Sorting Transcriptomics Immune Information from Tumor Molecular Features Allows Prediction of Response to Anti-PD1 Therapy in Patients with Advanced Melanoma.

Immunotherapy based on anti-PD1 antibodies has improved the outcome of advanced melanoma. However, prediction of response to immunotherapy remains an unmet need in the field. Tumor PD-L1 expression, mutational burden, gene profiles and microbiome profiles have been proposed as potential markers but are not used in clinical practice. Probabilistic graphical models and classificatory algorithms were used to classify melanoma tumor samples from a TCGA cohort. A cohort of patients with advanced melanoma treated with PD-1 inhibitors was also analyzed. We established that gene expression data can be grouped in two different layers of information: immune and molecular. In the TCGA, the molecular classification provided information on processes such as epidermis development and keratinization, melanogenesis, and extracellular space and membrane. The immune layer classification was able to distinguish between responders and non-responders to immunotherapy in an independent series of patients with advanced melanoma treated with PD-1 inhibitors. We established that the immune information is independent than molecular features of the tumors in melanoma TCGA cohort, and an immune classification of these tumors was established. This immune classification was capable to determine what patients are going to respond to immunotherapy in a new cohort of patients with advanced melanoma treated with PD-1 inhibitors Therefore, this immune signature could be useful to the clinicians to identify those patients who will respond to immunotherapy.

Genetic Profile and Functional Proteomics of Anal Squamous Cell Carcinoma: Proposal for a Molecular Classification.

Anal squamous cell carcinoma is a rare tumor. Chemo-radiotherapy yields a 50% 3-year relapse-free survival rate in advanced anal cancer, so improved predictive markers and therapeutic options are needed. High-throughput proteomics and whole-exome sequencing were performed in 46 paraffin samples from anal squamous cell carcinoma patients. Hierarchical clustering was used to establish groups de novo Then, probabilistic graphical models were used to study the differences between groups of patients at the biological process level. A molecular classification into two groups of patients was established, one group with increased expression of proteins related to adhesion, T lymphocytes and glycolysis; and the other group with increased expression of proteins related to translation and ribosomes. The functional analysis by the probabilistic graphical model showed that these two groups presented differences in metabolism, mitochondria, translation, splicing and adhesion processes. Additionally, these groups showed different frequencies of genetic variants in some genes, such as ATM, SLFN11 and DST Finally, genetic and proteomic characteristics of these groups suggested the use of some possible targeted therapies, such as PARP inhibitors or immunotherapy.

Combining deep learning with SUPPOSe and compressed sensing for SNR-enhanced localization of overlapping emitters.

We present gSUPPOSe, a novel, to the best of our knowledge, gradient-based implementation of the SUPPOSe algorithm that we have developed for the localization of single emitters. We study the performance of gSUPPOSe and compressed sensing STORM (CS-STORM) on simulations of single-molecule localization microscopy (SMLM) images at different fluorophore densities and in a wide range of signal-to-noise ratio conditions. We also study the combination of these methods with prior image denoising by means of a deep convolutional network. Our results show that gSUPPOSe can address the localization of multiple overlapping emitters even at a low number of acquired photons, outperforming CS-STORM in our quantitative analysis and having better computational times. We also demonstrate that image denoising greatly improves CS-STORM, showing the potential of deep learning enhanced localization on existing SMLM algorithms. The software developed in this work is available as open source Python libraries.

Supervised Machine Learning Methods and Hyperspectral Imaging Techniques Jointly Applied for Brain Cancer Classification.

Hyperspectral imaging techniques (HSI) do not require contact with patients and are non-ionizing as well as non-invasive. As a consequence, they have been extensively applied in the medical field. HSI is being combined with machine learning (ML) processes to obtain models to assist in diagnosis. In particular, the combination of these techniques has proven to be a reliable aid in the differentiation of healthy and tumor tissue during brain tumor surgery. ML algorithms such as support vector machine (SVM), random forest (RF) and convolutional neural networks (CNN) are used to make predictions and provide in-vivo visualizations that may assist neurosurgeons in being more precise, hence reducing damages to healthy tissue. In this work, thirteen in-vivo hyperspectral images from twelve different patients with high-grade gliomas (grade III and IV) have been selected to train SVM, RF and CNN classifiers. Five different classes have been defined during the experiments: healthy tissue, tumor, venous blood vessel, arterial blood vessel and dura mater. Overall accuracy (OACC) results vary from 60% to 95% depending on the training conditions. Finally, as far as the contribution of each band to the OACC is concerned, the results obtained in this work are 3.81 times greater than those reported in the literature.

Super-resolved edge detection in optical microscopy images by superposition of virtual point sources.

A new approach to the edge detection problem is presented which is specially designed to achieve high accuracy detection, below instrumental resolution (super resolution) in microscopy images. The method is based in a modified version of a recently published algorithm known as SUPPOSe, which performs a numerical reconstruction of an image as a superposition of virtual point sources. The method was tested in simulated and experimental optical microscopy images and compared to the standard Laplacian of Gaussian algorithm, showing huge differences when the size of the object is smaller than the lateral resolution provided by the instrument.

Computational models applied to metabolomics data hints at the relevance of glutamine metabolism in breast cancer.

BACKGROUND: Metabolomics has a great potential in the development of new biomarkers in cancer and it has experiment recent technical advances. METHODS: In this study, metabolomics and gene expression data from 67 localized (stage I to IIIB) breast cancer tumor samples were analyzed, using (1) probabilistic graphical models to define associations using quantitative data without other a priori information; and (2) Flux Balance Analysis and flux activities to characterize differences in metabolic pathways. RESULTS: On the one hand, both analyses highlighted the importance of glutamine in breast cancer. Moreover, cell experiments showed that treating breast cancer cells with drugs targeting glutamine metabolism significantly affects cell viability. On the other hand, these computational methods suggested some hypotheses and have demonstrated their utility in the analysis of metabolomics data and in associating metabolomics with patient's clinical outcome. CONCLUSIONS: Computational analyses applied to metabolomics data suggested that glutamine metabolism is a relevant process in breast cancer. Cell experiments confirmed this hypothesis. In addition, these computational analyses allow associating metabolomics data with patient prognosis.

An Archetype Query Language interpreter into MongoDB: Managing NoSQL standardized Electronic Health Record extracts systems.

The fast development of today's healthcare and the need to extract new medical knowledge from exponentially-growing volumes of standardized Electronic Health Records data, as required by studies in Precision Medicine, brings up a challenge that may probably only be addressed using NoSQL DBMSs, due to the non-optimal performance of traditional relational DBMSs on standardized data; and these database systems operated by semantic archetype-based query languages, because of the expected generalized extension of standardized EHR systems. An AQL into MongoDB interpreter has been developed to its first version. It translates system-independent AQL queries posed on ISO/EN 13606 standardized EHR extracts into the NoSQL MongoDB query language. The new interpreter has the advantages of both the archetype-based system-independent AQL queries and the dual-model-based standardized EHR extracts stored on document-centric NoSQL DBMSs, such as MongoDB. AQL queries are independent of applications, programming languages and system environments due to the use of the dual model, but EHR extracts featuring this model are best persisted on document-based NoSQL databases. Consequently, the interpreter allows us to query standardized EHR extracts semantically, and also affording optimal performance.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats.

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension.

Biological molecular layer classification of muscle-invasive bladder cancer opens new treatment opportunities.

BACKGROUND: Muscle-invasive bladder tumors are associated with a high risk of relapse and metastasis even after neoadjuvant chemotherapy and radical cystectomy. Therefore, further therapeutic options are needed and molecular characterization of the disease may help to identify new targets. The aim of this study was to characterize muscle-invasive bladder tumors at the molecular level using computational analyses. METHODS: The TCGA cohort of muscle-invasive bladder cancer patients was used to describe these tumors. Probabilistic graphical models, layer analyses based on sparse k-means coupled with Consensus Cluster, and Flux Balance Analysis were applied to characterize muscle-invasive bladder tumors at a functional level. RESULTS: Luminal and Basal groups were identified, and an immune molecular layer with independent value was also described. Luminal tumors showed decreased activity in the nodes of epidermis development and extracellular matrix, and increased activity in the node of steroid metabolism leading to a higher expression of the androgen receptor. This fact points to the androgen receptor as a therapeutic target in this group. Basal tumors were highly proliferative according to Flux Balance Analysis, which makes these tumors good candidates for neoadjuvant chemotherapy. The Immune-high group showed a higher degree of expression of immune biomarkers, suggesting that this group may benefit from immune therapy. CONCLUSIONS: Our approach, based on layer analyses, established a Luminal group candidate for therapy with androgen receptor inhibitors, a proliferative Basal group which seems to be a good candidate for chemotherapy, and an immune-high group candidate for immunotherapy.

Computational metabolism modeling predicts risk of distant relapse-free survival in breast cancer patients.

Aim: Differences in metabolism among breast cancer subtypes suggest that metabolism plays an important role in this disease. Flux balance analysis is used to explore these differences as well as drug response. Materials & methods: Proteomics data from breast tumors were obtained by mass-spectrometry. Flux balance analysis was performed to study metabolic networks. Flux activities from metabolic pathways were calculated and used to build prognostic models. Results: Flux activities of vitamin A, tetrahydrobiopterin and ß-alanine metabolism pathways split our population into low- and high-risk patients. Additionally, flux activities of glycolysis and glutamate metabolism split triple negative tumors into low- and high-risk groups. Conclusion: Flux activities summarize flux balance analysis data and can be associated with prognosis in cancer.

Three-dimensional confocal Raman temperature characterization of electrokinetically pumped microchannels.

A novel method for noninvasive, three-dimensional temperature characterization in microfluidic devices is presented. A specially designed confocal microscope was built and used to measure water temperature by sensing the Raman spectrum variations of the liquid. This is achieved by splitting the spectrum in the isosbestic point and detecting it with two photon counters. The difference between the signals of each detector divided by their sum shows a linear dependence with temperature. A fiber-coupled laser beam is used to pump the sample with 25 mW of optical power at 405 nm. This allows a 0.8 K temperature precision and a 9 µm axial resolution using a 1 s integration time. These features make temperature profiling in all dimensions possible, in contrast with previous methods, where the information present in the height of the channel is lost and the whole spectrum needs to be recovered before computing the sample temperature. Using this technique, different geometries of polydimethylsiloxane microchannels sealed with a 150 µm thick glass coverslip were studied, showing that heat flow through the glass is the dominating dissipation mechanism and defines the maximum temperature in the channel. The results show good agreement with previous work found in the literature.

Increased size and cellularity of advanced atherosclerotic lesions in mice with endothelial overexpression of the human TRPC3 channel.

In previous in vitro studies, we showed that Transient Receptor Potential Canonical 3 (TRPC3), a calcium-permeable, nonselective cation channel endowed with high constitutive function, is an obligatory component of the inflammatory signaling that controls expression of the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion to coronary artery endothelial cells. Also, TRPC3 expression in these cells was found to be up-regulated by proatherogenic factors, which enhanced inflammation and VCAM-1 expression. However, it remained to be determined whether these in vitro findings were of relevance to atherosclerotic lesion development in vivo. To answer this important question in the present work, we generated mice with endothelial-specific overexpression of human TRPC3 in an Apoe knockout background (TgEST3ApoeKO) and examined lesions in the aortic sinus following 10 and 16 wk on a high-fat diet. No significant differences were found in size or complexity of early stage lesions (10 wk). However, advanced plaques (16 wk) from TgEST3ApoeKO mice exhibited a significant increase in size and macrophage content compared with nontransgenic littermate controls. Remarkably, this change was correlated with increased VCAM-1 and phospho-IkBα immunoreactivity along the endothelial lining of lesions from transgenic animals compared with controls. These findings validate the in vivo relevance of previous in vitro findings and represent, to our knowledge, the first in vivo evidence for a proatherogenic role of endothelial TRPC3.

Is Latin America Ready to Identify Anaplastic Large Cell Lymphoma in Breast Implants Patients? Regional Encounter During the National Plastic Surgery Meeting in Cancun, Mexico.

INTRODUCTION: Anaplastic large cell lymphoma associated with breast implants is receiving increased attention. Most cases have been reported in Europe, North America (USA and Canada), Australia and New Zealand. Fewer cases have been reported in Latin America (including Mexico), Africa and Asia. METHODS: This report was delivered during our national plastic surgery meeting in Cancun in May 2017. Before the meeting, two participants reviewed the literature. The review was performed using the following information sources: PubMed, Embase, Cochrane, Fisterra, Google Scholar and LILACS, with entries from 1980 to August 2015 in several languages (English, Spanish, French and Portuguese). The results were revealed during the meeting to the other participants. The consensus was divided into two parts. The first part included an open-ended question regarding the incidence and prevalence of the problem. The second part included clinical scenarios with different items that were rated by the participants. After this activity, accordance among the responses was evaluated. RESULTS: Seven cases were reported during the meeting (3 from Mexico, 3 from Chile and 1 from Argentina). Fifty percent of the participants reported consulting with guidelines and clinical centers to help with potential cases. Most agreed that further studies must be done in cases of chronic seroma where the capsule plays an important role. DISCUSSION: A current debate exists about the incidence of this problem in Latin America because we did not report the same number of cases as Europe, Australia or North America. More studies are required to determine the differences among reports in Latin America. CONCLUSION: Most representatives agreed that further studies must be done. Concern is increasing, and the problem is known. Other factors involved may be considered, and the problem must not be ignored. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Evidence for constitutive bone morphogenetic protein-2 secretion by M1 macrophages: Constitutive auto/paracrine osteogenic signaling by BMP-2 in M1 macrophages.

Mechanisms mediating vascular calcification recapitulate osteogenic processes encompassing bone formation and imply participation of bone related proteins such as bone morphogenetic protein-2 (BMP-2). Macrophages are amongst the cells that contribute to vascular ossification by releasing cytokines that induce an osteogenic program in vascular smooth muscle cells, and also by becoming themselves osteoclast-like cells. In inflammatory vascular disease, the macrophage population in the vascular wall is diverse, with the M1 or inflammatory, and the M2 or anti-inflammatory macrophage types being dominant. Yet, the osteogenic potential of M1 and M2 macrophages remains unknown. Prompted by recent studies from our laboratory showing that in macrophages the Transient Receptor Potential Canonical 3 (TRPC3) channel contributes to endoplasmic reticulum (ER) stress-induced apoptosis in M1, but not in M2 macrophages, and given the strong relationship between ER stress and vascular calcification, we wished to examine whether TRPC3 would play a role in the osteogenic signaling of polarized macrophages. The findings reported here indicate that a constitutive BMP-2-dependent signaling operates in M1 macrophages, which is not affected by deletion of Trpc3 and is not subject to regulation by ER stress. Our studies suggest operation of an auto/paracrine mechanism by which BMP-2 secreted by M1 macrophages maintains constitutive activation of a BMP-2 receptor/SMAD1/5 signaling axis.

Early steatohepatitis in hyperlipidemic mice with endothelial-specific gain of TRPC3 function precedes changes in aortic atherosclerosis.

Nonalcoholic fatty liver disease (NAFLD) and its more advanced form nonalcoholic steatohepatitis (NASH) are the most common chronic liver diseases in developed countries. Moreover, NAFLD and NASH are considerable risk factors for atherosclerosis, the most frequent vascular pathology in these and other metabolic diseases. Despite this strong connection, current knowledge of the relationship between NAFLD/NASH and atherosclerosis is scarce. Recently, we studied hyperlipidemic Apoe knockout mice with endothelial-specific gain of transient receptor potential canonical 3 channel function (TgESTRPC3/ApoeKO) and found that these animals had increased burden of advanced aortic atherosclerosis (16 wk on high-fat diet) compared with nontransgenic ApoeKO littermate controls (non-Tg/ApoeKO), whereas early lesions (10 wk on high-fat diet) were not different. Here, we report that at the early stage when differences in aortic atherosclerosis are not yet manifest, the livers of TgESTRPC3/ApoeKO mice show steatosis, fibrosis, and altered hepatic enzymes compared with non-Tg/ApoeKO animals. Because differences in liver pathology were noticeable long before differences in atherosclerosis were evident, our studies suggest that TRPC3-related endothelial mechanisms that promote steatohepatitis may also contribute to atherosclerosis progression. In vitro, downregulation of TRPC3 in liver sinusoid endothelial cells reduces their susceptibility to endoplasmic reticulum stress-induced apoptosis, suggesting that a proapoptotic effect of TRPC3 may add to other fibrogenic factors in vivo. These novel findings show a positive association between augmented expression of an endothelial TRPC channel, development of early steatohepatitis, and atherosclerotic burden in a hyperlipidemic mouse model of NAFLD fed conventional Western-type diet.

On the Roles of the Transient Receptor Potential Canonical 3 (TRPC3) Channel in Endothelium and Macrophages: Implications in Atherosclerosis.

In the cardiovascular and hematopoietic systems the Transient Receptor Potential Canonical 3 (TRPC3) channel has a well-recognized role in a number of signaling mechanisms that impact the function of diverse cells and tissues in physiology and disease. The latter includes, but is not limited to, molecular and cellular mechanisms associated to the pathogenesis of cardiac hypertrophy, hypertension and endothelial dysfunction. Despite several of these functions being closely related to atherorelevant mechanisms, the potential roles of TRPC3 in atherosclerosis, the major cause of coronary artery disease, have remained largely unexplored. Over recent years, a series of studies from the authors' laboratory revealed novel functions of TRPC3 in mechanisms related to endothelial inflammation, monocyte adhesion to endothelium and survival and apoptosis of macrophages. The relevance of these new TRPC3 functions to atherogenesis has recently began to receive validation through studies in mouse models of atherosclerosis with conditional gain or loss of TRPC3 function. This chapter summarizes these novel findings and provides a discussion of their impact in the context of atherosclerosis, in an attempt to delineate a framework for further exploration of this terra incognita in the TRPC field.

Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages.

Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe(-/-) mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3(+/+) bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3(-/-)Apoe(-/-) animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages.

Structured Excitation Energy Transfer: Tracking Exciton Diffusion below Sunlight Intensity.

With the increasing demand for new materials for light-harvesting applications, spatiotemporal microscopy techniques are receiving increasing attention as they allow direct observation of the nanoscale diffusion of excitons. However, the use of pulsed and tightly focused laser beams generates light intensities far above those expected under sunlight illumination, leading to photodamage and nonlinear effects that seriously limit the accuracy and applicability of these techniques, especially in biological or atomically thin materials. In this work, we present a novel spatiotemporal microscopy technique that exploits structured excitation in order to dramatically decrease the excitation intensity, up to 10,000-fold when compared with previously reported spatiotemporal photoluminescence microscopy experiments. We tested our method in two different systems, reporting the first exciton diffusion measurement at illumination conditions below sunlight, both considering average power and peak exciton densities in an organic photovoltaic sample (Y6), where we tracked the excitons for up to five recombination lifetimes. Next, nanometer-scale energy transport was directly observed for the first time in both space and time in a printed monolayer of the light-harvesting complex 2 from purple bacteria.

Ceacam1 deletion causes vascular alterations in large vessels.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Molecular diagnosis of Chagas disease: a systematic review and meta-analysis.

BACKGROUND: The complexity of the Chagas disease and its phases is impossible to have a unique test for both phases and a lot of different epidemiological scenarios. Currently, serology is the reference standard technique; occasionally, results are inconclusive, and a different diagnostic technique is needed. Some guidelines recommend molecular testing. A systematic review and meta-analysis of available molecular tools/techniques for the diagnosis of Chagas disease was performed to measure their heterogeneity and efficacy in detecting Trypanosoma cruzi infection in blood samples. METHODS: A systematic review was conducted up to July 27, 2022, including studies published in international databases. Inclusion and exclusion criteria were defined to select eligible studies. Data were extracted and presented according to PRISMA 2020 guidelines. Study quality was assessed using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). A random-effects model was used to calculate pooled sensitivity, specificity, and diagnostic odds ratio (DOR). Forest plots and a summary of the receiving operating characteristics (SROC) curves displayed the outcomes. Heterogeneity was determined by I2 and Tau2 statistics and P values. Funnel plots and Deek's test were used to assess publication bias. A quantitative meta-analysis of the different outcomes in the two different clinical phases was performed. RESULTS: We identified 858 records and selected 32 papers. Studies pertained to endemic countries and nonendemic areas with adult and paediatric populations. The sample sizes ranged from 17 to 708 patients. There were no concerns regarding the risk of bias and applicability of all included studies. A positive and nonsignificant correlation coefficient (S = 0.020; P = 0.992) was obtained in the set of studies that evaluated diagnostic tests in the acute phase population (ACD). A positive and significant correlation coefficient (S = 0.597; P < 0.000) was obtained in the case of studies performed in the chronic phase population (CCD). This resulted in high heterogeneity between studies, with the master mix origin and guanidine addition representing significant sources. INTERPRETATION/CONCLUSIONS AND RELEVANCE: The results described in this meta-analysis (qualitative and quantitative analyses) do not allow the selection of the optimal protocol of molecular method for the study of Trypanosoma cruzi infection in any of its phases, among other reasons due to the complexity of this infection. Continuous analysis and optimization of the different molecular techniques is crucial to implement this efficient diagnosis in endemic areas.