Design and construction of a single tube, quantitative endpoint, LATE-PCR multiplex assay for ventilator-associated pneumonia.

AIMS: The goal of this study was to develop a molecular diagnostic multiplex assay for the quantitative detection of microbial pathogens commonly responsible for ventilator-associated pneumonia (VAP) and their antibiotic resistance using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHOD AND RESULTS: This multiplex assay was designed for the quantitative detection and identification of pathogen genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Pseudomonas aeruginosa, plus a control target from Lactococcus lactis. After amplification, the single-stranded amplicons were detected simultaneously in the same closed tube by hybridization to low-temperature molecular beacon probes labelled with four differently coloured fluorophores. The resulting hybrids were then analysed by determining the fluorescence intensity of each of the four fluorophores as a function of temperature. CONCLUSIONS: This LATE-PCR single tube multiplex assay generated endpoint fluorescent contours that allowed identification of all microbial pathogens commonly responsible for VAP, including MRSA. The assay was quantitative, identifying the pathogens present in the sample, no matter whether there were as few as 10 or as many 100 000 target genomes. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay is rapid, reliable and sensitive and is ready for preclinical testing using samples recovered from patients suffering from ventilator-associated pneumonia.

Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis.

AIMS: The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. METHODS AND RESULTS: The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons. CONCLUSIONS: All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.

Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates.

AIMS: To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates. METHODS AND RESULTS: Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (µl) of each culture suspension (1 × 10(8) CFU ml(-1) ) were added to 20 µl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates. CONCLUSIONS: Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities ≥ 97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism.

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Behavior of submicrometer particles in periodic alveolar airflows.

Here, we report a numerical experiment in which submicrometer particle entrainment in a periodic flow that matches those existing in the alveolus in the human lung was simulated for both sedentary and light activity. A spherical cavity with a prescribed velocity profile at the inlet was used to simulate the time-dependent periodical flow of air in the alveolus. Expansion and contraction of the alveolus were simulated by setting a conceptual permeable wall as the outer surface of the model and adjusting the boundary conditions in order to match the continuity of the flow. The simulations were conducted for breathing periods of 5 and 3 s, which match sedentary and light activity conditions, respectively, and the results were extrapolated to the real lung. It was found that, most of the particles mainly followed a straightforward path and reached the opposite side of the alveolar wall in both breathing conditions. The concentration patterns obtained are consistent with the fact that the flow within the alveolus is mainly diffusive and does not greatly depend on the flow velocity. It was found that the particles which are heavier than air move out of phase with the periodic airflow that crosses the alveolus entrance, and that these particles are significantly caught within the alveolus. Particle entrapment increases with breathing rate in accordance with experimental values and indicates that increase in breathing frequency in environments with high concentration of submicrometer particles has the consequence of increasing particle entrapment by several times with respect to normal breathing rate.

Constructal theory of flow architecture of the lungs.

Here we explain the reasons why we have a bronchial tree with 23 levels ofbifurcation. Based on Bejan's Constructal Principle we found that the best oxygen access to the alveolar tissues as well as carbon dioxide removal is provided by a flow structure composed of ducts with 23 levels of bifurcation (bronchial tree) that ends with spaces (alveolar sacs) from where oxygen diffuses into the tissues. The model delivers the dimensions of the alveolar sac, the total length of the airways, the total alveolar surface area, and the total resistance to oxygen transport inthe respiratory tree. A constructal law also emerges: the length defined by the ratio of the square of the airway diameter to its length is constant for all individuals of the same species and is related to the characteristics of the space allocated to the respiratory process and determines univocally thebranching level of the respiratory tree.