New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

On Fractional Orthonormal Polynomials of a Discrete Variable

A fractional analogue of classical Gram or discrete Chebyshev polynomials is introduced. Basic properties as well as their relation with the fractional analogue of Legendre polynomials are presented

Detection of parametric changes in the Peyrard-Bishop- Dauxois model of DNA using nonlinear Kalman filtering

The derivative-free nonlinear Kalman filter is proposed for state estimation and fault diagnosis in distributed parameter systems of the wave-type and particularly in the Peyrard-Bishop-Dauxois model of DNA dynamics. At a first stage, a nonlinear filtering approach is introduced for estimating the dynamics of the Peyrard-Bishop-Dauxois 1D nonlinear wave equation, through the processing of a small number of measurements. It is shown that the numerical solution of the associated partial differential equation results in a set of nonlinear ordinary differential equations. With the application of a diffeomorphism that is based on differential flatness theory it is shown that an equivalent description of the system is obtained in the linear canonical (Brunovsky) form. This transformation enables to obtain local estimates about the state vector of the DNA model through the application us of the standard Kalman filter recursion. At a second stage, the local statistical approach to fault diagnosis is used to perform fault diagnosis for this distributed parameter system by processing with statistical tools the differences (residuals) between the output of the Kalman filter and the measurements obtained from the distributed parameter system. Optimal selection of the fault threshold is succeeded by using the local statistical approach to fault diagnosis. The efficiency of the proposed filtering approach in the problem of fault diagnosis for parametric change detection, in nonlinear wave-type models of DNA dynamics, is confirmed through simulation experiments