Evolution of bacterial meningitis diagnosis in Sao Paulo State-Brazil and future challenges

Bacterial meningitis (BM) is a severe disease and still represents a serious public health problem with high rates of morbidity and mortality. The most common cases of BM around the world, mainly in Brazil, have been caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae type b. Bacterial culture is the gold-standard technique for BM confirmation, but approximately 50% of suspected cases are not culture-confirmed, due to problems related to improper transportation and seeding or previous antibiotic treatment. Immunological methods present low sensitivity and have possibility of cross-reactions. Real time PCR (qPCR) is a molecular technique and has been successful used for BM diagnosis at Instituto Adolfo Lutz in São Paulo State, Brazil, since 2007. The incorporation of qPCR in the Public Health surveillance routine in our state resulted in diminishing 50% of undetermined BM cases. Our efforts are focused on qPCR implementation in the BM diagnostic routine throughout Brazil

High-Precision, In Vitro Validation of the Sequestration Mechanism for Generating Ultrasensitive Dose-Response Curves in Regulatory Networks

Our ability to recreate complex biochemical mechanisms in designed, artificial systems provides a stringent test of our understanding of these mechanisms and opens the door to their exploitation in artificial biotechnologies. Motivated by this philosophy, here we have recapitulated in vitro the “target sequestration” mechanism used by nature to improve the sensitivity (the steepness of the input/output curve) of many regulatory cascades. Specifically, we have employed molecular beacons, a commonly employed optical DNA sensor, to recreate the sequestration mechanism and performed an exhaustive, quantitative study of its key determinants (e.g., the relative concentrations and affinities of probe and depletant). We show that, using sequestration, we can narrow the pseudo-linear range of a traditional molecular beacon from 81-fold (i.e., the transition from 10% to 90% target occupancy spans an 81-fold change in target concentration) to just 1.5-fold. This narrowing of the dynamic range improves the sensitivity of molecular beacons to that equivalent of an oligomeric, allosteric receptor with a Hill coefficient greater than 9. Following this we have adapted the sequestration mechanism to steepen the binding-site occupancy curve of a common transcription factor by an order of magnitude over the sensitivity observed in the absence of sequestration. Given the success with which the sequestration mechanism has been employed by nature, we believe that this strategy could dramatically improve the performance of synthetic biological systems and artificial biosensors

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

Background: The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings: A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells

Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T>C (L444P)

BACKGROUND: Gaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann-Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay. METHODS: Biotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin-conjugated fluorophore, the reactions were analyzed by Luminex IS100. Clinical evaluations were conducted using de-identified patient DNA samples. RESULTS: We evaluated a multiplexed suspension array assay that includes wild-type and mutant genetic determinations for Gaucher disease allele c.1448T>C. Two percent of samples reported to be wild-type by conventional methods were observed to be c.1448T>C heterozygous using our assay. Sequence analysis suggested that this phenomenon was due to co-amplification of the functional gene and a paralogous pseudogene (ΨGBA) due to a polymorphism in the primer-binding site of the latter. Primers for the amplification of this allele were then repositioned to span an upstream deletion in the pseudogene, yielding a much longer amplicon. Although it is widely reported that long amplicons negatively impact amplification or detection efficiency in recently adopted multiplex techniques, this assay design functioned properly and resolved the occurrence of false heterozygosity. CONCLUSION: Although previously available sequence information suggested GBA gene/pseudogene discrimination capabilities with a short amplified product, we identified common single-nucleotide polymorphisms in the pseudogene that required amplification of a larger region for effective discrimination

Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays

Rapid pyrazinamide drug susceptibility testing using a closed-tube PCR assay of the entire pncA gene

The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis. Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2–98.6) and specificity of 97.9% (95% CI: 96.1–99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5–100) but lower specificity of 91.8% (95% CI: 87.9–95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility