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.

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.

Detection of Porphyromonas gingivalis in the amniotic fluid in pregnant women with a diagnosis of threatened premature labor.

BACKGROUND: Epidemiologic and randomized controlled studies have shown that periodontal diseases may be associated with preterm labor and delivery of infants with low birth weights. The purpose of the present study was to determine the presence of microbial invasion of the amniotic cavity by periodontopathic bacteria in pregnant women with a diagnosis of threatened premature labor. METHODS: A periodontal examination and collection of amniotic fluid and subgingival plaque samples were performed on women identified as having threatened premature labor (preterm premature rupture of membranes without clinical infection or labor and preterm labor with intact membranes) and a gestational age ranging between 24 and 34 weeks. Samples collected from amniotic fluid and from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid and cultured. Porphyromonas gingivalis was identified primarily by colony morphology under stereoscopic microscope and rapid biochemical tests. Amniotic fluid or plaque samples were homogenized, DNA was extracted, and polymerase chain reaction (PCR) amplification of 16S rRNA with specific and universal primers was carried out. RESULTS: Twenty-six women with threatened premature labor were included: eight with preterm premature rupture of membranes and 18 with preterm labor with intact membranes. Eight women presented with gingivitis, 12 with chronic periodontitis, and six without periodontal disease. Microbial invasion of the amniotic cavity as detected by P. gingivalis PCR was 30.8% (eight of 26 patients). In these eight patients, P. gingivalis was present in both the subgingival samples and the respective amniotic fluid sample. CONCLUSION: The presence of microbial invasion of the amniotic cavity by P. gingivalis could indicate a role for periodontal pathogenic bacteria in pregnant women with a diagnosis of threatened premature labor.

Prevalence of periodontopathic bacteria in aggressive periodontitis patients in a Chilean population.

BACKGROUND: Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis (AgP). Other periodontopathic bacteria such as Porphyromonas gingivalis are also suspected of participating in aggressive periodontitis although the evidence to support this is controversial. The aim of the present study was to determine the prevalence of eight periodontopathic bacteria in Chilean patients with AgP. METHODS: Subgingival plaque samples were collected from 36 aggressive, 30 localized, and six generalized periodontitis patients. Samples from 17 advanced chronic periodontitis (CP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid (RTF) and cultured. Periodontal bacteria were primarily identified by colony morphology under stereoscopic microscope and rapid biochemical tests. The identity of some bacterial isolates was confirmed by colony polymerase chain reaction (PCR). RESULTS: AgP showed a significatively higher prevalence of C. rectus than CP (P = 0.036). The only statistical difference found was for C. rectus. Patients with AgP showed a higher, but not statistically significant, prevalence of P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. A similar prevalence in both groups of patients was observed for F. nucleatum and P. intermedia/nigrescens, and A. actinomycetemcomitans was less prevalent in AgP than CP patients. In localized AgP, P. intermedia/nigrescens, E. corrodens, F. nucleatum, and P. micros were the more prevalent pathogens in contrast to generalized AgP patients who harbored A. actinomycetemcomitans, P. gingivalis, and Capnocytophaga sp. as the most prevalent bacteria. CONCLUSIONS: C. rectus, P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. were the most predominant periodontopathic bacteria of AgP in this Chilean population, but the only statistical difference found here between AgP and CP was for C. rectus, suggesting that the differences in clinical appearance may be caused by factors other than the microbiological composition of the subgingival plaque of these patients. In this study, the prevalence of A. actinomycetemcomitans was much lower than that of P. gingivalis.

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.

[Relationship between periodontal diseases and ascending bacterial infection with preterm delivery]. / Relación entre enfermedad periodontal, infección bacteriana ascendente y patología placentaria con parto prematuro.

BACKGROUND: There is an association between periodontal diseases and preterm delivery. AIM: To assess the relationship between periodontal diseases, ascending bacterial infection and placental pathology with preterm delivery. PATIENTS AND METHODS: A periodontal examination and collection of amniotic fluid and subgingival plaque samples were performed in women with preterm labor with intact membranes, without an evident clinical cause or preterm premature rupture of membranes, without clinical chorioamnionitis or labor and a gestational age between 24 and 34 weeks. Microbial invasion of the amniotic cavity was defined as the presence of a positive amniotic fluid culture. Cervicovaginal infection was defined as a bacterial vaginosis or positive culture of cervix or vagina with a high neutrophil count. Ascending bacterial infection was diagnosed as the microbial invasion of the amniotic cavity by ascending bacteria or cervicovaginal infection. Corioamnionitis, funisitis or vellositis were diagnosed. RESULTS: Fifty-nine women were included: forty-two with preterm labor with intact membranes and seventeen with preterm premature rupture of membranes. The prevalence of periodontal diseases was 93.2%. Microbial invasion of the amniotic fluid was detected in 27.1% of patients. periodontal pathogenic bacteria were isolated in 18.6% of amniotic fluid samples and 71.2% of subgingival plaque samples. The prevalence of ascending bacterial infection was 83.1% and in 72.9% of women it was associated with periodontal disease. Preterm delivery (<37 weeks) occurred in 64.4% of patients and was significantly associated with generalized periodontal disease and with the association of ascending bacterial infection and periodontal diseases. Patients with preterm delivery and generalized periodontal disease had a higher frequency of chorioamnionitis and funisitis. CONCLUSIONS: Generalized periodontal disease and its association with ascending bacterial infection are related to preterm delivery and placental markers of bacterial ascending infection.

Cytotoxicity and trypanocidal activity of nifurtimox encapsulated in ethylcyanoacrylate nanoparticles.

The aim of the present study was to study the trypanocidal activity of nanoparticles loaded with nifurtimox in comparison with the free drug against Trypanosoma cruzi, responsible for Chagas' disease. Ethylcyanoacrylate nanoparticles acted as the delivery system into cells. As the obligate replicative intracellular form is amastigote, in vitro studies were performed on this form of parasite as well as on cell culture derived trypomastigotes. The fluorescence method used here was very useful as it allowed for the simultaneous study of trypanocide activity and cytotoxicity by determining living or dead parasites within living or dead host cells. According to these results, the greatest trypanocide activity on cell culture-derived trypomastigotes was recorded for nifurtimox-loaded nanoparticles with a 50% inhibitory concentration (IC50) twenty times less than that of the free drug. The cytotoxicity of unloaded nanoparticles at low concentrations was similar to that obtained by free drug when evaluated on Vero cells. Furthermore, nifurtimox-loaded nanoparticles showed increased trypanocide activity on intracellular amastigotes with an IC50 thirteen times less than that of nifurtimox. We also observed that the unloaded nanoparticles possess the previously-described trypanocide activity, similar to the standard solution of nifurtimox, although the mechanism for this has not yet been elucidated. In conclusion, it was possible to establish in vitro conditions using nifurtimox encapsulated nanoparticles in order to decrease the doses of the drug and thus to obtain high trypanocidal activity on both free trypomastigotes and intracellular amastigotes with low cytotoxicity for the host cell.

[Immunophenotyping of peripheral blood lymphocytes from Chilean chronic chagasic patients by flow cytometry]. / 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.

Relación entre enfermedad periodontal, infección bacteriana ascendente y patología placentaria con parto prematuro / Relationship between periodontal diseases and ascending bacterial infection with preterm delivery

Background: There is an association between periodontal diseases and preterm delivery. Aim: To assess the relationship between periodontal diseases, ascending bacterial infection and placental pathology with preterm delivery. Patients and methods: A periodontal examination and collection of amniotic fluid and subgingival plaque samples were performed in women with preterm labor with intact membranes, without an evident clinical cause or preterm premature rupture of membranes, without clinical chorioamnionitis or labor and a gestational age between 24 and 34 weeks. Microbial invasion of the amniotic cavity was defined as the presence of a positive amniotic fluid culture. Cervicovaginal infection was defined as a bacterial vaginosis or positive culture of cervix or vagina with a high neutrophil count. Ascending bacterial infection was diagnosed as the microbial invasion of the amniotic cavity by ascending bacteria or cervicovaginal infection. Corioamnionitis, funisitis or vellositis were diagnosed. Results: Fifty-nine women were included: fortytwowith preterm labor with intact membranes and seventeen with preterm premature rupture of membranes. The prevalence of periodontal diseases was 93.2%. Microbial invasion of the amniotic fluid was detected in 27.1% of patients. Periodontal pathogenic bacteria were isolated in 18.6% of amniotic fluid samples and 71.2% of subgingival plaque samples. The prevalence of ascending bacterial infection was 83.1% and in 72.9% of women it was associated with periodontal disease. Preterm delivery (<37 weeks) occurred in 64.4% of patients and was significantly associated with generalized periodontal disease and with the association of ascending bacterial infection and periodontal diseases. Patients with preterm delivery and generalized periodontal disease had a higher frequency of chorioamnionitis and funisitis...

Cytotoxicity and trypanocidal activity of nifurtimox encapsulated in ethylcyanoacrylate nanoparticles

The aim of the present study was to study the trypanocidal activity of nanoparticles loaded with nifurtimox in comparison with the free drug against Trypanosoma cruzi, responsible for Chagas' disease. Ethylcyanoacrylate nanoparticles acted as the delivery system into cells. As the obligate replicative intracellular form is amastigote, in vitro studies were performed on this form of parasite as well as on cell culture derived trypomastigotes. The fluorescence method used here was very useful as it allowed for the simultaneous study of trypanocide activity and cytotoxicity by determining living or dead parasites within living or dead host cells. According to these results, the greatest trypanocide activity on cell culture-derived trypomastigotes was recorded for nifurtimox-loaded nanoparticles with a 50% inhibitory concentration (IC50) twenty times less than that of the free drug. The cytotoxicity of unloaded nanoparticles at low concentrations was similar to that obtained by free drug when evaluated on Vero cells. Furthermore, nifurtimox-loaded nanoparticles showed increased trypanocide activity on intracellular amastigotes with an IC50 thirteen times less than that of nifurtimox. We also observed that the unloaded nanoparticles possess the previously-described trypanocide activity, similar to the standard solution of nifurtimox, although the mechanism for this has not yet been elucidated. In conclusion, it was possible to establish in vitro conditions using nifurtimox encapsulated nanoparticles in order to decrease the doses of the drug and thus to obtain high trypanocidal activity on both free trypomastigotes and intracellular amastigotes with low cytotoxicity for the host cell.