adverse Events 
 Incidence of Grade 3-5 Laboratory Adverse Events 
 Incidence of premature study drug/placebo 
discontinuation 
 Prevalence of depression by PHQ-9 score  
Inclusion Criteria 
1. 
HIV-infected 
2. 
Cryptococcal antigen (CrAg) positive in blood 
3. 
Age >=18 years and <65 years 
4. 
Written informed consent 
5. 
Women of childbearing potential who are participating in 
sexual activity that could lead to pregnancy must agree 
to use one reliable method of contraception while 
receiving fluconazole >=400mg/day 
Exclusion Criteria 
1. 
Prior history of cryptococcal meningitis 
2. 
Suspected meningitis or mania 
3. 
Suspected/known cirrhosis, jaundice, or ALT >5x ULN  
4. 
Receiving an antidepressant medicine  
5. 
Receiving antifungal therapy, >1 week 
6. 
Breastfeeding / Pregnancy  
7. 
Contraindication to Sertraline or fluconazole  
8. 
Current rifampin use or other prohibited medication 
9. 
EKG QTc >450ms 
Safety Monitoring  
1. In person visits will occur at baseline, 2 weeks, 4 weeks, 
and every 4 weeks thereafter. 
2. Phone calls will be conducted for the first ~100 participants 
in the Phase II trial at weeks 1, 3, and 5 to assess wellbeing 
and any thoughts of self harm. 
3. Participants will be provided contact numbers for study 
staff. If ill, transport will be provided to return to clinic/hospital 


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for appropriate diagnostic evaluation with medical care 
provided to treat new adverse events. 
3. Enhanced safety monitoring will occur with monitoring at 
baseline, 4, 8, and 12 weeks of: 
 Complete blood count 
 Serum Na+, K+, creatinine, ALT, total-bilirubin 
 EKG rhythm and QTc interval 
 Depression and suicidal ideation screening via PHQ-9 
survey instrument 
Safety oversight is via the NIH NIAID HIV Complications & 
Co-infections data and safety monitoring board (DSMB).  
Sertraline is an FDA approved medication (since 1991) which 
is prescribed without any monitoring. Unlike other older 
antidepressants, there is a wide safety margin for SSRIs.  In 
overdoses, consensus guidelines recommend no medical 
evaluation at sertraline doses <1250mg with at home 
observation (Protocol Section 8.1.8). 
 
Among 218 persons with cryptococcal meningitis who 
received sertraline 400mg/day for 2 weeks then 200mg for 8 
weeks in the 2015-2017 ASTRO-CM trial, no participant had 
a sertraline-related grade 3-5 or serious adverse event. 
 
Phase II Trial 
Activities for 
enhanced safety 
oversight 
 Contact with participants will occur weekly for the first 5 
weeks after randomization. Clinical symptoms will be 
assessed as will any concerns for self-harm (suicide). 
 A Trial Safety Committee will review all unexpected 
adverse events as they occur or any potential sertraline-
related adverse events (Section 12.7.4). 
 Early Safety Independent Reviews will occur after every 
40 subjects with a trigger for action of either: 
o >50% aggregate mortality + SAE incidence which is 
higher than ~40% expectation. (Section 12.7.5.)  
o >5% incidence of sertraline-related adverse event (i.e. 
1 out of each 40 randomized = 1 in 20 on sertraline) 
 Safety reviews will continue every 40 participants until 
DSMB assumes responsibility for monitoring safety. 
 The Trial Safety Committee will review these safety 
reports and shall be empowered to:  
1) Pause further enrollment;  
2) Request early DSMB review; and/or  
3) Request further interim safety reviews. 


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FDA Data on SSRIs 
and risk of suicide 
The U.S. FDA mandates a black box warning on all selective 
serotonin reuptake inhibitors (SSRIs) that use in adolescents 
and adults <25 years old with depression may increase the 
risk of suicidality. “Suicidality” is defined as: thoughts of self 
harm, suicide preparation, suicide attempts, or suicides.  This 
is based on a FDA safety report on 16 Nov 2006 which 
analyzed 372 anti-depression trials with 99,839 subjects. Any 
antidepressant medicine was associated with 2.3 fold higher 
odds of suicidality in adults <25 years, with SSRIs have a 
statistically significant result for the drug class. 
When sertraline was consider individually, the risk of 
suicidality was four-fold statistically lower (Odds Ratio = 0.25, 
95%CI, 0.29 to 0.90, P=0.03) for subjects given sertraline 
relative to placebo for suicide preparation, attempts, or suicide 
among adults with psychiatric disorders. In young adults <25 
years old, sertraline had non-statistically lower Odds Ratio of 
0.61 (95%CI, 0.15 to 253, P=0.50).  
Among 57 sertraline trials, there were no suicides in the 2126 
subjects assigned to sertraline but three suicides among 1733 
placebo subjects and six suicides among the 2143 subjects 
assigned to other antidepressant drugs. FDA report is at  
www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4272b1-01-FDA.pdf 
Combined Trial 
activities 
Phase II activities for the first ~100 participants are informative 
for trial continuation. If unexpected adverse events occur with 
sertraline use, the trial may be halted by trial safety committee 
or DSMB, but such events are unlikely to be statistically 
significant based on a ~100 participant sample size.  
All participants (Phase II and Phase III) will contribute data for 
the overall primary event.  The overall trial has adequate 
sample size and statistical power to detect clinically relevant 
differences in trial endpoints.   
 
 
3.1. 
Participating Sites  
1) 
Infectious Diseases Institute (IDI) / Mulago Hill complex, Kampala, Uganda.  
2) 
Joint Clinic Research Center (JCRC) – Mbarara Regional Centre of Excellence 
3) 
Medical Research Council (MRC) / Uganda Virus Research Institute (UVRI) Masaka 
Field Station 
A current list of study sites and the site lead investigator and/or points of contact at those 
sites is contained in study Appendix C. Minor additions, deletions, or substitutions from 
this list may occur over time and hence this list is potentially subject to revision. Appendix 
C will be updated with the most current list annually at the time of the Continuing Review 


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(if there are any updates), rather than submitting any interim updates in the form of formal 
protocol amendments. DAIDS approval will occur prior to any sites participating. 


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4. INTRODUCTION 
4.1. 
Background on Cryptococcal Antigen Screening  
In resource-limited settings, despite major efforts to expand ART access, the median 
CD4 T cell counts of patients initiating ART remain low with 20-25% of persons 
presenting with CD4<100 cells/µL.  Cryptococcal disease is distinct from many other 
infectious diseases in that asymptomatic persons with subclinical disease can be 
detected 3 weeks to >3 months prior to the development of clinical, symptomatic 
cryptococcal meningitis.1, 2  Asymptomatic persons living with AIDS and cryptococcal 
antigen (CrAg) in blood are common throughout Africa (Figure 1) averaging 7.2% (95% 
CI, 6.8-7.6%) in 38 studies of 14,815 persons from low and middle income countries 
(Figure 1).1, 3-10  This is not a historical legacy, as 21 of 38 CrAg prevalence studies have 
been reported in 2013-14.  
Cryptococcal antigen (CrAg) is a strong predictor of development of cryptococcal 
meningitis.  ART alone is insufficient for asymptomatic CrAg+ patients with CD4<100; 
both antifungal therapy and ART are needed to reduce mortality.  
 
 
Figure 1. Cryptococcal Antigen Prevalence (+95%CI) 


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A potential public health strategy to reduce AIDS-related mortality is to screen at-risk 
persons, using CrAg testing of serum or plasma, followed by targeted pre-emptive 
antifungal therapy for those testing positive.3, 4, 11 Recent work from South Africa reported 
that a positive serum or plasma CrAg <2 weeks prior to ART initiation was 100% 
sensitive for detecting the development of meningitis in the first year of ART.4 
CrAg+ patients have substantial risk of cryptococcal disease and mortality despite 
ART.1, 3, 4 Drs. Meya and Boulware (co-PIs) reported from a Ugandan cohort that all 
CrAg+ persons with a CD4 <100 cells/L at time of ART initiation who received ART but 
not anti-fungal therapy died within 3 months of ART initiation (median CD4 = 30 
cells/L).3  This 100% mortality without fluconazole was also found in Ifakara, Tanzania.12 
Conversely, there is 100% negative predictive value for CrAg-negative persons who 
receive ART to not develop cryptococcal meningitis.  
  
Pre-ART CrAg screening is recommended by WHO and PEPFAR.13, 14  
Our team has reported CrAg screening to be a highly cost-effective intervention in 
Africa, using the WHO Commission on Macroeconomics and Health criteria, costing only 
$21 per disability-adjusted life year (DALY) saved (95%CI: $15-$32), based on a 8.8% 
CrAg+ prevalence and 3-year outcomes in Uganda but not including avoided 
hospitalization costs.3 This estimate used the high cost CrAg latex agglutination test 
($16) versus the new $2 CrAg lateral flow assay (LFA). Based on South Africa cost data, 
Dr. Joseph Jarvis (Advisory Committee) et al estimated that CrAg screening saves 
$156 (95%CI: $119 to $197) for every person with a CD4<100/µL,15 and preemptive 
CrAg+ therapy results in 40-80% better 5-year survival.3, 16 Jarvis et al’s cost savings 
estimate is not just among those CrAg+, but every person with CD4<100/L, resulting in 
major cost savings to the health system.  
CrAg screening with fluconazole preemptive therapy has proven to be efficacious in 
one published randomized clinical trial in Zambia and Tanzania (Figure 2).17 In this trial, 
a 28% relative reduction in mortality with CrAg screening and ART adherence support 
occurred overall in persons with CD4<200 cells/L. 


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4.2. 
Problem  
Yet, the optimal management of those with asymptomatic cryptococcal 
antigenemia is unclear.  ART alone is NOT sufficient to prevent clinical cryptococcal 
disease or death.3, 4, 18, 19 In Kampala, our group prospectively demonstrated that 
preemptive fluconazole therapy is effective at improving short term and long term survival 
when starting ART.3  However, in this observational, small, prospective cohort (n=33 
CrAg+), fluconazole preemptive therapy was not standardized.  Varying doses and 
durations were used between 200-400 mg for 2-4 weeks.3 Of those CrAg+ with CD4 
<100 cells/L who received fluconazole, 15% developed meningitis and 29% died during 
follow up. 3 Longley et al reported higher fluconazole doses of 800-1200 mg have greater 
fungicidal activity than 400mg.20  In 2011, the WHO guidelines for cryptococcal disease 
recommended CrAg screening in those with a CD4 <100, followed by preemptive therapy 
for those who were asymptomatic CrAg+. 
WHO recommended preemptive therapy: 800mg fluconazole daily for 2 weeks, 
followed by 400mg daily for 8 weeks, “and continued maintenance with fluconazole 200 
mg/day is recommended. The optimal antifungal regimen in this population remains to be 
determined.”13, 21  This is now standard of care per Ugandan national HIV guidelines, and 
is standard of care.  
 
Notably, this is a conditional recommendation based on low quality evidence.22  
 
Six-month mortality for those asymptomatic CrAg+ persons treated with this regimen, 
however, has remained high at 32% (Figure 3). We seek to provide better evidence on 
the treatment and risk factors for asymptomatic CrAg+ populations. 
 
Figure 2. CrAg screening improves survival 
 


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4.2.1. Standard of care CrAg screening in Uganda 
Per Ugandan National HIV Guidelines, all ART-naïve persons with HIV-infection and a 
CD4<100 cells/μL should be screened for CrAg in the serum or plasma, irrespective of 
symptoms. Preemptive antifungal therapy is recommended for those asymptomatic 
CrAg+. The dosing of fluconazole is outlined above.  
Ugandan national guidelines do not currently include secondary fluconazole 
prophylaxis for asymptomatic CrAg+ persons. WHO guidelines are equivocal regarding 
secondary prophylaxis. South African guidelines recommend 200mg of fluconazole daily 
for 6 months for those with asymptomatic cryptococcal antigenemia, after completing the 
above WHO recommended regimen. The benefit of this lengthened therapy is not known. 
To be released, 2016 Ugandan HIV guidelines will recommend fluconazole 200mg/day 
secondary prophylaxis through 6 months. In this trial we will follow Ugandan national 
guidelines. Thus, we will follow the above regimen, however if guidelines are changed 
during the course of this trial, we will follow the updated recommended regimen.  
 
4.2.2. Evidence gaps in management of asymptomatic CrAg+ 
a) Optimal preemptive antifungal therapy is unknown 
Although CrAg+ persons receiving fluconazole generally do better than if not treated 
at all, 25-30% go on to develop meningitis or die despite the WHO recommended 
antifungal therapy and ART. A 2.9-fold worse survival was observed in asymptomatic 
CrAg+ vs. CrAg- persons with CD4<200 in Tanzania and Zambia (Figure 3 right).17  
 
One of the major, unresolved problems with CrAg screening is that CrAg+ individuals 
still have 2 to 3-fold higher mortality, despite receiving WHO recommended preemptive 
Figure 3. Survival by CrAg status in Uganda & REMSTART Trial in Tanzania & Zambia
 


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fluconazole therapy (Figure 3). Despite preemptive therapy, CrAg+ individuals accounted 
for 12% of deaths. As cryptococcosis accounts for ~15% of HIV-related mortality,1, 2, 23-26 
this is a significant problem.  
b) Potential additional therapies include sertraline and amphotericin. Flucytosine is 
another antifungal of interest, but flucytosine is not currently available in most low 
and middle income countries and is cost prohibitive in the U.S. ($2000/day).27 
Timing of ART in asymptomatic CrAg+ has not been studied 
In general, earlier initiation of ART results in earlier viral suppression, reduced risk 
of HIV transmission, and enhanced clinical outcomes. In other opportunistic 
infections, including TB, ART is also initiated as soon as possible. However, in 
cryptococcal meningitis a randomized control trial demonstrated excess mortality 
associated with early ART, likely due to immune reconstitution inflammatory 
syndrome (IRIS). Given this exception to the rule, an interesting area of future 
study is the timing of ART in asymptomatic CrAg+ persons. For this study, 
participants will be recommended to initiate ART at approximately 2-4 weeks after 
entry into care in line with Ugandan national guidelines.  
c) Risk factors for progression to fulminant meningitis 
Asymptomatic CrAg+ are at increased risk of meningitis and death. But who 
progresses to meningitis and the expected time frame of this progression has not 
previously been studied.  
Lumbar punctures have been proposed as a valuable tool to investigate potential 
CNS involvement. However, lumbar punctures are generally very poorly accepted 
in African populations, and understandably, in a notably asymptomatic population. 
Furthermore, the majority of clinics do not have the capacity or healthcare worker 
resources to perform lumbar punctures on a number of asymptomatic outpatients. 
Thus this is not currently part of the Ugandan guidelines. One group in Tanzania 
performed lumbar punctures in 31/32 CrAg+ persons, and identified 2 out of 12 
patients without any neurologic symptoms who had meningitis in CSF. Thus, the 
majority of persons with meningitis do have symptoms. 
CrAg titers have been found to predict risk of meningitis and death. Other risk 
factors have not yet been evaluated.  
d) Need for secondary prophylaxis 
After initial therapy with fluconazole 800mg daily x 2 weeks, then 400mg daily x 8 
weeks, it is unknown if there is additional benefit of giving patients 200mg daily of 
fluconazole thereafter. After fulminant meningitis, patients are given secondary 
prophylaxis with 200mg/day.  
WHO, South Africa, US, Uganda, and other guidelines differ. Specifically, the 


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WHO 2010 Rapid Advice conflicts itself.  The 2016 WHO guidelines and revised 
‘to be released’ 2016 Ugandan guidelines recommend secondary fluconazole 
prophylaxis, based on no data, but some ongoing mortality after 10 weeks.  
The large public health importance is that if secondary prophylaxis is 
necessary, then the cost of fluconazole preemptive therapy for CrAg+ increases 
by ~40%.  We hypothesize based on timing of mortality (Figure 3) that secondary 
prophylaxis has minimal benefit when giving ART.3, 17 
 
4.3. 
 Study Hypothesis  
We hypothesize cryptococcal-free survival will be improved by an enhanced package 
of CrAg+ preemptive care with the addition of adjunctive sertraline. 
4.4. 
Rationale: Antifungal Effect of Sertraline 
Sertraline.  Sertraline, the selective serotonin reuptake inhibitor (SSRI) anti-
depressant, has in vitro and in vivo activity against Cryptococcus neoformans.28, 29 
Sertraline’s mechanism of action is interference with Cryptococcus protein synthesis via 
eukaryotic translational initiation factor 3 (Tif3).29  
The first indication that sertraline exhibits antifungal properties came during 
observations from a clinical setting in which patients were observed to have resolution of 
recurrent vulvovaginal candidiasis during periods in which they were concurrently being 
treated with sertraline for premenstrual dysphoric disorder.30  Subsequent in vitro studies 
demonstrated that sertraline was fungicidal against both Candida and Aspergillus 
species,30, 31 though at minimum inhibitory concentrations (MICs) higher than what would 
likely be achievable in serum using standard human doses of sertraline.  More recently, 
Zhai et al, by using an approach of broad screening of known clinical compounds, have 
confirmed the modest inhibitory effects of sertraline against Aspergillus nidulans in vitro.29  
When in vitro susceptibility testing was extended to pathogenic yeasts, particularly potent 
antifungal activity was observed against Cryptococcus. The recognition of sertraline as 
an antifungal subsequently led to additional studies, including further in vitro studies 
using combination therapy with fluconazole, in vivo studies using a murine model, and 
preliminary studies to investigate the antifungal mechanism of sertraline.29  The methods 
and specific findings are summarized in Table 1. 
Table 1: Major Findings of Sertraline’s Anti-Cryptococcal Properties  
Major Conclusions 
Methods 
Specific Findings 29 
1. Sertraline is fungicidal 
against C. 
neoformans in vitro 
 Susceptibility testing of 24 distinct 
cryptococcal strains 
 Time course assay comparing fungal growth 
in nutrient- rich vs nutrient-depleted media 
 MIC90 ranged between 2-6 μg/mL  
 MFC ranged between 6-10 μg/mL 
 Fungal killing was independent of cell 
proliferation 
2. Fungicidal activity of 
sertraline is additive 
 Comparison of sertraline and fluconazole 
susceptibilities 
 Time course assay comparing monotherapy 
vs combo therapy 
 Combination therapy accelerated clearance  
 Larger and clearer halo in disks containing 
combo therapy  


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when combined with 
fluconazole 
 Quantification of additive effects by 
calculating FFCI 
 Effects of combo therapy were synergistic 
(FFCI < 0.5) or additive (FFCI 0.5 - 1.0) in 
all 24 strains tested 
3. Sertraline is effective 
against C. 
neoformans in vivo 
in a murine model 
 Mice were assigned to 1 of 4 treatment 
arms† and intravenously infected 
 Quantitative cultures obtained from 
suspensions of homogenized tissue 4 
days after being infected 
 Sertraline reduced fungal burden in brain 
with efficacy similar to fluconazole 
 The most potent anti-fungal effects were 
observed with combination therapy 
4. Sertraline interferes 
with translation in 
fungal cells 
 Whole-genome deletion screening (using 
collection of mutant Saccharomyces 
cerevisiae) for sertraline-sensitive or 
sertraline-resistant mutants 
 Gene ontology to identify processes 
affected by sertraline 
 Genes related to protein synthesis were 
highly enriched in sertraline-resistant strains 
 Mutant that was most sensitive to sertraline 
contained disrupted gene for the translation 
initiation factor Tif3 
5. Protein synthesis is 
inhibited in a C. 
neoformans cell-free 
system 
 In vitro translation assays utilizing C. 
neoformans cell extract as translation 
machinery for luciferase mRNA 
 Sertraline added to mixture in dose-
dependent manner 
 Sertraline inhibited translation efficiency in a 
dose- dependent manner 
 Translation activity 50% when at 30.6 μg/mL 
 No translation when at 122.4 μg/mL 
MIC = minimum inhibitory concentration; MFC = minimum fungicidal concentration; FFCI = fractional fungicidal concentration index 
†Experimental arms of in vivo murine model: 1. Control group (no drug); 2. Sertraline (15 mg/kg/day, started 7 days prior to infection) 
monotherapy; 3. Fluconazole (15 mg/kg/day, started 24 hours after infection) monotherapy; 4. Combination of sertraline (#2) and 
fluconazole (#3) above. 
In the study summarized above (Table 1), sertraline inhibited C. neoformans with an 
MIC between 2-6 g/mL.29  In addition, sertraline appeared to be fungicidal since killing 
was independent of cell proliferation (Figure 4A).  The combination of sertraline and 
fluconazole led to lower MICs and accelerated fungal clearance at a greater rate than 
either drug alone.  Quantifying these effects using the fractional fungicidal concentration 
index (FFCI), the combination of sertraline and fluconazole was either additive or 
synergistic in all strains tested.  When pretreated for 7 days at a dose of 15 mg/kg/day, 
sertraline was also effective against C. neoformans in an in vivo model of experimentally 
infected mice.  The inhibitory effect of sertraline was particularly potent in the brain of 
infected mice, with efficacy similar to fluconazole (Figure 4B).  The most potent anti-
fungal effects were observed in mice treated with sertraline + fluconazole combination 
therapy. 
Additional studies to ascertain the antifungal mechanism of sertraline suggest that 
translation may be inhibited in Cryptococcus.  In whole-genome deletion screening of 
Saccharomyces cerevisiae for instance, genes related to protein synthesis were highly 
enriched in sertraline-resistant strains (Figure 4C).   


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Figure 4. Antifungal in vitro and in vivo Activity of Sertraline 29 
 
4.5. 
Validation: Antifungal Effect of Sertraline 
Our research group has validated the in vitro sertraline activity among 198 clinical 
Cryptococcus isolates from Uganda collected during the COAT trial (2010-2012),32 128 clinical 
isolates from Uganda during 2013-201433 and 153 clinical isolates from Mexico (Trevino-
Rangel).34  Additionally, Trevino-Rangel et al also completed a murine model of cryptococcal 
infection, replicating the findings of Zhai et al. 29, 34  
Table 2. Cryptococcus in vitro Susceptibility to Sertraline 
Isolate Location 
N 
Concentration MIC (µg/mL) 
1 
2 
3 
4 
5 
6 
8 
12 
Global 29 
24 
 
2 
(8.3%) 
4 
(25%) 
10 
(67%) 
5 
(88%) 
3 
(100%) 
 
 
Uganda 2010-12 32 
198 
19 
(10%) 
35 
(27%) 
 
95 
(75%) 
 
16 
(83%) 
32 
(99%) 
1 
(100%) 
Mexico 34 
153 
62 
(40.5%) 
62 
(81%) 
 
26 
(97.4%) 
 
 
3 
(100%) 
 
Uganda 2013-14 33 
128 
10 
(7.8%) 
25 
(27%) 
 
73  
(84.4%) 
 
9 
(91.4%)
11 
(100%) 
 
Pooled Results 
503 
91 
(18%) 
124 
(43%) 
4 
(44%) 
204 
(84%) 
5 
(85%) 
28 
(91%) 
46 
(99.8%)
1 
(100%) 
Numbers represent N (cumulative percent). 
The therapeutic target concentration in brain is 4 g/mL for ~84% of Cryptococcus. 


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In our open label ASTRO trial for cryptococcal meningitis, when sertraline was added to 
amphotericin + fluconazole 800mg/day, the rate of CSF yeast clearance was 23% faster 
with adjunctive sertraline than historical cohort of the COAT trial who received amphotericin 
and fluconazole 800mg/day (Table 3, P=0.05).35, 36  
   
Table 3: Early Fungicidal Activity (EFA) of the CSF Cryptococcus Clearance Rate by Regimen 
Trial 
Treatment Regimen 
N 
-EFA Mean (95% CI) 
log10 CFU/ml/day 
COAT 
Amphotericin B 0.7-1 mg/kg/day + Fluconazole 800 mg/day 
189 0.30 (0.28 to 0.32) 
ASTRO 
Pilot 
Ampho B + Flu800 + sertraline 100-400mg/day, pooled doses 128 0.37 (0.33 to 0.42) 
EFA values are population means estimated with longitudinal mixed models with 95% CI 
 
 
Skepticism regarding sertraline as an antifungal.   
 
As displayed in Table 3, 
sertraline when added 
to standard antifungal 
therapy resulted in a 
23% faster rate of 
CSF clearance. 
Figure 5 displays the 
early fungicidal 
activity (EFA) plots of 
this increased 
fungicidal activity. The 
EFA plots display the 
quantitative CSF 
culture data (log10 
CFU/mL) vs. days of 
induction therapy.  
Somewhat noteworthy 
is comparing days 5-9 when CFU counts plummet with adjunctive sertraline, near when 
sertraline is reaching steady state levels (Day ~7). The comparison is the COAT trial which 
received the same background antifungal regimen of amphotericin B + fluconazole 
800mg/day, where clearance is linear.35   
Unlike fluconazole, sertraline has fungicidal in vitro activity against quiescent 
yeasts, which are not proliferating (Figure 6).29  This may have an important clinical effect. 
Figure 5.  CSF Cryptococcus Clearance Rate 


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In our open label ASTRO study 
(n=172), sertraline at >200mg/day 
eliminated cryptococcal relapse 
(0%) and greatly decreased 
paradoxical IRIS (<5% suspected 
IRIS) in comparison with COAT 
trial experience of 5% relapse and 
17% paradoxical IRIS incidence.35  
We hypothesize sertraline’s 
activity against quiescent yeasts in 
brain tissue and/or fluconazole 
synergy resulted in reduced 
relapse. This effect may be very 
important in subclinical infection in CrAg+ persons to eliminate the reservoir of quiescent 
yeasts.  
In summary, the data supporting antifungal activity of sertraline include: 1) in vitro activity 
on clinical isolates; 2) in vivo murine model; 3) mechanism of action (post-translation 
protein inhibition); 4) human data in persons with cryptococcal meningitis on increased 
rate of CSF clearance, less relapse, and less paradoxical IRIS.  
Sertraline is a generic medication with >25 manufacturers worldwide, >30 million U.S. 
annual prescriptions (with an excellent safety record), and the U.S. wholesale cost of 
300mg/day is $2.15 per week. This is less expensive than flucytosine (5FC) 
($4350/week), and unlike 5FC, sertraline is available in pharmacies in Africa. Efavirenz 
decreases sertraline levels by 25-50%; however, there is no sertraline effect on 
antiretroviral medicines.37, 38  
 
4.6   Experience in Higher Dose Sertraline 
A variety of early trials and studies investigated higher dose sertraline. In general, higher 
dose sertraline was well tolerated but did not have increased anti-depressant effects. 
1) The ASTRO pilot study enrolled 172 HIV-infected participants with cryptococcal 
meningitis of whom 95 participants received sertraline starting at 300-400 mg/day. 
AEs were common in this population, but there incidence of AEs did not differ 
compared to sertraline at 100-200 mg/day.33 
2) The ASTRO-CM randomized trial has enrolled 320 HIV-infected Ugandan 
participants with symptomatic cryptococcal meningitis (~50% randomized to 
sertraline 400mg/day) and 43 with cryptococcal relapse consented to open-label 
sertraline for compassionate care as of 19 October 2016. Overall the incidence of 
adverse events with a background therapy of amphotericin B deoxycholate and 
fluconazole 800mg/day is within expectations, being similar and slightly less than 
the prior COAT trial (Appendix D).35 No adverse events have been attributed to 


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study medicine. Tolerability has been good, as only three participants (0.8%) 
have discontinued the ASTRO-CM study medicine (blinded sertraline/placebo). 
3) In a trial of 66 participants with obsessive-compulsive disorder, high dose 250-
400mg/day (mean 357mg) for 16 weeks of 36 persons had similar safety profile as 
200mg/day.39 Two persons discontinued therapy due to adverse events at the 
200mg/day dose and none at the 400mg/day dose.39 At high dose, the most 
common adverse events were insomnia (20%), diarrhea (16.7%), nausea (13.3%), 
and somnolence (13.3%).39 These are similar (and lower) than listed in the 
package insert.40 
4) EEG studies testing 400mg showed dose-dependent change in CNS activation 
and a widening of the pupillary size.41 
5) Current Pfizer trial investigating sertraline at 400mg/day for lack of effect of 
sertraline on EKG QTc (n=54) has been completed as of 11 Oct 2016 
(ClinicalTrials.gov: NCT02651623) without data available. 
6) There is substantial experience in over-dose attempts using SSRIs.42-44  Among 
48,000 U.S. Poison Center calls regarding SSRI overdoses/exposures in 2004, 
consensus evidence based guidelines recommend for acute SSRI ingestions of 
sertraline of up 1250mg for observation at home among asymptomatic patients or 
those with mild effects.44 Among an Australian SSRI overdose cohort, among 103 
sertraline overdoses (median 4800mg, IQR, 3200-6000mg) ingestion, non-severe 
serotonin syndrome occurred in 20% and the EKG QTc was 6ms longer than a 
comparison control cohort (429 vs. 423ms) with 6% (6/103) of sertraline overdoses 
having a QTc >500ms.43  
 


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5. OBJECTIVES 
 
5.1. 
Primary Objective(s) 
 To determine if adjunctive sertraline improves 6-month cryptococcal meningitis 
free survival with retention-in-care over fluconazole preemptive monotherapy, 
which has a 25-30% risk of death with current standard of care. 
 
5.2. 
Secondary Objective(s) 
 To evaluate the safety of sertraline in asymptomatic CrAg+ persons. 
 To identify incidence and risk factors for meningitis or death in asymptomatic 
CrAg+ persons.  
 
5.3 Study Hypothesis 
We hypothesize cryptococcal-free survival will be improved by an enhanced    
package of CrAg+ preemptive care with the addition of adjunctive sertraline. 
5.4 
Primary Endpoint 
1) 6-month cryptococcal meningitis free survival with retention-in-care 
 
5.5 
Secondary Endpoints 
1) 6-month survival time 
2) Incidence of symptomatic cryptococcal meningoencephalitis 
3) Incidence of Clinical Adverse Events (Grade 3-5) 
4) Incidence of Grade 3-5 adverse laboratory abnormalities  
5) Incidence of all-cause premature study drug discontinuation 
6) Prevalence of depression by Patient Health Questionnaire (PHQ-9) 
 
Symptomatic meningoencephalitis is defined as clinical symptoms consistent with 
meningitis AND  
 CSF Cryptococcus culture positivity; OR  
 CSF CrAg positivity; OR 
 Cryptococcoma(s) by neuroimaging or post mortem exam. 
 
 
 


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6. STUDY DESIGN 
This will be a randomized controlled trial of WHO standard of care with fluconazole vs. 
standard of care plus sertraline amongst asymptomatic CrAg+ persons in Uganda. There 
will be enhanced safety monitoring beyond usual standard of care with an integrated 
phase II / III study design.  The first ~80 subjects enrolled will have additional real time 
safety monitoring and adjudication of unexpected adverse events or any potentially 
sertraline-related adverse events. 
Study Design 
Placebo 
Sertraline x 16 
weeks 
Fluconazole 800mg/day x2 
weeks, 400mg x 10 weeks; 
   200mg through 6 months 
(standard of care) 
~300 subjects 
~300 subjects 
 
Outcomes of cryptococcal meningitis and survival will be assessed through 6 months 
from time of CrAg testing. 
Participants may receive reminders via phone calls or text messages, to encourage them 
to return for appointments. Initially, during the phase II activities with enhanced safety 
monitoring, participants will have weekly contact through 6 weeks either in person (weeks 
0, 2, 4, and 6) or by phone (weeks 1, 3, and 5). 
 
Objective 2: Identify risk factors for meningitis and death 
The purpose of objective 2 is to define high risk populations that may require customized, 
intensive therapy in the future. We hypothesize that high initial CrAg titers in serum 
(>1:160), high CRP, and other biomarkers are risk factors for meningitis and death. 
Preliminary data from the original ORCAS study in Uganda from 2012 to 2014 suggests 
that CrAg titer is a risk factor for death (see Figure 7).  
In order to identify potential risk factors associated with treatment failure, we will perform 
CrAg titers at screening and among a subset at 4 and 8 weeks.  A subset of samples 
(based on sample availability) will be stored and testing for biomarkers will be done 
Asymptomatic CrAg+ persons
n=600
Fluconazole + Placebo
n=300
Fluconazole + Sertraline
n=300


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retrospectively in batches. Such biomarkers will include CRP, which we have previously 
demonstrated to be associated with IRIS and/or death. We anticipate that identifying 
biomarkers to predict meningitis and death, and validating titers as another risk factor, we 
will be able to use markers in the future to risk stratify patients prospectively, counsel 
them appropriately, and potentially customize therapy according to risk. Analysis may be 
performed retrospectively in batches.  
Figure 7: Survival among CrAg+ Persons by Baseline Plasma CrAg Titer 
. 
 
As part of exploring risk factors for meningitis and/or death, pathogenesis studies may 
include the following:.   
1) Serum/plasma storage for CrAg titers, serum cytokines, and biomarker analysis; 
2) Case-control exploratory sub-study on RNA transcriptome (gene expression) 
profiling will be performed on cases that fail preemptive therapy (i.e. develop 
cryptococcal meningitis or die), compared with controls with uneventful immune 
recovery with receipt of preemptive therapy. This case-control study will match 
controls with cases based on CrAg titer (+/- one two-fold dilution) and CD4 count 
(within 25 cells/µL). Participation in this sub-study would require an additional 
PaxGene RNA tube blood draw for storage at the enrollment visit with volume of 
2.5 mL.  Participants could decline participation in this sub-study and still 
participate in the trial. 
3) PBMC collection and/or antigen stimulation experiments will occur at selected 
sites.  This will be determined by site capacity, distance from central lab, and 
number of CrAg+ subjects at sites. 


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7. STUDY POPULATION 
7.1. 
Participant Eligibility Criteria 
This clinical trial will enroll HIV-infected CrAg+ persons at participating healthcare 
centers, who fulfill the inclusion/exclusion criteria outlined below. Cryptococcal 
infection is an AIDS-defining condition. 
7.1.1. Inclusion Criteria 
o HIV-infection, documented by any locally licensed rapid HIV test. 
o Cryptococcal antigen positive test (CrAg+) in blood 
o Age >=18 years and <65 years 
o Provision of informed consent 
o Female participants of childbearing potential who are participating in sexual 
activity that could lead to pregnancy must agree to use one reliable method 
of contraception: a barrier method of contraception (condoms or cervical cap) 
together with another reliable form of contraception (condoms, with a 
spermicidal agent; a diaphragm or cervical cap with spermicide; an intra-
uterine device (IUD); or hormonal-based contraception) while receiving 
fluconazole >400mg/day.  
 
7.1.2. Exclusion Criteria 
o 
Prior history of cryptococcal meningitis 
o 
Suspected meningitis (this is a clinical decision based on the patient’s history 
and clinical findings, but typically is a combination of headache, neck 
stiffness, and/or altered mental status) or mania 
o 
Suspected/known cirrhosis or serious hepatic comorbidities, transaminitis 
(ALT>5x upper limit of normal), jaundice, who should not receive fluconazole 
in the opinion of the study investigator 
o 
Receiving an antidepressant medicine  
o 
Currently receiving systemic antifungal therapy for >1 week 
o 
Breastfeeding or Pregnancy (a negative urine (or serum) pregnancy test 
before study entry is required for women of childbearing potential). 
o 
Contraindication to sertraline or fluconazole  
o 
Currently receiving rifampicin (Rifampin®) or other prohibited medicine (Refer 
to Section 8.4) 
o 
EKG QTc interval >450ms 
 
Comments on exclusion criteria: 
 Female participants of “childbearing potential” are defined as women who have 
reached menarche or who have not been post-menopausal for at least 24 


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consecutive months (i.e., who have had menses within the preceding 24 months) or 
have not undergone surgical sterilization (e.g., hysterectomy, or bilateral 
oophorectomy or salpingotomy).  Abstinence is acceptable as a contraceptive 
method. Women are required to use at least one form of contraception while 
receiving fluconazole >400mg/day and are recommended to use contraception and 
avoid pregnancy while receiving 200mg/day.  
 Persons with suspected symptomatic meningitis will be referred to hospital for 
diagnostic lumbar puncture and if CSF reveals cryptococcal meningitis (i.e. positive 
CSF CrAg+, culture, and/or India ink) amphotericin-based treatment will be initiated 
in accordance with WHO recommendations, outside of this study.   
 Mania may be a presentation of cryptococcal meningitis (or bipolar disorder) and is 
an exclusion criterion. 
 Prior ART is not an exclusion. ART-defaulters (i.e. prior receipt of ART who were 
lost to follow up and are now re-entering care) are not excluded.  Persons with 
immunologic/virologic failure who may be switching to second line ART are eligible.  
 Persons with a prior history of cryptococcal meningitis are excluded, as they may 
remain CrAg+ for months/years. In clinical practice, these persons should already 
be receiving fluconazole 200mg/day secondary prophylaxis if their CD4 is <100 
cells/µL.  
 CrAg positivity in “blood” includes: plasma, serum, whole blood, or by capillary 
fingerstick.45, 46 
 ALT measurement within 30 days of randomization must occur for persons to be 
eligible. The ALT ideally could be run on a specimen collected at screening. If the 
result does not exist, potential participants should have an ALT collected prior to 
randomization. This may delay therapy and necessitate an extra return visit to the 
clinic. This is a directive from the NIH study sponsor. 
 Rifampicin (rifampin) induces metabolism of sertraline via acting as an inducer 
hepatic enzymes of: CYP2B6, CYP2C9, CYP2C19, CYP3A4 (all relevant), as well 
as CYP2C8, CYP3A5, and CYP3A7 (not relevant to sertraline metabolism). In 9 
Ugandans, mean sertraline levels with rifampin of 208 ng/mL (95%CI, 130 to 285) 
were 50% lower than sertraline levels without rifampin of 416 ng/mL (95%CI, 350 to 
484; P<.001). Conversely fluconazole moderately inhibits CYP2C9 and CYP2C19 
pathways.  Refer to Table 10 in Section 8.6 for details on drug-drug interactions.  
o Rifapentine induces metabolism of CYP2B6, CYP3A4 (relevant) and 
CYP2C8 (not relevant). 
o Conversely, rifabutin is a weak inducer of CYP3A4 metabolism (relevant). 
Isoniazid is a CYP3A4 inhibitor.  
 Prohibited medications are listed in Section 8.4. These medicines are not generally 
available in Uganda. Documentation of concomitant medicines will occur at study 


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entry, and updates will occur at study visits, as appropriate. 
 A CD4 threshold is not an inclusion criterion as this is a trial of asymptomatic CrAg+ 
persons.  The absolute CD4 count affects the probability of being CrAg+, but CD4 is 
immaterial once someone has early disseminated cryptococcal infection, as 
evidenced by a CrAg+ result in blood. 
 EKG QTc >450ms exclusion is per the directive of the Funder.  Refer to Section  
8.1.7 on the published data on the negative “thorough QTc study” conducted on 
high dose sertraline as per FDA guidance.33 Sertraline does not cause EKG QTc 
prolongation (FDA Package Insert). 
 
7.2. 
Recruitment Process 
CrAg screening in CD4<100 is recommended as per national HIV guidelines and is 
generally performed as a reflex-laboratory test whenever the CD4 is <100 cells/µL. 
Additional recommendations are to perform CrAg testing among persons with 
suspected virologic failure or WHO clinical stage III/IV disease.  
In Kampala, the CrAg prevalence in persons with CD4<100 is 8-9% during 2015-
2016.  An estimated ~8000 persons will likely require CrAg screening to enroll a target 
of 600 asymptomatic CrAg+ persons. This protocol aims to enroll at approx. 125 
participants per year irrespective of their CD4 count. 
The protocol will not specify how clinics implement CrAg screening, as the 
screening method may vary by clinic based on clinic operations. SOPs will specify 
details of CrAg screening implementation. The probable methods of CrAg screening 
implementation are: 
1) Lab-based reflex test using remnant plasma left over after CD4 testing 
2) Provider initiated lab order  
3) Point-of-care CrAg test in combination with point-of-care CD4 testing 
4) Universal CrAg screening in the absence of prompt CD4 testing  
The predominant method is likely to be lab-based reflex lab screening after CD4 
testing at the CD4 laboratories for the sites. Refer to Appendix C for the overview of 
specimen collection and flow of laboratory testing by site.  
 
Positive CrAg tests are considered a critical laboratory value, which results in a 
phone call from the laboratory to the ordering provider. Study staff notified of positive 
CrAg tests will contact CrAg-positive patients to help facilitate persons returning to 
clinic expeditiously.  CrAg-positive persons identified by lab-reflex screening by clinic 
staff (through point-of-care testing), or via referral from outside clinics will be reviewed 
for eligibility and approached for informed consent by study personnel (typically a study 
nurse).  Asymptomatic CrAg+ persons who meet inclusion criteria will be offered 
enrollment in this study. 


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Persons referred from outside, non-study clinics must have a CrAg test repeated by 
the study laboratory or by trained study personnel prior to randomization (refer to the 
Delegations and Responsibility log as to which investigators have been trained).  
All participants must be verified for CrAg positivity at a DAIDS-approved 
facility prior to randomization. 
 


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8. INTERVENTIONS (STUDY PRODUCT) 
With the exception of the investigational drug, sertraline to be used in this study, all 
medications are considered standard of care.  Sertraline and/or placebo will be provided 
by the study.  No other medications will be provided by the study. 
8.1. 
Biomedical Interventions 
Sertraline is a selective serotonin reuptake inhibitor 
(SSRI) for oral administration. Sertraline is supplied as 
scored tablets containing sertraline hydrochloride 
equivalent to 25, 50 and 100 mg of sertraline.    
Ample clinical data supports the safety of sertraline at doses between 50-200mg.47-
49  Few drug-drug interactions of clinical significance exist50, and it is relatively safe in 
over-dosage.42, 43  Common adverse effects include nausea (25% vs. 11% for placebo), 
male ejaculation failure (14% vs. 1% for placebo), insomnia (21% vs. 11% for placebo), 
diarrhea (20% vs. 10% for placebo), dry mouth (14% vs. 8% for placebo), drowsiness 
(13% vs. 7% for placebo), dizziness (12% vs. 7% for placebo), tremor (8% vs. 2% for 
placebo) and decreased libido (6% vs. 1% for placebo).51  These adverse events are 
often transient and resolve spontaneously without dosage adjustment.  Sertraline also 
appears to be associated with microscopic colitis, a rare condition of unknown 
etiology.52  Akathisia caused by sertraline was observed in 16% of patients in a case 
series.53  Akathisia typically begins several hours after the initiation of treatment or a 
dose increase and usually disappears after being stopped or decreased.  Prolonged 
QT interval is considered a class effect seen with SSRIs, particularly with citalopram.  
However, of the SSRIs, sertraline has one of the least effects on QT interval.43  
Nevertheless, the QT interval was monitored in all patients enrolled into Phase I open 
label pilot, via serial (weekly) EKGs from baseline through the day 14 visit. 
In healthy volunteers over a 2-week period, sertraline slightly improved verbal 
fluency but did not affect word learning, short-term memory, vigilance, flicker fusion 
time, choice reaction time, memory span, or psychomotor coordination.54  No clinically 
relevant differences were observed in objective cognitive performance in a group of 
people treated for depression with sertraline for 1.5 years as compared to healthy 
controls.55 
All antidepressants, including sertraline, carry an FDA black box warning stating 
that antidepressants may increase the risk of suicide in persons with depression 
younger than 25 years.  This warning is based on pooled analyses of short-term 
placebo-controlled trials of antidepressant drugs (SSRI and others) that found a 1.5-
fold increase of suicidal behavior in young adults (aged 18-24) with major depressive 
disorder.56  Considered separately, sertraline use in adults decreased the odds of 


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suicidal behavior with a marginal statistical significance by 37% or 49% depending on 
the statistical technique used.56 
The development of a potentially life-threatening serotonin syndrome has been 
reported with SSRIs, including sertraline, alone but particularly with concomitant use of 
other serotonergic drugs.  The concomitant use of sertraline with the MAOI class of 
psychiatric drugs is contraindicated. 
Abrupt interruption of sertraline may result in withdrawal or a so-called 
discontinuation syndrome.  This syndrome occurred in 60% of subjects in a blind 
discontinuation study where sertraline was temporarily replaced by placebo.57  
Frequent symptoms reported include irritability, agitation, dizziness, headache, 
nervousness, crying, emotional labiality, bad dreams and anger.  This withdrawal 
syndrome was completely avoided when sertraline was gradually discontinued over 
three weeks.58  
In the 2013-2014 pilot study of the “Adjunctive Sertraline for the Treatment of 
Cryptococcal Meningitis” (ASTRO-CM) open-label pilot study in treating patients with 
symptomatic cryptococcal meningitis, the incidence of adverse events was not 
increased with higher dose sertraline.33  Table 4 compares the incidence of AEs 
among persons with cryptococcal meningitis who were treated with a background 
regimen of amphotericin B deoxycholate 0.7-1.0 mg/kg/day and fluconazole 800mg/day 
(COAT Trial 2010-2012 in Uganda) and that background regimen plus sertraline at 
doses 100-400mg/day.   
 
 
 
 
 
 
 
 
 
 
 
 
 


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Table 4: Safety Endpoints of Adjunctive Sertraline used for cryptococcal meningitis 
Clinical Outcomes 
Historical 
COAT trial 
cohort 35 
ASTRO Pilot 
Sertraline 100 
and 200mg/day33 
ASTRO Pilot 
Sertraline 
300 and 
400mg/day33 
Risk 
Difference 
(95%CI) a 
P-value 
N enrolled 
208 
77 
95 
 
 
Adverse Events 
Total number Grade 4 AEs 
119 
20 
27 
 
 
Grade 4 AEs, cumulative 
incidence 
88 (50%) 
15 (19%) 
22 (23%) 
0.04 
(-0.09, 0.16) 
0.56 
Total number Grade 5 AEs 
 
3 
9 
 
 
Grade 5 AE, cryptococcal related 
N/A 
1 (1%) 
3 (3%) 
0.02 
(-0.02, 0.06) 
0.42 
Grade 5 AE, non-cryptococcal 
related 
25 (12%) 
2 (2.6%) 
5 (5.2%) 
0.03 
(-0.03, 0.08) 
0.38 
Overall 12-week Mortality 
Cryptococcal-related mortality 
60 (29%) 
19 (25%) 
23 (24%) 
0.0 
(-0.13, 0.12) 
0.94 
Non-cryptococcal related 
mortality 
33 (16%) 
11 (14%) 
16 (17%) 
0.03 
(-0.08, 0.13) 
0.65 
Cryptococcal Relapse 
3 (1.7%) 
0 (0%) 
0 (0%) 
-- 
-- 
Cryptococcal Paradoxical IRIS 
Cumulative Incidence* 
15 (9.6%) 
1 (6.7%)* 
1 (4.3%)* 
1.27 
(0.69, 2.32)§ 
0.45§ 
Nausea, Vomiting, or Diarrhea, 
≥1 event 
142 (68%) 
59 (77%) 
61 (64%) 
-0.12 
(-0.26, 0.01) 
0.08 
Serotonin Syndrome 
0 (0%) 
0 (0%) 
1 (1%)** 
-- 
-- 
ALT >5x ULN (Grade 3) 
3 (1.7%) 
2 (2.6%) 
0 (0%) 
 
 
T-Bilirubin >5x ULN (Grade 3) 
1 (0.6%) 
 
 
 
 
Premature sertraline dose 
reduction, all cause 
-- 
0 (0%) 
1 (1%) 
-- 
-- 
Premature sertraline 
discontinuation 
-- 
1 (1%) 
5 (5%) 
0.04 
(-0.01, 0.09) 
0.16 
Lost to Follow up 
0% 
3 (4%) 
2 (2%) 
-0.02 
(-0.07, 0.03) 
0.49 
After two weeks of sertraline induction therapy in combination with amphotericin and fluconazole 800-
1200mg/day, all participants received 200mg/day until week 8 and then tapered through week 11.  The 
historical 2010-2012 cohort only included ART-naïve persons who received amphotericin and fluconazole 
800mg/day of whom 31 died before randomization, and 177 were randomized into the COAT trial.35  
a Risk differences of 300 and 400mg/day versus 200 and 100mg/day. No statistical comparisons to the 
historical cohort were performed. 
* Percentages calculated from competing risk model for paradoxical cryptococcal-IRIS among patients 
without prior history of cryptococcal meningitis, who were ART-naïve and who survived to initiate ART 
during the trial period. (N=15 for 100 and 200mg/day; N=23 for 300 and 400mg/day.) Passive follow up 


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after 12 weeks identified 1 case of relapse in a 100mg/day participant after sertraline discontinuation. In 
COAT trial, 7 additional relapse episodes and 11 additional IRIS events occurred after 12 weeks through 
48 weeks 
§ Hazard ratio and 95% CI from competing risks regression and corresponding p-value. 
** Protocol deviation by a participant taking 800mg/day for three days. 
8.1.1. 
Regimen (dose, schedule, route, administration) 
Study administration: Patients enrolled in this study will receive sertraline or 
placebo, as outlined in Section 6.  This trial will use sertraline 100 mg tabs or masked 
placebo (Table 5).  
Table 5: Schedule of study medicine to be dispensed, tablets per day. 
Study Week  
1 
2 
3 
4-12 
13 
14 
15 
16 
Placebo Control, tabs 
1 
2 
3 
4 
3 
2 
1 
0 
Sertraline, tabs 
1 
2 
3 
4 
3 
2 
1 
0 
Note: Each sertraline tab is 100mg tablet 
The use of sertraline (or placebo) is divided into three phases.  During the 
escalation phase (the first 3 weeks), subjects will initially receive 100mg (1 tab) dose of 
sertraline/placebo for days 1-7 increasing to 200mg/day (2 tabs) for days 8-14.  After 
receiving 14 days, subjects will escalate to sertraline/placebo of 300mg/day (3 tabs) for 
days 15-21. After 21 days, subjects will increase to 400mg/day (4 tabs) on day 22.  
This escalation in dose will be facilitated via a blister pack of study medications with 
doses of placebo/sertraline apportioned for the first ~30 days. The 4 tabs daily will be 
continued through the week 12 visit. Sertraline (or masked placebo) will begin to be 
tapered off starting after week 12.  The taper will entail taking 300mg (3 tabs) daily for 1 
week, then 2 tablets daily for 1 week, and finally 100mg (one tablet) daily for 1 week. 
This taper will also be apportioned via a blister pack of study medications. Participants 
will be off of all study drugs after week ~15.  The tapered discontinuation is intended to 
avoid a withdrawal syndrome. 
Drug-drug interactions:  There are no drug interactions from sertraline with 
amphotericin, fluconazole, or first-line ART, with the exception of efavirenz, which is 
reported to lower serum sertraline levels by 39%.37 ART will be initiated at ~2-4 weeks 
and/or second line ART will be changed at 2-4 weeks.  
8.1.2. 
Rationale for Sertraline Dose Selection 
Sertraline is typically administered at doses of 50mg-200mg for depression. Doses 
above this threshold do not have any further efficacy for depression treatment, but 
there is not additional toxicity. Our group has studied sertraline in the treatment for 
symptomatic cryptococcal meningitis at doses of 100mg to 400mg.33 Notably, grade 4 


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or 5 adverse event risk did not differ between dosing of 100-200mg/day vs higher 
doses of 300-400mg/day.  
Prior studies have reported that efavirenz lowers serum sertraline levels by 39%.37  
In our Ugandan meningitis study, we found that the presence of any ART (e.g. 
efavirenz, nevirapine, or PIs) significantly increased sertraline clearance lowering the 
serum levels by ~50%.33 This effect is no longer seen at sertraline doses of 400mg/day. 
Thus ART-status does not appreciably affect sertraline levels at this 400mg daily dose.  
Table 6. Mean Steady State Sertraline Plasma Concentrations at Days 7-14. 
Dose Cohort 
Percentiles Sertraline Concentration (ng/mL) 
5 
10 
25 
50 
75 
90 
95 
200mg on ART (n=26) 
22 
26 
64 
101 
214 
336 
675 
200mg ART naïve (n=24) 
108 
122 
198 
257 
362 
512 
532 
300mg on ART (n=23) 
43 
67 
111 
194 
305 
367 
458 
300mg ART naïve (n=23) 
43 
126 
201 
298 
392 
577 
673 
400mg on ART (n=13) 
139 
146 
262 
395 
486 
722 
 
400mg ART-naïve (n=18) 
118 
128 
278 
414 
583 
679 
 
Figure 8. The Distribution of Sertraline Plasma Concentrations by ART Status 
Figure 8 provides a visual distribution of 
Table 6 data based on steady state plasma 
concentrations measured between day 7 and 
day 14.  This is based on 423 total 
measurements among 140 research 
participants of whom 127 received sertraline 
doses of 200-400mg/day in conjunction with 
fluconazole 800mg/day. 
 
 
 
 
 
Figure 9 displays the projected proportion achieving therapeutic levels of sertraline in 
the brain by dose and ART status based on Cryptococcus organism susceptibility.33 
Notably those on ART achieved lower brain concentrations compared to those not on 
ART. 33 At 400mg per day, highest brain concentrations of sertraline are achieved.  In 
general, there is bi-directional synergy between sertraline and fluconazole, with a two-
fold reduction in MIC (i.e. sertraline MIC=4 g/mL in the presence of fluconazole is 


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lowered to ~2 g/mL as well as the fluconazole MIC being lowered 2-fold). As per Table 
2, ~85% of Cryptococcus isolates have a MIC <4 g/mL. 
 
 
Figure 9. Projected Proportion with Therapeutic Sertraline Levels in Brain based on 
Sertraline Dose, ART Status, and Cryptococcus MIC Susceptibility.  
 
Based on the distribution of Cryptococcus MICs and tissue penetration,33, 59 we project in 
the presence of 2-fold additive/synergistic effects of fluconazole, 92% of participants 
receiving sertraline at 200mg/day without ART and 81% of participants receiving 
300mg/day with ART will achieve therapeutic concentrations of sertraline in the brain. With 


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400mg daily of sertraline, an estimated 95% will achieve therapeutic drug levels in the 
brain, regardless of ART status. Steady state levels in blood are achieved by 7 days of 
sertraline dosing.33 
Based on the average fold-change increase over blood, based on post-mortem studies,59 
therapeutic sertraline levels should be achieved by 100% of participants in liver, lung, 
spleen, and bile (Table 6). 
 
Table 6. Fold-Change Increase in Sertraline Tissue Concentration over Blood  
 
Liver 
Lung 
Kidney 
Spleen 
Muscle 
Brain 
Heart 
Bile 
Average 
86.2 
68.6 
7.4 
45.8 
2.1 
21.7 
8.9 
32.0 
 +SD 
92.5 
49.8 
5.3 
47.0 
1.3 
14.5 
7.8 
28.4 
 
 
 
 
 
 
 
 
 
Min 
12.5 
11.0 
3.7 
7.0 
0.9 
10.0 
1.6 
0.0 
25th Percentile 
26.9 
22.8 
4.3 
17.3 
1.0 
13.0 
3.8 
7.8 
Median 
46.4 
64.7 
5.2 
34.6 
1.5 
16.5 
5.3 
20.8 
75th Percentile 
102.7 
102.4 
6.8 
48.0 
3.5 
21.3 
13.0 
52.4 
Max 
322.3 
157.8 
18.4 
167.2 
4.1 
57.3 
22.6 
78.3 
 
 
8.1.3. 
Study Product Formulation and Preparation 
A U.S. FDA-approved sertraline manufacturer will be used (e.g. NorthStarRx, 
Memphis, TN) to provide Sertraline 100mg film-coated tablets.  
A matched placebo will be prepared in Uganda by Kampala Pharmaceuticals 
Industries, Ltd. located at Plot M444B / P.O Box 7551 Kampala, Uganda. A finished 
product certificate of analysis will be provided to certify assurance testing that the 
placebo matches in physical appearance, size, average weight, disintegration time, 
and that the placebo lacks sertraline. 
 
8.1.4. 
Study Product Supply and Accountability 
Product Storage and Stability:  Sertraline will be stored at room temperature 
(25ºC) with excursions permitted to 15ºC-30ºC (59ºF to 86ºF) according to 
manufacturer guidelines. Placebo may be stored at room temperature with 
excursions permitted to 15ºC-35ºC. 
Clinical personnel used in the dispensation and administration of study drugs 
(sertraline and placebo) or antifungals involved in the study will adhere to Good 
Clinical Practice (GCP) guidelines.  Pharmacies utilized in the study will maintain 
the supply and record keeping of all study drug and antifungal dispensing.  The 
pharmacist of record at the Infectious Diseases Institute will be the central 
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Compliance with the randomization will be assessed via ongoing quality 
assurance monitoring via CRF reporting of the dispensed medication (e.g. unique 
identifier) and the DataFax management system.  
 
8.1.5. 
Sertraline Side Effect Profile 
The most common treatment-emergent adverse events associated with the use of 
sertraline in placebo-controlled clinical trials in adult patients with major depressive 
disorder, obsessive-compulsive disorder, panic disorder, posttraumatic stress 
disorder, premenstrual dysphoric disorder, and social anxiety disorder (FDA 
Package Insert) are generally minor and not dose dependent. Table 7. 
 
BODY SYSTEM/ADVERSE EVENT 
ZOLOFT 
(N=2799) 
PLACEBO 
(N=2394) 
  
  
  
Autonomic Nervous System  
  
  
Ejaculation Failure (males) 
14 % 
1 % 
Mouth Dry  
14 % 
8 % 
Sweating Increased  
7 % 
2 % 
Central & Peripheral Nervous 
System  
  
  
Somnolence  
13 % 
7 % 
Insomnia  
21 % 
11 % 
Dizziness  
12 % 
7 % 
Headache  
25 % 
23 % 
Paresthesia  
2 % 
1 % 
Tremor  
8 % 
2 % 
Disorders of Skin 
  
  
Rash  
3 % 
2 % 
Gastrointestinal  
  
  
Anorexia  
6 % 
2 % 
Constipation  
6 % 
4 % 
Diarrhea / Loose Stools  
20 % 
10 % 
Dyspepsia  
8 % 
4 % 
Nausea  
25 % 
11 % 
Vomiting  
4 % 
2 % 
General  
  
  
Fatigue  
12 % 
7 % 
Psychiatric Disorders  
  
  
Agitation  
5 % 
3 % 
Anxiety  
4 % 
3 % 
Libido Decreased  
6 % 
2 % 
Nervousness  
5 % 
4 % 
Special Senses 
 
 
Vision Abnormal  
3 % 
2 % 
All other adverse events were <2% and/or of equal or greater frequency with placebo. 


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8.1.6. 
Sertraline Precautions  
Liver Disease. Sertraline is metabolized by the liver. As per the FDA package 
insert, “Liver impairment can affect the elimination of sertraline. In patients with 
chronic mild liver impairment (N=10, 8 patients with Child-Pugh scores of 5-6 and 2 
patients with Child-Pugh scores of 7-8) who received 50 mg sertraline per day 
maintained for 21 days, sertraline clearance was reduced resulting in approximately 
3-fold greater exposure compared to age-matched volunteers with no hepatic 
impairment (N=10).”  “The use of sertraline in patients with liver disease must be 
approached with caution. If sertraline is administered to patients with liver impairment, a 
lower or less frequent dose should be used.” 
Hepatotoxicity.  The FDA package insert lists hepatotoxicity, from double-blind placebo 
controlled trials as a “rare event,” defined as <1 in 1000 persons.51  
Specifically in regards to hepatotoxicity, during the ASTRO-CM pilot study (n=177) in 
Uganda,33 two participants (1%) developed new ALT/AST elevation while receiving 
sertraline 200mg/day and fluconazole 400mg/day (and no ART). The first participant at 6 
weeks had a serum ALT = 152 U/L (grade 2) and AST = 217 U/L (grade 3), which was 
attributed to alcohol.  The patient was counseled, and re-assessment 4 and 6 weeks later 
revealed resolution (ALT = 67 then 26 U/L, AST =70 then 29 U/L). No medications were 
changed.  A second participant, at 4 weeks had ALT = 402 U/L (Grade 3) and AST = 346 
U/L (Grade 3). Follow up re-check 5 days later (ALT = 100 U/L and AST 44 U/L) and one 
month later (ALT = 33, AST = 56) demonstrated resolution. No medications were 
changed.  
Sertraline plasma concentrations at 4 weeks were 433 and 228 pg/mL, respectively for 
these two participants, equating to ~80th and ~35th percentile for sertraline dosed at 
200mg/day.  
Outcomes among persons with baseline liver transaminase elevation have also been 
documented in HIV-infected Ugandans living with AIDS during the ASTRO-CM pilot. One 
participant had baseline asymptomatic Grade 4 ALT/AST elevations (ALT = 544, AST = 
918 U/L), which steadily declined over two weeks while receiving sertraline 400mg/day 
and fluconazole 800mg/day. After 14 days, the ALT = 92 and AST = 117 U/L. For this 
participant, plasma steady state levels between days 10-14 averaged 157 pg/mL (~20th 
percentile for 400mg/day dose group).  
Thus, baseline or new liver transaminase elevations did not correspond to elevated 
plasma sertraline levels.  Elevation of transaminases is not diagnostic of cirrhosis or liver 
impairment. 
Renal Disease. As per the FDA package insert, excretion of unchanged drug in 
urine is a minor route of elimination. “In volunteers with mild to moderate (CLCR=30-
60 mL/min), moderate to severe (CLCR=10-29 mL/min) or severe (receiving 
hemodialysis) renal impairment (N=10 each group), the pharmacokinetics and 
protein binding of 200 mg sertraline per day maintained for 21 days were not altered 


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compared to age-matched volunteers (N=12) with no renal impairment. Thus 
sertraline multiple dose pharmacokinetics appear to be unaffected by renal 
impairment.”  
Substantial Adverse Reactions listed in the FDA Package insert with <1 per 1000 
incidence.  
Hyponatremia.  “Hyponatremia may occur as a result of treatment with SSRIs and 
serotonin–norepinephrine reuptake inhibitors, including <sertraline>. In many cases, 
this hyponatremia appears to be the result of the syndrome of inappropriate 
antidiuretic hormone secretion (SIADH). Cases with serum sodium lower than 110 
mmol/L have been reported. Elderly patients may be at greater risk of developing 
hyponatremia with SSRIs and serotonin–norepinephrine reuptake inhibitor. Also, 
patients taking diuretics or who are otherwise volume depleted may be at greater 
risk. Discontinuation of <sertraline> should be considered in patients with 
symptomatic hyponatremia.”51 
Platelet Function. “There have been rare reports of altered platelet function and/or 
abnormal results from laboratory studies in patients taking <sertraline>. While there 
have been reports of abnormal bleeding or purpura in several patients taking 
<sertraline>, it is unclear whether <sertraline> had a causative role.51 
Serotonin Syndrome. “Patients should be cautioned about the risk of serotonin 
syndrome with the concomitant use of serotonin–norepinephrine reuptake inhibitors 
and SSRIs, including <sertraline>, and triptans, tramadol, or other serotonergic agents,” 
such as tricyclic antidepressants, fentanyl, lithium, tryptophan, buspirone, and St. 
John’s Wort. These medications are listed as prohibited medications in Protocol Section 
8.4. Manifestations of serotonin syndrome include: confusion, agitation, reduced level of 
consciousness, seizures, myoclonus/clonus, hyperreflexia, tremors, muscle rigidity, 
ataxia, akathisia, hyperthermia, hypertension, tachycardia, diaphoresis, lacrimation, 
mydriasis, shivering, and/or diarrhea.   
Acute Angle Closure Glaucoma is an acute event associated with nausea, 
vomiting, and acute eye pain. The more common pre-existing glaucoma would be 
open-angle glaucoma.  The incidence of angle-closure glaucoma increases with 
age, is more common in women, and has a greater prevalence in Asians and East 
Indians than in Europeans and African. The prevalence of angle-closure glaucoma 
in the South Africa Black population was 2 per 100,000 per year. Most cases of 
primary angle closure glaucoma occur in the 6th to 7th decade of life. 
8.1.7. 
Electrocardiogram QT Interval Safety – Published Data  
Prolonged QT interval is considered a class effect seen with some SSRIs, 
particularly with citalopram (Celexa) and escitalopram (Lexapro).  However, of the 
SSRIs, sertraline has the least effect on QT interval.43  The FDA package insert states, 
“the electrocardiograms of 774 patients who received ZOLOFT in double-blind trials 


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were evaluated and the data indicate that ZOLOFT is not associated with the 
development of significant EKG abnormalities.”  A meta-analysis of 4292 adults 
receiving any SSRI, reported an average 3ms increase in QTc interval among persons 
receiving sertraline. 
In Uganda, during electrocardiogram QT monitoring of 53 research participants over 
2 weeks with cryptococcal meningitis in Uganda receiving sertraline + fluconazole 
800mg/day + amphotericin + ART, there was no evidence for QT prolongation (Table 
8).  
The mean change in QTc is -23ms (95% CI +0.75 to -45.2ms, P=0.043 by paired t-
test) from baseline to day 14 of fluconazole 400mg/day and various doses of sertraline 
(n=29 pairs). Among those receiving 300-400mg/day, there was no dose-dependent 
effect (P=0.56), and overall the mean QTc interval had a greater shortening with the 
higher doses (i.e. -28ms change with 300-400mg/day vs. -14ms change with 100-
200mg/day).     
 
Table 8. Mean electrocardiogram QTc Interval with adjunctive sertraline therapy 
and fluconazole 800mg/day in persons with cryptococcal meningitis at Mulago 
Hospital, Uganda.  
 
N 
Day 1 
QTc (ms) 
N 
Day 7 
QTc (ms) 
N 
Day 14 
QTc (ms) 
P-value 
Sertraline Dose 
100–200mg/day 25 
381 
(367, 395) 
23 
391 
(383, 399) 
18 
367 
(343, 390) 
0.56 
300–400mg/day 29 
383 
(372, 394) 
26 
392 
(368, 416) 
18 
355 
(331, 380) 
300mg/day 
13 
384 
(367, 401) 
11 
393 
(381, 406) 
8 
340 
(288, 393) 
0.33 
400mg/day 
16 
382 
(366, 399) 
15 
390 
(347, 434) 
10 
367 
(343, 391) 
Sex 
Men 
39 
383 
(373, 394) 
35 
389 
(371, 407) 27 
369 
(352, 385) 
0.35 
Women 
15 
379 
(363, 395) 14 
396 
(386, 406) 
9 
337 
(291, 383) 
ART at Diagnosis 
Receiving ART 
24 
377 
(361, 393) 
23 
399 
(373, 424) 
13 
365 
(340, 391) 
0.50 
Not receiving 
ART 
30 
386 
(377, 396) 26 
385 
(373, 396) 
23 
358 
(336, 381) 
Data displayed as mean (95%CI). P-values testing for interaction between follow-up 
time and sertraline dose group, sex, and ART at diagnosis, from linear mixed model. 


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No ASTRO-CM trial participants developed a new QTc >450ms (Grade 1 DAIDS 
Toxicity) while on sertraline, fluconazole 800mg/day, and amphotericin. 
 
8.1.8. 
 Sertraline Safety in Overdose 
SSRIs have a wide therapeutic margin of safety.43, 44  
Among a published case series of 469 SSRI intentional overdoses among 156 
persons taking sertraline alone, overdosing on a median of 24x daily dose (IQR 16-
30) of 200mg/day, toxicity (i.e. median ingestion = 4800mg).43 Among this sertraline 
group: 2% (n=3) developed self-limited seizures and 20% (n=31) serotonin 
syndrome. There were no cases of severe serotonin syndrome requiring ICU 
admission. Another 3% (n=4) presented with a GCS<9, but this associated with co-
ingestion of sedative medications. Of 310 overdoses, solely of SSRI medicines, with 
EKGs obtained, the following table demonstrates cardiac toxicities observed as 
median (IQR).43  
Table 9. Cardiovascular Changes with SSRI Overdoses 
Parameters With 
Overdose 
Sertraline 
(Zoloft®) 
Paroxetine 
(Paxil®) 
Fluoxetine 
(Prozac®) 
Citalopram 
(Celexa®) 
ASTRO 
Pilot 33 
N 
103 
78 
42 
57 
26 
Age, years 
28 
(20-37) 
31 
(23-28) 
35 
(23-41) 
30 
(22-41) 
37 
(31-42) 
Daily Defined Dose 
ingested of 200mg 
24x 
(16-30) 
15x 
(10-28) 
16x 
(10-26) 
16x 
(10-25) 
1.5-2x 
Heart Rate 
82 
(68-97) 
78 
(68-90) 
82 
(71-100) 
77 
(68-90) 
 
Hypotension, 
systolic <90 mmHg 
0 
0 
0 
2 (3.5%) 
0 
Arrhythmia 
0 
0 
0 
1 (1.8%) 
0 
EKG QT, ms 
380 
(360-400) 
375 
(350-400) 
380 
(360-400) 
400 
(360-440) 
302 
(261-337) 
EKG QTc, ms 
429 
(413-456) 
427 
(409-454) 
432 
(412-466) 
450 
(436-484) 
382 
(319-397) 
EKG QTc >500ms 
6 (6%) 
1 (1%) 
4 (10%) 
7 (12%) 
0 (0%) 
In a comparison group (n=309) ingesting non-cardiac toxic medications at the same site, 
the median QTc was 423ms (IQR, 404-447) versus the QTc of 429 (IQR, 413-456) in 
sertraline overdose.43  
The wide therapeutic safety margin is evidenced by the existence of evidence-based 
consensus guideline for out-of-hospital management for SSRI poisonings, based on the 
review of 48,000 SSRI exposures at U.S. Poison Centers in the year 2004.44  
For asymptomatic patients or those with mild effects following acute SSRI ingestions of 
sertraline of up 1250mg can be observed at home without any medical evaluation.44  Mild 


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effects were defined as vomiting, mydriasis, diaphoresis, or somnolence (but arousable with 
speaking voice or light touch).44 
 
 
 


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8.2. 
Fluconazole 
Fluconazole will be provided via the clinic as standard of care.13 Interruptions in 
care resulting from supply stock outs of standard therapies will be recorded and 
taken into account in the analyses. 
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 200 mg 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%, and CSF concentrations 
are ~100% of plasma levels. 
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:  Fluconazole is a standard of care medicine which may be 
obtained from the Ministry of Health, directly from the Diflucan Partnership Program 
(which in 2016 has a very intermittent supply), or any nationally registered or WHO 
prequalified fluconazole supply is acceptable. 
Product Storage and Stability:  Fluconazole needs to be stored below 86°F 
(30°C) and above 41°F (5°C). Storage shall be per the manufacturer’s 
recommendation. 
Study administration:  Fluconazole is an oral medication, used for the 
treatment of many fungal infections including cryptococcosis.  Dosing in this study 
will be per Ugandan and WHO standard of care.13  
Possible side effects of the medication are rare, but include headache, rash, 
nausea, vomiting, diarrhea, and abdominal pain.  Patients will be warned of 
potential side effects at study entry, and closely monitored for adverse events.  It is 
recommended that fluconazole doses of ≥800 mg/day be divided into at least twice 
daily administration to decrease potential GI side effects (e.g. nausea). 
Side Effects:  FDA Diflucan® label states: “Clinical adverse events were 
reported more frequently in HIV infected patients (21%) than in non-HIV infected 
patients (13%); however, the patterns in HIV infected and non-HIV infected patients 
were similar. The proportions of patients discontinuing therapy due to clinical 
adverse events were similar in the two groups (1.5%).  The following treatment-
related clinical adverse events occurred at an incidence of 1% or greater in 4048 


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patients receiving DIFLUCAN for 7 or more days in clinical trials: nausea 3.7%, 
headache 1.9%, skin rash 1.8%, vomiting 1.7%, abdominal pain 1.7%, and 
diarrhea 1.5%.” 
8.3. 
Assessment of Participant Adherence  
Pharmacy will record the dispensing history for the study medicine.  
Reconciliation of all study medicines will be performed by pharmacy staff (or their 
designee) via pill counts at time of dispensing and/or collection of returned study 
medicines.  
Study medical officers will query self-reported adherence at outpatient visits to 
clarify participant understanding of the correct dosing and/or the number of missed 
study medicines since the last visit.  Similar self-reported assessment of fluconazole 
and ART adherence will occur.  
Adherence to the exact taper will not be assessed at week 16 as no study 
medicines should be left. Pharmacy reconciliation will however occur if there are 
leftover study medicines at the week 16 visit. 
8.4. 
Prohibited Medications 
Monoamine oxidase inhibitors (MAOIs) and pimozide are contraindicated. These 
are not registered nor available in Uganda.  These are antidepressants and an 
exclusion criterion. 
Other prohibited medications include serotonergic drugs: sumatriptan, zolmitriptan, 
tricyclic antidepressants, fentanyl, lithium, tramadol, tryptophan, buspirone, and St. 
John’s Wort.  None of these medications are commonly available in Uganda (i.e. not 
available nor prescribed).  
A medication log will record all medications/substances taken by participants 
Below is a list of precautions taken by clinicians, per standard of care.  
8.5. 
Concomitant Medications and Procedures 
Standard HIV care shall be prescribed as part of routine care outside of this study. 
1. There is an interaction between sertraline and efavirenz, whereby sertraline levels 
are reported to be lowered by 39% by efavirenz, without an effect on efavirenz.37  
Efavirenz is first line ART.  In Uganda, we found a ~50% reduction in the area under 
the curve (AUC) exposure at lower doses of sertraline 100-300mg/day.33  However, 
at sertraline doses of 400mg daily, this effect was no longer seen, with non-
statistical differences in plasma levels.33  Thus, there will be no dose adjustment 
based on antecedent ART-status. 


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2. Darunavir/ritonavir combination also decreases the sertraline AUC by ~49% 
(90%CI, 42%-54%) and Cmax by ~44% (90%CI, 37%-51%) (Prezista FDA Package 
insert).  There is a paucity of published data on other protease inhibitors, but with 
the similar effect observed with darunavir/ritonavir, persons on second-line ART 
using protease inhibitors will also receive the same dosing schedule of the study 
medicine (sertraline vs. placebo). The lower AUC is likely due to the induction of 
metabolism by ritonavir of hepatic P450 enzymes of CYP2B6 (33-50%), CYP2CP 
(33-40%), and CYP2C19.62 
3. If steroids are to be prescribed, prednisolone would be preferred with avoidance of 
dexamethasone which induces the metabolism of sertraline.  
4. Rifampin enhances the metabolism of concurrently administered fluconazole, 
decreasing the AUC by 23%.  
FDA Package Insert: “Administration of a single oral 200 mg dose of 
DIFLUCAN after 15 days of rifampin administered as 600 mg daily in eight 
healthy male volunteers resulted in a significant decrease in fluconazole AUC 
and a significant increase in apparent oral clearance of fluconazole. There 
was a mean ± SD reduction in fluconazole AUC of 23% ± 9% (range: –13 to 
–42%). Apparent oral clearance of fluconazole increased 32% ± 17% (range: 
16 to 72%). Fluconazole half-life decreased from 33.4 ± 4.4 hours to 26.8 ± 
3.9 hours.”  
U.S. DHHS HIV OI Guidelines recommend: “Monitor for antifungal efficacy; 
may need to increase fluconazole dose.”63 
5.  Rifampin may also enhance the metabolism of sertraline, but this is not clearly 
known. During the ASTRO pilot using sertraline,33 minimal drug-drug interaction 
was observed. Rifampin at time of study entry is an exclusion criterion; however, if 
persons are diagnosed with TB after study entry and require TB therapy, 
participants shall remain in the C-ASSERT trial. 
6. Co-administration of other drugs known to prolong the QT interval and which are 
metabolized via enzyme CYP3A4 such as quinidine are contraindicated in patients 
receiving fluconazole.  If malaria treatment is necessary, an artesunate-based 
regimen should be used, which is in accordance to national guidelines. 
Prolonged QT interval is considered a class effect seen with SSRIs, particularly with 
citalopram.  However, of the SSRIs, sertraline has one of the least effects on QT 
interval.43  Nevertheless, a “thorough QT/QTc study” has been conducted to assure 
safety, as per FDA guidance.64 Among participants in the ASTRO-CM pilot study 
who received sertraline up to 400mg/day and fluconazole 800mg/day. The mean 
QTc interval decreased by -23ms (95%CI, +0.75 to -43ms, P=0.04) overall.33 There 
was no difference in QTc change between 100-200mg/day doses and 300-


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400mg/day doses (P=0.56) with higher doses trending toward greater decrease.33 
This is a “negative thorough QT/QTc study” based on the upper 95%CI <10ms by 
FDA criteria.64 In the ASTRO-CM study, the upper 95%CI was +0.75ms.33  At time 
of higher sertraline doses of 300-400mg/day, fluconazole will be dosed at 
400mg/day, which does not have an appreciable QTc effect. 
7. Zidovudine (AZT):  Fluconazole increases Cmax and AUC of zidovudine by 84% and 
74%, respectively, due to an approximately 45% decrease in oral zidovudine 
clearance.  The half-life of zidovudine was likewise prolonged by approximately 
128% following combination therapy with fluconazole.  Patients receiving this 
combination will be monitored for the development of AZT-related adverse reactions 
with the repeated scheduled CBCs at 4, 8, and 12 weeks.  
8. Protease Inhibitors:  Fluconazole may increase the serum concentration of some 
protease inhibitors.  Thus, it is not recommended to administer high dose 
fluconazole with protease inhibitors.  Participants in our study will not be started on 
protease inhibitors while still receiving high-dose fluconazole. If participants are to 
be started on second line ART, participants should be started on protease inhibitors 
after reduction to <=400mg/day of fluconazole after 2 weeks (i.e. switching to 
second line ART should occur at/after 2 weeks). 
9. Nevirapine:  Fluconazole is known to increase the concentration of Nevirapine.65  If 
administered together, participants will be closely monitored for nevirapine-
associated adverse events.  Healthcare providers will be encouraged to prescribe 
an efavirenz-based ART regimen if possible, given drug interactions between 
nevirapine and fluconazole. Nevirapine is no longer a first line ART medicine.  The 
fluconazole dose is expected to be 400 mg/day at the time of ART initiation.  This 
dosage of fluconazole used in conjunction with nevirapine is safe and well tolerated. 
66 Thus nevirapine is not contraindicated; however, efavirenz remains preferred. 
 
8.5.1. 
Rescue Medications 
Persons developing symptomatic meningitis shall be referred to hospital for 
appropriate management, which should include amphotericin B based combination 
therapy for culture-positive meningitis.  


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8.6. 
Pharmacokinetic Interactions between Sertraline, Antiretrovirals, and Fluconazole.  
Sertraline has relatively few drug-drug interactions as it is metabolized by five different cytochrome p450 pathways. Antiretrovirals 
appear to be the exception to the rule as both NNRTI and PIs induce multiple pathways of metabolism (Table 10). 
Drug 
Table 10. Sertraline Hepatic P450 Metabolism Pathways 
(% of sertraline metabolism) 
CYP2B6 
(36-40%) 
CYP2C9 
(15-17%) 
CYP2C19 
(4-14%) 
CYP3A4 
(15-18%) 
CYP2D6 
(16%) 
Efavirenz 
Induction varies by genetic 
polymorphism,67 ~55% at 
400 mg.68 
Moderate Inhibitor, 
increase EFV 1.7-2 fold 
at steady state 69 
Inducer,70 dose 
dependent, ~20% at 3 
mcg/mL EFV69 
Inducer 70 
~20%69 
Negligible 71 
Nevirapine 
Inducer,72 with 20-50% 
decrease AUC 73 
No effect 71 
No effect 71 
Weak Inducer 72, 
74 
Negligible 71 
Dolutegravir 
No effect 75 
No effect 75 
No effect 75 
No effect 75 
No effect 75 
Lopinavir/r 
Negligible 76, 77 
29% induction by 
LPV/r78 
100% induction by 
LPV/r78 
CYP3A 
Substrate / 
Weak inhibitor78 
Negligible at 
clinically relevant 
LPV/r levels79 
Atazanavir 
No effect 80 
Negligible Inhibition at 
clinically relevant 
doses80, 81 
Negligible Inhibition at 
clinically relevant 
doses 80, 81 
Substrate / 
Weak inhibitor82 
No effect80 
Darunavir/r* 
Induction 80 
Induction 80 
Induction 80 
Inhibition by 
DRV/r 80 
No effect 80 
Ritonavir 
Induction in vivo 62 
33-50% 
Induction62 
~33-40% 
Induction 62 
Strong 
Inhibitor76 
Inhibitor62 
Cobicistat 
Negligible 83 
Negligible 83 
Negligible83 
Strong 
Inhibitor84 
Inhibitor85 
Fluconazole  
(at 400mg) 
Negligible 86 
Modest Inhibitor73, 87 
~40% in vitro 
Moderate Inhibitor73, 87 
~70% in vitro 
Negligible 86, 87 
Negligible 86 
* darunavir/ritonavir reduces sertraline AUC by 49%.88 
Existing Ugandan sertraline PK data33 were collected with concomitant fluconazole 800mg/day. There is dose dependent inhibition of hepatic P450 
CYP2C9 and CYP2C19 pathways by fluconazole which accounts for 20-30% of sertraline metabolism.  Ritonavir also induces and inhibits pathways 
of sertraline metabolism. Overall, ritonavir induces pathways accounting for 70% of sertraline metabolism and inhibits pathways accounting for 40% 
of sertraline metabolism.  The net effect when studied with Darunavir/ritonavir was a 49% reduction in the sertraline AUC.88 This is similar as to the 
PK effect observed in Uganda with NNRTI regimens of efavirenz or nevirapine.33  Thus, we would expect a similar PK effect on sertraline with any 
ART regimen, most likely.  


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9. STUDY PROCEDURES / EVALUATIONS 
 
9.1. 
Schedule of Events  
 
*Pathogenesis studies including CrAg titer, peripheral blood mononuclear cell (PBMC) collection, 
RNA collection (2.5mL), and/or antigen stimulation may occur at selected clinical sites with the 
capacity to store these samples (Refer to Appendix C). Approximately 40mL of blood will be 
drawn in total for research purposes during the study, after consent.  Refer to laboratory SOPs 
for further details on collection and processing. CrAg titers may be performed on stored 
specimens, in batch. 
† Lab monitoring will consist of complete blood count (CBC) with platelets, Na+, K+, 
creatinine, ALT, and total-bilirubin.  
9.2. 
CrAg Screening 
CrAg screening may occur at time of CD4 testing with predominantly lab-
based CrAg testing or by provider initiated testing. This CrAg screening is 
standard of care in accordance with national and WHO guidelines. This CrAg 
testing will occur at the CD4 referral laboratories for each site (Refer to Appendix 
C for site description). Point-of-care CrAg testing is also acceptable, if not 
previously tested (e.g. insufficient specimen for CrAg testing, discarded specimen) 
Event 
CRAG 
Screen 
Pre-
Entry 
Enroll 
2w 
4w 
8w 
12w 16w 20w 24w Sick 
Visit 
CD4 Count 
Routine 
Care 
 
 
 
 
 
 
 
 
 
 
CrAg 
X 
 
 
 
 
 
 
 
 
 
 
Review Eligibility 
 
X 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
 
Detailed Study Visit 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Mental Health PHQ-9 
 
 
X 
 
X 
X 
X 
 
 
 
 
Assess for AEs 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CrAg Titer 
X 
 
+* 
 
 
+* 
 
 
 
 
 
Pregnancy Test 
 
♀X 
 
 
 
 
 
 
 
 
+♀ 
Lab Monitoring † 
 
ALT 
X  
 
X 
X 
X 
 
 
 
X 
EKG Monitoring 
 
X 
 
 
X 
X 
X 
 
 
 
 
Fluconazole, mg 
 
 
800 
400 
400 
400 
200 
200 
200 
 
 
Randomization 
 
 
X 
 
 
 
 
 
 
 
 
Study medicine, tabs 
 
 
1 → 2 3→4 
4 
4 
Taper 
0 
0 
0 
 
ART Initiation/Switch 
 
 
 
Target Target 
 
 
 
 
 
 
Specimen Storage* 
X 
 
X 
 
X 
X 
X 
 
 
 
X 


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when performed by a trained research staff member, as per the Delegation of 
Responsibilities log.  A positive CrAg result is generally considered a critical lab 
value. Upon notification of the CrAg result, clinic staff and/or research study staff 
will attempt to contact the person to have them return to clinic on an urgent basis. 
Contact may be by telephone, alternative listed contact in their clinic medical 
record, or active patient tracking.  
If leftover specimens exist, it is acceptable to test for ALT and pregnancy 
using this specimen.  
9.3. 
Pre-Entry Visit 
Pre-Entry Visit will assess eligibility for study enrollment by research staff. The 
research staff will perform the symptom-screen for meningitis, to confirm that the 
patient has no signs or symptoms concerning for meningitis.  This is based on the 
healthcare provider’s clinical impression.  
If the patient is otherwise asymptomatic, and there are no contraindications to 
study participation, the patient will be asked to provide informed consent for 
possible study participation (if eligible). HIV care (prescription of ART and 
fluconazole) from this point on will be performed by research staff until completion 
of 24 weeks of the study, or if the participant withdraws consent for further 
participation. Informed consent will be obtained by study personnel.  Women of 
reproductive potential must have a pregnancy test performed on either blood or 
urine. This may be performed as point of care testing or at the local site 
laboratory. If they are pregnant at time of enrollment, they are not eligible for the 
study.   
 Activities shall include: 
 Clinical Assessment for Symptomatic Meningitis 
 Informed Consent 
 Pertinent Medical History 
 Checklist of Inclusion/Exclusion Criteria 
 Pregnancy Testing (if not already obtained on CRAG screening sample) 
 ALT Testing (if not already obtained within 30 days of pre-entry visit) 
 EKG with QTc measurement.  
o QTc must be <450ms for participation. 
If the patient has signs or symptoms concerning for symptomatic meningitis, as 
suggested by meningitis symptoms as per the clinician’s judgment, the 
provider will refer the patient for further care, including consideration of a 
lumbar puncture if thought to be necessary. Referral will be made to the 
appropriate healthcare facility, which can perform a diagnostic lumbar 
puncture. This is routine care and considered best clinical practice. Our study 
will adhere to this routine standard of care practice. If 1) lumbar puncture does 
not suggest features of meningitis, 2) CSF is CrAg-negative, and 3) there is no 
longer a suspicion for meningitis, the patient can be eligible, at provider 
discretion.  


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If not already prescribed by another healthcare provider, fluconazole should be 
prescribed as soon as possible. CrAg+ persons may receive up to 7 days of 
fluconazole prior to randomization (at the enrollment visit) and still be eligible.  
 
9.4. 
Enrollment Visit (Week 0) 
If the participant is eligible for study participation, they will be randomized into 
the C-ASSERT trial. This may occur immediately after the Pre-entry visit (i.e. 
same clinic visit), or may require a second visit if all the inclusion/exclusion criteria 
results are not available (e.g. ALT lab result).  Randomization is stratified by site. 
The day of randomization is Day Zero. 
The following evaluations will be performed at the enrollment visit:  
 Labs: CBC (with platelets), Na+, K+, Creatinine, ALT, and Total 
bilirubin; 
 Patient Health Questionaire-9 (PHQ-9) questionnaire; 
 Documenting concomitant medications; 
 Research laboratory collections, as available at the sites, which may 
include: PaxGene RNA, DNA, PBMC, serum, and plasma – with 
subject written consent for specimen storage. 
At enrollment, plasma specimens will be collected / stored for CrAg titer and 
pathogenesis investigations. A PAXGene RNA tube (2.5mL) may be collected per 
site with storage capacity depending on study site and available funding for 
storage and to perform further testing.  
Study medicine will be dispensed by the study pharmacy.  The initial 
escalation phase of dosing will be provided in a blister-pack with a numbered 
sequence of up to 30 days study medication.  
Research staff will be responsible for refilling HIV medications and 
fluconazole for the duration of participation in the research study.  
9.5. 
Follow-Up Detailed Study Visits 
Follow up HIV care will occur with routine visits as per the standard of care.  
Specific detailed study visits will occur as per the Schedule of Events (Section 
9.1). Detailed study visits will have completion of a visit CRF at weeks 2, 4, and 
every 4 weeks thereafter through study termination at 24 weeks.  The study visit 
windows are +2 weeks.  These detailed follow up visits shall document and 
assess for: 
1) Symptoms consistent with cryptococcal disease and/or meningitis 
2) Clinically significant serious adverse events 
3) Medication adherence, problems, or discontinuation for study medicine, 
fluconazole, and ART 
4) For women, last menstrual period while receiving >400mg/day 
fluconazole. 


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A checklist will be used to query for symptoms of serious adverse events, which 
shall include: meningitis, CNS symptoms, serotonin syndrome, acute angle 
closure glaucoma, bleeding episodes, and other episodes of clinical importance. 
A mental health screen will be performed using Patient Health Questionaire-9 
(PHQ-9) at enrollment, week 4, week 8, and week 12.  After 16 weeks, the PHQ-9 
will be performed as clinically indicated. The PHQ-9 has nine questions which 
assess for depression as well as suicidal ideations and self-harm. Refer to Manual 
of Operations (MOP) for more details on the PHQ-9. 
In the event patients have transferred care, a telephone visit is acceptable for 
collection of data, per our intention to treat analysis. This is preferable to a missed 
visit and no data collection. The telephone visit will be used to assess for vital 
status (alive, dead) and for symptoms of meningitis. A telephone visit is not 
preferred but is preferable to no assessment.   
9.5.1. 
Week 2 Visit: ART initiation  
Pre-ART counseling and initiation of ART will occur per standard clinic 
protocols. ART initiation will be recommended at approximately 2-4 weeks after 
study entry.  The exact timing of ART initiation will not be mandated by the 
protocol, as this will be performed per standard of care in clinics and at provider 
discretion. There is insufficient evidence to guide exact timing of ART initiation 
among patients with asymptomatic cryptococcal antigenemia. Similarly, 
participants with virologic failure will be recommended for switch to second line 
therapy at 2-4 weeks. At follow up visits, ART adherence will be assessed via 
participant interview.  If there are concerns, pill counts and/or repeat ART 
counselling should be conducted.  
9.5.2. 
Week 4 and 8 visits 
A checklist will be used to query for symptoms of serious adverse events, which 
shall include: meningitis, CNS symptoms, serotonin syndrome, acute angle 
closure glaucoma (i.e. acute eye pain), bleeding episodes, and other episodes of 
clinical importance. Assessments will include: 
 
Symptoms consistent with cryptococcal disease and/or meningitis 
 
Clinically significant serious adverse events 
 
Medication adherence, problems, or discontinuation 
 
Patient Health Questionaire-9 (PHQ-9) 
 
Laboratory monitoring of: CBC (with platelets), Na+, K+, Creatinine, 
ALT, and Total bilirubin. 
 
EKG 
 
In the event patients have transferred care, a telephone visit is acceptable for 
collection of data, per the intention to treat analysis. A telephone visit is not 
preferred but is preferable to no assessment.  


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At the week 4 visit, the initially prescribed blister pack of study medicine will be 
returned with pharmacy conducting a reconciliation of returned medications. 
  
9.5.3. 
Week 12 Visit 
A checklist will be used to query for symptoms of serious adverse events, which 
shall include: meningitis, CNS symptoms, serotonin syndrome, acute angle 
closure glaucoma (i.e. acute eye pain), bleeding episodes, and other episodes of 
clinical importance. Assessments will include: 
 
Symptoms consistent with cryptococcal disease and/or meningitis 
 
Clinically significant serious adverse events 
 
Medication adherence, problems, or discontinuation 
o Pill count of returned study medications. 
 
Patient Health Questionaire-9 (PHQ-9) 
 
Laboratory monitoring of: CBC (with platelets), Na+, K+, Creatinine, 
ALT, and Total bilirubin. 
 
EKG 
 
At the week 12 visit, the participants shall be instructed to begin tapering by 
one study medicine tablet per week (3, 2, 1, and then stop). Study 
medications will be dispensed in a blister pack for the taper.  
As the patient will be an outpatient, the exact taper will not be monitored. 
Please refer to Pharmacy SOP for further details about how study drug will be 
supplied and monitored. 
  
9.5.4. 
Week 16 Visit  
A checklist will be used to query for symptoms of serious adverse events, which 
shall include: meningitis, CNS symptoms, serotonin syndrome, acute angle 
closure glaucoma (i.e. acute eye pain), bleeding episodes, and other episodes of 
clinical importance. Assessments will include: 
 
Symptoms consistent with cryptococcal disease and/or meningitis 
 
Clinically significant serious adverse events 
 
Any remaining study medicine should be returned at the week 16 study visit or 
termination visit, for destruction by the pharmacy.  No remaining study medication 
is the expectation. 
Laboratory monitoring shall be performed for follow up of any Grade >3 
abnormality to document resolution. EKG monitoring shall be performed for follow 
up of any Grade >3 abnormality (i.e. QTc >500ms). 
 


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9.5.5. 
Week 20 Visit  
A checklist will be used to query for symptoms of serious adverse events, which 
shall include: meningitis, CNS symptoms, serotonin syndrome, acute angle 
closure glaucoma (i.e. acute eye pain), bleeding episodes, and other episodes of 
clinical importance. Assessments will include: 
 
Symptoms consistent with cryptococcal disease and/or meningitis 
 
Clinically significant serious adverse events 
For follow up of any unresolved Grade >3 abnormality, laboratory monitoring shall 
be performed to document resolution. EKG monitoring shall be performed for 
follow up of any unresolved Grade >3 abnormality (i.e. QTc >500ms). 
 
9.5.6. 
Other Routine / Unscheduled Clinic Visits 
At other routine clinic visits for HIV care being conducted for medication refills, if 
persons are asymptomatic, no CRF need be completed.  If participants are ill, 
refer to Section 9.5.8 regarding sick visits. 
 
9.5.7. 
Pregnancy Visit 
At time of a concern for suspected pregnancy,  
1) Pregnancy test shall be performed 
If pregnant: 
2) Site PI (or designee) must be notified within 24 hours. 
3) CrAg titer on serum/plasma shall be performed. 
4) Fluconazole dose reduction should potentially be dose reduced as 
considered per discussion between the site PI, clinical provider, and the 
participant. See discussion in section 11.5.  
5) Sertraline/Placebo should be tapered as rapidly as is feasible, but with 
recognition that abrupt discontinuation can be associated with 
withdrawal may be elected to be continued or discontinued per 
discussion between the clinical provider and the participant. Sertraline is 
a FDA Category C, and SSRIs are generally thought to be a low risk. In 
general, study med tablets should be tapered approximately one per 
week.  Refer to Section 11.5 
9.5.8. 
Other Sick Visits 
Study personnel shall evaluate participants who present for sick visits as 
clinically appropriate and refer for hospitalized care, as needed. All sick visits 
must have an assessment for serious AEs. The study shall provide an 
appropriate diagnostic evaluation for any new AE. Participants will not need to 
pay for this evaluation. Specimens shall be collected as clinically appropriate 
(e.g. CSF, serum, plasma, etc.) based on physician discretion of the illness 
present. Women with a concern for pregnancy should have a pregnancy test 
checked. A visit CRF shall be completed for sick visits, even if the ultimate 
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9.5.9. Study Termination Visit 
The final study visit shall be targeted at 24 +2 weeks. The Termination CRF 
shall be completed.  If the participant has transferred care, a telephone call is 
acceptable to document vital status and document any relevant serious adverse 
events (e.g. meningitis, hospitalization, etc.).   
If persons are transiently lost to follow up, but return after 24 weeks, the 
Termination CRF shall be completed/updated with the known alive vital status.  
Similarly, if a participant is initially deemed as lost to follow up, but is later known 
to have died before 24 weeks, the CRF shall be updated. 
For persons lost to follow up, tracking is highly encouraged to assess their vital 
status and whether symptomatic meningitis has occurred. For deaths, the 
proximal contributing cause(s) of death shall be documented, if known.  If a death 
occurs in a healthcare facility a post-mortem exam may be offered to the next-of-
kin, as per routine medical practice. 
For participants with ongoing clinical AEs (Grade 3-4) at time of 24 weeks, 
they should be continued to be followed until AE resolution and/or final outcome 
(Refer to Section 10.3 on AE reporting). Laboratory monitoring shall be performed 
for follow up of any Grade >3 abnormality to document resolution, as clinically 
relevant. EKG monitoring shall be performed for follow up of any unresolved 
Grade >3 abnormality (i.e. QTc >500ms).  
 
9.6. 
Laboratory Evaluations 
9.6.1. Cryptococcal Antigen lateral flow assay (CrAg LFA) 
In July 2011, a lateral flow immunoassay was approved by the U.S. FDA for 
detection of cryptococcal antigen (Immy, Inc., Norman, Oklahoma). The CrAg 
lateral flow assay (LFA) is a rapid diagnostic dipstick immunochromatographic 
assay that provides a definitive result for presence of CrAg within 15 minutes.  
The procedure requires a drop (40μL) of serum, plasma, urine, or CSF specimen 
to be placed in a test tube, and the LFA dipstick is then placed into the same 
reservoir.  If CrAg glucuronoxylomannan capsular polysaccharide is present in the 
specimen, once the lateral flow dipstick comes in contact with the specimen, the 
LFA will bind to the gold-conjugated, anti-cryptococcal antibodies on the test strip 
causing a visible line. 
 


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The LFA can be run individually using an Eppendorf tube (or similar) as a 
reservoir or can be batched on 96-well plates.  
The CrAg LFA is an ideal point of care test, as persons with minimal training can 
perform it, without any additional laboratory equipment other than test tubes to 
hold the specimen.  Only 1 drop of bodily fluid (~0.05 mL) is required to detect 
CrAg, and the CrAg LFA assay can be performed at room temperature, not 
requiring refrigeration or heat inactivation. Fingerstick CrAg testing is generally 
equivalent to serum or plasma CrAg testing.45 CrAg titers will be performed by 
serial dilution at baseline using the screening or enrollment plasma specimen. 
Refer to lab SOP. CrAg titers may be performed on stored specimens.  
9.6.2. 
Routine Laboratory Monitoring 
Routine lab monitoring will be conducted at: Enrollment, 4, 8, and 12 weeks. This 
monitoring shall comprise of:  
 
Complete blood count (CBC) with platelets 
 
Serum electrolytes of Na+, K+ 
 
Serum creatinine  
 
Serum hepatic tests of:  
o Alanine aminotransferase (ALT) 
o Total bilirubin (t-bili).  
9.6.3. 
Additional Laboratory Monitoring  
Persons with grade >3 lab abnormalities should continue to have 
monitoring conducted at a 2-4 week interval until the abnormality resolves 
to Grade 2 or less. For further details on clinical management, please see 
Manual of Operations.  
Lab Test 
Ref Range 
Ugandan Adult 
DAIDS Grade >3 
Threshold 
Expected 
Event Rate 
Hemoglobin, g/dL 
M: 14.4 – 18.9 
F: 10.9 – 16.7 
M: <9 
F: <8.5 
50% 
WBC x109/L 
2.8 – 7.7 
<1.5 
5% 
Platelet x109/L 
M: 156 – 358 
F: 125 – 445 
<50 
<5% 
Sodium, mEq/L 
138 – 150 
<125 or >154 
<2% 
Potassium, mEq/L 
M: 3.7 – 5.4 
F:3.6 – 5.3 
<2.5 or >6.5 
<2% 
Creatinine, mg/dL 
M: 0.50 – 1.30 
F: 0.50 – 1.20 
M: >2.34 
F: >2.26 
<5% 
ALT, U/L 
M: 0 – 45 
F: 0 – 35 
M: >225 
F: >175 
<5% 
T-bilirubin, mg/dL 
M: 0.0 – 1.4 
F: 0.0 – 1.3 
>3.6 
>3.4 
<5% 
 
9.6.4. 
Specimen Preparation, Handling and Shipping 
All protocol specimens will be shipped using packaging that meets 
requirements specified by the International Air Transport Association Dangerous 


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Goods Regulations for UN 3373, Biological Substance, Category B, and Packing 
Instruction 650. Culture isolates, if obtained in this study, are to be shipped as 
specified for UN 2814 Category A Infectious Substances. 
Biohazard Containment. Transmission of HIV and other blood borne 
pathogens can occur through contact with contaminated needles, blood, and 
blood products. Respiratory pathogens such Mycobacterium tuberculosis are 
transmitted by inhalation of droplet nuclei. Appropriate blood, secretion, and 
respiratory precautions will be employed by all personnel in the collection of 
clinical samples and the shipping and handling of all clinical samples and isolates 
for this study, as currently recommended by the Centers for Disease Control and 
Prevention in the United States, the WHO internationally, and the NIH. 
 
9.7. 
EKG Monitoring  
EKG monitoring will be performed with measurement of QTc interval at baseline, 4, 8, 
12 weeks.  Relevant drug dosing at these time will be: 
Medicine 
Enrollment 
4 weeks 
8 weeks 
12 weeks 16 weeks 
Fluconazole 
0 * 
400mg 
400mg 
400mg 
200mg 
Sertraline/Placebo 
0 
400mg 
400mg 
400mg 
0 mg 
* Persons may receive up to 7 days of antifungal therapy prior to trial enrollment. 
The corrected QT interval (i.e. QTc) should be preferably calculated via either: 
 Fridericia’s method ܳܶܿൌ
ொ்
∛ோோ  , or (default QTc on GE Mac® 1200 EKG Instrument) 
 Framingham method ܳܶܿൌܳܶ൅0.154 ݔ ሺ1 െܴܴሻ 
CrAg+ persons with a baseline EKG QTc >450 at the pre-entry visit are not eligible for 
trial enrollment, per the directive of the Funder.  Per NIH rules, persons not eligible for 
trial participation may not have study personnel or study resources used to provide 
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10. ASSESSMENT OF SAFETY 
This is a clinical trial investigating whether adjunctive sertraline will lead to improved 
survival outcomes in HIV-infected persons with AIDS and cryptococcal antigenemia.  
Fluconazole and sertraline have a long track record of safety with >30 million U.S. 
prescriptions of sertraline annually.  
 
10.1. Safety Assessment Overview 
Serious Adverse Event 
A Serious AE is any adverse event, without regard to causality, that is or causes: 
o life-threatening; 
o death; 
o hospitalization; 
o persistent or significant disability or incapacity; or 
o congenital anomaly or birth defect.   
Any other medical event that, in the medical judgment of the Investigator, may 
jeopardize the subject or may require prompt medical or surgical intervention to 
prevent one of the outcomes listed above is also considered a Serious AE. A 
planned medical or surgical procedure is not, in itself, a Serious AE.   
Routine outpatient medical therapy for a common, expected illness in the study 
population (e.g. malaria, TB) is not a Serious AE.  
All AEs that do not meet any of the criteria for serious will be classified as non-
serious adverse events.   
Expected Adverse Events include: 
 Death due to Cryptococcal infection; 
o Comment: This is the primary endpoint and expected in up to 20-
25% of participants. 
 Symptoms due to cryptococcosis and/or meningitis 
 Hospitalization due to cryptococcosis (up to 30%) 
o Comment: This is a Serious AE due to hospitalization, but is expected. 
 Common ART side effects (e.g. anemia) 
 Immune reconstitution inflammatory syndrome 
 Tuberculosis (5-10%) 
o Comment: If hospitalization is required, this is a Serious AE. 
 AIDS-related opportunistic infections. 
 ART-related liver injury (4%)89 
These expected events will be reported to the IRB in aggregate via interim 
reviews. The goal of aggregate reporting is to continue to assure subject safety 
and to perform reporting in a meaningful manner which individual reporting cannot 
provide. The DSMB will monitor the primary endpoint of cryptococcal meningitis + 
death for overall safety/efficacy.  


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Unexpected Adverse Events 
All unexpected serious AEs will be reported, regardless of causality to sertraline to 
the DAIDS medical officer.  Examples would include severe hepatotoxicity while 
taking fluconazole and sertraline (without ART), which would be unexpected. 
Clinically significant serotonin syndrome is unexpected. Unexpected AEs will be 
adjudicated by the Trial Safety Committee (Section 12.7.4) and if deemed to be 
potentially-related to sertraline, reported to the FDA (Section 10.6). 
10.2. Grading Adverse Event Severity 
Grading clinical severity of AEs will occur via the Division of AIDS Table for Grading 
the Severity of Adult and Pediatric Adverse Events (version Nov 2014).   
 Grade 3: Severe – “Severe symptoms causing inability to perform usual social 
& functional activities with hospitalization indicated.” 
o Any AE which results in necessary hospitalization is at least Grade 3 
o Any AE of sufficient severity which prompts immediate study medicine 
discontinuation is at least a Grade 3 event. 
 An AE resulting in a dose decrease or early taper is not 
necessarily a Grade 3, unless it meets Grade 3 criteria. 
 Grade 4: Potentially Life-Threatening – “Potentially life-threatening symptoms 
causing inability to perform basic self-care functions with urgent intervention 
indicated to prevent permanent impairment, persistent disability, or death.” 
o Standard treatment for illness, which if not treated is potentially life 
threatening (e.g. malaria, TB), is not considered Grade 4 AE unless the 
present symptoms are potentially life threatening. For example, 
uncomplicated malaria is not a Grade 4 AE but cerebral malaria is a 
Grade 4 AE. Severe malaria requiring hospitalization is at least a 
Grade 3 AE. 
 Grade 5: Death 
10.3. Recording of Serious AEs  
All serious AEs occurring from study entry through study termination will be recorded.   
 Non-fatal AEs will be recorded on the ‘AE CRF.’   
 Fatal AEs, dependent on the time course of the death, may be recorded on an 
‘AE CRF,’ but must be recorded on the ‘Termination CRF.’  
The clinical course of each event should be followed until resolution, stabilization, or 
until it has been determined that the study treatment or participation is not the cause 
(e.g. motor vehicle accident, illicit drug use).  Serious AEs that are still ongoing at the 
end of the 6-month reporting period must be followed up to determine the final 
outcome. All unresolved serious AEs at 6-months should be followed until the events 
are stabilized or otherwise explained.   


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Cryptococcal-related deaths and incident cryptococcal meningitis will not be included 
in the analysis of AEs, but these will be analyzed as separate endpoints. 
Cryptococcal-free survival is the primary endpoint of the trial.  
When selecting a primary AE term to report, sites should select the term that best 
describes what occurred to the participant. For example, a participant may present 
with itching, urticaria, flushing, angioedema of the face, and dyspnea. If the 
underlying diagnosis is determined to be an acute allergic reaction, sites should 
report “Acute Allergic Reaction” as the primary AE term.  
 
10.3.1. Laboratory AEs 
Laboratory abnormalities do not need to be individually reported on an ‘AE CRF’ 
unless the event results in a Serious AE.  All laboratory 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 version Nov 
2014.  These laboratory AEs will be summarized separately from clinical AEs.  
 
10.4. Adverse Event Procedures and Reporting Requirements 
To comply with national regulations, reports of all serious unexpected adverse 
events, related or potentially related to the study medication will be submitted to the 
IRB of record and sponsor within 3 working days of the study site awareness of the 
AE. 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. 
At the time of the initial report, the following information will be provided: 
 Study identifier (C-ASSERT) 
 Study Site 
 Medical record number 
 Date of event  
 A description of the event 
 Medical treatments given / stopped  
 Working diagnosis 
 Current vital status 
Within the 7 working days following the event, the investigator will provide 
further information on the AE Reporting CRF 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 serious AEs will 
be provided promptly to DAIDS and IRB of record.  
10.5. Expedited AE Reporting Requirements for the Study to NIH 
The Suspected Unexpected Serious Adverse Reaction (SUSAR) Reporting 
Category, as defined in Version 2.0 of the DAIDS Expedited AE Manual, will be used 
for this study. The study products for which expedited reporting are required are: 
sertraline/ placebo. Unblinding will not routinely occur for event reporting, unless as 
requested by the IRB, FDA, or Sponsor.  
The Adverse Events are not reported under the DAIDS SUSAR system, which must 
be reported to the DAIDS medical officer are: 
 
Serious AEs; AND 
 
Unexpected; AND 


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 
Related or Potentially related to Sertraline 
Individual reporting of each SAE is not recommended as the expected event 
rate is 30-35% with the standard of care therapy.  The primary endpoint is a 
composite endpoint of death and/or cryptococcal meningitis (which requires 
hospitalization which is an SAE).  The DSMB is monitoring this primary endpoint 
for subject safety.  Individual reporting of primary endpoint events in a blinded trial 
provides no additional safety for subjects. 
Unexpected AEs or SAEs with potential relationship to sertraline would be 
important for safety monitoring. 
A Trial Safety Committee will review each unexpected SAE and perform an early 
independent review of trial safety (mortality + SAE incidence) after 75 subjects 
have been enrolled and accrued 3 months follow up. Refer to Section 12.7.4. 
 
10.6. FDA IND and NDA Safety Reporting 
For unexpected SAEs, reporting to FDA and NDA will be as follows: 
“FDA believes that the sponsor is better positioned than the individual investigator 
to assess the overall safety of the investigational drug because the sponsor has 
access to serious adverse event reports from multiple study sites and multiple 
studies and is able to aggregate and analyze these reports. Moreover, the 
sponsor is more familiar with the drug’s mechanism of action, class effects, and 
other information. For these reasons, investigators must immediately report any 
serious adverse event to the sponsor, whether or not the investigator considers 
the event to be drug related (21 CFR 312.64(b)).” 
According to 21 CFR 312.32(c)(4), a sponsor of a clinical study of a drug 
marketed or approved in the United States that is conducted under an IND must 
submit IND safety reports for unexpected suspected adverse reactions that are 
observed in the study 
Unanticipated / unexpected SAEs will be individually reported using FDA Form 
3500A as a ’15-day IND safety report’ or ‘7-day IND safety report.’   
 15 days: “The time frame for submitting an IND safety report to FDA and all 
participating investigators is no later than 15 calendar days after the 
sponsor determines that the suspected adverse reaction or other 
information qualifies for reporting (21 CFR 312.32©(1)).”  
 7 days: “The requirement for reporting any unexpected fatal or life-
threatening suspected adverse reaction to FDA is no later than 7 calendar 
days after the sponsor’s initial receipt of the information (21 CFR 
312.32©(2)).” 
This can be sent to FDA as an Electronic Common Technical Document (eCTD) 
format (e.g. PDF) via the FDA Electronic Submission Gateway. 


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Annual Reporting of Aggregate AEs will occur with annual FDA IND reports, in 
addition to expedited unexpected SAE reporting, based on the adjunction of the 
Trial Safety Committee (Section 12.4.7). 
All reports submitted to the FDA will also be submitted to the Uganda NDA. 
10.7 Safety Reporting Summary 
The summary of Adverse Event reporting and timeline is as follows.  
Grade 
Serious Unexpected To whom 
to Report 
What is reported? 
Timeline † 
1-2 
No 
Yes / No 
None 
None 
N/A 
3-4 
No 
No 
None 
Clinical AE CRF 
DSMB & Annual IRB 
Reporting 
1-3 
No 
Yes 
None 
Clinical AE CRF if Grade 
>3  
DSMB & Annual IRB 
Reporting 
Any 
Yes 
No 
DAIDS MO 
Clinical AE CRF or 
Termination CRF 
Trial Safety 
Committee review 
after each 40 
subjects have 
accrued 3 months 
Any 
Yes 
Yes 
IRB 
FDA, NDA 
DAIDS MO 
Clinical AE CRF 
FDA Form 3500A 
Clinical AE CRF 
< 3 working days 
< 7 calendar days 
< 3 working days 
Lab 
Value* 
No 
No 
None 
Blood Results CRF 
DSMB & Annual IRB 
Reporting 
*Lab abnormalities not requiring clinical intervention to not need to be individually 
reported on the Clinical AE CRF. These will be summarized by DAIDS toxicity via the 
trial’s statistical database for IRB reporting and DSMB monitoring. Lab abnormality AEs 
necessitating clinical intervention require reporting as a Clinical AE and reporting as 
above.  
† Additionally, monthly reporting on AE incidence will occur with progress reports to NIH 
NIAID DAIDS medical officer (Refer to Section 12.7.2). All AEs will be summarized for 
annual reporting to IRBs, DSMB, U.S. FDA, and Uganda NDA. 
 


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11. CLINICAL MANAGEMENT 
 
11.1. Clinical Management of Adverse Events 
Unanticipated and anticipated toxicities will be graded according to the Division of 
AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Version 
2014.  AEs assessed as related to non-study drugs (concomitant medicines) should 
be handled according to the relevant package inserts and the best medical judgment 
of the site investigator. 
11.2. Serotonin Syndrome 
Serotonin syndrome does not generally occur with SSRIs alone. Even in overdose, 
the incidence of serotonin syndrome with sertraline is 6% when the  
Physical presentation of serotonin syndrome includes:  
 Confusion, agitation, reduced level of consciousness, seizures, 
 Clonus/myoclonus, hyperreflexia, tremors, muscle rigidity,  
 Ocular clonus (e.g. -slow, continuous, horizontal eye movements) 
 Ataxia (lack of voluntary coordination), akathisia (e.g. restlessness/constant motion),  
 Hyperthermia (>38⁰C),  
 Hypertension, tachycardia,  
 Diaphoresis, lacrimation, mydriasis, shivering, and/or diarrhea. 
Hunter Criteria for Serotonin Syndrome - a patient must have taken a serotonergic 
agent and meet ONE of the following conditions: 
 Spontaneous clonus; 
 Inducible clonus PLUS agitation or diaphoresis; 
 Ocular clonus PLUS agitation or diaphoresis; 
 Tremor PLUS hyperreflexia; 
 Hypertonia PLUS temperature above 38ºC PLUS ocular clonus or inducible clonus. 
Clonus (spontaneous, inducible and ocular) is the most important sign in the 
Hunter Serotonin Toxicity Criteria. Clonus is rare in cryptococcosis. Serotonin 
syndrome is a diagnosis of exclusion. Metabolic, toxic, and infectious causes should be 
ruled out based on the patient's history and presentation. Differential diagnoses are: 
 Meningitis/encephalitis (headache uncommon with serotonin syndrome, 
clonus not present in cryptococcal meningitis) 
 Delirium tremens from alcohol withdrawal (assess by history), 
 Malignant hyperthermia or intoxications with adrenergic or anticholinergic 
meds (assess by concomitant medication history). 
Medical Management of Serotonin Syndrome 
o Discontinuation of all serotonergic agents.  
 Review concomitant medicines. SSRIs alone do not cause serotonin 
syndrome but can be caused via interactions with other prohibited 
medicines such as MAO inhibitors, pimozide, and serotonergic drugs: 


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sumatriptan, zolmitriptan, tricyclic antidepressants, fentanyl, lithium, 
tramadol, tryptophan, buspirone, and St. John’s Wort. 
o Supportive care aimed at normalization of vital signs 
 Intravenous fluids 
 Oxygen if hypoxia present 
o Sedation with benzodiazepines 
The Site Principal Investigator and C-ASSERT Principal investigator(s) must 
be contacted upon site awareness of a possible serotonin syndrome event. 
Participants with severe serotonin syndrome (involving altered mental status) require 
hospitalization and must have their Sertraline/Placebo held for 24 hours, concomitant 
medicines evaluated, and have a repeat clinical evaluation within 1 day. This is an 
unexpected Serious Adverse Event. Serotonin syndrome symptoms should generally 
dissipate in 24 hours.  Worsening symptoms beyond 24 hours should prompt 
consideration of alternative diagnoses, including lumbar puncture for CSF analysis.  
For persons with mild serotonin syndrome (e.g. without mental status changes), holding 
medication for 48 hours, and dose reduction by >25% is appropriate. 
11.3. Hepatic Dysfunction 
 
ALT >5x upper limit of normal is exclusion criteria for enrollment. Participants who develop 
hepatic dysfunction while on study drug should potentially have study medicine dose 
reduced and more frequent checking of liver function tests (see below). The incidence of 
hepatotoxicity among persons taking sertraline is <1 in 1000 (FDA Package Insert). 
Hepatotoxicity due to antiretroviral therapy is 2-18%, dependent on the population.89  
Based on historical experience in Uganda among persons with HIV/AIDS, the expectation 
is an approximately 4-6% incidence of liver function test abnormalities after starting ART, 
related to the immune reconstitution towards liver pathogens (e.g. hepatitis B). 
 
Liver Function Test Abnormality 
Study Medicine Action Next Lab Recheck 
ALT <2.5x ULN 
Continue 
As per protocol 
ALT 2.5-5x ULN 
Continue 
4 weeks 
ALT >5x ULN 
Dose reduce by ~25% 
<2 weeks 
Bilirubin <2.5x ULN 
Continue 
As per protocol 
Bilirubin 2.6-5x ULN  
Continue 
4 weeks 
Bilirubin 2.6-5x ULN & ALT 2.5-5x ULN 
Dose reduce by ~25% 
< 2 weeks 
Bilirubin >5x ULN 
Dose reduce by ~25% 
< 2 weeks 
 
 
 
 


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11.4. EKG QTc Abnormalities 
Clinical management actions for prolonged QTc are based on FDA E14 guidance for 
Safety Monitoring and Discontinuation Criteria (2.1.2).64 
DAIDS 
Toxicity 
Grade 
QTc 
Threshold 
Study Drug Action 
Repeat 
EKG * 
1 
450-470 ms 
Continue without interruption, at the discretion 
of the site investigator. 
< 4 weeks 
2 
471-500 ms 
Continue without interruption, at the discretion 
of the site investigator. 
< 2 weeks 
3 
>500 ms 
Reduce study drug dose by at least 25% and 
have an EKG repeated within <2 weeks. 
< 2 weeks 
4 
Torsade de 
pointes, etc.  
Discontinue fluconazole and sertraline study 
drug immediately. 
< 1 day 
* Repeat EKGs will be performed beyond the routine duration of EKGs. A QTc change 
>60ms which remains within normal QTc range <450ms will not prompt additional action.  
After a dose reduction, based on repeat EKG QTc, investigators may resume the prior 
study drug dose at their discretion. Sertraline does not typically have an EKG QTc effect; 
however, other concomitant medications may cause QTc effects. 
11.5. Pregnancy 
Women who become pregnant while receiving fluconazole must have:  
A) CrAg titer performed to gauge the current burden of cryptococcal infection.  
B) CD4 count measured. 
C) PI or designee must be contacted within 24 hours of site awareness of a 
pregnancy (preferably immediately).   
D) Study Medicine Tapered at 1 tablet per week. 
E) Referral for prenatal care, outside of the study  
Based on these results and in consultation with the site PI, consideration of 
altering fluconazole dosing should be one of the following:  
1) Dose reduction to <200 mg/day or  
2) Discontinuation if they have received >8 weeks of therapy.   
3) Discontinuation if their CrAg Titer <1:80 AND CD4 >50 cells/L. 
In August 2011, high dose fluconazole (400-800 mg/day) was labeled as FDA 
pregnancy category D, meaning that there is positive evidence of human fetal risk 
based on human data.  In humans, there are a total of nine case reports that suggest 
that dose-dependent teratogenicity during the first trimester of pregnancy may be 
associated with a rare and distinct set of birth defects in infants.90-97  These skeletal 
defects are similar to those described in animal studies.  Adverse events have been 
observed in some animal reproduction studies. When used in high doses, 
fluconazole is teratogenic in animal studies. FDA stated guidance is:  


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“Use of long-term, high-dose (400-800 mg/day) fluconazole during the 
first three months of pregnancy (first trimester) may be associated with a 
rare and distinct set of birth defects in infants.” 
High dose fluconazole is potentially teratogenic in the first trimester of pregnancy. 
This risk must be weighed against the risk of maternal death due to cryptococcal 
meningitis. Most azole antifungals, including fluconazole, are recommended to be 
avoided during pregnancy. 
Conversely, CrAg+ persons with CD4<100 who do not receive antifungal 
therapy have 100% mortality in two studies of CrAg+ persons,3, 12 so the risk vs. 
benefit of removing life-saving therapy to avoid a potential teratogenic effect is 
complicated and best discussed with the PI(s) with customized therapy plan.  
Lower doses (150 mg as a single dose) do not suggest an increase risk to the fetus. 
If the participant continues her pregnancy, the site or patient are encouraged to 
prospectively register her pregnancy in the "Antiretroviral  Pregnancy Registry” (In 
US and Canada: 1-800-258-4263, international: +1-910-256-0238). 
Table 7.  Five case reports of fluconazole-related teratogenicity  
Case 
(Ref) 
Indication 
Dose of 
fluconazole Weeks 
Timing in 
pregnancy 
Outcome 
Case 1 
90 
Coccidioidal 
meningitis 
400mg daily 
27 
Weeks 0-27 
Mother: premature rupture of 
membranes at 27 weeks. 
Infant: Multiple skeletal abnormalities; 
facial deformities including cleft 
palate. Died after birth. 
Case 2 
91 
Coccidioidal 
meningitis 
800mg daily 
37 
Weeks 0-7 
Weeks 9-38 
Infant: Skeletal, facial, cardiac 
abnormalities 
Case 3 
91 
Coccidioidal 
meningitis 
400mg daily 
16 
Weeks 0-16 
Infant: Skeletal abnormalities, cleft 
palate, pulmonary and cardiac 
complications. Died at 3 months.  
Case 4 
92 
Coccidioidal 
meningitis 
400mg daily 
800mg daily 
1200mg daily 
19 
Weeks 0 - 4 
Weeks 5 - 9 
Weeks 22-31 
Mother: Premature rupture of 
membranes at 31 weeks.  
Infant: Skeletal & facial abnormalities 
Case 5 
93 
HIV and vaginal 
candidiasis 
800mg daily 
34 
Weeks 0-23 
Weeks 27-37 
Infant: Skeletal abnormalities, 
abnormal facies 
Case 6 
94 
Candida 
parapsilosis 
fungemia 
200mg daily 
N/A 
Unspecified 
(1st trimester 
start) 
Infant: No abnormalities 
Case 7 
95 
Candida albicans 
fungemia 
400mg daily 
4 
Weeks 10-14 
Not specified 
Case 8 
96 
Candida albicans 
fungemia 
400mg daily 
7  
Weeks 16-23 
Mother: No abnormalities 
Infant: No abnormalities 
Case 9 
97 
Candida glabrata 
fungemia 
600mg daily 
3 
Weeks 14-17 
Infant: No abnormalities 
 
 
 


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11.6. Failure of Preemptive Treatment 
Persons with failure of preemptive antifungal therapy who develop symptomatic 
cryptococcal meningitis must be referred to the local/regional hospital for meningitis 
management. The study should facilitate this transportation. Participants will 
otherwise remain in the study, unless consent is withdrawn. 
11.7. Criteria for Discontinuation of Trial Participation 
A study subject will be discontinued from participation in the study if: 
 
Subject wishes to voluntarily withdraw. 
Subjects are free to withdraw from participating in the study at any time upon 
request.  Every effort will be made to undertake protocol-specific study procedures.  
If voluntary withdrawal occurs, the subject will be asked to continue their HIV care, 
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 they withdraw from the study.  The 
participants will be offered HIV medicines and fluconazole, per Ugandan standard of 
care. If the patient is on high dose sertraline (>200mg daily), the medication will be 
tapered slowly to avoid adverse side effects. 
11.7.1. 
Criteria for Premature Study Medicine Discontinuation 
A study subject shall discontinue the blinded study medicine if: 
 Any clinical adverse event (AE), laboratory abnormality, concurrent illness, or 
other medical condition or situation occurs such that continued 
participation in the study would not be in the best interest of the subject. 
 Withdraw of informed consent with a request by participant to withdraw.   
 Pregnant women should have their study medicine rapidly tapered by 1 
tablet per week and dose of fluconazole reduced to <200mg/day.  
Participants may elect to discontinue the study medicine and remain in the 
trial, per the intention to treat analysis. Vital status and signs/symptoms of 
meningitis will be obtained at 24 weeks post-enrollment. If a participant is 
voluntarily terminating antifungal therapy before 8 weeks, the site PI must be 
notified immediately, as the result may be ~100% mortality. 
 
11.7.2. 
Criteria for Study Medicine Dose Reduction/Resumption 
The study medicine/placebo may be dose reduced for participant safety in the 
event of an AE which in the opinion of the investigator is temporarily and causally 
associated with sertraline.  A prime example would be serotonin syndrome with 
clear causality. Other toxicities with possibility causality shall have management 
based on the best judgment of the clinical investigator.   
Dose reduction should be at least 25% (i.e. from 4 to 3 tablets/day) but may be 
a higher dose reduction based on the timing and severity of the toxicity. Once the 
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their discretion. If the toxicity recurs, sertraline should not be further dose 
escalated. In general, drug-related toxicities are more likely to be caused from 
other concomitant medicines, such as ART; however, investigator judgement is 
paramount. 
 
 


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12. STATISTICAL CONSIDERATIONS 
 
12.1. Study Endpoints 
All participants (Phase II and Phase III) will contribute data for the primary and 
secondary endpoints. 
 
12.1.1. 
Primary Endpoint 
1) 6-month cryptococcal meningitis free survival with retention-in-care 
o Those who die of any cause are failures 
o Those developing symptomatic cryptococcal meningitis are failures 
o Those lost to follow up and unable to be tracked are considered failures 
12.1.2. 
Secondary Endpoints 
2) 6-month survival time 
3) Incidence of symptomatic cryptococcal meningoencephalitis 
4) Incidence of Clinical Adverse Events (Grade 3,4,5) 
5) Incidence of Laboratory Grade 3-5 Adverse Events 
6) Incidence of premature study drug/placebo discontinuation 
7) Prevalence of Depression by PHQ-9 scale over time. 
12.1.3. 
Exploratory Endpoints 
8) Medication adherence by blood levels 
9) Risk factors for cryptococcal-free treatment failure 
10) Adherence with the cascade of HIV and antifungal care (e.g. time to: return to 
clinic, antifungal therapy, ART initiation) 
11) Use of text message or other reminders to minimize loss to follow up 
12.2. Analysis Plan 
We will compare survival by time-to-event analysis for cryptococcal-free survival 
using Log-rank test and survival displayed with a Kaplan-Meier survival curve with an 
indicator variable for treatment assignment.  Analysis will be by intention-to-treat.  
All asymptomatic CrAg+ persons will be included (i.e. denominator). Persons 
symptomatic at baseline (i.e. pre-existing meningitis) would not be enrolled but 
referred to hospital for cryptococcal meningitis care. The CrAg-negative 6-month 
survival rate among persons with CD4<100 and ART is ~89% in Uganda (e.g. 
Figure 3), thus if cryptococcal infection is fully negated, approximately 89% 6-month 
survival would be the target survival. 
12.3. Analysis Plan Secondary and Other Endpoints 
 6-month Survival Time 
Survival time will be analyzed using Log-rank test and displayed via Kaplan-Meier 
survival curve.  Persons lost to follow up will be considered failures. All 
participants will be censored at 6 months.  
 Incidence of Symptomatic Cryptococcal Meningoencephalitis  


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The cumulative incidence function will compare the randomized groups for 
incidence of symptomatic cryptococcal-related meningitis/encephalitis, which will 
account for the competing risk of death.31    
Symptomatic meningoencephalitis will be defined as meningitis symptoms plus:  
1) Cryptococcus culture positive meningitis,  
2) CSF CrAg+,  or  
3) Cryptococcoma(s) by neuroimaging or post mortem exam.98  
 Incidence of Adverse Events 
The cumulative incidence function will compare the randomized groups for 
incidence of Grade 3, 4, 5, and Serious AEs, which will account for the competing 
risk of death.31    
Cryptococcal-related deaths and incident cryptococcal meningitis will not be 
included in the analysis of AEs, but these will be analyzed as separate endpoints 
(as above).  
 Incidence of Laboratory Adverse Events 
The cumulative incidence function will compare the randomized groups for 
incidence of laboratory adverse events of Grade 3, 4, 5, which will account for the 
competing risk of death.31 A laboratory value which results in clinical action 
meeting the definition of a Serious AEs (e.g. hospitalization), is a Serious AE and 
will be also included above. Asymptomatic / minimally symptomatic lab 
abnormalities which do not result in hospitalization will be summarized herein. 
Based on the lab monitoring, this includes hematologic, elctrolyte, renal, and liver 
lab testing labnormalities.    
The frequency of EKG QTc abnormalities by Grade will be separately reported 
and do not need to be reported on the Clinical AE CRF unless symptomatic.  
 Incidence of Premature Study Drug/Placebo Discontinuation 
The cumulative incidence function will compare the randomized groups for all-
cause incidence of study drug/placebo discontinuation.31   Premature 
discontinuation of the study medicine/placebo may occur from withdrawal of 
consent, intolerability, non-adherencce, adverse event, or other potential reasons.  
 Prevalence of Depression 
Prevalence of Depression as per PHQ-9 questionnaire will be collected at 
baseline, 4 weeks, 8 weeks, and 12 weeks routinely. A mixed effects regression 
model with a random intercept for individual will be used to account for the intra-
subject correlation induced by repeated PHQ-9 measures over time. An 
interaction indicator variable of time and treatment groups will assess differences 
in PHQ-9 depression scores. Additionally, we will summarize the prevalence of 
depression (by severity) from baseline through 12 weeks by randomized 
group.PHQ-9 Total Score Depression Severity Scoring: 
1-4  
Minimal depression 
5-9  
Mild depression 
10-14 Moderate depression 
15-19 Moderate severe depression 


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20-27 Severe depression 
 
 Adherence 
Adherence: We will perform a 1:1 case: control study measuring fluconazole and 
sertraline plasma levels in those with treatment failure vs. controls to objectively 
assess non-adherence (i.e. absence of sertraline) or malabsorption or partial 
adherence (i.e. lower levels).  Those with treatment failure are those who develop 
cryptococcal meningitis and/or death during the 6 month follow up period. 
Controls will be randomly selected amongst the group who achieved cryptococcal-
free survival at 6 months. Controls will be matched to cases by clinic site and time 
of enrollment (+/- 6 months). If non-adherence is common in treatment failure, 
then improved patient education should be part of an enhanced package of care 
for the future. Plasma fluconazole and sertraline levels will be measured 
retrospectively at the University of Minnesota Clinical Pharmacology Analytical 
Services Laboratory, which has validated assays for both compounds.33 
 Biomarker Analysis 
First, we plan to conduct a nested case-control as a sub-study to measure 
plasma biomarkers on CrAg+ persons at screening and again at 4 weeks versus 
controls. Cases will be those with the primary outcome status of cryptococcal 
meningitis or death. Controls will be CrAg+ survivors who do not develop 
cryptococcal meningitis through 24 weeks. Controls will be randomly selected with 
matching based on site. We will test both routine clinical markers (e.g. CRP, d-
dimer). As we have reported in two prior HIV cohorts with AIDS that elevated pre-
ART plasma CRP is associated with risk of unmasking IRIS events and/or 
death.99, 100 Specifically, in a cryptococcal meningitis survivor cohort, pre-ART 
serum CRP >32 mg/L was associated with 8-fold higher risk of death on ART.100 
In a separate HIV cohort with CD4<200 cells/L, every two-fold increase in pre-
ART plasma CRP was associated with a 2.1-fold increased odds of IRIS and 2.1-
fold increase in odds of new AIDS event or death. 
 
Second, we plan to measure ~17 plasma cytokines / chemokines via Luminex 
multiplex assay (Bio-Rad, Hercules, CA) in order to generate an immune 
signature associated with future meningitis and/or death. Specimens will be stored 
with measurements in batch.  Analysis will compare mean levels of log2 
transformed biomarkers compare cases (meningitis or death) vs. controls 
(survivors) via logistic regression with and without adjustment for demographic or 
other variables (e.g. CrAg titer) associated with treatment failure. 
 
Third, we will compare cell phenotype and activation using flow cytometry 
among a subset of cases and controls to determine the cellular phenotype and 
activation pattern that are associated with development of cryptococcal meningitis 
and/or death. 
 
We hypothesize CrAg titer >1:160 and plasma CRP will also be predictive 
herein for the Aim 2 analysis of CrAg+ persons determining increased risk of 
failure of preemptive therapy (i.e. cryptococcal meningitis or death). The 
biomarker analyses are primarily exploratory, but with hypotheses similar to the 
published experience with cryptococcal meningitis.99, 100 


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Lastly, we will collect RNA from whole blood in PAXGene tubes at selected 
sites to potentially perform a future exploratory case:control transcriptome 
analysis of the gene signature associated with cryptococcal disease progression 
and/or death.  
 
12.4. Sample Size Considerations 
To enroll 600 people in total (Phase II and Phase III), with a CrAg prevalence rate 
of approximately 5%, we anticipate screening ~12,000 persons with a CD4<100 
cells/µL.  
With 300 subjects enrolled per group (n=600 in total), a two-sided log-rank test 
achieves 80% power at a 0.05 significance level to detect a hazard ratio of 0.60 
when the proportion with cryptococcal-free survival with retention-in-care in the 
control group is 75%, equating to an approximate survival of 84% in the 
intervention group.101 There is 90% power to detect a hazard ratio of 0.55 
equating to 85% cryptococcal-free survival. 
Approximately 120 events are expected during the course of the study.  For the 
biomarker case/control study if there are 120 participants per group then we can 
detect a 0.36 standard deviation difference in a biomarker level between cases 
and controls with 80% power and a 0.05 significance level. Similarly, if there are 
100 participants per group we can detect a 0.40 standard deviation difference, 
and with 80 participants per group we can detect a 0.44 standard deviation 
difference.   
The number of specimens to be measured for biomarker analyses will depend on 
the event rate and financial resources available. Ideally, at least a 1:1 case:control 
study would be performed comparing all cases of death or meningitis vs. equal 
number of randomly selected controls of the survivors without meningitis through 
24 weeks. A 1:2 case:control study would be better, or testing the entire cohort 
would be most ideal. The number of specimens tested will depend on the financial 
resources available. 
 
12.5. Randomization / Stratification 
We will use a permutated block randomization in a 1:1 allocation (n=300 per arm).  
Randomization will be stratified by district (e.g. Kampala, Mbarara, Masaka).  
Randomization codes will be developed and held by the trial biostatistician at the 
University of Minnesota.   
Randomization schedules will be supplied to the pharmacy at each clinical trial 
site, with notations for which dose to take during the induction phase and which 
study drug to take during the outpatient phase. Only the central pharmacy at IDI 
and the study biostatistician will have access to both the participant study 
identification number and the randomization group. Detailed instructions for study 
drug processing, labeling and dispensing are included in the Pharmacy SOP. 
12.6. Blinding Procedures/ Unblinding Procedures 


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This is a double-blind, placebo controlled study. Study medicine and placebo will 
be blinded and matched.  Sertraline is a known medicine with an extensive safety 
record. In the event of Serious AE, the study medicine may be discontinued at the 
providers’ decision. Due to the safety record of sertraline, unblinding will not occur 
for the participant or investigators. The treatment for a serious AE related to the 
study medicine is discontinuation of the medicine. Unblinding would not change 
clinical management and has the potential to introduce unintended bias. The data 
and safety monitoring board will be unblinded to serious AEs.  
Participants may be switched to open label sertraline at clinician discretion if they 
are diagnosed with severe depression after week 12. For persons with perceived 
intolerance or toxicity, dose reduction may occur at provider discretion. 
12.7. Data and Safety Monitoring 
Safety oversight.  Safety oversight will be under the direction of an independent 
data and safety monitoring board (DSMB). A standing NIH NIAID DSMB will have 
oversight with use the: “Charter for the Data and Safety Monitoring Boards of 
DAIDS, NIAID.”  The most current version (presently 24 June 2015) is available 
at: https://www.niaid.nih.gov/sites/default/files/dsmb_charter.pdf  
The NIAID Complications and Co-infections (CC) DSMB will have oversight 
responsibilities. The executive secretary for NIAID-CC-DSMB contact is: 
Sally Hunsberger, PhD 
Mathematical Statistician 
5601 Fishers Lane, rm 4D13 
Rockville MD, 20892 
e-mail:  sallyh@niaid.nih.gov 
Phone:  +1.240.669.5257 
 
The NIAID CC DSMB is scheduled to conduct an initial protocol review on 
September 6, 2017. Thereafter, the DSMB will conduct reviews after every 6-12 
months of enrollment and will meet at least annually. Based on the accumulating 
C-ASSERT trial data, the DSMB may meet more frequently. A Lan-DeMets 
spending function analog of the O’Brien-Fleming boundaries will guide early 
stoppage.  A formal sample size re-estimation will occur at time of the second 
DSMB report to be considered by the DSMB. This sample size estimation will 
assess the baseline assumptions of 75% incidence of cryptococcal free survival 
with retention in care versus the current observed experience to date. 
DSMB Reports will include the information contained in the Progress Reports and 
Clinical Quality Management Plan reports as well as an analysis for the primary 
endpoint.   
Open DSMB reports will be prepared with pooled demographic and trial progress 
data. This open report will be blinded to randomization allocation.  
Closed DSMB reports will have data prepared by randomization group for 
demographics of enrolled participants and the primary outcome.  


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Baseline demographic features of each study arm will be summarized, with 
statistical testing as appropriate for nominal and continuous variables to assure 
adequacy of randomization. Any parameters found to be potentially different 
between randomization groups (P<0.1) and associated with outcome may be 
included in a multivariable adjusted Cox regression analysis. 
12.7.1. 
Planned Interim Analyses and Stopping Guidelines 
DSMB will monitor the study for safety endpoints, accrual issues, and general 
implementation of the study beginning approximately 6 months after trial initiation.   
The first DSMB interim analysis will be prepared after approximately 100-150 
subjects are enrolled and have endpoint data, and the DSMB will review accruing 
data at least annually thereafter. The exact timing is based on the DSMB 
schedule. An early DSMB review may be called for by the Trial Safety Committee 
if there are initial safety concerns which occur in the Phase II trial. DSMB 
members can also request additional more frequent reviews.  
A Lan-DeMets spending function analog of the O’Brien-Fleming boundaries will be 
provided at each DSMB report for the stopping guidelines for 6-month 
cryptococcal-free survival outcome.  
The O’Brien-Fleming boundaries will be truncated at alpha=0.002 (|Z|>3.09). The 
provided Table 8 assumes three interim analyses and a final analysis with an 
overall two-sided cumulative alpha=0.05. 
Interim 
Analysis 
Sample Size 
| Z | 
P-value 
Cumulative 
Alpha 
1 
~150   (25%) 
> 3.09 
0.002 
0.0020 
2 
~300   (50%) 
> 3.09 
0.002 
0.0037 
3 
~450   (75%) 
> 2.38 
0.016 
0.0193 
Final 
   ~600  (100%) 
> 2.02 
0.031 
0.0500 
Sample Size Re-estimation:  For the second DSMB review, when approximately 
50% of the participants have been enrolled, a formal sample size re-estimation will 
occur to assess whether the initial assumptions for the primary endpoint (25% in the 
control group) were correct. The sample size re-estimation will be based on the 
pooled (and blinded) event rate and the 9% absolute difference in mortality 
expected between the groups. The sample size re-estimation will not take into 
account the interim treatment effect.   Based on the results of the sample size re-
estimation the DSMB may recommend that the study continue as planned or to 
increase the sample size, if the event rate is lower than a priori expectations.  The 
investigators will not receive this report. 
Futility Analysis:  The expected accrual rate is 125-150 participants per year. 
Enrollment in year 1 will likely be lower due to staggered starting of clinical sites, 
each of which is dependent on Funder permission to initiate. At the second DSMB 
the accrual is less than 75% of what is expected, the DSMB committee may ask 
the study team to submit a formal plan to increase enrollment. If the conditional 
power is <20% after the third interim analysis (or when approximately 75% of 
participants have been enrolled) or thereafter, trial discontinuation may be 


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considered by the DSMB. The DSMB will be given conditional power under both 
the design and the current data for their review.  Ultimately, the DSMB will make 
their own decision, irrespective of any stopping guidelines.   
 
12.7.2. 
Interim Monitoring and Safety Review 
A comprehensive Study Monitoring and Statistical Analysis Plan describes the types, 
content, and distribution schedules of study reports, and to define the statistical 
analysis plan to be implemented at the end of the study for the primary and secondary 
endpoints, including the specification of pre-defined subgroups. The purpose of the 
monitoring portion of the plan is to: 
o Protect and ensure the safety of the subjects; 
o Ensure the validity and integrity of the data for the clinical trial; 
o Ensure that the clinical trial is monitored appropriately; 
o Ensure that the data collected will be usable to monitor safety and address the 
protocol objectives; and 
o Ensure that the trial steering committee and sponsor are aware of the 
schedule of monitoring for the clinical trial 
 
The Monitoring Plan includes: 
1. Progress Reports, with submission to NIAID DAIDS medical officer at 
approximately monthly intervals.  
2. Quarterly Clinical Quality Management Plan (CQMP) Report 
3. Unexpected Serious Adverse Event Reports 
4. Open DSMB Report 
5. Closed DSMB Report 
 
The Progress Reports will include:  
Table 1: Participant Accrual 
Table 2: Follow-up Status for Enrolled Participants 
Table 3: Participant Baseline Characteristics 
Table 4: Study Drug Administration Timing and Proper Randomization 
Table 5: Summary of Grade 3, 4, and Grade 5 Adverse Events and Serious AEs 
Table 6: Line Listing of Cumulative Clinical Adverse Events by Participant ID 
Table 7: Summary of Laboratory Abnormalities by Visit 
 
The DSMB Reports will additionally include: 
Table 8: Summary of EKG QTc Findings 
 
12.7.3. 
Quality Control and Quality Assurance 
Ongoing data quality control (QC) and quality assurance (QA) activities will occur 
via data management system. Clinical Quality Management Plan (CQMP) 
reports will include: 
Table 9: Cumulative Withdrawn Consent and Protocol Violations 
Table 9a: Line Listing of Protocol Violations by Participant ID 
Table 10: Summary of Data Completeness 


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Table 11: Case Report Form (CRF) Delinquency Report 
Table 11a: Delinquent CRFs by Participant ID 
 
12.7.4. 
Trial Safety Committee 
A Trial Safety Committee will be assembled to review: 
o Unexpected Adverse Events 
o Potential Sertraline-related Adverse Events 
o Early Mortality + SAE Independent Review (Section 12.7.5).  
The composition of the Trial Safety Committee shall include three physicians with 
experience in clinical trials and HIV medicine: 
o Noeline Nakasujja – Chair, Department of Psychiatry, Makerere University 
o Jason Baker – Hennepin County Medical Center, MN 
o Tihana Bicanic, University of London - St. George’s, UK  
A quorum of 3 members is required for the early safety independent review.  For 
adjudication of unexpected adverse events or potential sertraline-related AEs, 
adjudication by at least two members is necessary with consensus agreement. 
The safety committee may request the unblinding of unexpected AE events 
or possible sertraline-related AEs from the trial biostatistician, which would 
be particularly relevant if the event is potentially related to sertraline. Any 
individual participant unblinding would occur after the committee’s adjudication, 
so as to not influence their adjudication. 
The Trial Safety Committee shall be empowered to: 1) pause or halt further 
enrollment; 2) request an early DSMB review; and/or 3) request further interim 
safety reviews.  The Trial Safety Committee shall be empowered until the DSMB 
assumes the responsibility for ongoing trial monitoring. The Trial Safety 
Committee may also choose to make recommendations to the DSMB for safety 
monitoring, based on their expertise in cryptococcosis, psychiatry, and clinical 
trials in Africa.  If a replacement Trial Safety Committee member is needed, the 
NIH sponsor will be notified.  
 
12.7.5. 
Early Safety Independent Review (Phase II activity) 
To address possible concerns that there may be unanticipated safety concerns 
with adjunctive sertraline in addition to the standard of care, an external Trial 
Safety Committee will conduct enhanced safety reviews as part of an integrated 
Phase II trial design. There will be two a priori triggers of: 
o >5% sertraline-related probable/definite adverse event (Grade 3-5 or SAE) 
This trigger would occur at 1 or more participants in the first 40 randomized 
(n=20 on sertraline), >2 participants in the first 80, or >3 participants in the 
first 120. Sertraline related Grade 3-5 adverse events are not expected.  
Potentially sertraline-related adverse events will be adjudicated on an 
ongoing basis. 
o Cumulative incidence of deaths + SAEs combined over both treatment 


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groups of >50%. The expected incidence is 40% with the current standard 
of care. This composite safety outcome will be calculated by the trial 
biostatistician after every 40 subjects have been enrolled and accrued 3 
months of follow up data. A pooled summary report shall be provided to the 
Trial Safety Committee for their review.  
 If either trigger is reached, options for the Trial Safety Committee would be a 
recommendation for pausing further enrollment, requesting an early DSMB 
review (by randomization group), and/or interim re-assessment prior to the 
DSMB (based on the timetable of the DSMB).   
These safety reviews will continue every 40 subjects until the DSMB assumes 
responsibility for safety monitoring. After ~100 subjects have been enrolled, 
sufficient subject outcomes should allow the DSMB to judge the frequency of 
further reviews by the DSMB and/or Trial Safety Committee.  
At the end of the phase II activities, the DSMB will be asked to make a 
recommendation regarding safety. Specifically, 1) should the phase III trial 
continue onwards; 2) is the safety monitoring adequate? and 3) whether the 
frequency of safety monitoring should be increased, remain the same, or 
decreased based on the accumulating data.  
This trigger is meant for subject safety; however, there is a chance of a false 
positive trigger because of early anomalous results caused by random probability 
and/or the unequal distribution of the first few subjects.  At the first review after 80 
subjects, there is a 6.3% chance of a false-positive review being triggered if the 
true mortality + SAE incidence is 40% as anticipated, and a 59% chance of review 
if the underlying true mortality + SAE incidence is 50%. At reviews after 100 
subjects, there is a 2.7% chance of a review being anomalously triggered if the 
true mortality + SAE incidence is 40%. 
13. Study Monitoring, Auditing, and Inspecting 
13.1.  Study Monitoring Plan 
Site monitors under contract to the National Institute of Allergy and Infectious 
Diseases (NIAID) and NIAID staff (or designee) will visit participating clinical 
research sites to review participants records, including consent forms, CRFs, 
medical records (e.g., physicians’ progress notes, nurses’ notes, individuals’ 
hospital charts), and laboratory records to ensure protection of study 
participants, compliance with the IRB-approved protocol / amendments, and 
accuracy and completeness of records. Remote monitoring via internet-based 
review of CRFs is also a possible monitoring strategy. The monitors will inspect 
sites’ regulatory files to ensure that local regulatory requirements, in addition 
to U.S. Federal regulations, are being followed. They will also inspect sites’ 
pharmacies to review product storage and management. 
An internal study team “Clinical Quality Monitoring Plan” (CQMP) will be 
implemented for QC/QA of CRFs to monitor correct completion of CRFs, missing 
CRFs, and remote review of digitally scanned source documents.  Refer to 
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13.2.  Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by 
the IRB, NIH NIAD DAIDS and DAIDS-affiliated personnel, government regulatory 
bodies, and University compliance and quality assurance groups of all study 
related documents (e.g. source documents, regulatory documents, data collection 
instruments, study data etc.).  The investigator will ensure the capability for 
inspections of applicable study-related facilities (e.g. pharmacy, diagnostic 
laboratory, etc.). 
Participation as an investigator in this study implies acceptance of potential 
inspection by government regulatory authorities and applicable University 
compliance and quality assurance offices. The NIH NIAID DAIDS Monitors will be 
used for monitoring. 
 
13.3. Laboratory Quality Control 
All protocol specific laboratory monitoring will be performed in a DAIDS-approved 
laboratory adhering to GCLP standards.  Inclusion/Exclusion criteria of ALT and 
pregnancy tests may be performed via FDA-approved assays which are CLIA-
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14. DATA HANDLING AND RECORDKEEPING 
This trial will use the DataFax system for multisite data collection. The DataFax 
System is a data collection system used for all data collection and data management 
purposes.  The DataFax unit is located at the Infectious Disease Institute in Kampala. 
The DataFax system relies on paper CRFs to collect data during patient visits and 
internet-enabled fax machines to send data into a central NIH server with recognition 
of the CRF data via optical character recognition. The server resides at the NIH in 
Bethesda. Data from the CRFs are collected, reviewed, and processed by the 
DataFax central office, housed at the IDI in Kampala, Uganda. The DataFax system is 
used to assess errors on the CRFs and perform additional quality control and quality 
assurance procedures. The DataFax system is also utilized by data managers to 
develop study-specific reports.  This enables real time quality assurance for the multi-
site study. Individual sites receive approximately weekly error queries for missing 
CRFs or labs, protocol deviations, and overdue visits.  The system incorporates real 
time error checking such that weekly error reports enable ongoing quality assurance 
measures. 
14.1. Data Management Responsibilities 
A data manager at the University of Minnesota will work with the trial statistician and 
DataFax unit to conduct Quality Assurance activities and prepare reports.  
14.2. 
Essential/Source Documents  
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. 
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, hospital records, clinical and office charts, laboratory notes, pharmacy 
dispensing records, recorded data from automated instruments, digital media, 
radiographs, and subject files and records kept at the pharmacy, at the laboratories. 
For this trial, the source documents will be the CRFs completed by study staff.  
It is acceptable to use CRFs as source documents, when the study personnel are 
making their original observations and recording on CRFs. In the event of 
transcription from another data source, e.g. laboratory report or hospitalization 
records, these records are the source document. A copy of any external record 
should be obtained and stored in the study chart.   
Routine clinic records completed by non-study staff are not source documents, 
unless they are being used to transcribe data.   
 
 
 


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15. HUMAN SUBJECTS PROTECTIONS 
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 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 certification prior to human subject 
involvement.  Study personnel involved in collecting clinical data should have current 
Good Clinical Practice (GCP) training. 
15.1. Institutional Review Board/Ethics Committee 
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 IRB of record.  Likewise, any future amendments to the study 
protocol will be approved by each site IRB.  Please refer to Appendix A for informed 
consent documents. 
This protocol and any amendments will undergo review and approval by the 
institutional human subjects review boards (IRBs) at the Joint Clinical Research 
Centre (FWA00009772), and the Uganda National Council of Science and 
Technology (UNCST) under FWA00001293, University of Minnesota 
(FWA00000312), and other local IRBs and ethics committees as appropriate. 
15.2. Vulnerable Participants 
o Illiterate participants: Eligible with 3rd person impartial witness  
o Pregnant women and fetuses: Not eligible to participate. 
o Prisoners: Not eligible to participate. 
o Children <18 years of age:  Not eligible to participate. 
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. 
15.3. Informed Consent 
Informed consent is a process that is initiated prior to the individual’s agreeing to 
participate in the study and continuing throughout the individual’ study participation. 
Upon clinic entry, persons provide a written consent for HIV treatment, which includes 
passive aggregate summary data collection for clinic reporting purposes. As the 
intervention of CrAg screening is a WHO recommendation, this would be considered 
standard of care – thus this alone would not require informed consent. 
The regulatory purpose of obtaining informed consent would be for participation in 
an experimental randomized clinical trial of the preemptive treatment of CrAg+ 
persons with sertraline vs. placebo. 


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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 starting 
intervention/administering 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. The subjects should have the 
opportunity to discuss the study with their surrogates or think about it prior to agreeing 
to participate. We will assess participant comprehension during the consent process.  
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.   
If subjects decline to participate in the study or withdraw consent, they will still be 
recommended to have preemptive therapy as per WHO treatment recommendations 
(i.e. the fluconazole preemptive treatment regimen). 
15.3.1. 
Documentation of Informed Consent 
Informed consent is a process that is initiated prior to an individual’s agreeing to 
participate in the study and continuing throughout the individual’s study participation. 
After CrAg+ diagnosis and eligibility criteria are confirmed by a study investigator, the 
study investigator will obtain full informed consent for the study by providing potential 
subjects with an approved consent form in English, Luganda (e.g. Kampala), 
Runyankole (e.g. Mbarara). Informed consent must occur within 7 calendar days of 
the potential study participant receiving their first dose of fluconazole.  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 that he or she may have about 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 be recommended to receive standard fluconazole 
preemptive therapy as per WHO and national guidelines. 
If, in the opinion of the investigator, potential participants 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 pre-existing meningitis. 
If the potential subject is deemed in the opinion of the investigator to be unable to 


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give informed consent, the investigator may choose to delay informed consent and/or 
refer the patient to hospital for a diagnostic lumbar puncture. 
15.3.2. 
Literacy / Proxy Consent 
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. 
15.4. Waiver of Informed Consent 
Cryptococcal antigen screening is considered routine care as recommended by 
national and WHO guidelines.  Thus CrAg screening is not considered a research 
activity, and specific consent will not be obtained.  
Two activities are requested for a waiver of informed consent.  First, a waiver of 
consent is requested for sample storage of the initial plasma specimens from CD4 
testing among persons who are CrAg+ but who do not return to enroll in the study. 
These previously collected specimens would otherwise be discarded. As untreated 
cryptococcal antigenemia has ~100% mortality, these persons most likely will have 
died. The purpose of storing such specimens is to investigate potential biomarkers, 
which are associated with high risk of acute mortality and thereby would constitute a 
critical value and medical emergency. Persons who decline informed consent would 
not have their baseline plasma stored. 
Second, a waiver of informed consent is requested for testing the initial 
plasma/serum specimen among CrAg+ persons for ALT, which is an exclusion 
criterion. The purpose to do this testing is to streamline the enrollment process and 
to expedite initiating therapy for this life threatening cryptococcal infection. In the 
absence of this ALT testing, CrAg+ persons will need to return to clinic, consent for 
the study, have their ALT drawn, and then return to clinic for a second visit to enroll 
in the study and be randomized and initiate therapy. This delay would likely increase 
the risk of death for CrAg+ participants.  
15.5. Stored Samples and Associated Data Considerations 
Long term storage of specimens beyond the duration of the project requires 
additional written informed consent for receiving blood draws or other sample 
collection 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. 
Alternatively, subjects may give limited consent to the collection, testing and 
storage of samples for the purposes of this research only.  This does not require 
additional consent, and all samples will be destroyed when the study is completed.  
Patients giving this limited consent will still be able to participate in the study. 


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15.6. Risks 
Persons who are CrAg+ with CD4<100 have an expected 20-30% 6-month 
mortality with preemptive treatment as per international standard of care as 
recommended by the Ugandan, WHO, and U.S. DHHS Guidelines.  Comparatively, 
the risk of additional sertraline vs. placebo is minimal.  
SSRIs are currently among the most prescribed drug classes worldwide, and 
sertraline in particular remains one of the most prescribed antidepressants on the 
U.S. retail market, with nearly 30 million prescriptions in 2007.102 Sertraline has 
minimal inhibitory effects on the major cytochrome P450 enzymes, and few drug-
drug interactions of clinical significance have been documented (Section 6.4).50  
Abundant clinical data supports the safety of long-term use of this as an 
antidepressant at doses between 50-200mg.47-49  Like other SSRI, sertraline is 
relatively safe in over-dosage.42, 43 Efavirenz decreases sertraline levels by 25-50% 
at lower doses of sertraline; however, there is no sertraline effect on antiretroviral 
medicines.37, 38  
Common adverse effects include nausea (25% vs. 11% for placebo), male 
ejaculation failure (14% vs. 1% for placebo), insomnia (21% vs. 11% for placebo), 
drowsiness (13% vs. 7% for placebo), diarrhea (20% vs. 10% for placebo), 
constipation (6% vs 4% for placebo), dry mouth (14% vs. 8% for placebo), dizziness 
(12% vs. 7% for placebo), tremor (8% vs. 2% for placebo) and decreased libido (6% 
vs. 1% for placebo).51  These adverse events are often transient and resolve 
spontaneously without dosage adjustment.  Sertraline also appears to be associated 
with microscopic colitis, a rare condition of unknown etiology.52  Akathisia (i.e. 
restlessness) caused by sertraline was observed in 16% of patients in a case 
series.53  Akathisia typically begins several hours after the initiation of treatment or a 
dose increase and usually disappears after being  
15.7. Benefits 
Research Participants will receive more oversight of their cryptococcal infection than 
in usual care. Participants who develop symptomatic meningitis will receive prompt 
referral for further expert care for cryptococcal meningitis in accordance with 
international standards of care.13   
Participants who receive sertraline, if effective, may receive a survival benefit.  There 
may additionally be a therapeutic benefit of more rapid resolution of depressive 
symptoms among persons with depression; however, this has unknown efficacy in 
this population with advanced HIV/AIDS and early disseminated cryptococcal 
infection. 
15.8. Compensation 
Participants will receive reimbursement of transportation expenses for unscheduled 
or scheduled study visits.  If participants are too ill to attend clinic an unmarked 
vehicle (e.g. special hire) will be sent to retrieve the participant to bring them to clinic 
or hospital.  Clinical trial insurance exists to compensate participants if they suffer 
bodily injury as a consequence of sertraline therapy via Lion Assurance Company, 
Ltd. P.O. Box 7658, Kampala. 


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15.9. Participant Privacy and Confidentiality 
All participant-related information including case report forms, laboratory 
specimens, evaluation forms, reports, etc., will be kept strictly confidential. All 
records will be kept in a secure, double locked location and only research staff will 
have access to the records. Participants will be identified only by means of a 
coded number specific to each participant.  
HIV clinic records will kept in the local HIV clinic as per local practice.  
All computerized databases will identify participants by numeric codes only, and 
will be password-protected. Upon request, participant records will be made 
available to the study sponsor, the sponsor’s monitoring representative, 
representatives of a participating pharmaceutical sponsor and applicable 
regulatory entities, including the UNCST, Uganda National Drug Authority, US FDA, 
JCRC IRB, or University of Minnesota. 
15.10. New / Incidental Findings 
Relevant new findings regarding CrAg screening will be incorporated by amendment 
into the protocol. 
15.11. Study Discontinuation 
The study may be discontinued at any time by the IRB, NIAID, FDA, Uganda 
National Drug Authority, or other government entities as part of their duties to 
ensure that research participants are protected.  
15.12. Post-Trial Access 
Participants with severe depression may elect to switch to open-label sertraline after 
16 weeks at provider discretion, and participants continued beyond the end of the 
study period.  Sertraline is a licensed / registered medicine in Uganda and is 
available in retail pharmacies.  
15.13. Community Advisory Board and Other Relevant Stakeholders 
The local Community Advisory Board(s) (e.g. “IDI Friend’s Council”) will be 
approached for this study to inform them of the national guidelines regarding pre-
ART CrAg screening and this randomized clinical trial. 
 
 


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16. ADMINISTRATIVE PROCEDURES 
16.1. Protocol Registration 
Prior to implementation of this protocol, and any subsequent full version 
amendments, each site must have the protocol and the protocol consent form(s) 
approved, as appropriate, by their local institutional review board (IRB)/ethics 
committee (EC) and any other applicable regulatory entity (RE). Upon receiving final 
approval, sites will submit all required protocol registration documents to the DAIDS 
Protocol Registration Office (PRO) at the Regulatory Support Center (RSC). The 
DAIDS PRO will review the submitted protocol registration packet to ensure that all 
of the required documents have been received. 
Site-specific informed consent forms (ICFs) WILL be reviewed and approved by the 
DAIDS PRO, and sites will receive an Initial Registration Notification from the DAIDS 
PRO that indicates successful completion of the protocol registration process. A 
copy of the Initial Registration Notification should be retained in the site's regulatory 
files. 
Upon receiving final IRB/EC and any other applicable RE approvals for an 
amendment sites, should implement the amendment immediately. Sites are required 
to submit an amendment registration packet to the DAIDS PRO at the RSC.  The 
DAIDS PRO will review the submitted protocol registration packet to ensure that all 
the required documents have been received.  Site-specific ICF(s) WILL NOT be 
reviewed and approved by the DAIDS PRO, and sites will receive an Amendment 
Registration Notification when the DAIDS PRO receives a complete registration 
packet. A copy of the Amendment Registration Notification should be retained in the 
site's regulatory files. 
For additional information on the protocol registration process and specific 
documents required for initial and amendment registrations, refer to the current 
version of the DAIDS Protocol Registration Manual. 
For additional information, refer to the protocol registration documents located at 
www.niaid.nih.gov/LabsAndResources/resources/DAIDSClinRsrch/Documents/proto
colregpolicy.pdf.  For questions regarding protocol registration, contact the Protocol 
Registration Office via e-mail at protocol@tech-res.com, fax (800-418-3544 or 301-
897-1701), or phone (301-897-1707). 
16.2. Regulatory Oversight 
The local IRB of record is the JCRC IRB in Kampala, Uganda for oversight and 
reporting of unexpected SAEs. Expected SAEs (e.g. meningitis, death) will be 
reported with annual reporting. 
16.3. ClinicalTrials.gov 
This protocol is subject to the Food and Drug Administration (FDA) Amendments 
Act of 2007 (FDAAA) and is registered in ClinicalTrials.gov with annual updates to be 
provided.  FDA Investigational New Drug (IND) application number is: 133772. 
16.4. Conflict of Interest 


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No conflict of interest exists by any investigator.  Any investigator who develops a 
new conflict of interest will disclose this to their relevant University oversight board, 
to the executive committee, and to the Study Sponsor. Sertraline is a generic 
medicine, so this is not expected.  
 
 
 


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18. APPENDICES 
APPENDIX A: Informed Consent 
Study Title: Cryptococcal Antigen Screening plus Sertraline 
The lead investigators of this research study are Dr. David Meya of the Infectious Disease Institute, 
and Dr. Radha Rajasingham 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 that 
requires hospitalization for treatment for 14 days and then continued therapy.  However, this 
infection can be detected before one develops symptoms and becomes ill.  People can be 
screened for infection by a blood test to detect “cryptococcal antigen,” (called CrAg), which is part 
of the fungus, in your blood.  We have tested your blood and found this cryptococcal antigen 
(CrAg).  This means that you recommended to receive early treatment now.  
 
However, It is unknown how best to treat people with cryptococcal antigen in their blood, who don’t 
otherwise yet have symptoms of infection around their brain.  It is unknown how best to treat 
people with a positive blood test for “Crypto.” If no treatment is given, most people will develop 
infection of the brain and/or die. Ugandan guidelines suggest using both HIV medicines and an 
anti-fungal medicine, called fluconazole, to treat this early infection. However, despite this 
treatment 1 in 4 people may get sick and/or die.  
Researchers have recently begun to look at another medicine that may be helpful in treating 
crypto. This medicine is called Sertraline, and it is actually a medicine that has been used for more 
than 25 years to treat depression (sadness).  Sertraline is one of the most commonly used 
medicines worldwide and is a registered, approved medicine in Uganda. Because sertraline is so 
widely used, much is known about this drug. It appears to be a very safe medicine that has few 
interactions with other medicines, and it is given as a pill. However, the ability of sertraline to treat 
Crypto is still being tested. The sertraline dose needed for Crypto is higher than commonly used to 
treat depression (sadness) at a dose of 400mg per day.  However in a recent study of infection of 
the brain with Crypto, adding sertraline 400mg for two-weeks did not work better than standard 


 
100 
therapy alone.   Sertraline has not been used in people without symptoms to treat early Crypto 
infection.  We do not know if Sertraline will work in early Crypto infection. 
The purpose of this study is to determine if usual therapy plus a high dose of Sertraline (400mg per 
day), will be better than usual therapy alone for treating early Crypto infection. Usual therapy is 
antifungal (fluconazole) and HIV medicines. We hope to have 600 people in Uganda participate. 
We will start by asking a number of questions to see if you are eligible for this research study as 
well as check your heart rate and rhythm with an electrocardiogram (EKG) for a baseline 
measurement. 
You confirm that the following has been explained to you, and you have had a chance to 
ask questions: 
1. The benefit of Sertraline (the experimental medicine) to treat Crypto infection in people is 
unknown. There may or may not be any benefit.  
2. Sertraline is usually given to treat depression (sadness) at doses of 50-200mg per day. This 
study will use a dose of 400mg per day.  
3. You will receive the usual antifungal medicine (fluconazole). 
4. The purpose of this study is to find out if Sertraline in addition to usual medicine will be 
better treatment to improve survival. You will therefore be selected to be treated with: 
A. placebo (empty pill), or  
B. Sertraline (experimental medicine) 
5. Whether you receive a or b (above) will be selected by chance (such as flipping a coin). 
6. You will be required to take all of your medications faithfully. 
7. You will be required to return for clinic visits approximately every 2 weeks x 3 visits, then 
every 4 weeks for 6 months. 
8. You may be contacted by phone or text message as a reminder of clinic appointments, to 
check how you are doing, and see if you are having any problems with your medicines. 
9. You will undergo further blood tests for this study today, in 1 month, in 2 months, and in 3 
months. These tests will check your liver and kidney function. 
10. If you agree, some samples may be collected and stored for future research tests to better 
understand Crypto infection and treatment. This may benefit future people in Uganda and 
around the world, but you will not benefit directly from this.  
11. You can choose not to have your samples stored and still participate in this study. 
12. Study doctors will review your medical records. 
13. You will not be paid for participating in this study, but you will be given up to 20,000 Uganda 
shillings to refund your transport expenses for each study related visit. 
14. Any information about you shall be kept private, and your data will be used without your 
name, picture, or identity. 
15. 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 HIV medicines free at the clinic. You will continue to be treated 
with HIV medicines and fluconazole, as this is standard of care.  
16. You will not be giving up any of your legal rights by signing this consent form.  
17. You are going to be given a copy of this form.  


 
101 
18. If you develop a bad headache or become sick during the study, you should return to clinic 
right away or contact the study doctor by phone. If you are too sick to travel to clinic, we will 
help you get to hospital and provide the needed medical care. 
Risks 
It is recommended by the Ugandan national guidelines and your doctors that you start antifungal 
treatment along with antiretroviral medicines to treat the Crypto infection and your HIV infection. 
The study has the following risks:  
There is a risk that you could be receiving an extra medicine that is not needed. 
Sertraline is a relatively safe drug, but side effects can still occur, as with any medicine. Common 
mild side effects can include: 
 Nausea (14%) 
 Diarrhea (10%) 
 Increased sweating (5%) 
 Dizziness (5%) 
 Tremor/Shakes (6%) 
 Dry mouth (6%) 
 Sleepiness (6%) 
 Trouble falling asleep (10%) 
 Restlessness 
 Other side effects occur in 1 in 100 people 
 
Most of these side effects are fairly mild and go away over 1-2 weeks on the medicine or with 
stopping the medicine. Severe adverse reactions occur very rarely (less than 1 in 1000 people). 
These rare events include sudden eye pain and bleeding. Your doctors will check blood tests for 
your liver and kidney function to make sure no severe problems happen. Your doctors will also 
check your heart rate and rhythm. Side effects may or may not be more common because of the 
higher sertraline dose.  Until the study is completed, we will not know if sertraline is more helpful, or 
if more side effects occur. Stopping the Sertraline suddenly can cause side effects. Thus, if 
planning to stop the Sertraline, you must tell your doctor. Your doctor will tell you how to slowly 
decrease the amount of Sertraline by taking a little bit less each day. 
Some similar drugs can cause increased thoughts of harming oneself (suicide) in adults with 
sadness who are <25 years old, but that this is not common with Sertraline. This can happen in 
about 1 in 500 people with sadness.  However, we will ask you about any harmful thoughts you 
may have and refer you to a counsellor if this is a problem for you. 
 
This study requires that a small amount of blood be taken through a needle from a vein in your arm 
several times during the study. This blood will be taken to check your liver and kidney function. If 
there are problems found, your medicine may be decreased. This is about 60mL (12 teaspoons) 
more blood in total than would have been taken over the next 6 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.  
Benefits of participating in the study 


 
100 
You may not benefit directly from participating in this research study. However, participation will 
provide important information about whether Sertraline is an effective treatment for cryptococcal 
meningitis. This may benefit other patients in the future.  
Pregnancy 
Pregnant and breastfeeding women cannot be in this research study. Pregnancy must be 
avoided when taking the usual medicine (fluconazole) for Crypto infection, due to risks to the 
unborn child (fetus). This risk is not unique to the study. If you are a woman and sexually active, 
you will be required to use at least one method(s) to prevent getting pregnant, such as condoms, 
oral contraceptive pills, etc. If you later desire to become pregnant or think you may be pregnant, 
please notify the study doctor right away, so that your medicines can be adjusted.  
 
Alternatives to Study Participation 
If you do not participate in this research study, you would be recommended to receive the usual 
fluconazole antifungal medicine and standard HIV medicines.   
 
Sample Storage 
If you agree to participate in this study, we will collect some samples of your blood for your medical 
care and leftover blood for research during the study. These stored samples will be identified by a 
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 Crypto infection, HIV, the immune system, and to measure 
medicine levels in your blood. Some of the tests will be done locally in Uganda, and some may be 
done in the United States. With your permission, we would like to collect a small volume of blood 
(up to 20mL) along with your other routine blood draws for research testing (50mL in total). If you 
prefer to not have any extra blood drawn, you may still participate in the study. Many research 
tests will be performed later and will not change your treatment, thus you will not receive the test 
results. The results may help future persons by better knowing about Crypto infection.  This may 
include de-identified genetic testing, which will not be identified back to you individually. 
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 get 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. 
Research Related Injury 
In the event that you become sick during the study, let us know right away.  We will help you get 
the care you need.  It is very important to let us know right away if you develop a severe headache 
which does not go away. In this case, you may need a different treatment.  
If you experience problems related to Crypto infection of the brain or HIV-infectionyou 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. There is no 


 
101 
program for compensation either through the local clinic, local University, or the U.S. National 
Institutes of Health (NIH). The NIH is a sponsor of this study. 
Confidentiality 
Your study information will be kept private, but will be included in your medical records. Your name, 
picture of you, or identity will not be shared in any publications or presentations. Your records for 
the study may be reviewed by representatives of the U.S. National Institutes of Health, the 
University of Minnesota, the US Federal Drug Administration, and Ugandan authorities with the 
appropriate regulatory oversight to assure the accuracy and quality of the records and the correct 
conduct of the research study. 
 
Voluntary Nature of the Study 
Participation in this study is voluntary and standard care is available. Your choice to or not to be in 
this study will not affect your current or future relations with the clinic.  If you choose to participate, 
you are free to stop at any time without affecting those relationships. If you want to stop being in 
the study, you will continue to receive HIV medicines from the clinic, but without the additional 
research medicine and safety blood tests provided by the research study. If you stop being in the 
study, we ask to be able to call you in 6 months by telephone to see how you are doing. 
 
Study approval 
Approval to conduct this study will be granted by the Uganda National Council for Science and 
Technology as well as Uganda National Drug Authority. The National Institutes of Health (NIH) is 
the United States study funder, and the University of Minnesota is the sponsor. 
Contacts and Questions 
You may ask any questions you have now. Please ask.  If you have questions later or any urgent 
health concerns, you are encouraged to contact:  
 
Dr. David Meya in Kampala on phone number 077-254-3730 
 
Dr. Conrad Muzoora in Mbarara on phone number 077-254-7175 
 
Dr. Fred Kibengo in Masaka on phone number 077-243-5251 
 
You will be given a clinic appointment card with the phone number before you leave. In case of any 
questions regarding Welfare and rights of participants, you should contact: 
Dr. Jesse Kagimba, the Chairperson Joint Clinic Research Centre Ethics Review 
Committee on telephone number 0414201148/7 or the Executive Secretary Uganda 
National Council for Science and Technology, Plot 6 Kimera Road, Ntinda on telephone 
041-4750-500. 
 
 


 
102 
Informed consent 
I hereby consent to participate in this research: “Cryptococcal Antigen Screening plus Sertraline” 
 
Participant Name: _______________________________________ Date _______________ 
 
Participant Signature: 
 
 
____________________________________________________  
    ________________ 
    OR Thumbprint 
 
 
Signature of Witness ……………………………………………….Date…………………………… 
 
 
 
   (If consent read aloud to the participant)  
 
 
Name of Witness…………………………………………………………………………………. 
 
Description of who the Witness is: ………………………………………………………………… 
 
Name / Signature of the Person who administered this informed consent: 
 
 
____________________________________________ Date/Time: _________________ 
 
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. 
 
 


 
103 
SAMPLE STORAGE INFORMED CONSENT FORM 
STUDY TITLE: Cryptococcal Antigen Screening plus Sertraline 
Purpose of Sample Storage  
The purpose of this research study is to find out if a high dose of Sertraline in addition to 
standard therapy, will be tolerated, safe, and more effective than standard therapy alone for 
treating early Crypto infection to increase survival. We request your permission to store 
samples for future research, so that we may better understand early Crypto infection for 
others in the future. This storage of your samples will not benefit you directly, but this may 
benefit other Ugandans in the future. 
You understand the following: 
1. If you agree to participate in this study, we will collect some samples of your blood for your 
medical care and leftover blood for research during the study. 
2. We would like to store these samples. These samples will be identified by a number, not 
your name or other personal information, so that only people working on the study can 
identify you.  
3. As part of the study, we would like to keep any left-over stored samples for research testing 
about Crypto infection, HIV, the immune system, and to measure medicine levels in your 
blood.  
4. In addition, we would like to keep the left-over stored samples for future possible research.  
5. With your permission, we would like to collect a small volume of blood (up to 20mL) along 
with your other routine blood draws for research testing (50mL in total). If you prefer to not 
have any extra blood drawn, you may still participate in the study.  
6. Many research tests will be performed later and will not change your treatment, thus you 
will not receive the test results. The results may help future persons by better knowing 
about Crypto infection.  This may include anonymous genetic testing, which will not be 
identified back to you or your name.  
 
Duration of storage 
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.   Samples may be 
stored in Uganda or in the United States.   
 
Ownership 
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. 
 
Study approval  


 
104 
Approval to conduct this study in Uganda has been granted by Uganda National Council for 
Science and Technology.  
 
Welfare of participants 
You will not be paid to have your samples stored for future biological tests.  
 
Confidentiality  
Your privacy will be protected as your samples will not have your name and any genetic 
information will not be put in your medical record. The identity and results from your samples 
will be kept private. Your identity will not be disclosed in any publications or presentations. 
 
Voluntary Nature of the Study 
You may refuse to donate your left-over samples for future research, or refuse to have 
additional samples collected for such future research.  Both of these things are optional.  Your 
decision will not affect your participation in this research study or your medical care in any way.  
If you do not wish to donate your left-over samples for future research, your samples will be 
destroyed when this study is finished.   
 
Risks 
Once the samples are collected, there are no additional risks to you from testing and 
storage for purposes of this study or future research.   
 
 
Contacts and Questions 
You may ask any questions you have now. If you have questions later, you are encouraged to contact:  
 
Dr. David Meya in Kampala on phone number 077-254-3730, or  
 
Dr. Conrad Muzoora in Mbarara on phone number 077-254-7175.  
 
Dr. Fred Kibengo in Masaka on phone number 077-243-5251 
In case of  any questions regarding the Welfare and rights of participants, you should contact Dr. 
Jesse Kagimba, the Chairperson Joint Clinical Research Centre Ethics Review Committee on 
telephone number 0414201148/7 or the HIV/AIDS Research Committee Secretariat at Uganda 
National Council for Science and Technology, Plot 6, Kimera Road, Ntinda PO Box 6884; on 
telephone 041-4750-500. 
If you have any questions about the use of your samples, you can ask them now or contact the 
above individuals later.   
 
 


 
105 
Please indicate your choice regarding storage of your samples below.   
 
I consent to the collection, testing and storage of my samples (blood, spinal fluid, and other 
samples) for the purposes of this research, as well as for future research.  Samples may be 
stored indefinitely, unless I change my mind and ask for them to be destroyed. This may 
include genetic testing. 
 
I consent to the collection, testing and storage of my samples (blood and spinal fluid) for the 
purposes of this research only.  Samples will be destroyed when the study is finished. 
 
Participant / Representative / 
Parent / Guardian Signature _____________________________________OR  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date ______________________________ Participant Name ________________________________ 
 
 
Signature of Witness (if needed): ______________________________________________________   
(If consent read aloud to the participant) 
 
Name of Witness……………………………………………………………………………………………… 
 
 
Description of who the Witness is: …………………………………………………………………………. 
 
 
 
 
Name/Signature of who administered informed consent: __________________________________ 
 
 
 
   
 
 
  Date/Time: __________________________________ 
 
 
 
 


 
106 
Appendix B: Study Statistical Plan 
Appendix B: Study Monitoring and Statistical Analysis Plan  
Table of Contents 
1. 
PURPOSE 
106 
2. 
STUDY OVERVIEW 
107 
3. 
SUMMARY OF PROGRESS AND MONITORING REPORTS 
108 
4. 
STUDY PROGRESS REPORTS 
109 
4.1 
ACCRUAL REPORT 
110 
4.2 
FOLLOW-UP FOR ENROLLED PARTICIPANTS 
110 
4.3 
BASELINE CHARACTERISTICS REPORT 
110 
4.4 
STUDY DRUG ADMINISTRATION TIMING AND PATIENT CARE INDICATORS 
110 
4.5 
ADVERSE EVENT 
110 
4.6 
CUMULATIVE CLINICAL ADVERSE EVENTS 
111 
4.7 
SUMMARY OF LABORATORY ABNORMALITIES 
111 
5. 
CLINICAL QUALITY MANAGEMENT REPORT (CQMP) 
112 
5.1 
PROTOCOL VIOLATIONS 
112 
5.2 
DELINQUENCY REPORT 
112 
6. 
UNEXPECTED SERIOUS ADVERSE EVENT REPORTING 
113 
7. 
PREGNANCY REPORTING 
113 
8. 
DSMB REPORTING 
114 
8.1 
OPEN REPORT TO THE DSMB 
114 
8.2 
CLOSED REPORT TO THE DSMB 
114 
9. 
EARLY STOPPING GUIDELINES 
115 
9.1 
SAMPLE SIZE RE-ESTIMATION 
115 
9.2 
FUTILITY ANALYSIS 
116 
10. 
STATISTICAL ANALYSIS PLAN 
117 
11. 
REFERENCES 
118 
 
 
Purpose 
The purpose of this Study Monitoring and Statistical Analysis Plan (SMSAP) is to describe 
the types, content, and distribution schedules of study progress and safety monitoring 
reports required for NIH NIAID sponsored studies, and to define the statistical analysis 
plan that will be implemented at the end of the study.  
 


 
107 
The purpose of the monitoring portion of the SMSAP is to: 
1) Protect and ensure the safety of the subjects; 
2) Ensure the validity and integrity of the data for the clinical trial; 
3) Ensure that the clinical trial is monitored appropriately; 
4) Ensure that the data collected can monitor safety and address protocol objectives;  
5) Ensure that the executive committee and sponsor are aware of the schedule of 
monitoring for the clinical trial 
The purpose of the statistical analysis portion of the SMSAP is to define a priori the 
analyses that will be completed for the primary and secondary endpoints, including the 
specification of pre-defined subgroups.  
Study Overview 
This Cryptococcal Antigen Screening plus Sertraline (C-ASSERT) Trial is a randomized 
trial comparing adjunctive sertraline versus placebo added to standard care for 
asymptomatic CrAg+ patients in Uganda . Study entry will occur within 1 week of initiating 
fluconazole antifungal treatment. Subjects will be randomized in a 1:1 allocation ratio to 
adjunctive placebo or sertraline. The adjunctive sertraline to be used in this trial is dosed at 
100-200mg/day for two weeks, then 300-400mg/day for two weeks as induction therapy, 
followed by 400mg/day for 8 weeks, and then tapered over 4 weeks.  
  
HIV-infected persons who are CrAg + 
receiving standard therapy, randomized
n=600
Placebo 1->2->3->4 
tabs/d escelation over 4 
weeks
n=300
Placebo 4 tabs/day 
x 8 wks
Placebo Taper 
over 4 weeks
Followed another 8 
weeks through 24-
weeks
Sertraline 100 -> 200 -> 
300-> 400 mg/d 
escelation over 4 weeks
n=300
Sertraline 400 mg/day 
x 8 wks
Sertraline taper  
over 4 weeks
Followed another 8 
weeks through 24-
weeks


 
108 
The primary endpoint is improved 6-month meningitis-free survival in CrAg+ HIV-
infected persons using adjunctive sertraline when compared to standard therapy alone. 
Secondary endpoints include comparisons of the following outcomes across trial arms:  
1. 6-month Survival Time 
2. Incidence of Symptomatic Cryptococcal Meningitis  
3. Incidence of Clinical Grade 3,4,5, and/or Serious Adverse Events 
4. Incidence of Grade 3-5 Laboratory Adverse Events 
5. Incidence of premature study drug/placebo discontinuation 
6. Prevalence of depression by PHQ-9 score 
The study will enroll 600 participants over a 4 year period. Once enrolled, each 
participant will be followed for 18 weeks for the primary endpoint, which allows for 
appropriate follow-up time for all secondary endpoints. Participants with a prior history of 
cryptococcal meningitis will not be enrolled in this trial. 
 
 
 
Summary of Progress and Monitoring Reports 
Table 1. Type of Reports and their distribution 
 
Reports 
Prepared By: 
Frequency 
Study 
Team 
Safety 
Committee 
IRB 
DSMB 
DAIDS  
Medical Officer 
Study Progress 
Reports 
Data Manager 
Monthly 
 
 
 
 
 
Clinical Quality 
Management Report 
Study 
Coordinator/ 
Data Manager 
Quarterly 
 
 
 
 
 
Unexpected Serious 
Adverse Event 
Reports 
Site PI 
As needed 
<72 hours 
 
 
 
 
 
Early Safety 
Independent Review 
Statistician 
After every 
40 subjects  
 
 
 
 
 
Sertraline-related or 
unexpected SAEs 
Statistician 
< 7 days 
 
 
 
 
 
New Pregnancy 
Site PI 
As Needed 
< 48 hours 
 
 
 
 
 
Open DSMB Report 
Statistician 
At least 
Annual 
 
 
 
 
 
Closed DSMB 
Report 
Statistician 
At least 
Annual 
 
 
 
 
 


 
109 
 
The Trial Safety Committee consists of Drs. Jason Baker, Noeline Nakasujja, and Tihana 
Bicanic, and they will review after every 40 subjects have accrued 3 months follow up data 
until the DSMB assumes responsibility for oversight. Refer to Protocol Section 12.7.5. 
The site biostatistician preparing the Progress, Monitoring and DSMB reports is Kathy 
Huppler Hullsiek PhD in conjunction with the Data Manager, Ms. Ananta Bangdiwala. 
DSMB reports are scheduled to be prepared after each ~25% of the trial population is 
enrolled, and at least annually. 
Study Progress Reports 
Study Progress Reports include reports on accrual, adherence to study protocol, baseline 
characteristics, and data completeness at each study site. Reports are pooled across study 
groups. 
Purpose:   The purpose of the Study Progress Report is to monitor enrollment to ensure that 
accrual goals are met in a timely manner, to inform sites of the number of subjects enrolled, 
and to inform sites of whether accrual is meeting specified target goals for each study site. 
The purpose of the Delinquency Report is to ensure a current database and to make Sites, 
Site PI, and trial PI aware of specific problems regarding missing clinical data for rapid 
resolution. 
Responsibility for Preparation: Data Manager / Statistician 
 
Frequency of Preparation: Monthly  
 
Distribution The Study Progress Report is distributed as follows:  
 Study Team  
 NIH NIAID Representative 
 
Contents: The contents of the Study Progress Reports are as follows: 
Table 1: Participant Accrual 
Table 2: Follow-up Status for Enrolled Participants 
Table 3: Participant Baseline Characteristics 
Table 4: Study Drug Administration Timing and Proper Randomization 
Table 5: Summary of Grade 3, 4, and Grade 5 Adverse Events and Serious AEs 
Table 6: Line Listing of Cumulative Clinical Adverse Events by Participant ID 
Table 7: Summary of Laboratory Abnormalities by Visit 


 
110 
18.1. 
Accrual Report 
The following reports will be provided: 
 Enrollment by month and study site 
 Cumulative enrollment versus goal, by study site 
 Cumulative enrollment, overall 
Line listing of study PID number with their randomization assignment is not proposed to assure 
that blinding is maintained and that the randomization sequence is not deciphered. 
18.2. 
Follow-up for Enrolled Participants 
Summary of participants currently:  
 Receiving induction therapy (1-4 weeks) 
 Receiving consolidation therapy (5-12 weeks) 
 Receiving maintenance therapy (13-24 weeks) 
 Terminated study 
18.3. 
Baseline Characteristics Report 
 The following baseline data will be cumulatively summarized: 
 Age 
 Sex 
 Baseline laboratory values 
o CD4+ counts 
o Creatinine 
o Potassium 
o Total hemoglobin 
o ALT 
o Total bilirubin 
 Antiretroviral therapy (ART) status 
 Tuberculosis medication status 
 PHQ-9 depression score (Q9) and total score 
18.4. 
Study Drug Administration Timing and Patient Care Indicators 
Purpose: The Study Drug Administration Timing and Patient Care Indicators Report will 
monitor the timeliness of study and standard drug regiments relative to time of CrAg 
testing. It also monitors timeliness of diagnosis and enrollment in the trial relative to 
hospital admission. 
The following indicators will be summarized by site:  
 
Time from CrAg+ to Sertraline 
 
Time from Informed Consent to Enrollment 
18.5. 
Adverse Event  
Adverse Events (AEs) will be summarized by; 


 
111 
 Number of new and cumulative number of AEs overall by Grade 3-5. 
 Serious AEs 
18.6. 
Cumulative Clinical Adverse Events 
 Line listing by Participant ID 
 
18.7. 
Summary of Laboratory Abnormalities 
 By laboratory abnormality by visit and by DAIDS Grading 
 
 


 
112 
Clinical Quality Management Report (CQMP) 
 
Purpose: To assure regulatory compliance and ongoing quality control and quality assurance 
of the clinical data collection. 
Activities: Refer to CQMP for all details, in brief, activities will involve: 
 Quality Control of CRF data for entry criteria, protocol adherence, and Good Clinical 
Practice (GCP) adherence 
 Quality Assurance of data recorded 
 Regulatory review 
 
Responsibility for Preparation: Study Coordinators and Data Manager  
Frequency:   1) Continuous activities 
2) Quarterly summary reports 
Contents (expanded from the monthly Progress Reports):    
 Table 8: Line Listing of Protocol Violations by Participant ID 
 Table 9: Summary of Data Completeness by Visit 
 Table 10: Case Report Form (CRF) Delinquency Report 
 Table 11: Delinquent CRFs by Participant ID 
 
18.8. 
Protocol Violations 
Purpose:  The purpose of the Protocol Adherence Reports is to identify on an ongoing 
basis the adherence to the protocol and the incidence of protocol violations. The following 
reports will be provided: 
 Consent withdrawn 
 Inclusion criteria violations 
 Exclusion criteria violations 
 Line listing of PID by protocol violation  
18.9. 
Delinquency Report 
The Delinquency Report will include: 
 Count of missing CRFs by study site, which are: 
o 4 – 8 weeks overdue 
o >8 – 12 weeks overdue 
o >12 weeks overdue  
 Delinquency Summary by PID and site, which will include: 
o Number/type of delinquent CRF 
o Expected visit number of delinquent CRFs 


 
113 
Unexpected Serious Adverse Event Reporting 
Purpose: To comply with national guidelines, all serious unexpected adverse events must be 
reported to the IRB within 3 working days. An adverse event related to cryptococcal 
meningitis (a pre-existing condition) will not be considered to be an unexpected event.  
Expected Serious Adverse Events (SAEs) will be reported at the frequency requested by the 
local IRB of record. 
 
Refer to Protocol Section 10 on Adverse Event Reporting, and the Division of AIDS (DAIDS) 
Table for Grading the Severity of Adult and Pediatric Adverse Events (version 2014) and 
Adverse Event CRF.  
Responsibility for Preparation: Site PI 
Frequency of Reporting: As serious unexpected adverse events occur 
The site PI will report serious unexpected adverse events to the IRB, Trial Safety Committee, 
and DAIDS medical officer within 72 hours of the awareness of the event 
  
The Trial Safety Committee will review each unexpected SAE and perform an early 
independent review of trial safety (mortality + SAE incidence) after 40 subjects have been 
enrolled and accrued 3 months follow up.  They will adjudicate any potentially sertraline-
related AEs or unexpected SAEs. 
Pregnancy Reporting 
In the event that a subject becomes pregnant after enrolling in the study, she must be 
referred to the local antenatal clinic. ART will be guided per national guidelines in the 
antenatal clinic. 
 
All pregnancies that occur during the study must be reported to the executive committee 
within 48 hours, regarding determination of continued fluconazole usage. Fluconazole is a 
known potential teratogen. Dependent on the duration of antifungal therapy and consultation 
with cryptococcal expert consultants (Drs. J. Perfect and T. Harrison), a recommendation will 
be made for the pregnant patient on the risks/benefits of fluconazole continuation, 
discontinuation, or dose reduction. This will be a non-binding recommendation. The decision 
to continue/discontinue/dose reduce fluconazole will be the exclusive choice of the research 
participant. The pregnant subjects may remain in the study regardless of their choice. 
 
A review of fluconazole teratogenicity of is available at: drugsafetysite.com/fluconazole. All 
pregnancies should be reported to the Antiretroviral Pregnancy Registry by fax at +44 1895 


 
114 
825 005. More information is available at www.apregistry.com. 
DSMB Reporting 
18.10. 
Open Report to the DSMB 
 Study progress and baseline data will be reviewed after each approximately 25% of 
subjects are randomized, and at least annually with any additional specified times as 
requested by the NIH DSMB. 
 Open DSMB reports will be prepared for the overall randomized group and will 
include: 
 Study progress reports: accrual, completeness of study follow-up, adherence to 
study protocol, data completeness 
 Baseline data: participant characteristics, medical history, laboratory measures 
 Baseline treatment status and regimens for CrAg + and HIV treatment 
 Safety data (Grade 3-5 adverse events) 
18.11. 
Closed Report to the DSMB 
Closed reports will report by treatment group. The closed reports will include: 
 Tables from the Open Report (by treatment arms) and Safety data (Grade 3-5 
adverse events) 
 Primary and secondary outcomes, including: 
o  6-month cryptococcal-free survival 
o incidence of symptomatic cryptococcal meningitis 
o incidence of adverse events 
o incidence of laboratory adverse events 
o incidence of premature study drug/placebo discontinuation 
 Other noteworthy significant clinical events 
 
Treatment groups will be formally statistically tested for: 
 6-month cryptococcal-free survival 
 
 


 
115 
Early Stopping Guidelines 
A Lan-DeMets spending function analog of the O’Brien-Fleming boundaries will be provided 
at each DSMB report for the stopping guidelines for 6-month cryptococcal-free survival 
outcome. The O’Brien-Fleming boundaries will be truncated at alpha=0.002 (|Z|>3.09). The 
provided table assumes three interim analyses, and a final analysis with an overall two-sided 
cumulative alpha=0.05. 
  
Interim 
Analysis 
Sample Size 
| Z | 
P-value 
Cumulative 
Alpha 
1 
~150   (25%) 
> 3.09 
0.002 
0.0020 
2 
~300   (50%) 
> 3.09 
0.002 
0.0037 
3 
~450   (75%) 
> 2.38 
0.016 
0.0193 
Final 
   ~600  (100%) 
> 2.02 
0.031 
0.0500 
 
The study was designed assuming 25% and 16% with a primary event in the control and 
treatment groups, respectively.  At the first DSMB review, the stopping boundary is unlikely to 
be crossed:  if the event rate in the control and treatment groups was 25% and 37%, 
respectively, then the Z-statistic would be 3.2 and the stopping boundary would have been 
crossed.  Thus for a consistent 25% event rate in the control arm, the event rate would need 
to increase from 16% to 32% in the treatment arm to stop the study at the first DSMB.  The 
difference necessary to trigger early stopping will converge toward an approximate 9% 
absolute mortality difference by study conclusion. 
The purpose of the early DSMB reviews is to assess the trends for safety and efficacy and 
allow for the DSMB to determine if more frequent than annual DSMB reviews are appropriate. 
If more interim analyses are desired by the DMSB, the Lan-DeMets spending function will be 
recalculated using O’Brien-Fleming boundaries and provided with each closed report. 
We recommend that the DSMB consider early termination or protocol modification 
only when the O’Brien-Fleming boundary is crossed for the difference in 6-month 
cryptococcal-free survival. 
Should a stopping boundary be crossed, we would recommend a sub-group analysis to 
determine whether the entire study should be stopped or a pre-specified subgroup excluded 
from further enrollment. Subgroups to consider include clinical site, ART use at baseline, and 
TB-status. 
18.12. 
Sample Size Re-estimation 
When approximately 300 participants (50%) have been randomized, a formal sample size re-


 
116 
estimation will be conducted to assess whether the initial assumptions for 6-month 
cryptococcal meningitis-free survival were correct. This sample size re-estimation will be 
included as part of the closed report to the DSMB. The sample size re-estimation will be 
based on the pooled (and blinded) event rate and the 9% absolute difference in mortality 
expected between the groups. The sample size re-estimation will not take into account the 
interim treatment effect. 
Potential options for the DSMB to recommend may include: 
 Continuing the study as planned 
 Increasing the sample size, if the interim-observed treatment effect size is smaller 
than had been anticipated but is still clinically relevant. 
 Closing the study to enrollment.  
18.13. 
Futility Analysis 
The expected accrual rate is 125-150 participants per year. Enrollment in year 1 will likely be 
lower due to staggered starting of clinical sites, each of which is dependent on Funder 
permission to initiate.  
At the second DSMB if the accrual is less than 75% of what is expected, the DSMB 
committee may ask the study team to submit a formal plan to increase enrollment.  
If the conditional power is <25% at the time of any interim analysis, discontinuation may be 
considered as a possible recommendation by the DSMB. The DSMB will be given conditional 
power under both the design and the current data for their review.  Ultimately, the DSMB will 
make their own decision, irrespective of any stopping guidelines.  
Stopping for futility is always a possible DSMB recommendation.  
 
 


 
117 
Statistical Analysis Plan 
For the primary and secondary outcomes one comparison will be done:   
 Sertraline versus placebo 
All analyses will be performed as intention-to-treat. Persons lost to follow up will be 
considered failures. All participants will be censored at 6 months. 
Null Hypothesis: H0: Sertraline has no survival benefit 
Alternative Hypothesis: H1: Sertraline has a survival benefit 
6-month cryptococcal-free survival, the primary outcome, will be statistically tested via a Log-
rank test with an indicator for treatment arm. Time to event analyses will also be summarized 
using Kaplan-Meier curves covering the time from enrollment to 6 months. 
Secondary endpoints that can be analyzed with time-to-event methods (incidence of 
symptomatic cryptococcal meningitis, incidence of adverse events, incidence of laboratory 
adverse events, incidence of premature study drug/placebo discontinuation) will be 
summarized with cumulative incidence (to account for competing risk of mortality).1  
 Symptomatic meningoencephalitis will be defined as meningitis symptoms plus:  
1) Cryptococcus culture positive meningitis,  
2) CSF CrAg+,  or  
3) Cryptococcoma(s) by neuroimaging or post mortem exam.  
The secondary outcome of prevalence of depression as per PHQ-9 questionnaire will be 
tested using a mixed effects regression model with a random intercept for individual to 
account for the intra-subject correlation induced by repeated PHQ-9 measures over time. An 
interaction indicator variable of time and treatment groups will assess differences in PHQ-9 
depression scores. Additionally, we will summarize the prevalence of depression (by severity) 
from baseline through 12 weeks by randomized group.PHQ-9 Total Score Depression 
Baseline demographic features of each study arm will be summarized, with statistical testing 
as appropriate for nominal and continuous variables to assure adequacy of randomization. If 
baseline variables differ between randomization groups, consideration of adjustment for such 
variable will occur. 
Baseline characteristics known to be associated with developing cryptococcal meningitis (e.g. 
CrAg titer) and baseline characteristics known to be associated with mortality (e.g. CD4, 
anemia) among those who are CrAg + will be queried as to whether they are equally 
distributed among the two randomized treatment arms.  If these are differentially distributed 
(not expected), a multivariable analysis will be conducted adjusting for the differentially 
distributed variables.  
 


 
118 
References 
1. 
Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a 
competing risk. Ann Stat 1998;16:1141–54. 
 
 
 
 
 


 
119 
Appendix C: Current Study Site List and Process Overview 
 
Appendix C is a separate document from the protocol and will be updated so as to remain current. 
 
C-ASSERT Trial Clinical Sites: 
1) Infectious Disease Institute (IDI), P.O. Box 22418, Kampala, Uganda.  
Contact: Dr. Elizabeth Nalintya enalintya@yahoo.com  
2) Joint Clinic Research Center (JCRC) – Mbarara Regional Centre of Excellence, Mbarara 
University of Science and Technology, 1 Hospital Road, Mbarara, Uganda 
Dr. Conrad Muzoora conradmuzoora@gmail.com  
3) Medical Research Council (MRC) / Uganda Virus Research Institute (UVRI) Masaka Field Station, 
P.O. Box 1603, Masaka, Uganda 
Dr. Freddie Kibengo freddie.kibengo@mrcuganda.org  
 
 
Overview of Laboratory Testing: 
CRAG Screening: CrAg screening will be performed as per standard of care in accordance with 
national and WHO guidelines.  In general, this screening will be predominantly performed as a lab-
based reflex test at time of CD4 testing, whenever the CD4 count is <100 cells/L. This will be 
performed at the local site CD4 referral laboratory.  These CD4 laboratories are on-site for clinics of: 
IDI-Kampala, TASO-Kampala, MRC-Masaka, and JCRC-Mbarara. In Kampala, the KCCA network of 
primary care clinics send CD4s to MUJHU-affiliated laboratory.  Additional outside clinics may also 
send CD4 specimens to TASO, MRC, and JCRC laboratories. Ugandan guidelines also recommend 
CrAg testing among individuals with suspected ART failure or WHO clinical stage III/IV illness, 
irrespective of the CD4 count. 
The initial CrAg positive plasma specimen tested at any non-DAIDS approved laboratory shall be 
transported for verification of CrAg-positivity at a DAIDS-approved laboratory (e.g. MU-JHU lab) or 
repeated at time of enrollment.  Hepatic ALT (exclusion criterion) may be run on collected serum or 
plasma specimens collected at this time point at the DAIDS-approved laboratory.   
 
Pre-Entry: Will assess for trial eligibility. Pregnancy testing for women of child-bearing potential 
will be tested at the local site laboratory on urine or serum. Pregnancy testing is required within 14 
days of enrollment for potentially childbearing women. If the exclusion ALT laboratory was not able to 
be run on a specimen collected at screening (i.e. insufficient volume), ALT must be run prior to 
randomization. The ALT can be run via ‘stat’ sample from the local laboratory or via a Piccolo point-of-
care chemistry instrument with a FDA-approved, CLIA waived panel. ALT testing is required within 30 
days of enrollment. 
If plasma was not stored from the screening visit (e.g. insufficient volume), CrAg status will be verified 
on a pre-entry specimen at a DAIDS-approved facility. This CrAg testing may be by fingerstick or 
blood draw. Any external lab result from a non-DAIDS approved facility will need to be repeated for 
verification. Only the DAIDS-approved lab facility lab result is official.   


 
120 
Enrollment: Immediately after the pre-entry visit (i.e. same day), persons who are eligible will be 
randomized. Enrollment visit blood draw will occur. Additional pre-ART laboratories may be drawn at 
this visit for purposes of routine HIV care in accordance with local clinic policy (e.g. hepatitis B 
screening, creatinine, hemoglobin).  Additional leftover serum and plasma shall be stored at -80⁰C for 
future pathogenesis studies.  PBMCs may be collected in Kampala depending on the time of day of 
the clinic visit. PAXGene RNA tubes may be collected at sites, based on financial resources available.  
 
Cerebrospinal Fluid (CSF) Testing: Persons with suspected meningitis shall be referred for a 
diagnostic lumbar puncture (LP). Such LPs may occur in the clinic, referral center, or hospital as 
appropriate. CSF specimens shall be tested locally for CSF CrAg and routine CSF profile. CSF 
cultures shall be performed at the Makerere University Clinical Microbiology laboratory or Mbarara 
University Microbiology laboratory.  Specimens from Masaka shall be sent to Kampala.  Refer to 
quantitative CSF culture SOP for methodology. All collected CSF shall be stored and cryopreserved at 
-80⁰C.   
A participant symptomatic for meningitis with a positive CSF CrAg or CSF culture = cryptococcal 
meningitis primary endpoint. CSF CrAg is more sensitive than culture (e.g. sensitivity >99% CrAg LFA 
vs. ~94% CSF culture not on ART, ~85% CSF culture when on ART).  
 
Follow Up Visits: 
There is routine laboratory monitoring specified by the protocol at 4, 8, and 12 weeks although the 
safety profile of sertraline (and fluconazole) are excellent. In the event of a serious adverse event, 
relevant specimens should be collected as appropriate by physician discretion. For example, in the 
event of jaundice, collection of liver function tests (e.g. AST, ALT, total bilirubin) would be appropriate. 
Such specimens may be run locally for purposes of immediate clinical care. However, all specimens 
for a serious adverse event (SAE) shall be stored and transported to a DAIDS-approved lab for 
confirmatory testing.  The laboratory values at the DAIDS-approved laboratory shall be used for 
official study-related documentation.  
 
Total Test Volume of Protocol Specified Tests  
Test 
N 
Volume (mL) 
Total Volume (mL) 
CBC 
4 
4-6 
16-24 
Electrolytes, Creatinine 
4 
4 
16 
Liver Function Tests 
5 
4 
20 
Total 
 
 
52-60 
 
 
 
 


 
121 
Pharmacy Overview:  
This is a randomized, double-blind placebo controlled trial.  Study medicine (sertraline or matched 
placebo) will be prepared at the central pharmacy at the IDI in Kampala. The pharmacist of record is 
Eva Laker.  Study medicine bottles will be labeled with a unique identifier. Biostatisticians at UMN will 
generate labels for the pill bottles. Each label will be pre-printed with only a unique drug ID number.  
There will be two sets of labels for Sertraline or Placebo. The labels will be delivered to the pharmacy 
staff at IDI in Kampala.  These unique identifiers will be in random sequences, such that there is no 
numeric pattern to identify sertraline or placebo bottles. In Kampala, medicines will be dispensed from 
the IDI pharmacy.  In Masaka and Mbarara, a small supply will be provided from the IDI pharmacy 
(e.g. sufficient for ~20 participants) and replenished as needed from the satellite pharmacies. 


 
122 
KAMPALA SITE OVERVIEW 
1) Infectious Diseases Institute (IDI) / College of Health Sciences, Makerere University, 
Kampala, Uganda for enrolment and follow up with referrals from health facilities 
including:  
 The AIDS Support Organisation (TASO), Kampala 
 Kampala Capital City Authority (KCCA) Clinics, Kampala, Uganda 
 Kawaala 
 Kawempe 
 Kiruddu 
 Kisenyi 
 Kiswa 
 Kitebi 
 Komamboga 
 Kisugu 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IDI Pharmacy will be the pharmacy of record for the C-ASSERT trial with Eva Laker as 
pharmacist of record. IDI pharmacy will perform the dispensing for all Kampala sites. With 
couriers displacing medicines to KCCA study sites.  
* CrAg LFA testing for TASO clinic will initially be performed at TASO. All follow up research-
related labs performed at MUJHU.  
CSF Analysis 
(as needed) 
Makerere 
Microbiology 
MU-JHU 
Laboratory 
Storage of 
Cryptococcal 
isolates & CSF 
CrAg 
Testing* 
Hematology & 
Chemistry 
Specimen Storage of 
Serum, Plasma, or 
PaxGene tubes 
CrAg Screening from IDI and KCCA 
will be performed as a lab-based reflex 
screening at time of CD4 testing with 
samples sent to MUJHU lab.  
TASO CrAg screening to be performed 
at the TASO lab. Confirmation will 
occur at MUJHU 
MU-JHU 
Laboratory 
PBMC 
(MUWRP) 
CrAg Titers 
MU-JHU 


 
123 
MBARARA SITE - OVERVIEW OF LABORATORY TESTS 
Lab-based reflex RAG screening will be implemented at local CD4 labs (e.g. MSF Epicentre). 
Outside clinics may refer patients with the CrAg testing repeated at the study site.  
  
 
 
 
 
 
 
      Courier 
 
 
 
 
 
 
 
Mbarara Satellite Pharmacy:  Will maintain a small stock of blinded study medication as prepared by 
the IDI pharmacy in Kampala for local dispensing.  
 
MASAKA SITE OVERVIEW 
Outside clinics may perform CrAg testing with referral of potential research participants to MRC-
Masaka, where repeat CrAg testing will be performed. The MRC-Masaka laboratory is GCLP 
compliance and participates in EQA. 
  
 
 
  
 
 
 
       
 
 
 
 
 
MRC-Masaka Satellite Pharmacy:  Will maintain a small stock of blinded study medication as 
prepared by the IDI pharmacy in Kampala for local dispensing. 
 
 
CSF Analysis 
(as needed) 
MUST 
Microbiology 
Storage of 
Cryptococcal 
isolates & CSF 
CrAg 
Testing 
Hematology & 
Chemistry 
MSF EpiCentre 
Lab 
Endpoint Lab 
Masaka or 
MUJHU 
Specimen 
Storage of 
Serum, Plasma,
CrAg Titers 
MU-JHU 
CSF Analysis 
(as needed) 
CSF CrAg 
UK MRC Lab 
CSF Culture 
Makerere Micro 
CrAg 
Testing 
Hematology & 
Chemistry 
Masaka MRC 
Lab 
Specimen Storage 
of Serum, Plasma


 
124 
Appendix D. Adverse Events observed in COAT and ASTRO-
CM sertraline Trial. 
 
Cryptococcal Optimal ART Timing (COAT) Trial in Uganda used amphotericin and fluconazole with a 
randomized timing of ART initiation. No sertraline was given. Data below are as published in the 
manuscript’s supplemental appendix.35  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 therapy.  
 
 
 
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. 


 
125 
 
 
Randomized ASTRO-CM Trial Participants Adverse Events: 
 
This reports on the ongoing Grade 4, 5, and SAEs observed during the randomized trial for 
cryptococcal meningitis with amphotericin + fluconazole 800 mg/day +/- sertraline 400mg/day. As of 
May 6, 2017, n=427 randomized participants. There has been no statistical difference in the incidence 
of adverse events.  There have been no sertraline-related Grade 4-5 AEs or SAEs among n=218 
participants randomized to 400mg/day of sertraline.  
 
Table A1 
Placebo 
Sertraline 
P-
value2 
 
 
 
 
Number Randomized 
209 
218 
 
 
 
 
 
Number of AEs per person, N (%) 
 
 
0.26 
None 
165 (78.9%) 
176 (80.7%) 
 
One 
32 (15.3%) 
37 (17.0%) 
 
Two 
10 (4.8%) 
5 (2.3%) 
 
Three or more 
2 (1.0%) 
0 (0.0%) 
 
Overall 
209 (100.0%) 
218 
(100.0%) 
 
 
 
 
 
Number of AEs by initial grade, N (%) 
 
 
0.60 
Grade 4 AE 
50 (86.2%) 
38 (80.9%) 
 
Grade 5 AE 
8 (13.8%) 
9 (19.1%) 
 
Total 
58 (100.0%) 
47 (100.0%) 
 
 
 
 
 
Number of AEs by final grade, N (%) 
 
 
0.77 
Grade 3 AE 
8 (13.8%) 
8 (17.0%) 
 
Grade 4 AE 
29 (50.0%) 
20 (42.6%) 
 
Grade 5 AE 
20 (34.5%) 
19 (40.4%) 
 
Unknown (ongoing follow up) 
1 (1.7%) 
0 (0.0%) 
 
Total 
58 (100.0%) 
47 (100.0%) 
 
Grade 3 = Severe, Grade 4 = Life Threatening, Grade 5 = Non-CM related Death. 
 
 


 
126 
 
Table A2. Adverse Events - Relations and Outcomes 
 
Placebo 
Sertraline 
P-value 
 
 
 
 
Number Randomized 
209 
218 
 
 
 
 
 
Relationships1, N (%) 
 
 
 
To Sertraline 
0 (0.0%) 
0 (0.0%) 
 
To Cryptococcus 
22 (37.9%) 
21 (45.7%) 
0.55 
To Antifungal therapy 
42 (72.4%) 
28 (60.9%) 
0.29 
To Antiretroviral drugs 
18 (31.0%) 
10 (21.7%) 
0.37 
To HIV/AIDS 
46 (79.3%) 
38 (82.6%) 
0.80 
 
 
 
 
Outcome, N (%) 
 
 
0.32 
Recovered/resolved 
26 (44.8%) 
19 (41.3%) 
 
Recovered/resolved with sequelae 
0 (0.0%) 
3 (6.5%) 
 
Severity worsened to Grade 5 
12 (20.7%) 
12 (26.1%) 
 
Death, unrelated 
16 (27.6%) 
10 (21.7%) 
 
Chronic, not expected to resolve 
0 (0.0%) 
0 (0.0%) 
 
Persistent, expected to resolve 
2 (3.4%) 
2 (4.3%) 
 
Unknown 
2 (3.4%) 
0 (0.0%) 
 
OVERALL 
1 (1.7%) 
47 (102.2%) 
 
 
 
 
 
1The relationships are NOT mutually exclusive. Multiple relationships could be recorded. 
Relationships could be possible, probable, or definite. 
 
Below is a line listing of Grade 4, 5, and SAEs.  The majority of AEs have been related to sequelae of 
AIDS, cryptococcal meningitis, and/or amphotericin B deoxycholate.  None have been deemed as an 
AE “related” to sertraline, in the opinion of the investigator. 
 
 


 
127 
 
Table A3. Types of Adverse Events by DAIDS Toxicity Table Category 
 
Placebo 
Sertraline 
 
 
 
Number Randomized 
209 
218 
 
 
 
Fever 
0 
4 
Infection 
1 
4 
Localized injection site reaction 
0 
1 
Hypotension 
1 
1 
Thrombosis/embolism 
0 
1 
Alteration in personality 
1 
1 
Headache 
1 
1 
Seizure (new onset) 
2 
2 
Dyspnea or respiratory distress 
4 
1 
Hemoglobin <8.5 g/L 
27 
17 
Decreased platelets <50 x109/L 
1 
0 
Acidosis 
2 
0 
ALT (SGPT), high 
0 
2 
Decreased serum bicarbonate 
1 
0 
Bilirubin (total), high 
0 
1 
Creatinine, high 
6 
3 
Potassium, high 
0 
2 
Potassium, low 
6 
2 
Sodium, low 
1 
1 
Miscellaneous  
4 
2 
 

SAP_001.pdf: 
C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-1 
Version: 6 Sept 2017 
Appendix B: Study Monitoring and Statistical Analysis Plan  
Table of Contents 
1. 
Purpose .................................................................................................................... 1 
2. 
Study Overview ........................................................................................................ 2 
3. 
Summary of Progress and Monitoring Reports ........................................................ 3 
4. 
Study Progress Reports ........................................................................................... 4 
4.1 
Accrual Report ....................................................................................................... 4 
4.2 
Follow-up for Enrolled Participants ......................................................................... 4 
4.3 
Baseline Characteristics Report ............................................................................. 5 
4.4 
Study Drug Administration Timing and Patient Care Indicators.............................. 5 
4.5 
Adverse Event ........................................................................................................ 5 
4.6 
Cumulative Clinical Adverse Events ....................................................................... 5 
4.7 
Summary of Laboratory Abnormalities ................................................................... 5 
5. 
Clinical Quality Management Report (CQMP) .......................................................... 5 
5.1 
Protocol Violations ................................................................................................. 6 
5.2 
Delinquency Report ................................................................................................ 6 
6. 
Unexpected Serious Adverse Event Reporting ........................................................ 7 
7. 
Pregnancy Reporting................................................................................................ 7 
8. 
DSMB Reporting ...................................................................................................... 8 
8.1 
Open Report to the DSMB ..................................................................................... 8 
8.2 
Closed Report to the DSMB ................................................................................... 8 
9. 
Early Stopping Guidelines ........................................................................................ 9 
9.1 
Sample Size Re-estimation .................................................................................... 9 
9.2 
Futility Analysis .................................................................................................... 10 
10. 
Statistical Analysis Plan ......................................................................................... 11 
11. 
References ............................................................................................................. 12 
 
1. Purpose 
The purpose of this Study Monitoring and Statistical Analysis Plan (SMSAP) is to describe 
the types, content, and distribution schedules of study progress and safety monitoring 
reports required for NIH NIAID sponsored studies, and to define the statistical analysis 
plan that will be implemented at the end of the study.  
 
The purpose of the monitoring portion of the SMSAP is to: 
1) Protect and ensure the safety of the subjects; 
2) Ensure the validity and integrity of the data for the clinical trial; 
3) Ensure that the clinical trial is monitored appropriately; 
4) Ensure that the data collected can monitor safety and address protocol objectives;  


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-2 
Version: 6 Sept 2017 
5) Ensure that the executive committee and sponsor are aware of the schedule of 
monitoring for the clinical trial 
The purpose of the statistical analysis portion of the SMSAP is to define a priori the 
analyses that will be completed for the primary and secondary endpoints, including the 
specification of pre-defined subgroups.  
2. Study Overview 
This Cryptococcal Antigen Screening plus Sertraline (C-ASSERT) Trial is a randomized 
trial comparing adjunctive sertraline versus placebo added to standard care for 
asymptomatic CrAg+ patients in Uganda . Study entry will occur within 1 week of initiating 
fluconazole antifungal treatment. Subjects will be randomized in a 1:1 allocation ratio to 
adjunctive placebo or sertraline. The adjunctive sertraline to be used in this trial is dosed at 
100-200mg/day for two weeks, then 300-400mg/day for two weeks as induction therapy, 
followed by 400mg/day for 8 weeks, and then tapered over 4 weeks.  
  
The primary endpoint is improved 6-month meningitis-free survival in CrAg+ HIV-infected 
persons using adjunctive sertraline when compared to standard therapy alone. Secondary 
endpoints include comparisons of the following outcomes across trial arms:  
1. 6-month Survival Time 
2. Incidence of Symptomatic Cryptococcal Meningitis  
3. Incidence of Clinical Grade 3,4,5, and/or Serious Adverse Events 
4. Incidence of Grade 3-5 Laboratory Adverse Events 
HIV-infected persons who are CrAg + receiving 
standard therapy, randomized
n=600
Placebo 1->2->3->4 tabs/d 
escelation over 4 weeks
n=300
Placebo 4 tabs/day 
x 8 wks
Placebo Taper 
over 4 weeks
Followed another 8 weeks 
through 24-weeks
Sertraline 100 -> 200 -> 
300-> 400 mg/d escelation 
over 4 weeks
n=300
Sertraline 400 mg/day 
x 8 wks
Sertraline taper  
over 4 weeks
Followed another 8 weeks 
through 24-weeks


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
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Version: 6 Sept 2017 
5. Incidence of premature study drug/placebo discontinuation 
6. Prevalence of depression by PHQ-9 score 
The study will enroll 600 participants over a ≈4 year period. Once enrolled, each 
participant will be followed for 18 weeks for the primary endpoint, which allows for 
appropriate follow-up time for all secondary endpoints. Participants with a prior history of 
cryptococcal meningitis will not be enrolled in this trial. 
3. Summary of Progress and Monitoring Reports 
Table 1. Type of Reports and their distribution 
 
Reports 
Prepared By: 
Frequency 
Study 
Team 
Safety 
Committee 
IRB 
DSMB 
DAIDS  
Medical Officer 
Study Progress 
Reports 
Data Manager 
Monthly 
 
 
 
 
 
Clinical Quality 
Management Report 
Study 
Coordinator/ 
Data Manager 
Quarterly 
 
 
 
 
 
Unexpected Serious 
Adverse Event 
Reports 
Site PI 
As needed 
<72 hours 
 
 
 
 
 
Early Safety 
Independent Review 
Statistician 
After every 
40 subjects  
 
 
 
 
 
Sertraline-related or 
unexpected SAEs 
Statistician 
< 7 days 
 
 
 
 
 
New Pregnancy 
Site PI 
As Needed 
< 48 hours 
 
 
 
 
 
Open DSMB Report 
Statistician 
At least 
Annual 
 
 
 
 
 
Closed DSMB 
Report 
Statistician 
At least 
Annual 
 
 
 
 
 
 
The Trial Safety Committee consists of Drs. Jason Baker, Noeline Nakasujja, and Tihana 
Bicanic, and they will review after every 40 subjects have accrued 3 months follow up data 
until the DSMB assumes responsibility for oversight. Refer to Protocol Section 12.7.5. 
 
The site biostatistician preparing the Progress, Monitoring and DSMB reports is Kathy 
Huppler Hullsiek PhD in conjunction with the Data Manager, Ms. Ananta Bangdiwala. 
DSMB reports are scheduled to be prepared after each ~25% of the trial population is 
enrolled, and at least annually. 
 
 


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-4 
Version: 6 Sept 2017 
4. Study Progress Reports 
Study Progress Reports include reports on accrual, adherence to study protocol, 
baseline characteristics, and data completeness at each study site. Reports are pooled 
across study groups. 
Purpose:   The purpose of the Study Progress Report is to monitor enrollment to ensure 
that accrual goals are met in a timely manner, to inform sites of the number of subjects 
enrolled, and to inform sites of whether accrual is meeting specified target goals for 
each study site. The purpose of the Delinquency Report is to ensure a current database 
and to make Sites, Site PI, and trial PI aware of specific problems regarding missing 
clinical data for rapid resolution. 
Responsibility for Preparation: Data Manager / Statistician 
 
Frequency of Preparation: Monthly  
 
Distribution The Study Progress Report is distributed as follows:  
• Study Team  
• NIH NIAID Representative 
 
Contents: The contents of the Study Progress Reports are as follows: 
Table 1: Participant Accrual 
Table 2: Follow-up Status for Enrolled Participants 
Table 3: Participant Baseline Characteristics 
Table 4: Study Drug Administration Timing and Proper Randomization 
Table 5: Summary of Grade 3, 4, and Grade 5 Adverse Events and Serious AEs 
Table 6: Line Listing of Cumulative Clinical Adverse Events by Participant ID 
Table 7: Summary of Laboratory Abnormalities by Visit 
4.1 Accrual Report 
The following reports will be provided: 
• Enrollment by month and study site 
• Cumulative enrollment versus goal, by study site 
• Cumulative enrollment, overall 
Line listing of study PID number with their randomization assignment is not proposed to 
assure that blinding is maintained and that the randomization sequence is not deciphered. 
4.2 Follow-up for Enrolled Participants 
Summary of participants currently:  
• Receiving induction therapy (1-4 weeks) 
• Receiving consolidation therapy (5-12 weeks) 
• Receiving maintenance therapy (13-24 weeks) 
• Terminated study 


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-5 
Version: 6 Sept 2017 
4.3 Baseline Characteristics Report 
• The following baseline data will be cumulatively summarized: 
• Age 
• Sex 
• Baseline laboratory values 
o CD4+ counts 
o Creatinine 
o Potassium 
o Total hemoglobin 
o ALT 
o Total bilirubin 
• Antiretroviral therapy (ART) status 
• Tuberculosis medication status 
• PHQ-9 depression score (Q9) and total score 
4.4 Study Drug Administration Timing and Patient Care Indicators 
Purpose: The Study Drug Administration Timing and Patient Care Indicators Report 
will monitor the timeliness of study and standard drug regiments relative to time of 
CrAg testing. It also monitors timeliness of diagnosis and enrollment in the trial 
relative to hospital admission. 
The following indicators will be summarized by site:  
• 
Time from CrAg+ to Sertraline 
• 
Time from Informed Consent to Enrollment 
4.5 Adverse Event  
Adverse Events (AEs) will be summarized by; 
• Number of new and cumulative number of AEs overall by Grade 3-5. 
• Serious AEs 
4.6 Cumulative Clinical Adverse Events 
• Line listing by Participant ID 
 
4.7 Summary of Laboratory Abnormalities 
• By laboratory abnormality by visit and by DAIDS Grading 
5. Clinical Quality Management Report (CQMP) 
 
Purpose: To assure regulatory compliance and ongoing quality control and quality 
assurance of the clinical data collection. 


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-6 
Version: 6 Sept 2017 
 
Activities: Refer to CQMP for all details, in brief, activities will involve: 
• Quality Control of CRF data for entry criteria, protocol adherence, and Good 
Clinical Practice (GCP) adherence 
• Quality Assurance of data recorded 
• Regulatory review 
 
Responsibility for Preparation: Study Coordinators and Data Manager  
 
Frequency:   1) Continuous activities 
2) Quarterly summary reports 
 
Contents (expanded from the monthly Progress Reports):    
• Table 8: Line Listing of Protocol Violations by Participant ID 
• Table 9: Summary of Data Completeness by Visit 
• Table 10: Case Report Form (CRF) Delinquency Report 
• Table 11: Delinquent CRFs by Participant ID 
 
5.1 Protocol Violations 
Purpose:  The purpose of the Protocol Adherence Reports is to identify on an 
ongoing basis the adherence to the protocol and the incidence of protocol violations. 
The following reports will be provided: 
• Consent withdrawn 
• Inclusion criteria violations 
• Exclusion criteria violations 
• Line listing of PID by protocol violation  
 
5.2 Delinquency Report 
The Delinquency Report will include: 
• Count of missing CRFs by study site, which are: 
o 4 – 8 weeks overdue 
o >8 – 12 weeks overdue 
o >12 weeks overdue  
• Delinquency Summary by PID and site, which will include: 
o Number/type of delinquent CRF 
o Expected visit number of delinquent CRFs 
 
 


C-ASSERT Study Monitoring and Statistical Analysis Plan (SMSAP) 
C-ASSERT Trial 
B-7 
Version: 6 Sept 2017 
6. Unexpected Serious Adverse Event Reporting 
Purpose: To comply with national guidelines, all serious unexpected adverse events 
must be reported to the IRB within 3 working days. An adverse event related to 
cryptococcal meningitis (a pre-existing condition) will not be considered to be an 
unexpected event.  Expected Serious Adverse Events (SAEs) will be reported at the 
frequency requested by the local IRB of record. 
 
Refer to Protocol Section 10 on Adverse Event Reporting, and the Division of AIDS 
(DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events (version 
2014) and Adverse Event CRF.  
Responsibility for Preparation: Site PI 
Frequency of Reporting: As serious unexpected adverse events occur 
The site PI will report serious unexpected adverse events to the IRB, Trial Safety 
Committee, and DAIDS medical officer within 72 hours of the awareness of the event 
  
The Trial Safety Committee will review each unexpected SAE and perform an early 
independent review of trial safety (mortality + SAE incidence) after 40 subjects have 
been enrolled and accrued 3 months follow up.  They will adjudicate any potentially 
sertraline-related AEs or unexpected SAEs. 
7. Pregnancy Reporting 
In the event that a subject becomes pregnant after enrolling in the study, she must be 
referred to the local antenatal clinic. ART will be guided per national guidelines in the 
antenatal clinic. 
 
All pregnancies that occur during the study must be reported to the executive committee 
within 48 hours, regarding determination of continued fluconazole usage. Fluconazole is 
a known potential teratogen. Dependent on the duration of antifungal therapy and 
consultation with cryptococcal expert consultants (Drs. J. Perfect and T. Harrison), a 
recommendation will be made for the pregnant patient on the risks/benefits of 
fluconazole continuation, discontinuation, or dose reduction. This will be a non-binding 
recommendation. The decision to continue/discontinue/dose reduce fluconazole will be 
the exclusive choice of the research participant. The pregnant subjects may remain in 
the study regardless of their choice. 
 
A review of fluconazole teratogenicity of is available at: drugsafetysite.com/fluconazole. 
All pregnancies should be reported to the Antiretroviral Pregnancy Registry by fax at 
+44 1895 825 005. More information is available at www.apregistry.com. 


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8. DSMB Reporting 
8.1 Open Report to the DSMB 
• Study progress and baseline data will be reviewed after each approximately 
25% of subjects are randomized, and at least annually with any additional 
specified times as requested by the NIH DSMB. 
• Open DSMB reports will be prepared for the overall randomized group and will 
include: 
• Study progress reports: accrual, completeness of study follow-up, adherence to 
study protocol, data completeness 
• Baseline data: participant characteristics, medical history, laboratory measures 
• Baseline treatment status and regimens for CrAg + and HIV treatment 
• Safety data (Grade 3-5 adverse events) 
8.2 Closed Report to the DSMB 
Closed reports will report by treatment group. The closed reports will include: 
• Tables from the Open Report (by treatment arms) and Safety data (Grade 3-5 
adverse events) 
• Primary and secondary outcomes, including: 
o  6-month cryptococcal-free survival 
o incidence of symptomatic cryptococcal meningitis 
o incidence of adverse events 
o incidence of laboratory adverse events 
o incidence of premature study drug/placebo discontinuation 
• Other noteworthy significant clinical events 
 
Treatment groups will be formally statistically tested for: 
• 6-month cryptococcal-free survival 
 
 


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9. Early Stopping Guidelines 
A Lan-DeMets spending function analog of the O’Brien-Fleming boundaries will be 
provided at each DSMB report for the stopping guidelines for 6-month cryptococcal-free 
survival outcome. The O’Brien-Fleming boundaries will be truncated at alpha=0.002 
(|Z|>3.09). The provided table assumes three interim analyses, and a final analysis with 
an overall two-sided cumulative alpha=0.05. 
  
Interim 
Analysis 
Sample Size 
| Z | 
P-value 
Cumulative 
Alpha 
1 
~150   (25%) 
> 3.09 
0.002 
0.0020 
2 
~300   (50%) 
> 3.09 
0.002 
0.0037 
3 
~450   (75%) 
> 2.38 
0.016 
0.0193 
Final 
   ~600  (100%) 
> 2.02 
0.031 
0.0500 
 
The study was designed assuming 25% and 16% with a primary event in the control 
and treatment groups, respectively.  At the first DSMB review, the stopping boundary is 
unlikely to be crossed:  if the event rate in the control and treatment groups was 25% 
and 37%, respectively, then the Z-statistic would be 3.2 and the stopping boundary 
would have been crossed.  Thus for a consistent 25% event rate in the control arm, the 
event rate would need to increase from 16% to 32% in the treatment arm to stop the 
study at the first DSMB.  The difference necessary to trigger early stopping will 
converge toward an approximate 9% absolute mortality difference by study conclusion. 
The purpose of the early DSMB reviews is to assess the trends for safety and efficacy 
and allow for the DSMB to determine if more frequent than annual DSMB reviews are 
appropriate. If more interim analyses are desired by the DMSB, the Lan-DeMets 
spending function will be recalculated using O’Brien-Fleming boundaries and provided 
with each closed report. 
We recommend that the DSMB consider early termination or protocol 
modification only when the O’Brien-Fleming boundary is crossed for the 
difference in 6-month cryptococcal-free survival. 
Should a stopping boundary be crossed, we would recommend a sub-group analysis to 
determine whether the entire study should be stopped or a pre-specified subgroup 
excluded from further enrollment. Subgroups to consider include clinical site, ART use 
at baseline, and TB-status. 
9.1 Sample Size Re-estimation 
When approximately 300 participants (50%) have been randomized, a formal sample 
size re-estimation will be conducted to assess whether the initial assumptions for 6-
month cryptococcal meningitis-free survival were correct. This sample size re-estimation 


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will be included as part of the closed report to the DSMB. The sample size re-estimation 
will be based on the pooled (and blinded) event rate and the 9% absolute difference in 
mortality expected between the groups. The sample size re-estimation will not take into 
account the interim treatment effect. 
Potential options for the DSMB to recommend may include: 
• Continuing the study as planned 
• Increasing the sample size, if the interim-observed treatment effect size is 
smaller than had been anticipated but is still clinically relevant. 
• Closing the study to enrollment.  
9.2 Futility Analysis 
The expected accrual rate is 125-150 participants per year. Enrollment in year 1 will 
likely be lower due to staggered starting of clinical sites, each of which is dependent on 
Funder permission to initiate.  
At the second DSMB if the accrual is less than 75% of what is expected, the DSMB 
committee may ask the study team to submit a formal plan to increase enrollment.  
If the conditional power is <25% at the time of any interim analysis, discontinuation may 
be considered as a possible recommendation by the DSMB. The DSMB will be given 
conditional power under both the design and the current data for their review.  
Ultimately, the DSMB will make their own decision, irrespective of any stopping 
guidelines.   
 
Stopping for futility is always a possible DSMB recommendation. 
 


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10. 
Statistical Analysis Plan 
For the primary and secondary outcomes one comparison will be done:   
• Sertraline versus placebo 
All analyses will be performed as intention-to-treat. Persons lost to follow up will be 
considered failures. All participants will be censored at 6 months. 
Null Hypothesis: H0: Sertraline has no survival benefit 
Alternative Hypothesis: H1: Sertraline has a survival benefit 
6-month cryptococcal-free survival, the primary outcome, will be statistically tested via a 
Log-rank test with an indicator for treatment arm. Time to event analyses will also be 
summarized using Kaplan-Meier curves covering the time from enrollment to 6 months. 
Secondary endpoints that can be analyzed with time-to-event methods (incidence of 
symptomatic cryptococcal meningitis, incidence of adverse events, incidence of 
laboratory adverse events, incidence of premature study drug/placebo discontinuation) 
will be summarized with cumulative incidence (to account for competing risk of 
mortality).1  
• Symptomatic meningoencephalitis will be defined as meningitis symptoms 
plus:  
1) Cryptococcus culture positive meningitis,  
2) CSF CrAg+,  or  
3) Cryptococcoma(s) by neuroimaging or post mortem exam.  
The secondary outcome of prevalence of depression as per PHQ-9 questionnaire will 
be tested using a mixed effects regression model with a random intercept for individual 
to account for the intra-subject correlation induced by repeated PHQ-9 measures over 
time. An interaction indicator variable of time and treatment groups will assess 
differences in PHQ-9 depression scores. Additionally, we will summarize the prevalence 
of depression (by severity) from baseline through 12 weeks by randomized group.PHQ-
9 Total Score Depression 
Baseline demographic features of each study arm will be summarized, with statistical 
testing as appropriate for nominal and continuous variables to assure adequacy of 
randomization. If baseline variables differ between randomization groups, consideration 
of adjustment for such variable will occur. 
Baseline characteristics known to be associated with developing cryptococcal 
meningitis (e.g. CrAg titer) and baseline characteristics known to be associated with 
mortality (e.g. CD4, anemia) among those who are CrAg + will be queried as to whether 
they are equally distributed among the two randomized treatment arms.  If these are 
differentially distributed (not expected), a multivariable analysis will be conducted 
adjusting for the differentially distributed variables.  
 


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11. 
References 
1. 
Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a 
competing risk. Ann Stat 1998;16:1141–54.