ABSTRACT
Leishmania RNA virus (LRV) is a double-stranded RNA virus belonging to the Totiviridae family detected as cytoplasmic inclusions in some strains of the human parasite Leishmania spp. Experimental evidence supports the hypothesis that human coinfection with Leishmania spp.-LRV triggers an exacerbated immune response in the host that can be responsible for the observed complicated outcomes in cutaneous leishmaniasis (CL), such as mucosal leishmaniasis (ML) and treatment failure of CL. However, the reported frequencies of LRV associated with complicated outcomes in patient's series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. To assess whether or not the inconsistent information about the frequency of LRV associated with CL complicated outcomes could be related to the virus detection approach, the present study evaluated the LRV presence in clinical samples using a diagnostic algorithm according to the type of the sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and six samples with non-Leishmania spp. infection, the LRV presence was assessed by RT-PCR, RT-qPCR, and nested RT-PCR. Viral load was estimated in parasite clinical isolates. By combining the methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 37.5% (6/16) of the incisional biopsies. Remarkably, in some cases (4/8), LRV1 was undetectable in the isolates but present in their respective biopsies, and less frequently, the opposite was observed (1/8), suggesting the possibility of loss of parasites harboring LRV1 during the in vitro growth.
Subject(s)
Leishmania/virology , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/virology , Leishmaniavirus/genetics , RNA, Viral/isolation & purification , Humans , Leishmania/classification , Leishmaniavirus/isolation & purification , Polymerase Chain Reaction/methods , Viral LoadABSTRACT
BACKGROUND: American cutaneous leishmaniasis (ACL) is a complicated disease producing about 67.000 new cases per year. The severity of the disease depends on the parasite species; however in the vast majority of cases species confirmation is not feasible. WHO suggestion for ACL produced by Leishmania braziliensis, as first line treatment, are pentavalent antimonial derivatives (Glucantime or Sodium Stibogluconate) under systemic administration. According to different authors, pentavalent antimonial derivatives as treatment for ACL show a healing rate of about 75% and reasons for treatment failure are not well known. METHODS: In order to characterise the clinical and parasitological features of patients with ACL that did not respond to Glucantime, a cross-sectional observational study was carried out in a cohort of 43 patients recruited in three of the Colombian Army National reference centers for complicated ACL. Clinical and paraclinical examination, and epidemiological and geographic information were recorded for each patient. Parasitological, histopathological and PCR infection confirmation were performed. Glucantime IC50 and in vitro infectivity for the isolated parasites were estimated. RESULTS: Predominant infecting Leishmania species corresponds to L. braziliensis (95.4%) and 35% of the parasites isolated showed a significant decrease in in vitro Glucanatime susceptibility associated with previous administration of the medicament. Lesion size and in vitro infectivity of the parasite are negatively correlated with decline in Glucantime susceptibility (Spearman: r = (-)0,548 and r = (-)0,726; respectively). CONCLUSION: A negative correlation between lesion size and parasite resistance is documented. L. braziliensis was found as the main parasite species associated to lesion of patients that underwent treatment failure or relapse. The indication of a second round of treatment in therapeutic failure of ACL, produced by L. braziliensis, with pentavalent antimonial derivatives is discussable.
Subject(s)
Antiprotozoal Agents/therapeutic use , Leishmania braziliensis/drug effects , Leishmaniasis, Cutaneous/drug therapy , Meglumine/therapeutic use , Organometallic Compounds/therapeutic use , Adult , Antiprotozoal Agents/pharmacology , Cohort Studies , Cross-Sectional Studies , Humans , Inhibitory Concentration 50 , Leishmania braziliensis/physiology , Male , Meglumine/pharmacology , Meglumine Antimoniate , Organometallic Compounds/pharmacology , Recurrence , Treatment Failure , U937 Cells , Young AdultABSTRACT
The discrimination of Leishmania species from patient samples has epidemiological and clinical relevance. In this study, different gene target PCR-restriction fragment length polymorphism (RFLP) protocols were evaluated for their robustness as Leishmania species discriminators in 61 patients with cutaneous leishmaniasis. We modified the hsp70-PCR-RFLP protocol and found it to be the most reliable protocol for species identification.
Subject(s)
DNA, Protozoan/genetics , Leishmania/genetics , Leishmania/isolation & purification , Leishmaniasis, Cutaneous/parasitology , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , Leishmania/classification , Species SpecificityABSTRACT
BACKGROUND: African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive. RESULTS: We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5. CONCLUSIONS: Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.
Subject(s)
Centromere/genetics , Tandem Repeat Sequences/genetics , Trypanosoma brucei brucei/genetics , Genome, Protozoan , Oligonucleotide Array Sequence Analysis , Restriction MappingABSTRACT
Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIß, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity.
Subject(s)
Antigens, Neoplasm/metabolism , Centromere/enzymology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Antigens, Neoplasm/chemistry , DNA Cleavage , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Molecular Sequence Data , RNA Interference , Sumoylation , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & development , Trypanosoma cruzi/enzymologyABSTRACT
El objetivo del presente estudio consistió en comparar la concordancia entre los resultados obtenidos por las técnicas IFI, ELISA y Western Blot en 72 sueros caninos procedentes de siete municipios de la zona endémica de leishmaniasis visceral zoonótica (LVZ) del departamento del Tolima (Colombia). Se utilizó como antígeno la cepa colombiana de Leishmania infantum MHOM/CO/CL044B para IFI, ELISA y WB, y el antígeno rK39 para una prueba de ELISA disponible comercialmente. Se encontró que la concordancia entre las diferentes técnicas comparadas fue menor del 16 por ciento (k<16 por ciento), lo que sugiere que las pruebas no son consistentes y por lo tanto, no son aceptables como método de diagnóstico en el presente estudio. La baja asociación de las pruebas serológicas utilizadas en el diagnóstico de L. infantum sugiere que es necesario desarrollar estudios que permitan establecer un algoritmo de pruebas diagnósticas en el país para confirmar el estado real de la infección en los animales y de esta forma orientar eficientemente los recursos de salud pública destinados para el control de la enfermedad.
The goal of present study was to compare the agreement between the results obtained by ELISA, IFI and WB tests in 72 canine serums from the South of the Tolima Department (a visceral leishmaniasis endemic area). The Colombian Leishmania infantum MHOM/CO/CL044B strain was used as antigen for ELISA, IFI and WB test, and the rK39 antigen for the commercial ELISA test. The agreement among the compared techniques was smaller than 16 percent (k<16 percent), suggesting that the tests are not consistent and therefore not acceptable as a diagnostic tool in this study. The low association between the serological tests used in diagnosing Leishmania infantum suggested the need for further studies aimed at establishing an algorithm for diagnostic tests in Colombia for confirming the real state of the animals´ infection and thereby efficiently orientating public health resources allocated for controlling visceral leishmaniasis.
Subject(s)
Animals , Dogs , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Serologic Tests/methods , Blotting, Western/veterinary , Veterinary MedicineABSTRACT
Objetivos Establecer la capacidad de las pruebas serológicas de Inmunofluorescencia indirecta (IFAT) e Inmunoensayo ligado a enzimas (ELISA), para detectar el estado real de la infección en leishmaniasis visceral canina (LVC). Métodos Un total de 211 perros criollos localizados en el sur del Departamento del Tolima, zona endémica para la leishmaniasis visceral, fueron evaluados por medio de examen clínico y serológico, usando como antígeno la cepa colombiana de Leishmania infantum (infantum) MHOM/COL/CL044B. Así mismo, con la finalidad de establecer reacciones cruzadas o coinfecciones, se evaluó la reactividad de los sueros ante antígenos específicos de Trypanosoma cruzi, por medio de la prueba Western Blot (WB). La frecuencia de LVC fue del 44,1 por ciento (93/211) y 50,2 por ciento (103/211) mediante IFAT y ELISA respectivamente. Resultados La seroreactividad ante antígenos específicos de T. cruzi fue del 1,42 por ciento (3/211). La concordancia de las técnicas serológicas fue baja (K=12,1 por ciento) y a pesar de la presencia de signos clínicos en los animales evaluados, las razones de prevalencia halladas, demostraron que no hay asociación entre la ocurrencia de las manifestaciones clínicas y la seropositividad a las pruebas diagnósticas. Conclusiones La baja asociación de las pruebas serológicas utilizadas en el diagnóstico de Leishmania infantum, sugiere que es necesario desarrollar estudios que permitan establecer un algoritmo de pruebas diagnósticas en el país para confirmar el estado real de la infección de los animales y de esta forma orientar eficientemente los recursos de Salud Pública destinados al control de la enfermedad.
Objectives Establishing indirect immunofluorescence (IFAT) and enzyme-inked immunoassay (ELISA) serological tests' ability to detect canine visceral leishmaniasis (CVL) infection. Methods 211 cross-bred dogs from the south of the Tolima department (a visceral leishmaniasis endemic area) were evaluated by clinical and serological exams, using the Colombian Leishmania infantum (infantum) MHOM/COL/CL044B strain as antigen. Sera reactivity to Trypanosoma cruzi- specífic antigens was evaluated by Western blot (WB) for establishing crossed-reactions or coinfections. CVL frequency was 44 ,1 percent (93/211) by IFAT and 50,2 percent (103/211) by ELISA. Results Seroreactivity to T. cruzi-specific antigens was 1 ,42 percent (3/211). Agreement between the serological techniques was low (K=12 ,1 percent) and, in spite of clinical signs being present in the animals being evaluated, the prevalence ratios found demonstrated the lack of association between the occurrence of the clinical manifestations and the diagnostic tests' seropositivity. Conclusions The low association between the serological tests used in diagnosing Leishmania infantum suggested the need for further studies aimed at establishing an algorithm for diagnostic tests in Colombia for confirming the real state of the animals' infection and thereby efficiently orientating public health resources allocated for controlling canine visceral leishmaniasis.
Subject(s)
Animals , Dogs , Female , Male , Dog Diseases/diagnosis , Leishmaniasis, Visceral/veterinary , Antibodies, Protozoan/immunology , Blotting, Western , Colombia , Data Interpretation, Statistical , Dog Diseases/epidemiology , Dog Diseases/immunology , Dog Diseases/prevention & control , Enzyme-Linked Immunosorbent Assay , Leishmania infantum/immunology , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/epidemiology , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/prevention & controlABSTRACT
Se estudiaron 13 mujeres con desnutrición proteico-calórica crónica global, (DNPC) cuyas edades oscilaron entre 8 y 14 años con un promedio de 11.8, hospitalizadas en una sala de Medicina Interna del Hospital Universitario San Vicente de Paúl y 7 mujeres controles, con edades similares. En ambos grupos, se practicaron exámenes exhaustivos clínico, nutricional y de laboratorio; el primero realizado por los médicos del grupo investigador y el segundo por la nutricionista. El eje hipotálamo-hipófisis-tiroideo de las pacientes y de los controles se estudió midiendo triyodotironina (T3), tetrayodotironina (T4) y la hormona estimulante de la tiroides (TSH) en condiciones basales y después de estímulo con hormona liberadora de tirotropina (TRH). También se practicó captación tiroidea a las 4 y 24 horas. No se encontró diferencia estadísticamente significativa entre las pacientes y los controles en la concentración de albúmina y de hemoglobina, el hematocrito, en los porcentajes de circunferencia muscular del brazo o del pliegue de grasa, ni en el conteo de linfocitos por mm3. Tampoco en la captación tiroidea, en la concentración de T3, T4 y TSH basales, en la relación T3/T4 en ayunas o después del estímulo con TRH, ni en el incremento de la T3 y la T4 después de la inyección de la hormona estimulante de la TSH. No hubo correlación entre el grado de desnutrición y la TSH basal, o después del estímulo con TRH, pero sí se demostró una diferencia estadísticamente significativa en la respuesta de la TSH a la TRH a los 10 y 20 minutos (P < 0.01) y en menor proporción a los 30 minutos. Se revisó la literatura relacionada con el eje hipotálamo-hipófisis-tiroideo en DNPC y se tratan de explicar las diferencias de los resultados entre este trabajo de investigación y los obtenidos por otros autores en la anorexia nerviosa y en el Kwashiorkor
