Prospective, Parallel-group, Open-label Randomized Controlled Trial of Four Treatment Regimes for Trichuriasis in Pediatric Patients
Phase: PHASE2
Conditions: Trichuris Infection, Helminthiasis
Sponsor: Alejandro Krolewiecki

Protocol version 1.0, 29Nov2018 
 
 
 
 
HI4T trial 
 
 
Prospective, parallel-group, open-label randomized 
controlled trial of four treatment regimens for trichuriasis 
in pediatric patients 
 
NCT04041453 
 
Protocol version 1.0 
29NOV2018 
 
 
 
 
 
 
 
 
 
 

Protocol version 1.0, 29Nov2018 
 
 
HI4T Trial - PROTOCOL SYNOPSIS 
 
STUDY IDENTIFICATION 
Prospective, parallel-group, open-label randomized controlled 
trial of four treatment regimens for trichuriasis in pediatric 
patients. HI4T trial. 
 
TYPE AND 
DEVELOPMENT 
CLINICAL PHASE 
 
Phase II non-inferiority randomized clinical trial 
 
SPONSOR 
Fundacion Mundo Sano, Spain 
INVESTIGATOR 
Dr. Lilian Sosa, Principal Invetigator 
Alejandro J. Krolewiecki, MD, PhD. General Coordination 
STUDY CENTERS 
• 
Universidad Autonoma de Honduras 
Laboratory centers: 
• 
Genetics Research Center. UNAH, Tegucigalpa, 
Honduras 
• 
Lanusse lab. CIVETAN, UNCPBA - Tandil, Argentina 
(pk). 
• 
Cimino lab, IIET, UNSa – Orán, Argentina 
(resistance) 
 
ETHICAL COMMITTEE 
APPROVAL  
CEI-MEIZ, UNAH, HON & Brock University, Canada.  
OBJECTIVES 
Primary endpoints 
Cure rate (CR) (conversion from being egg positive pre-treatment to 
egg negative post-treatment) for Trichuris infection will be assessed 
using the quadruple Kato-Katz method at 14 to 21 days post-
treatment. 
 
Secondary endpoints 
• 
To evaluate the safety and tolerability of the interventions. 
• 
To measure the egg reduction rate of the different 
treatment arms against T. trichiura.  
• 
To validate the multi-parallel qPCR for the determination of 
efficacy outcomes. 
• 
To determine the presence of isolates with resistance to 
the study drugs in the pre and post treatment isolates. 
• 
To determine population pk parameters of IVM through a 
population pk approach. 
 
DESIGN AND 
METHODOLOGY 
Phase II, open label randomized, controlled, parallel groups clinical 
trial in patients infected with at T. trichiura.  

Protocol version 1.0, 29Nov2018 
 
 
 
TREATMENT ARMS 
 
• 
Group 1: single dose of ALB 400 mg.  (active control arm). 
N:39 
• 
Group 2: single dose ALB 400mg + IVM 600µg/Kg. N: 57 
• 
Group 3: daily dose ALB 400mg for 3 consecutive days. N:24 
• 
Group 4: daily dose ALB 400mg + IVM 600µg for 3 
consecutive days. N:57 
 
Total Study Population: 177 
INCLUSION AND  
EXCLUSION CRITERIA  
Inclusion Criteria 
The study population will consist of participants who meet the 
following selection criteria:  
1. Individuals of both sexes infected with T. trichiura.  
2. Weight ≥15 kg.  
3. Age: 2 to 14 years old. 
4. Free acceptance to participate in the study by obtaining 
signed informed consent form approved by the EC. 
Exclusion Criteria 
Participants meeting the following criteria cannot be selected: 
1. Intake ALB, mebendazole and/or IVM within the previous 3 
months.  
2. Background of allergy, idiosyncrasy or hypersensitivity to 
drugs or its excipients.  
3. Acute clinical conditions (including diarrhea). 
4. Participation in another clinical trial during the 3 months 
preceding the drug administration. 
5. Pregnancy. 
SAMPLES COLLECTION 
Fresh stool sample in a 60ml plastic container at screening.  
One sample 14 to 21 days post treatment 
ANALYTICAL METHODS 
Efficacy outcomes: 
• 
Single Kato Katz. 
• 
Multiparallel qPCR 
Safety: frequency, distribution and severity of AEs and SAEs. 
Resistance: molecular biology 
PK: HPLC 
Study medication 
Albendazole: Nematel, ELEA Argentina. 400mg tabs 
Ivermectin: IVER P, ELEA Argentina. 6mg tablets 
 
Introduction - Justification 
Infection with T. trichiura is highly prevalent in Honduras and principally affects pediatric 
populations leading to growth and development impairment. 
Various medications have been approved for the treatment of T. trichiura infection, including 
albendazole (ALB) and mebendazole (MBZ), both belonging to the benzimidazole family (BZ). 
These treatments (especially ALB) are used in Honduras during mass-drug administration 
campaigns. 
Currently, the recommended treatment regimen is a single-dose of 400 mg of albendazole 
(ALB); however, the efficacy of this drug is considerably limited against T. trichiura infection 
(17%, CI of 95% 8 – 31%). This limited efficacy has raised concerns due to an increase in the 

Protocol version 1.0, 29Nov2018 
 
 
proportion of patients with this infection in areas where treatment is regularly administered. 
Several explanations for this limited efficacy have been explored, including lack of treatment 
compliance, the amount of time the parasite is exposed to the drug in the intestine, and more 
recently, the emergence of parasitic resistance to ALB. 
Due to ALB low efficacy as well as its limited spectrum, other countries have started using 
ivermectin (IVM) for the treatment of helminths. Trials using ivermectin have demonstrated its 
high tolerability and safety. Studies involving adults using high dosages of ivermectin showed 
good safety profile (doses of up to 10 times (10x) the 200µg/kg). 
Higher IVM doses didn’t result in differences of adverse events in comparison with a placebo. 
Ivermectin has also been utilized for treating Pediculosis capitis in pediatric populations. The 
approved dose of 400 µg/kg in these clinical trials was well tolerated with only 1.8% of patients 
(7 of 398) suspending the treatment due to adverse effects (a similar proportion to the group 
that received 0.5% malathion lotion).  
It has been demonstrated, especially in parasitic infections of veterinary importance, that 
using a single medication to treat parasitic infections can contribute to the emergence of 
anthelminthic resistance.  
The combination of two different drugs is currently considered the best route for avoidance of 
resistance. This also helps in reaching target levels of prevalence set out by control campaigns.  
In accordance with preliminary information, the combination of albendazole/ivermectin 
(ALB/IVM) could have increased benefits in terms of efficacy without increasing adverse 
effects. 
The present study is proposing the use of a high dose of IVM (600 µg/kg of weight) in children, 
with the purpose of determining the safety, tolerability and efficacy of this treatment co-
administered with albendazole. 
The results of this study will help determine if the co-administration of a simplified dose of 
IVM/ALB is a viable treatment for T. trichiura infection among endemic populations. 
The study communities of La Hicaca and surrounding villages are located in Honduras. Previous 
studies performed by our research group in these communities have found a prevalence of 
>60% in children up to 14 years of age. 
 
Methodology 
We propose a prospective clinical trial, with parallel control groups, randomized and 
incorporating four different treatment regimens (dose-ranging arms), as follows: 
• 
Arm 1: Single dose of ALB* 400mg (Active control arm) 
• 
Arm 2: Single dose of ALB 400mg + IVM** 600µg/Kg 
• 
Arm 3: Daily dose ALB 400mg / 3 consecutive days 
• 
Arm 4: Daily dose ALB 400mg + IVM 600µg/kg 3 consecutive days 
*ALB (ALBENDAZOL): Nematel® 400mg tablets 

Protocol version 1.0, 29Nov2018 
 
 
**IVM (IVERMECTINA): Iver P® 6mg tablets 
Pair comparisons for efficacy: 
• 
(P1) Arm 1 vs Arm 2 (ALB vs ALB/IVM600) 
• 
(P2) Arm 1 vs Arm 3 (ALB vs ALB x 3) 
• 
(P3) Arm 1 vs Rama 4 (ALB vs ALB/IVM600 x3) 
• 
(P4) Arm 2 vs Arm 4 (ALB x 3 vs ALB/IVM600 x3) 
 
Sample size calculation 
Sample Size calculation was based on the following: 
Hnull p1 = p2 = p3 = p4  
HAlternative: p2 > p1 
 
p3 > p1 
 
p4 > p1 
 
p4 > p2 
 
 
 
 
 
 
 
 
 
 
With this information, expected efficacy and size of the population, we have calculated a 
required sample size of 177 between 2 and 14 years of age children infected with T. trichiura.  
 
Population - Study procedures 
A parasitological survey will be conducted including at least 295 children within the community 
of La Hicaca and surroundings villages to identify infected children. 
ARM 
Expected 
Cure Rate 
Lost-to-
follow-up 
Sample 
Size 
Inflated lost 
to follow up 
ALB  
17% 
10% 
35 
39 
IVM-ALB  
55% 
10% 
51 
57 
(IVM-ALB) x3  
85% 
10% 
51 
57 
ALB x3  
60% 
10% 
21 
24 
Total 
177 

Protocol version 1.0, 29Nov2018 
 
 
After attending an informational meeting and obtaining parental consent and children’s 
assent, all participants will be required to provide information for a short enrollment form. 
They will then undergo clinical physical exam to determine their health status. Anthropometric 
measurements (height and weight) will also be collected to estimate standard nutritional 
indications, according to the following international regulations:  
• 
Body Mass Index (BMI): (BMI for ages 5 – 19) 
• 
Height-for-age: (5-19 years of age) 
• 
Weight-for-age: (5-10 years of age)  
Stunted growth, low weight and thinness will be determined using the Anthroplus 2007 
software (version 1.0.4) and will be defined as being -2 standard deviations (SD) away from the 
mean height-for-age, weight-for-age and BMI for age, respectively. 
As a precaution, girls who report having commenced menarche (first menstrual period), will be 
requested to provide a urine sample to rule out pregnancy. 
Exclusion Criteria: 
• 
Weight less than 15 kg 
• 
Having taken antiparasitic treatment (ALB, MBZ or IVM) in the past 3 months 
• 
History of allergies, idiosyncrasy or hypersensitivity to the medications (IVM and ALB) 
used in this study. 
• 
Acute clinical conditions (including diarrhea), based on the clinician investigator 
judgement 
• 
Participation in another clinical trial within the last 3 months prior to administration of 
the treatment 
• 
Pregnancy 
 
Once participants are enrolled, a stool sample will be requested to obtain baseline information 
regarding intestinal parasitic infections. 
 
Those participants who are parasitized only by T. trichiura will be enrolled in the clinical trial. 
Prior to the treatment administrations, blood samples will be collected to establish baseline 
measurements of hepatic enzymes Alanine transaminase (ALT) and aspartate transaminase 
(AST) and hemoglobin. Following, participants will be randomly divided into the following 
groups: 
• 
Arm 1: Single dose ALB 400mg (control group) 
• 
Arm 2: Single dose of ALB 400mg + IVM 600µg/kg 
• 
Arm 3: Daily dose of ALB 400mg for 3 consecutive days 
• 
Arm 4: Daily dose of ALB 400mg + IVM 600µg/kg for 3 consecutive days 
Four hours after treatment has initiated, a capillary blood sample will be collected via finger 
puncture (finger prick) from every participant in Arms 2 and 4 to determine the 
pharmacokinetics of the IVM (plasma concentration at 4, 24, and 72 hours following 
administration). Once the assigned regimen has been completed, each participant will undergo 
a clinical evaluation and a measurement of hepatic enzymes to monitor treatment safety 
(safety tests).  

Protocol version 1.0, 29Nov2018 
 
 
 
Anemia Diagnosis 
Hemoglobin concentration will be determined by the HemoCue method (HemoCue AB, 
Sweden. www.hemocue.com) which requires a capillary blood sample (by finger prick). 
 
 
Hepatic Enzymes (Transaminases) (Safety Test) 
Alanine transaminase (ALT) and aspartate transaminase (AST) levels will be determined by a 
private clinical laboratory, which will receive the anonymized samples (bearing only the 
participant identification code). The reference values will be determined by the laboratory but 
the most common ones are of the following ranges:  
ALT = 0 – 40 U/L 
AST = 0 – 40 U/L 
Should there be any participant with altered hepatic enzymes, they will be examined clinically 
and undergo another test at the end of the study. 
 
Establishing Parasitic Infection 
Stool samples from study participants will be collected and kept refrigerated in coolers. Same 
day analysis will be done via the Kato-Katz method, following WHO recommendations. A total 
egg count for each helminthic species will be done and expressed as the number of eggs per 
gram (EPG) of stool. Based on the EPG, an intensity of infection will be determined, including 
the categories light, moderate or heavy. Aliquots of each sample using each 95% ethanol and 
10% formalin will be preserved for molecular analysis and diagnosis of protozoan infection, 
respectively. 
 

Protocol version 1.0, 29Nov2018 
 
 
Treatment Efficacy 
To determine treatment efficacy, a method determining “cure rate” (CR) will be used. Cure 
rate is defined the percentage of subjects who no longer pass eggs after treatment (with a 0-
egg count per gram of stool at the end of the treatment period). The formula is as follows: 
 
 
Comparison of Kato Katz vs multi-panel qPCR for evaluating treatment efficacy 
A quantitative qPCR analysis will be done for the determination of intensity of infection in the 
stool samples provided by the participants, with the purpose being to compare these results 
with those of the Kato Katz technique. The agreement between both methods will be 
estimated using the Kappa statistic, which sets out to measure level of agreement between 
techniques. Values of κ = 0, 0.01-0.20, 0.21-0.4, 0.41-0.60, 0.61-0.80, 0.81-1 are interpreted as 
poor agreement, light agreement, moderate agreement, considerable agreement and 
excellent agreement, respectively.  
 
Identification of genetic mutations associated with BZ resistances before and after 
treatment 
DNA from T. trichiura will be extracted from the eggs found in stool samples provided by 
participants. This DNA will then be analyzed via the qPCR technique, using specific primers for 
the detection of single-nucleotide polymorphisms (SNPs) associated with resistance to 
benzimidazoles. The results will be presented using descriptive statistics. 
 
The research participants will be involved in the following procedures: 
1. Attend informational meeting where they will receive an invitation letter (Appendix 1) 
(duration: 30 min)  
2. Provide with informed consent: Parents or legal guardians will sign the informed 
consent (Appendix 2) and children older than 9 years will provide assent (Appendix 3) 
3. Fill the enrollment format (10 min) (Appendix 4)  
4. Clinical history format and physical examination (15 min) (Appendix 5)  
5. Anthropometric measurements (5 min) (Appendix 6) 
6. Venipuncture (3 min) (protocol can be found here: https://brocku.ca/research-at-
brock/wp-content/uploads/sites/73/REB-Guideline-Phlebotomy.pdf) 
7. Finger prick (5 min) (protocol can be found here: https://brocku.ca/research-at-
brock/wp-content/uploads/sites/73/SOP02-Finger-Prick-Blood-Sampling-edit.pdf) 
8. Provide fecal samples before receiving treatment and again at 14-21 days after 
receiving treatment (Appendix 7). 
 
 

Protocol version 1.0, 29Nov2018 
 
 
 
 
 
Summary of participation 
 
a. Applicable only to Arms with one-day treatment 
b. Applicable only to Arms with 3-day treatment 
c. Applicable only to research participants with abnormal liver enzymes in previous visits 
d. Applicable only to female participants that experienced menstruation already 
 
Adverse effects  
Treatment used in the study 
Oral administration of albendazole is safe but may cause rare mild and reversible adverse 
effects such as headache, vomit, diarrhea, abdominal pain, gastrointestinal discomfort, 
dizziness, hepatic function alterations, fever, leukopenia and very rarely pancytopenia. 
Similarly, ivermectin is a safe drug but may cause mild and reversible adverse effects such as 
headaches, dizziness, diarrhea, vomit, muscle pain, ocular irritation, drowsiness, hepatic 
function alteration and rarely postural hypertension. 
Stool Sample Collection 
Participants will have to collect their own stool samples, which may expose them to pathogens 
present in their own sample. 
 
Pre-
enrollment Enrollment Baseline 
Day 2 
Day 3 
Day 14-21 
Day 
- 30 to -2 
-7 to -1 
0 
1 
2 
14-23 
Stool sample 
X 
 
 
 
 
X 
Eligibility to RTC 
 
X 
 
 
 
 
Consent/Assent 
X 
 
 
 
 
 
Clinical 
evaluation 
Safety 
 
X 
X 
X 
X 
X 
Anthropometry 
 
X 
 
 
 
 
Randomization 
 
 
X 
 
 
 
Treatment* 
 
 
X 
Xb 
Xb 
 
Sample for PK* 
 
 
X 
 
Xb 
 
Safety tests 
(Liver enzymes) 
 
 
X 
 
Xb 
Xc 
Urine 
pregnancy test 
 
Xd 
 
 
 
 

Protocol version 1.0, 29Nov2018 
 
 
Venipuncture 
To measure hepatic enzyme levels, 2 blood draws will be done in all participants across the 
four study arms. This may cause transient pain, hematoma, vasovagal attack and in extremely 
rare cases, infection of the puncture site. 
Finger prick 
To determine hemoglobin levels and IVM pharmacokinetics, various finger punctures will take 
place, which may cause transient pain, slight bruise, sometimes vasovagal attack and in 
extremely rare cases, infection of the puncture site.  
 
References 
• 
[De Onis M., A.W. Onyango, Borghi E., Siyam A., C. Nishida, Siekmann J. (2007) 
Development of a WHO growth reference for school-aged children and adolescents. 
Bull World Health Organ 85:660-7. DOI: S0042-96862007000900010]. 
• 
WHO 2011. Haemoglobin concentrations for the diagnosis of anemia and its severity. 
Geneva, World Health Organization, 2011 
https://www.who.int/vmnis/indicators/haemoglobin.pdf.  
• 
WHO 2008. Monitoring Anthelmintic Efficacy for Soil Transmitted Helminths (STH). 
Available at: 
who.int/neglected_diseases/preventive_chemotherapy/Chemotherapy_anthelminthic
_drug_WHO_WB_final.pdf 
• 
Vercruysse, Jozef, et al. Is anthelmintic resistance a concern for the control of human 
soil-transmitted helminths?. International Journal for Parasitology: Drugs and Drug 
Resistance 1.1 (2011): 14-27. 
• 
Sanchez AL, Gabrie JA, Canales M, Rueda MM, Fontecha GA, Mason PW, et al. Soil-
Transmitted Helminths , Poverty , and Malnutrition in Honduran Children Living in 
Remote Rural Communities. Hum Parasit Dis. 2016;8:27–35. 
• 
Moser W, Schindler C, Keiser J. Efficacy of recommended drugs against soil transmitted 
helminths: systematic review and network meta-analysis. BMJ [Internet]. BMJ 
Publishing Group; 2017 Sep 25 [cited 2017 Oct 21];358:j4307. Available from: 
http://www.ncbi.nlm.nih.gov/pubmed/28947636 
• 
Echazú A, Juarez M, Vargas PA, Cajal SP, Cimino RO, Heredia V, et al. Albendazole and 
ivermectin for the control of soil-transmitted helminths in an area with high 
prevalence of Strongyloides stercoralis and hookworm in northwestern Argentina: A 
community-based pragmatic study. Jones M, editor. PLoS Negl Trop Dis  Available 
from: http://www.ncbi.nlm.nih.gov/pubmed/28991899  
• 
Ceballos L, Krolewiecki A, Juárez M, Moreno L, Schaer F, Alvarez LI, et al. Assessment of 
serum pharmacokinetics and urinary excretion of albendazole and its metabolites in 
human volunteers. PLoS Negl Trop Dis. 2018;12(1):1–15.