As a continuation of the project aimed at searching for new chemotherapeuticagents against Chagas disease or American trypanosomiasis, new selenocyanate derivatives are designed, synthesized, and biologically evaluated against the clinically more relevant dividing amastigote form of Trypanosoma cruzi, the etiologic agent of this illness. Furthermore, as all the title compounds are fluorine-containing molecules, it seemed to be reasonable to explore the role of fluorine atoms in the aromatic system and to determine the optimal position at the terminal phenoxy group, and therefore, various regioisomers are prepared. The conformationally restricted selenocyates structurally related to WC-9Se exhibited improved antiparasitic activity compared to the lead drugs, Out to be extremely potent inhibitors of T. cruzi growth. In particular, (±)-5-(3-fluorophenoxy)-2-(selenocyanatomethyl)-2,3-dihydrobenzofuran exhibited an EC50 value of 0.032 µM, which resulted in the most potent selenocyanate developed in the laboratory. The presence of the fluorine atom together with the rigidity of the molecules are beneficial for the anti-T. cruzi effect. The resulting antiparasitic activity provides further insight into the role of the selenocyanate group in its effective and putative anti-T. cruzi action.
Hugo S Steingrüber, Mayara S Bertolini, Margarita M Vallejos, Sergio H Szajnman, Roberto Docampo, Juan B Rodriguez. ChemMedChem. 2025 Nov 11:e202500658. doi: 10.1002/cmdc.202500658.
New test protocol can detect low levels of Trypanosoma cruzi, the parasite that causes Chagas disease
Researchers at the University of Georgia’s Center for Tropical and Emerging Global Diseases have developed the first test to determine whether treatment for Chagas disease was effective.
An estimated 6 million to 8 million people worldwide are infected with Trypanosoma cruzi, the parasite that causes Chagas disease.
“Currently during drug trials, we can only determine if a drug fails,” said Rick Tarleton, Regents’ Professor in the UGA Franklin College of Arts and Sciences. “A test of cure can indicate if a drug succeeds in clearing the infection.”
Rick Tarleton of the Center for Tropical and Emerging Global Diseases
Part of the problem in determining if T. cruzi infection is cured by treatment is that the immune system is often very good at controlling the infection. Current tests are not sensitive enough to detect low levels of parasites.
“If you have a cup a tea with a little bit of tea leaf in it you may not get a tea leaf in every sip,” Tarleton said. “When there are so few parasites in the blood stream, it decreases the chances that a blood draw will contain any.
“We’ve taken two samples from the same individual at the same time—one sample tests positive, and the other tests negative. Which is right?”
Thousands die from Chagas each year
Chagas disease kills more than 10,000 people every year, mainly in Central and South America. But it is also a concern in the United States, where the Centers for Disease Control and Prevention estimates there are 280,000 people living with this disease.
And it’s not only humans that suffer from this disease. Many mammals, including wildlife and dogs, can also become infected.
The Tarleton group conducted large-scale PCR testing of samples from naturally infected macaque monkeys, dogs and humans. The team also fragmented the DNA to more evenly distribute it within the sample. Standard PCR testing doesn’t fragment DNA.
“If we go back to Rick’s tea leaf example, it’s like taking the whole tea leaf, breaking it up into tiny bits and then stirring the tea before taking a sip,” said Brooke White, lead researcher of the study. “This increases the chances of detecting DNA.”
New test accurately detects parasite infection in monkeys, dogs and humans
The naturally infected macaques were monitored over 12 months with monthly blood tests. A subset also had blood samples drawn seven times over four weeks. In addition to the exhaustive PCR testing, the researchers grew T. cruzi cultures from the blood samples, which confirmed that the new protocol accurately detects infection even when the parasite number is very low.
“Since the macaques acquired the infection in the same way as humans and dogs and their disease progression is the same, we are confident that this test will work in other species,” said Tarleton.
Collaborators at Texas A&M and in Argentina also provided naturally infected dog and human samples, respectively. The researchers saw similar results to the macaques.
A need for better drug treatment for Chagas disease
There is a need for new drug treatments for Chagas disease. But without a true test of cure, researchers only know what does not work. While the new protocol is effective, the researchers noted that it is also labor intensive and time consuming, which translates into being costly.
As part of the study, the researchers sought out technologies that could make the process faster and cheaper.
“This test of cure is a real game changer for drug treatment studies,” said White. “We are already working with other research groups in hopes of creating a quicker and cheaper method of testing parasite load in their drug treatment studies in macaques, dogs and humans.”
The researchers began collaborating with Countable Labs whose new technology allows for larger samples to be assayed faster. Reducing costs and increasing efficiency makes it much more likely for this test to be used in a clinical setting.
“Our goal now is to move this test out of the research lab and into a clinical diagnostic lab where it will be widely accessible for detecting human and dog infections and tracking treatment outcomes,” said Tarleton.
T. cruzi-specific CD8+ T cells cross-react with multiple flagellar peptides.
CD8+ T cells are key effectors in immune control of Trypanosoma cruzi infection. Within C57BL/6 mice, the T. cruzi-specific CD8+ T cell response is largely comprised of T cells recognizing trans-sialidase (TS)- and mucin-encoded epitopes. Despite their immunodominance, these epitope-specific CD8+ T cells are entirely dispensable for immune control. In a screen for epitopes inducing “protective” CD8+ T cells, we uncovered a high level of cross-reactivity within the TSKb20-specific CD8+ T cell response. This cross-reactivity was driven by the TSKb20 epitope itself and not the infection. TCR sequencing defined key characteristics of the TSKb20 repertoire including biased TRBV12-1/12-2 and TRBJ2-1/2-7 gene usage and a highly dominant CDR3β motif. The dispensability of the TSKb20 response in the control of T. cruzi infection along with the broad reactivity of this T cell population prompted us to assess the relative effector capacity of TSKb20 T cells at the site of reinfection. Similar to other activated CD8+ T cells at the site, TSKb20-specific CD8+ T cells expressed transcriptional patterns associated with effector function, suggesting that TSKb20 T cells are capable and likely participants in parasite control. These results indicate that broad TCR reactivity does not compromise the ability of TSKb20-specific T cells to develop into phenotypically functional effectors. Additionally, the failure to identify individual parasite epitopes capable of driving a protective CD8+ T cell response challenges the paradigm that individual T. cruzi epitopes, including highly immunodominant ones, are critical to or exploitable for the potent recognition of T. cruzi-infected host cells and infection control.
Human infections by Trypanosoma cruzi propagate via its blood-feeding triatomine vector. Investigating parasite-vector interactions depends upon robust techniques to rear insects and analyze infections. Here, we present a protocol for laboratory rearing and infection tracking of Rhodnius prolixus. We describe steps for housing, feeding, and sorting strategies using 3D-printable designs. We also detail procedures for gut dissection, fecal collection, and parasite re-isolation. This protocol describes techniques that support efforts to understand and mitigate vector-mediated Chagas disease transmission.
Dormant and metabolically active T. cruzi amastigotes.
Trypanosoma cruzi typically establishes a life-long infection in its mammalian hosts, causing the destruction of muscle tissues and ultimately resulting in potentially fatal Chagas disease. In this review, we consider the array of avoidance mechanisms that allow for T. cruzi persistence, many of which are unconventional among protozoan pathogens but which collectively are highly effective in the face of otherwise potent host immune responses. We also reflect on the phenomenon of dormancy in T. cruzi amastigotes, which is likely not involved in the long-term persistence of infection. Lastly, we consider how these phenomena of persistence and dormancy complicate the effectiveness of potential therapeutic interventions to prevent Chagas disease.
The parasite that causes Chagas disease can cause inflammation in the heart, as shown in white here in a mouse model. (Image courtesy of Fernando Sanchez)
Supported by almost $4 million in new funding, researchers at the University of Georgia and Texas A&M are using improved detection and treatment methods to understand Chagas disease, a serious, often overlooked illness affecting both dogs and humans.
A team of researchers at the University of Georgia and Texas A&M University has received more than $4 million from federal and nongovernmental organizations to support research on Chagas disease.
The research will consist of multiple projects focused on the disease’s prevalence, diagnostics to detect the parasite that causes the disease, and treatment protocols to prevent infection and disease in dogs. The ultimate goal is to use the findings to help people as well.
Funded by a $3 million grant from the National Institutes of Health, UGA’s Rick Tarleton will co-lead a project focused on strategies to detect, treat and monitor treatment outcomes in dogs in Texas. The goal is to establish the best practices that prevent the development of cardiac disease, one severe potential side effect of Chagas disease, and to establish resistance to possible future infection.
The researchers will work with dogs that were naturally infected with Chagas disease. Because the disease presents similarly in dogs as in humans, dogs are a good model for examining the effectiveness of the treatment.
Rick Tarleton of the Center for Tropical and Emerging Global Diseases
“There are a number of important questions related to treatment efficacy and the protection that cured subjects have from future infection that cannot be easily addressed in humans but can be in these dog populations that are under intense transmission pressure in Texas,” said Tarleton, Regents Professor in UGA’s Franklin College of Arts and Sciences.
A growing threat to dog, human health
Tens of millions of people across the Americas have Chagas disease.
Chagas disease is a largely neglected disease. The parasite that causes it, Trypanosoma cruzi, is spread by blood-sucking insects known as kissing bugs, which can be found throughout North, Central and South America.
The disease, which commonly develops in humans and dogs, as well as many other mammals, often goes unnoticed in early stages. But a chronic infection can lead to serious heart and digestive system problems, making early diagnosis and prompt treatment important.
Although most human cases of Chagas disease are reported from South and Central America and Mexico, the parasite and its insect vector are found in abundance in the southern United States. Outdoor pets — particularly working dogs — face especially high risks of infection.
“These areas we are working in have 20% to 30% rates of new infections per year,” Tarleton said. “Those tend to be severe infections where the dogs either die or develop a disease that makes them unable to work.”
Kissing bugs can carry the parasite that causes Chagas disease, and they’re particularly abundant in Texas and the southern U.S. (Getty Images)
Texas has become a hotspot of kissing bugs.
“Unfortunately, Texas has emerged as a hotspot of infected kissing bugs, infected wildlife and infected dogs across the landscape,” said Dr. Sarah Hamer, a professor in the Texas A&M College of Veterinary Medicine and a primary investigator on the projects.
“These projects will advance Chagas disease research to understand the process of natural infections, disease and effect of treatments,” Hamer said. “These projects combine many aspects of biomedical research. We’re conducting field and laboratory research, treating dogs, measuring clinical outcomes and studying ecological factors. It’s truly a ‘One Health’ approach.”
A One Health approach to Chagas disease research
Diagnosing Chagas is complicated — in both people and canines. False negatives aren’t unheard of, leading people to not know they or their pets are infected. And that delays treatment.
Even when the disease is diagnosed promptly, treating the condition can be challenging.
The go-to medications used to treat Chagas, as currently applied, are not reliably effective. But they’re currently the only treatment option. Tarleton’s previous work in mice and other species show that their effectiveness can be improved by altering the dosing regimen.
To address these issues, the researchers will track infected canines using a combination approach with sensitive tests to detect both the parasite DNA and the body’s response to infection. The team will simultaneously test a revised dosing strategy for the current antiparasitic treatment, providing fewer but high-level doses and extending the administration period to improve effectiveness.
Recording health information from such a large population of dogs will hopefully help us understand why the disease develops in different ways.” —Dr. Sarah Hamer, Texas A&M
“The drug we’re using is an existing treatment for Chagas disease in humans,” said Dr. Ashley Saunders, a professor in the Texas A&M College of Veterinary Medicine and a primary investigator on the projects. “But Dr. Tarleton has shown that the parasites aren’t susceptible to this drug when they’re dormant. By changing the drug delivery protocol to dosing over a longer period of time, when the dormant parasites become active again, they are killed by the drug.”
In a related study funded by the United States Department of Homeland Security, the researchers will also monitor DHS-owned working dogs that are often trained in areas where Chagas disease is prevalent. The goal is to understand how the dogs are exposed to the disease as well as the impacts it can have on the canines’ heart health, as well as to develop monitoring and treatment strategies for these working dogs.
“One of the reasons that monitoring dogs is so helpful is because Chagas disease can produce so many different subsets of health problems,” Saunders said. “Some dogs end up with a heart abnormality, but a large number continue living and working happily for many years. Others will die quite suddenly, before anyone knew they had the disease.”
“Recording health information from such a large population of dogs will hopefully help us understand why the disease develops in different ways,” Hamer said.
Advancing canine Chagas disease management
With continued support from the American Kennel Club Canine Health Foundation, the team will treat and monitor individual pet dogs brought to Texas A&M’s Small Animal Teaching Hospital while developing a staging system for Chagas disease in dogs.
“The staging system we develop will help us to categorize the severity of disease, making it easier to determine which dogs will benefit most from drug treatment,” Saunders said. “This scoring system will work hand-in-hand with our improved diagnostic and treatment plan.”
Fig 1. Conditional knockout of TcPiezo1 and subcellular localization.
Trypanosoma cruzi, the causative agent of Chagas disease, is a parasitic protist that affects millions of people worldwide. Currently there are no fully effective drugs or vaccines available. Contact of T. cruzi infective forms with their host cells or with the extracellular matrix increases their intracellular Ca2+ concentration suggesting a mechano-transduction process. We report here that T. cruzi possesses two distinct mechanosensitive Piezo channels, named TcPiezo1 and TcPiezo2, with different subcellular localizations but similarly essential for normal proliferation, differentiation, and infectivity. While TcPiezo1 localizes to the plasma membrane, TcPiezo2 localizes to the lysosomes. Downregulation of TcPiezo1 expression by a novel ligand-regulated hammerhead ribozyme (HHR) significantly inhibited Ca2+ entry in cells expressing a genetically encoded Ca2+ indicator while downregulation of TcPiezo2 expression inhibited Ca2+ release from lysosomes, which are now identified as novel acidic Ca2+ stores in trypanosomes. The channels are activated by contact with extracellular matrix and by hypoosmotic stress. The results establish the essentiality of Piezo channels for the life cycle and Ca2+ homeostasis of T. cruzi and a novel lysosomal localization for a Piezo channel in eukaryotes.
Infection with the protozoan parasite Trypanosoma cruzi is generally well-controlled by host immune responses, but appears to be rarely eliminated. The resulting persistent, low-level infection results in cumulative tissue damage with the greatest impact generally in the heart in the form of chagasic cardiomyopathy. The relative success in immune control of T. cruzi infection usually averts acute phase death but has the negative consequence that the low-level presence of T. cruzi in hosts is challenging to detect unequivocally. Thus, it is difficult to identify those who are actively infected and, as well, problematic to gauge the impact of treatment, particularly in the evaluation of the relative efficacy of new drugs. In this study, we employ DNA fragmentation and high numbers of replicate PCR reaction (‘deep-sampling’) and to extend the quantitative range of detecting T. cruzi in blood by at least three orders of magnitude relative to current protocols. When combined with sampling blood at multiple time points, deep sampling of fragmented DNA allowed for detection of T. cruzi in all infected hosts in multiple host species, including humans, macaques, and dogs. In addition, we provide evidence for a number of characteristics not previously rigorously quantified in the population of hosts with naturally acquired T. cruzi infection, including, a >6 log variation between chronically infected individuals in the stable parasite levels, a continuing decline in parasite load during the second and third years of infection in some hosts, and the potential for parasite load to change dramatically when health conditions change. Although requiring strict adherence to contamination-prevention protocols and significant resources, deep-sampling PCR provides an important new tool for assessing therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease.
Brooke E White, Carolyn L Hodo, Sarah Hamer, Ashley B Saunders, Susana A Laucella, Daniel B Hall, Rick L Tarleton. Elife. 2025 Apr 15:14:RP104547. doi: 10.7554/eLife.104547.
