Rapid Detection and Differentiation of Clinically Relevant Candida Species Simultaneously from Blood Culture by Use of a Novel Signal Amplification Approach.

Fungal bloodstream infections are a significant problem in the United States, with an attributable mortality rate of up to 40%. An early diagnosis to direct appropriate therapy has been shown to be critical to reduce mortality rates. Conventional phenotypic methods for fungal detection take several days, which is often too late to impact outcomes. Herein, we describe a cost-effective multiplex assay platform for the rapid detection and differentiation of major clinically relevant Candida species directly from blood culture. This approach utilizes a novel biotin-labeled polymer-mediated signal amplification process combined with targeting rRNA to exploit phylogenetic differences for sensitive and unambiguous species identification; this assay detects seven pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, and C. guilliermondii) simultaneously with very high specificity to the species level in less than 80 min with the limits of detection at 1 × 103 to 10 × 103 CFU/ml or as few as 50 CFU per assay. The performance of the described assay was verified with 67 clinical samples (including mixed multiple-species infections as well), with an overall 100% agreement with matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based reference results. By providing a species identity rapidly, the clinician is aided with information that may direct appropriate therapy sooner and more accurately than current approaches, including PCR-based tests.

A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing.

We describe here a strategy that can distinguish between Staphylococcus species truly present in a clinical sample from contaminating Staphylococcus species introduced during the testing process. Contaminating Staphylococcus species are present at low levels in PCR reagents and colonize lab personnel. To eliminate detection of contaminants, we describe an approach that utilizes addition of sufficient quantities of either non-target Staphylococcal cells (Staphylococcus succinus or Staphylococcus muscae) or synthetic oligonucleotide templates to helicase dependent isothermal amplification reactions to consume Staphylococcus-specific tuf and mecA gene primers such that contaminating Staphylococcus amplification is suppressed to below assay limits of detection. The suppressor template DNA is designed with perfect homology to the primers used in the assay but an internal sequence that is unrelated to the Staphylococcal species targeted for detection. Input amount of the suppressor is determined by a mathematical model described herein and is demonstrated to completely suppress contaminating levels of Staphylococcus while not negatively impacting the appropriate clinical assay limit of detection. We have applied this approach to improve the specificity of detection of Staphylococcus species present in positive blood cultures using a chip-based array that produces results visible to the unaided eye.

Detection of rpoB gene mutations using helicase-dependent amplification.

For patients infected with tuberculosis, detection of rpoB gene mutations is critical for diagnosing drug-resistant strains of the causative pathogen, Mycobacterium tuberculosis (MTB). Traditional approaches to drug resistance include culture, which is very slow. Recently described real-time polymerase chain reaction approaches have addressed turnaround time but at relatively high cost. Here, we describe a novel amplification method, termed blocked-primer helicase-dependent amplification, for amplifying rpoB gene sequences in MTB. Resultant amplicon is hybridized to a probe set arrayed on a modified silicon-based chip to determine if there is any mutation in that region. Using this method, we could detect the majority of clinically relevant mutations in rpoB gene.

Rapid detection of rpoB gene mutations conferring rifampin resistance in Mycobacterium tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.

A gene-centered C. elegans protein-DNA interaction network.

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using "TF-centered" methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used "gene-centered" high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for approximately 10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.

Environmentally induced foregut remodeling by PHA-4/FoxA and DAF-12/NHR.

Growth and development of the Caenorhabditis elegans foregut (pharynx) depends on coordinated gene expression, mediated by pharynx defective (PHA)-4/FoxA in combination with additional, largely unidentified transcription factors. Here, we used whole genome analysis to establish clusters of genes expressed in different pharyngeal cell types. We created an expectation maximization algorithm to identify cis-regulatory elements that activate expression within the pharyngeal gene clusters. One of these elements mediates the response to environmental conditions within pharyngeal muscles and is recognized by the nuclear hormone receptor (NHR) DAF-12. Our data suggest that PHA-4 and DAF-12 endow the pharynx with transcriptional plasticity to respond to diverse developmental and physiological cues. Our combination of bioinformatics and in vivo analysis has provided a powerful means for genome-wide investigation of transcriptional control.

Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo.

The Caenorhabditis elegans UNC-45 protein contains tetratricopeptide repeats and a domain with similarity to fungal proteins, and it differentially colocalizes with myosin heavy chain B in the body wall muscles of adult worms. Although it is essential for normal myosin filament assembly in body wall muscle development, strong mutants show a previously unexplained maternal effect. We show here that the UNC-45 protein is maternally contributed and is present in all cells of the early embryo whereas zygotic UNC-45 expression is only detected in the developing muscle cells. Embryos produced from adults with reduced germline expression of UNC-45 exhibit cytokinesis defects suggesting that UNC-45 has a novel role in the early embryo in addition to muscle development. Yeast two-hybrid screens show that UNC-45 can directly interact with NMY-2, a non-muscle type II myosin, and UNC-45 and NMY-2 colocalize at cell boundaries in early embryos. Localization of UNC-45 at these boundaries is dependent upon the presence of NMY-2. Our results suggest that UNC-45 interacts with more than one type of myosin and functions in the embryo to regulate cytoplasmic myosin assembly and/or stability during cytokinesis.