This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the ...highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In mammals, chronic diseases resulting from infectious agents have been associated with functional T cell response deficiency, a high frequency of terminally differentiated T cells, the presence of ...monofunctional Ag-specific T cells, and increased expression of inhibitory receptors. Similar to other chronic diseases, the progressive loss of certain functional activities during Trypanosoma cruzi infection might result in the inability to control replication of this parasite. To examine this hypothesis, we evaluated the differentiation and cell effector function of CD8(+) T cells and characterized the expression of inhibitory receptors and the presence of the parasite in the bloodstream of chagasic patients. The results showed that patients at an advanced severe disease stage had a higher frequency of terminally differentiated CD8(+) T cells than patients at an early stage of the disease. A monofunctional CD8(+) T cell response was observed in patients at an advanced stage, whereas the coexpression of markers that perform three and four functions in response to parasite Ags was observed in patients at a less severe disease stage. The frequency of CD8(+) T cells producing granzyme B and perforin and those expressing inhibitory receptors was higher in symptomatic patients than in asymptomatic patients. Taken together, these findings suggest that during the course of Chagas disease, CD8(+) T cells undergo a gradual loss of function characterized by impaired cytokine production, the presence of advanced differentiation, and increased inhibitory receptor coexpression.
Background: Chikungunya virus (CHIKV) diagnosis has become a challenge for primary care physicians in areas where the Zika virus and/or Dengue virus are present. Case definitions for the three ...arboviral infections overlap. Methods: A cross-sectional analysis was carried out. A bivariate analysis was made using confirmed CHIKV infection as the outcome. Variables with significant statistical association were included in an agreement consensus. Agreed variables were analyzed in a multiple regression model. The area under the receiver operating characteristic (ROC) curve was calculated to determine a cut-off value and performance. Results: 295 patients with confirmed CHIKV infection were included. A screening tool was created using symmetric arthritis (4 points), fatigue (3 points), rash (2 points), and ankle joint pain (1 point). The ROC curve identified a cut-off value, and a score >= 5.5 was considered positive for identifying CHIKV patients with a sensibility of 64.4% and a specificity of 87.4%, positive predictive value of 85.5%, negative predictive value of 67.7%, area under the curve of 0.72, and an accuracy of 75%. Conclusion: We developed a screening tool for CHIKV diagnosis using only clinical symptoms as well as proposed an algorithm to aid the primary care physician.
CD8+ T cells have been shown to play a crucial role in Trypanosoma cruzi infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as ...follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control T. cruzi in the chronic phase of the infection are unknown.
To characterise the CD8+ T cell subsets that could be participating in the control of T. cruzi infection, in this study, we compared total and T. cruzi-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells.
These findings suggest that CD8+ TSCM cells may be associated with the immune response to T. cruzi and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Insufficient and irregular data reports on Leishmaniasis, issuing from the developing world, have left much to be desired in terms of understanding the molecular signatures producing distinct ...infectious phenotypes of the disease. Herein, we report on the complete genome sequencing of Leishmania naiffi and Leishmania guyanensis, sampled from patients in regions of Colombia and Venezuela. In this study, the isolates of cutaneous lesions from both species presented limited structural variation at the chromosomal level, low gene copy number variation, and high genetic heterogeneity. We compared these sequences to the reference genomes hitherto related from Brazil and French Guyana. Although of the same species, we note a consequential genomic disparity between the Venezuelan and French Guyanese isolates of L. guyanensis. Although less significant on the global schema of cutaneous and mucosal disease, such genomic studies of L. naiffi and L. guyanensis substantiate the gaps in understanding of the molecular architecture and multivariate clinical pictures of Leishmaniasis, on an international scale.
Knowledge of the prevalence and risk factors associated with maternal infection is the first step to develop a surveillance system for congenital transmission of Chagas disease. We conducted a ...cross-sectional study in Casanare, a disease-endemic area in Colombia. A total of 982 patients were enrolled in the study. A global prevalence of Trypanosoma cruzi infection of 4.0% (95% confidence interval CI = 2.8-5.3%) was found. Multivariate analysis showed that the most important risk-associated factors were age > 29 years (adjusted odds ratio aOR = 3.4, 95% CI = 0.9-12.4), rural residency (aOR = 2.2, 95% CI = 1.0-4.6), low education level (aOR = 10.2, 95% CI = 1.6-82.7), and previous knowledge of the vector (aOR = 2.2, 95% CI = 1.0-4.9). Relatives and siblings of infected mothers showed a prevalence of 9.3%. These findings may help physicians to investigate congenital cases, screen Chagas disease in siblings and relatives, and provide early treatment to prevent the chronic complications of Chagas disease.
Objective
To determine the prevalence and risk factors associated with Chagas disease in pregnant women in an endemic area of Santander, Colombia.
Methods
Cross‐sectional study included 23 ...municipalities of Santander, Colombia. Serological IFAT and ELISA tests were undertaken to detect IgG anti‐ Trypanosoma cruzi. A questionnaire was conducted for assessing the risk factors of each participant. Newborns were evaluated at birth and followed up to 1 year of age to determine congenital infection.
Results
An overall prevalence of 3.2% (95% CI 2.4–4.2) among 1518 pregnant women was detected. Prevalences by provinces were as follows: Guanentina: 6.0% (95% CI 4.1–8.5), García Rovira: 2.9% (95% CI: 1.5–4.8) and Comunera: 0.4% (0.4–2.3). The main risk factors identified were age >32 years old (OR: 2.1; 95% CI: 1.1–3.9); currently having a thatched roof (OR: 11.8; CI95% 2.2–63.2) and a thatched roof during childhood (OR: 3.0; 95% CI: 1.4–6.6); having below primary school education level (OR: 4.6; 95% CI: 2.2–9.5); and a history of a close contact with the vector (triatomine bugs) at least once during their lifetime (OR: 6.9; 95% CI: 3.7–12.9). No congenital cases were detected by parasitological or serological techniques.
Conclusions
Prevalence of Chagas disease in pregnant women is a potential source of infection in this Colombian endemic area. The main risk factors associated with seropositivity were related to conditions favouring the contact with the vector. The results show that it is necessary to continue an active surveillance in order to offer diagnosis and treatment to mothers and their newborns in addition to screening to pregnant women from endemic areas.
Objectif
Déterminer la prévalence et les facteurs de risque associés à la maladie de Chagas chez les femmes enceintes dans une zone endémique de Santander, en Colombie.
MéthodesEtude transversale couvrant 23 municipalités de Santander, en Colombie. Les tests sérologiques IFAT et ELISA ont été effectués pour détecter les anticorps IgG de T. cruzi. Un questionnaire a été administré pour évaluer les facteurs de risque de chaque participant. Les nouveau‐nés ont été évalués à la naissance et suivis jusqu’à un an pour déterminer l'infection congénitale.
Résultats
Une prévalence globale de 3,2% (IC95%: 2,4 à 4,2) chez 1518 femmes enceintes a été déterminée. Les prévalences par provinces étaient: Guanentina: 6,0% (IC95%: 4,1 à 8,5), García Rovira: 2,9% (IC95%: 1,5 à 4,8) et Comunera: 0,4% (IC95%: 0,4 à 2,3). Les principaux facteurs de risque identifiés étaient l’âge > 32 ans (OR: 2,1; IC95%: 1,1 à 3,9); avoir en cours un toit de chaume (OR: 11,8; IC95%: 2,2 à 63,2) et avoir eu un toit de chaume pendant l'enfance (OR: 3,0; IC95%: 1,4 à 6,6); avoir un niveau d’éducation en dessous de celui de l’école primaire (OR: 4,6; IC95%: 2,2 à 9,5) et une histoire d'un contact étroit avec le vecteur (les triatomes) au moins une fois au cours de sa vie (OR: 6,9; IC95%: 3,7 à 12,9). Aucun cas congénital n'a été détecté par les techniques parasitologiques ou sérologiques.
Conclusions
La prévalence de la maladie de Chagas chez les femmes enceintes est une source potentielle d'infection dans cette zone endémique Colombienne. Les principaux facteurs de risque associés à la séropositivité étaient liés à des conditions favorisant le contact avec le vecteur. Les résultats montrent que la surveillance active dans le but d'offrir un diagnostic et un traitement aux mères et à leurs nouveau‐nés et le dépistage des femmes enceintes dans les zones endémiques doit être poursuivie.
Objetivo
Determinar la prevalencia y los factores de riesgo asociados con la enfermedad de Chagas entre mujeres embarazadas de un área endémica de Santander, Colombia.
Métodos
Estudio crosseccional que incluyó 23 municipios de Santander, Colombia. Se realizaron las pruebas de IFAT y ELISA en muestras de suero para detectar IgG anti‐T. cruzi. Se pasó un cuestionario para evaluar los factores de riesgo de cada participante. Los recién nacidos se evaluaron en el momento del parto y se les realizó un seguimiento hasta que cumplieron el primer año, con el fin de determinar la presencia de una infección congénita.
Resultados
Se detectó una prevalencia total del 3.2% (IC 95% 2.4 – 4.2) entre 1,518 mujeres embarazadas. Las prevalencias por provincias eran: Guanentina: 6.0% (IC 95% 4.1 – 8.5), García Rovira: 2.9% (IC 95%: 1.5 – 4.8) y Comunera: 0.4% (0.4‐2.3). Los principales factores de riesgo identificados eran tener >32 años (OR: 2.1; IC 95%: 1.1 – 3.9); vivir en un hogar con un techo de paja (OR: 11.8; IC 95% 2.2 – 63.2) y haber vivido en un hogar con techo de paja durante la infancia (OR: 3.0; IC 95%: 1.4 – 6.6); tener un nivel de estudios menor que la primaria (OR: 4.6; IC 95%: 2.2 – 9.5) y una historia de contacto cercano con el vector (insectos triatominos) de al menos una vez durante el trascurso de su vida (OR: 6.9; 95% CI: 3.7 – 12.9). No se detectaron casos congénitos mediante técnicas parasitológicas o serológicas.
Conclusiones
La prevalencia de la enfermedad de Chagas entre mujeres embarazadas es una fuente potencial de infección en esta área endémica de Colombia. Los principales factores de riesgo asociados con la seropositividad estaban relacionados con condiciones que favorecían el contacto con el vector. Estos resultados muestran que se debe continuar con una vigilancia activa que ofrezca diagnóstico y tratamiento a las madres y a sus recién nacidos, así como el cribado de mujeres embarazadas pertenecientes a áreas endémicas.
Introducción. El diagnóstico de la enfermedad de Chagas es fundamental para brindar un tratamiento oportuno y mejorar el pronóstico del paciente. La capacidad discriminatoria de las pruebas ...serológicas para el diagnóstico varía de acuerdo con la prevalencia de la enfermedad y el antígeno utilizado en la prueba. Objetivo. Evaluar la capacidad discriminatoria de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (NovaTec Immunodiagnostica GmbH) en un grupo de individuos colombianos utilizando la prueba de inmunofluorescencia indirecta (IFI) y el ensayo de inmunoabsorción enzimática (ELISA) como referencia. Materiales y métodos. Se incluyeron 78 muestras de pacientes crónicos (36 asintomáticos y 42 sintomáticos) y 21 de controles sanos. También se analizaron 17 individuos no infectados con riesgo epidemiológico para la enfermedad de Chagas, siete con leishmaniasis y nueve con enfermedad cardiaca. Se evaluaron por PCR en tiempo real cuatro individuos cuyos resultados variaron entre pruebas. Resultados. Se encontraron diferencias significativas a una densidad óptica de 450 nm (p<0,0001) al comparar la mediana de la absorbancia entre los controles sanos (0,143) y los asintomáticos (2,401) o sintomáticos (2,776), entre los asintomáticos y sintomáticos (p=0,0408), entre los seronegativos con riesgo (0,232), individuos con enfermedades cardiacas (0,367) o con leishmaniasis (0,337) y los pacientes con enfermedad de Chagas (p<0,0001), y entre los controles sanos y los pacientes seronegativos con riesgo (p=0,0264), con enfermedades cardiacas (p=0,0015) o con leishmaniasis (p=0,002). La PCR en tiempo real fue positiva en tres de los cuatro casos. Conclusiones. Esta prueba comercial de ELISA permitió discriminar a los pacientes con Chagas de los controles. Se requieren estudios de fase II para determinar las características operativas de la prueba.
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► Repetitive SIRE element is present in the genome of all Trypanosoma cruzi DTUs. ► SIRE could be an active element in the parasite genome. ► SIRE Southern blot profiles differentiate ...DTU-I from the other DTUs. ► Given its features SIRE sequence is a good target for parasite DNA amplification.
Repetitive sequences constitute an important proportion of the Trypanosoma cruzi genome; hence, they have been used as molecular markers and as amplification targets to identify the parasite presence via PCR. In this study, a molecular characterization of the SIRE repetitive element was performed in the six discrete typing units (DTUs) of T. cruzi. The results evidenced that this element, located in multiple chromosomes, was interspersed in the genome of all DTUs of the parasite. The presence of several motifs implicated in element insertion, duplication, and functionality suggests that SIRE could be an active element in the parasite genome. Of interest, there were SIRE specific Alu I fragments that allowed to discriminate DTU I from the others DTUs. Moreover, an UPGMA phenetic tree constructed from fragment sharing Southern blot data showed that T. cruzi I isolates conform a cluster separated from the T. cruzi II–VI isolates. When the relative number of SIRE copies was determined, a variation from 105 to 2,000 copies per haploid genome was observed among the different isolates without kept a DTU-relationship. In all, these findings suggest that SIRE sequence is a good target for parasite DNA amplification.
Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and ...developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2′ substitutions defined 2′-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2′-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.
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