Risk Factors for Bartonella species Infection in Blood Donors from Southeast Brazil

Bacteria from the genus Bartonella are emerging blood-borne bacteria, capable of causing long-lasting infection in marine and terrestrial mammals, including humans. Bartonella are generally well adapted to their main host, causing persistent infection without clinical manifestation. However, these organisms may cause severe disease in natural or accidental hosts. In humans, Bartonella species have been detected from sick patients presented with diverse disease manifestations, including cat scratch disease, trench fever, bacillary angiomatosis, endocarditis, polyarthritis, or granulomatous inflammatory disease. However, with the advances in diagnostic methods, subclinical bloodstream infection in humans has been reported, with the potential for transmission through blood transfusion been recently investigated by our group. The objective of this study was to determine the risk factors associated with Bartonella species infection in asymptomatic blood donors presented at a major blood bank in Southeastern Brazil. Five hundred blood donors were randomly enrolled and tested for Bartonella species infection by specialized blood cultured coupled with high-sensitive PCR assays. Epidemiological questionnaires were designed to cover major potential risk factors, such as age, gender, ethnicity, contact with companion animals, livestock, or wild animals, bites from insects or animal, economical status, among other factors. Based on multivariate logistic regression, bloodstream infection with B. henselae or B. clarridgeiae was associated with cat contact (adjusted OR: 3.4, 95% CI: 1.1–9.6) or history of tick bite (adjusted OR: 3.7, 95% CI: 1.3–13.4). These risk factors should be considered during donor screening, as bacteremia by these Bartonella species may not be detected by traditional laboratory screening methods, and it may be transmitted by blood transfusion.     

Electrocardiographic Abnormalities in Trypanosoma cruzi Seropositive and Seronegative Former Blood Donors

Background

Blood donor screening leads to large numbers of new diagnoses of Trypanosoma cruzi infection, with most donors in the asymptomatic chronic indeterminate form. Information on electrocardiogram (ECG) findings in infected blood donors is lacking and may help in counseling and recognizing those with more severe disease.

Objectives

To assess the frequency of ECG abnormalities in T.cruzi seropositive relative to seronegative blood donors, and to recognize ECG abnormalities associated with left ventricular dysfunction.

Methods

The study retrospectively enrolled 499 seropositive blood donors in São Paulo and Montes Claros, Brazil, and 483 seronegative control donors matched by site, gender, age, and year of blood donation. All subjects underwent a health clinical evaluation, ECG, and echocardiogram (Echo). ECG and Echo were reviewed blindly by centralized reading centers. Left ventricular (LV) dysfunction was defined as LV ejection fraction (EF)<0.50%.

Results

Right bundle branch block and left anterior fascicular block, isolated or in association, were more frequently found in seropositive cases (p<0.0001). Both QRS and QTc duration were associated with LVEF values (correlation coefficients −0.159,p<0.0003, and −0.142,p = 0.002) and showed a moderate accuracy in the detection of reduced LVEF (area under the ROC curve: 0.778 and 0.790, both p<0.0001). Several ECG abnormalities were more commonly found in seropositive donors with depressed LVEF, including rhythm disorders (frequent supraventricular ectopic beats, atrial fibrillation or flutter and pacemaker), intraventricular blocks (right bundle branch block and left anterior fascicular block) and ischemic abnormalities (possible old myocardial infarction and major and minor ST abnormalities). ECG was sensitive (92%) for recognition of seropositive donors with depressed LVEF and had a high negative predictive value (99%) for ruling out LV dysfunction.

Conclusions

ECG abnormalities are more frequent in seropositive than in seronegative blood donors. Several ECG abnormalities may help the recognition of seropositive cases with reduced LVEF who warrant careful follow-up and treatment.     

Cost-Effectiveness of Blood Donation Screening for Trypanosoma cruzi in Mexico

An estimated 2 million inhabitants are infected with Chagas disease in Mexico, with highest prevalence coinciding with highest demographic density in the southern half of the country. After vector-borne transmission, Trypanosoma cruzi is principally transmitted to humans via blood transfusion. Despite initiation of serological screening of blood donations or donors for T. cruzi since 1990 in most Latin American countries, Mexico only finally included mandatory serological screening nationwide in official Norms in 2012. Most recent regulatory changes and segmented blood services in Mexico may affect compliance of mandatory screening guidelines. The objective of this study was to calculate the incremental cost-effectiveness ratio for total compliance of current guidelines from both Mexican primary healthcare and regular salaried worker health service institutions: the Secretary of Health and the Mexican Institute for Social Security. We developed a bi-modular model to analyze compliance using a decision tree for the most common screening algorithms for each health institution, and a Markov transition model for the natural history of illness and care. The incremental cost effectiveness ratio based on life-years gained is US$ 383 for the Secretary of Health, while the cost for an additional life-year gained is US$ 463 for the Social Security Institute. The results of the present study suggest that due to incomplete compliance of Mexico’s national legislation during 2013 and 2014, the MoH has failed to confirm 15,162 T. cruzi infections, has not prevented 2,347 avoidable infections, and has lost 333,483 life-years. Although there is a vast difference in T. cruzi prevalence between Bolivia and Mexico, Bolivia established mandatory blood screening for T.cruzi in 1996 and until 2002 detected and discarded 11,489 T. cruzi -infected blood units and prevented 2,879 potential infections with their transfusion blood screening program. In the first two years of Mexico’s mandated program, the two primary institutions failed to prevent due to incomplete compliance more potential infections than those gained from the first five years of Bolivia’s program. Full regulatory compliance should be clearly understood as mandatory for the sake of blood security, and its monitoring and analysis in Mexico should be part of the health authority’s responsibility.     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Ecological,Social and Biological Risk Factors for Continued Trypanosoma cruzi Transmission by Triatoma dimidiata in Guatemala

Background

Chagas disease transmission by Triatoma dimidiata persists in Guatemala and elsewhere in Central America under undefined ecological, biological and social (eco-bio-social) conditions.

Methodology

Eco-bio-social risk factors associated with persistent domiciliary infestation were identified by a cross-sectional survey and qualitative participatory methods. Quantitative and qualitative data were generated regarding Trypanosoma cruzi reservoirs and triatomine hosts. Blood meal analysis and infection of insects, dogs and rodents were determined. Based on these data, multimodel inference was used to identify risk factors for domestic infestation with the greatest relative importance (>0.75).

Principal Findings

Blood meal analysis showed that 64% of 36 bugs fed on chickens, 50% on humans, 17% on dogs; 24% of 34 bugs fed on Rattus rattus and 21% on Mus musculus. Seroprevalence among 80 dogs was 37%. Eight (17%) of 46 M. musculus and three (43%) of seven R. rattus from households with infected triatomines were infected with T. cruzi Distinct Typing Unit I. Results from interviews and participatory meetings indicated that vector control personnel and some householders perceived chickens roosting and laying eggs in the house as bug infestation risk factors. House construction practices were seen as a risk factor for bug and rodent infestation, with rodents being perceived as a pest by study participants. Multimodel inference showed that house infestation risk factors of high relative importance are dog density, mouse presence, interior wall plaster condition, dirt floor, tile roofing and coffee tree presence.

Conclusions/Significance

Persistent house infestation is closely related to eco-bio-social factors that maintain productive T. dimidiata habitats associated with dogs, chickens and rodents. Triatomine, dog and rodent infections indicate active T. cruzi transmission. Integrated vector control methods should include actions that consider the role of peridomestic animals in transmission and community memberś level of knowledge, attitudes and practices associated with the disease and transmission process.     

High Fat Diet Modulates Trypanosoma cruzi Infection Associated Myocarditis

Background

Trypanosoma cruzi, the causative agent of Chagas disease, has high affinity for lipoproteins and adipose tissue. Infection results in myocarditis, fat loss and alterations in lipid homeostasis. This study was aimed at analyzing the effect of high fat diet (HFD) on regulating acute T. cruzi infection-induced myocarditis and to evaluate the effect of HFD on lipid metabolism in adipose tissue and heart during acute T. cruzi infection.

Methodology/Principal Findings

CD1 mice were infected with T. cruzi (Brazil strain) and fed either a regular control diet (RD) or HFD for 35 days following infection. Serum lipid profile, tissue cholesterol levels, blood parasitemia, and tissue parasite load were analyzed to evaluate the effect of diet on infection. MicroPET and MRI analysis were performed to examine the morphological and functional status of the heart during acute infection. qPCR and immunoblot analysis were carried out to analyze the effect of diet on the genes involved in the host lipid metabolism during infection. Oil red O staining of the adipose tissue demonstrated reduced lipolysis in HFD compared to RD fed mice. HFD reduced mortality, parasitemia and cardiac parasite load, but increased parasite load in adipocytes. HFD decreased lipolysis during acute infection. Both qPCR and protein analysis demonstrated alterations in lipid metabolic pathways in adipose tissue and heart in RD fed mice, which were further modulated by HFD. Both microPET and MRI analyses demonstrated changes in infected RD murine hearts which were ameliorated by HFD.

Conclusion/Significance

These studies indicate that Chagasic cardiomyopathy is associated with a cardiac lipidpathy and that both cardiac lipotoxicity and adipose tissue play a role in the pathogenesis of Chagas disease. HFD protected mice from T. cruzi infection-induced myocardial damage most likely due to the effects of HFD on both adipogenesis and T. cruzi infection-induced cardiac lipidopathy.     

无偿献血者中隐匿性乙型肝炎病毒感染及表面抗原突变分析

采用多种免疫学检测和核酸检测相结合的方法调查了我国南方某城市无偿献血者中隐匿性乙型肝炎病毒(HBV)感染的存在情况。结果在9023例乙肝表面抗原(HBsAg)阴性的无偿献血者中,共发现17例HBV DNA阳性,隐匿性HBV感染者的发生率为0.19%(95%CI:0.11～0.30%)。序列分析显示其中6例在HBsAg"a"表位(aa124～aa147)存在不同程度氨基酸突变,突变发生率为42.9%(6/14,有3例未扩增出"a"表位片段序列),G145R突变是该地区隐匿性HBV携带者中发生频率最高的突变(4/6,66.7%)。隐匿性HBV感染者中基因型C的比例(10/17)显著高于HBsAg阳性的HBV感染者(0/15,P<0.01)。     

The Trypanosoma cruzi Diamine Transporter Is Essential for Robust Infection of Mammalian Cells

Trypanosoma cruzi is incapable of synthesizing putrescine or cadaverine de novo, and, therefore, salvage of polyamines from the host milieu is an obligatory nutritional function for the parasite. A high-affinity diamine transporter (TcPOT1) from T. cruzi has been identified previously that recognizes both putrescine and cadaverine as ligands. In order to assess the functional role of TcPOT1 in intact parasites, a Δtcpot1 null mutant was constructed by targeted gene replacement and characterized. The Δtcpot1 mutant lacked high-affinity putrescine-cadaverine transport capability but retained the capacity to transport diamines via a non-saturable, low-affinity mechanism. Transport of spermidine and arginine was not impacted by the Δtcpot1 lesion. The Δtcpot1 cell line exhibited a significant but not total defect in its ability to subsist in Vero cells, although initial infection rates were not affected by the lesion. These findings reveal that TcPOT1 is the sole high-affinity diamine permease in T. cruzi, that genetic obliteration of TcPOT1 impairs the ability of the parasite to maintain a robust infection in mammalian cells, and that a secondary low-affinity uptake mechanism for this key parasite nutrient is operative but insufficient for optimal infection.     

青海地区献血者HBV感染隐匿风险与基因型分析

目的:探讨青海地区献血者乙型肝炎病毒(hepatitis b virus,HBV)感染隐匿风险与基因型的相关性。方法:采用回顾性研究方法,选择2014年2月-2018年1月在我院进行无偿献血的青海地区人群750例,采用聚合酶链式反应-限制性内切酶片段法(PCR-RFLP)检测HBV DNA基因的多态性,并进行HBV感染隐匿风险分析。结果:在750例人群中,检出HBV隐匿性感染8例,检出率为1.1%,其中窗口期感染3例,一过性感染5例;基因C型6例,基因B型2例,基因B型患者的都为窗口期感染,核酸定量都≤20 IU/m L,与基因C型患者对比差异有统计学意义(P0.05)。多因素Logistic回归分析显示基因C型、核酸定量、家属病史、吸烟为导致HBV隐匿性感染的独立危险因素(P0.05)。结论:青海地区献血者HBV感染隐匿风险相对比较低,多为基因C型,基因C型为导致HBV隐匿性感染的独立危险因素。     

Global Metabolomic Profiling of Acute Myocarditis Caused by Trypanosoma cruzi Infection

Chagas disease is caused by Trypanosoma cruzi infection, being cardiomyopathy the more frequent manifestation. New chemotherapeutic drugs are needed but there are no good biomarkers for monitoring treatment efficacy. There is growing evidence linking immune response and metabolism in inflammatory processes and specifically in Chagas disease. Thus, some metabolites are able to enhance and/or inhibit the immune response. Metabolite levels found in the host during an ongoing infection could provide valuable information on the pathogenesis and/or identify deregulated metabolic pathway that can be potential candidates for treatment and being potential specific biomarkers of the disease. To gain more insight into those aspects in Chagas disease, we performed an unprecedented metabolomic analysis in heart and plasma of mice infected with T. cruzi. Many metabolic pathways were profoundly affected by T. cruzi infection, such as glucose uptake, sorbitol pathway, fatty acid and phospholipid synthesis that were increased in heart tissue but decreased in plasma. Tricarboxylic acid cycle was decreased in heart tissue and plasma whereas reactive oxygen species production and uric acid formation were also deeply increased in infected hearts suggesting a stressful condition in the heart. While specific metabolites allantoin, kynurenine and p-cresol sulfate, resulting from nucleotide, tryptophan and phenylalanine/tyrosine metabolism, respectively, were increased in heart tissue and also in plasma. These results provide new valuable information on the pathogenesis of acute Chagas disease, unravel several new metabolic pathways susceptible of clinical management and identify metabolites useful as potential specific biomarkers for monitoring treatment and clinical severity in patients.     

Trypanosoma cruzi Infection of Cultured Adipocytes Results in an Inflammatory Phenotype

Infection with Trypanosoma cruzi, the etiologic agent of Chagas disease is accompanied by an intense inflammatory reaction. Our laboratory group has identified adipose tissue as one of the major sites of inflammation during disease progression. Because adipose tissue is composed of many cell types, we were interested in investigating whether the adipocyte per se was a source of inflammatory mediators in this infection. Cultured adipocytes were infected with the Tulahuen strain of T. cruzi for 48–96 h. Immunoblot and quantitative PCR (qPCR) analyses demonstrated an increase in the expression of proinflammatory cytokines and chemokines, including interleukin (IL)‐1β, interferon‐γ, tumor necrosis factor‐α, CCL2, CCL5, and CXCL10 as well as an increase in the expression of Toll‐like receptors‐2 and 9 and activation of the notch pathway. Interestingly, caveolin‐1 expression was reduced while cyclin D1 and extracellular signal‐regulated kinase (ERK) expression was increased. The expression of PI3kinase and the activation of AKT (phosphorylated AKT) were increased suggesting that infection may induce components of the insulin/IGF‐1 receptor cascade. There was an infection‐associated decrease in adiponectin and peroxisome proliferator‐activated receptor‐γ (PPAR‐γ). These data provide a mechanism for the increase in the inflammatory phenotype that occurs in T. cruzi‐infected adipocytes. Overall, these data implicate the adipocyte as an important target of T. cruzi, and one which contributes significantly to the inflammatory response observed in Chagas disease.     

Factors from Trypanosoma cruzi Interacting with AP-1 Sequences

Interaction between factors from Trypanosoma cruzi extracts and AP-1 sequences was studied by electrophoretic mobility shift assays. Using a double-stranded probe carrying the AP-1 sequence from the SV40 promoter, three specific complexes designated A, B, and C were detected. Complexes A and C were formed when using single-stranded probes. The relative amount of complex B, specific for double-stranded DNA, increased as a function of probe length. Complexes were stabilized by cross-linking with UVC irradiation and resolved on denaturing SDS-PAGE. Complex A generated bands of 60- and 39 kDa; complex B produced two bands of 46- and 43 kDa; and complex C generated one band of 43 kDa. The AP-1 binding activity was much higher in purified nuclear preparations than in soluble fractions, and was detected in crude extracts from the three forms of the parasite. The binding signal, however, was much stronger in amastigote and trypomastigote than in the epimastigote forms. Specific binding was increased by oxidative stress. Antibodies raised against peptides corresponding to conserved domains of mammalian c-Jun and c-Fos detected bands of 40- and 60 kDa, respectively, in a nuclear epimastigote preparation.     

Some Factors Inducing Formation of Metacyclic Stages of Trypanosoma cruzi

ABSTRACT Epimastigotes of Trypanosoma cruzi , Peru strain, incubated in Contreras'artificial triatomine urine transformed into metacyclic trypomastigotes within 48 h at 28° C when 10 mM L-proline or L-glutamate was added to the medium. Smaller numbers of metacyclic stages were induced in the presence of glucose or L-alanine. The L-leucine and L-isoleucine, 2 amino acids known to inhibit proline catabolism, inhibited proiine-induced metacyclogenesis. Cells gassed with 5% CO₂ showed significantly faster rates and higher levels of transformation than those not gassed, therefore indicating the additional importance of CO₂ for transformation.     

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.     

TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.