The trypanocidal activity of amidine compounds does not correlate with their binding affinity to Trypanosoma cruzi kinetoplast DNA.

Due to limited efficacy and considerable toxicity, the therapy for Chagas' disease is far from being ideal, and thus new compounds are desirable. Diamidines and related compounds such as arylimidamides have promising trypanocidal activity against Trypanosoma cruzi. To better understand the mechanism of action of these heterocyclic cations, we investigated the kinetoplast DNA (kDNA) binding properties and trypanocidal efficacy against T. cruzi of 13 compounds. Four diamidines (DB75, DB569, DB1345, and DB829), eight arylimidamides (DB766, DB749, DB889, DB709, DB613, DB1831, DB1852, and DB2002), and one guanylhydrazone (DB1080) were assayed in thermal denaturation (T(m)) and circular dichroism (CD) studies using whole purified T. cruzi kDNA and a conserved synthetic parasite sequence. The overall CD spectra using the whole kDNA were similar to those found for the conserved sequence and were indicative of minor groove binding. Our findings showed that some of the compounds that exhibited the highest trypanocidal activities (e.g., DB766) caused low or no change in the T(m) measurements. However, while some active compounds, such as DB766, induced profound alterations of kDNA topology, others, like DB1831, although effective, did not result in altered T(m) and CD measurements. Our data suggest that the strong affinity of amidines with kDNA per se is not sufficient to generate and trigger their trypanocidal activity. Cell uptake differences and possibly distinct cellular targets need to be considered in the final evaluation of the mechanisms of action of these compounds.

Efficacy of an indirect immunofluorescence test in the diagnosis of canine leishmaniosis.

Of 146 dogs from a visceral leishmaniosis-endemic area that tested seronegative by indirect immunofluorescence (IIF) on blood samples collected on filter paper (IIFp), 51 (34.9%) and 10 (6.8%) tested positive by IIF on serum samples (IIFs) and enzyme immunoassay, respectively. Three samples (2.0%) tested positive by PCR. Leishmania chagasi was isolated from the skin of five (3.4%) dogs. Amastigote forms were identified in two of these five animals following histopathological and immunohistochemical examination. The findings highlight that detection methods such as IIFp can permit dogs infected with L. chagasi to remain undetected in endemic areas with attendant consequences for the epidemiology of infection both in the canine and human populations.

Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: evaluation by two molecular markers.

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.

Genetic polymorphism in Trypanosoma cruzi I isolated from Brazilian Northeast triatomines revealed by low-stringency single specific primer-polymerase chain reaction.

Different molecular markers have been employed for typing Trypanosoma cruzi strains from endemic areas of Chagas disease. The low-stringency single specific primer-polymerase chain reaction (LSSP-PCR) has been a sensitive and informative technique that uses the variable region of kinetoplast DNA minicircles as a genetic marker, allowing detection of DNA sequence variation. In the present study, we analyzed the intra-lineage genetic variability of the T. cruzi strains obtained from triatomine feces collected on filter paper FTA card by LSSP-PCR. The hybridization of the PCR products with a probe for the subgenus Schizotrypanum and a clone-specific probe from Dm28c confirmed the subgenus as T. (S.) cruzi and respective lineages as T. cruzi I. Phenetic analysis showed the presence of three clusters that diverged by different coefficients of similarity. Thirteen T. cruzi I genotypes were observed circulating among Triatoma pseudomaculata and Rhodnius nasutus from peridomiciliary and natural environments in five peri-urban and urban localities of Jaguaruana, Ceará, Brazil. These data indicate the importance of the circulation of T. cruzi I genotypes among T. pseudomaculata and R. nasutus in different environments and the possible risk of Chagas disease domestic transmission.

Genetic variability of Triatoma rubrovaria (Reduviidae: Triatominae) from Brazil, Argentina and Uruguay as revealed by two different molecular markers.

Randomly amplified polymorphic DNA (RAPD) and nuclear ribosomal DNA sequence analyses were used to assess the genetic population structure of the South American triatomine species Triatomo rubrovario throughout its geographical distribution. To investigate the genetic variability at both intraspecific and intrapopulational levels the RAPD profiles and the nucleotide sequences of the rDNA intergenic spacers, ITS-1 and ITS-2, were analysed and compared. The phenetic analysis based on RAPD profiles show three distinct clusters diverging by similarity coefficients ranging from 0.62 to 0.96. The ITS-1 and ITS-2 sequence variability detected may be considered very high, suggesting reproductive isolation between populations. A total of seven composite haplotypes (CH) were found, among which three are specific for Brazil, other three for Uruguay, and the last one common for the three countries studied. The population studied in Argentina does not represent an independent CH. Sequence analyses proved that the five populations studied are easily differentiable and that there is heterogeneity within each one. True mutations and indels are the responsible of sequence differences between haplotypes and populations, suggesting that divergence processes may presently go on within this species. The large intraspecific variability detected may underlie the known plasticity of T. rubrovaria, making it a potential intradomiciliary invader and consequently an appropriate vector for Chagas disease transmission. Therefore, this triatomine species must be continuously monitored throughout.

Chagas' disease and HIV co-infection: genotypic characterization of the Trypanosoma cruzi strain.

In the past few years, new aspects of the immunopathology of Chagas' disease have been described in immunosuppressed patients, such as fatal central nervous system lesions related to the reactivation of the parasite. This article is the first description of the genotypic characterization, at the strain level, of Trypanosoma cruzi isolated from a patient with Chagas' disease/AIDS co-infection. The presence of four hypodense lesions was observed in the cranial compute tomographic scan. the diagnosis of AIDS was assessed by the detection of anti-HIV antibodies using enzyme-linked immunosorbent assay (ELISA) and Western blot techniques. The CD4+ lymphocyte counts were maintained under 200 cells/mm3 during one year demonstrating the severity of the state of immunosuppression. Chagas' disease was confirmed by serological and parasitological methods. Trypomastigote forms were visualized in a thick blood smear. The parasite isolated is genotypically similar to the CL strain. The paper reinforces that cerebral Chagas' disease can be considered as another potential opportunistic infection in AIDS resulting from the reactivation of a dormant T. cruzi infection acquired years earlier.

Parasite genotypically related to a monoxenous trypanosomatid of dog's flea causing opportunistic infection in an HIV positive patient.

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a panel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea.

Isoenzymes detect variation in populations of Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae).

Triatoma brasiliensis is one of the most important vectors of Chagas disease in the semiarid zone of the northeast of Brazil. Intraspecific morphological and behavioural variation has been reported for different populations. Results for four distinct populations using eight isoenzymes are reported here. The literature describes three subspecies: T. brasiliensis brasiliensis Neiva, 1911; T. brasiliensis melanica Neiva & Lent, 1941 and T. brasiliensis macromelasoma Galvão, 1956. These subspecies differ mainly in their cuticle colour pattern and were regarded as synonyms by Lent and Wygodzinsky (1979). In order to evaluate whether the chromatic pattern is a morphological variation of different melanic forms within T. brasiliensis or due to interspecific variation, field collections were performed in localities where these three subspecies have been described: Caicó (Rio Grande do Norte), the type-locality for T. b. brasiliensis; Petrolina (Pernambuco) for T. b. macromelasoma and Espinosa (Minas Gerais) for T. b. melanica. A fourth distinct chromatic pattern was found in Juazeiro (Bahia). A total of nine loci were studied. Values of Nei's genetic distance (D) were calculated. T. b. brasiliensis and T. b. macromelasoma are the closest populations with a D = 0.295. T. b. melanica had a D > or = 0.537 when compared to the others, a distance in the range of interspecific variation for other triatomine species.

[Cases of american cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in the towns of Cosmópolis and Indaiatuba-region of Campinas, in São Paulo, Brazil]. / Casos de leishmaniose tegumentar americana por Leishmania (Viannia) braziliensis nos Municípios de Cosmópolis e Indaiatuba-região de Campinas, Estado de São Paulo, Brasil.

A study was carried out to identify Leishmania species involved in skin lesions of patients from Cosmópolis and Indaiatuba, State of São Paulo, Brazil. The epidemiological data of cutaneous leishmaniasis in two cities suggested a epidemic situation in 1994. The lesions were clinically characteristic of cutaneous leishmaniasis and five out six patients responded positively to Montenegro's intradermal test. The histopathology of skin lesions were characterized by two patterns: exudative-cellular reaction and exudative granulomatous reaction. The clinical and histopathological parameters suggested Leishmania (Viannia) braziliensis as the possible etiologic agent. In agreement, it was difficult to isolate and maintain the parasite in the laboratory. Characterization by in situ hybridization with kDNA amastigotes from lesions fragments confirmed that Leishmania (Viannia) braziliensis was the parasite responsible for the studied cutaneous lesions.

Restriction endonucleases from microorganisms isolated in Brazil: an isoschizomer of HaeIII from a thermophilic Bacillus sp.

1. The isolation and characterization of a restriction endonuclease from a thermophilic strain of Bacillus is described. 2. The enzyme recognizes the palindromic sequence 5'...GGCC...3' as determined by PEI-cellulose chromatography of pancreatic DNAse and snake venom phosphodiesterase digestion products of labelled DNA fragments, analysis of restriction digests and direct sequence analysis. 3. The enzyme, denominated BspBR, is an isoschizomer of HaeIII and BspRI.

Restriction endonucleases from microorganisms isolated in Brazil: an isoschizomer of Hae III from a thermophilic Bacillus SP

The isolation and characterization of a restriction endonuclease from a thermophilic strain of Bacillus is described. The enzyme recognizes the palindromic sequence 5'...GGCC...3' as determined by PEI-cellulose chromatography of pancreativc DNAse and snake venom phosphodieterase digestion products of labelled DNA fragments, analysis of restriction digests and direct sequence analysis. The enzyme, denominated BspBR, is an isoschizomer of HaeIII and BspRI

An Sau3 AI restriction endonuclease isoschizomer from Bacillus cereus.

The isolation and characterization of a restriction endonuclease from Bacillus cereus IOC 243 are described. The enzyme recognizes the palindromic sequence 5'-G(met-A,A)TC-3' as determined by PEI chromatography of pancreatic DNase, snake venom phosphodiesterase digestion products of labelled fragments, analysis of restriction digests from normal and N6-methyladenine-free DNA and direct sequence analysis of cloned fragments. The staggered cleavage products with 5' -terminal pGATC extensions are efficiently labelled with polynucleotide kinase and are easily cloned into BamHI sites. The enzyme, denoted Bce243, is thus an isoschizomer of Sau3AI. Its use and potential advantages in substituting Sau3AI. are discussed.