A comparative analysis of preprocessing techniques of cardiac event series for the study of heart rhythm variability using simulated signals

In the present study, using noise-free simulated signals, we performed a comparative examination of several preprocessing techniques that are used to transform the cardiac event series in a regularly sampled time series, appropriate for spectral analysis of heart rhythm variability (HRV). First, a group of noise-free simulated point event series, which represents a time series of heartbeats, was generated by an integral pulse frequency modulation model. In order to evaluate the performance of the preprocessing methods, the differences between the spectra of the preprocessed simulated signals and the true spectrum (spectrum of the model input modulating signals) were surveyed by visual analysis and by contrasting merit indices. It is desired that estimated spectra match the true spectrum as close as possible, showing a minimum of harmonic components and other artifacts. The merit indices proposed to quantify these mismatches were the leakage rate, defined as a measure of leakage components (located outside some narrow windows centered at frequencies of model input modulating signals) with respect to the whole spectral components, and the numbers of leakage components with amplitudes greater than 1%, 5% and 10% of the total spectral components. Our data, obtained from a noise-free simulation, indicate that the utilization of heart rate values instead of heart period values in the derivation of signals representative of heart rhythm results in more accurate spectra. Furthermore, our data support the efficiency of the widely used preprocessing technique based on the convolution of inverse interval function values with a rectangular window, and suggest the preprocessing technique based on a cubic polynomial interpolation of inverse interval function values and succeeding spectral analysis as another efficient and fast method for the analysis of HRV signals

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7%) and a specificity of 94.7% (95% CI, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.