Open access Protocol New chemotherapy regimens and biomarkers for Chagas disease: the rationale and design of the TESEO study, an open-­label, randomised, prospective, phase-­2 clinical trial in the Plurinational State of Bolivia Cristina Alonso-­Vega,1 Julio A Urbina ‍ ‍,2 Sergi Sanz,3,4,5 María-­Jesús Pinazo,1,6 Jimy José Pinto ‍ ‍,7 Virginia R Gonzalez,8 Gimena Rojas,7 Lourdes Ortiz,9,10 Wilson Garcia,11,12 Daniel Lozano,7 Dolors Soy,13,14 Rosa A Maldonado,8 Rana Nagarkatti,15 Alain Debrabant,15 Alejandro Schijman,16,17 M Carmen Thomas,18 Manuel Carlos López,18 Katja Michael,19 Isabela Ribeiro,20 Joaquim Gascon,1,6 Faustino Torrico,7 Igor C Almeida ‍ ‍8 To cite: Alonso-­Vega C, ABSTRACT Urbina JA, Sanz S, et al. New Strengths and limitations of this study Introduction Chagas disease (CD) affects ~7 million chemotherapy regimens people worldwide. Benznidazole (BZN) and nifurtimox and biomarkers for Chagas ► This study will evaluate the safety and efficacy of (NFX) are the only approved drugs for CD chemotherapy. new regimens of benznidazole and nifurtimox, with disease: the rationale and design of the TESEO study, Although both drugs are highly effective in acute and lower frequency of dosing combined with reduced an open-­label, randomised, paediatric infections, their efficacy in adults with chronic or extended treatment duration, vis-à-vis the stan- prospective, phase-­2 clinical CD (CCD) is lower and variable. Moreover, the high dards of care of both drugs. trial in the Plurinational incidence of adverse events (AEs) with both drugs has ► The assessment of parasitic load reduction will State of Bolivia. BMJ Open hampered their widespread use. Trials in CCD adults be performed during treatment and 36 months of 2021;11:e052897. doi:10.1136/ showed that quantitative PCR (qPCR) assays remain bmjopen-2021-052897 systematic follow-­up by quantitative PCR (qPCR), negative for 12 months after standard-­of-­care (SoC) conventional serology, and novel host-­derived and ► Prepublication history and BZN treatment in ~80% patients. BZN pharmacokinetic parasite-­derived biomarkers. additional supplemental material data and the nonsynchronous nature of the proliferative ► Basic population pharmacokinetics of both benzni- for this paper are available mammal-­dwelling parasite stage suggested that a lower dazole and nifurtimox will be performed in all treat- online. To view these files, BZN/NFX dosing frequency, combined with standard or ment arms. please visit the journal online extended treatment duration, might have the same or ► Parasite genotyping will be performed in all patients (http://dx.doi.org/10.1136/​ bmjopen-2021-052897). better efficacy than either drug SoC, with fewer AEs. before treatment and in patients with positive qPCR Methods and analysis New ThErapies and Biomarkers results during the 36 months of follow-­up. Received 27 April 2021 for ChagaS infEctiOn (TESEO) is an open-­label, ► One of limitations is that the study will only include Accepted 22 November 2021 randomised, prospective, phase-­2 clinical trial, with six adult chronic Chagas disease patients in the inde- treatment arms (75 patients/arm, 450 patients). Primary terminate or early cardiac compromised (Kuschnir objectives are to compare the safety and efficacy of two stage I), looking for the effectiveness of the different new proposed chemotherapy regimens of BZN and NFX aetiological treatments on the progression to severe in adults with CCD with the current SoC for BZN and cardiac and/or gastrointestinal disease stages. NFX, evaluated by qPCR and biomarkers for 36 months © Author(s) (or their posttreatment and correlated with CD conventional employer(s)) 2021. Re-­use serology. Recruitment of patients was initiated on 18 Plurinational State of Bolivia and the Ethics Committees of permitted under CC BY-­NC. No December 2019 and on 20 May 2021, 450 patients (study the participating institutions. The results will be disseminated commercial re-­use. See rights goal) were randomised among the six treatment arms. via publications in peer-­reviewed journals, conferences and and permissions. Published by The treatment phase was finalised on 18 August 2021. reports to the NIH, FDA and participating institutions. BMJ. Secondary objectives include evaluation of population Trial registration number NCT03981523. For numbered affiliations see pharmacokinetics of both drugs in all treatment arms, the end of article. INTRODUCTION incidence of AEs, and parasite genotyping. Correspondence to Ethics and dissemination The TESEO study was approved Chagas disease (CD) or American trypanoso- Professor Igor C Almeida; by the National Institutes of Health (NIH), U.S. Food and miasis is a tissue and blood parasitic disease ​icalmeida@​utep.​edu Drug Administration (FDA), federal regulatory agency of the caused by the kinetoplastid protozoan Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 1 Open access parasite Trypanosoma cruzi. It can be transmitted by the of qPCR) would occur in 60%–90% of the treated cases, faeces of reduviid bugs, congenital transmission, blood at 12 months of follow-­up.7 21 However, the Pan-­American transfusion, tissue transplant and oral transmission.1 Health Organization (PAHO) guidelines for the diag- CD reportedly affects ~7 million people mostly in Latin nosis and treatment of CD state that only the seroconver- America and is responsible for an estimated 14 000 deaths sion by conventional serology (CS) can be interpreted as yearly. It is also currently considered an emerging world- an indicator of parasitological cure,22 but it is well known wide public health problem due to increasing interna- that in chronically infected adults with successful parasi- tional migrations from endemic regions.2 The economic tological cure, CS seroconversion can take 10–20 years impact of CD is also very significant. According to a study to be confirmed following chemotherapy.23–25 Thus, the conducted in 2013, the global costs for CD are US$7– lack of biomarkers (BMKs) of early response to treatment US$19 billion per year, similar or higher to those of other and eventual parasitological cure is a main roadblock to widespread diseases, such as rotavirus infection.3 Despite evaluate the true efficacy of currently available and novel its important regional and international health impact, chemotherapeutic approaches.26 27 CD is considered by WHO one of the most neglected Recent studies suggest that the current BZN dose of tropical diseases (NTDs) associated with socioeconomical 5 mg/kg/day divided into two doses (SoC) can lead to inequality and thus exclusion and stigmatisation, both in an overdosing of the patients while using half of the daily endemic and nonendemic countries.4 Currently, no safe dose could be enough to reach and sustain anti-T. cruzi and efficacious vaccines for this disease are available, and therapeutic plasma levels.28 In the case of NFX, a recent the existing drugs and regimens for the aetiological treat- study showed that 3 mg/kg two times per day given for ment of this condition have toxicity and efficacy issues.5 60 days showed an efficacy of 70%, which is comparable Moreover, the aetiological treatment of CD in its chronic to the results with the SoC.29 A fundamental insight in stage was questioned for decades by the hypothesis that this respect was provided by a study in a murine model autoimmunity was the major mechanism leading to of the disease,30 which showed that reducing the dosing chronic CD (CCD) pathology.6 This hypothesis contrib- frequency of BZN or NFX from daily (continuous) to uted negatively to the development of aetiological treat- every 5 days (intermittent) provided the same parasiticidal ments for the chronic disease until very recently.7 As a efficacy, using a much lower total dose of the drug. Such result, several generations of health professionals were findings were reported by authors to indicate that both trained in the belief that CCD had no possible treatment.8 drugs act on the parasite through a critical peak serum Consequently, it is estimated that currently less than 1% concentration (Cmax effect), rather than a continuous of confirmed CD patients receive aetiological treatment.9 exposure (area under the curve-­AUC effect).31 However, However, the role of the persistence of T. cruzi as a trigger carefully designed translational pharmacokinetics–phar- for tissue damage and the pathophysiology of CD is now macodynamic modelling and population pharmacoki- recognised, providing the rationale for antiparasitic treat- netics (popPK) studies of both drugs in humans, as those ment for all seropositive patients.7 10–14 to be performed in the New ThErapies and Biomarkers To date, only two drugs have been approved for the for ChagaS infEctiOn (TESEO) study and future clinical treatment of CD, the nitroheterocyclic compounds studies, will be critical to define the precise pharmacody- benznidazole (BZN) and nifurtimox (NFX).13 Such treat- namic drivers for these drugs. ments are currently indicated for acute cases, congen- Based on these antecedents, our first hypothesis is that ital infections, reactivations and patients in the chronic a lower frequency of BZN or NFX dosing, with standard phase without symptomatology or with mild cardiac or or extended treatment duration, might have the same or digestive involvement. The dosing regimens currently better efficacy than the SoC of either drug, with fewer recommended (standard of care, SoC) are 5 mg/kg/day AEs. Our second hypothesis is that, in those patients divided into two doses for 60 days for BZN, and 8 mg/ who respond to BZN or NFX treatment, the serum levels kg/day divided into two or three doses for 60 days for of one or more potential BMKs proposed in this study NFX. Both treatments are known to be associated with will be significantly reduced or become negative within adverse events (AEs) up to 70% of the patients and with 3 years of post-­treatment follow-­up. The 3-­year follow-­up 10%–27% of serious AEs (SAEs), leading to permanent is designed to investigate whether the high level of para- treatment discontinuation in 9%–31% of the cases.15–20 sitaemia suppression induced by BZN 1 year after the end Moreover, the efficacy of these treatments is highly vari- of treatment (EOT) found in recent clinical studies32 33 is able, and it has been shown that it depends on multiple sustained after longer follow-­up times, or whether parasi- factors: age of the patient, disease stage, drug dose and taemia relapse occurs, as reported in the BENEFIT trial,34 treatment duration, and the infecting T. cruzi strain or previous observational studies ≥2 years after the EOT,35–40 genotype, among other factors. and in a canine model of CCD.41 On the other hand, for patients in the chronic stage Therefore, the TESEO study aims to assess the safety the efficacy of the treatment is difficult to assess. Using and efficacy of new dosing regimens of BZN and NFX for quantitative PCR (qPCR) as an efficacy parameter, it can the treatment of CCD patients, combining a reduction be estimated that parasitological clearance (defined as of the frequency of dosage with reduced or extended the parasitic load in the blood below the detectable limit treatment duration, as well as a 3-­ year follow-­up, and 2 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access regimens, the aim is to reduce AEs, while maintaining or Table 1 Treatment arms in TESEO study increasing antiparasitic efficacy. The detailed clinical trial Arm Drug* Regimen Arm abbreviation design is shown in figure 1. 1 BZN 150 mg b.i.d., 60 days BZN-­60 (SoC) In this study, the basic popPK parameters of BZN 2 BZN 150 mg q.d., 30 days BZN-­30 and NFX for all treatment regimens, including the SoC 3 BZN 150 mg q.d., 90 days BZN-­90 with both drugs, will also be evaluated. Patients will be followed up for 36 months. This extended follow-­up is 4 NFX 240 mg b.i.d., 60 days NFX-­60 (SoC) aimed at evaluating the sustainability of the antiparasitic 5 NFX 240 mg b.i.d., 30 days NFX-­30 effect, when compared with the results of recent clinical 6 NFX 240 mg q.d., 90 days NFX-­90 trials with a 12-­month follow-­up.32 33 42 The study also aims *Both drugs are taken orally. to generate information concerning qPCR and novel b.i.d, two times a day; BZN, benznidazole; NFX, nifurtimox; q.d., potential BMKs for the early assessment of antiparasitic once a day; SoC, standard of care; TESEO, New ThErapies and response to the current and novel chemotherapeutic Biomarkers for ChagaS infEctiOn. interventions proposed in this study. The study design was approved by the National Institutes evaluation by qPCR plus a panel of novel potential host-­ of Health (NIH) and US Food and Drug Administration derived and parasite-­derived BMKs of early assessment of (FDA) without requiring a placebo arm, based on proving therapeutic response to aetiological treatment and even- the non-­inferiority of the novel treatment schemes when tual parasitological cure. compared with the SoC and the use of a historical placebo from a recent study in the same geographical locations and populations in the Plurinational State of Bolivia.32 METHODS AND ANALYSIS Study design Objectives This clinical trial is an open-­ label, blinded allocation, Primary objectives randomised, prospective, phase-­ 2, observational study. A The primary objectives of the TESEO study are to deter- total of 450 patients will be randomly assigned to one of the mine the safety and efficacy of new proposed chemo- six treatment arms (75 in each arm) (table 1). The treat- therapy regimens of BZN or NFX in adults with CCD are ment arms include the SoC of BZN (150 mg two times per comparable or superior to SoC (table 1), evaluating the day for 60 days) and NFX (240 mg two times per day for 60 relevant time response of qPCR and novel BMKs for the days), while the four experimental groups will test a lower assessment of parasitaemia and systemic parasite clear- frequency of dosing, combined with shorter or extended ance. Parasitic loads (by qPCR) and levels of T. cruzi-­ treatment duration (table 1). With the proposed new specific BMKs will be evaluated at EOT and at 4, 6, 12, 18, Figure 1 TESEO clinical trial design. AEs, adverse events; BMKs, biomarkers; BZN, benznidazole; CD, Chagas disease; EOT, end of treatment; NFX, nifurtimox; popPK, population pharmacokinetic; qPCR, quantitative PCR; SAEs, serious AEs; SoC, standard-­of-­care; TESEO, New ThErapies and Biomarkers for ChagaS infEctiOn. Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 3 Open access 24 and 36 months of follow-­up, as compared with histor- have demonstrated the ubiquitous presence of high ical placebo control for BZN in the same population.32 levels of Ch anti-α-Gal Abs in CCD patients from different The EOTs are defined by the duration of the treatment endemic regions in Latin America,43 44 46 60 67–69 75–79 and arm (table 1, figure 1). in a non-­endemic country, Spain.74 80 We have previously shown that tGPI-­mucins81 (also known as TcMUC II, F2, BMKs for early assessment of therapeutic outcomes F2/3, or AT antigens),44 46 60 76 82 in a CL-­ELISA (or AT To address the need of BMKs for early assessment of CL-­ELISA), could be used to evaluate lytic Ch anti-α-Gal therapeutic responses and eventual parasitological cure, Ab titres to confirm the efficacy of BZN treatment in chil- various host-­derived and parasite-­derived BMKs, which dren and adolescents with recent chronic infection, in a have shown promising results in recent preclinical or placebo-­controlled randomised trial in Brazil.46 76 After clinical studies, will be evaluated in the TESEO study. a 3-­year follow-­up, 58% (37/64) of BZN-­ treated chil- The selected BMKs belong to two different classes: host-­ dren were considered cured by intention-­to-­treat (ITT) derived and parasite-­derived BMKs.26 The host-­derived analysis, as measured by negative seroconversion with BMKs include: (1) lytic, protective CD-­specific anti-α-ga- AT CL-­ELISA, although all CS tests remained positive.46 lactopyranosyl antibodies (Ch anti-α-Gal Abs),43–46 as After a 6-­year follow-­up of that trial, 65% and 85% of evaluated by chemiluminescent ELISA (CL-­ ELISA), the patients were considered cured by ITT and by per-­ using as antigens purified glycosylphosphatidylinositol protocol (PP) analysis, respectively, as measured by AT (GPI)-­anchored trypomastigote-­ derived mucins (tGPI-­ CL-­ELISA.76 In fact, these studies were the first to confirm mucins),44 47 or synthetic α-Gal-­containing neoglycopro- the efficacy of BZN chemotherapy for CCD, by using Ch teins (α-Gal-­NGPs)47–50 and (2) antibodies to the specific anti-α-Gal Abs as BMKs of parasitological cure. Based parasite-­ derived recombinant proteins KMP11, HSP70 on those successful studies46 76 and a subsequent clinical and PFR2, and the synthetic peptide 3973, as assessed trial in children in Argentina with similar BZN treatment by ELISA.51–55 The parasite-­ derived BMK involves the outcomes using the T. cruzi recombinant protein F29 detection of trypomastigote excreted/secreted antigens (also known as flagellar calcium-­binding protein, FCaBP) (TESA) by an aptamer-­based assay.56–58 BMKs will be eval- as a BMK for early assessment of therapeutic efficacy,83 uated at baseline, during treatment, at EOT, and through PAHO and WHO recommended BZN for the treatment a 3-­year follow-­up interval at the times indicated above of early CCD in children up to 12 years of age in 1999.81 in Primary Objectives and figure 1. Changes in BMKs’ In 2017, the FDA approved the BZN treatment in the U.S. levels will be correlated with parasitic load clearance, as for children (2–12 years of age), based on those clinical measured by qPCR. studies in Brazil and Argentina.46 76 83 84 In a recent clin- ical study with 56 adult patients with CCD subjected to Rationale for the selection of host-derived and parasite- BZN SoC treatment, we observed a significant decrease derived BMKs (p=0.0052) in the titres of lytic anti-α-Gal Abs using tGPI-­ Host-derived BMKs mucins in the AT CL-­ELISA, starting at 12 months and Lytic anti-α-Gal antibodies to tGPI-mucins and α-Gal-NGPs up to 36 months (EOT).80 Taken together, such findings The major lytic, protective antibodies (Abs) in CD patients strongly justify the use of Ch anti-α-Gal Abs as host BMKs are produced against highly immunogenic, immunodom- for early assessment of chemotherapeutic outcomes in the inant α-Gal epitopes abundantly expressed on O-­glycans of TESEO clinical trial. In this study, we also aim to validate tGPI-­mucins of the infective mammalian host-­dwelling or a series of synthetic α-Gal-­NGPs47–50 to eventually replace tissue-­culture-­derived trypomastigote (TCT) stage,44 59–63 the tGPI-­mucins as antigens in the AT CL-­ELISA. These and on complex phosphoglycans (P-­ glycans) of gp72 synthetic α-Gal-­NGPs will overcome technical difficulties glycoprotein of the infective insect-­ derived metacyclic in purifying and obtaining high quantities of consistently trypomastigote (MT) stage64; such glycans are absent in homogenous parasite-­ derived tGPI-­ mucins, for large-­ humans and nonhuman primates.65 66 Thus, very high scale application in clinical settings.47 levels of trypanolytic anti-α-Gal Abs are found in both acute (IgM) and chronic (IgG) stages of CD,43 45 67–69 Antibodies to T. cruzi-derived recombinant proteins KMP11, Hsp70, which induce complement-­ independent lysis of TCT and PFR2, and peptide 3973 stage,44 59 61 67 or complement-­ dependent lysis of MT Although most conventional serological tests are very stage.45 tGPI-­Mucins (in TCTs) and gp72 glycoprotein (in sensitive for the diagnosis of CD, the evaluation of patients MTs) are the main molecular targets of lytic anti-α-Gal under or following treatment is ambiguous, since some Abs from CCD patients (Ch anti-α-Gal Abs).44 59 60 63 70 anti-T. cruzi antibodies are long-­lasting and a significant Although tGPI-­mucins exhibit intraspecies polymorphism seroconversion occurs only decades following chemo- in their O-­linked glycans, the expression of highly immu- therapy.24 25 85 Remarkably, a significant decrease in the nogenic, nonreducing, terminal α-Gal residues seems reactivity of sera from CCD patients in the indeterminate to be very conserved on tGPI-­mucins from at least four (asymptomatic) phase against T. cruzi-­derived recombi- major T. cruzi discrete typing units (DTUs) or geno- nant proteins KMP11, HSP70, and PFR2, and peptide types71 72 causing infections in humans (TcI, TcII, TcV and 3973 was detected 9 months after BZN treatment in 67%, TcVI).44 60 73 74 Corroborating these results, we and others 50%, 34% and 49% of the patients, respectively.26 52 55 4 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access Moreover, these T. cruzi antigens were recognised with Endpoints (outcome measures) high sensitivity (90%, 70%, 75% and 90%, respectively) Efficacy endpoints and specificity (85%, 85%, 92% and 98%, respectively) The primary efficacy endpoint is sustained parasitolog- by sera from CCD patients,52 86 while they were not ical clearance determined by negative blood qPCR at recognised by sera from healthy donors and patients EOT and along the 3 years of follow-­up. The secondary with heterologous infections. It was also reported that efficacy endpoints are changes in the circulating parasitic the decrease in the reactivity of sera of CCD patients to load during treatment and along the follow-­up interval by a set of 14 recombinant T. cruzi proteins of the parasite, qPCR, as well as CS and NCS responses, and changes in included in a multiplex serologic assay,87 was associated the levels of the BMKs. with a substantial reduction in the parasitic load and with an improvement in the clinical status of treated CCD Safety endpoints patients.88 Recently, it has been reported that patients The safety endpoints will consider the following evalua- with demonstrated treatment efficacy based on anal- tions: incidence, severity, and seriousness of AEs, either ysis of these four serological BMKs as a set, showed an clinical, laboratory or ECG, and incidence of AEs leading enhanced antigen-­specific CD8+ T cell multifunctional to treatment discontinuation. All safety analyses will be responsiveness.54 performed blind to the treatment allocation. The AEs reporting period for the TESEO study begins on adminis- Parasite-derived BMKs tration of the first dose of trial medication for non-­serious Detection of TESA by aptamers events and after the signature of informed consent for T. cruzi is an intracellular parasite and was shown to secrete serious events. The reporting period concludes at the end proteins (ie, TESA) into the host milieu.89 The detection of patient participation in the study. of circulating parasite antigens can demonstrate that live organisms are present in the host even if direct detec- popPK endpoints tion of trypomastigotes in blood is negative.90 TESA BMK The popPK parameters (clearance–CL and volume of distribution–Vd) will be obtained by non-­linear mixed-­ levels were also shown to significantly decrease in CCD effects modelling (NONMEM) (ICON, Cambridge, Massa- patients following BZN SoC treatment.58 It was recently chusetts, USA). Other PK measures, such as AUC, Cmax, reported that an aptamer-­ based, non-­serological, non-­ and Cmin, will also be calculated, as previously described PCR assay could detect TESA BMKs circulating in the for BZN.28 Blood samples will be drawn from patients blood of infected mice.56 57 RNA aptamers selected to receiving aetiological treatment with BZN or NFX at bind with high specificity and affinity to TESA were used predose and postdose during the treatment, and at EOT. in enzyme-­linked aptamer assays to detect TESA BMKs in BZN and NFX popPK data will be correlated with the effi- plasma from infected mice, including chronically infected cacy and safety endpoints, parasitological response; the mice that failed BZN treatment. These treated animals frequency and timing of AEs will be correlated with the still contained significant levels of TESA, as compared serum levels of the drugs. with controls.56 Parasite genotyping Secondary objectives At the end of the 3-­year follow-­up period, T. cruzi geno- The secondary objectives will be the evaluation of the typing and restriction fragment length polymorphism parasitic load reduction during the treatment and 36 (RFLP)-­ PCR fingerprint of mitochondrial kinetoplast months of follow-­up by qPCR and serological response, as DNA (kDNA) products of parasite populations will be measured by CS with commercial ELISA kits (as described determined in the processed blood samples of all patients below), and non-­ CS (NCS) with the BMKs described prior to the study treatment, and in samples from patients above. with positive qPCR result(s) during the 3-­year follow-­up.91 The basic popPK parameters of all BZN and NFX treat- ment arms will be characterised and correlated with para- Patient eligibility and exclusion criteria sitological response as measured by qPCR. Adult patients (18–50 years old), infected with T. cruzi, The incidence of AEs, especially SAEs, AEs of Special as diagnosed by CS (two positive tests with different anti- Interest (AESI) and AEs leading to discontinuation of gens) and a positive qPCR result during the screening treatment are evaluated and correlated with the levels of period, will be eligible. The patient must be in the indeter- the drug in the moment of appearance of the AE(s). minate form (no clinical manifestations) or early cardiac Parasite genotyping will be performed at the end of stage (Kuschnir stage I) of CCD (table 2).92 93 To have a the follow-­up period in all patients prior to treatment homogeneous population without pathologies linked to and in patients with positive qPCR result(s). Such geno- age, that could be confounders, patients older than 50 typing will provide important information concerning years old were excluded from participating in this study. the T. cruzi genotypes72 and strains circulating in the Additionally, the patients must comply with all inclusion study region and their potential correlation with treat- criteria and none of the exclusion criteria, summarised ment response. in box 1. Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 5 Open access Table 2 Chagas disease cardiomyopathy classification for TESEO study, following the Kuschnir criteria* Kuschnir classification Kuschnir stage I Kuschnir stage II Kuschnir stage III Kuschnir stage IV Symptoms Asymptomatic Asymptomatic or mild NYHA I/II NYHA III/IV symptomatology Chest Rx Normal Normal Mild cardiomegaly Moderate or severe cardiomegaly ECG ► Non-­specific findings ► Complete RBBB±upper ► Q waves ► Atrial flutter. ► Incomplete right branch bundle LAH ► Third degree AV ► Atrial fibrillation block ► AV block (first and second block ► Upper left RBBB grade) ► Severe ► Non-­severe bradycardia ► Low-­voltage QRS bradycardia ► Long PR interval, without AV ► Diffuse changes in ST-­T. ► Polymorphic PVC block criteria. ► Monomorphic isolated ► Minor alterations of ST-­T waves ventricular extrasystoles *Modified from Rassi et al.93 AV, atrioventricular; LAH, left bundle branch hemiblock; NYHA, New York Heart Association; PVC, premature ventricular contractions; RBBB, right branch bundle block; TESEO, New ThErapies and Biomarkers for ChagaS infEctiOn. Recruitment, randomisation, treatment and follow-up of at each visit. Pharmacokinetic data with documentation patients of patient exposure will also be available at the end of the Potential participants will be recruited from the trial. Available data on the use of BZN and NFX suggest surrounding communities and the usual patients an overall compliance rate of 75%. If adherence falls visiting the Platform for the Comprehensive Care of below the suggested rate of 75%, which might inhibit the Patients with CD (Chagas Platforms; https://www.​ ability of the study to test its primary hypotheses, the clin- ceadesbolivia.org/plataformaChagas.aspx), Plurina- ical coordinator will suggest a conference call for study tional State of Bolivia, in three study sites: Cochabamba, PIs, co-­investigators (Co-­ Is) and consultants to discuss Tarija and Sucre. Review of the rate of patient accrual methods for improving adherence. and compliance with inclusion/exclusion criteria will Once the patient starts the study treatment, scheduled occur monthly during the recruitment phase to ensure follow-­up visits will occur on day 8 and then, biweekly until that enough participants are being enrolled and that the EOT, according to treatment arm, and up to 4, 6, 12, they meet eligibility criteria and the targeted goals 18, 24, 30 and 36 months after treatment initiation. In the outlined in study protocol. study visits, the patient will undergo a physical exam, ECG The eligible patients will be allocated in equal and pregnancy test (in selected visits); blood samples will proportions to one of the six treatment arms on day be drawn to assess safety and efficacy according to the 1 (table 1 and figure 1). Treatment allocation will be scheme of the clinical study design in figure 1. Inclusion designated by a computer-­generated randomised list and exclusion criteria for the study are given in box 1. produced by an independent statistician. Randomis- If a patient does not return to a scheduled visit, all ation envelopes are provided to the designated study necessary steps must be taken to contact the patient team members to be opened at the time of recruit- and document the patient’s situation. Before declaring ment. Master randomisation list will be kept stored and ‘follow-­up loss’, the medical investigator must do every- sealed by the study’s Steering Committee until the end thing possible to contact him/her, to establish the reason of the study. This study is an open-­label trial, thus, both for the interruption of the treatment, and all contact patients and field study teams will be aware of treat- attempts must be properly documented. To minimise lost ment allocation. However, clinical and safety assess- to follow-­up, during the screening the detailed contact ments and laboratory assessments will be performed information will be recorded, including the patient’s blinded to treatment allocation. mobile and/or landline phone number and address and/ Adherence to treatment will be monitored with the or the contact information of a family member. This infor- Morisky-­Green test94 and pill count, and AEs will be mation will remain in each study site (Chagas Platform). monitored by clinical and laboratory controls (basic If the patient does not appear at the Chagas Platform and haematology and biochemistry) pursuant to the partic- the telephone number is not available, the field team will ular protocols of each of the three study sites. Data on follow up with the patient or their family directly. It will adherence to the treatment protocol will be collected be considered lost to follow-­up of a patient only if he/she weekly by research staff and reviewed quarterly by the does not return to the follow-­up visit of 36 months. three PIs and the study clinical coordinator. Adherence As part of the study, we will evaluate if after the treat- of participants will be evaluated by performing pill counts ment the participant is free of the parasite that causes 6 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access this study. Although not a guarantee, these alternative Box 1 Inclusion and exclusion criteria of TESEO clinical treatments can be successful when the previous treatment trial. has failed, with lower side effects. Inclusion criteria 1. Adults, 18–50 years old. Study organisation 2. Weight: 40–90 kg. The TESEO clinical trial is being conducted in the Pluri- 3. Individuals diagnosed with CD by conventional serology (two posi- national State of Bolivia (for simplicity, henceforth also tive tests with different antigens) and with at least one positive qual- referred as Bolivia), in the Chagas Platforms of Coch- itative PCR assay out of three during the screening. 4. Patients classified as being in the indeterminate form (without abamba, Tarija, and Sucre. The Chagas Platforms are clinical manifestations) or early cardiac form (Kuschnir I) of CCD part of a collaborative project between the Fundación (table 2). Ciencia y Estudios Aplicados para el Desarrollo en Salud 5. Signed informed consent form. y Medio Ambiente (CEADES, Cochabamba, Bolivia) and Exclusion criteria the Barcelona Institute for Global Health (ISGlobal, 1. Clinical signs of dilated cardiomyopathy: Dyspnoea, legs’ oede- Barcelona, Spain).95 Bolivia was chosen as the study site ma, syncope, pulmonary crackles). Patients with an electrocardi- because it has the highest relative percentage of chron- ogram (ECG or EKG) showing the following characteristics: sinus ically infected, asymptomatic individuals of any endemic tachycardia or atrial fibrillation, ventricular, arrhythmias, left atrial enlargement, left bundle-­branch block accompanied by right axis country in the world, representing approximately 10% deviation, and/or patients with Fridericia’s corrected QT interval of the urban population and up to 30%–40% of the >450 ms, a formula for calculating the QT interval on an ECG.118 rural population.96 97 Moreover, the selected sites in 2. History of CD treatment with BZN or NFX or any triazole drug(s) in Bolivia have the necessary expertise in phase-­2 clinical the last 5 years trials,32 33 including high level of care to CD patients 3. Clinical signs and/or symptoms of a digestive form of CCD, which and the required infrastructure and personnel for the is characterised by the presence of two or more of the following TESEO study. The three sites also have facilities to carry criteria:* (A) Excessive exertion in at least 25% of bowel move- out all tests proposed in the study (ECG, biochemistry, ments. (B) Hard stools in at least 25% of stools (Bristol types 1 and 2). (C) Feeling of incomplete evacuation in at least 25% of bowel haematology and qPCR), as well as adequate logistics movements. (D) Feeling of obstruction or anorectal block in at least infrastructure to properly collect and store samples and 25% of bowel movements. (E) Manual manoeuvres to facilitate def- send them to laboratories in the USA and Spain for ecation in at least 25% of bowel movements. (F) Less than three further analyses. complete spontaneous stools per week. *Criteria must be met for The biochemistry, haematology and CS tests are carried at least the last 3 months and symptoms must have been started out at each study site. qPCR analysis is centralised at for at least 6 months before diagnosis. the BioMol Laboratory at CEADES. To that end, blood 4. Hypersensitivity to the active substances (BZN or NFX) or the samples collected in 6 M guanidine hydrochloride, 0.2 M excipient. 5. Previous diagnosis of porphyria. EDTA, pH 8.098 99 are sent from each site to the CEADES 6. Any other acute or chronic health conditions that, in the opinion BioMol Laboratory. At the end of the 3-­year follow-­up, of the study’s principal investigators (PIs), may interfere with the coded and frozen serum samples for analysis of BMKs efficacy and/or safety evaluation of the study drug. will be sent from each site to the CEADES laboratory and 7. Formal contraindication to BZN or NFX. stored at −20°C until shipment to UTEP. UTEP will carry 8. Any concomitant or anticipated use of drugs that are contraindicat- out the analysis of BMKs for lytic anti-α-Gal Abs and geno- ed with the use of BZN or NFX, as defined by the study’s Manual typing of the parasite populations and will forward the of Operations. serum samples to FDA (Silver Spring, Maryland, USA), 9. Individuals are currently known to abuse alcohol and/or drugs. 10. Pregnancy or breastfeeding. and ‘Instituto de Parasitología y Biomedicina López 11. Not using a highly effective contraceptive method (only for women Neyra’ (IPBLN, Granada, Spain), for specific BMK anal- in reproductive age). ysis (TESA detection by aptamers and NCS by ELISA, 12. Laboratory parameters outside the normal range or the acceptable respectively). range for the following parameters: (A) Transaminase levels (ala- nine transaminase, and aspartate transaminase) must be within Laboratory procedures the acceptable margin of 25% above the upper limit of normality. In this study, blood and urine samples are collected from (B) Creatinine levels must be within the acceptable margin of 10% above the upper limit of normality. (C) Total bilirubin must be within patients for the laboratory procedures described below the acceptable margin of 15% above the upper limit of normality. and future BMK studies. The maximal quantity of blood (D) Haemoglobin, platelets and leucocytes must be within the ac- collected per patient during each visit will be 18–25 mL. ceptable margin of ±5% of the normal range. Each urine sample to be collected per visit will be ~10 mL. Blood samples are separately processed for qPCR and CD. In the case of persistence of T. cruzi, an alternative parasite genotyping analysis, biochemical and safety treatment and post-­study follow-­up care will be offered, laboratory parameters, pregnancy test, popPK analysis at no cost to the individual by the regular patient care and conventional and NCS (BMK analysis) (figure 1 and programme of the three Chagas Platforms involved in appendix table 1). Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 7 Open access Conventional serology Box 2 Rules for the permanent interruption of the study For patient eligibility, two different CS tests based on drug different antigens are used. To assess the changes over time by CS, the specific kits Chagatek ELISA (Lemos 1. ALT or AST >8 times the upper limit of normal (ULN) Laboratory, Buenos Aires, Argentina) and Chagatest 2. ALT or AST >5 times the ULN for more than 2 weeks ELISA recombinante 3.0 (Wiener Lab., Rosario, Argen- 3. ALT or AST >3 times the ULN and total bilirubin >2 times the ULN or tina) are used. According to the kit inserts, the speci- the international normalised ratio >1.5 ficity and sensitivity of Chagatest ELISA recombinante 4. ALT or AST >3 times the ULN with the appearance of any one of the 3.0 are 98.3% and 99.3%, respectively; the specificity following symptoms: fatigue, nausea, vomiting, pain or sensitivity in and sensitivity of Chagatek ELISA are >99% and 100%, the upper right quadrant, fever, rash and/or eosinophilia (>500 cells/ mL) respectively. To avoid variability due to the technique, 5. An adverse event or any other condition that, in the opinion of the the serum samples collected at the various time points CoI(s) and/or PI(s), may place the patient at severe risk if he/she described in the protocol are kept frozen at −20°C and continues with the study treatment. will be processed in parallel at the end of the study (3-­year 6. Any condition that the investigators or PIs deem medically nec- follow-­up). essary to interrupt the treatment such as: (A) Significant leucope- nia (<2500 cells/mm3). (B) Severe gastrointestinal symptoms. (C) Safety laboratory parameters Severe allergic dermopathy. (D) Peripheral sensory neuropathy. Safety clinical laboratory parameters include blood cell 7. Pregnancy counts, and liver and kidney function tests. These anal- yses are regularly performed in each laboratory site, in such a way that results will be available daily, facilitating a to the study team and to the patients until the end of the close follow-­up of the patients. patients’ study participation. qPCR assays BZN and NFX serum concentrations For the evaluation of the effect of the different treatment Serum concentration of BZN and NFX will be quanti- protocols on the patients’ circulating parasitic load, a fied by ultrahigh performance high-­performance liquid qPCR method, based on TaqMan technology and vali- chromatography coupled to tandem mass spectrometry dated by an international panel for the standardisation at UTEP, following the previously described methodology and validation of T. cruzi PCR, supported by the PAHO/ for BZN.28 101 The same methodology will be validated for WHO/Special Programme for Research and Training NFX. in Tropical Diseases (PAHO/WHO/TDR), is used.98 100 The qPCR assays are performed at the CEADES BioMol Safety management Central Laboratory, according to the procedures previ- The study physician will evaluate all the AEs and classify ously described.98 99 Briefly, at each PCR time point, them as serious/nonserious and related/non-­related to 5 mL of blood is collected in triplicate in EDTA blood the study drugs. The study treatment can be interrupted sample tubes and mixed with equal parts of 6 M guani- temporarily or permanently according to the study physi- dine hydrochloride, 0.2 M EDTA, pH 8.0, at each Chagas cian evaluation and/or according to the defined protocol Platform. The processed blood samples are sent to the rules (box 2). The interruption of the treatment does CEADES BioMol Lab and stored at room temperature not imply withdrawal from the study. In such cases, the until processing. High-­throughput automated isolation treatment may be temporarily interrupted; therefore, of DNA from the processed blood samples is performed it will be considered incomplete or delayed. Treatment in the KingFisher Duo Prime system (Thermo Fisher can be resumed, following evaluation by the site medical Scientific), using the MagMax DNA Multi-­ Sample investigator and the study PI(s). In case of temporary Ultra 2.0 kit, according to the supplier’s instructions or permanent treatment interruption, patient should (https://assets.thermofisher.com/TFS-Assets/LSG/​ continue with the study visits and evaluations as planned, manuals/MAN0017325_MagMAX_MultiSampleUltra2_​ but the reasons for the treatment interruption should be WholeBlood_UG.pdf, Applied Biosystems, Thermo Fisher recorded in the appropriate original documentation and Scientific). The limit of detection (LOD) for T. cruzi satel- case report form (CRF). lite DNA is 0.69 parasite equivalents/mL, and the limit of The AEs’ reporting period starts upon administration quantification (LOQ) is 1.5 parasite equivalents/mL.98 100 of the first dose of study drug for non-­serious events and The PCR amplification is carried out in triplicate from after the signature of informed consent form (ICF) for each of the extracted samples, using a T. cruzi satellite SAEs and ends at the end of patient participation in the DNA region and the RNAse P as an endogenous ampli- trial. The AEs that are considered related to the study fication control. A PCR time point is considered positive drug or the study procedures, will be followed until if at least one of the 9 PCR amplifications results positive. resolved or considered stable by the study physicians, or if The qPCR analyses will be performed during the study needed, the patient care will be delegated to an adequate follow-­up; nevertheless, the results will not be disclosed health facility. 8 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access The reporting procedures for the AEs vary depending Sample size on the serious/nonserious classification of the event. All The sample size was determined following the gener- SAEs must be reported within 24 hours of awareness of alised estimating equations (GEE)-­ based approach the event by the investigator to the study coordinator and of Pan,102 involving the following considerations: our the two pharmacovigilance (PVG) teams working in this preliminary studies yielded an average clearance rate study: Division of Microbiology and Infectious Disease (0.91+0.81)/2=86% for standard BZN (as opposed to (DMID)-­ CROMS PVG (Safety and Pharmacovigilance (0.255+0.085)/2=17% for the historical placebo control Technical Resources International, Bethesda, Maryland, group),32 corresponding to an odds ratio (OR) of 29.99. USA) and Lat Research (Clinical Research Organisation, We assume an first-­ order autoregressive AR(1) model Buenos Aires, Argentina). If the SAE is considered related correlation structure among repeated measures on each to the study drug and unexpected, it meets the category patient with rho=0.2; a total of four non-­inferiority testing of Suspected Unexpected Serious Adverse Reaction (new BZN/NFX regimens vs BZN/NFX SoC) lead to a (SUSAR). A SUSAR must be informed promptly to the Bonferroni adjusted significance level α=0.05/4=0.0125. FDA, the Agencia Estatal de Medicamentos y Tecnologías According to the formula in Pan,102 a total of 75 patients en Salud (AGEMED), the Data and Safety Monitoring per arm allows us to establish non-­inferiority for all four Board (DSMB) and the Ethics Committees (ECs) of the comparisons with a margin as small as 9.5% in clearance participating institutions, within seven calendar days if the rate difference (corresponding to 1.887 in OR) at the event is fatal or life-­threatening and less than 15 days for joint significance level of α=0.05 with a power of 90%. any other SUSAR. The SAEs that do not meet the SUSAR A total of 450 patients in the trial gives sufficient characteristics, will be reported to the regulatory agencies power to establish the efficacy of all therapies against the in the annual study report, along with other safety data. historical placebo control, based on the results from our The study Steering Committee, composed by the three previous study.32 Additionally, this number of patients also PIs (ICA, FT and JG) and one of the coinvestigators (IR), allows at least a 90% probability of observing at least one can determine whether to terminate the trial at any time event of peripheral neuropathy or paraesthesia, transam- prior to inclusion of the intended number of patients. inase increase and hypersensitivity. However, it intends to exercise this right only for valid scientific or administrative reason(s). In terminating Data management and statistical analyses the trial, the study Steering Committee will assure that Clinical and epidemiological data are collected from adequate consideration is given to the protection of the patients’ visits during the study planned visits and patients’ interests. Reasons for early termination may entered the Data Management Programme. Laboratory data are uploaded to the system from records gener- include but not be limited to: (1) low enrolment rate; (2) ated in the sites’ laboratories. OpenClinica Community high frequency of protocol violations; (3) inaccurate or V.3.14 (OpenClinica, Waltham, Massachusetts, USA, incomplete data; (4) unsafe or unethical practices; (5) www.OpenClinica.com), an open-­source web-­based appli- following the recommendation of the DSMB or the ECs cation designed to support the data capture from study of the participating institutions; (6) administrative deci- research, will be used in the TESEO study. The study PIs sion; (7) insufficient time or resource to conduct the trial and the Biostatistics and Data Management Unit (DMU) and (8) lack of eligible patients. at ISGlobal, Barcelona, Spain, are responsible for: (1) study-­wide data management; (2) metadata creation; (3) Data and safety monitoring board data security and (4) quality assurance of data. A DSMB composed of a minimum of four members All analyses will be performed on the per-­ protocol (including one statistician) independent of the research (PP) and intention-­to-­treat (ITT) strategies. ITT’s anal- team and drug manufacturers, ELEA (BZN) and Bayer ysis will include all randomised patients and PP analysis (NFX), was set up prior to trial initiation (18 December will use an ITT population excluding people with any 2019). The DSMB for this protocol is convened by major protocol deviations such as patients who did not authority of the DMID, National Institute of Allergy receive their assigned treatment, patients who substan- and Infectious Diseases (NIAID), NIH, and is advisory tially violated screening, inclusion, or exclusion criteria to DMID and the study team. The DSMB will monitor (boxes 1 and 2), or patients with a permanently discon- the trial to ensure that harm is minimised, and benefits tinued treatment. Furthermore, the trial has a dataset maximised for the trial patients. The DSMB will review that includes all randomised patients having received at efficacy and safety data on an ongoing basis and at prede- least one dose of study therapy, to perform safety analyses. termined intervals, review all information related to the Data will be described according to their characteris- occurrence of SAEs and AEs leading to treatment discon- tics. The categorical data will be presented as frequen- tinuation, and issue recommendations about the trial if cies, and we will list the number of missing and will be the existing benefit/risk of the patients in the trial seems compared using the χ2 test or Fisher’s Exact test. More- compromised. The first DSMB meeting occurred on 10 over, the continuous data will be described as mean, stan- June 2020, once 20% of recruited patients had completed dard deviation (SD) or median, and interquartile ranges the treatment. (IQRs) in agreement with the data, following or not the Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 9 Open access theoretical normal distribution. In this case, the data will statistics according to the variable. Moreover, the number be compared using analysis of variance or Kruskal-­Wallis of patients with normal and abnormal, and with clinically at the 0.05 two-­ sided significance level. Time-­ to-­ event significant abnormalities will be summarised. data will be presented using Kaplan-­Meier curves with a ECG abnormalities will be described per treatment arm log-­rank test (two-­sided 0.05 significance level). The anal- as the proportion of patients per type of ECG finding and ysis of covariates on time-­to-­event data will be performed changes over time. using a Cox regression mode.103 Longitudinal data will The significance level for any comparison is α=0.05 and be analysed using generalised linear mixed-­effects models the analysis will be carried out using different statis- (GLMM) or GEE, using both Stata (release 16, 2019; tical packages, such us Stata (https://www.stata.com/​ https://www.stata.com/company/, StataCorp) and R company/, StataCorp) and R Studio (https://rstudio.​ Studio (https://rstudio.com/, Boston, Massachusetts, com/). USA). Interim analyses will be performed when all patients For the treatment compliance, a full course of treatment have completed treatment. The interim analysis results is defined by a minimum of 75% of prescribed treatment will not be disclosed, under any circumstances, to the days. The primary efficacy endpoint is a binary (cured or Steering Committee, the PIs and trial personnel at the non-­cured) variable based on a total of eight qPCR time- study sites managing day-­to-­day activities of the trial prior points from EOT until 36 months (figure 1). Each of the to the end of the trial. The analysis will be performed timepoints includes qPCR examinations of three samples, by an independent statistician. A futility stopping rule is by triplicate assays. A patient will be considered ‘cured’ if defined as no difference from the placebo (a historical he/she shows a total of 24 negative qPCR results. placebo,32 as to be used in this study), in sustained pblood The primary non-­inferiority assessment will be done parasitic load clearance at EOT. Regarding efficacy, with with a GLMM or GEE. clearance of blood parasitic load at EOT as the param- To assess the efficacy of each treatment group vs the eter of interest and cut-­off of 60%, the conditional power historical placebo control,32 we will apply Gibbons and (CP) at the time of the interim analysis will be calculated Bock’s method104 to test no trend among these correlated so that, if the probability that CP of rejecting the alterna- proportions, with a mean equal to 0.082 sustained nega- tive hypothesis (active arm superior to placebo) is higher tive clearance at 12 months in historical placebo control. than 60%, the treatment arm should be stopped. Patients To depict the time to reappearance of parasitaemia for will be considered as early treatment failures. This rule is patients who have cleared parasitaemia at EOT, Kaplan-­ rigid and if not followed in the DSMB recommendations, Meier survival curves will be drawn across treatment the type I error will be inflated as a result. For safety, on groups. completion of treatment of 20% of recruited patients, To determine how the different tested BMKs affect a safety interim analysis will be performed. The interim binary outcomes of parasite clearance, GLMM will be analysis results/outcome will not interfere with the trial performed. All the BMKs variables available will be procedures unless they meet the criteria defined for the included in the model from the baseline, during treat- harm and futility stopping rules. ment and during the follow-­up. This modelling analysis The Steering Committee and coinvestigators of each will allow us to examine which BMK variables are signifi- participating institution will have password-­ protected cantly associated with blood parasitic load evolution. This access to temporary and final trial datasets, housed at latter analysis will be presented for only the PP popula- the DMU/ISGlobal. Password-­protected access to tempo- tion, since randomised (ITT) analysis is not considered rary, individual and final datasets will also be provided to to be pertinent for this exploratory analysis. consultants, collaborators, and study investigators after The popPK analysis will be performed by NONMEM prior authorisation from the Steering Committee. To (ICON, Cambridge, Massachusetts, USA) through a three-­step strategy: (1) basic population model selection, safeguard confidentiality, data disseminated to autho- (2) covariate selection, and final population model selec- rised study investigators will be blinded to any identifying tion; and (3) model validation. patient information. For safety analyses, AEs will be classified according to the MedDRA dictionary (V.17.0) and summarised for Resources and data availability each treatment group by seriousness, severity, causality The investigators of this study are aware of and agreed and action taken with the study drug. Besides, the total to abide by the principles for sharing research resources, number of AEs, the number and the proportion of patients as described by NIH in the ‘Principles and Guidelines experiencing at least one AE during the treatment period for Recipients of NIH Research Grants and Contracts will be summarised for each treatment group by body on Obtaining and Disseminating Biomedical Research system and preferred term. In the same way, the propor- Programmes.’ Accordingly, resources developed in this tion of patients in each treatment group withdrawn due study will be available to the scientific community as soon to an AE and/or an SAE will be described. Safety labo- as the intellectual property of these resources and/or ratory parameters (haematology and biochemistry) research tools have been protected or disclosed in publi- and vital signs will be described using the summarised cations. If a specific research tool is requested from the 10 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access TESEO investigators and is available, it will be shared with personnel (coinvestigators, collaborators, consultants, members of the scientific community. staff and students), following the Recommendations Data sharing not applicable as no datasets generated for the Conduct, Reporting, Editing and Publication of and/or analysed for this study. However, once the data- Scholarly Work in Medical Journals from the Interna- sets resulting from this study are available, they will be tional Committee of Medical Journal Editors (http:// disseminated via publications in peer-­reviewed journals, www.icmje.org/icmje-recommendations.pdf). national and international conferences, and reports to the NIH, FDA and participating institutions. Patient and public involvement Patients or the public were not involved in the design, Ethics statement, patient confidentiality, and dissemination or conduct, or reporting, or dissemination plans of our policy research. This study is conducted according to the Declaration of Helsinki-­ Ethical Principles for Medical Research Standard Protocol Items: Recommendations for Interventional Involving Human Subjects, by the World Medical Associa- Trials Reporting guidelines tion. The study was registered in ​ClinicalTrials.​gov on July This manuscript was elaborated following the Standard 11, 2019. The study protocol has been approved by the Protocol Items: Recommendations for Interventional following institutions, Institutional Review Boards (IRBs) Trials reporting guidelines (https://www.spirit-statement.​ or Ethics Committees (ECs): FDA, Investigational New org/).105 Drug (IND), IND-­143169; NIH/NIAID/DMID, protocol number 18-­0015; UTEP, IRB protocol version number 743474-­ 1, originally approved on 20 April 2015, and DISCUSSION amended on 17 June 2021 (version number 743474-­20); It is well established that there is an urgent need to find Bolivian federal regulatory authority, AGEMED, protocol safer and more efficacious drugs or improved therapeutic number MS/AGEMED/AUMyT/AAAyC/CE/41/2019, regimens with the clinically available drugs for the aetio- originally approved on 8 February 2019, and amended logical treatment of CCD. Three key objectives remain: on 30 August 2021 (MS/AGEMED/AUMyT/AAAyC/ confirm the 20% treatment failure of parasitological CE/385/2021); CEADES, protocol number 743474-­ 7, ‘cure’ (sustained blood parasitic load clearance by qPCR) originally approved on 21 December 2019, and amended observed up to a 12-­month follow-­up in previous studies, on 16 August 2021 (version 743474-­20); and Comité Ético evaluate the parasitological efficacy on an extended (36-­ de Investigación Clínica, Hospital Clinic de Barcelona, month) interval, and reduce the frequent AEs associated originally approved on 7 November 2019, and amended with available drugs and current treatment schemes. In on 27 July 2021. The other participating institutions (FDA addition, the study aims to evaluate BMKs for early assess- and IPBLN) will follow the protocol approved UTEP IRB, ment of therapeutic outcomes, which remain as one of through an Inter-­Institutional Agreement (IIA). the main roadblocks in the development of new thera- Documented informed consent will be obtained by peutic options and also an important need for routine the assigned study site investigator only after the poten- management of patients. tial participant has been fully informed about all aspects Posaconazole and the E1224 (a ravuconazole prodrug) of the study: its duration, study treatments, study proce- were the most promising experimental anti-T. cruzi drugs, dures, potential risk and benefices, participant responsi- but they both failed when tested in clinical trials at the bilities and patient confidentiality. The participants will dose and duration of treatment used in these studies.32 42 confirm their voluntary participation in the study after Efforts have been made in different fields, from in vitro he/she has understood the explanation provided by assays (using high throughput screening, HTS) to drug the investigator, and any doubts or questions have been repurposing, but plausibly it still will take considerable satisfactorily answered. A copy of the informed consent time and effort to register a new drug for the aetiological form (ICF) will be provided to the participant. To assure treatment of CD.106–109 the patient confidentiality, the patients will be iden- The fact that CD belongs to the group of NTDs could tified by an identification code on the CRFs and in all explain that the accepted dosing treatments with BZN documents submitted to ethical committees, DSMB and and NFX, which were based on non-­randomised studies manufacturers. carried out in the ‘60s, have not been revised since their All modifications in the study IRB protocol and ICF will introduction. Lately, there is a trend in the study of be reported and approved by the ECs of the participating reduced antiparasitic regimens. An observational non-­ institutions, NIH/NIAID/DMID and FDA. randomised study, using the standard BZN dose (5 mg/ The data generated in this project will be disseminated kg/day) administered for 30 days in adults with CCD, via publications in peer-­reviewed journals, presentations showed that treated patients developed fewer ECG abnor- at national or international conferences, as well as in malities when compared with untreated patients along a reports to the NIH, FDA and participating institutions. median follow-­up of 9.8 years.85 Moreover, in a pilot study Publication authorship will be determined based on using an intermittent BZN scheme of 5 mg/kg/day every the relative scientific contributions of the PIs and key 5 days for a total of 60 days in 20 CCD patients, showed a Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 11 Open access good efficacy profile 1 week after the EOT and in 14 of pathological traits in vitro and in vivo murine models of the 17 patients (82%) at the end of follow-­up (12 to 48 CD.72 In the TESEO study we will determine the geno- months).110 111 However, none of these studies compared type of the parasites infecting all patients before the treat- the effectiveness and safety of the shorter regimen with ment, and in patients that exhibit positive qPCR result(s) the BZN SoC. during the 3-­year follow-­up. The results of genotyping in Following the concept of dose reduction, the BENDITA patients with therapeutic failure will allow us to evaluate trial (​ClinicalTrials.​gov, NCT03378661), was a placebo-­ the impact of polyclonal infections and possible drug controlled study that compared five new dose regimens tolerance on the outcome of aetiological treatments. of BZN alone or combined with E1224 with the BZN When completed, this study could be included in a meta-­ SoC. The BENDITA study results have shown that the analysis, as those currently published.23–25 115 116 dose regimen of 2 weeks had similar efficacy and fewer Since the TESEO trial is being performed in Bolivia, side effects than the 8 weeks of BZN SoC, as assessed by we have been using the CS tests Chagatek ELISA and sustained qPCR negative results at 6-­month and 12 month Wiener Chagatest ELISA recombinante 3.0, which that follow-­up.34 The BETTY (“Short-­ Course Benznidazole have previously shown high sensitivity and specificity for Treatment to Reduce Trypanosoma cruzi Parasitic Load the diagnosis of CD in that population.32 If other popu- in Women of Reproductive Age”) study (​ClinicalTrials.​ lations in North, Central, or South America, or other gov, NCT03672487) will compare the BZN SoC to a short region(s) (eg, Europe) undergo a protocol similar to course of 150 mg/day/30 days, in women of reproductive that of TESEO trial, the aforementioned tests and other age; the efficacy will be assessed by qPCR and endpoint commercial diagnostic tests for CD should be evaluated to PCR tests at the EOT and 10 months of follow-­up, and determine those with highest sensitivity and specificity for the safety will be assessed by the occurrence of SAEs in each specific population. From our experience and from both treatment arms. The EQUITY (“CHICAMOCHA 3 - that of other research groups in South America, Central Equivalence of Usual Interventions for Trypanosomiasis”)​ America and Mexico, there is a considerable variation study(​ ClinicalTrials.​gov, NCT02369978) is a placebo-­ in the diagnostic parameters with different kits, which controlled trial that will evaluate half standard doses of is mainly due to the parasite genotype and strain diver- NFX and BZN for 120 days (240 mg/day for NFX and sity.72 A recent article has described highly discordant 150 mg/day for BZN) in comparison with the SoC of both results when three CS tests (including Wiener Chagatest drugs (480 mg/day/60 days for NFX and 300 mg/day/60 ELISA recombinante V.3.0 and Chagatek ELISA used in days for BZN). The efficacy will be evaluated by PCR at this study) and two in-­house tests were evaluated with 196 12–18 months of follow-­up and by changes in the levels of sera from Veracruz, Mexico.117 That study underscores B-­type natriuretic peptides, a suggested surrogated BMK the need of a careful prescreening of the target popula- for CD cure,27 and also by CS.112 The MULTIBENZ (“Eval- tion and use of a well-­defined panel of positive (ideally, uation of Different Benznidazole Regimens for the Treat- confirmed by PCR) and negative endemic controls from ment of Chronic Chagas Disease”) study (​ClinicalTrials.​ the studied region, prior to the adoption of any CS test as gov, NCT03191162) aims to compare the SoC of BZN reliable diagnostic tool in any clinical trial. with two lower dose regimens: 150 mg/day/60 days and In conclusion, the TESEO study aims to attain a wide 400 mg/day/15 days; the efficacy will be assessed by PCR landscape of results that could provide the basis for a during the 12-­month follow-­up and by CS. new approach in the treatment of CCD. In comparing A key strength of the TESEO study is the long-­term eval- the safety and efficacy of four alternative regimens to the uation of antiparasitic efficacy by qPCR and novel poten- SoC of the two approved drugs for CD, BZN and NFX, tial BMKs. The 3-­year follow-­up will allow us to detect the study turns out to be more comprehensive than past therapeutic failure with six different BZN/NFX schemes or ongoing clinical trials in terms of both its breath and in the medium term, beyond the 12 months used in recent duration of follow-­up, using novel potential BMKs for CD clinical trials.32 33 Moreover, no published popPK for early assessment of therapeutic response and eventual NFX is available and we will use a previously described parasitological cure in direct comparison to qPCR and methodology for BZN28 101 to validate the popPK for CS, as well as the genotypification of the infecting T. cruzi NFX.113 The new data will provide useful information on populations. Moreover, an improved knowledge of the the requirements of follow-­up duration to assess the true efficacy of the currently available anti-T. cruzi drugs will efficacy of chemotherapy in CCD patients. Such efficacy allow us to better assess the potential of combining these will be evaluated using standard methodologies for CS with novel anti-T. cruzi drug candidates. and qPCR, as well as NCS, such as lytic anti-α-Gal anti- bodies,43–48 60 76 antibodies against KMP11, HSP70, PFR2 Trial status and its adaptation to the COVID-19 pandemic and peptide 3973,51–55 and parasite-­derived BMK TESA, situation using an aptamer-­based assay,56–58 as described above. It The recruitment of patients was initiated on 18 December is known that the efficacy of CCD treatment depends, 2019, and the first patient was randomised on 7 January among other factors, on the infecting T. cruzi strain or 2020. However, due to the SARS-­ CoV-­2/COVID-­ 19 genotype.72 114 In fact, T. cruzi genotypes or (DTUs, TcI-­ pandemic, the recruitment was interrupted between VII) exhibit marked differences regarding virulence and March and September 2020, following the quarantine 12 Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 Open access restrictions established by the Bolivian Ministry of Health. lost during the treatment and/or follow-­up as a direct Per the FDA Guidance on Conduct of Clinical Trials of consequence of COVID-­19. As an additional pandemic Medical Products During the COVID-­19 Public Health mitigation measure, the same COVID-­ 19 immunoas- Emergency (https://www.fda.gov/media/136238/down- says were implemented to regularly screen the medical load) and the Ministry of Health of Bolivia, a set of miti- personnel, nurses, and laboratory and administration gation measures were taken to safeguard the safety of staff in the three study sites. the patients and the study personnel. During the strict lockdown, the study team made efforts to assure that all Author affiliations 1 the patients randomised had continued with the study Barcelona Institute for Global Health (ISGLOBAL), Barcelona, Spain 2 Center for Biochemistry and Biophysics, Venezuelan Institute for Scientific treatment and the follow-­up visits; however, the sched- Research (IVIC), Caracas, Distrito Capital, Venezuela, Bolivarian Republic of uled visits had to be adapted according to the regional 3 Biostatistics and Data Management Unit, Barcelona Institute for Global Health, lockdown policies. On 20 April 2020, 20% of recruited Barcelona, Spain 4 patients completed their assigned treatment regimen. Consorcio de Investigación Biomédica en Red de Epidemiología y Salud Pública As stated in the study protocol, a DSMB virtual meeting (CIBERESP), Madrid, Spain 5 Department of Basic Clinical Practice, Universitat de Barcelona, Barcelona, Spain took place to review the study status and the safety 6 CIBER de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain summary report. No safety concerns were identified but 7 Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio considering the current COVID-­19 pandemic, the DSMB Ambiente (CEADES), Cochabamba, Bolivia, Plurinational State of recommended to consider testing the study subjects for 8 Department of Biological Sciences, The University of Texas at El Paso, El Paso, COVID-­19 at screening and during the study follow-­up. Texas, USA 9 After the strict lockdown measures in Bolivia were tempo- Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente (CEADES), Tarija, Bolivia, Plurinational State of rarily suspended, the DSMB recommendation was imple- 10 Universidad Autónoma Juan Misael Saracho, Tarija, Bolivia, Plurinational State of mented and the recruitment was resumed on 18 September 11 Centro Plataforma Chagas Sucre, Fundación Ciencia y Estudios Aplicados para el 2020, with the screening of patients for COVID-­19 before Desarrollo en Salud y Medio Ambiente (CEADES), Sucre, Bolivia, Plurinational State randomisation and during treatment and follow-­up. To of 12 that end, we developed a serological testing algorithm, Programa Departamental de Chagas Chuquisaca, Servicio Departamental de Salud de Chuquisaca, Chuquisaca, Bolivia, Plurinational State of which included initial laboratory screening using lateral 13 Pharmacy Service, Division of Medicines, Hospital Clinic de Barcelona, Barcelona, flow assay (LTA) commercial kits (approved by FDA for Spain emergency use during the pandemic) for anti-­SARS-­CoV-­2 14 Institut de Investigació Biomèdica Agustí Pi i Sunyer (IDIBAPS), Universitat de IgM and IgG antibodies (CTK Biotech, Poway, California, Barcelona, Barcelona, Spain 15 USA). If the patient was positive for LTA-­IgM and/or Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration (FDA), Silver Spring, LTA-­IgG, confirmatory ELISA tests (NovaLisa, NovaTec, Maryland, USA Immunodiagnostic, Dietzenbach, Germany) for both 16 Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de antibody classes were performed. In the screening phase, Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina patients with positive anti-­SARS-­CoV-­2 IgM antibodies by 17 National Scientific and Technical Research Council (CONICET), Buenos Aires, LTA and ELISA were not randomised into the study, even Argentina 18 if they met all inclusion criteria. They were put on hold Consejo Superior de Investigaciones Científicas, Instituto de Parasitología y Biomedicina López-­Neyra, Granada, Spain until they become positive only for anti-­SARS-­CoV-­2 IgG 19 Department of Chemistry and Biochemistry, The University of Texas at El Paso, El antibodies (by ELISA) and showed no COVID-­19 symp- Paso, Texas, USA toms, indicating that they were in the chronic phase of 20 Dynamic Portfolio Unit, Drugs for Neglected Diseases initiative, Geneva, the disease and represented no transmission risk. The Switzerland serological algorithm for COVID-­ 19 is summarised in figure 1 (bottom left). Acknowledgements We are very grateful to Marcelo Abril, Fundación Mundo Sano, Buenos Aires, Argentina, and Dr. Sergio Sosa-­Estani, DNDi, Rio de Janeiro, The study sites were successfully able to meet the Brazil, for their continuous support during the elaboration and implementation recruitment goal of 450 participants with the final rando- of this trial; Dr. Martin Springsklee (Medical Affairs Anti-­Infectives), Dr. Ulrich-­ misation occurring on 20 May 2021. On 18 August 2021, Dietmar Madeja (Head, Neglected Tropical Disease Programmes), and Dr. Maria-­ the treatment phase was concluded in 86% (n=387) of the Luisa Rodriguez (Global Project Leader) at Bayer AG, Berlin, Germany, and this company for the kind donation of the nifurtimox to be used in this study; Dr. Pedro randomised patients. Of the remaining 14% (n=63), 13% Albajar Viñas, WHO, for the support to the study through the kind advancement (n=58) had to interrupt the treatment during early stages of nifurtimox from the WHO stockpile; Ernesto Palma (Business Development and due to AEs. One per cent (n=5) withdrew the informed External Markets Manager) and Luis Ferrero (former ELEA’s Especial Business consent. Another DSMB Review Meeting was held virtu- Manager), at Laboratorio ELEA Phoenix S.A., Buenos Aires, Argentina, and this ally on 22 September 2021, after the study participants company for the generous donation of the benznidazole to be used in the TESEO study. We also thank Dr. Soyoung Jeon (currently at the New Mexico State had finalised their treatment phase. No significant safety University) and Dr. Xiaogang Su, Dept. of Mathematical Sciences, Border Biomedical concerns were identified. The DSMB requested follow-­up Research Center (BBRC), University of Texas at El Paso, for the statistical analyses information regarding participants who were COVID-­19 performed during the TESEO project evaluation by NIH. We are very thankful to seropositive for the next DSMB meeting, expected to take all the medical, supporting (nurses, social workers, and laboratory staff) and administrative personnel of the three Chagas Platforms in Bolivia for their technical place in September 2022. assistance and dedication in the recruitment, treatment, and follow-­up of the CCD Despite the considerable negative impact of the patients in this study. We would also like to thank all the staff (postdoctoral fellows, pandemic in the recruitment, thus far no patient has been technicians, and administrative personnel) and graduate and undergraduate Alonso-­Vega C, et al. BMJ Open 2021;11:e052897. doi:10.1136/bmjopen-2021-052897 13 Open access students of the participating institutions involved in this clinical trial and part of the 11 Machado FS, Dutra WO, Esper L, et al. Current understanding TESEO Study Group. of immunity to Trypanosoma cruzi infection and pathogenesis of Chagas disease. Semin Immunopathol 2012;34:753–70. Contributors JG, FT and ICA conceived the TESEO Study. CA-­V, JAU, SS, M-­JP, 12 Urbina JA, Docampo R. Specific chemotherapy of Chagas disease: JJP, VRG, GR, LO, WG, DL, DS, RAM, RN, AD, AS, MCT, MCL, KM, IR, JG, FT and ICA controversies and advances. Trends Parasitol 2003;19:495–501. substantially contributed to the design of the study reported in this protocol. SS 13 Urbina JA. Recent clinical trials for the etiological treatment of designed the statistical analyses of the study. CA-­V, JAU, SS, AD, MCT, MCL and ICA chronic Chagas disease: advances, challenges and perspectives. J drafted the manuscript. All authors have reviewed and approved the current version Eukaryot Microbiol 2015;62:149–56. of the manuscript for publication. 14 Tarleton RL. Parasite persistence in the aetiology of Chagas disease. Int J Parasitol 2001;31:550–4. Funding The source of financial support for research described in this article is 15 Aldasoro E, Posada E, Requena-­Méndez A, et al. What to expect the National Institutes of Health (NIH)/National Institute of Allergy and Infectious and when: benznidazole toxicity in chronic Chagas' disease Diseases (NIAID) grant entitled 'New Chemotherapy Regimens and Biomarkers treatment. J Antimicrob Chemother 2018;73:1060–7. for Chagas Disease' (U01AI129783, to ICA, FT and JG). NIH/NIAID/DMID provided 16 Jackson Y, Alirol E, Getaz L, et al. Tolerance and safety of nifurtimox in patients with chronic Chagas disease. Clin Infect Dis guidance in the design of this study. 2010;51:e69–75. Disclaimer The opinions expressed in this article do not necessarily reflect those 17 Pérez-­Molina JA, Sojo-­Dorado J, Norman F, et al. Nifurtimox of the NIH. This article reflects the views of the authors and should not be construed therapy for Chagas disease does not cause hypersensitivity to represent FDA’s views or policies. reactions in patients with such previous adverse reactions during benznidazole treatment. Acta Trop 2013;127:101–4. Competing interests The TESEO study principal investigators declare no financial 18 Pinazo M-­J, Muñoz J, Posada E, et al. Tolerance of benznidazole and competing interests for the overall trial and each study site. in treatment of Chagas' disease in adults. Antimicrob Agents Chemother 2010;54:4896–9. Patient consent for publication Not applicable. 19 Regueiro A, García-Álvarez A, Sitges M, et al. Myocardial Provenance and peer review Not commissioned; externally peer reviewed. involvement in Chagas disease: insights from cardiac magnetic resonance. Int J Cardiol 2013;165:107–12. Supplemental material This content has been supplied by the author(s). It has 20 Viotti R, Vigliano C, Lococo B, et al. Side effects of benznidazole as not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been treatment in chronic Chagas disease: fears and realities. Expert Rev peer-­reviewed. Any opinions or recommendations discussed are solely those Anti Infect Ther 2009;7:157–63. of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and 21 Sulleiro E, Muñoz-­Calderon AQrturo, Schijman AG. Role of responsibility arising from any reliance placed on the content. Where the content nucleic acid amplification assays in monitoring treatment includes any translated material, BMJ does not warrant the accuracy and reliability response in Chagas disease: usefulness in clinical trials. Acta Trop 2019;199:105120. of the translations (including but not limited to local regulations, clinical guidelines, 22 PAHO. Guidelines for the diagnosis and treatment of Chagas terminology, drug names and drug dosages), and is not responsible for any error disease. secondary guidelines for the diagnosis and treatment of and/or omissions arising from translation and adaptation or otherwise. Chagas disease, 2019. Available: https://www.who.int/publications/​ Open access This is an open access article distributed in accordance with the i/item/9789275120439 23 Ciapponi A, Barreira F, Perelli L, et al. Fixed vs adjusted-­dose Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which benznidazole for adults with chronic Chagas disease without permits others to distribute, remix, adapt, build upon this work non-­commercially, cardiomyopathy: a systematic review and meta-­analysis. PLoS Negl and license their derivative works on different terms, provided the original work is Trop Dis 2020;14:e0008529. properly cited, appropriate credit is given, any changes made indicated, and the use 24 Sguassero Y, Roberts KN, Harvey GB, et al. Course of serological is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. tests in treated subjects with chronic Trypanosoma cruzi infection: A systematic review and meta-­analysis of individual participant data. ORCID iDs Int J Infect Dis 2018;73:93–101. Julio A Urbina http://orcid.org/0000-0003-0736-0378 25 Fabbro DL, Streiger ML, Arias ED, et al. 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