High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potential.

A new time-domain narrowband velocity estimation technique for Doppler ultrasound flow imaging. I. Theory.

A significant improvement in blood velocity estimation accuracy can be achieved by simultaneously processing both temporal and spatial information obtained from a sample volume. Use of the spatial information becomes especially important when the temporal resolution is limited. By using a two-dimensional sequence of spatially sampled Doppler signal "snapshots" an improved estimate of the Doppler correlation matrix can be formed. Processing Doppler data in this fashion addresses the range-velocity spread nature of the distributed red blood cell target, leading to a significant reduction in spectral speckle. Principal component spectral analysis of the "snapshot" correlation matrix is shown to lead to a new and robust Doppler mode frequency estimator. By processing only the dominant subspace of the Doppler correlation matrix, the Cramer-Rao bounds on the estimation error of target velocity is significantly reduced in comparison to traditional narrowband blood velocity estimation methods and achieves almost the same local accuracy as a wideband estimator. A time-domain solution is given for the velocity estimate using the root-MUSIC algorithm, which makes the new estimator attractive for real-time implementation.

A new time-domain narrowband velocity estimation technique for Doppler ultrasound flow imaging. II. Comparative performance assessment.

For pt.I see ibid., vol.45, no.4, pp.939-54 (1998). The statistical performance of the new 2-D narrowband time-domain root-MUSIC blood velocity estimator described previously is evaluated using both simulated and flow phantom wideband (50% fractional bandwidth) ultrasonic data. Comparisons are made with the standard 1-D Kasai estimator and two other wideband strategies: the time domain correlator and the wideband point maximum likelihood estimator. A special case of the root-MUSIC, the "spatial" Kasai, is also considered. Simulation and flow phantom results indicate that the root-MUSIC blood velocity estimator displays a superior ability to reconstruct spatial blood velocity information under a wide range of operating conditions. The root-MUSIC mode velocity estimator can be extended to effectively remove the clutter component from the sample volume data. A bimodal velocity estimator is formed by processing the signal subspace spanned by the eigenvectors corresponding to the two largest eigenvalues of the Doppler correlation matrix. To test this scheme, in vivo common carotid flow complex Doppler data was obtained from a commercially available color flow imaging system. Velocity estimates were made using a reduced form of this data corresponding to higher frame rates. The extended root-MUSIC approach was found to produce superior results when compared to both 1- and 2-D Kasai-type estimators that used initialized clutter filters. The results obtained using simulated, flow phantom, and in vivo data suggest that increased sensitivity as well as effective clutter suppression can be achieved using the root-MUSIC technique, and this may be particularly important for wideband high frame rate imaging applications.

A nonstationary signal simulation model for continuous wave and pulsed Doppler ultrasound.

A computer simulation model of the nonstationary Doppler ultrasound signals arising from pulsatile blood flow is presented. The model uses sinusoidal components that are modulated by a power spectral density function that varies over the cardiac cycle. An empirical model consisting of two exponential functions is used to represent both the continuous wave and pulsed Doppler power spectral density for normal carotid arteries. It is shown that the spectrogram speckle patterns of the synthesized Doppler signals compare very well with those clinically recorded.