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The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T . cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome–largely a result of these same extensive families of genes–have prevented a full understanding of the extent of gene diversity and its maintenance in T . cruzi . In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T . cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T . cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.

Trypanosoma cruzi infection and Chagas disease remains among the most neglected of the neglected tropical diseases. Despite this, studies of the immune response to T. cruzi have provided new insights in immunology and guidance for approaches for prevention and treatment of the disease. T. cruzi represents one of the very best systems in which to study CD8 + T cell biology; mice, dogs, and primates (and many other mammals) are all natural hosts for this parasite, the robust T cell responses generated in these hosts can be readily monitored using the full range of cutting edge techniques, and the parasite can be easily modified to express (or not) a variety of tags, reporters, immune enhances, and endogenous or model antigens. The infection in most hosts is characterized by vigorous and largely effective immune responses, including CD8 + T cells capable of controlling T. cruzi at the level of the infected host cells. However, this immune control is only partially effective and most hosts maintain a low level infection for life. This review addresses the interplay of highly effective CD8 + T cell responses with elaborate pathogen immune evasion mechanisms, including the generation and simultaneous expression of highly variant CD8 + T cell targets and a host cell invasion mechanisms that largely eludes innate immune detection.

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches. Chagas disease is one of the most harmful tropical diseases in the Americas. It affects millions of people, predominantly in Latin America. It is usually spread by kissing bugs infected with Trypanosoma cruzi parasites. It is considered a neglected tropical disease because few effective treatments and preventive methods are routinely used. Several drugs can kill T. cruzi parasites, but they often fail to cure the infection. Many people with Chagas disease go on to have life-long infections and eventually develop heart failure. The reason for the high rate of treatment failure is not known. It does not appear to be the result of the parasites developing resistance to the drugs. One possibility is that the parasites can hide in a dormant state in the body, dodging the toxic drugs and living to reproduce another day. Now, Sánchez-Valdéz et al. identify a dormant form of the T. cruzi parasite that allows the infection to persist after treatment. In the experiments, a non-reproducing form of the so-called amastigote stage of the T. cruzi parasite inside the host cells was observed in infected mice and human cells. While some of the amastigote parasites continue multiplying, a few stop even without drug treatment – but can resume multiplication at a later time. They may also be able to change into the trypomastigote form of the parasite, which can infect new cells. These non-multiplying amastigotes can survive drug treatment for as long as 30 days, whereas the multiplying amastigotes are killed by such drugs. However, the surviving amastigotes then reestablish active infections after treatment has stopped. The experiments explain why treatment so often fails to cure Chagas disease. This suggests new treatment strategies are needed, including using existing drugs for a longer time perhaps with less frequent doses. New therapies that kill the dormant amastigotes may also help. Treatments that overcome the parasite’s ability to hide, could stop the progression of the disease and prevent heart-related deaths in those with persistent T. cruzi infections.

Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi , demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi , enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. IMPORTANCE Trypanosoma cruzi , the causative agent of human Chagas disease, is the leading worldwide cause of infectious myocarditis. Diagnostics for the infection are relatively poor, treatment options are limited and of variable effectiveness, and suitable vaccines are nonexistent. The T. cruzi genome is replete with genes of unknown function and greatly expanded gene families with hundreds of members. The absence of facile genetic engineering tools, including RNA interference, for T. cruzi has prevented elucidation of gene and gene family function and the development of better infection prevention and control measures. In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi , bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts. Trypanosoma cruzi , the causative agent of human Chagas disease, is the leading worldwide cause of infectious myocarditis. Diagnostics for the infection are relatively poor, treatment options are limited and of variable effectiveness, and suitable vaccines are nonexistent. The T. cruzi genome is replete with genes of unknown function and greatly expanded gene families with hundreds of members. The absence of facile genetic engineering tools, including RNA interference, for T. cruzi has prevented elucidation of gene and gene family function and the development of better infection prevention and control measures. In this study, we demonstrate that the CRISPR-Cas9 system is a versatile and powerful tool for genome manipulations in T. cruzi , bringing new opportunities for unraveling the functions of previously uncharacterized genes and how this human pathogen engages its large families of genes encoding surface proteins to interact with human and animal hosts.

Trypanosoma cruzi infection is characterized by chronic parasitism of non-lymphoid tissues and is rarely eliminated despite potent adaptive immune responses. This failure to cure has frequently been attributed to a loss or impairment of anti-T. cruzi T cell responses over time, analogous to the T cell dysfunction described for other persistent infections. In this study, we have evaluated the role of CD8+ T cells during chronic T. cruzi infection (>100 dpi), with a focus on sites of pathogen persistence. Consistent with repetitive antigen exposure during chronic infection, parasite-specific CD8+ T cells from multiple organs expressed high levels of KLRG1, but exhibit a preferential accumulation of CD69+ cells in skeletal muscle, indicating recent antigen encounter in a niche for T. cruzi persistence. A significant proportion of CD8+ T cells in the muscle also produced IFNγ, TNFα and granzyme B in situ, an indication of their detection of and functional response to T. cruzi in vivo. CD8+ T cell function was crucial for the control of parasite burden during chronic infection as exacerbation of parasite load was observed upon depletion of this population. Attempts to improve T cell function by blocking PD-1 or IL-10, potential negative regulators of T cells, failed to increase IFNγ and TNFα production or to enhance T. cruzi clearance. These results highlight the capacity of the CD8+ T cell population to retain essential in vivo function despite chronic antigen stimulation and support a model in which CD8+ T cell dysfunction plays a negligible role in the ability of Trypanosoma cruzi to persist in mice.

Trypanosoma cruzi , the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T . cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU’s) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T . cruzi infections were established.

There is significant heterogeneity in reported sensitivities and specificities of diagnostic serological assays for Chagas disease, as might be expected from studies that vary widely according to setting, research design, antigens employed, and reference standard. The purpose of this study is to summarize the reported accuracy of serological assays and to identify sources of heterogeneity including quality of research design. To avoid associated spectrum bias, our analysis was limited to cohort studies. We completed a search of PubMed, a bibliographic review of potentially relevant articles, and a review of articles identified by a study author involved in this area of research. Studies were limited to prospective cohort studies of adults published since 1985. Measures of diagnostic accuracy were pooled using a Der Simonian Laird Random Effects Model. A subgroup analysis and meta regression were employed to identify sources of heterogeneity. The QUADAS tool was used to assess quality of included studies and Begg's funnel plot was used to assess publication bias. Eighteen studies and 61 assays were included in the final analysis. Significant heterogeneity was found in all pre-determined subgroups. Overall sensitivity was 90% (95% CI: 89%-91%) and overall specificity was 98% (95% CI: 98%-98%). Sensitivity and specificity of serological assays for the diagnosis of Chagas disease appear less accurate than previously thought. Suggestions to improve the accuracy of reporting include the enrollment of patients in a prospective manner, double blinding, and providing an explicit method of addressing subjects that have an indeterminate diagnosis by either the reference standard or index test.

Abstract Trypanosoma cruzi infection in dogs can cause heart failure and sudden death with few treatment options available. A litter of 4 dogs living in a T cruzi endemic area were randomized to prophylaxis and nonprophylaxis groups as part of a study evaluating a modified benznidazole dosing regimen administered twice weekly to prevent T cruzi infection during a vector transmission season. The 2 dogs that received prophylaxis remained healthy without T cruzi infection or cardiac disease for >2 years. One dog that did not receive prophylaxis died unexpectedly with acute T cruzi–induced pancarditis, and the second dog tested positive for T cruzi and developed complex arrhythmias with markedly increased cardiac troponin I and improved with a higher benznidazole treatment dose. Although the small sample size precludes definitive conclusions, we describe the potential clinical benefit of prophylactic and early treatment with modified benznidazole dosing regimens for dogs with T cruzi infection.

The authors discuss the key challenges that undermine the control of Chagas disease and that must be urgently addressed to ensure long-term, sustainable control.

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei , (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro -transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi , as well as in T. brucei and Leishmania major . RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens. IMPORTANCE Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades. Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.