PubMed Health⌕ Search

Biomedical subjects

P Wincker

Publications and source records attributed to P Wincker.

51 records · Page 3Linked to original sources
PubMed1995

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.

Animals↗
PubMed1995

A polymorphic minisatellite sequence in the subtelomeric regions of chromosomes I and V in Leishmania infantum.

A minisatellite DNA sequence is described for the first time in Leishmania infantum. It is borne by four chromosomes and consists of an 81-bp repeat unit organised in several clusters. On chromosomes I and V of L. infantum, the clusters are tightly located in the size-variable subtelomeric regions. The organisation of this sequence may be related to that of the subtelomeric interspersed repeat sequences identified in the human genome. The sequencing of seven repeat units, some subcloned from the same cluster, allowed the definition of a consensus sequence of 81 bp, particularly G/C rich (73%). Two subfamilies were clearly defined: one exhibits a 91-95% homology with the consensus sequence; the second one comprises two monomers sharing a 91% homology but only 77% homology with the consensus sequence. The two types of monomers can be found in the same cluster. These data suggest interactions between monomers and a possible role of this sequence in the instability of these regions. Finally, restriction fragment length polymorphisms were revealed by this sequence among various strains of L. infantum. Besides allowing the detection of recombination events in the unstable regions of the chromosomes, this new marker may become a useful tool in the study of the parasite population dynamics in leishmaniasis foci.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Structural organisation of microsatellite families in the Leishmania genome and polymorphisms at two (CA)n loci.

In the present study, we have analysed the frequency and distribution of several microsatellite DNAs [(CA)n, (GGT)n and (GCA)n] in the genome of Leishmania. Hybridisation analysis on the molecular karyotypes of different Leishmania strains showed the presence of these three microsatellites on all chromosomes of the parasite. The number of microsatellite clusters appeared grossly similar among strains from different Old World complexes. However, these three microsatellite families showed an uneven distribution among heterologous chromosomes of the same strain. Moreover, restriction analysis of chromosome I in various strains of Leishmania infantum showed a strong clustering of these microsatellites in the same chromosomal region. A partial genomic library was screened with a (CA)n probe, and 21 positive clones were isolated. The sequencing of these clones confirmed the association of various microsatellites such as (CA)n, (CT)n, and (GCA)n. Finally, specific polymerase chain reaction amplification of two cloned (CA)n loci demonstrated allelic size polymorphisms among strains within L. infantum and Leishmania donovani. Most of the 34 strains analysed were found to be monoallelic, while two alleles were found in a small number of strains. The interest of these sequences for studies on ploidy and population genetics of the parasite is discussed.

Animals↗
PubMed1994

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.

Animals↗
PubMed1990

Characterization of a Trypanosoma cruzi specific nuclear repeated sequence.

We report the isolation of three different clones from a Trypanosoma cruzi genomic library bearing a common repeated sequence. This sequence is not tandemly repeated, and is dispersed on many chromosomes. All of the T. cruzi strains tested share this element. On the other hand, it is absent from the genome of other Kinetoplastida. The size of this element is about 10-12 kb, and its copy number is 220 in the T. cruzi Dm 28c genome. A transcript homologous to this sequence is detected in epimastigote forms of the parasite.

Animals↗
PubMed1989

Chromosome size and number polymorphisms in Leishmania infantum suggest amplification/deletion and possible genetic exchange.

We have studied the molecular karyotypes of 21 strains and 14 clones of Leishmania infantum using pulsed field gel electrophoresis (PFGE). We detected a high degree of polymorphism within this species, with 'strain-specific' patterns for most isolates, even within a restricted endemic area. Variations relate to both the size of chromosomes (270-2600 kb) and their number, which can vary from 24 to 31 between closely related isolates. This polymorphism does not correlate with isoenzyme analysis. Small size variations between homologous chromosomes of different strains are suggestive of DNA amplification/deletion events. Strains are also shown to be multiclonal, with slight differences between most clones, but with a predominant clone concealing the others in PFGE analysis. The analysis of these data leads to the hypothesis of occasional genetic exchange by nuclear fusion in Leishmania, as recently shown in the related protozoan Trypanosoma brucei.

Animals↗
PubMed1987

Unrelated sequences at the 5' end of mouse LINE-1 repeated elements define two distinct subfamilies.

Some full length members of the mouse long interspersed repeated DNA family L1Md have been shown to be associated at their 5' end with a variable number of tandem repetitions, the A repeats, that have been suggested to be transcription controlling elements. We report that the other type of repeat, named F, found at the 5' end of a few L1 elements is also an integral part of full length L1 copies. Sequencing shows that the F repeats are GC rich, and organized in tandem. The L1 copies associated with either A or F repeats can be correlated with two different subsets of L1 sequences distinguished by a series of variant nucleotides specific to each and by unassociated but frequent restriction sites. These findings suggest that sequence replacement has occurred at least once in 5' of L1Md, and is related to the generation of specific subfamilies.

Animals↗
PubMed1987

The relationships between the 5' end repeats and the largest members of the L1 interspersed repeated family in the mouse genome.

Analysis of a few large L1 elements has revealed two types of tandem repeats at the 5' end: A and F. In this study, the relationships between these repeats and a series of large L1 elements has been analysed. Most of cloned L1 repeats were shown to lack either A or F sequences at their 5' ends. F sequences are found less frequently associated than A sequences to the 5' ends of L1 and an evolutionary comparison shows that the A type was introduced more recently during the evolution of the mouse genome than the F type.

Animals↗
PubMedDate not supplied

Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil.

The sensitivities for Chagas disease diagnosis of haemoculture, xenodiagnosis, and polymerase chain reaction (PCR) amplification of Trypanosoma cruzi kinetoplast deoxyribonucleic acid (DNA) were compared for 101 patients living in an endemic region who were serologically positive for T. cruzi. PCR gave 60 positive results (59.4%), while a haemoculture was positive in 26 cases (25.7%) and xenodiagnosis in 36 (35.6%). Four xenodiagnosis-positive but PCR-negative patients were examined in detail. The discrepancies were not due to inhibition of the PCR reactions, as the samples were used successfully to amplify a human sequence. Nor were they due to a variation in kinetoplast DNA sequences, as the kinetoplast DNA of the parasite strains isolated from these patients after xenodiagnosis gave rise to the expected product when amplified by the PCR. We concluded that no parasite was present in the 5 mL of blood used for PCR, while probably a single T. cruzi cell was present in the blood volume ingested by the insects during xenodiagnosis (about 3 mL). This suggests that the total blood quantity collected for the PCR may be important with patients with low parasitaemia.

Base Sequence↗
PubMedDate not supplied

Immune response to Trypanosoma cruzi shed acute phase antigen in children from an endemic area for Chagas' disease in Bolivia.

A field study of the immune response to the shed acute phase antigen (SAPA) of Trypanosoma cruzi was carried out in the locality of Mizque, Cochabamba department, Bolivia. Schoolchildren (266), with an average of 8.6 +/- 3.6 years, were surveyed for parasitological and serological diagnosis, as well as antibodies directed against SAPA using the corresponding recombinant protein in ELISA. The antibodies against SAPA were shown in 82% of patients presenting positive serological diagnosis (IgG specific antibodies). The positive and negative predictive values were 0.88. Antibodies anti-SAPA were shown in 80.8% of the chagasic patients in the initial stage of the infection (positive IgM serology and/or positive buffy coat (BC) test) and in 81.4% of the patients in the indeterminate stage of the infection (positive IgG serology with negative BC and IgM tests). These results show that the anti-SAPA response is not only present during the initial stage of the infection (few months) but extends some years after infection.

Animals↗