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Biomedical subjects

J M Costa

Publications and source records attributed to J M Costa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Myotonic dystrophy protein kinase gene expression in skeletal muscle from congenitally affected infants.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by marked variability of its clinical manifestations. The mutational basis of DM is an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). We used quantitative RT-PCR to determine DMPK mRNA levels in muscular biopsies from three congenitally affected (CDM) and two control infants. The CDM infants had increased DMPK mRNA levels, which were not correlated to increased expression of the mutant allele. This increase may be the consequence of a maturational muscular arrest, which may maintain an elevated level of DMPK mRNA until birth.

Case-Control Studies↗
PubMed1996

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients.

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.

AIDS-Related Opportunistic Infections↗
PubMed1995

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Blotting, Southern↗
PubMed1995

A comparison of three interferon alfa-2b regimens for the long-term treatment of chronic non-A, non-B hepatitis. Multicenter Study Group.

BACKGROUND: We studied the effects of long-term treatment with interferon on histologic features of the liver and serum alanine aminotransferase concentrations in patients with chronic non-A, non-B hepatitis. METHODS: Consecutive patients who met the inclusion criteria were enrolled in the study. The diagnosis of chronic non-A, non-B hepatitis was established on the basis of the liver-biopsy findings and an abnormal serum alanine aminotransferase value (greater than 1.5 times the normal value) for at least one year. All patients were treated for six months with 3 million units of interferon alfa-2b given subcutaneously three times a week and were then randomly assigned to the same treatment for an additional 12 months (group 1), a regimen of 1 million units three times a week for 12 months (group 2), or no further treatment (group 3). Patients in group 3 who had elevated serum alanine aminotransferase concentrations for three consecutive months underwent the initial regimen once again. Follow-up continued for two years after the discontinuation of treatment. Histologic improvement was defined as a decrease of at least one grade in the score for necroinflammatory activity (0, no activity; 1, mild; 2, moderate; or 3, severe) between the first liver biopsy and a biopsy performed at 18 months. RESULTS: Of the 329 patients initially treated, 303 were randomized: 103 to group 1, 101 to group 2, and 99 to group 3. Of the 286 patients tested, 252 (88.1 percent) had antibodies to hepatitis C virus. In an intention-to-treat analysis, 46 of the patients in group 1 (44.7 percent) had normal serum alanine aminotransferase values at 18 months, as compared with 27 of the patients in group 2 (26.7 percent, P = 0.008) and 30 of those in group 3 (30.3 percent, P = 0.04). Between 19 and 42 months, 23 of the patients in group 1 (22.3 percent) continued to have normal serum alanine aminotransferase values (measured every six months), as compared with 10 of the patients in group 2 (9.9 percent, P = 0.02) and 8 of those in group 3 (8.1 percent, P = 0.005). Among the 176 patients with repeated liver biopsies at 18 months, more patients in group 1 had improved histologic-activity scores (69.6 percent) than in group 2 (47.6 percent, P = 0.02) or group 3 (38.6 percent, P < 0.001). CONCLUSIONS: Among patients with chronic non-A, non-B hepatitis, a regimen of 3 million units of interferon alfa-2b given three times a week for 18 months produced better histologic findings and serum alanine aminotransferase values than regimens involving a lower dose or a shorter duration of treatment.

Alanine Transaminase↗
PubMed1995

Polar and subpolar diffuse cutaneous leishmaniasis in Brazil: clinical and immunopathologic aspects.

BACKGROUND: Diffuse cutaneous leishmaniasis (DCL) is a rare manifestation of human leishmaniasis, characterized by multiple, slowly progressive nodules or plaques without ulceration, involving almost the entire body. It has been suggested, that DCL results from a lack of cell-mediated immunity to leishmanial antigen, leading to uncontrolled parasite growth. METHODS: We have performed detailed clinical, histopathologic, and immunologic investigations in six patients with DCL. Biopsies were taken from the nodules, processed, and examined for determination of the macrophagic pattern present, based on the intensity of vacuolation and the frequency of vacuolated cells, the parasite index, and the presence of eosinophils. Immunologically, patients were evaluated by their response to intradermal skin test to PPD or leishmania antigen, determination of antileishmania antibodies by immunofluorescent assay, and lymphocyte proliferation assay. RESULTS: There seemed to be a negative relation between nodules and skin ulcerations, whereas the highest number of parasites were observed in patients with the greatest number of vacuolated macrophages. The delayed hypersensitivity skin test to leishmanial antigen was negative, and antileishmania IgG antibodies were positive in all patients. CONCLUSIONS: Although all cases fulfill the criteria for being classified as DCL, they present a wide spectrum. Three cases were clearly at the unresponsive pole, and three other cases belonged to the subpolar form of DCL, exhibiting varying weak signs of antiparasite responsiveness.

Adolescent↗
PubMed1995

Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR.

A competitive PCR assay involving the use of bronchoalveolar lavage (BAL) samples for the diagnosis of invasive pulmonary aspergillosis (IPA) was developed. For this purpose, a 1-kb mitochondrial DNA fragment of Aspergillus fumigatus was sequenced. The primers used allowed amplification of A. fumigatus, A. flavus, A. terreus, and A. niger DNAs but not DNAs of other fungi and yeasts. BAL samples from 55 consecutively enrolled patients were tested. Three samples were excluded because of failure of correct amplification of the internal competitive control. Of 28 immunocompromised patients, 6 were PCR positive; 3 died of IPA and their BAL cultures yielded A. fumigatus; and 3 were culture negative and did not develop IPA. Of 15 human immunodeficiency virus-positive patients and 9 immunocompetent patients, 5 and 4, respectively, were both PCR positive and culture negative, and none developed aspergillosis. Thus, PCR confirmed IPA in three patients but gave positive results for 25% (12 of 49) of the patients who did not develop aspergillosis. The predictive value of PCR-positive results seems low for patients at risk for aspergillosis. Moreover, the risk of contamination of reaction buffers or biological samples with Aspergillus conidia seems high and has to be weighed in regard to the potential diagnostic benefit of PCR testing as a routine procedure.

AIDS-Related Opportunistic Infections↗
PubMed1995

Quantitative competitive PCR with bronchoalveolar lavage fluid for diagnosis of toxoplasmosis in AIDS patients.

Of 121 AIDS patients, 12 (10%) had Toxoplasma gondii DNA detected by competitive PCR in their bronchoalveolar lavage samples. Quantitation of the PCR results showed a correlation between the parasite burden and the serum lactic dehydrogenase titer. Quantitative competitive PCR could be useful to assess the diagnosis and the severity of pulmonary toxoplasmosis.

AIDS-Related Opportunistic Infections↗
PubMed1995

Late toxoplasmosis evidenced by PCR in a marrow transplant recipient.

We report a case of disseminated toxoplasmosis occurring 12 months after allogeneic BMT. The patient was seropositive for Toxoplasma gondii, and the donor was seronegative, so the patient was given anti-Toxoplasma prophylaxis. One year after BMT, he developed fever and muscle pain without other clinical symptoms. PCR amplification for T. gondii performed on blood was positive. Toxoplasma were found in bronchoalveolar lavage by PCR and in the marrow by special stains. With treatment, the PCR signal disappeared in 3 days while clinical symptoms resolved over 15 days. This case emphasizes the possibility of late toxoplasmosis after BMT despite prophylatic treatment, and the value of PCR in making the diagnosis.

Adult↗
PubMed1994

Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid.

BACKGROUND: Congenital infection with Toxoplasma gondii can produce serious sequelae. However, there is little consensus about screening during pregnancy, and the tests used to establish a prenatal diagnosis of toxoplasmosis are complex and slow. We evaluated a simpler approach that is based on a polymerase-chain-reaction (PCR) test. METHODS: Prenatal diagnostic tests, including ultrasonography, amniocentesis, and fetal-blood sampling, were performed in 2632 women with T. gondii infection acquired during pregnancy. In 339 consecutive women, a competitive PCR test for T. gondii was performed on amniotic fluid, and its results were compared with those of conventional diagnostic tests. The PCR test targets the B1 gene of T. gondii, uses an internal control, and can be completed in a day. Positive tests were confirmed by serologic testing of newborns or by autopsy in terminated pregnancies. RESULTS: Overall, the risk of fetal infection was 7.4 percent, but it increased sharply with gestational age. Congenital infection was demonstrated in 34 of 339 fetuses by conventional methods, and the PCR test was positive in all 34. In three other fetuses, only the PCR test gave positive results, and follow-up testing confirmed the presence of congenital toxoplasmosis. The PCR test gave one false negative result but no false positive results. The PCR test performed better than conventional parasitologic methods (sensitivity, 97.4 vs. 89.5 percent; negative predictive value, 99.7 vs. 98.7 percent). CONCLUSIONS: For the prenatal diagnosis of congenital T. gondii infection, an approach based on a PCR test performed on amniotic fluid is rapid, safe, and accurate.

Amniotic Fluid↗
PubMed1994

Clinical and immunopathological aspects of disseminated cutaneous leishmaniasis.

The clinico-pathological and immunological findings in eight patients from Bahia, Brazil with disseminated cutaneous leishmaniasis are described. This condition differs from anergic diffuse cutaneous leishmaniasis (DCL) and from classical American cutaneous leishmaniasis (ACL). The number of lesions in these patients ranged from 75 to 800 and were characterized by papules and an acneiform type of lesion with a few ulcers rather than nodules that are the main characteristic of DCL. On the other hand the high incidence of mucosal disease (38%) in patients with disseminated cutaneous leishmaniasis make the prevalence of mucosal involvement in this condition higher than that observed in ACL. L. amazonensis (five cases) and L. braziliensis (two cases) were the causal agents in the patients where the infecting agent was characterized. Antibody titers in disseminated cutaneous leishmaniasis were higher than those observed in ACL and patients with the highest antibody titers had mucosal involvement. Abnormalities in cellular immunity that are not observed in ACL such as decrease in CD4+ cells and absence of T cell response to leishmania antigen were observed in several patients with disseminated cutaneous leishmaniasis but restoration of these abnormalities occurred after treatment. In spite of the great number of lesions, the therapeutic response was good in six patients with disappearance of the lesions in a period shorter than that observed in ACL. In the two patients that presented therapeutic failure the causal agent was L. amazonensis. In such patients there was a predominance of ulcerated lesions, and a high titer of antibody was detected.

Adult↗
PubMed1994

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot.

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out mutants of E. carotovora subsp. carotovora. Out mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out mutants of Ecb168 were complemented to the Out phenotype by introduction of the corresponding cloned HindIII fragment. Out mutants of Ecb168 were less virulent than the Out parental strain on potato tubers. Strain Ecb168 and Out derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.

Journal Article↗
PubMed1994

[Chronic phase of Chagas disease. Clinical aspects and course of the disease].

PURPOSE: To evaluate the clinical and evolutive aspects of chronic chagasic patients. METHODS: Three hundred chronic chagasic patients, 180 females, with age ranging from 19 to 81 years (55.6 +/- 13.1) were retrospectively studied. Patients were divided according to the following clinical types: indeterminated, cardiac (with the subtypes: arrhythmogenic, dilated and mixed), digestive isolated and digestive plus cardiac involvement. The following variables were analysed: prevalence of each clinical forms, symptoms, electrocardiographic pattern and clinical outcome. RESULTS: At the start of the study, 73 (24.3%) patients were in indetermined type, 106 (35.3%) in cardiac arrhythmogenic, 95 (31.6%) in mixed, 7 (2.3%) in dilated, 16 (5.3%) in digestive plus cardiac type and 3 (1%) in the pure digestive type. The most prevalent symptoms were dyspnea on efforts (57%), palpitations (41.33%) and chest pain (33%). The most frequent electrocardiographic pattern was right bundle branch block plus antero-superior fascicular block, in 30% of the patients. The average follow-up time was 7.8 +/- 6.1 years and the outcome was considered good in 20 patients (6.6%), stable in 214 (71.3%) and bad in 66 (23%). At the end of the follow-up, 9 patients have evaluated from the indeterminated to the cardiac and digestive types, and 19 (17.92%), from the arrhythmogenic to mixed cardiac subtype. The follow-up was lost in 79 patients (26.3%), most of them, probably dead. CONCLUSION: With a mean time of 7.8 years, 12.3% of the patients in the indeterminated type evolved to the cardiac and/or digestive type; right brundle branch block with antero-superior fascicular block was the most prevalent electrocardiographic pattern; the outcome was stable or good in the majority of these patients.

Adult↗
PubMed1994

Chemiluminescent detection of amplified pseudorabies virus gp50 DNA with immobilized probes on microtiter wells.

A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 has been developed for the detection of PRV-DNA sequences. Primer pairs from PRV gp50 gene were used with the enzyme uracil N-glycosylase and dUTP instead dTTP to prevent contamination due to PCR product carry-over. Biotinylated PCR products were captured in microtiter wells by specific oligonucleotide covalently linked to the polystyrene wells. After recognition of the biotinylated PCR product with a streptavidin phosphatase conjugate, a chemiluminescent detection was realised. Different factors influencing the binding, the hybridization and the detection efficiency have been tested.

Base Sequence↗
PubMed1993

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling.

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0 U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G(-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5' region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patient's mother and a grandmother. The different phenotypes associated with the G(-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.

Adult↗