Biomedical subjects
C M Morel
Publications and source records attributed to C M Morel.
Present and future control of malaria.
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Reaching maturity - 25 years of the TDR.
In its first 25 years of existence, TDR has become a key player in the development of new tools for the control of tropical diseases and the training of researchers from disease-endemic countries. In order to maintain its leading position, cope with new health challenges and profit from new avenues opened by science and technology breakthroughs, a new strategic vision is now being implemented. It aims at a closer interaction with health systems and disease control programmes, capacity strengthening based on selected research initiatives and full exploitation of scientific and technological advances in the biomedical, social and information sciences, as discussed here by Carlos Morel.
The complexity of the sylvatic cycle of Trypanosoma cruzi in Rio de Janeiro state (Brazil) revealed by the non-transcribed spacer of the mini-exon gene.
American trypanosamiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection in natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, the mini-exon and the 24S alpha rRNA gene loci have shown that T. cruzi stocks can be clustered in 2 major phylogenetic groups: lineage 1 and lineage 2. In this report, 68 recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical areas in Rio de Janeiro, Brazil, have been typed based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, 26 stocks were from golden-lion tamarins, 31 from opossums, 2 from rodents and 1 from a three-toed sloth. Thirty (44%-30/68) isolates were typed as lineage 1, while 36 (53%-36/68) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, 3% (2/68) of the isolates were typed as lineage 1 + lineage 2. Using these geographical regions as models of sylvatic environments, it was observed that 96% of the Didelphis marsupialis were infected by lineage 2 isolates, while all 26 golden-lion tamarins were infected by lineage 1. The results show preferential association of the 2 lineages of T. cruzi with different hosts, composing the complexity of the sylvatic cycle.
Chagas disease, from discovery to control - and beyond: history, myths and lessons to take home.
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Polymerase chain reaction detection: new insights into the diagnosis of chronic Chagas disease.
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Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.
Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.
PCR-based diagnosis for Chagas' disease in Bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis.
A large field study has been performed in the Cochabamba region of Bolivia with the aim of comparing the polymerase chain reaction (PCR) with other diagnostic methods for Chagas' disease. The amplification of Trypanosoma cruzi-specific kinetoplast DNA sequences in blood samples was compared with classical serological methods, specific IgM detection and direct parasite visualization for 268 school children in a single village where Chagas' disease transmission is active. Of 113 children positive by classical serology or buffy coat examination, 106 were detected by PCR (sensitivity: 93.8%). We did not observe any significant difference of PCR sensitivity between initial (IgM and/or buffy coat positive) and indeterminate stage (only IgG positive) patients. Among the remaining 155 children unconfirmed as chagasic (who were either only IgM positive, IgG-, IgM-, and buffy coat-negative) only 1 case was PCR positive. This case may be due to DNA contamination, or to a very recent infection not detected otherwise, or to specific immune depression. These results show that PCR is a very sensitive parasitological test for Chagas' disease in active transmission regions. The future follow-up of the possibly infected patients who were only IgM-positive should clarify the interest of PCR and IgM tests in the detection of starting infections.
Characterization of two isolates of Trypanosoma cruzi obtained from the patient Berenice, the first human case of Chagas' disease described by Carlos Chagas in 1909.
Two isolates of Trypanosoma cruzi were obtained from the patient Berenice, the first human case of Chagas' disease (Chagas 1909), when she was 55 and 71 years old, respectively. The isolates were characterized on the basis of their epimastigote-trypomastigote differentiation in liquid media and of the electrophoretic pattern of EcoR1 digestion products of kinetoplast DNA (k-DNA) minicircles (schizodeme) and isoenzyme patterns (zymodeme). Clear differences were found between the isolates, suggesting the occurrence of a heterogeneous population of T. cruzi in the infection of this patient.
Experimental paleoparasitology: identification of Trypanosoma cruzi DNA in desiccated mouse tissue.
The polymerase chain reaction (PCR) has been used for clinical diagnosis of infectious disease and to research ancient animal and microbiological DNA from a wide range of tissues. PCR was used to study the possibility of Trypanosoma cruzi kinetoplast DNA (kDNA) extraction from experimentally desiccated mouse tissue (heart, skeletal muscle, liver, spleen, and pancreas). The results obtained suggest the application of this technique to T. cruzi detection in archaeological material.
Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization.
Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive. In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no cross-hybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.
Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation.
Trypanosoma cruzi specific sequences were amplified by the polymerase chain reaction from total blood of human chagasic patients and normal individuals. A 330 bp fragment originating from kinetoplast DNA was specifically detected in most chagasic individuals. We tested the sensitivity and specificity of this method in normal and affected individuals attending the Evandro Chagas Hospital, Rio de Janeiro. The results of these tests were compared with serological diagnosis performed using standard techniques, and in some cases with xenodiagnosis. We found that none of the serologically negative individuals gave any specific amplification product, whereas 55 out of 61 patients previously serodiagnosed as chagasic were positive using the PCR method (sensitivity: 90%). Xenodiagnosis, which is currently considered to be the most sensitive parasitological technique for Chagas' disease diagnosis, detected only 12 out of 28 serologically positive patients (sensitivity: 43%). The usefulness of the PCR method was further investigated with chagasic patients who had received anti-parasite treatment with benznidazole. It has always been difficult to evaluate the incidence of cure in such cases by serology, since a humoral response against T. cruzi antigens may remain for years even in the absence of the parasite. We observed a positive amplification result in only 9 out of 32 treated patients who remained reactive when tested using classical serology. These observations suggest that PCR is the most sensitive technique available for direct detection of T. cruzi in chagasic patients and that it can be a very useful instrument for the follow-up of patients after specific treatment.
High correlation between Chagas' disease serology and PCR-based detection of Trypanosoma cruzi kinetoplast DNA in Bolivian children living in an endemic area.
The detection of Trypanosoma cruzi kinetoplast DNA by polymerase chain reaction (PCR) amplification is a potentially powerful tool for the parasitological diagnosis of Chagas' disease. We have applied this technique in a field situation in Bolivia, where 45 children from a primary school were subjected to serological testing, buffy coat analysis and PCR diagnosis. 26 of the 28 serology-positive individuals were also positive by PCR. In addition, two serology-negative children gave a positive result by PCR, including one who was positive in the buffy coat test. These results suggest that PCR detection of T. cruzi DNA in blood can be a very useful complement to serology in Chagas' disease diagnosis in Bolivia.
Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area.
The feasibility of using DNA amplification by the polymerase chain reaction (PCR) for specific detection of Trypanosoma cruzi in human blood specimens was investigated. One hundred blood samples were collected in an endemic area of Minas Gerais, Brazil. They were submitted to DNA extraction and PCR amplification with kinetoplast DNA-specific primers using a simplified boiling procedure that linearized most minicircle molecules without the aid of chemical reagents. Samples that gave negative results were checked for possible inhibition of amplification using primers derived from a human-specific sequence, and those showing some level of inhibition were retested after a new DNA extraction. Of 86 patients previously diagnosed as chagasic by serologic techniques, 83 were positive in our PCR test (sensitivity = 96.5%), including all the xenodiagnosis-positive patients and 21 (87.5%) of 24 xenodiagnosis-negative individuals. In addition, four of six patients with doubtful serologic results were confirmed as positive by PCR. Our results suggest that the PCR may be a useful complement to serology in the diagnosis of Chagas' disease, and that it is the most powerful technique available for parasite detection in patients with chronic disease.
Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis.
A panel of 114 blood samples from chronic chagasic patients and nonchagasic patients was screened for Trypanosoma cruzi by xenodiagnostic, serologic, and polymerase chain reaction (PCR) amplification tests. Blood samples were preserved in a guanidine-EDTA buffer, and total blood DNA was isolated after chemical nuclease cleavage with 1,10-phenanthroline-copper ion and used as a template for PCR amplification of the conserved and variable regions of T. cruzi minicircle molecules. The PCR products were screened by Southern blot hybridization with a digoxigenin-labeled oligonucleotide probe specific for the conserved region of the minicircle. The method showed a sensitivity of 100% compared with the serologic test. In addition, all of the serology-positive, xenodiagnosis-negative samples were positive by PCR. This demonstrates that PCR amplification of T. cruzi kinetoplast minicircle DNA could replace xenodiagnosis for evaluation of parasitemia in chronic chagasic patients and could serve as a complement for serologic testing in the screening of blood bank donors.
Changes in the isoenzyme and kinetoplast DNA patterns of Trypanosoma cruzi strains induced by maintenance in mice.
Culture forms of thirteen Trypanosoma cruzi strains from 4 zymodemes and 9 schizodemes were inoculated and kept by successive passages in C3H mice. The strains were initially from the following zymodemes: 3 from A, 3 from B, 4 from C and 2 from D and 1 from AB mixed zymodemes. After approximately 18 months maintenance the parasites were isolated by hemoculture and again typed according to their isoenzyme and kinetoplast DNA patterns. The zymodeme A strains kept their initial patterns; from the 3 zymodeme B strains, two kept the initial patterns and one changed to zymodeme A; from the 4 zymodeme C, two kept the initial pattern and two changed to zymodeme B; from the 2 zymodeme D strains, one kept the initial pattern and one changed to zymodeme A. The strain from AB mixed zymodeme was reduced to zymodeme. A. The zymodeme changes were accompanied by schizodeme changes. Although not simultaneously, in one T. cruzi strain the parasitemia change was followed by zymodeme and schizodeme changes. The results showed that prolonged maintenance of T. cruzi in mice by successive passages alters the isoenzyme and k-DNA patterns of some strains and that these alterations tend to move towards zymodeme A, suggesting a selective effect of mice over these T. cruzi populations.
Population heterogeneity among clones of New World Leishmania species.
Cell cloning techniques and schizodeme analysis were used to detect mixtures of subpopulations in Leishmania parasites, isolated from humans and a reservoir host. Clones were obtained by plating promastigotes, at limited dilution, on solid medium. The resultant colonies were analysed by the restriction profile of kinetoplast DNA minicircles (schizodeme analysis). The efficiency of plating was around 40-60%, and subpopulations of different schizodemes, in stocks of Leishmania isolated from 2 cases of human cutaneous leishmaniasis were detected. The presence of different schizodemes in one isolate from a sylvatic animal suggested the possibility of a mixed natural infection.
Characterization of tubulin genes in Trypanosoma rangeli.
Tubulin genes in Trypanosoma rangeli, the only trypanosome besides T. cruzi to infect humans in America, are organized in homogeneous, alternate alpha and beta gene tandem repeats of 3.8 kb. The basic repeat was cloned, mapped and partially sequenced. In contrast to most other eukaryotes, where tubulin genes are scattered throughout the genome, trypanosomatids so far studied are characterized by tandem arrangements of these genes with the genus Trypanosoma displaying an alternating alpha- and beta-tubulin tandem repeat.