PREreview of "Urban Leishmaniasis in the Amazon: A One Health Framework Integrating Diagnostics, Microdiversity, Ultrastructure, and Extracellular Vesicles"

Abstract

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/22955348. Reviewers: Diptarup Mallick, Hala Taha Elbashir, Lukman Boyi Adamu,Prerna Mehta, Shubham Kalamkar, Joseph Odunayo Igbokwe, Egide KAYIRANGA, Deepak Vitthal Sawant, Suhagiya Jinay Jayantibhai, Muhammad Murtala Yusuf , T Susitha, Dr. Mwafaq Ramzi Haji, Pavithra J., Gliday Yuka, Rita Ofosuaa Agyemang Ntim, Madhubala Kumari, Mekuriaw Dereje Tekele, Dongsogo Julius (PhD) Brief summary of the study The manuscript investigates urban visceral leishmaniasis in the Brazilian Amazon using an integrated One Health framework that combines serological/parasitological diagnosis, molecular detection, SSU rDNA sequencing, parasite microdiversity, ultrastructural analysis, and extracellular vesicle (EV) biology. The principal dataset includes 1,499 dogs from Parauapebas, supplemented by 130 dogs from Belém, Marabá, and Colares for molecular and phylogenetic analyses. The study reports substantial diagnostic discordance, complementary detection by blood and conjunctival swabs, low-level SSU rDNA polymorphisms, ultrastructural changes, and increased EV release under thermal stress. Overall, the manuscript addresses an important One Health problem and brings together several complementary biological dimensions. However, the current version requires substantial clarification of experimental design, analytical methods, quantitative results, and the distinction between directly demonstrated findings and mechanistic interpretations. In particular, several conclusions concerning EV cargo, immune modulation, diagnostic discordance, parasite adaptation, and genetic stability appear stronger than what is directly demonstrated by the described experiments. Major comments The study design and integration of the different datasets require much clearer explanation. The manuscript combines 1,499 dogs from Parauapebas with a separate dataset of 130 dogs from Belém, Marabá, and Colares collected during different periods (2015–2018 versus 2019–2020). The authors should explicitly explain whether these datasets represent the same study population, independent cohorts, or previously collected samples. The inclusion/exclusion criteria, sampling strategy, demographic characteristics, clinical status, and number of samples available for each assay should be presented separately. The diagnostic analysis needs a complete 2 × 2 contingency table. The manuscript reports 60.8% TR DPP® positivity, 45.7% parasitological positivity, κ = 0.6483, and 269 discordant results. However, the exact numbers of true-positive, true-negative, false-positive, and false-negative observations should be provided. This is particularly important because Cohen's kappa depends on the underlying contingency table. Sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence intervals should also be considered, provided an appropriate reference standard is defined. The interpretation of diagnostic discordance is currently too mechanistic. The authors attribute TR DPP®-positive/parasitology-negative results to different biological windows of infection and subsequently suggest that EV-mediated mechanisms may explain serological positivity in the absence of detectable tissue parasitism. However, the study does not appear to directly demonstrate that EVs caused or contributed to the discordance. This should be presented as a hypothesis rather than an experimentally established mechanism. Alternative explanations, including differences in diagnostic sensitivity, sampling limitations, parasite burden, and persistence of antibodies, should be explicitly discussed. The molecular methodology is insufficiently detailed for reproducibility. The manuscript states that kDNA and two SSU rDNA regions were amplified but does not provide sufficient PCR information, including primer sequences, reaction volumes, concentrations, cycling conditions, controls, expected amplicon sizes with sufficient methodological detail, and criteria for calling a sample positive. These details are essential for a molecular epidemiological study. The microdiversity analysis requires stronger methodological justification. The manuscript interprets C→T substitutions and Y/R ambiguities as evidence of intraregional microdiversity. However, chromatogram ambiguity can also arise from mixed templates, sequencing quality, PCR artifacts, or insufficiently resolved base calls. The authors should provide sequence quality criteria, chromatograms or supplementary evidence for representative polymorphisms, sequence accession numbers, coverage/sequence quality information, and criteria used to distinguish genuine variants from ambiguous sequencing signals. The statement that the parasite population is "genetically stable" should be moderated. The phylogenetic analysis is based on SSU rDNA, a relatively conserved marker, and appears to involve a limited number of sequences. Uniformly short branches and high bootstrap values do not by themselves establish genome-wide genetic stability. The conclusion should therefore be restricted to the SSU rDNA sequences analyzed rather than generalized to the overall L. infantum population. The phylogenetic methodology should be expanded. The manuscript reports neighbor-joining analysis with Kimura-2-parameter distances and 1,000 bootstrap replicates. The authors should state the software and version used, sequence alignment parameters, reference/outgroup sequences, accession numbers, treatment of ambiguous positions, and rationale for selecting neighbor-joining. A complementary maximum-likelihood analysis could provide stronger support for the phylogenetic conclusions. The EV experiments are inadequately described and currently support conclusions beyond the reported data. The manuscript states that promastigotes were exposed to baseline, mild-stress, and heat-stress conditions and that EV concentrations were measured by NTA. The exact temperatures, exposure durations, culture conditions, biological and technical replicates, EV isolation/purification method, particle-size distributions, instrument settings, and statistical comparisons are not reported. These details are necessary to evaluate the validity of the EV findings. EV characterization should be strengthened according to contemporary EV research standards. The manuscript discusses EV cargo consisting of proteins, lipids, and RNAs, but the presented methodology does not indicate that cargo was actually experimentally characterized in this study. If these cargo categories are derived from previous literature rather than measured experimentally, this distinction must be made explicit. Similarly, morphological observations alone should not be interpreted as definitive evidence of specific EV populations without appropriate characterization. The ultrastructural observations need quantitative analysis. TEM findings are described qualitatively as lipid droplets, vesicle-like structures, vacuolar lipid inclusions, and lysosomal-related organelles. The manuscript would be substantially stronger if the authors reported the number of cells examined, number of independent preparations, criteria for identifying each structure, representative micrographs with scale bars, and quantitative comparisons between experimental groups or parasite stages. The statistical analysis is too briefly described. The statistical section lists kappa, chi-square tests, logistic regression, contingency tables, and descriptive statistics but does not specify which variables were entered into each model, how continuous variables were handled, model assumptions, variable-selection strategy, adjustment for confounders, treatment of missing observations, or model diagnostics. These details are essential, particularly for the claimed association between infection and ecological/clinical variables. The manuscript should address clustering and spatial dependence. Dogs were sampled across administrative zones and municipalities, meaning observations may not be statistically independent. If logistic regression was used for risk-factor assessment, the authors should explain whether municipality/administrative zone clustering was considered. A multilevel or spatially informed approach may be appropriate depending on the underlying sampling structure. The ecological interpretation is not adequately supported by measured environmental variables. The manuscript repeatedly attributes differences in infection to ecological gradients, vector distribution, urban structure, and environmental heterogeneity. However, the methods do not describe direct measurement of vector abundance, land use, socioeconomic variables, vegetation, climate, or other environmental predictors. The discussion should distinguish between observed spatial heterogeneity and hypothesized ecological explanations. The One Health framing should be better operationalized. The manuscript uses the One Health concept throughout, but the actual study appears primarily focused on canine infection and parasite biology. A stronger One Health interpretation would explicitly connect animal findings with vector, environmental, and human-health surveillance implications while clearly identifying which components were actually measured and which are proposed implications. Several causal statements should be revised. Statements such as EVs "influencing host responses," contributing to diagnostic discordance, or ultrastructural signatures demonstrating specific adaptive mechanisms go beyond the direct experimental evidence presented. The authors should consistently use language such as "is consistent with," "may indicate," or "supports the hypothesis that" where direct mechanistic experiments were not performed. Minor comments The title is informative but very broad because it combines epidemiology, diagnostics, molecula