Molina-Morant et al. Trials (2020) 21:328 https://doi.org/10.1186/s13063-020-4226-2 STUDY PROTOCOL Open Access Efficacy and safety assessment of different dosage of benznidazol for the treatment of Chagas disease in chronic phase in adults (MULTIBENZ study): study protocol for a multicenter randomized Phase II non- inferiority clinical trial D. Molina-Morant1, M. L. Fernández2, P. Bosch-Nicolau1, E. Sulleiro3, M. Bangher4, F. Salvador1, A. Sanchez-Montalva1, A. L. P. Ribeiro5, A. M. B. de Paula6, S. Eloi7,8, R. Correa-Oliveira9, J. C. Villar10,11, S. Sosa-Estani12,13 and I. Molina1* Abstract Background: Chagas disease (CD) continues to be a neglected infectious disease with one of the largest burdens globally. Despite the modest cure rates in adult chronic patients and its safety profile, benznidazole (BNZ) is still the drug of choice. Its current recommended dose is based on nonrandomized studies, and efficacy and safety of the optimal dose of BNZ have been scarcely analyzed in clinical trials. Methods/design: MULTIBENZ is a phase II, randomized, noninferiority, double-blind, multicenter international clinical trial. A total of 240 patients with Trypanosoma CD in the chronic phase will be recruited in four different countries (Argentina, Brazil, Colombia, and Spain). Patients will be randomized to receive BNZ 150 mg/day for 60 days, 400 mg/day for 15 days, or 300 mg/day for 60 days (comparator arm). The primary outcome is the efficacy of three different BNZ therapeutic schemes in terms of dose and duration. Efficacy will be assessed according to the proportion of patients with sustained parasitic load suppression in peripheral blood measured by polymerase chain reaction. The secondary outcomes are related to pharmacokinetics and drug tolerability. The follow-up will be 12 months from randomization to end of study participation. Recruitment was started in April 2018. Conclusion: This is a clinical trial conducted for the assessment of different dose schemes of BNZ compared with the standard treatment regimen for the treatment of CD in the chronic phase. MULTIBENZ may help to clarify which is the most adequate BNZ regimen in terms of efficacy and safety, predicated on sustained parasitic load suppression in peripheral blood. Trial registration: ClinicalTrials.gov, NCT03191162. Registered on 19 June 2017. Keywords: Chagas disease, Benznidazole, Therapeutic, Multicenter study, Clinical trial * Correspondence: imolina@vhebron.net 1 Infectious Diseases Department, Vall d’Hebron University Hospital, PROSICS Barcelona, Universitat Autònoma de Barcelona, P° Vall d’Hebron 119, Edifici Mediterrània, VHIR, 08035 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Molina-Morant et al. Trials (2020) 21:328 Page 2 of 10 Introduction cannot be used to define cure, because a negative result Chagas disease (CD) is a neglected parasitic infection does not mean absence of the infection; however, in recent caused by the protozoan Trypanosoma cruzi. It is en- years, it has become an important tool used to identify demic in the American continent, and according to the therapeutic failure when the result remains positive after latest estimates, it affects around 6 million people. Thir- completed treatment [8]. teen percent of the Latin American population remains Antitrypanosomal treatment is always recommended for at risk of contracting the infection, which is transmitted acute and congenital CD, reactivated CD infections, and to humans by Triatominae insects [1]. CD has also be- chronic CD in individuals younger than 18 years of age [3, come a rising health problem in nonendemic countries 12]. Despite the limitations of treatment of chronic CD in because of international migration, and nonvectorial adults, international guidelines recommend treatment with transmission can occur trhough blood transfusion, organ either BNZ or nifurtimox in patients under 50 years old with transplant, and congenital infection [2, 3]. In addition, nonestablished cardiac complications [13, 14]. This is based orally transmitted CD has been detected in endemic mainly on the lower long-term clinical progression observed areas because of food carrying either infected Triatomi- in patients treated with BNZ after a mean follow-up of 10 nae insects or their feces [4]. years, the parasite persistence and concomitant chronic in- After malaria and schistosomiasis, CD represents the flammation underlying CCM, and the prevention of vertical third largest parasitic disease burden globally, with more transmission to children born by infected women and than 15,000 deaths attributed directly to chronic Chagas treated before pregnancy [3, 15]. Results of a systematic re- cardiomyopathy (CCM) annually. CCM is the main view and meta-analysis showed little benefit of the treatment, complication in the chronic phase of CD, and it develops and the BENEFIT (Evaluation of the Use of Antiparasital in approximately 30% of patients chronically infected Drug [Benznidazole] in the Treatment of Chronic Chagas’ with T. cruzi [1]. Moreover, it is the most common form Disease) trial found no statistically significant reduction of of nonischemic cardiomyopathy in Latin America [5, 6]. cardiac clinical impairment in patients with moderate to se- Currently, there are only two available drugs to treat CD: vere cardiomyopathy [16, 17]. Treatment should be individu- nifurtimox and benznidazole (BNZ). Of these two, BNZ is the alized for patients older than 50 years of age and for patients one most studied and most often used as a treatment. How- with comorbidities [3]. ever, current schemes of this treatment have some limitations. On the one hand, it has a limited efficacy based on serocon- BNZ dosing and duration version (around 50–80% in the acute phase of the disease and Currently, the recommended BNZ dosage and duration 8–20% in the chronic phase) [7]. Another important limitation regimen for CD treatment is 5–7 mg/kg/day for 60 days. is the high rate of adverse events (AEs) when using these This recommendation is based on studies carried out in drugs. The incidence of AEs related to BNZ varies from 40– the 1970s [18]. However, nowadays, both the dose and 50% up to 98%, and around 15% of these patients have to de- duration of treatment are under discussion, predicated finitively stop the treatment for this reason, with the rate even in findings from CD murine models, pharmacokinetic higher in patients treated with nifurtimox [8–10]. The most (PK)/pharmacodynamic studies, and studies in patients commonly observed AEs are hypersensitivity (rash, fever, gen- who discontinued the treatment. On this basis, it seems eralized edema, lymphadenopathy, myalgia, and arthralgia), clear that BNZ dose may be optimized. gastrointestinal disorders, bone marrow toxicity (neutropenia and thrombocytopenic purpura), and peripheral polyneurop- Lower dose athy [9]. Current knowledge about the BNZ toxicity mecha- Two population PK studies have shown through mathem- nisms is scarce because the main studies have focused on the atical models that lower dosage with the same duration clinical aspects of these AEs [10]. Our group recently carried would have the same efficacy [19, 20]. One of them [19] out an analysis of the cytokine profile and human leukocyte was carried out in children and the other in adults [20]. In antigen (HLA) classes I and II of patients who were treated the pediatric study, children were treated with a standard with BNZ, and we found a higher treatment discontinuation dose of BNZ. Although significantly lower concentrations rate due to skin hypersensitivity AEs in patients who had the of the drug were achieved compared with those reported HLA-B*3505 allele [11]. in adults, the treatment was effective in all patients who Moreover, another drawback of the studies assessing the completed the treatment course. Moreover, data from a efficacy of BNZ in chronic CD is the lack of a biomarker second study carried out in adults revealed that a dose of to define the cure of disease. Currently, the cure criteria 5 mg/kg/day might lead to overexposure in the majority of are negative seroconversion of two serologic assays against patients and that a BNZ dose of 2.5 mg/kg/day is enough different antigens, but it usually takes several years after to adequately keep BNZ trough plasma concentrations an effective treatment, precluding its use in clinical trials. within the recommended target range according to previ- In addition, detection of T. cruzi DNA in peripheral blood ous PK studies [21, 22]. Molina-Morant et al. Trials (2020) 21:328 Page 3 of 10 Higher dose levels with the therapeutic response and AEs, to correl- Recent in vitro assays that quantify the time necessary to ate the presence of HLA-B*3505 with the presence of se- eliminate the parasites (time-to-kill assays) showed that vere AEs, and to correlate the different discrete typing nitroheterocyclic compounds such as BNZ are dose- units of T. cruzi with the therapeutic response. dependent [23]. In fact, treatment schemes with a higher- than-standard dose of BNZ (400 mg daily) with the same Primary endpoints duration already have been used (STOP-CHAGAS [A The primary endpoint is parasitologic response, defined Study of the Use of Oral Posaconazole in the Treatment as maintained negative qualitative PCR results during of Asymptomatic Chronic Chagas Disease] study), without the 12-month follow-up period. For efficacy assessments, observing a higher proportion of side effects [24]. Further- the end of treatment of each treatment arm will be de- more, another study of 54 patients treated with BNZ tried fined in accordance with the duration of the treatment to establish a correlation between the serum concentra- regimen. Incidence and severity of AEs and those lead- tions of the drug and the appearance of AEs. Fifty-three ing to treatment discontinuation will also be recorded. patients (98%) experienced at least one AE during follow- up, but no relationship was found between the drug serum Secondary endpoints concentration and the occurrence of AEs [25]. Secondary endpoints are parasitic clearance at weeks 1, 2, 4, and 8 during the treatment period and at 4, 6, and Shorter regimens 8 months during the follow-up period, measured by Finally, regarding the duration of treatment, recent stud- qualitative PCR. Serological response will be assessed by ies in animal models have shown that shorter schemes conventional serology at 12-month follow-up. The pro- (25% of standard duration) achieve the same cure rate portion of HLA-B*3505 carriers among the patients who [26]. This is under assessment in other clinical trials [27, experience SAEs will be recorded, as will blood concen- 28], but findings of one study showed an important cure trations of BNZ at 15 d and by the end of treatment. rate in patients who had to abandon the treatment due to severe adverse events (SAEs) [29]. Patient eligibility Methods/design Patients aged ≥ 18 years having any combination of at The MULTIBENZ study (Evaluation of Different Benzni- least two positive serologic test results against T. cruzi dazole Regimens for the Treatment of Chronic Chagas (indirect immunofluorescence, indirect Disease; ClinicalTrials.gov, NCT03191162) is a phase II, hemagglutination, or ELISA) and not having previously noninferiority, parallel-arm, randomized, double-blind, received treatment with BNZ or nifurtimox (either com- multicenter international clinical trial assessing the effi- pletely or partially) will be eligible. Inclusion and exclu- cacy and safety of three different BNZ dose schemes for sion criteria are summarized in Tables 1 and 2, the treatment of CD in chronic phase. It will be carried respectively. out in four different countries: Argentina, Brazil, Colombia, and Spain (protocol version V1/05-12-2016). Randomization and follow-up Patients are randomly assigned to receive BNZ 150 mg/ Outcomes and endpoints day for 60 days, 400 mg/day for 15 days, or the standard The primary objective of MULTIBENZ is to evaluate the ef- scheme of 300 mg/day for 60 days. Because this is a ficacy of different BNZ regimens at 12 months after double-blind trial, investigators and all sponsor staff will randomization in patients with CD in the chronic phase. not be aware of the treatment allocation and The primary efficacy outcome is defined as the proportion of randomization list until the end of the trial. Double- patients with sustained parasitic load suppression in periph- blinding will be adopted for all trial arms. Patients will eral blood measured by polymerase chain reaction (PCR) be randomized 1:1:1, and randomization will be done via during the first 12 months of follow-up after randomization. a remote and interactive response system, according to a The secondary objectives are to evaluate the parasitic predefined list. To avoid bias, randomization will be cen- kinetics by detecting parasitic DNA measured by PCR in tralized and concealed. The treatment groups will be al- peripheral blood at different time points (weeks 1, 2, 4, located on day 1 on the basis of a balanced block and 8 during the treatment period and in the fourth, randomization, taking into account the country. Each sixth, and eighth months after the start of treatment), to patient will be assigned an identification code that will evaluate the serological response by enzyme-linked im- correspond to the trial kit number allocated to the pa- munosorbent assay (ELISA) methods at the end of tient. The label will indicate the trial number and the follow-up (month 12), to assess the tolerability and number of the kit, but it will not indicate the treatment safety of the different BNZ regimens, to correlate BNZ designation. Molina-Morant et al. Trials (2020) 21:328 Page 4 of 10 Table 1 Inclusion criteria Scheduled follow-up visits will occur at 7, 15, 28, and Inclusion criteria (patients should meet all criteria) 60 days and up to 4, 6, 8, and 12 months after initiation • Adults ≥ 18 years old of treatment. More information is provided in Fig. 1. • Have been diagnosed with Chagas disease by two positive serological tests using different antigens Sample size and data analysis • Have detectable Trypanosoma cruzi DNA in peripheral blood through a For the sample size calculation, we considered a nonin- qualitative interpretation of the polymerase chain reaction technique feriority design for two-sample comparison of propor- • Written informed consent provided tions. Assumption of a noninferiority margin of 10% in • Weight ≥ 50 kg and ≤ 80 kg relation to standard treatment, which represents a re- duction of 50% of the total number of patients who ei- • Ability to comply with all tests and specified protocol visits and have a permanent address ther have a positive PCR result during follow-up or have to discontinue the treatment due to AEs, has been taken • Patients must be residents of areas free of vector transmission (Triatoma infestans), defined by local health programs or by the Pan into account as a hypothesis. It is estimated that in the American Health Organization/World Health Organization definition. standard arm of treatment, 40% of patients will be evalu- • Women of childbearing age should have a urine or serum negative ated as treatment failures according to the intention-to- pregnancy test at the moment of the baseline visit. Breastfeeding treat principle. should not be allowed, and a barrier method of contraception should be used during the treatment phase. For a unilateral type I error (p = 0.025) and a power of 80%, a comparison between four groups, an effect size of 0.067, and an average proportion of 0.75 with a variance of 0.013, 204 individuals would be needed. Considering a rate of 15% defaulted patients, the total number of pa- tients that should be included in the study is 240, which will comprise 60 participants per country. The categorical data will be presented as absolute numbers and proportions, and the continuous variables will be expressed as means and standard deviations when normal distribution is demonstrated (using the Table 2 Exclusion criteria Kolmogorov-Smirnov test) or as medians and interquar- Exclusion criteria tile ranges when it is not. • Having previously received treatment with benznidazole or nifurtimox (either completely or incompletely) For comparison of the distribution of categorical vari- ables, the χ2 test or Fisher’s exact test will be used, and • Signs and/or symptoms of severe cardiac form of Chagas disease (as confirmed by local national guidelines) the Mann-Whitney U test or Student’s t test will be used for continuous variables, respectively, depending on the • Impossibility to complete the specified protocol follow-up visits presence or not of normal distribution. • Acute or chronic health problems that, in the opinion of the principal investigator, may interfere with the evaluation of the efficacy and/or A comparative analysis of the main clinicoepidemiologi- safety related to the drug (for example, acute infections, human cal variable among the three groups will be carried out. immunodeficiency virus infection, liver or kidney disease) The primary efficacy analysis will be the comparison of • History of alcohol abuse the proportion of patients with sustained parasitologic • Known hypersensitivity to metronidazole drugs clearance of each treatment arm compared with the stand- • Concomitant use or history of use of allopurinol, antimicrobial, ard arm of treatment. The time until the first positive PCR antiparasitic, or antifungal agents result for T. cruzi will be evaluated by Kaplan-Meier sur- • Having laboratory parameters outside the range of normal or that are vival analysis with the log-rank test for significance. considered clinically relevant by the responsible physician: The proportion of patients with SAEs and/or AEs Total leukocyte count must be within the normal range, with an leading to treatment discontinuation will be described acceptable range of ± 5%. Total platelet count must be within the normal range up to 550, per trial arm and by System Organ Class (using pre- 000/mm3 or 550 × 109/L. ferred terms defined by MedDRA 13.1), according to the Total bilirubin must be within the normal range. National Cancer Institute’s Common Terminology Cri- Transaminase levels must be within the normal range, with an acceptable range of 25% above the upper limit of normal (ULN). teria for Adverse Events (version 4.03). Incidence rate Total creatinine level must be within the normal range, with an and 95% confidence interval will be presented per trial acceptable variation of 10% above the ULN. arm for SAEs and AEs per category, along with the most Alkaline phosphatase level must be within the normal range up to < 2.5× ULN. frequent AEs. Gamma glutamyl transferase level must be within the normal Safety laboratory parameters (hematology and bio- range up to 2× ULN. chemistry) will also be described individually per trial Fasting glucose must be within the normal range. arm, showing the proportion of patients by degree of Molina-Morant et al. Trials (2020) 21:328 Page 5 of 10 Fig. 1 Clinical trial design. BNZ Benznidazole, BV Baseline visit, EoF End of follow-up, FV Follow-up visit, SV Screening visit, TV Treatment visit elevation relative to upper limit of normal and baseline PCR values, as well as blood level changes over time. Laboratories included in the project will carry out the same Safety data will be correlated to efficacy and treatment reverse transcriptase (RT)-PCR protocol following the in- compliance data and to PK parameters. Differences in structions included in the laboratory manual agreed among the rate of absorption in serology will be estimated with all of them. To carry out this technique, 5 ml of whole blood t test pairs or the Wilcoxon test, depending on their dis- will be collected and mixed with 5 ml of guanidine hydro- tribution. All safety analyses will be carried out on all chloride 6 M–ethylenediaminetetraacetic acid 0.2 M for a patients treated, understood as all patients who receive minimum of 72 h at room temperature. Three DNA extrac- at least one dose of treatment. Finally, the sensitivity, tions will be performed using the manual column method specificity, and positive and negative predictive values of (High Pure PCR Template Preparation Kit; Roche Diagnos- the presence of HLA-B*3505 and their relationship with tics, Mannheim, Germany), except in Spanish centers (Vall the occurrence of SAEs will be calculated. d´Hebron University Hospital and Ramón y Cajal University The efficacy analysis will be carried out according to the Hospital) where DNA extraction will also be done in tripli- intention-to-treat principle. The group of patients analyzed cate using an automated extraction method (NucliSens easy- will be all randomized patients in each of the treatment MAG, bioMèrieux, Marcy l’Etoile, France).The consensual branches. Cases lost during follow-up and dropouts will be PCR protocol consists of a real-time multiple PCR (Duffy considered treatment failures.In addition, a per-protocol et al. 2013) [30] that allows the amplification of a T. cruzi sat- analysis will be defined as all patients receiving randomized ellite DNA region and a linearized recombinant plasmid treatment who meet the main criteria, have not perman- used as an internal amplification control. The RT-PCR will ently left the administration of the treatment, and have no be carried out in duplicate from each of the extractions. At other protocol deviation. Patients lost to follow-up will be least one amplification of the six performed with an amplifi- excluded unless they present a positive PCR result, in which cation cycle (cycle threshold) of T. cruzi below 40 and a cor- case, they will be included in the analysis. The results will rect value of Internal Amplification Control (IAC) will be be analyzed using IBM SPSS Statistics version 19.0 software interpreted as positive. To be correct, the values of the IAC (IBM Corp., Armonk, NY, USA). must meet Tukey’s criteria. To assess the homogeneity of the results obtained by the different laboratories, a harmonization panel consisting of Laboratory procedures ten tubes containing blood with uninfected guanidine and Serology infected with 1, 10, and 100 parasitic equivalents per milliliter Two different anti–T. cruzi serologic tests based on dif- of T. cruzi strains TcV and TcVI was processed. The samples ferent antigens were used for assessing patient eligibility. were processed blindly by the different laboratories, and the To avoid interlaboratory variability, serum samples col- results were evaluated by an external center in charge of lected at the times indicated in the protocol will be sent providing the panel and analyzing the results (Instituto de at the end of the study to a centralized laboratory that Investigaciones en Ingeniería Genética y Biología Molecular, will process them using two techniques in parallel: Buenos Aires, Argentina). On the other hand, and following Architect Chagas (Abbott Laboratories, Wiesbaden, the same work scheme, four external quality control panels Germany) and ORTHO Trypanosoma cruzi ELISA Test will be analyzed during the study period. System (Ortho Clinical Diagnostics, Raritan, NJ, USA). In order to give greater robustness to the results, sam- HLA typing ples from external quality control sent by the National The typing of HLA-B alleles is carried out from the Program of Quality Control of Brazil will be included. dried blood samples on paper (dried blood spots [DBS]). Molina-Morant et al. Trials (2020) 21:328 Page 6 of 10 For this purpose, DNA is extracted using DNA Elution will be withdrawn from the study. The screening proced- Solution reagent (catalog no. 159994; Qiagen, Carpin- ure must occur up to 90 days or less before the initiation of teria, CA, USA), and the concentration and quality are treatment. Serology and DNA determination by PCR will be evaluated by measuring the absorbance at 260 and 280 accepted as valid and will not need to be repeated if a posi- nm using the Colibri microvolume spectrometer (Titer- tive result was obtained in a previous period of 3 months. tek-Berthold, Pforzheim, Germany). The characterization All patients will undergo a clinical history and physical of the HLA-B alleles is carried out by PCR sequence- examination. Peripheral blood extraction will be per- specific oligonucleotide (SSO) (Luminex Corp., Austin, formed for analysis: hemogram, biochemistry assays, and TX, USA), following the instructions of the Lifecodes HLA study, and a negative pregnancy test (either in HLA typing kit (Immucor; Diagnóstica Longwood, Zara- urine or blood) is mandatory in case of women in child- goza, Spain). Briefly, the PCR-SSO/Luminex consists of bearing age. The evaluation of the visceral involvement amplification with biotinylated primers of the most poly- of CD will be completed with a chest x-ray and electro- morphic regions of the HLA-B gene, followed by cardiogram. The performance of other complementary hybridization of the amplified product with specific tests will be carried out according to the clinical investi- probes for each allele located on the surface of Luminex gator’s decision, but they will not be considered neces- microspheres and revealed with conjugated streptavidin sary for the inclusion of the patient. with phycoerythrin. Finally, it is analyzed using an xMAP100 fluoroanalyzer (Luminex Corp.). 3. Treatment phase BNZ serum concentration In this phase, the patient will be randomized to one of the Quantification of BNZ is done from dried blood samples three arms of treatment, whose duration will be 60 days in- on paper (DBS). The quantification is performed by li- dependently of the arm assigned. Initially, a baseline visit will quid chromatography (ACQUITY ultra performance li- be performed in which the patient will be trained on how to quid chromatography high-strength silica T3 C18, 2.1 × take the drug and identify AEs. After that, a total of four 50 mm; Waters, Milford, MA, USA) coupled to triple- scheduled visits will occur during the treatment period, at 7, quadrupole mass spectrophotometry (Xevo TQ; Waters). 15, 28, and 60 days after treatment initiation. A summary of these visits is presented in Table 3. Study organization During this phase, any patient may consult spontan- The MULTIBENZ study network includes four countries eously for the eventual occurrence of any AE. The deci- and seven centers, with the Spanish coordinating center sion to interrupt the treatment will be according to the located at Vall d’Hebron University Hospital in Barce- discretion of the clinical researcher treating the patient, lona, Spain. The study contemplates a total of three taking into account the severity, intensity, and extent of stages, which are outlined below. these AEs. In addition, the patient may decide unilat- erally to suspend or not the medication at any time dur- 1. Recruitment sites ing the treatment period. Countries involved in the recruitment will be Spain 4. Follow-up phase (University Hospital Vall d’Hebron, Barcelona; University Hospital Ramón y Cajal, Madrid), Argentina (Instituto Once the patient has taken the last dose of treatment, Nacional de Parasitología Dr. Mario Fatala Chaben, Buenos which may be at the end of the therapeutic scheme (day Aires; Instituto de Cardiología Juana Francisca Cabral, 60), when a severe AE that obliges the patient to sus- Corrientes), Brazil (Centro de Pesquisas René Rachou – pend the medication occurs, or when the patient unilat- Fundação Oswaldo Cruz, Belo Horizonte; Hospital Univer- erally decides to suspend the medication, this phase will sitário Clemente de Faria, Montes Claros), and Colombia begin, and it will last up to 12 months after (Fundación Cardioinfantil – Instituto de Cardiología). randomization. The visit schedule of this phase is pre- sented in Table 3. 2. Selection phase Patients with CD in the chronic phase who come to SAEs and unblinding: care of patients with AEs the study centers will be evaluated in order to assess if All AEs and other study outcomes in the randomization, they meet the inclusion criteria. For those who agree to treatment, and follow-up periods will have to be re- participate and sign the informed consent, detection of ported. In case of mild or moderate AEs, the administra- parasitic DNA by PCR in peripheral blood will be per- tion of BNZ will be suspended temporarily according to formed. Patients with a PCR result negative for T. cruzi the clinical researcher’s decision. Symptomatic treatment Molina-Morant et al. Trials (2020) 21:328 Page 7 of 10 Table 3 Visit schedule Selection visit Baseline Treatment Treatment Treatment End of Side effect Follow-up visit Final (from − 3 months visit visit visit visit treatment visit visit (months 4, 6, visit to day 0) (day 0) (day 7 ± 2) (day 15 ± (day 28 ± (day 60 ± 7) (not and 8) (month 3) 4) scheduled) 12) Informed consent x Demographic data x Pathological x background Toxic habits x Chagas disease x history Concomitant x x x x x x x x x medication Directed anamnesis x x x x x x x x x Physical x x x x x x x x x examination Pregnancy test x Hematology x x x x x x Biochemistry x x x x x x HLA x Chest x-ray x EKG x PK x x x Serology x x PCR x x x x x x x Other x complementary tests Adverse events x x x x x x Randomization x Study medication x x x x x Abbreviations: EKG Electrocardiogram, HLA Human leukocyte antigen, PCR Polymerase chain reaction, PK Pharmacokinetics will be provided to the patient according to the treating whereby the researcher will explain to each patient the na- physician. ture of the study; its purposes, procedures, expected dur- It will be considered that a patient has finished the ation, and the potential risks and benefits related to study when he or she has completed the treatment and participating; and any inconvenience that may occur. follow-up phase (per-protocol principle). The study may be interrupted in case of voluntary decision of the pa- Discussion tient at any time if an AE forces interruption of the CD continues to be an infectious disease with one of the treatment, if significant protocol violations occur, or if highest disease burdens worldwide. Despite the modest the treating physician deems it would benefit the patient cure rates in adult chronic patients and its safety profile, (intention-to-treat principle). BNZ remains the best treatment option against the dis- ease due to the lack of therapeutic alternatives [7]. Con- Ethics and patient confidentiality ventional diagnostic methods for establishing cure rates The protocol has been approved by national regulatory in chronic CD have marked inherent limitations; how- agencies (in accordance with the ethical standards laid ever, the use of more sensitive methods for parasite de- down in the Declaration of Helsinki as revised in 2013), by tection, such as PCR, could provide a suitable tool for the institutional review boards for clinical research of all follow-up assessment of treatment in patients with CD, participating institutions, and by the national ethics review because detectable T. cruzi DNA in peripheral blood committees of the countries involved in the study (when samples after treatment end is considered a therapeutic applicable). All patients provide written informed consent, failure [31]. Molina-Morant et al. Trials (2020) 21:328 Page 8 of 10 The currently used dose of BNZ is based on nonran- with prolonged exposure. Unfortunately, due to the lack of domized studies that were carried out more than 50 clinical published experience and the potential risk of go- years ago, and they have never been analyzed in clinical nadal toxicity and its effect over the pituitary–testicular axis, trials. Moreover, there are no data regarding the rela- that treatment arm was discarded [34, 35]. tionship between dose and efficacy, because the great Another aspect that will be assessed is the genetic variabil- majority of published experience (clinical trials and ob- ity of the parasite over the treatment response. The great servational studies) has been with the standard dose. majority of randomized clinical trials have been conducted Recent studies have brought to light interesting results in Argentinean or Bolivian patients, and scarce information that point out different possibilities of treatment is available in other geographical regions. In any case, the schemes. On the basis of two PK studies, authors sug- few existing data suggest an important effect of parasitic di- gested that current treatment regimens could be over- versity in the treatment efficacy and response [17, 36]. dosed [19, 20]. One them extrapolated the idea Therefore, this clinical trial will evaluate the efficacy according to the lower concentration observed in treated and safety of different dosing regimens of BNZ com- children that finally achieved cure. The other based the pared with the standard treatment scheme. Experimental conclusion on dosage regimen simulation under steady- dosing regimens have been chosen on the basis of evi- state conditions comparing the estimated concentration dence derived from previous studies, in which it has with the optimal therapeutic accepted range. This hy- been found that shorter-duration schemes and/or lower pothesis has also been observed preclinically using an dose with the same duration could achieve the same in vivo model under the framework of the Berenice Pro- cure rate. Moreover, higher doses could be used without ject (Benznidazol and Triazol Research Group for Nano- entailing a higher rate of AEs. The results of this clinical medicine and Innovation on Chagas Disease). Results trial will help to better identify the most adequate BNZ obtained from the murine model led researchers to con- regimen in terms of efficacy and safety for the treatment clude that a dose of 40% of the total of the version used of CD in the chronic phase. as standard is comparable in efficacy. The MULTIBENZ study is included within the Bere- At the same time, there is evidence of the opposite. nice Project, founded by the European Commission and According to mechanism of action of BNZ, it seems that initiated in September 2012. The aim of the Berenice the efficacy of the drug (and all nitroderivative drugs) is Project is to provide a new and cost-effective solution to concentration-dependent. To include an arm with higher treat patients with CD in chronic phase and to develop dose in a clinical trial could pose an increased risk of new drug formulations with trypanocidal activity. Its toxicity. The clinical experience obtained through clin- main objective is to obtain a more effective, better toler- ical trials with higher doses of BNZ (400 mg/day) led to ated, and cheaper treatment to cure CD. The results ob- the belief that higher dose is not associated with higher tained in the Berenice Project will upgrade European frequency of AEs [23, 24]. competitiveness through the transformation of research In addition, the mechanisms underlying BNZ toxicity in the field of neglected infectious diseases. are still not well understood. The production of several metabolites in the enzymatic reduction of the drug, their Trial status accumulation, and their interactions with cellular con- Recruitment was started in April 2018, and it is estimated to stituents could be the main reasons for producing these be completed in all countries by August 2020. Protocol ver- AEs [32, 33]. Some AEs of the drug have a certain tem- sion V1/ 05-12-2016. poral pattern. For example, dermatological and digestive Acknowledgements manifestations usually tend to occur around day 10 of Authors from the Drugs for Neglected Diseases initiative (DNDi) thank its donors, treatment, while neurological events and arthritis appear public and private, who have provided funding to DNDi since its inception in 2003. after day 40 of treatment, probably because they are re- A full list of DNDi donors can be found at https://www.dndi.org/donate/donors/. lated to the cumulative total dose and not to the serum Authors’ contributions concentration [9]. Age and female sex have also been DMM, PBN, FS, and ASM wrote the original draft of the manuscript and analyzed and considered as classic risk factor for AEs. However, there interpreted data. MLF, MB, ALPR, AMBP, SE, RCO, JCV, and SSE wrote, reviewed, and edited the manuscript. ES analyzed data. IM wrote the original draft of the manuscript, remains an important lack of knowledge about the analyzed and interpreted data, and designed the work. All authors read and approved mechanisms of toxicity [9, 10]. the final manuscript. According to the duration of the treatment, two ap- Funding proaches were considered. Taking into account the cure This clinical trial is supported by the European Berenice Project, a rates of patients with CD treated with shorter courses of collaborative project funded under the European Community’s 7th BNZ (those who had to interrupt the treatment because of Framework Program (grant agreement HEALTH-305937). The funder had no role in the clinical trial design; in the collection, analysis, and interpretation AEs), a shortened regimen has been incorporated. Finally, re- of data; in the writing of the report; or in the decision to submit the article searchers explored the possibility of including a new arm for publication. Molina-Morant et al. Trials (2020) 21:328 Page 9 of 10 Availability of data and materials 9. Molina I, Salvador F, Sánchez-Montalvá A, Treviño B, Serre N, Sao Avilés A. The datasets generated and analyzed during the study will be available from Toxic profile of benznidazole in patients with chronic Chagas disease: risk the corresponding author on reasonable request. factors and comparison of the product from two different manufacturers. Antimicrob Agents Chemother. 2015;59(10):6125–31. Ethics approval and consent to participate 10. Hasslocher-Moreno AM, do Brasil PEAA, de Sousa AS, Xavier SS, Chambela The protocol has been approved by national regulatory agencies (in MC, Sperandio da Silva GM. Safety of benznidazole use in the treatment of accordance with the ethical standards laid down in the Declaration of chronic Chagas’ disease. J Antimicrob Chemother. 2012;67(5):1261–6. Helsinki as revised in 2013), by the institutional boards for clinical research of 11. Salvador F, Sánchez-Montalvá A, Martínez-Gallo M, Sala-Cunill A, Viñas L, all participating institutions, and by the national ethics review committees of García-Prat M, et al. Evaluation of cytokine profile and HLA association in the countries involved in the study (when applicable). Referral ethical board: benznidazole related cutaneous reactions in patients with Chagas disease. Vall d’Hebron University Hospital Ethical Committee on Clinical Research with Clin Infect Dis. 2015;61(11):1688–94. Drugs (EUDRACT no. 2016-003789-21). All patients provide written informed 12. Coura JR, Borges-Pereira J. Chronic phase of Chagas disease: why should it be consent, whereby the researcher will explain to each patient the nature of treated? A comprehensive review. Mem Inst Oswaldo Cruz. 2011;106(6):641–5. the study; its purposes, procedures, and expected duration; the potential risks 13. Sguassero Y, Roberts KN, Harvey GB, Comandé D, Ciapponi A, Cuesta CB, and benefits related to participating; and any inconvenience that may occur. et al. Course of serological tests in treated subjects with chronic Trypanosoma cruzi infection: a systematic review and meta-analysis of Consent for publication individual participant data. Int J Infect Dis. 2018;73:93–101. Not applicable. 14. Pan American Health Organization (PAHO). Guidelines for the diagnosis and treatment of Chagas disease. Washington, DC: PAHO; 2018. http://iris.paho. Competing interests org/xmlui/handle/123456789/49653. 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