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A J Romanha

Publications and source records attributed to A J Romanha.

At least 19 recordsLinked to original sources
PubMed2006

Genetic filtering and optimal sampling of Schistosoma mansoni populations.

Allelic variation in 6 microsatellite markers was compared between frozen Schistosoma mansoni eggs and laboratory-passaged worms originating from the same 5 fecal samples obtained from Brazilian residents. Based on allelic richness values, the number of alleles detected per locus did not differ between egg and worm DNA templates. However, our ability to score loci differed between these DNA templates, with worms providing more scored loci per individual than eggs. Differences also existed between the worms and eggs in the identity of the specific alleles that were detected. Additionally, we observed a reduction in homozygous genotypes among laboratory-passaged worms relative to the eggs. Allelic diversity curves were calculated by genotyping all worms from a representative host sample to determine the relationship between the number of alleles detected at a locus and the number of worms genotyped. Curves for the 5 residents' worm infrapopulations for each of the loci were very similar. The equation y=19.55 x ln(x) + 9.992 explained the association between sampling effort (x) and number of alleles detected (y) with an R(2) of 0.775. In conclusion, egg DNA templates and allelic diversity curves can benefit efforts to discern the sociological, ecological and evolutionary forces impacting the genetic diversity and disease epidemiology of human schistosomes.

Alleles↗
PubMed2006

Leishmania identification by PCR of Giemsa-stained lesion imprint slides stored for up to 36 years.

This study examined the ability of PCR to amplify Leishmania DNA, stored on Giemsa-stained slides, from American cutaneous leishmaniasis (ACL) patients. In total, 475 slides stored for up to 36 years were obtained from an outpatient clinic in a Brazilian ACL-endemic region, and Leishmania DNA was amplified from 395 (83.2%) of the DNA samples using primers specific for the minicircle kinetoplast DNA. Restriction fragment length polymorphism analysis of these amplicons demonstrated that Leishmania (Viannia) braziliensis was the only species present in these samples. The results demonstrated that archived Giemsa-stained slides can provide a Leishmania DNA source for performing clinical and epidemiological studies of leishmaniasis.

Animals↗
PubMed2005

Influence of the long-term Trypanosoma cruzi infection in vertebrate host on the genetic and biological diversity of the parasite.

The influence of the long-term Trypanosoma cruzi infection in vertebrate host on the biological and genetic properties of the parasite was evaluated. Four T. cruzi isolates obtained from different chronic chagasic dogs infected with Berenice-78 T. cruzi strain during 2 and 7 years were comparatively analyzed. The long-term T. cruzi infection has led to alterations in parasitemia, virulence and pathogenicity of Be-78 strain for mice. These biological parameters varied from low to high in realation to the parental strain. Randomly amplified polymorphic DNA and isoenzyme profiles detected two distinct genetic groups of parasites. The first group included the parental strain and two T. cruzi isolates, and the second group the two other isolates. Interestingly, the isolates of the second group showed a reversibility of the genetic profile to the parental strain after 25 passages in mice. No correlation between the genetic groups and biological properties of the isolates was observed. Our findings confirmed the population heterogeneity of the Be-78 strain, and showed how differently it responds to the long-term infection in the same vertebrate hosts.

Animals↗
PubMed2004

An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells.

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 microl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 microl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100 degrees C for 45 min. After cooling, 100 microl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r(2) > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.

Biomarkers↗
PubMed2004

Clinical and laboratory evaluation of compliance in acute lymphoblastic leukaemia.

AIM: To evaluate compliance in children with acute lymphoblastic leukaemia (ALL). METHODS: Compliance was assessed through specific interviews, annotations from medical charts, and erythrocytic determination of 6-mercaptopurine metabolites. RESULTS: A total of 39 patients who had concluded maintenance phase of chemotherapy were included in the study. Mothers were responsible for delivering 6-MP in 87% of cases. Thirty five interviewees said that medical prescription was well understood and that the main reason for non-compliance was forgetfulness. Non-compliance was detected through interviews (33.3% of the cases), reports from medical charts (30.7%), and drug determination (16.6%); 53.8% of children were found to be non-compliant. Non-compliance was significantly associated with chronic undernourishment. Although not statistically significant, there was a trend for the group of non-compliant children to be associated with low per capita family income. No significant associations of non-compliance with age at diagnosis, gender, parents' schooling level, number of family members, power consumption, and medians of absolute leucocyte or neutrophil blood counts were detected. A short follow up period precluded valid analysis on outcome. In the non-compliant group (n = 21), seven children relapsed, contrasting with three relapses in the compliant group (n = 18). CONCLUSIONS: Results suggest that non-compliance is one of the mechanisms which underlies the adverse influence of socioeconomic factors on the outcome of children with ALL. Additional studies are necessary to confirm this hypothesis. Comprehensive approaches to the problem of non-compliance are urgently needed.

Adolescent↗
PubMed2003

[Relationships between Trypanosoma cruzi transmission cycles in the county of Bambuí, Minas Gerais, Brazil].

This study examines recent relationships between domestic and sylvatic transmission cycles of T. cruzi in the county of Bambuí, MG, Brazil. In the late l930s, Panstrongylus megistus was found in 75% of houses. Subsequently, Triatoma infestans became the predominant species, found in 20% of urban households and in more than 60% of periurban homes. With intense insecticide control campaigns between l956 and l969, T. infestans was eradicated from the county, transmission of Chagas' disease to man was interrupted, and P. mesgistus appeared in rural residences. Samples of T. cruzi isolated by xenodiagnosis and hemoculture from 43 opossums (Didelphis albiventris) captured in both peridomiciliary and sylvatic areas were characterized by isoenzyme analysis and--regardless of isolation method--were found to present the Z1 zymodeme profile. Through the "Chagas' Disease Epidemiological Surveillance Program", from August l986 to December l988, 154 specimens of P. megistus were captured by the local population in both peridomiciliary and intradomiciliary environments, of which 9.8% were infected with T. cruzi. In isoenzyme analyses of l3 T. cruzi strains isolated from these triatomines, six were found to be of the Z1 zymodeme (sylvatic transmission cycles) and seven were found to be of the Z2 zymodeme (domestic transmission cycle). The capture of P. megistus specimens in intradomiciliary environments that were naturally infected with parasites of both cycle types indicates an overlap of transmission cycles of Chagas' disease in the county of Bambuí. Further evidence for the interrelationship of the two cycles was provided by the isolation of T. cruzi of the Z2 zymodeme from a cat and the participation of the dog as a reservoir of Z1 T. cruzi. The presence of P. megistus in the peridomiciliary environment represents an important link between the sylvatic and intradomestic environments serving as a carrier of Z1 T. cruzi and maintaining the transmission cycles of Z2 T. cruzi in the peridomestic and intradomestic environments thus providing the potential for a gradual reinfestation of the county if the "Epidemiological Surveillance" is interrupted.

English Abstract↗
PubMed2002

Populational structure of Schistosoma mansoni assessed by DNA microsatellites.

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.

Animals↗
PubMed2002

Genetic variability of the main intermediate host of the Schistosoma mansoni in Brazil, Biomphalaria glabrata (Gastropoda: Planorbidae) assessed by SSR-PCR.

The genetic variability of Brazilian Biomphalaria glabrata populations was studied using SSR-PCR. This technique is a variant of the polymerase chain reaction (PCR), which consists of using a single primer directed towards microsatellite regions under high stringency reaction conditions. Twenty snails of each population from eight distant Brazilian localities were analyzed. Morphology and PCR-RFLP were used for previous specific identification of the snails. Bands generated after gel electrophoresis of the SSR-PCR products of each snail were used to study intra- and interpopulation genetic variability. Fifty-five prominent bands were considered in a pairwise band comparison for the determination of genetic variability. Genetic variability was greater between populations than within populations. Snail populations from the field and the laboratory presented almost no genetic differences. No relationship between genetic variability and geographic distance was found. SSR-PCR proved to be a good alternative molecular tool for the population study of B. glabrata.

Animals↗
PubMed2002

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries.

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Animals↗
PubMed2001

IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma.

The ability of up-regulatory [recombinant (r) IFN-gamma, rIFN-beta and rTNF-alpha] and down-regulatory (rIL-4, rIL-10 and rIL-13) cytokines to control the expression of indoleamine 2,3-dioxygenase (INDO) and anti-Toxoplasma activity in the human fibrosarcoma cell line 2C4 was evaluated. Activation of fibroblasts with rIFN-gamma, rIFN-beta and rTNF-alpha resulted in augmentation of INDO expression and activity leading to 40.0, 25.0 and 27.0 % inhibition of tachyzoite growth, respectively. An additive effect was observed when host cells were incubated with rIFN-gamma plus rTNF-alpha. With regard to the down-regulatory cytokines we observed that IL-4 as well as IL-13, but not IL-10, induced significant inhibition of IFN-gamma-induced control of parasite replication, INDO mRNA expression and tryptophan catabolism. Similarly, IL-4 but not IL-10 inhibited the cell surface expression of HLA-DR and CD2 induced by IFN-gamma. Consistent with these findings we were able to detect by reverse transcription-PCR the expression of mRNA for different chains of IL-4 and IL-13 receptors (IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2) but not for IL-10 receptor in the 2C4 and other human lung fibroblast cell lines (LL24 and MRC5). Together our results indicate that IL-4 and IL-13, but not IL-10, are implicated in the negative regulation of IFN-gamma-induced anti-Toxoplasma activity in human cells from fibroblast lineage.

Animals↗
PubMed2001

Attempt to differentiate Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis using the SSR-PCR technique.

The ability to differentiate reference strains of Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis was evaluated using the simple sequence repeat polymerase chain reaction (SSR-PCR) technique. This technique differentiates the Leishmania species, generating distinct DNA amplicon profiles. The SSR-PCR profiles were similar to but more reproducible than those produced by RAPD. SSR-PCR is presented as an alternative to other molecular methods for the differentiation of Leishmania species or strains.

Animals↗
PubMed2001

Genetic studies of Psammolestes tertius (hemiptera: reduviidae: triatominae) using male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA.

Two Brazilian populations of Psammolestes tertius (Ceará and Minas Gerais) collected from thornbird nests (Furnariidae) were compared by male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA (RAPD). Male genitalia showed no difference between the populations. In contrast, morphometry, isoenzyme, and RAPD clearly distinguished the two populations. Possible mechanisms of dispersal and the origin of Psammolestes are discussed.

Animals↗
PubMed2001

Anti-Trypanosoma cruzi immunoglobulin G1 can be a useful tool for diagnosis and prognosis of human Chagas' disease.

Two functionally distinct antibodies, categorized as conventional serology antibodies (CSA) and lytic antibodies (LA) have been described in Chagas' disease, based on their ability to bind to fixed epimastigotes (EPI) or live trypomastigotes (TRYPO), respectively. In this study, the profile of immunoglobulin G (IgG) subclasses of CSA and LA were analyzed by flow cytometry using serum samples from chronic chagasic patients with the indeterminate (IND), cardiac (CARD), and digestive (DIG) clinical forms of the disease. The results were expressed as percentage of positive fluorescent parasites (PPFP) for each sample. CSA showed a higher PPFP than LA for all samples. At serum dilutions between 1:256 and 1:2,048, IgG1 anti-EPI was able to distinguish chagasic from nonchagasic individuals. Different profiles of IgG subclasses were observed for CSA and LA. IgG1 and IgG2 were the main subclasses in CSA, whereas IgG1 and IgG3 were the predominant ones in LA. The reactivity of IgG2 anti-EPI was greater in IND and CARD than in DIG patients. Furthermore, a low level of IgG1 and IgG3 LA was associated with most of the CARD patients. On the other hand, a high level of IgG1 LA was associated with most of the IND patients. In summary, our findings indicate the potential of IgG1 anti-EPI for serological diagnosis of Chagas' disease, providing further evidence for a protective role of LA, and show that IgG1 anti-live Trypanosoma cruzi TRYPO may be used to predict the risk of cardiac damage in Chagas' disease.

Animals↗
PubMed2001

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

Animals↗
PubMed2001

A study of experimental reinfection by Trypanosoma cruzi in dogs.

The role of reinfection in the evolution of Chagas' disease was evaluated in dogs alternately infected with the 147 and SC-1 strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain 147, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas' disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas' disease. All infected dogs developed this form of the disease, which was independent of the number of infections.

Animals↗
PubMed2000

Trypanosoma cruzi: parasitaemia produced in mice does not seem to be related to in vitro parasite-cell interaction.

The interaction of Trypanosoma cruzi strains producing subpatent or high parasitaemia in mice with mouse macrophages, Vero and L929 cells was evaluated using tissue culture trypomastigotes. Macrophages were the cells most readily infected while Vero cells presented the highest parasite intracellular multiplication rates. Subpatent strains were equal or more infective than the high parasitaemia. Due to the small number of strains, no correlation could be established between the zymodemes and parasitaemia or parasite-cell interaction in vitro. However parasitaemia in mice does not seem to be related to in vitro parasite-cell interaction.

Animals↗
PubMed2000

Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil.

Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Adolescent↗