Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

In vitro susceptibility of Stenotrophomonas maltophilia isolates: comparison of disc diffusion, Etest and agar dilution methods.

The disc diffusion, Etest and agar dilution techniques were compared to evaluate the antimicrobial susceptibility profile of 70 Stenotrophomonas maltophilia isolates to seven antimicrobial agents. The S. maltophilia isolates were consecutively collected from May 2000 to May 2002 from individual patients, who were hospitalized in a private Brazilian hospital. The antimicrobial susceptibility tests were carried out and interpreted according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. The Etest was carried out according to the manufacturer's instructions. There was good agreement among the distinct susceptibility testing results for chloramphenicol, doxycycline, gatifloxacin, trimethoprim-sulfamethoxazole and ticarcillin-clavulanate, suggesting that the disc diffusion and Etest methods are reliable for testing this group of antimicrobials against S. maltophilia. In contrast, a weak correlation was found between the disc diffusion and agar dilution techniques for testing polymyxin B and colistin with unacceptable very major error rates (18.1% and 22.7% for polymyxin B and colistin, respectively). Trimethoprim- sulfamethoxazole (MIC50, 0.06 mg/L; 98.5% susceptible) and gatifloxacin (MIC50, 0.12 mg/L; 98.5% susceptible) were the most potent antimicrobial agents tested against S. maltophilia isolates. In contrast, the worst in vitro activity was found for ticarcillin-clavulanate (MIC50, 16 mg/L; 59.1% susceptible). Although our results confirm that trimethoprim-sulfamethoxazole, gatifloxacin and doxycycline have an excellent in vitro activity against S. maltophilia, further clinical studies are necessary to evaluate the clinical efficacy of these compounds for the treatment of S. maltophilia infections, since no randomized controlled trials have been carried out and no correlation between the clinical response and susceptibility testing results has been reported.

Body weight of rats following area postrema ablation: effect of early force-feeding.

This study examined the effect of intragastric force-feeding of a milk diet on body weights of rats with lesions of the area postrema/caudal medial nucleus of the solitary tract (AP/cmNTS). Force-feeding was conducted over the first 10 days after the ablation. Body weight was monitored both during and after force-feeding. Food intake was measured during all ad libitum feeding periods. During force-feeding, rats with AP/cmNTS lesions gained weight at the same rate as force-fed sham-lesioned rats or sham-lesioned rats that voluntarily ingested an equal amount of the milk. When returned to ad libitum feeding, lesioned rats that had been force-fed were not hypophagic and did not lose weight. Body weights of such rats remained above those of lesioned rats that were not force-fed and similar to those of nonlesioned rats throughout this study. Despite their normal weights, preliminary analysis indicated that body fat of the force-fed lesioned rats may have been reduced. These findings suggest that the effects of AP/cmNTS ablation are multiple and that reduction of body weight need not be the primary effect of such lesions.