p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil.

Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7%) of 230 unrelated CF alleles. Fifteen (13.0%) patients were homozygous for this mutation, while 20 (17.4%) were heterozygous; the remaining 80 (69.6%) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2%) patients, 21 (18.3%) had the sequence variation 4521G/A, 11 (9.6%) had a not yet described sequence variation 4407T/A and 8 (7.0%) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

p.F508del in a heterogeneous cystic fibrosis population from Minas Gerais, Brazil

Cystic fibrosis (CF) is the most common autosomal recessive disease of the Caucasian population. Among the various CF mutations, p.F508del is the most frequent, accounting for two-thirds of the global CF chromosomes, although showing great variability among populations. We have studied 115 unrelated CF patients from a mixed population of Minas Gerais (Brazil). To evaluate part of the DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, blood DNA was obtained and PCR was performed using two pairs of primers that anneal to exons 10 and 24 of the CFTR gene. The PCR product was then submitted to automatic sequencing using the ABI PRISM 310 Genetic Analyzer. The p.F508del mutation was found in 50 (21.7 percent) of 230 unrelated CF alleles. Fifteen (13.0 percent) patients were homozygous for this mutation, while 20 (17.4 percent) were heterozygous; the remaining 80 (69.6 percent) patients did not carry the p.F508del mutation. Exon 24 sequence had no change in 75 (65.2 percent) patients, 21 (18.3 percent) had the sequence variation 4521G/A, 11 (9.6 percent) had a not yet described sequence variation 4407T/A and 8 (7.0 percent) patients had both sequence variations (4521G/A and 4407T/A). The polymorphism 4407T/A results in an amino acid modification from aspartic acid to glutamic acid, which will probably have no function effect in CFTR. This low p.F508del prevalence can be due to the variable ethnic origin of this population from Minas Gerais, which may have a high diversity of CF rare mutations.

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5 percent, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3 percent for IgG and from 4.8 to 1.1 percent for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7 percent for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1 percent, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6 percent, respectively. The specificity of positive IgG or IgA was 93.5 percent in children without enteropathies and 78.7 percent in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9 percent. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country.

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5%, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3% for IgG and from 4.8 to 1.1% for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7% for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1%, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6%, respectively. The specificity of positive IgG or IgA was 93.5% in children without enteropathies and 78.7% in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9%. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption.

Pathogenic aspects of pyogenic liver abscess associated with experimental schistosomiasis.

Schistosomiasis mansoni infection that occurs concurrently with Staphylococcus aureus bacteremia favors the formation of pyogenic liver abscess. The present experimental study in mice evaluated the following aspects of the relationship between infection with Schistosoma mansoni and liver abscess caused by S. aureus: a) the role of the eggs of S. mansoni in the genesis of the abscesses; b) the influence of different phases of schistosomiasis in the development of liver abscesses; and c) the effect of the treatment of schistosomiasis on the development of the abscesses. Macroscopic and histopathological study showed multiple liver abscesses around granulomas of S. mansoni in the acute and chronic phases of schistosomiasis. Treatment of acute schistosomiasis before experimentally-induced bacteremia did not prevent the formation of liver abscess. The study findings indicate that granulomas around S. mansoni eggs and worms lodged in the liver provide a focus and substrate for pyogenic abscesses caused by S. aureus.

Whipple's disease. Report of five cases with different clinical features.

Whipple's disease (WD) is a rare systemic disease of infectious etiology which involves the small intestine but can virtually affect any organ. We present here five cases (four males and one female) ranging in age from 20 to 59 years. All patients had intestinal involvement associated or not with clinical manifestations linked to this organ. Vegetation in the tricuspid valve was observed in one patient, suggesting endocarditis caused by Tropheryma whippelii, with disappearance of the echocardiographic alterations after treatment. In one of the male patients the initial clinical manifestation was serologically negative spondylitis, with no diarrhea occurring at any time during follow-up. Ocular involvement associated with intestinal malabsorption and significant weight loss were observed in one case. In the other two cases, diarrhea was the major clinical manifestation. All patients were diagnosed by histological examination of the jejunal mucosa and, when indicated, of extraintestinal tissues by light and electron microscopy. After antibiotic treatment, full remission of symptoms occurred in all cases. A control examination of the intestinal mucosa performed after twelve months of treatment with sulfamethoxazole-trimethoprim revealed the disappearance of T. whippelii in four patients. The remaining patient was lost to follow-up.

Congenital cytomegalovirus infection in a neonatal intensive care unit in brazil evaluated by PCR and association with perinatal aspects.

Cytomegalovirus (CMV) infection is the most common congenital infection, affecting 0.4% to 2.3% newborns. Most of them are asymptomatic at birth, but later 10% develop handicaps, mainly neurological disturbances. Our aim was to determine the prevalence of CMV shed in urine of newborns from a neonatal intensive care unit using the polymerase chain reaction (PCR) and correlate positive cases to some perinatal aspects. Urine samples obtained at first week of life were processed according to a PCR protocol. Perinatal data were collected retrospectively from medical records. Twenty of the 292 cases (6.8%) were CMV-DNA positive. There was no statistical difference between newborns with and without CMV congenital infection concerning birth weight (p=0.11), gestational age (p=0.11), Apgar scores in the first and fifth minutes of life (p=0.99 and 0. 16), mother's age (p=0.67) and gestational history. Moreover, CMV congenital infection was neither related to gender (p=0.55) nor to low weight (<2,500 g) at birth (p=0.13). This high prevalence of CMV congenital infection (6.8%) could be due to the high sensitivity of PCR technique, the low socioeconomic level of studied population or the severe clinical status of these newborns.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay.

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 10(6) leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 10(6) leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100 per cent of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100 per cent, specificity of 92 per cent, a positive predictive value of 43 per cent and a negative predictive value of 100 per cent for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

[Study of intestinal malabsorption diseases as cause of monosymptomatic short stature] / Estudo de doenças de má absorção intestinal como causa de baixa estatura monossintomática.

OBJECTIVE: This study was carried out in order to evaluate the etiology of monosymptomatic childhood short stature (below the third percentile or with growth rate of less than 5 cm/year) with emphasis on causes due to intestinal malabsorption. METHODS: Each patient was submitted to endocrinological, biochemical and hematological investigation. Determination of serum anti-gliadin antibodies, fecal fat, chloride levels in sweat, jejunal biopsy and bone age was also obtained.RESULTS: A total of 51 children was studied, most of them belonging to the group of normal variants. Four children had abnormally high sweat chloride, compatible with the diagnosis of cystic fibrosis. These children were asymptomatic regarding respiratory and gastrointestinal tract. CONCLUSIONS: We conclude that cystic fibrosis, besides celiac disease, must be included in the differential diagnosis of short stature in childhood.

Schistosomiasis and associated infections.

In hospital-based series viral hepatitis B has been frequently described in association with schistosomiasis whilst in field-based studies the association has not been confirmed. The association between schistosomiasis and Salmonella bacteraemia has been well documented. More recently, acute schistosomiasis has been shown to be a facilitating factor in the genesis of pyogenic liver abscesses caused by Staphylococcus aureus. New evidences indicate an interaction between the acquired immunodeficiency syndrome (AIDS) and schistosomiasis. In this paper, data on the association of schistosomiasis with other infections are updated.

Visceral larva migrans and tropical pyomyositis: a case report.

We report a case of tropical pyomyositis in a boy who presented with a severe febrile illness associated with diffuse erythema, and swelling in many areas of the body which revealed on operation extensive necrotic areas of various muscles that required repeated débridement. The patient gave a history of contact with dogs, and an ELISA test for Toxocara canis was positive. He also presented eosinophilia and high serum IgE levels. Staphylococcus aureus was the sole bacteria isolated from the muscles affected. We suggest that tropical pyomyositis may be caused by the presence of migrating larvae of this or other parasites in the muscles. The immunologic and structural alterations caused by the larvae, in the presence of concomitant bacteremia, would favour seeding of the bacteria and the development of pyomyositis.

Morphological changes of the jejunal mucosa in protracted diarrhea and their correlation with disease duration, weight loss and serum albumin levels

The pathogenesis of protracted diarrhea is multifactorial. In developing countries, intestinal infectious processes seem to play an important role in triggering the syndrome. Thirty-four children aged 1 to 14 months, mean 6.5 months, with protracted diarrhea were studied clinically and in terms of small intestinal mucosal morphology. Mild, moderate or severe hypotrophy of the jejunal mucosa was detected in 82 percent of cases, and mucosal atrophy was observed in 12 percent. The intensity of the morphological changes of the jejunal mucosa correlated negatively with serum albumin levels. No correlation was detected between mucosal grading and duration of diarrhea or between mucosal grading and weight reported as percentile. After nutritional support was instituted, serial jejunal biopsies were obtained from 12 patients: five patients submitted to parenteral nutrition for 7 to 38 days, mean 17 days, and 7 patients reveiving a hypoallergenic oral diet (semi-elemental formula, 3; chicken formula, 3; human milk, 1). In seven cases (58 percent) a progressive increase in villus height and a decrease in the number of inflammatory cells were noted. Recovery of the morphologic pattern was accompanied by clinical improvement in all patients.

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil.

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in tumor suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra- and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70% of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing.

Utilization of microsatellites for the analysis of genomic alterations in colorectal cancers in Brazil

Two different pathogenetic mechanisms are proposed for colorectal cancers. One, the so-called "classic pathway", is the most common and depends on multiple additive mutational events (germline and/or somatic) in suppressor genes and oncogenes, frequently involving chromosomal deletions in key genomic regions. Methodologically this pathway is recognizable by the phenomenon of loss of heterozygosity. On the other hand, the "mutator pathway" depends on early mutational loss of the mismatch repair system (germline and/or somatic) leading to accelerated accumulation of gene mutations in critical target genes and progression to malignancy. Methodologically this second pathway is recognizable by the phenomenon of microsatellite instability. The distinction between these pathways seems to be more than academic since there is evidence that the tumors emerging from the mutator pathway have a better prognosis. We report here a very simple methodology based on a set of tri-, tetra-and pentanucleotide repeat microsatellites allowing the simultaneous study of microsatellite instability and loss of heterozygosity which could allocate 70 per cent of the colorectal tumors to the classic or the mutator pathway. The ease of execution of the methodology makes it suitable for routine clinical typing.

Morphological changes of the jejunal mucosa in protracted diarrhea and their correlation with disease duration, weight loss and serum albumin levels.

The pathogenesis of protracted diarrhea is multifactorial. In developing countries, intestinal infectious processes seem to play an important role in triggering the syndrome. Thirty-four children aged 1 to 14 months, mean 6.5 months, with protracted diarrhea were studied clinically and in terms of small intestinal mucosal morphology. Mild, moderate or severe hypotrophy of the jejunal mucosa was detected in 82% of cases, and mucosal atrophy was observed in 12%. The intensity of the morphological changes of the jejunal mucosa correlated negatively with serum albumin levels. No correlation was detected between mucosal grading and duration of diarrhea or between mucosal grading and weight reported as percentile. After nutritional support was instituted, serial jejunal biopsies were obtained from 12 patients: five patients submitted to parenteral nutrition for 7 to 38 days, mean 17 days, and 7 patients receiving a hypoallergenic oral diet (semi-elemental formula, 3; chicken formula, 3; human milk, 1). In seven cases (58%) a progressive increase in villus height and a decrease in the number of inflammatory cells were noted. Recovery of the morphologic pattern was accompanied by clinical improvement in all patients.

Correlaçäo cito-histológica na punçäo aspirativa da tiróide com agulha fina / Hystocytological correlation in fine needle aspiration biopsy of the thyroid gland

A punçao aspirativa da tiróide com agulha fina (PAAF) tem sido empregada em freqüência crescente para diagnóstico de muitas doenças tiroidianas. OBJETIVO. Correlacionar os diagnósticos citológia com os anatomopatológicos para avaliar a eficácia do exame citológico como teste diagnóstico. CASUISTICA E MÉTODO. Foram comparados os diagnósticos de 328 exames citológicos de punçao aspirativa com agulha fina da tiróide com os achados anatomopatológicos das peças cirúrgicas correspontes. RESULTADOS. Em todas as doenças diagnosticadas, acurácia do exame citopatológico superou 95 por cento. Entre as doenças mais freqüentes, a sencibilidade do teste foi de 98 por cento, 69 por cento, 81 por cento, 85 por cento e 89 por cento, respectivamente, para bócio colóide nodular, neoplasia de células foliculares, tiroidite de Hashimoto, carcinoma papilar e neoplasia de células de Hürthle; em todas elas, a especificidade foi igual ou superior a 89 por cento. CONCLUSAO. Com base nesses resultados e dada sua relativa simplicidade, fica mais uma vez documentada a utilidade da PAAF como método propedêutico das doenças da tiróide.

[Histocytological correlation in fine needle aspiration biopsy of the thyroid gland]. / Correlação cito-histológica na punção aspirativa da tiróide com agulha fina.

PURPOSE: To correlate cytological diagnosis with the anatomicopathological ones to assess the accuracy of cytological examination as a diagnostic test. CASES AND METHOD: The results of 328 cytological diagnoses were compared with the corresponding anatomicopathological results from surgical specimens. RESULTS: In all thyroid diseases the accuracy of the method was above 95%. Among the more frequent diseases, the sensitivity of the test was 98%, 69%, 81%, 85% and 89%, respectively for nodular colloid goiter, follicular neoplasms, Hashimoto's thyroiditis, papillary carcinoma and Hürthle-cell neoplasia; in all of them the test specificity was at least 89%. CONCLUSION: Based on these results and owing to its relative simplicity, this study also documents the FNAB usefulness as a diagnostic method for thyroid diseases.

Changes in pentobarbital induced sleeping-time in mice infected with Schistosoma mansoni and immunosuppressed.

To evaluate whether the intensity of the hepatic granulomatous response induced by S. mansoni eggs plays a role in drug metabolism, mice were infected with 40 cercariae and tested to assess the sodic pentobarbital induced sleeping-time. To decrease the inflammatory reaction the animals were irradiated with 400 Rad or received azathioprine, 20 mg/kg, 3 times a week, for 4 weeks, respectively in or beginning in the 33th post-infection day. In infected animals receiving azathioprine the area of the hepatic granulomas was smaller and the sleeping-time was similar to that of non-infected ones (controls). In mice infected and irradiated the granuloma dimensions were similar to those of animals only infected, in these two latter groups of animals, the sleeping-time was more prolonged than that of the control animals. These results show that: 1) mice with unaltered hepatic granulomatous reaction show reduction in metabolism of sodic pentobarbital; 2) granulomatous response diminished by azathioprine does not interfere with the capacity of metabolism of the anesthetic drug.