Octavio Fernandes

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specific probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Molecular epidemiology of American trypanosomiasis in Brazil based on dimorphisms of rRNA and mini-exon gene sequences

American trypanosomiasis is transmitted in nature via a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, or via a domestic cycle where the parasite comes into contact with humans through domiciliated triatomines. The pool of T. cruzi isolates consists of sub-populations presenting a broad genetic diversity. In contrast to the heterogeneity suggested by isoenzyme analysis, PCR amplification of sequences from the 24S alpha rRNA gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from 12 Brazilian states were analysed by the 24S alpha RNA and mini-exon typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally. Molecular typing of human parasite isolates from three well-characterised endemic regions of Chagas disease (Minas Gerais, Paraiba and Piaui) and from Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage-1 stocks.

Detection of Leishmania DNA by Polymerase Chain Reaction in Scars of Treated Human Patients

Leishmaniasis is an endemic disease in developing countries. The efficacy of therapy is usually evaluated through clinical parameters. To define the parasitologic cure, 20 patients were biopsied before and 1 month to 8 years after treatment. Paraffin-embedded tissue was used for DNA isolation. All patients had a positive polymerase chain reaction (PCR) result before therapy, except 1, for whom no histopathologic material was available. The causative agent was identified as belonging to the Leishmania (Viannia) subgenus by hybridization. Despite clinical healing and absence of reactivation or development of mucosal lesions, PCR was positive in scars of 16 patients (80%). The results suggest that parasites persist in the skin for many years despite treatment. Depending on specific pathogenetic features of the parasite and the immune status of the host, this phenomenon might result in mucosal lesions. Alternatively, it could have a role in the maintenance of immunologic memory in patients living in areas in which leishmaniasis is endemic.

Chagas disease: from bush to huts and houses. Is it the case of the Brazilian amazon?

Two of the major problems facing the Amazon - human migration from the other areas and uncontrolled deforestation - constitute the greatest risk for the establishment of endemic Chagas disease in this part of Brazil. At least 18 species of triatomines had been found in the Brazilian Amazon, 10 of them infected with Trypanosoma cruzi, associated with numerous wild reservoirs. With wide-range deforestation, wild animals will perforce be driven into other areas, with tendency for triatomines to become adapted to alternative food sources in peri and intradomicilies. Serological surveys and cross-sectional studies for Chagas disease, carried out in rural areas of the Rio Negro, in the Brazilian Amazon, showed a high level of seropositivity for T. cruzi antibodies. A strong correlation of seroreactivity with the contact of gatherers of piaçava fibers with wild triatomines could be evidenced.

Populational heterogeneity of Brazilian Trypanosoma cruzi isolates revealed by the mini-exon and ribosomal spacers

Departamento de Medicina Tropical **Departamento de Protozoologia ***Departamento de Imunologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil *Departamento de Patologia, Uerj, Rio de Janeiro, RJ, Brasil ****Department of Microbiology & Immunology, School of Medicine, University of California, Los Angeles, USA *****Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Sao Paulo, SP, Brasil

Chagas' disease in the Brazilian Amazon: III. a cross-sectional sutdy

Dois inqueritos sorologicos para a infeccao chagasica foram realizados, respectivamente em 1991 e 1993, em amostras por conglomerado familiar na populacao da cidade de Barcelos, utilizando-se a reacao de imunofluorescencia indireta para anticorpos anti-T. cruzi. No primeiro inquerito de 628 amostras de sangue de residentes em 142 domicilios, 12,7% foram positivas para anticorpos anti-T. cruzi; no segundo inquerito de 658 amostras de sangue de residentes em 171 domicilios, 13,7% foram positivas, confirmando os resultados anteriores. De 170 individuos com sorologia positiva para infeccao chagasica 112 (66%) compareceram para entrevista e para exame clinico e eletrocardiografico. Destes 82 (73,2%) submeteram-se ao xenodiagnostico. Dos 112 entrevistados, 52 (46,4%) reconheceram o triatomineo como piolho da piacava, 48 (42,8%) disseram existir em seus locais de trabalho, geralmente em piacavais na area rural e 19(16,9%) disseram ja terem sido picados pelo inseto. Dos 82 xenodiagnosticos aplicados, 2 (2,4%) foram positivos para T. cruzi. Apenas 9 (8%) dos 112 ECG realizados tinham alteracoes compativeis com a doenca de Chagas. As evidencias demonstram que a infeccao chagasica na area tem caracteristicas profissionais, e transmitida pelo contato com triatomineos silvestres e que a cepa de T. cruzi circulante e de baixa virulencia e patogenicidade, possivelmente por ser silvestre e ainda nao adaptada ao homem.Two serological surveys for Chagas infection were carried out, in 1991 and 1993, respectively, using a conglomerate family samples from the residents in the town of Barcelos (in the northern part of the State of Amazonas, on the right bank of the Rio Negro, 490 Km up-river from Manaus), using indirect immunofluorescent tests for anti-T. cruzi antibodies. In the first survey (1991), 628 blood samples from the residents of 142 dwellings were tested, showing positive in 12.7% for anti-T. cruzi antibodies and in 1993 an other 658 samples from residents of 171 dwellings showed positive in 13.7% of the tests, thus confirming the previous results. From 170 individuals with positive serology for T. cruzi antibodies, 112 (66%) were interviewed and submitted to electrocardiographic and clinical examinations; 82 (73.2%) of them gave consent for xenodiagnosis. From the 112 interviewed 52 (46.4%) recognized the triatomines as piaçavas lice, 48 (42.8%) knew the bugs from their work places being gatherers of piaçava fibers in rural areas and 19 (16.9%) said that have been bitten by bugs in their huts. Only 2 (2.4%) of 82 xenodiagnosis applied were positive for T. cruzi and 9 (8%) of the ECG had alterations compatible with Chagas disease.

Enteric parasites and HIV infection: occurrence in AIDS patients in Rio de Janeiro, Brazil

The occurrence of intestinal parasites, its relation with the transmission mechanism of HIV, and the clinical state of the AIDS patients, were analyzed in 99 Group IV patients (CDC, 1986), treated at Hospital Universitário Pedro Ernesto (HUPE), between 1986 and 1988. The group consisted of 79 (79.8%) patients whose HIV transmission mechanism took place through sexual contact and of 16 (20.2%) who were infected through blood. Feces samples from each patient were examined by four distincts methods (Faust et al., Kato-Katz, Baermann-Moraes and Baxby et al.). The most occurring parasites were: Cryptosporidium sp., Entamoeba coli and Endolimax nana (18.2%), Strongyloides stercoralis and Giardia lamblia (15.2%), E. histolytica and/or E. hartmanni (13.1%), Ascaris lumbricoides (11.1%) and Isospora belli (10.1%). Furthermore, 74.7% of the patients carried at least one species. Intestinal parasites were found in 78.5% of the patients who acquired the HIV through sexual intercourse and in 56.3% of those infected by blood contamination. The difference, was not statistically significant (p greater than 0.05). In the group under study, the increase of the occurrence of parasitic infections does not seem to depend on the acquisition of HIV through sexual contact. It appears that in developing countries, the dependency is more related to the classic mechanisms of parasites transmission and its endemicity.

An oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia)

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

The genetic diversity of Brazilian Trypanosoma cruzi isolates and the phylogenetic positioning of zymodeme 3, based on the internal transcribed spacer of the ribosomal gene

There is a high level of genetic diversity among the clinical isolates of the taxon Trypanosoma cruzi. The results of recent studies on well defined gene loci have revealed that T. cruzi can be divided into two major phylogenetic lineages, designated T. cruzi I and T. cruzi II. Further intra-lineage polymorphisms were explored, in clinical isolates from five representative regions of Brazil, using the internal transcribed spacers that flank the 5.8S ribosomal RNA gene. These regions were amplified in a PCR and the products were digested with six restriction enzymes before being subjected to RFLP analysis in polyacrylamide gels. The results were used to construct a complex evolutionary tree, in which the isolates were clustered into two major phylogenetic lineages with a low level of similarity. To investigate the evolutionary relationships between the isolates, the PCR products were cloned and sequenced. The results of the subsequent phylogenetic analysis confirmed the presence of the two major groups of T. cruzi and showed that zymodeme 3, a third iso-enzymatic sub-group, is closer to T. cruzi I than to T. cruzi II.

Biodemas de cepas do Trypanosoma cruzi isoladas de humanos de três áreas endêmicas de Minas Gerais

In order to study the biological behavior of T. cruzi strains and to determine a putative association between their biodeme and the clinical forms of Chagas disease, 14 strains isolated from humans were evaluated. The individuals were from the municipalities of Pains, Iguatama and Berilo (Minas Gerais State). The biological behavior was evaluated in albino swiss mice, weighing 10 to 15 grams, which were infected with 1x10(4) blood tripomastigotes. The infectivity, parasitemia, tripomastigote morphology, virulence and the tissue distribution of the protozoan were analyzed. A behavior similar to biodeme II (São Felipe strain) was observed in 9 strains, while 5 stocks were characterized as belonging to biodeme III. It was not possible to establish a relationship between the biodeme strain and the severity of the disease in the patients.In order to study the biological behavior of T. cruzi strains and to determine a putative association between their biodeme and the clinical forms of Chagas disease, 14 strains isolated from humans were evaluated. The individuals were from the municipalities of Pains, Iguatama and Berilo (Minas Gerais State). The biological behavior was evaluated in albino swiss mice, weighing 10 to 15 grams, which were infected with 1x104 blood tripomastigotes. The infectivity, parasitemia, tripomastigote morphology, virulence and the tissue distribution of the protozoan were analyzed. A behavior similar to biodeme II (Sao Felipe strain) was observed in 9 strains, while 5 stocks were characterized as belonging to biodeme III. It was not possible to establish a relationship between the biodeme strain and the severity of the disease in the patients.