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

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.

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 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.

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.