There is still an ongoing demand for a simple broad-spectrum molecular diagnostic assay for pathogenic bacteria. For this purpose, we developed a single-plex High Resolution Melt (HRM) assay that generates complex melt curves for bacterial identification. Using internal transcribed spacer (ITS) region as the phylogenetic marker for HRM, we observed complex melt curve signatures as compared to 16S rDNA amplicons with enhanced interspecies discrimination. We also developed a novel Naïve Bayes curve classification algorithm with statistical interpretation and achieved 95% accuracy in differentiating 89 bacterial species in our library using leave-one-out cross-validation. Pilot clinical validation of our method correctly identified the etiologic organisms at the species-level in 59 culture-positive mono-bacterial blood culture samples with 90% accuracy. Our findings suggest that broad bacterial sequences may be simply, reliably and automatically profiled by ITS HRM assay for clinical adoption.

The gene responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPMI) is located on human chromosome 21q22.3 in a region defined by recombination breakpoints and linkage disequilibrium. As part of an effort to clone the EPM1 gene on the basis of its chromosomal location, we have constructed a 753-kb bacterial clone contig that encompasses the region containing the gene. Because DNA markers from the region did not identify intact yeast artificial chromosome (YAC) clones after screening several libraries, we built the contig from cosmid clones and used bacterial artificial chromosome (BAC) and bacteriophage PI clones to fill gaps. In addition to constructing the clone contig, we determined the locations of the E¢oRI, Sadl, EaBI, and Nod restriction sites in the clones, resulting in a high-resolution restriction map of the region. Most of the contig is represented by a level of redundancy that allows the orders of most restriction sites to be determined, provides multiple data points supporting the clone orders and orientations, and allows a set of clones with a minimum degree of overlap to be chosen for efficient additional analysis. The clone and restriction maps are in excellent agreement with maps generated of the region by other methods. These ordered bacterial clones and the mapping information obtained from them provide valuable reagents for isolating candidate genes for EPMI, as well as for determining the nucleotide sequence of a 750 kb region of the human genome.Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is inherited as an autosomal recessive disorder and is characterized by severe stimulus-sensitive myoclonus, tonicclonic seizures, and progressive neural degeneration. The age of onset is typically between 6 and 15 years. This condition is one of a group of five different subtypes of progressive myoclonus epilepsy, all of which show progressive neurodegeneration and variable degrees of severity. Unlike the other progressive myoclonus epilepsies, EPM1 is not characterized by biochemical markSCorresponding author. E-MAIL myers@shgc.stanford.edu; FAX (415) 725-9689.

Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.

With the advancement of microbiological discovery, it is evident that many infections, particularly bloodstream infections, are polymicrobial in nature. Consequently, new challenges have emerged in identifying the numerous etiologic organisms in an accurate and timely manner using the current diagnostic standard. Various molecular diagnostic methods have been utilized as an effort to provide a fast and reliable identification in lieu or parallel to the conventional culture-based methods. These technologies are mostly based on nucleic acid, proteins, or physical properties of the pathogens with differing advantages and limitations. This review evaluates the different molecular methods and technologies currently available to diagnose polymicrobial infections, which will help determine the most appropriate option for future diagnosis.

BackgroundTimely identification of a causative pathogen and its antimicrobial susceptibility profile is important for effective therapy. This is especially true in the case of bloodstream infections caused by the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) pathogens where inappropriate antibiotic prescription often leads to higher mortality and increased selection for multi-drug resistant strains. However, current standard protocols for pathogen identification (ID) and antimicrobial susceptibility testing (AST) take days to complete and despite the advancement of molecular diagnostics, none can concurrently provide reliable ID and AST information.MethodsWe developed a method of direct ID and AST of ESKAPE pathogens using real-time PCR-HRM (high resolution melt) as the end-point analysis coupled with whole blood sample preparation. Our assay utilizes blood cell lysis, removal of background human DNA and protein, pathogen enrichment, antibiotic exposure, and broad-range PCR-HRM analysis targeting bacterial internal transcribed spacer region to determine ID and AST in less than 10 hours. We then assessed antimicrobial
susceptibility/resistance by calculating the difference in threshold cycle compared with untreated sample (ΔCt), and determined species identity by its melt curve using an automated computer algorithm against a reference curve database of 89 bacterial species.ResultsOur assay was able to reach a limit of detection of 10 CFU/mL for all tested ESKAPE organisms except for E. faecium (100 CFU/mL). Using a final concentration of 100 CFU/mL and a calculated ΔCt cutoff value of 1.5, we observed significant ΔCt changes with antibiotic treatment and established minimum inhibitory concentrations (MICs) for each strain. Comparison of the observed values to reference MICs showed overall similar results. Furthermore, our automated machine-learning based computer algorithm was able to correctly identify each organism based on its melt curve.ConclusionOur results suggest that reliable rapid bacterial ID and AST information can be simultaneously obtained directly from whole blood using our combined ID-AST assay.Disclosures All authors: No reported disclosures.