Final Technical Report 09 LW 112

Since the development of new antibiotics is out-paced by the emergence of bacterial resistance to existing antibiotics, it is crucial to understand the genetic mechanisms underlying resistance existing antibiotics. At the center of this mystery is a poorly understood phenomenon, heteroresistance: the coexistence of multiple subpopulations with varying degrees of antibiotic resistance. A better understanding of the fundamental basis of heteroresistance could result in sorely needed breakthroughs in treatment options. This project proposed to leverage a novel microfluidic (microchemostat) technology to probe the heteroresistance phenomenon in bacteria, with the aim of restoring the efficacy of existing {beta}-lactam antibiotics. The clinically important bacteria Methicillin Resistant S. aureus (MRSA) was used as the test case of bacteria that exhibits antibiotic heteroresistance. MRSA is difficult to treat because it is resistant to all {beta}-lactam antibiotics, as well as other classes of antimicrobials. Whereas {beta}-lactams such as methicillin and oxacillin are the preferred antibiotics to treat S. aureus infections due to their efficacy and low side effects, accurate determination and use of oxacillin/methicillin dosage is hampered by heteroresistance. In fact, invasive MRSA infections now account for about 95,000 deaths per year, a number that exceeds the deaths due to either influenza or HIV (12). In some MRSA strains, two subpopulations of cells may coexist: both populations carry the mecA gene that confers resistance, but mecA is differentially expressed so that only a small number of cells are observed during in vitro testing. Why this occurs is not understood. Prior experiments have sought to explain this phenomenon with conflicting results, with technology being the primary barrier to test the system sufficiently. This is the final report on work accomplished under the Lab-wide LDRD project 09-LW-112. This project was awarded to Frederick Balagadde who has left LLNL for a position at Stanford University. This report is prepared by Raymond Lenhoff who assumed the role of PI on the project for the remaining two months in August of 2010. The project accomplished most of its original objectives despite the fact that numerous biosafety related approvals not envisioned in the original proposal had to be obtained. In addition, the original PI left prior to the last two months of the project. A microfluidic device capable of the culture and optical data collection on microcultures of S. aureus was developed. A simpler chip design was developed and produced. New chip-interface and optical-analysis software was written and tested. S. aureus was successfully cultured and preliminary data (fluorescence and bright field) was collected. The project has provided valuable expertise in microfluidic culture that can be leveraged for host pathogen interaction studies and has been used in a new $9M DARPA proposal which is now being written for submission by Jan 4, 2011

LLNL Genomic Assessment: Viral and Bacterial Sequencing Needs for TMTI, Task 1.4.2 Report

Good progress has been made on both bacterial and viral sequencing by the TMTI centers. While access to appropriate samples is a limiting factor to throughput, excellent progress has been made with respect to getting agreements in place with key sources of relevant materials. Sharing of sequenced genomes funded by TMTI has been extremely limited to date. The April 2010 exercise should force a resolution to this, but additional managerial pressures may be needed to ensure that rapid sharing of TMTI-funded sequencing occurs, regardless of collaborator constraints concerning ultimate publication(s). Policies to permit TMTI-internal rapid sharing of sequenced genomes should be written into all TMTI agreements with collaborators now being negotiated. TMTI needs to establish a Web-based system for tracking samples destined for sequencing. This includes metadata on sample origins and contributor, information on sample shipment/receipt, prioritization by TMTI, assignment to one or more sequencing centers (including possible TMTI-sponsored sequencing at a contributor site), and status history of the sample sequencing effort. While this system could be a component of the AFRL system, it is not part of any current development effort. Policy and standardized procedures are needed to ensure appropriate verification of all TMTI samples prior to the investment in sequencing. PCR, arrays, and classical biochemical tests are examples of potential verification methods. Verification is needed to detect miss-labeled, degraded, mixed or contaminated samples. Regular QC exercises are needed to ensure that the TMTI-funded centers are meeting all standards for producing quality genomic sequence data

Development and validation of a computational model of the knee joint for the evaluation of surgical treatments for osteoarthritis

A three-dimensional (3D) knee joint computational model was developed and validated to predict knee joint contact forces and pressures for different degrees of malalignment. A 3D computational knee model was created from high-resolution radiological images to emulate passive sagittal rotation (full-extension to 658-flexion) and weight acceptance. A cadaveric knee mounted on a six-degree-of-freedom robot was subjected to matching boundary and loading conditions. A ligamenttuning process minimised kinematic differences between the robotically loaded cadaver specimen and the finite element (FE) model. The model was validated by measured intra-articular force and pressure measurements. Percent full scale error between FE-predicted and in vitro-measured values in the medial and lateral compartments were 6.67% and 5.94%, respectively, for normalised peak pressure values, and 7.56% and 4.48%, respectively, for normalised force values. The knee model can accurately predict normalised intra-articular pressure and forces for different loading conditions and could be further developed for subject-specific surgical planning

Heterologous Replacement of the Supposed Host Determining Region of Avihepadnaviruses: High In Vivo Infectivity Despite Low Infectivity for Hepatocytes

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22–90 (Du-He4) or its subsegments 22–37 and 37–90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 108 viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants

CI2 for creating and comparing confidence-intervals for time-series bivariate plots

Currently no method exists for calculating and comparing the confidence-intervals (CI) for the time-series of a bivariate plot. The study’s aim was to develop ‘CI2′ as a method to calculate the CI on time-series bivariate plots, and to identify if the CI between two bivariate time-series overlap. The test data were the knee and ankle angles from 10 healthy participants running on a motorised standard-treadmill and non-motorised curved-treadmill. For a recommended 10+ trials, CI2 involved calculating 95% confidence-ellipses at each time-point, then taking as the CI the points on the ellipses that were perpendicular to the direction vector between the means of two adjacent time-points. Consecutive pairs of CI created convex quadrilaterals, and any overlap of these quadrilaterals at the same time or ±1 frame as a time-lag calculated using cross-correlations, indicated where the two time-series differed. CI2 showed no group differences between left and right legs on both treadmills, but the same legs between treadmills for all participants showed differences of less knee extension on the curved-treadmill before heel-strike. To improve and standardise the use of CI2 it is recommended to remove outlier time-series, use 95% confidence-ellipses, and scale the ellipse by the fixed Chi-square value as opposed to the sample-size dependent F-value. For practical use, and to aid in standardisation or future development of CI2, Matlab code is provided. CI2 provides an effective method to quantify the CI of bivariate plots, and to explore the differences in CI between two bivariate time-series

Comparison of two normative paediatric gait databases

The availability of age-matched normative data is an essential component of clinical gait analyses. Comparison of normative gait databases is difficult due to the high-dimensionality and temporal nature of the various gait waveforms. The purpose of this study was to provide a method of comparing the sagittal joint angle data between two normative databases. We compared a modern gait database to the historical San Diego database using statistical classifiers developed by Tingley et al. (2002). Gait data were recorded from 60 children aged 1–13 years. A six-camera Vicon 512 motion analysis system and two force plates were utilized to obtain temporal-spatial, kinematic, and kinetic parameters during walking. Differences between the two normative data sets were explored using the classifier index scores, and the mean and covariance structure of the joint angle data from each lab. Significant differences in sagittal angle data between the two databases were identified and attributed to technological advances and data processing techniques (data smoothing, sampling, and joint angle approximations). This work provides a simple method of database comparison using trainable statistical classifiers

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays