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Officials say it can't be moved Read More. November , Newsletters Never miss a beat Sign Up Now Subscribe now to get the latest news delivered right to your inbox. Newest Slideshows. Special Issues City Guide. Holiday Guide. Summer Guide. Contrary to bacterial infections, in which laboratory assays to measure antibiotic susceptibility are well established, a procedure to evaluate BZ susceptibility of T.
The early diagnosis of drug resistance would improve the clinical management of Chagas disease, dictating alternative therapies with a combination of existing drugs or new anti- T. In this study, we have developed a methodology to quantify BZ susceptibility in haemoculture-derived isolates from patients in the chronic phase of Chagas disease.
The procedure was applied to parasites obtained before and after BZ treatment. Comparison of the in vitro BZ resistance of the pre-treatment parasites from patients considered cured and from those experiencing therapeutic failure showed no correlation with therapeutic outcome. Parasite isolates and cultivation - Human isolates were obtained by haemoculture as described by Chiari et al. In order to minimize parasite selection, LIT-FCS medium was added every two days to each culture for a maximum of eight weeks.
Parasites were cryopreserved in liquid nitrogen. The characteristics of T. The recently proposed nomenclature for the strains is used, where discrete typing units DTU are defined as sets of stocks that are genetically more related to each other than to any other stock and that are identifiable by common genetic, molecular or immunological markers Zingales et al.
Total parasite DNA was obtained as described Macedo et al. Amplification of microsatellites by PCR was achieved as previously described Oliveira et al. Patients and treatment scheme - Since , one of us EDG , in conjunction with researchers of the Clinics Hospital of the Federal University of Minas Gerais UFMG , has been conducting a prospective study to test the hypothesis that BZ treatment can impact the evolution of the disease, preventing or retarding the development of defined clinical forms.
Further details on the prospective study will be published elsewhere. Signed informed consent was obtained from the participants. In the present study, only seven patients of the original cohort were included. Treatment was started after haemocultures produced positive scores. All the patients completed treatment. During follow-up, individuals lived in an urban area and did not travel to endemic areas. The characteristics of the patients are summarized in Table II. After this period, the number of living parasites was counted in a Neubauer chamber.
Drug activity was determined by calculating the percentage of growth inhibition in the treated samples against the control in the absence of BZ. Three independent assays with duplicates in each assay were performed on separate occasions. Standardization of the assay for screening BZ susceptibility - The in vitro assay was standardized with epimastigote forms, as this developmental stage is predominant in haemocultures obtained from infected individuals.
The IC 50 value obtained from three separate assays with duplicates in each assay was Because the stock solution of BZ was diluted in DMSO, we analyzed the effect of different concentrations of this solvent on the viability of VL10 epimastigotes.
Final DMSO concentrations of 0. To validate the assay, susceptibility to BZ was assessed for 11 additional T. The IC 50 to BZ varied from 7. These observations suggest a correlation between in vitro BZ susceptibility and the treatment outcome reported in the experimental model. The IC 50 values determined monthly did not show any considerable variation, falling within the limits of the SD data not shown. BZ susceptibility of T. The test for BZ susceptibility was applied to parasites obtained from seven chronic patients 4 females and 3 males , between years of age Table II.
Six patients presented the indeterminate form of Chagas disease and one patient presented mild cardiac damage. The patients were submitted to BZ therapy and, at different times after treatment, haemocultures were performed and the presence of parasites scored Table II.
The titre of anti- T. All of the haemocultures derived from patients 7, 22 and 36 were negative for parasites, as were the PCR assays data not shown. Negativation of anti- T. According to these criteria, the patients were considered cured Gontijo et al.
The IC 50 of the pre-treatment parasites derived from the non-cured patients 3, 12, 19 and 50 varied from These data indicate that the IC 50 values determined in our assay do not predict the outcome of BZ treatment.
It is remarkable that the IC 50 to BZ of the pre-treatment isolate of the non-cured patient We also determined the BZ susceptibility of the epimastigotes derived from post-treatment parasites from the four non-cured patients Table III. Molecular typing of the isolates - In Chagas disease, the individual may be infected with more than one parasite population Macedo et al. Contemplating the hypothesis that variation in the IC 50 of the isolates obtained before and after treatment could be related to the selection of a particular parasite population, we proceeded to genotype parasites based on the analysis of nine polymorphic microsatellite loci containing di, tri and tetra-nucleotide repetition motifs Table IV.
Using this set of microsatellites, it was possible to differentiate pre-treatment parasite populations for all four evaluated patients. Interestingly, when examining pre and post-treatment isolates from a given patient, we concluded that the population was monoclonal and identical, as indicated by the presence of only one or two different alleles per locus. Accordingly, and within the sensitivity of the microsatellite assay, we ruled out parasite selection or re-infection during or after treatment.
The three stages of the T. Although the intracellular amastigote model is considered the most consistent indicator of in vivo activity, a number of tests showed a strong correlation between the activity of compounds against epimastigote cultures and their activity in mice Croft , Urbina Nevertheless, differences of several orders of magnitude of the IC 50 values of epimastigotes and amastigotes were reported.
Because the goal of this study was to develop a procedure to measure the BZ susceptibility of clinical isolates with the aim of predicting the response to treatment, rather than correlating BZ susceptibility of the epimastigotes with the treatment outcome, we standardized the assay with haemoculture-derived epimastigotes.
We have not attempted to develop the assay using intracellular amastigotes because the process is time consuming and can lead to the selection of a particular parasite population. The amastigote model involves the differentiation of haemoculture-derived epimastigotes into metacyclic trypomastigotes in order to establish infection in mammalian cell monolayers.
We have made an effort to obtain metacyclic trypomastigotes from the clinical isolates in differentiation media. We have not used bloodstream trypomastigotes because their numbers are low in the chronic phase of Chagas disease. Standardization of the assay to measure BZ susceptibility was performed with T. These values are of the same order of magnitude of IC 50 of epimastigotes in stocks belonging to different DTU Villarreal et al. Our data support the latter conclusion. We observed that the IC 50 value is a stable characteristic that is maintained during prolonged epimastigote sub-culturing up to 4 months.
This observation agrees with previous reports showing that drug-resistant T. While a serious, well-designed anti-crime strategy in Mexico is badly needed and overdue, any application of a "hotspot strategy" will not be without its challenges, as the successes of such strategies have often occurred in settings of a far smaller scale than Mexico, explains Vanda Felbab-Brown.
This piece was originally published by Mexico Today. A serious well-designed anti-crime strategy is badly needed and overdue, and a hotspot strategy is well-tested and widely-embraced. But its successes have often occurred in settings of a far smaller scale than Mexico. Criminal violence in Mexico remains devastatingly high. These changes are almost entirely driven by the ebb and flow of turf wars between criminal groups; they are a manifestation of the natural fluctuation in operational tempo of violent criminal markets or warzones.
The 3. Moreover, almost 17, homicides in the first six months of should be unacceptable in any year. But the approach left unaddressed how these youth programs and cultural efforts were to interact with local and national policing.
Later on, numerous socio-economic initiatives were included—from distributing glasses to school children to various vocational programs.
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