Comparison of methods for urinary albumin determination in patients with type 1 diabetes

We tested the correlation of the albumin-to-creatinine ratio (A/C) in an early-morning urine sample, measured with a commercial kit (DCA 2000®), with the conventional immunoturbidimetric determination in the laboratory and with overnight albumin excretion rate (reference method). Fifty-five type 1 diabetic adolescents had their first-morning urine collected on the 1st and 8th day of the period. Urinary albumin and creatinine were determined immediately using the DCA 2000® kit. Samples were also stored for laboratory analysis. To evaluate the correlation between early-morning urinary A/C ratio and overnight albumin excretion rate, 16 subjects had a timed overnight urine collection. A/C ratios determined with the DCA 2000® kit and by the laboratory method were 13.1 ± 20.5 and 20.4 ± 46.3 mg/g, respectively. A/C results by both methods proved to be strongly correlated (r = 0.98, P<0.001). DCA 2000®-determined A/C showed 50% sensitivity and 100% specificity when compared to the reference method. Spot urinary A/C of the subset of 16 subjects significantly correlated with their overnight albumin excretion rate (r = 0.98, P<0.001). Intraindividual variation ranged from 17 to 32% and from 9 to 63% for A/C and overnight albumin excretion rate, respectively. In conclusion, an early-morning specimen should be used instead of timed overnight urine and the A/C ratio is an accurate, reliable and easily determined parameter for the screening of diabetic nephropathy. Immediate measurement of the A/C ratio is feasible using the DCA 2000® kit. Intraindividual variability indicates the need for repeated determinations to confirm microalbuminuria and the diagnosis of incipient diabetic nephropathy.

Newborn screening for biotinidase deficiency in Brazil: biochemical and molecular characterizations

Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10% of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30% of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09%). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Changes in clinical and laboratory findings at the time of diagnosis of primary hyperparathyroidism in a University Hospital in São Paulo from 1985 to 2002

In contrast to most developed countries, most patients with primary hyperparathyroidism in Brazil are still symptomatic at diagnosis. However, we have been observing a change in this pattern, especially in the last few years. We evaluated 104 patients, 77 females and 27 males aged 11-79 years (mean: 54.4 years), diagnosed between 1985 and 2002 at a University Hospital. Diagnosis was made on the basis of clinical findings and of high total and/or ionized calcium levels, high or inappropriate levels of intact parathyroid hormone and of surgical findings in 80 patients. Patients were divided into three groups, i.e., patients diagnosed from 1985 to 1989, patients diagnosed from 1990 to 1994, and patients diagnosed from 1995 to 2002. The number of new cases diagnosed/year increased from 1.8/year in the first group to 6.0/year in the second group and 8.1/year in the third group. The first group comprised 9 patients (mean serum calcium ± SD, 13.6 ± 1.6 mg/dl), 8 of them (88.8%) defined as symptomatic. The second group comprised 30 patients (mean calcium ± SD, 12.2 ± 1.63 mg/dl), 22 of them defined as symptomatic (73.3%). The third group contained 65 patients (mean calcium 11.7 ± 1.1 mg/dl), 34 of them symptomatic (52.3%). Patients from the first group tended to be younger (mean ± SD, 43.0 ± 15 vs 55.1 ± 14.4 and 55.7 ± 17.3 years, respectively) and their mean serum calcium was significantly higher (P < 0.05). All of symptomatic patients independent of group had higher serum calcium levels (12.4 ± 1.53 mg/dl, N = 64) than asymptomatic patients (11.4 ± 1.0 mg/dl, N = 40). Our data showed an increase in the percentage of asymptomatic patients over the years in the number of primary hyperparathyroidism cases diagnosed. This finding may be due to an increased availability of diagnostic methods and/or to an increased awareness about the disease.

Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification

The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler), honey, Vitis vinifera (grape), Pyrus communis (pear), Malus sp. (apple) and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone), 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid), 41 in enrichment medium (De Ley and Swings) and later in the GYC medium (glucose, yeast extract and calcium carbonate). 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP) in Assis, stored in malt extract at -196 ºC.

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.

Efficiency of methods to detect Sclerotinia sclerotiorum in commercial soybean seed lots

Most soybean pathogens are seed transmitted, deserving emphasis the fungus Sclerotinia sclerotiorum, which has been presenting worrying levels of field incidence in some soybean cropping areas in several Brazilian states. The objective of this study was to verify the efficiency of different methods for detecting S. sclerotiorum on soybean seeds artificially infected in the laboratory and from field production areas with a historical disease incidence. Seed samples of seven different cultivars collected from naturally infested fields, and one seed sample artificially inoculated in the laboratory were used. The following detection methods recommended in the literature were compared: Blotter test at 7 ºC, 14 ºC, and 21 ºC; Rolled Paper; and Neon-S. Results demonstrated that these methods showed no repeatability and had a low sensitivity for detecting the pathogen in seeds from areas with disease incidence. They were effective, however, for its detection on artificially inoculated seeds. In the Blotter test method at 7 ºC, there was a lower incidence of other fungi considered undesirable during seed analysis.