Clinical trial NCT02625974
Conditions: Chagas Disease
Sponsor: Bayer
Start date: 2016-01-27; Completion date: 2021-08-10
Enrollment: 330 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Researchers are looking for a better way to treat children who have an infectious disease caused by the parasite Trypanosoma cruzi (Chagas' disease).

Chagas'disease is an inflammatory, infectious disease caused by the parasite Trypanosoma cruzi. This parasite is mainly spread by insects called triatomine bug. If Chagas' disease is left untreated, it can later cause serious heart and digestive problems.

The study treatment nifurtimox has been used for more than 50 years to treat Chagas' disease. When used early after infection, it kills the parasite. In people who have long-term Chagas' disease, it's no longer possible to kill the parasite. However, nifurtimox may help slow the progression of the disease and its most serious complications.

Nifurtimox was developed for use in adults only, but has also been used in children (off-label) for over 40 years. Currently it is available for doctors to give to adults and to children. However, there are not enough data about nifurtimox in children.

The main purpose of this study is to learn how well nifurtimox works in children aged 8 months to less than 18 years with Chagas' disease. To answer this, the researchers will compare the amount of antibodies against the parasite Trypanosoma cruzi in the serum (fluid from blood without the clotting factors) between children treated with nifurtimox for 60 days with untreated children from the past (control group):

* 12 months and
* 4 years after the end of treatment. The data for the control group will come from 2 previous studies conducted in children.

Detailed description:

Arms: arm 1: Nifurtimox tablets administered three times daily for 60 days (Days 1 - 60, active nifurtimox treatment) arm 2: Nifurtimox tablets administered three times daily for 30 days, followed by placebo administered three times daily for 30 days (Days 1 - 30, active nifurtimox treatment; Days 31 - 60, placebo)
Interventions: intervention 1: Nifurtimox (Lampit, BAYA2502) intervention 2: Placebo
Intervention descriptions: intervention 1: For pediatric participants with body weight ≤ 40 kg: dosage 10 to 20 mg/kg/day in three divided doses.

For pediatric participants with body weight \> 40 kg: 8 - 10 mg/kg/day in three divided doses.

60 days or 30 days of nifurtimox treatment intervention 2: Matching placebo

Protocol documents:
Prot_002.pdf: 


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Prospective, historically controlled study to evaluate the efficacy and safety of a new 
pediatric formulation of nifurtimox in children aged 0 to 17 years with Chagas’ disease
This protocol version is an integration of the following documents / sections:
Original protocol, Version 1.0, dated 04 NOV 2014
Amendment no. 1 (described in Section 15.1)
forming integrated protocol Version 2.0, dated 02 APR 2015
Amendment no. 2 (described in Section 15.2)
forming integrated protocol Version 3.0, dated 19 OCT 2015
Amendment no. 3 (described in Section 15.3)
forming integrated protocol Version 4.0, dated 07 APR 2016
Amendment no. 4 (described in Section 15.4)
forming integrated protocol Version 5.0, dated 15 NOV 2016
Amendment no. 5 (described in Section 15.5)
forming integrated protocol Version 6.0, dated 12 OCT 2017
Amendment no. 6 (described in Section 15.6)
forming integrated protocol Version 7.0, dated 20 DEC 2017
Amendment no. 7 (described in Section 15.7) 
forming integrated protocol Version 8.0, dated 11 MAY 2018
Amendment no. 8 (described in Section 15.8) 
forming integrated protocol Version 9.0, dated 23 JUL 2019
Amendment no. 9 (described in Section 15.9) 
forming integrated protocol Version 10.0, dated 18 OCT 2019
Amendment no. 10 (global)
forming integrated protocol Version 11.0, dated 03 NOV 2020
Amendments not included in the consecutive numbering of amendments are 
local amendments not forming part of this integrated global protocol.


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1.
Title page
Study title: Prospective, historically controlled study to evaluate the efficacy and safety 
of a new pediatric formulation of nifurtimox in children aged 0 to 17 years 
with Chagas’ disease
Short title: Prospective study of  a pediatric nifurtimox formulation for Chagas’ disease
Test drug:
BAY No. A2502/Nifurtimox 
Study purpose:
To evaluate efficacy and safety, and characterize the 
pharmacokinetics, of nifurtimox in children with Chagas’ 
disease using 30-day and 60-day treatment durations
Clinical study phase:
3
Date:
03 NOV 2020
Registration:
EudraCT:  NA
Version no.:
11.0
Sponsor’s study no.:
16027
Sponsor:
Bayer AG, D-51368 Leverkusen, Germany
Sponsor’s medical expert:
 18F Citi Group Tower, No.33 Huayuan 
Shiqiao Rd, Shanghai, 200120, P.R. China
Phone: 
The study will be conducted in compliance with the protocol, ICH-GCP and any applicable 
regulatory requirements.
This is an electronically generated document that does not bear any sponsor signatures. The 
signature of the sponsor’s medically responsible person is filed in the TMF and available on 
request.
Confidential
The information provided in this document is strictly confidential and is intended solely for 
the guidance of the clinical investigation. Reproduction or disclosure of this document 
whether in part or in full to parties not associated with the clinical investigation, or its use for 
any other purpose, without the prior written consent of the sponsor is not permitted.
Throughout this document, symbols indicating proprietary names (, TM) may not be displayed.  Hence, the 
appearance of product names without these symbols does not imply that these names are not protected.
PPD
PPD


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Signature of principal investigator
The signatory agrees to the content of the final clinical study protocol as presented.
Name:
Affiliation:
Date:
Signature:
Signed copies of this signature page are stored in the sponsor’s study file and in the respective 
center’s investigator site file.


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2.
Synopsis
Title
Prospective, historically controlled study to evaluate the efficacy and safety of 
a new pediatric formulation of nifurtimox in children aged 0 to 17 years with 
Chagas’ disease
Short title
Prospective study of  a pediatric nifurtimox formulation for Chagas’ disease
Clinical study phase
3
Study objective(s)
This study consists of two parts: 

Part 1 comprises the treatment with nifurtimox including the 1-year 
follow up (CHICO)

Part 2 comprises the 3-year long-term follow-up (LTFU) of all 
subjects who were randomized and received at least one dose of their 
assigned nifurtimox treatment regimen in CHICO (CHICO SECURE)
Part 1 (CHICO)
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to 
historical untreated control at the 12-month follow-up (360 days from 
end of treatment [EOT]) as sero-reduction (defined as a ≥20% 
reduction in optical density [OD] measured by conventional enzyme-
linked immune sorbent assay [ELISA]) compared to baseline in 
subjects ≥8 months to < 18 years of age at randomization; or, sero-
conversion (defined as negative Immunoglobulin G concentration in 
all subjects)1
Secondary objectives of the study are:2

To assess the comparability of a 30-day regimen of nifurtimox to a 
60-day regimen of nifurtimox as sero-reduction or sero-conversion at 
the 12-month follow-up (360 days from EOT)3

To evaluate the safety/tolerability profile of nifurtimox by laboratory 
parameters (hematology, blood chemistry, urinalysis), 
electrocardiogram (ECG) monitoring, vital sign measurements (blood 
pressure, heart rate, respiratory rate, temperature), adverse event (AE) 
monitoring, and physical examinations, including neurological 
examinations

To evaluate the pharmacokinetics (PK)/pharmacodynamics (PD) of 
nifurtimox in children receiving the drug for treatment of Chagas’ 
disease (to be described in a separate report)
Exploratory objectives of the study are:4

To assess the comparability of a 60-day regimen of nifurtimox to
historical active control (benznidazole)5 as sero-reduction or 
sero-conversion at the 12-month follow-up (360 days from EOT)6
                                                
1 Text modified as per amendment 2
2 Text of first 3 secondary objectives modified, and fourth secondary objective was added, per Modification 2 (Amendment 
1)
3 Text modified per Modification 2 (Amendment 1)
4 Exploratory objectives added as per amendment 2.
5 Sero-conversion for the historical controls will be estimated from cure rates as presented in two 
publications.[15][16]  See Section 5, Study Design, Justification of the design.  


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
To assess the comparability of a 30-day regimen of nifurtimox to a 
60-day regimen of nifurtimox using quantitative polymerase chain 
reaction (qPCR) at the 12-month follow-up (360 days from EOT)7

To evaluate the relationship of conventional serology (as sero-
reduction or sero-conversion) to qPCR using frequencies of matches 
and mismatches to assess agreement8

To evaluate the relationship of non-conventional serology to 
conventional serology9
Part 2 (CHICO SECURE)
The primary objective of the LTFU part of the study is: 

Assess the incidence of seronegative conversion as confirmed by two 
types of assay, recombinant ELISA, and indirect hemagglutination 
assay (IHA), in subjects who were randomized and received at least 
one dose of the 60-day nifurtimox treatment regimen, compared to an
external control group of untreated patients with Chagas’ disease at 
the 4-year follow-up. 
The secondary objectives are:

Assess the incidence of seronegative conversion, as confirmed by two 
types of assay (recombinant ELISA and IHA), in subjects who were
randomized and received at least one dose of the 30-day nifurtimox 
treatment regimen at the 4-year follow-up.

Evaluate the proportion of responders who show both seronegative 
conversion as confirmed by two types of assay (recombinant ELISA 
and IHA) and no evidence of established cardiomyopathy as 
evaluated in electrocardiogram (ECG) recordings.

Assess serial reduction in OD measured by recombinant ELISA and 
total purified antigen ELISA, in subjects who were randomized and 
received at least one dose of either the 60- or 30-day nifurtimox 
treatment regimens compared to Visit 1 in Part 1.
The exploratory objectives are:

Assess the incidence of seronegative conversion as confirmed by two 
types of assay (recombinant ELISA and IHA) in subjects by age 
categories (≤2 years, >2 years to ≤6 years, >6 to ≤12 years, >12 to 
<18 years; age is defined as subject’s age at randomization). 

Assess the proportion of responders who show seronegative 
conversion and the incidence of seronegative conversion as confirmed 
by non-conventional serology in subjects who were randomized and 
received at least one dose of either 60- or 30-day nifurtimox treatment 
regimens.

Assess the occurrence of congenital infection with T. cruzi in children 
                                                                                                                                                        
6 Text modified per Modification 2 (Amendment 1)
7 Text modified per Modification 2 (Amendment 1)
8 Text modified per Modification 2 (Amendment 1)
9 Fourth objective added per Modification 2 (Amendment 1)


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born of female subjects who were randomized and received at least 
one dose of the 60- or 30-day nifurtimox treatment regimen. The 
congenital infection will be confirmed by parasitological method in 
children ≤8 months of age and by serological method in children 
>8 months of age.
Test drug(s)
Nifurtimox 30-mg and 120-mg tablets with score lines to be divided into two 
equal halves to allow for 15-mg and 60-mg dose increments, respectively.
Name of active ingredient
Nifurtimox
Dose(s)
For pediatric subjects with body weight ≤40 kg: 10-20 mg/kg/day in three 
divided doses 
For pediatric subjects with body weight > 40 kg: 8 – 10 mg/kg/day in three 
divided doses. The planned mg/kg per day dosages are as in the Company 
Core Data Sheet.
Route of administration
Oral tablets, to be administered with food
Duration of treatment
60 days of nifurtimox (Treatment Group 1), or 30 days of nifurtimox followed 
by 30 days of placebo (Treatment Group 2)
Reference drug(s)
None
Indication
Treatment of Chagas’ disease in children younger than 18 years of age
Diagnosis and main 
criteria for inclusion 
/exclusion
Part 1 (CHICO)

Male and female pediatric subjects aged 0 days to younger than 18 years

Chagas’ disease diagnosed/confirmed by:
o Subjects < 8 months of age at randomization must demonstrate direct 
observation of Trypanosoma cruzi by concentration test
o Subjects ≥8 months to < 18 years of age at randomization must 
demonstrate a positive conventional ELISA result for both of the 
following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test and indirect hemagglutination 
assay (IHA) will be obtained; however, a positive result will not be required 
for diagnosis.10,11
Subjects will also be eligible if there is acceptable documentation of positive 
Chagas’ disease within three months prior to screening and they have not had 
prior anti-trypanocidal or anti-parasitic treatment.12
Written informed consent by the subject and/or parent(s) or legally authorized 
representative(s) according to the age established per local regulations must be 
obtained from all subjects prior to screening for the study.  In addition, 
depending on the subject’s age, subject’s assent is required as applicable by 
local laws and regulations (varies in each country).
Females of childbearing potential (i.e., female subjects who have experienced 
menarche) and male subjects must agree to use adequate contraception if 
sexually active from the time of signing the informed consent/assent form until 
3 months after the last study drug administration.  Depending on the country, 
                                                
10 IFA test added per Amendment 5
11 Replaced IFA with IHA per Amendment 6.
12 Text modified/added to clarify test required for Chagas’ disease diagnosis per Modification 2, text “at randomization” 
added for clarity (Amendment 1)


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study site personnel may dispense condoms at the time that study drug is 
dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.
Subjects aged 0 to 27 days who, at birth, were pre-term (i.e., gestational age 
less than 37 weeks), weighed less than 2500 g, or had a maximum Apgar score 
< 7 at 5 minutes will be excluded from the study.  Subjects with 
contraindications/warnings to nifurtimox administration (or with conditions 
that may increase the risk of the undesirable effects of nifurtimox), are 
immuno-compromised, or have had previous treatment with trypanocidal 
agents or an accepted indication for anti-parasitic therapy (e.g., reactivation of 
Chagas' infection due to immunosuppression by several diseases or treatment 
with prolonged oral steroids) will also be excluded from the study.
Part 2 (CHICO SECURE)
Inclusion criteria
1.
Male and female subjects who were randomized and received at least one 
dose of  their assigned 60- or 30-day regimen of nifurtimox treatment
2.
Written informed consent by the subject and/or parent(s) or legally 
authorized representative(s) according to the age established per local 
regulations must be obtained from all subjects who are screened for the 
study. In addition, depending on the subject’s age, subject’s assent is 
required as applicable by local laws and regulations (varies in each 
country)
Exclusion criteria
1.
Subjects with acute or chronic health conditions or congenital disorders 
which, in the opinion of the investigator, would make them unsuitable for 
participation in the clinical study
2.
Subjects living in housing conditions where there is no active or effective 
vector-control to T. cruzi reinfection as determined by Ministry of Health 
guideline of the respective country and the judgement of the investigator
3.
Subjects with clinical manifestations of Chagas’ disease, such as:

Known evidence of Chagas’ disease-related gastrointestinal 
dysfunction (e.g., megaesophagus, megacolon, or both) or 
Chagas’ digestive disease

Serious manifestations of acute Chagas’ disease, including 
myocarditis, meningoencephalitis, or pneumonitis
4.
Immuno-compromised subjects (e.g., those with human 
immunodeficiency virus infection, primary immunodeficiency, or 
prolonged treatment with corticosteroids or other immunosuppressive 
drugs)
5.
Subjects who the investigator considers unlikely to adhere to the protocol, 
or complete the long-term follow-up
6.
Subjects with close affiliation with the investigational site; e.g., a close 
relative of the investigator, dependent person (e.g., employee or student of 
the investigational site)


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Study design
Part 1 (CHICO)
Prospective, randomized (to dosing regimen), age-stratified, double-blind, 
parallel-group study. Subjects will be randomized into four strata as follows:
Stratum 1:
0 to 27 days
Stratum 2:
28 days to younger than 8 months
Stratum 3:
8 months to younger than 2 years
Stratum 4:
2 years to younger than 18 years
A minimum of 38 subjects in each age stratum is targeted, but not 
required, in order to be able to derive meaningful safety conclusions.13
Part 2 (CHICO SECURE)
Prospective, long-term follow-up to evaluate seronegative conversion 
and serial reduction in OD. Subjects will be followed up for a total of 4 
years after end of nifurtimox treatment. The first year of follow-up 
period is included in part 1 and 3 years of follow-up are included in part 
2 of the study.  
The following age categories will be considered for part 2 of the study:
≤2 years, >2 years to ≤6 years, >6 to ≤12 years, >12 to <18 years (age is 
defined as subject’s age at randomization).
Methodology
Part 1 (CHICO)
No study-specific procedures will be performed before the subject/legally 
authorized representative has signed the informed consent form, or a minor 
subject has provided assent, including asking a potential subject to fast (if 
applicable, depending on the age of the subject at the discretion of the 
investigator) prior to the screening blood samples. Informed consent/assent for 
optional PK blood sampling will also be obtained. After the consents/assents
have been obtained, screening assessments will be performed from 1 to 14 
days prior to randomization at Visit 2.14
After study eligibility has been confirmed and safety assessments have been 
performed at Visit 2 (Day 1), subjects will be randomized via Interactive Voice 
Response System/Interactive Web Response System in a 2:1 ratio (60-day 
regimen vs. 30-day regimen) to one of two treatment groups:15
Nifurtimox tablets administered three times daily for 60 days (Days 1 – 60, 
active nifurtimox treatment, Treatment Group 1) or 
Nifurtimox tablets administered three times daily for 30 days, followed by 
nifurtimox placebo administered three times daily for 30 days (Days 1 – 30, 
active nifurtimox treatment; Days 31 – 60, placebo; Treatment Group 2).  
The first dose of study drug will be administered at Visit 2. Pre- and post-dose 
PK blood samples will be obtained at specified time points from those subjects 
consenting to PK assessments. At the 2 – 4 hour post-dose time point of PK 
blood sampling (i.e., at the time of maximum concentration), an ECG (optional 
for subjects < 5 years of age at the discretion of the investigator) will be 
obtained to allow for PK/PD investigations. Study drug will be dispensed and 
instructions for study drug administration will be provided to all subjects.16
                                                
13 Text modified as of amendment 2
14 Text added for clarity (Amendment 1)
15 Text modified for clarity (Amendment 1)
16 Text modified/added for clarity and per recommendations per Modification 1 (Amendment 1)


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Subjects will return to the investigational site for efficacy and safety 
assessments at Visits 3 (Day 7±1) and 6 (Day 30).  Subjects who have not 
consented to PK assessments will take that day’s doses of study drug as 
instructed.  Subjects consenting to PK assessments must hold the morning dose 
of study drug; for these subjects, a pre-dose PK blood sample will be obtained, 
then the morning dose of study drug will be administered.  Post-dose PK blood 
samples will be obtained at specified time points, and ECGs will be obtained at 
the 2 – 4 hour post-dose time point.  A pre-paid phone card will be provided to 
all subjects to facilitate contact between study site personnel and the 
subjects/subjects’ authorized representatives.17
At Visit 6 (Day 30), subjects will return all remaining study drug and empty 
packaging, and study drug for the remaining 30 days of treatment will be 
dispensed.18  
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments 
where study site personnel will contact the subject/legally authorized 
representative via telephone to assess the occurrence of AEs, use of 
concomitant medications, and compliance with study drug administration.  A 
Phone Contact Form for the telephone assessments will be provided to all 
sites.19
Subjects will return to the investigational site for efficacy and safety 
assessments on Day 60 (Visit 8), which will be the EOT for both treatment 
groups.  Subjects who have not consented to PK assessments will take that 
day’s doses of study drug as instructed.  Subjects consenting to PK 
assessments must hold the morning dose of study drug.  A pre-dose PK blood 
sample will be collected, then the morning dose of study drug will be 
administered. Post-dose PK blood samples will be obtained at specified time 
points, and ECGs will be obtained at the 2 – 4 hour post-dose time point.  
Study drug will be collected, and no additional study drug will be dispensed.20
After the EOT visit (Visit 8), subjects will return to the site on Days 90
(Visit 9), 240 (Visit 10), and 420 (Visit 11) for additional efficacy and safety 
assessments.  The total duration of each subject’s participation is expected to 
be approximately 14 months.21  
Safety will be assessed via laboratory parameters (hematology, blood 
chemistry, urinalysis), vital sign measurements (blood pressure [optional in 
subjects < 5 years of age at the discretion of the investigator], heart rate, 
respiratory rate, temperature), monitoring of ECGs (optional in subjects 
< 5 years of age at the discretion of the investigator), monitoring of AEs, and 
physical examinations, including neurological examinations.22  
Subjects who discontinue prematurely from study drug administration will
continue to return to the investigational site for study assessments at Visit 3, 6, 
and 8 (EOT), and undergo telephone assessments as described for Visits 4, 5, 
and 7.  If subjects are unable/unwilling to do so, they must return to the 
investigational site 30 (±3) days after the last dose of study drug for EOT 
(Visit 8) assessments, and undergo telephone assessments as described for 
Visits 4, 5, and 7.  If the subject is unable/unwilling to return to the clinic for 
                                                
17 Text modified/added for clarity and per recommendations of Modification 1 (Amendment 1)
18 Text added for clarity (Amendment 1)
19 Text added to describe telephone assessments per Modification 8 (Amendment 1)
20 Text modified/added for clarity and per ECG assessments at PK sampling per Modification 1 (Amendment 1)
21 Text modified for clarity (Amendment 1)
22 Text modified for clarity and to include neurological examinations per Modification 10 (Amendment 1)


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the EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 
5, and 7 may be performed in lieu of Visit 8 assessments.23
Part 2 (CHICO SECURE)
No study-specific procedures will be performed before the subject and/or 
parent(s) or legally authorized representative(s) has signed the informed 
consent form, or a minor subject has provided assent for part 2 of the study.  
After the consents/assents have been obtained, the assessments scheduled for
follow up (FU) Visit 1 including eligibility assessments will be performed. 
The first visit should be performed as soon as possible after availability of 
regulatory and ethics committee approval for all subjects who have already 
completed part 1 of the study for 1 year 6 weeks or longer.  Subjects who are 
entering the study beyond the time window for FU Visit 1 (2 years6 weeks) 
will have their eligibility assessments as soon as possible provided that 
consents/assents have been obtained. 
Subjects will have annual visits at the investigational site for efficacy and 
safety assessments for 3 years.  Timing of study visits is relative to end of 
treatment in part 1 of the study.
At each investigational site visit, blood samples for serology tests 
(recombinant ELISA, total purified antigen ELISA, IHA and a non-
conventional ELISA test) will be collected.  Blood samples will be taken under 
fasting conditions, if applicable, depending on the age of the subject, at the 
discretion of the investigator.  Subjects will undergo a physical examination, 
body weight, height/length and vital signs will be measured and protocol 
specified AEs will be recorded.  A 12-lead electrocardiogram (ECG) will be 
obtained at each annual visit.  The subject’s concomitant medications will be 
recorded.  Signs and symptoms of Chagas’ disease will be assessed and blood 
will be taken for detection of T. cruzi’s deoxyribonucleic acid (DNA) using 
quantitative polymerase chain reaction (qPCR).  If during the course of the 
study, conventional ELISA OD increases, the subject has positive qPCR 
results and shows signs or symptoms of Chagas’ disease, it is up to the 
investigator’s discretion to initiate antitrypanosomal treatment.
Whenever an AE considered at least possibly related to nifurtimox or caused 
by protocol-required procedures occurs, the subject should report it to the 
investigator.
Female subjects who gave birth before end of part 2 of the study will be 
invited to test their child for the presence or absence of infection with T. cruzi.
Between the annual visits at the investigational site, subjects and/or his or her 
parent(s) or legally authorized representative(s) will be contacted by qualified 
personnel by a method agreed (e.g., phone call) about 6 months after a visit at 
the investigational site to identify the occurrence of potential clinical 
symptoms of Chagas’ disease or any pathology mentioned in the exclusion 
criteria using a structured interview.  In case of suspected findings, an 
unscheduled visit at the investigational site to initiate appropriate diagnostic 
evaluations will be requested.  
Type of control
For primary objective: historical
For secondary objectives: historical and 30-day vs. 60-day treatment
                                                
23 Text added to include assessments for subjects who prematurely discontinued study drug per Modification 11 
(Amendment 1)


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Data Monitoring 
Committee
An independent Data Monitoring Committee will meet periodically to review 
the safety data of enrolled subjects, as well as the continuing scientific merit of 
the trial.  (Not applicable to part 2)
Number of subjects
Approximately 300 pediatric subjects will be randomized (2:1 randomization, 
60-day regimen vs. 30-day regimen) in part 1 (CHICO); all subjects who were 
randomized and received at least one dose of their assigned 60- or 30-day 
nifurtimox treatment regimen will be assessed for eligibility for part 2 (CHICO
SECURE) providing the consent has been obtained.  
Primary variable(s)
Part 1 (CHICO)
The primary efficacy variable will be sero-reduction or sero-conversion at 
12 months post-treatment using two conventional ELISA serology tests as the 
measure of efficacy.24
Part 2 (CHICO SECURE)
The primary efficacy variable will be the incidence rate of seronegative 
conversion in subjects who were randomized and received at least one dose of 
the 60-day nifurtimox treatment, measured and confirmed by two types of 
assay (recombinant ELISA and IHA).  Both test results should be negative for 
the subject to be considered as seroconverted.
Time point/frame of 
measurement for primary 
variable(s)
Part 1 (CHICO): 12 months post-treatment
Part 2 (CHICO SECURE): 48 months post-treatment 
                                                
24 Text modified per Modification 2 (Amendment 1)


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Plan for statistical analysis Part 1 (CHICO)
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with sero-reduction or 
sero-conversion (60-day regimen) and the proportion estimated from historical 
data will be tested using a 2-sided 95% confidence interval (CI) for a single 
proportion.25
Superiority will be confirmed if the lower limit of the CI for the proportion of 
nifurtimox subjects (60-day regimen) with sero--reduction or sero-conversion 
is greater than the proportion of historical placebo subjects with 
sero-conversion.26
Secondary efficacy analysis
A secondary analysis will be done to compare the proportion of subjects with 
sero-reduction or sero-conversion for the 60-day and 30-day nifurtimox 
regimens. This will be performed using a 2-sided 95% CI for the difference of 
two independent proportions.27
The analyses specified above will also be done using data from the EOT visit, 
and 3 and 6 months after EOT. 
The relationship of conventional serology results to qPCR results will be done 
using frequencies of matches and mismatches on the determination of disease 
status, and phi-correlation and kappa coefficient to assess the degree of 
agreement.  The relationship of non-conventional serology to conventional 
serology will be analyzed in the same way.28
The primary efficacy analysis will be done using the full analysis set, which is 
the set of subjects who received at least one dose of study drug.  Analyses will 
also be done using the per-protocol set, which is comprised of subjects treated 
with study drug who have no major protocol deviations.
According to Guhl et al. (2004), nifurtimox 12 months post- treatment cure 
rate is about 55%, with a sample size of 200 for the 60-day regimen, the power 
is 99% for the lower limit of the 95% CI to be greater than 16%, a performance 
goal based on historical study. 29
Part 2 (CHICO SECURE)
Primary efficacy analysis 
The difference in the incidence rate of nifurtimox subjects with seronegative 
conversion (60-day regimen) and the rate estimated from historical data will be 
tested using a Poisson two-sided 95% confidence interval (CI).
The incidence rate of seronegative conversion for the primary efficacy variable 
will be modelled using a Poisson distribution with a 2-sided 95% CI.  The rate 
will be estimated as the number of seronegative conversion divided by the time 
at risk of event (person-year) during the study. 
This study uses an external control group of untreated patients with Chagas’ 
disease in the 4-year follow-up period presented in Sosa et al. [15, 35].  In the 
reference, seroconversion to a negative result in the placebo group after 4-year 
follow-up was detected in 2 subjects with conventional serology and in 0 
subjects with IHA test.  Thus, none of the subjects in the placebo group was 
                                                
25 Text modified per Modification 2 (Amendment 1)
26 Text modified per Modification 2 (Amendment 1)
27 Text modified per Modification 2 (Amendment 1)
28 Text modified/added per Modification 2 (Amendment 1)
29 Modified as per amendment 2


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considered as seroconverted by both test results.  The person time at risk was 
on average 44 * 4 = 176 person years.  The incidence rate estimate was 0% (0 / 
176).
Superiority will be confirmed if the lower limit of the CI for the incidence rate 
of seronegative conversion for subjects in 60-day regimen is greater than the 
rate of historical placebo subjects with sero-conversion (i.e. 0 %).
Secondary efficacy analysis
The OD values from recombinant ELISA and total purified antigen ELISA 
will be analyzed descriptively.  Changes from baseline will be summarized to 
show any serial reduction in OD.  Baseline is defined as the OD values from 
the same ELISA tests measured at Visit 1 in part 1 of the study.
The proportion of subjects who show both seronegative conversion as 
confirmed by two assays (recombinant ELISA and IHA) and no evidence of 
established cardiomyopathy as measured by ECG will be calculated overall 
and by treatment regimen. 
The same method for calculating incidence rate and its 2-sided 95% CI will be 
applied on seronegative conversion in subjects who were randomized and 
received at least one dose of 30-day nifurtimox treatment.


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Protocol amendment summary of changes table
Amendment no. 10 (03 NOV 2020)
Overall rationale for the amendment
This amendment was prepared to incorporate feedback received from the FDA on 
10 JUL 2020 The Agency requested inclusion of non-conventional ELISA in part 2 (CHICO 
SECURE) of the study and that antibody titers by serial dilution be obtained for all available 
serum samples. None of the changes implemented in this amendment impact the benefit-risk 
evaluation of the study.
Section # and name
Description of change
Section 1 Title page
Updated to indicate that this is an electronically generated 
document that does not bear any sponsor signatures. 
Removed page for Signature of the sponsor’s medically 
responsible person.
Section 2 Synopsis
Section 4.2 Objectives for part 2 (CHICO 
SECURE)
Exploratory objective added to assess the proportion of 
responders who show seronegative conversion and the
incidence of seronegative conversion as confirmed by non-
conventional serology in subjects who were randomized and 
received at least one dose of either 60- or 30-day nifurtimox 
treatment regimens.
Section 9.4 Efficacy
Section 10.3.1.2 part 2 (CHICO SECURE)
Addition of efficacy variable: proportion of responders who 
show seronegative conversion and the incidence of 
seronegative conversion as confirmed by non-conventional 
ELISA in subjects who were randomized and received at 
least one dose of either 60- or 30-day nifurtimox treatment 
regimens.
Section 2 Methodology
Section 5.2 Part 2 (CHICO SECURE)
Section 9.1 Table 9-2: Schedule of procedures 
and assessments in Part 2 – Study 16027
Section 9.2.4.2 Annual study visits (FU Visits 1, 
3, and 5)
Section 9.4.2 Serological Tests
Addition of non-conventional serological test (ELISA F-29).
Section 10.3.2.2 Part 2 (CHICO SECURE)
Analysis of the secondary and exploratory efficacy variables 
was updated to include non-conventional serology.
Typographical, grammatical, and other minor edits are not identified.


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Table of contents
1.
Title page............................................................................................................................ 2
Signature of principal investigator......................................................................................... 3
2.
Synopsis.............................................................................................................................. 4
3.
Introduction..................................................................................................................... 21
4.
Study objectives............................................................................................................... 24
4.1
Objectives for part 1 (CHICO) ...................................................................................... 24
4.2
Objectives for part 2 (CHICO SECURE)...................................................................... 25
5.
Study design..................................................................................................................... 26
5.1
Part 1 (CHICO).............................................................................................................. 26
5.2
Part 2 (CHICO SECURE).............................................................................................. 34
6.
Study population ............................................................................................................. 36
6.1
Inclusion criteria ............................................................................................................ 36
6.2
Exclusion criteria ........................................................................................................... 37
6.3
Withdrawal of subjects from study................................................................................ 39
6.3.1
Withdrawal ................................................................................................................. 39
6.3.2
Replacement ............................................................................................................... 41
6.4
Subject identification ..................................................................................................... 41
7.
Treatment(s) .................................................................................................................... 42
7.1
Treatments to be administered....................................................................................... 42
7.2
Identity of study treatment............................................................................................. 42
7.3
Treatment assignment.................................................................................................... 42
7.4
Dosage and administration............................................................................................. 43
7.5
Blinding ......................................................................................................................... 44
7.6
Drug logistics and accountability................................................................................... 44
7.7
Treatment compliance.................................................................................................... 45
7.8
Treatment in part 2 (CHICO SECURE) ........................................................................ 45
8.
Non-study therapy........................................................................................................... 45
8.1
Prior and concomitant therapy....................................................................................... 45
8.2
Post-study therapy.......................................................................................................... 46
9.
Procedures and variables ............................................................................................... 46
9.1
Tabular schedule of evaluations .................................................................................... 46
9.2
Visit description............................................................................................................. 51
9.2.1
Screening .................................................................................................................... 51
9.2.1.1
Visit 1 – screening visit (Days -14 to -1) ................................................................ 51
9.2.2
Treatment phase.......................................................................................................... 52
9.2.2.1
Visit 2 – baseline (treatment phase, Day 1) ............................................................ 52
9.2.2.2
Visit 3 (Day 7 ± 1 days) .......................................................................................... 53
9.2.2.3
Visit 4 (Day 14 ± 1 days) ........................................................................................ 54
9.2.2.4
Visit 5 (Day 21 ± 3 days) ........................................................................................ 54
9.2.2.5
Visit 6 (Day 30 ± 3 days) ........................................................................................ 54
9.2.2.6
Visit 7 (Day 42 ± 3 days) ........................................................................................ 56
9.2.2.7
Visit 8 (Day 60 ± 3 days)/End of Treatment Visit .................................................. 56
9.2.3
Follow-up phase ......................................................................................................... 57


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9.2.3.1
Visit 9 (Day 90 ± 7)................................................................................................. 57
9.2.3.2
Visit 10 (Day 240 ± 7)............................................................................................. 58
9.2.3.3
Visit 11 (Day 420 ± 7)............................................................................................. 58
9.2.4
Long-Term Follow-up phase...................................................................................... 59
9.2.4.1
Eligibility procedures (FU Visit 1).......................................................................... 59
9.2.4.2
Annual study visits (FU Visits 1, 3, and 5)............................................................. 59
9.2.4.3
Interim phone contact visits (FU Visits 2 and 4) .................................................... 60
9.2.4.4
Unscheduled study visits......................................................................................... 60
9.3
Population characteristics .............................................................................................. 60
9.3.1
Demographic .............................................................................................................. 60
9.3.2
Medical history........................................................................................................... 60
9.4
Efficacy.......................................................................................................................... 61
9.4.1
Polymerase chain reaction test ................................................................................... 62
9.4.2
Serological tests.......................................................................................................... 62
9.4.3
Concentration tests for T. cruzi .................................................................................. 63
9.4.4
Assessment of Chagas’ disease signs and symptoms................................................. 63
9.5
Pharmacokinetics/pharmacodynamics........................................................................... 65
9.6
Safety ............................................................................................................................. 67
9.6.1
Adverse events............................................................................................................ 67
9.6.1.1
Definitions............................................................................................................... 67
9.6.1.2
Classifications for adverse event assessment.......................................................... 68
9.6.1.2.1
Seriousness........................................................................................................... 68
9.6.1.2.2
Intensity................................................................................................................ 69
9.6.1.2.3
Causal relationship............................................................................................... 69
9.6.1.2.4
Action taken with study treatment ....................................................................... 70
9.6.1.2.5
Other specific treatment(s) of adverse events...................................................... 70
9.6.1.2.6
Outcome............................................................................................................... 71
9.6.1.3
Assessments and documentation of adverse events ................................................ 71
9.6.1.4
Reporting of serious adverse events........................................................................ 71
9.6.1.5
Expected adverse events.......................................................................................... 72
9.6.1.6
Adverse events of special safety interest................................................................. 72
9.6.2
Pregnancies................................................................................................................. 73
9.6.3
Further safety.............................................................................................................. 73
9.6.3.1
Laboratory assessments........................................................................................... 73
9.6.3.2
Total volume of blood............................................................................................. 74
9.6.3.3
Physical examination............................................................................................... 75
9.6.3.4
Vitals signs, height/length, and weight ................................................................... 75
9.6.3.5
12-lead electrocardiogram....................................................................................... 76
9.6.3.6
Urine pregnancy test................................................................................................ 76
9.7
Other procedures and variables...................................................................................... 76
9.8
Appropriateness of procedures/measurements .............................................................. 76
10. Statistical methods and determination of sample size ................................................. 77
10.1
General considerations................................................................................................... 77
10.2
Analysis sets .................................................................................................................. 77
10.3
Variables and planned statistical analyses..................................................................... 78
10.3.1
Variables..................................................................................................................... 78
10.3.1.1
Part 1 (CHICO) ....................................................................................................... 78
10.3.1.2
Part 2 (CHICO SECURE)....................................................................................... 78


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10.3.2
Statistical and analytical plans.................................................................................... 79
10.3.2.1
Part 1 (CHICO) ....................................................................................................... 79
10.3.2.2
Part 2 (CHICO SECURE)....................................................................................... 81
10.4
Determination of sample size......................................................................................... 83
10.5
Planned interim analyses ............................................................................................... 84
11. Data handling and quality assurance ............................................................................ 84
11.1
Data recording................................................................................................................ 84
11.2
Monitoring ..................................................................................................................... 85
11.3
Data processing.............................................................................................................. 86
11.4
Missing data................................................................................................................... 86
11.5
Audit and inspection ...................................................................................................... 86
11.6
Archiving ....................................................................................................................... 87
12. Premature termination of the study .............................................................................. 87
13. Ethical and legal aspects................................................................................................. 88
13.1
Investigator(s) and other study personnel...................................................................... 88
13.2
Funding and financial disclosure ................................................................................... 89
13.3
Ethical and legal conduct of the study........................................................................... 89
13.4
Subject information and consent.................................................................................... 90
13.5
Publication policy and use of data ................................................................................. 91
13.6
Compensation for health damage of subjects/insurance................................................ 91
13.7
Confidentiality ............................................................................................................... 91
14. Reference list.................................................................................................................... 92
15. Protocol amendments...................................................................................................... 95
15.1
Amendment 1................................................................................................................. 95
15.1.1
Overview of changes .................................................................................................. 96
15.1.2
Changes to the protocol text..................................................................................... 101
15.2
Amendment 2............................................................................................................... 167
15.2.1
Overview of changes ................................................................................................ 167
15.2.2
Changes to the protocol text..................................................................................... 168
15.3
Amendment 3............................................................................................................... 194
15.3.1
Overview of changes ................................................................................................ 195
15.3.2
Changes to the protocol text..................................................................................... 195
15.4
Amendment 4............................................................................................................... 195
15.4.1
Overview of changes ................................................................................................ 195
15.4.2
Changes to the protocol text..................................................................................... 196
15.5
Amendment 5............................................................................................................... 204
15.5.1
Overview of changes ................................................................................................ 204
15.5.2
Changes to the protocol text..................................................................................... 205
15.6
Amendment 6............................................................................................................... 207
15.6.1
Overview of changes ................................................................................................ 207
15.6.2
Changes to the protocol text..................................................................................... 208
15.7
Amendment 7............................................................................................................... 211
15.7.1
Overview of changes to the study............................................................................. 211
15.8
Amendment 8............................................................................................................... 212
15.8.1
Overview of changes to the study............................................................................. 212
15.9
Amendment 9............................................................................................................... 213


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15.9.1
Overview of changes to the study............................................................................. 213
16. Appendices..................................................................................................................... 215
16.1
Phone Contact Form for telephone assessments.......................................................... 215
16.1.1
Part 1 (CHICO)......................................................................................................... 215
16.1.2
Part 2 (CHICO SECURE) ........................................................................................ 217
16.2
Schwartz formula and mean (range) eGFR for infants and children of various 
ages .............................................................................................................................. 221
16.3
PK Subject diary and Subject diary ............................................................................ 222
16.4
Procedure for multiplex real time polymerase chain reaction to Trypanosoma 
cruzi detection in patients with Chagas’ disease.......................................................... 249
16.5
Pharmacokinetic blood sample handling ..................................................................... 256
16.6
Maximum blood volumes for pediatric patients – Clinical and Laboratory 
Standards Institute........................................................................................................ 259
Table of tables
Table 7–1:  Total daily dosage of nifurtimox oral tablets based on body weight.................... 43
Table 7–2:   Individual dosages of nifurtimox oral tablets based on body weight .................. 43
Table 9–1:  Schedule of procedures and assessments in part 1 – Study 16027 ....................... 47
Table 9–2:  Schedule of procedures and assessments in part 2 – Study 16027 ....................... 50
Table 9–3:  Pharmacokinetic sampling time points and windows........................................... 66
Table 9–4:  Description of laboratory parameters (safety) ...................................................... 74
Table 9–5:  Total volume of blood by age ............................................................................... 74
Table of figures
Figure 5–1: Study Design of Part 1 – Protocol 16027 ............................................................. 27


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List of abbreviations30
AE
adverse event
ALP
alkaline phosphatase
ALT
alanine aminotransferase
AST
aspartate aminotransferase
β-hCG
beta-human chorionic gonadotropin  
BP
blood pressure
CI
confidence interval
Cmax
maximum concentration
CCDS
company core data sheet
CRO
Contract Research Organization
DMC
Data Monitoring Committee
DNA
deoxyribonucleic acid
ECG
electrocardiogram
eCRF
electronic case report form
EDTA
ethylenediaminetetra-acetic acid
eGFR
estimated glomerular filtration rate
ELISA
enzyme-linked immune sorbent assay
EOT
end-of-treatment
FAS
full analysis set
FDA
US Food and Drug Administration
FU
follow up
GCP
Good Clinical Practice
GEB
Guanidine-EDTA-Blood
GMP
Good Manufacturing Practice
IAC
internal amplification control
ICH
International Conference on Harmonisation
IEC
independent ethics committee
                                                
30 Abbreviations added or deleted based on changes to text throughout Synopsis and body of protocol; qualitative PCR 
changed to quantitative PCR (Amendment 1) and Amendment 6.


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List of abbreviations30
IHA
Indirect hemagglutination assay
IgG
Immunoglobulin G
INR
international normalized ratio
IRB
institutional review board
IVRS/IWRS
Interactive Voice Response System/Interactive Web Response System
LLN
lower limit of normal
LTFU
long-term follow-up
LSLV
Last subject last visit
MCH
mean corpuscular hemoglobin
MCHC
mean corpuscular hemoglobin concentration
MCV
mean corpuscular volume
OD
optical density
PAHO
Pan American Health Organization
PD
pharmacodynamics
PK
pharmacokinetics
PPS
Per protocol set
PT
prothrombin time
PTT
partial thromboplastin time
QC
quality control
qPCR
quantitative polymerase chain reaction
RBC
red blood cell
RPM
revolutions per minute
RR
respiratory rate per minute
SAE
serious adverse event
SAP
statistical analysis plan
SAS
Statistical Analysis System
SUSAR
suspected, unexpected, serious adverse reactions
T. cruzi
Trypanosoma cruzi
WBC
white blood cell
WHO
World Health Organization


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3.
Introduction
Background
American trypanosomiasis (Chagas’ disease), first described by Carlos Chagas in 1909, is 
caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) and is a vector-borne disease 
transmitted to humans by large, blood-sucking reduviid bugs (e.g., Triatoma infestans, 
Rhodnius prolixus, and Triatoma dimidiata) infected with the parasite.  Other possible 
mechanisms of transmission include blood transfusions, congenitally-transmitted or acquired 
infection, infected solid organ or bone marrow transplants, ingestion of contaminated food or 
drink, or laboratory accidents.  
In 2006, the Pan American Health Organization (PAHO) estimated the prevalence of Chagas’ 
disease in 21 endemic Latin American countries to be approximately 8 million infected 
persons, with nearly 100,000 persons at risk for infection.  The number of new cases per year 
was calculated to be 55,185, of which 40,800 (74%) were vector-related infections and 
14,385 (26%) were congenitally acquired infection.[1]  Long considered to be endemic in the 
Latin American and Caribbean region, there is increasing evidence to suggest that Chagas’ 
disease is becoming a more global disease.  Some estimate the prevalence of T. cruzi infection 
in Latin American-born persons living in the United States to be just over 300,000 infected 
individuals (1.31%)[2], while others suggest that the prevalence may be even higher 
(1 million cases) with an especially high burden of disease in Texas and along the Gulf of 
Mexico coast.[3]  Further evidence of the globalization of Chagas’ disease and confirmation 
that the T. cruzi parasite can travel from endemic to non-endemic countries is the data from 
PAHO that estimates the number of infected persons to be more than 80,000 in Europe, 5,500 
in Canada, 3,000 in Japan, and 1,500 in Australia.  In North America (which includes Canada, 
Mexico, and the United States), researchers have estimated the number of seropositive 
mothers and newborns infected with T. cruzi to be approximately 40,000 and 2,000, 
respectively.[4]  Clearly, Chagas’ disease can no longer be considered just a zoonotic disease 
that affects only poor rural communities in Latin America.  It has become a worldwide 
concern with severe public health consequences over the long term.
The natural history of the disease following exposure to the parasite begins with an acute 
phase that may be completely asymptomatic.  If symptoms occur, they are usually a 
self-limiting febrile illness that occurs 1 to 2 weeks after exposure to triatomine bugs, or up to 
a few months after transfusion of infected blood.  Manifestations of the acute disease resolve 
spontaneously in about 90% of infected individuals even if the infection is not treated with 
trypanocidal drugs.  Death may occur during the acute phase in 5% to 10% of symptomatic 
patients, usually the result of severe myocarditis and/or meningoencephalitis.  About 60% to 
70% of these individuals will never develop clinically apparent disease.  These patients have 
the indeterminate form of chronic Chagas’ disease.  The remaining 30% to 40% of patients 
will go on to develop a determinate form of chronic disease manifested by cardiac disorders 
(e.g., conduction abnormalities, bradyarrhythmias and tachyarrhythmias, apical aneurysms, 
heart failure, thromboembolism and sudden death) or digestive disorders (e.g., 
megaesophagus, megacolon), or a combination of cardiodigestive disorders 10 to 30 years 
after the initial infection.  Gastrointestinal dysfunction develops in 10% to 15% of chronically 
infected patients.  The cardiac form of the disease is the most serious, and develops in about 
20% to 30% of patients.  
Most new infections occur in children younger than 15 years of age, either by vector or 
vertical transmission, with the highest frequency in young children aged 1 to 5 years.  


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Unfortunately, in most endemic areas, neither pregnant women nor newborns are routinely 
tested for T. cruzi infection at present.  Congenital cases are frequently asymptomatic and, 
unless specific tests are ordered, the disease passes unnoticed.  The acute phase of Chagas’ 
disease can be particularly severe in children, with up to 10% of these patients dying of 
meningoencephalitis or myocarditis.  Early diagnosis of congenital disease is essential for 
improving treatment outcomes, and research has shown that the disease is curable if treatment 
is initiated soon after infection.[5][6]  Some of the clinical manifestations of congenital 
Chagas’ disease are prematurity, low birth weight, low Apgar scores, hepatosplenomegaly, 
jaundice, edema, respiratory distress syndrome, and meningoencephalitis.  
Comprehensive information on the study drug can be found in the latest available version of 
the Investigators’ Brochure.31
Rationale of the study
Currently, only nifurtimox and benznidazole are approved in selected South American 
countries for the etiological treatment of Chagas’ disease in children and adults.  Nifurtimox 
was first introduced in 1965.  It is approved in Argentina, Chile, El Salvador, Guatemala, 
Uruguay, and Honduras. 
Recently, benznidazole has been approved in the United States for treatment of Chagas’ 
disease in children aged 2-12 years.  These medications were developed in the late 1960s 
(nifurtimox) and early 1970s (benznidazole).  Nifurtimox, a nitrofuran compound, acts 
through production of reduced oxygen metabolites (e.g., superoxide and hydrogen peroxide), 
for which the parasites have lower detoxification capacity, as compared with vertebrate cells.  
Benznidazole, a nitroimidazole derivative, is thought to act through covalent binding of 
nitroreduction intermediates to parasite molecules.  Based on clinical study data, anti-
trypanosomal drug treatment in children with chronic T. cruzi infection was accepted as the 
standard of care throughout Latin America by the late 1990s, followed in recent years by a 
growing movement to offer treatment to older patients.  Because treatment is expected to 
reduce the probability of congenital transmission, stronger consideration may be warranted 
for women of reproductive age.
Bayer was granted Orphan Drug Designation of nifurtimox for treatment of Chagas’ disease 
(American Trypanosomiasis) caused by T. cruzi pursuant to Section 526 of the Federal Food, 
Drug, and Cosmetic Act (21 U.S.C. 360bb) on 05 AUG 2010.
Nifurtimox has been classified by the World Health Organization (WHO) as a vitally 
important medicine and is included on the WHO Model List of Essential Medicines for 
Chagas’ disease.
This study consists of two parts: 

Part 1 comprises the treatment with nifurtimox including the 1-year follow up 
(CHICO)

Part 2 comprises the 3-year long-term follow-up (LTFU) of all subjects who were 
randomized and received at least one dose of their assigned nifurtimox treatment
regimen in CHICO (CHICO SECURE) 
                                                
31 Revised since Investigators’ Brochure became available to serve as the reference document for nifurtimox (Amendment 1)


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Part 1 (CHICO) was designed to develop a better understanding of the efficacy, 
safety/tolerability, and pharmacokinetics (PK) (absorption, distribution, metabolism, and 
elimination) of nifurtimox in children with a diagnosis of Chagas’ disease using pediatric 
formulations.  Results from this study will demonstrate parasitological cure using 
conventional serological results to allow optimization of treatment of Chagas’ disease in this 
most vulnerable population.32  In turn, this will improve the outlook for children by reducing 
mortality and long-term complications.  This knowledge will allow for better and more 
appropriate approaches to the treatment of Chagas’ disease.  It will also provide more up-to-
date information on the use of nifurtimox, and has the potential to increase our understanding 
of treatment response in children.
Part 2 (CHICO SECURE) was designed, at the request of the US Food and Drug 
Administration (FDA), to assess the incidence of seronegative conversion in subjects who 
were randomized and received at least one dose of their assigned 60- or 30-day nifurtimox 
treatment regimen.  In part 2 (CHICO SECURE), subjects will be followed up for 3 years 
totaling to a follow-up period of 4 years after end of nifurtimox treatment, the first year of 
follow up is included in the design of part 1 (CHICO). 
Conversion to negative serology is currently the only test available to assess parasitological
cure.  However, this seronegative conversion can take years to decades after treatment.  There 
is evidence suggesting that the time to seronegative conversion is known to be inversely 
proportional to the pretreatment duration of infection.  In the chronic stage serological 
conversion from positive to negative can be slow and can take years to decades [29, 30].
Benefit-risk assessment
Since 1991, the estimated global prevalence of T. cruzi infection has decreased from 
18 million to 8 million due to intensive vector control and blood bank screening.  The PAHO 
estimates that approximately 60,000 new T. cruzi infections occur each year [1].  As other 
transmission routes have diminished, the proportion of infections attributable to congenital 
infection has increased.  An estimated 26% of incident infections now occur through 
mother-to-child transmission.  Mortality among infected infants is significantly higher than in 
uninfected infants, ranging from less than 5% to 20% in published studies [7][8].  However, 
even severe congenital Chagas’ disease may not be recognized because signs are often 
nonspecific, or because the diagnosis is not considered.  Children younger than 2 years of age 
appear to be at higher risk of severe manifestations than older individuals.  Severe acute 
Chagas’ disease carries a substantial risk of mortality [8].
In T. cruzi infection, treatment with nifurtimox reduces the severity of symptoms, and 
shortens the clinical course and duration of detectable parasitemia.  Parasitological cure is 
reported in 60% to 85% of patients treated in the early phase.  Furthermore, published 
evidence suggests that if the duration of infection is less than 1 year, the percentage of 
patients cured (i.e., elimination of parasites) approaches 100% [5][6].  
Nifurtimox is well tolerated in children, but its pharmacology in the pediatric population is 
still not well defined.  The currently available marketed product was developed for adults, and 
there is a significant need for a pediatric formulation particularly for infants and newborns to 
support weight-adjusted dosing.33  The safety profile of the drug is relatively well-
                                                
32 Text modified for clarity (Amendment 1)
33 Text modified for clarity, and per changes as a result of Amendment 1


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characterized.  Adverse drug reactions are moderately common in adults, but significantly less 
in children.  Nifurtimox causes gastrointestinal side effects in 50% to 75% of adult patients.  
These side effects include anorexia, nausea, vomiting, and abdominal discomfort.  Mild 
neurologic toxicity is common (occurring in up to 50% of adult patients), including mainly 
headache, but also (less commonly) irritability, insomnia, disorientation, mood changes, 
paresthesias, and tremors.  Peripheral neuropathy is a rare side effect in adults.  Leukopenia is
a very rare side effect [9].
Safety of nifurtimox in the pediatric population is well established.  The drug has been used 
off-label in children for over 40 years.  Adverse drug reactions are commonly milder and less 
frequent than those observed in adults.  Studies performed to evaluate the effect of nifurtimox 
in pediatric oncological disease suggest safety even at super-therapeutic doses (e.g., 
30 mg/kg/day) [10].  Case series of pediatric Chagas’ patients support the safety of the drug
[11].
Consensus documents from the WHO, and the majority of Latin American countries (i.e., 
Argentina, Brazil, Bolivia) [12][13][14] strongly recommend anti-trypanosomal treatment for 
acute, congenital, and reactivated T. cruzi infection, and for children with chronic infection.  
Given that Chagas’ disease is an infection commonly acquired in childhood that leads to 
significant morbidity in adults, the benefits of treatment with nifurtimox in infants and 
children (to prevent long-term complications) far outweigh the small risks of AEs associated 
with the medication in children.  
For the monitoring of efficacy of antitrypanosomal treatment, the criterion of cure usually 
depends on serologic conversion of anti-T. cruzi antibody response to negative.  Sero-
conversion in children and adolescents may occur several years after treatment, requiring 
long-term follow-up.  Therefore, subjects who were randomized and received at least one 
dose of their assigned nifurtimox treatment and did not receive additional treatment for 
Chagas’ disease after completion of nifurtimox treatment will be followed up for a total of 4 
years after end of nifurtimox treatment.
The overall benefit-risk assessment for the LTFU is considered favorable based on the 
available data.  It is expected that the information will help to gain further knowledge about 
the serological response after treatment with nifurtimox in Chagas’ disease.
4.
Study objectives
4.1
Objectives for part 1 (CHICO)
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated 
control at the 12-month follow-up (360 days from end of treatment [EOT]) as sero-
reduction (defined as a ≥20% reduction in optical density [OD] measured by 
conventional enzyme-linked immune sorbent assay [ELISA]) compared to baseline in 
subjects ≥8 months to <18 years of age at randomization; or sero-conversion 
(defined as negative Immunoglobulin G [IgG] concentration) in all subjects34
Secondary objectives of the study are:
                                                
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
To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction or sero-conversion at the 12-month follow-up (360 days 
from EOT)35

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital 
sign measurements (blood pressure, heart rate, respiratory rate, temperature), adverse 
event (AE) monitoring, and physical examinations, including neurological 
examinations36

To evaluate the pharmacokinetics (PK)/pharmacodynamics (PD) of nifurtimox in 
children receiving the drug for treatment of Chagas’ disease (to be described in a 
separate report)
Exploratory objectives of the study are:37

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole)38 as sero-reduction or sero-conversion at the 12-month follow-
up (360 days from EOT)39

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox using quantitative polymerase chain reaction (qPCR) at the 12-month 
follow-up (360 days from EOT)40

To evaluate the relationship of conventional serology (as sero-reduction or 
sero-conversion) to qPCR using frequencies of matches and mismatches to assess 
agreement41

To evaluate the relationship of non-conventional serology to conventional serology42

To evaluate the relationship of conventional serology to IHA43,44
4.2
Objectives for part 2 (CHICO SECURE)
The primary objective of the LTFU part of the study is: 

Assess the incidence of seronegative conversion as confirmed by two types of assay,
recombinant ELISA, and indirect hemagglutination assay (IHA), in subjects who were 
randomized and received at least one dose of the 60-day nifurtimox treatment regimen, 
compared to an external control group of untreated patients with Chagas’ disease at 
the 4-year follow-up. 
                                                
35 Text modified per Modification 2 (Amendment 1)
36 Text added to include neurological examinations per Modification 10 (Amendment 1)
37 Exploratory objectives added as per Amendment 2
38 Sero-conversion for the historical controls will be estimated from cure rates as presented in two 
publications.[15][16]  See Section 5, Study Design, Justification of the design.  
39 Text modified per Modification 2 (Amendment 1)
40 Text modified per Modification 2 (Amendment 1)
41 Text modified per Modification 2 (Amendment 1)
42 Fourth objective added per Modification 2 (Amendment 1)
43 IFA test added per Amendment 5.
44 Replaced IFA with IHA per Amendment 6.


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The secondary objectives are:

Assess the incidence of seronegative conversion, as confirmed by two types of assay 
(recombinant ELISA and IHA), in subjects who were randomized and received at least 
one dose of the 30-day nifurtimox treatment regimen at the 4-year follow-up.

Evaluate the proportion of responders who show both seronegative conversion as 
confirmed by two types of assay (recombinant ELISA and IHA) and no evidence of 
established cardiomyopathy as evaluated in electrocardiogram (ECG) recordings.

Assess serial reduction in OD measured by recombinant ELISA and total purified 
antigen ELISA, in subjects who were randomized and received at least one dose of 
either the 60- or 30-day nifurtimox treatment regimen compared to Visit 1 in part 1 of 
the study.
The exploratory objectives are:

Assess the incidence of seronegative conversion as confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects by age categories (≤2 years, >2 years to ≤6 
years, >6 to ≤12 years, >12 to <18 years; age is defined as subject’s age at 
randomization). 

Assess the proportion of responders who show seronegative conversion and the
incidence of seronegative conversion as confirmed by non-conventional serology in 
subjects who were randomized and received at least one dose of either 60- or 30-day 
nifurtimox treatment regimens.

Assess the occurrence of congenital infection with T. cruzi in children born of female 
subjects who were randomized and received at least one dose of 60- or 30-day 
nifurtimox treatment regimen.  The congenital infection will be confirmed by 
parasitological method in children ≤8 months of age and by serological method in 
children >8 months of age.
5.
Study design
5.1
Part 1 (CHICO)
Design overview
This is a Phase 3, prospective, randomized (to dosing regimen), age-stratified, double-blind, 
parallel-group study to evaluate the efficacy, safety/tolerability, and PK of oral administration 
of nifurtimox in children with a diagnosis of Chagas’ disease. 
A schematic of part 1 (CHICO) of the study design is presented in Figure 5–1.


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Figure 5–1: Study Design of Part 1 – Protocol 1602745
This study will enroll pediatric subjects aged 0 to younger than 18 years.  Subjects < 8 months 
of age at randomization with a diagnosis of Chagas’ disease must demonstrate direct 
observation of T. cruzi by concentration test, and subjects ≥8 months to < 18 years of age at 
randomization with a diagnosis of Chagas’ disease must demonstrate a positive conventional 
ELISA result for both of the following tests to confirm diagnosis:46

Recombinant ELISA 

Total purified antigen ELISA   
Additionally, a non-conventional ELISA test and IHA 47,48 will be obtained; however, a 
positive result will not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ disease
within three months prior to screening.49
                                                
45 Figure modified as a result of changes made to the protocol per Amendment 1; Visit 4 changed to 14 ± 1 day; Visit 5 was 
made a telephone assessment at 21 ± 3 days; visits renumbered from that point forward; footnote b deleted; footnote c 
re-lettered to footnote b; text to new footnote b changed per Modification 11 (Amendment 1)
46 Text modified for clarity (Amendment 1)
47 IFA test added per Amendment 5.
48 Replaced IFA with IHA per Amendment 6.
49 Text modified/added per Modification 2 (Amendment 1)


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Approximately 300 pediatric subjects will be randomized (2:1 randomization, 60-day regimen 
vs. 30-day regimen).50 Subjects will be stratified by age at randomization into four strata as 
specified below:51
Stratum 1:52
0 to 27 days
Stratum 2:
28 days to younger than 8 months
Stratum 3:
8 months to younger than 2 years
Stratum 4:
2 years to younger than 18 years
A minimum of 38 subjects in each age stratum is targeted, but not required, in order to be able 
to derive meaningful safety conclusions.  Enrollment may53 continue until this specification is
met, unless it is determined that such a target would be unlikely to be reached in a reasonable 
time.54
No study-specific procedures will be performed before the subject/legally authorized 
representative has signed the informed consent form, or a minor subject has provided assent,  
including asking a potential subject to fast (if applicable, depending on the age of the subject 
at the discretion of the investigator) prior to the screening blood samples.  Informed 
consent/assent for optional PK blood sampling will also be obtained. After the 
consents/assents have been obtained, screening assessments will be performed from 1 to 14 
days prior to randomization at Visit 2.55
After study eligibility has been confirmed and safety assessments have been performed at 
Visit 2 (Day 1),56 subjects will be randomized via Interactive Voice Response 
System/Interactive Web Response System (IVRS/IWRS) in a 2:1 ratio (60-day regimen vs. 
30-day regimen) to one of two treatment groups: 

Nifurtimox tablets administered three times daily for 60 days (Days 1 – 60, active 
nifurtimox treatment, Treatment Group 1) or 

Nifurtimox tablets administered three times daily for 30 days, followed by nifurtimox 
placebo administered three times daily for 30 days (Days 1 – 30, active nifurtimox 
treatment; Days 31 – 60, placebo; Treatment Group 2).  
The first dose of study drug will be administered at Visit 2.  Pre- and post-dose PK blood 
samples will be obtained at specified time points from those subjects consenting to PK 
assessments (see Table 9–1).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the 
time of maximum concentration [Cmax]), an ECG (optional for subjects < 5 years of age at the 
discretion of the investigator) will be obtained to allow for PK/PD investigations. Study drug 
                                                
50 Modified as per amendment 2
51 Text modified for clarity (Amendment 1)
52 Cohort replaced with stratum throughout body of protocol as per amendment 2
53 Text modified as per amendment 4
54 Text added as per amendment 2
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will be dispensed, instructions for study drug administration will be provided, and diaries with 
instructions for completion will be given57, to all subjects.58
Subjects will return to the investigational site for efficacy and safety assessments at Visits 3 
(Day 7±1) and 6 (Day 30). Subjects who have not consented to PK assessments will take that 
day’s doses of study drug as instructed.  Subjects consenting to PK assessments must hold the 
morning dose of study drug; for these subjects, a pre-dose PK blood sample will be obtained, 
then the morning dose of study drug will be administered with food and the PK diary 
completed.59  Post-dose blood samples will be obtained at specified time points, and ECGs 
will be obtained at the 2 – 4 hour post-dose time point (see Table 9–1).  A pre-paid phone 
card will be provided to all subjects to facilitate contact between study site personnel and the 
subjects/subjects’ authorized representatives.60  
At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and 
study drug for the remaining 30 days of treatment, along with subject diaries, will be 
dispensed.61  
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site 
personnel will contact the subject/legally authorized representative via telephone to assess the 
occurrence of AEs, use of concomitant medications, and compliance with study drug 
administration, and completion of subject diary.  A Phone Contact Form for the telephone 
assessments will be provided to all sites (see Section 16.1).62
Subjects will return to the investigational site for efficacy and safety assessments on Day 60 
(Visit 8), which will be the EOT for both treatment groups.  Subjects who have not consented 
to PK assessments will take that day’s doses of study drug as instructed.  Subjects consenting 
to PK assessments must hold the morning dose of study drug. A pre-dose PK blood sample 
will be collected, then the morning dose of study drug will be administered with food, and the
subject will complete the PK diary. Post-dose PK blood samples will be obtained at specified 
time points, and ECGs will be obtained at the 2 – 4 hour post-dose time point (see Table 9–1).  
Study drug will be collected, and no additional study drug will be dispensed.63  
After the EOT visit (Visit 8), subjects will return to the site on Days 90 (Visit 9), 240 (Visit 
10), and 420 Visit 11) for additional efficacy and safety assessments.  The total duration of 
each subject’s participation is expected to be approximately 14 months.64  
Assessments of efficacy and PK will be performed at specified time windows.  In order to 
minimize the burden of blood loss due to PK sampling, age-appropriate sparse sampling 
strategies combined with (micro-)bioanalysis techniques will be applied (see Table 9–5).  At 
                                                
57 Text modified as per amendment 4
58 Text added for clarity and per Modification 1 (Amendment 1)
59 Text modified as per amendment 4
60 Text modified/added for clarity and per Modification 1 (Amendment 1)
61 Text added for clarity (Amendment 1)
62 Text added for clarity and per Modifications 6 and 8 (Amendment 1)
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least 10 subjects per age stratum should ideally be recruited for PK assessments.65  
Recruitment of less than 10 subjects per age group is not considered as a protocol violation.66
Safety will be assessed via laboratory parameters (hematology, blood chemistry, urinalysis), 
vital sign measurements (blood pressure [optional in subjects < 5 years of age at the discretion 
of the investigator], heart rate, respiratory rate, temperature), monitoring of ECGs (optional in
subjects < 5 years of age at the discretion of the investigator), monitoring of AEs, and 
physical examinations, including a neurological examination (see Section 9.6.3.3).67  
Subjects who discontinue prematurely from study drug administration will continue to return 
to the investigational site for study assessments at Visits 3, 6, and 8 (EOT), and undergo
telephone assessments as described for Visits 4, 5, and 7.  If subjects are unable/unwilling to 
do so, they must return to the investigational site 30 (±3) days after the last dose of study drug 
for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 
5, and 7.  If the subject is unable/unwilling to return to the clinic for the EOT Visit (Visit 8), 
then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of
Visit 8 assessments.68
Also, for reasons of patient safety and at the discretion of the investigator, a subject may be 
asked to take a “drug holiday” [interruption of study drug administration] or a reduced daily 
dosage may be requested by the investigator from the depot.  A decision by the investigator 
for drug holiday or reduced dosage will be based on the occurrence and/or severity of adverse 
effects, and/or the clinical status of the subject.  Any and all changes to study drug 
administration or daily dosage must be recorded within the subject’s study chart, as well as, 
the electronic case report form (eCRF).69
Primary variable(s)
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months 
post-treatment using two conventional ELISA serology tests as the measure of efficacy.70
Justification of the design
This study uses an historical untreated-controlled design, as it is unethical to have a 
prospective placebo-controlled superiority study design.  Only two benznidazole
placebo-controlled studies have been conducted in children with Chagas’ disease.[15][16] 
Direct comparisons between these studies cannot be made due to differences in study 
populations (Argentina vs. Brazil), medication dosage (5 mg/kg vs. 7.5 mg/kg), outcome 
measures, and time of follow-up (4 years vs. 3 years).  Data derived from these studies will, 
however, be used as the comparative historical placebo control to be utilized in the analysis of
the study endpoints.  Hence, a comparison of sero-reduction and/or sero-conversion from 
baseline to 12-month follow-up for both primary and secondary endpoint evaluations (versus 
                                                
65 Text deleted/added for clarity (Amendment 1)
66 Modified as per amendment 4
67 Text modified/deleted for clarity, and per Modifications 9 and 10 (Amendment 1)
68 Text added per Modification 11 (Amendment 1)
69 Text modified as per amendment 4
70 Text modified per Modification 2 (Amendment 1)


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historical placebo) will be made as well as for a comparison with benznidazole. Details are 
presented in Section 10.71
There are several choices for diagnosis of Chagas’ disease and monitoring of drug efficacy 
after anti-parasitic treatment.  Traditional parasitological methods, such as direct microscopic 
observation, xenodiagnoses, and hemoculture, show low sensitivities, limiting their 
usefulness.  Microhematocrit is the method of choice to identify congenital infection because 
of its heightened sensitivity and the small amount of blood needed.  Microscopic examination 
of cord blood or peripheral blood of the neonate by this technique is strongly recommended 
during the first month of life.  For the monitoring of drug efficacy, the criterion of cure 
usually depends on serologic conversion of the anti-T. cruzi antibody response to negative.  
Sero-conversion in children and adolescents may occur several years after treatment, 
requiring long-term follow-up. 
Sero-reduction (measured as a ≥20% reduction of the ELISA absorbance [OD] value) at 
12 months post-treatment will be considered equivalent to historical information on 
sero-conversion at subsequent time points after treatment.72
Both primary and secondary study endpoints are based upon a target of ≥ 20% decrease in 
antibody concentration (sero-reduction) to T. cruzi in conventional serological tests or 
negativization of serology. This is consistent with one previous study in children 6 to 12 
years of age infected with T. cruzi in the intermediate (early chronic) phase of Chagas’ 
disease [15].  Subjects were administered 5mg/kg benznidazole or placebo in a randomized 
fashion for 60 days, and followed for 48 months.  At 12 months, a 21% reduction in 
concentration of Chagas’ disease-specific antibodies compared to baseline (ELISA mean OD
0.467 [± 0.099] vs. 0.369 [± 0.107]) was observed in subjects who received benznidazole.  
For the subsequent time points of 18, 24, and 48 months, percent concentration reductions in 
antibody titer were reported as 23%, 29%, and 27%, respectively.  After a 48-month follow-
up, negative sero-conversion was detected in 11.3% (P<0.05) of the benznidazole group and 
only 4.5% of the placebo group.73
The above data are comparable to a study in early chronic Chagas’ patients treated with 
7.5 mg/kg benznidazole or placebo for 60 days, which also showed approximately 20% 
antibody concentration reduction at 12 months [16]. Analysis of concentration over time 
indicated a consistent decrease in antibody concentrations in the benznidazole group, and an 
initial decrease followed by a progressive increase in the placebo group.  At the end of the 
3-year follow-up, children who received benznidazole had a significantly lower mean 
antibody concentration than placebo-treated children (P<0.00001).  As persistence of positive 
results by conventional serology for years after treatment is common, these tests may or may 
not revert to sero-negative (sero-conversion) after many years or even decades.  Thus, 
reduction in T. cruzi antibody concentration has been used in clinical practice as a surrogate 
indicator for parasitological cure (sero-conversion) following anti-trypanosomal treatment.74
While a decrease ≥20% in antibody concentration has been demonstrated across two 
independent clinical trials in pediatric patients with Chagas’ disease, neither author has 
                                                
71 Text modified for clarity (Amendment 1)
72Text modified for clarity (Amendment 1)
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published extended follow-up data which include serological results for the patients in these 
studies.  Since signs and symptoms are usually lacking in early Chagas’ disease, it is not 
possible to demonstrate immediate meaningful clinical benefit to active treatment. However, 
significant reductions in T. cruzi antibody concentration (sero-reduction) by conventional 
serology has been accepted as a predictor of future sero-conversion, and early treatment cure 
of infection will ultimately reduce the risk of developing visceral abnormalities, as well as
contribute to the decrease in T. cruzi transmission.75
Alternative and more rapid approaches as an early indicator of negative sero-conversion in 
treated patients have been suggested.  Using non-conventional serological techniques, i.e., 
highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote 
glycoconjugate antigen and an epimastigote complex [20], the time to negativization was 
significantly sooner for the non-conventional ELISA than for the conventional ELISA.  The 
chemiluminescent ELISAs provide tests that are highly sensitive and specific for Chagas' 
disease diagnosis.  They can be used in blood bank screening and to monitor the treatment of 
patients undergoing chemotherapy [21].76
Non-conventional ELISA antigens, such as those used in the Sosa-Estani and deAndrade 
trials (i.e., F29 and F 2/3), [15][16] are often considered as early markers for effective 
Chagas’ disease therapy.  In the study by Altcheh, et al, the kinetics of disappearance of 
conventional serology and anti-F2/3 antibodies were compared in 21 patients with congenital 
Chagas’ disease after receiving benznidazole treatment. Patients were divided into two 
groups: (A) age < 8 months at diagnosis and (B) age > 9 months at diagnosis.  Group A 
presented negative outcome for conventional serology at 6.6 months (CI 95 3.4-9.8 months) 
and for anti-F2/3 at 4 months (CI 95 0.9-7.1 months, p = 0.18).  Group B exhibited non-
reactive conventional serology at 63.1 months (CI 95 42.1-84.2 months) whereas anti-F2/3 
antibody determination became negative at 21.9 months (CI 95 5.7-38.1 months, p = 0.0025).  
In patients belonging to Group A, antibodies were undetectable by both conventional serology
and anti-F2/3 ELISA soon after receiving chemotherapy.  In infants included in Group B, a 
negative result for anti-F2/3 antibody detection occurred significantly in advance of negative 
conventional serology reactivity.  Consequently, the anti-F2/3 antibody assay becoming 
negative should be considered as a surrogate endpoint for assessment of cure or positive 
response to treatment, particularly in those patients with prolonged time of infection [18].
A new perspective on Chagas’ disease diagnosis was opened by the use of the PCR as a 
measure of treatment response.  Over the past few years, the availability of PCR to detect 
T. cruzi DNA in blood samples has opened new possibilities for the evaluation of response to 
trypanocidal chemotherapy.77
Clearance of parasitemia (evaluated most commonly by xenodiagnoses or PCR) combined 
with disappearance of antibodies (seronegative conversion) has been proposed as cure criteria 
by some authors.[19]  The main limitations in evaluating treatment response for Chagas’ 
disease using conventional serology stems from the need for long-term follow-up (years to 
decades) to observe negativization of conventional serological tests.78
                                                
75 Term “titer(s)” replaced with “concentration(s)”; text modified for clarity (Amendment 1)
76 Paragraph added to include updated information (Amendment 1)
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In the experience of Parasitology service, Ricardo Gutierrez Children´s Hospital, Buenos 
Aires unpublished data (presented at European Society of Pediatric Infectious Disease 2014 
and meeting of Food and Drug Administration) by 
, a correlation between 
negativization of conventional serology (whole antibody against T. cruzi measured by ELISA 
or hemoagglutination inhibition) and PCR results in children 2 years of age and younger is 
fairly high; the correlation between these two tests in children older than 3 years is lower, as 
sero-conversion (i.e., becoming negative) may take several years, while PCR becomes 
negative within 60 days of treatment, and remains negative in this age group also.  In the 
whole population, at least 20% sero-reduction of conventional serology was observed at 12 
months.  This percentage was higher in the younger children than in the older children.79
By comparing 48 PCR methods from 26 laboratories, it was shown that the four best 
performing methods showed a high sensitivity (10 fg/µl DNA and 0.5 parasites/mL blood),
and an overall accuracy of 87% to 90% [22]. More recent publications showed comparable 
sensitivity [23][24].
Several studies concluded that PCR has a higher sensitivity for detecting therapeutic failures 
compared to usual parasitological and conventional serologic methods. However, only two 
demonstrated specificity and showed that PCR could be used as a tool for early determination
of cure [25][26].
Furthermore, real-time PCR (qPCR) protocols for detection of T. cruzi’s DNA were 
developed.  The development of qPCR assays have several advantages compared with 
conventional PCR. First, the introduction of closed-tube qPCR systems has resulted in the 
development of rapid microbial diagnostics with low contamination risk. Also, qPCR has a 
shorter time-to-result, and allows quantitative assays and automation [27].80
Studies have demonstrated the substantial value of qPCR, in terms of sensitivity and 
specificity, in the direct parasitological diagnosis of Chagas’ disease and follow-up 
assessment of chemotherapy.  However, in this Phase 3 study, parasitological cure based on 
serological tests for T. cruzi infection (ELISA) will be used as the primary efficacy endpoint 
to demonstrate efficacy of nifurtimox due to lack of historical PCR data from untreated 
children with Chagas’ disease.81 Other parasitological methods (direct microscopic) and 
qPCR tests will comprise other variables to demonstrate drug efficacy.82
The 30-day and 60-day treatment regimens were selected with the intention of providing the 
most effective treatment within the shortest possible duration.  During the 1st Chagas Platform 
Technical meeting (September 1, 2009, Rio de Janeiro)[28] it was concluded that the 
minimum length of treatment for nifurtimox should be 30 days which is based on good results 
with fewer than 60 days of treatment.  Hence, the 30-day arm is included to evaluate the 
potential of this shorter regimen.83 In a recent study from Bianchi et al, 62 patients with 
Chagas’ disease confirmed by different serological tests were treated with nifurtimox 
(Lampit), and followed for 30 months post-treatment.  All children were treated during 60 
days according to protocols established by the WHO.  Monitoring was performed every 20 
                                                
79 Paragraph moved from previous page to here; text deleted (Amendment 1)
80 Text modified for clarity (Amendment 1)
81 Text added for clarity (Amendment 1)
82 Text deleted for clarity as of amendment 2
83 Text added as per amendment 2
PPD


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days to evaluate treatment safety.  Results indicated that both parasite load (measured through 
qPCR) and antibodies (ELISA absorbance) showed a significant median reduction 6 months 
after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 0.6 to 0.2 absorbance units 
respectively (p<0.001).  Further reductions were evidenced by the 30-month post-treatment 
time point.  Sixty days of treatment with nifurtimox was very well tolerated and successfully 
reduced parasite load and antibody titers.  The results show for the first time the therapeutic 
response and safety of nifurtimox treatment for T. cruzi infection in a population of school-
aged children in the asymptomatic chronic phase of Chagas’ disease in Colombia [29].84
End of part 1 of the study
The end of part 1 of the study as a whole will be reached as soon as the last visit of the last 
subject has been reached in all centers in all participating countries.85
5.2
Part 2 (CHICO SECURE)
Design overview
This is a prospective, long-term follow-up of subjects, who were randomized and received at 
least one dose of their assigned 60- or 30-day nifurtimox treatment.  The study is designed to 
evaluate seronegative conversion and serial reduction in OD measured by ELISA.
Subjects will be followed up for a total of 4 years after end of nifurtimox treatment.  The first 
year of follow-up period is included in part 1 and 3 years of follow-up are included in part 2 
of the study.  
The following age categories will be considered for part 2 of the study: ≤2 years, >2 years to 
≤6 years, >6 to ≤12 years, >12 to <18 years (age is defined as subject’s age at randomization).
Subjects enrolled will be followed annually with visits to the investigational site.  
Seronegative conversion will be measured by two types of assay, recombinant ELISA, IHA, 
and by non-conventional ELISA.
For assessment of serial reduction compared to baseline defined as screening visit of part 1 
(Visit 1), OD will be measured by recombinant ELISA and total purified antigen ELISA. 
No study-specific procedures will be performed before the subject and/or parent(s) or legally 
authorized representative(s) has signed the informed consent form, or a minor subject has 
provided assent for part 2 of the study.  After the consents/assents have been obtained, the 
assessments scheduled for FU Visit 1 including eligibility assessments will be performed.  
(FU Visit 1 according to Table 9–2; for details please refer to Section 9.2). 
The first visit should be performed as soon as possible after availability of regulatory and 
ethics committee approval for all subjects who have already completed part 1 of the study for 
1 year 6 weeks or longer.  Subjects who are entering the study beyond the time window for 
FU Visit 1 (2 years6 weeks) will have their eligibility assessments as soon as possible 
provided that consents/assents have been obtained (see Table 9–2; for details please refer to 
Section 9.2). 
Subjects will have annual visits at the investigational site for efficacy and safety assessments 
for 3 years.  Timing of study visits is relative to end of treatment in part 1 of the study.
                                                
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At each investigational site visit, blood samples for serology tests (recombinant ELISA, total 
purified antigen ELISA, IHA, and non-conventional ELISA) will be collected.  Blood 
samples will be taken under fasting conditions, if applicable, depending on the age of the 
subject, at the discretion of the investigator.  Subjects will undergo a physical examination, 
body weight, height/length and vital signs will be measured and AEs considered at least 
possibly related to nifurtimox and AEs caused by protocol-required procedures will be 
recorded.  A 12-lead electrocardiogram (ECG) will be obtained at each annual visit.  The
subject’s concomitant medications will be recorded.  Signs and symptoms of Chagas’ disease 
will be assessed and blood will be taken for detection of T. cruzi’s deoxyribonucleic acid 
(DNA) using quantitative polymerase chain reaction (qPCR).  If during the course of the 
study, OD measured by ELISA increases, the subject has positive qPCR results and shows 
signs or symptoms of Chagas’ disease, it is up to the investigator’s discretion to initiate 
antitrypanosomal treatment while the subject continues in the LTFU.
Whenever an AE considered at least possibly related to nifurtimox or caused by protocol-
required procedures occurs, the subject should report it to the investigator.
Female subjects who give birth before end of part 2 of the study will be invited to test their 
child for the presence or absence of infection with T. cruzi. 
Between the annual visits at the investigational site, subjects and/or his or her parent(s) or 
legally authorized representative(s) will be contacted by qualified personnel by a method 
agreed (e.g., phone call) about 6 months after a visit at the investigational site to identify the 
occurrence of potential clinical symptoms of Chagas’ disease or any pathology mentioned in 
the exclusion criteria using a structured interview.  In case of suspected findings, an 
unscheduled visit at the investigational site to initiate appropriate diagnostic evaluations will 
be requested. 
The schedule of procedures/assessments to be performed during part 2 of the study is 
provided in Section 9.1. 
Primary variable
The primary efficacy variable will be the incidence rate of seronegative conversion measured 
by two types of assay (recombinant ELISA and IHA) in subjects who were randomized and 
received at least one dose of the 60-day nifurtimox treatment regimen.  Both test results 
should be negative for the subject to be considered as seroconverted.
End of part 2 of the study
The end of part 2 of the study as a whole will be reached as soon as the last visit of the last 
subject has been reached in all centers in all participating countries.
Primary completion
The primary completion event for part 2 of the study is last subject last visit (LSLV).
Justification of the design
For the monitoring of efficacy of antitrypanosomal treatment, the criterion of cure usually 
depends on serologic conversion of the anti-T. cruzi antibody response to negative.  
Seronegative conversion in children and adolescents may occur several years after treatment, 
requiring long-term follow-up.  Conversion of serological responses to negative, 
demonstrated by more than one type of assay (ELISA and either IHA or IFA) is the FDA-
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Annual visits at the investigational site are planned for efficacy and safety measurements and 
subjects will be closely monitored for signs and symptoms of Chagas’ disease.  Criteria for 
withdrawal of individual subjects or termination of the entire study are described in 
Section 6.3 and Section 12, respectively.
It is well known, that mothers with Chagas’ disease can transmit T. cruzi to their fetuses, who 
become carriers of the infection and are then at risk of developing severe cardiac disease later 
in the course of their lives.  If identified early enough after birth, the infected newborns can be 
treated and cured. 
6.
Study population
6.1
Inclusion criteria 
Inclusion criteria for part 1 (CHICO)
1. Male and female pediatric subjects aged 0 days to younger than 18 years
2. Chagas’ disease diagnosed/confirmed by:

Subjects < 8 months of age at randomization must demonstrate direct observation of 
T. cruzi by concentration test86

Subjects ≥ 8 months to < 18 years of age at randomization must demonstrate a positive 
conventional ELISA result for both of the following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA87   
Additionally, a non-conventional ELISA test and IHA 88,89 will be obtained; however, a 
positive result will not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation (two ELISA tests) of 
positive Chagas’ disease within three months prior to screening and they have not had 
prior anti-trypanocidal or anti-parasitic treatment.90 (The subject’s parent or legally 
acceptable representative must agree to the collection of a baseline blood specimen for 
ELISA testing later during the study).91
3. Written informed consent by the subject and/or parent(s) or legally authorized 
representative(s) according to the age established per local regulations must be obtained 
from all subjects who are screened for the study.  In addition, depending on the subject’s 
age, subject’s assent is required as applicable by local laws and regulations (varies in each 
country).
4. Females of childbearing potential (i.e., female subjects who have experienced menarche) 
and male subjects must agree to use adequate contraception if sexually active from the 
time of signing the informed consent/assent form until 3 months after the last study drug 
                                                
86 Text modified for clarity (Amendment 1)
87 Text modified/added for clarity and per Modification 2 (Amendment 1)
88 IFA test added per Amendment 5
89 Replaced IFA with IHA per Amendment 6.
90 Text added per Modification 2 (Amendment 1)
91 Text added as per amendment 2.


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administration.  Depending on the country, study site personnel may dispense condoms at 
the time that study drug is dispensed to either the subject or the subject’s parent/legal 
guardian, with an explanation of rationale and risks.
Inclusion criteria for part 2 (CHICO SECURE)
1. Male and female subjects who were randomized and received at least one dose of their 
assigned 60- or 30-day regimen of nifurtimox treatment
2. Written informed consent by the subject and/or parent(s) or legally authorized
representative(s) according to the age established per local regulations must be obtained 
from all subjects who are screened for the study.  In addition, depending on the subject’s 
age, subject’s assent is required as applicable by local laws and regulations (varies in each 
country)
6.2
Exclusion criteria
Exclusion criteria for part 1 (CHICO)
1. Subjects aged 0 to 27 days who, at birth, were pre-term (i.e., gestational age less than 
37 weeks), weighed less than 2500 g, or had a maximum Apgar score <7 at 5 minutes  
2. Subjects with any of the following conditions that is associated with Chagas’ disease, such 
as:

Known evidence of Chagas’ disease-related cardiomyopathy/Chagas’ heart disease  

Known evidence of Chagas’ disease-related gastrointestinal dysfunction (e.g., 
megaesophagus, megacolon, or both) or Chagas’ digestive disease

Serious manifestations of acute Chagas’ disease, including myocarditis, 
meningoencephalitis, or pneumonitis

Known evidence of Chagas’ disease-related damage to the peripheral nervous 
system or peripheral neuropathy (i.e., affected subjects show a combination of 
sensory impairment and diminished tendon reflexes that mainly involve the lower 
limbs)

Clinically significant psychiatric disorder (e.g., moderate to severe depression, 
severe anxiety, or psychosis) or epilepsy
3. Subjects with contraindications/warnings to nifurtimox administration, or with conditions 
that may increase the risk of the undesirable effects of nifurtimox, including:

Hypersensitivity to nifurtimox or any hydantoin, or to any of the excipients

Suspected or known porphyria

Severe renal impairment defined by the following:
o
For subjects < 1 year of age at randomization, estimated glomerular filtration 
rate (eGFR) < 100% of the lower limit of normal (LLN) appropriate for age 
(eGFR should be calculated according to Schwartz formula; see Section
16.2)92  
                                                
92 Text revised for clarity; cross-reference renumbered to 16.2 (Amendment 1)


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o
For subjects ≥ 1 year to < 18 years of age at randomization, eGFR < 80% of 
LLN appropriate for age 

Severe hepatic impairment (alanine aminotransferase [ALT] or aspartate 
aminotransferase [AST] considered clinically significant by the investigator)

History of brain injury, predisposition to seizures or epilepsy, psychiatric disease, 
or serious behavioral alteration 

Severe or significant gastrointestinal disorders, or metabolism and nutrition 
disorders 
4. Subjects with a history of malignancy in the previous 5 years 
5. Subjects who are chronic abusers or current users of alcohol or recreational drugs, or the 
newborn infants of mothers who are chronic abusers or current users of alcohol or 
recreational drugs
6. Subjects with any condition that would prevent him/her from taking oral medication
7. Immuno-compromised subjects (e.g., those with human immunodeficiency virus 
infection, primary immunodeficiency, or prolonged treatment with corticosteroids or other 
immunosuppressive drugs)
8. Subjects with any other acute or chronic health conditions or congenital disorders which, 
in the opinion of the investigator, would make them unsuitable for participation in a 
clinical study or may interfere with the efficacy, safety, and/or PK evaluation of the study 
drug 
9. Subjects who have had previous treatment with trypanocidal agents or an accepted 
indication for antiparasitic therapy (e.g., reactivation of Chagas' infection due to 
immunosuppression by several diseases or treatment with steroids)  
10. Subjects who have had treatment with any investigational medicinal product within 
30 days before the first dose of study drug or93 have previously received the study 
treatment
11. Subjects who are pregnant or breastfeeding 
12. Subjects of breastfeeding mothers under treatment with trypanocidal agents
13. Subjects who the investigator considers unlikely to adhere to the protocol, comply with
study drug administration, or complete the clinical study and follow-up
14. Subjects with close affiliation with the investigational site; e.g., a close relative of the 
investigator, dependent person (e.g., employee or student of the investigational site)
15. Subjects living in housing conditions where there is no active or effective vector-control 
to T. cruzi reinfection as determined by Ministry of Health guidelines in each country
Exclusion criteria for part 2 (CHICO SECURE)
1. Subjects with acute or chronic health conditions or congenital disorders which, in the 
opinion of the investigator, would make them unsuitable for participation in the clinical 
study
                                                
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2. Subjects living in housing conditions where there is no active or effective vector-control 
to T. cruzi reinfection as determined by Ministry of Health guideline of the respective 
country and the judgement of the investigator
3. Subjects with clinical manifestations of Chagas’ disease, such as:

Known evidence of Chagas’ disease-related gastrointestinal dysfunction (e.g., 
megaesophagus, megacolon, or both) or Chagas’ digestive disease

Serious manifestations of acute Chagas’ disease, including myocarditis, 
meningoencephalitis, or pneumonitis
4. Immuno-compromised subjects (e.g., those with human immunodeficiency virus 
infection, primary immunodeficiency, or prolonged treatment with corticosteroids or other 
immunosuppressive drugs)
5. Subjects who the investigator considers unlikely to adhere to the protocol, or complete the 
long-term follow-up
6. Subjects with close affiliation with the investigational site; e.g., a close relative of the 
investigator, dependent person (e.g., employee or student of the investigational site)
Justification of selection criteria for part 2 (CHICO SECURE)
This LTFU has been designed at the request of the US FDA to assess the incidence of 
seronegative conversion in subjects who were randomized and received at least one dose of 
nifurtimox treatment for 4 years after end of nifurtimox treatment. 
For assessment of conversion of serological response to negative, 2 distinct serologic tests are 
recommended that use different techniques and/or detect antibodies to different antigens. 
Two commonly used techniques are ELISA and IHA. 
The exclusion criteria are valid for known or suspected conditions and are chosen to ensure 
that subjects with conditions which may have an effect on the aims of the follow-up are 
excluded.
Considering that the follow-up period is evaluating long-term seronegative conversion and 
OD reduction measured by ELISA after nifurtimox treatment, subjects who required 
antitrypanosomal treatment, including any investigational treatment for Chagas’ disease after 
nifurtimox regimens will be analyzed separately. 
To reduce the risk of reinfection as a confounding factor in the study, subjects residing in 
areas where vector control has not been instituted are excluded as well. 
6.3
Withdrawal of subjects from study
In general, the criteria, rules, and procedures in this section apply to part 1 and part 2 of the 
study.  Criteria, rules, and procedures not applicable to either part of the study are otherwise 
marked.
6.3.1
Withdrawal
Withdrawal criteria
Subjects must be withdrawn from the study if any of the following occurs:
At their own request or at the request of their parents’ or legally acceptable 
representative.  At any time during the study and without giving reasons, a subject may 


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decline to participate further.  The subject will not suffer any disadvantage as a result 
of this decision.
Pregnancy. If any urine pregnancy test is positive, a serum pregnancy test must be 
performed to confirm the result.  See Section 9.6.2.94 (not applicable to part 2)
Subjects, with positive  parasitology test at the 30-day visit for whom (1) it has been 
determined that the drug was administered correctly and (2) the daily dosage given was 
correct, must be discontinued from the study and treated with an alternative 
trypanocidal medication, selected by the investigator.95 (not applicable to part 2)
Subjects may be withdrawn from the study if any of the following occurs:
If, in the investigator's opinion, continuation of the study would be harmful to the 
subject's well-being
At the specific request of the sponsor and in liaison with the investigator (e.g., obvious 
non-compliance, safety concerns)

For the following situations that are regarded by the investigator to be related to the 
use of study drug (nifurtimox) (not applicable to part 2):

Severe nervous system disorders such as seizure, vertigo/dizziness,   
polyneuropathy, or peripheral neuropathy

Severe gastrointestinal disorders, severe decreased appetite

Significant loss of body weight in the judgment of the investigator 

Severe hepatic impairment 

Severe renal impairment

Psychic disorders such as disorientation, psychotic behavior

Development of malignant disease

Hematological abnormalities such as severe leukopenia
Depending on the time point of withdrawal, a withdrawn subject is referred to as either
“screening failure” or “dropout” as specified below:
Screening failure
A subject who, for any reason (e.g., failure to satisfy the selection criteria), terminates the 
study before the time point used for the definition of “dropout” (see below) is regarded as a 
“screening failure.”
Re-starting the defined set of screening procedures to enable the “screening failure” subject’s 
participation at a later time point is allowed only in the following situations (not applicable to 
part 2):
                                                
94 Third bullet point and text re: positive microscopy deleted (Amendment 1)
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The subject had successfully passed the screening procedures, but could not start 
subsequent treatment on schedule.
Initial screening occurred too early to complete the required washout period (Day -14 
to Day -1) after prior therapy.
The in-/exclusion criteria preventing the subject’s initial attempt to participate have 
been changed (via protocol amendment).
Subjects not meeting inclusion/exclusion criteria at screening may be re-screened once.  At
the discretion of the investigator and in consultation with Bayer, the screening period (or re-
screening period) may be extended up to 3 days to allow for receipt of test results, to evaluate 
changes in a subject’s condition, or for reasons not under the subject’s control.96 In any case, 
the investigator has to ensure that the repeated screening procedures do not expose the subject 
to an unjustifiable health risk.  Also, for re-screening, the subject has to re-sign the informed 
consent form, even if it was not changed after the subject’s previous screening.  Only the re-
screening will be captured in the eCRF.97 (Not applicable to part 2)
Dropout
A subject who discontinues study participation prematurely for any reason is defined as a 
“dropout” if the subject has already been randomized, assigned to treatment, and administered 
at least one dose of study drug.
General procedures
In all cases, the reason for withdrawal must be recorded in the electronic case report form 
(eCRF) and in the subject's medical records. 
The subject may object to the generation and processing of post-withdrawal data as specified 
in Section 13.4.
Any subject removed from the study will remain under medical supervision until discharge or 
transfer is medically acceptable.
Details for the premature termination of the study as a whole (or components thereof) are 
provided in Section 12.
6.3.2
Replacement
A subject who is withdrawn from the study will not be replaced.
6.4
Subject identification
After documentation of consent/assent (informed consent/assent form) by the parent(s) and 
assent by the child in accordance with local regulations (varies in each country), each subject 
will be assigned a 9-digit number for unambiguous identification throughout the study 
constructed as follows:
                                                
96 Text modified as per amendment 4
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Digits 1 to 2 
=  Country code
Digits 3 to 5
=  Center number within the country
(Digits 1 to 5
=  Trial unit)
Digits 6 to 9
=  Current subject number within the center
Subject identification numbers must be used in sequence, and no number should be skipped, 
substituted, or reused.
7.
Treatment(s)
7.1
Treatments to be administered
Each subject will receive nifurtimox oral tablets three times daily for 60 days, or for 30 days 
followed by nifurtimox placebo administered three times daily for 30 days.98
Each nifurtimox tablet contains either 30 mg or 120 mg of nifurtimox as the active ingredient, 
and the following excipients:  calcium hydrogen phosphate dehydrate, maize starch, silica 
colloidal anhydrous, and sodium lauryl sulfate.
7.2
Identity of study treatment
Nifurtimox 30-mg and 120-mg oral tablets and matching placebo tablets for each dosage form 
will be provided by Bayer HealthCare AG. The tablets for both dosage forms are yellow, 
round, and biconvex.  The matching placebo tablets for each dosage (30-mg and 120-mg 
formulations) will bear the same appearance and packaging as the active study drug tablets.99
All study drugs will be labeled according to the requirements of local law and legislation.  
Label text will be approved according to the sponsor’s agreed procedures, and a copy of the 
labels will be made available to the study site upon request.
For all study drugs, a system of numbering in accordance with all requirements of Good 
Manufacturing Practice (GMP) will be used, ensuring that each dose of study drug can be 
traced back to the respective bulk batch of the ingredients.  Lists linking all numbering levels 
will be maintained by the sponsor’s clinical supplies Quality Assurance group. 
A complete record of batch numbers and expiry dates of all study treatment as well as the 
labels will be maintained in the sponsor’s study file.
7.3
Treatment assignment
Subjects will be randomized via IVRS/IWRS to one of two treatment groups in a 2:1 ratio
(60-day regimen vs. 30-day regimen) as follows: 

Nifurtimox tablets administered three times daily for 60 days (Days 1 – 60, active 
nifurtimox treatment, Treatment Group 1), or 

Nifurtimox tablets administered three times daily for 30 days, followed by nifurtimox 
placebo administered three times daily for 30 days (Days 1 – 30, active nifurtimox 
treatment; Days 31 – 60, placebo; Treatment Group 2).  
                                                
98 Last sentence deleted since it appears later in Section 7.4 (Amendment 1)
99 Text modified for clarity (Amendment 1) 


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7.4
Dosage and administration
Nifurtimox oral tablets will be administered in recommended doses by body weight according 
to the company core data sheet (CCDS; Table 7–1).  For infants and children weighing more 
than 6.0 kg and < 40kg, the daily dosage will be 10-20 mg/kg.  Total daily dosages for 
adolescents weighing > 40 mg will be 8-10 mg/kg, slightly lower than that recommended in 
the CCDS, to minimize the likelihood of adverse experiences.  
Table 7–1:  Total daily dosage of nifurtimox oral tablets based on body weight
Body weight group
Total daily dose of nifurtimox [mg / kg body weight]
Adolescents (40 – 60 kg)
8-10
Infants and children (<40 kg)
10-20
Study drug will be dispensed at Visits 2 (Day 1) and 6 (Day 30), with instructions for 
administration.100 Dosages of study drug dispensed at Visits 2 and 6 will be the same even if a 
different dosage is indicated based on a patient’s weight change.101 Nifurtimox will be 
administered three times a day, in the morning, at noon, and at night, with food.  The 30-mg 
and 120-mg tablets have score lines and can be divided into two equal halves to allow for 15-
mg or 60 mg dose increments, respectively.  The tablet is manufactured for quick 
disintegration in order to allow administration to subjects < 6 years old who are not able to 
swallow tablets.  Before administration, the tablet should be dissolved in enough water to fill 
a teaspoon (approximately 5 mL) to form a soft slurry102 that should be given immediately 
with food.  Nifurtimox will be dispensed based on body weight at randomization, as presented 
in Table 7–2.103
Table 7–2:   Individual dosages of nifurtimox oral tablets based on body weight104
Age Group
Body Weight (kg)105
Number of 30-mg 
Tablets
Number of 120-mg 
Tablets
2.5 – 4.5
3 x daily ½ tablet
4.6 - <9
3 x daily 1 tablet
---
9 - <13
3 x daily 1 ½ tablets
---
13 - <18
3 x daily 2 tablets
---
18 - <22
3 x daily 2 ½ tablets
---
22 - <27
3 x daily 3 tablets
---
27 - <35
3 x daily 3 ½ tablets
---
35 - <41
---
3 x daily 1 ½  tablets
≥12 to < 18 years
41 - <51
---
3 x daily 1 tablet
51 - < 71 
---
3 x daily 1 ½ tablets
71 - < 91
---
3 x daily 2 tablets
91 or greater
---
3 x daily 2  ½ tablets
                                                
100 First sentence added for clarity (Amendment 1)
101 Text modified as per amendment 4
102 Bioequivalence testing of the slurry has determined it to be comparable to 4 x 30-mg tablets and the 120-mg tablet.
103 Text modified for clarity (Amendment 1) 
104 Table 7-1, Table 7-2 and associated text in dosage and administration modified as of amendment 2
105 In this column, upper limit of weight ranges in each category changed per the most current dosing information 
(Amendment 1) 


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At Visits 2 and 6, when study drug is dispensed, subjects/legally authorized representatives 
will be provided with a diary on which to document the date, exact time, and doses (morning, 
noon, and night) of study drug, and comments (see Section 1).  Should a subject vomit or spit 
out study drug, the occurrence will be recorded on the diary along with the time of the 
event.106 Subjects/legally authorized representatives will be instructed to bring their diaries 
with them to each in-clinic study visit.  The diaries will be collected and reviewed at each 
study visit to perform drug accountability and assess treatment compliance.107
Riboflavin is included as a colorant in the matching placebo tablets for study drug blinding 
purposes. A common side effect of riboflavin ingestion is bright yellow urine. Subjects 
and/or their legally authorized representatives will be advised of the possibility of change in 
the subject’s urine color within the informed consent form, and when study drug is 
administered and dispensed at the investigational site.108  
Subjects consenting to optional PK assessments must withhold taking the morning dose of 
study drug on the day of Visits 3, 6, and 8, when pre- and post-dose PK blood samples will be 
obtained. Subjects not consenting to optional PK assessments may take their study drug on 
the mornings of Visits 3, 6, and 8 as instructed.109
7.5
Blinding
In compliance with applicable regulations, in the event of a suspected, unexpected, serious 
adverse reaction (SUSAR; see Section 9.6.1.5) related to the blinded treatment, the subject’s 
treatment code will usually be unblinded before reporting to the health authorities, ethic 
committees and investigators (see Section 9.6.1.4).
Emergency unblinding by the investigator110
In case of emergency unblinding for a serious adverse event (SAE), investigators will use the 
IVRS/IWRS to unblind a subject. 
7.6
Drug logistics and accountability
At Visit 6 (Day 30), subjects will return their diaries, all remaining study drug, and empty 
packaging.  The diaries will be reviewed to assess study drug accountability, and study drug 
for the remaining 30 days of treatment will be dispensed.  
At Visit 8 (Day 60), subjects will return their diaries, all remaining study drug, and empty 
packaging.  The diaries will be reviewed to assess study drug accountability, and no 
additional study drug will be dispensed.111
Details regarding storage conditions for nifurtimox are provided in the Investigators’ 
Brochure.112  Additional details regarding storage conditions may be provided separately.  
Nifurtimox and the matching placebo are not to be stored above 25°Celsius.
                                                
106 As per amendment 2
107 Diaries capturing information on daily dosing to be provided to subjects; diary added as appendix in Section 16 
(Amendment 1)
108 Text re: possible discoloration of urine due to riboflavin added per Modification 4 (Amendment 1)
109 Text added for clarity regarding study drug dosing in relation to PK assessments (Amendment 1)
110 Emergency unblinding added as of amendment 2
111 Text added for clarity regarding diaries and return of study drug and packaging (Amendment 1)
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All study drugs will be stored at the investigational site in accordance with Good Clinical 
Practice (GCP) and GMP requirements and the instructions given by the clinical supplies 
department of the sponsor (or its affiliate/Contract Research Organization [CRO]), and will be 
inaccessible to unauthorized personnel.  Special storage conditions and a complete record of 
batch numbers and expiry dates can be found in the sponsor’s study file; the site-relevant 
elements of this information will be available in the investigator site file.  On the day of 
receipt, the responsible site personnel will confirm receipt of study drug via IVRS/IWRS.  
The site personnel will use the study drug only within the framework of this clinical study and 
in accordance with this protocol.  Receipt, distribution, return and destruction (if any) of the 
study drug must be properly documented according to the sponsor’s agreed and specified 
procedures. 
Written instructions on medication destruction will be made available to affected parties as 
applicable.
If performing drug accountability implies a potential risk of contamination, a safety process/ 
guidance for handling returned drug will be provided.
7.7
Treatment compliance
Study site personnel will review diaries and113 perform a tablet count of all returned study 
medication to determine treatment compliance.  Any discrepancies between actual and 
expected amount of returned study medication must be discussed with the subject at the time 
of the visit, and any explanation must be documented in the source records.
Treatment compliance will also be assessed during the telephone contacts at Visits 4, 5, and 7
(see Section 16.1).114
7.8
Treatment in part 2 (CHICO SECURE)
No study drug will be administered in the LTFU.
At their discretion, investigators may choose the appropriate drug for subjects assessed as 
requiring antitrypanosomal treatment during the course of LTFU.  This treatment is not 
considered as study drug. 
8.
Non-study therapy
8.1
Prior and concomitant therapy
Information on prior and concomitant medications used during the study is useful to support 
PK evaluations as planned in this study in order to evaluate/exclude potential drug-drug 
interactions (part 1).
All prior and concomitant medications administered from the time the informed 
consent/assent is signed until the last follow-up visit will be recorded in the subject’s source 
documentation file and reported in the eCRF.  Information on concomitant medication use 
will also be recorded on the Phone Contact Form which will be completed during the 
telephone assessments at Visits 4, 5, and 7 in part 1 of the study and at FU Visits 2 and 4 in 
                                                
113 Text added for clarity re: diaries (Amendment 1)
114 Sentence per Modification 12 (Amendment 1)


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part 2 of the study (see Section 16.1).115 Antitrypanosomal treatment required during the 
course of LTFU at the discretion of the investigator is considered as concomitant medication.  
The following information will be entered in the eCRF: generic name of the medication, 
indication, dose, unit, frequency, route of administration, and start and stop date (if stopped 
before the end of study).
The ingestion of alcohol may increase the incidence and severity of undesirable effects of 
nifurtimox.  Therefore, chronic abuse of alcohol and intake of alcohol or other 
alcohol-containing preparations during the study period are prohibited (applicable to part 1 
only).
8.2
Post-study therapy
Study medication will not be continued after the study.  Subsequent treatment should be 
discussed between the subject and/or the subject’s legally authorized representative and the 
investigator during the final visit.  At the end of this study, it is expected that the subject will 
receive standard treatment according to local clinical guidance.
9.
Procedures and variables
9.1
Tabular schedule of evaluations
The schedule of procedures/assessments to be performed during part 1 (CHICO) of the study 
is presented in Table 9–1.116
The schedule of procedures/assessments to be performed during part 2 (CHICO SECURE) of 
the study is presented in Table 9–2.
                                                
115 Text modified for clarity, and sentence regarding concomitant medication assessment per Phone Contact Form added per 
Modifications 6 and 12 (Amendment 1)
116 Table revised to include a telephone assessment at Visit 4, and an assessment (Visit 5) prior to the Day 30 Visit as 
recommended (Modifications 6 and 8); wording of assessments expanded for clarity; diary dispense and collection added; 
coagulation added to serum laboratory assessments, and neurological assessment added per Modification 10; concomitant 
medications moved to appear earlier in table.  Table footnotes revised and re-lettered according to changes made in text per 
Amendment 1 (Amendment 1). In addition, replaced IFA with IHA per Amendment 6.


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Table 9–1:  Schedule of procedures and assessments in part 1 – Study 16027
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
Initiation procedures
Informed consent/assent

Demographics (sex, age)

Medical/surgical history

Inclusion/exclusion criteria


Efficacy
Assessment of Chagas’ disease signs and symptoms d







Concentration test for T. cruzi (subjects < 8 months of age)





Conventional and non-conventional serologic testing for 
Chagas’ diseasee






qPCRe






Indirect hemagglutination assay (IHA) *


Safety
Adverse events











Concomitant medication











Physical examination f







Neurological examination g




Vital signs (heart rate, RR, BP, temperature), height/length, 
and weight h








12-lead ECG  i








Serum hematology, chemistry, and coagulation j
k




Urinalysis (if specimen can be obtained)
k




Urine pregnancy test  l






Pharmacokinetics
PK blood sampling (optional) m




Study Treatment
Study drug administration n


Study drug and diary dispensed o

p
Study drug and diary collected

p

Study drug accountability/review compliance






Dispense pre-paid phone card q


Collect pre-paid phone card




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Table 9–1:  Schedule of procedures and assessments in part 1 – Study 16027
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
BP = blood pressure, ECG = electrocardiogram, EOT = end-of-treatment, IHA = indirect hemagglutination assay, qPCR = quantitative polymerase chain reaction, PK = 
pharmacokinetic, RR = respiratory rate per minute.
a  At Days 14, 21, and 42, study site personnel will contact the subject/legally authorized representative via telephone to assess for the occurrence of adverse events, use of 
concomitant medications, and compliance with study drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and compliance information will be 
provided to all sites (see Section 16.1).  If any safety concern arises, subjects may return to the study site for an Unscheduled Visit. 
b  Subjects who discontinue prematurely from study drug administration will continue to return to the investigational site for study assessments at Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7.  If subjects are unable/unwilling to do so, they must return to the investigational site 30 (±3) days after the last dose of 
study drug for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 5, and 7.  If the subject is unable/unwilling to return to the clinic for the 
EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of Visit 8 assessments.
c  All assessments are to be obtained pre-treatment on Day 1 except for post-dose PK blood sampling.
d To be performed during physical examinations; see Section 9.4.4
e Specimen taken for non-conventional ELISA, qPCR and stored (as per amendment 2)
f  A complete physical examination of body systems will be performed at the Screening Visit.  A brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will be performed at all subsequent designated time points.  Abnormal findings 
on physical examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).
g  A neurological examination including assessments of mental status and cognition, cranial nerves, motor function, deep tendon reflexes, sensation, and coordination and gait will be 
performed.  Abnormal findings on neurological examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see 
Section 9.6.1.1).
h Blood pressure is optional in subjects < 5 years of age at the discretion of the investigator.  Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.  
i  12-lead ECG is optional in subjects < 5 years of age at the discretion of the investigator.  At the 2 – 4 hour time point for PK blood sampling (i.e., at the time of maximum 
concentration), the ECG will be obtained to allow for PK/PD investigations. 
j  Fasting of subjects for serum laboratory assessments is dependent on the age of the subject at the discretion of the investigator.
k Test conducted by local laboratories (as per amendment 2)
l Urine pregnancy tests will be performed on all females of childbearing potential (i.e., all female subjects who have experienced menarche).  Any subject with a positive urine 
pregnancy test will have a serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be discontinued from the study and undergo all study assessments 
as described in the EOT Visit.
m   Blood samples for pharmacokinetic parameters will be obtained prior to administration of study drug, and at designated time points thereafter (see Table 9–1).  They are optional and 
require consent/assent of the subject/subject’s legally authorized representative(s).
n Subjects consenting to optional PK assessments must withhold taking the morning dose of study drug on the day of Visits 3, 6, and 8.  Subjects not consenting to optional PK 
assessments may take their study drug on the mornings of Visits 3, 6, and 8 as instructed.
o  Depending on the country, study site personnel may dispense condoms at the time that study drug is dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.


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Table 9–1:  Schedule of procedures and assessments in part 1 – Study 16027
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
p At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and study drug for the remaining 30 days of treatment will be dispensed.  
q  A pre-paid phone card will be provided at Visits 3 and 6 to facilitate contact between study site personnel and the subjects/subjects’ authorized representatives at Visits 4, 5, and 7.  
* IHA will be performed at Visit 1 and Visit 11 only, if adequate specimens are available


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Table 9–2:  Schedule of procedures and assessments in part 2 – Study 16027
FU Visit 1
Yeara 2
(± 6 weeks)
FU Visit 2 
Phone 
Follow-up
Year 2.5 
(± 6 weeks)
FU Visit 3
Yeara 3
(± 6 weeks )
FU Visit 4
Phone 
Follow-up
Year 3.5 
(± 6 weeks)
FU Visit 5
Yeara 4
(± 6 weeks)
Informed consent/assent

Inclusion/exclusion criteria

Concomitant medications





Recombinant ELISA, total purified antigen ELISA, non-
conventional ELISA



IHA



qPCR test

d

d

Physical examination



Clinical signs or symptoms of Chagas’ disease





Body weight and height/length



Vital signs (systolic blood pressure, diastolic blood pressure, 
pulse)



12-lead ECGc



Adverse eventsb





Inquiry of housing conditions
(exclusion criterion #3)





a The year of each visit will be relative to the end of treatment in part 1 (CHICO).
b Adverse events considered at least possibly related to nifurtimox and adverse events caused by protocol-required procedures. 
c In case of abnormalities suspicious for cardiac involvement, further diagnostic examinations will be performed.
d At the discretion of the investigator in case of suspected reinfection or reactivation of Chagas’ disease (unscheduled study visit).


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9.2
Visit description
9.2.1
Screening
9.2.1.1 Visit 1 – screening visit (Days -14 to -1)
No study-specific procedures will be performed before the subject/legally authorized 
representative signs the informed consent form, or a minor subject provides assent, including 
asking a potential subject to fast (if applicable, depending on the age of the subject at the 
discretion of the investigator) prior to the screening blood samples.  Informed consent/assent 
for optional PK blood sampling will also be obtained.  After the consents/assents have been 
obtained, the following assessments will be performed:

Collection of demographic data

Medical and surgical history, including date of menarche for female subjects, and 
method of contraception

Evaluation of inclusion and exclusion criteria

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects <8 months of age at randomization) and
conventional serologic testing for Chagas’ disease (subjects ≥8 months < 18 years of 
age) conducted in local laboratory (or acceptable documentation of positive Chagas’ 
disease within 3 months prior to screening and no prior anti-trypanocidal or anti-
parasitic treatment).  Specimens taken for conventional serology, non-conventional 
serology, IHA, and qPCR will be frozen and stored at -20oC prior to shipment to the 
central laboratory where they will be stored at -70oC.117,118

Assessment of pretreatment AEs  and concomitant medications

A complete physical examination of body systems.  Abnormal findings will be 
documented as medical history (see Section 9.6.1.1).

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as medical history (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age  at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.
                                                
117 Modified as per Amendment 2 and Amendment 5.
118 Replaced IFA with IHA per Amendment 6.


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
Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
excluded from study participation.119
9.2.2
Treatment phase
9.2.2.1 Visit 2 – baseline (treatment phase, Day 1)
Visit 2 will take place after all laboratory test results and assessments performed at the 
Screening Visit are available.  The following assessments will be performed:

Re-evaluation of inclusion and exclusion criteria 

Assessment of pretreatment AEs and concomitant medications  

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects <5 years of age at the discretion of the investigator).  

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
excluded from study participation.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9–1).

Administer first dose of study drug to all subjects

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9–1).  At the 2 – 4 hour time point of PK blood sampling (i.e., at 
the time of Cmax), an ECG will be obtained to allow for PK/PD investigations.120
Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3 and to complete the PK subject diary, according to the instructions 
provided on the PK subject diary .121; subjects not consenting to optional PK 
assessments may take their study drug on the day of Visit 3 as instructed.  The PK 
subject diary has been added in order for the PK subjects to record all foods they 
consume when taking each dose of study drug, along with the times they eat each 
meal.  Examples of these entries, based on foods typically consumed in South 
American countries, will be given to each subject at the PK study sites.122
                                                
119 Text added/deleted/modified for clarity and to reflect changes made per Amendment 1
120 Added as per amendment 2.
121 Text modified as per amendment 4
122 Text modified as per amendment 4


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
Subjects and/or their legally authorized representatives will be advised of the 
possibility of change in the subject’s urine color due to the ingredient riboflavin.  
Depending on the country, study site personnel may dispense condoms at the time that 
study drug is dispensed to either the subject or the subject’s parent/legal guardian, 
with an explanation of rationale and risks.123  
9.2.2.2 Visit 3 (Day 7 ± 1 days)

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease, and qPCR 

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator). 

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
discontinued from the study and undergo all study assessments as described in the 
EOT Visit.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9–1).
                                                
123 Text added/deleted/modified for clarity and to reflect changes made per Amendment 1


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
Administer study drug with food to subjects consenting to PK assessments (who 
withheld their morning dose of study drug) and instruct subject on completion of PK 
diary, according to the instructions provided on the PK subject diary.124  

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9–1).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Collect study drug and diary.  Additional diaries may be provided if necessary.

Drug accountability and review of diaries for study drug compliance

Dispense pre-paid phone card to facilitate contact between study site personnel and the 
subjects/subjects’ authorized representatives at Visit 4.125  
9.2.2.3 Visit 4 (Day 14 ± 1 days)
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary or PK subject diary126. A Phone Contact 
Form for the telephone contacts to obtain safety and compliance information will be provided 
to all sites (Section 16.1).  If any safety concern arises, subjects may return to the study site 
for an Unscheduled Visit.127
9.2.2.4 Visit 5 (Day 21 ± 3 days)
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary or PK subject diary.  A Phone Contact 
Form for the telephone contacts to obtain safety and compliance information will be provided 
to all sites (see Section 16.1).  If any safety concern arises, subjects may return to the study 
site for an Unscheduled Visit.
Subjects consenting to optional PK assessments will be reminded to withhold taking the 
morning dose of study drug on the day of Visit 6; subjects not consenting to optional PK 
assessments may take their study drug on the day of Visit 6 as instructed.128
9.2.2.5 Visit 6 (Day 30 ± 3 days)

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease and qPCR 
                                                
124 Text modified as per Amendment 4
125 Text added/deleted/modified for clarity and to reflect changes made per Amendment 1
126 Text modified as per amendment 4
127 Text modified for clarity and per Modifications 6 and 8 (Amendment 1)
128 Text modified for clarity and per Modifications 6 and 8 (Amendment 1)


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
Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, the 
skin for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator)

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
discontinued from the study and undergo all study assessments as described in the 
EOT Visit.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9–1).

Administer study drug with food to subjects consenting to PK assessments (who 
withheld their morning dose of study drug) and instruct subject on completion of PK 
diary, according to the instructions provided on the PK subject diary.129  

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9–1).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Collect and dispense study drug and diary.  At this visit, subjects will return all 
remaining study drug and empty packaging, and study drug for the remaining 30 days 
of treatment will be dispensed.  

Drug accountability and review of diaries for study drug compliance

Collect and dispense pre-paid phone card to facilitate contact between study site 
personnel and the subjects/subjects’ authorized representatives at Visit 7.130  
                                                
129 Text modified as per amendment 4
130 Visit 6 assessments added per changes made per Amendment 1


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9.2.2.6 Visit 7 (Day 42 ± 3 days)
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary.131 A Phone Contact Form for the 
telephone contacts to obtain safety and compliance information will be provided to all sites
(see Section 16.1).  If any safety concern arises, subjects may return to the study site for an 
Unscheduled Visit.
Subjects consenting to optional PK assessments will be reminded to withhold taking the 
morning dose of study drug on the day of Visit 8; subjects not consenting to optional PK 
assessments may take their study drug on the day of Visit 8 as instructed.132
9.2.2.7 Visit 8 (Day 60 ± 3 days)/End of Treatment Visit

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and 
non-conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Neurological examination (see Section 9.6.3.3). Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator. 

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
                                                
131 Text modified as per amendment 4
132 Text modified/added for clarity and per Modification 6 (Amendment 1)


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discontinued from the study and undergo all study assessments as described in the 
EOT Visit.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9–1).

Administer study drug with food to subjects consenting to PK assessments (who 
withheld their morning dose of study drug), and instruct subject on completion of PK 
diary, according to the instructions provided on the PK subject diary.133

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9–1).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained.

Collect study drug, diary, and pre-paid phone card

Drug accountability and review of diaries for study drug compliance and for PK 
subjects, documentation of meals during study drug administration.134,135
9.2.3
Follow-up phase
9.2.3.1 Visit 9 (Day 90 ± 7)

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age)

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1). 

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.
                                                
133 Text modified as per amendment 4
134 Text modified as per amendment 4
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
Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results.  See Section 9.6.2.136
9.2.3.2 Visit 10 (Day 240 ± 7)

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator)137

Conventional and non-conventional serologic testing for Chagas’ disease and
qPCR138

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1). 

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight139
9.2.3.3 Visit 11 (Day 420 ± 7)

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator)140

Conventional and non-conventional serologic testing and IHA for Chagas’ disease and 
qPCR 141,142

Assessment of AEs

Concomitant medications
                                                
136 Text added/deleted/modified for clarity and to reflect changes made per Amendment 1
137 Modified as per amendment 2.
138 Modified as per amendment 2.
139 Text added/deleted/modified for clarity and to reflect changes made per Amendment 1
140 Modified as per amendment 2
141 IFA test added per amendment 5.
142 Replaced IFA with IHA per Amendment 6.


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
Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1). 

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight143
9.2.4
Long-Term Follow-up phase
Subjects will have an annual visit for 3 years (see Table 9–2).
Female subjects who give birth by the end of part 2 of the study will be invited to test their 
child for the absence or presence of infection with T. cruzi.  The congenital infection will be 
confirmed by parasitological method in children ≤8 months of age and by serological method 
in children >8 months of age.  Refusal to test the child will not influence the subject’s 
enrollment and participation in the LTFU.
9.2.4.1 Eligibility procedures (FU Visit 1)
After the consent/assent has been obtained, eligibility assessments will be performed at the 
first visit.  The first visit should be performed as soon as possible after availability of 
regulatory and ethics committee approval for all subjects who have already completed part 1 
of the study for 1 year 6 weeks or longer.  Subjects who completed part 1 of the study 10.5 
months or less prior to the start of the LTFU will be informed about part 2 of the study and 
will have their eligibility assessments according to the visit schedule provided that 
consents/assents have been obtained.
The subject’s update on medical history and concomitant medication will be recorded.  
9.2.4.2 Annual study visits (FU Visits 1, 3, and 5)
At each investigational site visit, blood samples for serology tests (recombinant ELISA, total 
purified antigen ELISA, IHA, and non-conventional ELISA) and for detection of T. cruzi’s
DNA using qPCR will be collected.  Blood samples will be taken under fasting conditions, if 
applicable, depending on the age of the subject, at the discretion of the investigator. 
Subjects will have a physical examination, including measurement of weight and 
height/length, measurement of vital signs and assessment of signs and symptoms of Chagas’ 
disease.  A 12-lead ECG will be obtained at each annual visit.  AEs considered at least 
possibly related to nifurtimox and AEs caused by protocol-required procedures will be 
collected and any concomitant medications since their last visit will be recorded.  The subjects 
will be asked about their housing conditions.  Females of childbearing potential will be asked 
if the subject is pregnant or delivered a baby.  In case of pregnancy, the pregnancy will be 
followed as described in Section 9.6.2. 
                                                
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9.2.4.3 Interim phone contact visits (FU Visits 2 and 4)
As shown in Table 9–2, between annual study visits, all subjects will be contacted to assess 
clinical signs and symptoms of Chagas’ disease or any exclusion criteria and will be asked
about their housing conditions.  Any update in concomitant medications, and any protocol-
specified AEs experienced since the last study visit will be recorded.  Subjects may be asked 
to return prior to their next annual visit to the investigational site, if, in the opinion of the 
investigator, it is clinically necessary.
In addition, subjects will receive a reminder contact approximately 2 weeks prior to their 
scheduled next annual visit at the discretion of the investigator. 
9.2.4.4 Unscheduled study visits
If in the opinion of the investigator, the subject needs to be evaluated clinically between the 
annual visits, the subject will be asked to return for an unscheduled study visit.  At this visit, 
subjects will have a physical examination, including clinical signs and symptoms of Chagas’ 
disease.  Blood will be taken for detection of T. cruzi’s DNA using qPCR, if deemed 
necessary based on the judgement of the investigator.
Subjects in whom reinfection or reactivation of Chagas’ disease is confirmed and, in the 
opinion of the investigator, require antitrypanosomal treatment will continue to be followed 
up. 
9.3
Population characteristics
9.3.1
Demographic
At screening, each subject’s date of birth144, age, and sex will be recorded.
9.3.2
Medical history
At screening, medical history findings (i.e., previous diagnoses, diseases or surgeries) meeting 
all criteria listed below will be collected as available to the investigator:145

Not pertaining to the study indication 

Start before signing of the informed consent 

Considered relevant for the subject’s study eligibility
Detailed instructions on the differentiation between (i) medical history and (ii) AEs can be 
found in Section 9.6.1.1.
                                                
144 Text modified since full date of birth will be collected (rather than year of birth) (Amendment 1)
145 Text modified for clarity (Amendment 1)


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9.4
Efficacy 
Part 1 (CHICO)
Efficacy assessments will be obtained at designated study visits (see Table 9–1).  The primary 
efficacy variable will be sero-reduction or sero-conversion at 12 months post-treatment using 
two conventional ELISA serology tests as the measure of efficacy.
A quality control program will be implemented for all tests according to the laboratory 
QC/QA department SOPs.146  
Other efficacy variables include disease state determined by qPCR, concentration tests for
T. cruzi (subjects < 8 months of age at randomization), conventional and non-conventional 
serologic testing, and clinical signs/symptoms of Chagas’ disease.147
The complete list of variables to be analyzed for this study will be provided in the statistical 
analysis plan (SAP).
Part 2 (CHICO SECURE)
Efficacy assessments will be obtained at designated study visits (see Table 9–2).  The primary 
efficacy variable will be seronegative conversion as confirmed by two types of assay using 
recombinant ELISA and IHA, in subjects who were randomized and received at least one 
dose of the 60-day nifurtimox treatment regimen.
Other efficacy variables include:

Incidence of seronegative conversion, as confirmed by two types of assays, 
recombinant ELISA and IHA, in subjects who were randomized and received at least 
one dose of the 30-day nifurtimox treatment regimen.

Proportion of responders who show seronegative conversion and the incidence of 
seronegative conversion as confirmed by non-conventional ELISA in subjects who 
were randomized and received at least one dose of either 60- or 30-day nifurtimox 
treatment regimens.

Proportion of responders who show both seronegative conversion as confirmed by two 
types of assay (recombinant ELISA and IHA) and no evidence of established 
cardiomyopathy as evaluated in ECG recordings.

Serial reduction of OD measured by recombinant ELISA and total purified antigen 
ELISA, in subjects who were randomized and received at least one dose of either the 
60- or 30-day nifurtimox treatment regimen compared to Visit 1 in part 1of the study.
In the LTFU period, all assessments will follow the same procedures as in part 1 (CHICO) of 
the study.  The same quality control program will also be implemented for all tests according 
to the laboratory QC/QA department SOPs. 
                                                
146 Modified as per amendment 2
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The complete list of variables for LTFU to be analyzed will be provided in the statistical 
analysis plan (SAP).
9.4.1
Polymerase chain reaction test
A qPCR test will be performed at the Screening Visit, Visits 3, 6, and 8, and Follow-up Visits 
10 and 11 in part 1 of the study and at FU Visits 1, 3, and 5 in part 2 of the study.  Additional 
qPCR test may be required for unscheduled visits, if applicable.
Details describing a suggested collection, processing, storage and shipment for qPCR blood 
samples, and a suggested qPCR test protocol, are described in Section 1.  The study central 
laboratory procedures and qPCR protocol will be followed.148
9.4.2
Serological tests
At screening, subjects must have the following serological tests to confirm a diagnosis of 
Chagas’ disease:

Subjects < 8 months of age at randomization must demonstrate direct observation of 
T. cruzi by concentration test149

Subjects ≥8 months to < 18 years of age at randomization must demonstrate a positive 
conventional ELISA result for both of the following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA150   
Additionally, a non-conventional ELISA test and IHA will be obtained; however, a positive 
result will not be required for diagnosis151,152.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ disease 
within 3 months prior to screening and they have had no prior anti-trypanocidal or anti-
parasitic treatment.153
After randomization, conventional and non-conventional serological tests will be performed at 
Visits 3, 6, and 8 (EOT), and Visits 10 and 11 (Follow-up).154
All subjects ≥8 months to < 18 years of age at randomization will have two baseline 
specimens collected for serologic testing.  One sample will be used to determine the initial 
diagnosis of Chagas’ disease while the other sample will be frozen and retained to be assayed 
later as an internal control.  Subjects < 8 months of age at randomization will have one 
specimen obtained, which will be frozen and retained.  Total blood volume to be taken for the 
                                                
148 Modified as per amendment 2
149 Text modified for clarity (Amendment 1)
150 Text modified for clarity and per Modification 2 (Amendment 1)
151 IFA test added per Amendment 5.
152 Replaced IFA with IHA per Amendment 6.
153 Text modified/added for clarity and per Modification 2 (Amendment 1)
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required three serological and PCR tests is 4.0-5.0 mL, depending on the subject’s age.  Care 
will be taken to minimize sample volumes by using a micro-sampling collection technique, 
when possible.155
Details describing the collection, processing, storage and shipment of blood samples, and the 
serologic test protocols, will be described in a separate serologic test manual from the central 
laboratory.
For part 2 of the study, conventional serological tests (recombinant ELISA and total purified 
antigen ELISA), IHA, and a non-conventional serological test (ELISA F-29) will be 
performed at FU Visits 1, 3, and 5.
9.4.3
Concentration tests for T. cruzi
Concentration tests for T. cruzi will be performed on all subjects < 8 months of age at 
randomization at the screening visit, and at Visits 3, 6, 8 and 9.
Details describing the collection, processing, storage and shipment of blood samples, and the 
concentration test protocol, will be described in a separate manual.  Concentration tests for
T. cruzi will be performed by a local laboratory according to the standard of care for each 
country.156
Concentration test for T. cruzi is not applicable for subjects in part 2 of the study.  However, 
concentration test for T. cruzi may be performed in children born of female study subjects to 
detect congenital infection (see Section 9.6.2).
9.4.4
Assessment of Chagas’ disease signs and symptoms
Assessments of the presence of Chagas’ disease signs and symptoms will be performed 
during physical examinations at Screening, Visits 3, 6, and 8 (EOT), and Visits 9, 10, and 11 
(follow-up) in part 1 and at FU Visits 1, 3, and 5 in part 2 of the study, and will be entered 
into the eCRF. Signs and symptoms of Chagas’ disease include but are not limited to the 
following:157
Acute Chagas’ disease:

Fever: usually prolonged 

malaise

lymphadenopathy

hepatomegaly

splenomegaly

subcutaneous edema (localized or generalized)
                                                
155 Text added per Modification 3 (Amendment 1)
156 Text modified for clarity and per Modification 2 (Amendment 1)
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
signs of portal of entry of T. cruzi:
o
through the skin - chagoma 
o
via the ocular mucous membranes - Romaña sign

hypotonicity

anemia

myocarditis

meningoencephalitis

pneumonitis

ECG abnormality, may include but not limited to:
o
sinus tachycardia
o
first-degree atrioventricular block
o
low QRS voltage
o
primary T-wave changes

Chest radiograph abnormality, include variable degrees of cardiomegaly
Chronic Chagas’ disease:
Digestive manifestations:

megaesophagus

megacolon
Cardiac manifestations:

abnormalities of the conduction system 

bradyarrhythmias and tachyarrhythmias

apical aneurysms

cardiac failure

thromboembolism (systemic or pulmonary)

sudden death

ECG abnormalities, e.g.:
o
right bundle branch block
o
left anterior fascicular block
o
ventricular premature beats
o
ST-T changes
o
abnormal Q waves
o
low voltage of QRS


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o
various degrees of AV block
o
sick sinus syndrome
o
low QRS voltage
o
others
In part 2, progression of clinical Chagas’ disease will be defined as:

presence of positive serological tests for T. cruzi

clinical (e.g. progressive dyspnea, fatigue, asthenia) and electrocardiographic 
alterations that are characteristic of chronic Chagas’ cardiomyopathy (e.g., new or 
worsening signs of cardiac conduction disorders, sustained ventricular tachycardia, 
etc.)

alterations that are characteristic of chronic Chagas’ gastrointestinal disease (e.g., 
megaesophagus, megacolon)
9.5
Pharmacokinetics/pharmacodynamics
If the subject/subject’s legally authorized representative(s) provide(s) consent/assent to do so, 
plasma concentrations of nifurtimox will be evaluated at Visit 2 (baseline) and Visits 3, 6, and 
8 (EOT) on at least 10 subjects per age stratum should be selected from all participating PK
centers.  Plasma nifurtimox concentrations will be determined using a sparse sampling 
approach.158  
At Visits 2, 3, 6, and 8, the subjects should present themselves to the investigational site 
without taking the morning dose of study medication.  Blood samples for PK will be obtained 
in-clinic prior to the administration of study drug with food and at specific time points and 
time windows as presented in Table 9–1.  At the 2 – 4 hour time point of PK blood sampling 
(i.e., at the time of Cmax), an ECG (optional for subjects < 5 years of age at the discretion of 
the investigator) will be obtained to allow for PK/PD investigations.159  All subjects are to 
complete the PK subject diary according to instructions provided..160
PK/PD investigation is not applicable in part 2 of the study.
                                                
158 Text modified for clarity and per Amendment 1
159 Text modified for clarity and per Modification 1 (Amendment 1)
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Table 9–3: Pharmacokinetic sampling time points and windows161
Visits  (Days)
Time point
Time window
2, 3, 6, 8  (1, 7, 30, 60)
Pre-dose (pre-treatment)
---
Post-dose
5-10 minutes post-dose
10-120 minutes post-dose
2-4 hours post-dose a
4-8 hours post-dose
a  An ECG will be obtained at this time point (optional in subjects < 5 years of age at the 
discretion of the investigator)
PK blood samples are to be obtained from a heel prick (subjects up to 1 month of age), finger 
stick, or ear lobe stick at the discretion of the investigator. For all subjects throughout the 
study, the selected specimen handling technique will be via microsampling in order to provide 
uniform analytical samples while maintaining total blood volumes within the limitations for 
safety. 
Samples of a blood volume of 2 x 75 = 150 μL at predefined time points should be taken 
according to Table 9–3. If samples cannot be taken due to other reasons (e.g., sampling 
deemed not feasible by the investigator due to safety) or if the samples are taken outside the 
defined time windows (e.g., > 8 hours post-dose), the subject will still remain valid for PK 
analysis. At least one sample within the specified timeframe should be taken during the 
course of the study. 162 That means that, at the end of the study, at least one sample from each 
time window will be available, e.g., if the 4-8 hour post-dose sample was not collected until 
the last visit, then a sample at this time window should in any case be drawn at that time 
point. This must be documented by the investigator.
The PK samples should be obtained within the planned sampling time windows unless 
important or unforeseen mandatory interventions coincide (e.g., for medical reasons).163  In 
these exceptional cases, the corresponding sample may be obtained outside the predefined 
windows without losing its validity for subsequent PK analysis.  This will not be considered 
as a protocol violation.  If a subject fails to provide the number of samples defined in the 
protocol, this will also not render him/her invalid for the subsequent PK analysis.
Details describing the procedures for obtaining and handling PK blood samples are provided 
in Section 16.5.
Analysis of PK samples
Nifurtimox concentrations in plasma will be measured by a validated assay using high 
performance liquid chromatography followed by tandem mass spectrometric detection.  
Quality control (QC) and calibration samples will be analyzed concurrently with study 
samples.  The results of QC samples will be reported together with concentrations in the study 
samples in the clinical study report.
                                                
161 Visit numbers changed to reflect changes made per Amendment 1; footnote a added per Modification 1 (Amendment 1)
162 Text modified for clarity (Amendment 1)
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The bioanalyst will remain unblinded for analysis of study samples.  Concentrations are 
calculated from the chromatographic raw data in accordance with current Bayer guidelines
[31].
Pharmacokinetic evaluation
Plasma concentration time courses will be analyzed and reported within the study report.  In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 
report.  Population PK methods will be used to provide parameter estimates describing the PK 
behavior of nifurtimox and to identify possible covariates with significant impact on 
pharmacokinetics (e.g., age or body weight).164,165
9.6
Safety
9.6.1
Adverse events
9.6.1.1 Definitions
Definition of adverse event (AE)
In a clinical study, an AE is any untoward medical occurrence (i.e., any unfavorable and 
unintended sign [including abnormal laboratory findings], symptom, or disease) in a patient or 
clinical investigation subject after providing written informed consent for participation in the 
study.  Therefore, an AE may or may not be temporally or causally associated with the use of 
a medicinal (investigational) product.
A surgical procedure that was planned prior to the start of the study by any physician treating 
the subject should not be recorded as an AE (however, the condition for which the surgery is 
required may be an AE).
In the following differentiation between medical history and AEs, the term “condition” may 
include abnormal physical examination findings, symptoms, diseases, laboratory, or ECG.

Conditions that started before signing of informed consent and for which no symptoms 
or treatment are present until signing of informed consent are recorded as medical 
history (e.g., seasonal allergy without acute complaints).

Conditions that started before signing of informed consent and for which symptoms or 
treatment are present after signing of informed consent, at unchanged intensity, are 
recorded as medical history (e.g., allergic pollinosis).

Conditions that started or deteriorated after signing of informed consent will be 
documented as AEs.  This includes intercurrent illnesses.
                                                
164 Text modified for clarity (Amendment 1)
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Definition of serious adverse event (SAE)
An SAE is classified as any untoward medical occurrence that, at any dose, meets any of the 
following criteria (a – f):
a.
Results in death
b. Is life-threatening
The term ‘life-threatening’ in the definition refers to an event in which the subject 
was at risk of death at the time of the event, it does not refer to an event which 
hypothetically might have caused death if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization
A hospitalization or prolongation of hospitalization will not be regarded as an SAE 
if at least one of the following exceptions is met:
The admission results in a hospital stay of less than 12 hours
The admission is pre-planned 
(e.g., elective or scheduled surgery arranged prior to the start of the study; 
admission is part of the study procedures as described in Section 9.2)
The admission is not associated with an AE 
(e.g., social hospitalization for purposes of respite care).
However, it should be noted that invasive treatment during any hospitalization may 
fulfill the criterion of ‘medically important’ and as such may be reportable as an 
SAE dependent on clinical judgment.  In addition, where local regulatory 
authorities specifically require a more stringent definition, the local regulation 
takes precedence.
d. Results in persistent or significant disability/incapacity
Disability means a substantial disruption of a person’s ability to conduct normal 
life’s functions.
e.
Is a congenital anomaly/birth defect
f.
Is another serious or important medical event as judged by the investigator
9.6.1.2 Classifications for adverse event assessment
In part 1 of the study, all AEs will be assessed and documented by the investigator according 
to the categories detailed below. 
In part 2 of the study, AEs considered at least possibly related to nifurtimox and AEs caused 
by protocol-required procedures will be assessed and documented by the investigator 
according to the categories detailed below.
9.6.1.2.1
Seriousness
For each AE, the seriousness must be determined according to the criteria given in 
Section 9.6.1.1.


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9.6.1.2.2
Intensity
The intensity of an AE is classified according to the following categories:
Mild:  a type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention.  The event does not generally interfere with 
usual activities of daily living.
Moderate:  a type of AE that is usually alleviated with additional specific 
therapeutic intervention.  The event interferes with usual activities of daily 
living, causing discomfort, but poses no significant or permanent risk of harm to 
the subject.
Severe:  a type of AE that requires intensive therapeutic intervention.  The event 
interrupts usual activities of daily living, or significantly affects clinical status.  
The event possesses a significant risk of harm to the subject and hospitalization 
may be required.166
9.6.1.2.3
Causal relationship 
The assessment of the causal relationship between an AE and the administration of treatment 
is a decision to be made by the investigator, who is a qualified physician, based on all 
information available at the time of the completion of the eCRF.
Causality should be assessed separately for each study treatment as detailed in the eCRF.  If 
the investigator feels that the event cannot be firmly attributed to one of the study treatments 
(e.g., owing to a suspected underlying interaction), the same assessment will be documented 
for each study treatment.
The assessment is based on the question whether there was a “reasonable causal relationship” 
to the study treatment in question.
Possible answers are “yes” or “no”
An assessment of “no” would include:
1.
The existence of a highly likely alternative explanation, e.g., mechanical bleeding at 
surgical site.
or
2.
Non-plausibility, e.g., the subject is struck by an automobile when there is no 
indication that the drug caused disorientation that may have caused the event; cancer 
developing a few days after the first drug administration.
An assessment of “yes” indicates that that the AE is reasonably associated with the use of the 
study treatment.
Important factors to be considered in assessing the relationship of the AE to study treatment 
include:
                                                
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
The temporal sequence from drug administration:  The event should occur after the 
drug is given.  The length of time from drug exposure to event should be evaluated in 
the clinical context of the event.

Recovery on drug discontinuation (de-challenge), recurrence on drug re-introduction 
(re-challenge): Subject’s response after de-challenge or re-challenge should be 
considered in view of the usual clinical course of the event in question.

Underlying, concomitant, intercurrent diseases: 
Each event should be evaluated in the context of the natural history and course of the 
disease being treated and any other disease the subject may have.

Concomitant medication or treatment: 
The other drugs the subject is taking or the treatment the subject receives should be 
examined to determine whether any of them might have caused the event in question.

Known response pattern for this class of drug: Clinical/preclinical

Exposure to physical and/or mental stresses: The exposure to stress might induce 
adverse changes in the recipient and provide a logical and better explanation for the 
event

The pharmacology and PK of the study treatment: 
The PK properties (absorption, distribution, metabolism and excretion) of the study 
treatment, coupled with the individual subject’s PD should be considered.

The assessment is not possible
9.6.1.2.4
Action taken with study treatment 
Any action on study treatment to resolve the AE is to be documented using the categories 
listed below.
The study treatment action should be recorded separately for each study treatment as detailed 
in the eCRF.

Drug withdrawn

Drug interrupted

Dose reduced

Dose not changed

Dose increased

Not applicable

Unknown
9.6.1.2.5
Other specific treatment(s) of adverse events

None

Remedial drug therapy


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
Other
9.6.1.2.6
Outcome
The outcome of the AE is to be documented as follows: 

Recovered/resolved

Recovering/resolving

Recovered/resolved with sequelae

Not recovered/not resolved

Fatal

Unknown
9.6.1.3 Assessments and documentation of adverse events
Once informed consent/assent is obtained during part 1, all AEs occurring through the end of 
the part 1 follow-up phase will be recorded on the applicable eCRF pages. After the part 1 
follow-up phase through the end of the part 2 follow-up phase, only AEs considered at least 
possibly related to nifurtimox and those caused by protocol-related procedures should be 
recorded. 
No study drug will be administered during part 2. For subjects requiring further treatment 
with antitrypanosomal drugs, including retreatment with nifurtimox during part 2, the 
applicable national reporting system should be followed for any spontaneous non-serious and 
serious adverse reaction considered to be not related to the study medication administered in 
part 1 (nifurtimox and/or placebo).
After the end of the part 2 follow-up phase, there is no requirement to actively collect AEs, 
including those having an outcome of “death.” The type of information that should be 
assessed and recorded by the investigator for each AE is listed in Section 9.6.1.2. 
“Death” should not be recorded as an AE on the AE page.  Instead, “death” is the outcome of 
underlying AE(s).
For all SAEs, the sponsor has to carry out a separate assessment for expectedness, 
seriousness, and causal relationship to study drug.
9.6.1.4 Reporting of serious adverse events
The definition of SAEs is given in Section 9.6.1.1.  Each SAE must be followed up until 
resolution or stabilization by submission of updated reports to the designated recipient in part 
1 of the study.  In part 2, only SAEs considered at least possibly related to nifurtimox or 
caused by protocol-related procedures must be reported.  These events must be followed up 
until resolution or stabilization by the submission of updated reports to the designated 
recipient and in accordance with local regulations.


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Investigator’s notification of the sponsor
All investigators will be thoroughly instructed and trained on all relevant aspects of the 
investigator’s reporting obligations for SAEs.  This information, including all relevant contact 
details, is summarized in the investigator site file.  This information will be updated as 
needed.
The investigator must report immediately (within 24 hours of the investigator’s awareness) all 
SAEs occurring during the observation period defined in Section 9.6.1.3 to the recipient 
detailed in the instructions for SAE reporting included in the Investigator File.  For this, an 
AE page in the eCRF as well as the complementary pages provided in the Investigator File 
must be completed for each SAE. 
SAEs occurring after the protocol-defined observation period will be processed by the 
sponsor according to all applicable regulations.
Notification of the IECs/IRBs
Notification of the International Ethics Committees (IECs)/Institutional Review Boards 
(IRBs) about all relevant events (e.g., SAEs, SUSARs) will be performed by the sponsor 
and/or by the investigator according to all applicable regulations and IEC/IRB procedures.
Notification of the authorities
The processing and reporting of all relevant events (e.g., SAEs, SUSARs) to the authorities 
will be done by the sponsor according to all applicable regulations.
Sponsor’s notification of the investigational site
The sponsor will inform all investigational sites about reported relevant events (e.g., 
SUSARs) according to all applicable regulations.
9.6.1.5 Expected adverse events
For this study, the applicable reference document is the most current Investigators’ Brochure
for nifurtimox.167
If relevant new safety information is identified, the information will distributed to all 
participating sites.
The expectedness of AEs will be determined by the sponsor according to the applicable 
reference document and according to all local regulations.
9.6.1.6 Adverse events of special safety interest
In this study, all AEs of weight loss of > 20%, severe rash, and severe polyneuropathy will be 
considered AEs of special safety interest.168 Adverse events of special safety interest will be 
assessed, monitored, and reported as described in Section 9.6.1.4.
                                                
167 Revised since Investigators’ Brochure became available to serve as the reference document for nifurtimox 
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9.6.2
Pregnancies
Part 1 (CHICO)
Any subject who becomes pregnant during the course of the study will discontinue study 
treatment.  Pregnancy must be confirmed by serum pregnancy test.  The investigator must 
report to the sponsor any pregnancy occurring in a female study subject during her 
participation in this study.  The outcome of the pregnancy should be followed up carefully, 
and any outcome of the mother and the child at delivery should be reported.
For a pregnancy in the partner of a male study subject, all efforts will be made to obtain 
similar information on course and outcome, subject to the partner’s consent and any abnormal 
outcome of the child should be reported. 
For all reports, the forms provided are to be used.  The investigator should submit them within 
the same timelines as an SAE. 
Part 2 (CHICO SECURE)
At each visit in part 2, female subjects will be asked if she is pregnant or gave birth to a baby 
after the first dose of nifurtimox treatment regimen or since the last visit.
The investigator must report to the sponsor any pregnancy occurring in a female study subject 
during her participation in this study.  The outcome of the pregnancy should be followed up 
carefully, and any outcome of the mother and the child at delivery should be reported. 
Female subjects who give birth by the end of part 2 of the study will be invited to test their 
child for the absence or presence of infection with T. cruzi, except if the child was already 
tested according to the standards of the country or has already received treatment for Chagas’ 
disease.  The congenital infection will be confirmed by parasitological method in children 
≤8 months of age and by serological method in children >8 months of age.  Efforts should be 
made to obtain the results of congenital infection testing when already done according to the 
standards of the country.  The result of congenital infection will be documented in the 
applicable eCRF form.
9.6.3
Further safety
9.6.3.1 Laboratory assessments
Safety laboratory assessments will be performed at screening, Visits 3, 6, 8 (EOT), and 9
(follow-up). Fasting is dependent on the age of the subject at the discretion of the investigator.  
If any abnormal laboratory values occur at the EOT Visit (Visit 8), these determinations will 
be followed until normal or stable.169
Laboratory parameters will be analyzed by a local laboratory according to the schedule 
specified in Table 9–4:  .
                                                                                                                                                        
168 Sentence corrected as per amendment 3
169 Heading 9.6.3.1 added; text modified for clarity and to reflect changes made per Amendment 1.


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Table 9–4:  Description of laboratory parameters (safety)
Hematology
Hemoglobin, hematocrit, RBC count, WBC count and 
differential (including lymphocytes, monocytes, eosinophils, 
basophils, neutrophils), MCV, MCH, MCHC, platelets
Blood chemistry
Albumin, total protein, BUN, creatinine, glucose, total bilirubin, 
ALP, AST, ALT, uric acid
Coagulation
INR, PT, PTT
Urinalysis (urine dipstick) a
RBCs, WBCs, pH, specific gravity, glucose, protein, bilirubin, 
urobilinogen, nitrite, ketones
Urine pregnancy test b
Urine β-hCG
ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, 
β−hCG = beta-human chorionic gonadotropin, BUN = blood urea nitrogen, INR = international 
normalized ratio, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, 
MCHC = mean corpuscular hemoglobin concentration, PT = prothrombin time, PTT = partial 
thromboplastin time, RBC = red blood cell, WBC = white blood cell.
a  If abnormalities are identified by the dipstick, the samples will be sent for confirmatory testing170.  
Cell/sediment microscopy should be performed only if positive dipstick results are obtained for 
WBC, RBC/hemoglobin, or protein.
b  To be performed at Visits 1, 2, 3, 6, 8, and 9  on females of childbearing potential (i.e., female 
subjects who have experienced menarche).  If any urine pregnancy test is positive, a serum 
pregnancy test will be performed to confirm the result.171
Safety laboratory assessments are not applicable in part 2 of the study.
9.6.3.2 Total volume of blood
In order to minimize the burden of blood loss due to PK sampling, age-appropriate sparse 
sampling strategies combined with (micro-)bioanalysis techniques will be applied.  The total 
volume of blood obtained at each visit (for serology, qPCR, safety, and PK specimens) will be 
a maximum of approximately 8.1 mL 15.6 mL depending on the child’s age and weight as 
per standards of Clinical Laboratory and Standards Institute; see Section 16.6 and Table 9–5
below.172
Table 9–5:  Total volume of blood by age
Age
Weight
Circulating blood 
volume
Maximum allowable every 
two weeks
Pre-term neonate
500 grams
40 mL
1.5 mL
Full-term neonate
4 kilograms
320 mL
12 mL
Infant (3 years)
15 kilograms
1.2 L
45 mL
Child (12 years)
40 kilograms
3.2 L
120 mL
                                                
170 Modified as per amendment 2
171 Text modified to reflect changes made per Amendment 1
172 Text modified for clarity and to expand the maximum amount of blood required (Amendment 1)


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9.6.3.3 Physical examination
Part 1 (CHICO)
Physical examinations will be performed by a medically qualified person.  A complete 
physical examination of body systems will be performed at the Screening Visit.  A brief 
physical examination, including assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will 
be performed at all subsequent designated time points (see Table 9–1). Abnormal findings on 
physical examination at Screening will be documented as medical history; abnormal findings 
thereafter will be documented as AEs (see Section 9.6.1.1).173
A neurological examination will be performed at Screening, and Visits 3, 6, and 8 (EOT). 
The examination will include assessments of mental status and cognition, cranial nerves, 
motor function, deep tendon reflexes, sensation, and coordination and gait.  Abnormal 
findings on neurological examination at Screening will be documented as medical history; 
abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).174
Assessments of Chagas’ disease signs and symptoms will be performed at the time of physical 
examinations (see Section 9.4.4).175
Part 2 (CHICO SECURE)
Physical examinations will be performed by a medically qualified person.  A complete 
physical examination of body systems will be performed at FU Visit 1, 3, and 5 (see Table 9–
2).  Abnormal findings on physical examination before signing informed consent will be 
documented as medical history; abnormal findings thereafter will be documented as AEs (see 
Section 9.6.1.2).  Assessments of Chagas’ disease signs and symptoms will be performed at 
the time of physical examinations.
9.6.3.4 Vitals signs, height/length, and weight
In part 1 of the study, vital signs (systolic and diastolic blood pressure [optional in subjects < 
5 years of age at the discretion of the investigator], heart rate, respiratory rate, and oral or 
rectal body temperature) will be measured at screening and Visits 2, 3, 6, 8 (EOT), 9, 10, and 
11.  After a 5-minute rest in a sitting position, vital signs will be measured using non-invasive 
equipment.  In part 2 of the study, vital signs (systolic blood pressure, diastolic blood 
pressure, pulse) will be performed at FU Visits 1, 3, and 5.
Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.176  
                                                
173 Text modified/added for clarity (Amendment 1)
174 Paragraph added per Modification 10 (Amendment 1)
175 Text added for clarity (Amendment 1)
176 Text modified to reflect changes made per Amendment 1; last sentence moved from previous section (9.6.3.3) to this 
section (Amendment 1)


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9.6.3.5 12-lead electrocardiogram
A resting 12-lead ECG will be performed at screening and Visits 2, 3, 6, 8 (EOT), and 9, 10, 
11 (Follow-up) on all subjects ≥ 5 years of age, and at the discretion of the investigator on all 
subjects < 5 years of age.  For subjects consenting to PK assessments, an ECG will be 
obtained at the 2 – 4 hour time point of PK blood sampling (i.e., at the time of Cmax), to allow 
for PK/PD investigations. 
The ECG will be performed at the site according to procedure, and with the subject in the 
supine position after he/she has rested for 10 minutes.177
A resting 12-lead ECG will be performed at FU Visits 1, 3, and 5 in part 2 of the study on all 
subjects.  The ECG will be performed at the site according to standard procedures, and with 
the subject in the supine position after he/she has rested for at least 10 minutes.
ECG recordings will be evaluated by the investigator and a cardiologist associated with the 
site.  In case of ECG abnormalities suspicious for cardiac involvement of Chagas’ disease, 
further diagnostic examinations may be required at the discretion of the investigator, (e.g.,
echocardiography, chest X-ray, 24-hour ECG). 
9.6.3.6 Urine pregnancy test 
A urine pregnancy test on all female subjects of childbearing potential (i.e., all females who 
have experienced menarche) will be performed at screening and Visits 2, 3, 6, 8 (EOT), and 9
(Follow-up).  If any urine pregnancy test is positive, a serum pregnancy test will be performed 
to confirm the result. If pregnancy is confirmed at Visits 1 or 2, the subject is to be excluded 
from study participation.  If pregnancy is confirmed at Visits 3 or 6, the subject will be 
discontinued from the study (See Section 9.6.2) and undergo all study assessments as 
described for the EOT Visit (Section 9.2.2.7).178
There will be no urine pregnancy test in part 2 of the study.
9.7
Other procedures and variables
In part 1 of the study, no other procedures will be performed, and no other variables will be 
measured. 
In part 2 of the study, the housing conditions, especially with regard to active or effective 
vector-control to T. cruzi reinfection as determined by Ministry of Health guidelines in each 
country and the judgement of the investigator, will be inquired at FU Visits 1, 2, 3, 4 and 5.
9.8
Appropriateness of procedures/measurements
The efficacy and safety measurements scheduled for this study are widely used and generally 
accepted as reliable, accurate, and relevant. 
The safety variables evaluated in this study are routine clinical parameters that allow risk 
assessment of the investigational drug.
                                                
177 Text modified for clarity and per Modifications 1 and 9 (Amendment 1)
178 Text modified/added for clarity and to reflect changes made per Amendment 1 


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10.
Statistical methods and determination of sample size
10.1
General considerations
This study has been designed to focus on the efficacy and safety of treatment with nifurtimox 
in children diagnosed with Chagas’ disease.  In this study, approximately 300 pediatric 
subjects will be randomized (2:1 randomization, 60-day regimen vs. 30-day regimen).  
Subjects will be stratified by age into four strata.  A minimum of 38 subjects in each age 
stratum is targeted, but not required, in order to be able to derive meaningful safety 
conclusions.  Enrollment will continue until this specification is met, unless it is determined 
that such a target would be unlikely to be reached in a reasonable time.179
Unless otherwise specified, all significance tests will be conducted using a 2-sided alpha level 
of 0.05, and CIs will be 2-sided 95% intervals.  All variables collected in this study will be 
summarized with descriptive statistics at each assessment time.  Subject disposition and 
demographic and baseline characteristics will be summarized descriptively for each treatment 
group.  For continuous variables, descriptive statistics will include means, standard 
deviations, medians, minimums, and maximums.  For categorical variables, frequency counts 
and percentages will be provided.  These will be calculated for each age stratum by dose, as 
well as the overall set of study subjects.
No imputations will be made for missing values occurring in the safety and background 
variables.  Treatment of missing values for efficacy variables is addressed later in this 
section.180 Additional analyses may be specified in the SAP if missing data patterns observed 
during blinded review of the data suggest any possible bias. 
Statistical analysis will be performed using Statistical Analysis System (SAS); the version 
used will be specified in the SAP.
10.2
Analysis sets
Part 1 (CHICO)
The primary efficacy analysis will be done using the full analysis set (FAS), which is the set 
of subjects who received at least one dose of study drug.  Analyses will also be done using the 
per-protocol set, which is comprised of subjects treated with study drug who have no major 
protocol deviations.  Complete specifications will be provided in the SAP.
Analyses of safety and background data will be performed on the FAS.
Part 2 (CHICO SECURE)
The primary efficacy analysis will be done using the full analysis set (FAS) as defined in part 
1.  Subjects will be analyzed as randomized in part 1 (CHICO) of the study.  Complete 
specifications will be provided in the SAP.
                                                
179 Modified as per amendment 2
180 Text modified/added for clarity (Amendment 1)


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Analyses of safety and background data will be performed on the FAS.  For safety analyses, 
subjects will be analyzed as treated in part 1 of the study.
10.3
Variables and planned statistical analyses
10.3.1
Variables
10.3.1.1Part 1 (CHICO)
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months 
post-treatment using two conventional ELISA serology tests as the measure of efficacy.  This 
sero-reduction or sero-conversion is considered cure, and the primary variable is binary (cure, 
no cure).  
In the event of discordancy between the two conventional ELISA test results, the following 
will be considered: 

For sero-reduction, the average percentage of OD reductions will be used (e.g., Test 
#1 = 15% and Test #2 = 25%; average = 20% and, hence, cure). 

For sero-conversion, only when both test results are negative the subject is considered 
as negative (cure).181
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease, 
concentration test for T. cruzi (subjects < 8 months of age at randomization), conventional and 
non-conventional serologic testing, and disease state determined by qPCR. 
Background variables include demographics (sex, age), height/length, weight, 
medical/surgical history, and medication history.
Safety variables include AEs, physical examination abnormalities, vital signs, ECG 
abnormalities, hematology and blood chemistry, coagulation, and urinalysis.182
10.3.1.2Part 2 (CHICO SECURE)
Efficacy assessments will be obtained at designated study visits (see Table 9–2). 
Primary efficacy variable 
The primary efficacy variable will be the incidence rate of seronegative conversion measured 
and confirmed by two types of assay (recombinant ELISA and IHA) in subjects who were 
randomized and received at least one dose of the 60-day nifurtimox treatment regimen.  Both 
test results should be negative for the subject to be considered as seroconverted.  Incidence 
rate is the number of new cases of seronegative conversion over the study period (i.e., 4 years 
after end of nifurtimox treatment) divided by the person-time at risk. [33]
Secondary and other exploratory efficacy variables
Secondary efficacy variables include:
                                                
181 Modified as per amendment 2
182 Text modified/added for clarity and to reflect changes per Amendment 1


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
Incidence rate of seronegative conversion measured and confirmed by two types of 
assay (recombinant ELISA and IHA) in subjects who were randomized and received 
at least one dose of the 30-day nifurtimox treatment regimen

Proportion of responders who show both seronegative conversion by two types of 
assays and no evidence of established cardiomyopathy as measured by ECG

Serial reduction of OD measured by recombinant ELISA and total purified antigen 
ELISA
Other exploratory efficacy variables include:

Incidence rate of seroconversion measured and confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects by age categories (≤2 years, >2 years to ≤6 
years, >6 to ≤12 years, >12 to <18 years; age is defined as subject’s age at 
randomization)

Proportion of responders who show seronegative conversion and the incidence rate of 
seronegative conversion as confirmed by non-conventional serology in subjects who 
were randomized and received at least one dose of either 60- or 30-day nifurtimox 
treatment regimens

Proportion of presence of parasite or positive serology in babies born of mothers who 
were randomized and received at least one dose of either the 60- or 30-day nifurtimox 
treatment regimen
Safety variables
Safety variables include AEs and vital signs.
10.3.2
Statistical and analytical plans 
10.3.2.1Part 1 (CHICO)
Efficacy
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with sero-reduction183 or 
sero-conversion (60-day regimen) and the proportion estimated from historical data will be 
tested using a 2-sided 95% CI for a single proportion.
The null hypothesis H0 is pnifurtimox  =  pplacebo
The alternative hypothesis H1 is pnifurtimox  ≠  pplacebo, where pt is the sero-conversion rate for 
treatment t.
                                                
183 Text modified for clarity (Amendment 1)


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Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox 
subjects (60-day regimen) with sero-reduction or sero-conversion is greater than the 
proportion of historical placebo subjects with sero-conversion.184
Sero-conversion for the historical placebo control is estimated as 16% from cure rates as 
presented in two publications [15][16], using a number greater than the larger of the upper 
limits of the 95% CIs from the two studies.  Further details about the derivation of these rates 
are provided in Section 10.4.185
Secondary and exploratory efficacy analyses 186
A secondary analysis will be done to compare the proportion of subjects with sero-reduction
or sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be performed 
using a 2-sided 95% CI for the difference of two independent proportions.
The analyses specified above will also be done using data from the EOT visit, and 3 and 6 
months after EOT. 
The relationship of conventional serology results to qPCR results will be done using 
frequencies of matches and mismatches on the determination of disease status, and 
phi-correlation and kappa coefficient to assess the degree of agreement.  The relationship of 
non-conventional serology to conventional serology will be analyzed in the same way.187 The 
results from IHA and conventional serology will also be compared.  The details of secondary 
and exploratory efficacy analyses will be further specified in the SAP.188,189
Demographics and baseline characteristics
Demographics and baseline characteristics will be summarized by treatment and total 
population, using descriptive statistics and frequency tables as appropriate.
Safety 
Safety variables will be summarized by means of descriptive statistics and/or frequency tables
as appropriate.  Summaries will be given by treatment regimen.  All post-treatment AEs and 
SAEs, as well as drug-related AEs and SAEs, will be summarized by MedDRA terms.190
AEs occurring between the time of consent and the first dose of study drug will be 
summarized separately.  Subjects experiencing urine discoloration from riboflavin ingestion
will be listed also, although these incidences will not necessarily be considered AEs.
                                                
184 Text deleted per Modification 2 (Amendment 1)
185 Text modified as of amendment 2
186 “Exploratory” added per Amendment 5.
187 Text modified/added for clarity (Amendment 1)
188 Sentence added per Amendment 5.
189 Replaced IFA with IHA per Amendment 6.
190 Text modified as of amendment 2


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Missing data/drop outs 
No imputations will be made for missing data due to dropouts or other causes.  For the 
primary efficacy analysis done on the FAS, a patient without an assessment at the primary 12 
month time point will be considered a treatment failure.  For the secondary analysis using the 
PPS, patients who do not have serology determinations to assess the primary endpoint will be 
excluded from the analysis.
The following sensitivity analyses for the primary efficacy variable will be performed:

Missing values due to the following reasons treated as treatment failures and other 
missing values excluded from analysis:191
      1. the reason for discontinuation is known or suspected to be treatment ineffectiveness or 
an AE
       2. a subject  has not had a negative serology at any visit and has no serology 
determination for the primary analysis visit  
       3. an indeterminate or incorrectly performed serology determination occurs at the primary 
analysis visit.192

Observed data only

Analysis on PPS
Pharmacokinetic data 
Specifications for PK data analysis will be provided in a separate document.  Plasma 
concentration vs time data documented within the study report will be analyzed descriptively 
using univariate statistics.193
10.3.2.2Part 2 (CHICO SECURE)
Analysis of the primary efficacy variable
The difference in the incidence rate of nifurtimox subjects with seronegative conversion (60-
day regimen) and the rate estimated from historical data will be tested using a Poisson two-
sided 95% exact confidence interval (CI).
The incidence rate of seronegative conversion for the primary efficacy variable will be 
modelled using a Poisson distribution with a 2-sided 95% exact CI.  The rate will be 
estimated as the number of seronegative conversion divided by the time at risk of event 
(person-year) during the study.  The rate of seronegative conversion () and a 2-sided limits (, ) 
of the 95% CI for the rate can be calculated using the following formula [34]:  
                                                
191 Modified as per amendment 2 
192 Text modified/added for clarity as per Modification 2 (Amendment 1)
193 Modified as per amendment 4


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where n is the number of seronegative conversion observed , N the total subjects at risk in 
annual visit , Vi the th annual visit,   the total number of visits,   and  are lower and upper 
confidence limits for the rate of seronegative conversion respectively,  is the chi-square 
quantile for upper tail probability on v degrees of freedom.
This study uses an external control group of untreated patients with Chagas’ disease in the 4-
year follow-up period presented in Sosa et al. [15, 35].  In the reference, seroconversion to a 
negative result in the placebo group after 4-year follow-up was detected in 2 subjects with 
conventional serology and in 0 subjects with IHA test.  Thus, none of the subjects in the 
placebo group was considered as seroconverted by both test results.  The person time at risk 
was on average 44 * 4 = 176 person years.  The incidence rate estimate was 0% (0 / 176).
Superiority will be confirmed if the lower limit of the CI for the incidence rate of seronegative 
conversion for subjects in 60-day regimen is greater than the rate of historical placebo 
subjects with sero-conversion (i.e. 0 %).
No imputations will be made for missing data due to dropouts or other causes, as person-year 
is considered in the calculation of incidence rate. 
Frequency counts and percentages of seronegative conversion will be provided by visit. 
Missing data/drop outs
No imputations will be made for missing data due to dropouts or other causes.  For the 
primary efficacy analysis done on the FAS, drop-outs will be included in the analysis using a 
Poisson distribution with person-year.
Analysis of the secondary and exploratory efficacy variables
The OD values from recombinant ELISA and total purified antigen ELISA will be analyzed 
descriptively.  Changes from baseline will be summarized to show any serial reduction of OD.  
Baseline is defined as the OD values from the same ELISA tests measured at Visit 1 in part 1 
of the study.
The proportion of subjects who show both seronegative conversion as confirmed by two 
assays (recombinant ELISA and IHA) and no evidence of established cardiomyopathy as 
measured by ECG will be calculated overall and by treatment regimen. 
The proportion of subjects who show seronegative conversion as confirmed by non-
conventional serology will be calculated overall and by treatment regimen.
The same method for calculating incidence rate and its 2-sided 95% CI will be applied on the 
following variables:

Seronegative conversion as confirmed by two types of assay (recombinant ELISA and 
IHA) in subjects who were randomized and received at least one dose of the 30-day 
nifurtimox treatment regimen

Seronegative conversion as confirmed by two types of assay (recombinant ELISA and 
IHA) in all subjects by age categories.  The age categories are grouped as ≤2 years, >2 


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years to ≤6 years, >6 to ≤12 years, >12 to <18; age is defined as subject’s age at 
randomization 

Seronegative conversion as confirmed by non-conventional serology in subjects who 
were randomized and received at least one dose of either 60- or 30-day nifurtimox 
treatment regimen
Analysis of the safety variables 
Safety variables will be summarized by means of descriptive statistics and/or frequency tables 
as appropriate by treatment group. 
AEs will be assessed by the investigator regarding their causal relationship to the 
administration of treatment and to any study procedure of the LTFU based on all information 
available.  The results will be displayed in summary tables and listings as descriptive 
statistics.
10.4
Determination of sample size
Part 1 (CHICO)
This study uses historical controls estimated from cure rates presented in two publications 
[15][16].  The historical cure rates are as follows.
Publication
Age 
range
Sero-conversion rate 
(95%CI) 
in placebo patients
Andrade et al 
1996
7-12
3/65 = 5% (1%,13%)
Sosa et al 1998
6 -12
2/44= 5% (1%,16%)
For the primary objective, superiority over placebo will be confirmed if the lower limit of the 
95% CI for the nifurtimox (60-day regimen) cure rate is greater than 16%, the larger of the 
upper limits of the 95% CIs from the two publications.194
According to Guhl et al [32], nifurtimox 12 month post treatment cure rate is about 55%, with 
a sample size of 200 for the 60-day regimen, the power is 99% for the lower limit of the 95% 
CI to be greater than 16%.195
The number of subjects in the 30-day treatment regimen is based on the width of a 95% CI for 
the difference of two proportions. A sample size of 100 subjects for the 30-day subjects 
together with 200 subjects for the 60-day subjects will produce a CI with a half-width of 
approximately 0.12.
Part 2 (CHICO SECURE)
                                                
194 Modified as per amendment 2
195 Modified as per amendment 2


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According to Sosa-Estani [15, 35], the 48 months post treatment cure rate for benznidazole is 
about 5% as confirmed by two types of assay.  For the primary objective, assuming a drop-out 
rate of 15%, the expected number of patients to be observed at the end of Part 2 
(i.e., 48 months post treatment) in the 60-day regimen would be 170 out of the 200 patients in 
Part 1.  Using the assumptions of 170 nifurtimox 60-day regimen patients in Part 2, a 5% cure 
rate, and that seronegative conversion events follow Poisson distribution, the estimated 
incidence rate is calculated to be 1.32% and the 95% CI of the incidence rate is (0.61%, 
1.51%).
10.5
Planned interim analyses
No interim analysis is planned for the entire study.
11.
Data handling and quality assurance
In general, the criteria, rules, and procedures in this section apply to part 1 and part 2 of the 
study.  Criteria, rules, and procedures not applicable to either part of the study are otherwise 
marked.
11.1
Data recording
The data collection tool for this study will be a validated electronic data capture system.  
Subject data necessary for analysis and reporting will be entered/transmitted into a validated 
database or data system (SAS). 
Source documentation
It is the expectation of the sponsor that key data entered into the eCRF is supported by source 
documentation available at the site. 
Study-specific data not needed for the subject’s routine medical care (e.g., scores or 
questionnaires) may be entered directly into the eCRF, without availability of corresponding 
source documentation.
The site must implement processes to ensure availability of all required source 
documentation.  A source document checklist (not part of this protocol) will be used at the 
site to identify the source data for key data points collected and the monitor will work with the 
site to complete this. 
ECG tracings will be maintained with the subject’s source documentation, and ECG findings 
will be entered into the eCRF.
Completed subject diaries will be collected by site personnel at specific study visits (see Table 
9–1).  Information entered onto the subject diaries will be used to assess drug accountability 
and treatment compliance, and entered into the eCRF.  The completed diaries will be 
maintained with the subject’s source documentation.196 (Not applicable to part 2)
                                                
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On the days of PK blood sampling, the investigator should document if the patient was on 
stable dose (stable dose = last 6 doses taken).  The actual sampling date and sampling time 
should be documented.  Failure to comply with the documented sampling windows or missing 
samples does not invalidate the subject or the sample for the PK evaluation.  (Not applicable 
to part 2)
For subjects consenting to undergo PK blood sampling, blood samples will be sent to the 
sponsor, and the following data will be entered into the SAS after database lock for part 1 of 
the study.
Administered dose and exact times of two medication intakes during the visit, exact times of 
blood sampling, time of first dose after blood sampling, and samples collected within time 
window will be recorded in the eCRF.197 (Not applicable to part 2)
Data recorded from screening failures
Data of 'only screened subjects' will be recorded at least as source data, as far as the reason for 
the premature discontinuation is identifiable.  In addition to the applicable inclusion and/or 
exclusion criteria, the following data should be recorded in the eCRF:
Demographic information (subject number; year of birth/age; sex; if applicable 
race/ethnicity)
Date of informed consent
Reason for premature discontinuation
Date of last visit  
These data will be transferred to the respective database.
For screening failures with an SAE, the following data should be collected in the eCRF in 
addition to the data specified above:
All information related to the SAE such as: 
o Concomitant medication
o Medical history
o Other information needed for SAE complementary page
11.2
Monitoring
In accordance with applicable regulations, GCP, and sponsor’s/CRO’s procedures, monitors 
will contact the site prior to the start of the study to review with the site staff the protocol, 
study requirements, and their responsibilities to satisfy regulatory, ethical, and sponsor’s 
requirements.  When reviewing data collection procedures, the discussion will also include 
identification and documentation of source data items.
                                                
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The sponsor/designee will monitor the site activity to verify that the:
Data are authentic, accurate, and complete.  
Supporting data may be requested (example: blood glucose readings to support a 
diagnosis of diabetes). 
Safety and rights of subjects are being protected
Study is conducted in accordance with the currently approved protocol 
(including study treatment being used in accordance with the protocol)
Any other study agreements, GCP, and all applicable regulatory requirements are met.
The investigator and the head of the medical institution (where applicable) agrees to allow the 
monitor direct access to all relevant documents.
11.3
Data processing
Data will be collected as described in Section 11.1.  Clinical data management will be 
performed in accordance with applicable sponsor’s/CRO’s standards and data cleaning 
procedures.  This is applicable for data recorded on eCRF as well as for data from other 
sources (e.g., IVRS/IWRS, laboratory, ECG, ePRO, adjudication committees).
For data coding (AEs, medication), The Medical Dictionary for Regulatory Activities will be 
used for AEs, and the WHO Drug Dictionary will be used for prior and concomitant 
medications.198
After its initial release for biometrical analysis, the clinical database is planned to be 
re-opened for the inclusion of the PK data.199 (Not applicable to part 2)
11.4
Missing data 
In general, no imputations will be made for missing data other than those described in 
Section 10.200  More details will be provided in the SAP.
11.5
Audit and inspection
To ensure compliance with GCP and regulatory requirements, a member of the sponsor’s (or a 
designated CRO’s) quality assurance unit may arrange to conduct an audit to assess the 
performance of the study at the study site and of the study documents originating there.  The 
investigator/institution will be informed of the audit outcome.
In addition, inspections by regulatory health authority representatives and IEC(s)/IRB(s) are 
possible.  The investigator should notify the sponsor immediately of any such inspection.
The investigator/institution agrees to allow the auditor or inspector direct access to all relevant 
documents and allocate his/her time and the time of his/her staff to the auditor/inspector to 
                                                
198 Text modified for clarity (Amendment 1)
199 Text deleted for clarity (Amendment 1)
200 Text added for clarity (Amendment 1)


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discuss findings and any issues.  Audits and inspections may occur at any time during or after 
completion of the study.
11.6
Archiving
Essential documents shall be archived safely and securely in such a way that ensures that they 
are readily available upon authorities’ request.
Patient (hospital) files will be archived according to local regulations and in accordance with 
the maximum period of time permitted by the hospital, institution, or private practice.  Where 
the archiving procedures do not meet the minimum timelines required by the sponsor, 
alternative arrangements must be made to ensure the availability of the source documents for 
the required period.
The investigator/institution notifies the sponsor if the archival arrangements change (e.g., 
relocation or transfer of ownership).
The investigator site file is not to be destroyed without the sponsor’s approval.
The contract with the investigator/institution will contain all regulations relevant for the study 
center.
12.
Premature termination of the study
All criteria, rules, and procedures in this section apply to part 1 and part 2 of the study.
The sponsor has the right to close this study (or, if applicable, individual segments thereof 
[e.g., treatment arms; dose steps; centers]) at any time, which may be due but not limited to 
the following reasons: 
If risk-benefit ratio becomes unacceptable owing to, for example,
Safety findings from this study (e.g., SAEs)
Results of any interim analysis
Results of parallel clinical studies
Results of parallel animal studies 
(e.g., toxicity, teratogenicity, carcinogenicity or reproduction toxicity).
If the study conduct (e.g., recruitment rate; drop-out rate; data quality; protocol 
compliance) does not suggest a proper completion of the trial within a reasonable time 
frame.
The investigator has the right to close his/her center at any time.
For any of the above closures, the following applies:
Closures should occur only after consultation between involved parties.  Final decision 
on the closure must be in writing.
All affected institutions (e.g., IEC(s)/IRB(s); competent authority(ies); study center; 
head of study center) must be informed as applicable according to local law.


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All study materials (except documentation that has to remain stored at site) must be 
returned to the sponsor.  The investigator will retain all other documents until 
notification is given by the sponsor for destruction.
In the event of a partial study closure, ongoing subjects, including those in post study 
follow-up, must be taken care of in an ethical manner.
Details for individual subject's withdrawal can be found in Section 6.3.1.
13.
Ethical and legal aspects
In general, the criteria, rules, and procedures in this section apply to part 1 and part 2 of the 
study.  Criteria, rules, and procedures not applicable to either part of the study are otherwise 
marked.
13.1
Investigator(s) and other study personnel
This study will be conducted at investigational study centers in Latin America and Mexico.
The medical expert for this study is 
 5F Citi Group Tower, No. 33 Huayuan 
Shiqiao Rd, Shanghai, 200120, P.R. China.
The Study Manager and the Medical Expert will assign the coordinating investigator 
responsible for signing the clinical study report. 
All other study personnel not included in this section are identified in a separate personnel list 
(not part of this clinical study protocol) as appropriate.  This list will be updated as needed; an 
abbreviated version with personnel relevant for the centers will be available in each center’s 
investigator site file.
Whenever the term ‘investigator’ is noted in the protocol text, it may refer to either the 
principal investigator at the site, or an appropriately qualified, trained and delegated 
individual of the investigational site.
The principal investigator of each center must sign the protocol signature page and must 
receive all required external approvals (e.g., health authority, ethics committee, sponsor) 
before subject recruitment may start at the respective center.  Likewise, all amendments to the 
protocol must be signed by the principal investigator and must have received all required 
external approvals before coming into effect at the respective center.
A complete list of all participating centers and their investigators, as well as all required 
signature documents, will be maintained in the sponsor’s study file.
The global sponsor of this study is identified on the title page of this protocol.  If required by 
local law, local co-sponsors will be nominated; they will be identified on the respective 
country-specific signature pages.
External data evaluation bodies
An independent Data Monitoring Committee (DMC) will meet periodically to review the 
safety data of enrolled subjects, as well as the continuing scientific merit of the trial.
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The DMC may recommend stopping enrollment in the case of a negative risk/benefit 
assessment.  The DMC will be comprised of a minimum of two clinicians with expertise in 
relevant clinical specialties and at least one statistician knowledgeable about statistical 
methods for clinical trials.  Each committee member will be screened for evidence of an 
absence of serious conflicts of interest.  The operation of the DMC will be governed by a 
charter that will describe the group’s frequency of meetings, procedures (including, but not 
limited to, periodic safety monitoring), and requirements for reporting its observations to the 
Sponsor.  (Not applicable to part 2)
13.2
Funding and financial disclosure
Funding
This study will be funded by its sponsor.
Financial disclosure 
Each investigator (including principal and/or any sub investigators) who is directly involved 
in the treatment or evaluation of research subjects has to provide a financial disclosure 
according to all applicable legal requirements.  All relevant documentation will be filed in the 
trial master file.
13.3
Ethical and legal conduct of the study
The procedures set out in this protocol, pertaining to the conduct, evaluation, and 
documentation of this study, are designed to ensure that the sponsor and investigator abide by 
GCP guidelines and the guiding principles detailed in the Declaration of Helsinki.  The study 
will also be carried out in keeping with applicable local law(s) and regulation(s).
Documented approval from appropriate IEC(s)/IRBs will be obtained for all participating 
centers/countries before start of the study, according to GCP, local laws, regulations, and 
organizations.  When necessary, an extension, amendment, or renewal of the IEC/IRB 
approval must be obtained and also forwarded to the sponsor.  The responsible unit (e.g., 
IEC/IRB, head of the study center/medical institution) must supply to the sponsor, upon 
request, a list of the IEC/IRB members involved in the vote and a statement to confirm that 
the IEC/IRB is organized and operates according to GCP and applicable laws and regulations.
Strict adherence to all specifications laid down in this protocol is required for all aspects of 
study conduct; the investigator may not modify or alter the procedures described in this 
protocol. 
Modifications to the study protocol will not be implemented by either the sponsor or the 
investigator without agreement by both parties.  However, the investigator or the sponsor may 
implement a deviation from, or a change of, the protocol to eliminate an immediate hazard(s) 
to the trial subjects without prior IEC/IRB/sponsor approval/favorable opinion.  As soon as 
possible, the implemented deviation or change, the reasons for it and if appropriate the 
proposed protocol amendment should be submitted to the IEC/IRB/head of medical 
institution/sponsor.  Any deviations from the protocol must be explained and documented by 
the investigator.
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13.4
Subject information and consent
All relevant information on the study will be summarized in an integrated subject information 
sheet and informed consent form provided by the sponsor or the study center.  A sample 
subject information and informed consent form is provided as a document separate to this 
protocol.
Based on this subject information sheet, the investigator OR DESIGNEE will explain all 
relevant aspects of the study to each subject/legal representative or proxy consenter (if the 
subject is under legal protection) prior to his/her entry into the study (i.e., before any 
examinations and procedures associated with the selection for the study are performed or any 
study-specific data is recorded on study-specific forms).
The investigator OR DESIGNEE will also mention that written approval of the IRB/IEC has 
been obtained.
Each subject/legal representative or proxy consenter will be informed about the following 
aspects of premature withdrawal:
Each subject has the right to withdraw from the study at any time without any 
disadvantage and without having to provide reasons for this decision. 
The subject’s consent covers end-of-study examinations as specified in the visit 
description described in Section 13.4 to be conducted after withdrawal of consent. 
The subject’s data that have been collected until the time of withdrawal will be retained 
and statistically analyzed in accordance with the SAP. 
Subject-specific data on the basis of material obtained before withdrawal may be 
generated after withdrawal (e.g., image reading, analysis of biological specimen such 
as blood, urine, or tissues); these data would also be retained and statistically analyzed 
in accordance with the SAP.  The subject has the right to object to the generation and 
processing of this post-withdrawal data.  For this, he/she needs to sign a corresponding 
declaration of objection; alternatively, the subject’s oral objection may be documented 
in the subject’s source data.
Each subject/legal representative or proxy consenter will have ample time and opportunity to 
ask questions.
Only if the subject/legal representative or proxy consenter voluntarily agrees to sign the 
informed consent form and has done so, and assent is obtained from minor subjects, may 
he/she enter the study.  Additionally, the investigator OR DESIGNEE will personally sign 
and date the form.  The subject/legal representative or proxy consenter will receive a copy of 
the signed and dated form.  
The signed informed consent statement is to remain in the investigator site file or, if locally 
required, in the patient’s note/file of the medical institution.
In the event that informed consent is obtained on the date that baseline study procedures are 
performed, the study record or subject´s clinical record must clearly show that informed 
consent was obtained prior to these procedures.


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If the subject is not capable of providing a signature, a verbal statement of consent can also be 
given in the presence of an impartial witness (independent of the sponsor and the 
investigator).  This is to be documented in the subject’s source document and signed by the 
informing investigator OR DESIGNEE as well as the witness.
For minors or adults under legal protection, consent shall be given by the legal guardian(s).  
The consent of a minor or adult under legal protection shall also be requested where such a 
person is able to express his/her own will.  His/her refusal or the withdrawal of his/her 
consent may not be disregarded.
The informed consent form and any other written information provided to subjects/legal 
representatives or proxy consenters will be revised whenever important new information 
becomes available that may be relevant to the subject’s consent, or there is an amendment to 
the protocol that necessitates a change to the content of the subject information and/or the 
written informed consent form.  The investigator OR DESIGNEE will inform the 
subject/legal representative or proxy consenter of changes in a timely manner and will ask the 
subject to confirm his/her participation in the study by signing the revised informed consent 
form.  Any revised written informed consent form and written information must receive the 
IEC/IRB`s approval/favorable opinion in advance of use.
13.5
Publication policy and use of data
The sponsor has made the information regarding the study protocol publicly available on the 
internet at www.clinicaltrials.gov. 
All data and results and all intellectual property rights in the data and results derived from the 
study will be the property of the sponsor who may utilize them in various ways, such as for 
submission to government regulatory authorities or disclosure to other investigators.
Regarding public disclosure of study results, the sponsor will fulfill its obligations according 
to all applicable laws and regulations.  The sponsor is interested in the publication of the 
results of every study it performs. 
The sponsor recognizes the right of the investigator to publish the results upon completion of 
the study.  However, the investigator, whilst free to utilize study data derived from his/her 
center for scientific purposes, must obtain written consent of the sponsor on the intended 
publication manuscript before its submission.  To this end, the investigator must send a draft 
of the publication manuscript to the sponsor within a time period specified in the contract.  
The sponsor will review the manuscript promptly and will discuss its content with the 
investigator to reach a mutually agreeable final manuscript. 
13.6
Compensation for health damage of subjects/insurance
The sponsor maintains clinical trial insurance coverage for this study in accordance with the 
laws and regulations of the country in which the study is performed.
13.7
Confidentiality
All records identifying the subject will be kept confidential and, to the extent permitted by the 
applicable laws and/or regulations, will not be made publicly available.


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Subject names will not be supplied to the sponsor.  Only the subject number will be recorded 
in the eCRF, and if the subject name appears on any other document (e.g., pathologist report), 
it must be obliterated before a copy of the document is supplied to the sponsor.  Study 
findings stored on a computer will be stored in accordance with local data protection laws.  As 
part of the informed consent process, the subjects will be informed in writing that 
representatives of the sponsor, IEC/IRB, or regulatory authorities may inspect their medical 
records to verify the information collected, and that all personal information made available 
for inspection will be handled in strictest confidence and in accordance with local data 
protection laws.
If the results of the study are published, the subject’s identity will remain confidential.
The investigator will maintain a list to enable subjects to be identified.
14.
Reference list 
1. Organización Panamericana de la Salud. Estimación cuantitativa de la enfermedad de 
Chagas en las Americas. Organización Panamericana de la Salud, Montevideo, 
Uruguay 2006. (Spanish).
2. Bern C, Montgomery SP. An estimate of the burden of Chagas’ disease in the United 
States. Clin Infect Dis. 2009 Sep 1;49(5):e52-4. doi: 10.1086/605091.
3. Hotez PJ, Dumonteil E, Woc-Colburn L, Serpa JA, Bezek S, Edwards MS, et al. 
Chagas’ disease: “the new HIV/AIDS of the Americas”. PLoS Negl Trop Dis. 
2012;6(5):e1498. doi: 10.1371/journal.pntd.0001498. Epub 2012 May 29.
4. Buekens P, Almendares O, Carlier Y, Dumonteil E, Eberhard M, Gamboa-Leon R, et 
al. Mother-to-child transmission of Chagas’ disease in North America: why don’t we 
do more? Matern Child Health J. 2008 May;12(3):283-6. Epub 2007 Jun 30.
5. Moya PR, Paolasso RD, Blanco S, Lapasset M, Sanmartino C, Basso B, et al. 
Treatment of Chagas’ disease with nifurtimox during the first months of life. (Article 
in Spanish). Medicina (B Aires). 1985;45(5):553-8.
6. Generalitat de Catalunya. Departament de Salut. Direcció General de Planificació i 
Avaluació. Protocol for screening and diagnosing Chagas’ disease in pregnant Latin 
American women and their newborns. Barcelona 2010.
7. Bittencourt AL. Possible risk factors for vertical transmission of Chagas’ disease. 
Rev Inst Med Trop Sao Paulo. 1992 Sep-Oct;34(5):403-8.
8. Torrico F, Alonso-Vega C, Suarez E, Rodriguez P, Torrico MC, Dramaix M, et al. 
Maternal Trypanosoma cruzi infection, pregnancy outcome, morbidity, and mortality 
of congenitally infected and non-infected newborns in Bolivia. Am J Trop Med Hyg. 
2004 Feb;70(2):201-9.
9. Bern C. Antitrypanosomal therapy for chronic Chagas’ disease. N Engl J Med. 
2011 Jun 30;364(26):2527-34. doi: 10.1056/NEJMct1014204.


Integrated Clinical Study Protocol
BAY A2502/ 16027
03 NOV 2020
Version 11.0
Page:  93 of 259
10. Saulnier Sholler GL, Bergendahl GM, Brard L, Singh AP, Heath BW, Bingham PM, 
et al.  J Pediatr Hematol Oncol. 2011 Jan;33(1):25-30.
11. Freilij H, Altcheh J. Congenital Chagas' disease: diagnostic and clinical aspects. Clin
Infect Dis. 1995 Sep;21(3):551-5.
12. Bern C, Montgomery SP, Herwaldt BL, Rassi A Jr, Marin-Neto MA, Dantos RO, et 
al. Evaluation and treatment of Chagas’ disease in the United States: a systematic 
review. JAMA. 2007 Nov 14;298(18):2171-81.
13. Control of Chagas disease. Second report of a WHO expert committee. World Health 
Organ Tech Rep Ser. 2002;905.
14. Ministério da Saúde. Secretaria de Vigilância em Saúde. Brazilian consensus on 
Chagas’ disease. Rev Soc Bras Med Trop. 2005;38 Suppl 3:7-29. (Portuguese).
15. Sosa Estani S, Segura EL, Ruiz AM, Velazquez E, Porcel BM, Yampotis C. Efficacy 
of chemotherapy with benznidazole in children in the indeterminate phase of Chagas’ 
Disease. Am J Trop Med Hyg. 1998 Oct;59(4):526-529.
16. de Andrade AL, Zicker F, de Oliveira RM, Almeida Silva S, Luquetti A, Travassos 
LR, et al.  Randomised trial of efficacy of benznidazole in treatment of early 
Trypanosoma cruzi infection. Lancet. 1996 Nov 23;348(9039):1407–13.
17. Almeida IC, Ferguson MA, Schenkman S, Travassos LR. Lytic anti-alpha-galactosyl 
antibodies from patients with chronic Chagas' disease recognize novel O-linked 
oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins 
of Trypanosoma cruzi. Biochem J. 1994 Dec 15:304 (Pt 3):793-802.
18. Altcheh J, Corral R, Biancardi MA, Freilij H. Anti-F2/3 antibodies as cure marker in 
children with congenital Trypanosoma cruzi infection. Medicina (B Aires). 
2003;63(1):37-40.
19. World Health Organization (WHO). Control of  Chagas’ disease. Second report of the 
WHO Expert Committee. WHO Tech rep ser905, World Health Organization:120.
20. Porcel BM, Bonrempi EJ, Henriksson J, Rydaker M, Aslund L, Segura EL, et al. 
Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes 
encoding putative calcium-binding proteins, highly conserved in trypanosomatids. 
Exp Parasitol. 1996 Dec:84(3):387-99.
21. Piron M, Fisa R, Casamitjana N, Lopez-Chejade P, Puig L, Verges M, et al. 
Development of a real-time PCR assay for Trypanosoma cruzi detection in blood 
samples. Acta Trop. 2007 Sep;103(3):195-200. Epub 2007 Jun 23.
22. Schijman AG, Bisio M, Orellana L, Sued M, Duffy T, Mejia Jaramillo A, et al. 
International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA 
in blood samples from Chagas’ disease patients. PLoS Negl Trop Dis. 2011 Jan 
11;5(1):e931. doi: 10.1371/journal.pntd.0000931.
23. Moreira OC, Ramírez JD, Velázquez E, Melo MF, Lima-Ferreira C, Guhl F, et al. 
Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma 


Integrated Clinical Study Protocol
BAY A2502/ 16027
03 NOV 2020
Version 11.0
Page:  94 of 259
cruzi parasitemia in patients with chronic Chagas’ disease cardiomyopathy: a substudy 
from the BENEFIT trial. Acta Trop. 2013 Jan;125(1):23-
31. doi: 10.1016/j.actatropica.2012.08.020. Epub 2012 Sep 12.
24. Duffy T, Cura CI, Ramirez JC, Abate T, Cayo NM, Parrado R, et al. Analytical 
performance of a multiplex Real-Time PCR assay using TaqMan probes for 
quantification of Trypanosoma cruzi satellite DNA in blood samples. PLoS Negl Trop 
Dis. 2013;7(1):e2000. doi: 10.1371/journal.pntd.0002000. Epub 2013 Jan 17.
25. Schijman AG, Altcheh J, Burgos JM, Biancardi M, Bisio M, Levin MJ, et al. 
Aetiological treatment of congenital Chagas' disease diagnosed and monitored by the 
polymerase chain reaction. J Antimicrob Chemother. 52(3):441-9.
26. Solari A, Ortiz S, Soto A, Arancibia C, Campillay R, Contreras M, et al.
Treatment of Trypanosoma cruzi-infected children with nifurtimox: a 3-year follow-
up by PCR. J Antimicrob Chemother. 2001 Oct;48(4):515-9.
27. Jannes G, De Vos D. A review of current and future molecular diagnostic tests for use 
in the microbiology laboratory. Methods Mol Biol. 2006;345:1-21.
28. First Chagas Paltform Technical Meeting Report, September 1, 2009, Rio de 
Janeiro201
29. Bianchi F, Cucunuba Z, Guhl F, Gonzalez NL, Freilij H, Nicholls RS, et al. Follow-up 
of an Asymptomatic Chagas’ Disease Population of Children after Treatment with 
Nifurtimox (Lampit) in a Sylvatic Endemic Transmission Area of Colombia. 
PLoS Negl Trop Dis. 2015 Feb 27;10(2):e0003465.
30. Bern C. Chagas' Disease. N Engl J Med. 2015;373(5):456-66.
31. Voith B, Beckermann B, Brendel E, Zimmer D. (2000) Recommendation for the 
Harmonization of Analytical Methodology in Pharmacokinetic Investigations – A 
Task Force Report. Bayer Product Report No. PH-30516.202
32. Guhl F, Nicholls R, Montoya R, Rosas F, Velasco VM, et al. (2004) Rapid 
negativization of serology after treatment with benznidazole for chagas disease in a 
group of colombian schoolchildren. Medimond International Proceedings. Spain, Vol. 
1. pp. 107–114203
33. Selvin S. Statistical analysis of epidemiologic data. Oxford University Press; 2004 
May 13.
34. Ulm K. Simple method to calculate the confidence interval of a standardized mortality 
ratio (SMR). American journal of epidemiology. 1990;131(2):373-5.
35. FDA. 2017. Benznidazole tablets. Company: Chemo Research, S.L. Available from 
https://www.accessdata.fda.gov/drugsatfda_docs/nda/2017/209570Orig1s000TOC.cfm
                                                
201 Reference 28 added as per amendment 2
202 References 17, 19, 20, and 28 modified/added based on changes made to text in Section 5 (Amendment 1) 
203 Reference added as per amendment 2


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15.
Protocol amendments
The Protocol Amendment Summary of Changes Table for the current amendment is located 
directly before the Table of Contents.
15.1
Amendment 1
Description of Amendment
On 30 DEC 2014, Bayer HealthCare Pharmaceuticals Inc. received a “study may proceed” 
letter for Investigational New Drug 109901 (BAY A2502 - nifurtimox), detailing 
recommended revisions to the protocol.  The primary purpose of Amendment 1 is to update 
the protocol in response to these regulatory recommendations, as described below:
ECGs will be obtained at the time PK samples are collected when nifurtimox has reached 
Cmax or peak steady-state concentrations (i.e., at the 2 – 4 hour time point).  The number of
evaluable PK subjects will be at least 10 per age cohort.  
The reduction in the ELISA OD value for subjects over 8 months of age at enrollment as an 
indicator of the decrease in antibody titer from study enrollment to 12 months post-treatment 
(percentage change over time in the study test sample baseline OD value minus the OD of the 
sample at 12 months divided by the baseline OD) was added to the protocol.  A 20% or 
greater reduction in OD at 12 months is regarded as a clinically meaningful indicator of cure 
and will indicate positive treatment response (cure) for the primary endpoint.   In subjects 
who are < 8 months old at study entry, an antibody titer below the level of detection of the 
ELISA (i.e., negative antibody titer) at 12-months post-treatment will be considered a positive 
treatment response.  For placebo historical data, sero-reduction data from two publications 
will be used.  For secondary endpoints, ELISA OD values will be measured at the 1 and 
2 months during treatment, and at 3, 6, and 12 months post-treatment.  To confirm the 
diagnosis of Chagas’ disease in subjects > 8 months old, a conventional ELISA test, plus 
another supplemental test as confirmation, will be utilized. The results from both tests must be 
positive in order for a patient to be considered eligible for study enrollment. For subjects 
under 8 months of age, direct microscopic observation of T. cruzi by concentration test will be 
required for diagnosis.  New tests for confirming the diagnosis of Chagas’ disease were 
added: recombinant ELISA and total purified antigen ELISA.  These changes were also 
reflected in the statistical methods section of the protocol (Section 10).
Since indirect immunofluorescence and indirect hemagglutination tests are not quantitative 
tests, which is required for evaluation of the primary endpoint, the ELISA tests for diagnosis 
were changed to recombinant ELISA and total purified antigen ELISA in the protocol.  For 
evaluation of the primary endpoint at 12-months post-treatment, only one ELISA assay (the
same one used for diagnosis) will be used.  Additionally, two subject baseline specimens will 
be collected. One will be used for the initial diagnostic test; the other will be frozen and 
retained to assay as an internal subject control along with the subject’s 12-month specimen. 
The use of riboflavin in the placebo tablets in the 30-day treatment group has the potential for 
unblinding subjects and investigators if coloring of the urine is observed.  This is unlikely to 
significantly affect the safety evaluation or the primary efficacy endpoint; however, it may 
cause compliance issues and/or dropouts.  Since the urine color change does not occur in all 


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subjects, this possibility will be reinforced to each subject during randomization and when 
study drug is dispensed.
Information about laboratory procedures, assay variability and cutoffs, and facilities to be 
used for all assays was added.  Chemistry, hematology, and urinalysis will be done in the 
local labs of the selected sites.  Relevant information and documents regarding laboratory 
procedures, assay variability and cutoffs, and facilities to be used, will be provided to the 
agencies in a separate IND information amendment after site selection in the participating 
countries.  Serological testing will be done in a central lab.  
A safety and compliance questionnaire/script (Phone Contact Form) for use by site staff when 
conducting the telephone assessments at Visits 4, 5, and 7 was developed and included in the 
protocol appendices. 
For standardization of adverse event reporting, the terms “mild”, “moderate”, and “severe” 
were defined within the protocol.
A telephone assessment at Day 14 was added to obtain safety and compliance information.  A 
telephone assessment was selected so as not to impose the burden of a sixth study site visit on 
the subject and/or guardian, who typically resides in remote areas with transportation 
challenges.
Measurement of ECGs will be optional assessments for subjects < 5 years of age, and 
required for subjects 5 years of age and older.
A neurological examination to be performed at the time of each physical examination in the 
study was added.
Detailed procedures regarding follow-up on all subjects who prematurely discontinue study 
drug was added.
Monitoring of treatment compliance was included in the Visit 6 (Day 30) study assessments 
to be conducted via telephone; this is reflected in the Phone Contact Form.
15.1.1
Overview of changes
This section provides a conceptual overview of all modifications to the amended protocol, as 
introduced by this amendment.  The associated changes to the protocol text are detailed in 
Section 15.1.2.
Modification 1:  Text added to indicate that ECGs will be obtained at the time PK samples are 
collected when nifurtimox has reached Cmax or peak steady-state concentrations (i.e., at the 
2 – 4 hour time point)  
Rationale:  Regulatory recommendation to assess efficacy and pharmacokinetics

Synopsis, Methodology

Section 5, Study design

Section 9.1, Tabular schedule of evaluations

Table 9-1, Schedule of procedures and assessments – Study 16027


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
Section 9.2.2, Treatment Phase

Section 9.2.3, Follow-up Phase

Section 9.5, Pharmacokinetic/pharmacodynamics assessments

Table 9–2:  Pharmacokinetic sampling time points and windows

Section  9.6.3.5, 12-lead electrocardiogram
Modification 2:  Clarification of study objectives and efficacy variables regarding 
sero-reduction and sero-conversion; change in tests required for confirmation of Chagas’ 
disease (recombinant ELISA and total purified antigen ELISA)
Rationale:  Regulatory recommendation for efficacy and inclusion criteria

Synopsis, Study objective(s), Diagnosis and main criteria for inclusion/exclusion, 
Primary efficacy variable(s), and Plan for statistical analysis

Section 4, Study objectives

Section 5, Study design

Section 6.1, Inclusion criteria

Section 9.1, Tabular schedule of evaluations

Section 9.2.1, Screening

Section 9.2.2, Treatment phase

Section 9.2.3, Follow-up phase

Section 9.4, Efficacy

Section 9.4.2, Serological tests

Section 9.4.3, Concentrations tests for T. cruzi

Section 10.3, Variables and planned statistical analyses

Section 10.4, Determination of sample size
Modification 3: Baseline specimens for serological tests to be collected for diagnosis, and 
frozen as a subject control.
Rationale:  Regulatory recommendation

Serological tests, Section 9.4.2
Modification 4:  Change in urine color to be reinforced to subjects at the visits where study 
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Rationale:  Regulatory recommendation to avoid issues with treatment compliance

Section 7.4, Dosage and administration

Section 9.2.2.1, Visit 2 – baseline (treatment phase, Day 1)
Modification 5:  Information about laboratory procedures, assay variability and cutoffs, and 
facilities to be used for all assays was added.  
Rationale:  Regulatory recommendation

Section 9.4.2, Serological tests
Modification 6:  A safety and compliance questionnaire/script (Phone Contact Form) for use 
by site staff when conducting the telephone assessments was added. 
Rationale:  Regulatory recommendation

Synopsis, Methodology

Section 5, Study design

Section 8.1, Prior and concomitant therapy

Section 9.1, Tabular schedule of evaluations

Table 9-1, Schedule of procedures and assessments – Study 16027

Section 9.2.2.3, Visit 4 (Day 14 ± 3 days)

Section 9.2.2.4, Visit 5 (Day 21 ± 3 days)

Section 9.2.2.6, Visit 7 (Day 42 ± 3 days)

Section 16.1, Phone Contact Form for telephone assessments
Modification 7:  For standardization of adverse event reporting, the terms “mild”, “moderate”, 
and “severe” were defined within the protocol.
Rationale:  Regulatory recommendation for safety

Section 9.6.1.2.2,Intensity
Modification 8:  A telephone assessment at Day 14 was added.  
Rationale:  Regulatory recommendation to obtain safety and compliance information

Synopsis, Methodology

Section 5, Study design

Section 7.7, Treatment compliance


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
Section 9.1, Tabular schedule of evaluations

Table 9-1, Schedule of procedures and assessments – Study 16027

Section 9.2.2.3, Visit 4 (Day 14 ± 3 days)

Visit 5 (Day 30 ± 3 days), Section 9.2.2.4
Modification 9:  Measurement of ECGs will be optional assessments for subjects < 5 years of 
age, and required for subjects 5 years of age and older.
Rationale:  Regulatory recommendation for safety

Synopsis, Methodology

Section 5, Study design

Section 9.1, Tabular schedule of evaluations

Table 9-1, Schedule of procedures and assessments – Study 16027

Section 9.2.1, Screening

Section 9.2.2, Treatment phase

Section 9.2.3, Follow-up phase

Section 9.5, Pharmacokinetics/pharmacodynamics

Section 9.6.3.4, Vital signs, height/length, and weight

Section 9.6.3.5, 12-lead electrocardiogram
Modification 10:  A neurological examination to be performed at the time of each physical 
examination in the study was added.
Rationale: Regulatory recommendation for safety and efficacy

Synopsis, Methodology

Section 4, Study objectives

Section 5, Study design

Section 9.1, Tabular schedule of evaluations

Table 9-1, Schedule of procedures and assessments – Study 16027

Section 9.2.1, Screening

Section 9.2.2, Treatment phase

Section 9.6.3.3, Physical examination

Section 9.6.3, Further safety


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Modification 11:  Detailed procedures regarding follow-up on all subjects who prematurely 
discontinue study drug was added.
Rationale: Regulatory recommendation for safety

Synopsis, Methodology

Section 5, Study design

Table 9-1, Schedule of procedures and assessments – Study 16027

Section 9.1, Tabular schedule of evaluations
Modification 12:  Monitoring of treatment compliance was included in the Visit 6 (Day 30) 
study assessments to be conducted via telephone; this is reflected in the Phone Contact Form.
Rationale: Regulatory recommendation for safety and efficacy

Synopsis, Methodology

Section 5, Study design

Section 7.7, Treatment compliance

Section 8, Non-study therapy

Section 9.2.2.5, Visit 6 (Day 30 ± 3 days)

Section 16.1, Phone Contact Form for telephone assessments
Modification 13: A Phone Contact Form was developed and added as an appendix (16.1) to 
the protocol.
Rationale: To assess incidence of adverse events, use of concomitant medications, and 
treatment compliance

Section 16.1
Modification 14: A Subject Diary was developed and added as an appendix (16.3) to the 
protocol.
Rationale: To assess treatment compliance

Section 16.3


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15.1.2
Changes to the protocol text
Editorial note
In the sections on changes to the protocol text, all protocol sections affected by the respective 
amendment are detailed; the sequence of the sections follows the structure of the most recent 
protocol version. As applicable, changes to the protocol text are highlighted as follows:

Addition of a whole new portion
Brief identification of the new portion

Removal of a whole portion
Complete display of the removed portion,  formatted 
as crossed out

Editing of an existing portion
Comparative presentation of “old text” versus “new 
text”, with “old text” referring to the most recent 
previous protocol version. Deletions are crossed out 
in the “old text”.  Additions are underlined in the 
“new text”.

Terminological changes
Brief specification of the terminological change
Editorial changes, correction of typographical errors, punctuation revisions, addition of 
references, updates to all cross-referenced field codes, and minor revisions of language were 
made to ensure clarity and consistency throughout the document.
The following terminological changes were made globally throughout the document:

The term “titer(s)” was changed to “concentration(s)”

Age groups in the study population were modified as follows: “subjects older than 
8 months of age” to “subjects > 8 months of age at randomization”; “[subjects] 
younger than 18 years of age” to “[subjects] < 18 years of age at randomization”; 
“subjects 8 months of age and younger” to “subjects ≤8 months of age at 
randomization”

“qualitative PCR” was changed to “quantitative PCR”
Synopsis, Study Objectives
This section was changed as a result of Modifications 2 and 10.
Old text:
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated control as 
seroreduction (≥20% sero-reduction compared to baseline in subjects older than 8 months of age and 
younger than 18 years of age), or sero-conversion (in subjects 8 months of age and younger) at the 
12month follow-up (360 days from end of treatment [EOT]) 


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New text:
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated control at the 
12month follow-up (360 days from end of treatment [EOT]) as seroreduction (defined as a ≥20%
reduction in optical density measured by conventional enzymelinked immune sorbent assay [ELISA]) 
compared to baseline in subjects ≥8 months to <18 years of age at randomization), or sero-conversion
(defined as negative Immunoglobulin G concentration for all subjects).
Old text:
Secondary objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active control 
(benznidazole) as sero-reduction (≥20% seroreduction compared to baseline in subjects older than 8 
months of age and younger than 18 years of age) or seroconversion (in subjects 8 months of age and 
younger) and to qualitative polymerase chain reaction (qPCR) at the 12-month followup (360 days from 
EOT) 

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of nifurtimox as 
seroreduction (≥20% sero-reduction compared to baseline in subjects older than 8 months of age and 
younger than 18 years of age), or sero-conversion (in subjects 8 months of age and younger) and 
qualitative polymerase chain reaction (qPCR) at the 12-month follow-up (360 days from EOT) 

To evaluate the relationship of conventional serology (≥20% seroreduction in subjects older than 8 
months of age and younger than 18 years of age, or seroconversion in subjects 8 months of age and 
younger) to qPCR using frequencies of matches and mismatches to assess agreement

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters (hematology, blood 
chemistry, urinalysis), electrocardiogram (ECG) monitoring (optional depending on the age of the 
subject, at the discretion of the investigator), vital sign measurements (blood pressure [optional 
depending on the age of the subject, at the discretion of the investigator], heart rate, respiratory rate, 
temperature), adverse event (AE) monitoring, and physical examinations

…
New text:

To assess the comparability of a 60-day regimen of nifurtimox to historical active control 
(benznidazole) as sero-reduction or seroconversion at the 12-month followup (360 days from EOT) 

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of nifurtimox as 
seroreduction or sero--conversion and to quantitative polymerase chain reaction (qPCR) at the 
12-month follow-up (360 days from EOT) 

To evaluate the relationship of conventional serology (as sero-reduction or sero-conversion) to qPCR 
using frequencies of matches and mismatches to assess agreement

To evaluate the relationship of non-conventional serology to conventional serology

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters (hematology, blood 
chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital sign measurements (blood pressure, 
heart rate, respiratory rate, temperature), adverse event (AE) monitoring, and physical examinations, 
including neurological examinations

…


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Synopsis, Diagnosis and main criteria for inclusion/exclusion
This section was changed for clarity and as a result of changes made per Modification 2.
Old text:

Male and female pediatric subjects aged 0 days to younger than 18 years

Chagas’ disease diagnosed/confirmed by:
o Subjects younger than 8 months of age must demonstrate direct observation of Trypanosoma cruzi by 
concentration test
o Subjects 8 months of age and older and younger than 18 years must demonstrate a positive enzyme-
linked immune sorbent assay (ELISA) plus a positive result for one of the following tests to confirm 
diagnosis:

Alternate ELISA (e.g., Chagatest ELISA recombinate v3.0)

Hemoagglutination (e.g., Chagatest hemoagglutination inhibition)  

Direct Agglutination (e.g., SERODIA-Chagas) 
Written informed consent by the subject and/or parent(s) or legally authorized representative(s) …
…
New text:

Male and female pediatric subjects aged 0 days to younger than 18 years

Chagas’ disease diagnosed/confirmed by:
o
Subjects < 8 months of age at randomization must demonstrate direct observation of Trypanosoma cruzi
by concentration test
o
Subjects > 8 months to < 18 years of age at randomization must demonstrate a positive conventional
ELISA result for both of the following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA 
Additionally, a non-conventional ELISA test will be obtained; however, a positive result will not be 
required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ disease within three 
months prior to screening and they have not had prior anti-trypanocidal or anti-parasitic treatment.
Written informed consent by the subject and/or parent(s) or legally authorized representative(s) …
…
Synopsis, Methodology
This section was changed for clarity and as a result of changes made per Modifications 1, 8, 
10, and 11.
Old text:
After informed consent/assent has been obtained at screening and study eligibility has been confirmed at Visit 2, 
subjects will be randomized via Interactive Voice Response System/Interactive Web Response System in a 2:1 
ratio (60-day regimen vs. 30-day regimen) to one of two treatment groups:


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…
The first dose of study drug will be administered at Visit 2. 
Subjects will return to the investigational site for efficacy and safety assessments at Visits 3 (Day 7±1).  A 
pre-paid phone card will be provided to facilitate contact between study site personnel and the subjects/subjects’ 
authorized representatives.  
Subjects will return to the investigational site for efficacy and safety assessments on Days 30 (±3 days) and 60 
(±3 days).  Day 60 will be the EOT for both treatment groups.  On Days 21 (±3 days) and 42 (±3 days), study 
site personnel will contact the subject’s parent or legal guardian via telephone to assess the occurrence of AEs 
and concomitant medications. 
Subjects will then return to the site on Days 90, 240, and 420 (±7 days) after the start of treatment for the 
collection of additional efficacy and safety data.  The total duration of each subject’s participation is expected to 
be approximately 14.5 months.  
Assessments of efficacy and PK will be performed at specified time windows.  PK blood sampling will be 
optional on consent/assent of the subject/subject’s legally authorized representative(s).  In order to minimize the 
burden of blood loss due to PK sampling, ageappropriate sparse sampling strategies combined with (micro-
)bioanalysis techniques will be applied.  
Safety will be assessed at specified time points via laboratory parameters (hematology, blood chemistry, 
urinalysis), vital sign measurements (blood pressure [optional depending on the age of the subject, at the 
discretion of the investigator], heart rate, respiratory rate, temperature), monitoring of ECGs (optional depending 
on the age of the subject, at the discretion of the investigator), monitoring of AEs, and physical examinations.  
As an additional safety precaution, all subjects with a positive microscopy or qPCR test result at Day 30 (±3 
days) will be considered a treatment failure, discontinued from the study, and treated with an alternative 
antitrypanosomal therapy.
New text:
No study-specific procedures will be performed before the subject/legally authorized representative has signed 
the informed consent form, or a minor subject has provided assent, including asking a potential subject to fast (if 
applicable, depending on the age of the subject at the discretion of the investigator) prior to the screening blood 
samples.  Informed consent/assent for optional PK blood sampling will also be obtained.  After the 
consents/assents have been obtained, screening assessments will be performed from 1 to 14 days prior to 
randomization at Visit 2.
After study eligibility has been confirmed and safety assessments have been performed at Visit 2 (Day 1), 
subjects will be randomized via Interactive Voice Response System/Interactive Web Response System in a 2:1 
ratio (60-day regimen vs. 30-day regimen) to one of two treatment groups:
…
The first dose of study drug will be administered at Visit 2.  Pre- and post-dose PK blood samples will be 
obtained at specified time points from those subjects consenting to PK assessments. At the 2 – 4 hour post-dose 
time point of PK blood sampling (i.e., at the time of maximum concentration), an ECG (optional for subjects 
< 5 years of age at the discretion of the investigator) will be obtained to allow for PK/PD investigations.  Study 
drug will be dispensed and instructions for study drug administration will be provided to all subjects.
Subjects will return to the investigational site for efficacy and safety assessments at Visits 3 (Day 7±1) and 6 
(Day 30).  Subjects who have not consented to PK assessments will take that day’s doses of study drug as 
instructed.  Subjects consenting to PK assessments must hold the morning dose of study drug; for these subjects, 
a pre-dose PK blood sample will be obtained, then the morning dose of study drug will be administered.  Post-
dose PK blood samples will be obtained at specified time points, and ECGs will be obtained at the 2 – 4 hour 
post-dose time point.  A pre-paid phone card will be provided to all subjects to facilitate contact between study 
site personnel and the subjects/subjects’ authorized representatives.  


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At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and study drug for the 
remaining 30 days of treatment will be dispensed.  
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site personnel will 
contact the subject/legally authorized representative via telephone to assess the occurrence of AEs, use of 
concomitant medications, and compliance with study drug administration.  A Phone Contact Form for the 
telephone assessments will be provided to all sites.
Subjects will return to the investigational site for efficacy and safety assessments on Day 60 (Visit 8), which will 
be the EOT for both treatment groups.  Subjects who have not consented to PK assessments will take that day’s 
doses of study drug as instructed.  Subjects consenting to PK assessments must hold the morning dose of study 
drug. A pre-dose PK blood sample will be collected, then the morning dose of study drug will be administered.  
Post-dose PK blood samples will be obtained at specified time points, and ECGs will be obtained at the 2 – 4 
hour post-dose time point. Study drug will be collected, and no additional study drug will be dispensed.    
After the EOT visit (Visit 8), subjects will return to the site on Days 90 (Visit 9), 240 (Visit 10), and 420 (Visit 
11) for additional efficacy and safety assessments.  The total duration of each subject’s participation is expected 
to be approximately 14 months.  
Safety will be assessed via laboratory parameters (hematology, blood chemistry, urinalysis), vital sign 
measurements (blood pressure [optional in subjects < 5 years of age at the discretion of the investigator], heart 
rate, respiratory rate, temperature), monitoring of ECGs (optional in subjects < 5 years of age at the discretion of 
the investigator), monitoring of AEs, and physical examinations, including neurological examinations.  
Subjects who discontinue prematurely from study drug administration will continue to return to the 
investigational site for study assessments at Visit 3, 6, and 8 (EOT), and undergo telephone assessments as 
described for Visits 4, 5, and 7. If subjects are unable/unwilling to do so, they must return to the investigational 
site 30 (±3) days after the last dose of study drug for EOT (Visit 8) assessments, and undergo telephone 
assessments as described for Visits 4, 5, and 7. If the subject is unable/unwilling to return to the clinic for the 
EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of 
Visit 8 assessments.
Synopsis, Primary variable(s)
This section was changed as a result of Modification 2.
Old text:
The primary efficacy variable will be a ≥20% sero-reduction (subjects older than 8 months of age and younger 
than 18 years of age) or sero-conversion (subjects 8 months of age and younger) in antibody Immunoglobulin G 
titer at 12 months post-treatment using conventional ELISA serology as the measure of efficacy.
New text:
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months post-treatment using two
conventional ELISA serology tests as the measure of efficacy.
Synopsis, Plan for statistical analysis
This section was changed per Modification 2.
Old text:
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with ≥20% seroreduction or sero-conversion (60-day 
regimen) and the proportion estimated from historical data will be tested using an exact 2-sided 95% confidence 
interval (CI) for a single proportion.


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Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox subjects (60day 
regimen) with ≥20% sero-reduction or seroconversion is greater than the proportion of historical placebo 
subjects with ≥20% sero-reduction or sero-conversion.  This same CI will also be used for comparison with the 
historical benznidazole subjects with ≥20% seroreduction or sero-conversion.
Secondary efficacy analysis
A secondary analysis will be done to compare the proportion of subjects with ≥20% sero-reduction or sero-
conversion for the 60-day and 30-day nifurtimox regimens. This will be performed using a standard, symmetric, 
asymptotic 2sided 95% CI for the difference of two independent proportions.
…
The relationship of serological results to qPCR results will be done using frequencies of matches and 
mismatches on the determination of disease status, and phicorrelation and kappa coefficient to assess the degree 
of agreement.  
…
Sample size is based on the primary endpoint, the difference in the proportion of nifurtimox subjects with ≥20%
sero-reduction (subjects older than 8 months of age and younger than 18 years of age) or sero-conversion
(subjects 8 months of age and younger) (60-day regimen) and the proportion of historical placebo subjects with 
≥20% seroreduction or sero-conversion for the two age groups. The test of superiority over placebo will be 
powered based on the detection of a difference in proportion of nifurtimox subjects with ≥20% sero-reduction 
or seroconversion of 0.10.  This is considered a clinically meaningful difference according to several experts in 
the field.  Given that the nifurtimox (60day) proportion of subjects with ≥20% seroreduction or seroconversion 
is 0.60, 220 evaluable subjects in this group will provide at least 80% power for an exact binomial test. This 
sample size will provide an exact 95% CI with a width of ≤0.15 (e.g., [0.53, 0.67]) for use in comparing 
nifurtimox to the proportion of historical benznidazole subjects with ≥20% sero-reduction or sero-conversion.
New text:
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with seroreduction or sero-conversion (60-day regimen) 
and the proportion estimated from historical data will be tested using an exact 2-sided 95% confidence interval 
(CI) for a single proportion. 
Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox subjects (60day 
regimen) with sero-reduction or seroconversion is greater than the proportion of historical placebo subjects with 
seroconversion.
…
Secondary efficacy analysis
The same CI for the primary endpoint analysis will also be used for comparison with the historical benznidazole 
subjects with sero-conversion to assess whether or not their cure rates are comparable.
…
The relationship of conventional serology results to qPCR results will be done using frequencies of matches and 
mismatches on the determination of disease status, and phicorrelation and kappa coefficient to assess the degree 
of agreement.  The relationship of non-conventional serology to conventional serology will be analyzed in the 
same way.
…
Sample size is based on the primary endpoint, the difference in the proportion of nifurtimox subjects with 
seroreduction or sero-conversion (60-day regimen) and the proportion of historical placebo subjects with sero-


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conversion. The test of superiority over placebo will be powered based on the detection of a difference in 
proportion of 0.10. This quantity represents a difference between rates, such as 0.60-0.50=0.10, rather than the 
difference that would result from subtracting 10% of 0.60 (that is, 0.06) from 0.60.  Given that the nifurtimox 
(60day) proportion of subjects with seroreduction or seroconversion is 0.60, 220 evaluable subjects in this group 
will provide at least 80% power for an exact binomial test. This sample size will provide an exact 95% CI with a 
width of ≤0.15 (e.g., [0.53, 0.67]) for use in comparing nifurtimox to the proportion of historical benznidazole 
subjects with sero-conversion.
…
List of abbreviations
This section was changed as a result of changes made to the body of the protocol.
Old text:
CCDS
Company Core Data Sheet
HAI
hemoagglutination inhibition
qPCR
qualitative polymerase chain reaction
New text:
Cmax
maximum concentration
OD
optical density 
qPCR
quantitative polymerase chain reaction
Introduction, Section 3
This section was changed since an Investigators’ Brochure became available as a reference 
document for nifurtimox.
Background
Old text:
…
Further details can be found in the latest available versions of the Company Core Data Sheets 
(CCDS), which contain comprehensive information on the study drug.
New text:
…
Comprehensive information on the study drug can be found in the latest available version of 
the Investigators’ Brochure.
Rationale of the study
This section was changed for clarity.
Old text:
…


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This study was designed to develop a better understanding of the efficacy, safety/tolerability, 
and pharmacokinetics (PK) (absorption, distribution, metabolism, and elimination) of 
nifurtimox in children with a diagnosis of Chagas’ disease.  Results from this study will 
demonstrate parasitological cure (using serological results) to allow optimization of treatment 
of Chagas’ disease in this most vulnerable population.  In turn, this will improve the outlook 
for children by reducing mortality and longterm complications.  This knowledge will allow 
for better and more appropriate approaches to the treatment of Chagas’ disease.  It will also 
provide more up-to-date information on the use of nifurtimox, and has the potential to 
increase our understanding of treatment response in children.
New text:
…
This study was designed to develop a better understanding of the efficacy, safety/tolerability, 
and pharmacokinetics (PK) (absorption, distribution, metabolism, and elimination) of 
nifurtimox in children with a diagnosis of Chagas’ disease using pediatric formulations.  
Results from this study will demonstrate parasitological cure using conventional serological 
results to allow optimization of treatment of Chagas’ disease in this most vulnerable 
population.  In turn, this will improve the outlook for children by reducing mortality and 
long-term complications.  This knowledge will allow for better and more appropriate 
approaches to the treatment of Chagas’ disease.  It will also provide more up-to-date 
information on the use of nifurtimox, and has the potential to increase our understanding of 
treatment response in children.
Benefit-risk assessment
This section was changed for clarity and as a result of Amendment 1.
Old text:
…
Nifurtimox is well tolerated in children, but its pharmacology in the pediatric population is 
still not well defined. The currently available formulation was developed for adults, and there 
is a significant need for a pediatric formulation particularly for infants and newborns.  The 
safety profile of the drug is relatively well-characterized…  
…
New text:
…
Nifurtimox is well tolerated in children, but its pharmacology in the pediatric population is 
still not well defined. The currently available marketed product was developed for adults, and 
there is a significant need for a pediatric formulation particularly for infants and newborns to 
support weightadjusted- dosing.  The safety profile of the drug is relatively 
well-characterized…  
…
Study objectives, Section 4


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This section was changed as a result of Modifications 2 and 10.
Old text:
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated 
control as seroreduction (≥20% sero-reduction compared to baseline in subjects older 
than 8 months of age and younger than 18 years of age), or sero-conversion (in 
subjects 8 months of age and younger) at the 12month follow-up (360 days from end 
of treatment [EOT]) 
Secondary objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole) as sero-reduction (≥20% seroreduction compared to baseline 
in subjects older than 8 months of age and younger than 18 years of age), or 
seroconversion (in subjects 8 months of age and younger) and qualitative polymerase 
chain reaction (qPCR) at the 12-month follow-up (360 days from EOT) 

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction (≥20% sero-reduction compared to baseline in subjects 
older than 8 months of age and younger than 18 years of age), or sero-conversion (in 
subjects 8 months of age and younger) and qPCR at the 12-month follow-up (360 days 
from EOT) 

To evaluate the relationship of conventional serology (≥20% sero-reduction in 
subjects older than 8 months of age and younger than 18 years of age, or 
seroconversion in subjects 8 months of age and younger) to qPCR using frequencies 
of matches and mismatches to assess agreement

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring 
(optional depending on the age of the subject and at the discretion of the investigator), 
vital sign measurements (blood pressure [optional depending on the age of the subject 
and at the discretion of the investigator], heart rate, respiratory rate, temperature), 
adverse event (AE) monitoring, and physical examinations

…
New text:
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated 
control at the 12month follow-up (360 days from end of treatment [EOT]) as 
seroreduction (defined as a ≥20% reduction in optical density [OD] measured by 
conventional enzymelinked immune sorbent assay [ELISA]) compared to baseline in 
subjects > 8 months to <18 years of age at randomization or sero-conversion (defined 


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as negative Immunoglobulin G [IgG] concentration) in subjects ≤8 months of age at 
randomization
Secondary objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole)27 as sero-reduction or sero-conversion at the 12-month 
follow-up (360 days from EOT)

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction or sero-conversion and to quantitative polymerase chain 
reaction (qPCR) at the 12-month follow-up (360 days from EOT) 

To evaluate the relationship of conventional serology (as sero-reduction or 
seroconversion) to qPCR using frequencies of matches and mismatches to assess 
agreement

To evaluate the relationship of non-conventional serology to conventional serology

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital 
sign measurements (blood pressure, heart rate, respiratory rate, temperature), adverse 
event (AE) monitoring, and physical examinations, including neurological 
examinations

…
Footnote 27 was added to the bottom of the page for clarity, new text:
27 Sero-conversion for the historical controls will be estimated from cure rates as presented in two 
publications.[15][16]  See Section 5, Study Design, Justification of the design.   
Study design, Section 5
This section was changed for clarity and as a result of Modifications 1, 2, 6, 8, 9, 10, 11, and 
12.
Figure 5-1 was replaced with a new figure as a result of changes made per Amendment 1.


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Old figure:
New figure:


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Old text:
This study will enroll pediatric subjects aged 0 to younger than 18 years.  Subjects younger 
than < 8 months of age with a diagnosis of Chagas’ disease must demonstrate direct 
observation of T. cruzi by concentration test, and subjects 8 months of age and older and 
younger than 18 years of age with a diagnosis of Chagas’ disease must demonstrate a positive 
enzymelinked immune sorbent assay (ELISA) plus a positive result for one of the following 
tests to confirm diagnosis:

Alternate ELISA (e.g., Chagatest ELISA recombinate v3.0)

Hemagglutination (e.g., Chagatest hemoagglutination inhibition [HAI])  

Direct Agglutination (e.g., SERODIA-Chagas) 
Subjects will be stratified by age into four cohorts as specified below:
…
…
Subjects will return to the investigational site for efficacy and safety assessments on Days 30 
(±3 days) and 60 (±3 days).  Day 60 will be the EOT for both treatment groups.  On Days 21 
(±3 days) and 42 (±3 days), study site personnel will contact the subject’s parent or legal 
guardian via telephone to assess the occurrence of AEs and concomitant medications.  
Subjects will then return to the site on Days 90, 240, and 420 (±7 days) after the start of 
treatment for the collection of additional efficacy and safety data.  The total duration of each 
subject’s participation is expected to be approximately 14.5 months.  
Assessments of efficacy and PK will be performed at specified time windows.  PK blood 
sampling will be optional on consent/assent of the subject/subject’s legally authorized 
representative(s). In order to minimize the burden of blood loss due to PK sampling,
ageappropriate sparse sampling strategies combined with (micro-)bioanalysis techniques will 
be applied.  
Safety will be assessed at specified time points via laboratory parameters (hematology, blood 
chemistry, urinalysis), vital sign measurements (blood pressure [optional depending on the 
age of the subject, at the discretion of the investigator], heart rate, respiratory rate, 
temperature), monitoring of ECGs (optional depending on the age of the subject and at the 
discretion of the investigator), monitoring of AEs, and physical examinations.  As an 
additional safety precaution, all subjects with a positive microscopy or qPCR test result at 
Day 30 (3 days) will be considered a treatment failure, discontinued from the study, and 
treated with an alternative antitrypanosomal therapy.  This condition implies that the decision 
criteria for determining the presence of Chagas’ disease use both serology and qPCR.
New text:
This study will enroll pediatric subjects aged 0 to younger than 18 years.  Subjects < 8 months 
of age at randomization with a diagnosis of Chagas’ disease must demonstrate direct 
observation of T. cruzi by concentration test, and subjects > 8 months to < 18 years of age at 


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randomization with a diagnosis of Chagas’ disease must demonstrate a positive conventional
ELISA result for both of the following tests to confirm diagnosis:

Recombinant ELISA

Total purified antigen ELISA 
Additionally, a non-conventional ELISA test will be obtained; however, a positive result will 
not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ disease 
within three months prior to screening. 
Subjects will be stratified by age at randomization into four cohorts as specified below:
…
No study-specific procedures will be performed before the subject/legally authorized 
representative has signed the informed consent form, or a minor subject has provided assent,  
including asking a potential subject to fast (if applicable, depending on the age of the subject 
at the discretion of the investigator) prior to the screening blood samples.  Informed 
consent/assent for optional PK blood sampling will also be obtained.  After the 
consents/assents have been obtained, screening assessments will be performed from 1 to 14 
days prior to randomization at Visit 2.
After study eligibility has been confirmed and safety assessments have been performed at 
Visit 2 (Day 1), subjects will be randomized via Interactive Voice Response 
System/Interactive Web Response System (IVRS/IWRS) in a 2:1 ratio (60-day regimen vs. 
30-day regimen) to one of two treatment groups: 
…
The first dose of study drug will be administered at Visit 2.  Pre- and post-dose PK blood 
samples will be obtained at specified time points from those subjects consenting to PK 
assessments (see Table 9-2).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the 
time of maximum concentration [Cmax] ), an ECG (optional for subjects < 5 years of age at the 
discretion of the investigator) will be obtained to allow for PK/PD investigations.  Study drug 
will be dispensed, and instructions for study drug administration will be provided to all 
subjects.
Subjects will return to the investigational site for efficacy and safety assessments at Visits 3 
(Day 7±1) and 6 (Day 30).  Subjects who have not consented to PK assessments will take that 
day’s doses of study drug as instructed.  Subjects consenting to PK assessments must hold the 
morning dose of study drug; for these subjects, a pre-dose PK blood sample will be obtained, 
then the morning dose of study drug will be administered.  Post-dose blood samples will be 
obtained at specified time points, and ECGs will be obtained at the 2 – 4 hour post-dose time 
point (see Table 9-2).  A pre-paid phone card will be provided to all subjects to facilitate 
contact between study site personnel and the subjects/subjects’ authorized representatives.  
At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and 
study drug for the remaining 30 days of treatment will be dispensed.  


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Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site 
personnel will contact the subject/legally authorized representative via telephone to assess the 
occurrence of AEs, use of concomitant medications, and compliance with study drug 
administration.  A Phone Contact Form for the telephone assessments will be provided to all 
sites (see Section 16.1).
Subjects will return to the investigational site for efficacy and safety assessments on Day 60 
(Visit 8), which will be the EOT for both treatment groups.  Subjects who have not consented 
to PK assessments will take that day’s doses of study drug as instructed.  Subjects consenting 
to PK assessments must hold the morning dose of study drug.  A pre-dose PK blood sample 
will be collected, then the morning dose of study drug will be administered.  Post-dose PK 
blood samples will be obtained at specified time points, and ECGs will be obtained at the 2 4 
hour post-dose time point (see Table 9-2).  Study drug will be collected, and no additional 
study drug will be dispensed.  
After the EOT visit (Visit 8), subjects will return to the site on Days 90 (Visit 9), 240 
(Visit 10), and 420 (Visit 11) for additional efficacy and safety assessments.  The total 
duration of each subject’s participation is expected to be approximately 14 months.  
Assessments of efficacy and PK will be performed at specified time windows.  In order to 
minimize the burden of blood loss due to PK sampling, ageappropriate sparse sampling 
strategies combined with (micro-)bioanalysis techniques will be applied (see Table 94).  At 
least 10 subjects per age cohort will be recruited for PK assessments.   
Safety will be assessed via laboratory parameters (hematology, blood chemistry, urinalysis), 
vital sign measurements (blood pressure [optional in subjects < 5 years of age at the discretion 
of the investigator], heart rate, respiratory rate, temperature), monitoring of ECGs (optional in 
subjects < 5 years of age at the discretion of the investigator), monitoring of AEs, and 
physical examinations, including a neurological examination (see Section 9.6.3.3).
Subjects who discontinue prematurely from study drug administration will continue to return 
to the investigational site for study assessments at Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7.  If subjects are unable/unwilling to 
do so, they must return to the investigational site 30 (±3) days after the last dose of study drug 
for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 
5, and 7.  If the subject is unable/unwilling to return to the clinic for the EOT Visit (Visit 8), 
then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of 
Visit 8 assessments.
Primary variable(s)
This section was changed as a result of Modification 2.
Old text:
The primary efficacy variable will be a ≥20% sero-reduction (subjects older than 8 months 
of age and younger than 18 years of age) or sero-conversion (subjects 8 months of age and 
younger) in antibody Immunoglobulin G (IgG) titer at 12 months posttreatment using 
conventional ELISA serology as the measure of efficacy.


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New text:
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months 
posttreatment using two conventional ELISA serology tests as the measure of efficacy. 
Justification of the design
This section was changed for clarity and to provide updated study information.
Old text:
This study uses an historical untreated-controlled design as it is unethical to have a 
prospective placebo-controlled superiority study design.  Only two benznidazole 
placebocontrolled studies have been conducted in children with Chagas’ disease.[15][16]  
Direct comparisons between these studies cannot be made due to differences in study 
populations (Argentina vs. Brazil), medication dosage (5 mg/kg vs. 7.5 mg/kg), outcome 
measures, and time of follow-up (4 years vs. 3 years).  Data derived from these studies will, 
however, be used as the comparative historical placebo control to be utilized in the analysis of 
the study endpoints.  Hence, a comparison of sero-reduction from baseline to 12-month 
follow-up for both primary and secondary endpoint evaluations (versus historical placebo) 
will be made as well as for a non-inferiority analysis versus benznidazole.  Details are 
presented in Section 10.
…
Sero-reduction of 20% at 12 months post-treatment will be considered equivalent to historical 
information on seroconversion at subsequent time points.
Both primary and secondary study endpoints are based upon a target of ≥20% decrease in 
IgG antibody titers (sero-reduction) to T. cruzi in conventional serological tests or 
negativization of serology.  This is consistent with one previous study, which showed a 20% 
sero-reduction at 12 months in children 6 to 12 years of age infected with T. cruzi in the 
intermediate (early chronic) phase of Chagas’ disease.[15]  Subjects were administered 
5mg/kg benznidazole or placebo in a randomized fashion for 60 days, and followed for 
48 months.  Sero-conversion to a negative result after the 48-month post-treatment follow-up 
was detected by conventional serology in 11.3 % (P < 0.05) of the benznidazole groups and 
only 4.5% of the placebo group.  In the benznidazole group, a significant decrease in the mean 
antibody titer against T. cruzi as measured by three different assays including ELISA was 
shown over the course of the study.  No significant changes were observed in the placebo 
group.  At 12 months, a 21% sero-reduction titer compared to baseline (ELISA mean OD 
0.467 [± 0.099] vs. 0.369 [± 0.107]) was observed in subjects who received benznidazole.  
For the subsequent time points of 18, 24, and 48 months, percent titer reductions were 23%, 
29%, and 27%, respectively.  At the end of the 48-month follow-up period, nearly 62% of 
benznidazole-treated subjects and none of the placebotreated subjects were sero-negative for 
T. cruzi.  Xenodiagnosis performed at that time was positive in 51.2% of the placebo group 
and 4.7% of the benznidazole group (P<0.001).  The low frequency of positive xenodiagnosis 
would be indicative of a low parasitemia and, therefore, low antigenic load which would 
prevent development of chronic disease.
…


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While a decrease ≥20% in antibody titer has been demonstrated across two independent 
clinical trials in pediatric patients with Chagas’ disease, neither author has published extended 
follow-up data which include serological results for the patients in these studies.  Since signs 
and symptoms are usually lacking in early Chagas’ disease, it is not possible to demonstrate 
immediate meaningful clinical benefit to active treatment.  However, it is presumed that 
significant reductions in T. cruzi antibody titer (seroreduction) by conventional serology is an 
early predictor of future sero-conversion, and early treatment and cure of infection will 
ultimately reduce the risk of developing visceral abnormalities, as well as contribute to the 
decrease in T. cruzi transmission.
In the experience of Parasitology service, Ricardo Gutierrez Children´s Hospital, Buenos 
Aires unpublished data (presented at European Society of Pediatric Infectious Disease 2014 
and meeting of Food and Drug Administration) by 
, a correlation between 
negativization of conventional serology (whole antibody against T. cruzi measured by ELISA 
or HAI) and PCR results in children 2 years of age and younger is fairly high; the correlation 
between these two tests in children older than 3 years is lower, as sero-conversion (i.e., 
becoming negative) may take several years, while PCR becomes negative within 60 days of 
treatment, and remains negative in this age group also.  In the whole population, at least 20% 
sero-reduction of conventional serology was observed at 12 months.  This percentage was 
higher in the younger children than in the older children.
Clearance of parasitemia (evaluated most commonly by xenodiagnoses or PCR) combined 
with disappearance of antibodies (seronegative conversion) has been proposed as cure criteria 
by some authors. The main limitations in evaluating treatment response for Chagas’ disease 
using conventional serology stems from the need for long-term follow-up (years to decades) 
to observe negativization of conventional serological tests.  (lytic antibodies measured by 
chemiluminescent ELISA with purified trypomastigote), rapid serological response (i.e., 
negative titers) in a shorter time period were demonstrated.[16][18]  
These non-conventional serological techniques are not standardized, have showed poor 
reproducibility, and are expensive and difficult to perform, and the preparation of the antigens 
is complex.  Furthermore, there are no commercial reagents available for these tests, which 
means that the techniques must be prepared in-house with the consequent reproducibility 
issues among centers, and even within batches in the same center.
A new perspective on Chagas’ disease diagnosis was opened by the use of the PCR as a 
measure of treatment response.  Over the past few years, the availability of PCR to detect 
T. cruzi DNA in blood samples has opened new possibilities for the evaluation of response to 
trypanocidal chemotherapy.  Furthermore, real-time PCR (qPCR) protocols for detection of 
T. cruzi’s DNA were developed.
There was no generally accepted and commercially available PCR method or kit available 
until recently.  Many protocols were available differing with respect to 1) the method of DNA 
isolation, 2) the PCR conditions, i.e., conventional endpoint or real-time quantitative PCR, 
3) use or lack of use of a Taqman probe, which increases the specificity of the test[19], and 
4) inclusion of an internal standard, which can be employed to prove the validity of the data.
…
PPD


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Subsequent development of qPCR assays have several advantages compared with 
conventional PCR.  First, the introduction of closed-tube qPCR systems has resulted in the 
development of rapid microbial diagnostics with low contamination risk.  Also, qPCR has a 
shorter time-to-result, and allows quantitative assays and automation.[2725]
Studies have demonstrated the substantial value of qPCR, in terms of sensitivity and 
specificity, in the direct parasitological diagnosis of Chagas’ disease and follow-up 
assessment of chemotherapy.  However, in this Phase 3 study, parasitological cure based on 
serological tests for T. cruzi infection (ELISA) will be used as the primary efficacy endpoint 
to demonstrate efficacy of nifurtimox.  Other parasitological methods (direct microscopic) 
and qPCR tests will be secondary efficacy variables…
The 30-day and 60day treatment regimens were selected with the intention of providing the 
most effective treatment within the shortest possible duration.  
…
New text:
This study uses an historical untreated-controlled design as it is unethical to have a 
prospective placebo-controlled superiority study design.  Only two benznidazole 
placebocontrolled studies have been conducted in children with Chagas’ disease.[15][16]  
Direct comparisons between these studies cannot be made due to differences in study 
populations (Argentina vs. Brazil), medication dosage (5 mg/kg vs. 7.5 mg/kg), outcome 
measures, and time of follow-up (4 years vs. 3 years).  Data derived from these studies will, 
however, be used as the comparative historical placebo control to be utilized in the analysis of 
the study endpoints.  Hence, a comparison of sero-reduction and/or sero-conversion from 
baseline to 12-month follow-up for both primary and secondary endpoint evaluations (versus 
historical placebo) will be made as well as for a comparison with benznidazole.  Details are 
presented in Section 10.
…
Sero-reduction (measured as a ≥20% reduction of the ELISA absorbance [OD] value) at 
12 months post-treatment will be considered equivalent to historical information on 
seroconversion at subsequent time points.
Both primary and secondary study endpoints are based upon a target of ≥20% decrease in 
antibody concentration (sero-reduction) to T. cruzi in conventional serological tests or 
negativization of serology.  This is consistent with one previous study in children 6 to 
12 years of age infected with T. cruzi in the intermediate (early chronic) phase of Chagas’ 
disease.[15]  Subjects were administered 5mg/kg benznidazole or placebo in a randomized 
fashion for 60 days, and followed for 48 months.  Sero-conversion to a negative result after 
the 48-month post-treatment follow-up was detected by conventional serology in 11.3 % 
(P < 0.05) of the benznidazole groups and only 4.5% of the placebo group.  In the 
benznidazole group, a significant decrease in the mean antibody concentration against 
T. cruzi as measured by three different assays including ELISA was shown over the course of 
the study.  No significant changes were observed in the placebo group.  At 12 months, a 21% 
reduction in concentration of Chagas’ diseasespecific antibodies compared to baseline 


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(ELISA mean OD 0.467 [± 0.099] vs. 0.369 [± 0.107]) was observed in subjects who received 
benznidazole.  For the subsequent time points of 18, 24, and 48 months, percent concentration
reductions were 23%, 29%, and 27%, respectively.  At the end of the 48-month follow-up 
period, nearly 62% of benznidazole-treated subjects and none of the placebotreated subjects 
were sero-negative for T. cruzi.  Xenodiagnosis performed at that time was positive in 51.2% 
of the placebo group and 4.7% of the benznidazole group (P<0.001).  The low frequency of 
positive xenodiagnosis would be indicative of a low parasitemia and, therefore, low antigenic 
load which would prevent development of chronic disease.
…
While a decrease ≥20% in antibody concentration has been demonstrated across two 
independent clinical trials in pediatric patients with Chagas’ disease, neither author has 
published extended follow-up data which include serological results for the patients in these 
studies.  Since signs and symptoms are usually lacking in early Chagas’ disease, it is not 
possible to demonstrate immediate meaningful clinical benefit to active treatment.  However, 
it is presumed that significant reductions in T. cruzi antibody concentration (seroreduction) by 
conventional serology is a predictor of future sero-conversion, and early treatment and cure of 
infection will ultimately reduce the risk of developing visceral abnormalities, as well as 
contribute to the decrease in T. cruzi transmission.
Alternative and more rapid approaches as an early indicator of negative sero-conversion in 
treated patients have been suggested.  Using non-conventional serological techniques, i.e., 
highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote 
glycoconjugate antigen and an epimastigote complex[20], the time to negativization was 
significantly sooner  for the nonconventional ELISA than for the conventional ELISA.  The 
chemiluminescent ELISAs provide tests that are highly sensitive and specific for Chagas' 
disease diagnosis.  They can be used in blood bank screening and to monitor the treatment of 
patients undergoing chemotherapy.[21]
Non-conventional ELISA antigens, such as those used in the Sosa-Estani and deAndrade
trials (i.e., F29 and F 2/3),[15][16] are often considered as early markers for effective Chagas’ 
disease therapy.  In the study by Altcheh, et al, the kinetics of disappearance of conventional 
serology and anti-F2/3 antibodies were compared in 21 patients with congenital Chagas’ 
disease after receiving benznidazole treatment.  Patients were divided into two groups: (A) 
age < 8 months at diagnosis and (B) age > 9 months at diagnosis.  Group A presented 
negative outcome for conventional serology at 6.6 months (CI 95 3.4-9.8 months) and for 
anti-F2/3 at 4 months (CI 95 0.9-7.1 months, p = 0.18).  Group B exhibited non-reactive 
conventional serology at 63.1 months (CI 95 42.1-84.2 months) whereas anti-F2/3 antibody 
determination became negative at 21.9 months (CI 95 5.7-38.1 months, p = 0.0025).  In 
patients belonging to Group A, antibodies were undetectable by both conventional serology 
and anti F2/3 ELISA soon after receiving chemotherapy.  In infants included in Group B, a 
negative result for anti-F2/3 antibody detection occurred significantly in advance of negative 
conventional serology reactivity.  Consequently, the  antiF2/3 antibody assay becoming 
negative should be considered as a surrogate endpoint for assessment of cure or positive 
response to treatment, particularly in those patients with prolonged time of infection.[18]


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A new perspective on Chagas’ disease diagnosis was opened by the use of the PCR as a 
measure of treatment response.  Over the past few years, the availability of PCR to detect 
T. cruzi DNA in blood samples has opened new possibilities for the evaluation of response to 
trypanocidal chemotherapy.  
Clearance of parasitemia (evaluated most commonly by xenodiagnoses or PCR) combined 
with disappearance of antibodies (seronegative conversion) has been proposed as cure criteria 
by some authors.[19]  The main limitations in evaluating treatment response for Chagas’ 
disease using conventional serology stems from the need for long-term follow-up (years to 
decades) to observe negativization of conventional serological tests.  
In the experience of Parasitology service, Ricardo Gutierrez Children´s Hospital, Buenos 
Aires unpublished data (presented at European Society of Pediatric Infectious Disease 2014 
and meeting of Food and Drug Administration) by 
, a correlation between 
negativization of conventional serology (whole antibody against T. cruzi measured by ELISA 
or hemoagglutination inhibition) and PCR results in children 2 years of age and younger is 
fairly high; the correlation between these two tests in children older than 3 years is lower, as 
sero-conversion (i.e., becoming negative) may take several years, while PCR becomes 
negative within 60 days of treatment, and remains negative in this age group also.  In the 
whole population, at least 20% sero-reduction of conventional serology was observed at 
12 months.  This percentage was higher in the younger children than in the older children.
…
Furthermore, real-time PCR (qPCR) protocols for detection of T. cruzi’s DNA were 
developed.  The development of qPCR assays have several advantages compared with 
conventional PCR.  First, the introduction of closed-tube qPCR systems has resulted in the 
development of rapid microbial diagnostics with low contamination risk.  Also, qPCR has a 
shorter time-to-result, and allows quantitative assays and automation.[27]
Studies have demonstrated the substantial value of qPCR, in terms of sensitivity and 
specificity, in the direct parasitological diagnosis of Chagas’ disease and follow-up 
assessment of chemotherapy.  However, in this Phase 3 study, parasitological cure based on 
serological tests for T. cruzi infection (ELISA) will be used as the primary efficacy endpoint 
to demonstrate efficacy of nifurtimox due to lack of historical PCR data from untreated 
children with Chagas’ disease.  Other parasitological methods (direct microscopic) and qPCR 
tests will be secondary efficacy variables…
The 30-day and 60day treatment regimens were selected with the intention of providing the 
most effective treatment within the shortest possible duration.  In a recent study from Bianchi 
et al, 62 patients with Chagas’ disease confirmed by different serological tests were treated 
with nifurtimox (Lampit), and followed for 30 months post-treatment.  All children were 
treated during 60 days according to protocols established by the WHO.  Monitoring was 
performed every 20 days to evaluate treatment safety.  Results indicated that both parasite 
load (measured through qPCR) and antibodies (ELISA absorbance) showed a significant 
median reduction 6 months after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 
0.6 to 0.2 absorbance units respectively (p<0.001).  Further reductions were evidenced by the 
30-month post treatment time point.  Sixty days of treatment with nifurtimox was very well 
PPD


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tolerated and successfully reduced parasite load and antibody titers.  The results show for the 
first time the therapeutic response and safety of nifurtimox treatment for T. cruzi infection in a 
population of school-aged children in the asymptomatic chronic phase of Chagas’ disease in 
Colombia.[28]
End of study
This section was changed to revise the template language.
Old text:
For this study, important data will be collected after last-patient last-visit; the end of the study 
as a whole will be the date when the clean database is available.
New text:
The end of the study as a whole will be reached as soon as the last visit of the last subject has 
been reached in all centers in all participating countries (EU and non-EU). 
Study population, Section 6
This section was changed for clarity and as a result of Modification 2.
Inclusion criteria, Section 6.1
Old text:
1. Male and female pediatric subjects aged 0 days to younger than 18 years
2. Chagas’ disease diagnosed/confirmed by:

Subjects younger than 8 months of age must demonstrate direct observation of T. cruzi
by concentration test

Subjects 8 months of age and older and younger than 18 years of age must 
demonstrate a positive ELISA plus a positive result for one of the following tests to 
confirm diagnosis:

Alternate ELISA (e.g., Chagatest ELISA recombinate v3.0)

Hemoagglutination (e.g., Chagatest HAI)  

Direct Agglutination (e.g., SERODIA-Chagas) 
…
New text:
1. Male and female pediatric subjects aged 0 days to younger than 18 years
2. Chagas’ disease diagnosed/confirmed by:

Subjects < 8 months of age at randomization must demonstrate direct observation of 
T. cruzi by concentration test


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
Subjects ≥8 months to < 18 years of age at randomization must demonstrate a 
positive conventional ELISA result for both of the following tests to confirm 
diagnosis:

Recombinant ELISA

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test will be obtained; however, a positive result 
will not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ 
disease within three months prior to screening and they have not had prior 
anti-trypanocidal or anti-parasitic treatment.
…
Exclusion criteria, Section 6.2
This section was changed for clarity.
Old text:
3. Subjects with contraindications/warnings to nifurtimox administration, or with conditions 
that may increase the risk of the undesirable effects of nifurtimox, including:

…

…

Severe renal impairment defined by the following:
o
For subjects < 1 year of age, estimated glomerular filtration rate (eGFR) 
< 100% of the lower limit of normal (LLN) appropriate for age (eGFR should 
be calculated according to Schwartz formula; see Section 16.1) 
o
For subjects ≥1 year to < 18 years of age, eGFR < 80% of LLN appropriate 
for age 
New text:
3. Subjects with contraindications/warnings to nifurtimox administration, or with conditions 
that may increase the risk of the undesirable effects of nifurtimox, including:

…

…

Severe renal impairment defined by the following:
o
For subjects < 1 year of age at randomization, estimated glomerular filtration 
rate (eGFR) < 100% of the lower limit of normal (LLN) appropriate for age 
(eGFR should be calculated according to Schwartz formula; see Section 16.2) 
o
For subjects ≥1 year to < 18 years of age at randomization, eGFR < 80% of 
LLN appropriate for age 


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…
Withdrawal of subjects from study, Section 6.3
This section was changed for clarity.
Withdrawal, Section 6.3.1
Withdrawal criteria
Old text:
Subjects must be withdrawn from the study if any of the following occurs:

At their own request or at the request of their legally acceptable representative…

Pregnancy.  If any urine pregnancy test is positive, a serum pregnancy test must be 
performed to confirm the result.  See Section 9.6.2.

Positive microscopy or qPCR at Visit 5 (Day 30).  All subjects with a positive 
microscopy or qPCR test result at Visit 5 will be considered a treatment failure, 
discontinued from the study, and treated with an alternative antitrypanosomal therapy.
New text:
Subjects must be withdrawn from the study if any of the following occurs:

At their own request or at the request of their legally acceptable representative…

Pregnancy.  If any urine pregnancy test is positive, a serum pregnancy test must be 
performed to confirm the result.  See Section 9.6.2.
Screening failure
This section was changed for clarity.
Old text:
…
Re-starting the defined set of screening procedures to enable the “screening failure” subject’s 
participation at a later time point is not allowed with the following exceptions:

The subject had successfully passed the screening procedures, but could not start 
subsequent treatment on schedule.

Initial screening occurred too early to complete the required washout period after prior 
therapy.

The in-/exclusion criteria preventing the subject’s initial attempt to participate have 
been changed (via protocol amendment).
Subjects not meeting inclusion/exclusion criteria at screening may be re-screened once.  In 
any case, the investigator has to ensure that the repeated screening procedures do not expose 
the subject to an unjustifiable health risk.  Also, for re-screening, the subject has to re-sign the 
informed consent form, even if it was not changed after the subject’s previous screening.  


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…
New text:
…
Re-starting the defined set of screening procedures to enable the “screening failure” subject’s 
participation at a later time point is allowed only in the following situations:

The subject had successfully passed the screening procedures, but could not start 
subsequent treatment on schedule.

Initial screening occurred too early to complete the required washout period (Day -14 
to Day -1) after prior therapy.

The in-/exclusion criteria preventing the subject’s initial attempt to participate have 
been changed (via protocol amendment).
Subjects not meeting inclusion/exclusion criteria at screening may be re-screened once. At 
the discretion of the investigator and in consultation with Bayer, the screening period may be 
extended up to 3 days to allow for receipt of test results or to evaluate changes in a subject’s 
condition.  In any case, the investigator has to ensure that the repeated screening procedures 
do not expose the subject to an unjustifiable health risk.  Also, for re-screening, the subject 
has to re-sign the informed consent form, even if it was not changed after the subject’s 
previous screening.  The second re-screening will be captured in the eCRF.
…
Treatment(s), Section 7
Treatments to be administered, Section 7.1
This section was changed for clarity.
Old text:
Each subject will receive nifurtimox oral tablets  three times daily for 60 days, or for 30 days 
followed by nifurtimox placebo administered three times daily for 30 days.  Nifurtimox 30mg 
and 120-mg tablets have score lines and can be divided into two equal halves to allow for 
15mg and 60-mg dose increments, respectively.
Each nifurtimox tablet…
New text:
Each subject will receive nifurtimox oral tablets  three times daily for 60 days, or for 30 days 
followed by nifurtimox placebo administered three times daily for 30 days
Each nifurtimox tablet...
Identity of study treatment, Section 7.2
This section was changed for clarity.
Old text:


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Nifurtimox 30-mg and 120-mg oral tablets and matching placebo tablets for each dosage form 
will be provided by Bayer HealthCare AG.  The tablets for both dosage forms are yellow, 
round, and biconvex. The matching placebo tablets for the 30-mg and 120-mg formulations 
will bear the same appearance and packaging as the active study drug tablets.
Riboflavin is included as a colorant in the matching placebo tablets for study drug blinding 
purposes. A common side effect of riboflavin ingestion is bright yellow urine. Reports of 
urine color changes will not be considered as AEs but they will be entered in the eCRF.
…
New text:
Nifurtimox 30-mg and 120-mg oral tablets and matching placebo tablets for each dosage form 
will be provided by Bayer HealthCare AG.  The tablets for both dosage forms are yellow, 
round, and biconvex. The matching placebo tablets for each dosage (30-mg and 120-mg 
formulations) will bear the same appearance and packaging as the active study drug tablets.
…
Dosage and administration, Section 7.4
This section was changed for clarity and per Modification 4.
Old text:
…
Nifurtimox will be administered three times a day, preferably in the morning, at noon, and at 
night, with food.  The 30-mg and 120-mg tablets have score lines and can be divided into two 
equal halves to allow for 15-mg or 60 mg dose increments, respectively.  The tablet will be
manufactured for quick disintegration in order to allow administration to subjects < 6 years 
old who are not able to swallow tablets.  Before administration, the tablet should be dissolved 
in a small amount (5 mL) of water on a teaspoon to form a soft slurry1 that should be given 
immediately with food.  Nifurtimox will be administered based on body weight as presented 
in Table 7-2.
New text:
…
Study drug will be dispensed at Visits 2 (Day 1) and 6 (Day 30), with instructions for 
administration.  Nifurtimox will be administered three times a day, in the morning, at noon, 
and at night, with food.  The 30-mg and 120-mg tablets have score lines and can be divided 
into two equal halves to allow for 15-mg or 60 mg dose increments, respectively.  The tablet 
is manufactured for quick disintegration in order to allow administration to subjects < 6 years 
old who are not able to swallow tablets.  Before administration, the tablet should be dissolved 
in enough water to fill a teaspoon (approximately 5 mL) to form a soft slurry69 that should be 
given immediately with food.  Nifurtimox will be administered based on body weight as 
presented in Table 7-2.
Footnote 1 was renumbered to 69, and revised for clarity as follows:


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Old text:
1 Bioequivalence testing of the slurry is being evaluated in an ongoing study.  Preliminary results indicate PK 
comparable to a tablet; final results expected by end of 2014.
New text:
70 Bioequivalence testing of the slurry has determined it to be comparable to 4 x 30mg tablets and the 120mg 
tablet.
Table 7-2 was modified to change upper limit of body weight in each category in Column 2.  
Old Table 7-2:
Table 7-2:  Dosage of nifurtimox oral tablets by body weight
Age Group
Body Weight (kg)
Number of 30-mg 
Tablets
Number of 120-mg 
Tablets
< 12 years
3 - 5
3 x daily ½ tablet
---
6 - 9
3 x daily 1 tablet
---
10 - 15
3 x daily 1 ½ tablets
---
16 - 18
3 x daily 2 tablets
---
19 - 21
3 x daily 2 ½ tablets
---
22 - 26
3 x daily 3 tablets
---
27 - 34
3 x daily 3 ½ tablets
---
35 - 40
---
3 x daily 1 ½  tablets
12 to ≤18 years
41 - 45
---
3 x daily 1 tablet
46 - 50
---
3 x daily 1 tablet
51 - 55
---
3 x daily 1 ½ tablets
56 - 60
---
3 x daily 1 ½ tablets
61 - 70
---
3 x daily 1 ½ tablets
71 - 80
---
3 x daily 2 tablets
81 - 90
---
3 x daily 2 tablets 
    > 90
---
3 x daily 2  ½ tablets


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New Table 7-2:
Table 7-2:  Dosage of nifurtimox oral tablets by body weight 
Age Group
Body Weight (kg)
Number of 30-mg 
Tablets
Number of 120-mg 
Tablets
< 12 years
3 - <6
3 x daily ½ tablet
---
6 - <10
3 x daily 1 tablet
---
10 - <16
3 x daily 1 ½ tablets
---
16 - <19
3 x daily 2 tablets
---
19 - <22
3 x daily 2 ½ tablets
---
22 - <27
3 x daily 3 tablets
---
27 - <35
3 x daily 3 ½ tablets
---
35 - <41
---
3 x daily 1 ½  tablets
≥12 to < 18 years
41 - <46
---
3 x daily 1 tablet
46 - <51
---
3 x daily 1 tablet
51 - <56
---
3 x daily 1 ½ tablets
56 - <61
---
3 x daily 1 ½ tablets
61 - <71
---
3 x daily 1 ½ tablets
71 - <81
---
3 x daily 2 tablets
81 - <91
---
3 x daily 2 tablets 
    91 or greater
---
3 x daily 2  ½ tablets
New text added beneath table:
At Visits 2 and 6, when study drug is dispensed, subjects/legally authorized representatives 
will be provided with a diary on which to document the date and doses (morning, noon, and 
night) of study drug, and comments (see Section 16.3).  Subjects/legally authorized 
representatives will be instructed to bring their diaries with them to each in-clinic study visit.  
The diaries will be collected and reviewed at each study visit to perform drug accountability 
and assess treatment compliance.
Riboflavin is included as a colorant in the matching placebo tablets for study drug blinding 
purposes. A common side effect of riboflavin ingestion is bright yellow urine. Subjects 
and/or their legally authorized representatives will be advised of the possibility of change in 
the subject’s urine color within the informed consent form, and when study drug is 
administered and dispensed at the investigational site.
Subjects consenting to optional PK assessments must withhold taking the morning dose of 
study drug on the day of Visits 3, 6, and 8, when pre- and post-dose PK blood samples will be 
obtained.  Subjects not consenting to optional PK assessments may take their study drug on 
the mornings of Visits 3, 6, and 8 as instructed.
Drug logistics and accountability, Section 7.6
This section was changed for clarity.
Old text:


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Details regarding storage conditions for nifurtimox are provided in the CCDSs.  Additional 
details regarding storage conditions may be provided separately.  Nifurtimox and the 
matching placebo are not to be stored above 25°Celsius.
…
New text:
At Visit 6 (Day 30), subjects will return their diaries, all remaining study drug, and empty 
packaging.  The diaries will be reviewed to assess study drug accountability, and study drug 
for the remaining 30 days of treatment will be dispensed.  
At Visit 8 (Day 60), subjects will return their diaries, all remaining study drug, and empty
packaging.  The diaries will be reviewed to assess study drug accountability, and no 
additional study drug will be dispensed. 
Details regarding storage conditions for nifurtimox are provided in the Investigators’ 
Brochure.  Additional details regarding storage conditions may be provided separately.  
Nifurtimox and the matching placebo are not to be stored above 25°Celsius.
…
Treatment compliance, Section 7.7
This section was changed for clarity and per Modification 12.
Old text:
Study site personnel will perform a tablet count of all returned study medication to determine 
treatment compliance. Any discrepancies between actual and expected amount of returned 
study medication must be discussed with the subject at the time of the visit, and any 
explanation must be documented in the source records.
New text:
Study site personnel will review diaries and perform a tablet count of all returned study 
medication to determine treatment compliance. Any discrepancies between actual and 
expected amount of returned study medication must be discussed with the subject at the time 
of the visit, and any explanation must be documented in the source records.
Treatment compliance will also be assessed during the telephone contacts at Visits 4, 5, and 7 
(see Section 16.1).
Non-study therapy, Section 8
This section was changed for clarity and per Modifications 6 and 12.
Prior and concomitant therapy, Section 8.1
Old text:
…
All prior and concomitant medications administered from the time the informed 
consent/assent is signed during the study will be recorded in the subject’s source 


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documentation file and reported in the eCRF.  The following information will be entered in 
the eCRF: generic name of the medication, indication, dose, unit, frequency, route of 
administration, and start and stop date (if stopped before the end of study).
…
New text:
…
All prior and concomitant medications administered from the time the informed 
consent/assent is signed until the last follow-up visit will be recorded in the subject’s source 
documentation file and reported in the eCRF.  Information on concomitant medication use 
will also be recorded on the Phone Contact Form which will be completed during the 
telephone assessments at Visits 4, 5, and 7 (see Section 16.1).  The following information will 
be entered in the eCRF: generic name of the medication, indication, dose, unit, frequency, 
route of administration, and start and stop date (if stopped before the end of study).
…
Procedures and variables, Section 9
Tabular schedule of evaluations, Section 9.1
This section was changed as a result of Modifications 1, 2, 6, 8, 9, 10, and 11.
Table 9-1 was modified to reflect changes made per Amendment 1.  
Old table:


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                           Table 9-1:  Schedule of procedures and assessments – Study 16027
Phase Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5
6 a
7 (EOT) b
8
9
10
Day
-14 to -1
1 c
7 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
Initiation procedures
Informed consent/assent

Demographics (sex, age)

Medical/surgical history

Inclusion/exclusion criteria


Height/length d and weight 








Efficacy
Presence of Chagas’ disease symptoms








Clinical evaluation






Microscopy (subjects < 8 months of age)

e
f
Serologic testing for Chagas’ disease

e




qPCR

e
f



Safety
Adverse events



a

a




Physical examination g








Vital signs (heart rate, RR, BP [optional], 
temperature) h








12-lead ECG (optional) h

e




Hematology and blood chemistry i

e




Urinalysis (local lab) (if specimen can be 
obtained)

e




Urine pregnancy test j






Pharmacokinetics
PK blood sampling (optional) k




Medication
Concomitant medication



a

a


Study drug administration 



Study drug accountability



Study drug dispensed and/or collected l




Review of study drug compliance



Dispense/collect pre-paid phone card m




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BP = blood pressure, ECG = electrocardiogram, EOT = end-of-treatment, qPCR = qualitative polymerase chain reaction, RR = respiratory rate per minute.
a  At Days 21 and 42, study site personnel will contact the subject’s parent or legal guardian via telephone to assess for the occurrence of adverse events and concomitant 
medications. If any safety concern arises, subjects may return to the study site for an Unscheduled Visit.
b  Subjects discontinuing from the study early are to undergo all study assessments as described in the EOT Visit.
c  All assessments are to be obtained pre-treatment on Day 1.
d  Height/length and weight will be obtained with the subject wearing minimal clothing and without shoes.
e  May be done at Visit 2 (Day 1) if not performed at screening or if the results are not available.
f   Subjects with a positive microscopy or qPCR test result will be considered a treatment failure, discontinued from the study, and treated with an alternative antitrypanosomal therapy.
g  A complete physical examination, including a complete review of body systems, will be performed at the Screening Visit.  A directed physical examination, including assessments of 
heart, lungs, and abdomen, skin for the presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will be performed at all subsequent visits.  
h Blood pressure and 12-lead ECG are optional depending on the age of the subject and at the discretion of the investigator. Whenever possible, ECGs should be obtained in close 
proximity to the 10- to 120-minute PK sample collection time window.
i  Serum laboratory assessments are to be performed with the subject in a fasting state.
J Urine pregnancy tests will be performed on all females of childbearing potential (i.e., all female subjects who have experienced menarche).  Any subject with a positive urine 
pregnancy test will have a serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be discontinued from the study and undergo all study assessments as 
described in the EOT Visit.
k   Fasting blood samples for pharmacokinetic parameters are optional on consent/assent of the subject/subject’s legally authorized representative(s).
l  Depending on the country, study site personnel may dispense condoms at the time that study drug is dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.
m  A pre-paid phone card will be provided to facilitate contact between study site personnel and the subjects/subjects’ authorized representatives at Days 21 and 42.  


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New table:
Table 9-1:  Schedule of procedures and assessments – Study 16027 
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
Initiation procedures
Informed consent/assent

Demographics (sex, age)

Medical/surgical history

Inclusion/exclusion criteria


Efficacy
Assessment of Chagas’ disease signs and symptoms d







Concentration test for T. cruzi (subjects < 8 months of age)






Conventional and non-conventional serologic testing for 
Chagas’ disease






qPCR






Safety
Adverse events











Concomitant medication











Physical examination e







Neurological examination f




Vital signs (heart rate, RR, BP, temperature), height/length, 
and weight g








12-lead ECG h






Serum hematology, chemistry, and coagulation i





Urinalysis (if specimen can be obtained)





Urine pregnancy test  j






Pharmacokinetics
PK blood sampling (optional) k




Study Treatment
Study drug administration l



Study drug and diary dispensed m


n
Study drug and diary collected

n

Study drug accountability/review compliance








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Dispense pre-paid phone card o


Collect pre-paid phone card


BP = blood pressure, ECG = electrocardiogram, EOT = end-of-treatment, qPCR = quantitative polymerase chain reaction, PK = pharmacokinetic, RR = respiratory rate per minute.
a  At Days 14, 21, and 42, study site personnel will contact the subject/legally authorized representative via telephone to assess for the occurrence of adverse events, use of
concomitant medications, and compliance with study drug administration. A Phone Contact Form for the telephone contacts to obtain safety and compliance information will be 
provided to all sites (see Section 16.1). If any safety concern arises, subjects may return to the study site for an Unscheduled Visit. 
b  Subjects who discontinue prematurely from study drug administration will continue to return to the investigational site for study assessments at  Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7. If subjects are unable/unwilling to do so, they must return to the investigational site 30 (±3) days after the last dose of 
study drug for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 5, and 7. If the subject is unable/unwilling to return to the clinic for the 
EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of Visit 8 assessments.
c  All assessments are to be obtained pre-treatment on Day 1 except for post-dose PK blood sampling.
d  To be performed during physical examinations; see Section 9.4.4
e A complete physical examination of body systems  will be performed at the Screening Visit.  A brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will be performed at all subsequent designated time points. Abnormal findings 
on physical examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).
f  A neurological examination including assessments of mental status and cognition, cranial nerves, motor function, deep tendon reflexes, sensation, and coordination and gait will be 
performed. Abnormal findings on neurological examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see 
Section 9.6.1.1).
g Blood pressure is optional in subjects < 5 years of age at the discretion of the investigator. Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.  
h  12-lead ECG is optional in subjects < 5 years of age at the discretion of the investigator. At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of maximum 
concentration), an ECG will be obtained to allow for PK/PD investigations. 
i  Fasting of subjects for serum laboratory assessments is dependent on the age of the subject at the discretion of the investigator.
j  Urine pregnancy tests will be performed on all females of childbearing potential (i.e., all female subjects who have experienced menarche).  Any subject with a positive urine 
pregnancy test will have a serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be discontinued from the study and undergo all study assessments 
as described in the EOT Visit.
k   Blood samples for pharmacokinetic parameters will be obtained prior to administration of study drug, and at designated time points thereafter (see Table 9-2). They are optional and 
require consent/assent of the subject/subject’s legally authorized representative(s).
l  Subjects consenting to optional PK assessments must withhold taking the morning dose of study drug on the day of Visits 3, 6, and 8. Subjects not consenting to optional PK 
assessments may take their study drug on the mornings of Visits 3, 6, and 8 as instructed.
m  Depending on the country, study site personnel may dispense condoms at the time that study drug is dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.
n   At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and study drug for the remaining 30 days of treatment will be dispensed.  
o A pre-paid phone card will be provided at Visits 3 and 6 to facilitate contact between study site personnel and the subjects/subjects’ authorized representatives at Visits 4, 5, and 7.  


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Visit description, Section 9.2
Screening, Section 9.2.1
Visit 1 – screening visit (Days -14 to -1), Section 9.2.1.1
This section was changed for clarity and to reflect changes made Modifications 2, 9, and 10.
Old text:
No study-specific procedures, including asking a potential subject to fast prior to the 
screening blood samples, may be performed before the subject/legally authorized 
representative signs the informed consent form, or a minor subject provides assent. After the 
consent/assent is obtained, the following evaluations will be performed:

…

Presence of Chagas’ disease symptoms 

Microscopy (subjects younger than 8 months of age), serologic testing for Chagas’ 
disease, and qPCR

Assessment of pretreatment AEs 

Physical examination including height/length and weight

Vital signs (blood pressure [optional depending on the age of the subject and at the 
discretion of the investigator], heart rate, respiratory rate, and temperature)

12-lead ECG (optional depending on the age of the subject and at the discretion of the 
investigator)

Fasting serum hematology and chemistry, and urinalysis (if urine specimen can be 
obtained)

…

Concomitant medications
New text:
No study-specific procedures will be performed before the subject/legally authorized 
representative signs the informed consent form, or a minor subject provides assent, including 
asking a potential subject to fast (if applicable, depending on the age of the subject at the 
discretion of the investigator) prior to the screening blood samples.  Informed consent/assent 
for optional PK blood sampling will also be obtained.  After the consents/assents have been
obtained, the following assessments will be performed:

…

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age at randomization),
conventional and non-conventional serologic testing for Chagas’ disease (or 
acceptable documentation of positive Chagas’ disease within 3 months prior to 
screening and no prior anti-trypanocidal or anti-parasitic treatment), and qPCR


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
Assessment of pretreatment AEs and concomitant medications

A complete physical examination of body systems. Abnormal findings will be 
documented as medical history (see Section 9.6.1.1). 

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as medical history (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age), height/length, and 
weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

…
Treatment Phase, Section 9.2.2
Visit 2 – baseline (treatment phase, Day 1), Section 9.2.2.1
This section was changed for clarity and to reflect changes made per Modifications 1, 2, 4, 9, 
and 10.
Old text:
Visit 2 will take place after all laboratory test results and assessments are available.  The 
following assessments will be performed prior to study drug administration:

Re-evaluation of inclusion and exclusion criteria 

Presence of Chagas’ disease symptoms

Microscopy (subjects younger than 8 months of age), serologic testing for Chagas’ 
disease, and qPCR (if not performed at screening or the results are not available)

Assessment of pretreatment AEs  

Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy 

Vital signs (blood pressure [depending on the age of the subject and at the discretion 
of the investigator], heart rate, respiratory rate, and temperature), height/length, and 
weight

12-lead ECG (if not performed at screening or the results are not available; optional
depending on the age of the subject and at the discretion of the investigator).  
Whenever possible, ECGs should be obtained in close proximity to the 10- to 
120minute PK sample collection time window.

Fasting serum hematology and chemistry, and urinalysis (if urine specimen can be 
obtained) (if not performed at screening or the results are not available)


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
…

Pharmacokinetic blood samping (optional with consent/assent of the subject/subject’s 
legally authorized representative)

Concomitant medications

Administer first dose of study drug 

Dispense study drug with instructions for use.  Depending on the country, study site 
personnel may dispense condoms at the time that study drug is dispensed to either the 
subject or the subject’s parent/legal guardian, with an explanation of rationale and 
risks.
New text:
Visit 2 will take place after all laboratory test results and assessments performed at the 
Screening Visit are available.  The following assessments will be performed:

Re-evaluation of inclusion and exclusion criteria 

Assessment of pretreatment AEs  and concomitant medications

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of 
Cmax), an ECG will be obtained to allow for PK/PD investigations.  

…

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9-2)

Administer first dose of study drug to all subjects

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9-2).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3; subjects not consenting to optional PK assessments may take their 
study drug on the day of Visit 3 as instructed.
Subjects and/or their legally authorized representatives will be advised of the possibility of 
change in the subject’s urine color due to the ingredient riboflavin.  Depending on the 
country, study site personnel may dispense condoms at the time that study drug is dispensed 
to either the subject or the subject’s parent/legal guardian, with an explanation of rationale 
and risks.
Visit 3 (Day 7 ± 1 days), Section 9.2.2.2
This section was changed for clarity and to reflect changes made per Amendment 1


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Old text:

Presence of Chagas’ disease symptoms 

Clinical evaluation

Assessment of AEs

Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy

Vital signs (blood pressure is optional depending on the age of the subject and at the 
discretion of the investigator), height/length, and weight

12-lead ECG (optional depending on the age of the subject and at the discretion of the 
investigator).  Whenever possible, ECGs should be obtained in close proximity to the 
10- to 120minute PK sample collection time window.

Fasting serum hematology and chemistry, and urinalysis (if urine specimen can be 
obtained)

Urine pregnancy test…

Pharmacokinetic blood sampling (optional on consent/assent of the subject/subject’s 
legally authorized representative)

Concomitant medications

Drug accountability and review of study drug compliance

Administer and dispense study drug  

Provide pre-paid phone card to facilitate contact between study site personnel and the 
subjects/subjects’ authorized representatives at Visits 4 and 6.  
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease, and qPCR 

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).


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
Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of 
Cmax), an ECG will be obtained.

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

Urine pregnancy test…

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9–2).

Administer study drug to subjects consenting to PK assessments (who withheld their 
morning dose of study drug)  

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9–2).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained.

Collect study drug and diary.  Additional diaries may be provided if necessary.

Drug accountability and review of diaries for study drug compliance

Dispense pre-paid phone card to facilitate contact between study site personnel and the 
subjects/subjects’ authorized representatives at Visit 4  
Visit 4 (Day 21 ± 3 days), Section 9.2.2.3
This section was changed for clarity and per Modifications 6 and 8.
Old heading: Visit 4 (Day 21 ± 3 days)
New heading: Visit 4 (Day 14 ± 3 days)
Old text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs and concomitant medications.  If any safety concern arises, 
subjects may return to the study site for an Unscheduled Visit.
New text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 
compliance information will be provided to all sites (see Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.
Visit 5 (Day 30 ± 3 days), Section 9.2.2.4
This section was changed for clarity and per Modifications 6 and 8.
Old heading:  Visit 5 (Day 30 ± 3 days)


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New heading:  Visit 5 (Day 21 ± 3 days)
Old text:
The bulleted list of assessments appearing in this section was deleted in its entirety.
New text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 
compliance information will be provided to all sites (see Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.
Subjects consenting to optional PK assessments will be instructed to withhold taking the 
morning dose of study drug on the day of Visit 6; subjects not consenting to optional PK 
assessments may take their study drug on the day of Visit 6 as instructed.
Visit 6 (Day 42 ± 3 days), Section 9.2.2.5
This section was changed as a result of Modification 12.
Old heading:  Visit 6 (Day 42 ± 3 days)
New heading:  Visit 6 (Day 30 ± 3 days)
Old text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs and concomitant medications. If any safety concern arises, 
subjects may return to the study site for an Unscheduled Visit.
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease and qPCR. 

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1)

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator)


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
12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of 
Cmax), an ECG will be obtained. 

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
discontinued from the study and undergo all study assessments as described in the 
EOT Visit.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9-2).

Administer study drug to subjects consenting to PK assessments (who withheld their 
morning dose of study drug) 

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9-2).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Collect and dispense study drug and diary.  At this visit, subjects will return all 
remaining study drug and empty packaging, and study drug for the remaining 30 days 
of treatment will be dispensed.  

Drug accountability and review of diaries for study drug compliance

Collect and dispense pre-paid phone card to facilitate contact between study site 
personnel and the subjects/subjects’ authorized representatives at Visit 7.  
Visit 7 (Day 60 ± 3 days)/End of Treatment Visit, Section 9.2.2.6
This section was changed for clarity and per Modification 6.
Old heading:  Visit 7 (Day 60 ± 3 days)/End of Treatment Visit
New heading:  Visit 7 (Day 42 ± 3 days)
Old text:
The bulleted list of assessments appearing in this section was deleted in its entirety.
New text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 
compliance information will be provided to all sites (see Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.


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Subjects consenting to optional PK assessments will be instructed to withhold taking the 
morning dose of study drug on the day of Visit 8; subjects not consenting to optional PK 
assessments may take their study drug on the day of Visit 8 as instructed.
Visit 7 (Day 60 ± 3 days)/End of Treatment Visit, Section 9.2.2.7
This section was revised for clarity and as a result of changes per Amendment 1.
Old heading: Visit 7 (Day 60 ± 3 days)/End of Treatment Visit
New heading: Visit 8 (Day 60 ± 3 days)/End of Treatment Visit
Old Text:

Presence of Chagas’ disease symptoms 

Serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy.  

Vital signs (blood pressure is optional depending on the age of the subject and at the 
discretion of the investigator), height/length, and weight

12-lead ECG (optional depending on the age of the subject and at the discretion of the 
investigator).   

Fasting serum hematology and chemistry, and urinalysis (if urine specimen can be 
obtained).   

Urine pregnancy test …

Pharmacokinetic blood sampling (optional with consent/assent of the subject/subject’s 
legally authorized representative)

Concomitant medications

Collect study drug and pre-paid phone card

Drug accountability and review of study drug compliance
New Text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 


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peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Neurological examination (see Section 9.6.3.3).  Abnormal findings will be 
documented as AEs (see Section 9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the 
investigator).  At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of 
Cmax), an ECG will be obtained.  

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator. 

Urine pregnancy test …

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9-2). 

Administer study drug to subjects consenting to PK assessments (who withheld their 
morning dose of study drug.

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9-2).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Collect study drug, diary, and pre-paid phone card

Drug accountability and review of diaries for study drug compliance
Follow-up phase, Section 9.2.3
This section was changed for clarity and to reflect changes made per Modifications 1, 2, and 
9.
Visit 8 (Day 90 ± 7), Section 9.2.3.1
This section was changed for clarity and to reflect changes made per Amendment 1.
Old heading: Visit 8 (Day 90 ± 7)
New heading: Visit 9 (Day 90 ± 7)
Old text:

Presence of Chagas’ disease symptoms 

Clinical evaluation

Assessment of AEs

Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy 


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
Vital signs (blood pressure is optional depending on the age of the subject and at the 
discretion of the investigator), height/length, and weight

12-lead ECG (optional depending on the age of the subject and at the discretion of the 
investigator)

Fasting serum hematology and chemistry, and urinalysis (if urine specimen can be 
obtained).  

Urine pregnancy test…

Concomitant medications
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age)

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Serum hematology and chemistry, coagulation, and urinalysis (if urine specimen can 
be obtained).  Fasting is dependent on the age of the subject at the discretion of the 
investigator.

Urine pregnancy test…
Visit 9 (Day 240 ± 7), Section 9.2.3.2
This section was changed for clarity and to reflect changes made per Amendment 1.
Old heading: Visit 9 (Day 240 ± 7)
New heading: Visit 10 (Day 240 ± 7)
Old text:

Presence of Chagas’ disease symptoms

Clinical evaluation

Serologic testing for Chagas’ disease and qPCR

Assessment of AEs


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
Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy

Vital signs (blood pressure is optional depending on the age of the subject and at the 
discretion of the investigator), height/length, and weight
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1). 

Vital signs (blood pressure is optional in subjects< 5 years of age at the discretion of 
the investigator), height/length, and weight
Visit 10 (Day 420 ± 7), Section 9.2.3.3
This section was changed for clarity and to reflect changes made per Amendment 1.
Old heading: Visit 10 (Day 420 ± 7)
New heading: Visit 11 (Day 420 ± 7)
Old text:

Presence of Chagas’ disease symptoms 

Clinical evaluation

Serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Physical examination, and assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and clinical assessments for the presence of peripheral 
neuropathy  

Vital signs (blood pressure is optional depending on the age of the subject and at the 
discretion of the investigator), height/length, and weight
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Conventional and non-conventional serologic testing for Chagas’ disease and qPCR


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
Assessment of AEs

Concomitant medications

Brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and clinical assessments for the presence of 
peripheral neuropathy.  Abnormal findings will be documented as AEs (see Section 
9.6.1.1).  

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight
Population characteristics, Section 9.3
Demographic, Section 9.3.1
This section was changed for clarity.
Old text:  At screening, each subject’s year of birth, age, and sex will be recorded.
New text:  At screening, each subject’s date of birth, age, and sex will be recorded.
Medical history, Section 9.3.2
This section was changed for clarity.
Old text:
Medical history findings (i.e., previous diagnoses, diseases or surgeries) meeting all criteria 
listed below will be collected as available to the investigator…
New text:
At screening, medical history findings (i.e., previous diagnoses, diseases or surgeries) meeting 
all criteria listed below will be collected as available to the investigator…
Efficacy, Section 9.4
This section was changed for clarity and to reflect changes made per Modification 2.
Old text:
The primary efficacy variable will be a ≥20% sero-reduction (subjects older than 8 months 
of age and younger than 18 years of age) or sero-conversion (subjects 8 months of age and 
younger) in antibody IgG titer at 12 months posttreatment using conventional ELISA serology 
as the measure of efficacy.
A quality control program will be implemented for the primary efficacy variable (i.e., ELISA 
titers).  At least 20% of measurements, chosen at random, will be confirmed by a reference 
laboratory (Chagas Service, Buenos Aires Children’s Hospital “Ricardo Gutierrez”).
Other efficacy variables include disease state determined by qPCR, microscopy (subjects 
younger than 8 months of age), clinical signs/symptoms of Chagas’ disease, and clinical 
evaluation.
…
New text:


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Efficacy assessments will be obtained at designated study visits (see Table 9-1).  The primary 
efficacy variable will be sero-reduction or sero-conversion at 12 months posttreatment using 
two conventional ELISA serology tests as the measure of efficacy.
A quality control program will be implemented for the primary efficacy variable (i.e., ELISA 
concentrations).  At least 20% of measurements, chosen at random, will be confirmed by a 
reference laboratory (Chagas Service, Buenos Aires Children’s Hospital “Ricardo Gutierrez”).
Other efficacy variables include disease state determined by qPCR, concentrations tests for 
T. cruzi (subjects < 8 months of age at randomization), conventional and non-conventional 
serologic testing, and clinical signs/symptoms of Chagas’ disease.
…
Polymerase chain reaction test, Section 9.4.1
This section was changed for clarity and to reflect changes made per Amendment 1.
Old text:
A qPCR test will be performed at the Screening Visit, Visit 2 (if not done at screening or the 
results are not available), Visits 5 and 7, Visit 9 and 10 (Followup).
Details describing the collection, processing, storage and shipment of qPCR blood samples, 
and the qPCR test protocol, are described in Section 16.2.  
New text:
A qPCR test will be performed at the Screening Visit, Visits 3, 6, and 8, and Follow-up Visits
10 and 11.
Details describing the collection, processing, storage and shipment of qPCR blood samples, 
and the qPCR test protocol, are described in Section 16.4.  
Serological tests, Section 9.4.2
This section was changed for clarity and per Modifications 2, 3, and 5.
Old text:
At screening, subjects must have the following serological tests to confirm a diagnosis of 
Chagas’ disease:

Subjects younger than 8 months of age must demonstrate direct observation of T. cruzi by 
concentration test

Subjects 8 months of age and older and younger than 18 years of age must demonstrate a 
positive ELISA plus a positive result for one of the following tests to confirm diagnosis:

Alternate ELISA (e.g., Chagatest ELISA recombinate v3.0)

Hemagglutination (e.g., Chagatest HAI)  

Direct Agglutination (e.g., SERODIA-Chagas) 
After enrollment, serological tests will be performed at Visit 2 (if not done at screening, or the 
results are not available), 5, and 7, and Visits 9 and 10 (Follow-up).


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Details describing the collection, processing, storage and shipment of blood samples, and the 
serologic test protocol, will be described in a separate serologic test manual.  Serologic tests 
will be performed by a local laboratory according to the standard of care for each country.  
New text:
At screening, subjects must have the following serological tests to confirm a diagnosis of 
Chagas’ disease:

Subjects < 8 months of age at randomization must demonstrate direct observation of 
T. cruzi by concentration test

Subjects ≥8 months of age to < 18 years of age at randomization must demonstrate a 
positive conventional ELISA result for both of the following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA 
Additionally, a non-conventional ELISA test will be obtained; however, a positive result will 
not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ disease 
within 3 months prior to screening and they have had no prior anti-trypanocidal or 
anti-parasitic treatment.
After randomization, conventional and non-conventional serological tests will be performed at 
Visits 3, 6, and 8, and Visits 10 and 11 (Follow-up).
All subjects ≥8 months to < 18 years of age at randomization will have two baseline 
specimens collected for serologic testing.  One sample will be used to determine the initial 
diagnosis of Chagas’ disease while the other sample will be frozen and retained to be assayed 
along with the 12-month post-treatment specimen as an internal control.  Subjects < 8 months 
of age at randomization will have one specimen obtained which will be frozen and retained.  
Total blood volume to be taken for the required for three serological and PCR tests is 
4.0-5.0 mL, depending on the subject’s age.  Care will be taken to minimize sample volumes 
by using a micro-sampling collection technique.
Details describing the collection, processing, storage and shipment of blood samples, and the 
serologic test protocol, will be described in a separate serologic test manual.  Initial serologic 
tests will be performed by a local laboratory according to the standard of care for each 
country.  Subsequent tests will be performed by a central laboratory.
Microscopy, Section 9.4.3
This section was changed for clarity and per Modification 2.
Old heading: Microscopy
New heading: Concentration tests for T. cruzi
Old text:
Microscopy tests will be performed on all subjects younger than 8 months of age at the 
screening visit, and at Visits 2 (if not done at screening or the results are not available), and 5.


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Details describing the collection, processing, storage and shipment of blood samples, and the 
microscopy test protocol, will be described in a separate microscopy test manual.  Microscopy
will be performed by a local laboratory according to the standard of care for each country.
New text:
Concentration tests for T. cruzi will be performed on all subjects < 8 months of age at 
randomization at the screening visit, and at Visits 3 and 6.
Details describing the collection, processing, storage and shipment of blood samples, and the 
concentration test protocol, will be described in a separate manual.  Concentration tests for 
T. cruzi will be performed by a local laboratory according to the standard of care for each 
country.
Clinical manifestations of Chagas’ disease, Section 9.4.4
This section was changed for clarity and per changes made per Amendment 1.
Old heading:  Clinical manifestations of Chagas’ disease 
New heading:  Assessment of Chagas’ disease signs and symptoms
Old text:
Assessment of clinical manifestations of Chagas’ disease will be performed at designated 
study visits.  Subjects will be assessed for the following:
Acute Chagas’ disease:
…
New text:
Assessments of the presence of Chagas’ disease signs and symptoms will be performed
during physical examinations at Screening, Visits 3, 6, and 8 (EOT), and Visits 9, 10, and 11 
(follow-up), and will be entered into the eCRF.  Signs and symptoms of Chagas’ disease 
include but are not limited to the following:
Acute Chagas’ disease:
…
Pharmacokinetics/pharmacodynamics, Section 9.5
This section was changed for clarity, per Amendment 1, and per Modifications 1 and 9.
Old text:
If the subject/subject’s legally authorized representative(s) provide(s) consent/assent to do so, 
plasma concentrations of nifurtimox will be performed at Visit 2 (baseline) and Visits 3, 5, 
and 7 (EOT) on at least 10 subjects per cohort selected from all participating centers.  Plasma 
nifurtimox concentrations will be determined using a sparse sampling approach.  
The time points and time windows for PK blood sampling is presented in Table 9-2.  
New text:
If the subject/subject’s legally authorized representative(s) provide(s) consent/assent to do so, 
plasma concentrations of nifurtimox will be evaluated at Visit 2 (baseline) and Visits 3, 6, and 


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8 (EOT) on at least 10 subjects per age cohort selected from all participating centers.  Plasma 
nifurtimox concentrations will be determined using a sparse sampling approach.  
At Visits 2, 3, 6, and 8, the subjects should present themselves to the investigational site 
without taking the morning dose of study medication.  Blood samples for PK will be obtained 
in-clinic prior to the administration of study drug and at specific time points and time 
windows for PK blood sampling as presented in Table 9-2.  At the 2 – 4 hour time point of 
PK blood sampling (i.e., at the time of Cmax), an ECG (optional for subjects < 5 years of age at 
the discretion of the investigator) will be obtained to allow for PK/PD investigations.
Table 9-2 was modified to clarity the visits at which PK sample will be drawn; footnote a 
added per Modification 1.
Old table:
Table 9-2:  Pharmacokinetic sampling time points and windows
Visits  (Days)
Time point
Time window
2, 3, 5, 7  (1, 7, 30, 60)
Pre-dose (pre-treatment)
---
2, 3, 5, 7  (1, 7, 30, 60)
Post-dose
5-10 minutes post-dose
10-120 minutes post-dose
2-4 hours post-dose 
4-8 hours post-dose
New table:
Table 9-2:  Pharmacokinetic sampling time points and windows 
Visits  (Days)
Time point
Time window
2, 3, 6, 8  (1, 7, 30, 60)
Pre-dose (pre-treatment)
---
Post-dose
5-10 minutes post-dose
10-120 minutes post-dose
2-4 hours post-dose a
4-8 hours post-dose
a  An ECG will be obtained at this time point (optional in subjects < 5 years of age at the 
discretion of the investigator)
Old text:
Samples of a blood volume of 2 x 75 = 150 μL at predefined time points should be taken 
according to Table 9-2. If samples cannot be taken due to other reasons (e.g., sampling 
deemed not feasible by the investigator due to safety) or if the samples are taken outside the 
defined time windows (e.g., > 8 hours post-dose), the sample will still remain valid for PK 
analysis. At least one sample per period within the specified timeframe should be taken 
during the course of the study. That means that, at the end of the study, at least one sample 
from each time window will be available, e.g., if the 4-8 hour post-dose sample was not 
collected until the last visit, then a sample at this time window should in any case be drawn at 
that time point. This must be documented by the investigator.
Details describing the procedures for obtaining and handling PK blood samples are provided 
in Section 16.3.


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At these study visits, the subjects should present themselves to the hospital without taking the 
morning dose of study medication.  During these visits, the PK samples should be obtained 
within the planned sampling time windows…
Analysis of PK samples
…
Pharmacokinetic evaluation
Plasma concentration time courses will be analyzed and reported within the study report.  In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 
report.  Nonparametric and parametric PK estimation methods will be used to provide 
parameter estimates describing the PK behavior of nifurtimox and to identify possible 
covariates related to age.
New text:
Samples of a blood volume of 2 x 75 = 150 μL at predefined time points should be taken 
according to Table 9-2. If samples cannot be taken due to other reasons (e.g., sampling 
deemed not feasible by the investigator due to safety) or if the samples are taken outside the 
defined time windows (e.g., > 8 hours post-dose), the subject will still remain valid for PK 
analysis. At least one sample should be taken during the course of the study. That means 
that, at the end of the study, at least one sample from each time window will be available, e.g., 
if the 4-8 hour post-dose sample was not collected until the last visit, then a sample at this 
time window should in any case be drawn at that time point. This must be documented by the 
investigator.
The PK samples should be obtained within the planned sampling time windows…
Details describing the procedures for obtaining and handling PK blood samples are provided 
in Section 16.3.
Analysis of PK samples
…
Pharmacokinetic evaluation
Plasma concentration time courses will be analyzed and reported within the study report.  In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 
report.  Population PK methods will be used to provide parameter estimates describing the PK 
behavior of nifurtimox and to identify possible covariates related to age.
Intensity, Section 9.6.1.2.2
This section was changed as a result of Modification 7.
Old text:
The intensity of an AE is classified according to the following categories:

Mild

Moderate

Severe


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New text:
The intensity of an AE is classified according to the following categories:

Mild:  a type of AE that is usually transient and may require only minimal 
treatment or therapeutic intervention.  The event does not generally interfere with 
usual activities of daily living.

Moderate:  a type of AE that is usually alleviated with additional specific 
therapeutic intervention.  The event interferes with usual activities of daily living, 
causing discomfort, but poses no significant or permanent risk of harm to the 
subject.

Severe:  a type of AE that requires intensive therapeutic intervention.  The event 
interrupts usual activities of daily living, or significantly affects clinical status. The 
event possesses a significant risk of harm to the subject and hospitalization may be 
required.
Expected adverse events, Section 9.6.1.5
This section was changed since an Investigators’ Brochure for nifurtimox became available.
Old text:
For this study, the applicable reference documents are the most current, locally approved 
versions of the CCDSs for nifurtimox.
New text:
For this study, the applicable reference documents is the most current Investigators’ Brochure
for nifurtimox.
Further safety, Section 9.6.3
This section was changed for clarity and to reflect changes made per Modification 10.
New section heading, 9.6.3.1 Laboratory assessments, was added.  All Heading 4 titles in 
Section 9.6.3 were renumbered from this point forward.
Table 1 was revised to clarity visits at which urine pregnancy will be obtained per changes 
made via Amendment 1.
Old text and table:
Safety laboratory assessments will be performed with the subject in a fasting state at 
screening, Visits 2 (if not done at screening or the results are not available), 3, 5, 7, 8, and 8
(follow-up), and the EOT Visit.  If any abnormal laboratory values occur at the EOT Visit 
(Visit 7), these determinations will be followed until normal or stable.
Laboratory parameters will be analyzed by a local laboratory according to the schedule 
specified in Table 9-3.


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Table 9-3:  Description of laboratory parameters (safety)
Hematology
Hemoglobin, hematocrit, RBC count, WBC count and 
differential (including lymphocytes, monocytes, eosinophils, 
basophils, neutrophils), MCV, MCH, MCHC, platelets
Blood chemistry
Albumin, total protein, BUN, creatinine, glucose, total bilirubin, 
ALP, AST, ALT, uric acid
Coagulation
INR, PT, PTT
Urinalysis (urine dipstick) a
RBCs, WBCs, pH, specific gravity, glucose, protein, bilirubin, 
urobilinogen, nitrite, ketones
Urine pregnancy test b
Urine β-hCG
ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase,
βhCG = beta-human chorionic gonadotropin, INR = international normalized ratio, MCV = mean 
corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular 
hemoglobin concentration, PT = prothrombin time, PTT = partial thromboplastin time, RBC = red 
blood cell, WBC = white blood cell.
a  If abnormalities are identified by the local laboratory, the samples will be sent to a central 
laboratory. Cell/sediment microscopy should be performed only if positive dipstick results are 
obtained for WBC, RBC/hemoglobin, or protein.
b  To be performed at Visits 2 (if not performed at screening), 3, 5, 7, and 8 on females of childbearing 
potential (i.e., female subjects who have experienced menarche).  If any urine pregnancy test is 
positive, a serum pregnancy test will be performed to confirm the result.
New text/table:
Safety laboratory assessments will be performed at screening, Visits 3, 6, 8 (EOT), and 9
(follow-up).  Fasting is dependent on the age of the subject at the discretion of the investigator.  
If any abnormal laboratory values occur at the EOT Visit (Visit 8), these determinations will 
be followed until normal or stable.
Laboratory parameters will be analyzed by a local laboratory according to the schedule 
specified in Table 9-3.


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Table 9-3:  Description of laboratory parameters (safety) 
Hematology
Hemoglobin, hematocrit, RBC count, WBC count and 
differential (including lymphocytes, monocytes, eosinophils, 
basophils, neutrophils), MCV, MCH, MCHC, platelets
Blood chemistry
Albumin, total protein, BUN, creatinine, glucose, total bilirubin, 
ALP, AST, ALT, uric acid
Coagulation
INR, PT, PTT
Urinalysis (urine dipstick) a
RBCs, WBCs, pH, specific gravity, glucose, protein, bilirubin, 
urobilinogen, nitrite, ketones
Urine pregnancy test b
Urine β-hCG
ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, 
βhCG = beta-human chorionic gonadotropin, INR = international normalized ratio, MCV = mean 
corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular 
hemoglobin concentration, PT = prothrombin time, PTT = partial thromboplastin time, RBC = red 
blood cell, WBC = white blood cell.
a  If abnormalities are identified by the local laboratory, the samples will be sent to a central 
laboratory. Cell/sediment microscopy should be performed only if positive dipstick results are 
obtained for WBC, RBC/hemoglobin, or protein.
b  To be performed at Visits 1, 2, 3, 6, 8, and 9  on females of childbearing potential (i.e., female 
subjects who have experienced menarche).  If any urine pregnancy test is positive, a serum 
pregnancy test will be performed to confirm the result.
Total volume of blood, Section 9.6.3.2
This section was changed for clarity.
Old text:
The total volume of blood obtained at each visit will be as per standards of Clinical 
Laboratory and Standards Institute; see Section 16.4 and Table 9-4 below.
New text:
In order to minimize the burden of blood loss due to PK sampling, ageappropriate- sparse 
sampling strategies combined with (micro-)bioanalysis techniques will be applied.  The total 
volume of blood obtained at each visit will be a maximum of approximately 
7.8 mL - 14.6 mL depending on the child’s age and weight as per standards of Clinical 
Laboratory and Standards Institute; see Section 16.6 and Table 9-4 below.
Physical examination, Section 9.6.3.3
This section was changed for clarity and per Modification 10.
Old text:
Physical examinations will be performed by a medically qualified person.  A complete 
physical examination, including a complete review of body systems will be performed at the 
screening visit.  A brief physical examination, including weight and assessments of heart, 
lungs, and abdomen, the skin for signs of severe dermatitis, and assessments for the presence 
of peripheral neuropathy will be performed at designated time points (see Table 9-1). 
Height/length and weight will be obtained with the subject wearing minimal clothing and 
without shoes.  


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New text:
Physical examinations will be performed by a medically qualified person.  A complete 
physical examination of body systems will be performed at the screening visit.  A brief 
physical examination, including assessments of heart, lungs, and abdomen, skin for the 
presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will 
be performed at all subsequent designated time points (see Table 9-1). Abnormal findings on 
physical examination at Screening will be documented as medical history; abnormal findings 
thereafter will be documented as AEs (see Section 9.6.1.1).
A neurological examination will be performed at Screening, and Visits 3, 6, and 8 (EOT).  
The examination will include assessments of mental status and cognition, cranial nerves, 
motor function, deep tendon reflexes, sensation, and coordination and gait.  Abnormal 
findings on neurological examination at Screening will be documented as medical history; 
abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).
Assessments of Chagas’ disease signs and symptoms will be performed at the time of physical 
examinations (see Section 9.4.4).
Vitals signs, Section 9.6.3.4
This section was changed for clarity and as a result of changes made per Modification 9.
Heading 9.6.3.4 was revised:  Vital signs, height/length, and weight
Old text:
Vital signs (systolic and diastolic blood pressure [optional depending on the age of the subject 
and at the discretion of the investigator], heart rate, respiratory rate, and oral or rectal body 
temperature) will be measured at screening and Visits 2, 3, 5, 7 (EOT), 8, 9, and 10.  After a 
5-minute rest in a sitting position, vital signs will be measured using non-invasive equipment.
New text:
Vital signs (systolic and diastolic blood pressure [optional in subjects < 5 years of age at the 
discretion of the investigator], heart rate, respiratory rate, and oral or rectal body temperature) 
will be measured at screening and Visits 2, 3, 6, 8 (EOT), 9, 10, and 11.  After a 5-minute rest 
in a sitting position, vital signs will be measured using non-invasive equipment.
Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.  
12-lead electrocardiogram, Section 9.6.3.5
This section was changed for clarity and per Modifications 1 and 9.
Old text:
Depending on the age of the subject and at the discretion of the investigator, a resting 12-lead 
ECG will be performed at screening and Visits 2, 3, 5, 7 (EOT), and 8 (Follow-up). 
The ECG will be performed at the site according to procedure.  The ECG will be obtained
with the subject in the supine position after he/she has rested for 10 minutes.  Whenever 
possible, ECGs should be obtained in close proximity to the 10- to 120-minute PK sample 
collection time window.


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New text:
A resting 12-lead ECG will be performed at screening and Visits 2, 3, 6, 8 (EOT), and 9
(Follow-up) on all subjects ≥5 years of age, and at the discretion of the investigator on all 
subjects  < 5 years of age.  For subjects consenting to PK assessments, an ECG will be 
obtained at the 2 – 4 hour time point of PK blood sampling (i.e, at the time of Cmax), to allow 
for PK/PD investigations. 
The ECG will be performed at the site according to procedure, and with the subject in the 
supine position after he/she has rested for 10 minutes. 
Urine pregnancy test, Section 9.6.3.6
This section was changed for clarity and to reflect changes made per Amendment 1.
Old text:
A urine pregnancy test on all female subjects of childbearing potential (i.e., all females who 
have experienced menarche) will be performed at screening and Visits 2, 3, 5, 7 (EOT), and 8
(Follow-up).  If any urine pregnancy test is positive, a serum pregnancy test will be performed 
to confirm the result.  
New text:
A urine pregnancy test on all female subjects of childbearing potential (i.e., all females who 
have experienced menarche) will be performed at screening and Visits 2, 3, 6, 8 (EOT), and 9
(Follow-up).  If any urine pregnancy test is positive, a serum pregnancy test will be performed 
to confirm the result.  If pregnancy is confirmed at Visits 1 or 2, the subject is to be excluded 
from study participation.  If pregnancy is confirmed at Visits 3 or 6, the subject will be 
discontinued from the study and undergo all study assessments as described for the EOT 
Visit.  See Section 9.6.2.
Statistical methods and determination of sample size, Section 10
General considerations, Section 10.1
This section was changed for clarity.
Old text:
This study has been designed to focus on the efficacy and safety of treatment with nifurtimox 
in children diagnosed with Chagas’ disease.  In this study, approximately 390 pediatric 
subjects will be randomized to ensure 330 evaluable pediatric subjects (2:1 randomization, 
60-day regimen vs. 30-day regimen).  Subjects will be stratified by age into four cohorts...  
…
No imputations will be made for missing values.  Additional analyses may be specified in the 
SAP if missing data patterns observed during blinded review of the data suggest any possible 
bias. 
…


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New text:
This study has been designed to focus on the efficacy and safety of treatment with nifurtimox 
in children diagnosed with Chagas’ disease.  In this study, approximately 390 pediatric 
subjects will be randomized to ensure 330 evaluable pediatric subjects (2:1 randomization, 
60-day regimen vs. 30-day regimen).  A subject is considered evaluable if there is sufficient 
information to determine whether or not he or she is cured.   Subjects will be stratified by age 
into four cohorts...  
…
No imputations will be made for missing values occurring in the safety and background 
variables.  Treatment of missing values for efficacy variables is addressed later in this section.  
Additional analyses may be specified in the SAP if missing data patterns observed during 
blinded review of the data suggest any possible bias. 
…
Variables and planned statistical analyses, Section 10.3
This section was changed for clarity and to reflect changes made per Modification 2.
Old text:
Variables
The primary efficacy variable will be a ≥20% sero-reduction (subjects older than 8 months 
of age and younger than 18 years of age) or seroconversion (subjects 8 months of age and 
younger) in antibody IgG titer at 12 months posttreatment using conventional ELISA serology 
as the measure of efficacy.  
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease, clinical 
evaluation, microscopy (subjects younger than 8 months of age), and disease state determined 
by qPCR. 
…
Safety variables include AEs, physical examination abnormalities, vital signs, ECG 
abnormalities, hematology and blood chemistry, and urinalysis.
New text:
Variables
The primary efficacy variable will be sero-reduction or seroconversion at 12 months 
posttreatment using two conventional ELISA serology tests as the measure of efficacy.  This 
sero-reduction or sero-conversion is considered cure, and the primary variable is binary (cure, 
no cure).  In the event of discordancy between the two conventional ELISA test results for 
subjects ≥8 months of age, the average percentage sero-reduction will be used (e.g., Test #1 
= 15% and Test #2 = 25%; average = 20% and, hence, cure). The methodology for calculating 
the average percentage sero-reduction will be specified in the SAP.
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease, 
concentration test for T. cruzi (subjects < 8 months of age at randomization), conventional and 
non-conventional serologic testing, and disease state determined by qPCR. 


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…
Safety variables include AEs, physical examination abnormalities, vital signs, ECG 
abnormalities, hematology and blood chemistry, coagulation, and urinalysis.
Statistical and analytical plans 
Old text:
Efficacy
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with ≥20% sero-reduction or 
seroconversion (60-day regimen) and the proportion estimated from historical data will be 
tested using an exact 2-sided 95% CI for a single proportion.
…
Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox 
subjects (60day regimen) with ≥20% sero-reduction or sero-conversion is greater than the 
proportion of historical placebo subjects with ≥20% sero-reduction or sero-conversion.  This 
same CI will also be used for comparison with the historical benznidazole subjects with ≥
20% sero-reduction or seroconversion.[15]
Sero-conversion for the historical controls will be estimated from cure rates as presented in 
two publications.[15][16]  The sero-conversion rates used for placebo and benznidazole are 
15% and 60%, respectively…
New text:
Efficacy
Primary efficacy analysis
The difference in the proportion of nifurtimox subjects with sero-reduction or seroconversion 
(60-day regimen) and the proportion estimated from historical data will be tested using an 
exact 2-sided 95% CI for a single proportion.
…
Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox 
subjects (60day regimen) with sero-reduction or sero-conversion is greater than the proportion 
of historical placebo subjects with sero-conversion.  
Sero-conversion for the historical controls will be estimated from cure rates as presented in 
two publications.[15][16]  The sero-conversion rates used for placebo and benznidazole are 
16% and 58%, respectively….
Old text:
Secondary efficacy analyses
A secondary analysis will be done to compare the proportion of subjects with ≥20%
seroreduction or sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be 


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performed using a standard, symmetric, asymptotic 2-sided 95% CI for the difference of two 
independent proportions.
…
The relationship of serological results to qPCR results will be done using frequencies of 
matches and mismatches on the determination of disease status, and phicorrelation and kappa 
coefficient to assess the degree of agreement.  
…
New text:
Secondary efficacy analyses
The same CI for the primary endpoint analysis will also be used for comparison with the 
historical benznidazole subjects with sero-conversion to assess whether or not their cure rates 
are comparable.
A secondary analysis will be done to compare the proportion of subjects with seroreduction or 
sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be performed 
using a standard, symmetric, asymptotic 2-sided 95% CI for the difference of two independent 
proportions.
…
The relationship of conventional serology results to qPCR results will be done using 
frequencies of matches and mismatches on the determination of disease status, and 
phicorrelation and kappa coefficient to assess the degree of agreement.  The relationship of 
non-conventional serology to conventional serology will be analyzed in the same way.
…
Missing data/drop outs 
Old text:
No imputations will be made for missing data due to dropouts or other causes.  Statistical 
analyses will be based on subjects with data necessary for the technique used.  
…
New text:
No imputations will be made for missing data due to dropouts or other causes. For the 
primary efficacy analysis done on the FAS, a patient without an assessment at the primary 12 
month time point will be considered a treatment failure. For the secondary analysis using the 
PPS, patients who do not have serology determinations to assess the primary endpoint will be 
excluded from the analysis.
Sensitivity analyses will be performed treating only missing values due to the following 
reasons as treatment failures:
      1. the reason for discontinuation is known or suspected to be treatment ineffectiveness or 
an AE


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       2. a subject  has not had a negative serology at any visit and has no serology 
determination for the primary analysis visit  
       3. an indeterminate or incorrectly performed serology determination occurs at the primary 
analysis visit.204
…
Determination of sample size, Section 10.4
This section was changed for clarity and per Modification 2.
Old text:
Sample size is based on the primary endpoint, the difference in the proportion of nifurtimox 
subjects with ≥20% sero-reduction (subjects older than 8 months of age and younger than 
18 years of age) or sero-conversion (subjects 8 months of age and younger) (60-day regimen) 
and the proportion of historical placebo subjects with ≥20% sero-reduction or sero-
conversion for the two age groups.  The test of superiority over placebo will be powered 
based on the detection of a difference in proportion of nifurtimox subjects with ≥20% sero-
reduction or sero-conversion of 0.10.  This is considered a clinically meaningful difference 
according to several experts in the field. Given that the nifurtimox (60day) proportion of 
subjects with ≥20% sero-reduction or sero-conversion is 0.60, 220 evaluable subjects in this 
group will provide at least 80% power for an exact binomial test. This sample size will 
provide an exact 95% CI with a width of ≤0.15 (e.g., [0.53,0.67]) for use in comparing 
nifurtimox to the proportion of historical benznidazole subjects with ≥20% sero-reduction or
sero-conversion.
…
New text:
Sample size is based on the primary endpoint, the difference in the proportion of nifurtimox 
subjects with sero-reduction or sero-conversion (60-day regimen) and the proportion of 
historical placebo subjects with sero-conversion.  The test of superiority over placebo will be 
powered based on the detection of a difference in proportion of 0.10.  This quantity represents 
a difference between rates, such as 0.600.50=0.10, rather than the difference that would result 
from subtracting 10% of 0.60 (that is, 0.06) from 0.60.  Given that the nifurtimox (60day) 
proportion of subjects with sero-reduction or sero-conversion is 0.60, 220 evaluable subjects 
in this group will provide at least 80% power for an exact binomial test. This sample size will 
provide an exact 95% CI with a width of ≤0.15 (e.g., [0.53,0.67]) for use in comparing 
nifurtimox to the proportion of historical benznidazole subjects with sero-conversion.
…
Data recording, Section 11.1
This section was changed for clarity.
                                                
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New paragraph added:
Completed subject diaries will be collected by site personnel at specific study visits (see Table 
9-1),  Information entered onto the subject diaries will be used to assess drug accountability 
and treatment compliance, and entered into the eCRF.  The completed diaries will be 
maintained with the subject’s source documentation,
Data processing, Section 11.3
This section was changed for clarity.
Old text:
…
For data coding (e.g., AEs, medication), internationally recognized and accepted dictionaries 
will be used.
After its initial release for biometrical analysis, the clinical database is planned to be re-
opened for the inclusion of the PK data.  If PK data are not available prior to database lock for 
this study, a planned database unlock/re-opening will be required to load the data.
New text:
…
For data coding (AEs, medication), The Medical Dictionary for Regulatory Activities will be 
used for AEs, and the WHO Drug Dictionary will be used for prior and concomitant 
medications.
After its initial release for biometrical analysis, the clinical database is planned to be re-
opened for the inclusion of the PK data.
Missing data, Section 11.4
This section was changed for clarity.
Old text:
In general, no imputations will be made for missing data.  More details will be provided in the 
SAP.
New text:
In general, no imputations will be made for missing data other than those described in 
Section 10.  More details will be provided in the SAP.
Reference list, Section 14
The following references were added to the reference list per changes made as a result of 
Amendment 1, and the cross-references were renumbered in the list and within the protocol 
body accordingly.
18. Altcheh J, Corral R, Biancardi MA, Freilij H. Anti-F2/3 antibodies as cure marker in 
children with congenital Trypanosoma cruzi infection. Medicina (B Aires). 2003;63(1):37-40.
Old footnote 18


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18. Almeida IC, Gazzinelli R, Ferguson MA, Travassos LR. Trypanosoma cruzi mucins: 
potential functions of a complex structure. Mem Inst Oswaldo Cruz. 1999;94 Suppl 1:173-
6
New footnote 17
17. Almeida IC, Ferguson MA, Schenkman S, Travassos LR. Lytic antialpha--galactosyl 
antibodies from patients with chronic Chagas' disease recognize novel O-linked 
oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of 
Trypanosoma cruzi. Biochem J. 1994 Dec 15:304 (Pt 3):793-802.
20. Porcel BM, Bonrempi EJ, Henriksson J, Rydaker M, Aslund L, Segura EL, et al. 
Trypanosoma rangeli and Trypanosoma cruzi: molecular characterization of genes encoding 
putative calcium-binding proteins, highly conserved in trypanosomatids. Exp Parasitol. 1996 
Dec:84(3):387-99.
28. Bianchi F, Cucunuba Z, Guhl F, Gonzalez NL, Freilij H, Nicholls RS, et al. Follow-up of 
an Asymptomatic Chagas’ Disease Population of Children after Treatment with Nifurtimox 
(Lampit) in a Sylvatic Endemic Transmission Area of Colombia. PLoS Negl Trop Dis. 2015 
Feb 27;10(2):e0003465.
Appendices, Section 16
Appendix 16.1, Phone Contact Form for telephone assessments was added per Modifications 
6, 12, and 13.


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Appendix 16.1, Schwartz formula and mean (range) eGFR for infants and children of various 
ages was renumbered to Appendix 16.2.
The table of normal GFR in children up to 12 years of age was replaced with a table of normal 
GFR in Children and Young Adults as follows:
Old table:


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New table and text:
Normal GFR in Children and Young Adults
Age (Sex)
Mean GFR ± SD
(mL/min/1.73 m2)
1 week (males and females)
40.6 ± 14.8
2-8 weeks (males and females)
65.8 ± 24.8
>8weeks (males and females)
95.7 ± 21.7
2-12 years (males and females)
133.0 ± 27.0
13-21 years (males)
140.0 ± 30.0
13-21 years (females)
126.0 ± 22.0
Data based on three studies.[0][5][6]
GFR, glomerular filtration rate; SD, standard deviation.
1. Heilbron DC, Holliday MA, al-Dahwi A, Kogan BA. Expressing glomerular filtration 
rate in children. Pediatr Nephrol. 1991 Jan;5(1):5-11.
2. Coulthard MG. Maturation of glomerular filtration in preterm and mature babies. 
Early Hum Dev. 1985 Sep;11(3-4):281-92.
3. Schwartz GJ, Feld LG, Lanford DJ. A simple estimate of glomerular filtration rate in 
full-term infants during the first year of life. J Pediatr. 1984 Jun;104(6):849-54.
The estimated GFR may be calculated using serum creatinine results at screening, and using 
the calculator in the following link:
http://www-users.med.cornell.edu/~spon/picu/calc/crclschw.htm


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Appendix 16.3 Subject diary was added per Modification 14.


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Appendix 16.2  Procedure for multiplex real time polymerase chain reaction to Trypanosoma 
cruzi detection in patients with Chagas’ disease was renumbered to 16.4.
Appendix 16.3 Pharmacokinetic blood sample handling was changed renumbered to 16.5.
Appendix 16.4 Maximum blood volumes for pediatric patients – Clinical and Laboratory 
Standards Institute was renumbered to 16.6.


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15.2
Amendment 2
There have been 2 global amendments to the study protocol.  Changes made to the study 
protocol by Amendment 1 are described in detail in Section 15.1 and by Amendment 2 in 
Section 15.2.
15.2.1
Overview of changes
This section provides a conceptual overview of all modifications to the amended protocol, as 
introduced by this amendment.  The reasons for the protocol changes included in Amendment 
2 are: 1) to address FDA comments regarding handling of the analyses of data for the primary 
efficacy endpoint, 2) to provide textual clarifications resulting from comments received from 
Ethics Committees and ex-US Health Authorities, and 3) overall editorial corrections and 
clarifications. 
An important modification is that a minimum number of 38 subjects in each age stratum will 
be targeted, in order to obtain an adequate number of completed subjects per group for 
analyses. Enrollment will continue until this specification is met, unless it is determined that 
such a target would be unlikely to be reached in a reasonable time.  
The associated changes to the protocol text are detailed in Section 15.2.2.
Sections affected include:

Synopsis and Study objectives, Section 4

Synopsis and Study design, Section 5

Justification of the design, Section 5

Inclusion criteria, Section 6.1

Exclusion criteria, Section 6.2

Withdrawal of subjects from study, Section 6.3

Dosage and administration, Section 7.4

Blinding, Section 7.5

Procedures and variables, Section 9

Tabular schedule of evaluations, Section 9.1

Visit description, Section 9.2

Screening, Section 9.2.1

Treatment Phase, Section 9.2.2

Visit 10 (Day 420 ± 7), Section 9.2.3.3

Visit 11  (Day 420 ± 7), 9.3.2.2

Efficacy, Section 9.4

Polymerase chain reaction test, Section 9.4.1


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
Serological tests, Section 9.4.2

Statistical methods and determination of sample size, Section 10

General considerations, Section 10.1

Analysis sets, Section 10.2

Variables and planned statistical analyses, Section 10.3

Determination of sample size, Section 10.4

Data recording, Section 11.1

Reference list, Section 14

Subject diary, Appendix 16.3
15.2.2
Changes to the protocol text
In this section, deletions are crossed out, while additions are underlined.
Synopsis and Section 4:  Study Objectives
Old text:
The primary objective of this Phase 3 clinical study is:
To assess the superiority of a 60-day regimen of nifurtimox to historical untreated control at 
the 12-month follow-up (360 days from end of treatment [EOT]) as sero-reduction  (defined 
as a ≥20% reduction in optical density [OD] measured by conventional 
enzymelinked- immune sorbent assay [ELISA]) compared to baseline in subjects > 8 months 
to <18 years of age at randomization; or sero-conversion (defined as negative 
Immunoglobulin G [IgG] concentration)205 in subjects ≤ 8 months of age at randomization.
Secondary objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole) as sero-reduction or seroconversion at the 12-month followup 
(360 days from EOT) 

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction  or sero-conversion and to quantitative polymerase chain 
reaction (qPCR) at the 12-month follow-up (360 days from EOT) 

To evaluate the relationship of conventional serology (as sero-reduction or sero-
conversion) to qPCR using frequencies of matches and mismatches to assess 
agreement

To evaluate the relationship of non-conventional serology to conventional serology 

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital 
                                                
205 Text modified per Modification 2 (Amendment 1)


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sign measurements (blood pressure, heart rate, respiratory rate, temperature), adverse 
event (AE) monitoring, and physical examinations, including neurological 
examinations

To evaluate the pharmacokinetics (PK)/pharmacodynamics of nifurtimox in children 
receiving the drug for treatment of Chagas’ disease
New text:
The primary objective of this Phase 3 clinical study is:
To assess the superiority of a 60-day regimen of nifurtimox to historical untreated control at 
the 12-month follow-up (360 days from end of treatment [EOT]) as sero-reduction  (defined 
as a ≥20% reduction in optical density [OD] measured by conventional 
enzymelinked- immune sorbent assay [ELISA]) compared to baseline in subjects ≥8 months 
to <18 years of age at randomization; or sero-conversion (defined as negative 
Immunoglobulin G [IgG] concentration)206 in all subjects.
Secondary objectives of the study are:

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction or sero-conversion at the 12-month follow-up (360 days 
from EOT) 

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital 
sign measurements (blood pressure, heart rate, respiratory rate, temperature), adverse 
event (AE) monitoring, and physical examinations, including neurological 
examinations

To evaluate the pharmacokinetics (PK)/pharmacodynamics (PD) of nifurtimox in 
children receiving the drug for treatment of Chagas’ disease
Exploratory objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole) as sero-reduction or seroconversion at the 12-month follow-up 
(360 days from EOT)

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox using quantitative polymerase chain reaction (qPCR) at the 12-month 
follow-up (360 days from EOT)

To evaluate the relationship of conventional serology (as sero-reduction or 
seroconversion) to qPCR using frequencies of matches and mismatches to assess 
agreement 

To evaluate the relationship of non-conventional serology to conventional serology
                                                
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Synopsis, and Study design, Section 5
Old text:
Subjects will be stratified by age at randomization into four cohorts as specified below:
Cohort 1:
0 to 27 days
Cohort 2:
28 days to younger than 8 months
Cohort 3:
8 months to younger than 2 years
Cohort 4:
2 years to younger than 18 years
New text:
Approximately 390 pediatric subjects will be randomized (2:1 randomization, 60-day regimen 
vs. 30-day regimen). Subjects will be stratified by age at randomization into four strata as 
specified below:
Stratum 1:
0 to 27 days
Stratum 2:
28 days to younger than 8 months
Stratum 3:
8 months to younger than 2 years
Stratum 4:
2 years to younger than 18 years
A minimum of 38 subjects in each age stratum is targeted, but not required, in order to be able 
to derive meaningful safety conclusions.  Enrollment will continue until this specification is 
met, unless it is determined that such a target would be unlikely to be reached in a reasonable 
time.
Justification of the design, Section 5
Old text:
…
At 12 months, a 21% reduction in concentration of Chagas’ diseasespecific antibodies 
compared to baseline (ELISA mean OD 0.467 [± 0.099] vs. 0.369 [± 0.107]) was observed in
subjects who received benznidazole.  For the subsequent time points of 18, 24, and 48 
months, percent concentration reductions were 23%, 29%, and 27%, respectively.  At the end 
of the 48-month follow-up period, nearly 62% of benznidazole -treated subjects and none of 
the placebotreated subjects were sero-negative for T. cruzi.  Xenodiagnosis performed at that 
time was positive in 51.2% of the placebo group and 4.7% of the benznidazole group 
(P<0.001).  The low frequency of positive xenodiagnosis would be indicative of a low 


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parasitemia and, therefore, low antigenic load which would prevent development of chronic 
disease.
The above data are comparable to a study in early chronic Chagas’ patients treated with 
7.5 mg/kg benznidazole or placebo for 60 days, which also showed approximately 20% 
antibody concentration reduction at 12 months.[16]  Analysis of concentration over time 
indicated a consistent decrease in antibody concentrations in the benznidazole group, and an 
initial decrease followed by a progressive increase in the placebo group.  A significant 
difference between the groups was apparent 6 months after completion of treatment.  At the 
end of the 3-year follow-up, children who received benznidazole had five-fold lower 
geometric mean concentration by indirect immunofluorescence than placebo-treated children 
(196 [147-256] vs. 1068 [809-1408], P<0.00001).  The intent-to-treat analysis based on 
ELISA showed that treatment was successful in 37 (58%) of 64 children in the benznidazole 
group compared with 3 (5%) of 65 children in the placebo group.  As persistence of positive 
results by conventional serology for years after treatment is common, these tests may or may 
not revert to seronegative (sero--conversion) after many years or even decades.  Thus, 
reduction in T. cruzi antibody concentration has been used in clinical practice as a surrogate 
indicator for parasitological cure (sero-conversion) following anti-trypanosomal treatment. 
While a decrease ≥20% in antibody concentration has been demonstrated across two 
independent clinical trials in pediatric patients with Chagas’ disease, neither author has 
published extended follow-up data which include serological results for the patients in these 
studies.  Since signs and symptoms are usually lacking in early Chagas’ disease, it is not 
possible to demonstrate immediate meaningful clinical benefit to active treatment.  However, 
it is presumed that significant reductions in T. cruzi antibody concentration (sero-reduction) 
by conventional serology is a predictor of future sero-conversion, and early treatment and
cure of infection will ultimately reduce the risk of developing visceral abnormalities, as well 
as contribute to the decrease in T. cruzi transmission.
…
Studies have demonstrated the substantial value of qPCR, in terms of sensitivity and 
specificity, in the direct parasitological diagnosis of Chagas’ disease and follow-up 
assessment of chemotherapy.  However, in this Phase 3 study, parasitological cure based on 
serological tests for T. cruzi infection (ELISA) will be used as the primary efficacy endpoint 
to demonstrate efficacy of nifurtimox due to lack of historical PCR data from untreated 
children with Chagas’ disease. Other parasitological methods (direct microscopic) and qPCR 
tests will be secondary efficacy variables to demonstrate drug efficacy.  Significant progress 
has occurred over recent years on the standardization and validation of PCR method in 
Chagas’ disease.  Studies in pediatric and adult Chagas’ patients have been performed 
demonstrating the high sensitivity and specificity of qPCR.  Meanwhile, it is well-recognized 
as the method of choice for the evaluation of treatment response especially in children with 
acute or early chronic Chagas’ disease.  Information on the correlation of PCR results in 
patients with Chagas’ reactivation and also with micro-hematocrit findings in acute Chagas’ 
disease provide evidence to support its use.
The 30-day and 60day treatment regimens were selected with the intention of providing the 
most effective treatment within the shortest possible duration.  In a recent study from Bianchi 
et al, 62 patients with Chagas’ disease confirmed by different serological tests were treated 


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with nifurtimox (Lampit), and followed for 30 months post-treatment.  All children were 
treated during 60 days according to protocols established by the WHO.
…
New text:
…
At 12 months, a 21% reduction in concentration of Chagas’ diseasespecific antibodies 
compared to baseline (ELISA mean OD 0.467 [± 0.099] vs. 0.369 [± 0.107]) was observed in 
subjects who received benznidazole.  For the subsequent time points of 18, 24, and 48 
months, further reductions in antibody titer were reported as 23%, 29%, and 27%, 
respectively. After a 48-month follow-up, negative sero-conversion was detected in 11.3% 
(P<0,05) of the benznidazole group and only 4.5% of the placebo group.
The above data are comparable to a study in early chronic Chagas’ patients treated with 
7.5 mg/kg benznidazole or placebo for 60 days, which also showed approximately 20% 
antibody concentration reduction at 12 months.[16]  Analysis of concentration over time 
indicated a consistent decrease in antibody concentrations in the benznidazole group, and an 
initial decrease followed by a progressive increase in the placebo group.    At the end of the 
3-year follow-up, children who received benznidazole had a significantly lower mean 
antibody concentration than placebo-treated children (P<0.00001).    As persistence of 
positive results by conventional serology for years after treatment is common, these tests may 
or may not revert to seronegative (sero--conversion) after many years or even decades.  Thus, 
reduction in T. cruzi antibody concentration has been used in clinical practice as a surrogate 
indicator for parasitological cure (sero-conversion) following anti-trypanosomal treatment. 
While a decrease ≥20% in antibody concentration has been demonstrated across two 
independent clinical trials in pediatric patients with Chagas’ disease, neither author has 
published extended follow-up data which include serological results for the patients in these 
studies.  Since signs and symptoms are usually lacking in early Chagas’ disease, it is not 
possible to demonstrate immediate meaningful clinical benefit to active treatment.  However, 
significant reductions in T. cruzi antibody concentration (sero-reduction) by conventional 
serology has been accepted  as a predictor of future sero-conversion, and early treatment with
cure of infection will ultimately reduce the risk of developing visceral abnormalities, as well 
as contribute to the decrease in T. cruzi transmission.
…
Studies have demonstrated the substantial value of qPCR, in terms of sensitivity and 
specificity, in the direct parasitological diagnosis of Chagas’ disease and follow-up 
assessment of chemotherapy.  However, in this Phase 3 study, parasitological cure based on 
serological tests for T. cruzi infection (ELISA) will be used as the primary efficacy endpoint 
to demonstrate efficacy of nifurtimox due to lack of historical PCR data from untreated 
children with Chagas’ disease. Other parasitological methods (direct microscopic) and qPCR 
tests will comprise other variables to demonstrate drug efficacy.  
The 30-day and 60-day treatment regimens were selected with the intention of providing the 
most effective treatment within the shortest possible duration. During the 1st Chagas Platform 
Technical meeting (September 1, 2009, Rio de Janeiro) [28] it was concluded that the 


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minimum length of treatment for nifurtimox should be 30 days which is based on good results 
with fewer than 60 days of treatment. Hence, the 30-day arm is included to evaluate the 
potential of this shorter regimen.  In a recent study from Bianchi et al, 62 patients with 
Chagas’ disease confirmed by different serological tests were treated with nifurtimox 
(Lampit), and followed for 30 months post-treatment.  All children were treated during 60 
days according to protocols established by the WHO.
…  
Old text:
End of study
The end of the study as a whole will be reached as soon as the last visit of the last subject has 
been reached in all centers in all participating countries (EU and non-EU).
New text:
End of study
The end of the study as a whole will be reached as soon as the last visit of the last subject has 
been reached in all centers in all participating countries.
Inclusion criteria, Section 6.1
Old text:
…

Subjects ≥8 months to < 18 years of age at randomization must demonstrate a 
positive conventional ELISA result for both of the following tests to confirm 
diagnosis:

Recombinant ELISA 

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test will be obtained; however, a positive result 
will not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation of positive Chagas’ 
disease within three months prior to screening and they have not had prior 
antitrypanocidal or anti-parasitic treatment.
…
New text:
…

Subjects ≥8 months to < 18 years of age at randomization must demonstrate a 
positive conventional ELISA result for both of the following tests to confirm 
diagnosis:

Recombinant ELISA 


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
Total purified antigen ELISA   
Additionally, a non-conventional ELISA test will be obtained; however, a positive result 
will not be required for diagnosis.
Subjects will also be eligible if there is acceptable documentation (two ELISA tests) of 
positive Chagas’ disease within three months prior to screening and they have not had 
prior antitrypanocidal or anti-parasitic treatment. (The subject’s parent or legally 
acceptable representative must agree to the collection of a baseline blood specimen for 
ELISA testing later during the study).
…
Exclusion criteria, Section 6.2
Old text:
…
9. Subjects who have had previous treatment with trypanocidal agents or an accepted 
indication for antiparasitic therapy (e.g., reactivation of Chagas' infection due to 
immunosuppression by several diseases or treatment with steroids)  
10. Subjects who have had treatment with any investigational medicinal product within 
30 days before the first dose of study drug, and have previously received the study 
treatment
11. Subjects who are pregnant or breastfeeding
...
New text:
…
9. Subjects who have had previous treatment with trypanocidal agents or an accepted 
indication for antiparasitic therapy (e.g., reactivation of Chagas' infection due to 
immunosuppression by several diseases or treatment with steroids)  
10. Subjects who have had treatment with any investigational medicinal product within 
30 days before the first dose of study drug, or have previously received the study 
treatment
11. Subjects who are pregnant or breastfeeding
...
Withdrawal of subjects from study, Section 6.3
Old text:
Withdrawal criteria
Subjects must be withdrawn from the study if any of the following occurs:


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At their own request or at the request of their legally acceptable representative.  At any 
time during the study and without giving reasons, a subject may decline to participate 
further.  The subject will not suffer any disadvantage as a result of this decision.
Pregnancy.  If any urine pregnancy test is positive, a serum pregnancy test must be 
performed to confirm the result.  See Section 9.6.2.
New text: 
Withdrawal criteria
Subjects must be withdrawn from the study if any of the following occurs:
At their own request or at the request of their parents’ or legally acceptable 
representative.  At any time during the study and without giving reasons, a subject may 
decline to participate further.  The subject will not suffer any disadvantage as a result 
of this decision.
Pregnancy.  If any urine pregnancy test is positive, a serum pregnancy test must be 
performed to confirm the result.  See Section 9.6.2.
Subjects,with positive  parasitology test at the 30-day visit for whom (1) it has been 
determined that the drug was administered correctly and (2) the daily dosage given was 
correct, must be discontinued from the study and treated with an alternative 
trypanocidal medication, selected by the investigator.
Dosage and administration, Section 7.4
Old text:
Nifurtimox oral tablets will be administered in recommended doses by body weight as per 
WHO guidelines as presented in Table 7-1.
Table 7-1:  Dosage of 30-mg nifurtimox oral tablets by body weight
Drug
Body weight
Dose and duration a
Nifurtimox
< 40 kilograms
10-12 mg/kilogram per day in 3 divided doses (maximum 
dosage 15 mg/kilogram per day) 
> 40 kilograms
8 – 10 mg/kilogram per day in 3 divided doses
a The planned mg/kg per day dosages as per WHO guidelines are less than the recommended 
daily dosages in the Company Core Data Sheet.
…
New text:
Nifurtimox oral tablets will be administered in recommended doses by body weight according 
to the CCDS (Table 7-1). For infants and children weighing more than 6.0 kg and < 40kg, the 
daily dosage  will be 10 - 20 mg/kg.  Total daily dosages for adolescents weighing > 40 mg 
will be 8-10mg/kg, slightly lower than that recommended in the CCDS, to minimize the 
likelihood of adverse experiences.  


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Table 7-1:  Total daily dosage of nifurtimox oral tablets  based on body weight
Body weight group
Total daily dose of nifurtimox [mg / kg body weight]
Adolescents ( 40 – 60 kg )
8-10
Infants and children ( < 40 kg )
10-20  
…
Old text:
Table 7-2:  Dosage of nifurtimox oral tablets by body weight 
Age Group
Body Weight (kg)
Number of 30-mg 
Tablets
Number of 120-mg 
Tablets
< 12 years
3 - <6
3 x daily ½ tablet
---
6 - <10
3 x daily 1 tablet
---
10 - <16
3 x daily 1 ½ tablets
---
16 - <19
3 x daily 2 tablets
---
19 - <22
3 x daily 2 ½ tablets
---
22 - <27
3 x daily 3 tablets
---
27 - <35
3 x daily 3 ½ tablets
---
35 - <41
---
3 x daily 1 ½  tablets
≥12 to < 18 years
41 - <46
---
3 x daily 1 tablet
46 - <51
---
3 x daily 1 tablet
51 - <56
---
3 x daily 1 ½ tablets
56 - <61
---
3 x daily 1 ½ tablets
61 - <71
---
3 x daily 1 ½ tablets
71 - <81
---
3 x daily 2 tablets
81 - <91
---
3 x daily 2 tablets 
91 or greater
---
3 x daily 2  ½ tablets
At Visits 2 and 6, when study drug is dispensed, subjects/legally authorized representatives 
will be provided with a diary on which to document the date and doses (morning, noon, and 
night) of study drug, and comments (see Section 1). Subjects/legally authorized 
representatives will be instructed to bring their diaries with them to each in-clinic study visit.  
The diaries will be collected and reviewed at each study visit to perform drug accountability 
and assess treatment compliance.
…
New text: 
…


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Table 7-2:   Individual dosages of nifurtimox oral tablets based on body weight
Age Group
Body Weight (kg)207
Number of 30-mg 
Tablets
Number of 120-mg 
Tablets
2.5 – 4.5
3 x daily ½ tablet
4.6 - <9
3 x daily 1 tablet
---
9 - <13
3 x daily 1 ½ tablets
---
13 - <18
3 x daily 2 tablets
---
18 - <22
3 x daily 2 ½ tablets
---
22 - <27
3 x daily 3 tablets
---
27 - <35
3 x daily 3 ½ tablets
---
35 - <41
---
3 x daily 1 ½  tablets
≥12 to < 18 years
41 - <51
---
3 x daily 1 tablet
51 - < 71 
---
3 x daily 1 ½ tablets
71 - < 91
---
3 x daily 2 tablets
91 or greater
---
3 x daily 2  ½ tablets
At Visits 2 and 6, when study drug is dispensed, subjects/legally authorized representatives 
will be provided with a diary on which to document the date, exact time, and doses (morning, 
noon, and night) of study drug, and comments (see Section 1). Should a subject vomit or spit 
out study drug, the occurrence will be recorded on the diary along with the time of the event. 
Subjects/legally authorized representatives will be instructed to bring their diaries with them 
to each in-clinic study visit.  The diaries will be collected and reviewed at each study visit to 
perform drug accountability and assess treatment compliance.
…
Blinding,  Section 7.5
Old text:
Blinding
In compliance with applicable regulations, in the event of a suspected, unexpected, serious 
adverse reaction (SUSAR) (see Section 9.6.1.5) related to the blinded treatment, the subject’s 
treatment code will usually be unblinded before reporting to the health authorities, ethic 
committees and investigators (see Section 9.6.1.4).
New text:
                                                
207 In this column, upper limit of weight ranges in each category changed per the most current dosing information 
(Amendment 1) 


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Blinding
In compliance with applicable regulations, in the event of a suspected, unexpected, serious 
adverse reaction (SUSAR) (see Section 9.6.1.5) related to the blinded treatment, the subject’s 
treatment code will usually be unblinded before reporting to the health authorities, ethic 
committees and investigators (see Section 9.6.1.4).


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Emergency unblinding by the investigator
In case of emergency unblinding for a SAE investigators will use the IVRS/IWRS to unblind a subject.
Procedures and variables, Section 9
Tabular schedule of evaluations, Section 9.1
Old table:
Table 9-1:  Schedule of procedures and assessments – Study 16027 
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
Initiation procedures
Informed consent/assent

Demographics (sex, age)

Medical/surgical history

Inclusion/exclusion criteria


Efficacy
Assessment of Chagas’ disease signs and symptoms d







Concentration test for T. cruzi (subjects < 8 months of age)






Conventional and non-conventional serologic testing for 
Chagas’ disease






qPCR






Safety
Adverse events











Concomitant medication











Physical examination e







Neurological examination f




Vital signs (heart rate, RR, BP, temperature), height/length, 
and weight g








12-lead ECG h






Serum hematology, chemistry, and coagulation i







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Urinalysis (if specimen can be obtained)





Urine pregnancy test  j






Pharmacokinetics
PK blood sampling (optional) k




Study Treatment
Study drug administration l



Study drug and diary dispensed m


n
Study drug and diary collected

n

Study drug accountability/review compliance






Dispense pre-paid phone card o


Collect pre-paid phone card




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BP = blood pressure, ECG = electrocardiogram, EOT = end-of-treatment, qPCR = quantitative polymerase chain reaction, PK = pharmacokinetic, RR = respiratory rate per minute.
a  At Days 14, 21, and 42, study site personnel will contact the subject/legally authorized representative via telephone to assess for the occurrence of adverse events, use of 
concomitant medications, and compliance with study drug administration. A Phone Contact Form for the telephone contacts to obtain safety and compliance information will be 
provided to all sites (see Section 16.1).  If any safety concern arises, subjects may return to the study site for an Unscheduled Visit. 
b  Subjects who discontinue prematurely from study drug administration will continue to return to the investigational site for study assessments at  Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7. If subjects are unable/unwilling to do so, they must return to the investigational site 30 (±3) days after the last dose of 
study drug for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 5, and 7. If the subject is unable/unwilling to return to the clinic for the 
EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of Visit 8 assessments.
c  All assessments are to be obtained pre-treatment on Day 1 except for post-dose PK blood sampling.
d  To be performed during physical examinations; see Section 9.4.4
e  A complete physical examination of body systems  will be performed at the Screening Visit.  A brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will be performed at all subsequent designated time points. Abnormal findings 
on physical examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).
f A neurological examination including assessments of mental status and cognition, cranial nerves, motor function, deep tendon reflexes, sensation, and coordination and gait will be 
performed. Abnormal findings on neurological examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see 
Section 9.6.1.1).
g Blood pressure is optional in subjects < 5 years of age at the discretion of the investigator. Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.  
h  12-lead ECG is optional in subjects < 5 years of age at the discretion of the investigator. At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of maximum 
concentration), an ECG will be obtained to allow for PK/PD investigations. 
i  Fasting of subjects for serum laboratory assessments is dependent on the age of the subject at the discretion of the investigator.
j  Urine pregnancy tests will be performed on all females of childbearing potential (i.e., all female subjects who have experienced menarche).  Any subject with a positive urine 
pregnancy test will have a serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be discontinued from the study and undergo all study assessments 
as described in the EOT Visit.
k   Blood samples for pharmacokinetic parameters will be obtained prior to administration of study drug, and at designated time points thereafter (see Table 9-2). They are optional and 
require consent/assent of the subject/subject’s legally authorized representative(s).
l  Subjects consenting to optional PK assessments must withhold taking the morning dose of study drug on the day of Visits 3, 6, and 8. Subjects not consenting to optional PK 
assessments may take their study drug on the mornings of Visits 3, 6, and 8 as instructed.
m  Depending on the country, study site personnel may dispense condoms at the time that study drug is dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.
n   At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and study drug for the remaining 30 days of treatment will be dispensed.  
o  A pre-paid phone card will be provided at Visits 3 and 6 to facilitate contact between study site personnel and the subjects/subjects’ authorized representatives at Visits 4, 5, and 7.  


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New table:
Table 9-1:  Schedule of procedures and assessments – Study 16027
Phase
Screening
Treatment Phase
Follow-up Phase
Visit
1
2
3 
4 a
5 a
6
7 a
8 (EOT) b
9
10
11 
Day
-14 to -1
1 c
7 ± 1
14 ± 1
21 ± 3
30 ± 3
42 ± 3
60 ± 3
90 ± 7
240 ± 7
420 ± 7
Initiation procedures
Informed consent/assent

Demographics (sex, age)

Medical/surgical history

Inclusion/exclusion criteria


Efficacy
Assessment of Chagas’ disease signs and symptoms d







Concentration test for T. cruzi (subjects < 8 months of age)





Conventional and non-conventional serologic testing for 
Chagas’ diseasee






qPCR






Safety
Adverse events











Concomitant medication











Physical examination f







Neurological examination g




Vital signs (heart rate, RR, BP, temperature), height/length, 
and weight h








12-lead ECG  i








Serum hematology, chemistry, and coagulation j
k




Urinalysis (if specimen can be obtained)
k




Urine pregnancy test  l






Pharmacokinetics
PK blood sampling (optional) m




Study Treatment
Study drug administration n


Study drug and diary dispensed o

p
Study drug and diary collected

p

Study drug accountability/review compliance








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Dispense pre-paid phone card q


Collect pre-paid phone card


BP = blood pressure, ECG = electrocardiogram, EOT = end-of-treatment, qPCR = quantitative polymerase chain reaction, PK = pharmacokinetic, RR = respiratory rate per minute.
a  At Days 14, 21, and 42, study site personnel will contact the subject/legally authorized representative via telephone to assess for the occurrence of adverse events, use of 
concomitant medications, and compliance with study drug administration. A Phone Contact Form for the telephone contacts to obtain safety and compliance information will be 
provided to all sites (see Section 16.1).  If any safety concern arises, subjects may return to the study site for an Unscheduled Visit. 
b  Subjects who discontinue prematurely from study drug administration will continue to return to the investigational site for study assessments at Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7. If subjects are unable/unwilling to do so, they must return to the investigational site 30 (±3) days after the last dose of 
study drug for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 5, and 7. If the subject is unable/unwilling to return to the clinic for the 
EOT Visit (Visit 8), then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of Visit 8 assessments..
c  All assessments are to be obtained pre-treatment on Day 1 except for post-dose PK blood sampling.
d To be performed during physical examinations; see Section 9.4.4
e Specimen taken for non-conventional ELISA, qPCR  and stored (as per amendment 2)
f  A complete physical examination of body systems will be performed at the Screening Visit.  A brief physical examination, including assessments of heart, lungs, and abdomen, skin 
for the presence of severe dermatitis, and assessments for the presence of peripheral neuropathy will be performed at all subsequent designated time points. Abnormal findings 
on physical examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see Section 9.6.1.1).
g  A neurological examination including assessments of mental status and cognition, cranial nerves, motor function, deep tendon reflexes, sensation, and coordination and gait will be 
performed. Abnormal findings on neurological examination at Screening will be documented as medical history; abnormal findings thereafter will be documented as AEs (see 
Section 9.6.1.1).
h Blood pressure is optional in subjects < 5 years of age at the discretion of the investigator. Height/length and weight will be obtained with the subject wearing minimal clothing and no 
shoes.  
i  12-lead ECG is optional in subjects < 5 years of age at the discretion of the investigator. At the 2 – 4 hour time point  for PK blood sampling (i.e., at the time of maximum 
concentration), the  ECG will be obtained to allow for PK/PD investigations. 
j  Fasting of subjects for serum laboratory assessments is dependent on the age of the subject at the discretion of the investigator.
k Test conducted by local laboratories (as per amendment 2)
l Urine pregnancy tests will be performed on all females of childbearing potential (i.e., all female subjects who have experienced menarche).  Any subject with a positive urine 
pregnancy test will have a serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be discontinued from the study and undergo all study assessments 
as described in the EOT Visit.
m   Blood samples for pharmacokinetic parameters will be obtained prior to administration of study drug, and at designated time points thereafter (see Table 9−2). They are optional and 
require consent/assent of the subject/subject’s legally authorized representative(s).
n Subjects consenting to optional PK assessments must withhold taking the morning dose of study drug on the day of Visits 3, 6, and 8. Subjects not consenting to optional PK 
assessments may take their study drug on the mornings of Visits 3, 6, and 8 as instructed.
o  Depending on the country, study site personnel may dispense condoms at the time that study drug is dispensed to either the subject or the subject’s parent/legal guardian, with an 
explanation of rationale and risks.
p At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and study drug for the remaining 30 days of treatment will be dispensed.  
q  A pre-paid phone card will be provided at Visits 3 and 6 to facilitate contact between study site personnel and the subjects/subjects’ authorized representatives at Visits 4, 5, and 7.  


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Visit description, Section 9.2
Screening, Section 9.2.1
Visit 1 – screening visit (Days -14 to -1), Section 9.2.1.1
Old text:
…

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age at randomization), 
conventional and non-conventional serologic testing for Chagas’ disease (or 
acceptable documentation of positive Chagas’ disease within 3 months prior to 
screening and no prior anti-trypanocidal or anti-parasitic treatment), and qPCR

Assessment of pretreatment AEs  and concomitant medications
…
New text:
…

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. cruzi (subjects < 8 months of age at randomization) and 
conventional serologic testing for Chagas’ disease (subjects ≥8 months < 18 years of 
age) conducted in local laboratory (or acceptable documentation of positive Chagas’ 
disease within 3 months prior to screening and no prior anti-trypanocidal or anti-
parasitic treatment).  Specimens taken for conventional serology, non-conventional
serology and qPCR will be frozen and stored at -20oC prior to shipment to the central 
laboratory where they will be stored at -70oC .

Assessment of pretreatment AEs  and concomitant medications
…
Treatment Phase, Section 9.2.2
Visit 2 – baseline (treatment phase, Day 1), Section 9.2.2.1
This section was changed for clarity and to reflect changes made per Modifications 1, 2, 4, 9, 
and 10.
Old text:
…

Assessment of pretreatment AEs and concomitant medications  

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight


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
12-lead ECG (optional in subjects <5 years of age at the discretion of the investigator).  
At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of Cmax), an ECG 
will be obtained to allow for PK/PD investigations.

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
excluded from study participation.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9−1).

Administer first dose of study drug to all subjects

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9−1).  At the 2 – 4 hour time point (i.e., at the time of Cmax), an 
ECG will be obtained. 

Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3; subjects not consenting to optional PK assessments may take their 
study drug on the day of Visit 3 as instructed.
…
New text:
…

Assessment of pretreatment AEs and concomitant medications  

Vital signs (blood pressure is optional in subjects < 5 years of age at the discretion of 
the investigator), height/length, and weight

12-lead ECG (optional in subjects <5 years of age at the discretion of the investigator).  

Urine pregnancy test on all females of childbearing potential (i.e., those who have 
experienced menarche).  Any subject with a positive urine pregnancy test will have a 
serum pregnancy test to confirm results; if pregnancy is confirmed, the subject is to be 
excluded from study participation.

For subjects consenting to PK assessments, a pre-dose PK blood sample will be 
obtained (see Table 9−1).

Administer first dose of study drug to all subjects

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9−1) At the 2 – 4 hour time point of PK blood sampling (i.e., at 
the time of Cmax), an ECG will be obtained to allow for PK/PD investigations. 
Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3; subjects not consenting to optional PK assessments may take their 
study drug on the day of Visit 3 as instructed.


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…
Section 9.2.2.3
Old text: 
9.2.2.3 Visit 4 (Day 14 ± 3 days)
New text: 
9.2.2.3 Visit 4 (Day 14 ± 1 days)
Visit 10 (Day 420 ± 7), Section 9.2.3.2
Old text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Concentration test for T. Cruzi (subjects < 8 months of age), conventional and non-
conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Concomitant medications
…
New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Conventional and non-conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs

Concomitant medications
…
9.3.2.2 Visit 11  (Day 420 ± 7)
Old text: 

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

Conventional and non-conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs


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New text:

Assessment of Chagas’ disease signs and symptoms (to be performed during physical 
examinations); see Section 9.4.4

12-lead ECG (optional in subjects < 5 years of age at the discretion of the investigator)

Conventional and non-conventional serologic testing for Chagas’ disease and qPCR

Assessment of AEs
…
Efficacy, Section 9.4
Old text:
Efficacy assessments will be obtained at designated study visits (see Table 9−1).  The primary 
efficacy variable will be sero-reduction or sero-conversion at 12 months post-treatment using 
two conventional ELISA serology tests as the measure of efficacy.
A quality control program will be implemented for the primary efficacy variable (i.e., ELISA 
concentration).  At least 20% of measurements, chosen at random, will be confirmed by a 
reference laboratory (Chagas Service, Buenos Aires Children’s Hospital “Ricardo Gutierrez”).
Other efficacy variables include disease state determined by qPCR, concentration tests for
T. cruzi (subjects < 8 months of age at randomization), conventional and non-conventional 
serologic testing, and clinical signs/symptoms of Chagas’ disease.
…
New text:
Efficacy assessments will be obtained at designated study visits (see Table 9−1).  The primary 
efficacy variable will be sero-reduction or sero-conversion at 12 months post-treatment using 
two conventional ELISA serology tests as the measure of efficacy.
A quality control program will be implemented for all tests according to the laboratory 
QC/QA department SOPs. 
Other efficacy variables include disease state determined by qPCR, concentration tests for
T. cruzi (subjects < 8 months of age at randomization), conventional and non-conventional 
serologic testing, and clinical signs/symptoms of Chagas’ disease.
…
Polymerase chain reaction test, Section 9.4.1
Old text:
A qPCR test will be performed at the Screening Visit, Visits 3, 6, and 8, and Follow-up Visits 
10 and 11.
Details describing the collection, processing, storage and shipment of qPCR blood samples, 
and the qPCR test protocol, are described in Section 1. 
New text:


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A qPCR test will be performed at the Screening Visit, Visits 3, 6, and 8, and Follow-up Visits 
10 and 11.  
Details describing a suggested  collection, processing, storage and shipment for qPCR blood 
samples, and a suggested qPCR test protocol, are described in Section 1. The study central 
laboratory procedures and qPCR protocol will be followed.
Serological tests, Section 9.4.2
Old text:
…
All subjects ≥8 months to < 18 years of age at randomization will have two baseline 
specimens collected for serologic testing.  One sample will be used to determine the initial 
diagnosis of Chagas’ disease while the other sample will be frozen and retained to be 
assayed along with the 12-month post-treatment specimen as an internal control.  Subjects < 8 
months of age at randomization will have one specimen obtained which will be frozen and 
retained.  Total blood volume to be taken for the required for three serological and PCR tests 
is 4.05.0 mL, depending on the subject’s age.  Care will be taken to minimize sample volumes 
by using a micro-sampling collection technique.
Details describing the collection, processing, storage and shipment of blood samples, and the 
serologic test protocol, will be described in a separate serologic test manual.  Initial serologic 
tests will be performed by a local laboratory according to the standard of care for each 
country.  Subsequent tests will be performed by a central laboratory.
…
New text:
…
All subjects ≥8 months to < 18 years of age at randomization will have two baseline 
specimens collected for serologic testing.  One sample will be used to determine the initial 
diagnosis of Chagas’ disease while the other sample will be frozen and retained to be 
assayed later  as an internal control.  Subjects < 8 months of age at randomization will have 
one specimen obtained which will be frozen and retained.  Total blood volume to be taken for 
the required  three serological and PCR tests is 4.05.0 mL, depending on the subject’s age.  
Care will be taken to minimize sample volumes by using a micro-sampling collection 
technique, when possible.
Details describing the collection, processing, storage and shipment of blood samples, and the 
serologic test protocols, will be described in a separate serologic test manual from the central
laboratory.
…
Statistical methods and determination of sample size, Section 10
General considerations, Section 10.1
Old text:


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This study has been designed to focus on the efficacy and safety of treatment with nifurtimox 
in children diagnosed with Chagas’ disease.  In this study, approximately 390 pediatric 
subjects will be randomized to ensure 330 evaluable pediatric subjects (2:1 randomization, 
60-day regimen vs. 30-day regimen).  A subject is considered evaluable if there is sufficient 
information to determine whether or not he or she is cured.  Subjects will be stratified by age 
into four cohorts. Enrollment will continue until there are a total of at least 330 evaluable 
subjects.  A minimum of 38 subjects in each age stratum is targeted, as this would include an 
expected 25 subjects randomized to the 60-day dosing regimen. This is nearly 10% of the 
study sample size, and should provide a maximum CI half-width of approximately 0.20.  
Enrollment will continue until this specification is met, unless it is determined that such a 
target would be unlikely to be reached in a reasonable time.  
…
New text:
This study has been designed to focus on the efficacy and safety of treatment with nifurtimox 
in children diagnosed with Chagas’ disease.  In this study, approximately 390 pediatric 
subjects will be randomized (2:1 randomization, 60-day regimen vs. 30-day regimen).  
Subjects will be stratified by age into four strata. A minimum of 38 subjects in each age 
stratum is targeted, but not required, in order to be able to derive meaningful safety 
conclusions. Enrollment will continue until this specification is met, unless it is determined 
that such a target would be unlikely to be reached in a reasonable time.
…
10.2 Analysis sets
Old text:
The primary efficacy analysis will be done using the full analysis set (FAS), which is the set 
of subjects who received at least one dose of study drug.  This set is sometimes referred to as 
the modified intent-to-treat set.  Analyses will also be done using the per-protocol set, which 
is comprised of subjects treated with study drug who have no major protocol deviations.  
Complete specifications will be provided in the SAP.
Analyses of safety and background data will be performed on the FAS.
New text: 
The primary efficacy analysis will be done using the full analysis set (FAS), which is the set 
of subjects who received at least one dose of study drug. Analyses will also be done using the 
per-protocol set, which is comprised of subjects treated with study drug who have no major 
protocol deviations.  Complete specifications will be provided in the SAP.
Analyses of safety and background data will be performed on the FAS.
Variables and planned statistical analyses, Section 10.3
Old text:
Variables


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The primary efficacy variable will be sero-reduction or sero-conversion at 12 months 
posttreatment using two conventional ELISA serology tests as the measure of efficacy. This 
sero-reduction or sero-conversion is considered cure, and the primary variable is binary (cure, 
no cure).  In the event of discordancy between the two conventional ELISA test results for 
subjects ≥8 months of age, the average percentage sero-reduction will be used (e.g., Test #1 
= 15% and Test #2 = 25%; average = 20% and, hence, cure).  The methodology for 
calculating the average percentage sero-reduction will be specified in the SAP.
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease, 
concentration test for T. cruzi (subjects < 8 months of age at randomization), conventional and 
nonconventional serologic testing, and disease state determined by qPCR.
…
New text:
Variables
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months 
posttreatment using two conventional ELISA serology tests as the measure of efficacy. This 
sero-reduction or sero-conversion is considered cure, and the primary variable is binary (cure, 
no cure).  
In the event of discordancy  between the two conventional ELISA test results, the following 
will be considered: 

For sero-reduction, the average percentage of OD reductions will be used (e.g., Test 
#1 = 15% and Test #2 = 25%; average = 20% and, hence, cure). 

For sero-conversion, only when both test results are negative the subject is considered 
as negative (cure). 
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease, 
concentration test for T. cruzi (subjects < 8 months of age at randomization), conventional and 
nonconventional serologic testing, and disease state determined by qPCR. 
…
Old text:
Statistical and analytical plans 
…
Sero-conversion for the historical control will be estimated from cure rates as presented in 
two publications .[15][16  The sero-conversion rates used for placebo and benznidazole are 
16% and  45%, respectively.  The placebo value was derived by using a number greater than 
the larger of the upper limits of the 95% CIs from the two studies.  Further details about the 
derivation of these rates will be given in the SAP.
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The same CI for the primary endpoint analysis will also be used for comparison with the 
historical benznidazole subjects with sero-conversion to assess whether or not their cure rates 
are comparable. Further details about the comparison will be given in the SAP.
A secondary analysis will be done to compare the proportion of subjects with seroreduction or 
sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be performed 
using a standard, symmetric, asymptotic 2-sided 95% CI for the difference of two independent 
proportions.
New text:
Statistical and analytical plans 
…
Sero-conversion for the historical placebo control is estimated as 16% from cure rates 
presented in two publications [15][16], taking a number greater than the larger of the upper 
limits of the 95% CIs from the two studies. Further details about the derivation of these rates
are provided in Section10.4.
Secondary efficacy analyses
A secondary analysis will be done to compare the proportion of subjects with seroreduction or 
sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be performed 
using a 2-sided 95% CI for the difference of two independent proportions.
…
Safety 
Old text:
Safety variables will be summarized by means of descriptive statistics and/or frequency tables 
as appropriate.  Summaries will be given by treatment regimen. All post-treatment AEs and 
hematological/biochemical toxicities based on laboratory measurements, as well as drug-
related AEs and SAEs, will be summarized by category and worst grade. AEs occurring 
between the time of consent and the first dose of study drug will be summarized separately.  
Subjects experiencing urine discoloration from riboflavin ingestion will be summarized also, 
although these incidences will not necessarily be considered AEs.
New text:
Safety variables will be summarized by means of descriptive statistics and/or frequency tables 
as appropriate.  Summaries will be given by treatment regimen. All post-treatment AEs and 
SAEs, as well as drugrelated AEs and SAEs, will be summarized by category.  AEs occurring 
between the time of consent and the first dose of study drug will be summarized separately.  
Subjects experiencing urine discoloration from riboflavin ingestion will be summarized also, 
although these incidences will not necessarily be considered AEs.
Missing data/drop outs 
Old text:


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No imputations will be made for missing data due to dropouts or other causes. For the 
primary efficacy analysis done on the FAS, a patient without an assessment at the primary 12 
month time point will be considered a treatment failure. For the secondary analysis using the 
PPS, patients who do not have serology determinations to assess the primary endpoint will be 
excluded from the analysis.
Sensitivity analyses will be performed,  treating only missing values due to the following 
reasons as treatment failures:
      1. the reason for discontinuation is known or suspected to be treatment ineffectiveness or 
an AE
       2. a subject  has not had a negative serology at any visit and has no serology 
determination for the primary analysis visit  
       3. an indeterminate or incorrectly performed serology determination occurs at the primary 
analysis visit.
…
New text:
No imputations will be made for missing data due to dropouts or other causes. For the 
primary efficacy analysis done on the FAS, a patient without an assessment at the primary 12 
month time point will be considered a treatment failure. For the secondary analysis using the 
PPS, patients who do not have serology determinations to assess the primary endpoint will be 
excluded from the analysis.
The following sensitivity analyses for the primary efficacy variable will be performed:

Missing values due to the following reasons treated as treatment failures and other 
missing values excluded from analysis:
      1. the reason for discontinuation is known or suspected to be treatment ineffectiveness or 
an AE
       2. a subject has not had a negative serology at any visit and has no serology 
determination for the primary analysis visit  
       3. an indeterminate or incorrectly performed serology determination occurs at the primary 
analysis visit.

Observed data only.

Analysis on PPS.
Synopsis and Determination of sample size, Section 10.4
Old text:
Sample size is based on the primary endpoint, the difference in the proportion of nifurtimox 
subjects with sero-reduction or seroconversion (60-day regimen) and the proportion of 
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powered based on the detection of a difference in proportion of 0.10.  This quantity represents 
a difference between rates, such as 0.60-0.50=0.10, rather than the difference that would 
result from subtracting 10% of 0.60 (that is, 0.06) from 0.60.  Given that the nifurtimox 
(60day) proportion of subjects with sero-reduction or sero-conversion is 0.60, 220 evaluable 
subjects in this group will provide at least 80% power for an exact binomial test. This sample 
size will provide an exact 95% CI with a width of ≤0.15 (e.g., [0.53,0.67]) for use in 
comparing nifurtimox to the proportion of historical benznidazole subjects with 
seroconversion.
The number of subjects in the 30-day treatment regimen is based on the width of a 95% 
asymptotic symmetric CI for the difference of two proportions. A sample size of 110 subjects 
for the 30-day subjects together with 220 subjects for the 60-day subjects will produce a CI 
with a half-width of approximately 0.20.
…
New text:
This study uses historical controls estimated from cure rates presented in two publications 
[15][16]. The historical cure rates are as follows.
Publication
Age 
range
Sero-conversion rate 
(95%CI) 
in placebo patients
Andrade et al 
1996
7-12
3/65 = 5% (1%,13%)
Sosa et al 1998
6 -12
2/44= 5% (1%,16%)
For the primary objective, superiority over placebo will be confirmed if the lower limit of the 
95% CI for the nifurtimox (60day regimen) cure rate is greater than 16%,  the larger of the 
upper limits of the 95% CIs from the two publications. 
According to Guhl et al [31], nifurtimox 12 month post treatment cure rate is about 55%, with 
a sample size of 260 for the 60-day regimen, the power is 99% for the lower limit of the 95% 
CI to be greater than 16%.
The number of subjects in the 30-day treatment regimen is based on the width of a 95% CI for 
the difference of two proportions. A sample size of 130 subjects for the 30-day subjects 
together with 260 subjects for the 60-day subjects will produce a CI with a half-width of 
approximately 0.20.
…
Data recording, Section 11.1
Old text:
…


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For subjects consenting to undergo PK blood sampling, blood samples will be sent to the 
sponsor, and the data will be entered into the SAS after database lock.  The following will be 
documented in the eCRF: Times of the two most recent medication intake prior to the visit, 
administered dose and exact time of dosing at the visit, exact time of blood sampling , time of 
first dose after blood sampling , samples sampled within time window.
New text:
…
For subjects consenting to undergo PK blood sampling, blood samples will be sent to the 
sponsor, and the data will be entered into the SAS after database lock.
Administered dose and exact times of two medication intakes during the visit, exact times of 
blood sampling, time of first dose after blood sampling, and samples collected within time 
window will be recorded in the eCRF. 
Reference list, Section 14
The following references were added to the reference list per changes made as a result of 
Amendment 2, and the cross-references were renumbered in the list and within the protocol 
body accordingly.
28. First Chagas Paltform Technical Meeting Report, September 1, 2009, Rio de Janeiro
31. Guhl F, Nicholls R, Montoya R, Rosas F, Velasco VM, et al. (2004) Rapid negativization 
of serology after treatment with benznidazole for chagas disease in a group of colombian 
schoolchildren. Medimond International Proceedings. Spain, Vol. 1. pp. 107–114.
Subject diary, Appendix 16.3
The subject diary was updated.
15.3
Amendment 3
There have been 3 global amendments to the study protocol.  Changes made to the study 
protocol by Amendment 1 are described in detail in Section 15.1, by Amendment 2 in Section 
15.2 and by Amendment 3 in Section 15.3.


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15.3.1
Overview of changes
This section provides a conceptual overview of all modifications to the amended protocol, as 
introduced by this amendment.  The reason for the protocol change included in Amendment 3 
is to delete a phrase “and ECG abnormalities” which was added in error to amendment 2.  
The associated changes to the protocol text are detailed in Section 15.3.2.
Sections affected include:

Adverse events of special safety interest, Section 9.6.1.6
15.3.2
Changes to the protocol text
In this section, deletions are crossed out, while additions are underlined.
Section 9.6.1.6:  Adverse events of special safety interest
Old text:
In this study, all AEs of weight loss of > 20%, severe rash, severe polyneuropathy, and ECG 
abnormalities208 will be considered AEs of special safety interest. Adverse events of special 
safety interest will be assessed, monitored, and reported as described in Section 9.6.1.4
New text:
In this study, all AEs of weight loss of > 20%, severe rash and severe polyneuropathy will be 
considered AEs of special safety interest. Adverse events of special safety interest will be 
assessed, monitored, and reported as described in Section 9.6.1.4
15.4
Amendment 4
There have been 4 global amendments to the study protocol.  Changes made to the study 
protocol by Amendment 1 are described in detail in Section 15.1, by Amendment 2 in Section 
15.2, by Amendment 3 in Section 15.3 and by Amendment 4 in Section 15.4.
15.4.1
Overview of changes
This section provides a conceptual overview of all modifications to the amended protocol, as 
introduced by this amendment.  The primary reason for amendment 4 is to implement a PK 
subject diary which will allow PK subjects to record the meals consumed with study drug 
administration. Additionally, a few clarifying editorial changes were made.
Modification 1: text was added to Section 5- Study design - “Also, for reasons of patient 
safety and at the discretion of the investigator, a subject may be asked to take a “drug 
holiday” [interruption of study drug administration] or a reduced daily dosage may be 
requested from the depot. A decision by the investigator for drug holiday or reduced dosage 
will be based on the occurrence and/or severity of adverse effects, and/or the clinical status of 
                                                
208 Added as per amendment 2


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the subject. Any and all changes to study drug administration or daily dosage must be 
recorded within the subject’s study chart, as well as, the eCRF.” 
Rationale:  Decision for “drug holiday” would be made by the investigator based on the 
nature and severity of AE’s (eg, nausea, vomiting loss of appetite, etc) and clinic al status of 
the subject.  A “holiday” has been reported to allow the subject to resume and complete 
treatment.
Modification 2: text was added to 6.3 withdrawal of subjects from study. “Only the re-
screening will be captured in the eCRF.”
Rationale:  This textural change was made to provide clarification regarding how data was 
captured for patients who are re-screened.  In this study, only one number is assigned per 
patient.  This same number is used for screening and randomization. If a subject fails initial 
screening and is re-screened, the same subject number is used for the re-screen and ultimately 
for the randomization.  Hence, only the data from the re-screen visit will be captured in the 
eCRF.  Data from the initial screening visit will be captured in the source document at the 
study site.
Modification 3: text was added to 7.4. “Dosages of study drug dispensed at Visits 2 and 6 
will be the same even if a different dosage is indicated based on a patient’s weight change.”
Rationale:  Modification was done as a clarification study drug was assigned. Keeping the 
dosages the same throughout the treatment period was a decision made for consistency.
The associated changes to the protocol text are detailed in Section 15.4.2.
Sections affected include:

Study design – Section 5

Withdrawal of subjects from study – Section 6.3

Dosage and administration – Section 7.4

Treatment phase – Section 9.2.2

Pharmacokinetics/pharmacodynamics – Section 9.5

Statistical methods and determination of sample size – Section 10

Subject diary, Appendix 16.3
15.4.2
Changes to the protocol text
In this section, deletions are crossed out, while additions are underlined.
Section 5: Study design
Old text:
…
A minimum of 38 subjects in each age stratum is targeted, but not required, in order to be able 
to derive meaningful safety conclusions. Enrollment will continue until this specification is 
met, unless it is determined that such a target would be unlikely to be reached in a reasonable 
time.
…


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Study drug will be dispensed, and instructions for study drug administration will be provided  
to all  subject
…
Subjects consenting to PK assessments must hold the morning dose of study drug; for these 
subjects, a pre-dose PK blood sample will be obtained, then the morning dose of study drug 
will be administered.  Post-dose blood samples will be obtained at specified time points, and 
ECGs will be obtained at the 2 – 4 hour post-dose time point (see Table 9−1). A pre-paid 
phone card will be provided to all subjects to facilitate contact between study site personnel 
and the subjects/subjects’ authorized representatives.  
At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and 
study drug for the remaining 30 days of treatment will be dispensed.  
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site 
personnel will contact the subject/legally authorized representative via telephone to assess the 
occurrence of AEs, use of concomitant medications, and compliance with study drug 
administration.  A Phone Contact Form for the telephone assessments will be provided to all 
sites (see Section 16.1).
…
A pre-dose PK blood sample will be collected, then the morning dose of study drug will be 
administered.  Post-dose PK blood samples will be obtained at specified time points, and 
ECGs will be obtained at the 2 – 4 hour post-dose time point (see Table 9−1)
…
In order to minimize the burden of blood loss due to PK sampling, ageappropriate- sparse 
sampling strategies combined with (micro-)bioanalysis techniques will be applied (see Table 
9−4).  At least 10 subjects per age stratum will be recruited for PK assessments.
…
New text:
…
A minimum of 38 subjects in each age stratum is targeted, but not required, in order to be able 
to derive meaningful safety conclusions. Enrollment may continue until this specification is 
met, unless it is determined that such a target would be unlikely to be reached in a reasonable 
time.
…
Study drug will be dispensed, instructions for study drug administration will be provided, and 
diaries with instructions for completion will be given, to all subjects.
…
Subjects consenting to PK assessments must hold the morning dose of study drug; for these 
subjects, a pre-dose PK blood sample will be obtained, then the morning dose of study drug 


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will be administered with food and the PK diary completed.  Post-dose blood samples will be 
obtained at specified time points, and ECGs will be obtained at the 2 – 4 hour post-dose time 
point (see Table 9−1). A pre-paid phone card will be provided to all subjects to facilitate 
contact between study site personnel and the subjects/subjects’ authorized representatives. 
At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and 
study drug for the remaining 30 days of treatment, along with subject diaries, will be 
dispensed.  
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site 
personnel will contact the subject/legally authorized representative via telephone to assess the 
occurrence of AEs, use of concomitant medications, and compliance with study drug 
administration, and completion of subject diary.  A Phone Contact Form for the telephone 
assessments will be provided to all sites (see Section 16.1).
…
A pre-dose PK blood sample will be collected, then the morning dose of study drug will be 
administered with food, and the subject will complete the PK diary.  Post-dose PK blood 
samples will be obtained at specified time points, and ECGs will be obtained at the 2 – 4 hour 
post-dose time point (see Table 9−1)
…
In order to minimize the burden of blood loss due to PK sampling, ageappropriate- sparse 
sampling strategies combined with (micro-)bioanalysis techniques will be applied (see Table 
9−4).  At least 10 subjects per age stratum should ideally be recruited for PK assessments.
Recruitment of less than 10 subjects per age group is not considered as a protocol violation.    
…
Also, for reasons of patient safety and at the discretion of the investigator, a subject may be 
asked to take a “drug holiday” [interruption of study drug administration] or a reduced daily 
dosage may be requested by the investigator from the depot. A decision by the investigator for 
drug holiday or reduced dosage will be based on the occurrence and/or severity of adverse 
effects, and/or the clinical status of the subject. Any and all changes to study drug 
administration or daily dosage must be recorded within the subject’s study chart, as well as, 
the eCRF. 
Section 6.3: Withdrawal of subjects from study
Screening failure:
Old text:
Subjects not meeting inclusion/exclusion criteria at screening may be re-screened once.  At 
the discretion of the investigator and in consultation with Bayer, the screening period may be 
extended up to 3 days to allow for receipt of test results or to evaluate changes in a subject’s 
condition. In any case, the investigator has to ensure that the repeated screening procedures do 
not expose the subject to an unjustifiable health risk.  Also, for re-screening, the subject has to 


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resign the informed consent form, even if it was not changed after the subject’s previous 
screening.  The second re-screening will be captured in the eCRF.
New text:
Subjects not meeting inclusion/exclusion criteria at screening may be re-screened once.  At 
the discretion of the investigator and in consultation with Bayer, the screening period (or re-
screening period) may be extended up to 3 days to allow for receipt of test results, to evaluate 
changes in a subject’s condition, or for reasons not under the subject’s control. In any case, 
the investigator has to ensure that the repeated screening procedures do not expose the subject 
to an unjustifiable health risk.  Also, for re-screening, the subject has to resign the informed 
consent form, even if it was not changed after the subject’s previous screening.  Only the re-
screening will be captured in the eCRF.
Section 7.4: Dosage and administration
Old text: 
Study drug will be dispensed at Visits 2 (Day 1) and 6 (Day 30), with instructions for 
administration. Nifurtimox will be administered three times a day, in the morning, at noon, 
and at night, with food.  The 30-mg and 120-mg tablets have score lines and can be divided 
into two equal halves to allow for 15-mg or 60 mg dose increments, respectively. The tablet is
manufactured for quick disintegration in order to allow administration to subjects < 6 years 
old who are not able to swallow tablets.  Before administration, the tablet should be dissolved 
in enough water to fill a teaspoon  (approximately 5 mL)  to form a soft slurry that should be 
given immediately with food.  Nifurtimox will be administered based on body weight as 
presented in Table 7−2.
New text: 
Study drug will be dispensed at Visits 2 (Day 1) and 6 (Day 30), with instructions for 
administration. Dosages of study drug dispensed at Visits 2 and 6 will be the same even if a 
different dosage is indicated based on a patient’s weight change. Nifurtimox will be 
administered three times a day, in the morning, at noon, and at night, with food.  The 30-mg 
and 120-mg tablets have score lines and can be divided into two equal halves to allow for 15-
mg or 60 mg dose increments, respectively.  The tablet is manufactured for quick 
disintegration in order to allow administration to subjects < 6 years old who are not able to 
swallow tablets.  Before administration, the tablet should be dissolved in enough water to fill 
a teaspoon (approximately 5 mL) to form a soft slurry that should be given immediately with 
food.  Nifurtimox will be dispensed based on body weight at randomization, as presented in 
Table 7−2.
Section 9.2.2:  Treatment phase
Old text:
…

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9−1) At the 2 – 4 hour time point of PK blood sampling (i.e., at 


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the time of Cmax), an ECG will be obtained to allow for PK/PD investigations.
Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3; subjects not consenting to optional PK assessments may take their 
study drug on the day of Visit 3 as instructed.
New text:

For subjects consenting to PK assessments, post-dose PK blood samples will be 
obtained (see Table 9−1) At the 2 – 4 hour time point of PK blood sampling (i.e., at 
the time of Cmax), an ECG will be obtained to allow for PK/PD investigations. 
Dispense study drug and diary with instructions for use.  Subjects consenting to 
optional PK assessments will be instructed to withhold taking study drug on the 
morning of Visit 3 and to complete the PK subject diary, according to the instructions 
provided on the PK subject diary; subjects not consenting to optional PK assessments 
may take their study drug on the day of Visit 3 as instructed. The PK subject diary has 
been added in order for the PK subjects to record all foods they consume when taking 
each dose of study drug, along with the times they eat each meal. Examples of these 
entries, based on foods typically consumed in South American countries, will be given 
to each subject at the PK study sites.
Visit 3 (Day 7 ± 1 days)
Old text:
…
Administer study drug to subjects consenting to PK assessments (who withheld their morning 
dose of study drug)
... 
New text: 
…
Administer study drug with food to subjects consenting to PK assessments (who withheld 
their morning dose of study drug) and instruct subject on completion of PK diary.
... 
Visit 4 (Day 14 ± 1 days)
Old text:
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 


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compliance information will be provided to all sites (Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.
New text: 
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary.  A Phone Contact Form for the 
telephone contacts to obtain safety and compliance information will be provided to all sites 
(Section 16.1).  If any safety concern arises, subjects may return to the study site for an
unscheduled visit
Visit 5 (Day 21 ± 3 days)
Old text: 
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 
compliance information will be provided to all sites (see Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.
…
New text: 
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary.  A Phone Contact Form for the 
telephone contacts to obtain safety and compliance information will be provided to all sites 
(see Section 16.1).  If any safety concern arises, subjects may return to the study site for an 
Unscheduled Visit.
…
Visit 6 (Day 30 ± 3 days)
Old text:
…
Administer study drug to subjects consenting to PK assessments (who withheld their morning 
dose of study drug)
New text: 
…
Administer study drug with food to subjects consenting to PK assessments (who withheld 
their morning dose of study drug) and instruct subject on completion of PK diary.  
Visit 7 (Day 42 ± 3 days)
Old text: 


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Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, and  compliance with study 
drug administration.  A Phone Contact Form for the telephone contacts to obtain safety and 
compliance information will be provided to all sites (see Section 16.1).  If any safety concern 
arises, subjects may return to the study site for an Unscheduled Visit.
…
New text: 
Study site personnel will contact the subject/legally authorized representative via telephone to 
assess for the occurrence of AEs, use of concomitant medications, compliance with study 
drug administration, and completion of subject diary.  A Phone Contact Form for the 
telephone contacts to obtain safety and compliance information will be provided to all sites 
(see Section 16.1).  If any safety concern arises, subjects may return to the study site for an 
Unscheduled Visit.
…
Visit 8 (Day 60 ± 3 days)/End of Treatment Visit
Old text: 
Administer study drug to subjects consenting to PK assessments (who withheld their morning 
dose of study drug).  
…
Drug accountability and review of diaries for study drug compliance
New text: 
Administer study drug to subjects consenting to PK assessments (who withheld their morning 
dose of study drug), and instruct subject on completion of PK diary.  
…
Drug accountability and review of diaries for study drug compliance and for PK subjects, 
documentation of meals during study drug administration.
Section 9.5: Pharmacokinetics/pharmacodynamics
Old text: 
If the subject/subject’s legally authorized representative(s) provide(s) consent/assent to do so, 
plasma concentrations of nifurtimox will be evaluated at Visit 2 (baseline) and Visits 3, 6, and 
8 (EOT) on at least 10 subjects per age stratum selected from all participating  centers.  
Plasma nifurtimox concentrations will be determined using a sparse sampling approach.  
At Visits 2, 3, 6, and 8, the subjects should present themselves to the investigational site 
without taking the morning dose of study medication.  Blood samples for PK will be obtained 
in-clinic prior to the administration of study drug and at specific time points and time 
windows as presented in Table 9–1. At the 2 – 4 hour time point of PK blood sampling (i.e., 
at the time of Cmax), an ECG (optional for subjects < 5 years of age at the discretion of the 
investigator) will be obtained to allow for PK/PD investigations.


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New text: 
If the subject/subject’s legally authorized representative(s) provide(s) consent/assent to do so, 
plasma concentrations of nifurtimox will be evaluated at Visit 2 (baseline) and Visits 3, 6, and 
8 (EOT) on at least 10 subjects per age stratum should be selected from all participating PK 
centers.  Plasma nifurtimox concentrations will be determined using a sparse sampling 
approach.  
At Visits 2, 3, 6, and 8, the subjects should present themselves to the investigational site 
without taking the morning dose of study medication.  Blood samples for PK will be obtained 
in-clinic prior to the administration of study drug with food and at specific time points and 
time windows as presented in Table 9−1. At the 2 – 4 hour time point of PK blood sampling 
(i.e., at the time of Cmax), an ECG (optional for subjects < 5 years of age at the discretion of 
the investigator) will be obtained to allow for PK/PD investigations.  All subjects are to 
complete the PK subject diary according to instructions provided.
Pharmacokinetic evaluation
Old text:
Plasma concentration time courses will be analyzed and reported within the study report.  In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 
report.  Population PK methods will be used to provide parameter estimates describing the PK 
behavior of nifurtimox and to identify possible covariates related to age.
New text:
Plasma concentration time courses will be analyzed and reported within the study report.  In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 
report.  Population PK methods will be used to provide parameter estimates describing the PK 
behavior of nifurtimox and to identify possible covariates with significant impact on 
pharmacokinetics (e.g. age or body weight etc.).
Section 10: Statistical methods and determination of sample size
Pharmacokinetic data 
Old text: 
Specifications for PK data analysis will be provided in a separate document.
New text: 
Specifications for PK data analysis will be provided in a separate document. Plasma 
concentration vs time data documented within the study report will be analyzed descriptively 
using univariate statistics. 
Subject diary, Appendix 16.3
The subject diary was modified and PK subject diary was created from it.


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15.5
Amendment 5
There have been 5 global amendments to the study protocol.  Changes made to the study 
protocol by Amendment 1 are described in detail in Section 15.1, by Amendment 2 in Section 
15.2, by Amendment 3 in Section 15.3, by Amendment 4 in Section 15.4 and by Amendment 
5 in Section 15.5.
15.5.1
Overview of changes
The reason for Amendment 5 is to introduce an additional test (Immunofluorescent antibody, 
IFA), which would validate the current ELISA test results, as requested by FDA.  This test 
will be carried out at Visit 1 and Visit 11 using random back-up samples from the study 
subjects.
The associated changes to the protocol text are detailed in Section 15.5.2.
Sections affected include:

Synopsis, Diagnosis and main criteria for inclusion/exclusion

Study objectives, Section 4

Study design, Section 5

Inclusion criteria, Section 6.1

Tabular schedule of evaluations, Section 9.1

Visit 1 – screening visit (Days -14 to -1), Section 9.2.1.1

Visit 11 (Day 420 ± 7), Section 9.2.3.3

Serological tests (Section 9.4.2)

Variables and planned statistical analyses, Section 10.3


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15.5.2
Changes to the protocol text
In this section, all additions are underlined.
Section 2:  Synopsis
Added text:
Diagnosis and main criteria for 
inclusion /exclusion

Chagas’ disease diagnosed/confirmed by:
…
o Subjects ≥8 months to < 18 years of age at randomization must 
demonstrate a positive conventional ELISA result for both of the 
following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test and Immunofluorescent antibody 
(IFA) test will be obtained; however, a positive result will not be required for 
diagnosis.
…
List of abbreviations
Added text:
IFA
Immunofluorescent antibody
Section 4: Study objectives
Added text:
Exploratory objectives of the study are:
…

To evaluate the relationship of conventional serology to IFA test
Section 5: Study design
Added text:
… Additionally, a non-conventional ELISA test and IFA test will be obtained; however, a 
positive result will not be required for diagnosis.
Section 6.1: Inclusion criteria
Added text:
… Additionally, a non-conventional ELISA test and IFA test will be obtained; however, a 
positive result will not be required for diagnosis.
Section 9.1 Tabular schedule of evaluations
Added text (at Visits 1 and 11):
Immunofluorescent antibody (IFA) test *


[… ] IFA = immunofluorescent antibody,  […]
* IFA will be performed at Visit 1 and Visit 11 only, if adequate specimens are available


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Section 9.2.1.1 Visit 1 – screening visit (Days -14 to -1)
Added text:
[…] Specimens taken for conventional serology, non-conventional serology, IFA and qPCR 
will be frozen and stored at -20oC prior to shipment to the central laboratory where they will 
be stored at -70oC.
Section 9.2.3.3: Visit 11 (Day 420 ± 7)
Added text:
Conventional and non-conventional serologic testing and IFA for Chagas’ disease and qPCR
Section 9.4.2: Serological tests
Added text:
[…] Additionally, a non-conventional ELISA test and IFA will be obtained; however, a 
positive result will not be required for diagnosis.
Section 10.3 Variables and planned statistical analyses
Added text:
Secondary and exploratory efficacy analyses
Added text:
The results from IFA test and conventional serology will also be compared. The details of 
secondary and exploratory efficacy analyses will be further specified in the SAP.


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15.6
Amendment 6
There have been 6 global amendments to the study protocol.  Changes made to the study 
protocol by Amendment 1 are described in detail in Section 15.1, by Amendment 2 in Section 
15.2, by Amendment 3 in Section 15.3, by Amendment 4 in Section 15.4, by Amendment 5 in 
Section 15.5 and by Amendment 6 in Section 15.6.
15.6.1
Overview of changes
The reason for Amendment 6 is to replace the immunofluorescent antibody (IFA) test 
introduced via Amendment 5 by the indirect hemagglutination assay (IHA).
Use of another serological test such as IFA or IHA was requested by FDA to validate the 
current ELISA test results.  This test will be carried out at Visit 1 and Visit 11 using random 
back-up samples from the study subjects.
Rationale: Given that another type of assay has to be performed and the FDA recommended 
either IFA or IHA, the IHA is considered the more suitable assay compared to the IFA taking 
into account the number of samples to be analyzed and the time frame for performing the 
analyses.
Furthermore, the sponsor’s responsible Global Clinical Leader has changed and the name was 
updated.
The associated changes to the protocol text are detailed in Section 15.6.2.
Sections affected include:

List of abbreviations

Signature of the sponsor’s medically responsible person

Synopsis, Diagnosis and main criteria for inclusion/exclusion

Study objectives, Section 4

Study design, Section 5

Inclusion criteria, Section 6.1

Tabular schedule of evaluations, Section 9.1

Visit 1 – screening visit (Days -14 to -1), Section 9.2.1.1

Visit 11 (Day 420 ± 7), Section 9.2.3.3

Serological tests (Section 9.4.2)

Variables and planned statistical analyses, Section 10.3


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15.6.2
Changes to the protocol text
In this section, deletions are crossed out, while additions are underlined.
Signature page of the sponsor’s medically responsible person
Old text:
Name: 
New text:
Name: 
Section 2:  Synopsis
Old text:
Diagnosis and main criteria for 
inclusion /exclusion

Chagas’ disease diagnosed/confirmed by:
…
o Subjects ≥8 months to < 18 years of age at randomization must 
demonstrate a positive conventional ELISA result for both of the 
following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test and Immunofluorescent antibody 
(IFA) test will be obtained; however, a positive result will not be required for 
diagnosis.
…
New text:
Diagnosis and main criteria for 
inclusion /exclusion

Chagas’ disease diagnosed/confirmed by:
…
o Subjects ≥8 months to < 18 years of age at randomization must 
demonstrate a positive conventional ELISA result for both of the 
following tests to confirm diagnosis:

Recombinant ELISA 

Total purified antigen ELISA  
Additionally, a non-conventional ELISA test and indirect hemagglutination 
assay (IHA) will be obtained; however, a positive result will not be required 
for diagnosis.
…
PPD
PPD


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List of abbreviations
Old text:
IFA
Immunofluorescent antibody
New text:
IHA
Indirect hemagglutination assay
Section 4: Study objectives
Old text:
Exploratory objectives of the study are:
…

To evaluate the relationship of conventional serology to IFA test
New text:
Exploratory objectives of the study are:
…

To evaluate the relationship of conventional serology to IHA 
Section 5: Study design
Old text:
… Additionally, a non-conventional ELISA test and IFA test will be obtained; however, a 
positive result will not be required for diagnosis.
New text:
… Additionally, a non-conventional ELISA test and IHA will be obtained; however, a 
positive result will not be required for diagnosis.
Section 6.1: Inclusion criteria
Old text:
… Additionally, a non-conventional ELISA test and IFA test will be obtained; however, a 
positive result will not be required for diagnosis.
New text:
… Additionally, a non-conventional ELISA test and IHA will be obtained; however, a 
positive result will not be required for diagnosis.


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Section 9.1 Tabular schedule of evaluations
Old text (at Visits 1 and 11): 
Immunofluorescent antibody (IFA) test *


[… ] IFA = immunofluorescent antibody,  […]
* IFA will be performed at Visit 1 and Visit 11 only, if adequate specimens are available
New text (at Visits 1 and 11):
Indirect hemagglutination assay (IHA)*


[…] IHA = indirect hemagglutination assay […]
* IHA will be performed at Visit 1 and Visit 11 only, if adequate specimens are available
Section 9.2.1.1 Visit 1 – screening visit (Days -14 to -1)
Old text:
… Specimens taken for conventional serology, non-conventional serology, IFA and qPCR 
will be frozen and stored at -20oC prior to shipment to the central laboratory where they will 
be stored at -70oC.
New text:
… Specimens taken for conventional serology, non-conventional serology, IHA and qPCR 
will be frozen and stored at -20oC prior to shipment to the central laboratory where they will 
be stored at -70oC.
Section 9.2.3.3: Visit 11 (Day 420 ± 7)
Old text:
Conventional and non-conventional serologic testing and IFA for Chagas’ disease and qPCR
New text:
Conventional and non-conventional serologic testing and IHA for Chagas’ disease and qPCR
Section 9.4.2: Serological tests
Old text:
… Additionally, a non-conventional ELISA test and IFA will be obtained; however, a 
positive result will not be required for diagnosis.
New text:
… Additionally, a non-conventional ELISA test and IHA will be obtained; however, a 
positive result will not be required for diagnosis.


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Section 10.3 Variables and planned statistical analyses
Old text:
Secondary and exploratory efficacy analyses
…
The results from IFA test and conventional serology will also be compared. The details of 
secondary and exploratory efficacy analyses will be further specified in the SAP.
New text:
Secondary and exploratory efficacy analyses
…
The results from IHA and conventional serology will also be compared. The details of 
secondary and exploratory efficacy analyses will be further specified in the SAP.
15.7
Amendment 7
Integrated protocol Version 8.0, dated 11 MAY 2018.
15.7.1
Overview of changes to the study
Overall rationale for the amendment
This amendment is prepared to satisfy FDA’s request to “further describe and verify the 
clinical benefit of nifurtimox” according to the FDA meeting minutes of the Type C meeting 
in August 2017 by following the subjects in Study 16027 for a reasonable amount of time to 
demonstrate reversion of serology to negative. 
Changes include extending the current Study 16027 with the long-term follow-up (LTFU), 
and detailed study design and procedures for this LTFU considering FDA’s recommendations 
to the detailed study concept for the LTFU received in March 2018.
Section # and name
Description of change
Brief rationale
Synopsis
Section 3 Introduction
Defined the current ongoing study and 
the LTFU as part 1 and part 2; 
provided the rationale and benefit-risk 
assessment for the LTFU. 
To satisfy FDA’s request to “further 
describe and verify the clinical benefit 
of nifurtimox”, and to implement 
FDA’s recommendations to the 
detailed study concept for the LTFU
Synopsis
Section 4 Study objectives
Provided study objectives for the 
LTFU
Provided updated information on 
secondary objective pharmacokinetic 
(PK)/pharmacodynamic (PD) 
evaluation
To satisfy FDA’s request to “further 
describe and verify the clinical benefit 
of nifurtimox”, and to implement 
FDA’s recommendations to the 
detailed study concept for the LTFU
A pharmacokinetic model needs to be 
established to perform PK/PD 
evaluations.


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Section # and name
Description of change
Brief rationale
Synopsis
Section 5 Study design
Provided detailed study design for the 
LTFU 
To satisfy FDA’s request to “further 
describe and verify the clinical benefit 
of nifurtimox”, and to implement 
FDA’s recommendations to the 
detailed study concept for the LTFU
Synopsis
Section 6.1 Inclusion criteria
Section 6.2 Exclusion criteria
Provided selection criteria for the 
LTFU
Synopsis
Section 9.1 Tabular schedule 
of evaluations
Section 9.2 Visit description
Provided the schedule of 
procedures/assessments and the 
detailed visits description for the 
LTFU
Section 9.4 Efficacy
Provided efficacy assessments for the 
LTFU
Section 9.6 Safety
Provided safety assessments for the 
LTFU
Synopsis
Section 10 Statistical methods 
and determination of sample 
size
Provided analysis sets, variables, 
statistical and analytical plan for the 
LTFU;
Provided updated information on 
sample size
To satisfy FDA’s request to “further 
describe and verify the clinical benefit 
of nifurtimox”, and to implement 
FDA’s recommendations to the 
detailed study concept for the LTFU;
Sample size recalculation based on 
clarification of study objective in 
Amendment 2
Section 16.2 Form for interim 
phone contacts
Provided phone contact form for 
phone assessments in the LTFU
To satisfy FDA’s request to “further 
describe and verify the clinical benefit 
of nifurtimox”, and to implement 
FDA’s recommendations to the 
detailed study concept for the LTFU
Typographical, grammatical, and other minor edits are not identified.
15.8
Amendment 8
Integrated protocol version 9.0, dated 23 JUL 2019.
15.8.1
Overview of changes to the study
Overall rationale for the amendment
This amendment was prepared to incorporate feedback received from study site personnel at 
the investigator meeting for part 2 of the study in APR 2019.  None of the changes 
implemented in this amendment impact the benefit-risk evaluation of the study.
Section # and name
Description of change
Brief rationale


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Section # and name
Description of change
Brief rationale
Synopsis
Section 4.2  Objectives for 
part 2 (CHICO SECURE)
Section 5.2 Part 2 (CHICO 
SECURE)
Section 6.2 Exclusion criteria
Section 10.3 Variables and 
planned statistical analyses
Clarified measurement of optical 
density for recombinant ELISA and 
total purified antigen ELISA 
Ensure all serology collected in part 2 
(CHICO SECURE) will be the same 
as in part 1 (CHICO), as requested by 
FDA
Synopsis
Section 6.2 Exclusion criteria
Section 9.7 Other procedures 
and variables
Clarified evaluation of housing 
conditions as judged by the 
investigator
Consideration of clinical standards 
established in the study countries
Section 9.6.2 Pregnancies
Clarified evaluation of 
presence/absence of congenital 
infection with T. cruzi in babies born 
by female study participants
Consideration of clinical standards 
established in the study countries and 
to avoid unnecessary blood sampling 
in babies born by female study 
participants
Section 9.4.4 Assessment of 
Chagas’ disease
Definition of progression of clinical 
Chagas’ disease modified to include 
alterations that are characteristic of 
the chronic form of the disease
Allow for a more comprehensive 
evaluation of progression of clinical 
Chagas’ disease in part 2 of the study
Section 9.6.1.4 Reporting of 
serious adverse events
Guidance updated to note that SAEs 
in part 2 must also be reported in 
accordance with local regulations
Consideration of clinical standards 
established in the study countries
Typographical, grammatical, and other minor edits are not identified.
15.9
Amendment 9
Integrated protocol version 10.0, dated 18 OCT 2019.
15.9.1
Overview of changes to the study
Overall rationale for the amendment
This amendment was prepared to incorporate feedback received from the FDA on 
19 SEP 2019.  It will allow subjects who were randomized and received at least one dose of 
their assigned nifurtimox treatment regimen and who are otherwise eligible to participate in 
the long-term follow-up portion of the study (Part 2, CHICO SECURE), regardless of 
subsequent treatment for Chagas’ disease.  None of the changes implemented in this 
amendment impact the benefit-risk evaluation of the study.
Section # and name
Description of change


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Section # and name
Description of change
Synopsis
Section 5.2  Design overview of Part 2 
Section 6.2  Exclusion criteria for Part 2
Part 2 exclusion criterion 1 removed
Synopsis
Section 5.2  Design overview of Part 2
Section 6.3.1  Withdrawal
Section 9.2.4.4  Unscheduled study visits
Subjects initiating antitrypanosomal treatment are no longer 
required to discontinue from the study
Section 7.8  Treatment in Part 2
Any antitrypanosomal treatment chosen by the investigator 
during LTFU is not considered as study drug.
Typographical, grammatical, and other minor edits are not identified.


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16.
Appendices
16.1
Phone Contact Form for telephone assessments209
16.1.1
Part 1 (CHICO)
A medically qualified site staff member should use this form during the phone calls with 
the study subject’s parent/s at days 14, 21 and 42. This form is designed to find out how 
the treated child has been feeling and whether his/her parent/legal guardian has 
contacted a doctor.
Please obtain information about any symptoms or medical events that are NEW or 
CHANGED and about any NEW concomitant medication or DOSE CHANGE since the 
last patient’s visit.
Visit Number
Visit Date
Form completion date
Medical Care
Has the health condition of the child changed since last study visit?
Yes / No
-
If Yes, please specify all relevant information:
Has your child experienced any GI problem since last study visit?
Yes / No
-
If Yes, please specify the medical event, start/stop date, action taken and outcome:
Has your child experienced any CNS problems since last study visit?
Yes / No
-
If Yes, please specify the medical event, start/stop date, action taken and outcome:
Has your child experienced any respiratory problems since last study visit?
Yes / No
-
If Yes, please specify the medical event, start/stop date, action taken and outcome:
Has the child visited a Physician or attended to the emergency room due to a 
medical condition since last study visit?
Yes / No
-
If yes, did the child’s doctor provide any diagnose?
                                                
209 Section heading and Form added per Amendment 1; Heading 2 renumbered from this point forward (Amendment 1)


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Has your child experienced any weight loss since last study visit?
Yes / No
-
If yes, please indicate the current weight:
Other medical events (not listed above)
-
If Yes, please specify the medical event, start/stop date, action taken and outcome:
-
If Yes, please specify the medical event, start/stop date, action taken and outcome:
Study Drug Intake
Has your child taken the study drug three times a day as required?
Yes/No
-
If not, please explain
Has your child taken the study drug with food?
Yes/No
-
If not, please explain
Concomitants Medication
Did your child start taking any NEW concomitant medication since last 
study visit?
Yes / No
If Yes, please specify the medication, start/stop date, dose and the reason:
  
Did your child change the dose of or stop taking any of the concomitant 
medications since last study visit?
Yes / No
If Yes, please specify the medication, dose change/stop date, new dose and the reason:
  
If any safety concern arises, subjects may return to the study site for an 
Unscheduled visit.
Name / Signature:
Date:


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16.1.2
Part 2 (CHICO SECURE)
Between annual study visits, all patients will be contacted to assess clinical signs and 
symptoms of potential reinfection or reactivation of Chagas disease or any exclusion criteria, 
to record any update in concomitant medications, and any AEs experienced since the last 
study visit. Patients may be asked to return prior to their next annual visit to the study center, 
if, in the opinion of the investigator, it is clinically necessary. A medically qualified site staff 
member will use this form when contacting the patient and/or his or her parent(s) or legally 
authorized representative about 6 months after the last annual site visit.
Visit 2   
Form completion date:
Visit 4   
Form completion date:
Has the health condition changed since last annual study visit?
Yes 
No
-
If YES, please specify:
Assessment of clinical signs and symptoms of potential Chagas’ disease (mark all that apply)

bug’s bite 

localized swelling at the site of bug’s bite

eyelid swelling

rash

fever

malaise

fatigue

body aches

headache

loss of appetite

difficulty swallowing

abdominal pain

nausea

vomiting

diarrhea

swollen glands

irregular heartbeat

other, please specify:


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Only to be completed if any sign or symptom of Chagas’ disease is ticked:
Was a physician visited?
-
If YES, did the physician provide any diagnose (please specify, if 
applicable)?
Yes
No
Has any weight loss occurred since the last annual study visit?
Yes
No
-
If YES, please indicate the current weight:  __________ kg
Other acute or chronic health conditions (not listed above)
Yes
No
-
If YES, please specify:
Health condition:
Start date:
Stop date:
Action taken:
Outcome:
Health condition:
Start date:
Stop date:
Action taken:
Outcome:
For female patients of childbearing age only
Is the patient currently pregnant?
If YES, are you willing to bring the baby to the next visit to have the baby 
tested for Chagas’ disease?
If NO, please specify reason:
Yes
No
Yes
No


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Have you moved your residence since the last annual study visit?
Yes
No
If YES, is your new residence included in enhanced domestic vector 
control?
Yes
No
Are you currently taking any medication for treatment of Chagas’ disease?
Yes
No
If YES, please specify:

Nifurtimox
Dose:
Start date:
Stop date:
Reason:

Benznidazole
Dose:
Start date:
Stop date:
Reason:

Other, please specify:
Dose:
Start date:
Stop date:
Reason:
   
  
Is there any change in concomitant medication since the last annual study 
visit?
If YES, please specify:
Change in dose of concomitant medication:
Drug:
New dose:
Reason:
Drug:
New dose:
Yes
No


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Reason:
Stop taking concomitant medication:
Drug:
Dose:
Stop date:
Reason:
Drug:
Dose:
Stop date:
Reason:
New concomitant medication:
Drug:
Dose:
Start date:
Stop date (if applicable):
Reason:
Drug:
Dose:
Start date:
Stop date (if applicable):
Reason:
In case of suspected findings, an unscheduled visit at the study center has to be requested.
Unscheduled visit recommended?
Yes
No
Name / Signature:
Date:


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16.2
Schwartz formula and mean (range) eGFR for infants and children of various 
ages210
Normal GFR in Children and Young Adults
Age (Sex)
Mean GFR ± SD
(mL/min/1.73 m2)
1 week (males and females)
40.6 ± 14.8
2-8 weeks (males and females)
65.8 ± 24.8
>8weeks (males and females)
95.7 ± 21.7
2-12 years (males and females)
133.0 ± 27.0
13-21 years (males)
140.0 ± 30.0
13-21 years (females)
126.0 ± 22.0
Data based on three studies.[0][5][6]
GFR, glomerular filtration rate; SD, standard deviation.
4. Heilbron DC, Holliday MA, Al-Dahwi A, Kogan BA. Expressing glomerular filtration 
rate in children. Pediatr Nephrol 5:5-11, 1991.
5. Coultard MG. Maturation of glomerular filtration in preterm and mature babies. Early 
Hum Dev 11:281-292, 1985.
6. Schwartz GJ, Feld LG, Lanford DJ. A simple estimate of glomerular filtration rate in 
full-term infants during the first year of life. J Pediatr 104:849-854, 1984.
The estimated GFR may be calculated using serum creatinine results at screening, and using 
the calculator in the following link: 211
http://www-users.med.cornell.edu/~spon/picu/calc/crclschw.htm
                                                
210 National Kidney Foundation. K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, 
Classification, and Stratification. Am J Kidney Dis 39:S1-S266, 2002 (suppl 1)
211 Old table removed and new table/text added change in determination of eGFR per Amendment 1


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16.3
PK Subject diary and Subject diary 212
PK Subject Diary
Patient N°
Weight at 
Randomization
Treatment 
Start Date
Formulation 
assigned
Please return study drug at every study 
visit
Number of tablets for each administration
(Investigator: fill the boxes according to the assigned dose)
Day of 
Treatment
Date
Time of 
Morning 
dose
Time of 
Noon 
dose
Time of 
Night 
dose
Meal Ingested
Comments
-1
N/A
N/A
N/A
Night Meal:                                                  Time___ : ___
1
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___      
Night Meal:                                                   Time___ : ___    
                                                
212 A subject diary and PK subject diary were created as per amendment 4. 


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
3
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
4
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
5
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
7
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
8
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                                     Time___ : ___
Noon Meal:                                                          Time___ : ___        
Night Meal:                                                          Time___ : ___        
9
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
11
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
12
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
13
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
14
Morning Meal:                                             Time___ : ___


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
15
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
16
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
17
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
18
___ : ___
___ : ___
___ : ___
Morning Meal:                                             Time___ : ___


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not taken
not taken
not taken
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
19
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
20
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
21
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
23
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
24
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
26
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
27
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
29
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
30
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
32
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
33
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
35
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
36
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
38
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
39
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
41
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
42
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
44
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
45
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
47
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
48
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
50
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
51
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
53
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
54
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
56
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
57
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    


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___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
59
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
60
___ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Morning Meal:                                             Time___ : ___
Noon Meal:                                                  Time___ : ___     
Night Meal:                                                   Time___ : ___    
Instructions on how to use this Patient Diary:

Please document the date and time of each study drug intake exactly. In case the dose was not taken, please
check the corresponding box .

If dose reduced, please mention in the comment field and provide the new dose the patient took.

If dose not taken at all, please mention in the comment field with the reason.

When taking the study drug, if the child vomits or spits out the medication, please mention in the comment field 
along with the time.


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
If you have vomiting, please record it, along with the time, under the “Comments” column.

Please record all foods ingested with the morning, noon and night doses of the study drug.

Please remember to return this diary with ALL bottles of study drug dispensed (including empty ones) to the

Investigator.


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Subject Diary 
Patient N°
Weight at 
Randomization
Treatment 
Start Date
Formulation 
assigned
Please return study drug at every study 
visit
Number of tablets for each administration
(Investigator: fill the boxes according to the assigned dose)
Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
1
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
2
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
3
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
4
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
5
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
6
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
7
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
8
__ : ___
___ : ___
___ : ___


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Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
not taken
not taken
not taken
9
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
10
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
11
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
12
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
13
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
14
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
15
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
16
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
17
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
18
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
19
__ : ___
___ : ___
___ : ___


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Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
not taken
not taken
not taken
20
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
21
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
22
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
23
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
24
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
25
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
26
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
27
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
28
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
29
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
30
__ : ___
___ : ___
___ : ___


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Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
not taken
not taken
not taken
31
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
32
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
33
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
34
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
35
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
36
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
37
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
38
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
39
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
40
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
41
__ : ___
___ : ___
___ : ___


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Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
not taken
not taken
not taken
42
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
43
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
44
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
45
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
46
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
47
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
48
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
49
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
50
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
51
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
52
__ : ___
___ : ___
___ : ___


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Day of 
Treatment
Date
Time of 
Morning dose
Time of 
Noon dose
Time of Night 
dose
Comments
not taken
not taken
not taken
53
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
54
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
55
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
56
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
57
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
58
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
59
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
60
__ : ___
not taken
___ : ___
not taken
___ : ___
not taken
Instructions on how to use this Patient Diary:

Please document the date and time of each study drug intake exactly. In case the dose was not taken, please
check the corresponding box .

If dose reduced, please mention in the comment field and provide the new dose the patient took.

If dose not taken at all, please mention in the comment field with the reason.


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
When taking the study drug, if the child vomits or spits out the medication, please mention in the comment field 
along with the time.

If you have vomiting, please record it, along with the time, under the “Comments” column.

Please remember to return this diary with ALL bottles of study drug dispensed (including empty ones) to the

Investigator.


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16.4
Procedure for multiplex real time polymerase chain reaction to Trypanosoma 
cruzi detection in patients with Chagas’ disease
Blood samples collection
1. Material

Ethanol 70%

Latex gloves, without talc

Disposable syringe and needle

Conical tubes with the appropriate volume of Guanidine 6M-ethylenediaminetetra-
acetic acid (EDTA) 0.2M buffer

Permanent marker

Stretcher 

Container and bags for disposal of biological material
2. Implementation
Collected peripheral blood samples will be immediately mixed with an equal volume of 
Guanidine EDTA buffer pursuing the following volume consideration: 

Babies 1 day to 12 months of age: minimum 1 mL of blood – Use 15 mL tubes with 
red tag

Children from 1 to 14 years old: minimum 2 mL of blood –Use 15 mL tubes with 
yellow tag

Children ≥15 years of age: minimum 5 mL of blood – Use 50 mL tubes.
3. Sample collection procedure 
NOTE: The collection of blood must be performed by personnel qualified by training to draw 
blood. 
Label the conic tube with patient´s full name and date of collection
Collect the appropriate volume of whole blood in the conic tube and close the screw cap.
Thoroughly mix conic tubes containing blood and buffer by gently inverting the tube 6-8 
times. 
The specimens can be stored at room temperature until and during shipment to the laboratory.  
Samples must be received in the laboratory within 1 month of collection.
NOTE: Excessive exposure to heat results in the breakdown of deoxyribonucleic acid (DNA). 
Guanidine-EDTA-Blood (GEB) specimens must be refrigerated at 5° ± 3°C if room 
temperature could exceed 30°C.


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Genetic material (DNA) extraction
1.1 Material
-
Sodium hypochlorite 0.5%-1% freshly diluted
-
Ethanol 70%
-
Dry bath
-
Automatic variable volume micropipette 0-10 µL
-
Automatic variable volume micropipette 20-200 µL
-
Automatic variable volume micropipette 100-1000 µL
-
Latex gloves, without talc
-
Racks (1.5/2 mL tubes)
-
Racks (15/50 mL conical tubes)
-
High Pure PCR Template Preparation Kit, Roche (catalogue number: 11796828001)
-
Absolute ethanol
-
Distilled water (PCR use)
-
Microcentrifuge
-
Vortex
-
Permanent marker
-
Container and bags for disposal of biological material
-
Tubes eppendorf like (1,5 mL)
-
Tubes (2 mL)
-
Filter tips (10 µL, 200 µL y 1000 µL)
1.2 Sample reception
The sample reception will be conducted verifying samples codes, subject initials, date of birth 
and date of collection.
Samples are acceptable when they meet the requirements of homogeneity, absence of clots 
and adequate code.
Once received in the laboratory, and until analyzed, the specimens will be stored at 5°C ± 3°C 
or at room temperature if it does not exceed 30°C.
1. Pre-analytical procedure
Once collected, the samples will be preserved at least 24 hours before heating.  GEB samples 
will be heated to 100°C during 15 minutes in a dry bath.  Gently open the screw cap before 
heating. 
Cool to room temperature and let stand at least 12 hours at 4°C before processing.


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2. Implementation
The extraction procedure will be carried out with a maximum of 12 samples or controls each 
run.  All runs must include one negative control (seronegative GEB sample) and one weak 
positive control.  It will be performed up to 2 times a day (1 in the morning and 1 in the 
afternoon) to avoid pipetting mistakes and contaminations.
Before starting the DNA extraction procedure from GEB samples, pursue the following 
instructions:

Disinfect work area, cleaning surfaces with damp cloth with sodium hypochlorite 
0,51%: bench, pipettes, equipment, etc.  

Illuminate the work area with ultraviolet light (sterilization) for 15 minutes 

Verify samples code and the number of samples to be processed.

Verify the selection of appropriate positive and negative controls.

Aliquot the amount of extraction reagents and tubes that will be used during the 
procedure.

Label tubes and columns with date and the relevant code seated in the workbook 
using permanent marker 

Turn on the dry bath at 70°C.
3. Extraction procedure 
Before starting the purification procedure:

Heat the required amount of elution buffer at 70ºC in a dry bath

Homogenize the samples by vortex during 15 to 30 seconds.
1. Add 5 µL de IAC (40 pg/µL) and 40 μL of Proteinase K in a 2 mL tube DNAse free
2. Add 300 μL of GEB sample (blood – guanidine EDTA buffer).
3. Add 100 μL of Binding Buffer and mix by pulse-vortexing for 15 seconds
4. Mix by pulse-vortexing for 3 seconds, spin down tubes.
5. Incubate for 10 min at 70°C in a dry bath, spin tubes before opening.
6. Add 100 μL of isopropyl alcohol and mix by pulse-vortexing for 15 seconds.  After 
mixing, briefly spin down tubes to remove drops from the inside of the lid spin tubes.
7. Carefully apply the mixture from Step 6 to the High Pure Filter column (in a 2 mL 
collection tube) without wetting the rim, and close the cap. 
8. Centrifuge at 8,000 revolutions per minute (RPM) for 1 minute.  Place the High Pure 
Filter column in a clean 2 mL collection tube, and discard the tube containing the 
filtrate.
9. Carefully open the High Pure Filter column and add 500 μL of inhibitor removal 
buffer without wetting the rim, and close the cap.


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10. Centrifuge at 8,000 RPM for 1 minute.  Place the High Pure Filter column in a clean 2 
mL collection tube, and discard the collection tube containing the filtrate.
11. Carefully open the High Pure Filter column and add 500 μL Wash Buffer without 
wetting the rim, and close the cap. 
12. Centrifuge at 8,000 RPM for 1 minute.  Place the High Pure Filter column in a clean 2 
mL collection tube, and discard the collection tube containing the filtrate.
13. Repeat Steps 11 and 12.
14. Place the High Pure Filter column in a new 2 mL collection tube and discard the old 
collection tube with the filtrate.  Centrifuge at full speed for 10 seconds.
15. Place the High Pure Filter column in a clean 1.5 mL microcentrifuge tube labeled with 
the sample code seated in the workbook, and discard the collection tube containing the 
filtrate. 
16. Carefully open the High Pure Filter column and add 100 μL of elution buffer 
(pre-incubate at 70°C) and centrifuge at 8,000 RPM for 1 minute. 
17. For long-term storage of DNA, store at –20°C.
Procedure for multiplex real time polymerase chain reaction (qPCR)
1. Materials 

Automatic variable volume micropipette 0-10 µL

Automatic variable volume micropipette  0-20 µL

Automatic variable volume micropipette 20-200 µL

Automatic variable volume micropipette 100-1000 µL

Filter tips (10 µL, 20 µL, 200 µL y 1000 µL)

Latex gloves without talc

Racks (0.2 mL and 1.5 mL tubes)

Microcentrifuge

PCR cabinet with ultraviolet radiation

Real time PCR thermo cycler Applied Biosystems, StepOne

Vortex

Permanent marker

Container and bags for disposal of biological material

Real time PCR reagents and master mix (Master mix Roche 2X: catalog number 
04913949001; sterile distilled water PCR quality) 

Primers and probes to T. cruzi: 
cruzi 1: 5´-ASTCGGCTGATCGTTTTCGA- 3´


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cruzi 2: 5´-AATTCCTCCAAGCAGCGGATA- 3´
cruzi 3 (probe): 5´-CACACACTGGACACCAA- 3´

Primers and probes IAC:
IAC Tq Fw: 5´-ACCGTCATGGAACAGCACGTA- 3´
IAC Tq Rv: 5´-CTCCCGCAACAAACCCTATAAAT- 3´
IAC (sonda): 5´-AGCATCTGTTCTTGAAGGT- 3´

Plates and adhesive films to qPCR (Applied biosystems catalog number 4375816 
and 4375928)
2. Implementation 
The qPCR may be performed twice a day, making a run in the morning and one in the 
afternoon, taking into account the time required for each run is 4 hours (reagents pipetting and 
cycles)
The number of samples per run may be at most 43, plus 5 quality controls, making a total of 
48 wells, maximum capacity of qPCR thermo cycler.
Before start the mix preparation pursue the following instructions: 

Defrost DNA samples, controls and reagents, and hold at 4ºC until use. Will be use 
on each plate two positive controls in duplicate (CL Brener 10 fg/µL, 1 fg/µL) and 
1 white control (distilled water used to mix preparation)

Prepare the reaction protocol (see related documents) with the amount and order of 
samples and controls in the PCR plate.

Turn on ultraviolet light in the PCR cabinet during 15 minutes 

Enter reaction protocol data in the StepOne software using the template “POE 
qPCRMtq Chagas” and save the file.
3. qPCR procedure 

Pipette the reaction mixture according to the following detail (example for N reactions):


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Reagents
Final 
concentration
Volume
(x 1 reaction)
Volume
(x N reactions)
Roche Fast Start Universal 
Probe Master Mix (Rox) 
(2X)
1X
10 μL
N x 10
cruzi 1 (Fw) (50 μM)
0,75 μM
0,3 μL
N x 0,3
cruzi 2 (Rv) (50 μM)
0,75 μM
0,3 μL
N x 0,3
cruzi 3 (probe) (5 μM) 
0,05 μM
0,2 μL
N x 0,2
IAC Tq Fw (5 μM)
0,1 μM
0,4 μL
N x 0,4
IAC Tq Rv (5 μM)
0,1 μM
0,4 μL
N x 0,4
IAC probe (5 μM)
0,05 μM
0,2 μL
N x 0,2
H2O PCR quality
-
3,2 μL
N x 3,2
Total volume  (mix) x well
-
15 μL
15 μL
DNA sample volume
5 μL
5 μL
Final volume 
20 μL
20 μL

Distribute 15 µL of the reaction mixture per well.

Add 5 µL of DNA sample or controls in the wells following the reaction protocol.

Seal the plate with the adhesive film without touch the surface.

Confirm that the samples have been applied in the reaction mixture and not in the edges of 
the tube.

Start the reaction
4. Cycles and reaction conditions
First step: 10 min at 95ºC.
Second step: 40 cycles of 15 sec at 95ºC and 1 min at 58ºC, fluorescence data collection in the 
yellow (IAC-VIC) and green (T. cruzi-FAM) channel.
5. Interpretation of results
Establish the threshold in 0.02 to T. cruzi and 0.01 to the internal amplification control (IAC).
Analyze the results of PCR negative and positive controls
Run valid:

Positive controls are detectable to T. cruzi analysis (green channel). The 
fluorescent curve crosses the threshold leading to a Ct appropriate in relation to the 
concentration of T. cruzi DNA in the respective positive control (assess with 
control chart).

White controls are not detectable to T. cruzi analysis. The fluorescent curve does 
not cross the threshold leading to absence of Ct.


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Valid sample:

The internal amplification control (yellow channel) amplified with and efficient 
signal 
Positive sample or detectable to T. cruzi:

The fluorescent curve in the green channel crosses the threshold leading to a Ct 
value.
Negative sample or not detectable to T. cruzi: 

The fluorescent curve does not cross the threshold leading to absence of Ct value.


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16.5
Pharmacokinetic blood sample handling 
Sample Handling Sheet
Version 1.0
Study Identifier:
BAY a2502 (Nifurtimox) / 16027
Analyte Name:
BAY a2502 Nifurtimox
Author:
Name:
Department:
GDD-GED-DMPK-BANP-BAW
Date:
2014-10-06
Matrix:
Plasma for bioanalysis of PK samples 
Precautions:

Samples degrade in the presence of light. Light protection measures have to be 
considered during all steps of sample handling. Avoid exposure to direct daylight or 
neon light. Please transfer the samples immediately into the refrigerator after blood 
sampling and after centrifugation. Reduce the time when samples are exposed to light 
as much as possible.
Materials:

Polypropylentube with a length of at least 90 mm, e.g., Sarstedt 100 x 15.7 mm REF 
55.539

Push caps to close the polypropylentube mentioned above, e.g., Sarstedt REF 
65.803.512

Micro Haematocrit tubes, lithium heparinized, ca.75 µL, e.g., Vitrex Medical (DK) 
REF 161813

Haematocrit sealing wax, e.g. Brand GmbH (Germany) REF 749510
Sample collection, handling and storage:
The total duration between blood sample collection at room temperature and final storage 
of the plasma sample must not exceed 90 minutes. 
PPD


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
Please avoid exposure to direct daylight or neon light during sample processing.

Please refer to study protocol for sampling time points.

The blood samples have to be collected into two Micro Haematocrit tubes from the 
heel prick (subjects up to 1 month of age), fingerstick or ear lobe stick at the discretion 
of the investigator.

Seal the lower side of the Micro Haematocrit tubes with wax.

Transfer every Micro Haematocrit tube into one labelled polypropylene tube and lock 
with push cap. 
Caution: Please put the wax sealed lower side downwards.

Transfer the blood samples immediately into the refrigerator (protected from light) until 
centrifugation or proceed with centrifugation immediately as described below.


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
Centrifuge blood samples at 2000 g (not higher) for approximately 10 minutes at room 
temperature.

Immediately after centrifugation transfer the centrifuged samples into the freezer.  In case 
samples cannot be frozen immediately, please store the samples temporarily in the 
refrigerator. Please sort samples according to subject ID 

Freeze the samples in an upright position until shipment at < -15 °C
Sample stability

Analyte of interest is stable at ≤- 15 °C for at least 557 days 
Sample shipment:

Please inform the responsible Analytical Laboratory and Biosample Operations Manager 
in time (2 to 3 days before shipment) regarding sample shipment.

Packaging of samples on sufficient dry ice

Preferably use World Courier as courier service or use the central lab preferred provider 

Sample arrival is preferred on Monday to Thursday from 8:00 am to 16:00 pm CET.
Shipment Address:
Laboratory 
Bayer Pharma AG
GDD-GED-DMPK-BANP
Aprather Weg, Geb. 468
42096 Wuppertal
Phone: 
Fax: 
Email:
PPD
PPD
PPD
PPD


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16.6
Maximum blood volumes for pediatric patients – Clinical and Laboratory 
Standards Institute

SAP_003.pdf: 
 
Document Type: 
Statistical Analysis Plan 
Official Title: 
Prospective, historically controlled study to evaluate the efficacy 
and safety of a new pediatric formulation of nifurtimox in children 
aged 0 to 17 years with Chagas’ disease 
NCT Number: 
NCT02625974  
Document Date: 
26 JAN 2020 
 


Statistical Analysis Plan
BAY No. A2502/IMPACT No. 16027
Page:
1 of
30
Reference Number: BPD-SOP-060
Supplement Version: 6
Prospective, historically controlled study to evaluate the efficacy and safety 
of a new pediatric formulation of nifurtimox in children aged 0 to 17 years 
with Chagas’ disease
Prospective study of a pediatric nifurtimox formulation for Chagas’ disease
Bayer study drug
BAY No. A2502/Nifurtimox
Study purpose:
To evaluate efficacy and safety, and characterize the 
pharmacokinetics, of nifurtimox in children with Chagas’ disease 
using 30-day and 60-day treatment durations
Clinical study 
phase:
3
Date:
26 JAN 2020
Study No.:
16027
Version:
3.0
Author:
Confidential
The information provided in this document is strictly confidential and is intended solely 
for the guidance of the clinical investigation.  Reproduction or disclosure of this
document, whether in part or in full, to parties not associated with the clinical 
investigation or its use for any other purpose without the prior written consent of the 
sponsor is not permitted.
Throughout this document, symbols indicating proprietary names (®, TM) are not displayed.  Hence, the 
appearance of product names without these symbols does not imply that these names are not protected.
This Statistical Analysis Plan is produced on a word-processing system and bears no signatures.
The approval of the Statistical Analysis Plan is documented in a separate Signature Document.
PPD


Statistical Analysis Plan
BAY No. A2502/IMPACT No. 16027
Page:
2 of
30
Reference Number: BPD-SOP-060
Supplement Version: 6
Table of Contents
Abbreviations............................................................................................................................ 3
1.
Introduction....................................................................................................................... 4
2.
Study Objectives................................................................................................................ 4
2.1
Objectives for part 1 (CHICO) ........................................................................................ 4
2.2
Objectives for part 2 (CHICO SECURE)........................................................................ 5
3.
Study Design ...................................................................................................................... 6
3.1
Part 1 (CHICO)................................................................................................................ 6
3.2
Part 2 (CHICO SECURE)................................................................................................ 9
4.
General Statistical Considerations ................................................................................ 11
4.1
General Principles.......................................................................................................... 11
4.2
Handling of Dropouts .................................................................................................... 11
4.3
Handling of Missing Data.............................................................................................. 12
4.4
Interim Analyses and Data Monitoring ......................................................................... 12
4.5
Data Rules...................................................................................................................... 13
4.6
Validity Review ............................................................................................................. 13
5.
Analysis Sets .................................................................................................................... 14
5.1
Assignment of analysis sets ........................................................................................... 14
6.
Statistical Methodology .................................................................................................. 15
6.1
Population characteristics .............................................................................................. 15
6.1.1
Disposition.................................................................................................................. 15
6.1.2
Demographic and Other Baseline Characteristics...................................................... 15
6.1.3
Medical History.......................................................................................................... 15
6.1.4
Extent of Exposure and Compliance .......................................................................... 15
6.2
Efficacy.......................................................................................................................... 16
6.2.1
Part 1 (CHICO)........................................................................................................... 16
6.2.2
Part 2 (CHICO SECURE) .......................................................................................... 22
6.3
Safety ............................................................................................................................. 25
6.4
Pharmacokinetics / pharmacodynamics......................................................................... 27
6.5
Determination of sample size......................................................................................... 28
7.
Document history and changes in the planned statistical analysis............................. 28
8.
References ........................................................................................................................ 29
9.
Appendices....................................................................................................................... 29
9.1
Assessment of Chagas’ disease signs and symptoms .................................................... 29


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Abbreviations
AE
Adverse event
ATC
Anatomical Therapeutic Chemical
CI
Confidence interval
Cmax
maximum concentration
D
Day
DMC
Data Monitoring Committee
ECG
electrocardiogram
ELISA
Enzyme-linked immunosorbent assay
EOT
End of treatment
FAS
Full analysis set
IgG
Immunoglobulin G
IHA
Indirect hemagglutination assay
IVRS/IWRS
Interactive Voice Response System/Interactive Web Response System
MedDRA
Medical Dictionary for Regulatory Activities
OD
Optical density
PD
pharmacodynamics
PK
pharmacokinetics
PPS
Per-protocol set
qPCR
Quantitative polymerase chain reaction
SAE
Serious adverse event
SAP
Statistical Analysis Plan
SAS
Statistical Analysis System
T. cruzi
Trypanosoma cruzi
TEAE
Treatment-emergent adverse event
V
Visit
WHO
World Health Organization
WHO-DD
World Health Organization – Drug dictionary


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1.
Introduction
This statistical analysis plan (SAP) describes the study objectives, study design, study 
population, efficacy and safety variables, statistical analysis methods, and study tables to be 
used in this study.  The original protocol, Version 1.0, is dated 04 NOV 2014. This SAP v3.0 , 
dated 26 JAN 2020, is based on the integrated clinical study protocol Version 11.0, dated 03 
NOV 2020, which includes the CHICO SECURE part of the study. 
This study consists of two parts:

Part 1 comprises the treatment with nifurtimox including the 1-year follow up
(CHICO)

Part 2 comprises the 3-year long-term follow-up (LTFU) of all subjects who were
randomized and received at least one dose of their assigned nifurtimox treatment
regimen in CHICO (CHICO SECURE)
Part 1 (CHICO) was designed to develop a better understanding of the efficacy, 
safety/tolerability, and pharmacokinetics (PK) of nifurtimox in children with a diagnosis of 
Chagas’ disease using pediatric formulations. Results from this study will demonstrate 
parasitological cure using conventional serological results to allow optimization of treatment 
of Chagas’ disease in this most vulnerable population. In turn, this will improve the outlook 
for children by reducing mortality and long-term complications. This knowledge will allow 
for better and more appropriate approaches to the treatment of Chagas’ disease. It will also 
provide more up-to-date information on the use of nifurtimox, and has the potential to 
increase our understanding of treatment response in children.
Part 2 (CHICO SECURE) was designed, at the request of the US Food and Drug
Administration (FDA), to assess the incidence of seronegative conversion in subjects who
were randomized and received at least one dose of their assigned 60- or 30-day nifurtimox
treatment regimen. In part 2 (CHICO SECURE), subjects will be followed up for 3 years
totaling to a follow-up period of 4 years after end of nifurtimox treatment, the first year of
follow up is included in the design of part 1 (CHICO).
2.
Study Objectives
2.1
Objectives for part 1 (CHICO)
The primary objective of this Phase 3 clinical study is:

To assess the superiority of a 60-day regimen of nifurtimox to historical untreated 
control at the 12-month follow-up (360 days from end of treatment [EOT]) as sero-


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reduction (defined as a ≥ 20% reduction in optical density [OD] measured by 
conventional enzyme-linked immunosorbent assay [ELISA]) compared to baseline in 
subjects ≥8 months to < 18 years of age at randomization or sero-conversion (defined 
as negative Immunoglobulin G [IgG] concentration) in all subjects
Secondary objectives of the study are:

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox as sero-reduction or sero-conversion at the 12-month follow-up (360 days 
from EOT)

To evaluate the safety/tolerability profile of nifurtimox by laboratory parameters 
(hematology, blood chemistry, urinalysis), electrocardiogram (ECG) monitoring, vital 
sign measurements (blood pressure, heart rate, respiratory rate, temperature), adverse 
event (AE) monitoring, and physical examinations, including neurological 
examinations

To evaluate the pharmacokinetics (PK)/pharmacodynamics (PD) of nifurtimox in 
children receiving the drug for treatment of Chagas’ disease
Exploratory objectives of the study are:

To assess the comparability of a 60-day regimen of nifurtimox to historical active 
control (benznidazole) as sero-reduction or sero-conversion at the 12-month follow-up 
(360 days from EOT)

To assess the comparability of a 30-day regimen of nifurtimox to a 60-day regimen of 
nifurtimox using qualitative polymerase chain reaction (qPCR) at the 12-month 
follow-up (360 days from EOT)

To evaluate the relationship of conventional serology (as sero-reduction or sero-
conversion) to qPCR using frequencies of matches and mismatches to assess 
agreement

To evaluate the relationship of non-conventional serology to conventional serology

To evaluate the relationship of conventional serology to indirect haemagglutination 
assay (IHA)
2.2
Objectives for part 2 (CHICO SECURE)
The primary objective of the long-term follow-up (LTFU) part of the study is:


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
To assess the incidence of seronegative conversion as confirmed by two types of 
assay, recombinant ELISA and indirect hemagglutination assay (IHA), in subjects 
who were randomized and received at least one dose of the 60-day nifurtimox 
treatment regimen, compared to an external control group of untreated patients with 
Chagas’ disease at the 4-year follow-up.
The secondary objectives are:

To assess the incidence of seronegative conversion, as confirmed by two types of 
assay (recombinant ELISA and IHA), in subjects who were randomized and received 
at least one dose of the 30-day nifurtimox treatment regimen at the 4-year follow-up.

To evaluate the proportion of responders who show both seronegative conversion as 
confirmed by two types of assay (recombinant ELISA and IHA) and no evidence of 
established cardiomyopathy as evaluated in electrocardiogram (ECG) recordings.

To assess serial reduction of antibody titers, as measured by recombinant ELISA and 
total purified antigen ELISA, in subjects who were randomized and received at least 
one dose of either the 60- or 30-day nifurtimox treatment regimen compared to Visit 1 
in part 1 of the study
The exploratory objectives are:

To assess the incidence of seronegative conversion as confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects by age categories (≤2 years, >2 years to ≤6 
years, >6 to ≤12 years, >12 to <18 years; age is defined as subject’s age at 
randomization).

Assess the proportion of responders and the incidence of seronegative conversion as 
confirmed by non-conventional serology in subjects who were randomized and 
received at least one dose of either 60- or 30-day nifurtimox treatment regimens.

To assess the occurrence of congenital infection with T. cruzi in children born of 
female subjects who were randomized and received at least one dose of 60- or 30-day 
nifurtimox treatment regimen. The congenital infection will be confirmed by 
parasitological method in children ≤8 months of age and by serological method in 
children >8 months of age.
3.
Study Design
3.1
Part 1 (CHICO)
Design overview


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This is a Phase 3, prospective, randomized (to dosing regimen), age-stratified, double-blind, 
parallel-group study to evaluate the efficacy, safety/tolerability, and PK of oral administration 
of nifurtimox in children with a diagnosis of Chagas’ disease. A schematic of the study design 
is presented in Figure 3–1. 
Figure 3–1. Study Design of Part 1
In this study, approximately 300 pediatric subjects will be randomized (2:1 randomization,
60-day regimen vs. 30-day regimen). Subjects will be stratified by age at randomization into 
four strata as specified below:
Stratum 1:
0 to 27 days
Stratum 2:
28 days to younger than 8 months
Stratum 3:
8 months to younger than 2 years
Stratum 4:
2 years to younger than 18 years
A minimum of 38 subjects in each age stratum is targeted, but not required, in order to be able 
to derive meaningful safety conclusions. Enrollment will continue until this specification is 


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met, unless it is determined that such a target would be unlikely to be reached in a reasonable 
time.
After study eligibility has been confirmed and safety assessments have been performed at 
Visit 2 (Day 1), subjects will be randomized via Interactive Voice Response 
System/Interactive Web Response System (IVRS/IWRS) in a 2:1 ratio (60-day regimen vs. 
30-day regimen) to one of two treatment groups:
-
Nifurtimox tablets administered three times daily for 60 days (Days 1 – 60, active
nifurtimox treatment, Treatment Group 1) or
-
Nifurtimox tablets administered three times daily for 30 days, followed by nifurtimox
placebo administered three times daily for 30 days (Days 1 – 30, active nifurtimox
treatment; Days 31 – 60, placebo; Treatment Group 2).
The study is composed of 3 periods: 
-
Screening period
-
Treatment period
-
Follow-up period
Treatment period
The first dose of study drug will be administered at Visit 2. Pre- and post-dose PK blood 
samples will be obtained at specified time points from those subjects consenting to PK 
assessments. At the 2 – 4 hour time point of PK blood sampling (i.e., at the time of maximum
concentration [Cmax] ), an ECG (optional for subjects < 5 years of age at the discretion of the
investigator) will be obtained to allow for PK/PD investigations. Study drug will be 
dispensed, and instructions for study drug administration will be provided to all subjects. 
Subjects will return to the investigational site for efficacy and safety assessments at Visit 3 
(Day 7±1) and Visit 6 (Day 30). Subjects who have not consented to PK assessments will take 
that day’s doses of study drug as instructed. Subjects consenting to PK assessments must hold 
the morning dose of study drug; for these subjects, a pre-dose PK blood sample will be 
obtained, then the morning dose of study drug will be administered. Post-dose blood samples 
will be obtained at specified time points, and ECGs will be obtained at the 2 – 4 hour post-
dose time point. A pre-paid phone card will be provided to all subjects to facilitate contact
between study site personnel and the subjects/subjects’ authorized representatives. 
At Visit 6 (Day 30), subjects will return all remaining study drug and empty packaging, and 
study drug for the remaining 30 days of treatment will be dispensed.
Visits 4 (Day 14), 5 (Day 21), and 7 (Day 42) will be telephone assessments where study site 
personnel will contact the subject/legally authorized representative via telephone to assess the 


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occurrence of AEs, use of concomitant medications, and compliance with study drug 
administration. A Phone Contact Form for the telephone assessments will be provided to all 
sites.
Subjects will return to the investigational site for efficacy and safety assessments on Day 60 
(Visit 8), which will be the EOT for both treatment groups. Subjects who have not consented 
to PK assessments will take that day’s doses of study drug as instructed. Subjects consenting 
to PK assessments must hold the morning dose of study drug. A pre-dose PK blood sample 
will be collected, then the morning dose of study drug will be administered. Post-dose PK 
blood samples will be obtained at specified time points, and ECGs will be obtained at the 2 –
4 hour post-dose time point. Study drug will be collected, and no additional study drug will be 
dispensed. 
Follow-up period
After the EOT visit (Visit 8), subjects will return to the site on Days 90 (Visit 9), 240 (Visit 
10), and 420 (Visit 11) for additional efficacy and safety assessments. The total duration of 
each subject’s participation is expected to be approximately 14 months. 
Primary variable(s)
The primary efficacy variable will be sero-reduction or sero-conversion at 12 months post-
treatment using two conventional ELISA serology tests as the measure of efficacy.
End of part 1 of the study
The end of part 1 of the study as a whole will be reached as soon as the last visit of the last 
subject has been reached in all centers in all participating countries.
3.2
Part 2 (CHICO SECURE)
Design overview
This is a prospective, long-term follow-up of subjects, who were randomized and received at 
least one dose of their assigned 60- or 30-day nifurtimox treatment. The study is designed to 
evaluate seronegative conversion and serial reduction in OD measured by ELISA.
Subjects will be followed up for a total of 4 years after end of nifurtimox treatment. The first 
year of follow-up period is included in part 1 and 3 years of follow-up are included in part 2 
of the study.
The following age categories will be considered for part 2 of the study: ≤2 years, >2 years to 
≤6 years, >6 to ≤12 years, >12 to <18 years (age is defined as subject’s age at randomization).


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Subjects enrolled will be followed annually with visits to the investigational site. 
Seronegative conversion will be measured by two types of assay, recombinant ELISA and 
IHA. For assessment of serial reduction compared to baseline defined as screening visit of 
part 1 (Visit 1), OD will be measured by recombinant ELISA and total purified antigen 
ELISA.
The first visit should be performed as soon as possible after availability of regulatory and 
ethics committee approval for all subjects who have already completed part 1 of the study for 
1 year ± 6 weeks or longer. Subjects who are entering the study beyond the time window for 
FU Visit 1 (2 years ± 6 weeks) will have their eligibility assessments as soon as possible 
provided that consents/assents have been obtained. Subjects will have annual visits at the 
investigational site for efficacy and safety assessments for 3 years. Timing of study visits is 
relative to end of treatment in part 1 of the study.
Between the annual visits at the investigational site, subjects and/or his or her parent(s) or
legally authorized representative(s) will be contacted by qualified personnel by a method
agreed (eg. phone call) about 6 months after a visit at the investigational site to identify the
occurrence of potential clinical symptoms of Chagas’ disease or any pathology mentioned in
the exclusion criteria using a structured interview. In case of suspected findings, an
unscheduled visit at the investigational site to initiate appropriate diagnostic evaluations will
be requested.
Primary variable
The primary efficacy variable will be the incidence rate of seronegative conversion measured 
by two types of assay (recombinant ELISA and IHA) in subjects who were randomized and 
received at least one dose of the 60-day nifurtimox treatment regimen. Both test results should 
be negative for the subject to be considered as seroconverted.
End of part 2 of the study
The end of part 2 of the study as a whole will be reached as soon as the last visit of the last 
subject has been reached in all centers in all participating countries.
Primary completion
The primary completion event for part 2 of the study is last subject last visit (LSLV).


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4.
General Statistical Considerations
4.1
General Principles
The statistical evaluation will be performed using the software package SAS release 9.2 or 
higher (SAS Institute Inc., Cary, NC, USA). Unless otherwise specified, all significance tests 
will be conducted using a 2-sided alpha level of 0.05, and confidence intervals (CIs) will be 2-
sided 95% intervals. All variables collected in this study will be summarized with descriptive 
statistics at each assessment time. For continuous variables, descriptive statistics will include
the number of data available and missing data, means, standard deviations, medians, 
minimums, and maximums. For categorical variables, frequency counts and percentages will 
be provided. These will be calculated for each age stratum by treatment regimen, as well as 
the overall set of study subjects.
4.2
Handling of Dropouts
A subject who discontinues study participation prematurely for any reason is defined as a
“dropout” if the subject has already been randomized, assigned to treatment, and administered 
at least one dose of study drug.
A subject who, for any reason (e.g., failure to satisfy the selection criteria), terminates the 
study before the time point used for the definition of “dropout” is regarded as a “screening 
failure”.
Depending on the time point of withdrawal, a withdrawn subject is referred to as either 
“screening failure” or “dropout”. A subject who is withdrawn from the study will not be 
replaced.
Subjects who discontinue prematurely from study drug administration will continue to return 
to the investigational site for study assessments at Visits 3, 6, and 8 (EOT), and undergo 
telephone assessments as described for Visits 4, 5, and 7. If subjects are unable/unwilling to 
do so, they must return to the investigational site 30 (±3) days after the last dose of study drug 
for EOT (Visit 8) assessments, and undergo telephone assessments as described for Visits 4, 
5, and 7. If the subject is unable/unwilling to return to the clinic for the EOT Visit (Visit 8), 
then a telephone assessment as described for Visits 4, 5, and 7 may be performed in lieu of 
Visit 8 assessments.
Premature discontinuations from study drug will be summarized by treatment regimen, both 
for any type of discontinuation, and for each specific reason for discontinuation.  


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4.3
Handling of Missing Data
Part 1 (CHICO)
No imputations will be made for missing values occurring in the safety and background
variables. Background variables include demographics (sex, age), height/length, weight, 
medical/surgical history, and medication history.
All efforts will be taken to collect all data. However, despite best efforts, it may be inevitable 
that missing data are reported. In the primary efficacy analysis done on the full analysis set 
(FAS), subjects who have missing conventional serology results at the 12 month time point 
will be treated as failures (i.e. no cure). 
For the secondary analysis using the per-protocol set (PPS), all subjects who do not have both 
conventional serology determinations to assess the 12-month time point will be excluded from 
the analysis. 
Part 2 (CHICO SECURE)
No imputations will be made for missing data due to dropouts or other causes. For the 
primary efficacy analysis done on the FAS, drop-outs will be included in the analysis using a 
Poisson distribution with person-year.
4.4
Interim Analyses and Data Monitoring
No interim analysis is planned.
Part 1 (CHICO)
An independent Data Monitoring Committee (DMC) will meet periodically to review the 
safety data of enrolled subjects, as well as the continuing scientific merit of the trial.
The DMC may recommend stopping enrollment in the case of a negative risk/benefit 
assessment. The DMC will be comprised of a minimum of two clinicians with expertise in 
relevant clinical specialties and at least one statistician knowledgeable about statistical 
methods for clinical trials. Each committee member will be screened for evidence of an 
absence of serious conflicts of interest. The operation of the DMC will be governed by a 
charter that will describe the group’s frequency of meetings, procedures (including, but not 
limited to, periodic safety monitoring), and requirements for reporting its observations to the 
Sponsor. 
Part 2 (CHICO SECURE)
No DMC is planned.


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4.5
Data Rules
Baseline
Baseline is defined as the last measurements performed prior to the first study drug 
administration, which is either in Visit 1 (e.g. parameters of serum hematology, chemistry, 
coagulation, and urinalysis) or Visit 2 (e.g. vital signs and ECG), unless otherwise specified.
If Visit 2 is missing, use data from Visit 1 instead.
Repeated measures
If there are repeated measurements per time point (e.g. laboratory values, vital signs, etc.), the 
following rules will be used:
•
Before the start of the study drug administration (i.e., for screening and baseline 
value), the latest measurement will be used, in the event these latest measurements are 
measurements at scheduled visits. Unscheduled visits will be used, if there are no 
measurements at scheduled visits. If the latter is the case, the last unscheduled visit will be 
used. 
•
In case of repeated measurements at any post-screening time point, the first value of 
the scheduled measurements at that time point will be used. No unscheduled measurements
will be used for any time points beside screening / baseline even where measurements at
scheduled visits are missing.
In case of non-adherence to time windows, data will be assigned to the nearest nominal visit 
according to assessment schedule.
4.6
Validity Review
The results of the validity review meeting will be documented in the Validity Review Report 
and may comprise decisions and details relevant for statistical evaluation. Any changes to the 
statistical analysis prompted by the results of the validity review meeting will be documented 
in an amendment and, if applicable, in a supplement to this SAP.


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5.
Analysis Sets
5.1
Assignment of analysis sets
Part 1 (CHICO)
Full analysis set (FAS)
The efficacy analyses will be done using the full analysis set (FAS), which is the set of 
randomized subjects who received at least one dose of study drug. 
Analyses of safety and background data will be performed on the FAS.
Per protocol set (PPS)
The primary analysis will also be done using the per-protocol set, which is comprised of 
randomized subjects treated with study drug who have no major protocol deviations.  Major 
protocol deviations defined in this study will include:

Subjects who have both conventional serology results missing at the 12-month time 
point

Subjects who do not meet in-/exclusion criteria but entered treatment phase

Subjects who take less than 80% or more than 120% of total assigned doses of study 
drug

Subjects whose treatment codes are unblinded during the study

Subjects who have taken prohibited medicine during the study
Part 2 (CHICO SECURE)
Full analysis set (FAS)
The efficacy analyses will be done using the full analysis set (FAS), as defined in part 1.
Subjects will be analyzed as randomized in part 1 (CHICO) of the study. 
Analyses of safety and background data will be performed on the FAS. For safety analyses, 
subjects will be analyzed as treated in part 1 of the study.


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6.
Statistical Methodology
6.1
Population characteristics
6.1.1
Disposition
Disposition will be summarized descriptively for the study phases of screening, treatment, and 
follow-up for FAS. The number of subjects discontinuing the screening phase together with 
the primary reason for discontinuation will be presented overall. The number of subjects 
discontinuing the treatment and follow-up phases together with the primary reason for 
discontinuation will be presented overall and by treatment regimen. In addition, the number of
subjects with major/important protocol deviations will be presented overall and by treatment
regimen. Subjects discontinuing the study due to COVID-19 and any protocol deviation 
caused by COVID-19 will be summarized accordingly.
6.1.2
Demographic and Other Baseline Characteristics
Demographics (sex, ethnicity, race, age at randomization, weight, height/length) and baseline 
characteristics (i.e., ELISA, IHA and qPCR test results, Chagas’ disease signs and symptoms)
will be summarized overall and by treatment regimen. The same summary will also be 
performed for each age stratum.
6.1.3
Medical History
For data coding, Medical Dictionary for Regulatory Activities (MedDRA) [version 18.0 or 
most recent version] will be used for medical history. Medical history (i.e., previous 
diagnoses, diseases or surgeries) not pertaining to the study indication, start before signing of 
the informed consent and considered relevant to the study will be presented for each 
MedDRA primary system organ class and high level term by treatment regimen and overall
using frequency counts. 
Concomitant medications will be tabulated by treatment regimen using ATC codes (WHO-
DD); frequencies of subjects having received each drug category will be provided.
6.1.4
Extent of Exposure and Compliance
Part 1 (CHICO)


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All summaries related to intake of study medication will be by treatment regimen based on 
FAS.
The treatment duration will be summarized descriptively overall and for each age stratum. It 
will be calculated as follows:
Treatment duration =  (drug stop date - drug start date + 1 day).
If the end date of the first drug distribution and the start date of the second drug distribution 
are the same, the calculation is:
Treatment duration = (drug stop date– drug start date for the 1st distribution) + (drug stop 
date– drug start date for the 2nd distribution + 1)
The number of tablets and corresponding extent of exposure (by dose) will be summarized 
descriptively. Compliance is defined as 100 * number of tablets taken / number of tablets per 
protocol. The compliance will be summarized descriptively by treatment regimen and overall.  
In addition, compliance will be categorized into three groups (<80%, 80-120%, >120%) and 
summarized by treatment regimen and overall.
Part 2 (CHICO SECURE)
Not applicable.
6.2
Efficacy
6.2.1
Part 1 (CHICO)
6.2.1.1
Primary efficacy variable and analysis
The primary efficacy variable will be sero-reduction (defined as a ≥ 20% reduction in optical
density [OD]) or sero-conversion (defined as negative Immunoglobulin G [IgG] 
concentration) in the 60-day regimen group at 12 months post-treatment using two 
conventional ELISA serology tests. This sero-reduction (in subjects ≥ 8 months to < 18 years 
of age at randomization) or sero-conversion (in all subjects) is considered cure, and the 
primary variable is binary (cure, no cure). In the event of discordancy between the two 
conventional ELISA test results, the following will be considered:

For sero-reduction, the average percentage of OD reductions for the two conventional 
ELISA tests will be used (e.g., Test #1 = 15% and Test #2 = 25%; average = 20% and, 
hence, cure).


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
For sero-conversion, only when both test results are negative that the subject is 
considered as negative (i.e., cure). 

If one of the test results is missing, the subject is considered as “no cure”.
Derivation of the rate for sero-conversion for the historical controls 
Historical controls are estimated from cure rates presented in two publications [1][2]. The 
historical placebo cure rate used is shown in Table 6–1:
Table 6–1: Historical Cure Rates for Placebo
Publication
Age range
(in years)
Sero-conversion rate (95%CI)
in placebo patients
de Andrade et al 1996
7-12
3/65 = 5% (1%,13%)
Sosa et al 1998
6 -12
2/44= 5% (1%,16%)
Both publications show a 5% cure rate for the placebo group. For the primary objective in our 
study, superiority over placebo will be confirmed if the lower limit of the 95% CI for the 
nifurtimox (60-day regimen) cure rate is greater than 16%, the larger of the upper limits of the 
95% CIs from the two publications.
Primary analysis of the primary efficacy variable 
The difference in the proportion of nifurtimox subjects with sero-reduction or sero-conversion 
(60-day regimen) and the proportion estimated from historical data will be tested using an 
asymptotic 2-sided 95% CI with a continuity correction of 1/2n for a single proportion.
The null hypothesis H0 is pnifurtimox = pplacebo
The alternative hypothesis H1 is pnifurtimox ≠pplacebo, where pt is the cure rate for treatment t.
Superiority will be confirmed if the lower limit of the CI for the proportion of nifurtimox
subjects (60-day regimen) with sero-reduction or sero-conversion is greater than 16%. The
proportion and asymptotic 2-sided limits (pl, pu) of the 95% CI for pnifurtimox can be computed 
by [3]


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where x denotes the number of nifurtimox subjects with sero-reduction or sero-conversion and 
n the total number of subjects in 60-day regimen group. The SAS code will be:
PROC FREQ; 
TABLES cured / BINOMIALC;
WEIGHT counts; 
RUN;
Secondary analysis of the primary efficacy variable
A secondary analysis will be done to compare the proportion of subjects with sero-reduction
or sero-conversion for the 60-day and 30-day nifurtimox regimens. This will be performed
using an asymptotic 2-sided 95% CI for the difference of two independent proportions.
The null hypothesis H0 is pnifurtimox, 60d = pnifurtimox, 30d
The alternative hypothesis H1 is pnifurtimox, 60d ≠pnifurtimox, 30d, where pt is the cure rate for 
treatment t.
Difference in efficacy between the two groups will be confirmed if the CI for the difference of 
two proportions of nifurtimox subjects (60-day regimen versus 30-day regimen) with sero-
reduction or sero-conversion does not include 0. The proportions and asymptotic 2-sided 
limits (ll, lu) of the 95% CI for pnifurtimox, 60d - pnifurtimox, 30d can be obtained by [3]
where pi, xi and ni denote the cure rate, the number of nifurtimox subjects with sero-reduction 
or sero-conversion, and the total number of subjects, for treatment i respectively. Treatment 
i=1, 2 represents 60-day and 30-day nifurtimox regimens, respectively. 
The SAS code will be:


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PROC FREQ; 
TABLES group*cured / RISKDIFFC;
WEIGHT counts; 
RUN;
The analyses specified above (the proportion of subjects with sero-reduction or sero-
conversion and an asymptotic 95% CI for the 60-day nifurtimox regimen, and the difference 
of two independent proportions for the 60-day and 30-day regimens and an asymptotic 95% 
CI) will also be done using data from Days 7 (Visit 3), Days 30 (Visit 6), the EOT visit (Visit 
8), and Days 240 (Visit 10). In the event of small sample sizes for the subgroups, an exact 
95% CI will be calculated instead of an asymptotic one. 
Sensitivity analyses of the primary efficacy variable
The following sensitivity analyses for the primary efficacy variable will be performed:

Missing values due to the following reasons treated as treatment failures and other 
missing values excluded from analysis:
1. the reason for discontinuation is known or suspected to be treatment ineffectiveness or 
an AE, 
2. a subject has not had a negative serology at any visit and has no serology 
determination for the primary analysis visit, 
3. an indeterminate or incorrectly performed serology determination occurs at the 
primary analysis visit (e.g. missing values due to lab procedure, not enough sample 
materials to perform the test, performance error, etc.). 

Observed data only

Analysis on PPS
Subgroup analyses of the primary efficacy variable
The proportion of nifurtimox subjects with sero-reduction or sero-conversion (60-day 
regimen) and an asymptotic 2-sided 95% CI for the proportion will be calculated by country
and for each age stratum.
The same proportion and an asymptotic 2-sided 95% CI for the difference of the 60-day and 
30-day nifurtimox regimens will also be summarized by country and for each age stratum.


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6.2.1.2
Secondary efficacy variables and analysis
Secondary efficacy variables include clinical signs/symptoms of Chagas’ disease (see 
Appendix 9.1), concentration test for T. cruzi (for subjects < 8 months of age ), non-
conventional serologic testing, and disease state determined by qPCR (“cure” defined as Not 
Detectable / “no cure” defined as Detectable).
All secondary efficacy variables, except signs/symptoms of Chagas’ disease, will be 
summarized overall and by treatment regimen at each assessment time for each age stratum.
Signs/Symptoms of Chagas’ disease will be summarized for each item (see Appendix 9.1). 
The relationship of conventional serology results to qPCR results will be done using
frequencies of matches and mismatches on the determination of disease status, and phi-
correlation and kappa coefficient to assess the degree of agreement. The relationship of non-
conventional serology to conventional serology will be analyzed in the same way. The 
following SAS code will be used:
PROC FREQ; 
TABLES group*cured / CHISQ AGREE;
WEIGHT counts; 
RUN;
6.2.1.3
Exploratory efficacy analysis and analysis
For the exploratory objective to assess the comparability of nifurtimox with benznidazole, the 
cure rate of 58% with a 95% CI (45%, 70%) from publication of de Andrade et al. in 1996 [2] 
will be referenced. If the lower limit of the same 95% CI for the primary efficacy variable (the 
60-day nifurtimox regimen cure rate) is greater than 45%, nifurtimox will be deemed as 
comparable to benznidazole.
The comparison of conventional serology to IHA results will be done using a 3-by-3 table 
(Table 6–2) with the following categories:

ELISA tests: sero-conversion and sero-positive (sub-categories: sero-reduction and 
others), where “others” refers to any sero-positive results that do not satisfy the definition 
of “sero-reduction”.

IHA test: negative and positive (sub-categories: titers decreasing and titers non-
decreasing).


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Table 6–2 Summary table of comparison between serology and IHA results
Conventional ELISA Tests
Sero-
negative
Sero-positive
Missing
Total
Sero-reduction
Others
IHA 
Testing
60-Day
(N=x)
Negative
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive
Decreasing in 
titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Non-decreasing
in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
30-Day
(N=x)
Negative
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive
Decreasing in 
titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Non-decreasing
in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
In addition, McNemar’s test will be performed by treatment regimen for all patients in FAS 
(Table 6–3) with the following categories:

ELISA tests: cure (sero-conversion and sero-reduction) versus no cure;

IHA test: negative or positive but decreasing in titers versus positive but non-decreasing 
in titers.
Table 6–3 McNemar’s test on comparison between serology and IHA results for all 
patients in FAS
Conventional ELISA Tests
Cure (sero-
negative or sero-
reduction)
No cure
Missing
Total
2
P-Value
IHA 
Testing
60-Day
(N=x)
Negative or positive 
but decreasing in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive but non-
decreasing in titers  
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
x.xx
x.xx
30-Day
(N=x)
Negative or positive 
but decreasing in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive but non-
decreasing in titers  
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
x.xx
x.xx
The same test would also be performed by treatment regimen in sero-positive subjects (Table 
6–4). The categories for this test are


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
ELISA tests: sero-reduction versus others (sero-positive that are not sero-reduction);

IHA test: negative or positive but decreasing in titers versus positive but non-decreasing 
in titers.
Table 6–4 McNemar’s test on comparison between serology and IHA results for all 
sero-positive patients in FAS
Conventional ELISA Tests
Sero-reduction
Others
Missing
Total
2
P-Value
IHA 
Testing
60-Day
(N=x)
Negative or positive 
but decreasing in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive but non-
decreasing in titers  
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
x.xx
x.xx
30-Day
(N=x)
Negative or positive 
but decreasing in titers
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Positive but non-
decreasing in titers  
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Missing
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
Total
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
xx (xx.x%)
x.xx
x.xx
6.2.2
Part 2 (CHICO SECURE)
6.2.2.1
Primary efficacy variable and analysis
Primary efficacy variable
The primary efficacy variable will be the incidence rate of seronegative conversion measured 
and confirmed by two types of assay (recombinant ELISA and IHA) in subjects who were
randomized and received at least one dose of the 60-day nifurtimox treatment regimen. Both 
test results should be negative for the subject to be considered as seroconverted. Incidence 
rate is the number of new cases of seronegative conversion over the study period (i.e. 4 years 
after end of nifurtimox treatment) divided by the person-time at risk.
Subjects used antitrypanosomal drugs during the study before they were seroconverted will 
not be considered as sero-convertion, but their time of observation till when they took
antitrypanosomal drugs will be factored into the person time calculation.
Primary analysis of the primary efficacy variable 
The difference in the incidence rate of nifurtimox subjects with seronegative conversion (60-
day regimen) and the seronegative rate estimated from historical data will be tested using a 
Poisson two-sided 95% exact confidence interval (CI).


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The incidence rate of seronegative conversion for the primary efficacy variable will be
modelled using a Poisson distribution with a 2-sided 95% exact CI. The rate will be estimated
as the number of seronegative conversion divided by the total time at risk (person-year)
during the study. The rate of seronegative conversion (R) and a 2-sided limits (Rl, Ru) of the
95% CI for the rate can be calculated using the following formula [4]
where n is the number of seronegative conversion observed , N the total subjects at risk in 
annual visit , Vi the th annual visit,  
the total number of visits,  
and 
are lower and 
upper confidence limits for the rate of seronegative conversion respectively, 
is the chi-
square quantile for upper tail probability on v degrees of freedom.
This study uses an external control group of untreated patients with Chagas’ disease in the 4-
year follow-up period presented in Sosa et al. [7, 7]. In the reference, seroconversion to a 
negative result in the placebo group after 4-year follow-up was detected in 2 subjects with 
conventional serology and in 0 subjects with IHA test. Thus, none of the subjects in the 
placebo group was considered as seroconverted by both test results. The person time at risk 
was on average 44 * 4 = 176 person years. The incidence rate estimate was 0% (0 / 176).
Superiority will be confirmed if the lower limit of the CI for the incidence rate of seronegative 
conversion for subjects in 60-day regimen is greater than the rate of historical placebo 
subjects with sero-conversion (i.e. 0 %).
Frequency counts and percentages of seronegative conversion will be provided by visit.
6.2.2.2
Secondary efficacy variables and analysis
Secondary efficacy variables include:

Incidence rate of seronegative conversion measured and confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects who were randomized and received at least one 
dose of the 30-day nifurtimox treatment regimen


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
Proportion of responders who show both seronegative conversion by two types of assays 
(recombinant ELISA and IHA) and no evidence of established cardiomyopathy as 
measured by ECG

Serial reduction of OD measured by recombinant ELISA and total purified antigen ELISA
Analysis of secondary efficacy variables
The OD values from individual conventional ELISA tests (recombinant ELISA and total 
purified antigen ELISA) will be analyzed descriptively. Changes from baseline will be 
summarized to show any serial reduction of OD values. Baseline is defined as the OD values 
from the same ELISA tests measured at Visit 1 in part 1 of the study.
The proportion of subjects who show both seronegative conversion as confirmed by two 
assays (recombinant ELISA and IHA) and no evidence of established cardiomyopathy as 
measured by ECG will be calculated overall and by treatment regimen. 
The same method for calculating incidence rate and its 2-sided 95% CI of the primary 
efficacy variable will be applied on the following variable: 

Incidence rate of seronegative conversion measured and confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects who were randomized and received at least one 
dose of the 30-day nifurtimox treatment regimen
6.2.2.3
Exploratory efficacy analysis and analysis
Exploratory efficacy variables include:

Incidence rate of seroconversion measured and confirmed by two types of assay 
(recombinant ELISA and IHA) in subjects by age categories (≤2 years, >2 years to ≤6 
years, >6 to ≤12 years, >12 to <18 years; age is defined as subject’s age at 
randomization)

Proportion of presence of parasite or positive serology in babies born of mothers who
were randomized and received at least one dose of either the 60- or 30-day nifurtimox 
treatment regimen

Proportion of responders and incidence rate of seronegative conversion as confirmed 
by non-conventional serology in subjects who were randomized and received at least 
one dose of either 60- or 30-day nifurtimox treatment regimens
Analysis of exploratory efficacy variables
The same method for calculating incidence rate and its 2-sided 95% CI of the primary 
efficacy variable will be applied on the following variable:


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
Incidence rate of nifurtimox subjects with seronegative conversion in all subjects by 
age categories. The age categories are grouped as ≤2 years, >2 years to ≤6 years, >6 to 
≤12 years, >12 to <18; age is defined as subject’s age at randomization

Incidence rate of seronegative conversion as confirmed by non-conventional serology
in subjects who were randomized and received at least one dose of either 60- or 30-
day nifurtimox treatment regimens
The proportion of subjects with seronegative conversion as confirmed by non-conventional 
serology will be calculated overall and by treatment regimen. The numeric results of non-
conventional serology will be summarized overall and by treatment regimen. 
The proportion of presence of parasite or positive serology in babies born of mothers who 
were randomized and received at least one dose of either the 60- or 30-day nifurtimox 
treatment regimen will be calculated overall and by treatment regimen. 
The results from qPCR and IHA will be summarized overall and by treatment regimen. 
Signs/Symptoms of Chagas’ disease will be summarized for each item (see Appendix 9.1). 
6.3
Safety
Part 1 (CHICO)
Safety Variables
Safety variables include adverse events (AEs), physical examination abnormalities, vital 
signs, ECG, hematology and blood chemistry, coagulation, and urinalysis. All analyses of 
safety variables will be descriptive only, no formal testing will be performed. 
Adverse Events
All adverse events occurring during the study (i.e., from signing of informed consent until last 
visit) will be reported. The MedDRA coding system will be used to code the adverse events.
An overview of frequencies of subjects who died, experienced any adverse event, experienced 
any drug-related adverse event, experienced a serious adverse event (SAE), experienced a 
drug-related serious adverse event, or discontinued due to an adverse event will be 
summarized by treatment regimen. 


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Adverse events will be considered treatment emergent, if they first occurred or worsened at or 
after first application of study medication during the course of the study up to and including 7
days after last application of study medication.
Incidence of treatment emergent AEs (TEAEs), drug-related TEAEs, serious TEAEs, drug-
related serious TEAEs, as well as TEAEs leading to discontinuation will be summarized by
primary system organ class and high level term by treatment regimen.  
The definitions of incidence are as follows:

Incidence of all events:  # of subjects reporting the event with a start date during or after 
treatment/# of subjects valid for safety.  

Incidence of drug-related events:  # of subjects reporting a drug-related event with a start 
date during or after treatment/# of subjects valid for safety. 

Incidence of events by maximum intensity:  # of subjects reporting the event at the 
indicated intensity with a start date during or after treatment/# of subjects valid for safety.  
Adverse events leading to death, premature discontinuation and serious adverse events will be 
listed. In this study, all AEs of urticarial, weight loss of > 20%, severe rash, and severe 
polyneuropathy will be considered AEs of special safety interest. Adverse events of special 
safety interest will be summarized by treatment regimen.
AEs occurring between the time of consent and the first dose of study drug will be 
summarized separately.
Laboratory Data
Quantitative data will be summarized by means of descriptive statistics (arithmetic mean, 
standard deviation, median, minimum and maximum) by visit and treatment regimen for the 
original data as well as for the difference to baseline. Frequency tables will be provided for 
qualitative data. Summaries will be given by treatment regimen. The incidence of laboratory 
data outside the reference range (low, high) and clinically significant laboratory changes will 
be summarized by treatment regimen using frequency tables. The definitions of incidence are 
as follows:
Incidence of high lab abnormalities: # of subjects with at least one high laboratory 
assessment after the start of treatment who had a normal or lower than normal 
laboratory assessment at baseline / # of subjects at baseline with a normal or lower 
than normal laboratory assessment who also had at least one valid laboratory value 
after start of treatment. Subjects with missing or high abnormal values at baseline are 
not included in the denominator. 

Incidence of low lab abnormalities: # of subjects with at least one low laboratory 
assessment after the start of treatment who had a normal or higher than normal 


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laboratory assessment at baseline / # of subjects at baseline with a normal or higher 
than normal laboratory assessment who also had at least one valid laboratory value 
after start of treatment. Subjects with missing or low abnormal values at baseline are 
not included in the denominator. 
ECG and Vital Signs Data
Quantitative ECG data, both original values and change from baseline, will be summarized by 
visit and treatment regimen. ECG interpretations will also be summarized by visit and 
treatment regimen. ECG data will be analyzed for the FAS and by age stratum.
Vital signs data will be analyzed descriptively. The original values and changes from baseline 
will be summarized by visit and treatment regimen. 
Pregnancy Data
Pregnancy data will be listed by visit and treatment regimen.
Part 2 (CHICO SECURE)
Safety variables include AEs, physical examination abnormalities, ECG and vital signs. All 
analyses of safety variables will be descriptive only, no formal testing will be performed.
Adverse events will be recorded when considered at least possibly related to nifurtimox and
adverse events caused by protocol-required procedures.
Quantitative ECG data, both original values and change from baseline, will be summarized by 
visit and treatment regimen. ECG interpretations will also be summarized by visit and 
treatment regimen. ECG data will be analyzed for the FAS and by age stratum.
Vital signs data will be analyzed descriptively. The original values and changes from baseline 
will be summarized by visit and treatment regimen. 
Pregnancy data will be listed by visit and treatment regimen.
6.4
Pharmacokinetics / pharmacodynamics
Part 1 (CHICO)
Plasma concentration time courses will be analyzed and reported within the study report. In 
addition, a full PK evaluation of the data will be performed and reported in a separate study 


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report. Population PK methods will be used to provide parameter estimates describing the PK
behavior of nifurtimox and to identify possible covariates related to age.
Part 2 (CHICO SECURE)
Not applicable.
6.5
Determination of sample size 
Part 1 (CHICO)
According to Guhl et al. [5], nifurtimox 12 months post-treatment cure rate is about 55%, 
with a sample size of 260 for the 60-day regimen, the power is 99% for the lower limit of the 
95% CI to be greater than 16%.
The number of subjects in the 30-day treatment regimen is based on the width of a 95% CI for 
the difference of two proportions. A sample size of 130 subjects for the 30-day subjects 
together with 260 subjects for the 60-day subjects will produce a CI with a width of 
approximately 20%.
Part 2 (CHICO SECURE)
According to Sosa-Estani [7, 7], the 48 months post treatment cure rate for benznidazole is 
about 5% as confirmed by two types of assay. For the primary objective, assuming a drop-out 
rate of 15%, the expected number of patients to be observed at the end of Part 2 (i.e. 48 
months post treatment) in the 60-day regimen would be 170 out of the 200 patients in Part 1. 
Using the assumptions of 170 nifurtimox 60-day regimen patients in Part 2, a 5% cure rate,
and that seronegative conversion events follow Poisson distribution, the estimated incidence 
rate is calculated to be 1.32% and the 95% CI of the incidence rate is (0.61%, 1.51%).
7.
Document history and changes in the planned statistical analysis
Figure 7–1 Version History
Version
Date
Action
Version 1.0
21 OCT 2015
Signed initial version
Version 2.0
16 FEB 2018
Signed version 2.0


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Version 3.0
26 JAN 2020
Signed version 3.0
8.
References
1. Sosa Estani S, Segura EL, Ruiz AM, Velazquez E, Porcel BM, Yampotis C. Efficacy of 
chemotherapy with benznidazole in children in the indeterminate phase of 
Chagas’Disease. Am J Trop Med Hyg. 1998 Oct;59(4):526-529.
2. de Andrade AL, Zicker F, de Oliveira RM, Almeida Silva S, Luquetti A, TravassosLR, 
et al. Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma 
cruzi infection. Lancet. 1996 Nov 23;348(9039):1407–13.
3. Fleiss, J. L., Levin, B., and Paik, M. C. (2003), Statistical Methods for Rates and 
Proportions, Third Edition, New York: John Wiley & Sons.
4. Ulm K. Simple method to calculate the confidence interval of a standardized mortality 
ratio (SMR). American journal of epidemiology. 1990;131(2):373-5.
5. Guhl F, Nicholls R, Montoya R, Rosas F, Velasco VM, et al. (2004) Rapid 
negativization of serology after treatment with benznidazole for chagas disease in a 
group of colombian schoolchildren. Medimond International Proceedings. Spain, Vol. 1. 
pp. 107–114
6. Sosa Estani S, Segura EL, Ruiz AM, Velazquez E, Porcel BM, Yampotis C. Efficacy of 
chemotherapy with benznidazole in children in the indeterminate phase of 
Chagas’Disease. Am J Trop Med Hyg. 1998 Oct;59(4):526-529
7. FDA. 2017. Benznidazole tablets. Company: Chemo Research, S.L. Available from 
https://www.accessdata.fda.gov/drugsatfda_docs/nda/2017/209570Orig1s000TOC.cfm
9.
Appendices
9.1
Assessment of Chagas’ disease signs and symptoms
Assessments of the presence of Chagas’ disease signs and symptoms will be performed 
during physical examinations at Screening, Visits 3, 6, and 8 (EOT), and Visits 9, 10, and 11 
(follow-up) in part 1 and at FU Visits 1, 3, and 5 in part 2 of the study, and will be entered 
into the eCRF. Signs and symptoms of Chagas’ disease include but are not limited to the 
following:
Acute Chagas’ disease:

Fever: usually prolonged


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
malaise

lymphadenopathy

hepatomegaly

splenomegaly

subcutaneous edema (localized or generalized)

signs of portal of entry of T. cruzi:
o
through the skin – chagoma
o
via the ocular mucous membranes - Romaña sign

hypotonicity

anemia

myocarditis

meningoencephalitis

pneumonitis

ECG abnormality, may include but not limited to: 
o
sinus tachycardia
o
first-degree atrioventricular block
o
low QRS voltage
o
primary T-wave changes

Chest radiograph abnormality, include variable degrees of cardiomegaly