Marta Gajardo

Phylogenetic analysis of microsatellite markers further supports the two hybridization events hypothesis as the origin of the Trypanosoma cruzi lineages

To better understand the evolution of the etiologic agent of Chagas disease, we cloned and sequenced 25 alleles from five Tripanosoma cruzi microsatellite markers. The study of the sequences showed highly conserved alleles present in T. cruzi clones belonging to TCI, TCIIc, and TCIIe. This result was also confirmed by the phylogenetic analysis of MCLE01 allele sequences. The examination by capillary electrophoresis of six microsatellite markers from 19 T. cruzi clones showed a high proportion of the alleles found both in the TCI and TCII sublineages. The phylogenetic reconstruction of these 19 clones produced a tree with two major clusters with bootstrap support of 100% and 95%. The first cluster includes T. cruzi clones belonging to the TCI and TCIIa lineages. The second cluster is composed of TCI, TCIIc, TCIId, and TCIIe T. cruzi clones. The analysis of five microsatellite markers in the CLBrener genome showed that almost all the microsatellite markers are synteny; non-Esmeraldo and Esmeraldo haplotypes probably come from the TCIIc and TCIIb lineages. Taken together, our results are in agreement with the two hybridization events hypothesis as the origin of current T. cruzi lineages.

Microsatellite marker analysis shows differentiation among Trypanosoma cruzi populations of peripheral blood and dejections of Triatoma infestans fed on the same chronic chagasic patients: Microsatellite marker analysis and T. cruzi

To investigate whether Trypanosoma cruzi populations found in chagasic cardiopathic and non-cardiopathic patients are genetically differentiated, three molecular microsatellite markers were analysed. This analysis was also applied to compare T. cruzi samples from peripheral blood or dejections of Triatoma infestans fed on the blood of the same patients. In order to obtain the first objective, analyses of predominant T. cruzi genotypes were conducted using three approaches: a locus-by-locus analysis; a Fisher method across three loci; and analysis of molecular variance by Genepop and Arlequin programs. Only with one locus and on the blood samples was a significant differentiation detected among non-cardiopathic and cardiopathic groups, which was not confirmed by the other two methods. On the contrary, with the three approaches, it was found that T. cruzi clones present in the blood of patients are genetically differentiated from those detected in dejections of T. infestans fed on the same patients. Our results showed that the most frequent lineage both in blood as well as in triatomine dejection samples was TcI. No significant difference in T. cruzi lineage distribution was observed among chagasic cardiopathic and non-cardiopathic patients. The majority of the samples (50–60%) had only one T. cruzi clone (uniclonal) either in blood or dejection samples.

Geographical structuring of Trypanosoma cruzi populations from Chilean Triatoma infestans triatomines and their genetic relationship with other Latino American counterparts.

Abstract In order to obtain more information about the population structure of Chilean Trypanosoma cruzi, and their genetic relationship with other Latino American counterparts, we performed the study of T. cruzi samples detected in the midgut content of Triatoma infestans insects from three endemic regions of Chile. The genetic characteristics of these samples were analysed using microsatellite markers and PCR conditions that allow the detection of predominant T. cruzi clones directly in triatomine midgut content. Population genetic analyses using the Fisher’s exact method, analysis of molecular variance (AMOVA) and the determination of FST showed that the northern T. cruzi population sample was genetically differentiated from the two southern population counterparts. Further analysis showed that the cause of this genetic differentiation was the asymmetrical distribution of TcIII T. cruzi predominant clones. Considering all triatomines from the three regions, the most frequent predominant lineages were TcIII (38%), followed by TcI (34%) and hybrid (8%). No TcII lineage was observed along the predominant T. cruzi clones. The best phylogenetic reconstruction using the shared allelic genetic distance was concordant with the population genetic analysis and tree topology previously described studying foreign samples. The correlation studies showed that the lineage TcIII from the III region was genetically differentiated from the other two, and this differentiation was correlated with geographical distance including Chilean and mainly Brazilian samples. It will be interesting to investigate whether this geographical structure may be related with different clinical manifestation of Chagas disease.

Microsatellite loci-based distribution of Trypanosoma cruzi genotypes from Chilean chronic Chagas disease patients and Triatoma infestans is concordant with a specific host-parasite association hypothesis.

The objective of this study was to investigate if there is specific host-parasite association in Chilean populations of Trypanosoma cruzi. For this purpose, two groups of parasites were analyzed, one from chronic chagasic patients, and the other from Triatoma infestans triatomines in three regions of the country. The first group consisted of four types of samples: parasites from peripheral blood of non-cardiopathic T. cruzi infected patients (NB); parasites from their corresponding xenodiagnosis (NX); parasites from peripheral blood of T. cruzi infected cardiopathic patients (CB) and parasites from their xenodiagnostics (CX). The T. infestans sample in turn was from three regions: III, V and M (Metropolitan). The genetic differentiation by the Fisher exact method, the lineage distribution of the samples, the molecular phylogeny and the frequency of multiclonality were analysed. The results show that not only are the groups of T. cruzi clones from Chagas disease patients and vectors genetically differentiated, but also all the sub-groups (NB, NX, CB and CX) from the III, V and M regions. The analysis of lineage distribution was concordant with the above results, because significant differences among the percentages of TcI, TcIII and hybrids (TcV or TcVI) were observed. The phylogenetic reconstruction with these Chilean T. cruzi samples was coherent with the above results because the four chagasic samples clustered together in a node with high bootstrap support, whereas the three triatomine samples (III, V and M) were located apart from that node. The topology of the tree including published T. cruzi clones and isolates was concordant with the known topology, which confirmed that the results presented here are correct and are not biased by experimental error. Taken together the results presented here are concordant with a specific host-parasite association between some Chilean T. cruzi populations.

Relación entre enfermedad periodontal, infección bacteriana ascendente y patología placentaria con parto prematuro

Objetivo: Determinar la relacion entre enfermedad periodontal, infeccion bacteriana ascendente y patologia placentaria, con parto prematuro. Pacientes y Metodos: Participaron embarazadas entre 24 y 34 semanas de gestacion, con trabajo de parto prematuro sin causa clinica evidente y membranas intactas o con el diagnostico de rotura prematura de membranas (RPM), sin trabajo de parto y sin corioamnionitis clinica. Todas las embarazadas tuvieron estudio periodontal clinico y evaluacion microbiologica de la placa subgingival, del liquido amniotico (LA) y cervicovaginal. R ecibieron corticoesteroides, antibioticos, tocolisis (casos con membranas intactas) y manejo expectante hasta las 35 semanas (casos con RPM) . Las placentas se enviaron a estudio y se diagnostico corioamnionitis, funisitis y vellositis. Se definio invasion microbiana de la cavidad amniotica ( IMCA) el cultivo positivo del liquido amniotico. Infeccion cervicovaginal (ICV) se diagnostico con vaginosis bacteriana (VB) o cultivo positivo para bacteria patogena u oportunista en cervix o vagina, con incremento significativo de los leucocitos polimorfonucleares. Se considero como infeccion bacteriana ascendente (IBA) la presencia de IMCA por bacterias ascendentes y/o ICV. Resultados: Se incluyeron 59 pacientes, 42 con membranas intactas y 17 con RPM . La frecuencia de la enfermedad periodontal fue 93.2%. La IMCA fue 27.1% Se aislaron bacterias patogenas periodontales del LA en el 18.6% y de la placa subgingival en el 71.2% de los casos. La IBA fue 83.1%. La asociacion IBA con enfermedad periodontal fue 72.9%. El parto prematuro ( 82.1% p=0.03 y con la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal 74.4% p=0.03. Los casos con nacimiento prematuro y enfermedad periodontal generalizada tuvieron significativa mayor frecuencia de corioamnionitis y funisitis histologica 69.6% p=0.04. Conclusiones : L a enfermedad periodontal generalizada y la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal se asocian con parto prematuro . En estos casos, son frecuentes l os marcadores histologicos placentarios de infeccion ascendente .

Inmunofenotipificación de linfocitos en sangre periférica de pacientes chagásicos crónicos chilenos mediante citometría de flujo

Background: Cellular immune mechanisms of the resistance to infection by T cruzi as well as the pathogenesis of Chagas disease are still controversial. Aim: To quantify and analyse the peripheral blood immune cells from chagasic and non chagasic patients by flow cytometry. Patients and methods: Peripheral blood samples were taken from 21 individuals seropositive for Chagas disease, under no specific treatment. Control samples from 21 healthy blood donors were also obtained. To quantify immune cells populations by flow cytometry, antibodies against CD3, CD4, CD8, CD16/56, CD45/14, CD19 and HLA-DR markers were used. Results: The percentage of CD8+ cells was low and the CD4+/CD8+ ratio was high in chagasic patients, compared to their non infected counterparts. No statistically significant differences in the number of CD4+, NK, B, CD4+HLADR+ and CD8+HLADR+ cells, were observed within the two groups. Conclusions: These results show that Chilean chronic chagasic patients have lower percentage of CD8+ cells and higher CD4+/CD8+ ratio than non infected individuals (Rev Med Chile 2002; 130: 363-367