Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis

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With its epidemiological complexity, by difficulty to obtain appropriate diagnoses and poor treatment, the search for novel therapeutic options remains. Methods In this context, we conducted a systematic review and meta-analysis of preclinical studies employing animal models to verify the progress in CD treatment. We searched the PubMed database for CD treatment studies published between 1990 and 2023, adhering to the PRISMA guidelines. Results Twelve papers met the inclusion criteria. The findings indicate that the fifteen treatment alternatives examined, mainly between 2010 and 2014, demonstrated efficacy in experimental CD models, evidenced by significant parasitemia reduction. Bis-triazole DO870 and VNI were effective in the acute and chronic phases, respectively. However, of these emerging therapies, only posaconazole and fexinidazole have progressed to clinical trials, yielding unsatisfactory outcomes as CD monotherapies Conclusions This meta-analysis highlights the existence of promising new drug candidates for CD treatment, but most remain in the preclinical stages. Those that reached clinical trials did not demonstrate optimal results, underscoring the ongoing challenges in CD therapy. Collaborative efforts among the academic community, pharmaceutical industries, funding agencies, and government agencies are urgently needed to accelerate the development of more effective medications against CD. Inplasy registration INPLASY202430101 (25/03/2024) " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/13-885", "name": "Treatment options applied to the preclinical studies using animal..." } } ] } Home Browse Treatment options applied to the preclinical studies using animal... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Machaca-Luque LY, Candia-Puma MA, Roque-Pumahuanca BM et al. Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.12688/f1000research.150723.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Systematic Review Revised Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] Laura Yesenia Machaca-Luque 1,2 , Mayron Antonio Candia-Puma https://orcid.org/0000-0002-6328-3840 1,2 , Brychs Milagros Roque-Pumahuanca 1,2 , [...] Haruna Luz Barazorda-Ccahuana https://orcid.org/0000-0001-8791-0506 2 , Luis Daniel Goyzueta-Mamani https://orcid.org/0000-0003-0308-1160 2 , Alexsandro Sobreira Galdino 3 , Ricardo Andrez Machado-de-Ávila 4 , Rodolfo Cordeiro Cordeiro Giunchetti 5,6 , Eduardo Antonio Ferraz Coelho 7 , Miguel Angel Chavez-Fumagalli https://orcid.org/0000-0002-8394-4802 2 Laura Yesenia Machaca-Luque 1,2 , Mayron Antonio Candia-Puma https://orcid.org/0000-0002-6328-3840 1,2 , [...] Brychs Milagros Roque-Pumahuanca 1,2 , Haruna Luz Barazorda-Ccahuana https://orcid.org/0000-0001-8791-0506 2 , Luis Daniel Goyzueta-Mamani https://orcid.org/0000-0003-0308-1160 2 , Alexsandro Sobreira Galdino 3 , Ricardo Andrez Machado-de-Ávila 4 , Rodolfo Cordeiro Cordeiro Giunchetti 5,6 , Eduardo Antonio Ferraz Coelho 7 , Miguel Angel Chavez-Fumagalli https://orcid.org/0000-0002-8394-4802 2 PUBLISHED 16 May 2025 Author details Author details 1 Facultad de Ciencias Farmacéuticas, Bioquímicas y Biotecnológicas, Universidad Católica de Santa María, Arequipa, 04000, Peru 2 Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, Arequipa, 04000, Peru 3 Laboratório de Biotecnologia de Microrganismos, , MG e Instituto Nacional de Ciência e Tecnologia em Biotecnologia Industrial, Universidade Federal de Sao Joao del-Rei, Divinópolis, State of Minas Gerais, 35501-296, Brazil 4 Programa de Pós-Graduação em Ciências da Saúde, University of the Extreme South of Santa Catarina, Criciúma, State of Santa Catarina, 88806-000, Brazil 5 INCT-DT, Instituto Nacional de Ciencia e Tecnologia em Doencas Tropicais, Salvador, State of Bahia, 40015-970, Brazil 6 Laboratório de Biologia das Interações Celulares, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, 31270-901, Brazil 7 Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina,, Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, 31270-901, Brazil Laura Yesenia Machaca-Luque Roles: Data Curation Mayron Antonio Candia-Puma Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology Brychs Milagros Roque-Pumahuanca Roles: Data Curation Haruna Luz Barazorda-Ccahuana Roles: Investigation, Writing – Review & Editing Luis Daniel Goyzueta-Mamani Roles: Data Curation, Investigation, Writing – Review & Editing Alexsandro Sobreira Galdino Roles: Investigation, Writing – Review & Editing Ricardo Andrez Machado-de-Ávila Roles: Investigation, Writing – Review & Editing Rodolfo Cordeiro Cordeiro Giunchetti Roles: Investigation, Writing – Review & Editing Eduardo Antonio Ferraz Coelho Roles: Investigation, Writing – Review & Editing Miguel Angel Chavez-Fumagalli Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Neglected Tropical Diseases collection. Abstract Background Chagas disease (CD) is a neglected tropical disease endemic to Latin America, has emerged as a global health concern due to the migration of infected individuals. With its epidemiological complexity, by difficulty to obtain appropriate diagnoses and poor treatment, the search for novel therapeutic options remains. Methods In this context, we conducted a systematic review and meta-analysis of preclinical studies employing animal models to verify the progress in CD treatment. We searched the PubMed database for CD treatment studies published between 1990 and 2023, adhering to the PRISMA guidelines. Results Twelve papers met the inclusion criteria. The findings indicate that the fifteen treatment alternatives examined, mainly between 2010 and 2014, demonstrated efficacy in experimental CD models, evidenced by significant parasitemia reduction. Bis-triazole DO870 and VNI were effective in the acute and chronic phases, respectively. However, of these emerging therapies, only posaconazole and fexinidazole have progressed to clinical trials, yielding unsatisfactory outcomes as CD monotherapies Conclusions This meta-analysis highlights the existence of promising new drug candidates for CD treatment, but most remain in the preclinical stages. Those that reached clinical trials did not demonstrate optimal results, underscoring the ongoing challenges in CD therapy. Collaborative efforts among the academic community, pharmaceutical industries, funding agencies, and government agencies are urgently needed to accelerate the development of more effective medications against CD. Inplasy registration INPLASY202430101 (25/03/2024) READ ALL READ LESS Keywords Chagas disease, treatment, efficacy, systematic review, meta-analysis, parasitemia, preclinical studies Corresponding Author(s) Miguel Angel Chavez-Fumagalli ( [email protected] ) Close Corresponding author: Miguel Angel Chavez-Fumagalli Competing interests: No competing interests were disclosed. Grant information: This research was funded by Universidad Católica de Santa María (grants 27574-R-2020, and 28048-R-2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Machaca-Luque LY et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Machaca-Luque LY, Candia-Puma MA, Roque-Pumahuanca BM et al. Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.12688/f1000research.150723.3 ) First published: 05 Aug 2024, 13 :885 ( https://doi.org/10.12688/f1000research.150723.1 ) Latest published: 16 May 2025, 13 :885 ( https://doi.org/10.12688/f1000research.150723.3 ) Revised Amendments from Version 2 Several modifications have been made to the discussion section of the manuscript to address key concerns and improve clarity. Specifically, we have expanded the interpretation of drug efficacy in the chronic phase of Chagas disease, emphasizing the limitations of relying solely on peripheral parasitaemia due to its intermittent nature and low sensitivity in this stage. The mechanisms of action of benznidazole and nifurtimox have been clarified with additional details on their interaction with parasitic nitroreductase, while also incorporating a discussion on metabolically quiescent parasite forms and their potential role in treatment failure. Furthermore, we have strengthened the analysis of methodological heterogeneities and reporting inconsistencies across studies, underscoring the need for standardized approaches in future preclinical research. Several modifications have been made to the discussion section of the manuscript to address key concerns and improve clarity. Specifically, we have expanded the interpretation of drug efficacy in the chronic phase of Chagas disease, emphasizing the limitations of relying solely on peripheral parasitaemia due to its intermittent nature and low sensitivity in this stage. The mechanisms of action of benznidazole and nifurtimox have been clarified with additional details on their interaction with parasitic nitroreductase, while also incorporating a discussion on metabolically quiescent parasite forms and their potential role in treatment failure. Furthermore, we have strengthened the analysis of methodological heterogeneities and reporting inconsistencies across studies, underscoring the need for standardized approaches in future preclinical research. See the authors' detailed response to the review by Andrea Angheben See the authors' detailed response to the review by Juan Carlos Gabaldon READ REVIEWER RESPONSES Introduction Chagas Disease, caused by the protozoan parasite Trypanosoma cruzi , is a neglected tropical illness endemic to Latin America, predominantly impacting rural and impoverished communities. 1 Worldwide, the disease affects an estimated 6 to 7 million people, with 10,000 deaths each year. 2 Additionally, there are emerging reports indicating the spread of Chagas Disease to non-endemic countries, fueled by factors such as human migration, travel patterns, globalization, and climate change, thereby introducing new complexities to disease management and surveillance efforts. 3 The disease presents two distinct clinic phases: acute and chronic. Acute CD is often characterized by nonspecific symptoms such as fever, malaise, and local edema at the site of parasite entry, 4 while chronic infection potentially lead to serious cardiac and gastrointestinal problems such as megaesophagus, megacolon, arrhythmias, and cardiomyopathy. All of these complications further increase the risk of sickness and death. 5 The pathogenesis of Chagas disease is a complex process influenced by various factors, including transmission dynamics and environmental conditions. Effective disease control necessitates addressing factors such as poor sanitation, shared housing, and “epidemiological blind spots” that hinder surveillance and treatment strategies. 6 , 7 The current therapeutic options for CD mostly consist of two nitroheterocyclic compounds, such as benznidazole and nifurtimox, developed in the late 1960s and early 1970s, respectively. 8 , 9 While these treatments have been widely used for decades, they are associated with significant side effects and require prolonged treatment regimens, which can affect patient adherence. 10 Although traditionally considered to have limited efficacy in chronic CD, recent evidence suggests that treatment may still provide clinical benefits, particularly in slowing disease progression. 11 Traditionally, it was thought that CD only became clinically evident after prolonged exposure to the parasite. However, it is now understood that clinical symptoms can be triggered by various factors, including host immune responses and parasite load, not merely by continued exposure. 12 Given this evolving perspective, the treatment of infected patients is now widely recommended unless specific contraindications exist. However, existing therapies still fail to achieve parasitological clearance in a substantial number of patients, and since sterilizing cure remains the primary therapeutic goal, the development of new drugs continues to be a priority. 13 , 14 The challenges in drug development include the complex biology of T. cruzi and host immune responses, as well as the lack of financial incentives for pharmaceutical companies to invest in treatments for a disease primarily affecting impoverished populations. 15 , 16 Recently, fundamental research, particularly in the preclinical stage, has been the focal point of efforts aimed at developing new treatment strategies for CD. These endeavors predominantly utilize techniques derived from in vitro or animal investigations. 9 , 17 Notably, animal model-based preclinical research has emerged as indispensable for evaluating novel CD treatment approaches. 18 Such studies facilitate the exploration of fundamental mechanisms of action and pharmacokinetic characteristics, while also providing valuable insights into the safety and efficacy of innovative therapies. 19 An additional challenge in evaluating treatment outcomes in clinical trials is the absence of a reliable cure biomarker, making it difficult to assess treatment efficacy accurately. This limitation affects the design and interpretation of clinical studies. 20 This systematic review and meta-analysis aim to evaluate the efficacy of different treatment options for Chagas disease in preclinical studies using animal models. The study compares the effectiveness of various anti-parasitic compounds in reducing parasitemia and improving disease outcomes, providing a quantitative assessment to identify promising candidates for future research. Given the heterogeneity in therapeutic approaches, we aim to answer the following research question: What is the efficacy of different treatment options in reducing parasitemia in preclinical models of Chagas disease, and how do they compare in terms of therapeutic outcomes? Methods Study protocol This systematic review conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Table S1). 21 With registration number INPLASY202430101 and DOI: 10.37766/inplasy2024.3.0101 , the protocol for this systematic review was registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY) website. The entire protocol is accessible at inplasy.com ( https://inplasy.com/inplasy-2024-3-0101/ ). Information sources and search strategy The literature was searched for phrases about CD therapy using the MeSH (Medical Subject Headings) term “Chagas Disease”. Using the VOSviewer program (version 1.6.20), the findings were shown in a network diagram showing the co-occurrence of MeSH keywords. 22 We looked at clusters in the network map to choose phrases associated with CD therapy. Furthermore, a second round of searches was carried out by linking each MeSH term identified in the cluster analysis with the MeSH terms “Chagas Disease” and “Treatment Outcome”, which relate to assessing the outcomes of interventions used to combat diseases and determining their efficacy, 23 For the years 1990–2023, records were obtained from the bibliographic database PubMed ( https://pubmed.ncbi.nlm.nih.gov/ , last accessed 24 May 2023). Selection criteria and data extraction The procedure for selecting studies for this review comprised three separate phases. During the initial identification phase, only animal studies published between 1990 and 2023 were taken into account. Duplicate articles, non-English publications, reviews, and meta-analyses were excluded at this stage. The subsequent screening phase involved checking the titles and abstracts of the identified articles, and in the eligibility/qualification phase, full-text studies highly relevant to the research question were retrieved, specifically focusing on treatment options for CD. Data on the type of compound used for treatment, dosage, duration of treatment, total sample size, number and species of experimental animals infected with T. cruzi , phase of CD, T. cruzi strain, sample type, and description of the controls were extracted from each of the chosen studies. Studies with insufficient data were excluded. In contrast, those that assessed the effectiveness of therapy by disclosing parasitemia data were retained, considering a 70% decrease in parasitemia as a positive treatment for both the treated and control groups. Options for treatment that included a drug in addition to a conventional CD medication, like nifurtimox or benznidazole, weren’t included. Drugs used in monotherapy were emphasized to confirm which ones show higher effectiveness. WebPlotDigitizer was used for data analysis when the information was shown graphically. 24 , 25 WebPlotDigitizer version 5 is a semi-automatic tool that lets you manually plot two-dimensional charts and extract numerical data. It is free online ( https://automeris.io/WebPlotDigitizer/ ) or as desktop software that may be downloaded. 24 This instrument has been employed in several systematic reviews and meta-analyses, encompassing therapy efficacy assessment. 26 – 28 L.Y.M.-L. carried out the data extraction, and M.A.C.-P. Independently checked it. If there were any differences, M.A.C.-F. was consulted and discussed. Quality assessment and risk of bias The methodological quality and risk of bias of the included animal studies were assessed using SYRCLE’s risk of bias tool, 29 which evaluates key domains such as selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases specific to animal studies. Selection bias examines random allocation, performance bias ensures uniform treatment, detection bias checks for blinding, attrition bias looks at dropout management, and reporting bias ensures complete reporting of outcomes. Additional biases specific to animal studies are also considered. To evaluate the overall quality of evidence, the GRADE approach 30 was applied, which assesses certainty based on risk of bias, inconsistency, indirectness, imprecision, and publication bias, categorizing evidence into four levels: high, moderate, low, or very low certainty. Statistical analysis Results were entered into a Microsoft Excel (version 2108, Microsoft Corporation, Redmond, WA, USA) spreadsheet and analyzed in the R programming environment (version 4.2.3) using the “metafor” package https://www.metafor-project.org/doku.php/metafor (accessed on 21 February 2024). 31 Plotting the synthesis findings and estimating a random-effects model are just a few of the numerous tasks that may be performed by the user with the help of the “metafor” package. 31 , 32 The number of treated animals who tested positive or negative for CD (tpos and tneg, respectively), and the corresponding number of untreated animals (control) who tested positive or negative for CD (cpos and cneg, respectively), were evaluated independently for each therapeutic option available with the “metafor” package”. A Random Effects Model (RE model) was utilized in the meta-analysis process. According to this model, every study has a unique true effect that varies depending on the differences in animals, interventions, or circumstances between studies. 33 The Q value (Q), the I-squared (I 2 ), the tau-squared (T 2 ), and the risk ratio (RR) were also computed. One indicator of heterogeneity among the studies in the meta-analysis is the Q. The sum of the squares representing the discrepancies between the weighted overall effect and the observed effects are used to compute it. A high Q score indicates higher levels of study heterogeneity. 34 The overall percentage of variance in the estimated effects that results from heterogeneity between studies as opposed to random variation is measured by the I 2 statistic. It is represented as a percentage and computed as (Q - df ) /Q. 35 Excessive I 2 values (above 50%) suggest significant variability among the studies. 36 In a random effects model, the variation between studies is represented by the T 2 . A random-effects model was chosen over a fixed-effects model for several reasons. First, this model accounts for variability in true effects across studies, which is particularly relevant in preclinical research involving different animal models, experimental conditions, and treatment protocols. Even if a low I 2 suggests minimal observed heterogeneity, unmeasured differences in study design, strain susceptibility, infection models, or pharmacokinetic responses may still exist. 37 Second, the T 2 statistic estimates the variance of true effect sizes beyond sampling error, allowing for uncertainty in underlying biological mechanisms affecting treatment efficacy. 33 Finally, since preclinical studies often involve small sample sizes and diverse methodologies, a more conservative approach was adopted with the random-effects model, which provides more generalizable estimates by assuming a distribution of possible effects rather than a single fixed effect size. 38 As the RR of the event happening in the exposed or treated group compared to the unexposed or untreated group, the RR measures the connection between an exposure or treatment and an outcome of interest. 39 Compared to the reference group, an exposure or treatment is linked to a higher outcome risk if the relative risk (RR) is more significant than 1. Conversely, a lower risk is suggested when the RR is less than 1. If the relative risk (RR) is 1, then there is no variation in risk among the groups. Confidence intervals are a useful tool for assessing the accuracy of the estimate, in addition to the RR value. 40 In this study, the event considered for RR calculation is the negativization of parasitemia (TC-) at the end of the study. Therefore, an RR > 1 indicates a higher probability of parasite clearance in the treated group compared to the control group, reflecting greater treatment efficacy in reducing parasite burden. For all computations, a 99% confidence level was used, with a 0.1 continuity adjustment applied as necessary. Results Data sources and study selection In this work, we performed a systematic review and meta-analysis to evaluate the effectiveness of many treatment options for lowering parasitemia. We considered both established therapies and recently developed, potentially effective medicines for CD. Figure S1 shows a flowchart of the study approach. A search for the MeSH term “Chagas Disease” AND “Treatment Outcome” was performed in the PubMed database and a MeSH term co-occurrence network map was developed. Through the search, 323 scientific publications were published between 1990 and 2023. A network map with 1,020 keywords was produced, with the minimal number of keyword occurrences set at five ( Figure 1 ). Three major clusters were found to have formed during the network map study. Terms including “nitroimidazoles”, “nifurtimox”, “benznidazole”, and “antiprotozoal agents” were found in the cluster about the therapy of CD (highlighted in green). There were other similar denominators, including phrases like “treatment outcome,” “humans,” “Chagas disease,” “female,” “male,” and “ Trypanosoma cruzi ” ( Figure 1 ). Figure 1. A network diagram using VOSviewer and PubMed represented CD treatment results based on MeSH term. The terms found in the first analysis were used to perform a second search in the PubMed database. After the new phrases were associated with “Chagas Disease” and “Treatment Outcome”, the following new search strings were generated: (Chagas Disease [MeSH Terms]) AND (Treatment Outcome [MeSH Terms]) AND (nifurtimox [MeSH Terms]), (Chagas Disease [MeSH Terms]) AND (Treatment Outcome [MeSH Terms]) AND (nitroimidazoles [MeSH Terms]), and (Chagas Disease [MeSH Terms]) AND (Treatment Outcome [MeSH Terms]) AND (Therapeutics [MeSH Terms]) for commonly used treatments (nifurtimox and benznidazole) as well as recently developed treatments against CD. There were 40, 107, and 112 studies chosen for the first, second, and third search strings, respectively. We excluded 91, 90, and 66 articles in the identification, screening, and eligibility phases, respectively, based on the three-step selection criteria that we employed. This resulted in the selection of 12 studies for the meta-analysis, one published in 2001, and the others between 2010 and 2014, several of which discussed various treatment choices; as a result, 30 publications in total were included in the investigation. In these investigations, the following novel chemical compounds were examined: Posaconazole, AmBisome ® , Cyclopalladated complex 7a, Fexinidazole, Psilostachyin A, Cynaropicrin, Reversible cruzipain inhibitors Cz007 and Cz008, dehydroepiandrosterone-sulfate, VNI, (−)−hinokinin-loaded microparticles, allopurinol, clomipramine, GW788388, and Bis-triazole D0870 ( Table 1 ). The most significant number of studies utilizing animal models to investigate potential novel treatments for CD have been conducted in Brazil. In addition, albeit to a lesser degree, studies on this subject have also been done in several other nations, including Argentina, Canada, France, and the United States ( Figure 2 ). Table 1. Chemical compounds evaluated in preclinical studies using animal models of CD. Reference Molecule Chemical Structure Route, dose (mg/kg/day) 41 , 42 Posaconazole p.o., 20 41 AmBisome ® i.p., 25 45 Cyclopalladated complex 7a i.p., 0.12 46 Fexinidazole p.o., 300 47 Psilostachyin A i.p., 50 47 Cynaropicrin i.p., 50 44 Reversible cruzipain inhibitor Cz007 p.o., 50 44 Reversible cruzipain inhibitor Cz008 p.o., 50 48 Dehydroepiandrosterone-sulfate s.c., 40 49 VNI p.o., 50 50 (-)-Hinokinin s.c., 20 51 Allopurinol p.o., 15 51 Clomipramine p.o., 5 52 GW788388 p.o., 3 43 Bis-Triazole DO870 p.o., 5 i.v., 3 Figure 2. A demographic breakdown of international research on novel CD therapy options included in the meta-analysis. Risk of bias and certainty of evidence The risk of bias assessment using SYRCLE’s tool revealed a high overall risk across the included studies, with prevalent concerns regarding selection, performance, and detection biases. Issues such as random housing, allocation concealment, and incomplete reporting contributed to the high bias risk, primarily due to inadequate randomization, blinding procedures, and selective outcome reporting. These biases were common across the animal models used, raising concerns about the reliability of the findings. Despite some studies showing fewer issues, the overall risk of bias remained high, potentially affecting the interpretation of results (Table S2). The GRADE evaluation showed varying levels of evidence certainty. Several studies were rated with moderate certainty, suggesting promising efficacy of drug combinations and treatments in animal models of CD, but clinical validation in humans is necessary to confirm these findings. Some studies were rated with moderate-low certainty due to the lack of human validation, despite demonstrating superior effectiveness in preclinical settings. A few studies showed low certainty, especially when treatments exhibited limited or no efficacy. High certainty was attributed to studies demonstrating high efficacy in mice, suggesting potential for human application, though further validation is needed. Overall, the evidence was mixed, with promising results but significant gaps in clinical validation (Table S3). Meta-analysis of the treatment options for CD CD phase The acute phase of the disease and its treatment options posaconazole, AmBisome ® , Cyclopalladated complex 7a, Fexinidazole, Psilostachyin A, Cynaropicrin, Reversible cruzipain inhibitors Cz007 and Cz008, dehydroepiandrosterone-sulfate, VNI, (−)−hinokinin-loaded microparticles, allopurinol, clomipramine, GW788388, and Bis-triazole D0870 were also covered in twelve studies 41 – 52 that included a total of 741 animals. As Figure 3 shows, the treatment alternatives, particularly Bis-triazole D0870, proved to be effective compared to the control groups. (RR = 8.22, 95% CI [4.80, 14.06]). The tests showed that there was minimal heterogeneity (Q (df = 24) = 14.31, p = 0.94; I 2 = 0.0%; T 2 = 0.0%)). Two studies 41 , 49 with a total of 138 animals were examined that dealt with the chronic phase of the disease and its treatment choices (VNI, Benznidazole, Nifurtimox, Posaconazole AmBisome ® ). The treatment alternatives were effective compared to the control groups, especially VNI, as Figure 4 illustrates (RR = 11.29, 95% CI [3.32, 38.36]). There was minimal heterogeneity, according to the tests (Q (df = 4) = 0.33, p = 0.99; I 2 = 0.0%; T 2 = 0.0%). Figure 3. Forest plot comparing efficacy between treated and control groups according to CD phase. Error bars represent 95% CI. The square shapes represent the estimated RR. The vertical dashed line represents the no-effect line. Figure 4. Forest plot comparing efficacy between treated and control groups according to animal strain. Symbol meaning: * Acute phase, ** Chronic phase. Error bars represent 95% CI. The square shapes represent the estimated RR. The vertical dashed line represents the no-effect line. Experimental animal models - strains The scientific articles were categorized into smaller groups based on the type of animal model that received a T. cruzi inoculation. Only one study 48 employed Wistar strain rats. Other mice strains were employed in the other studies: Swiss, 41 , 42 , 44 , 46 , 47 BALB/c, 47 , 50 , 51 CD-1, 43 , 52 and BALB/cJ. 41 , 45 , 49 As can be observed in Figure 5 , CD−1 was the strain that responded to the treatment the best out of all the investigated strains. Its RR = 11.41, 95% CI [2.05, 63.36], indicates this. The tests showed that there was very little heterogeneity (Q (df = 2) = 1.94, p = 0.38; I2 = 0.0%; T2 = 0.0%)). On the other hand, the strain with the highest number of trials was the Swiss strain, which as shown in Figure 5 with an RR = 8.02, 95% CI [3.49, 18.46]) also showed a substantial response to the various treatment methods (Q (df = 12) = 8.21, p = 0.77; I 2 = 0.0%; T 2 = 0.0%) showed low heterogenicity. With an RR = 6.58, 95% CI [2.32, 18.69], and negligible heterogenicity (Q (df = 7) = 2.74, p = 0.91; I 2 = 0.0%; T 2 = 0.0%), BALB/cJ was the strain that responded to the treatment alternatives the least ( Figure 4 ). Figure 5. Forest plot comparing efficacy between treated and control groups by sex of animal models. Symbol meaning: * Acute phase, ** Chronic phase. Error bars represent 95% CI. The square shapes represent the estimated RR. The vertical dashed line represents the no-effect line. Experimental animal models - sex The sex of the animal model was used to categorize the scientific studies. RR = 9.36, 95% CI [4.83, 18.12] was the result of 7 studies including female experimental animals 41 – 43 , 45 , 46 , 49 , 50 ; Q (df = 18) = 7.91, p = 0.98; I 2 = 0.0%; T 2 = 0.0%, indicated a low level of heterogenicity ( Figure 5 ). The studies with the male sex of the experimental animals were 5, 44 , 47 , 48 , 51 , 52 showed a RR = 6.38, 95% CI [2.54, 16.04], with heterogenicity data of Q (df = 10) = 6.62, p = 0.76; I 2 = 0.0%; T 2 = 0.0% ( Figure 5 ). While analyzing the results, it is clear that female experimental animals are more susceptible than male counterparts to the various CD therapy approaches. Other research on CD treatments In the final analysis of the scientific publications, 53 one article about immunotherapy using DNA vaccines in mice was discovered. Immunotherapy boosts the body’s immune response, whereas conventional therapies utilize medications to treat a disease’s symptoms or underlying causes. 54 This study was left out of the meta-analysis because of their disparate biological objectives, which make them hard to evaluate in terms of efficacy and safety. Discussion Summary of main findings Most of the studies included in the meta-analysis were conducted between 2010 and 2014. Progress in CD research has been slow due to multiple interrelated challenges. Scientifically, the biological complexity of T. cruzi —including its genetic diversity and ability to evade the host immune system—has hindered the identification of effective therapeutic targets. 55 – 57 Additionally, limited funding for CD-specific research has constrained large-scale clinical trials and resource availability, delaying therapeutic innovation. 58 – 60 The pharmaceutical industry’s lack of interest, driven by the low profitability of treatments for diseases prevalent in low-income regions, has further slowed progress. 61 , 62 The rise of drug-resistant strains highlights the need for new therapies, yet regulatory barriers for neglected diseases prolong development timelines. 57 , 63 – 65 Another possible reason for the decline in new studies after this period is the shift toward computational drug discovery, particularly in silico models, which offer cost-efficient strategies for identifying drug targets. 66 However, translating these computational advances into practical treatments for CD remains challenging. 67 Together, these factors create a complex and difficult environment for developing novel CD therapies. A persistent lack of funding dedicated to CD research and treatment development is a significant obstacle to therapeutic progress. 58 – 60 The lack of financial resources limits researchers’ capacity to conduct robust clinical trials and acquire essential resources. Compounding this issue, the pharmaceutical industry shows limited interest due to the low commercial potential of medications targeting diseases prevalent in low-income regions. 61 , 62 Furthermore, the emergence of drug-resistant strains emphasizes the urgency of finding alternative treatments, yet regulatory hurdles associated with approving novel therapeutics for neglected diseases further contribute to delays. 57 , 63 – 65 Collectively, these factors create a complex and discouraging landscape that severely hinders the development of new CD treatments. In Brazil, researchers conducted three-quarters of the studies that comprise this meta-analysis. This country is a natural hub for study in this field since it is one of the nations most impacted by CD, has a robust research infrastructure, and an epidemiological database. 68 , 69 Research in the nation has also been enhanced by the presence of academic institutions, centers of excellence in tropical health, and financial and research resources. 70 On the other hand, even though other Latin American nations are also impacted by CD, scientific research and the health system face structural and financial obstacles that may hinder study. 71 Regarding North American countries like the United States of America and Canada, involvement in CD research may depend on the financing available for global health research initiatives and the interests of certain academics or groups. 72 , 73 The lack of urgency in researching and developing therapies may be attributed to the disease’s low incidence in this area. 73 , 74 Only one study from France was considered in the meta-analysis. However, it should be considered that, although non-endemic countries do not directly suffer from CD in their populations, they should consider globalization and human mobility, since these factors increase the possibility of transfer. of diseases to non-endemic countries. 75 – 77 Beyond the moral need to alleviate animals’ suffering, the worldwide scope of CD necessitates a cooperative, international effort to solve the problems this neglected tropical disease presents. The systematic review and meta-analysis of preclinical studies on CD treatment highlights the critical need for rigor in experimental designs, emphasizing the high risk of bias observed across the included studies. The assessment using SYRCLE’s tool revealed prevalent issues with selection, performance, and detection biases, notably due to inadequate randomization, blinding procedures, and incomplete reporting, which undermines the reliability of findings. 78 , 79 Despite these concerns, the GRADE evaluation presented promising efficacy of treatments, with moderate certainty assigned to several studies. These findings suggest that, while preclinical models indicate potential treatment effectiveness, clinical validation in humans remains essential for confirmation. 80 A few studies showed higher certainty due to demonstrated efficacy in animal models, particularly in mice, underscoring the importance of further validation. 17 , 81 This review is essential as it identifies both the potential and limitations of current treatment strategies, emphasizing the urgent need for more rigorous and clinically relevant preclinical research to bridge the gap between animal models and human applications in CD therapy. The results of this study, bis-triazole DO870 and VNI have demonstrated promise as treatments for CD in the acute and chronic stages, respectively. The assessment of these substances’ safety and effectiveness in actual clinical settings is unknown, as no published clinical trials have been performed too far. Similarly, only two of the treatment options included in this meta-analysis—posaconazole and fexinidazole—have undergone clinical trial evaluation. Posaconazole’s clinical research shows that, although exhibiting trypanostatic action during therapy, it was ineffective in treating asymptomatic T. cruzi carriers in the long term. By contrast, it was demonstrated that benznidazole monotherapy outperformed posaconazole, with high RT-PCR conversion rates lasting up to a year. However, in 32% of instances, posaconazole side effects resulted in medication termination. 82 The various regimens had an acceptable safety profile in the clinical research evaluating fexinidazole; nonetheless, they were ineffective in treating T. cruzi infection. This has led to discontinuing fexinidazole monotherapy development as a treatment for T. cruzi infection. 83 These results highlight the necessity of investigating alternative therapeutic approaches to treat CD as well as the significance of conducting thorough assessments of the safety and effectiveness of medicines in clinical trials. Both benznidazole and nifurtimox have been demonstrated to be more successful in lowering parasitemia during the acute phase of CD. These drugs work by preventing the parasite from synthesizing its DNA, which prevents it from multiplying and spreading throughout the host. 84 This effect is mediated by their activation via parasitic nitroreductases (e.g., TcNTR), which reduce their nitro groups into cytotoxic metabolites. These reactive intermediates form adducts with parasite DNA, proteins, and lipids, disrupting essential metabolic pathways. 85 Furthermore, it has been noted that these medications have the ability to cause oxidative damage in the parasite, through the generation of superoxide radicals and peroxynitrite during redox cycling of their nitro groups, which helps to eradicate it. 86 , 87 Nevertheless, in the chronic stage of CD, only a limited effectiveness of current treatments has been observed. This could be attributed to multiple factors, including the persistence of metabolically quiescent parasite forms in certain tissues, their limited accessibility to drugs, the host’s compromised immune response, and disease-induced tissue damage. 88 , 89 Consequently, overcoming these challenges, particularly the role of dormant parasite populations in therapeutic failure, has become a critical focus in preclinical research for chronic CD, a phase poorly managed by conventional medications. As was previously indicated, VNI has demonstrated encouraging outcomes for CD’s chronic phase. This compound inhibits the Trypanosomatidae enzyme CYP51, 90 which prevents the parasites from synthesizing vital sterols and ultimately compromises the integrity of their cell membranes, killing them. 91 It also lessens cardiac fibrosis and inflammation, which may indicate a possibility for stopping or healing heart damage brought on by CD. 49 With a wide range of activity against different strains of Trypanosoma infections, including resistant ones, and the potential for improved pharmacokinetics and reduced side effects, 92 – 94 this class of inhibitor may offer safer and more efficient alternatives for treating CD during its chronic stage. Within this species, the strains of Trypanosoma cruzi exhibit unique genetic variations. Regarding their genome, virulence, medication resistance, and capacity to elude the host immune system, each of these strains is distinct. 95 , 96 These variations may have an impact on how CD progresses and how affected individuals respond to treatment. 97 The main strains of Trypanosoma cruzi studied in the meta-analysis were Tulahuen and Y, for the chronic and acute phases, respectively. The Y strain, recognized for its high virulence, is preferably employed in models attempting to reproduce the acute phase of the disease, allowing the evaluation of therapy efficacy in decreasing parasitemia and initial infection symptoms. 98 On the other hand, research examining the long-term pathogenesis and progression of the disease, as well as the evaluation of therapeutic interventions targeted at lowering parasitic burden and preventing or reversing associated organic damage, utilize the Tulahuen strain, which is recognized for its adaptability and ability to induce a stable chronic infection. 99 , 100 Conversely, concerning the animal models utilized to assess novel medications against CD, we may bring up the instance of dogs, who serve as significant parasite reservoirs. 101 A canine model that accurately mimics human illness has been developed, which makes it easier to assess novel medicinal agents. Routine effectiveness trials are hampered by the high cost and extended lifespan of dogs. 102 , 103 Non-human primates have also been considered possible models, although their usage is restricted due to expense, lack of validation, and ethical concerns. 104 , 105 On the other hand, murine models remain the most popular because of their affordability, portability, and capacity to replicate several facets of human illness. 17 , 106 As demonstrated by the fact that all of the preclinical investigations in this review were carried out in mouse models, they are therefore invaluable resources for researching T. cruzi infection and assessing novel antiparasitic medications. Limitations and strengths The influence on the validity, generalization, and robustness of the results is the intrinsic limitation of the smaller number of scientific publications, 107 as is the case in this meta-analysis. A limited data set could not include all available evidence on the topic in question, requiring a cautious interpretation of the results. 108 Therefore, it is necessary to support additional research on new treatment options for CD to overcome this deficiency and increase the available scientific evidence. The current meta-analysis enables a thorough assessment of therapy alternatives in a standardized and controlled setting by concentrating on preclinical studies utilizing animal models, laying a strong platform for further research and therapeutic development. Additionally, complex statistical methods, such as those included in the “metafor” package, enable in-depth data analysis, making it easier to control study heterogeneity and accurately estimate treatment effects. 109 This helps identify patterns and trends in the effectiveness of treatments, which in turn helps direct decision-making and design of future clinical trials. Furthermore, it is important to acknowledge that a key limitation of this meta-analysis is its dependence on peripheral parasitemia as the primary efficacy measure for chronic Chagas disease, which may not adequately reflect the disease's complex pathophysiology characterized by tissue-specific parasite persistence and progressive organ damage. 110 While this metric provides standardization across preclinical studies, 111 its utility is constrained by chronic phase characteristics including low/intermittent parasitemia and poor correlation with tissue parasite burden. 112 The SYRCLE tool's identification of reporting inconsistencies for tissue-based endpoints further compounds this limitation. Although compounds like VNI show promising anti-parasitic and immunomodulatory activity, their tissue-level efficacy remains uncertain without standardized assessment of extravascular reservoirs. 82 Future preclinical research should incorporate multimodal endpoints including quantitative PCR of tissue parasitic load, histopathological analysis, and functional cardiac assessments to better predict clinical outcomes, while maintaining parasitemia as a practical initial screening parameter. Another critical limitation of this meta-analysis is the high risk of bias identified across the included studies, which stems from variability in experimental designs, reporting inconsistencies, and non-standardized conditions typical of preclinical research. The overall certainty of the evidence is further compromised by these methodological heterogeneities, limiting the generalizability of the findings. 113 While preclinical studies provide valuable initial insights, their translational relevance is constrained by the lack of uniform protocols and the inherent differences between animal models and human pathophysiology. 114 These challenges underscore the need for cautious interpretation of the results and highlight the importance of standardizing future preclinical research to enhance the reliability and applicability of the data. Implications for future research To identify potential compounds and therapeutic targets, extensive research, including drug discovery approaches, is necessary. 115 Similar to the studies included in this meta-analysis, potential therapeutic options should be evaluated in preclinical models, such as cell cultures and animal disease models, to determine their efficacy and safety. 116 Subsequently, substances that have potential in preclinical research may proceed to clinical trials. There are several important reasons why many therapeutic approaches that show promise in preclinical research are never tested in clinical trials. Initially, moving from preclinical research to clinical trials is an expensive and complex procedure that requires meticulous preparation and substantial monetary means. 117 Clinical studies to evaluate potential drugs can often be hampered by a lack of money or investment from the pharmaceutical industry or funding bodies. 118 , 119 Before starting clinical trials, regulatory and safety issues also need to be resolved. Failure to do so could cause further delays or obstacles in transitioning a promising therapy from preclinical to clinical development. 120 Additionally, the availability of resources and motivation to conduct clinical trials may be impacted by the lack of interest or priority of the scientific community or the pharmaceutical industry regarding a particular condition, such as CD. 121 , 122 Conclusions This meta-analysis has demonstrated that novel therapeutic options exist that are successful in treating CD; however, most of them are still in the preclinical development stage, and those that have advanced to the clinical trial stage have not demonstrated the best outcomes, meaning that CD treatment remains unresolved. With the primary goal of helping the CD population, the academic community and pharmaceutical companies must collaborate in creating new drugs, as well as continue and apply research that has produced promising results that could hasten the discovery and availability of more effective treatments. Ethics and consent Ethical approval and consent were not required. Author contributions Conceptualization: M.A.C.-P. and M.A.C.-F.; data curation: M.A.C.-P., L.Y.M.-L., B.M.R.-P., and L.D.G.M.; formal analysis: M.A.C.-P. and M.A.C.-F.; funding acquisition: M.A.C.-P., E.A.F.C., and M.A.C.-F.; investigation: L.D.G.M., H.L.B.C., A.S.G., R.A.M.D., R.C.G., and E.A.F.C.; methodology: M.A.C.-P. and M.A.C.-F.; writing—review and editing: L.D.G.M., H.L.B.C., A.S.G., R.A.M.D., R.C.G., and E.A.F.C. All authors have read and agreed to the published version of the manuscript. Data availability No data are associated with this article. Underlying data Extended data Figshare: Supplementary figure of results from systematic reviews and meta-analyses ( https://doi.org/10.6084/m9.figshare.26240111 ) 123 and Supplementary tables of results from systematic reviews and meta-analyses ( https://doi.org/10.6084/m9.figshare.28635869 ). 124 This project contains the following underlying data: • Figure S1. A flowchart representing the study selection process’s meta-analysis and systematic review. • Table S1. PRISMA checklist. • Table S2. SYRCLE’s risk of bias tool. • Table S3. GRADE approach. • Table S4. Data results of systematic review of CD treatment possibilities applied to preclinical research with animal model. • Table S5. Data results of meta-analysis of CD treatment possibilities applied to preclinical research with animal model. • Table S6. Reasons for exclusion of full-text reviewed articles. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgments The authors would thanks to CNPq, CAPES (code 001), UFSJ, UFMG, UESC. ASG, RAMA, RCG and EAFC thanks to CNPQ for their PQ/DT Fellowship. The authors also thank to the National Institute of Science and Technology in Industrial Biotechnology (INCT-BI), Brazil. References 1. Bonney KM: Chagas disease in the 21st Century: a public health success or an emerging threat? Parasite. 2014 Mar 10; 21 (11): 10–11. PubMed Abstract | Publisher Full Text | Free Full Text 2. Word Health Organization: Chagas’ Disease (American Trypanosomiasis) Fact Sheet.2023 [cited 2024 Feb 23]. Reference Source 3. Mackey TK, Liang BA, Cuomo R, et al. : Emerging and Reemerging Neglected Tropical Diseases: a Review of Key Characteristics, Risk Factors, and the Policy and Innovation Environment. Clin. Microbiol. Rev. 2014 Oct; 27 (4): 949–979. PubMed Abstract | Publisher Full Text | Free Full Text 4. 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Publisher Full Text Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 05 Aug 2024 ADD YOUR COMMENT Comment Author details Author details 1 Facultad de Ciencias Farmacéuticas, Bioquímicas y Biotecnológicas, Universidad Católica de Santa María, Arequipa, 04000, Peru 2 Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, Arequipa, 04000, Peru 3 Laboratório de Biotecnologia de Microrganismos, , MG e Instituto Nacional de Ciência e Tecnologia em Biotecnologia Industrial, Universidade Federal de Sao Joao del-Rei, Divinópolis, State of Minas Gerais, 35501-296, Brazil 4 Programa de Pós-Graduação em Ciências da Saúde, University of the Extreme South of Santa Catarina, Criciúma, State of Santa Catarina, 88806-000, Brazil 5 INCT-DT, Instituto Nacional de Ciencia e Tecnologia em Doencas Tropicais, Salvador, State of Bahia, 40015-970, Brazil 6 Laboratório de Biologia das Interações Celulares, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, 31270-901, Brazil 7 Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina,, Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, 31270-901, Brazil Laura Yesenia Machaca-Luque Roles: Data Curation Mayron Antonio Candia-Puma Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology Brychs Milagros Roque-Pumahuanca Roles: Data Curation Haruna Luz Barazorda-Ccahuana Roles: Investigation, Writing – Review & Editing Luis Daniel Goyzueta-Mamani Roles: Data Curation, Investigation, Writing – Review & Editing Alexsandro Sobreira Galdino Roles: Investigation, Writing – Review & Editing Ricardo Andrez Machado-de-Ávila Roles: Investigation, Writing – Review & Editing Rodolfo Cordeiro Cordeiro Giunchetti Roles: Investigation, Writing – Review & Editing Eduardo Antonio Ferraz Coelho Roles: Investigation, Writing – Review & Editing Miguel Angel Chavez-Fumagalli Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This research was funded by Universidad Católica de Santa María (grants 27574-R-2020, and 28048-R-2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 16 May 2025, 13:885 https://doi.org/10.12688/f1000research.150723.3 version 2 Revised Published: 15 Apr 2025, 13:885 https://doi.org/10.12688/f1000research.150723.2 version 1 Published: 05 Aug 2024, 13:885 https://doi.org/10.12688/f1000research.150723.1 Copyright © 2025 Machaca-Luque LY et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Machaca-Luque LY, Candia-Puma MA, Roque-Pumahuanca BM et al. Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.12688/f1000research.150723.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 3 VERSION 3 PUBLISHED 16 May 2025 Revised Views 0 Cite How to cite this report: Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.182003.r385243 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v3#referee-response-385243 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 28 May 2025 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain Approved VIEWS 0 https://doi.org/10.5256/f1000research.182003.r385243 Authors have adequately ... Continue reading READ ALL Authors have adequately addressed my major comments. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.182003.r385243 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v3#referee-response-385243 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 15 Apr 2025 Revised Views 0 Cite How to cite this report: Angheben A. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378071 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378071 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 May 2025 Andrea Angheben , IRCCS Sacro Cuore Hospital, Negrar, Verona, Italy Approved VIEWS 0 https://doi.org/10.5256/f1000research.179980.r378071 I've read author's answers to my review and I recognize that the paper and author's work has been planned with a good methodology but the limits I've highlighted still remain. Although I am not fully convinced that as it stands ... Continue reading READ ALL I've read author's answers to my review and I recognize that the paper and author's work has been planned with a good methodology but the limits I've highlighted still remain. Although I am not fully convinced that as it stands the paper is well fruible particularly by clinicians and I believe that methods could be improved (particularly with extension of database queries), authors do declare clearly their approach and the paper can be indexed to my opinion. Competing Interests: No competing interests were disclosed. Reviewer Expertise: My area of expertise covers epidemiological and clinical aspects of Chagas disease and other NTDs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Angheben A. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378071 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378071 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378072 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378072 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 26 Apr 2025 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.179980.r378072 Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, ... Continue reading READ ALL Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378072 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378072 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 24 Jun 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 24 Jun 2025 Author Response Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why ... Continue reading Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. Answer: Thank you for your positive feedback. We are glad that the revisions have significantly improved the manuscript, particularly from a methodological standpoint. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Answer: We sincerely appreciate your insightful comment regarding the interpretation of results during the chronic stage of infection. As you rightly pointed out, peripheral parasitaemia may not fully capture drug effectiveness in this phase due to its low and intermittent nature. While our meta-analysis focused on peripheral parasitaemia as a standardized outcome across studies, we acknowledge this limitation and have now explicitly discussed its implications in the limitations section, emphasizing that conclusions about efficacy in the chronic stage should be interpreted with caution and may require complementary outcomes in future research. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. Answer: We sincerely appreciate your comments, which have greatly improved our manuscript. In response to your observations, we have simplified the language in the first paragraph of the discussion to enhance clarity. Additionally, we have expanded the explanation of BNZ and NFX's mechanism of action, specifically detailing their effects on parasite nitroreductase. We have also discussed the potential role of metabolically quiescent parasite forms in therapeutic failure during the chronic stage and their implications for pre-clinical study design. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Answer: We sincerely appreciate your comments regarding the limitations of our meta-analysis. In response to your observation, we have thoroughly addressed the high risk of bias by incorporating a detailed discussion on methodological heterogeneity, variability in experimental designs, and reporting inconsistencies across the included studies. We have also emphasized the need for cautious interpretation of the results due to the translational constraints of preclinical research, as well as the importance of standardizing future studies to improve reliability and applicability. Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. Answer: Thank you for your positive feedback. We are glad that the revisions have significantly improved the manuscript, particularly from a methodological standpoint. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Answer: We sincerely appreciate your insightful comment regarding the interpretation of results during the chronic stage of infection. As you rightly pointed out, peripheral parasitaemia may not fully capture drug effectiveness in this phase due to its low and intermittent nature. While our meta-analysis focused on peripheral parasitaemia as a standardized outcome across studies, we acknowledge this limitation and have now explicitly discussed its implications in the limitations section, emphasizing that conclusions about efficacy in the chronic stage should be interpreted with caution and may require complementary outcomes in future research. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. Answer: We sincerely appreciate your comments, which have greatly improved our manuscript. In response to your observations, we have simplified the language in the first paragraph of the discussion to enhance clarity. Additionally, we have expanded the explanation of BNZ and NFX's mechanism of action, specifically detailing their effects on parasite nitroreductase. We have also discussed the potential role of metabolically quiescent parasite forms in therapeutic failure during the chronic stage and their implications for pre-clinical study design. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Answer: We sincerely appreciate your comments regarding the limitations of our meta-analysis. In response to your observation, we have thoroughly addressed the high risk of bias by incorporating a detailed discussion on methodological heterogeneity, variability in experimental designs, and reporting inconsistencies across the included studies. We have also emphasized the need for cautious interpretation of the results due to the translational constraints of preclinical research, as well as the importance of standardizing future studies to improve reliability and applicability. Competing Interests: No competing interests Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 24 Jun 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 24 Jun 2025 Author Response Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why ... Continue reading Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. Answer: Thank you for your positive feedback. We are glad that the revisions have significantly improved the manuscript, particularly from a methodological standpoint. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Answer: We sincerely appreciate your insightful comment regarding the interpretation of results during the chronic stage of infection. As you rightly pointed out, peripheral parasitaemia may not fully capture drug effectiveness in this phase due to its low and intermittent nature. While our meta-analysis focused on peripheral parasitaemia as a standardized outcome across studies, we acknowledge this limitation and have now explicitly discussed its implications in the limitations section, emphasizing that conclusions about efficacy in the chronic stage should be interpreted with caution and may require complementary outcomes in future research. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. Answer: We sincerely appreciate your comments, which have greatly improved our manuscript. In response to your observations, we have simplified the language in the first paragraph of the discussion to enhance clarity. Additionally, we have expanded the explanation of BNZ and NFX's mechanism of action, specifically detailing their effects on parasite nitroreductase. We have also discussed the potential role of metabolically quiescent parasite forms in therapeutic failure during the chronic stage and their implications for pre-clinical study design. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Answer: We sincerely appreciate your comments regarding the limitations of our meta-analysis. In response to your observation, we have thoroughly addressed the high risk of bias by incorporating a detailed discussion on methodological heterogeneity, variability in experimental designs, and reporting inconsistencies across the included studies. We have also emphasized the need for cautious interpretation of the results due to the translational constraints of preclinical research, as well as the importance of standardizing future studies to improve reliability and applicability. Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. Answer: Thank you for your positive feedback. We are glad that the revisions have significantly improved the manuscript, particularly from a methodological standpoint. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Answer: We sincerely appreciate your insightful comment regarding the interpretation of results during the chronic stage of infection. As you rightly pointed out, peripheral parasitaemia may not fully capture drug effectiveness in this phase due to its low and intermittent nature. While our meta-analysis focused on peripheral parasitaemia as a standardized outcome across studies, we acknowledge this limitation and have now explicitly discussed its implications in the limitations section, emphasizing that conclusions about efficacy in the chronic stage should be interpreted with caution and may require complementary outcomes in future research. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. Answer: We sincerely appreciate your comments, which have greatly improved our manuscript. In response to your observations, we have simplified the language in the first paragraph of the discussion to enhance clarity. Additionally, we have expanded the explanation of BNZ and NFX's mechanism of action, specifically detailing their effects on parasite nitroreductase. We have also discussed the potential role of metabolically quiescent parasite forms in therapeutic failure during the chronic stage and their implications for pre-clinical study design. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Answer: We sincerely appreciate your comments regarding the limitations of our meta-analysis. In response to your observation, we have thoroughly addressed the high risk of bias by incorporating a detailed discussion on methodological heterogeneity, variability in experimental designs, and reporting inconsistencies across the included studies. We have also emphasized the need for cautious interpretation of the results due to the translational constraints of preclinical research, as well as the importance of standardizing future studies to improve reliability and applicability. Competing Interests: No competing interests Close Report a concern COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 05 Aug 2024 Views 0 Cite How to cite this report: Angheben A. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355965 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355965 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Feb 2025 Andrea Angheben , IRCCS Sacro Cuore Hospital, Negrar, Verona, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.165317.r355965 The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, ... Continue reading READ ALL The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Another general comments refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)?. Probably authors should comment also taking into account this aspect in the discussion. Finally some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Yes Is the statistical analysis and its interpretation appropriate? Partly Are the conclusions drawn adequately supported by the results presented in the review? Yes If this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable Competing Interests: No competing interests were disclosed. Reviewer Expertise: My area of expertise covers epidemiological and clinical aspects of Chagas disease and other NTDs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Angheben A. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355965 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355965 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 15 Apr 2025 Author Response The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the ... Continue reading The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). Answer: Thank you for your valuable and insightful comments. Regarding the exclusive use of PubMed, we selected this database due to its comprehensive and rigorous coverage of peer-reviewed biomedical literature, which aligns with the focus of our review. While we acknowledge that other databases like Lilacs and Embase could provide additional studies, PubMed was sufficient to gather the most relevant and high-quality research for our systematic analysis. In response to your concern about the inclusion of studies involving drug combinations with nifurtimox and benznidazole, it is important to highlight that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapies in preclinical models of Chagas disease. While clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), the results of monotherapy are essential to establish a solid comparative baseline and avoid biases from uncontrolled pharmacological interactions. Furthermore, combination therapies present variability in dosage, duration, and potential synergy, which complicates the standardization of results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more accurate evaluation of the individual efficacy of each compound, which is crucial for future therapeutic strategies and the development of new molecules. However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Answer: Thank you for your valuable feedback. We appreciate your suggestion regarding the use of diamonds to represent the pooled effect in the graphical representation of the results. However, we chose not to include diamonds in this case due to the specific approach used in our meta-analysis, which was performed using the R programming environment (version 4.2.3) with the "metafor" package. In this package, the pooled effect is typically represented by point estimates and confidence intervals, rather than diamonds. While we understand that diamonds could offer a quick visual summary for clinicians, we prioritized a more precise representation of the data with confidence intervals, which we believe provides a clearer understanding of the variability in the effect sizes. Another general comment refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)? Probably authors should comment also taking into account this aspect in the discussion. Answer: We appreciate your comment and thank you for suggesting the inclusion of this aspect. We have added this point to the discussion section. Finally, some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover, in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact, they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Answer: Thank you for your valuable comments. We have revised the manuscript accordingly: the phrase "only becoming aware whenever patients are infected even further" has been clarified to better reflect the current understanding that CD typically becomes clinically evident in the later stages of infection. Additionally, we have addressed the issue of "therapeutic failure associated with existing therapies" by including the important point regarding the lack of a cure biomarker, which limits the ability to measure strong outcomes in clinical trials. The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). Answer: Thank you for your valuable and insightful comments. Regarding the exclusive use of PubMed, we selected this database due to its comprehensive and rigorous coverage of peer-reviewed biomedical literature, which aligns with the focus of our review. While we acknowledge that other databases like Lilacs and Embase could provide additional studies, PubMed was sufficient to gather the most relevant and high-quality research for our systematic analysis. In response to your concern about the inclusion of studies involving drug combinations with nifurtimox and benznidazole, it is important to highlight that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapies in preclinical models of Chagas disease. While clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), the results of monotherapy are essential to establish a solid comparative baseline and avoid biases from uncontrolled pharmacological interactions. Furthermore, combination therapies present variability in dosage, duration, and potential synergy, which complicates the standardization of results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more accurate evaluation of the individual efficacy of each compound, which is crucial for future therapeutic strategies and the development of new molecules. However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Answer: Thank you for your valuable feedback. We appreciate your suggestion regarding the use of diamonds to represent the pooled effect in the graphical representation of the results. However, we chose not to include diamonds in this case due to the specific approach used in our meta-analysis, which was performed using the R programming environment (version 4.2.3) with the "metafor" package. In this package, the pooled effect is typically represented by point estimates and confidence intervals, rather than diamonds. While we understand that diamonds could offer a quick visual summary for clinicians, we prioritized a more precise representation of the data with confidence intervals, which we believe provides a clearer understanding of the variability in the effect sizes. Another general comment refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)? Probably authors should comment also taking into account this aspect in the discussion. Answer: We appreciate your comment and thank you for suggesting the inclusion of this aspect. We have added this point to the discussion section. Finally, some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover, in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact, they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Answer: Thank you for your valuable comments. We have revised the manuscript accordingly: the phrase "only becoming aware whenever patients are infected even further" has been clarified to better reflect the current understanding that CD typically becomes clinically evident in the later stages of infection. Additionally, we have addressed the issue of "therapeutic failure associated with existing therapies" by including the important point regarding the lack of a cure biomarker, which limits the ability to measure strong outcomes in clinical trials. Competing Interests: No competing interests Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 15 Apr 2025 Author Response The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the ... Continue reading The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). Answer: Thank you for your valuable and insightful comments. Regarding the exclusive use of PubMed, we selected this database due to its comprehensive and rigorous coverage of peer-reviewed biomedical literature, which aligns with the focus of our review. While we acknowledge that other databases like Lilacs and Embase could provide additional studies, PubMed was sufficient to gather the most relevant and high-quality research for our systematic analysis. In response to your concern about the inclusion of studies involving drug combinations with nifurtimox and benznidazole, it is important to highlight that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapies in preclinical models of Chagas disease. While clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), the results of monotherapy are essential to establish a solid comparative baseline and avoid biases from uncontrolled pharmacological interactions. Furthermore, combination therapies present variability in dosage, duration, and potential synergy, which complicates the standardization of results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more accurate evaluation of the individual efficacy of each compound, which is crucial for future therapeutic strategies and the development of new molecules. However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Answer: Thank you for your valuable feedback. We appreciate your suggestion regarding the use of diamonds to represent the pooled effect in the graphical representation of the results. However, we chose not to include diamonds in this case due to the specific approach used in our meta-analysis, which was performed using the R programming environment (version 4.2.3) with the "metafor" package. In this package, the pooled effect is typically represented by point estimates and confidence intervals, rather than diamonds. While we understand that diamonds could offer a quick visual summary for clinicians, we prioritized a more precise representation of the data with confidence intervals, which we believe provides a clearer understanding of the variability in the effect sizes. Another general comment refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)? Probably authors should comment also taking into account this aspect in the discussion. Answer: We appreciate your comment and thank you for suggesting the inclusion of this aspect. We have added this point to the discussion section. Finally, some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover, in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact, they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Answer: Thank you for your valuable comments. We have revised the manuscript accordingly: the phrase "only becoming aware whenever patients are infected even further" has been clarified to better reflect the current understanding that CD typically becomes clinically evident in the later stages of infection. Additionally, we have addressed the issue of "therapeutic failure associated with existing therapies" by including the important point regarding the lack of a cure biomarker, which limits the ability to measure strong outcomes in clinical trials. The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). Answer: Thank you for your valuable and insightful comments. Regarding the exclusive use of PubMed, we selected this database due to its comprehensive and rigorous coverage of peer-reviewed biomedical literature, which aligns with the focus of our review. While we acknowledge that other databases like Lilacs and Embase could provide additional studies, PubMed was sufficient to gather the most relevant and high-quality research for our systematic analysis. In response to your concern about the inclusion of studies involving drug combinations with nifurtimox and benznidazole, it is important to highlight that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapies in preclinical models of Chagas disease. While clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), the results of monotherapy are essential to establish a solid comparative baseline and avoid biases from uncontrolled pharmacological interactions. Furthermore, combination therapies present variability in dosage, duration, and potential synergy, which complicates the standardization of results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more accurate evaluation of the individual efficacy of each compound, which is crucial for future therapeutic strategies and the development of new molecules. However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Answer: Thank you for your valuable feedback. We appreciate your suggestion regarding the use of diamonds to represent the pooled effect in the graphical representation of the results. However, we chose not to include diamonds in this case due to the specific approach used in our meta-analysis, which was performed using the R programming environment (version 4.2.3) with the "metafor" package. In this package, the pooled effect is typically represented by point estimates and confidence intervals, rather than diamonds. While we understand that diamonds could offer a quick visual summary for clinicians, we prioritized a more precise representation of the data with confidence intervals, which we believe provides a clearer understanding of the variability in the effect sizes. Another general comment refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)? Probably authors should comment also taking into account this aspect in the discussion. Answer: We appreciate your comment and thank you for suggesting the inclusion of this aspect. We have added this point to the discussion section. Finally, some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover, in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact, they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Answer: Thank you for your valuable comments. We have revised the manuscript accordingly: the phrase "only becoming aware whenever patients are infected even further" has been clarified to better reflect the current understanding that CD typically becomes clinically evident in the later stages of infection. Additionally, we have addressed the issue of "therapeutic failure associated with existing therapies" by including the important point regarding the lack of a cure biomarker, which limits the ability to measure strong outcomes in clinical trials. Competing Interests: No competing interests Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355966 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355966 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 08 Jan 2025 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.165317.r355966 The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team ... Continue reading READ ALL The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Are the rationale for, and objectives of, the Systematic Review clearly stated? No Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? Yes Are the conclusions drawn adequately supported by the results presented in the review? Partly If this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) No References 1. Hooijmans CR, Rovers MM, de Vries RB, Leenaars M, et al.: SYRCLE's risk of bias tool for animal studies. BMC Med Res Methodol . 2014; 14 : 43 PubMed Abstract | Publisher Full Text 2. Romantsik O, Bank M, Menon JML, Malhotra A, et al.: Value of preclinical systematic reviews and meta-analyses in pediatric research. Pediatr Res . 2024; 96 (3): 643-653 PubMed Abstract | Publisher Full Text 3. Rassi A, Grimshaw A, Sarwal A, Sah R, et al.: Impact of antiparasitic therapy on cardiovascular outcomes in chronic Chagas disease. A systematic review and meta-analysis. eClinicalMedicine . 2025; 79 . Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gabaldon JC. Reviewer Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355966 ) The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355966 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 15 Apr 2025 Author Response The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to ... Continue reading The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Answer: Thank you for your insightful comments and for pointing out the recent findings regarding the efficacy of BNZ and NFX in the chronic stage of Chagas disease. We have carefully revised the introduction to incorporate your suggestions. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. Answer: Thank you for your valuable comments and suggestions. We would like to clarify that our systematic review is registered in the INPLASY database. The registration details are available in the manuscript. Regarding the search strategies, they were developed based on a bibliometric analysis, which is why they are described as a result in the data sources and study selection section. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Answer: Thank you for your comment. The exclusion of articles in Spanish and Portuguese was due to methodological reasons to ensure consistency, replicability, and accessibility in data extraction and analysis. Although the team includes native speakers of these languages, incorporating multiple languages could introduce variability in the interpretation of technical terms and statistical data. Moreover, the most impactful scientific databases predominantly index studies in English, facilitating comparability with other meta-analyses and ensuring that our findings are easily verifiable and reusable. Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? Answer: Thank you for your valuable observation. In the data review, we have included a section on Parasitemia Measurement Methods , where we report that parasitemia was assessed using microscopy, PCR, and blood culture across different studies. The methodology used to measure parasitemia was not a criterion for inclusion or exclusion in our analysis. This decision was based on the need to ensure a comprehensive review of the available data, considering that different studies employ various techniques depending on their resources, study design, and diagnostic capabilities. By not restricting the analysis to a specific measurement method, we aimed to capture a broader range of findings, enhancing the generalizability and applicability of our conclusions. An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Answer: Thanks for the observation. The use of parasitemia data alone in this study is based on its relevance as a quantifiable and reproducible indicator of therapeutic efficacy in preclinical models of Chagas disease. Assessing parasitemia reduction allows for a direct measurement of the trypanocidal activity of drugs in peripheral circulation, facilitating comparisons between studies and treatments. Furthermore, since this study is a systematic review and meta-analysis of preclinical treatments in animal models, it focused on standardized data widely reported in the scientific literature, prioritizing studies that documented significant reductions in parasitemia as a criterion for therapeutic success. Although the presence of the parasite in deep tissues is relevant in the chronic phase, obtaining such data requires invasive techniques or specific markers that are not always available or standardized across all analyzed studies. Therefore, the selection of parasitemia as the variable of analysis was based on criteria of data availability, comparability, and methodological robustness within the scope of the study. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. Answer: Thank you for your observation. However, it is important to emphasize that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapy treatments in preclinical models of Chagas disease. Although clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), monotherapy results are essential to establish a solid comparative baseline and avoid biases arising from uncontrolled pharmacological interactions. Additionally, combination treatments exhibit variability in dosage, duration, and potential synergy, making it difficult to standardize results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more precise evaluation of each compound’s individual efficacy, which is essential for future therapeutic strategies and the development of new molecules. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Answer: Thank you for your valuable feedback and for taking the time to review our manuscript. In response to your concern regarding the risk of bias assessment, we have applied SYRCLE’s Risk of Bias Tool to assess the risk of bias for each of the studies included in our review. Additionally, we have incorporated the GRADE criteria to assess the certainty of the evidence, to ensure a robust evaluation of the evidence’s quality and applicability. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. Answer: We appreciate your observation and the opportunity to clarify our objective. Our meta-analysis does not simply aim to confirm that antiparasitic treatment reduces parasitemia but rather to compare the efficacy of different compounds in preclinical models of Chagas disease, identifying those with the highest therapeutic potential. Since few treatments have successfully progressed to clinical trials, our study provides a critical evaluation of emerging options and their differences in efficacy depending on the experimental model and disease phase. For the sake of clarity, we will reinforce the explicit formulation of our objective. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. Answer: Thank you for your valuable observation. We acknowledge the inconsistency in terminology in the statistical analysis section and throughout the text. We will replace “participants” and “patients” with “animals”, to ensure clarity and accuracy. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Answer: Thank you for your valuable observation. We appreciate your careful review of our methodological choices. We will provide a clearer justification for the use of the random-effects model in the meta-analysis, ensuring that our decision is well-supported despite the low reported heterogeneity. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. Answer: Thank you for your valuable comment. We have followed your recommendation and included the data extraction sheets for the articles that underwent full-text screening, along with an additional column in the table in the supplementary files to indicate the reasons for the exclusion of certain papers from the meta-analysis. We appreciate your feedback and believe it improves the clarity of the manuscript. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Answer: We appreciate your observation regarding the clarity of the RR calculation. We have revised the text to explicitly describe the comparison for which the RR is calculated, ensuring clearer interpretation and avoiding potential confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Answer: Thank you for your valuable feedback. We have made the necessary changes and now include the name of each compound used in each line of the Forest plot to improve readability and clarity. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Answer: The meta-analysis conducted in this study provides a quantitative assessment of the impact of various therapeutic options in animal models of Chagas disease, offering a comprehensive view of their preclinical efficacy. While it is true that some treated animals achieved parasitemia clearance, the value of our study lies in the systematic identification of efficacy patterns across different compounds and the comparative evaluation of their relative performance in different disease phases. Rather than merely confirming that some treatments can reduce parasitemia, this analysis allows us to discern which treatments demonstrate greater consistency and potential for future clinical research, helping to focus efforts on the most promising options for the development of new therapies​. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. Answer: We appreciate the reviewer’s observation regarding the complexity of systematic reviews of preclinical studies due to methodological variability compared to clinical trials. While many such reviews aim to prioritize compounds for clinical evaluation, our study also serves the critical purpose of identifying therapeutic response patterns and providing a quantitative assessment of treatment efficacy in animal models, which is particularly relevant for neglected diseases like Chagas. The inclusion of posaconazole, despite its unsatisfactory clinical outcomes, remains valuable for understanding its preclinical performance and benchmarking emerging therapies, while the absence of benzoxaboroles and cyanotriazoles was not due to bias but rather the lack of published preclinical studies meeting our inclusion criteria. Our systematic approach, following PRISMA guidelines and registered in INPLASY (INPLASY202430101), ensures transparency and reproducibility, making our findings a valuable contribution to guiding future research and identifying promising candidates for further investigation. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. Answer: We sincerely appreciate the reviewer’s thoughtful observation and encouragement regarding our work on Chagas disease therapeutics. While a well-structured scoping review could indeed provide a broad overview of compounds under preclinical evaluation, our decision to conduct a systematic review and meta-analysis was driven by the need to quantitatively assess treatment efficacy across multiple studies using standardized methodologies. By adhering to PRISMA guidelines, our study synthesizes available preclinical data, providing statistical insights into parasitemia reduction and the therapeutic potential of different compounds. This quantitative approach not only allows for a more rigorous comparison of treatment effects but also highlights trends, gaps, and inconsistencies in the current literature, which are critical for guiding future research and clinical translation. We believe that this level of analysis is essential for evidence-based decision-making and prioritization of drug candidates for Chagas disease treatment. Nonetheless, we acknowledge the value of a scoping review in complementing our findings by exploring a broader range of therapeutic strategies beyond efficacy metrics, and we appreciate the suggestion for future research directions. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Answer: Thank you for your valuable comments and suggestions. In response to your observation, we have made the necessary changes to the manuscript. We have refined the research question to ensure it is clear and concise, accurately reflecting the study's objective. Additionally, we have conducted a risk of bias assessment using the SYRCLE tool and addressed the certainty of the evidence using the GRADE approach, as you recommended. These improvements enhance the robustness of our analysis and ensure a more reliable interpretation of the results. The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Answer: Thank you for your insightful comments and for pointing out the recent findings regarding the efficacy of BNZ and NFX in the chronic stage of Chagas disease. We have carefully revised the introduction to incorporate your suggestions. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. Answer: Thank you for your valuable comments and suggestions. We would like to clarify that our systematic review is registered in the INPLASY database. The registration details are available in the manuscript. Regarding the search strategies, they were developed based on a bibliometric analysis, which is why they are described as a result in the data sources and study selection section. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Answer: Thank you for your comment. The exclusion of articles in Spanish and Portuguese was due to methodological reasons to ensure consistency, replicability, and accessibility in data extraction and analysis. Although the team includes native speakers of these languages, incorporating multiple languages could introduce variability in the interpretation of technical terms and statistical data. Moreover, the most impactful scientific databases predominantly index studies in English, facilitating comparability with other meta-analyses and ensuring that our findings are easily verifiable and reusable. Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? Answer: Thank you for your valuable observation. In the data review, we have included a section on Parasitemia Measurement Methods , where we report that parasitemia was assessed using microscopy, PCR, and blood culture across different studies. The methodology used to measure parasitemia was not a criterion for inclusion or exclusion in our analysis. This decision was based on the need to ensure a comprehensive review of the available data, considering that different studies employ various techniques depending on their resources, study design, and diagnostic capabilities. By not restricting the analysis to a specific measurement method, we aimed to capture a broader range of findings, enhancing the generalizability and applicability of our conclusions. An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Answer: Thanks for the observation. The use of parasitemia data alone in this study is based on its relevance as a quantifiable and reproducible indicator of therapeutic efficacy in preclinical models of Chagas disease. Assessing parasitemia reduction allows for a direct measurement of the trypanocidal activity of drugs in peripheral circulation, facilitating comparisons between studies and treatments. Furthermore, since this study is a systematic review and meta-analysis of preclinical treatments in animal models, it focused on standardized data widely reported in the scientific literature, prioritizing studies that documented significant reductions in parasitemia as a criterion for therapeutic success. Although the presence of the parasite in deep tissues is relevant in the chronic phase, obtaining such data requires invasive techniques or specific markers that are not always available or standardized across all analyzed studies. Therefore, the selection of parasitemia as the variable of analysis was based on criteria of data availability, comparability, and methodological robustness within the scope of the study. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. Answer: Thank you for your observation. However, it is important to emphasize that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapy treatments in preclinical models of Chagas disease. Although clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), monotherapy results are essential to establish a solid comparative baseline and avoid biases arising from uncontrolled pharmacological interactions. Additionally, combination treatments exhibit variability in dosage, duration, and potential synergy, making it difficult to standardize results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more precise evaluation of each compound’s individual efficacy, which is essential for future therapeutic strategies and the development of new molecules. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Answer: Thank you for your valuable feedback and for taking the time to review our manuscript. In response to your concern regarding the risk of bias assessment, we have applied SYRCLE’s Risk of Bias Tool to assess the risk of bias for each of the studies included in our review. Additionally, we have incorporated the GRADE criteria to assess the certainty of the evidence, to ensure a robust evaluation of the evidence’s quality and applicability. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. Answer: We appreciate your observation and the opportunity to clarify our objective. Our meta-analysis does not simply aim to confirm that antiparasitic treatment reduces parasitemia but rather to compare the efficacy of different compounds in preclinical models of Chagas disease, identifying those with the highest therapeutic potential. Since few treatments have successfully progressed to clinical trials, our study provides a critical evaluation of emerging options and their differences in efficacy depending on the experimental model and disease phase. For the sake of clarity, we will reinforce the explicit formulation of our objective. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. Answer: Thank you for your valuable observation. We acknowledge the inconsistency in terminology in the statistical analysis section and throughout the text. We will replace “participants” and “patients” with “animals”, to ensure clarity and accuracy. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Answer: Thank you for your valuable observation. We appreciate your careful review of our methodological choices. We will provide a clearer justification for the use of the random-effects model in the meta-analysis, ensuring that our decision is well-supported despite the low reported heterogeneity. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. Answer: Thank you for your valuable comment. We have followed your recommendation and included the data extraction sheets for the articles that underwent full-text screening, along with an additional column in the table in the supplementary files to indicate the reasons for the exclusion of certain papers from the meta-analysis. We appreciate your feedback and believe it improves the clarity of the manuscript. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Answer: We appreciate your observation regarding the clarity of the RR calculation. We have revised the text to explicitly describe the comparison for which the RR is calculated, ensuring clearer interpretation and avoiding potential confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Answer: Thank you for your valuable feedback. We have made the necessary changes and now include the name of each compound used in each line of the Forest plot to improve readability and clarity. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Answer: The meta-analysis conducted in this study provides a quantitative assessment of the impact of various therapeutic options in animal models of Chagas disease, offering a comprehensive view of their preclinical efficacy. While it is true that some treated animals achieved parasitemia clearance, the value of our study lies in the systematic identification of efficacy patterns across different compounds and the comparative evaluation of their relative performance in different disease phases. Rather than merely confirming that some treatments can reduce parasitemia, this analysis allows us to discern which treatments demonstrate greater consistency and potential for future clinical research, helping to focus efforts on the most promising options for the development of new therapies​. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. Answer: We appreciate the reviewer’s observation regarding the complexity of systematic reviews of preclinical studies due to methodological variability compared to clinical trials. While many such reviews aim to prioritize compounds for clinical evaluation, our study also serves the critical purpose of identifying therapeutic response patterns and providing a quantitative assessment of treatment efficacy in animal models, which is particularly relevant for neglected diseases like Chagas. The inclusion of posaconazole, despite its unsatisfactory clinical outcomes, remains valuable for understanding its preclinical performance and benchmarking emerging therapies, while the absence of benzoxaboroles and cyanotriazoles was not due to bias but rather the lack of published preclinical studies meeting our inclusion criteria. Our systematic approach, following PRISMA guidelines and registered in INPLASY (INPLASY202430101), ensures transparency and reproducibility, making our findings a valuable contribution to guiding future research and identifying promising candidates for further investigation. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. Answer: We sincerely appreciate the reviewer’s thoughtful observation and encouragement regarding our work on Chagas disease therapeutics. While a well-structured scoping review could indeed provide a broad overview of compounds under preclinical evaluation, our decision to conduct a systematic review and meta-analysis was driven by the need to quantitatively assess treatment efficacy across multiple studies using standardized methodologies. By adhering to PRISMA guidelines, our study synthesizes available preclinical data, providing statistical insights into parasitemia reduction and the therapeutic potential of different compounds. This quantitative approach not only allows for a more rigorous comparison of treatment effects but also highlights trends, gaps, and inconsistencies in the current literature, which are critical for guiding future research and clinical translation. We believe that this level of analysis is essential for evidence-based decision-making and prioritization of drug candidates for Chagas disease treatment. Nonetheless, we acknowledge the value of a scoping review in complementing our findings by exploring a broader range of therapeutic strategies beyond efficacy metrics, and we appreciate the suggestion for future research directions. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Answer: Thank you for your valuable comments and suggestions. In response to your observation, we have made the necessary changes to the manuscript. We have refined the research question to ensure it is clear and concise, accurately reflecting the study's objective. Additionally, we have conducted a risk of bias assessment using the SYRCLE tool and addressed the certainty of the evidence using the GRADE approach, as you recommended. These improvements enhance the robustness of our analysis and ensure a more reliable interpretation of the results. Competing Interests: No competing interests Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli , Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru 15 Apr 2025 Author Response The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to ... Continue reading The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Answer: Thank you for your insightful comments and for pointing out the recent findings regarding the efficacy of BNZ and NFX in the chronic stage of Chagas disease. We have carefully revised the introduction to incorporate your suggestions. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. Answer: Thank you for your valuable comments and suggestions. We would like to clarify that our systematic review is registered in the INPLASY database. The registration details are available in the manuscript. Regarding the search strategies, they were developed based on a bibliometric analysis, which is why they are described as a result in the data sources and study selection section. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Answer: Thank you for your comment. The exclusion of articles in Spanish and Portuguese was due to methodological reasons to ensure consistency, replicability, and accessibility in data extraction and analysis. Although the team includes native speakers of these languages, incorporating multiple languages could introduce variability in the interpretation of technical terms and statistical data. Moreover, the most impactful scientific databases predominantly index studies in English, facilitating comparability with other meta-analyses and ensuring that our findings are easily verifiable and reusable. Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? Answer: Thank you for your valuable observation. In the data review, we have included a section on Parasitemia Measurement Methods , where we report that parasitemia was assessed using microscopy, PCR, and blood culture across different studies. The methodology used to measure parasitemia was not a criterion for inclusion or exclusion in our analysis. This decision was based on the need to ensure a comprehensive review of the available data, considering that different studies employ various techniques depending on their resources, study design, and diagnostic capabilities. By not restricting the analysis to a specific measurement method, we aimed to capture a broader range of findings, enhancing the generalizability and applicability of our conclusions. An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Answer: Thanks for the observation. The use of parasitemia data alone in this study is based on its relevance as a quantifiable and reproducible indicator of therapeutic efficacy in preclinical models of Chagas disease. Assessing parasitemia reduction allows for a direct measurement of the trypanocidal activity of drugs in peripheral circulation, facilitating comparisons between studies and treatments. Furthermore, since this study is a systematic review and meta-analysis of preclinical treatments in animal models, it focused on standardized data widely reported in the scientific literature, prioritizing studies that documented significant reductions in parasitemia as a criterion for therapeutic success. Although the presence of the parasite in deep tissues is relevant in the chronic phase, obtaining such data requires invasive techniques or specific markers that are not always available or standardized across all analyzed studies. Therefore, the selection of parasitemia as the variable of analysis was based on criteria of data availability, comparability, and methodological robustness within the scope of the study. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. Answer: Thank you for your observation. However, it is important to emphasize that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapy treatments in preclinical models of Chagas disease. Although clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), monotherapy results are essential to establish a solid comparative baseline and avoid biases arising from uncontrolled pharmacological interactions. Additionally, combination treatments exhibit variability in dosage, duration, and potential synergy, making it difficult to standardize results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more precise evaluation of each compound’s individual efficacy, which is essential for future therapeutic strategies and the development of new molecules. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Answer: Thank you for your valuable feedback and for taking the time to review our manuscript. In response to your concern regarding the risk of bias assessment, we have applied SYRCLE’s Risk of Bias Tool to assess the risk of bias for each of the studies included in our review. Additionally, we have incorporated the GRADE criteria to assess the certainty of the evidence, to ensure a robust evaluation of the evidence’s quality and applicability. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. Answer: We appreciate your observation and the opportunity to clarify our objective. Our meta-analysis does not simply aim to confirm that antiparasitic treatment reduces parasitemia but rather to compare the efficacy of different compounds in preclinical models of Chagas disease, identifying those with the highest therapeutic potential. Since few treatments have successfully progressed to clinical trials, our study provides a critical evaluation of emerging options and their differences in efficacy depending on the experimental model and disease phase. For the sake of clarity, we will reinforce the explicit formulation of our objective. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. Answer: Thank you for your valuable observation. We acknowledge the inconsistency in terminology in the statistical analysis section and throughout the text. We will replace “participants” and “patients” with “animals”, to ensure clarity and accuracy. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Answer: Thank you for your valuable observation. We appreciate your careful review of our methodological choices. We will provide a clearer justification for the use of the random-effects model in the meta-analysis, ensuring that our decision is well-supported despite the low reported heterogeneity. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. Answer: Thank you for your valuable comment. We have followed your recommendation and included the data extraction sheets for the articles that underwent full-text screening, along with an additional column in the table in the supplementary files to indicate the reasons for the exclusion of certain papers from the meta-analysis. We appreciate your feedback and believe it improves the clarity of the manuscript. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Answer: We appreciate your observation regarding the clarity of the RR calculation. We have revised the text to explicitly describe the comparison for which the RR is calculated, ensuring clearer interpretation and avoiding potential confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Answer: Thank you for your valuable feedback. We have made the necessary changes and now include the name of each compound used in each line of the Forest plot to improve readability and clarity. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Answer: The meta-analysis conducted in this study provides a quantitative assessment of the impact of various therapeutic options in animal models of Chagas disease, offering a comprehensive view of their preclinical efficacy. While it is true that some treated animals achieved parasitemia clearance, the value of our study lies in the systematic identification of efficacy patterns across different compounds and the comparative evaluation of their relative performance in different disease phases. Rather than merely confirming that some treatments can reduce parasitemia, this analysis allows us to discern which treatments demonstrate greater consistency and potential for future clinical research, helping to focus efforts on the most promising options for the development of new therapies​. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. Answer: We appreciate the reviewer’s observation regarding the complexity of systematic reviews of preclinical studies due to methodological variability compared to clinical trials. While many such reviews aim to prioritize compounds for clinical evaluation, our study also serves the critical purpose of identifying therapeutic response patterns and providing a quantitative assessment of treatment efficacy in animal models, which is particularly relevant for neglected diseases like Chagas. The inclusion of posaconazole, despite its unsatisfactory clinical outcomes, remains valuable for understanding its preclinical performance and benchmarking emerging therapies, while the absence of benzoxaboroles and cyanotriazoles was not due to bias but rather the lack of published preclinical studies meeting our inclusion criteria. Our systematic approach, following PRISMA guidelines and registered in INPLASY (INPLASY202430101), ensures transparency and reproducibility, making our findings a valuable contribution to guiding future research and identifying promising candidates for further investigation. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. Answer: We sincerely appreciate the reviewer’s thoughtful observation and encouragement regarding our work on Chagas disease therapeutics. While a well-structured scoping review could indeed provide a broad overview of compounds under preclinical evaluation, our decision to conduct a systematic review and meta-analysis was driven by the need to quantitatively assess treatment efficacy across multiple studies using standardized methodologies. By adhering to PRISMA guidelines, our study synthesizes available preclinical data, providing statistical insights into parasitemia reduction and the therapeutic potential of different compounds. This quantitative approach not only allows for a more rigorous comparison of treatment effects but also highlights trends, gaps, and inconsistencies in the current literature, which are critical for guiding future research and clinical translation. We believe that this level of analysis is essential for evidence-based decision-making and prioritization of drug candidates for Chagas disease treatment. Nonetheless, we acknowledge the value of a scoping review in complementing our findings by exploring a broader range of therapeutic strategies beyond efficacy metrics, and we appreciate the suggestion for future research directions. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Answer: Thank you for your valuable comments and suggestions. In response to your observation, we have made the necessary changes to the manuscript. We have refined the research question to ensure it is clear and concise, accurately reflecting the study's objective. Additionally, we have conducted a risk of bias assessment using the SYRCLE tool and addressed the certainty of the evidence using the GRADE approach, as you recommended. These improvements enhance the robustness of our analysis and ensure a more reliable interpretation of the results. The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Answer: Thank you for your insightful comments and for pointing out the recent findings regarding the efficacy of BNZ and NFX in the chronic stage of Chagas disease. We have carefully revised the introduction to incorporate your suggestions. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. Answer: Thank you for your valuable comments and suggestions. We would like to clarify that our systematic review is registered in the INPLASY database. The registration details are available in the manuscript. Regarding the search strategies, they were developed based on a bibliometric analysis, which is why they are described as a result in the data sources and study selection section. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Answer: Thank you for your comment. The exclusion of articles in Spanish and Portuguese was due to methodological reasons to ensure consistency, replicability, and accessibility in data extraction and analysis. Although the team includes native speakers of these languages, incorporating multiple languages could introduce variability in the interpretation of technical terms and statistical data. Moreover, the most impactful scientific databases predominantly index studies in English, facilitating comparability with other meta-analyses and ensuring that our findings are easily verifiable and reusable. Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? Answer: Thank you for your valuable observation. In the data review, we have included a section on Parasitemia Measurement Methods , where we report that parasitemia was assessed using microscopy, PCR, and blood culture across different studies. The methodology used to measure parasitemia was not a criterion for inclusion or exclusion in our analysis. This decision was based on the need to ensure a comprehensive review of the available data, considering that different studies employ various techniques depending on their resources, study design, and diagnostic capabilities. By not restricting the analysis to a specific measurement method, we aimed to capture a broader range of findings, enhancing the generalizability and applicability of our conclusions. An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Answer: Thanks for the observation. The use of parasitemia data alone in this study is based on its relevance as a quantifiable and reproducible indicator of therapeutic efficacy in preclinical models of Chagas disease. Assessing parasitemia reduction allows for a direct measurement of the trypanocidal activity of drugs in peripheral circulation, facilitating comparisons between studies and treatments. Furthermore, since this study is a systematic review and meta-analysis of preclinical treatments in animal models, it focused on standardized data widely reported in the scientific literature, prioritizing studies that documented significant reductions in parasitemia as a criterion for therapeutic success. Although the presence of the parasite in deep tissues is relevant in the chronic phase, obtaining such data requires invasive techniques or specific markers that are not always available or standardized across all analyzed studies. Therefore, the selection of parasitemia as the variable of analysis was based on criteria of data availability, comparability, and methodological robustness within the scope of the study. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. Answer: Thank you for your observation. However, it is important to emphasize that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapy treatments in preclinical models of Chagas disease. Although clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), monotherapy results are essential to establish a solid comparative baseline and avoid biases arising from uncontrolled pharmacological interactions. Additionally, combination treatments exhibit variability in dosage, duration, and potential synergy, making it difficult to standardize results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more precise evaluation of each compound’s individual efficacy, which is essential for future therapeutic strategies and the development of new molecules. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Answer: Thank you for your valuable feedback and for taking the time to review our manuscript. In response to your concern regarding the risk of bias assessment, we have applied SYRCLE’s Risk of Bias Tool to assess the risk of bias for each of the studies included in our review. Additionally, we have incorporated the GRADE criteria to assess the certainty of the evidence, to ensure a robust evaluation of the evidence’s quality and applicability. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. Answer: We appreciate your observation and the opportunity to clarify our objective. Our meta-analysis does not simply aim to confirm that antiparasitic treatment reduces parasitemia but rather to compare the efficacy of different compounds in preclinical models of Chagas disease, identifying those with the highest therapeutic potential. Since few treatments have successfully progressed to clinical trials, our study provides a critical evaluation of emerging options and their differences in efficacy depending on the experimental model and disease phase. For the sake of clarity, we will reinforce the explicit formulation of our objective. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. Answer: Thank you for your valuable observation. We acknowledge the inconsistency in terminology in the statistical analysis section and throughout the text. We will replace “participants” and “patients” with “animals”, to ensure clarity and accuracy. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Answer: Thank you for your valuable observation. We appreciate your careful review of our methodological choices. We will provide a clearer justification for the use of the random-effects model in the meta-analysis, ensuring that our decision is well-supported despite the low reported heterogeneity. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. Answer: Thank you for your valuable comment. We have followed your recommendation and included the data extraction sheets for the articles that underwent full-text screening, along with an additional column in the table in the supplementary files to indicate the reasons for the exclusion of certain papers from the meta-analysis. We appreciate your feedback and believe it improves the clarity of the manuscript. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Answer: We appreciate your observation regarding the clarity of the RR calculation. We have revised the text to explicitly describe the comparison for which the RR is calculated, ensuring clearer interpretation and avoiding potential confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Answer: Thank you for your valuable feedback. We have made the necessary changes and now include the name of each compound used in each line of the Forest plot to improve readability and clarity. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Answer: The meta-analysis conducted in this study provides a quantitative assessment of the impact of various therapeutic options in animal models of Chagas disease, offering a comprehensive view of their preclinical efficacy. While it is true that some treated animals achieved parasitemia clearance, the value of our study lies in the systematic identification of efficacy patterns across different compounds and the comparative evaluation of their relative performance in different disease phases. Rather than merely confirming that some treatments can reduce parasitemia, this analysis allows us to discern which treatments demonstrate greater consistency and potential for future clinical research, helping to focus efforts on the most promising options for the development of new therapies​. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. Answer: We appreciate the reviewer’s observation regarding the complexity of systematic reviews of preclinical studies due to methodological variability compared to clinical trials. While many such reviews aim to prioritize compounds for clinical evaluation, our study also serves the critical purpose of identifying therapeutic response patterns and providing a quantitative assessment of treatment efficacy in animal models, which is particularly relevant for neglected diseases like Chagas. The inclusion of posaconazole, despite its unsatisfactory clinical outcomes, remains valuable for understanding its preclinical performance and benchmarking emerging therapies, while the absence of benzoxaboroles and cyanotriazoles was not due to bias but rather the lack of published preclinical studies meeting our inclusion criteria. Our systematic approach, following PRISMA guidelines and registered in INPLASY (INPLASY202430101), ensures transparency and reproducibility, making our findings a valuable contribution to guiding future research and identifying promising candidates for further investigation. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. Answer: We sincerely appreciate the reviewer’s thoughtful observation and encouragement regarding our work on Chagas disease therapeutics. While a well-structured scoping review could indeed provide a broad overview of compounds under preclinical evaluation, our decision to conduct a systematic review and meta-analysis was driven by the need to quantitatively assess treatment efficacy across multiple studies using standardized methodologies. By adhering to PRISMA guidelines, our study synthesizes available preclinical data, providing statistical insights into parasitemia reduction and the therapeutic potential of different compounds. This quantitative approach not only allows for a more rigorous comparison of treatment effects but also highlights trends, gaps, and inconsistencies in the current literature, which are critical for guiding future research and clinical translation. We believe that this level of analysis is essential for evidence-based decision-making and prioritization of drug candidates for Chagas disease treatment. Nonetheless, we acknowledge the value of a scoping review in complementing our findings by exploring a broader range of therapeutic strategies beyond efficacy metrics, and we appreciate the suggestion for future research directions. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Answer: Thank you for your valuable comments and suggestions. In response to your observation, we have made the necessary changes to the manuscript. We have refined the research question to ensure it is clear and concise, accurately reflecting the study's objective. Additionally, we have conducted a risk of bias assessment using the SYRCLE tool and addressed the certainty of the evidence using the GRADE approach, as you recommended. These improvements enhance the robustness of our analysis and ensure a more reliable interpretation of the results. Competing Interests: No competing interests Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 05 Aug 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 3 (revision) 16 May 25 read Version 2 (revision) 15 Apr 25 read read Version 1 05 Aug 24 read read Juan Carlos Gabaldon , Universitat de Barcelona, Barcelona, Spain Andrea Angheben , IRCCS Sacro Cuore Hospital, Negrar, Verona, Italy Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 May 2025 | for Version 3 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Authors have adequately addressed my major comments. Competing Interests No competing interests were disclosed. Reviewer Expertise Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Gabaldon JC. Peer Review Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.182003.r385243) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-885/v3#referee-response-385243 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Angheben A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 May 2025 | for Version 2 Andrea Angheben , IRCCS Sacro Cuore Hospital, Negrar, Verona, Italy 0 Views copyright © 2025 Angheben A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I've read author's answers to my review and I recognize that the paper and author's work has been planned with a good methodology but the limits I've highlighted still remain. Although I am not fully convinced that as it stands the paper is well fruible particularly by clinicians and I believe that methods could be improved (particularly with extension of database queries), authors do declare clearly their approach and the paper can be indexed to my opinion. Competing Interests No competing interests were disclosed. Reviewer Expertise My area of expertise covers epidemiological and clinical aspects of Chagas disease and other NTDs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Angheben A. Peer Review Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378071) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378071 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 26 Apr 2025 | for Version 2 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Competing Interests No competing interests were disclosed. Reviewer Expertise Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 24 Jun 2025 Miguel Angel Chavez-Fumagalli, Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru Authors have adequately addressed most of my comments, and the manuscript has generally improved significantly, particularly from a methodological point of view. I specially appreciated the insightful explanation on why a random effects model was selected despite apparently low heterogeneity, as well as the inclusion of a risk of bias assessment. Answer: Thank you for your positive feedback. We are glad that the revisions have significantly improved the manuscript, particularly from a methodological standpoint. The one significant thing I still believe should be corrected is the interpretation of results during the chronic stage of infection. I understand the reasons why authors decided to exclusively evaluate peripheral parasitaemia as an outcome of drug effectiveness in the animal models included in the meta-analysis. However, this is likely inadequate in the chronic stage of disease, when parasitaemia is known to be low and intermittent. Authors indicate that some drugs were found to be effective in the chronic stage of disease, which is also the one of most clinical interest, but such a statement might be inadequate if based exclusively on peripheral parasitaemia. If the effectiveness of compounds in the chronic stage wants to be evaluated, then other outcomes, such as the ones mentioned in my previous review should be included. If only peripheral parasitaemia will be considered, then this should be mentioned and thoroughly discussed in the limitations section. Answer: We sincerely appreciate your insightful comment regarding the interpretation of results during the chronic stage of infection. As you rightly pointed out, peripheral parasitaemia may not fully capture drug effectiveness in this phase due to its low and intermittent nature. While our meta-analysis focused on peripheral parasitaemia as a standardized outcome across studies, we acknowledge this limitation and have now explicitly discussed its implications in the limitations section, emphasizing that conclusions about efficacy in the chronic stage should be interpreted with caution and may require complementary outcomes in future research. Similarly, the discussion section could still be improved. The language in some parts feels artificially complicated, particularly in the first paragraph. On the other hand, the few lines discussing the mechanism of action of BNZ and NFX seem superficial and don’t really describe their effect on parasite nitroreductase, their demonstrated target. There is also no mention on the potential effect of metabolically-quiescent parasite forms on the high rates of therapeutic failure observed in the chronic stage, nor its implications for the design of pre-clinical studies. Answer: We sincerely appreciate your comments, which have greatly improved our manuscript. In response to your observations, we have simplified the language in the first paragraph of the discussion to enhance clarity. Additionally, we have expanded the explanation of BNZ and NFX's mechanism of action, specifically detailing their effects on parasite nitroreductase. We have also discussed the potential role of metabolically quiescent parasite forms in therapeutic failure during the chronic stage and their implications for pre-clinical study design. The high risk of bias, and the overall certainty of evidence described by the authors, and their implications for the interpretation of findings should also be discussed in the limitations section, as they severely affect the generalizability of the findings and highlight the difficulties of inferring data exclusively from pre-clinical experiments, which are often carried out in non-standardized conditions. Answer: We sincerely appreciate your comments regarding the limitations of our meta-analysis. In response to your observation, we have thoroughly addressed the high risk of bias by incorporating a detailed discussion on methodological heterogeneity, variability in experimental designs, and reporting inconsistencies across the included studies. We have also emphasized the need for cautious interpretation of the results due to the translational constraints of preclinical research, as well as the importance of standardizing future studies to improve reliability and applicability. View more View less Competing Interests No competing interests reply Respond Report a concern Gabaldon JC. Peer Review Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.179980.r378072) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-885/v2#referee-response-378072 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Angheben A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Feb 2025 | for Version 1 Andrea Angheben , IRCCS Sacro Cuore Hospital, Negrar, Verona, Italy 0 Views copyright © 2025 Angheben A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Another general comments refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)?. Probably authors should comment also taking into account this aspect in the discussion. Finally some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Yes Is the statistical analysis and its interpretation appropriate? Partly Are the conclusions drawn adequately supported by the results presented in the review? Yes If this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable Competing Interests No competing interests were disclosed. Reviewer Expertise My area of expertise covers epidemiological and clinical aspects of Chagas disease and other NTDs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli, Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru The paper entitled "Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis" is an interesting paper exploring the studies on the use of new compounds on animal models of Chagas disease, according to index literature. The paper may well interest F1000Research readers. Two compounds seem to be promising but have never reached the clinical phase of evaluation whereas those used on humans have demonstrated not to be superior of existing drugs. I have some comments to be addressed before taking a decision on the indexing of the paper. Authors follow a "systematic" process for their literature search but some limitations should be recognized: "only" PubMed has been explored (not Lilacs nor Embase have been explored) and studies with medication added to the well known nifurtimox and benzidazole were not included (why, taking into account control groups?). Answer: Thank you for your valuable and insightful comments. Regarding the exclusive use of PubMed, we selected this database due to its comprehensive and rigorous coverage of peer-reviewed biomedical literature, which aligns with the focus of our review. While we acknowledge that other databases like Lilacs and Embase could provide additional studies, PubMed was sufficient to gather the most relevant and high-quality research for our systematic analysis. In response to your concern about the inclusion of studies involving drug combinations with nifurtimox and benznidazole, it is important to highlight that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapies in preclinical models of Chagas disease. While clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), the results of monotherapy are essential to establish a solid comparative baseline and avoid biases from uncontrolled pharmacological interactions. Furthermore, combination therapies present variability in dosage, duration, and potential synergy, which complicates the standardization of results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more accurate evaluation of the individual efficacy of each compound, which is crucial for future therapeutic strategies and the development of new molecules. However, methodology for the other aspects is very well described and rigorous, to me. For the statistics, mainly for the graphical representation fo the results, I just ask authors if they can summarize the results of the meta-analysis with diamonds corresponding to the pooled effect of the drugs, or explaining why the prefer not to do it. For a clinician like me, this can be of healp in understanding results very rapidly. Answer: Thank you for your valuable feedback. We appreciate your suggestion regarding the use of diamonds to represent the pooled effect in the graphical representation of the results. However, we chose not to include diamonds in this case due to the specific approach used in our meta-analysis, which was performed using the R programming environment (version 4.2.3) with the "metafor" package. In this package, the pooled effect is typically represented by point estimates and confidence intervals, rather than diamonds. While we understand that diamonds could offer a quick visual summary for clinicians, we prioritized a more precise representation of the data with confidence intervals, which we believe provides a clearer understanding of the variability in the effect sizes. Another general comment refers to the facts (also commented by authors) that they identified studies just in a 5 years period and then for around ten years no new studies have been published on animal models. Can this fact be related to a new approach in the field of drugs discovery (in silico models appearance)? Probably authors should comment also taking into account this aspect in the discussion. Answer: We appreciate your comment and thank you for suggesting the inclusion of this aspect. We have added this point to the discussion section. Finally, some minor comments: in the introduction the phrase "only becoming aware whenever patients are infected even further" is not clear to me; as I understand it seems that CD become clinically evident for continuous exposure to parasites which is not corresponding to the current knowledge. Please clarify. Moreover, in the introduction, authors list the difficulties in patient management ending with the phrase "therapeutic failure associated with existing therapies". In fact, they do not mention and important factor which is also a limitation for the trials in the clinical phase, which is the lack of a cure biomarker and therefor of strong outcomes to be measured when conducting a clinical trial. Answer: Thank you for your valuable comments. We have revised the manuscript accordingly: the phrase "only becoming aware whenever patients are infected even further" has been clarified to better reflect the current understanding that CD typically becomes clinically evident in the later stages of infection. Additionally, we have addressed the issue of "therapeutic failure associated with existing therapies" by including the important point regarding the lack of a cure biomarker, which limits the ability to measure strong outcomes in clinical trials. View more View less Competing Interests No competing interests reply Respond Report a concern Angheben A. Peer Review Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355965) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355965 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 08 Jan 2025 | for Version 1 Juan Carlos Gabaldon , Barcelona Institute for Global Health, Universitat de Barcelona, Barcelona, Catalonia, Spain 0 Views copyright © 2025 Gabaldon J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Are the rationale for, and objectives of, the Systematic Review clearly stated? No Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? Yes Are the conclusions drawn adequately supported by the results presented in the review? Partly If this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) No References 1. Hooijmans CR, Rovers MM, de Vries RB, Leenaars M, et al.: SYRCLE's risk of bias tool for animal studies. BMC Med Res Methodol . 2014; 14 : 43 PubMed Abstract | Publisher Full Text 2. Romantsik O, Bank M, Menon JML, Malhotra A, et al.: Value of preclinical systematic reviews and meta-analyses in pediatric research. Pediatr Res . 2024; 96 (3): 643-653 PubMed Abstract | Publisher Full Text 3. Rassi A, Grimshaw A, Sarwal A, Sah R, et al.: Impact of antiparasitic therapy on cardiovascular outcomes in chronic Chagas disease. A systematic review and meta-analysis. eClinicalMedicine . 2025; 79 . Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Parasitology, tropical medicine, neglected tropical diseases I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 15 Apr 2025 Miguel Angel Chavez-Fumagalli, Computational Biology and Chemistry Research Group, Vicerrectorado de Investigación, Universidad Católica de Santa María, Pedro Vilcapaza, 04000, Peru The authors provide an interesting work comparing a number of different medications and compounds that have been tested in animal models of Chagas disease and carry out a meta-analysis to summarize the results observed. While I congratulate the author team for the effort put in this well-written work, and for highlighting an important issue, such as the limited availability of medications currently available for the treatment of Chagas disease, I feel that the objective and research question that they intend to answer are unclear, and there are some methodological limitations that prevent me from accepting the document in its current state. Introduction: The introduction summarizes the epidemiological landscape of Chagas disease adequately. However, the idea that BNZ and NFX are ineffective in the chronic stage of disease could soon be reconsidered, and I recommend reading the recent meta-analysis by Rassi et al (Ref 3) showing improved clinical outcomes upon parasitic treatment. It is also increasingly accepted that treatment of infected patients should be the norm, unless specific counter-indications exist. The authors could, in turn, mention that existing therapies still fail to clear parasitaemia in a substantial number of patients, and that since sterilizing cure remains the main objective of therapy, interest in the development of new medications remains justified. Answer: Thank you for your insightful comments and for pointing out the recent findings regarding the efficacy of BNZ and NFX in the chronic stage of Chagas disease. We have carefully revised the introduction to incorporate your suggestions. Methods: Authors might want to register the systematic review in the PROSPERO database as well, which includes systematic reviews of human and animal studies. Authors should also include the search strategy used in the different datasets as a supplementary file. Answer: Thank you for your valuable comments and suggestions. We would like to clarify that our systematic review is registered in the INPLASY database. The registration details are available in the manuscript. Regarding the search strategies, they were developed based on a bibliometric analysis, which is why they are described as a result in the data sources and study selection section. A significant body of Chagas disease literature comes from Latin America, as acknowledged in the review by the large number of works from Brazil, and is sometimes described in Spanish and Portuguese. Considering that the author team seems to have both native Spanish and Portuguese speakers, why were non-English articles excluded from the review and meta-analysis? Answer: Thank you for your comment. The exclusion of articles in Spanish and Portuguese was due to methodological reasons to ensure consistency, replicability, and accessibility in data extraction and analysis. Although the team includes native speakers of these languages, incorporating multiple languages could introduce variability in the interpretation of technical terms and statistical data. Moreover, the most impactful scientific databases predominantly index studies in English, facilitating comparability with other meta-analyses and ensuring that our findings are easily verifiable and reusable. Was blood parasitaemia the only outcome considered by the authors, how was it measured in the different studies, did all of them use PCR, microscopy, was this considered in the inclusion/exclusion criteria? Answer: Thank you for your valuable observation. In the data review, we have included a section on Parasitemia Measurement Methods , where we report that parasitemia was assessed using microscopy, PCR, and blood culture across different studies. The methodology used to measure parasitemia was not a criterion for inclusion or exclusion in our analysis. This decision was based on the need to ensure a comprehensive review of the available data, considering that different studies employ various techniques depending on their resources, study design, and diagnostic capabilities. By not restricting the analysis to a specific measurement method, we aimed to capture a broader range of findings, enhancing the generalizability and applicability of our conclusions. An important advantage of animal studies is that they allow a more comprehensive study of the presence of parasites in tissues, such as the digestive tract, the heart or the CNS. This can be done through biopsies, or non-invasive methods using live luminescence, if parasites expressing a luciferase marker are used. An outcome based on the capacity of drugs to clear parasites from deep organs (rather than only clearing peripheral parasitaemia) should be included, given the intermittent nature of parasitaemia in the chronic stages of T. cruzi infection. Answer: Thanks for the observation. The use of parasitemia data alone in this study is based on its relevance as a quantifiable and reproducible indicator of therapeutic efficacy in preclinical models of Chagas disease. Assessing parasitemia reduction allows for a direct measurement of the trypanocidal activity of drugs in peripheral circulation, facilitating comparisons between studies and treatments. Furthermore, since this study is a systematic review and meta-analysis of preclinical treatments in animal models, it focused on standardized data widely reported in the scientific literature, prioritizing studies that documented significant reductions in parasitemia as a criterion for therapeutic success. Although the presence of the parasite in deep tissues is relevant in the chronic phase, obtaining such data requires invasive techniques or specific markers that are not always available or standardized across all analyzed studies. Therefore, the selection of parasitemia as the variable of analysis was based on criteria of data availability, comparability, and methodological robustness within the scope of the study. Similarly, I disagree with excluding combination therapies. Extensive clinical data shows that the efficacy and safety profile of BNZ and NFX, while far from ideal, has not been beaten by any monotherapy evaluated, which makes it attractive to evaluate the potential role of combined schemes. This could also be included as a sub-analysis comparing combined vs individual treatment schemes. Answer: Thank you for your observation. However, it is important to emphasize that the exclusion of combination therapies in this systematic review is based on the need to rigorously assess the efficacy of monotherapy treatments in preclinical models of Chagas disease. Although clinical data exist on the combination of benznidazole (BNZ) and nifurtimox (NFX), monotherapy results are essential to establish a solid comparative baseline and avoid biases arising from uncontrolled pharmacological interactions. Additionally, combination treatments exhibit variability in dosage, duration, and potential synergy, making it difficult to standardize results in preclinical studies. By focusing exclusively on monotherapies, we ensure a more precise evaluation of each compound’s individual efficacy, which is essential for future therapeutic strategies and the development of new molecules. However, my major methodological concerns are related to the fact that the authors do not include any form of risk of bias assessment for any of the studies included in the review, or for the review as a whole. There are currently available tools to evaluate the risk of bias of preclinical studies, I recommend checking the SYRCLE’s risk of bias tool Hooijmans CR et.al., 2014 (Ref 1) , which is consistently used for this purpose. Additionally, there is no assessment of the certainty of the evidence provided by this review, which could be obtained by applying the widely used GRADE criteria, useful for both clinical and preclinical studies. I recommend the authors check reference Romantsik O et.al., 2024 (Ref 2), which provides clear guidelines on how to approach the different segments of a preclinical systematic review. No information on the way potential publication bias was addressed is provided. Answer: Thank you for your valuable feedback and for taking the time to review our manuscript. In response to your concern regarding the risk of bias assessment, we have applied SYRCLE’s Risk of Bias Tool to assess the risk of bias for each of the studies included in our review. Additionally, we have incorporated the GRADE criteria to assess the certainty of the evidence, to ensure a robust evaluation of the evidence’s quality and applicability. Beyond all of this, I don’t seem to completely understand the objective of the meta-analysis. By comparing animals treated with many different compounds, the research question that the authors seem to be wanting to answer is if anti-parasitic treatment (in general) leads to a higher probability of presenting a negative parasitaemia in animals, which seems to have an obvious answer. If this is not the case, I recommend the authors make the research question more explicit in the methods section. Answer: We appreciate your observation and the opportunity to clarify our objective. Our meta-analysis does not simply aim to confirm that antiparasitic treatment reduces parasitemia but rather to compare the efficacy of different compounds in preclinical models of Chagas disease, identifying those with the highest therapeutic potential. Since few treatments have successfully progressed to clinical trials, our study provides a critical evaluation of emerging options and their differences in efficacy depending on the experimental model and disease phase. For the sake of clarity, we will reinforce the explicit formulation of our objective. In the statistical analysis section, authors repeatedly refer to “participants” or “patients”, even though the study focuses exclusively on preclinical studies carried out in animals. The term “subjects” or “animals” should be used here, and consistently throughout the text, to avoid confusion. Answer: Thank you for your valuable observation. We acknowledge the inconsistency in terminology in the statistical analysis section and throughout the text. We will replace “participants” and “patients” with “animals”, to ensure clarity and accuracy. I don’t entirely understand the decision to use a random-effects model for the meta-analysis even though the reported heterogeneity in the different comparisons is consistently very low (reported as 0%). The authors should provide further justification for this decision. Answer: Thank you for your valuable observation. We appreciate your careful review of our methodological choices. We will provide a clearer justification for the use of the random-effects model in the meta-analysis, ensuring that our decision is well-supported despite the low reported heterogeneity. Results: Data extraction sheets of the articles that underwent full-text screening should be provided, along with a table indicating the reason why some of these papers were excluded from the meta-analysis. This could be included as an extra column of the table presented in the supplementary files. Answer: Thank you for your valuable comment. We have followed your recommendation and included the data extraction sheets for the articles that underwent full-text screening, along with an additional column in the table in the supplementary files to indicate the reasons for the exclusion of certain papers from the meta-analysis. We appreciate your feedback and believe it improves the clarity of the manuscript. The comparison for which the RR is calculated is not clearly described in the text. Based on my interpretation of the figures and text, authors are measuring the RR of presenting a negative parasitaemia (TC-) at the end of the study, which would explain the RRs > 1 being associated with more effective treatment (a higher "risk" of showing no parasites in blood). However, this is not disclosed in the text and favors confusion. Answer: We appreciate your observation regarding the clarity of the RR calculation. We have revised the text to explicitly describe the comparison for which the RR is calculated, ensuring clearer interpretation and avoiding potential confusion. Similarly, the nomenclature system used by the authors to represent the different compounds tested in each study is very confusing. I recommend including the name of the compound used in each line of the Forest plot to improve readability. Answer: Thank you for your valuable feedback. We have made the necessary changes and now include the name of each compound used in each line of the Forest plot to improve readability and clarity. Again, the benefit of seeing the aggregated effect of many different treatment schemes seems unclear to me, as it is just showing that some animals who received some anti-parasitic treatments cleared parasitaemia. Answer: The meta-analysis conducted in this study provides a quantitative assessment of the impact of various therapeutic options in animal models of Chagas disease, offering a comprehensive view of their preclinical efficacy. While it is true that some treated animals achieved parasitemia clearance, the value of our study lies in the systematic identification of efficacy patterns across different compounds and the comparative evaluation of their relative performance in different disease phases. Rather than merely confirming that some treatments can reduce parasitemia, this analysis allows us to discern which treatments demonstrate greater consistency and potential for future clinical research, helping to focus efforts on the most promising options for the development of new therapies​. Justification for the decision Systematic reviews of preclinical studies are complex due to the less stringent standardization of methods compared with clinical trials. These reviews often have the objective of prioritizing compounds to move forward for clinical evaluation, and are of value when a large numbers of compounds are being tested in different studies, which does not seem to be the case here. As the authors rightly point out, some compounds whose efficacy was discarded in clinical trials, such as posaconazaole are included in the meta-analysis, while others that show promise to progress to clinical stages, such as benzoxaboroles and cyanotriazoles are not, casting doubts on the applicability of the results. Answer: We appreciate the reviewer’s observation regarding the complexity of systematic reviews of preclinical studies due to methodological variability compared to clinical trials. While many such reviews aim to prioritize compounds for clinical evaluation, our study also serves the critical purpose of identifying therapeutic response patterns and providing a quantitative assessment of treatment efficacy in animal models, which is particularly relevant for neglected diseases like Chagas. The inclusion of posaconazole, despite its unsatisfactory clinical outcomes, remains valuable for understanding its preclinical performance and benchmarking emerging therapies, while the absence of benzoxaboroles and cyanotriazoles was not due to bias but rather the lack of published preclinical studies meeting our inclusion criteria. Our systematic approach, following PRISMA guidelines and registered in INPLASY (INPLASY202430101), ensures transparency and reproducibility, making our findings a valuable contribution to guiding future research and identifying promising candidates for further investigation. I applaud the authors interest in bringing attention to the limited availability of new therapeutic alternatives for Chagas disease, and invite them to keep working on the subject. However, If the authors just want to provide a high-level image of the compounds currently being evaluated in preclinical stages, perhaps a well-structured scoping literature review is a better format to do so, as would also allow them to describe the benefits and pitfalls of more drugs currently under study. Answer: We sincerely appreciate the reviewer’s thoughtful observation and encouragement regarding our work on Chagas disease therapeutics. While a well-structured scoping review could indeed provide a broad overview of compounds under preclinical evaluation, our decision to conduct a systematic review and meta-analysis was driven by the need to quantitatively assess treatment efficacy across multiple studies using standardized methodologies. By adhering to PRISMA guidelines, our study synthesizes available preclinical data, providing statistical insights into parasitemia reduction and the therapeutic potential of different compounds. This quantitative approach not only allows for a more rigorous comparison of treatment effects but also highlights trends, gaps, and inconsistencies in the current literature, which are critical for guiding future research and clinical translation. We believe that this level of analysis is essential for evidence-based decision-making and prioritization of drug candidates for Chagas disease treatment. Nonetheless, we acknowledge the value of a scoping review in complementing our findings by exploring a broader range of therapeutic strategies beyond efficacy metrics, and we appreciate the suggestion for future research directions. If the authors feel that a systematic review of preclinical studies is necessary, or the best way to present their work, then I encourage them to design a concise research question they want to answer, and guarantee that issues such as the risk of bias assessment and the certainty of evidence of the work are adequately addressed, as these are essential for the interpretation of the results. Answer: Thank you for your valuable comments and suggestions. In response to your observation, we have made the necessary changes to the manuscript. We have refined the research question to ensure it is clear and concise, accurately reflecting the study's objective. Additionally, we have conducted a risk of bias assessment using the SYRCLE tool and addressed the certainty of the evidence using the GRADE approach, as you recommended. These improvements enhance the robustness of our analysis and ensure a more reliable interpretation of the results. View more View less Competing Interests No competing interests reply Respond Report a concern Gabaldon JC. Peer Review Report For: Treatment options applied to the preclinical studies using animal models for Chagas Disease: a systematic review and meta-analysis [version 3; peer review: 2 approved] . F1000Research 2025, 13 :885 ( https://doi.org/10.5256/f1000research.165317.r355966) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-885/v1#referee-response-355966 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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