Enhancing Trypanosomatid Identification and Genotyping with Oxford Nanopore Sequencing: Development and Validation of an 18S rRNA Amplicon-Based Method.

Trypanosomatids, including Trypanosoma and Leishmania species, present significant medical and veterinary challenges, causing substantial economic losses, health complications, and even fatalities. Diagnosing and genotyping these species and their genotypes is often complex, involving multiple steps. This study aimed to develop an amplicon-based sequencing (ABS) method using Oxford Nanopore long-read sequencing to enhance Trypanosomatid detection and genotyping. The 18S rDNA gene was targeted for its inter-species conservation. The Trypanosomatid-ABS method effectively distinguished between 11 Trypanosoma species (including Trypanosoma evansi, Trypanosoma theileri, Trypanosoma vivax, and Trypanosoma rangeli) and 6 Trypanosoma cruzi discrete typing units (TcI to TcVI and TcBat), showing strong concordance with conventional methods (κ index of 0.729, P < 0.001). It detected co-infections between Trypanosomatid genera and T. cruzi, with a limit of detection of one parasite per mL. The method was successfully applied to human, animal, and triatomine samples. Notably, TcI predominated in chronic Chagas samples, whereas TcII and TcIV were found in the acute stage. Triatomine vectors exhibited diverse Trypanosomatid infections, with Triatoma dimidiata mainly infected with TcI and occasional TcBat co-infections, and Rhodnius prolixus showing TcI and TcII infections, along with T. rangeli co-infections and mixed TcII infections. Animals were infected with T. vivax, T. theileri, and T. evansi. The ABS method's high resolution, sensitivity, and accuracy make it a valuable tool for understanding Trypanosomatid dynamics, enhancing disease control strategies, and enabling targeted interventions.

Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. METHODS/PRINCIPLE FINDINGS: Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. CONCLUSIONS/SIGNIFICANCE: Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.

The Trypanosoma cruzi Antigen and Epitope Atlas: antibody specificities in Chagas disease patients across the Americas.

During an infection the immune system produces pathogen-specific antibodies. These antibody repertoires become specific to the history of infections and represent a rich source of diagnostic markers. However, the specificities of these antibodies are mostly unknown. Here, using high-density peptide arrays we examined the human antibody repertoires of Chagas disease patients. Chagas disease is a neglected disease caused by Trypanosoma cruzi, a protozoan parasite that evades immune mediated elimination and mounts long-lasting chronic infections. We describe a proteome-wide search for antigens, characterised their linear epitopes, and show their reactivity on 71 individuals from diverse human populations. Using single-residue mutagenesis we revealed the core functional residues for 232 of these epitopes. Finally, we show the diagnostic performance of identified antigens on challenging samples. These datasets enable the study of the Chagas antibody repertoire at an unprecedented depth and granularity, while also providing a rich source of serological biomarkers.

Incidencia de eventos vasculares mayores después de cirugía no cardiaca: impacto del monitoreo perioperatorio con troponina y electrocardiograma / Incidence of major vascular events after cardiac surgery: impact of preoperative monitoring with troponin and electrocardiogram

Introducción: los cambios demográficos recientes condujeron a un aumento del riesgo de eventos vasculares mayores después de cirugía no cardiaca. El monitoreo con troponina y electrocardiografía identificaría más de estos eventos. Métodos: de manera prospectiva se recolectaron datos de pacientes elegibles (mayores de 45 años no seleccionados sometidos a cirugía no cardiaca, bajo anestesia regional o general, con estancia hospitalaria prevista mayor o igual a 24 horas en dos hospitales de Bucaramanga), durante dos períodos de tiempo, antes y después de monitoreo diagnóstico post-operatorio (evaluaciones seriadas de troponina T y electrocardiogramas sin conocimiento de datos clínicos). Para el componente de tiempo anterior a la intervención (cuidado clínico convencional), se tomaron historias clínicas de todos los pacientes elegibles de una muestra aleatoria de tres meses correspondientes a 2005. Para el componente de tiempo posterior al monitoreo, se siguieron 100 pacientes elegibles consecutivos. El desenlace primario fue la incidencia de eventos vasculares mayores intrahospitalarios, incluyendo infarto del miocardio (definido como troponina elevada asociada a cambios electrocardiográficos sugestivos, independiente de los síntomas). Resultados: se incluyeron 534 historias clínicas y 100 pacientes quirúrgicos prospectivos (edad media 62,2 años, DE 12,9; 56% mujeres). El tipo de cirugía más frecuente fue la ortopédica (26,8%) seguida de la intra-abdominal (20,2%). La incidencia de eventos fue 2,8% en historias clínicas, en comparación con una incidencia de 7% en pacientes sometidos a monitoreo (p = 0,071). Los cuatro infartos del miocardio identificados en estos pacientes fueron silentes. Conclusión: el monitoreo diagnóstico post-operatorio con troponina y electrocardiografía, identificó una mayor proporción de eventos vasculares, principalmente infartos silentes del miocardio.

Introduction: recent demographic changes have led to an increased risk of major vascular events among patients undergoing non-cardiac surgery. Troponin and electrocardiogram monitoring would further identify these major vascular events. Methods: we prospectively collected data on elegible patients (non-selected individuals aged 45 or older undergoing non-cardiac surgery under general or regional anesthesia in two hospitals in Bucaramanga, with expected length of stay longer than 24 hours) during a time-interrupted series, before and after postoperative diagnostic monitoring (blinded assessment of troponin T and electrocardiograms ignoring clinical data). For the period before the intervention (usual clinical care), two independent reviewers extracted clinical information from clinical histories (of all eligible patients from 3 randomly-selected months of 2005). For the period after diagnostic monitoring, we followed 100 consecutive eligible patients. Primary outcome was a composite of major vascular events within hospital, including myocardial infarction (defined as any troponin elevation associated with electrocardiographic changes suggesting ischemia, regardless of symptoms). Results: we included 534 clinical charts and 100 prospective surgical patients (mean age 62.2, SD 12.9 years; 56% women). The more frequent surgical procedures were orthopedics (26.8%) followed by abdominal (20.2%). The incidence of major vascular events recorded in clinical charts was 2.8%, compared with 7% among monitored patients (p=0,071). All four myocardial infarctions identified among the later group were silent. Conclusion: postoperative monitoring with troponin and electrocardiography identified a higher proportion of major vascular events, mainly silent myocardial infarctions.

Early mobilisation for patients following acute myocardiac infarction: a systematic review and meta-analysis of experimental studies.

OBJECTIVE: To determine the impact of early mobilisation (EM) on total mortality and non-fatal re-infarction after acute myocardial infarction (AMI). DESIGN: Systematic review and meta-analysis. DATA SOURCES: MEDLINE, CINAHL, HealthStar, EMBASE, the Cochrane Library Controlled Trials Registry and experts. METHODS: Target studies included published and unpublished experimental, controlled studies in any language comparing AMI patients allocated to any in-hospital early mobilisation or a control/standard treatment. Two reviewers independently assessed study eligibility and quality and performed data extraction. We calculated relative risks (RRs) and 95% confidence intervals (CIs) using the random-effects model. OUTCOMES: All-cause mortality or re-infarction up to 1-year post-AMI. RESULTS: Out of 385 potentially relevant studies, 14 met our eligibility criteria (13 published before 1983). There were 149 deaths (9.3% of 1607) and 82 non-fatal re-infarctions (5.2% of 1580) among post-AMI patients receiving EM, compared with 179 deaths (11.6% of 1541) and 80 non-fatal re-infarctions (5.3% of 1518) among AMI patients receiving control treatment (RR=0.85, 95% CI 0.68, 1.05 and RR=1.02, 95% CI 0.75, 1.39 respectively). CONCLUSION: Our meta-analysis demonstrated a trend towards decreased mortality with EM after AMI. However, there is uncertainty about early mobilisation and more research should be developed having into account all kind of differences among patients receiving treatment after AMI worldwide.

Función diastólica en sujetos con serología positiva para enfermedad de Chagas procedentes del estudio CHICAMOCHA / Diastolic function in subjects with positive Chagas’ disease serology from the Chicamocha study

Se presenta un estudio ecocardiográfico en 430 donantes de banco de sangre, de los cuales 120 fueron negativos para Trypanosoma cruzi (controles), 231 fueron seropositivos sin cambios en el electrocardiograma (estadio I) y 79 fueron seropositivos con cambios en el electrocardiograma (estadio II). Se estudió la función diastólica y, a través del flujo mitral, se encontró un aumento significativo de la velocidad de la onda A con relación al grupo control (54 vs. 50,5 cm/seg). Con relación al flujo de las venas pulmonares, no hubo cambios significativos en las velocidades sistólicas pero sí aumento de las velocidades diastólicas de los sujetos estadio II con relación a los controles (48,7 vs. 46,7 cm/seg). El parámetro más significativo se halló en la duración de la onda A de las venas pulmonares, la cual aumentó en los sujetos estadio I y mucho más en los sujetos estadio II (0,13 seg para el grupo control, 0,14 seg para el grupo estadio I y 0,15 seg respectivamente). La diferencia entre la duración de la onda A mitral y la duración de la onda A de las venas pulmonares, mostró menor significancia en los sujetos estadio I y mayor aún (resultado negativo) en los sujetos estadio II, expresando así un aumento de la presión capilar pulmonar de estos últimos (control 0,012 seg, estadio I 0,009 seg y estadio II-0,007). La relación entre la duración de la onda A de las venas pulmonares y de la onda A mitral (Ap/Am), mostró, igualmente, un aumento progresivo con respecto a los controles (control 0,94, estadio I 0,96 y estadio II 1,08). El tiempo de relajación isovolumétrica aumentó significativamente en los sujetos estadio II con relación al grupo control (0,084 seg vs. 0,076 seg). En lo que concierne a las velocidades del anillo mitral, se encontró un aumento significativo en la velocidad de la onda A en los sujetos estadio II con respecto a los controles (17,9 cm/seg vs. 15,9 cm/seg). No hubo diferencias significativas en la velocidad sistólica ni en la velocidad de la...

An echographic study of 430 blood bank donors is presented. 120 were negative for Trypanosoma cruzi (controls), 231 were serum-positive without changes in the electrocardiogram (state I) and 79 were positive with electrocardiographic changes (state II). The diastolic function was studied and through the mitral flow a significant increase in the A wave velocity in relation to the control group, was found (54 vs. 50.5 cm/s). In relation to the pulmonary veins’ flow, there were no significant changes in the systolic velocities but there was an increase in the diastolic velocity in state II subjects (48.7 vs. 46.7 cm/s). The most signifying parameter was that of the pulmonary veins’ A wave duration, that increased in state I subjects and even more in state II subjects (0.13 s for the control group, 0.14 s for state I group and 0.15 s for state II, respectively). The difference between the duration of the mitral A wave and the duration of the pulmonary veins’ A wave showed less significance in state I subjects and even less (negative) in state II subjects, expressing in this way an increment in pulmonary capillary tension in these last ones (control: 0.012 s, state I: 0.09 s and state II: 1.08 s). The relation between the pulmonary veins’ A wave and that of the mitral A wave (Ap/Am) showed a progressive increment as well, in regard to the control group (17.9 cm/s vs. 15.9 cm/s). There were no significant differences in the systolic velocity, or in the velocity of the E ring wave. When observing the M-colour flow propagation behaviour, a significant decrease was noticed in state I subjects and even more in state II subjects in relation to the control group (72.7, 66.8 and 62.6 cm/s respectively).

How strong is the evidence for the use of perioperative blockers in non-cardiac surgery? Systematic review and meta-analysis of randomised controlled trials

Objective To determine the effect of perioperative blocker treatment in patients having non-cardiac surgery. Design Systematic review and meta-analysis.Data sources Seven search strategies, including searching two bibliographic databases and hand searching seven medical journals.Study selection and outcomes We included randomised controlled trials that evaluated blocker treatment in patients having non-cardiac surgery. Perioperative outcomes within 30 days of surgery included total mortality, cardiovascular mortality, non-fatal myocardial infarction, non-fatal cardiac arrest, non-fatal stroke, congestive heart failure, hypotension needing treatment, bradycardia needing treatment, and bronchospasm. Results Twenty two trials that randomised a total of 2437 patients met the eligibility criteria. Perioperative blockers did not show any statistically significant beneficial effects on any of the individual outcomes and the only nominally statistically significant beneficial relative risk was 0.44 (95% confidence interval 0.20 to 0.97, 99% confidence interval 0.16 to 1.24) for the composite outcome of cardiovascular mortality, non-fatal myocardial infarction, and non-fatal cardiac arrest. Methods adapted from formal interim monitoring boundaries applied to cumulative meta-analysis showed that the evidence failed, by a considerable degree, to meet standards for forgoing additional studies. The individual safety outcomes in patients treated with perioperative blockers showed a relative risk for bradycardia needing treatment of 2.27 (95% CI 1.53 to 3.36, 99% CI 1.36 to 3.80) and a nominally statistically significant relative risk for hypotension needing treatment of 1.27 (95% CI 1.04 to 1.56, 99% CI 0.97 to 1.66). Conclusion The evidence that perioperative blockers reduce major cardiovascular events is encouraging but too unreliable to allow definitive conclusions to be drawn...

Estudio electrocardiográfico en sujetos con serología positiva y negativa para Tripanosoma Cruzi. Pacientes asintomáticos procedentes del estudio CHICAMOCHA (Cardiovascular Health Investigation from Colombia to Assess the Markers and Outcomes of Chagas disease) / Electrocardiographic study in subjects with positive and negative serology for Tripanosoma Cruzi. Asymptomatic patients coming from the CHICAMOCHA STUDY (Cardiovascular Health Investigation from Colombia to Assess the Markers and Outcomes of Chagas Disease)

Se presenta un estudio electrocardiográfico, hecho en 2.130 donantes de bancos de sangre, en la ciudad de Bucaramanga, Colombia, entre los años 1999 y 2004. Se comparan los hallazgos entre 486 seropositivos T (+) y 1.644 seronegativos T (-) para Tripanosoma Cruzi. Se encontraron diferencias porcentuales estadísticamente significativas en la población T (+) con relación a los T (-) en los siguientes parámetros: bloqueo de rama derecha (6,1/2,6 p<0,001), hemibloqueo izquierdo anterosuperior (5,7/2,3 p<0,001), bloqueo aurículo-ventricular (2,7/1 p=0,006), extrasístoles ventriculares (2,5/0,9 p=0,009), aplanamiento o inversión de la onda T (16,4/10,8 p=0,001) y PR limítrofe (6,9/4,1 p=0,009). Del mismo modo, se encontraron diferencias significativas a favor del grupo T (-) en cuanto a patrón de bloqueo de rama derecha (9,9/5,9 p=0,007) y trastornos inespecíficos de la conducción (42,5/34,1 p=0,001). No se encontró diferencia estadísticamente significativa en cuanto a presentar extrasistolia supraventricular u ondas Q patológicas. Igualmente, si se asocian los trastornos de conducción y del ritmo, poseer al menos uno de ellos ocurre más en el grupo T (+) (16,8/11,3 p<0,001) y es aún más significativo si se presentan dos o más trastornos asociados (3,0/0,9 p<0,001).

Cardiovascular autonomic function testing in asymptomatic T. cruzi carriers: a sensitive method to identify subclinical Chagas' disease.

BACKGROUND: Although impaired cardio-vagal response characterizes full-blown Chagas' disease, this feature among otherwise healthy T. cruzi serology carriers (SERO[+]) requires confirmation. The purpose of this study was to determine whether abnormal cardio-vagal responses were different among SERO[+] subjects with varying ECG alterations. METHODS: We assessed cardio-vagal reflex response in 57 randomly selected healthy blood donors (36 SERO[+], 15 with ECG rhythm/conduction abnormalities). The following cardiac autonomic tests were performed: (1) short-term heart rate variability (HRV), (2) Deep breathing test (DBT), (3) cold face test, (4) cold pressor test (CPT), (5) Valsalva maneuver, and (6) baroreflex sensitivity after administration of nitroprusside (BRS-NTP) and phenylephrine (BRS-PNP). RESULTS: Overall, SERO[+] subjects had 161/324 (49.7%) abnormal responses, compared to 41/189 (21.7%) in SERO[-] (p<0.001). Similar rates were found in SERO[+] according to ECG status (68/135, 50.4% in ECG[+] and 93/189, 49.2% in ECG[-], p=0.836). Covariate-adjusted pooled odd ratios (95%CI) for abnormal responses compared to SERO[-] were: 2.73 (1.71-4.35) for SERO[+], and 2.63 (1.63-4.34) for SERO[+]/ECG[-] (p<0.001). BRS-NTP, CPT and DBT individually showed significant differences between SERO[-] and SERO[+] groups. Conversely, ECG changes among SERO[+] were not associated with a significant excess of autonomic abnormality either overall (OR=1.09, 95%CI: 0.67-1.78, p=0.719) or by any individual test. CONCLUSIONS: Early cardio-vagal dysfunction was documented in SERO[+] subjects regardless of ECG status. Cardiac autonomic evaluation may be useful for identification of subclinical disease in SERO[+] subjects.

Disfunción autonómica en sujetos seropositivos para T. cruzi asintomáticos

La enfermedad de Chagas crónica se caracteriza por daño cardiaco microvascular, contráctil y autonómico, sin que se comprenda completamente cuál de ellos determina el inicio de la enfermedad. Con el fin de edentificar alteraciones autonómicas en sujetos asintomáticos seropositivos para Tripanosoma cruzi, se evaluó la función autonómica cardíaca en 18 donantes de banco seropositivos (11 "Chagas 1") con electrocardiograma normal y siete "Chagas 2" con alteraciones del ritmo o de conducción) y 24 individuos seronegativos. Se realizaron tres pruebas de eferencia parasimpática (frecuencia cardíaca máxima/mínima en respiración profunda, frío en cara y respuesta presora al frío) y dos de eferencia simpática (respuesta cronotrópica y presora en la mesa inclinada). Durante el procedimiento se registró también el número variaciones de la frecuencia cardíaca (VFC) utilizando un oxímetro de pulso. Se encontraron diferencias significativas (p<0.033) en la frecuencia cardíaca basal y en las pruebas de respiración profunda, frío en cara y la respuesta presora a la mesa inclinada. La VFC fue menor en el grupo Chagas 2 en la mayoría de las pruebas. La respuesta parasimpática fue menor para el grupo Chagas 2. La función simpática se caracterizó por aumento del tono basal en el grupo Chagas 1. Estos hallazgos sugieren disautonomía en sujetos seropositivos asintomáticos. Se requieren estudios con mejores instrumentos y mayores poblaciones, para consolidar los conocimientos en este campo