Clinicopathological alterations in wild mammals from the reservoir system of Trypanosoma cruzi:a scoping review

Trypanosoma cruzi is the etiologic agent of Chagas disease.This flagellated protozoan is transmitted to humans as well as different species of domestic and wild animals via vectors from the Reduviidae family(known as"kissing bugs").Despite the fact that hundreds of species of wild mammals are part of the reservoir system,the morphologi-cal changes and clinical manifestations resulting from the pathogenesis of the infection have been largely neglected.The aim of this review is to systematically compile the available information regarding clinicopathological altera-tions in wild mammals due to natural infection by T.cruzi.Information was obtained from six online bibliographic data search platforms,resulting in the identification of 29 publications that met the inclusion criteria.Mortality was the most common clinical manifestation,cardiac damage was the main finding at necropsy,and lymphoplas-macytic inflammation was the most frequent microscopic injury.Thus,regardless of its role as a reservoir,T.cruzi has the potential to affect the health status of wild mammals,a situation that highlights the need for further research to analyze,measure,and compare its effects at both the individual and population levels.

Unlocking the in vitro anti-Trypanosoma cruzi activity of halophyte plants from the southern Portugal

Objective:To evaluate the in vitro anti-Trypanosoma cruzi（T.cruzi） activity of organic extracts prepared from halophyte species collected in the southern coast of Portugal（Algarve）,and chemically characterize the most active samples.Methods:Acetone,dichloromethane and methanol extracts were prepared from 31 halophyte species and tested in vitro against trypomastigotes and intracellular amastigotes of the Tulahuen strain of T.cruzi.The most active extract was fractionated by preparative HPLC-DAD,affording 11 fractions.The most selective fraction was fully characterized by 1H-NMR.Results:From 94 samples tested,one was active,namely the root dichloromethane extract of Juncus acutus(IC50 < 20 μg/mL).This extract was fractionated by HPLC,affording 11 fractions,one of them containing only a pure compound（juncunol）,and tested for anti-parasitic activity.Fraction 8(IC50 = 4.1 μg/mL) was the most active,and was further characterized by 1H-NMR.The major compounds were phenanthrenes,9,10-dihydrophenanthrenes and benzocoumarins.Conclusion:Our results suggest that the compounds identified in fraction 8 are likely responsible for the observed anti parasitic activity.Further research is in progress aiming to isolate and identify the specific active molecules.To the best of our knowledge,this is the first report on the in vitro anti T.cruzi activity of halophyte species.

Cardiac Lesions in Naturally Infected Dogs with Trypanosoma cruzi

With the purpose to determine the frequency and type of cardiac lesions in naturally exposed dogs to Trypanosoma cruzi, ninety one stray dogs, capture by the Canine and Feline Control Center （dog pound） from the municipality of Merida, were studied. Before euthanasia, blood samples were taken to detect 72 cruzi antigens by indirect immunofluorescence antibody test and Western Blot and to detect the genome of parasite by Polymerase Chain Reaction. Immediately after euthanasia, hearts were macroscopically evaluated and a sample of the middle right atrial wall of each dog was taken for histopathological analyses. DNA was also obtained from paraffin blocks of seropositives animals with microscopic lesions to detect 72 cruzi genome. Of ninety one dogs, thirteen were seropositive. All seropositive dogs showed an association （P 〈 0.05） with lymphocytoplasmatic myocarditis. The presence of the 72 cruzi genome was also detected by PCR in cardiac septum tissue of seropositive dogs and in all the cases with microscopic lesions indicating the high pathogenicity of the local circulating strain. No association with macroscopic lesions was observed in seropositive dogs. Also, the presence of Dirofilaria immitis （D. immitis） was found in 6% of dogs evaluated. This study demonstrates a high tropism to cardiac tissue and virulence of the strains of 72 cruzi circulating in the studied dog population.

Canova medication changes TNF-α and IL-10 serum levels in mice infected with Trypanosoma cruzi Y strain

Objective:To identify whether Canova medication changes TNF-α and IL-10 serum levels in mice infected with Trypanosoma cruzi Y strain.Methods:Animals were divided into five groups:non-treated infected animals(I); benznidazole-treated infected animals(Bz; 100 mg/kg body weight,single daily dose by gavage); Canova medication(CM) treated infected animals(CM;0.2 mL/animal,single daily dose by gavage); benznidazole- and Canova medication–treated infected animals with the above-mentioned dose(Bz+CM);and non-infected animals(C).TNF-α and IL-10 levels were determined in serum aliquots after 4,7,10,13,and 29 days of infection.An ELISA technique was employed with R&D System Inc.antibody pairs.Results:A high increase in TNF-α and IL-10 levels occurred in the infected and CM-treated groups within the treatment employed on the 10 th day after infection,coupled with a IL-10 decrease on the 13 th day after infection when compared with the other experimental groups.Conclusions:CM may change the balance between plasma cytokine levels(TNF-α and IL-10) in mice infected with Y strain T.cruzi,with important consequences leading towards a more severe infection.

Specific primers design based on the superoxide dismutase b gene for Trypanosoma cruzi as a screening tool:Validation method using strains from Colombia classified according to their discrete typing unit

Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.

Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained from Flowers of Anthemis Tinctoria, Against Trypanosoma Cruzi

In Brazil and several other Latin American countries, Chagas' disease still constitutes a serious medical and social problem, and there is a need to develop new, more-potent drugs with fewer side effects to effectively treat this disease. We investigated the antitrypanosomal effect of a crude extract, fractions, and a semi-purified subfraction rich in a mix- ture of isomeric labdane sesquiterpenes, obtained from flowers of Anthemis tinctoria, against Trypanosoma cruzi. In epimastigote forms, the aqueous crude extract, dichloromethane fraction, and semi-purified subfraction showed a dose-dependent inhibitory activity, with IC50 of 2.3 μg/ml, 1.8 μg/ml, and 0.2 μg/ml, respectively. In the interaction in- dex, the semi-purified subfraction showed a reduction in both the percentage of infected LLCMK2 cells and the mean number of amastigotes per infected cell. The cytotoxicity evaluation demonstrated that the cytotoxic concentrations of the semi-purified subfraction were higher for LLCMK2 cells than for the protozoans, with a selectivity index of 35.0. Epimastigote forms treated with the semi-purified subfraction showed ultrastructural and morphological alterations such as rounding of the cells and bleb formation in the flagellum and cytoplasmic membrane. These results show that the flowers from A. tinctoria may be a source of new drugs with antiprotozoal activity. However, additional in vitro and in vivo studies are needed to validate the use of A. tinctoria in the treatment of Chagas’ disease.

Antibody delivery into viable epimastigotes of <i>Trypanosoma cruzi</i>as a tool to study the parasite biology

American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, hence the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal (Trypano-soma cruzi). T. cruzi is an RNAi-negative parasite, therefore the silencing genes strategies by RNAi is not possible;for that reason, antibodies may be taken as a tool for studying the parasite proteins function by blocking these molecules with specific antibodies. The aim of this work was to establish a methodology for antibody delivery (antibody transfection) into viable parasites. We used anti-cyclin-A antibody (human origin) in western blot assay with epimastigote of T. cruzi proteins and this recognized a ~55 kDa polypeptide. Several methods for antibody transfection (electroporation, saponin permeabilization and a lipid-based formulation) were tested. The first two methods were unsuccessful. In electroporation was impossible to visualize the antibody inside parasites and with saponin permeabilization, antibodies were successfully introduced, but with loss of parasites viability. The lipid-based formulation method forms noncovalent complexes with antibodies. These complexes are internalized by cells and antibodies are released into the cytoplasm. With this method, a successful antibody delivery was achieved. Anti-cyclin antibodies were visualized in the cytoplasm from fixed transfected parasites (immunofluorescence assays). At 24 h post-transfection, parasites maintained their viability (90%) and were able to arrest the cell cycle in G0/G1-phase of cultured epimastigotes (cell population increased in G0/G1-phase from 50.5% to 66.2% and decreased in S-phase from 47.2% to 26%). It was also observed that anti-cyclin-A antibodies inhibit the parasite population doubling (p T. cruzi, with a simple and cheap technique, which will allows carrying out further studies of this protozoan.

Behavioral parameters of six populations of/Wecavs phyllosomus longipennis(Heteroptera:Reduviidae)from areas with high and low prevalences of Trypanosoma cruzi human infection

Three behaviors of epidemiological importance,namely feeding latency,feeding duration and defecation latency,for six populations of Meccus phyllosomus longipennis(Usinger)from areas of central,western and north-central Mexico with high(HP)and low(LP)prevalence of Trypanosoma cruzi(Chagas)human infection were evaluated in this study.The median feeding latency(the time taken to begin feeding)was highly variable between instars.Within-instar comparisons showed that at least 65%of the LP populations(N3 to adult)started to feed significantly(P<0.05)later than the HP population,with N1 showing no difference,and N2 from LP populations feeding sooner than those from HP populations.The six populations had similar median feeding durations within instars.A higher(P<0.05)percentage of the instars from HP populations defecated faster than the respective instars from the three LP populations.Approximately 25%of the young nymphs(N1 to N3)and females in the HP populations defecated<2 min postfeeding,compared with 4%-6%of the young nymphs and 1.3%-3%of females in the LP populations.Moreover,17.7%-38.8%of the older nymphs(N4 to N5)in the HP populations and 6.8%-13.4%in the LP populations defecated during or immediately after feeding.Our results indicate that the HP populations have a greater potential than the LP populations to transmit T.cruzi infections,which may underlie the differences in the prevalence of T.cruzi infection in some areas where M p.longipennis is currently distributed.

Re-emerging threat of Trypanosoma cruzi vector transmission in El Salvador, update from 2018 to 2020

Background:Since the late twentieth century,Chagas disease gained global attention to suppress the vector burden as a main control strategy in endemic countries.In Central America,multi-national initiative successfully achieved significant reduction in the estimated disease prevalence as well as elimination of the region's principal vector species at the time in 2012.While the last decade has witnessed significant changes in ecosystem-such as urbanization and replacement of the main vector species-that can possibly affect the vector's habitation and residual transmission,the up-to-date vector burden in the region has not been evaluated thoroughly due to the cessation of active vector surveillance.The aim of this study was to update the risk of vector-borne Trypanosoma cruzi infection in El Salvador,the top Chagas disease-endemic country in Central America.Methods:A nationwide vector survey was conducted in the domestic environment of El Salvador from September 2018 to November 2020.The selection of the houses for inspection was based on expert purposeful sampling.Infection for T.cruzi was examined by microscopic observation of the insects'feces,followed by a species confrmation using PCR.The data were analyzed using R software version 4.1.3.Proportion estimates with 95%confidence intervals were inferred using the Jeffrey's method provided under the epiR package.Results:A total of 1529 Triatoma dimidiata was captured from 107 houses(infestation rate,34.4%;107/311)in all the fourteen departments of the country visited within the period;prevalence of T.cruzi infection was as high as 10%(153/1529).In the country,domestic T.dimidiata infestation was distributed ubiquitously,while T.cruzi infection rates varied across the departments.Five out of fourteen departments showed higher infection rates than the average,suggesting sporadic high-risk areas in the country.Conclusions:Our comprehensive study revealed substantial T.cruzi infection of T.dimidiata across the country,indicating potential active transmission of the disease.Therefore,strengthened surveillance for both vector and human infection is required to truly eliminate the risk of T.cruzi transmission in Central America.

Seropositivity for Trypanosoma cruzi in domestic dogs from Sonora,Mexico

Background:Chagas disease is an important health problem in Latin America due to its incapacitating effects and associated mortality.Studies on seropositivity for Trypanosoma cruzi in Mexican dogs have demonstrated a direct correlation between seropositivity in humans and dogs,which can act as sentinels for the disease in this region.The objective of this study was to determine the seropositivity for T.cruzi infection in dogs from Sonora,a northern borderstate of Mexico.Methods:Responsible pet owners were selected at random from an urban area of Empalme municipality,Sonora,Mexico,and from there,180 dog samples were collected.Anti-T.cruzi antibodies were determined using the enzymelinked immunosorbent assay(ELISA)method.Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T.cruzi antibodies.For the statistical analysis,chi-square tests were conducted.Results:Dogs’sera showed a seropositivity rate of 4.44%.The rate of seropositivity was not associated with the dogs’age,sex,or socioeconomics pertaining to the geographical area.One sample(1/180,0.55%)showed the acute state of the disease.Conclusions:The study found a presence of anti-T.cruzi antibodies in dogs in this area,which suggests vector transmission.There is a need for active surveillance programs throughout the state of Sonora and vector control strategies should also be implemented in endemic regions.

Evaluation of parasitemia by qPCR in patients with chronic Chagas disease treated with benznidazole

Objective:To evaluate parasitemia by qPCR in patients undergoing etiological treatment and followed in a Brazilian reference center.Methods:Parasite load was quantified by qPCR in 32 participants with chronic Chagas disease who were treated with benznidazole.Serological analyses were performed before and after the treatment and parasite loads were compared prior and 12/18 months post the treatment.Results:Thirty-two participants were recruited and treated with benznidazole,and 20 were followed-up.Adverse events(AE)were observed in 22 out of 29 participants that had safety data(76%),and dermatological alterations were the most frequently observed AE.Of the 20 participants analyzed,13 and 7 completed 12 and 18 months follow-up after the treatment,respectively.12 Months after the final treatment,Trypanosoma cruzi was detectable in 3 patients by qPCR;18 months after the final treatment,Trypanosoma cruzi was detectable per qPCR in 4 of the 7 participants.Thus,between 12 and 18 months,7 participants of the 20 initial follow-up cases showed positive qPCR,indicating treatment failures.Conclusions:qPCR can be used as an alternative method for evaluating the effectiveness of the etiological treatment of CD,and can be applied to analyze early therapeutic failures.The study showed that benznidazole therapy had limited effectiveness in treating chronic CD patients,thus emphasizing the importance of conducting continued research for developing more effective therapies and diagnosis for CD.

美洲锥虫病——美洲新型艾滋病

美洲锥虫病,又称查加斯病,是由克氏锥虫引起的潜在致命的寄生虫性人兽共患病。除人外,在美洲200多种哺乳动物感染了克氏锥虫。人和动物感染的主要传播媒介为吸血昆虫——锥蝽,偶尔通过非吸血昆虫媒介机制传播,如输血、污染食品、母婴感染。该病的临床症状因感染阶段不同而异,早期急性期无症状或症状轻微,隐匿期亦无症状,慢性期的典型症状为渐进性慢性心脏和消化系统损害,持续终生,部分病例死亡。因克氏锥虫与人免疫缺陷病毒(HIV)的早期传播模式、漫长的潜伏期和难以治愈等方面极其相似,有专家将查加斯病称为"美洲的新艾滋病"。该病曾局限于美洲地区,但现已随人口迁移传播到欧洲等大陆,成为全球性疫病。在急性期,使用苄硝唑和硝呋替莫抗寄生虫药物治疗很有效;在慢性期,也可延迟或防止症状恶化,但仍有部分慢性病例最终发展成致命的心脏和消化道衰竭。查加斯病可通过病原鉴定、血清学和分子学技术进行诊断。目前尚无查加斯病疫苗,媒介昆虫控制是最有效的预防手段。论文详细介绍了美洲锥虫病的病原学、生活史、流行病学、临床症状、病理变化、诊断、治疗以及防控措施,以供参考。

Challenges in the chemotherapy of Chagas disease: Looking for possibilities related to the differences and similarities between the parasite and host

Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.

RIL—2对急性克氏锥虫感染小鼠的抗SRBC直接溶血空斑反应及抗虫保护性免疫影响的初步研究

为研究ＩＬ—２在克氏锥虫感染过程中出现的免疫抑制机制中的作用，８只克氏锥虫感染的Ｃ３Ｈ／ＨｅＪｎ小鼠每日皮下注射两次，每日总量５０００μ重组人ｒＩＬ－２；对照组相同数量小鼠皮下注射等量的无钙、镁离子的ＰＢＳ。实验结果表明ｒＩＬ－２注射组小鼠脾细胞对羊红细胞的反应能力由ＰＢＳ注射鼠的１５。２％提高到９３。５％（Ｐ＜０．０５）。相当接近于正常的未感染小鼠的空斑形成细胞数。ｒＩＬ－２注射组小鼠仅表现轻度降低的寄生虫血症，其动态变化在两组间未显示出差异。除ＰＢＳ组的１只小鼠在感染后第３６ｄ死亡外，ｒＩＬ－２以及ＰＢＳ注射组的其它动物都在克氏锥虫急性感染期存活。尽管ｒＩＬ－２注射并未明显地提高克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠的抗虫保护性免疫，本实验提示ＩＬ－２不足是克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠非特异性免疫抑制的原因之一。

Interferon-γ and other inflammatory mediators in cardiomyocyte signaling during Chagas disease cardiomyopathy

Chagas disease cardiomyopathy(CCC), the main consequence of Trypanosoma cruzi(T.cruzi) infection, is an inflammatory cardiomyopathy that develops in up to 30% of infected individuals. The heart inflammation in CCC patients is characterized by a Th1 T cell-rich myocarditis with increased production of interferon(IFN)-γ, produced by the CCC myocardial infiltrate and detected at high levels in the periphery. IFN-γ has a central role in the cardiomyocyte signaling during both acute and chronic phases of T.cruzi infection. In this review, we have chosen to focus in its pleiotropic mode of action during CCC, which may ultimately be the strongest driver towards pathological remodeling and heart failure. We describe here the antiparasitic protective and pathogenic dual role of IFN-γ in Chagas disease.

细菌脂多糖促进克氏锥虫抗原诱导的自身免疫性心肌炎

目的观察细菌脂多糖(LPS)对克氏锥虫抗原诱导自身免疫性心肌炎的影响。方法克氏锥虫抗原辅以完全弗氏佐剂免疫A/J小鼠,LPS经腹腔接种方式给药,其后加强免疫2次,28 d后,测定心肌肌球蛋白特异的迟发型超敏反应(DTH)及自身抗体,并取鼠心脏观察心肌炎症情况。结果LPS提高锥虫抗原诱导的心肌肌球蛋白特异性DTH及自身抗体,并且25μg LPS与锥虫抗原免疫的小鼠可见明显的心肌炎。结论LPS能够促进心肌肌球蛋白特异的自身免疫反应及锥虫抗原诱导的自身免疫性心肌炎。

萘酰肼类化合物的合成设计及其抑制克鲁斯氏锥体虫半胱氨酸蛋白酶的活性

目的卡格氏病 (Chagas′disease)是由克鲁斯氏锥体虫引起的一种寄生虫病 ,克鲁斯氏锥体虫半胱氨酸蛋白酶 (cruzain)是锥体虫生活周期中起重要作用的一种酶 ,研究该酶的抑制剂可望发展出一类治疗卡格氏病的新药。方法采用DOCK程序对前期研究中的抑酶活性最强的萘酰肼化合物z1与cruzain拟合的情况进行了研究 ,辅助指导药物分子设计和化学结构修饰 ,设计了 7个目标化合物z2～z8,并采用酶法体外筛选测定其抗克鲁斯氏锥体虫活性。结果以 2 ,3 二羟基萘为起始原料 ,经甲醚化保护、硝化、还原、氮甲基化、甲酰化、脱保护基和缩合等共 7步反应 ,制得目标化合物N′ (2 ,3 二羟基 4 二甲氨基 1 萘亚甲基 ) 3 羟基 2 萘甲酰肼 (z7) ,其抑制cruzain的IC50 值约为 0 2 0 μmol L ,其活性强度是化合物z1的两倍多。结论目标化合物经1 H NMR和MS确定了其化学结构。目标化合物z7的抑制酶的活性大大增强 。

N,N′-双取代脲类克鲁斯氏锥体虫半胱氨酸蛋白酶小分子抑制剂的合成及其活性研究

目的 :寻找克鲁斯氏锥体虫体半胱氨酸蛋白酶 (Cruzain)的小分子抑制剂。方法 :根据对Cruzain分子结构的计算机模拟设计结果 ,选用N ,N′ 双取代脲为先导结构 ,设计并合成了 2 1个未见文献报道的双取代脲衍生物 ,确证了它们的化学结构 ,测定了化合物的体外抑制Cruzain的IC50 值。结果 :生物活性测定结果显示所合成的化合物均有不同程度的抑制Cruzain的活性 ,其中化合物Ⅳ8、Ⅳ9和Ⅳ1 7的活性与对照药tf

Intra-Domiciliary Transmission of Chagas’ Disease in Rural Areas of Yucatan Mexico

With the objective to describe the relationship between T. cruzi infection in humans and sinantropic reservoir and vectors in the rural conditions a study was performed in municipalities of the Yucatan state in Mexico. Blood samples from humans, dogs and opossum were taken from the households. Vectors were also collected and the infection and infestation indexes were calculated. Serum samples were evaluated by indirect hemagglutination (IH) and indirect immunofluorescence (IFI) to determine IgG antibodies against Trypanosoma cruzi. The polymerase chain reaction (PCR) was also performed in the samples taken from humans and reservoirs. Blood smears were performed in reservoirs. Sampled humans underwent to an electrocardiographic study to identify functional and conduction alterations of the heart. Household building characteristics were noted. A total of 367 blood samples were collected from people. The presence of IgG antibodies was 16.6% and 71.0% at PCR. Electrocardiographic studies revealed that 31% of patients seropositive to T. cruzi have a medical condition. 20.0% of dogs were seropositive and 68.5 positive to PCR. At the direct detection 13.6% resulted positive. In opossum 29.1% were seropositive and 72.9% positive to PCR. A total of 269 triatomineos were captured and 52.7% were infected with T. cruzi. The infection and infestation index were 52.4% and 60.8% respectively. The presence of adult vectors and their nymph’s were more frequently found in households made with wattle roofs (45.2 and 73.9 % respectively) than other kind of building materials. Electrocardiographic abnormalities found in positive persons were: Incomplete right bundle branch block (55%), tachycardia (35%), old myocardial infarction (5%) and incomplete block of the left bundle (5%). This work highlights the importance of comprehensive studies on the transmission of T. cruzi in the Yucatan where the link between human infection in synanthropic reservoirs and vectors is established. This information is important to design programs for the epidemiological surveillance and control to break the chain of transmission of the disease.

Use of Essential Oils of the Genus Citrus as Biocidal Agents

In this study, the essential oils extracted from the peels of Citrus aurantifolia, Citrus limon and Citrus sinensis were chemically characterized and quantified. These essential oils and their standards limonene, citral and limonene + citral were evaluated (at concentrations ranging from 500 to 3.91 mL·mL-1) regarding their anti-trypanosome, antifungal and antibacterial activities. The chemical identification and quantification (by GC/FID and GC/MS) displayed the limonene as the major component of the three essential oils. It did not verified anti-trypanosome activity for all treatments. Regarding antimicrobial activity, the results were satisfactory against Gram-negative bacteria (Staphylococcus aureus, Listeria. monocytogenes and Salmonella cholerasuis) and funghi (Aspergillus niger, A. flavus and A. carbonarius), except for the bacterium Pseudomonas aeruginosa, which showed no inhibition for any treatment used.