New trends in infective endocarditis.

The present article is an update of the literature on endocarditis. A multidisciplinary group of Spanish physicians with an interest in cardiac infections selected the most important papers produced lately in the field. Two of the members of the group discussed the content of each of the selected papers, with a critical review by others members of the panel. After a review of the state of the art papers from the fields of epidemiology, new causative microorganisms (bacterial and fungal), clinical findings including those in special patients, laboratory diagnosis, prognostic factors, nosocomial endocarditis, prophylaxis, new drugs and guidelines for antibiotic treatment were discussed by the group.

Inducible nitric oxide synthase promoter polymorphism in human brucellosis.

Nitric oxide (NO), produced by the inducible nitric oxide synthase (NOS2) protein, is a defence mechanism against various pathogens, including bacteria of the genus Brucella. The aim of this study was to investigate the possible association between the NOS2 gene promoter polymorphism, TAAA(n) and CCTTT(n) microsatellites, and the predisposition to human brucellosis. We performed a case-control study in 85 patients with brucellosis and 100 healthy individuals, matched for age and sex, living in the same geographic area, in whom the NOS2 promoter was genotyped by using a polymerase chain reaction (PCR)-based method combined with fluorescent technology. No statistically significant differences were observed in the distribution of TAAA(n) alleles between the groups under study. When the overall NOS2 CCTTT(n) allele distribution of the brucellosis patients was compared with that of the control subjects, a significant skewing was observed (P = 0.04, by chi(2) test from 2 x 9 contingency table). Interestingly, we observed a trend towards Brucella infection protection with the 9 repeat (181 bp) allele (1.8% patients vs. 7.5% controls; P = 0.01, odds ratios = 0.22, 95% confidence interval = 0.05-0.83), which turned out to be non-significant after applying multiple testing. We concluded that the NOS2 microsatellite polymorphism might not have a major effect on brucellosis; nevertheless, the fact that a non-significant trend towards protection was detected in the CCTTT(n) alleles may be an indication for a follow-up study.

Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

[Prevalence of primary resistance mutations in patients with newly diagnosed HIV infection in the province of Málaga (Spain)]. / Prevalencia de mutaciones de resistencia primaria en los nuevos diagnósticos de infección por el VIH en la provincia de Málaga.

INTRODUCTION: The reported prevalence of primary resistance mutations differs between studies. An analysis was performed to determine the prevalence of primary resistance mutations and HIV subtypes in our area. METHODS: Prospective study performed in all patients diagnosed with HIV in the year 2005 in the province of Malaga (Spain). Plasma samples from these patients were tested for genotypic resistance (TruGene HIV-1 genotyping kit; Bayer Healthcare Diagnostics) and HIV subtype. RESULTS: A total of 172 cases were diagnosed, 6 of them recent seroconvertors. Genotype resistance testing disclosed resistance mutations in 7.8% (95% CI 3.5-12.0%) of 153 patients in which it was performed (6 to NNRTIs, 4 to NRTIs, and 3 to PIs). HIV subtype was B in 81.8% of patients, and non-B in 18.1% (51.8% of them of sub-Saharan origin, in whom the prevalence of this subtype was 73.6%). Among European patients, only those from Spain presented the non-B subtype (prevalence 7.4%). The only factor related with the presence of resistance mutations was seroconversion (OR 9.2; 95% CI 1.3-61.9; P < .02). CONCLUSIONS: There was a considerable prevalence of primary resistance mutations in patients with newly diagnosed HIV infection in Malaga province, with seroconversion being the only related factor. The high prevalence of the non-B HIV subtype in the Spanish population is noteworthy. Genotype resistance testing is recommendable in all newly diagnosed HIV patients in our area.

PCR-DIG ELISA with biotinylated primers is unsuitable for use in whole blood samples from patients with brucellosis.

In an attempt to avoid some of the inconveniences associated with conventional PCR, such as electrophoresis in ethidium bromide, we developed and analyzed the yield of a digoxigenin-based enzyme-linked immunosorbent PCR assay (PCR-DIG ELISA) for the detection of specific Brucella target DNA. During the DNA amplification process in healthy subjects and controls (Brucella abortus B-19) non-specific amplification of fragments was formed between genomic DNA and specific biotin-labeled primers. The labeled non-specific fragments bound to streptavidin-coated wells, saturating the solid phase streptavidin by biotin-streptavidin interaction. The formation of these non-specific PCR products was demonstrated by reduction in absorbance with hemin, a Taq polymerase inhibitor, and identified by use of a silver stained method which improves the sensitivity of nucleic acid visualization.